method for mass spectrometric quantification of analytes extracted from a microsampling device

专利摘要:
The present invention relates to mass spectrometric methods for determining the amount of analyte in a sample collected by a microsampling device. Provided herein are methods aimed at quantifying the amount of an analyte in a sample by extracting an analyte from a sample collected by a microsampling device, purifying the sample through liquid chromatography, ionizing the analyte to generate one or more detectable ions through mass spectrometry; and determining the amount of the one or more ions by mass spectrometry. The amount of analyte in the sample is related to the amount of analyte in the patient.
公开号:BR112017025097B1
申请号:R112017025097-7
申请日:2016-05-27
公开日:2021-05-11
发明作者:Diana Tran；Scott Goldman；Mildred Goldman；Leslie Edinboro；Julia Addiss；Darren Weber；Porus Mistry；Nigel Clarke
申请人:Quest Diagnostics Investments Llc；
IPC主号:

专利说明:

Cross Reference to Related Orders
[001] The present application claims priority to U.S. Patent Application No. 62/167,164, filed May 27, 2015, which is incorporated herein by reference in its entirety. Background of the Invention
[002] Mass spectrometric quantification of patient analytes requires collection of fluid samples in relatively large amounts. All samples require refrigeration on dry ice or freezing for transport, which is expensive and inconvenient for personnel handling the sample. Also fluid samples can be considered a biohazard that requires a special transport method.
[003] Collection of patient samples using dried blood cards requires less volume than the fluid collection described above. Dried blood species are collected by applying a few drops of blood obtained by piercing the heel or finger and putting it on filter paper, which is then perforated for extraction and analysis. However, dried blood cards present issues and inconsistency and variability in quantification due to the inherent separation of red blood cells and serum that occurs after placing the blood on filter paper. Also, variability in the location of the drill area to be mined and analyzed could significantly affect the quantification results.
[004] A reliable and accurate method for mass spectrometric quantification of analyte is needed. Invention Summary
[005] In one aspect, methods are provided herein for mass spectrometric quantification of analytes collected and extracted from a microsampling device.
[006] In certain embodiments, the methods provided herein are directed to quantifying the amount of an analyte in a sample comprising (a) extracting an analyte from a sample collected by a microsampling device; (b) ionizing the analyte to generate one or more detectable ions by mass spectrometry; and (c) determining the quantity of one or more ions by mass spectrometry. In some embodiments, the amount of the one or more ions determined is used to determine the amount of analyte in the sample. In some embodiments, the amount of analyte in the sample is related to the amount of analyte in the patient.
[007] In certain embodiments, the methods provided herein relate to quantifying the amount of an analyte in a capillary blood sample comprising (a) extracting an analyte from a capillary blood sample collected by a microsampling device; (b) ionizing the analyte to generate one or more detectable ions by mass spectrometry; and (c) determining the amount of the one or more ions by mass spectrometry. In some embodiments, the amount of the one or more ions determined is used to determine the amount of analyte in the sample. In some modalities, the amount of analyte in the sample is related to the amount of analyte in the patient.
[008] In some modalities, capillary blood is collected by a microsampling device. In some modalities, capillary blood is not collected by a smear of dried blood.
[009] In some embodiments, the methods provided herein comprise purification of samples prior to mass spectrometry. In some embodiments, the methods comprise purification of samples using liquid chromatography. In some embodiments, liquid chromatography comprises high performance liquid chromatography (HPLC) or high turbulence liquid chromatography (HTLC). In some embodiments, the methods comprise subjecting a sample to solid phase extraction (SPE). In some embodiments, the methods comprise submitting a sample to the reversed-phase analytical column.
[0010] In some embodiments, the methods provided herein are directed to quantifying the amount of an analyte in a sample comprising (a) extracting an analyte from a sample collected by a microsampling device, (b) purifying the sample through chromatography liquid, (c) ionizing the analyte to generate one or more ions detectable by mass spectrometry; and (d) determining the amount of the one or more ions by mass spectrometry. In some embodiments, the amount of the one or more ions determined is used to determine the amount of analyte in the sample. In some embodiments, the amount of analyte in the sample is related to the amount of analyte in the patient.
[0011] In some embodiments, mass spectrometry comprises tandem mass spectrometry. In some embodiments, mass spectrometry is high resolution mass spectrometry. In some embodiments, mass spectrometry is high resolution/high precision mass spectrometry.
[0012] In some embodiments, ionization is through atmospheric pressure chemical ionization (APCI). In some embodiments, ionization is through electrospray ionization (ESI). In some modalities, said ionization is in a positive mode. In some embodiments, said ionization is in negative ion mode.
[0013] In some embodiments, the microsampling device containing the sample is placed in a 96-well plate. In some embodiments, the microsampling device containing the sample is rack-96. In some embodiments, automation places the 96-rack on a 96-well plate. In some modalities, automation is HAMILTON® automation.
[0014] In some embodiments, the methods provided here comprise adding internal standards to the sample. In some modalities, the internal pattern is checked. In some embodiments, the internal pattern is deuterated or isotopically marked. In some embodiments, the internal standard is added with extraction buffer. In some embodiments, the microsampling device is pre-soaked with an internal pattern and dried.
[0015] In some embodiments, the extraction step comprises adding an extraction buffer to the sample collected by a microsampling device. In some embodiments, the extraction step comprises placing the microsampling device containing the sample into a 96-well plate containing an extracting solvent. In some modalities, the extraction step is automatic. In some embodiments, the 96-well plate is vortexed and then the absorbent tips of the microsampling device are removed. In some embodiments, the extraction step comprises drying under nitrogen. In some modalities, the extraction step comprises reconstituting the sample in solution. In some embodiments, reconstitution comprises adding an aqueous or acidic organic solution or both to the sample. In some embodiments, the reconstituted solution is filtered.
[0016] In some embodiments, the methods provided here comprise high-throughput automation of extraction and mass spectrometric analysis of multiple samples at the same time. In some embodiments, the methods provided herein comprise use of an apparatus that allows automation of extraction and mass spectrometric analysis of multiple samples at the same time. In some embodiments, an apparatus that allows automation comprises a microsampling device. In some embodiments, the microsampling device is configured in a high-performance apparatus.
[0017] In some modalities, the extracted sample is injected into a mass spectrometric system. In some modalities, the extracted sample is injected into liquid chromatography. In some embodiments, the extraction and mass spectrometry steps are performed in an online manner to allow automatic sample analysis. In some embodiments, extraction, purification and mass spectrometry steps are performed in an online manner to allow automatic sample analysis.
[0018] In some embodiments, the analyte is underivatized.
[0019] In some embodiments, the sample collected by the microsampling device does not require sample processing.
[0020] In some modalities, the sample collected by the microsampling device is whole blood. In some modalities, the sample collected by the microsampling device is urine. In some modalities, the sample collected by the microsampling device is saliva. In some embodiments, the sample collected by the microsampling device is serum or plasma.
[0021] In some embodiments, the microsampling device comprises an absorbent tip that collects the sample. In some embodiments, the sample collected by the microsampling device absorbs a fixed volume of fluid from the patient. In some modalities, the patient's fluid is capillary blood. In some embodiments, the sample collected by the microsampling device has a volume of less than or equal to 150 µL. In some embodiments, the sample collected by the microsampling device has a volume of less than or equal to 100 µL. In some embodiments, the sample collected by the microsampling device has a volume of less than or equal to 50 µL. In some modalities, the sample collected by the microsampling device has a volume between 5 μL and 150 μL, inclusive. In some modalities, the sample collected by the microsampling device has a volume between 10 μL and 100 μL, inclusive. In some modalities, the sample collected by the microsampling device has a volume of about 10 µL. In some modalities, the sample collected by the microsampling device has a volume of about 15 µL. In some modalities, the sample collected by the microsampling device has a volume of about 20 µL. In some modalities, the sample collected by the microsampling device has a volume of about 30 µL. In some modalities, the sample collected by the microsampling device has a volume of about 50 µL. In some modalities, the sample collected by the microsampling device has a volume of about 100 µL. In some modalities, the sample collected by the microsampling device absorbs a fixed volume of blood, regardless of the amount of hematocrit.
[0022] In certain embodiments, the methods provided here are directed towards quantifying the amount of an analyte in a low volume of sample. In some embodiments, the methods provided herein are directed to quantifying the amount of an analyte in a sample comprising (a) extracting an analyte from a sample of less than or equal to 100 µL; (b) ionizing the analyte to generate one or more ions detectable by mass spectrometry; and (c) determining the quantity of one or more ions by mass spectrometry. In some embodiments, the amount of the one or more ions determined is used to determine the amount of analyte in the sample. In some modalities, the amount of analyte in the sample is related to the amount of analyte in the patient.
[0023] In some embodiments, the sample is a capillary blood sample. In some modalities, the sample is not a venous blood sample.
[0024] In some embodiments, the methods provided here are directed to quantifying the amount of an analyte in a low volume capillary blood sample. In some embodiments, the methods provided herein are directed to quantifying the amount of an analyte in a sample comprising (a) extracting an analyte from a capillary blood sample of less than or equal to 100 µL; (b) sample purification by liquid chromatography; (c) ionizing the analyte to generate one or more ions detectable by mass spectrometry; and (d) determining the amount of the one or more ions by mass spectrometry. In some embodiments, the amount of the one or more ions determined is used to determine the amount of analyte in the capillary blood sample. In some modalities, the amount of analyte in the sample is related to the amount of analyte in the patient.
[0025] In some embodiments, the methods comprise extracting an analyte from a sample of less than or equal to 50 µL. In some embodiments, the methods comprise extracting an analyte from a sample of less than or equal to 30 µL. In some embodiments, the methods comprise extracting an analyte from a sample of less than or equal to 20 µL. In some embodiments, the methods comprise extracting an analyte from a sample of less than or equal to 15 µL. In some embodiments, the methods comprise extracting an analyte from a sample of less than or equal to 10 µL.
[0026] In some modalities, the sample collected by the microsampling device can be transported without refrigeration or freezing. In some modalities, the sample collected by the microsampling device can be transported without dry ice. In some modalities, the sample collected by the microsampling device can be transported at room temperature. In some modalities, the sample collected by the microsampling device can be transported without biohazard issues.
[0027] In some modalities, the sample collected by the microsampling device requires little training for collection. In some modalities, the sample collected by the microsampling device can be collected anywhere. In some modalities, the sample collected by the microsampling device can be dried at room temperature for transport.
[0028] In some embodiments, the microsampling device comprises apparatus that allows automation of extraction and mass spectrometric analysis. In some embodiments, the microsampling device comprises apparatus that allows high-throughput automation of extraction and mass spectrophotometric analysis of multiple samples at the same time. In some embodiments, the microsampling device is a MITRA® tip. In some embodiments, the microsampling device is enclosed in a cartridge designed for automation of mass spectrometric analysis and extraction.
[0029] In some modalities, the methods further comprise sample collection with a microsampling device. In some embodiments, the collection step comprises performing a finger prick and applying an absorbent tip of the microsampling device to the blood. In some modalities, the collection step comprises applying an absorbent tip to the patient's urine or saliva. In some embodiments, the sample collected in the microsampling device is air dried. In some modalities, the sample collected in the microsampling device is air dried for 1 to 2 hours before transport.
[0030] In some embodiments, the analyte is a steroid. In some embodiments, the steroid is cortisol, cortisone, progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, dehydroepiandrosterone, corticosterone, deoxycorticosterone, 11-desoxycortisol, pregnenolone, 17-hydroxypregnenolone, 18-hydroxycorticosterone, or 21-hydroxycorticosterone. In some modalities, the analyte is a steroid on a steroid panel for diagnosing congenital adrenal hyperplasia (CAH). In some embodiments, the steroid is selected from the group consisting of cortisol, cortisone, progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, dehydroepiandrosterone, corticosterone, deoxycorticosterone, 11-desoxycortisol, pregnenolone, 17-hydroxypregnenodeoxycorthione, and 21-hydroxysolcorthione. In some embodiments, the steroid is 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3.
[0031] In some embodiments, one or more ions comprise a cortisone precursor ion with a mass to charge (m/z) ratio of 361.4 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 121.2 ± 0.5 or 163.2 ± 0.5. In some embodiments, one or more ions comprise a cortisol precursor ion with a mass to charge (m/z) ratio of 363.4 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 121.1 ± 0.5 or 267.2 ± 0.5. In some embodiments, one or more ions comprise a 21-deoxycortisol precursor ion with a mass to charge (m/z) ratio of 347.3 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 121.1 ± 0.5 or 269.2 ± 0.5. In some embodiments, one or more ions comprise a corticosterone precursor ion with a mass to charge (m/z) ratio of 347.4 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 121.1 ± 0.5 or 311.3 ± 0.5. In some embodiments, one or more ions comprise an 11-deoxycortisol precursor ion with a mass to charge (m/z) ratio of 347.4 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 97.1 ± 0.5 or 109.1 ± 0.5. In some embodiments, one or more ions comprise an androstenedione precursor ion with a mass to charge (m/z) ratio of 287.4 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 97.1 ± 0.5 or 109.1 ± 0.5. In some embodiments, one or more ions comprise 11-deoxycorticosterone precursor ion with a mass to charge (m/z) ratio of 331.4 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 97.1 ± 0.5 or 109.1 ± 0.5. In some embodiments, one or more ions comprise testosterone precursor ion with a mass to charge (m/z) ratio of 289.4 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 97.1 ± 0.5 or 109.1 ± 0.5. In some embodiments, one or more ions comprise a 17-hydroxyprogesterone precursor ion with a mass to charge (m/z) ratio of 331.4 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 97.1 ± 0.5 or 109.1 ± 0.5. In some embodiments, one or more ions comprise a progesterone precursor ion with a mass to charge (m/z) ratio of 315.3 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 97.1 ± 0.5 or 109.1 ± 0.5. In some embodiments, one or more ions comprise cortisone-d7 precursor ion with a mass to charge (m/z) ratio of 369.4 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 169.2 ± 0.5. In some embodiments, one or more ions comprise cortisol-d4 precursor ion with a mass to charge (m/z) ratio of 367.4 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 121.0 ± 0.5. In some embodiments, one or more ions comprise corticosterone-d4 precursor ion with a mass to charge (m/z) ratio of 351.1 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 121.1 ± 0.5. In some embodiments, one or more ions comprise 11-deoxycortisol-13C3 precursor ion with a mass to charge (m/z) ratio of 350.4 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 100.1 ± 0.5. In some embodiments, one or more ions comprise a 13C3-androstenedione precursor ion with a mass to charge (m/z) ratio of 290.4 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 100.1 ± 0.5. In some embodiments, one or more ions comprise testosterone-13C3 precursor ion with a mass to charge (m/z) ratio of 292.4 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 112.1 ± 0.5. In some embodiments, one or more ions comprise a 17-hydroxyprogesterone-13C3 precursor ion with a mass to charge (m/z) ratio of 334.3 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 100.0 ± 0.5. In some embodiments, one or more ions comprise a progesterone-13C3 precursor ion with a mass to charge (m/z) ratio of 318.5 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 100.1 ± 0.5.
[0032] In some modalities, the analyte is an opiate. In some modalities, the opiate is cis-tramadol, O-desmethyl tramadol, tapentadol, N-desmethyltapentadol, codeine, morphine, oxymorphone, noridrocodone, oxycodone, noroxicodone, hydromorphone, hydrocodone, buprenorphine, norbuprenorphine, fentanyl, monoacetylmorphine, 6 6-MAM), methadone, dihydrocodeine, naloxone, naltrexone, 6β-naltrexol, nalorphine, nalbuphine or 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). In some embodiments, the opiate is selected from the group consisting of cis-tramadol, O-desmethyl tramadol, tapentadol, N-desmethyltapentadol, codeine, morphine, oxymorphone, norhydrocodone, oxycodone, noroxycodone, hydromorphone, hydrocodone, buprenorphine, norbuprenorphine, norhydrocodone, fentanyl , 6-monoacetylmorphine (6-MAM), methadone, dihydrocodeine, naloxone, naltrexon, 6β-naltrexol, nalorphine, nalbuphine and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). In some modalities, opiate is extracted from a whole blood sample, saliva, or urine.
[0033] In some embodiments, the analyte is benzodiazepine. In some embodiments, the benzodiazepine is oxazepam, temazepam, lorazepam, nordiazepam, diazepam, chlordiazepoxide, triazolam, midazolam, alprazolam, clonazepam, bromazepam, clobazam, nitrazepam, fenazepam, flunitepam, or flunitepam, medazepam. In some embodiments, the benzodiazepine is selected from the group consisting of oxazepam, temazepam, lorazepam, nordiazepam, diazepam, chlordiazepoxide, triazolam, midazolam, alprazolam, clonazepam, bromazepam, clobazam, nitrazepam, flunazepam, flunitazepam, fenazepam, and merazepam, prazepam. In some modalities, the benzodiazepine is extracted from a whole blood or urine sample.
[0034] In some embodiments, one or more ions comprise a bromazepam precursor ion with a mass to charge (m/z) ratio of 316 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 214 ± 0.5 or 270 ± 0.5. In some embodiments, one or more ions comprise an oxazepam precursor ion with a mass to charge (m/z) ratio of 287 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 104 ± 0.5 or 241 ± 0.5. In some embodiments, one or more ions comprise a clobazam precursor ion with a mass to charge (m/z) ratio of 300 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 224 ± 0.5 or 259 ± 0.5. In some embodiments, one or more ions comprise nitrazepam precursor ion with a mass to charge (m/z) ratio of 282 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 180 ± 0.5 or 236 ± 0.5. In some embodiments, one or more ions comprise an alprazolam precursor ion with a mass to charge (m/z) ratio of 309.1 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 165 ± 0.5 or 280.9 ± 0.5. In some embodiments, one or more ions comprise a triazolam precursor ion with a mass to charge (m/z) ratio of 343 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 206 ± 0.5 or 308 ± 0.5. In some embodiments, one or more ions comprise a clonazepam precursor ion with a mass to charge (m/z) ratio of 316 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 214 ± 0.5 or 270 ± 0.5. In some embodiments, one or more ions comprise a flurazepam precursor ion with a mass to charge (m/z) ratio of 388 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 287.9 ± 0.5 or 315 ± 0.5. In some embodiments, one or more ions comprise a precursor ion of lorazepam with a mass to charge (m/z) ratio of 321 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 229.1 ± 0.5 or 331 ± 0.5. In some embodiments, one or more ions comprise a flunitrazepam precursor ion with a mass to charge (m/z) ratio of 314 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 211 ± 0.5 or 268 ± 0.5. In some embodiments, one or more ions comprise a temazepam precursor ion with a mass to charge (m/z) ratio of 301.1 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 177 ± 0.5 or 255 ± 0.5. In some embodiments, one or more ions comprise a midazolam precursor ion with a mass to charge (m/z) ratio of 326 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 129 ± 0.5 or 244 ± 0.5. In some embodiments, one or more ions comprise a nordiazepam precursor ion with a mass to charge (m/z) ratio of 271 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 139.8 ± 0.5 or 165 ± 0.5. In some embodiments, one or more ions comprise a fenazepam precursor ion with a mass to charge (m/z) ratio of 351 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 185.9 ± 0.5 or 206 ± 0.5. In some embodiments, one or more ions comprise a chlordiazepam precursor ion with a mass to charge (m/z) ratio of 301 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 259 ± 0.5 or 224 ± 0.5. In some embodiments, one or more ions comprise a diazepam precursor ion with a mass to charge (m/z) ratio of 285 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 154 ± 0.5 or 193 ± 0.5. In some embodiments, one or more ions comprise a prazepam precursor ion with a mass to charge (m/z) ratio of 325 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 165 ± 0.5 or 271 ± 0.5. In some embodiments, one or more ions comprise a precursor medazepam ion with a mass to charge (m/z) ratio of 271 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 180 ± 0.5 or 207.1 ± 0.5.
