A high-throughput quantitative detection method for tacrolimus in blood based on acoustic excitation mass spectrometry

Abstract

This invention relates to a high-throughput quantitative detection method for tacrolimus in blood based on acoustic excitation mass spectrometry, belonging to the field of analytical technology. The method includes the following steps: taking a blood sample, adding an internal standard solution, mixing thoroughly to obtain a mixture; performing phase separation on the obtained mixture to obtain an extract containing tacrolimus; adding a volatile ammonium salt aqueous solution to the extract, mixing thoroughly to obtain the test solution; and performing acoustic excitation mass spectrometry detection on the test solution. This invention simplifies the sample pretreatment process and enables rapid, continuous sample injection and analysis. It features high detection speed, high throughput, and accurate quantification, making it suitable for rapid quantitative detection of tacrolimus in large batches of blood samples and for therapeutic drug monitoring.

Description

Technical Field

[0001] This invention relates to a high-throughput quantitative detection method for tacrolimus in blood based on acoustic excitation mass spectrometry, belonging to the field of analytical technology. Background Technology

[0002] Organ transplantation is a crucial treatment for patients with end-stage organ failure, significantly improving their survival rate and quality of life. However, successful transplantation depends not only on donor-recipient matching but also on long-term, standardized immunosuppressive therapy to prevent rejection. Among existing immunosuppressive regimens, tacrolimus has become one of the most commonly used drugs in organ transplant immunosuppressive therapy, playing a key role in suppressing rejection and improving graft survival. However, tacrolimus has a narrow therapeutic window and significant individual pharmacokinetic differences; insufficient dosage may increase the risk of rejection, while excessive dosage may lead to nephrotoxicity and infection risks. Therefore, strict monitoring of blood drug concentrations is essential in clinical practice to achieve personalized dosing for each patient.

[0003] Currently, the quantitative detection of tacrolimus in blood mainly relies on immunoassay and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Immunoassay is fast and highly automated, but antibodies are prone to cross-reaction with tacrolimus metabolites, leading to systematically high results and affecting clinical dosage adjustments. LC-MS / MS offers higher specificity, but typically requires complex pretreatment and chromatographic separation, severely limiting its application in high-volume, rapid clinical settings. Therefore, there is an urgent need to develop a detection method that balances high throughput, high specificity, and high accuracy to meet the pressing demand for efficient and precise monitoring of tacrolimus blood concentrations. This patented technology aims to overcome this technological bottleneck. Summary of the Invention

[0004] To address the shortcomings of existing methods, this invention employs a simple solvent extraction and separation pretreatment method combined with an Echo MS+ system to provide a high-throughput quantitative detection method for tacrolimus in blood based on acoustic excitation mass spectrometry. This method features simple sample pretreatment, high specificity, high throughput, high sensitivity, and high accuracy.

[0005] To achieve the objective of this invention, the technical solution adopted is as follows: a high-throughput quantitative detection method for tacrolimus in blood based on acoustic excitation mass spectrometry, comprising the following steps: (1) Take a blood sample, add internal standard solution, mix well to obtain a mixture; (2) The mixture obtained in step (1) is subjected to phase separation treatment to obtain an extract containing tacrolimus; (3) Add volatile ammonium salt aqueous solution to the extract, mix well to obtain the test solution; (4) Perform acoustic excitation mass spectrometry detection on the solution to be tested.

[0006] Preferably, in step (1), the blood sample is whole blood, plasma or serum.

[0007] Preferably, in step (1), tacrolimus is used. 13 C,d2( 13 The internal standard solution is prepared by using C and deuterated tacrolimus as internal standards, wherein the solvent of the internal standard solution is a mixture of acetonitrile and water in a volume ratio of 1:0.6-1.5.

[0008] More preferably, the volume ratio of acetonitrile to water is 1:0.8 to 1:1.3.

[0009] Preferably, in step (1), the volume ratio of blood to internal standard solution is 1:1-1000, and the concentration of internal standard is 0.1-10 ng / mL.

[0010] More preferably, in step (1), the volume ratio of blood to internal standard solution is 1:20-300, and the concentration of internal standard is 0.5-5 ng / mL.

[0011] Preferably, in step (1), the volume of the blood sample is 1-50 μL.

[0012] More preferably, in step (1), the volume of the blood sample is 1-10 μL.

[0013] Preferably, in step (2), the phase separation process is to take the supernatant after freezing treatment, with a freezing temperature of 0 ~ -80℃ and a freezing time of 1-30 min.

[0014] More preferably, in step (2), the freezing temperature is -20 ~ -80℃ and the freezing time is 5-10 min.

[0015] Preferably, in step (3), the concentration of the volatile ammonium salt aqueous solution is 0.1-10 mM, and the volume ratio of the volatile ammonium salt aqueous solution to the extract is 1:0.5-1.

[0016] More preferably, in step (3), the concentration of the volatile ammonium salt aqueous solution is 4 mM.

[0017] Preferably, in step (3), the sampling volume of the extract is 5-50 μL.

[0018] More preferably, in step (3), the sampling volume of the extract is 10-40 μL.

[0019] Preferably, in step (3), the volatile ammonium salt includes at least one of ammonium formate, ammonium acetate, and ammonium fluoride.

[0020] More preferably, in step (3), the volatile ammonium salt is ammonium acetate.

