Probe composition LC-MS / MS analysis method suitable for biological exemption permeability evaluation

Abstract

The invention discloses an analysis method development kit capable of being quickly applied to model database establishment. The development kit covers 10 model drugs with low permeability, medium permeability and high permeability, a liquid chromatogram tandem triple quadrupole mass spectrum system which is most popularized is applied, a simplest'one-step precipitation 'sample pretreatment method is used, only two specifications of chromatographic columns are used, and the model drugs with low permeability, medium permeability and high permeability are obtained. The real-time monitoring of the quantitative concentration of 10 model drugs in a biological sample can be achieved, and the lower quantitative limit is as low as 1.0-5.0 ng / mL; the sample concentration of the cell penetrating fluid is measured according to a method established by LC-MS / MS, the accumulated transport amount Q and papp of the medicine in a Caco-2 monolayer cell membrane are solved, and permeability evaluation is carried out according to the accumulated transport amount Q and papp; the analysis method disclosed by the invention is good in specificity, high in sensitivity and wide in dynamic linear range, and the accuracy, precision, reproducibility and quality control of the analysis method all meet biological sample analysis requirements.

Description

Technical Field

[0001] The present invention relates to the technical field of composition technology, and in particular to a probe composition LC-MS / MS analysis method package suitable for bio-waiver permeability evaluation. Background Art

[0002] Replacing in vivo bioequivalence studies with in vitro evaluation methods during generic drug research can reduce clinical resource consumption and speed up the drug launch process, which is the main reason for bioequivalence exemption based on the Biopharmaceutics Classification System (BCS). According to the basic theory of BCS, the main factors affecting the absorption rate and extent of active pharmaceutical ingredients (API) in common oral solid preparations are the BCS category of API and the dissolution characteristics and excipient composition of the drug preparation. Based on the water solubility and intestinal permeability of API, it is divided into four categories, namely Class I (high solubility, high permeability), Class II (low solubility, high permeability), Class III (high solubility, low permeability) and Class IV (low solubility, low permeability). For drugs of BCS Class I and Class III, as long as other excipient components in the prescription do not significantly affect the absorption of API, and it is not a drug with a narrow therapeutic window or oral absorption, it is not necessary to prove the possibility of bioavailability and bioequivalence of the drug in vivo, that is, bioequivalence exemption.

[0003] With the rapid development of generic drugs in my country, the demand for biowaiver applications based on BCS is becoming more and more urgent. When the drug preparation is a common oral preparation with systemic effect, and the active ingredient of the drug meets the solubility and permeability (BCS I and III) standards, and the dosage form and specifications of the test preparation are the same as those of the reference preparation, the BCS-based biowaiver can be applied. The U.S. Food and Drug Administration (FDA) is the first agency to propose bioequivalence exemption guidelines, and has continuously updated the guidelines based on development. The scope of exemption is mainly BCS I and III, and there are more restrictions on BCS III drugs. For BCS III drugs, permeability is the rate-limiting step for drug absorption in the body. For permeability requirements, EMA has given more specific requirements than FDA. The European Medicines Agency (EMA) emphasizes that permeability data should come from human studies and stipulates that data should be obtained based on reliable human studies and reasonable mass balance studies.

[0004] In May 2016, the former State Food and Drug Administration issued the "Guidelines for Exemption of Human Bioequivalence Testing", which was mainly formulated with reference to the FDA guidelines, and also referred to the 2015 edition of the "Chinese Pharmacopoeia", the 39th edition of the "United States Pharmacopoeia" and the relevant technical requirements of the World Health Organization (WHO) and EMA. Among them, for the detection of API intestinal permeability, three detection methods that do not involve human subjects are recommended: in vivo or in situ intestinal perfusion of suitable animal models, permeability detection of ex vivo intestinal tissue, and permeability detection of suitable monolayer epithelial cells. The research method is the same as that of the FDA. In many cases (for example, absolute bioavailability is equal to or greater than 85%; equal to or greater than 85% of the drug is excreted in the urine in the form of the prototype drug, etc.), a single detection method may be sufficient. However, when a single permeability test is not sufficient to fully describe the permeability category of the drug, it is recommended to use two different analytical methods. When there are contradictions in the results of studies of different experimental types, human data should be considered to replace in vitro or animal data.

[0005] To explain the practicality of a permeability test method for human biowaiver, sufficient model drugs should be used to illustrate the order relationship between the permeability test values ​​and drug absorption data obtained in vivo or in vitro. For in vivo intestinal perfusion test methods in humans, NMPA recommends 6 model drugs for validation. For in vivo or in situ perfusion studies in animals or in vitro cultured cell studies, 20 model drugs are recommended for validation. At the same time, based on the variability of the study, it should also be considered to use a sufficient number of subjects, animals, ex vivo tissue samples or monolayer cells to maximize the reliability of the evaluation data. At the same time, this order relationship should also take into account the precise distinction between high and low intestinal permeability APIs. However, when different laboratories conduct in vitro and in vivo permeability studies, the results of model drugs often have inter-laboratory differences. In addition, in the permeability test of monolayer epithelial cells, the experimental results will be affected by the differences in cell batches, resulting in experimental false positives. Therefore, the order relationship established for the same test method in different laboratories is different. In order to improve the reliability of the results, each laboratory will establish its own model drug database and judgment indicators, such as high and low permeability judgment values. The information requirements to support the high permeability of a tested API must include the research methods to determine the high permeability of the tested API. To establish such a model drug database, the first thing to do is to establish analytical methods for quantitative detection of these model drugs in biological matrices (eg. intestinal perfusion fluid, cell suspension).