[0035] In some embodiments, the analyte is an antiepileptic drug. In some modalities, the antiepileptic drug is valproic acid, tiagabine, topiramate, levitiracetum, lamotrigine, lacosamide, Ethosuximide, carbamazepine, eslicarbamazepine, 10,11- carbamazepine, phenobarbital, rufinamide, primidone, phenytoin, pentabaline, gabaline, zonisamide. In some embodiments, the antiepileptic drug is selected from the group consisting of valproic acid, tiagabine, topiramate, levitiracetum, lamotrigine, lacosamide, Ethosuximide, carbamazepine, eslicarbamazepine, 10,11-carbamazepine, phenobarbital, rufinamide, primidone, felytoin gabapentin and pregablin. In some embodiments, the antiepileptic drug is extracted from a whole blood sample.
[0036] In some embodiments, one or more ions comprise a felbamate precursor ion with a mass to charge (m/z) ratio of 339 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 117.3 ± 0.5 or 261 ± 0.5. In some embodiments, one or more ions comprise a felbamate precursor ion with a mass to charge (m/z) ratio of 117 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 115 ± 0.5 or 91 ± 0.5. In some embodiments, one or more ions comprise an Ethosuximide precursor ion with a mass to charge (m/z) ratio of 142 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 44.3 ± 0.5 or 39.3 ± 0.5. In some embodiments, one or more ions comprise a lacosamide precursor ion with a mass to charge (m/z) ratio of 251 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 91.2 ± 0.5 or 65.2 ± 0.5. In some embodiments, one or more ions comprise a precursor ion of lamotrigine with a mass to charge (m/z) ratio of 256 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 211 ± 0.5 or 145 ± 0.5. In some embodiments, one or more ions comprise a topiramate precursor ion with a mass to charge (m/z) ratio of 338.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 78.2 ± 0.5 or 96.2 ± 0.5. In some embodiments, one or more ions comprise a gabapentin precursor ion with a mass to charge (m/z) ratio of 172.3 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 91.2 ± 0.5 or 67.2 ± 0.5. In some embodiments, one or more ions comprise an eslicarbazepine precursor ion with a mass to charge (m/z) ratio of 297.1 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 194 ± 0.5 or 179 ± 0.5. In some embodiments, one or more ions comprise a primidone precursor ion with a mass to charge (m/z) ratio of 219.8 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 79 ± 0.5 or 135.2 ± 0.5. In some embodiments, one or more ions comprise a pregabalin precursor ion with a mass to charge (m/z) ratio of 160.1 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 55.2 ± 0.5 or 77.2 ± 0.5. In some embodiments, one or more ions comprise a carbamazepine precursor ion with a mass to charge (m/z) ratio of 237 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 194.1 ± 0.5 or 179 ± 0.5. In some embodiments, one or more ions comprise a phenobarbital precursor ion with a mass to charge (m/z) ratio of 231 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 44.2 ± 0.5 or 188.1 ± 0.5. In some embodiments, one or more ions comprise an epoxide precursor ion with a mass to charge (m/z) ratio of 236.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 141.2 ± 0.5 or 112.2 ± 0.5. In some embodiments, one or more ions comprise a zonisamide precursor ion with a mass to charge (m/z) ratio of 213.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 77.2 ± 0.5 or 102.1 ± 0.5. In some embodiments, one or more ions comprise a tiagabine precursor ion with a mass to charge (m/z) ratio of 376.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 111.1 ± 0.5 or 149.1 ± 0.5. In some embodiments, one or more ions comprise a phenytoin precursor ion with a mass to charge (m/z) ratio of 253.1 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 104.2 ± 0.5 or 182.2 ± 0.5. In some embodiments, one or more ions comprise a levetiracetam precursor ion with a mass to charge (m/z) ratio of 171.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 126.2 ± 0.5 or 69.2 ± 0.5. In some embodiments, one or more ions comprise a valproic acid precursor ion with a mass to charge (m/z) ratio of 143 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 143 ± 0.5. In some embodiments, one or more ions comprise a rufinamide precursor ion with a mass to charge (m/z) ratio of 239 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 127.2 ± 0.5 or 261 ± 0.5. In some embodiments, one or more ions comprise a primdone precursor ion with a mass to charge (m/z) ratio of 219 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 126 ± 0.5 or 141 ± 0.5. In some embodiments, one or more ions comprise a topiramate D12 precursor ion with a mass to charge (m/z) ratio of 350 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 78.2 ± 0.5. In some embodiments, one or more ions comprise a D3 epoxide precursor ion with a mass to charge (m/z) ratio of 256 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 77 ± 0.5. In some embodiments, one or more ions comprise a 13C3 lamotrigine precursor ion with a mass to charge (m/z) ratio of 259 ± 0.5. In some embodiments, the one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 214 ± 0.5. In some embodiments, one or more ions comprise a levetiracetam D6 precursor ion with a mass to charge (m/z) ratio of 177.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 132.2 ± 0.5.
[0037] In some embodiments, the analyte is an immunosuppressant. In some modalities, the immunosuppressant is cyclosporin A, sirolimus, tacrolimus and everolimus. In some embodiments, the immunosuppressant is selected from the group consisting of cyclosporine A, sirolimus, tacrolimus, and everolimus. In some modalities, the immunosuppressant is extracted from a whole blood sample.
[0038] In some modalities, the analyte is a bartiburate. In some embodiments, the barbiturate is phenobarbitol, amobarbitol, butalbital, pentobarbitol, secobarbitol, or thiopental. In some embodiments, the barbiturate is selected from the group consisting of phenobarbitol, amobarbitol, butalbital, pentobarbitol, secobarbitol, and thiopental. In some modalities, the barbiturate is taken from a whole blood sample.
[0039] In some embodiments, one or more ions comprise a secobarbital precursor ion with a mass to charge (m/z) ratio of 237.0 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 42.0 ± 0.5. In some embodiments, one or more ions comprise an amobarbital precursor ion with a mass to charge (m/z) ratio of 225.0 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 182.0 ± 0.5. In some embodiments, one or more ions comprise a pentobarbital precursor ion with a mass to charge (m/z) ratio of 225.6 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 42.0 ± 0.5. In some embodiments, one or more ions comprise a thiopental precursor ion with a mass to charge (m/z) ratio of 241.0 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 57.9 ± 0.5. In some embodiments, one or more ions comprise a phenobarbital precursor ion with a mass to charge (m/z) ratio of 231.0 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 42.0 ± 0.5. In some embodiments, one or more ions comprise a butalbital precursor ion with a mass to charge (m/z) ratio of 223.1 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 42.1 ± 0.5.
[0040] In some embodiments, the analyte is tamoxifen. In some embodiments, the analyte is a metabolite of tamoxifen. In some embodiments, said metabolite is norendoxifene. In some embodiments, said metabolite is endoxifene or N-Desmethyl-4-Hydroxy Tamoxifen. In some embodiments, said metabolite is 4'-Hydroxy Tamoxifen. In some embodiments, said metabolite is 4-Hydroxy Tamoxifen. In some embodiments, said metabolite is N-Desmethyl-4'-Hydroxy Tamoxifen. In some embodiments, said metabolite is N-Desmethyl Tamoxifen. In some embodiments, said metabolite is selected from the group consisting of norendoxifene, endoxifene, 4'-Hydroxy Tamoxifen, 4-Hydroxy Tamoxifen, N-Desmetol-4'-Hydroxy Tamoxifen, and N-Desmethyl-4'-Hydroxy Tamoxifen. In some embodiments, tamoxifen or its metabolite is extracted from a whole blood sample.
[0041] In some embodiments, one or more ions comprise a tamoxifen precursor ion with a mass to charge (m/z) ratio of 372.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 72.14 ± 0.5. In some embodiments, one or more ions comprise an endoxifen precursor ion with a mass to charge (m/z) ratio of 374.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 58.1 ± 0.5. In some embodiments, the one or more ions comprise a 4-hydroxy tamoxifen precursor ion with a mass to charge (m/z) ratio of 388.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 72.1 ± 0.5. In some embodiments, one or more ions comprise an N-desmethyl-4'-hydroxy tamoxifen precursor ion with a mass to charge (m/z) ratio of 374.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 58.1 ± 0.5. In some embodiments, one or more ions comprise a 4'-hydroxy tamoxifen precursor ion with a mass to charge (m/z) ratio of 388.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 72.1 ± 0.5. In some embodiments, one or more ions comprise an N-desmethyl-4'-hydroxy tamoxifen precursor ion with a mass to charge (m/z) ratio of 358.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 58.1 ± 0.5.
[0042] In some embodiments, the analyte is a drug for oncology. In some modalities, the analyte is anastrozole. In some embodiments, the analyte is letrozole. In some embodiments, the analyte is exemestane. In some embodiments, the analyte is selected from the group consisting of anastrozole, letrozole and exemestane. In some embodiments, the oncology drug is extracted from a whole blood sample.
[0043] In some embodiments, the analyte is tetrahydrocannabinol (THC) or its metabolite. In some modalities, THC is extracted from a urine sample.
[0044] In some embodiments, the extracted analyte is hydrolyzed. In some embodiments, the analyte is hydrolyzed prior to extraction.
[0045] In some embodiments, the collision energy is within the range of about 5 to 60 V. In some embodiments, the collision energy is within the range of about 5 to 60 V.
[0046] In another aspect, methods for diagnosing congenital adrenal hyperplasia in patients are provided herein. In some embodiments, the endogenous steroid quantification methods provided herein are used for diagnosing congenital adrenal hyperplasia.
[0047] In another aspect, methods for detecting or monitoring the use of THC in an individual are provided herein. In another aspect, methods are provided herein for detecting or monitoring barbiturate use in an individual. In another aspect, methods for detecting or monitoring opiate use in an individual are provided herein. In another aspect, methods for detecting or monitoring benzodiazepine use in an individual are provided herein.
[0048] In another aspect, methods are provided herein for detecting or monitoring antiepileptic drug use in an individual. In another aspect, methods for monitoring the effectiveness of an antiepileptic drug in an individual are provided herein.
[0049] In another aspect, methods are provided herein for detecting or monitoring the use of tamoxifen in an individual. In another aspect, methods are provided herein for monitoring the effectiveness of tamoxifen in an individual.
[0050] In another aspect, certain methods presented here use high resolution/high precision mass spectrometry to determine the amount of analyte in a sample. In some embodiments, using high-precision/high-resolution mass spectrometry, the methods include: (a) subjecting the analyte in a sample to an ionization source under conditions suitable for generating ions, where the ions are detectable through spectrometry. pasta; and (b) determine the quantity of one or more ions by high resolution/high precision mass spectrometry. In these modalities, the amount of one or more ions determined in step (b) is related to the amount of analyte in the sample. In some embodiments, high resolution/high accuracy mass spectrometry is conducted at a FWHM of 10,000 and a mass accuracy of 50 ppm. In some embodiments, high-resolution/high-precision mass spectrometry is conducted with a high-resolution/high-precision time-of-flight (TOF) mass spectrometer. In some embodiments, the ionization conditions comprise analyte ionization under acidic conditions. In some related embodiments, acidic conditions comprise treating said sample with formic acid prior to ionization.
[0051] In any of the methods described here, the sample may comprise a biological sample. In some embodiments, the biological sample can comprise a biological fluid such as urine, plasma or serum. In some embodiments, the biological sample can comprise a sample from a human; such as an adult male or female, or a youth male or female, where the youth is under 18, under 15, under 12, or under 10. The human sample can be analyzed to diagnose or monitor a disease state or condition or to monitor the therapeutic effectiveness of treating a disease state or condition. In some related embodiments, the methods described herein can be used to determine the amount of analyte in a biological sample when obtained from a human.
[0052] In embodiments using tandem mass spectrometry, tandem mass spectrometry can be conducted by any method known in the art, including, for example, multiple reaction monitoring, precursor ion scanning, or product ion scanning.
[0053] In some embodiments, tandem mass spectrometry comprises fragmentation of a precursor ion into one or more fragment ions. In embodiments where the amounts of two or more fragment ions are determined, the amount can be subjected to any mathematical manipulation known in the art to relate the measured ion amounts to the amount of analyte in the sample. For example, the amounts of two or more fragment ions can be added up as part of determining the amount of analyte in the sample.
[0054] In some embodiments, high-resolution/high-precision mass spectrometry is conducted at a power of resolution (FWHM) of greater than or equal to about 10,000, such as greater than or equal to about 15,000, such as more than or equal to about 20,000, such as more than or equal to about 25,000. In some embodiments, high resolution/high precision mass spectrometry is conducted at an accuracy of less than or equal to about 50 ppm, such as less than or equal to about 20 ppm, such as less than or equal to about 10 ppm, such as less than or equal to about 5 ppm; such as less than or equal to about 3 ppm. In some embodiments, high resolution/high precision mass spectrometry is conducted at a resolving power (FWHM) of greater than or equal to about 10,000 and an accuracy of less than or equal to about 50 ppm. In some embodiments, resolving power is greater than about 15,000 and accuracy is less than or equal to about 20 ppm. In some embodiments, resolving power is greater than or equal to about 20,000 and accuracy is less than or equal to about 10 ppm; preferably the resolving power is greater than or equal to about 20,000 and the accuracy is less than or equal to about 5 ppm, such as less than or equal to about 3 ppm.
[0055] In some embodiments, high-resolution/high-precision mass spectrometry can be conducted with an orbitrap mass spectrometer, a time-of-flight (TOF) mass spectrometer, or a transform ion cyclotron resonance mass spectrometer Fourier transform (sometimes known as a Fourier transform mass spectrometer).
[0056] Mass spectrometry (either tandem or high resolution/high precision) can be performed in positive ion mode. Alternatively, mass spectrometry can be performed in negative ion mode. Various sources of ionization, including, for example, atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI), can be used to ionize the analyte.
[0057] In any method presented here, a separately detectable internal standard may be provided in the sample, the amount of which is also determined in the sample. In modalities using a separately detectable internal standard, all or a portion of both the analyte of interest and the internal standard present in the sample is ionized to produce a plurality of detectable ions in a mass spectrometer and one or more ions produced from each are detected by mass spectrometry. In these modalities, the presence or amount of ions generated from the analyte of interest can be related to the presence of the amount of analyte of interest in the sample through comparison with the amount of internal standard ions detected.
[0058] Alternatively, the amount of analyte in a sample can be determined by comparison with one or more external reference standards. Exemplary external reference standards include blank plasma or serum spiked with human or non-human analyte, a synthetic analyte analog, or an isotopically labeled variant thereof.
[0059] The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following detailed description of the invention and the claims. Brief Description of Drawings
[0060] Figure 1 shows chromatogram of 14 steroids analyzed by mass spectrometry.
Figures 2-5 show normal levels of cortisol (Figure 2), cortisone (Figure 3), testosterone (Figure 4), and androstenedione (Figure 5) in a normal adult male as quantified by the present assay.
[0062] Figures 6-10 show normal levels of progesterone (Figure 6), cortisol (Figure 7), cortisone (Figure 8), androstenedione (Figure 9), 17-OH progesterone (Figure 10) in an adult female , quantified by this assay.
Figures 11-17 show levels of cortisol (Figure 11), cortisone (Figure 12), progesterone (Figure 12), androstenedione (Figure 14), testosterone (Figure 15), 21-deoxycortisol (Figure 16) and 17 -OH progesterone (Figure 17) in a child, quantified by the present assay.
[0064] Figure 18 shows standard linearity of testosterone between 50-10,000 ng/dL.
[0065] Figure 19 shows chromatogram of tamoxifen and its metabolites.
[0066] Figure 20 shows chromatogram of letrozole, exemestane and anastrozole.
[0067] Figure 21 shows exemplary opiate chromatograms (oxymorphone, hydromorphone and codeine) and corresponding internal standards.
[0068] Figure 22 shows exemplary opiate chromatograms (noroxycodone, oxycodone and nohydrocodone) and corresponding internal standards.
[0069] Figure 23 shows exemplary opiate chromatograms (morphine, hydrocodone and norfentanil) and corresponding internal standards.
[0070] Figure 24 shows exemplary opiate (fentanyl) chromatogram and corresponding internal pattern.
Figures 25 to 28 show data for morphine, codeine, hydromorphone and oxycodone (respectively) obtained from patient urine using a 20 uL MITRA® tip with glucuronidase hydrolysis.
[0072] Figure 29 shows oxycodone data obtained from patient saline using a 50 uL MITRA® tip.
[0073] Figures 30 and 31 show the results of hematocrit study of buprenorphine and norfentanil, respectively.
Figures 32 and 33 show the results of negative urine spattered with barbiturates (secobarbital, amobarbital, pentobarbital and thiopental).
[0075] Figures 34 and 38 show the results of several patient samples quantified for phenobarbital and butalbital.
[0076] Figure 39 shows the results of THC carboxy metabolite analysis in patient sample using 20 uL tip and glucuronidase hydrolysis.
[0077] Figure 40 shows the results of hematocrit study of gabapentin and rufinamide.
[0078] Figure 41 shows the chromatogram of the 25-hydroxyvitamin D analysis.
[0079] Figure 42 shows the calibration curve of 25-hydroxyvitamin D2 analysis.
[0080] Figure 43 shows the calibration curve of 25-hydroxyvitamin D3 analysis. Detailed Description of the Invention
[0081] As used herein, unless otherwise stated, the singular forms "a", "an" and "o" and "a" include plural reference. Thus, for example, a reference to "a protein" includes a plurality of protein molecules.
[0082] As used herein, the terms "purifying", "purifying" and "enriching" do not refer to the removal of all materials from the sample except the analyte(s) of interest. Rather, these terms refer to a procedure that enriches the amount of one or more analytes of interest relative to other components in the sample that interfere with the detection of the analyte of interest. Sample purification by various means can allow relative reduction of one or more interfering substances, for example, one or more substances that may or may not interfere with the detection of selected parent or offspring ions by mass spectrometry. Relative reduction as this term is used does not require that any substance, present with the analyte of interest in the material to be purified, be completely removed through purification.
[0083] As used herein, the term "immunopurification" or "immunopurify" refers to a purification procedure that uses antibodies, including polyclonal or monoclonal antibodies, to enrich the one or more analytes of interest. Immunopurification can be performed using any of the immunopurification methods well known in the art. Often the immunopurification procedure uses antibodies bound, conjugated or otherwise bound to a solid support, for example, a column, well, tube, gel, capsule, particle or the like. Immunopurification as used herein includes, without limitation, procedures often referred to in the art as immunoprecipitation, as well as procedures often referred to in the art as affinity chromatography or immunoaffinity chromatography.
[0084] As used herein, the term "immunoparticle" refers to a capsule, bead, gel particle or the like which has antibodies attached, conjugated or otherwise attached to its surface (or on and/or on the particle). In certain preferred embodiments, immunoparticles are Sepharose or agarose beads. In alternative preferred embodiments, immunoparticles comprise glass, plastic or silica or silica gel beads.
[0085] As used herein, the term "sample" refers to any sample that may contain an analyte of interest. As used herein, the term "body fluid" means any fluid that can be isolated from an individual's body. For example, "body fluid" can include blood, plasma, serum, bile, saliva, urine, tears, perspiration and the like. In preferred embodiments, the sample comprises a human body fluid sample, preferably plasma or serum.
[0086] As used herein, the term "solid phase extraction" or "SPE" refers to a process in which a chemical mixture is separated into components as a result of the affinity of components dissolved or suspended in a solution (i.e., mobile phase) with a solid through which or around which the solution is passed (ie solid phase). In some cases, as the mobile phase passes through or around the solid phase, undesirable components of the mobile phase can be retained by the solid phase resulting in a purification of the analyte in the mobile phase. In other cases, the analyte may be retained by the solid phase, allowing unwanted mobile phase components to pass through or around the solid phase. In such cases, a second mobile phase is then used to elute the retained analyte out of the solid phase for further processing or analysis. SPE, including TFLC, can operate via a unitary or mixed mode mechanism. Mixed-mode mechanisms utilize ion exchange and hydrophobic retention in the same column, for example, the solid phase of a mixed-mode SPE column may exhibit strong anion exchange and hydrophobic retention, or it may exhibit strong cation exchange and hydrophobic retention.
[0087] In general, the affinity of an SPE column packing material with an analyte can be due to any of a variety of mechanisms, such as one or more chemical interactions or an immunoaffinity interaction. In some embodiments, analyte SPE is conducted without the use of an immunoaffinity column packing material. That is, in some embodiments, analyte is purified from a sample through an SPE column that is not an immunoaffinity column.
[0088] As used herein, the term "chromatography" refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of chemical entities as they flow around or over a phase solid or liquid stationary.
