A method for quantitatively detecting mycophenolic acid in trace amounts of blood by high performance liquid chromatography-tandem mass spectrometry

Abstract

The present invention belongs to the field of detection of mycophenolic acid blood drug concentration, and specifically relates to a method for quantitatively detecting mycophenolic acid in trace blood by high performance liquid chromatography-tandem mass spectrometry, which comprises the following steps: after adding an internal standard solution to the plasma sample to be tested, adding a mixed solution of KH2PO4 and EDTA, the pH value of the mixed solution is 8-9, and mixing evenly; adding a propionic anhydride solution to the evenly mixed system, oscillating and reacting, centrifuging, and taking the supernatant for LC-MS / MS detection. Mycophenolic acid has a phenolic hydroxyl group, and under certain pH value and temperature, it undergoes an esterification reaction with propionic anhydride, and the derivative product is [M + CH3CH2CO] + peak, which simultaneously solves the problem that the sample volume is too low to affect the sensitivity, and reduces the interference of other substances.

Description

Technical Field

[0001] The invention belongs to the field of mycophenolic acid blood concentration monitoring, and specifically relates to a method for quantitatively detecting mycophenolic acid in trace amounts of blood by high performance liquid chromatography-tandem mass spectrometry. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Mycophenolate mofetil (MF) is a trade name for an immunosuppressant drug, also known as Seccoping. It is mainly used clinically to prevent and treat rejection reactions of kidney, liver, heart and bone marrow transplants and autoimmune diseases. Mycophenolate mofetil (MF) will be completely metabolized into mycophenolic acid (MPA) in the body to exert its pharmacological activity. Due to the large individual differences in the bioavailability of mycophenolic acid drugs in the human body, the blood drug concentration is closely related to the efficacy and toxicity, and the blood drug concentration is easily affected by many factors (genetic characteristics, transplant type, combined medication, comorbidities, age, etc.). Therefore, by regularly monitoring the blood drug concentration of mycophenolic acid in patients, an individualized dosing plan is formulated to provide auxiliary guidance for promoting the rational use of mycophenolic acid drugs in clinical practice.

[0004] However, organ transplant patients have to endure a double blow physically and psychologically after surgery. Irregular blood sampling to monitor drug concentrations is a long-term pain and harm that patients need to endure for a certain period of time. Each monitoring generally requires milliliters of whole blood, and the frequency of blood sampling is higher in the early stage after transplantation, because multiple blood sampling points are needed to estimate the relationship between mycophenolic acid blood concentration and AUC (area under the drug-time curve), so as to more accurately reflect the patient's drug exposure and more accurately predict the drug efficacy. However, multiple blood sampling increases the patient's physiological burden, which may be an unavoidable pain for adult patients, not to mention for special groups of younger age such as infants or children. Therefore, for organ transplant patients, when monitoring blood drug concentrations, minimally invasive micro-blood sampling should be the first choice.

[0005] At present, there are reports on the use of dried blood spots to monitor mycophenolic acid blood concentration. Compared with conventional blood collection methods, dried blood spot sampling is less invasive, the sample size is small, and patients are more likely to accept it. However, due to the small sample size, the conventional pre-treatment method has poor sensitivity; the stability of dried blood spots during room temperature drying; the uniformity of dried blood spots during preparation; individual differences in hematocrit will have a certain impact on the concentration measurement results; and the consistency of results with other types of samples remains to be investigated, etc. These uncertainties have affected the promotion and application of dried blood spot mycophenolic acid drug monitoring in clinical practice. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for quantitatively detecting trace amounts of mycophenolic acid in blood by high performance liquid chromatography-tandem mass spectrometry.

[0007] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0008] In a first aspect, the present invention provides a method for quantitatively detecting trace amounts of mycophenolic acid in blood by high performance liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0009] After adding the internal standard solution to the plasma sample to be tested, add a mixed solution of KH2PO4 and EDTA, the pH value of the mixed solution is 8-9, and mix well;

[0010] Add propionic anhydride solution to the mixed system, shake for reaction, centrifuge, and take the supernatant for LC-MS / MS detection.