[0006] For intestinal permeability detection methods that do not involve human subjects, NMPA recommends 20 model drugs to verify the reliability of the evaluation data and the accuracy of the established method in distinguishing low and high permeability. However, in actual operations, due to the lack of a unified standard in the selection of model drugs, different countries have different standards, so the principles of formulation are also very different. Most people's research is based on a certain class of drugs in the BCS classification system for in vivo and in vitro permeability studies, and even in the selection of a certain class of drugs, the selection is single and biased. A recognizable model drug library should include low permeability drugs (fa < 50%), medium permeability drugs (fa = 50% - 84%) and high permeability drugs (fa ≥ 85%) according to the permeability classification.

[0007] However, to be fully and comprehensively selected in the model drug database, it is unexpected that there are a large number of model drugs in the model drug database, that is, it is necessary to establish quantitative detection analysis methods for all these model drugs, which puts high demands on the detection and analysis. There are many researchers who use a single chromatographic analysis to complete the detection, but this requires a high level of professionalism from the researchers. In terms of the acidity coefficient (pKa), the pKa values ​​of different model drugs vary greatly. For example, the pKa of antipyrine (high permeability model drug) is 1.4, the pKa of minoxidil (high permeability model drug) is 4.61, and the pKa of metformin (medium permeability model drug) is 12.4. This means that in the chromatographic analysis, the composition of the mobile phase must be constantly adjusted to meet the pH value required for the current detection, which will make the establishment of the entire model database laborious and time-consuming. Although many researchers have avoided too much time spent on chromatographic conditions by applying chromatography-mass spectrometry technology and taking advantage of the high sensitivity and selectivity of mass spectrometry detection, the optimization of pretreatment methods and chromatographic columns still makes the entire analysis method complicated and diverse due to the diverse ester moisture coefficients of model drugs, and the large investment in chromatographic columns also causes a waste of resources. Some people also use magnetic permeability, turbidimetric solubility, etc. to predict the permeability of drugs, but these are not conventional detection methods and require specialized detection equipment. The processing process is complicated, the data analysis is cumbersome, and the calculation is large. Summary of the invention

[0008] In order to solve the problems raised in the above background technology, the purpose of the present invention is to provide an analytical method development kit that can be quickly applied to the establishment of a model database. The development kit covers 10 model drugs with low permeability, medium permeability, and high permeability, and applies the most popular liquid chromatography tandem triple quadrupole mass spectrometry system. With the simplest "one-step precipitation" sample pretreatment method, only two specifications of chromatographic columns are used to achieve real-time monitoring of the quantitative concentration of 10 model drugs in biological samples, and the quantitative lower limit is as low as 1.0-5.0ng / mL.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] The present invention provides a probe composition LC-MS / MS analysis method package suitable for biowaiver permeability evaluation, comprising the following steps:

[0011] S2-1. Selection of probe composition: According to the "Guidelines for Exemption of Human Bioequivalence Studies" issued by the former State Food and Drug Administration in May 2016, we selected five high permeability (human absorption fraction, fa ≥ 85%) model drugs, namely propranolol, antipyrine, metoprolol, carbamazepine, and minoxidil, three medium permeability (fa = 50-84%) model drugs, namely atenolol, chlorpheniramine, and amiloride, and two low permeability (fa < 50%) model drugs, namely famotidine and sulpiride.

[0012] Compound selection for detection of internal standard: Tamsulosin was selected as the internal standard for detection.

[0013] S2-2. Preparation of standard solution: weigh the standard of famotidine, amiloride, sulpiride or minoxidil, place it in a brown volumetric flask, first add DMSO to fully dissolve it, then dilute to the mark with acetonitrile, shake upside down to prepare a stock solution with a final concentration of 1.0 mg / mL, and refrigerate at 4°C away from light; weigh the standard of propranolol, antipyrine, metoprolol, carbamazepine, atenolol, or chlorpheniramine, place it in a brown volumetric flask, dilute to the mark with methanol, shake upside down to prepare a stock solution with a final concentration of 1.0 mg / mL, and refrigerate at 4°C away from light;

[0014] S2-3. Preparation of internal standard solution: Weigh the tamsulosin standard and place it in a transparent volumetric flask. First, add DMSO to fully dissolve it, then add acetonitrile to the scale, shake upside down to prepare a tamsulosin stock solution with a final concentration of 1.0 mg / mL, and refrigerate at 4°C away from light.

[0015] S2-4, preparation of protein precipitant: pipette the stock solution of tamsulosin prepared in S3, place it in a transparent volumetric flask, dilute to the mark with acetonitrile, shake upside down to prepare a tamsulosin working solution with a final concentration of 10 μg / mL; then take the tamsulosin working solution, place it in a transparent reagent bottle, add acetonitrile to dilute to the mark, shake well to obtain a protein precipitant, and the final concentration of tamsulosin in the protein precipitant is 5 ng / mL;

[0016] S2-5. Processing of biological samples: Take the cell permeate (the collected sample in step S2), add the protein precipitant, shake for 5 minutes to mix, centrifuge at 18000 rpm / min for 5 minutes, take 80 μL of the supernatant and transfer it to the injection bottle, and analyze it by LC-MS / MS

[0017] S2-6. Preparation of standard curve:

