• 专利附录
  • 专利说明
  • 权利要求
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  • 同族专利
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    • 专利摘要:
    An analysis method based on multiple sample injections for enrichments on a chromatographic column eliminates or reduces the overload of the chromatographic column caused by the mismatch of the sample solvent with the chromatographic mobile phase, and can be used for simplifying the sample preparation, improving the signal-to-noise ratio of the signal and the detection sensitivity, and can assist in solving the problem of enriching trace unstable or volatile tested substances in conventional sample preparation method.
    公开号:NL2029104A
    申请号:NL2029104
    申请日:2021-09-02
    公开日:2021-11-01
    发明作者:Shao Dongliang；Liu Niuniu；Gao Zhu；Liu Kun；Zhang Yang；Zhao Chengshi；Xu Deshuang；Zhou Fang；Yang Rong
    申请人:Anhui Guoke Testing Tech Co Ltd；
    IPC主号:
    专利说明:

    ANALYSIS METHOD BASED ON MULTIPLE SAMPLE INJECTIONS FORENRICHMENTS ON CHROMATOGRAPHIC COLUMN
    TECHNICAL FIELD The present disclosure relates to the technical field of chemical component analysis, and more specifically, to an analysis method based on multiple sample injections for enrichments on a chromatographic column.
    BACKGROUND ART The detection and analysis based on high performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC-MS) plays an important role in life science, product quality control, sports fair competition and other fields. In general, the main steps of sample preparation include selecting an appropriate solvent for liquid-phase/liquid-phase or liquid-phase/solid-phase extraction to achieve the purpose of enriching the target substances and removing impurities. Further treatments are required if the extraction working medium used does not match the liquid chromatography mobile phase or if the concentration of the substance to be tested in the extract liquid is too low. Otherwise, in the chromatographic analysis process, the former is easy to overload the chromatographic column and affect the separation effect, while the latter will make the detection fail to meet the requirements. Due to the large differences in physical properties, chemical properties and the like of the tested substances, especially in the case of unknown properties, it is sometimes difficult to find a suitable sample processing scheme. For example, the conventional method for solving the above problems is to remove the extraction medium by nitrogen blowing, heating, evaporation under reduced pressure, or the like, and then to redissolve the target detection substance in a suitable solvent. This step is labor- consuming and time-consuming, and easy to cause loss, isomerism, or decomposition of the substance to be tested, causing deviation. For the volatile substance to be tested, this method cannot be used. Therefore, in the actual detection process, analysts tend to adopt the measure of reducing the volume of sample injection or using a large column load chromatography column to detect the substance to be tested in the extraction medium directly. However, this method is often accompanied by insufficient sample injection, which leads to the failure of detection, and cannot solve the problem of low concentration of the substance to be detected.
    3
    SUMMARY The present disclosure aims to solve the above defects, and provides an analysis method based on multiple sample injections for enrichments on a chromatographic column. In order to achieve the above purpose, the technical scheme of the present disclosure is as follows: an analysis method based on multiple sample injections for enrichments on a chromatographic column, wherein: when being applied in a reverse chromatography, the method includes the following steps : step 1: performing a column equilibrium on a liquid phase by adopting an aqueous phase proportion higher than that of a mobile phase used for normal analysis, wherein the mobile phase used in the process of the column equilibrium includes a liquid A and a liquid B, a proportion of the liquid A is 0.00%-95.00%, and a balance is the liquid B; step 2: performing multiple sample injections on the liquid phase by adopting the aqueous phase proportion higher than that of the mobile phase used for normal analysis, and enriching a substance to be tested on the chromatographic column; step 3: performing a sample analysis after a normal solvent ratio is restored.
    Further, the liquid A is an organic solvent or water/organic mixed solvent with or without a buffer reagent, and the liquid B is an aqueous solvent or water/organic mixed solvent with or without a buffer reagent.
    Further, the number of the sample injections in the step 2 is 2-500 times.
