Method for simultaneously determining tacrolimus and metabolite 13-O-nortacrolimus thereof in whole blood by liquid chromatography-mass spectrometry

Abstract

The invention provides a method for simultaneously determining tacrolimus and a metabolite 13-O-demethylated tacrolimus in whole blood by liquid chromatography-mass spectrometry, which comprises the following steps: (1) pretreatment of a whole blood sample: taking the whole blood sample added with an anticoagulant, adding an internal standard working solution, uniformly mixing, adding a protein precipitator, and carrying out vortex oscillation to lyse red blood cells to precipitate protein; centrifuging, taking supernate, filtering by a filter membrane, and injecting a sample; (2) a sample to be detected is injected into the chromatographic system and eluted through a mobile phase, and the mobile phase A is an aqueous solution of 0.1% formic acid; a mobile phase B: 0.1% formic acid-acetonitrile; and (3) performing mass spectrometric detection after elution so as to detect the tacrolimus serving as the to-be-analyzed substance and the metabolite 13-O-nortacrolimus of the tacrolimus. The method has the advantages of rapidness, accuracy, high sensitivity, high efficiency, strong specificity and simplicity in operation, and can be suitable for clinical detection of medicines.

Description

[Technical field]

[0001] The invention relates to a method for simultaneously determining tacrolimus and its metabolite 13-O-nor-tacrolimus in whole blood by liquid chromatography-mass spectrometry. [Background technology]

[0002] Tacrolimus (FK506rolimus, FK506) is one of the most commonly used immunosuppressants in clinical practice and is widely used in immunosuppressive treatment of patients with solid organ transplants (such as liver, kidney, etc.). The "Expert Consensus on the Application of Immune Checkpoint Inhibitors in Liver Transplant Recipients with Liver Cancer in China (2024 Edition)" recommends calcineurin immunosuppressants (Calcineurin Inhibitor, CNI) including tacrolimus as basic immunotherapy drugs. In addition, tacrolimus is also the first choice drug for refractory nephrotic syndrome (NS) in children and the first-line treatment drug for membranous nephropathy (MN).

[0003] Tacrolimus has low oral bioavailability, incomplete and irregular absorption, large inter-individual pharmacokinetic variation, and poor correlation between dosage and blood concentration, which increases the difficulty of clinical use. In addition, tacrolimus is prone to rejection at low concentrations, and the incidence of adverse reactions related to nephrotoxicity and neurotoxicity increases significantly at high concentrations. The therapeutic window is narrow, and even if the drug concentration is within the therapeutic window, patients often experience adverse reactions related to nephrotoxicity and the digestive system. Therefore, it is a common practice in clinical practice to guide tacrolimus dose adjustment by monitoring blood concentration. Commonly used methods include high-performance liquid tandem mass spectrometry (LC-MS / MS), enzyme-linked immunosorbent assay (ELISA), chemiluminescent microparticle immunoassay (CMIA), etc.

[0004] More than 95% of tacrolimus is excreted through bile in the form of metabolites, and less than 5% of tacrolimus is excreted through the kidneys in the form of the original drug. Tacrolimus is mainly metabolized by CYP3A5 and CYP3A4 enzymes in the liver and intestinal mucosa. 13-O-desmethyltacrolimus (13-O-FK506, M1) is the main metabolite of tacrolimus, and its biological activity is 10% of the original drug. On the one hand, the different CYP3A enzyme activities caused by different genetic backgrounds of patients will significantly affect the metabolic rate of tacrolimus, and thus affect the dosage required by patients. For example, CYP3A5*1 carriers require higher dosages than CYP3A5*3 / *3 homozygotes; compared with CYP3A4*1G carriers, CYP3A4*1 / *1 carriers need a lower dose of tacrolimus to achieve the same therapeutic target concentration. On the other hand, the patient's CYP genotype also affects the concentration of tacrolimus and its metabolites, and then affects the incidence of adverse reactions. Studies in proximal tubular epithelial cells have found that CYP3A4 / A5 genetic polymorphisms are associated with calcineurin inhibitor-induced toxicity and profibrotic reactions. Large clinical studies have also found that renal transplant patients carrying at least one CYP3A5*1 allele mutation are more susceptible to calcineurin inhibitor-induced nephrotoxicity, which in turn leads to histological lesions and deterioration of transplant organ function. These studies suggest that the metabolites of tacrolimus can affect its therapeutic effects and toxic reactions. Monitoring the blood concentration of tacrolimus metabolites, especially the main metabolite M1, can avoid only considering the exposure of tacrolimus original drug and ignoring the pharmacological effects of its main metabolite M1, and better reduce adverse drug reactions caused by patient variability. However, at present, the blood concentration monitoring of tacrolimus in China is limited to the original drug, and there is no methodology for the simultaneous detection of tacrolimus and its metabolites.