[0018] Take several portions of the cell culture medium, add the stock solutions of propranolol, antipyrine, metoprolol, amiloride, minoxidil, carbamazepine, atenolol, chlorpheniramine, famotidine or sulpiride prepared in step S2-2 in sequence, shake for 30 seconds to mix, and prepare standard biological samples with a final concentration of propranolol, antipyrine, and metoprolol of 3-500 ng / mL; standard biological samples with a final concentration of chlorpheniramine and carbamazepine of 1-200 ng / mL; and standard biological samples with a final concentration of sulpiride and atenolol of 1-200 ng / mL. A standard biological sample with a final concentration of 5-500 ng / mL; a standard biological sample with amiloride and famotidine concentrations of 3-200 ng / mL; a standard biological sample with a final concentration of 5-500 ng / mL of minoxidil; the linearity of the standard curve is determined by plotting the relationship between the peak area ratio and the concentration of the drug to be tested, and the data are fitted using a least squares regression with a weight coefficient of 1 / x within the concentration range; the peak area ratio is the ratio of the peak area of ​​the analyte to the peak area of ​​the internal standard;

[0019] S2-7, LC-MS / MS determination: The final biological matrix concentration of the substance to be tested in the biological sample is obtained by liquid chromatography-tandem mass spectrometry, and the concentration of the drug to be tested is calculated according to the data obtained by injection and the obtained standard curve equation;

[0020] Chromatographic conditions: The chromatographic system selected a high performance liquid chromatography (LC-30A) of Shimadzu, Japan. The analytical columns used were Agilent brand ZORBAX Eclipse Plus C18 chromatographic column, with a specification of 1.8 μm, 100×2.1 mmcolumn; Agilent brand ZORBAX Eclipse Plus C18 chromatographic column, with a specification of 4.6 μm, 50×3.5 mmcolumn; the column temperature was 40°C; the chromatographic mobile phase adopted a unified composition, that is, the aqueous phase (A) was ultrapure water containing 0.1% formic acid and 2 mM ammonium formate, and the organic phase (B) was pure acetonitrile; the specific chromatographic conditions (chromatographic gradient setting, flow rate, analysis time) for the LC-MS / MS detection of the 10 model drugs are listed in Table 1, and the injection volume was 5 μL:

[0021] Mass spectrometry conditions: ABSciex mass spectrometry system (API5000) (ABSciex, USA) was used; ion source: ESI; positive ion mode, MRM: multiple reaction ion monitoring; spray voltage (IS) 5500V, auxiliary gas 1 (GS1) 55Arb, auxiliary gas 2 (GS2) 55Arb, auxiliary gas heating temperature (TEM) 550°C, curtain gas (CΜR) 40Arb, collision gas (CAD) 10Pa. MRM was selected in positive ion mode, Q0 entrance voltage (EP) was set to 10V, Q2 exit voltage (CXP) was set to 12V; MRM (mass spectrometry multiple reaction monitoring) detection parameters of ten model drugs and two detection internal standards are listed in Table 2.

[0022] Table 1 Main chromatographic conditions for LC-MS / MS detection of ten model drugs

[0023]

[0024] Table 2 Mass spectrometry conditions for LC-MS / MS detection of ten model drugs

[0025]

[0026] Furthermore, in step S2-5, the volume ratio of the sample to the protein precipitant is 1:5.

[0027] Further, in step S2-6, in the preparation of the standard curve, standard biological samples with final concentrations of propranolol, antipyrine, and metoprolol of 3, 5, 10, 20, 50, 100, 200, and 500 ng / mL are prepared; standard biological samples with final concentrations of chlorpheniramine and carbamazepine of 1, 2, 5, 10, 20, 50, 100, and 200 ng / mL; standard biological samples with final concentrations of sulpiride and atenolol of 5, 10, 20, 50, 100, 200, and 500 ng / mL; standard biological samples with final concentrations of amiloride and famotidine of 3, 5, 10, 20, 50, 100, and 200 ng / mL; and standard biological samples with final concentrations of minoxidil of 5, 10, 20, 50, 100, 200, and 500 ng / mL.

[0028] The present invention also provides an application of a probe composition LC-MS / MS analysis method in drug biowaiver permeability evaluation, comprising the following steps:

[0029] S1. After cell culture, wash the upper and lower channels of the chip with serum-free medium, equilibrate at 37°C for 15-20 minutes, discard the liquid in the chip and then administer the drug, and conduct drug transport experiments on the AP-BL side;

[0030] S2. During the drug transport experiment on the AP-BL side, samples were collected at different time points, and the sample concentrations were determined according to the established probe combination LC-MS / MS analysis method to calculate the cumulative transport amount Q and papp of the drug in the monolayer cell membrane, so as to evaluate the permeability;

[0031] Q=V R ×(C1+C2…+C (i-1) )+V R ×C i

[0032] Where V R is the volume of the test solution in the lower channel (0.2 ml); C i is the measured concentration of the lower channel at time i, μg / mL;

[0033]

[0034] Papp (cm / s) is the apparent permeability coefficient of the drug from the AP side to the BL side of the cell membrane, where A is the surface area of ​​the semipermeable membrane (0.69 cm 2 ), C0 is the initial drug concentration, μg / mL, dQ / dt is the amount of drug transported per unit time, and is the slope of the linear regression of the cumulative drug transport amount Q versus time t;

[0035] S3, import the Papp value of the drug obtained in step S2 and its published human absorption fraction Fa value into Graphpad software, select the Hyperbola equation for nonlinear fitting to perform curve fitting, and obtain the fitting equation eq. (1),

[0036]

[0037] Compared with the prior art, the beneficial effects of the present invention are: fast and simple. The determination of ten different test compounds adopts the same sample pretreatment method and the same chromatographic mobile phase composition. By presetting the chromatographic conditions and mass spectrometry conditions, continuous injection analysis can be achieved even if the test compounds are different. It is only necessary to select different preset methods for different test compounds when submitting the injection sequence. This method greatly simplifies the platform construction of bio-waiver, reduces the difficulty of platform construction, and reduces the time cost and economic cost of platform construction. It only needs to configure two chromatographic columns of different specifications and models, and adopts the sample pretreatment method of one-step precipitation with the least consumables consumption to achieve the evaluation of three permeability levels of low / medium / high.