[0011] Mycophenolic acid is also a phenolic substance in terms of molecular structure. Propionic anhydride reagents have been used in the derivatization of phenolic substances. Derivatization can not only improve the detection sensitivity but also improve the stability of the substance. However, there are no reports on the derivatization of mycophenolic acid. Therefore, the inventors tried to use propionic anhydride reagents to derivatize mycophenolic acid substances. After derivatization, no target substance was found in the original mycophenolic acid ion channel, which indicates that the mycophenolic acid substance itself has undergone a transformation during the derivatization process.

[0012] First, the parent ion scanning and product ion fragment scanning of the derivatized mycophenolic acid were performed on the mass spectrometer, and the sizes of the parent ion and characteristic daughter ion fragments of the derivatized mycophenolic acid were finally determined. The transformation process of the derivatized substance was determined by comparing the ion pair information before and after the derivatization of mycophenolic acid. Mycophenolic acid has a phenolic hydroxyl group. At a certain pH value and temperature, it undergoes an esterification reaction with propionic anhydride. The derivatized product is more ionized and more stable [M + CH3CH2CO] + The peak was obtained by analyzing the molecular weight of the sample from 320 to 376, and the increase in molecular weight also reduced the interference of other substances in the sample (in complex biological samples, the lower the molecular weight, the more susceptible it is to interference).

[0013] The specific derivatization reaction process is as follows Figure 3 As shown, the phenolic hydroxyl group in mycophenolic acid and the carboxyl group in propionic acid undergo an esterification reaction, and the dotted line in the figure indicates the location where the esterification reaction occurs.

[0014] In addition, the derivatization process needs to occur in an environment with a pH value greater than 7. As the reaction proceeds, other products such as propionic acid will be produced, causing the environmental pH value to change. The pH value gradually decreases, and at the same time, the protein precipitation process is promoted, which may be related to the change in the isoelectric point of the protein due to the decrease in pH. When the pH is lower than 7, the derivatization reaction automatically terminates, and the low pH is also conducive to improving the ionization efficiency during mass spectrometry detection. Therefore, the entire derivatization process does not require conventional steps such as organic solvent protein precipitation, nitrogen blowing, and termination of the derivatization reaction. The pretreatment operation is simple, time-saving, labor-saving, and low-cost, which is conducive to clinical promotion and use.

[0015] In some embodiments, the internal standard is mycophenolic acid-D3.

[0016] In some embodiments, the concentration of EDTA in the mixed system after mixing is 0.05-0.1 mol / L. EDTA is used to maintain the stability of the buffer system.

[0017] In some embodiments, after adding propionic anhydride to the mixed system, the concentration of propionic anhydride in the mixed system is 5%-10%. When the concentration of propionic anhydride is too high, propionic acid will be generated during the derivatization reaction. If the amount added is too large, too much propionic acid will be generated, and the pH value will immediately decrease, affecting the derivatization reaction. When the concentration of propionic anhydride is too low, the reaction efficiency of the derivatization reaction will be affected.

[0018] Preferably, the solvent of the added propionic anhydride solution is acetonitrile. The chemical properties of propionic anhydride are relatively active, and it is easy to react chemically with water or alcohols (such as methanol or ethanol) to generate propionic acid or esters, so acetonitrile is selected as the solvent, which is relatively stable and not easy to be oxidized or reduced.

[0019] In some embodiments, the shaking reaction time is 10-20 min.

[0020] In some embodiments, the volume of the plasma sample to be tested is 3-8 μL, preferably 5 μL.

[0021] In some embodiments, the detection conditions of liquid chromatography are: the chromatographic column is a C18 chromatographic column;

[0022] Mobile phase: Phase A is an aqueous solution containing 0.05-0.15% formic acid and 0.5-1.5 mM ammonium formate, and phase B is methanol.

[0023] The analytes were ionized in the positive ion mode in the mass spectrometer ion source, and formic acid was added to the mobile phase to improve the ionization efficiency and thus the detection signal.

[0024] The addition of ammonium formate is mainly to improve the chromatographic peak shape, making it more single, sharp and symmetrical.