[0005] In addition to the LC-MS / MS method, there are other methods such as immunoassays, including chemiluminescent microparticle immunoassays and enzyme amplification immunoassays, which all have the problem of making the measurement results biased. The LC-MS / MS method has the characteristics of high sensitivity and strong specificity. It combines the efficient separation ability of liquid chromatographs with the component identification ability of mass spectrometers. It is an effective means for separating and analyzing complex organic mixtures and is currently widely used in the detection of biological samples. A number of studies have shown that LC-MS / MS can simultaneously separate tacrolimus and other multiple drug components, but can only determine tacrolimus alone. The present invention has developed a LC-MS / MS method for simultaneous quantitative analysis of plasma tacrolimus and its main metabolite 13-O-desmethyltacrolimus. The sample pretreatment operation method is simple, and the whole blood sample can be sampled and analyzed after protein precipitation, centrifugation, and filtration. Compared with the conventional tacrolimus determination pretreatment multiple extraction and nitrogen blowing concentration method, it saves steps and time and is more suitable for actual inspection work. [Summary of the invention]

[0006] The technical problem to be solved by the present invention is to provide a method for simultaneously determining tacrolimus and its metabolite 13-O-desmethyltacrolimus in whole blood by liquid chromatography-mass spectrometry. The method can simultaneously determine tacrolimus and its metabolite 13-O-desmethyltacrolimus in whole blood, has the advantages of being fast and accurate, highly sensitive, efficient, highly specific, and simple to operate, and can be applied to clinical detection of drugs.

[0007] The present invention is achieved in that:

[0008] A method for simultaneously determining tacrolimus and its metabolite 13-O-desmethyltacrolimus in whole blood by liquid chromatography-mass spectrometry, the method comprising the following steps:

[0009] (1) Pretreatment of whole blood samples: take a whole blood sample with anticoagulant added, add internal standard working solution, mix well, add protein precipitant, vortex and lyse red blood cells to precipitate protein; take the supernatant after centrifugation, filter through a filter membrane, and inject;

[0010] (2) Inject the sample to be tested into the chromatographic system and elute it with the mobile phase. The chromatographic conditions are as follows: Chromatographic column: ACQUITY BEH C 18 ; Column temperature: 25°C; Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid-acetonitrile; Gradient elution, flow rate 0.4 mL . min -1 , injection volume was 10uL, analysis time was 6min;

[0011] (3) After the elution, mass spectrometry is performed to detect tacrolimus and its metabolite 13-O-nor-tacrolimus as the analytes; the mass spectrometry conditions are as follows:

[0012] Electrospray ionization source, positive ion scanning, multiple reaction monitoring (MRM) mode was used for detection. The desolvation tube temperature was 500°C and the desolvation gas flow rate was 1000L. . hr -1 , source temperature: 150℃, cone gas flow: 50L . hr -1 .

[0013] Furthermore, in step (1): EDTA-2K is used as an anticoagulant and hydroxychloroquine is used as an internal standard working solution;

[0014] The protein precipitant is methanol / 0.2 mol / L in a volume ratio of 80 / 20 . L -1 ZnSO4 solution, wherein the solution contains 0.1% formic acid.

[0015] Furthermore, in the step (2), the parameters of the gradient elution are: 0-2.8 min, A:B=95:5; 2.8-3.3 min, A:B=5:95; 3.3-4 min, A:B=5:95; 4-6 min, A:B=95:5; all are measured in v / v.

[0016] Furthermore, in the step (3), the ion reactions used for quantitative analysis are: tacrolimus: m / z 826.94→m / z 616.40; 13-O desmethyl tacrolimus: m / z 808.4-m / z 790.53; internal standard hydroxychloroquine m / z 336.32→m / z 247.15.

[0017] The present invention has the following advantages:

[0018] 1. The present invention can simultaneously determine tacrolimus and its active metabolites. Compared with the existing single determination of tacrolimus, the present invention can more comprehensively understand the in vivo exposure of the immunosuppressant and its active metabolites, is more conducive to the comprehensive estimation of its pharmacological effects, has greater guiding significance for clinical practice, and provides a more detailed and accurate basis for individualized drug therapy.