[0038] The analytical method established by the present invention has good specificity, high sensitivity, and a wide dynamic linear range. The accuracy, precision, reproducibility, and quality control of the analytical method all meet the requirements for biological sample analysis.

[0039] According to the methodological verification, the lower limit of quantification of the 10 probe model drugs in the analytical distribution package was as low as 1-5 ng / mL, the upper limit of quantification was 200-500 ng / mL, and the linearity was good within the quantitative range (r>0.99). The linear range and lower limit of quantification of the LC-MS / MS quantitative detection of the 10 model drugs are shown in Table 3. The intra-batch precision and intra-batch accuracy of the 10 model drugs at low, medium and high concentrations within three analytical batches, as well as the inter-batch precision and inter-batch accuracy between three analytical batches were investigated. The results are shown in Tables 4 and 5. The intra-batch / inter-batch precision deviations of the 10 model drugs at low, medium and high concentrations were all less than 15%, and the intra-batch / inter-batch precision deviations were all between ±15%, which met the relevant regulations for biological sample determination. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the correlation curve between the human absorption fraction (Fa) and Papp in the Caco-2 cell model. DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Example 1

[0043] The purpose of the present invention is to develop an analytical method development kit that can be quickly applied to the establishment of a model database. The development kit covers 10 model drugs with low permeability, medium permeability and high permeability, and uses a liquid chromatography-tandem mass spectrometry system to achieve real-time monitoring of the quantitative concentrations of the 10 model drugs in biological samples.

[0044] A probe composition LC-MS / MS analysis method kit suitable for biowaiver permeability evaluation comprises the following steps:

[0045] 1. Selection of probe composition: According to the "Guidelines for Exemption of Human Bioequivalence Studies" issued by the former State Food and Drug Administration in May 2016, we selected five high permeability (human absorption fraction, fa ≥ 85%) model drugs, namely propranolol, antipyrine, metoprolol, carbamazepine, and minoxidil, three medium permeability (fa = 50-84%) model drugs, namely atenolol, chlorpheniramine, and amiloride, and two low permeability (fa < 50%) model drugs, namely famotidine and sulpiride, from the table "Recommended drugs for illustrating the suitability of permeability tests in Appendix A".

[0046] 2. Selection of compounds for detection of internal standard: Tamsulosin was selected as the internal standard for detection.

[0047] 3. Preparation of standard solution: Accurately weigh 10 mg of famotidine, amiloride, sulpiride or minoxidil standard, place in a 10 mL brown volumetric flask, first add 1 mL DMSO to fully dissolve it, then dilute to 10 mL mark with acetonitrile, shake upside down to prepare a stock solution with a final concentration of 1.0 mg / mL, and refrigerate at 4°C away from light. Accurately weigh 10 mg of propranolol, antipyrine, metoprolol, carbamazepine, atenolol, or chlorpheniramine standard, place in a 10 mL brown volumetric flask, dilute to 10 mL mark with methanol, shake upside down to prepare a stock solution with a final concentration of 1.0 mg / mL, and refrigerate at 4°C away from light.

[0048] 4. Preparation of internal standard solution: Accurately weigh 10 mg of tamsulosin standard and place it in a 10 mL transparent volumetric flask. First, add 1 mL of DMSO to fully dissolve it, then dilute to 10 mL with acetonitrile, shake upside down to prepare a stock solution with a final concentration of 1.0 mg / mL, and refrigerate at 4°C away from light.

[0049] 5. Preparation of protein precipitant: Pipette 1 mL of tamsulosin stock solution (1.0 mg / mL) into a 100 mL transparent volumetric flask, dilute to 10 mL mark with acetonitrile, shake upside down to prepare an internal standard working solution with a final concentration of 10 μg / mL. Take 0.25 mL of tamsulosin working solution (10 μg / mL) into a 500 mL transparent reagent bottle, add 500 mL of acetonitrile, shake well to obtain the protein precipitant. The final concentration of the internal standard (tamsulosin) in the protein precipitant is 5 ng / mL.

[0050] 6. Processing of biological samples: Taking the Caco-2 cell model as an example, take 50 μL of cell permeate (samples collected at different time periods in the drug transport experiment), add 250 μL of protein precipitant, shake for 5 minutes to mix, centrifuge at 18000 rpm / min for 5 minutes, take 80 μL of supernatant and transfer it to the injection bottle for LC-MS / MS analysis.

[0051] 7. Preparation of standard curve:

[0052] Take several portions of 45 μL of cell culture medium (DMEM), add 5 μL of a series of working solutions of propranolol, antipyrine, metoprolol, amiloride, minoxidil, carbamazepine, atenolol, chlorpheniramine, famotidine or sulpiride respectively, oscillate for 30 seconds to mix, and prepare standard biological samples with a final concentration of propranolol, antipyrine, and metoprolol of 3-500 ng / mL; standard biological samples with a final concentration of chlorpheniramine and carbamazepine of 1-200 ng / mL; standard biological samples with a final concentration of sulpiride and atenolol of 5-500 ng / mL; standard biological samples with amiloride and famotidine concentrations of 3-200 ng / mL; standard biological samples with a final concentration of minoxidil of 5-500 ng / mL; determine the linearity of the standard curve by plotting the relationship between the peak area ratio and the concentration of the drug to be tested, and fit the data using least squares regression with a weight coefficient of 1 / x within the concentration range. The peak area ratio is the ratio of the peak area of ​​the analyte to the peak area of ​​the internal standard.