[0025] Preferably, the detection flow rate of the liquid chromatography is 0.3-0.5 mL / min; the gradient elution program is: 0-1.0 min, 10% B; 1.1-3.5 min, 98% B; 3.6-4.5 min, 10% B.

[0026] In some embodiments, the detection ion pairs of the derivatized mycophenolic acid and the internal standard (mycophenolic acid-D3) are m / z 377→207 and m / z 380→210, respectively; the lens voltages are 110 V and 115 V, respectively; the collision voltages are 13 V and 13 V, respectively; and the electrospray ESI source positive ion mode is adopted, with an ion source temperature of 365°C and a positive ion voltage of 4000 V.

[0027] The beneficial effects achieved by one or more embodiments of the present invention are as follows:

[0028] Mycophenolic acid has a phenolic hydroxyl group. At a certain pH value and temperature, it undergoes an esterification reaction with propionic anhydride. The derived product is more ionized and more stable [M + CH3CH2CO] + The peak was solved, and the problem of low sample volume affecting sensitivity was solved, thus greatly improving the sensitivity of the analysis (by nearly 20 times), and the molecular weight was changed from 320 to 376. The increase in molecular weight also reduced the interference of other substances in the sample (in complex biological samples, the lower the molecular weight, the easier it is to be interfered). At the same time, it made up for the shortcomings of the dry blood spot method.

[0029] The performance of the method was fully verified and the method had good stability. It was compared with the detection method with conventional sampling volume and the performance was equivalent. The results were consistent and there was no obvious deviation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0031] Figure 1 is a result correlation curve of an embodiment of the present invention;

[0032] Figure 2 1 is a comparison chart of the signal intensities of non-derivatized (A) and derivatized (B) mycophenolic acid in an embodiment of the present invention.

[0033] Figure 3 Detailed derivatization reaction diagram of mycophenolic acid in an embodiment of the present invention. DETAILED DESCRIPTION

[0034] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0035] The present invention will be further described below in conjunction with the embodiments.

[0036] Example

[0037] Mycophenolic acid (standard), purity: 98%; Mycophenolic acid-D3 (internal standard), chemical purity: 97%, isotopic abundance: 99.8%.

[0038] Instrument: YS EXT 9050MD high performance liquid chromatography tandem mass spectrometry detection system.

[0039] Kinetex C18 column (2.1 mm × 50 mm, 2.6 μm);

[0040] Mobile phase: Phase A was 0.1% formic acid in 1 mM ammonium formate in water, and phase B was methanol;

[0041] Column temperature: 40 °C; flow rate: 0.4 mL / min.

[0042] Gradient elution: 0~1.0 min, 10% B, 1.1~3.5 min, 98 % B, 3.6~4.5 min, 10% B.

[0043] The detection ion pairs of derivatized mycophenolic acid and internal standard (mycophenolic acid-D3) were m / z 377→207 and m / z380→210, respectively. The lens voltages were 110 V and 115 V, respectively. The collision voltages were 13 V and 13 V, respectively. The electrospray ESI source positive ion mode was used, with an ion source temperature of 365℃ and a positive ion voltage of 4000 V.

[0044] Preparation of mycophenolic acid standard stock solution: Take 10 mg of mycophenolic acid standard, add acetonitrile to dissolve and make up to volume in a 10 mL volumetric flask, shake well to obtain 1 mg / mL mycophenolic acid stock solution.

[0045] Preparation of mycophenolic acid-D3 internal standard solution: Take 1 mg of internal standard, add 1 mL of acetonitrile to dissolve and mix, and then obtain 1 mg / mL internal standard stock solution.

[0046] Preparation of mycophenolic acid series standard solutions: First, according to the therapeutic range of mycophenolic acid blood concentration and the expected clinical distribution level, the calibration curve range is determined to be 0.2-40 μg / mL. Take blank plasma and add different volumes of 1 mg / mL mycophenolic acid stock solution. The final concentrations of each point of the prepared calibration curve are 0.2 μg / mL, 0.4 μg / mL, 1.0 μg / mL, 3.2 μg / mL, 8.0 μg / mL, 16 μg / mL, and 40 μg / mL, for a total of 7 concentration points. The pretreatment method is the same as the plasma sample pretreatment.