[0019] 2. The pretreatment cost of the present invention is low and more convenient: compared with the existing solid phase extraction method for extracting tacrolimus, the cost of this method can be saved by about 50%, and the consumption of solid phase adsorption columns is eliminated; compared with the existing ether extraction, the reagents of the present invention are easy to obtain and more conducive to the labor protection of operators, shortening the concentration time and improving work efficiency. The protein precipitant used in the present invention is more economical, convenient and easy to obtain. Due to its simple operation, it can be applied to conventional determination

[0020] 3. The invention has strong specificity and adopts ACQUITY BEH C18 (2.1×100mm, 1.7μm) chromatographic column, aqueous phase: 0.1% formic acid in water; organic phase: 0.1% formic acid-acetonitrile, gradient elution, the retention time of tacrolimus is 3.68min, the retention time of hydroxychloroquine is 1.42min, and the retention time of 13-0-desmethyltacrolimus is 3.24min. The separation degrees are all greater than 1.7, with high separation efficiency and no mutual interference.

[0021] 4. The present invention has high sensitivity, and the quantitative limit of tacrolimus is 1 ng·mL -1 The limit of quantification of 13-O-desmethyltacrolimus is 10 ng·mL -1 (S / N>10)

[0022] In conclusion, the method of the present invention is rapid and accurate, highly sensitive, efficient, highly unique, and simple to operate, and can be applied to clinical drug testing.

Brief Description of the Drawings

[0023] The present invention will be further described below in conjunction with embodiments with reference to the accompanying drawings.

[0024] Figure 1 It is the mass spectrum of tacrolimus and its metabolite 13-O-tacrolimus and internal standard hydroxychloroquine in the examples of the present invention.

[0025] Figure 2 It is a chromatogram of the corresponding ion channels of human blank whole blood in an embodiment of the present invention.

[0026] Figure 3 This is the ion chromatogram after adding the standard substance to the blank human whole blood in the embodiment of the present invention, wherein the retention time of tacrolimus is 3.68 min, the retention time of 13-O desmethyl tacrolimus is 3.24 min, and the retention time of the internal standard hydroxychloroquine is 1.42 min.

[0027] Figure 4 It is a regression equation diagram of tacrolimus in an embodiment of the present invention.

[0028] Figure 5 It is a regression equation diagram of 13-O tacrolimus in an embodiment of the present invention. [Specific implementation method]

[0029] The following will be combined with the attached Figure 1-5 The technical scheme of the present invention is clearly and completely described in detail with specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0030] Example

[0031] 1. Experimental Materials and Instruments

[0032] 1.1 Drugs and reagents

[0033] UPLC-MS / MS method for tacrolimus and its active metabolites was established. Required materials: hydroxychloroquine sulfate reference substance, tacrolimus standard substance, acetonitrile (chromatographic grade), formic acid (chromatographic grade), ZnSO 4. 7H2O, methanol (chromatographic grade), deionized water, 13-O desmethyl tacrolimus

[0034] 1.2 Instruments

[0035] Instruments and equipment used to establish UPLC-MS / MS of tacrolimus and its active metabolites: ultra-high performance liquid chromatography, triple quadrupole mass spectrometer, data processing system, dual-range electroanalytical balance, high-speed refrigerated centrifuge, vortex mixer, pure water equipment

[0036] 2. Reagent Preparation

[0037] 2.1 Preparation of tacrolimus and 13-O-desmethyltacrolimus working solutions

[0038] Accurately weigh 2.1 mg of tacrolimus standard, add appropriate amount of acetonitrile to dissolve, transfer to a 10 mL volumetric flask, and dilute with acetonitrile to obtain 210 μg mL -1 The tacrolimus standard stock solution was stored at -80°C for future use. Accurately pipette 210 μg mL -1 476.2 μL of tacrolimus standard stock solution was added to a 10 mL volumetric flask and fixed to volume with acetonitrile: water (3:7, v / v) to make 10 μg mL -1 Tacrolimus standard stock solution.