[0053] 8.LC-MS / MS determination:

[0054] The final biological matrix concentration of the substance to be tested in the biological sample is obtained by liquid chromatography-tandem mass spectrometry, and the concentration of the drug to be tested is calculated according to the obtained standard curve equation based on the data obtained by injection;

[0055] 8.1 Chromatographic conditions: The chromatographic system selected was a high performance liquid chromatograph (LC-30A) produced by Shimadzu, Japan. The analytical columns used were Agilent ZORBAX Eclipse Plus C18 chromatographic columns with specifications of 1.8 μm, 100×2.1 mm column and Agilent ZORBAX Eclipse Plus C18 chromatographic columns with specifications of 4.6 μm, 50×3.5 mm column. The column temperature was 40°C. The chromatographic mobile phase used a uniform composition, i.e., the aqueous phase (A) was ultrapure water containing 0.1% formic acid and 2 mM ammonium formate, and the organic phase (B) was pure acetonitrile. The specific chromatographic conditions (chromatographic gradient settings, flow rates, and analysis time) for the LC-MS / MS detection of the 10 model drugs are listed in Table 1. The injection volume was 5 μL:

[0056] 8.2 Mass spectrometry conditions: ABSciex mass spectrometry system (API5000) (ABSciex, USA) was used; ion source: ESI; positive ion mode, MRM: multiple reaction ion monitoring; spray voltage (IS) 5500V, auxiliary gas 1 (GS1) 55Arb, auxiliary gas 2 (GS2) 55Arb, auxiliary gas heating temperature (TEM) 550°C, curtain gas (CΜR) 40Arb, collision gas (CAD) 10Pa. MRM was selected in positive ion mode, Q0 entrance voltage (EP) was set to 10V, Q2 exit voltage (CXP) was set to 12V; MRM (mass spectrometry multiple reaction monitoring) detection parameters of ten model drugs and two detection internal standards are listed in Table 2.

[0057] Table 1 Main chromatographic conditions for LC-MS / MS detection of ten model drugs

[0058]

[0059] Table 2 Mass spectrometry conditions for LC-MS / MS detection of ten model drugs

[0060]

[0061] Example 2

[0062] 1. Experimental Materials

[0063] 1.1 Drugs and reagents

[0064] Propranolol hydrochloride, antipyrine, metoprolol tartrate, chlorpheniramine maleate, atenolol, sulpiride, carbamazepine, famotidine, amiloride, and minoxidil were purchased from Selleck, formic acid and ammonium formate were commercially available at analytical grade, and acetonitrile and methanol (chromatographic grade) were purchased from Merck. DMEM culture medium (Gibco), dimethyl sulfoxide (DMSO), fetal bovine serum (Gibco), double antibody (Gibco), and 0.1% trypsin.

[0065] 1.2 Main instruments

[0066] High performance liquid chromatography-quadrupole tandem mass spectrometry (including Shimadzu high performance liquid chromatography system (LC-30A) from Japan), Sciex mass spectrometry system (API5000) from the United States, electrospray ionization source, and Analyst 1.6.3 workstation). GENIEVORTEX-2 vortex mixing device; Thermo Sorwall high-speed refrigerated centrifuge; Milli-Q GradientA1 ultrapure water machine (Millipore Inc, ΜSA); Eppendorf MinispinAG22331 desktop centrifuge.

[0067] 1.3 Cell lines

[0068] Caco-2 cell lines were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, and the cell lines used in the experiment were 9-40 generations.

[0069] 2. Treatment of biological samples: Take 50 μL of cell permeate, add 250 μL of protein precipitant, shake for 5 minutes to mix, centrifuge at 18000 rpm / min for 5 minutes, take 80 μL of supernatant and transfer to the injection bottle for LC-MS / MS analysis.

[0070] 3. Validation of analytical methods for LC-MS / MS quantitative detection

[0071] 3.1 Standard curve

[0072] Take several portions of 45 μL cell culture medium (DMEM), add 5 μL of the series of working solutions of the drugs to be tested, shake for 30 seconds, and prepare standard biological samples with final concentrations of 3, 5, 10, 20, 50, 100, 200, and 500 ng / mL for propranolol, antipyrine, and metoprolol; standard biological samples with final concentrations of 1, 2, 5, 10, 20, 50, 100, and 200 ng / mL for chlorpheniramine and carbamazepine; Standard biological samples with final concentrations of 5, 10, 20, 50, 100, 200, and 500 ng / mL for amiloride and atenolol; standard biological samples with final concentrations of 3, 5, 10, 20, 50, 100, and 200 ng / mL for amiloride and famotidine; standard biological samples with final concentrations of 5, 10, 20, 50, 100, and 200 ng / mL for minoxidil; LC-MS / MS analysis after treatment according to the method under "2". The peak area ratio (y) of the drug to be tested and the internal standard in the biological sample to be tested was used as the dependent variable, and the final concentration of the substance to be tested was used as the independent variable (x). The least squares method (weight coefficient is 1 / x) regression operation was performed to obtain the linear equation of the standard curve. The measured concentration of the substance to be tested in the test sample was calculated back according to the standard curve of the day. The standard curve equation described in Table 3 is the linear equation obtained by fitting the standard biological samples of the three standard curves, and the correlation coefficient r>0.99 indicates good linearity.