    Further, when the method is applied for testing low-concentration plant-derived anthocyanin, chromatographic conditions are: an Agilent PorousShell 120 EC-C 182.7um 3.0 x150mm is the chromatographic column, a column temperature is 35°C, a number of the sample injections is 10 times with a volume of 2.0 ul each time, a gradient elution condition is 0.00min-44.00min, increasing a proportion of the liquid A in the mobile phase from 0.00% to 90.00%, and then decreasing to 0.00%, decreasing a proportion of the liquid B in the mobile phase from 100.00%
    to 10.00%, and then increasing to 100.00%, a flow rate is 0.300ml/min-1.200ml/min, the liquid As a 100% acetonitrile, and the liquid B is a 1.00% aqueous formic acid. Further, when the method is applied for analyzing an N-isopentyl dendrobium in a dendrobium extract diluent, chromatographic conditions are: an Agilent Poroshell 120 EC-C18 2.7um 3.0 x150mm is the chromatographic column, a column temperature is 35°C, a number of the sample injection is 10 times with a volume of 3.0ul each time, a gradient elution condition is 0.00min -98.00min, increasing a proportion of the liquid A in the mobile phase from 0.00% to 95.00%, and then decreasing to 0.00%, decreasing a proportion of the liquid B in the mobile phase from
    100.00% to 5.00%, and then increasing to 100.00%, a flow rate is 0.600ml/min-1.000ml/min, the liquid A is a 100% acetonitrile, and the liquid B is a 0.100% aqueous formic acid. Further, when the method is applied for testing an N-isopentyl dendrobium in a dendrobium extract diluent, chromatographic conditions are: an Agilent Poroshell 120 EC-C18 2. 7um 3.0 x 150mm is the chromatographic column, a column temperature is 35°C, a number of the sample injections is 15 or 100 times with a volume of 3.0ul each time, a gradient elution condition is
    0.00min -98.00min, increasing a proportion of the liquid A in the mobile phase from 0.00% to
    95.00%, and then decreasing to 0.00%, decreasing a proportion of the liquid B in the mobile phase from 100.00% to 5.00%, and then increasing to 100.00%, a flow rate is 0.600 ml/min-
    1.000 ml/min, the liquid A is a 100% acetonitrile, and the liquid B is a 0.100% aqueous formic acid. Further, when being applied in a normal chromatography, the method includes the following steps : step 1: performing a column equilibrium firstly; wherein a mobile phase used in the process of the column equilibrium includes a liquid A and a liquid B, a proportion of the liquid A is 0.00%-
    95.00%, and a balance is the liquid B; step 2: performing multiple sample injections, and enriching a substance to be tested on the chromatographic column; step 3: performing a sample analysis. Further, the liquid A is an isopropyl alcohol, an ethyl acetate, a tetrahydrofuran, a methanol, a mixed solvent of a hexane and an isopropyl alcohol, a mixed solvent of a hexane and an ethyl acetate, a mixed solvent of a hexane and a tetrahydrofuran or a mixed solvent of a dichloromethane and a methanol. The liquid B is a hexane, a dichloromethane, a mixed solvent of a hexane and an isopropanol, a mixed solvent of a hexane and an ethyl acetate, a mixed solvent of a hexane and a tetrahydrofuran, or a mixed solvent of a dichloromethane and a methanol.
    Compared with the prior art, the disclosure has the following beneficial effects: the sample injection method disclosed by the present disclosure eliminates or reduces the overload of the chromatographic column caused by the mismatch of the sample solvent with the chromatographic mobile phase, thereby realizing the effective detection of the substance to be tested, simplifying the preparation of the sample, improving the signal-to-noise ratio of the signal and the detection sensitivity, and can assist in solving the problem of enriching trace unstable or volatile tested substances in a conventional sample preparation method.
    BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a comparison diagram of detection results of the first embodiment of the present disclosure. FIG. 2 is a comparison diagram of detection results of the second embodiment of the present disclosure. FIG. 3 is a comparison diagram of detection results of the second embodiment of the present disclosure. FIG. 4 is a comparison diagram of detection results of the third embodiment of the present disclosure. FIG. 5 is a comparison diagram of detection results of the third embodiment of the present disclosure. FIG. 6 is a graph showing a detection result of changing the number of sample injections according to the third embodiment of the present disclosure.
    DETAILED DESCRIPTION OF THE EMBODIMENTS The technical scheme in the embodiments of the present disclosure will be described clearly and completely with reference to the drawings in the embodiments of the present disclosure, and obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of them. The embodiments in this disclosure and the features in the embodiments can be combined with each other without conflicts. Based on the embodiments of the present disclosure, all other embodiments obtained by a person of ordinary skill in the art without creative works are within the scope of the present disclosure.