[0089] As used herein, the term "liquid chromatography" or "LC" means a process of selectively delaying one or more components of a fluid solution as the fluid percolates uniformly through a column of a finely divided substance or through passageways capillaries. The delay results in the distribution of mixture components between one or more stationary phases and the raw fluid (ie, mobile phase) as this fluid moves relative to the stationary phase(s). Examples of "liquid chromatography" include reversed phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC) and turbulent flow liquid chromatography (TFLC) (sometimes known as high turbulence liquid chromatography (HTLC) or liquid chromatography high yield).
[0090] As used herein, the term "high performance liquid chromatography) or "HPLC" (sometimes known as "high pressure liquid chromatography") refers to liquid chromatography in which the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase, typically a densely packed column.
[0091] As used herein, the term "turbulent flow liquid chromatography" or "TFLC" (sometimes known as high turbulent liquid chromatography or high performance liquid chromatography) refers to a form of chromatography that utilizes turbulent flow of material being tested by packing the column as the basis for carrying out the separation. TLC has been applied in the preparation of samples containing two unnamed drugs prior to analysis by mass spectrometry. See, for example, Zimmer et al., J Chromatogr A 854: 23-35 (1999); see also U.S. Patent Nos. 5,968,367, 5,919,368, 5,795,469 and 5,772,874, which explain further TFLC. Common versed in the art comprise "turbulent flow". When fluid flows slowly and smoothly, the fluid is called "laminar fluid". For example, fluid moving through an HPLC column at low flow rates is laminar. In laminar flow the movement of fluid particles is orderly with particles generally moving in substantially straight lines. At faster speeds, water inertia overcomes fluid frictional forces and turbulent flow results. Fluid not in contact with the irregular boundary "exits", which is slowed down by friction or deflected by a non-uniform surface. When a fluid is turbulently flowing, it flows in eddies or eddies (or vortices) with more "drag" than when the flow is laminar. Many references are available to assist in determining when fluid flow is laminar or turbulent (eg, Turbulent Flow Analysis: Measurement and Prediction, PS Bernard & JM Wallace, John Wiley & Sons, Inc., (2000); An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott, Cambridge University Press (2001)).
[0092] As used herein, the term "gas chromatography" or "GC" refers to chromatography in which the sample mixture is vaporized and injected into a stream of carrier gas (such as nitrogen or helium) moving through a column containing a stationary phase composed of a liquid or a solid particulate and is separated into its component compounds according to the affinity of the stationary phase compounds.
[0093] As used herein, the term "large particle column" or "extraction column" refers to a chromatography column having an average particle diameter greater than about 50 µm. As used in the present context, the term "about" means ± 10%.
[0094] As used herein, the term "analytical column" refers to a chromatography column having sufficient chromatographic plates to perform a separation of materials in a sample that elutes from the column sufficient to allow a determination of the presence or amount of a analyte. Such columns are often distinguished from "extraction columns", which have the general purpose of separating or extracting retained material from non-retained materials in order to obtain a purified sample for further analysis. As used in the present context, the term "about" means ± 10%. In a preferred embodiment, the analytical column contains particles of about 5 µm in diameter.
[0095] As used herein, the terms "online" and "in line", for example, as used in "automatically online" or "online extraction", refers to a procedure performed without the need operator intervention. In contrast, the term "off-line" as used herein refers to a procedure requiring manual intervention by an operator. In this way, if samples are subjected to precipitation and the supernatants are then manually loaded into an autosampler, the precipitation and loading steps are offline from subsequent steps. In various method modalities, one or more steps can be performed automatically online.
[0096] As used herein, the term "mass spectrometry" or "MS" refers to an analytical technique for identifying compounds by their mass. MS refers to methods of filtering, detecting and measuring ions based on their mass-to-charge or "m/z" ratio. MS technology generally includes (1) ionizing the compounds to form charged compounds and (2) detecting the molecular weight of the charged compounds and calculating a mass-to-charge ratio. Compounds can be ionized and detected by any suitable means. A "mass spectrometer" usually includes an ionizer, a mass analyzer, and an ion detector. In general, one or more molecules of interest are ionized and the ions are subsequently introduced into a mass spectrometric instrument where, due to a combination of magnetic and electric fields, the ions follow a course in space that is mass dependent ("m ") and charge ("z"). See, for example, U.S. Patent Nos. 6,204,500, entitled "Mass Spectrometry From Surfaces;" 6,107,623, entitled "Methods and Apparatus for Tandem Mass Spectrometry;" 6,268,144 entitled "DNA Diagnostics Based On Mass Spectrometry;" 6,124137 entitled "Surface-Enhanced Photolabile Attachment And Release For Desorption And Detection Of Analytes;" Wright et al., Prostate Cancer and Prostatic Diseases 1999, 2:264-76; and Merchant and Weinberger, Electrophoresis 2000, 21: 1164-67.
[0097] As used herein, "high resolution/high precision mass spectrometry" refers to mass spectrometry conducted with a mass analyzer capable of measuring the mass to charge ratio of a charge species with sufficient precision and accuracy to confirm a single chemical ion. Confirmation of a single chemical ion is possible for an ion when individual isotopic peaks of that ion are readily discernible. The particular resolving power and mass accuracy required to confirm a single chemical ion varies with the ion's state of mass and charge.
[0098] As used herein, the term "resolving power" or "resolving power (FWHM)" (also known in the art as "m/Δm50%") refers to a ratio of mass to observed charge divided by the width of the peak mass at maximum height 50% (Maximum Width at Half Height, "FWHM"). The effect of differences in resolution power is illustrated in Figures 1A-C, which show theoretical mass spectra of an ion with an m/z of about 1093. Figure 1A shows a theoretical mass spectrum of a mass analyzer with resolving power of about 3000 (a typical operating condition for a conventional quadrupole mass analyzer). As seen in Figure 1A, any individual isotopic peaks are discernible. By comparison, Figure 1B shows a theoretical mass spectrum of a mass analyzer with a resolving power of about 10,000, with individual isotopic peaks clearly discernible. Figure 1C shows a theoretical mass spectrum of a mass analyzer with a resolving power of about 12,000. At this higher resolving power, individual isotopic peaks contain less than 1% contribution from baseline.
[0099] As used herein, a "single chemical ion" with respect to mass spectrometry refers to a single ion with a single atomic formation. The single ion can be single or multiple charged.
[00100] As used herein, the term "accuracy" (or "mass accuracy") with respect to mass spectrometry refers to the potential deviation of the instrument response from the true m/z of the investigated ion. Accuracy is typically expressed in parts per million (ppm). The effect of differences in mass accuracy is illustrated in Figures 2A-D, which show the limits of potential differences between a detected m/z and the actual m/z for a theoretical peak in m/z of 1093.52094. Figure 2A shows the potential range of m/z detected at an accuracy of 120 ppm. In contrast, Figure 2B shows the potential range of m/z detected at an accuracy of 50 ppm. Figures 2C and 2D show the even narrower power ranges of m/z detected at accuracies of 20 ppm and 10 ppm.
[00101] High resolution/high precision mass spectrometry methods of the present invention can be conducted on instruments capable of performing mass analysis with FWHM of more than 10,000, 15,000, 20,000, 25,000, 50,000, 100,000 or even more. Likewise, methods of the present invention can be conducted on instruments capable of performing mass analysis with accuracy of less than 50 ppm, 20 ppm, 15 ppm, 10 ppm, 5 ppm, 3 ppm or even less. Instruments capable of these performance characteristics may incorporate certain orbitrap mass analyzers, time-of-flight ("TOF") mass analyzers, or Fourier transform ion cyclotron resonance mass analyzers. In preferred embodiments, the methods are performed with an instrument that includes an orbitrap mass analyzer or a TOF mass analyzer.
[00102] The term "orbitrap" describes an ion trap consisting of a barrel-type electrode and an internal coaxial electrode. Ions are injected tangentially into the electric field between the electrodes and trapped because electrostatic interactions between the ions and electrodes are balanced by centrifugal forces as the ions orbit the coaxial inner electrode. Once an ion orbits the coaxial inner electrode, the orbital path of a trapped ion oscillates along the axis of the central electrode at a harmonic frequency in relation to the ion's mass-to-charge ratio. Orbital oscillation frequency detection allows orbitrap to be used as a mass analyzer with high accuracy (minimum of 1-2 ppm) and high resolution power (FWHM) (up to about 200,000). An orbitrap-based mass analyzer is described in detail in Pat. U.S. No. 6,995,364, incorporated herein by reference in its entirety. Use of orbitrap analyzers has been reported for qualitative and quantitative analysis of various analytes. See, for example, U.S. Patent Application Pub. 2008/0118932 (filed November 9, 2007); Bredehoft et al., Rapid Commun. Mass Spectrom., 2008, 22:477-485; Le Breton et al., Rapid Commun. Mass Spectrom., 2008, 22:3130-36; Thevis et al., Mass Spectrom. Reviews, 2008, 27:35-50; Thomas et al., J. Mass Spectrom., 2008, 43:908-15; Schenk et al., BMC Medical Genomics, 2008, 1:41; and Olsen et al., Nature Methods, 2007, 4:709-12.
[00103] As used herein, the term "operating in negative ion mode" refers to those methods of mass spectrometry in which negative ions are generated and detected. The term "operating in positive ion mode", as used herein, refers to those methods of mass spectrometry in which positive ions are generated and detected. In preferred embodiments, mass spectrometry is conducted in positive ion mode.
[00104] As used herein, the term "ionizing" or "ionizing" refers to the process of generating an analyte ion having a net electrical charge equal to one or more electron units. Negative ions are those having a net negative charge of one or more electron units, while positive ions are those having a net positive charge of one or more electron units.
[00105] As used herein, the term "electron ionization" or "EI" refers to methods in which an analyte of interest in a gaseous or vapor phase interacts with a stream of electrons. Impact of electrons with the analyte produces analyte ions, which can then be subjected to a mass spectrometry technique.
[00106] As used herein, the term "chemical ionization" or "CI" refers to methods in which a reactant gas (eg ammonia) is subjected to electron impact and analyte ions are formed by the interaction of gas ions reagent and analyte molecules.
[00107] As used herein, the term "fast atom bombardment" or "FAB" refers to methods in which a beam of high energy atoms (often Xe or Ar) impacts a non-volatile sample, contained desorption and ionization molecules in the sample. Test samples are dissolved in a viscous liquid matrix such as glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-16-crown ether, 2-nitrophenyloctyl ether, sulfolane, diethanolamine and triethanolamine. Choosing an appropriate matrix for a compound or sample is an empirical process.
[00108] As used herein, the term "matrix-assisted laser desorption ionization" or "MALDI" refers to methods in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample by multiple ionization pathways, including photoionization, protonation, deprotonation, and cluster deterioration. For MALDI, the sample is mixed with an energy absorbing matrix, which facilitates the desorption of analyte molecules.
[00109] As used herein, the term "surface augmented laser desorption ionization" or "SELDI" refers to another method in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample through of various courses of ionization, including photoionization, protonation, deprotonation, and cluster decay. For SELDI, the sample is typically bound to a surface that preferably retains one or more analytes of interest. As with MALDI, this process can also employ an energy absorbing material to facilitate ionization.
[00110] As used herein, the term "electrospray ionization" or "ESI" refers to methods in which a solution is passed along a short course of capillary tube, at the end of which a positive or negative electrical potential is applied. . Solution reaching the end of the tube is vaporized (nebulized) into a jet or spray of very small droplets of solvent vapor solution. This mist of droplets flows through an evaporation chamber. As the droplets get smaller the electric surface charge density increases until such a moment that the natural repulsion between similar charges causes ions as well as neutral molecules to be released.
[00111] As used herein, the term "atmospheric pressure chemical ionization" or "APCI" refers to methods of mass spectrometry that are similar to ESI; however, APCI produces ions through ion molecule reactions that take place within a plasma at atmospheric pressure. The plasma is maintained by an electrical discharge between the spray capillary and a counter electrode. Then ions are typically extracted into the mass analyzer through the use of a set of differentially pumped skimmer stages. A preheated dry N2 gas counterflow can be used to improve solvent removal. Gas phase ionization in APCI may be more effective than ESI for analyzing less polar species.
[00112] The term "atmospheric pressure photoionization" or "APPI" as used herein refers to the form of mass spectrometry in which the mechanism for the ionization of molecule M is photon absorption and electron ejection to form the molecular ion M+ . Because the photon energy is typically slightly above the ionization potential, the molecular ion is less susceptible to dissociation. In many cases it may be possible to analyze samples without the need for chromatography, thus saving significant time and expense. In the presence of water vapor or protic solvents, the molecular ion can extract H to form MH+. This tends to occur if M has a high proton affinity. This does not affect the quantitation accuracy because the sum of M+ and MH+ is constant. Drug compounds in protic solvents are generally seen as MH+, while non-polar compounds such as naphthalene or testosterone generally form M+. See, for example, Robb et al., Anal. Chem. 2000, 72(15): 3653-3659.
[00113] As used herein, the term "inductively coupled plasma" or "ICP" refers to methods in which a sample interacts with a partially ionized gas at a sufficiently high temperature so that most elements are atomized and ionized.
[00114] As used herein, the term "field desorption" refers to methods in which a non-volatile test sample is placed on an ionizing surface and a strong electric field is used to generate analyte ions.
[00115] As used herein, the term "desorption" refers to the removal of an analyte from a surface and/or the entry of an analyte into a gas phase. Laser desorption Thermal desorption is a technique in which a sample containing the analyte is thermally desorbed in the gas phase by a laser pulse. The laser hits the back of a specially made 96-well plate with a metal base. The laser pulse hits the base and the heat causes the sample to transfer into the gas phase. The gas phase sample is then taken to the mass spectrometer.
[00116] As used herein, the term "selective ion monitoring" is a detection mode for a mass spectrometric instrument in which only ions within a relatively narrow mass range, typically about one mass unit, are detected.
[00117] As used herein, "multiple reaction mode", sometimes known as "selected reaction monitoring", is a detection mode for a mass spectrometric instrument in which a precursor ion and one or more fragment ions are selectively detected.
[00118] As used herein, the term "lower limit of quantification", "lower limit of quantification" or "LLOQ" refers to the point at which measurements become quantitatively significant. The analyte response in this LOQ is identifiable, distinct and reproducible with a relative standard deviation (%RSD) of less than 20% and an accuracy of 85% to 115%.
[00119] As used herein, the term "detection limit" or "LOD" is the point at which the measured value is greater than the uncertainty associated with it. LOD is the point at which a value is beyond the uncertainty associated with its measurement and is defined as three times the RSD of the mean at zero concentration.
[00120] As used herein, an "amount" of an analyte in a body fluid sample generally refers to an absolute value reflecting the mass of the detectable analyte in sample volume. However, an amount also comprises a relative amount compared to another amount of analyte. For example, an amount of an analyte in a sample may be an amount that is greater than a control or normal level of analyte normally present in the sample.
[00121] The term "about" as used herein in reference to quantitative measurements not including the measurement of the mass of an ion refers to the indicated value plus or minus 10%. Mass spectrometry instruments may vary slightly in determining the mass of a given analyte. The term "about" in the context of an ion's mass or an ion's mass/charge ratio refers to +/- 0.50 atomic mass units.
[00122] Collection of venous blood from the newborn can be problematic. Although the minimum serum volume required for the comparative steroid panel (or CAH panel) is minimal, at least 1-2 mL of whole blood is acquired through venipuncture. Use of a microsampling device (Mitra tip) requires only 20 uL of capillary blood and makes it easier and less invasive, especially for neonates, which eliminates the need to perform venipuncture.
[00123] In one aspect, methods are provided herein for mass spectrometric quantification of analytes collected and extracted from a microsampling device.
[00124] In certain embodiments, the methods provided herein are directed to quantifying the amount of an analyte in a sample comprising (a) extracting an analyte from a sample collected by a microsampling device; (b) ionizing the analyte to generate one or more ions detectable by mass spectrometry; and (c) determining the quantity of one or more ions by mass spectrometry. In some embodiments, the amount of the one or more ions determined is used to determine the amount of analyte in the sample. In some embodiments, the amount of analyte in the sample is related to the amount of analyte in the patient.
[00125] In some embodiments, the methods provided here comprise purification of samples prior to mass spectrometry. In some embodiments, the methods comprise purification of samples using liquid chromatography. In some embodiments, liquid chromatography comprises high performance liquid chromatography (HPLC) or high turbulence liquid chromatography (HTLC). In some embodiments, the methods comprise subjecting a sample to solid phase extraction (SPE). In some embodiments, the methods comprise submitting a sample to the reversed-phase analytical column.
[00126] In some embodiments, the methods provided herein are directed to quantifying the amount of an analyte in a sample comprising (a) extracting an analyte from a sample collected by a microsampling device, (b) purifying the sample through liquid chromatography; (c) ionizing the analyte to generate one or more ions detectable by mass spectrometry; and (d) determining the amount of the one or more ions by mass spectrometry. In some embodiments, the amount of the one or more ions determined is used to determine the amount of analyte in the sample. In some embodiments, the amount of analyte in the sample is related to the amount of analyte in the patient.
[00127] In some embodiments, mass spectrometry comprises tandem mass spectrometry. In some embodiments, mass spectrometry is high resolution mass spectrometry. In some embodiments, mass spectrometry is high resolution/high accuracy mass spectrometry.
[00128] In some embodiments, ionization is through chemical ionization under atmospheric pressure (APCI). In some embodiments, ionization is through electrospray ionization (ESI). In some embodiments, said ionization is in positive ion mode. In some embodiments, said ionization is in negative ion mode.
[00129] In some embodiments, the microsampling device containing the sample is placed in a 96-well plate. In some embodiments, the microsampling device containing the sample is placed on a 96-rack. In some embodiments, automation puts the 96-rack into a 96-well plate. In some modalities, automation is HAMILTON® automation.
[00130] In some embodiments, the methods provided here comprise adding internal standards to the sample. In some modalities, the internal pattern is checked. In some embodiments, the internal pattern is deuterated or isotopically marked. In some embodiments, the internal standard is added with extraction buffer. In some embodiments, the microsampling device is pre-soaked with an internal pattern and dried.
[00131] In some embodiments, the extraction step comprises adding an extraction buffer to the sample collected by a microsampling device. In some embodiments, the extraction step comprises placing the microsampling device containing the sample into a 96-well plate containing an extracting solvent. In some modalities, the extraction step is automatic. In some embodiments, the 96-well plate is vortexed and then the absorbent tips of the microsampling device are removed. In some embodiments, the extraction step comprises drying in nitrogen. In some modalities, the extraction step comprises reconstituting the sample in solution. In some embodiments, reconstitution comprises adding an aqueous or acidic organic solution or both to the sample. In some embodiments, the reconstituted solution is filtered.
[00132] In some modalities, the extracted sample is injected into a mass spectrometric system. In some modalities, the extracted sample is injected into liquid chromatography. In some embodiments, mass spectrometry extraction steps are performed in an online manner to allow automatic sample analysis. In some embodiments, extraction, purification and mass spectrometry steps are performed in an online manner to allow automatic sample analysis.
[00133] In some embodiments, the analyte is underivatized.
[00134] In some embodiments, the sample collected by the microsampling device does not require sample processing.
[00135] In some modalities, the sample collected by the microsampling device is whole blood. In some modalities, the sample collected by the microsampling device is urine. In some modalities, the sample collected by the microsampling device is saliva. In some embodiments, the sample collected by the microsampling device is serum or plasma.