[0039] Accurately pipette 1 mL of tacrolimus standard stock solution (10 μg mL -1 ) into a 10 mL volumetric flask and dilute to 1000 ng mL with acetonitrile: water (3:7, v / v). -1 High concentration tacrolimus standard working solution, accurately pipette 1000ng·mL -1 1 mL of high-concentration tacrolimus standard sample was added to a 10 mL volumetric flask and fixed to volume with acetonitrile: water (3:7, v / v) to obtain a medium-concentration tacrolimus standard working solution of 100 ng mL -1 ; Accurately pipette 1 mL of the medium concentration standard solution into a 10 mL volumetric flask and dilute to 10 ng mL with acetonitrile: water (3:7, v / v). -1 Tacrolimus standard working solution. The preparation process is shown in Table 1.

[0040] 2.213-O Desmethyl Tacrolimus Working Solution Preparation

[0041] Accurately weigh 1.6 mg of 13-O-desmethyltacrolimus standard, dissolve it in an appropriate amount of methanol, transfer it to a 10 mL volumetric flask, and dilute it with methanol to obtain 160 μg mL -1 The 13-O desmethyl tacrolimus standard stock solution was stored at -80°C for future use. Accurately pipette 160 μg mL -1 13-O desmethyl tacrolimus standard stock solution 625 μL was added to a 10 mL volumetric flask and fixed to volume with acetonitrile: water (3:7, v / v) to make 10 μg mL -1 13-O-desmethyltacrolimus standard stock solution. The preparation process is shown in Table 2.

[0042] Accurately pipette 1 mL of 13-O desmethyl tacrolimus standard stock solution (10 μg mL -1 ) into a 10 mL volumetric flask and dilute to 1000 ng mL with acetonitrile: water (3:7, v / v). -1 High concentration 13-O desmethyl tacrolimus standard working solution, accurately pipette 1000ng·mL -1 1 mL of high-concentration standard sample was added to a 10 mL volumetric flask and fixed to volume with acetonitrile: water (3:7, v / v) to obtain a medium-concentration 13-O desmethyl tacrolimus standard working solution of 100 ng mL -1 The preparation process is shown in Table 2.

[0043] Table 1 Preparation process of tacrolimus working solution

[0044]

[0045] Table 2 Preparation process of 13-O desmethyl tacrolimus working solution

[0046]

[0047] 2.3 Preparation of internal standard working solution

[0048] Accurately weigh 1.74 mg of hydroxychloroquine sulfate reference substance, add appropriate amount of acetonitrile to dissolve, transfer to a 10 mL volumetric flask, dilute to the mark with acetonitrile, and shake well to obtain 174 μg mL -1 Hydroxychloroquine sulfate reference stock solution, equivalent to 132 μg mL -1 The hydroxychloroquine reference stock solution was stored at -20°C for future use. Accurately pipette 50 μL of the hydroxychloroquine reference stock solution into a 10 mL volumetric flask and dilute to 660 ng mL with acetonitrile: water (3:7, v / v). -1 Hydroxychloroquine reference stock solution. Accurately pipette 660ng·mL -1 Add 834 μL of the hydroxychloroquine reference stock solution to a 10 mL volumetric flask and dilute to 55.044 ng mL with acetonitrile: water (3:7, v / v). -1 Hydroxychloroquine reference working solution.

[0049] 2.4 Preparation of standard curve working solution

[0050] After diluting the working solution with whole blood containing ethylenediaminetetraacetic acid dipotassium (EDTA-2K) as the anticoagulant, take 100uL of simulated whole blood sample, add 20uL of hydroxychloroquine internal standard working solution, mix well, add 200uL of precipitant, vortex mix for 3min, and rotate at 12000r / min at 4℃. -1Centrifuge for 10 min, take the supernatant, and filter it through a 0.22 μm filter membrane to obtain the standard curve gradient concentration samples. The preparation process of whole blood samples of tacrolimus and its active metabolites is shown in Table 3.

[0051] Table 3 Preparation process of whole blood samples of tacrolimus and its active metabolites

[0052]

[0053] 2.5 Preparation of protein precipitant:

[0054] 0.2mol . L -1 Preparation of ZnSO4 solution: Accurately weigh about 0.6 g of ZnSO4.7H2O, add appropriate amount of deionized water, transfer to a 10 mL volumetric flask, dilute to the mark with deionized water, and shake well to obtain about 0.2 mol . L -1 ZnSO4 solution.

[0055] Preparation of precipitant: methanol / 0.2 mol . L -1 ZnSO4, 80 / 20 (containing 0.1% formic acid) was used to obtain a protein precipitant, which was then stored at 4°C for future use.