    5 It should be noted that if there are directional indications (such as up, down, left, right, front, back, ...) in the embodiment of the present disclosure, the directional indications are only used to explain the relative position relationship and movement situation among components in a certain specific posture (as shown in the figure), and if the specific posture changes, the directional indications will change accordingly.
    In addition, if there are terms of ‘first’ and ‘second’ in the embodiments of the present disclosure, the terms of ‘first’ and ‘second’ are only used for description purposes, and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of indicated technical features. Therefore, the features defined with ‘first’ and ‘second’ can include at least one of the features explicitly or implicitly. In addition, the meaning of ‘and/ or’ appearing in the whole text includes three parallel schemes, taking ‘A and/or B’ as an example, including A scheme, B scheme, or a scheme that both A and B satisfy. In addition, ‘multiple’ means more than two. In addition, the technical schemes among the various embodiments can be combined with each other, but must be based on the implementation by the ordinary person skilled in the art, when the combination of the technical solutions appears to be contradictory to each other or cannot be realized, it should be considered that the combination of the technical scheme does not exist and is not within the scope of protection claimed by the present disclosure.
    The first Embodiment A multiple sample injections method for detecting plant-derived anthocyanin monomers by HPLC: Specific steps:
    1. Sample preparation Weighing delphinium pigment, cornflower pigment, petunia pigment, geranium pigment, paeoniflorin and mallow pigment accurately, dissolving them in a volumetric flask filled with 10% hydrochloric acid methanol solution (v/v, similarly hereinafter) at constant volume, and storing the single standard solution in a brown bottle at -18°C; preparing the anthocyanidin standard sample by diluting with 10% hydrochloric acid methanol after mixing single standard solution, the final concentration 1s 0.0663ug/ml and 3.3 lug/ml, and the standard sample was filtered by 0.22um Teflon membrane for later use.
    2. Configuration of HPLC The chromatographic column is Agilent Poroshell 120 EC-C18 2. 7um 3.0 x 150mm. The liquid phase A is 100% acetonitrile, and the liquid phase B is 1.00% formic acid aqueous solution (v/v, similarly hereinafter).
    3. Set up the sample injection procedure Liquid phase condition time (min) A (%) B (%) flow rate (mL/min) ow ow [ww [om ow ew [ww | ow Column temperature: 35°C Detection wavelength: 530nm
    4. Set up the sample analysis procedure Liquid phase condition Time A B flow rate (min) (%) (%) (mL/min) an on ww [ow an [ow ww [ow aa ow [ww | ow aw [ow ww | ow an ow [ww | ow Data acquisition (a) FIG. 1A curve obtained by conventional sample injection method by injecting 2.0uL anthocyanin standard sample with a concentration of 3.3 1ng/uL: using the sample analysis procedure to inject a 2.0uL anthocyanin standard sample (3.31ng/uL methanol solution) and performing an HPLC analysis. (b) FIG. 1B curve obtained by conventional sample injection method by injecting 2.0uL anthocyanin standard sample with a concentration of 0.0662ng/uL: using the sample analysis procedure to inject a 2.0ul anthocyanin standard sample (0.0662 ng/uL methanol solution) and performing an HPLC analysis. (€) FIG.
    IC curve obtained by conventional sample injection method by injecting 20.0uL anthocyanin standard sample with a concentration of 0.0662ng/uL: using the sample analysis procedure to inject a 20.0uL anthocyanin standard sample (0.0662ng/uL methanol solution) and performing an HPLC analysis. (d) FIG.
    ID curve obtained by multiple sample injections method by injecting 20.0uL anthocyanin standard sample with a concentration of 0.0662ng/uL:: using the sample injection procedure continuously for 9 times, injecting 2.0uL anthocyanin standard sample each time (0.0662ng/uL methanol solution), and then using the sample analysis procedure to inject a 2.0uL anthocyanin standard sample (0.0662 ng/uL methanol solution) and performing an HPLC analysis.