[00136] In some embodiments, the microsampling device comprises an absorbent tip that collects the sample. In some embodiments, the sample collected by the microsampling device absorbs a fixed volume of fluid from the patient. In some embodiments, the sample collected by the microsampling device has a volume of less than or equal to 150 µL. In some embodiments, the sample collected by the microsampling device has a volume of less than or equal to 100 µL. In some embodiments, the sample collected by the microsampling device has a volume of less than or equal to 50 µL. In some modalities, the sample collected by the microsampling device has a volume between 5 μL and 150 μL, inclusive. In some modalities, the sample collected by the microsampling device has a volume between 10 μL and 100 μL, inclusive. In some modalities, the sample collected by the microsampling device has a volume of about 10 µL. In some modalities, the sample collected by the microsampling device has a volume of about 15 µL. In some modalities, the sample collected by the microsampling device has a volume of about 20 µL. In some modalities, the sample collected by the microsampling device has a volume of about 30 µL. In some modalities, the sample collected by the microsampling device has a volume of about 50 µL. In some modalities, the sample collected by the microsampling device has a volume of about 100 µL. In some modalities, the sample collected by the microsampling device absorbs a fixed volume of blood, regardless of the amount of hematocrit.
[00137] In some embodiments, the methods provided herein are directed to quantifying the amount of an analyte in a sample comprising (a) extracting an analyte from a sample of less than or equal to 100 µL; (b) ionizing the analyte to generate one or more detectable ions by mass spectrometry; and (c) determining the amount of the one or more ions by mass spectrometry. In some embodiments, the amount of the one or more ions determined is used to determine the amount of analyte in the sample. In some modalities, the amount of analyte in the sample is related to the amount of analyte in the patient.
[00138] In some embodiments, the methods provided herein are directed to quantifying the amount of an analyte in a sample comprising (a) extracting an analyte from a sample of less than or equal to 100 µL; (b) sample purification by liquid chromatography; (c) ionizing the analyte to generate one or more ions detectable by mass spectrometry; and (d) determining the amount of the one or more ions by mass spectrometry. In some embodiments, the amount of the one or more ions determined is used to determine the amount of analyte in the sample. In some embodiments, the amount of analyte in the sample is related to the amount of analyte in the patient.
[00139] In some embodiments, the methods comprise extracting an analyte from a sample of less than or equal to 50 μL. In some embodiments, the methods comprise extracting an analyte from a sample of less than or equal to 30 µL. In some embodiments, the methods comprise extracting an analyte from a sample of less than or equal to 20 µL. In some embodiments, the methods comprise extracting an analyte from a sample of less than or equal to 15 µL. In some embodiments, the methods comprise extracting an analyte from a sample of less than or equal to 10 µL.
[00140] In some modalities, the sample collected by the microsampling device can be transported without refrigeration or freezing. In some modalities, the sample collected by the microsampling device can be transported without dry ice. In some modalities, the sample collected by the microsampling device can be transported at room temperature. In some modalities, the sample collected by the microsampling device can be transported without biohazard issues.
[00141] In some modalities, the sample collected by the microsampling device requires little training for collection. In some modalities, the sample collected by the microsampling device can be collected anywhere. In some embodiments, the sample collected by the microsampling device can be dried at room temperature for shipment.
[00142] In some embodiments, the microsampling device is a MITRA® tip. In some embodiments, the microsampling device is enclosed in a cartridge designed for automation of mass spectrometric analysis and extraction.
[00143] In some modalities, the methods further comprise sample collection with a microsampling device. In some embodiments, the collection step comprises making a tweezer with the fingers and applying an absorbent tip of the microsampling device to the blood. In some modalities, the collection step comprises applying an absorbent tip to the patient's urine or saliva. In some modalities, the sample collected in the microsampling device is air dried for one to two hours before transportation.
[00144] In some embodiments, the analyte is a steroid. In some embodiments, the steroid is cortisol, cortisone, progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, dehydroepiandrosterone, corticosterone, deoxycorticosterone, 11-desoxycortisol, pregnolone, 17-hydroxypregnenolone, 18-hydroxycorticosterone, or 21-desoxycortisol. In some modalities, the analyte is a steroid on a steroid panel for diagnosing congenital adrenal hyperplasia (CAH). In some embodiments, the steroid is selected from the group consisting of cortisol, cortisone, progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, dehydroepiandosterone, corticosterone, deoxycorticosterone, 11-deoxycortisol, pregnenolone, 17-hydroxypregnenodione, 18-hydroxycorticosterone, and 21-hydroxycorticosterone. In some embodiments, the steroid is 25-hydroxyvitamin D2 or 25-hydroxyvitamin D.
[00145] In some embodiments, the analyte is an opiate. In some modalities, the opiate is cis-tramadol, O-desmethyltramadol, tapentadol, N-desmethyltapentadol, codeine, morphine, oxymorphone, noridrocodone, oxycodone, noroxycodone, hydromorphone, hydrocodone, buprenorphine, norbuprenorphine, fentanyl, monoacetylmorphine, 6 6-MAM), methadone, dihydrocodeine, naloxone, naltrexone, 6β-naltrexol, nalorphine, nalbuphine or 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). In some embodiments, the opiate is selected from the group consisting of cis-tramadol, O-desmethyl tramadol, tapentadol, N-desmethyltapentadol, codeine, morphine, oxymorphone, noridrocodone, oxycodone, noroxycodone, hydromorphone, hydrocodone, buprenorphine, norbuprenorphine, norhydrocodone, fentanyl , 6-monoacetylmorphine (6-MAM), methadone, dihydrocodeine, naloxone, naltrexone, 6β-naltrexol, nalorphine, nalbuphine and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). In some modalities, opiate is extracted from a whole blood sample, saliva, or urine.
[00146] In some embodiments, the analyte is a benzodiazepine. In some embodiments, the benzodiazepine is oxazepam, temazepam, lorazepam, nordiazepam, diazepam, chlordiazepoxide, triazolam, midazolam, alprazolam, clonazepam, bromazepam, clobazam, nitrazepam, fenazepam, flunitepam, or flunitepam, medazepam. In some embodiments, the benzodiazepine is selected from the group consisting of oxazepam, temazepam, lorazepam, nordiazepam, diazepam, chlordiazepoxide, triazolam, midazolam, alprazolam, clonazepam, bromazepam, clobazam, nitrazepam, flunazepam, flunitazepam, fenazepam, and merazepam, prazepam. In some modalities, the benzodiazepine is extracted from a whole blood or urine sample.
[00147] In some embodiments, one or more ions comprise a bromazepam precursor ion with a mass to charge (m/z) ratio of 316 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 214 ± 0.5 or 270 ± 0.5. In some embodiments, one or more ions comprise an oxazepam precursor ion with a mass to charge (m/z) ratio of 287 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 104 ± 0.5 or 241 ± 0.5. In some embodiments, one or more ions comprise a clobazam precursor ion with a mass to charge (m/z) ratio of 300 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 224 ± 0.5 or 259 ± 0.5. In some embodiments, one or more ions comprise a nitrazepam precursor ion with a mass to charge (m/z) ratio of 282 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 180 ± 0.5 or 236 ± 0.5. In some embodiments, one or more ions comprise an alprazolam precursor ion with a mass to charge (m/z) ratio of 309.1 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 165 ± 0.5 or 280.9 ± 0.5. In some embodiments, one or more ions comprise a triazolam precursor ion with a mass to charge (m/z) ratio of 343 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 206 ± 0.5 or 308 ± 0.5. In some embodiments, one or more ions comprise a clonazepam precursor ion with a mass to charge (m/z) ratio of 316 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 214 ± 0.5 or 270 ± 0.5. In some embodiments, one or more ions comprise flurazepam precursor ion with a mass to charge (m/z) ratio of 388 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 287.9 ± 0.5 or 315 ± 0.5. In some embodiments, one or more ions comprise a precursor ion of lorazepam with a mass to charge (m/z) ratio of 321 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 229.1 ± 0.5 or 331 ± 0.5. In some embodiments, one or more ions comprise a flunitrazepam precursor ion with a mass to charge (m/z) ratio of 314 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 211 ± 0.5 or 268 ± 0.5. In some embodiments, one or more ions comprise a temazepam precursor ion with a mass to charge (m/z) ratio of 301.1 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 177 ± 0.5 or 255 ± 0.5. In some embodiments, one or more ions comprise a midazolam precursor ion with a mass to charge (m/z) ratio of 326 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 129 ± 0.5 or 244 ± 0.5. In some embodiments, one or more ions comprise a nordiazepam precursor ion with a mass to charge (m/z) ratio of 271 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 139.8 ± 0.5 or 165 ± 0.5. In some embodiments, one or more ions comprise a fenazepam precursor ion with a mass to charge (m/z) ratio of 351 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 185.9 ± 0.5 or 206 ± 0.5. In some embodiments, one or more ions comprise a chlordiazepam precursor ion with a mass to charge (m/z) ratio of 301 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 259 ± 0.5 or 224 ± 0.5. In some embodiments, one or more ions comprise a diazepam precursor ion with a mass to charge (m/z) ratio of 285 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 154 ± 0.5 or 193 ± 0.5. In some embodiments, one or more ions comprise a prazepam precursor ion with a mass to charge (m/z) ratio of 325 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 165 ± 0.5 or 271 ± 0.5. In some embodiments, one or more ions comprise a precursor medazepam ion with a mass to charge (m/z) ratio of 271 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 180 ± 0.5 or 207.1 ± 0.5,
[00148] In some embodiments, the analyte is an antiepileptic drug. In some modalities, the antiepileptic drug is valproic acid, tiagabine, topiramate, levitiracetum, lamotrigine, lacosamide, Ethosuximide, carbamazepine, eslicarbamazepine, 10,11- carbamazepine, phenobarbital, rufinamide, primidone, phenytoin, pentabaline, gabaline, zonisamide. In some embodiments, the antiepileptic drug is selected from the group consisting of valproic acid, tiagabine, topiramate, levitiracetum, lamotrigine, lacosamide, Ethosuximide, carbamazepine, eslicarbamazepine, 10,11-carbamazepine, phenobarbital, rufinamide, primidone, gabazapine, phenytoin and pregablin. In some embodiments, the antiepileptic drug is extracted from a whole blood sample.
[00149] In some embodiments, one or more ions comprise a felbamate precursor ion with a mass to charge (m/z) ratio of 339 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 117.3 ± 0.5 or 261 ± 0.5. In some embodiments, one or more ions comprise an ethosuximide precursor ion with a mass to charge (m/z) ratio of 142 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 44.3 ± 0.5 or 39.3 ± 0.5. In some embodiments, one or more ions comprise a lacosamide precursor ion with a mass to charge (m/z) ratio of 251 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 91.2 ± 0.5 or 65.2 ± 0.5. In some embodiments, one or more ions comprise a precursor ion of lamotrigine with a mass to charge (m/z) ratio of 256 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 211 ± 0.5 or 145 ± 0.5. In some embodiments, one or more ions comprise a topiramate precursor ion with a mass to charge (m/z) ratio of 338.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 78.2 ± 0.5 or 96.2 ± 0.5. In some embodiments, one or more ions comprise a gabapentin precursor ion with a mass to charge (m/z) ratio of 172.3 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 91.2 ± 0.5 or 67.2 ± 0.5. In some embodiments, one or more ions comprise an eslicarbazepine precursor ion with a mass to charge (m/z) ratio of 297.1 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 194 ± 0.5 or 179 ± 0.5. In some embodiments, one or more ions comprise a primidone precursor ion with a mass to charge (m/z) ratio of 219.8 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 79 ± 0.5 or 135.2 ± 0.5. In some embodiments, one or more ions comprise a pregabalin precursor ion with a mass to charge (m/z) ratio of 160.1 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 55.2 ± 0.5 or 77.2 ± 0.5. In some embodiments, one or more ions comprise a carbamazepine precursor ion with a mass to charge (m/z) ratio of 237 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 194.1 ± 0.5 or 179 ± 0.5. In some embodiments, one or more ions comprise a phenobarbital precursor ion with a mass to charge (m/z) ratio of 231 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 44.2 ± 0.5 or 188.1 ± 0.5. In some embodiments, one or more ions comprise an epoxide precursor ion with a mass to charge (m/z) ratio of 236.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 141.2 ± 0.5 or 112.2 ± 0.5. In some embodiments, one or more ions comprise a zonisamide precursor ion with a mass to charge (m/z) ratio of 213.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 77.2 ± 0.5 or 102.1 ± 0.5. In some embodiments, one or more ions comprise a tiagabine precursor ion with a mass to charge (m/z) ratio of 376.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 111.1 ± 0.5 or 149.1 ± 0.5. In some embodiments, one or more ions comprise a phenytoin precursor ion with a mass to charge (m/z) ratio of 253.1 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 104.2 ± 0.5 or 182.2 ± 0.5. In some embodiments, one or more ions comprise a levetiracetam precursor ion with a mass to charge (m/z) ratio of 171.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 126.2 ± 0.5 or 69.2 ± 0.5. In some embodiments, one or more ions comprise a valproic acid precursor ion with a mass to charge (m/z) ratio of 143 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 143 ± 0.5. In some embodiments, one or more ions comprise a rufinamide precursor ion with a mass to charge (m/z) ratio of 239 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 127.2 ± 0.5 or 261 ± 0.5. In some embodiments, one or more ions comprise a primdone precursor ion with a mass to charge (m/z) ratio of 219 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 126 ± 0.5 or 141 ± 0.5. In some embodiments, one or more ions comprise a topiramate D12 precursor ion with a mass to charge (m/z) ratio of 350 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 78.2 ± 0.5. In some embodiments, one or more ions comprise a D3 epoxide precursor ion with a mass to charge (m/z) ratio of 256 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 77 ± 0.5. In some embodiments, one or more ions comprise a precursor ion of lamotrigine with a mass to charge (m/z) ratio of 259 ± 0.5. In some embodiments, the one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 214 ± 0.5. In some embodiments, one or more ions comprise a levetiracetam D6 precursor ion with a mass to charge (m/z) ratio of 177.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 132.2 ± 0.5.
[00150] In some embodiments, the analyte is an immunosuppressant. In some modalities, the immunosuppressant is cyclosporin A, sirolimus, tacrolimus or everolimus. In some embodiments, the immunosuppressant is selected from the group consisting of cyclosporine A, sirolimus, tacrolimus, and everolimus. In some modalities, the immunosuppressant is extracted from a whole blood sample.
[00151] In some embodiments, the analyte is a barbiturate. In some embodiments, the barbiturate is phenobarbital, amobarbitol, butalbital, phenobarbitol, secobarbitol, or thiopental. In some embodiments, the barbiturate is selected from the group consisting of phenobarbital, amobarbital, butalbital, pentobarbitol, secobarbitol, and thiopental. In some modalities, the barbiturate is taken from a whole blood sample.
[00152] In some embodiments, one or more ions comprise a secobarbital precursor ion with a mass to charge (m/z) ratio of 237.0 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 42.0 ± 0.5. In some embodiments, one or more ions comprise an amobarbital precursor ion with a mass to charge (m/z) ratio of 225.0 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 182.0 ± 0.5. In some embodiments, one or more ions comprise a pentobarbital precursor ion with a mass to charge (m/z) ratio of 225.6 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 42.0 ± 0.5. In some embodiments, one or more ions comprise a thiopental precursor ion with a mass to charge (m/z) ratio of 241.0 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 57.9 ± 0.5. In some embodiments, one or more ions comprise a phenobarbital precursor ion with a mass to charge (m/z) ratio of 231.0 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 42.0 ± 0.5. In some embodiments, one or more ions comprise a butalbital precursor ion with a mass to charge (m/z) ratio of 223.1 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 42.1 ± 0.5.
[00153] In some embodiments, the analyte is tamoxifen. In some embodiments, the analyte is a metabolite of tamoxifen. In some embodiments, said metabolite is norendoxifene. In some embodiments, said metabolite is endoxifene or N-Desmethyl-4-Hydroxy Tamoxifen. In some embodiments, said metabolite is 4'-Hydroxy Tamoxifen. In some embodiments, said metabolite is 4-Hydroxy Tamoxifen. In some embodiments, said metabolite is N-Desmethyl-4'-Hydroxy Tamoxifen. In some embodiments, said metabolite is N-Desmethyl Tamoxifen. In some embodiments, said metabolite is selected from the group consisting of norendoxifene, endoxifene, 4'-Hydroxy Tamoxifen, 4-Hydroxy Tamoxifen, N-Desmethyl-4'-Hydroxy Tamoxifen, and N-Desmethyl-4'-Hydroxy Tamoxifen. In some embodiments, tamoxifen or its metabolite is extracted from a whole blood sample.
[00154] In some embodiments, one or more ions comprise a tamoxifen precursor ion with a mass to charge (m/z) ratio of 372.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 72.14 ± 0.5. In some embodiments, one or more ions comprise an endoxifen precursor ion with a mass to charge (m/z) ratio of 374.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 58.1 ± 0.5. In some embodiments, the one or more ions comprise a 4-hydroxy tamoxifen precursor ion with a mass to charge (m/z) ratio of 388.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 72.1 ± 0.5. In some embodiments, one or more ions comprise an N-desmethyl-4'-hydroxy tamoxifen precursor ion with a mass to charge (m/z) ratio of 374.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 58.1 ± 0.5. In some embodiments, one or more ions comprise a 4'-hydroxy tamoxifen precursor ion with a mass to charge (m/z) ratio of 388.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 72.1 ± 0.5. In some embodiments, one or more ions comprise an N-desmethyl-4'-hydroxy tamoxifen precursor ion with a mass to charge (m/z) ratio of 358.2 ± 0.5. In some embodiments, one or more ions comprise one or more fragment ions with a mass to charge (m/z) ratio of 58.1 ± 0.5.
[00155] In some embodiments, the analyte is a drug for oncology. In some modalities, the analyte is anastrozole. In some modalities, the analyte is lestrozole. In some embodiments, the analyte is exemestane. In some embodiments, the analyte is selected from the group consisting of anastrozole, letrozole and exemestane. In some embodiments, the oncology drug is extracted from a whole blood sample.
[00156] In some embodiments, the analyte is tetrahydrocannabinol (THC) or its metabolite. In some modalities, THC is extracted from a urine sample.
[00157] In some embodiments, the extracted analyte is hydrolyzed. In some embodiments, the analyte is hydrolyzed prior to extraction.
[00158] In some embodiments, the collision energy is within the range of about 5 to 60 V. In some embodiments, the collision energy is within the range of about 5 to 60 V.
[00159] In another aspect, methods for diagnosing congenital adrenal hyperplasia in patients are provided herein. In some embodiments, the endogenous steroid quantification methods provided herein are used for diagnosing congenital adrenal hyperplasia.
[00160] In another aspect, methods for detecting or monitoring THC use in an individual are provided herein. In another aspect, methods are provided herein for detecting or monitoring barbiturate use in an individual. In another aspect, methods for detecting or monitoring opiate use in an individual are provided herein. In another aspect, methods for detecting or monitoring benzodiazepine use in an individual are provided herein.
[00161] In another aspect, methods for detecting or monitoring antiepileptic drug use in an individual are provided herein. In another aspect, methods for monitoring the effectiveness of an antiepileptic drug in an individual are provided herein.
[00162] In another aspect, methods for detecting or monitoring tamoxifen use in an individual are provided herein. In another aspect, methods are provided herein for monitoring the effectiveness of tamoxifen in an individual.
[00163] In another aspect, certain methods presented here use high resolution/high precision mass spectrometry to determine the amount of analyte in a sample. In some embodiments, using high-precision/high-resolution mass spectrometry, the methods include: (a) subjecting the analyte of a sample to an ionization source under conditions suitable for generating ions, where the ions are detectable through spectrometry. pasta; and (b) determine the quantity of one or more ions by high resolution/high precision mass spectrometry. In these modalities, the amount of one or more ions determined in step (b) is related to the amount of analyte in the sample. In some embodiments, high resolution/high accuracy mass spectrometry is conducted at a FWHM of 10,000 and a mass accuracy of 50 ppm. In some embodiments, high-resolution/high-precision mass spectrometry is conducted with a high-resolution/high-precision time-of-flight (TOF) mass spectrometer. In some embodiments, the ionization conditions comprise analyte ionization under acidic conditions. In some related embodiments, acidic conditions comprise treating said sample with formic acid prior to ionization.
[00164] In any of the methods described here, the sample may comprise a biological sample. In some embodiments, the biological sample can comprise a biological fluid such as urine, plasma or serum. In some embodiments, the biological sample can comprise a sample from a human; such as an adult male or female, or a youth male or female, where the youth is under 18, under 15, under 12, or under 10. The human sample can be analyzed to diagnose or monitor a disease state or condition or to monitor the therapeutic effectiveness of treating a disease state or condition. In some related embodiments, the methods described herein can be used to determine the amount of analyte in a biological sample when obtained from a human.
[00165] In embodiments using tandem mass spectrometry, tandem mass spectrometry can be conducted by any method known in the art, including, for example, multiple reaction monitoring, precursor ion scanning, or product ion scanning.
[00166] In some embodiments, tandem mass spectrometry comprises fragmentation of a precursor ion into one or more fragment ions. In embodiments where the amounts of two or more fragment ions are determined, the amount can be subjected to any mathematical manipulation known in the art to relate the measured ion amounts to the amount of analyte in the sample. For example, the amounts of two or more fragment ions can be added up as part of determining the amount of analyte in the sample.