[0056] 3. Equipment conditions

[0057] 3.1 Chromatographic conditions

[0058] Column: ACQUITY BEH C 18 (2.1*100mm, 1.7μm); chromatographic column temperature: 25℃; mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid-acetonitrile; gradient elution, flow rate: 0.4mL . min -1 , injection volume was 10uL, and analysis time was 6min.

[0059] The parameters of gradient elution are: 0-2.8 min, A:B (95:5, v / v); 2.8-3.3 min, A:B (5:95, v / v); 3.3-4 min, A:B (5:95, v / v); 4-6 min, A:B (95:5, v / v); wherein A is 0.1% formic acid-water solution, and B is 0.1% formic acid-acetonitrile.

[0060] 3.2 Mass spectrometry conditions

[0061] Electrospray ionization (ESI) source, positive ion scanning, multiple reaction monitoring (MRM) mode were used for detection. The desolvation tube temperature was 500°C and the desolvation gas flow rate was 1000L.. hr -1 ; Source temperature: 150℃; Cone gas flow: 50L . hr -1 ; The ion reactions used for quantitative analysis are: tacrolimus: m / z 826.94→m / z 616.40; 13-O desmethyl tacrolimus: m / z 808.4-m / z 790.53; internal standard hydroxychloroquine: m / z 336.32→m / z 247.15. The mass spectrometry parameters of tacrolimus, 13-O desmethyl tacrolimus and their internal standard substances are shown in Table 4. Figure 1 The figure is the mass spectra of tacrolimus, its metabolite 13-O-tacrolimus and internal standard hydroxychloroquine obtained under the mass spectrometry conditions.

[0062] Table 4 Mass spectrometry parameters of tacrolimus, 13-O-nor-tacrolimus and internal standard

[0063]

[0064] 4. Biological specimen pretreatment

[0065] Take EDTA-2K as anticoagulant and hydroxychloroquine as internal standard working solution, take 100μL of whole blood sample, add 20μL of internal standard working solution, mix well, add 200μL of precipitant prepared under "2.5", vortex for 3min to lyse red blood cells and precipitate protein. 4℃12000r·min -1 Centrifuge for 10 minutes; after centrifugation, take the supernatant and filter it through a 0.22μm filter membrane, and inject 10μL.

[0066] 5. Methodological Validation

[0067] 5.1 Exclusivity

[0068] Take 100 μL of blank whole blood sample without adding internal standard working solution, and follow the operation under "4. Biological specimen pretreatment" to investigate the influence of endogenous matrix in blank whole blood sample, and obtain the blank whole blood chromatogram of the corresponding channel ( Figure 2 ); Take a simulated rat whole blood sample and follow the steps in “4. Biological specimen pretreatment” to examine the separation effect of each analyte in the sample and obtain a simulated whole blood sample chromatogram ( Figure 3 ). Under the established UPLC-MS / MS conditions, the retention time of tacrolimus is 3.68min, the retention time of 13-O desmethyl tacrolimus is 3.24min, and the retention time of internal standard hydroxychloroquine is 1.42min. It can be seen from the figure that there is no obvious impurity peak response at the peak position of each substance in the corresponding ion channel, indicating that the specificity of the experimental conditions is good.

[0069] 5.2 Standard curve

[0070] The gradient simulated blood sample prepared in "2.4" was treated with the method of "4. Biological specimen pretreatment", and the standard samples of different concentrations were measured using the chromatographic and mass spectrometric conditions of "3. Instrument conditions". The standard curve was fitted with the peak area of ​​the analyte / peak area of ​​the internal standard-concentration to obtain the linear regression equation:

[0071] Tacrolimus: f(x)=0.0160747x+0.05686, R 2 =0.9989(n=8)(see Figure 4 );

[0072] 13-O desmethyl tacrolimus: f(x) = 0.000147x + 0.009428, R 2 =0.9943(n=8)(see Figure 5 );

[0073] 5.3 Precision and Accuracy

[0074] Prepare the simulated whole blood to be tested according to step "4. Pretreatment of biological specimens", take the sample to be tested and detect it by liquid chromatography-mass spectrometry according to step "3. Instrument and equipment conditions", record the peak area corresponding to tacrolimus and 13-O desmethyl tacrolimus, substitute the peak area ratio of the peak area to the corresponding internal standard into the standard curve established in "5.2", and calculate the concentration of tacrolimus and 13-O desmethyl tacrolimus in the whole blood to be tested. The quality control concentration of the simulated whole blood sample is shown in Table 5.