    In order to realize multiple sample injections under the condition of a high proportion of aqueous phase, the existing anthocyanidin sample analysis procedure was modified from the original 11.00: 89.00 acetonitrile/1.00% formic acid aqueous solution (v/v, similarly hereinafter) for the liquid equilibrium to 0.00: 100.00 acetonitrile/ 1.00% formic acid aqueous solution; if necessary, perform multiple sample injections under this condition; at the start of the analysis, the mobile phase was rapidly restored to 11.00: 89.00 acetonitrile/1.00% formic acid aqueous solution and, on this basis, the desired liquid phase gradient was set and modified according to the results of the analysis.
    For the final liquid phase conditions, please refer to the experimental procedures in this section.
    The retention time of the 530nm absorption peak is usually used as the basis for the determination of each component in the anthocyanin detection by an HPLC.
    Therefore, a standard sample of 2.0uL with a relatively high concentration of anthocyanin (3.31 ng/uL methanol solution) was firstly injected by the conventional sample injection method, and chromatographic analysis was performed, the ordinate of the analysis results was reduced to a 5: 1 ratio and shown as curve A in FIG. 1, it can be seen that there is no significant column overload caused by this volume sample injection, and the chromatographic peaks of the obtained components are in good shape, so the analysis results are taken as reference, in the figure, swallowwort pigment, cornflower pigment, petunia pigment, geranium pigment, paeoniflorin, and mallow pigment are marked as 1, 2, 3, 4, 5 and 6, respectively.
    In order to compare the results of detecting low concentration anthocyanin standard sample using the multiple sample injections method with the conventional sample injection method, the standard sample concentration was diluted to 0.0662ng/uL of methanol, at this concentration, taking the previous sample injection volume, i.e. 2.0uL, the instrument used could only barely detect the cornflower pigment, and the result is shown as the curve B in FIG. 1. On the basis of the above reasons, the sample injection volume was increased to 20.0uL, and the result shows that the components of the cyanine could be distinguished, but the sample solvent causes the chromatographic column to be overloaded, thereby reducing the separation effect, increasing the difficulty of distinguishing each component, and the result is shown as the curve C in FIG. 1. In view of the fact that the chromatographic column is overloaded during the detection of actual samples with complex components, the problem of discrimination will become more prominent, the method described in the present disclosure was adopted, which is, 10 injections of 2.0uL per injection.
    The peak shape of the obtained chromatographic anthocyanidin components is good and the retention time is close to the control, and the result is shown as the curve D in FIG.L.
    In the process of multiple sample injections, the time required for each sample injection is mainly relate to the completion of all mechanical actions by the instrument.
    The average time for an automatic sample injector to complete a sample injection herein is about 50 seconds, and below is the same.
    Obviously, the sample injection at this rate does not overload the chromatographic column due to column equilibration. In view of the above, the detection sensitivity of anthocyanin by multiple injections method is increased compared with a single sample injection of 2.0uL. Compared with a single sample injection of 20.0uL, the results obtained by the multiple sample injections method are more favorable for the identification of anthocyanin components when the total sample injection volume is the same. The second Embodiment A multiple sample injections method for analyzing N-isopentenyl dendrobinium in dendrobium extracted diluent by liquid chromatography-secondary mass spectrometry Specific steps:
    1. Sample preparation Cleaning fresh dendrobium stem, cutting into sections (1-2cm), drying in oven (50-557C), pulverizing, sieving through 50 mesh, weighing 0.1471g of powder into an empty bottle, adding
    1.47mL of methanol for ultrasonic extraction for 40 min, passing through a 0.22um nylon membrane, and diluting with methanol as needed.
    2. Configuration of liquid phase mass spectrometry The chromatographic column is Agilent Poroshell 120 EC-C18 2.7um 3.0 x 150mm. The liquid phase A is 100% acetonitrile, and the liquid phase B is 0.100% formic acid aqueous solution.
