Method for rapidly and quantitatively detecting concentration of mycophenolic acid in serum by high performance liquid chromatography-tandem mass spectrometry

Abstract

The invention discloses a method for rapidly and quantitatively detecting mycophenolic acid (MPA) in a trace serum sample by high performance liquid chromatography-tandem mass spectrometry, which comprises the following steps: pretreating the trace serum sample by a protein precipitation method, separating MPA from interference components in a serum matrix by high performance liquid chromatography, detecting MPA by mass spectrometry, and quantifying by an isotope internal standard method. The horizontal axis is the concentration of the mycophenolic acid standard solution, and the longitudinal axis is the peak area ratio of the mycophenolic acid to the internal standard of the mycophenolic acid, constructing a calibration curve, obtaining a regression equation and a correlation coefficient, and calculating the content of MPA. The method for quantitatively detecting mycophenolic acid in serum, provided by the invention, has the advantages of low venous blood sampling amount, trace serum detection, low detection cost, simplicity and rapidness in operation and accurate result, and solves the problems of low removal efficiency of impurities such as protein and the like and unstable matrix effect or recovery rate.

Description

Technical Field

[0001] This invention belongs to the field of blood drug concentration monitoring technology, specifically relating to a method for rapid quantitative detection of trace amounts of mycophenolic acid in serum using high performance liquid chromatography-tandem mass spectrometry. Background Technology

[0002] Mycophenolic acid (MPA) is the active metabolite of the immunosuppressant mycophenolate mofetil (MMF) (trade name: mycophenolate mofetil), and is widely used in the treatment of post-organ transplant rejection and autoimmune diseases. MPA exerts its immunosuppressive effect by selectively inhibiting inosine mononucleotide dehydrogenase (IMPDH), thereby blocking the proliferation of T / B lymphocytes.

[0003] Mycophenolic compounds (MPA) plasma concentrations are closely related to efficacy and toxicity. However, the bioavailability and pharmacokinetics of MPA vary greatly among individuals, and plasma concentrations are affected by multiple factors. Concentrations below the therapeutic window may lead to rejection, while concentrations above the therapeutic window may cause adverse reactions such as bone marrow suppression and gastrointestinal toxicity. Therefore, therapeutic drug monitoring (TDM) of patients taking mycophenolic compounds is crucial for guiding individualized MPA administration in clinical practice.

[0004] After organ transplantation, patients taking mycophenolic compounds (MPCs) experience a significant psychological and physical burden due to prolonged and frequent drug concentration monitoring. MPC monitoring requires blood sampling at multiple time points, with each sampling point involving at least 2 mL of blood. Clinically, five sampling points are common, but in some special cases, up to eleven are used. Clinically, the area under the drug-time curve (AUC) is calculated based on the MPC blood concentration at each time point. The AUC reflects the patient's drug exposure after administration and helps predict treatment efficacy. Given this characteristic of MPC monitoring, reducing the amount of blood drawn at each monitoring point and using micro-samples can minimize the burden and harm caused by high-frequency drug concentration monitoring.

[0005] Currently, the mainstream clinical detection method for MPA is enzyme-amplified immunoassay (EMIT). With the development of detection technology, high performance liquid chromatography (HPLC), high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS), and ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) have begun to be used in clinical practice.

[0006] Enzyme-amplified immunoassay (EPI) offers high automation but suffers from antibody cross-reactivity, potentially leading to false positives (e.g., interference from metabolites like mycophenolate mofetil). Its accuracy is lower than chromatography. While high-performance liquid chromatography (HPLC) is reported to be cost-effective, it suffers from low sensitivity, susceptibility to matrix interference, and long analysis times (>10 min per sample). Liquid chromatography-mass spectrometry (LC-MS), while offering better sensitivity and specificity, often employs complex pretreatment (requiring derivatization, solid-phase extraction, etc.) or protein precipitation (acetonitrile or methanol) to replace solid-phase extraction. However, current methods still face challenges such as high precipitant usage, failure to meet continuous reagent consumption requirements, and susceptibility to solvent effects. Furthermore, many of the aforementioned methods utilize plasma samples, which, due to their complex composition and the use of anticoagulants, exhibit low clearance efficiency for proteins and other impurities, matrix effects (ion inhibition rate >20%), and unstable recovery rates. The use of buffer salts can also affect column life, making it difficult to meet the rapid and accurate testing needs of large clinical sample volumes and hindering clinical adoption. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for rapid quantitative detection of trace amounts of mycophenolic acid in serum using high-performance liquid chromatography-tandem mass spectrometry, comprising the following steps: Add protein precipitant (containing isotopic internal standard MPA-d3) to the trace serum sample to be tested, and vortex to mix; Centrifuge the mixed system, take the supernatant and dilute it in pure water, shake to mix, and take the diluted liquid for LC-MS / MS detection. The isotope internal standard method was used for quantification. The concentration of mycophenolic acid standard solution was used as the abscissa, and the peak area ratio of mycophenolic acid to its internal standard was used as the ordinate to construct a calibration curve, obtain the regression equation and correlation coefficient, and calculate the content of the analyte.