[0075] Table 5 Preparation process of quality control solution of tacrolimus and its active metabolites simulated whole blood samples

[0076]

[0077] After diluting the working solution with EDTA-2K as the anticoagulant, take 100uL of simulated whole blood sample, add 20uL of hydroxychloroquine internal standard working solution, mix well, add 200uL of precipitant, vortex mix for 3min, and rotate at 12000r / min at 4℃. -1 After centrifugation for 10 minutes, the supernatant was taken and filtered through a 0.22 μm filter membrane to prepare five portions of the corresponding lower limit of quantitation, low, medium and high concentration QC whole blood sample solutions. According to the operation under "4" Whole blood sample pretreatment", each concentration level was analyzed 5 times, and the measured concentration was obtained by the curve obtained from the standard curve item. The ratio of the measured concentration to the theoretical concentration was calculated to obtain the accuracy. The samples at the same concentration level were operated in parallel 5 times within one day and analyzed for 5 consecutive days to examine the intra-day and inter-day precision of the method. The precision of the lower limit of quantitation, low, medium and high concentration samples was expressed as relative standard deviation (RSD). The results are shown in Table 6.

[0078] Table 6 Accuracy of tacrolimus and active metabolites (n=5)

[0079]

[0080]

[0081] Table 7 Intra-day and inter-day precision of tacrolimus and its metabolites

[0082]

[0083]

[0084] It can be seen from Tables 6 and 7 that the lower limit of quantification, intra-day precision of low, medium and high concentration tacrolimus whole blood sample solutions are <5.89%, inter-day precision <9.23%, accuracy is within the range of 101.01-109.83%, and RSD <5.82%. The lower limit of quantification, low, medium and high concentration intra-day precision of 13-O desmethyl tacrolimus whole blood sample solutions are <7.67%, inter-day precision <9.65%, accuracy is within the range of 100.06-105.80%, and RSD <8.04%, all of which are lower than 15%, meeting the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0085] 5.4 Matrix Effects:

[0086] Blank whole blood samples from 6 different individuals were collected and pre-treated according to "4. Pre-treatment of whole blood samples". Low and high concentrations of tacrolimus standard solutions, 13-O desmethyl tacrolimus and internal standard working solution were added. After vortex mixing, the concentrations were 750 ng mL -1 , 3ng·mL -1 The high and low concentration samples were injected and analyzed, and 6 samples were measured at each concentration level to obtain the corresponding peak area A i The low and high concentration tacrolimus standard solutions, 13-O desmethyl tacrolimus and internal standard were diluted with acetonitrile: water (30:70, v / v) mobile phase and directly injected for analysis. Six samples were measured at each concentration level to obtain the corresponding peak area As. i / A s Calculate the matrix effect. Results The normalized matrix effect factors of the internal standards of HQC and LQC were 0.87-1.06, the RSDs of high and low concentrations of tacrolimus were 4.37% and 10.20%, respectively, both lower than 15%, and the RSDs of the matrix effects of high and low concentrations of 13-O desmethyl tacrolimus whole blood sample solutions were 9.97% and 12.28%, respectively, both lower than 15%, which met the requirements of the 2020 edition of the Chinese Pharmacopoeia. The results are shown in Table 8.

[0087] Table 8 Matrix effects of tacrolimus and metabolites (n=6)

[0088]

[0089] 5.5 Stability Study

[0090] Same as "2.1 Preparation of tacrolimus and 13-O-desmethyltacrolimus working solution", prepare QC simulated whole blood samples of the test substance at low and high concentration levels, and examine their stability after being placed under freezing conditions (-80℃) for 7 days and at -20℃ for 7 days, and after being repeatedly frozen and thawed for 3 times at -80℃ and at -20℃ for 3 times. Calculate the concentration in the whole blood sample with the standard curve obtained in "5.1 Standard curve", and evaluate the sample stability with the first measured concentration as 100%.

[0091] The RSD of the low- and high-concentration tacrolimus and 13-O-desmethyltacrolimus whole blood sample solutions placed in a -80°C freezer for seven days and repeatedly frozen and thawed three times were all <15.00%. The results are shown in Tables 9 and 10.