    3. Liquid phase mass spectrometry sample injection procedure Chromatographic condition an [ow [ow | om on ow [ow | om Column temperature: 35°C Secondary mass spectrometry parameters:
    Fragmentor (V) Precursor (m/z) Scan range (m/z) 50 - 400
    4. Liquid phase mass spectrometric sample analysis procedure Chromatographic condition:
    50.00 23.00 77.00 0.600
    82.00 95.00 0.600
    90.00 95.00 1.000
    91.00 000 100.00 0.600
    98.00 000 100.00 0.600 Column temperature: 35°C Secondary mass spectrometry parameters:
    5. Data acquisition (a) FIG. 2A curve and FIG. 3a spectrogram obtained by the multiple sample injections method: using the liquid phase mass spectrometric sample analysis procedure continuously for 9 times, injecting 3.0uL 1:100,000 dendrobium nobile extract diluent each time, and then using the liquid phase mass spectrometric sample analysis procedure to inject a 3.0uL sample and performing instrument analysis. (b) FIG. 2B curve and FIG. 3b spectrogram obtained by the conventional sample injection method: using the liquid phase mass spectrometric sample analysis procedure to inject 30.0uL 1:100,000 dendrobium nobile extract diluent and performing instrument analysis. (c) FIG. 2C curve and FIG. 3c spectrogram obtained by the conventional sample injection method: using the liquid phase mass spectrometric sample analysis procedure to inject 3.0uL 1:100,000 dendrobium nobile extract diluent and performing instrument analysis. (d) FIG. 3d spectrogram obtained by the conventional sample injection method: using the liquid phase mass spectrometric sample analysis procedure to inject 3.0uL 1:1000 dendrobium nobile extract diluent and performing instrument analysis.
    Results: In order to realize multiple sample injections under the condition of a high proportion of aqueous phase, the existing anthocyanidin sample analysis procedure was modified from the original 5.00:95.00 acetonitrile/0. 100% formic acid aqueous solution for the liquid equilibrium to 0.00:100.00 acetonitrile/0.100% formic acid aqueous solution.
    If necessary, multiple sample injections are performed under this condition.
    At the start of the analysis, the mobile phase was rapidly restored to 5.00: 95.00 acetonitrile /0.100% formic acid aqueous solution and, on this basis, the desired liquid phase gradient was set and modified according to the results of the analysis.
    For the final liquid phase conditions, please refer to the experimental procedures in this section.
    Using liquid chromatography-secondary mass spectrometry (LC-MS/MS) with different sample injection methods to analyze the methanol diluted dendrobium nobile extract (dendrobium extract: methanol ratio is 1:100,000), the secondary mass spectrometric total ion chromatogram (LC-MS/MS TIC, parent ion m/z 332.3) of N-Isopentenyl denorubium and the signal-to-noise ratio of the corresponding target peaks are shown in FIG. 2, wherein, the determination of the target peak has been confirmed by the corresponding secondary mass spectra (FIG. 3a-c) and the analysis results of higher concentration samples.
    Whether it is the total ion current intensity or the signal-to-noise ratio of the target peak, adopting multiple sample injections method (a total of 10 sample injections, 3.0uL per injection, and curve A in FIG. 2) has obvious advantages over conventional sample injection (a single injection of 4.5uL, curve C in FIG. 2, or a direct injection of 30.0uL, curve B in FIG. 2). It can also be seen that a single large volume sample injection of 30.0uL does not improve the signal-to-noise ratio of the target peaks compared to the conventional sample injection of 4.5uL, but rather reduces it because during the large volume injection, the sample solvent causes the column to be overloaded, which affects the target peak shape, and finally results in a decrease in signal-to- noise ratio.
    As shown in FIG. 3, in terms of the quality of the secondary mass spectrum, the advantages of the multiple sample injections method is also obvious.
    FIG. 3a is the secondary spectrum of N- isopentenyl dendrobidium obtained by the multiple sample injections method (10 injections of methanol diluent with a concentration of 3.0uL per injection), the characteristic peak basically conforms to the control spectrum (FIG. 3d, conventional sample injection of 3.0uL 1: 1,000 dilution). Compared with conventional sample injections of 30.0uL (FIG. 3b) and 4.5uL (FIG. 3c) diluted solution with the same concentration (1: 100,000 methanol), the baseline noise is obviously improved.
    In summary, it is easy to realize multiple sample injections by using independent sample injection procedure. Moreover, the multiple sample injections method can simplify the sample processing and increase the signal-to-noise ratio of liquid chromatography-secondary mass spectrometry when it is used for the direct detection of low concentration samples with methanol as solvent. The third Embodiment A multiple sample injections method for analyzing an N-isopentenyl dendrobinium in a dendrobium extracted diluent by liquid chromatography-secondary mass spectrometry Specific steps:
    1. Sample preparation Cleaning fresh dendrobium stem, cutting into sections (1-2cm), drying in oven (50-55°C), pulverizing, sieving with 50 mesh, weighing 0.1471g of powder into an empty bottle, adding
    1.47mL of methanol for ultrasonic extraction for 40 min, passing through a 0.22um nylon membrane, and diluting with methanol as needed.