[0008] In some embodiments, the volume of the serum sample to be tested is only 5-15 μL, and the amount of blood drawn from the patient's vein is no more than 1 mL.

[0009] In some embodiments, the serum is obtained by collecting blood from inert separation gel coagulation vacuum blood collection tubes or drying tubes, and centrifuging the blood after natural coagulation. Compared with plasma, this reduces the interference of other complex substances and anticoagulants in the sample.

[0010] In some embodiments, the amount of protein precipitant (containing the isotopic internal standard MPA-d3) used is significantly reduced to only 20-60 μL. Compared with the existing large-volume method (taking 780 μL as an example), a 4L bottle of methanol will theoretically increase the sample volume by about 13 times. The cost of methanol reagents and its consumption ratio are significantly reduced to negligible, which meets the current hospital requirements for continuous cost reduction in clinical testing.

[0011] In some embodiments, the protein precipitant is composed of methanol and a 50-100 mg / mL zinc sulfate heptahydrate solution mixed at a volume ratio of 7:3. This mixture promotes the protein precipitation process, significantly enhances the precipitation efficiency of soluble proteins and other impurities, and makes the extracted target analyte solution purer. Figure 1 As shown, tube A contains 15 μL of plasma with 60 μL of conventional protein precipitant (methanol) containing an isotope internal standard as a control; tube B contains 15 μL of serum with 60 μL of protein precipitant (methanol and zinc sulfate heptahydrate mixed at a volume ratio of 7:3). Compared to the control, when the sample is added, the precipitate in tube A is thin and loose, while that in tube B is a thick and compact clump. Figure 2 As shown, after vortexing and centrifugation of the mixing system, tube A has obvious unadsorbed precipitate residue on the wall, and the supernatant is pale yellow; tube B has a clean wall with only trace residue, and the supernatant is colorless and transparent. The precipitate obtained is more than that of tube A, showing a highly efficient precipitation effect. This can significantly reduce the interference of protein components on the detection of analytes, reduce matrix effects, and reduce the risk of impurities clogging the chromatographic column and instrument tubing.

[0012] In some embodiments, the isotopic internal standard protein precipitant is composed of a protein precipitant and an MPA-d3 internal standard working solution mixed at a volume ratio of 100:1.

[0013] In some embodiments, the mycophenolic acid D3 internal standard working solution is prepared by the following steps: MPA-d3 is dissolved in methanol to prepare a 1 mg / mL stock solution, and then 15 μL of the stock solution is transferred and added to 985 μL of methanol to obtain 1000 μL of internal standard working solution with a concentration of 15 μg / mL.

[0014] In some embodiments: the mixing system is vortexed at a speed of 2500 rpm for 5 minutes.

[0015] In some embodiments: the mixed system is centrifuged at 15,000 rpm for 5 min, and 10 μL of the supernatant is transferred and mixed with 300 μL of diluent.

[0016] In some embodiments, the diluent is pure water, which is inexpensive and can further effectively reduce matrix effects, alleviate ionization inhibition or enhancement effects, reduce cone blockage or plasma instability, and protect the ion source of the chromatographic column. The interference response detected in blank serum samples after the above pretreatment steps is significantly lower than that of undiluted plasma samples using conventional large-volume organic solvents (methanol) as precipitants; the matrix interference response intensity of the latter is approximately four times that of the former. Figure 3 Blank plasma matrix chromatogram and Figure 4 The matrix chromatogram of blank serum is shown.