[0073] 3.2 Lower limit of quantification

[0074] Take several portions of 45 μL cell culture medium (DMEM), add 5 μL of the working solution of the drug to be tested in turn, shake for 30 seconds, and prepare standard biological samples with a final concentration of 3 ng / mL for propranolol, antipyrine, metoprolol, amiloride, and famotidine; standard biological samples with a final concentration of 1 ng / mL for chlorpheniramine and carbamazepine; standard biological samples with a final concentration of 5 ng / mL for sulpiride, atenolol, and minoxidil; process according to the method under "2" and analyze by LC-MS / MS. Prepare 5 standard biological samples in parallel for each drug to be tested, and calculate the arithmetic mean (Mean) and standard deviation (SD) of the measurement results of the 5 samples.

[0075] The precision of the quantitative lower limit described in Table 3 is measured by the relative standard deviation (RSD). RSD is the ratio of the standard deviation to the arithmetic mean of the measurement result, usually expressed as a percentage. If RSD is less than or equal to 15%, it meets the relevant regulations for biological sample determination "9012 Guidelines for Validation of Quantitative Analysis Methods for Biological Samples". The accuracy of the quantitative lower limit described in Table 3 is measured by the relative error (RE). RE is the ratio of the absolute error to the true value, usually expressed as a percentage. If RE is between -15% and 15%, it meets the relevant regulations for biological sample determination "9012 Guidelines for Validation of Quantitative Analysis Methods for Biological Samples".

[0076] Table 3 Linearity and lower limit of quantification of ten model drugs by LC-MS / MS quantitative detection

[0077]

[0078] 3.3 Precision and Accuracy

[0079] Take several portions of 45 μL cell culture medium (DMEM), add 5 μL of the working solution of the drug to be tested in turn, shake for 30 seconds, and prepare standard biological samples with a final concentration of 3, 5, 50, and 400 ng / mL for propranolol, antipyrine, and metoprolol; standard biological samples with a final concentration of 1, 2, 20, and 150 ng / mL for chlorpheniramine and carbamazepine; standard biological samples with a final concentration of 5, 10, 50, and 400 ng / mL for sulpiride, atenolol, and minoxidil; and standard biological samples with a final concentration of 3, 5, 50, and 150 ng / mL for amiloride and famotidine; and analyze by LC-MS / MS according to the method under "2". For each drug to be tested, prepare 5 standard biological samples in parallel at each concentration. Calculate the arithmetic mean (Mean) and standard deviation (SD) of the measurement results of 5 samples of each concentration of each drug to be tested in an analytical batch. Three analytical batches were tested continuously, and the arithmetic mean (Mean) and standard deviation (SD) of the measurement results of 15 samples of each concentration of each drug to be tested among the three analytical batches were calculated.

[0080] Table 4 Intra-batch precision and accuracy of LC-MS / MS detection of ten model drugs (n=5)

[0081]

[0082] Table 5 Inter-batch precision and accuracy of LC-MS / MS detection of ten model drugs (n=5)

[0083]

[0084]

[0085] The intra-batch precision described in Table 4 is measured by the relative standard deviation (RSD) within an analytical batch, that is, the ratio of the standard deviation (SD) to the arithmetic mean (Mean) of the measurement results, expressed as a percentage. If RSD is less than or equal to 15%, it meets the relevant regulations for biological sample determination "9012 Guidelines for Validation of Quantitative Analysis Methods for Biological Samples". The intra-batch accuracy described in Table 4 is measured by the relative error (RE) within an analytical batch, that is, the ratio of the absolute error (the difference between the arithmetic mean and the true value) to the true value, expressed as a percentage. If RE is between -15% and 15%, it meets the relevant regulations for biological sample determination "9012 Guidelines for Validation of Quantitative Analysis Methods for Biological Samples".

[0086] The inter-batch precision described in Table 5 refers to the relative standard deviation (RSD) between the three analytical batches, and the inter-batch accuracy refers to the relative error (RE) between the three analytical batches. The algorithm and requirements are the same as the intra-batch precision and intra-batch accuracy.

[0087] Example 3

[0088] Taking the permeability detection method of monolayer epithelial cells as an example, we established our own model drug database and judgment indicators on Caco-2 cells.

[0089] Caco2 cell culture

[0090] 1. Caco-2 cells were cultured in DMEM high-glucose complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were cultured at a constant temperature of 37°C, 5% CO2, and 90% relative humidity. The medium was changed every other day, and the cells were passaged when they reached 90% confluence. The 9th to 40th generation cells were used for the experiment.

[0091] 2. Extracellular matrix coating: 100ul of serum-free DMEM containing rat type I collagen (50ug / ml) and Matrigel (300ug / ml) was injected into the upper channel of the chip. After the device was incubated at 37°C in a 5% CO2 incubator for 1-2 hours, the residual ECM solution was aspirated with a pipette; the channel was rinsed once with preheated complete culture medium, and the chip was kept in a 37°C carbon dioxide humidified incubator until the cells were seeded;

[0092] 3. Cell seeding: Passage the Caco-2 cells that have grown 80-90% of the vial, discard the original culture medium, add 1 mL of trypsin for pre-digestion, discard the culture medium, add 1 mL of trypsin for digestion for 5 min, then add 2 mL of 10% DMEM culture medium to terminate digestion, centrifuge at 1000 rpm for 5 min, discard the upper layer, add 1 mL of culture medium for resuspending, and adjust the cell density; Caco-2 cells are cultured at 0.5×105 / cm 2 The density of the cells was inoculated in the upper channel so that the cell suspension could just completely flow through the channel; the chip was placed in an incubator for 4 to 6 hours. After the cells adhered to the wall, the culture medium in the upper and lower channels was filled up, and the evaporation chamber was filled up with ultrapure water; the chip was placed in an incubator at 37°C and cultured for 21 days. The medium was changed every other day during the first week, and the culture medium was changed every day after one week.