[00167] In some embodiments, high-resolution/high-precision mass spectrometry is conducted at a resolving power (FWHM) of greater than or equal to about 10,000, such as greater than or equal to about 15,000, such as more than or equal to about 20,000, such as more than or equal to about 25,000. In some embodiments, high resolution/high precision mass spectrometry is conducted at an accuracy of less than or equal to about 50 ppm, such as less than or equal to about 20 ppm, such as less than or equal to about 10 ppm, such as less than or equal to about 5 ppm; such as less than or equal to about 3 ppm. In some embodiments, high resolution/high precision mass spectrometry is conducted at a resolving power (FWHM) of greater than or equal to about 10,000 and an accuracy of less than or equal to about 50 ppm. In some embodiments, resolving power is greater than about 15,000 and accuracy is less than or equal to about 20 ppm. In some embodiments, resolving power is greater than or equal to about 20,000 and accuracy is less than or equal to about 10 ppm; preferably the resolving power is greater than or equal to about 20,000 and the accuracy is less than or equal to about 5 ppm, such as less than or equal to about 3 ppm.
[00168] In some embodiments, high-resolution/high-precision mass spectrometry can be conducted with an orbitrap mass spectrometer, a time-of-flight (TOF) mass spectrometer, or a transform ion cyclotron resonance mass spectrometer Fourier transform (sometimes known as a Fourier transform mass spectrometer).
[00169] Mass spectrometry (either tandem or high resolution/high precision) can be performed in positive ion mode. Alternatively, mass spectrometry can be performed in negative ion mode. Various sources of ionization, including, for example, atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI), can be used to ionize the analyte.
[00170] In any method presented here, a separately detectable internal standard may be provided in the sample, the amount of which is also determined in the sample. In modalities using a separately detectable internal standard, all or a portion of both the analyte of interest and the internal standard present in the sample is ionized to produce a plurality of detectable ions in a mass spectrometer and one or more ions produced from each are detected by mass spectrometry. In these modalities, the presence or amount of ions generated from the analyte of interest can be related to the presence of the amount of analyte of interest in the sample through comparison with the amount of internal standard ions detected.
[00171] Alternatively, the amount of analyte in a sample can be determined by comparison with one or more external reference standards. Exemplary external reference standards include blank plasma or serum spiked with human or non-human analyte, a synthetic analyte analog, or an isotopically labeled variant thereof. Sample Preparation for Mass Spectrometry Analysis
[00172] One method of sample purification that can be used prior to mass spectrometry is applying a sample to a solid phase extraction (SPE) column under conditions where the analyte of interest is reversibly retained by the packing material. column, while one or more other materials are not retained. In this technique, the first mobile phase condition can be employed in which the analyte of interest is retained by the column, and a second mobile phase condition can subsequently be employed to remove retained material from the column once unretained materials are completely removed. with washing.
[00173] In some embodiments, analyte in a sample may be reversibly retained in an SPE column with a packing material comprising an alkyl bonded surface. For example, in some embodiments, a C-8 online SPE column (such as an Oasis HLB online SPE column/cartridge (2.1 mm x 20 mm) from Phenomenex, Inc. or equivalent) may be used to enrich analyte prior to mass spectrometric analysis. In some embodiments, use of an SPE column is conducted with HPLC Grade 0.2% aqueous formic acid as a wash solution and use of 0.2% formic acid in acetonitrile as an elution solution.
[00174] Another method of sample purification that can be used prior to mass spectrometry is liquid chromatography (LC). In liquid chromatography techniques, an analyte can be purified by applying a sample to a chromatographic analytical column under mobile phase conditions where the analyte of interest elutes at a differential rate compared to one or more other materials. Such procedures can enrich the amount of one or more analytes of interest relative to one or more other components of the sample.
[00175] Certain liquid chromatography methods, including HPLC, are based on relatively slow laminar flow technology. Traditional HPCL analysis is based on column packing where laminar flow of the sample through the column is the basis for separating the analyte of interest from the sample. One skilled in the art will understand that separation on such columns is a splitting process and can select LC, including HPLC, instruments and columns that are suitable for use with C-peptide. The chromatographic analytical column typically includes a medium (i.e., a material of packaging) to facilitate separation of chemical moieties (ie, fractionation). The medium can include small particles. The particles typically include a bonded surface that interacts with the various chemical moieties to facilitate separation of the chemical moieties. A suitable bonded surface is a hydrophobic bonded surface such as an alkyl bonded or cyano bonded surface. Alkyl bonded surfaces can include C-4, C-8, C-12 or C-18 bonded alkyl groups. In some embodiments, the chromatographic analytical column is a monolithic C-18 column. The chromatographic analytical column includes an inlet port for receiving a sample and an outlet portion for discharging an effluent that includes the fractionated sample. The sample can be delivered to the inlet port directly, or from an SPE column, such as an online SPE column or a TFLC column. In some embodiments, the on-line filter can be used in front of the SPE column and/or HPLC column to remove particulates and phospholipids in the samples before the samples reach the SPE and/or TFLC and/or HPLC columns.
[00176] In one embodiment, the sample can be applied to the HPLC column at the inlet port, eluted with a solvent or solvent mixture and discharged at the outlet port. Different solvent modes can be selected for elution of the analyte(s) of interest. For example, liquid chromatography can be performed using a gradient mode, an isocratic mode, or a polytypic (i.e., mixed) mode. During chromatography, the separation of materials is carried out by variables such as choice of eluent (also known as "mobile phase"), elution mode, gradient conditions, temperature, etc.
[00177] In some embodiments, analyte in a sample is enriched with HPLC. This HPLC can be conducted with a monolithic C-18 column chromatographic system, for example, an Onyx Monolithic C-18 column from Phenomenex Inc. (50 x 2.0 mm) or equivalent. In certain embodiments, HPCL is performed using 0.2% formic acid HPLC grade as solvent A and 0.2% formic acid in acetonitrile as solvent B.
[00178] Through careful selection of valves and connector piping, two or more chromatographic columns can be connected as needed so that material is passed from one to the other without the need for any manual steps. In preferred embodiments, valve and piping selection is controlled by a pre-programmed computer to carry out the necessary steps. More preferably, the chromatographic system is also connected in such a way online to the detector system, for example, an MS system. In this way, an operator can place a sample tray in an autosampler and the remaining operations are carried out under computer control, resulting in purification and analysis of all selected samples.
[00179] In some embodiments, TFLC can be used for analyte purification prior to mass spectrometry. In such modalities, samples can be extracted using a TFLC column that captures the analyte. The analyte is then eluted and transferred online to an analytical HPLC column. For example, sample extraction can be performed with a TFLC extraction cartridge with a large particle size (50 μm) packing. Sample eluted from this column can then be transferred online to an analytical HPLC column for further purification prior to mass spectrometry. Because the steps involved in these chromatography procedures can be linked automatically, the need for operator involvement during analyte purification can be minimized. This feature can result in time and cost savings and eliminate the opportunity for operator error.
[00180] In some embodiments, one or more of the above purification techniques can be used in parallel for analyte purification to allow simultaneous processing of multiple samples. Analyte Detection and Quantification through Mass Spectrometry
[00181] Mass spectrometry is performed using a mass spectrometer, which includes an anion source for ionizing the fractionated sample and creating charged molecules for further analysis. In various embodiments, analyte can be ionized by any method known to one of skill in the art. For example, analyte ionization can be accomplished through electron ionization, chemical ionization, electrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization (APPI), thermal desorption by diode laser (LDTD), fast atom bombardment (FAB), liquid secondary ionization (LSI), matrix aided laser desorption ionization (MALDI), field ionization, field desorption, thermospray/plasma spray ionization, ionization by surface augmented laser desorption (SELDI), inductively coupled plasma (ICP) and particle beam ionization. One skilled in the art will understand that the choice of ionization method can be determined based on the analyte to be measured, type of sample, type of detector, choice of positive versus negative mode, etc., analyte can be ionized in positive mode or negative. In preferred embodiments, analyte is ionized by ESI in positive ion mode.
[00182] In mass spectrometry techniques generally, after the sample has been ionized, the positively or negatively charged ions created in this way can be analyzed to determine a ratio of mass to charge (m/z). Various analyzers for determining m/z include quadrupole analyzers, ion trapping analyzers, time-of-flight analyzers, ion cyclotron resonance mass analyzers, and orbitrap analyzers. Some exemplary ion trapping methods are described in Bartolucci et al., Rapid Commun. Mass Spectrom. 2000, 14:967-73.
[00183] Ions can be detected using various detection modes. For example, selected ions can be detected, ie using a selective ion monitoring mode (SIM) or alternatively mass transitions resulting from collision-induced dissociation or neutral loss can be monitored, eg multiple reaction monitoring (MRM) or selected reaction monitoring (SRM). In some embodiments, the mass-to-charge ratio is determined using a quadrupole analyzer. In a "quadrupole" or "quadrupole ion trapping" instrument, ions in an oscillating radio frequency field experience a force proportional to the DC potential applied between the electrodes, the amplitude of the RF signal, and the mass/charge ratio. Voltage and amplitude can be selected so that only ions having a particular mass/charge ratio travel the length of the quadrupole, while all other ions are deflected. In this way, quadrupole instruments can act as both a "mass filter" and a "mass detector" for the ions injected into the instrument.
[00184] As the ions collide with the detector they produce a pulse of electrons which are converted into a digital signal. The acquired data is transmitted to a computer, which graphically represents collected ion counts versus time. The resulting mass chromatograms are similar to chromatograms generated in traditional HPLC-MS methods. The areas under the peaks corresponding to particular ions, or the amplitude of such peaks, can be measured and related to the amount of analyte of interest. In certain embodiments, the area under the curves, or peak amplitudes, for fragment ion(s) and/or precursor ions are measured to determine the amount of analyte. The relative abundance of a given ion can be converted to an absolute amount of the original analyte using standard calibration curves based on peaks of one or more ions from an internal or external molecular standard.
[00185] The resolution of MS techniques can be increased by employing certain mass spectrometric analyzers through "tandem mass spectrometry" or "MS/MS". In this technique, a precursor ion (also called a parent ion) generated from a molecule of interest can be filtered in an MS instrument and the precursor ion subsequently fragmented to provide one or more fragment ions (also called daughter ions or parent ions). product) which are then analyzed in a second MS procedure. Through careful selection of precursor ions, only ions produced by certain analytes are passed to the fragmentation chamber, where collisions with atoms of an inert gas produce the fragment ions. Because both precursor and fragment ions are produced in a reproducible manner under a given set of ionization/fragmentation conditions, the MS/MS technique can provide an extremely powerful analytical tool. For example, the combination of filtering/fragmentation can be used to eliminate interfering substances and can be particularly useful in complex samples such as biological samples. In certain embodiments, a mass spectrometric instrument with multiple quadrupole analyzers (such as a quadrupole triple instrument) is employed to conduct tandem mass spectrometric analysis.
[00186] In certain embodiments using an MS/MS technique, precursor ions are isolated for further fragmentation and collision activated dissociation (CAD) is used to generate fragment ions from precursor ions for further detection. In CAD, precursor ions gain energy through collisions with an inert gas, and subsequently fragment through a process referred to as "unimolecular decomposition". Sufficient energy must be deposited in the precursor ion so that certain bonds within the ion can be broken due to the increased vibrational energy.
[00187] In some embodiments, analyte in a sample is detected and/or quantified using MS/MS as follows. Analyte is enriched in a sample by first subjecting the sample to SPE, then liquid chromatography, preferably HPLC; the flow of liquid solvent from a chromatographic analytical column into the heated nebulizer interface of an MS/MS analyzer; and the solvent/analyte mixture is converted to steam in the interface heat-loaded tubing. During these processes, the analyte is ionized. The ions, eg precursor ions, pass through the instrument orifice and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters, allowing ion selection (ie, "precursor" and "fragment" ion selection in Q1 and Q3, respectively) based on their mass-to-charge ratio. (m/z). Quadrupole 2 (Q2) is the collision cell, in which ions are fragmented. The first quadrupole of the mass spectrometer (Q1) selects molecules with the m/z of an analyte ion. Precursor ions with the m/z current are allowed to pass into the collision chamber (Q2), while unwanted ions with any m/z collide with the sides of the quadrupole and are eliminated. Precursor ions of input Q2 collide with neutral gas molecules (such as Argon molecules) and fragment. The generated fragment ions are passed to quadrupole 3 (Q3), where the fragment ions are selected for detection.
[00188] Alternative modes of operation of a tandem mass spectrometric instrument that can be used in certain modalities include product ion scanning and precursor ion scanning. For a description of these modes of operation, see, for example, E. Michael Thurman et al., ChromatographicMass Spectrometric Food Analysis for Trace Determination of Pesticide Residues, Chapter 8 (Amadeo R. Fernandez-Alba, ed., Elsevier 2005) (387) .
[00189] In other embodiments, a high resolution/high accuracy mass analyzer can be used for quantitative analyte analysis according to methods of the present invention. To obtain acceptable accuracy for quantitative results, the mass spectrometer may be capable of displaying a resolution energy (FWHM) of 10,000 or more, with an accuracy of about 50 ppm or less for the ions of interest; preferably the mass spectrometer exhibits a resolving power (FWHM) of 18,000 or less, with an accuracy of about 5 ppm or less; such as a resolving power (FWHM) of 20,000 or better and accuracy of about 3ppm or less; such as a resolving power (FWHM) of 25,000 or better and accuracy of about 3ppm or less. Three exemplary analyzers capable of displaying the required level of performance for analyte ions are orbitrap mass analyzers, certain TOF mass analyzers, and Fourier transform ion cyclotron resonance mass analyzers.
[00190] Elements found in biological active molecules, such as carbon, oxygen and nitrogen, exist naturally in several different isotopic forms. For example, most carbon is present as 12C, but approximately 1% of naturally occurring carbon is present as 13C. In this way, some fraction of naturally occurring molecules containing at least one carbon atom will contain at least one 13C atom. Inclusion of naturally occurring elemental isotopes in molecules gives rise to multiple molecular isotopic forms. The difference in masses of molecular isotopic forms is at least 1 atomic mass unit (amu). This is because elementary isotopes differ by at least one neutron (mass of one neutron ~ 1 amu). When molecular isotopic forms are ionized to multiply charge states, the mass distinction between isotopic forms can become difficult to discern because mass spectrometric detection is based on the ratio of mass to charge (m/z). For example, two isotopic forms differing in mass by 1 amu that are both ionized to a 5+ state will exhibit differences in their m/z of only 0.2. High resolution/high accuracy mass spectrometers are able to discern between isotopic forms of highly multiply charged ions (such as ions with charges of ±2, ±3, ±4, ±5 or more).
[00191] Due to naturally occurring elemental isotopes, multiple isotopic forms typically exist for each molecular ion (each of which can give rise to a separately detectable spectrometric peak if analyzed with a sufficiently sensitive mass spectrometric instrument). The m/z ratios and relative abundances of multiple isotopic forms collectively comprise an isotopic signature for a molecular ion. In some embodiments, the m/z ratios and relative abundances for two or more molecular isotopic forms can be used to confirm the identity of a molecular ion under investigation. In some embodiments, the mass spectrometric peak of one or more isotopic forms is used to quantify a molecular ion. In some related embodiments, a single mass spectrometric peak of an isotopic form is used to quantify a molecular ion. In other related embodiments, a plurality of isotopic peaks are used to quantify a molecular ion. In these latter embodiments, the plurality of isotopic peaks can be subjected to any appropriate mathematical treatment. Various mathematical treatments are known in the art and include, but are not limited to, sum of area under multiple peaks or average of multiple peak response. However, the precise masses observed for isotopic variants of any ion may vary slightly due to instrumental variance.
[00192] In some embodiments, the relative abundance of one or more ions is measured with a high resolution/high accuracy mass spectrometer in order to qualitatively assess the amount of analyte in the sample. Use of high resolution orbitrap analyzers has been reported for qualitative and quantitative analysis of various analytes. See, for example, U.S. Patent Application Pub. No. 2008/0118932 (filed November 9, 2007); Bredehoft, et al., Rapid Commun. Mass Spectrom., 2008, 22:477-485; Le Breton et al., Rapid Commun. Mass Spectrom., 2008, 22:3130-36; Thevis et al., Mass Spectrom. Reviews, 2008, 27:35-50; Thomas et al., J. Mass Spectrom., 2008, 43:908-15; Schenk et al., BMC Medical Genomics, 2008, 1:41; and Olsen et al., Nature Methods, 2007, 4:70912.
[00193] The results of an analyte assay can be related to the amount of analyte in the original sample by various methods known in the art. For example, given that sampling and analysis parameters are carefully controlled, the relative abundance of a given ion can be compared to a table that converts the relative abundance into an absolute quantity of the original molecule. Alternatively, external standards can be administered with the samples, and a standard curve constructed based on ions generated from these standards. Using such a standard curve, the relative abundance of a given ion can be converted into an absolute quantity of the original molecule. In certain preferred embodiments, an internal standard is used to generate a standard curve for calculating the amount of analyte. Methods of generating and using such standard curves are well known in the art and one of ordinary skill in the art is able to select an appropriate internal standard. For example, in a preferred embodiment, one or more isotopically labeled analyte forms can be used as internal standards. Various other methods for relating the amount of an ion to the amount of the original molecule will be well known to those of ordinary skill in the art.
[00194] As used herein, an "isotopic marker" produces a mass shift in the tagged molecule relative to the untagged molecule when analyzed through mass spectrometric techniques. Examples of suitable labels include deuterium (2H), 13C and 15N. One or more isotopic labels can be incorporated at one or more positions on the molecule and one or more types of isotopic labels can be used on the same isotopically labeled molecule.
[00195] One or more steps of any of the methods described above can be performed using automatic machines. In certain embodiments, one or more purification steps are performed online and more preferably all of the purification steps and mass spectrometry can be performed in an online manner.
[00196] The following examples serve to illustrate the invention. These Examples are in no way intended to limit the scope of the methods. EXAMPLES Example 1: Steroid mass spectrometric assay
[00197] Patient samples were extracted directly from the 20 uL MITRA® tips. The tips were placed directly into the NUNC® 96-well plate. 500 uL extraction solvent (1M NH4OH in 50/50 Methanol/Ethyl Acetate) and 50 uL internal standard (containing the stable isotope) and extraction buffer were then added to each well. The plate was mixed at room temperature for one hour before drying under nitrogen. After the drying step, the samples were brought back into solution by adding an acidic and aqueous organic solution (0.1% FA 200 uL in Water/Methanol 50/50) to each well. The plate is mixed and then filtered. 100 uL of the filtrate was injected into the LC-MS/MS system with APCI source (chemical ionization under atmospheric pressure) in positive ion mode. The following reagents were used: Mobile Phase A - 0.1% Formic Acid in Water; Mobile Phase B - Methanol/Acetonitrile 80/20; Extraction Solvent: 1M Ammonium Hydroxide in 50/50 Methanol/Ethyl Acetate.
[00198] Thermo Scientific's ARIA® TX-4 System was used for liquid chromatography and separation was performed through an analytical column reversed-phase HPLC column (KINETEX® C18). The detector used was AB Sciex's QTRAP® 6500.
[00199] The following steroids were detected and quantified non-derivatized using a 20 uL MITRA® tip or two 6 mm DBS punctures: cortisol, cortisone, progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, dehydroepiandrosterone, corticosterone, deoxycorticosterone, 11-deoxycortisol, pregnenolone, 17-hydroxypregnenolone & 21-deoxycortisol. Figure 1. The following mass transitions were used for analysis by mass spectrometry.