[0047] Preparation of mycophenolic acid quality control solution: The concentration of quality control products is determined according to the Chinese Pharmacopoeia. The high, medium and low level quality controls are respectively set near 75% of the high value of the calibration product, near the middle point of the calibration curve, and near 3 times the lower limit of quantitation. Blank plasma is taken and different volumes of 1 mg / mL mycophenolic acid stock solution are added. The high, medium and low level quality control concentrations are 30 μg / mL, 3.2 μg / mL and 0.6 μg / mL respectively. The quality control product represents a quality control range to effectively control the quality of the detection link. The pretreatment method is the same as the plasma sample pretreatment.

[0048] Plasma sample derivatization treatment method: take 5 μL of plasma sample, add 2 μL of 10 μg / mL internal standard solution, mix, add 25 μL of 0.2 mol / L KH2PO40.1 mol / L EDTA solution (pH 8.5) and mix, then add 10 μL of acetonitrile solution containing 25% propionic anhydride (volume ratio 1:3), shake and mix for 15 min, centrifuge at 10000 rpm for 3 min, take 20 μL of supernatant, transfer to an injection vial, add 20 μL of water and mix, and perform LC-MS / MS analysis.

[0049] Sensitivity comparison: After derivatization, the signal of mycophenolic acid at the same concentration can be increased by 20 times, which greatly reduces the sample volume. The retention time of the derivatized and non-derivatized substances is consistent with that of the internal standard. The specific results are shown in Figure 2 .

[0050] The feasibility of the established high performance liquid chromatography-tandem mass spectrometry method for detecting mycophenolic acid concentration in trace blood was investigated by methodology, including detection limit, quantification limit, linearity, precision, extraction recovery, and comparison with conventional detection methodology to evaluate the consistency of the detection results of the two methods. In addition, while conducting the methodological investigation, the same 5 μL trace sample was pre-treated by conventional method and derivatization method, and the difference in the effects of the two pre-treatment methods was compared.

[0051] The above conventional detection method is: take 200 μL of plasma in a 1.5 mL centrifuge tube, add 800 μL of internal standard solution (containing acetonitrile, as above), vortex for 5 minutes, centrifuge at 14000 r / min for 10 minutes, and take the supernatant for sampling and analysis (liquid chromatography conditions and mass spectrometry conditions are the same as above).

[0052] Detection limit, quantification limit and linear range: The calibration curve was prepared according to the preparation method of the mycophenolic acid series standard solution, and the extraction method was the same as that of the plasma sample. The concentration of the mycophenolic acid standard solution was used as the horizontal axis, and the signal ratio of mycophenolic acid to its internal standard was used as the vertical axis to construct the calibration curve, and the regression equation and correlation coefficient were obtained. The concentration corresponding to the signal-to-noise ratio (S / N) ≥ 3 was used as the detection limit, and the concentration corresponding to (S / N) ≥ 10 and the result deviation within 15% was used as the quantification limit. The detection limit and quantification limit of mycophenolic acid were obtained, and the results are shown in Table 1. The derivatized product is represented by mycophenolic acid-PA. Compared with the underivatized mycophenolic acid, the performance of the derivatized mycophenolic acid is significantly better.

[0053] Table 1 Detection limit, quantification limit and linear range

[0054]

[0055] Precision: The quantitative lower limit sample and three levels of quality control samples were used as the test samples, with three analysis batches per day, each batch repeated three times, and tested continuously for 5 days to evaluate the intra-day precision and inter-day precision, and the coefficient of variation CV was used to express the precision. According to industry standards, the coefficient of variation CV must be less than 15%. The precision test results of mycophenolic acid are shown in Table 2. The intra-day and inter-day precision coefficients of variation CV of each level of the derivatized group were less than 15%. Due to sensitivity issues, two low-concentration samples in the non-derivative group were not detected and no valid data was found. Although the coefficient of variation CV of the other two concentration samples was less than 15%, the overall precision results were worse than those of the derivatized group.