[0093] Drug transport experiments:

[0094] 1. Before the drug transport experiment, wash the upper and lower channels of the chip three times with serum-free medium to remove residual medium and metabolites, equilibrate at 37°C for 15-20 minutes, discard the liquid in the chip and then administer the drug to conduct transport studies on the AP-BL side;

[0095] 2. Add 300 μL of drug donor solution to the upper channel of the chip, and immediately sample 100 μL (t=0); add 200 μL of blank serum-free culture medium to the lower channel, and sample 100 μL from the lower channel after 30, 60, 90, and 120 min, respectively, and add the same blank serum-free culture medium at the same time; the collected samples are measured for concentration according to the method established by LC-MS / MS in Example 1, and the cumulative transport amount Q and papp of the drug in the Caco-2 monolayer cell membrane are calculated to evaluate the permeability.

[0096] Q=V R ×(C1+C2…+C (i-1) )+V R ×C i ;

[0097] Where V R is the volume of the test solution in the lower channel (0.2 ml), C i is the measured concentration of the lower channel at time i (μg / mL).

[0098]

[0099] Papp (cm / s) is the apparent permeability coefficient of the drug from the AP side to the BL side of the cell membrane, where A is the surface area of ​​the semipermeable membrane (0.69 cm 2 ), C0 is the initial drug concentration (μg / mL), dQ / dt is the amount of drug transported per unit time, and is the slope of the linear regression of the cumulative drug transport amount Q versus time t.

[0100] The apparent permeability coefficients (P app ), listed in Table 6.

[0101] Table 6 Papp of ten model drugs in the AP-BP direction of Caco-2 cells

[0102] Drug Name Dosing concentration (μg / mL) Human body absorption fraction (Fa) <![CDATA[P app (×10 -6 cm / s)]]> Propranolol 10 93 25.7±0.247 Antipyrine 20 97 24.6±0.494 Metoprolol 40 92 17.2±0.197 Carbamazepine 10 98 38.2±0.150 Chlorpheniramine 20 100 20.7±0.262 Minoxidil 10 90 4.75±0.078 Amiloride 40 60 1.85±0.418 Atenolol 20 50 0.15±0.415 Famotidine 40 45 0.20±0.797 Sulpiride 40 40 0.16±0.471

[0103] The P values ​​of the 10 model drugs in Table 6 were app The value was correlated with the published human absorption fraction Fa value to obtain the fitting curve (see Appendix Figure 1 ) and the fitting equation eq.(1), the r of the fitting equation 2 >0.9(n=10), indicating that P app The values ​​correlate well with the Fa values. It is known that the absorption fraction range of the model drug we selected covers <50%, 50-84% and ≥85%. According to the literature, the permeability of metoprolol is very close to reaching the medium / high limit, so it is used as an internal standard. When the drug obtains P app Values ​​equal to or greater than that of metoprolol app When the value is ≥ 85%, the drug is considered to have high permeability. By using metoprolol as a high permeability internal standard, the model drug with Fa ≥ 85% can be classified more accurately.

[0104]

[0105] Here, the correlation analysis is to use the human body absorption fraction (Fa) in Table 6 as the independent variable (X), and the P in Table 6 app The arithmetic mean of the results was taken as the dependent variable (Y) and imported into Graphpad software, where the Hyperbola equation of nonlinear fitting was selected for curve fitting to obtain the fitting equation.

[0106] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0107] It should be noted that the above content only illustrates the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications all fall within the protection scope of the claims of the present invention.