[0017] In some embodiments, the liquid chromatography column is a C18 column, Phenyl-Hexyl® 2.6µm C1890 Å, 50x3mm.

[0018] In some embodiments: the mobile phase A of the liquid chromatography section is an aqueous solution containing 0.1% formic acid, and the mobile phase B is a methanol solution containing 0.1% formic acid. This is because the use of buffer salts can easily lead to their precipitation, clogging the pores between the stopper plate and the bonded phase particles, hindering mass transfer of the mobile phase, and causing an increase in column pressure. Salts within the column can also alter the retention time of the same compound. Buffer salts can penetrate deep into the bonded phase, damaging the silica matrix, leading to bonded phase loss from the column, a loosened column bed, or condensation on the surface of the bonded phase, making it difficult for the C18 carbon chains to extend. The reduced retention capacity of substances leads to decreased column efficiency. Therefore, the mobile phase of this invention does not contain any buffer salts to compensate for the above defects, extend the service life of the chromatographic column, reduce the growth of microorganisms in the mobile phase, and further reduce the risk of impurities entering the chromatographic system. To date, the laboratory has cumulatively analyzed more than 1,200 clinical samples of mycophenolic acid, exceeding the average number of injections of 500-1,000 per column. The chromatographic peak shape is normal, the retention time variation is still ≤±2.5%, and the column pressure is stable at 17.9±0.48 MPa with a CV of 2.68%.

[0019] In some embodiments: Chromatographic conditions: Gradient elution is performed using a mixture of mobile phase A and mobile phase B, with parameters shown in Table 1; flow rate is 0.5-0.8 mL / min, column temperature is 35-45℃, and injection volume is 1-2 μL.

[0020] Table 1 Gradient elution parameters of the mobile phase .

[0021] In some embodiments: mass spectrometry conditions: the ion source is in electrospray ionization (ESI+) mode, positive ion mode scanning, and multiple reaction monitoring (MRM) analysis; the mass spectrometry parameters are: ion source temperature (TEM): 550℃; curtain gas pressure (CUR): 20.00psi; ionization voltage (IS): 5000V; nebulizer pressure (GS1): 50.00psi; auxiliary heater pressure (GS2): 50.00psi; collision gas pressure (CAD): 9.00psi; and the quantitative ion pairs, declustering voltage (DP), collision voltage (CE), inlet voltage (EP), and outlet voltage (CXP) of the analyte are monitored simultaneously. The parameters are shown in Table 2.

[0022] Table 2 Mass Spectrometry Detection Parameters .

[0023] In some embodiments, the mycophenolic acid mixed working stock solution (MIX) is prepared by the following steps: the standard is dissolved in methanol to prepare a stock solution of 1 mg / mL, and then 100 μL of the stock solution is transferred to 900 μL of 78% methanol-water to obtain 1000 μL MIX working stock solution (the final solution system is 80% methanol-water), with a concentration of 100 μg / mL.

[0024] The working solution for the mycophenolic acid standard curve was prepared according to the following steps: The above mixed standard stock solution was prepared into calibrator solutions with six different concentration points using blank serum matrix. Taking 1.5 mL of each concentration point as an example (2 mL of STD5 needs to be prepared as stock solution for STD1 and STD2), the configuration parameters are shown in Table 3. Table 3. Preparation parameters of working solution for mycophenolic acid standard curve .

[0025] In some embodiments, the blank serum matrix is ​​blank healthy human serum that does not contain mycophenolic acid.

[0026] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows: This invention provides a method that requires minimal venous blood collection, detects trace amounts of serum, features a simple and rapid pretreatment process, uses minimal reagents, is low in cost, exhibits high sensitivity and specificity, and yields accurate results. It completes the separation and detection of mycophenolic acids in serum in approximately 2.4 minutes, overcoming the problems of low removal efficiency of impurities such as proteins, matrix effects, or unstable recovery rates. The method has undergone thorough performance validation and demonstrates good stability. It provides a clinically applicable method for mycophenolic acid monitoring that requires minimal blood volume, has low detection costs, is simple and rapid to operate, and yields accurate results. It is suitable for large-scale quantitative analysis of clinical samples and is conducive to clinical promotion. Attached Figure Description

[0027] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0028] Figure 1 A comparative experimental diagram showing the addition of mycophenolic plasma and serum samples to different precipitants.