[0056] Table 2 Precision

[0057]

[0058] Note: N / A means not detected

[0059] Extraction recovery rate: clinical patient plasma samples were selected and divided into 3 equal volumes. A blank reagent (methanol chromatography grade or above) without the analyte was added to one of the samples as the base sample; the same volume of mycophenolic acid standard solution with different concentrations was added to the other 2 samples. The concentration of the standard solution in the sample after addition is shown in Table 3. Two recovery samples with different added concentrations were prepared, and each sample was tested 3 times. It is required that the mycophenolic acid in the low-level sample is near the lower limit of the therapeutic range after the addition of the standard solution, and the mycophenolic acid concentration in the high-level sample is near the upper limit of the therapeutic range. The extraction recovery rate of mycophenolic acid at different concentrations is in the range of 85%-115%, indicating that the test results are accurate and good.

[0060] Table 3 Extraction recovery

[0061]

[0062] Comparison of consistency of results: 10 plasma samples from clinical patients were selected and tested by the derivatization method and the conventional detection method, respectively. The derivatization method required a sample size of 5 μL, and the conventional method required a sample size of 100 μL. Each sample was tested once, and the test results were subjected to a paired t-test using SPSS software to analyze whether there was a significant difference in the test results of the two methods. The results are shown in Table 4. For the comparison of the test results under different methods, the P value was > 0.05, which indicated that there was no significant difference in the quantitative results of the two detection methods, the test data were relatively similar, and they were equivalent in terms of quantitative results. Figure 1 The correlation between the two detection methods is shown in Figure 2. By looking at the regression coefficient and correlation coefficient, it can be seen that the two detection methods have a good correlation. Therefore, when the sample size is reduced by 20 times, the detection results similar to those of the conventional method can be obtained, such as Figure 1 As shown, the method can be directly used in clinical testing after being fully verified on a large number of samples without the need for additional calibration or therapeutic reference range shifting.

[0063] Table 4. Consistency of results

[0064]

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for quantitatively detecting trace amounts of mycophenolic acid in blood by high performance liquid chromatography-tandem mass spectrometry, characterized in that: The steps include: After adding the internal standard mycophenolic acid-D3 solution to the plasma sample to be tested, add a mixed solution of KH2PO4 and EDTA, the pH value of the mixed solution is 8-9, and mix well; Adding propionic anhydride solution to the mixed system, shaking the mixture to obtain derivatized mycophenolic acid, centrifuging, and taking the supernatant for LC-MS / MS detection; The chromatographic column for liquid chromatography was a C18 column; Mobile phases for liquid chromatography: Phase A is an aqueous solution containing 0.05-0.15% formic acid and 0.5-1.5 mM ammonium formate, and phase B is methanol; The detection flow rate of the liquid chromatography was 0.3-0.5 mL / min; the gradient elution program was: 0-1.0 min, 10% B, 1.1-3.5 min, 98% B, 3.6-4.5 min, 10% B.

2. The method for quantitatively detecting trace amounts of mycophenolic acid in blood by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: In the mixed system after mixing, the concentration of EDTA is 0.05-0.1 mol / L.

3. The method for quantitatively detecting trace amounts of mycophenolic acid in blood by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The concentration of propionic anhydride added to the mixed system after mixing is 5%-10%.

4. The method for quantitatively detecting trace amounts of mycophenolic acid in blood by high performance liquid chromatography-tandem mass spectrometry according to claim 3, characterized in that: The solvent of the added propionic anhydride solution is acetonitrile.

5. The method for quantitatively detecting trace amounts of mycophenolic acid in blood by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The oscillation reaction time is 10-20 minutes.

6. The method for quantitatively detecting trace amounts of mycophenolic acid in blood by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The volume of the plasma sample to be tested is 3-8 μL.

7. The method for quantitatively detecting trace amounts of mycophenolic acid in blood by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The detection ion pairs of derivatized mycophenolic acid and internal standard mycophenolic acid-D3 were m / z 377→207 and m / z 380→210, respectively; The lens voltages are 110 V and 115 V respectively; The collision voltages were 13 V, 13 V; The electrospray ESI source positive ion mode was used, with the ion source temperature at 365 °C and the positive ion voltage at 4000 V.

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