[00200] Figures 2-17 show levels of various steroids in adult males, adult females and children.
[00201] Table 1 shows distinguishing features of enzyme deficiencies of congenital adrenal hyperplasia.


[00202] Analytical sensitivity: the limit of quantification (LOQ) is the point at which measurements become quantitatively significant. Acceptability criteria for the LOQ are defined as the lowest reproducible concentration at which the coefficient of variation (CV) is < 20%. To determine the preliminary LOQ, several replicates of samples varying in concentration were administered over several days. Preliminary analytical measurable range was determined in a linear range study. Table 2:


[00203] Table 3 shows differential diagnosis of enzyme deficiencies causing classic congenital adrenal hyperplasia.

[00204] Figure 18 shows standard linearity of testosterone between 50-10,000 ng/dL. Example 2: oncology drugs
[00205] In this assay, 20 uL MITRA® tips were used to collect patient samples. The tips were pre-soaked in an internal standard and dried for 2-24 hours.
[00206] The tips were embedded in calibration standards. Samples were eluted in 500 µl of elution buffer and dried. Samples were then resuspended in 200 µl of loading buffer. 90 uL samples were injected into LC-MS/MS for quantification.
[00207] Figure 19 shows chromatogram of tamoxifen and its metabolites.
Figure 20 shows chromatogram of letrozole, exemestane and anastrozole. Example 3: opiates
[00209] In this assay, whole blood was centrifuged and sprinkled with opiate standards at different concentration levels to serve as assay calibrators.
[00210] MITRA® 10 uL and 15 uL tips were immersed in whole blood calibrators until fully saturated. Tips saturated with whole blood calibrators were allowed to dry at room temperature for at least 2.5 hours.
[00211] 400 μl of extraction buffer (internal standards of deuterated opiate in 65% ethyl acetate: 0.1% formic acid in methanol) was used to extract opiates from tips in a vortex for 40 minutes. Alternatively, 500 uL of ethyl acetate and methanol 60:40 with 1% formic acid was used to extract opiate from tips on a vortex for one hour at 850 rpm. The tips were then discarded and the extracted samples were completely dried under nitrogen gas at 60°C by Porvair. The samples were then resuspended in 30% MeOH:0.1% FA in water, vortexed. Alternatively, samples were resuspended in 230 uL of 50:50 methanol and water with 0.1% formic acid. The samples were then injected into LC-MS/MS for quantification in positive ion mode on a Thermo Ultra triple quadrupole mass spectrometer. For mobile phase A, 0.1% formic acid in water was used. For mobile phase B, 100% acetonitrile was used. Agilent 3 x 100 mm phenyl hexyl column was used. Runtime was 9 minutes.
[00212] Table 4 shows the linear range of each opiate in a 10 uL tip vs. 15 uL tip.