[0029] Figure 2 Comparative experimental results of mycophenolic plasma and serum samples after pretreatment with different precipitants.

[0030] Figure 3 Chromatograms showing the intensity of interfering signals after pretreatment of blank plasma samples with protein precipitant (large volume methanol).

[0031] Figure 4After pretreatment of blank serum samples according to the present invention, the chromatogram of interference signal intensity was detected.

[0032] Figure 5 Chromatogram of extracted ions from mycophenolic acid standard (retention time 2.21 min).

[0033] Figure 6 Chromatogram of mycophenolic acid internal standard extracted ions (retention time 2.21 min). Detailed Implementation

[0034] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0035] To enhance understanding of the present invention, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0036] Example 1. Materials 1.1 Serum and plasma samples: The samples collected in this invention were from healthy individuals who underwent health checkups in June 2024 at the Health Management Center of the First Affiliated Hospital of Jinan University. The samples used for cumulative column lifespan testing were from kidney transplant patients treated with mycophenolate mofetil from June 2024 to the present at the Department of Kidney Transplantation and Department of Pediatrics of the First Affiliated Hospital of Jinan University.

[0037] 1.2 Major Instruments and Equipment: High-performance liquid chromatography-mass spectrometry (Jasper HPLC-AB SCIEX Triple Quad 4500MD, Shanghai Aibocaisi Analytical Instrument Trading Co., Ltd.), nitrogen generator (PerMuta, Genius 1024, UK); fume hood (Shandong Boke Scientific Instrument Co., Ltd., FH1500(E)); centrifuge (Beijing Baiyang, BY-R16); multi-tube vortex mixer (Hangzhou Ruicheng, MS200); pipettes (Eppendorf, Germany, 0.5–10 μL, 10–100 μL, 100–1000 μL); bottle-top dispensers (BRAND, Germany, Dispensette S Organic 5–50 mL), 0.0001 g electronic balance (Sartorius, Germany, Secura), ultra-low temperature freezer, graduated cylinders, etc.

[0038] 1.3 Reagents and consumables: Chromatographic column (Phenomenex, USA, Phenyl-Hexyl® 2.6µm C18 90 Å, 50 x 3mm); methanol (Merck, Germany, chromatographic grade); formic acid (Merck, Germany, chromatographic grade); zinc sulfate heptahydrate (Huaguang Technology); ultrapure water (Watson's distilled water).

[0039] 1.4 Standards and internal standards: Mycophenolic acid standard, purity: 99.9% (Tianjin Alta Technology Co., Ltd., 10mg); MPA-d3 internal standard, chemical purity: 98.7%, isotopic purity: 99.9% (Tianjin Alta Technology Co., Ltd., 1mg).

[0040] 1.5 Quality control materials: third-party Siemens commercial mycophenolic acid (MPA) standard quality control materials (Siemens, Germany); self-prepared quality control materials (mixed human serum with added MPA standard).

[0041] 2. Methods 2.1 Preparation of protein precipitant 2.1.1 Preparation of zinc sulfate heptahydrate solution: Weigh 3.6g of zinc sulfate heptahydrate, dissolve it in 40mL of water to prepare a 90mg / mL zinc sulfate heptahydrate aqueous solution. The ratio can be adjusted as needed. Store at room temperature.

[0042] 2.1.2 Preparation of protein precipitant: The protein precipitant is prepared by mixing methanol and zinc sulfate heptahydrate solution at a volume ratio of 7:3 according to the amount to be tested, and stored at 4-8℃ for later use.

[0043] 2.2 Preparation of Mycophenolic Acid Isotope Internal Standard Working Solution 2.2.1 Dissolve MPA-d3 in methanol to prepare a stock solution of 1 mg / mL. The preparation parameters are shown in Table 4.

[0044] Table 4 Preparation of MPA-d3 isotope internal standard stock solution

[0045] 2.2.2 Take 15 μL of the stock solution from 2.2.1 and add 985 μL of methanol to obtain 1000 μL of internal standard working solution with a concentration of 15 μg / mL. Store at -80℃ for later use. The preparation parameters are shown in Table 5.