[0092] Table 9 Stability after 7-day cryopreservation (n=6)

[0093]

[0094] Table 10 Freeze-thaw stability (n=6)

[0095]

[0096] In summary, the present invention has the following advantages:

[0097] 1. The present invention can simultaneously determine tacrolimus and its active metabolites. Compared with the existing single determination of tacrolimus, the present invention can more comprehensively understand the in vivo exposure of the immunosuppressant and its active metabolites, is more conducive to the comprehensive estimation of its pharmacological effects, has greater guiding significance for clinical practice, and provides a more detailed and accurate basis for individualized drug therapy.

[0098] 2. The pretreatment cost of the present invention is low and more convenient: compared with the existing solid phase extraction method for extracting tacrolimus, the cost of this method can be saved by about 50%, and the consumption of solid phase adsorption columns is eliminated; compared with the existing ether extraction, the reagents of the present invention are easy to obtain and more conducive to the labor protection of operators, shortening the concentration time and improving work efficiency. The protein precipitant used in the present invention is more economical, convenient and easy to obtain. Due to its simple operation, it can be applied to conventional determination

[0099] 3. The invention has strong specificity and adopts ACQUITY BEH C18 (2.1×100mm, 1.7μm) chromatographic column, aqueous phase: 0.1% formic acid in water; organic phase: 0.1% formic acid-acetonitrile, gradient elution, the retention time of tacrolimus is 3.68min, the retention time of hydroxychloroquine is 1.42min, and the retention time of 13-0-desmethyltacrolimus is 3.24min. The separation degrees are all greater than 1.7, with high separation efficiency and no mutual interference.

[0100] 4. The present invention has high sensitivity, and the quantitative limit of tacrolimus is 1 ng·mL -1 The limit of quantification of 13-O-desmethyltacrolimus is 10 ng·mL -1 (S / N>10).

[0101] In conclusion, the method of the present invention is rapid and accurate, highly sensitive, efficient, simple to operate, and can be applied to clinical testing of drugs.

[0102] Although the specific implementation modes of the present invention are described above, those skilled in the art should understand that the specific implementation modes described are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A method for simultaneously determining tacrolimus and its metabolite 13-O-nor-tacrolimus in whole blood by liquid chromatography-mass spectrometry, characterized in that: The method steps are as follows: (1) Pretreatment of whole blood samples: take a whole blood sample with anticoagulant added, add internal standard working solution, mix well, add protein precipitant, vortex and lyse red blood cells to precipitate protein; take the supernatant after centrifugation, filter through a filter membrane, and inject; (2) Inject the sample to be tested into the chromatographic system and elute it with the mobile phase. The chromatographic conditions are as follows: Chromatographic column: BEH C 18 ; Column temperature: 25°C; Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid-acetonitrile; Gradient elution, flow rate 0.4 mL . min -1 , injection volume was 10uL, analysis time was 6min; (3) After the elution, mass spectrometry is performed to detect tacrolimus and its metabolite 13-O-nor-tacrolimus as the analytes; the mass spectrometry conditions are as follows: Electrospray ionization source, positive ion scanning, multiple reaction monitoring (MRM) mode was used for detection. The desolvation tube temperature was 500°C and the desolvation gas flow rate was 1000L. . hr -1 , source temperature: 150℃, cone gas flow: 50L . hr -1 .

2. The method for simultaneously determining tacrolimus and its metabolite 13-O-nortacrolimus in whole blood by liquid chromatography-mass spectrometry according to claim 1, characterized in that: In the step (1): EDTA-2K is used as an anticoagulant and hydroxychloroquine is used as an internal standard working solution; The protein precipitant is methanol / 0.2 mol / L in a volume ratio of 80 / 20 . L -1 ZnSO4 solution, wherein the solution contains 0.1% formic acid.

3. The method for simultaneously determining tacrolimus and its metabolite 13-O-nortacrolimus in whole blood by liquid chromatography-mass spectrometry according to claim 1, characterized in that: In the step (2), the parameters of the gradient elution are: 0-2.8 min, A:B=95:5; 2.8-3.3 min, A:B=5:95; 3.3-4 min, A:B=5:95; 4-6 min, A:B=95:5; all are measured in v / v.

4. The method for simultaneously determining tacrolimus and its metabolite 13-O-nortacrolimus in whole blood by liquid chromatography-mass spectrometry according to claim 1, characterized in that: In the step (3), the ion reactions used for quantitative analysis are: tacrolimus: m / z 826.94→m / z 616.40; 13-O desmethyl tacrolimus: m / z 808.4-m / z 790.53; internal standard hydroxychloroquine: m / z 336.32→m / z 247.15.

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