[00213] Figures 21 to 24 show exemplary opiate chromatogram and corresponding internal pattern.
Figures 25 to 28 show data for morphine, codeine, hydromorphone and oxycodone (respectively) obtained from patient urine using a 20 uL MITRA® tip with glucuronidase hydrolysis.
Figure 29 shows oxycodone data obtained from patient saliva using a 50 uL MITRA® tip.
[00216] Figures 30 and 31 show the results of hematocrit study of buprenorphine and norfentanil, respectively. Example 4: benzodiazepines
[00217] In this assay, whole blood was centrifuged and spiked with benzodiazepine standards at different concentration levels to serve as assay calibrators.
[00218] 10 uL and 20 uL MITRA® tips were immersed in whole blood calibrators until fully saturated. Tips saturated with whole blood calibrators were allowed to dry at room temperature for at least 2.5 hours.
[00219] 400 μl of extraction buffer (internal standards of deuterated benzodiazepine in 65% ethyl acetate: 0.1% formic acid in methanol) was used to extract opiates from the tips in a vortex for 40 minutes. Alternatively, 500 uL of ethyl acetate and methanol 60:40 with 1% formic acid (or alternatively 0.1% formic acid) was used to extract benzodiazepines from tips in a vortex for one hour at 85 rpm. The tips were then discarded and the extracted samples were completely dried under nitrogen gas at 60°C by Porvair. The samples were then resuspended in 10% MeOH:0.1% formic acid in water, vortexed. Alternatively, samples were resuspended in 230 uL of methanol and water 50:50 with 0.1% formic acid. Alternatively, samples were resuspended in 200 µl of 0.1% formic acid in 10% methanol and 90% water. Samples were vortexed at 1200 rpm for 5 to 30 minutes. The samples were then injected into LC-MS/MS for quantification in ESI positive mode on a Thermo Ultra triple quadrupole mass spectrometer. For mobile phase A, 0.1% formic acid in water was used. Alternatively, 20 mM ammonium acetate at pH 5.2 was used. For mobile phase B, 100% acetonitrile was used. Agilent 3x100 mm phenyl hexyl column was used. Alternatively, BDS Hypersil C18 column, 100x3 mm, 3 μ, was used. Runtime was 6 minutes.
[00220] A flow rate of 0.7 ml/minute was obtained: 0-60 sec-90% A: 10% B; 60-210 sec-increases to 30% B; 210-360 sec-increases to 65% B; 360-420 sec-increases to 100% B; 420-480 sec-step 100% B; 480-600 sec-step 90% A: 10% B.
[00221] Table 5 shows benzodiazepines analyzed in 20 µl tips.


[00222] Table 6 shows the linear range of each opiate in 10 uL tip vs. 20 uL tip.

Example 5: bartitutes
[00223] In this assay, urine samples negative for barbiturates were sprinkled with barbiturate standards at different concentration levels to serve as assay calibrators.
[00224] MITRA® 20 uL tips were dipped into the urine calibrators until fully saturated. Tips saturated with urine calibrators were allowed to dry at room temperature.
[00225] The samples were extracted in methanol for one hour. The extracted samples were hydrolyzed for one hour at 60°C in a thermomixer. Samples were then centrifuged and supernatant was injected into LC-MS/MS for quantification. The liquid chromatography run time was 5.75 minutes. The acquisition window was 2.5 minutes. The assay allowed 2 plex, given every 2.75 minutes. 0.03% NH4OH was used for mobile phase A. 90% CAN and 10% MP A were used for mobile phase B.
Figures 32 and 33 show the results of negative urine spattered with barbiturates (secobarbital, amobarbital, pentobarbital and thiopental).
[00227] Figures 34 to 38 show the results of several patient samples quantified for phenobarbital and bultabital. Example 6: THC
[00228] In this assay, patient urine samples were analyzed.
[00229] 20 uL MITRA® tips were dipped into the urine samples until fully saturated. Tips saturated with urine samples were allowed to dry at room temperature.
[00230] Samples were extracted in 100% methanol by vortexing at 900 rpm for one hour. The samples were dried with nitrogen air at 60°C until completely dry. Samples were resuspended in 200 µl of 20 mM sodium citrate buffer at pH 4.5. Glucuronidase was added to the sample and incubated in a thermomixer for 40 minutes at 60°C. Samples were centrifuged at 5500 rpm for 3 minutes and supernatant was injected into LC-MS/MS (ABI5500) for quantification.
[00231] Figure 39 shows the results of analysis of carboxy metabolite THC in patient sample using 20 uL tip and glucuronidase hydrolysis. Example 7: antiepileptic drugs
[00232] In this assay, whole patient blood samples were analyzed.
[00233] MITRA® 20 uL tips were immersed in whole blood samples until fully saturated. Tips saturated with whole blood samples were allowed to dry at room temperature.
[00234] Samples were extracted in 90% methanol and 10% water for one hour. The samples were dried with nitrogen air at 60°C until completely dry. Samples were resuspended in 0.1% formic acid in water and injected into LC-MS/MS for quantification. 5 mL were injected into Thermo Fisher Quantiva. Thermo Fisher Beta-Basic C18m 100x3mm analytical column was used. Mobile Phase A: 0.1% FA; Mobile Phase B: Methanol.
[00235] Table 7 shows mass transitions used in mass spectrometric analysis.