[0046] Table 5 Preparation of MPA-d3 isotope internal standard working solution

[0047] 2.3 Preparation of isotope-containing internal standard protein precipitant: The protein precipitant in 2.1 and the MPA-d3 internal standard working solution in 2.2 are mixed at a volume ratio of 100:1 according to the detection dosage, and stored at -20℃ for later use.

[0048] 2.4 Serum sample pretreatment: Take 15 μL of serum sample and add 60 μL of protein precipitant containing isotope internal standard. Vortex the mixture at 2500 rpm for 5 min. Centrifuge the mixture at 15000 rpm for 5 min. Transfer 10 μL of supernatant and mix it with 300 μL of diluent (pure water) for later use.

[0049] 2.5 Chromatographic conditions: A gradient elution was performed using a mixed mobile phase A (an aqueous solution containing 0.1% formic acid) and mobile phase B (a methanol solution containing 0.1% formic acid). The gradient elution program parameters are shown in Table 1. The flow rate was 0.6 mL / min, the column temperature was 40 °C, and the injection volume was 2 μL. The liquid chromatography column was a C18 column, Phenyl-Hexyl® 2.6µm C18 90 Å, 50x3mm.

[0050] Table 1. Gradient elution parameters of the mobile phase

[0051] 2.6 Mass spectrometry conditions: The ion source was in electrospray ionization (ESI+) mode, with positive ion mode scanning and multiple reaction monitoring (MRM) analysis. The mass spectrometry parameters were as follows: ion source temperature (TEM): 550℃; curtain gas pressure (CUR): 20.00 psi; ionization voltage (IS): 5000 V; nebulizer pressure (GS1): 50.00 psi; auxiliary heater pressure (GS2): 50.00 psi; collision gas pressure (CAD): 9.00 psi. The quantitative ion pairs, declustering voltage (DP), collision voltage (CE), inlet voltage (EP), and outlet voltage (CXP) of the analyte were monitored simultaneously. The parameters are shown in Table 2.

[0052] Table 2 Mass Spectrometry Detection Parameters

[0053] 2.7 Preparation of working solution for mycophenolic acid standard curve 2.7.1 Preparation of mycophenolic acid standard stock solution: Dissolve the standard in methanol to prepare a stock solution with a concentration of 2.00 mg / mL, as shown in Table 6. Then take 200 μL of the above mycophenolic acid stock solution and add 200 μL of methanol to prepare a stock solution of mycophenolic acid with a concentration of 1.0 mg / mL.

[0054] Table 6. Preparation parameters of mycophenolic acid standard stock solution

[0055] 2.7.2 Preparation of Mycophenolic Acid Mixed Working Stock Solution (MIX): Transfer 100 μL of the stock solution in 2.7.1 to 900 μL of 78% methanol-water to obtain 1000 μL MIX working stock solution (the final solution system is 80% methanol-water), with a concentration of 100 μg / mL. The parameters are shown in Table 7.

[0056] Table 7 Preparation parameters of mycophenolic acid mixed working stock solution (MIX)

[0057] 2.7.3 Preparation of working solution for mycophenolic acid standard curve: Based on the therapeutic range of mycophenolic acid blood concentration and the expected clinical distribution level, the calibration curve range was determined to be 0.5-15 μg / mL. Using the working stock solution from 2.7.2, six different concentration points (STD1-STD6) were prepared with blank serum matrix. Taking 1.5 mL of solution for each concentration point as an example (2 mL of solution was prepared for STD5 as stock solutions for STD1 and STD2), the preparation parameters are shown in Table 3; the pretreatment method is the same as described in 2.4.

[0058] Table 3. Preparation parameters of working solution for mycophenolic acid standard curve

[0059] 2.8 Preparation of Mycophenolic Acid Quality Control Solution: A blank serum matrix solution containing mycophenolic acid was prepared. The concentration of the quality control solution was determined according to the Chinese Pharmacopoeia. Three times the lower limit of quantitation, near the midpoint of the calibration curve, and near 75% of the high value of the calibrator were used as low, medium, and high quality control levels, respectively denoted as LQC, MQC, and HQC. Blank serum was taken, and different volumes of 1 mg / mL mycophenolic acid stock solution were added to prepare quality control concentrations of 0.8 μg / mL, 2.5 μg / mL, and 10 μg / mL. The preparation parameters are shown in Table 8. Quality control solutions are used to effectively control the quality of the testing process, continuously monitor and evaluate the testing process, and ensure the accuracy, stability, and reliability of the test results. The pretreatment method is the same as described in 2.4.