    2. Configuration of liquid phase mass spectrometry The chromatographic column is Agilent Poroshell 120 EC-C18 2.7um 3.0 x 150mm. The liquid phase A is 100% acetonitrile, and the liquid phase B is 0.100% formic acid aqueous solution.
    3. Liquid phase mass spectrometry sample injection procedure Chromatographic condition: ow ow [ww | om ow ew [ww | ow Column temperature: 35°C Secondary mass spectrometry parameters:
    Fragmentor (V) Precursor (m/z) 360.3 Scan range (m/z) 50 - 400
    4. Liquid phase mass spectrometry sample injection procedure Chromatographic conditions
    50.00 23.00 77.00 0.600
    90.00 95.00 1.000
    91.00 000 100.00 0.600
    98.00 000 100.00 0.600 Column temperature: 35°C Secondary mass spectrometry parameters: Fragmentor (V)
    5. Data acquisition (a) FIG. 4A curve and FIG. 5a spectrogram obtained by the multiple sample injections method: using the liquid phase mass spectrometric sample analysis procedure continuously for 14 times, injecting 3.0uL 1:100,000 dendrobium nobile extract diluent each time, and then using the liquid phase mass spectrometric sample analysis procedure to inject a 3.0uL sample and performing instrument analysis.
    (b) FIG. 4B curve and FIG. Sb spectrogram obtained by injecting of the conventional sample injection method: using the liquid phase mass spectrometric sample analysis procedure to inject 45.0uL 1:100,000 dendrobium nobile extract diluent and performing instrument analysis.
    (c) FIG. 4C curve and FIG. 5c spectrogram obtained by injecting of the conventional sample injection method: using the liquid phase mass spectrometric sample analysis procedure to inject 3.0uL 1:100,000 dendrobium nobile extract diluent and performing instrument analysis.
    (d) FIG. 5d spectrogram obtained by injecting of the conventional sample injection method: using the liquid phase mass spectrometric sample analysis procedure to inject 3.0uL 1:1,000,000 dendrobium nobile extract diluent and performing instrument analysis.
    (e) The curve A and the upper left corner spectrogram in FIG. 6 are obtained by the multiple sample injections method: using the liquid phase mass spectrometric sample analysis procedure continuously for 99 times, injecting 3.0uL 1:100,000 dendrobium nobile extract diluent each time, and then using the liquid phase mass spectrometric sample analysis procedure to inject a 3.0uL sample and performing instrument analysis.
    (f) The curve B in FIG. 6 obtained by injecting of the conventional sample injection method:
    using the liquid phase mass spectrometric sample analysis procedure to inject 3.0uL 1:1,000 dendrobium nobile extract diluent and performing instrument analysis.
    Results: In order to realize multiple sample injections under the condition of a high proportion of aqueous phase, the existing anthocyanidin sample analysis procedure was modified from the original 5.00:95.00 acetonitrile/0.100% formic acid aqueous solution for the liquid equilibrium to 0.00:100.00 acetonitrile/0.100% formic acid aqueous solution, if necessary, perform multiple sample injections under this condition; at the start of the analysis, the mobile phase was rapidly restored to 5.00:95.00 acetonitrile /0.100% formic acid aqueous solution and, on this basis, the desired liquid phase gradient was set and modified according to the results of the analysis. For the final liquid phase conditions, please refer to the experimental procedures in this section. The results of high performance liquid chromatography-secondary mass spectrometry (LC-MS/MS, parent ion: m/z 360.3) analysis of the target n-isopentyl dendroxinium are shown in FIGs. 4-6 respectively. The curve A in FIG. 4 is the secondary mass spectrometry total ion flow chromatography (LC-MS/MS TIC) obtained by multiple sample injections (15 sample injections, 3.0uL each time), it can be seen from the figure that the target peak can be identified, and verified by the corresponding secondary mass spectra (FIG. 5a) and analysis of the higher concentration extracts.