[00236] Table 8 shows the calibration standards used in the analysis.


[00237] Reproducibility: Acceptability criteria: CV% must be less than allowable < TEa/2. The Tea for this assay is determined to be 30%. Ten replicates of each quality control were analyzed within a single run in the order that follows; low, medium and high.
[00238] Table 9 shows the reproducibility of Ethosuximide. The CV% for Ethosuximide ranged from 5.16% to 2.23% at all three levels of quality control.


[00239] Table 10 shows the reproducibility of Gabapentin. The CV% for Gabapentin ranged from 7.01% to 3.61% at all three levels of quality control.

[00240] Table 11 shows the reproducibility of Levetiracetam. The CV% for Levetiracetam ranged from 8.46% to 4.17% at all three levels of quality control.

[00241] Table 12 shows the reproducibility of Pregabalin. CV% for Pregabalin ranged from 6.10% to 4.08% at all levels of quality control.


[00242] Table 13 shows the reproducibility of Zonisamide CV% to Zonisamide ranged from 6.35% to 4.87% at all three levels of quality control.

[00243] Table 14 shows the reproducibility of Lamotrigine. CV% for Lamotrigine ranged from 6.77% to 6.10% at all three levels of quality control.

[00244] Table 15 shows the reproducibility for Lacosamide. The CV% for Lacosamide ranged from 5.78% to 3.26% at all three levels of quality control.

[00245] Table 16 shows the reproducibility of Rufinamide. CV% for Rurfinamide ranged from 9.12% to 5.78% at all three levels of quality control.

[00246] Table 17 shows the reproducibility of Felbamato. The CV% for Felbamato ranged from 8.63% to 5.89% at all three levels of quality control.

[00247] Table 18 shows the reproducibility of 10.11 Carbamazepine Epoxide. The CV% for 10.11 Carbamazepine Epoxide ranged from 8.46% to 5.89% at all three levels of quality control.

[00248] Table 19 shows the reproducibility of Phenytoin. The CV% for Phenytoin ranged from 8.40% to 7.26% at all three levels of quality control.

[00249] Table 20 shows the reproducibility of Carbamazepine. CV% for Carbamazepine ranged from 9.45% to 4.93% at all three levels of quality control.

[00250] Table 21 shows the reproducibility of Eslicarbamazepine. The CV% for Eslicarbamazepine ranged from 10.65% to 3.74% at all three levels of quality control.

[00251] Table 22 shows the reproducibility of Tiagabine. The CV% for Tiagabine ranged from 13.18% to 7.05% at all three levels of quality control.

[00252] Total reproducibility: Acceptability criteria: unacceptable if Total SD > 1/2TEa or Total SD must be less than a defined maximum SD or CV. CV% must be less than allowable < TEa/2. The Tea for this assay is determined to be 30%.
[00253] The CV% for Ethosuximide ranged from 12.84% to 1.1% at all three levels of quality control.
[00254] The CV% for Gabapentin ranged from 10.43% to 3.05% at all three levels of quality control.
[00255] The CV% for Levetiracetam ranged from 8.48% to 2.28% at all three levels of quality control.
[00256] The CV% for Pregabalin ranged from 10.21% to 2.43% at all three levels of quality control.
[00257] The CV% for Zonisamide ranged from 12.44% to 1.44% at all three levels of quality control.
[00258] The CV% for Lamotrigine ranged from 12.17% to 3.80% at all three levels of quality control.
[00259] The CV% for Lacosamide ranged from 12.17% to 3.80% at all three levels of quality control.
[00260] The CV% for Rufinamide ranged from 12.01% to 2.50% at all three levels of quality control.
[00261] The CV% for Felbamato ranged from 7.92% to 2.03% at all three levels of quality control.
[00262] The CV% for 10.11 Carbamazepine Epoxide ranged from 12.44% to 1.76% at all three levels of quality control.
[00263] CV% for Phenytoin ranged from 10.92% to 2.55% at all three levels of quality control.
[00264] The CV% for Carbamazepine ranged from 12.64% to 2.05% at all three levels of quality control.
[00265] The CV% for Eslicarbamazepine ranged from 13.49% to 3.60% at all three levels of quality control.
[00266] The CV% for Tiagabine ranged from 16.11% to 0% at all three levels of quality control.
[00267] Analytical Sensitivity: Limit of Detection (LOD) - Calculation: LOD = blank mean + 4SD. The following are LODs: Ethosuximide - 3.24 ng/ml; Levetiracetam - 0.22 ng/ml; Pregabalin - 0.29 ng/ml; Lamotrigine - 0.17 ng/ml; Lacosamide - 0.47 ng/ml.
[00268] Accuracy: Known Pattern Recovery - Acceptability Criteria: Error due to lack of perfect recovery (amount recovered LESS amount added) must be < 2 SD or CV 15% when TEa is 30%. Three whole blood samples were spattered at the following concentrations: 10, 30 and 60 ug/ml, each spatter level assayed in triplicate. There is no dilution analysis due to the way whole blood is collected and dried on the Mitra microsampling device.
[00269] Table 23 shows accuracy of Ethosuximide.



[00270] Table 24 shows the accuracy of levetiracetam.



[00271] Table 25 shows the accuracy of pregabalin.



[00272] Table 26 shows accuracy of zonisamide.



[00273] Table 27 shows accuracy of lamotrigine.



[00274] Table 28 shows accuracy of lacosamide.



[00275] Table 29 shows accuracy of rufinamide.



[00276] Table 30 shows accuracy of felbamate.



[00277] Table 31 shows accuracy of carbamazepine.



[00278] Table 32 shows accuracy of phenytoin.



[00279] Table 33 shows accuracy of carbamazepine.



[00280] Table 34 shows accuracy of eslicarbamazepine.



[00281] Table 35 shows accuracy of tiagabine.



[00282] Figure 40 shows the results of hematocrit study of gabapentin and rufinamide. Example 8: 25OH Hydroxy Vitamin D
[00283] In this assay, patient whole blood samples were analyzed.
[00284] Vitamin D in human blood was extracted from Mitra 20 uL microsampling device by adding 10 uL of internal standard and 500 uL of extraction solvent (1M ammonium hydroxide solution in ethyl acetate and methanol 50:50) in a clean 96-well plate. Mitra tips were dripped into the wells with the IS/extraction solvent mixture. The plate was mixed in a heated plate mixer/vortexer at 80 rpm for one hour at 45°C (Eppendorf mixmate). The extraction solvent in the mixed sample plate was dried under heated nitrogen @ 60°C for ~15 minutes to concentrate the sample. When drying was complete, 100 μl of 0.1 ng/ml derivatization reagent (PTAD) in acetonitrile was added to the sample wells and incubated at room temperature for one hour. 100 uL of HPLC grade water was added to the wells to quench the reaction. The samples were then transferred to a 96-well filter plate (Captiva ND) with a 96-well collection plate attached under it. Positive pressure was applied to the filter plate to allow the filtrate to pass. 25 uL of sample was injected into the LC-MSMS system.
[00285] Separation was achieved using a C18 column of reverse phase and mobile phase consisting of 0.1% aqueous formic acid (mobile phase A) and methanol and acetonitrile 50:50 (mobile phase B). The Aria LX system equipped with Agilent SL pumps was coupled to a TSQ Quantum Ultra triple quadrupole mass spectrometer as a detector, with a heated electrospray (HESI) source. 25-Hydroxyvitamins D2 and D3 were detected and quantified in MRM/SEM positive ionization mode scan. The following parameters were used: Ionization Voltage 5000 V; Steaming Temperature 450°C; Wrapping Gas 20 Arb; Aux 20 Arb; Collision pressure: 1.0 mTorr; Collision Power 16-18 V.
[00286] The following mass transitions are monitored.

[00287] Figure 41 shows the chromatogram of the analysis of 25-hydroxyvitamin D. Figure 42 shows the calibration curve of the analysis of 25-hydroxyvitamin D2. Figure 43 shows the calibration curve for 25-hydroxyvitamin D3 analysis.
[00288] The linear range of analysis was 5-100 ng/ml. The limit of quantification (LOQ) was 4 ng/ml.
[00289] The contents of articles, patents and patent applications, and all other electronically available documents and information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication were specifically and individually indicated is incorporated by reference. Applicant reserves the right to physically incorporate into this application any or all materials and information in any such articles, patents, patent applications or other physical and electronic documents.
[00290] The methods described illustratively herein may be suitably practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. In this way, for example, the terms "comprising", "including", "containing", etc., should be read expansively and without limitation. Further, the terms and expressions used herein have been used as descriptive rather than limiting terms, and there is no intent in the use of such terms and expressions to exclude any equivalents of the shown and described features or portions thereof. It is recognized that various modifications are possible within the scope of the claimed invention. In this way, it is to be understood that although the present invention has been specifically disclosed through preferred embodiments and optional features, modification and variation of the embodied invention disclosed herein may be carried out by those skilled in the art, and that such modifications and variations are considered to be within the scope of the present invention.
[00291] The invention has been described broadly and generally herein. Each of the more limited species and subgeneric groupings that fit the generic description are also part of the methods. This includes the generic description of methods with a negative condition or limitation removing any subject matter of the genus, regardless of whether or not the excised material is specifically mentioned therein.
[00292] Other modalities are within the claims that follow. Further, where features or aspects of the methods are described in terms of Markush groups, those skilled in the art will recognize that the invention is also described in terms of any individual member or subgroup of members of the Markush group as well.

权利要求:
Claims (24)
[0001]
1. Method for determining the amount of an analyte in a sample through mass spectrometry, characterized in that it comprises: (a) extracting an analyte from a sample collected by a microsampling device, in which the sample collected by the device microsampling has a volume of less than or equal to 100 µL; (b) ionizing the analyte to generate one or more detectable ions by mass spectrometry; and (c) determining the amount of the one or more ions by mass spectrometry; wherein the amount of the one or more ions determined is used to determine the amount of analyte in the sample.
[0002]
2. Method according to claim 1, characterized in that the amount of analyte in the sample is related to the amount of analyte in the patient.
[0003]
3. Method according to claim 1, characterized in that said sample comprises a sample of whole blood, urine, saliva, plasma or serum.
[0004]
4. Method according to claim 1, characterized in that the extraction step comprises adding an extraction buffer to the sample collected by a microsampling device, optionally wherein the extraction step comprises drying under nitrogen gas, optionally in that the extraction step comprises reconstitution of the sample in solution.
[0005]
5. Method according to claim 1, characterized in that the microsampling device comprises an apparatus that allows automation of extraction and mass spectrometric analysis of multiple samples at the same time.
[0006]
6. Method according to claim 1, characterized in that the extraction and mass spectrometry steps are performed in an online manner to allow automatic sample analysis.
[0007]
7. Method according to claim 1, characterized in that the sample collected by the microsampling device has a volume of less than or equal to 50 μL, optionally wherein the sample collected by the microsampling device has a volume of about 10 µL, about 15 µL, or about 20 µL.
[0008]
8. Method according to claim 1, characterized in that the sample is hydrolyzed before quantification by mass spectrometry.
[0009]
9. Method according to claim 1, characterized in that it further comprises purifying the sample before mass spectrometry, optionally wherein said purification comprises submitting the sample to liquid chromatography, optionally wherein the liquid chromatography comprises liquid chromatography of high performance (HPLC) or high turbulence liquid chromatography (HTLC).
[0010]
10. Method according to claim 1, characterized in that the sample is capillary blood.
[0011]
11. Method according to claim 1, characterized in that mass spectrometry is tandem mass spectrometry.
[0012]
12. Method according to claim 1, characterized in that the ionization is atmospheric pressure chemical ionization (APCI), optionally wherein the ionization is in positive ion mode.
[0013]
13. Method according to claim 1, characterized in that an internal standard for said analyte is added to the sample, optionally wherein the internal standard is deuterated or isotopically labeled.
[0014]
14. Method according to claim 1, characterized in that the microsampling device is enclosed in a cartridge designed for automation of extraction and mass spectrometric analysis.
[0015]
15. Method according to claim 1, characterized in that the microsampling device is a MITRA® tip.
[0016]
16. Method according to claim 1, characterized in that the analyte is a steroid, optionally wherein the steroid is cortisol, cortisone, progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, dehydroepiandrosterone, corticosterone, deoxycorticosterone, 11-deoxycortisol , pregnenolone, 17-hydroxypregnenolone, 18-hydroxycorticosterone, 21-deoxycortisol, 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3.
[0017]
17. Method according to claim 1, characterized in that the analyte is an opiate, optionally wherein the opiate is cis-tramadol, O-desmethyl tramadol, tapentadol, N-desmethyltapentadol, codeine, morphine, oxymorphone, norhydrocodone, oxycodone, noroxycodone, hydromorphone, hydrocodone, buprenorphine, norbuprenorphine, fentanyl, norfentanyl, 6-monoacetylmorphine (6-MAM), methadone, dihydrocodeine, naloxone, naltrexone, 6β-naltrexol, nalorphine, nalbuphine or 2-ethylidene -dimethyl-3,3-diphenylpyrrolidine (EDDP).
[0018]
18. Method according to claim 1, characterized in that the analyte is a benzodiazepine, optionally wherein the benzodiazepine is oxazepam, temazepam, lorazepam, nordiazepam, diazepam, chlordiazepoxide, triazolam, midazolam, alprazolam, clonazepam, brobazaepam, , nitrazepam, fenazepam, prazepam, medazepam, flunitrazepam or flurazepam.
[0019]
19. Method according to claim 1, characterized in that the analyte is an antiepileptic drug, optionally wherein the antiepileptic drug is valproic acid, tiagabine, topiramate, levitiracetum, lamotrigine, lacosamide, Ethosuximide, carbamazepine, eslicarbamazepine, 10, 11-carbamazepine, phenobarbital, rufinamide, primidone, phenytoin, zonisamide, felbamate, gabapentin or pregablin.
[0020]
20. Method according to claim 1, characterized in that the analyte is an immunosuppressant, optionally in which the immunosuppressant is cyclosporine A, sirolimus, tacrolimus or everolimus.
[0021]
21. Method according to claim 1, characterized in that the analyte is barbiturate, optionally wherein the barbiturate is phenobarbitol, amobarbitol, butalbital, pentobarbitol, secobarbitol or thiopental.
[0022]
22. Method according to claim 1, characterized in that the analyte is tamoxifen or a metabolite thereof, optionally wherein the metabolite is norendoxifene, N-Desmethyl-4-Hydroxy Tamoxifen, 4'-Hydroxy Tamoxifen, 4- Hydroxy Tamoxifen, N-Desmethyl-4'-Hydroxy Tamoxifen, N-Desmethyl Tamoxifen.
[0023]
23. Method according to claim 1, characterized in that the analyte is a drug for oncology, optionally in which the analyte is anastrozole, letrozole or exemestane.
[0024]
24. Method according to claim 1, characterized in that the analyte is tetrahydrocannabinol (THC) or a metabolite thereof.

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法律状态:
2020-07-28| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-03-30| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-05-11| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 27/05/2016, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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US201562167164P| true| 2015-05-27|2015-05-27|

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US62/167,164|2015-05-27|

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PCT/US2016/034815|WO2016191738A1|2015-05-27|2016-05-27|Methods for mass spectrometric quantitation of analytes extracted from a microsampling device|

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