[0060] Table 8 Preparation of working solutions for quality control products

[0061] Note: The table above uses a 1.5 mL volume for each concentration point as an example. The volume can be adjusted appropriately while keeping the ratio unchanged.

[0062] 3. Method Validation The feasibility of the established high-performance liquid chromatography-tandem mass spectrometry method for rapid quantitative detection of trace amounts of mycophenolic acid in serum was investigated methodologically, including peak shape, limit of detection, limit of quantitation, linearity, precision, and extraction recovery.

[0063] 3.1 Extraction ion chromatogram: as shown Figure 5 , Figure 6 As shown, the peaks of the mycophenolic acid standard and internal standard are symmetrical, with a retention time of 2.21 min (±2.5%), and there is no interference from other peaks, indicating that the detection conditions are good.

[0064] 3.2 Limit of Detection, Limit of Quantification, and Linear Range: A calibration curve was prepared according to the standard solution preparation method in 2.7, with the pretreatment method as described in 2.4. Quantification was performed using the isotope internal standard method. A calibration curve was constructed with the concentration of mycophenolic acid standard solution as the abscissa and the peak area ratio of mycophenolic acid to its internal standard as the ordinate. The regression equation and correlation coefficient were obtained, and the concentration of MPA in serum was calculated. The limit of detection was defined as the concentration corresponding to a signal-to-noise ratio (S / N) ≥ 3, and the limit of quantification was defined as the concentration corresponding to (S / N) ≥ 10 with a result deviation within 15%. The linear fitting equation for mycophenolic acid within the concentration range was verified to be linear for three consecutive days, with a correlation coefficient above 0.999, meeting the quantitative requirements. The results are shown in Table 9.

[0065] Table 9. Limit of detection, limit of quantitation, linear regression equation and linear correlation coefficient of mycophenolic acid

[0066] 3.3 Extraction Recovery Rate: Serum samples from healthy clinical examinees were divided into four equal volumes. One sample contained a blank reagent (methanol) without the analyte, serving as the baseline sample. The other three samples each contained the same volume of mycophenolic acid standard solution at different concentrations. The concentrations of the standard solution in the samples after addition are shown in Table 10. Three recovery samples with different added concentrations were prepared. After adding the standard solution, the mycophenolic acid concentration in the low-level sample was near the lower limit of the therapeutic range, while the mycophenolic acid concentration in the high-level sample reached near the upper limit of the therapeutic range. The sample pretreatment method was the same as described in 2.4, and each sample was tested three times. The extraction recovery rate of mycophenolic acid at different concentrations was between 85% and 115%, indicating good accuracy of the detection results. The plasma sample extraction recovery rate was verified by comparison using the same preparation method as the serum sample, and conventional large-volume organic solvent protein precipitant (methanol) was used for pretreatment. The results are shown in Table 11.

[0067] Table 10 Results of mycophenolic acid extraction recovery in serum

[0068] Table 11 Results of mycophenolic acid extraction recovery from plasma

[0069] 3.4 Precision: Three levels of quality control samples from Siemens' commercially available mycophenolic acid control material were used as test samples. Three analytical batches were tested daily, with each batch repeated three times, for five consecutive days. Simultaneously, three levels of self-prepared quality control samples were used as test samples, with three analytical batches tested daily, with each batch repeated three times, for five consecutive days. This was to assess intra-day and inter-day precision, expressed as the coefficient of variation (CV). According to industry standards, the CV should be less than 15%. Based on the laboratory testing capabilities of the invented detection method, the laboratory quality targets for this project were set as follows: intra-day precision less than 1 / 4 of the industry standard (3.75%); inter-day precision less than 1 / 3 of the industry standard (5%). If the measured intra-batch imprecision (Sr) is less than the target intra-batch imprecision, the intra-batch imprecision is accepted; otherwise, further comparisons are made. If the measured total imprecision (S1) is less than the target total imprecision, the total imprecision is accepted; otherwise, further comparisons are made. The precision test results of the detection method are shown in Tables 12-17 (the units of the values ​​in the tables are μg / mL).