    For comparison, the peak of N-isopentyl dendroxinium could not be distinguished with a single injection of 3.0uL dilution by conventional sample injection method (curve C in FIG. 4); if an attempt is made to improve the detection of the sample by simply increasing the sample injection volume, a single sample injection of 45.0uL is the same as the total amount of samples injected by the previous multiple sample injection method, and the results also did not have any significant peak formation (curve B in FIG. 4).
    It can be seen that the method of multiple sample injections can effectively enhance the detection of low concentration target substances by secondary mass spectrometry total ion chromatography (LC-MS/ MS TIC).
    In order to provide a direct comparison of the secondary mass spectra obtained by the two sample injection methods, FIG. 5 shows respectively: a. 45.0uL sample injection by multiple sample injections method (3.0uL per injection, 15 times in total), b. 45uL conventional sample injection, and c. 3.0uL conventional sample injection of diluent to obtain N-isopentenyl dendrinium spectra; as a control, FIG. 5d is the result of a conventional sample injection of
    3.0uL 1:1,000 dilution; compared with the conventional sample injection method for analyzing low concentration diluent, the secondary mass spectrum obtained by multiple sample injections method retains the characteristic peak of N-isopentyl dendrinium in the control spectrum (FIG.
    5d), and the baseline noise of the spectrum is lower.
    A careful comparison of the secondary mass spectrogram obtained by the multiple sample injections method (FIG. 5a) with the control spectrogram (FIG. 5d) shows that for some low abundance peaks, such as m/z 74 and 89, it is difficult to determine independently whether it is characteristic peak of isopentenyl dendroxinium or noise.
    For this reason, on the basis of using the same volume of dendrobium nobile dilution (3.0uL) each time, we increased the number of sample injections significantly and make the total number up to 100; in order to avoid unnecessary loss of the automatic sample injection device, this example was only implemented once.
    The upper left corner of FIG. 6 shows the secondary mass spectrum obtained from 100 sample injections, and the quality of this spectrum is improved compared with the previous multiple sample injections (FIG. 5a, 15 sample injections in total, 3.0uL each time); besides, the difference of relative abundance of characteristic ions from that of the control spectrum is small. The curve A in FIG. 6 is the corresponding total ion flow chromatogram, and the retention time of the target peak is still very close to that of the control obtained by the conventional method (same as curve B in FIG. 6, 3.0uL 1:1000 dilution once injected), however, the peak shape of the former is broadened, and the full width at half maxima (FWHM) increased from 0.34 minutes (curve B) to about 0.65 minutes, which may be due to the appearance of sample diffusion on the column during the long sample injection period.
    In summary, this embodiment again shows that the sample processing can be simplified and the sensitivity of detection and analysis can be improved by using a separate sample injection procedure and multiple sample injections method.
    The above three embodiments show the use of multiple sample injection method in HPLC and LC-MS detection with independent sample injection procedures; without adding any devices and control software, and without significantly reducing the separation effect of the chromatographic column, the multiple sample injections method can enrich the samples on the column by several times, so as to eliminate or reduce the overload of the chromatographic column caused by the incompatibility of sample solvents, increase the total injection volume, improve the signal-to-noise ratio of detecting low-concentration targets, and enhance the sensitivity near the lower detection limit.
    After optimization of column equilibrium conditions, single sample injection volume and total sample injection times, this strategy can aid analysis based on other solvent samples.
    It can be seen that the multiple sample injections method is simple and easy, which can be used to simplify the sample preparation and help the analyzers avoid the difficulties in some conventional sample preparation methods.
    It will be apparent to those skilled in the art that the present disclosure is not limited to the details of the above-described exemplary embodiments, and that the present disclosure can be embodied in other specific forms without departing from the spirit or essential characteristics of the present disclosure.
    Accordingly, the embodiments should be considered exemplary and non-limiting in all respects, the scope of the disclosure being defined by the appended claims rather than the foregoing description, It is therefore intended that all variations falling within the meaning and scope of the equivalents of the claims be encompassed within the present disclosure.
    权利要求:
    Claims (8)
    [1]
    CONCLUSIONS Ll. An analysis method based on multiple test injections for enrichments on a chromatographic column and applied in a reverse chromatography, comprising: step 1: firstly performing a column equilibrium, where a mobile phase used in the column equilibrium process is a liquid A and comprises a liquid B; one wherein a ratio of liquid A is 0.00%-95.00% and a balance is liquid B; step 2: multiple test injections are performed and a substance is enriched to be tested on the chromatographic column; and step 3: consists in making a pilot analysis.