Claims

1. Application of a probe composition LC-MS / MS analysis method in drug biowaiver permeability evaluation, characterized in that: The following steps are involved: S1. After cell culture, wash the upper and lower channels of the chip with serum-free medium, equilibrate at 37°C for 15-20 minutes, discard the liquid in the chip and then administer the drug, and conduct drug transport experiments on the AP-BL side; S2. During the drug transport experiment on the AP-BL side, samples were collected at different time points, and the concentration of the collected samples was determined according to the established probe combination LC-MS / MS analysis method to calculate the cumulative transport amount Q and papp of the drug in the monolayer cell membrane, so as to evaluate the permeability; Q=V R ×(C1+C2…+C (i-1) )+V R ×C i Where V R is the volume of the test solution in the lower channel; C i is the measured concentration of the lower channel at time i, μg / mL; Papp (cm / s) is the apparent permeability coefficient of the drug from the AP side to the BL side of the cell membrane, where A is the surface area of ​​the semipermeable membrane, C0 is the initial drug concentration, μg / mL, dQ / dt is the amount of drug transported per unit time, and dQ / dt is the slope of the linear regression of the cumulative drug transport amount Q on time t; S3, import the Papp value of the drug obtained in step S2 and its published human absorption fraction Fa value into Graphpad software, select the Hyperbola equation for nonlinear fitting to perform curve fitting, and obtain the fitting equation eq. (1), 2. The use of a probe composition LC-MS / MS analysis method according to claim 1 in the evaluation of drug biowaiver permeability, characterized in that: In step S2, the establishment of the probe composition LC-MS / MS analysis method comprises the following steps: S2-1. Selection of probe composition: The probe composition includes model drugs with high permeability, model drugs with medium permeability, and model drugs with low permeability. Model drugs with high permeability include propranolol, antipyrine, metoprolol, carbamazepine, and minoxidil. Model drugs with medium permeability include atenolol, chlorpheniramine, and amiloride. Model drugs with low permeability include famotidine and sulpiride. S2-2. Preparation of standard solution: weigh the standard of famotidine, amiloride, sulpiride or minoxidil, place it in a brown volumetric flask, first add DMSO to fully dissolve it, then dilute to the mark with acetonitrile, shake upside down to prepare a stock solution with a final concentration of 1.0 mg / mL, and refrigerate at 4°C away from light; weigh the standard of propranolol, antipyrine, metoprolol, carbamazepine, atenolol, or chlorpheniramine, place it in a brown volumetric flask, dilute to the mark with methanol, shake upside down to prepare a stock solution with a final concentration of 1.0 mg / mL, and refrigerate at 4°C away from light; S2-3. Preparation of internal standard solution: Weigh the tamsulosin standard and place it in a transparent volumetric flask. First, add DMSO to fully dissolve it, then add acetonitrile to the scale, shake upside down to prepare a tamsulosin stock solution with a final concentration of 1.0 mg / mL, and refrigerate at 4°C away from light. S2-4, preparation of protein precipitant: pipette the stock solution of tamsulosin prepared in S3, place it in a transparent volumetric flask, dilute to the mark with acetonitrile, shake upside down to prepare a tamsulosin working solution with a final concentration of 10 μg / mL; then take the tamsulosin working solution, place it in a transparent reagent bottle, add acetonitrile to dilute to the mark, shake well to obtain a protein precipitant, and the final concentration of tamsulosin in the protein precipitant is 5 ng / mL; S2-5. Processing of biological samples: Take the sample cell permeate collected in step S2, add a protein precipitant, shake for 5 minutes to mix, centrifuge at 18000 rpm / min for 5 minutes, take 80 μL of the supernatant and transfer it to a sample injection bottle for LC-MS / MS analysis; S2-6, preparation of standard curve: take several portions of cell culture medium, add the stock solution of propranolol, antipyrine, metoprolol, amiloride, minoxidil, carbamazepine, atenolol, chlorpheniramine, famotidine or sulpiride prepared in step S2-2, shake for 30 seconds to mix, and prepare standard biological samples with a final concentration of 3-500 ng / mL for propranolol, antipyrine, and metoprolol; a final concentration of 1-200 ng / mL for chlorpheniramine and carbamazepine; and a standard biological sample with a final concentration of 1-200 ng / mL for sulpiride. Standard biological samples with a final concentration of 5-500 ng / mL for Priligy and atenolol; standard biological samples with a final concentration of 3-200 ng / mL for amiloride and famotidine; standard biological samples with a final concentration of 5-500 ng / mL for minoxidil; the linearity of the standard curve is determined by plotting the relationship between the peak area ratio and the concentration of the drug to be tested, and the data are fitted by least squares regression with a weight coefficient of 1 / x within the concentration range; the peak area ratio is the ratio of the peak area of ​​the analyte to the peak area of ​​the internal standard; S2-7, LC-MS / MS determination: The final biological matrix concentration of the substance to be tested in the biological sample is obtained by liquid chromatography-tandem mass spectrometry, and the concentration of the drug to be tested is calculated according to the data obtained by injection and the obtained standard curve equation; Chromatographic conditions: The analytical columns were Agilent ZORBAX Eclipse Plus C18 columns with specifications of 1.8 μm, 100×2.1 mm column and Agilent ZORBAX Eclipse Plus C18 columns with specifications of 4.6 μm, 50×3.5 mm column; the column temperature was 40°C; the chromatographic mobile phases were of uniform composition, i.e., aqueous phase A was ultrapure water containing 0.1% formic acid and 2 mM ammonium formate, and organic phase B was pure acetonitrile; gradient elution, injection volume was 5 μL: The specific chromatographic conditions for LC-MS / MS detection of the 10 model drugs are listed in Table 1 ; Table 1 Main chromatographic conditions for LC-MS / MS detection of ten model drugs Mass spectrometry conditions: ion source: ESI; positive ion mode, MRM: multiple reaction ion monitoring; spray voltage 5500V, auxiliary gas 155Arb, auxiliary gas 255Arb, auxiliary gas heating temperature 550℃, curtain gas 40Arb, collision gas 10Pa; MRM in positive ion mode was selected, Q0 inlet voltage was set to 10V, Q2 outlet voltage was set to 12V; MRM detection parameters of ten model drugs and two detection internal standards are listed in Table 2. Table 2 Mass spectrometry conditions for LC-MS / MS detection of ten model drugs 3. Application of a probe composition LC-MS / MS analysis method according to claim 2 in drug biowaiver permeability evaluation, characterized in that: In step S2-5, the volume ratio of the sample to the protein precipitant is 1:

5.

4. The use of a probe composition LC-MS / MS analysis method according to claim 2 in the evaluation of drug biowaiver permeability, characterized in that: In step S2-6, in the preparation of the standard curve, standard biological samples with final concentrations of propranolol, antipyrine and metoprolol of 3, 5, 10, 20, 50, 100, 200 and 500 ng / mL are prepared; standard biological samples with final concentrations of chlorpheniramine and carbamazepine of 1, 2, 5, 10, 20, 50, 100 and 200 ng / mL; standard biological samples with final concentrations of sulpiride and atenolol of 5, 10, 20, 50, 100, 200 and 500 ng / mL are prepared; standard biological samples with final concentrations of amiloride and famotidine of 3, 5, 10, 20, 50, 100 and 200 ng / mL; standard biological samples with final concentrations of minoxidil of 5, 10, 20, 50, 100, 200 and 500 ng / mL are prepared.

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