[0070] Table 12 Precision of low-value samples of self-prepared quality control materials (sample concentration: 0.8 μg / mL)

[0071] Table 13. Median sample precision of self-prepared quality control samples (sample concentration: 2.5 μg / mL)

[0072] Table 14 Precision of high-value self-prepared quality control samples (sample concentration: 10 μg / mL)

[0073] Table 15 Precision of low-value samples of Siemens quality control materials (concentration label: 1.00 μg / mL)

[0074] Table 16. Median sample precision of Siemens quality control products (concentration label: 8.00 μg / mL)

[0075] Table 17 Precision of high-value Siemens quality control samples (concentration label: 12.00 μg / mL)

[0076] 4. Discussion This invention provides a rapid quantitative detection method for mycophenolic acids in trace amounts of serum using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). The established serum sample pretreatment process is simple, employing a technique of dilution with only 20-60 μL of a small volume of high-strength protein precipitant to efficiently remove protein and other impurities from 5-15 μL of trace serum, significantly reducing matrix interference. This results in a more stable and sensitive mycophenolic acid signal, achieving good baseline separation between the compound and matrix interference. The peak elution time is 2.21 min (±2.5%), and the ion pair (m / z) changes from 321.1 to 207.3. Accurate quantification is achieved using an isotope internal standard method.

[0077] Through method validation, this invention provides a rapid quantitative detection method for trace amounts of mycophenolic acid in serum using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). The method exhibits good linearity (correlation coefficient > 0.99), a limit of quantification (LOQ) of approximately 0.25 μg / mL, and a limit of detection (LOD) of approximately 0.06 μg / mL. Spiking recovery tests evaluated the accuracy of the method. Results showed that the spiked recovery rate of mycophenolic acid was close to 100%, and the RSDs of three replicates at three different spiked concentrations were in the range of 0.00%–1.29%, indicating good accuracy. The RSDs of three replicates at three different spiked concentrations of mycophenolic acid in plasma were in the range of 1.80%–2.09%. Due to matrix or volume effects, the spiked recovery rate exceeded that of this invention by approximately 10%–12%. The precision results of the method showed that, with three analytical batches per day, each batch repeated three times, and continuous testing for five days, the intra-batch precision of the self-prepared quality control material mycophenolic acid was 1.33%-1.59%, and the inter-batch precision was 1.32%-2.40%. The intra-batch precision of the third-party Siemens mycophenolic acid quality control material was 1.23%-1.60%, and the inter-batch precision was 1.46%-2.75%. The detection method has good repeatability and reproducibility. Furthermore, the detection of the third-party quality control material further independently verified the detection quality of the present invention and the excellent performance of the entire detection system. The detection results are comparable to those of other laboratories.

[0078] In summary, the method of this invention requires a small amount of venous blood, detects trace amounts of serum, has a simple and rapid pretreatment process, uses minimal reagents, is low in cost, has high sensitivity and specificity, and provides accurate results. It completes the separation and detection of mycophenolic acids in serum in approximately 2.4 minutes, overcoming the problems of low removal efficiency of impurities such as proteins, matrix effects, or unstable recovery rates. The method has undergone thorough performance validation and exhibits good stability. It provides a rapid, trace-level, and quantitative serum detection method for monitoring the concentration of mycophenolic acid therapeutic drugs in clinical practice, suitable for large-scale clinical testing, greatly reducing the blood draw and economic burden for patients taking mycophenolic acid drugs, and facilitating clinical promotion.

[0079] The above embodiments are only used to illustrate the preferred embodiments of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that various modifications and changes may still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions or improvements may be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A method for rapid quantitative detection of mycophenolic acid in trace serum samples using high performance liquid chromatography-tandem mass spectrometry, characterized in that, The analyte is MPA, and the test includes the following steps: Add the protein precipitant containing the isotopic internal standard MPA-d3 to the trace serum sample to be tested, and shake to mix. Centrifuge the mixed system, take the supernatant and dilute it in pure water, shake to mix, and take the diluted liquid for LC-MS / MS detection. The isotope internal standard method was used for quantification. The concentration of mycophenolic acid standard solution was used as the abscissa and the peak area ratio of mycophenolic acid to its internal standard was used as the ordinate to construct a calibration curve, obtain the regression equation and correlation coefficient, and calculate the content of the analyte. The volume of the serum sample to be tested is 5-15 μL.