    [2]
    The method of claim 1, wherein liquid A is an organic solvent or a water/organic mixed solvent with or without a buffer reagent and liquid B is an aqueous solvent or water/organic mixed solvent with or without a buffer reagent.
    [3]
    The method of claims 1 or 2, wherein the number of test injections in step 2 is 2-500 times.
    [4]
    The method of claims 1 or 2, wherein when the method is used to test a low concentration vegetable anthocyanin, the chromatographic conditions are as follows: an Agilent Porousshell 120 EC-C18 2.7 Um 3.0 x 150 mm is the chromatographic column, a column temperature is 35°C, the number of test injections is 10 times with a volume of 2.0 µl each, a gradient elution state is 0.00 min -44.00 min, a ratio of liquid A in the mobile phase is increased from 0.00% to 90.00% and then the ratio is again decreased to 0.00%, decreasing a ratio of liquid B in the mobile phase from 100.00% to 10.00% and then to be increased again to 100.00%, a flow rate is 0.300 ml/min-1,200 ml/min, liquid A is a 100% acetonitrile and liquid
    B is a 1.00% hydrous formic acid.
    [5]
    The method of claims 1 or 2, wherein when the method is used to test an N-isopentyl dendrobium in a dendrobium extract based diluent and the chromatographic conditions are as follows: an Agilent Poroshell 120 EC-C18 2 .7 µm 3.0 x 150 mm is the chromatographic column, a column temperature is 35°C, the number of test injections is 10 times with a volume of 3.0 µl each, a gradient elution state is 0.00 min - 98.00 min, wherein the ratio of liquid A in the mobile phase is increased from 0.00% to 95.00% and then decreased to 0.00%, wherein a ratio of liquid B in the mobile phase is decreased from 100.00% to 5.00% and then the ratio is increased again to 100.00%, has a flow rate of 0.600 ml/min-1,000 ml/min, and wherein liquid A is a 100% acetonitrile and liquid B is a 0.100% hydrous formic acid.
    [6]
    The method of claims 1 or 2, wherein when the method is used to test an N-isopentyl dendrobium in a dendrobium extract based diluent, the chromatographic conditions are as follows: an Agilent Poroshell 120 EC-C18 2.7 µm is 3.0 * 150 mm is the chromatographic column, a column temperature is 35°C, the number of test injections is 15 or 100 times with a volume of 3.0 µl each, a gradient elution state 0.00 min - 98.00 min 1s, wherein the ratio of liquid A in the mobile phase is increased from 0.00% to 95.00% and then decreased to 0.00%, whereby a ratio of liquid B in the mobile phase is decreased from 100.00% to 5 0.00% and then the ratio is increased again to 100.00%, has a flow rate of 0.600 ml/min-1,000 ml/min, and wherein liquid A is a 100% acetonitrile and liquid B is a 0.100% hydrous formic acid.
    [7]
    The method according to claims 1 or 2, wherein, when the method is used in a normal chromatography, the method comprises the following steps:
    step 1: firstly, performing a column equilibrium, wherein a mobile phase used in the column equilibrium process comprises a liquid A and a liquid B; and wherein the ratio of liquid A is 0.00%-95.00% and a balance liquid B is 15; step 2: performing multiple test injections and enriching a substance to be tested on the chromatographic column; and step 3: consists in making a pilot analysis.
    [8]
    The method according to claim 7, wherein liquid A is an isopropyl alcohol, an ethyl acetate, a tetrahydrofuran, a methanol, a mixed solvent of a hexane and an isopropyl alcohol, a mixed solvent of a hexane and an ethyl acetate, a mixed solvent of a hexane and a tetrahydrofuran or a mixed solvent of a dichloromethane and a methanol; and liquid B is a hexane, a dichloromethane, a mixed solvent of a hexane and an isopropanol, a mixed solvent of a hexane and an ethyl acetate, a mixed solvent of a hexane and a tetrahydrofuran, or a mixed solvent of a dichloromethane and a methanol .
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    CN112083094A|2020-12-15|
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    CN202010921084.8A|CN112083094A|2020-09-04|2020-09-04|Analytical method for enrichment on chromatographic column based on multiple sample injection|
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