2. The method for rapid quantitative detection of mycophenolic acid in trace serum samples by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: Serum is obtained by collecting blood from inert gel-coagulated vacuum blood collection tubes or dried tubes, and centrifuging the blood after natural coagulation. The amount of blood collected from the vein should not exceed 1 mL.

3. The method for rapid quantitative detection of mycophenolic acid in trace serum samples by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The amount of protein precipitant containing the isotopic internal standard MPA-d3 is 20-60 μL.

4. The method for rapid quantitative detection of mycophenolic acid in trace serum samples by high performance liquid chromatography-tandem mass spectrometry according to claim 3, characterized in that: The protein precipitant is composed of methanol and zinc sulfate heptahydrate solution in a volume ratio of 7:3; the concentration of zinc sulfate heptahydrate solution is 50-100 mg / mL.

5. The method for rapid quantitative detection of mycophenolic acid in trace serum samples by high performance liquid chromatography-tandem mass spectrometry according to claim 3, characterized in that: The isotopic internal standard protein precipitant is composed of a protein precipitant and MPA-d3 internal standard working solution mixed at a volume ratio of 100:

1. The MPA-d3 internal standard working solution was prepared as follows: MPA-d3 was dissolved in methanol to prepare a 1 mg / mL stock solution, and then 15 μL of the stock solution was transferred and added to 985 μL of methanol to obtain 1000 μL of internal standard working solution with a concentration of 15 μg / mL.

6. The method for rapid quantitative detection of mycophenolic acid in trace serum samples by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: After mixing, the mixture was centrifuged at 15,000 rpm for 5 min. 10 μL of the supernatant was then transferred and mixed with 300 μL of diluent, which was pure water.

7. The method for rapid quantitative detection of mycophenolic acid in trace serum samples by high performance liquid chromatography-tandem mass spectrometry according to claim 6, characterized in that: In the liquid chromatography section, mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is a methanol solution containing 0.1% formic acid.

8. The method for rapid quantitative detection of mycophenolic acid in trace serum samples by high performance liquid chromatography-tandem mass spectrometry according to claim 7, characterized in that: Chromatographic conditions: Gradient elution was performed using a mixture of mobile phases A and B, with parameters shown in Table 1; the flow rate was 0.5-0.8 mL / min, the column temperature was 35-45℃, and the injection volume was 1-2 μL. Table 1. Gradient elution parameters of the mobile phase 。 9. The method for rapid quantitative detection of mycophenolic acid in trace serum samples by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: Mass spectrometry conditions: Ion source in electrospray ionization (ESI+) mode, positive ion mode scanning, multiple reaction monitoring (MRM) analysis; mass spectrometry parameters: ion source temperature (TEM): 550℃; curtain gas pressure (CUR): 20.00psi; ionization voltage (IS): 5000V; nebulizer pressure (GS1): 50.00psi; auxiliary heater pressure (GS2): 50.00psi. Collision air pressure (CAD): The test was conducted at 9.00 psi, simultaneously monitoring the quantitative ion pairs, declustering voltage (DP), collision voltage (CE), inlet voltage (EP), and outlet voltage (CXP) of the analyte. The parameters are shown in Table 2. Table 2 Mass Spectrometry Detection Parameters 。 10. The method for rapid quantitative detection of mycophenolic acid in trace serum samples by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: Mycophenolic acid mixed working stock solution (MIX) was prepared according to the following steps: the standard was dissolved in methanol to prepare a stock solution of 1 mg / mL, and then 100 μL of the stock solution was transferred to 900 μL of 78% methanol-water solution to obtain 1000 μL MIX working stock solution (the final solution system is 80% methanol-water solution) with a concentration of 100 μg / mL. The working solution for the mycophenolic acid standard curve was prepared according to the following steps: The above mixed standard stock solution (MIX) was prepared into calibrator solutions for six different concentration points using a blank serum matrix. Taking 1.5 mL of each concentration point as an example (2 mL of STD5 needs to be prepared as stock solutions for STD 1 and STD 2), the blank serum matrix is ​​healthy human or animal serum that does not contain mycophenolic acid, and the parameters are shown in Table 3. Table 3. Preparation parameters of working solution for mycophenolic acid standard curve 。

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