[0021] Preferably, in step (3), the mixing is carried out by vortexing, and the vortexing time is preferably 10-30 s.

[0022] Preferably, in step (4), the acoustic excitation mass spectrometer consists of an Echo MS+ system and a SCIEX Triple Quad 6500+ mass spectrometer (SCIEX, USA).

[0023] Preferably, in step (4), the solution to be tested is added to a microplate and detected by acoustic excitation mass spectrometry. Nano-level droplets are ejected from the wells of the microplate using an acoustic droplet ejection method. The droplets are introduced into an open interface, captured and diluted by a carrier liquid, and then transported to an electrospray ion source to enter the mass spectrometer. The ion signals of tacrolimus and internal standard are collected on the mass spectrometer in multiple reaction monitoring (MRM) mode of tandem mass spectrometry. The quantification is performed based on the response ratio (peak area or peak intensity) of tacrolimus and internal standard and the working curve to obtain the concentration of tacrolimus in the blood sample.

[0024] Preferably, the microplate is a 384-well plate or a 1536-well plate; more preferably, the microplate is a 384-well plate.

[0025] Preferably, in step (4), nano-level droplets are injected using an acoustic droplet injection method, and the single injection volume of the acoustic droplet injection is 2.5-1000 nL.

[0026] More preferably, the single injection volume of the acoustic droplet jet is 2.5-200 nL.

[0027] Preferably, in step (4), the carrier liquid for acoustic excitation mass spectrometry detection is selected from any of the following: acetonitrile: aqueous solution (7:3, v / v), methanol solution containing 0.1% formic acid, methanol solution containing 2 mM ammonium acetate, or methanol solution containing 2 mM ammonium fluoride.

[0028] Preferably, in step (4), the detection conditions for the acoustic excitation mass spectrometry are as follows: electrospray positive ion mode is used for acquisition, the ion spray voltage is 5500 V, the spray gas is 90 psi, the auxiliary heating gas is 45 psi, the curtain gas is 20 psi, the collision gas is 9 psi, and the ion source temperature is 600 ℃; the residence time of a single MRM channel is 15 ms, the pause time is 3 ms, and the total scan time is 0.054 s; the carrier liquid flow rate is 360 μL / min, and the excitation interval is 2500 ms.

[0029] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a high-throughput quantitative detection method for tacrolimus in blood based on acoustic excitation mass spectrometry. This method uses solvent extraction and phase separation to obtain the supernatant to be tested, which simplifies the sample pretreatment process and eliminates the need for additional protein precipitation and centrifugation steps, making it suitable for large-scale sample processing.

[0030] The method of this invention, combined with acoustic excitation mass spectrometry (Echo MS) for detecting tacrolimus in blood samples, achieves a sampling rate of 1-3 samples / second, significantly increasing throughput and meeting the needs of rapid testing of large batches of samples in clinical practice. Simultaneously, the addition of an internal standard during sample extraction helps reduce the impact of matrix effects and instrument fluctuations on quantitative results, thereby improving accuracy. This invention can be used for high-throughput quantitative detection of tacrolimus concentrations in the blood of organ transplant patients, providing support for therapeutic drug monitoring and personalized dosing.

[0031] In the method of this invention, by adding a volatile ammonium salt to the sample to be tested, the target analyte preferentially forms [M+NH4] under electrospray positive ion conditions. + It combines with ions and inhibits [M+Na] ions caused by trace amounts of sodium ions in the system. + The formation of adduct ions reduces signal fluctuations and background interference caused by metal adducts. Compared to the condition without ammonium salt addition, [M+NH4] + The signal strength can be increased by about 4 times and maintained with good stability, thereby improving the sensitivity and quantitative accuracy of tacrolimus detection. Attached Figure Description

[0032] Figure 1 This is a first-order mass spectrum of the tacrolimus sample in Example 1 of the present invention under conditions with and without the addition of ammonium acetate; Figure 2 The image shown is a secondary mass spectrum of tacrolimus in Example 1 of this invention; Figure 3 The images show the collected spectra of tacrolimus under different liquid loading conditions in Example 1 of the present invention (measured three times consecutively). Figure 4 The signal intensity of tacrolimus in Example 1 of this invention at injection volumes of 2.5-200 nL; Figure 5 This is the acoustic excitation mass spectrometry acquisition spectrum of the quality control sample in Example 2 of the present invention. Detailed Implementation

[0033] The present invention will now be described in detail with reference to the embodiments and accompanying drawings. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0034] Unless otherwise specified, the methods described in the following examples are conventional; the reagents and materials described are commercially available unless otherwise specified.

[0035] Example 1

[0036] A 1 μg / mL tacrolimus standard solution was prepared. Equal volumes of ammonium acetate aqueous solution (4 mM) and pure water were added to prepare sample solutions. These sample solutions were continuously injected into the ion source, and a first-stage full scan was performed in ESI positive ion mode to determine the main adduct ion forms and ionization behavior. The mass spectrometry results are shown below. Figure 1 As shown in the figure, adding volatile ammonium salts (such as ammonium acetate) to the test solution can provide sufficient ammonium ions, shifting the ionization equilibrium towards [M+NH4+]. + Directional movement. For example... Figure 1 As shown in Figure b, after adding ammonium acetate, [M + Na] + The peak signal was significantly suppressed, while [M+NH4] + The peak signal was significantly enhanced, with the signal intensity increasing approximately fourfold compared to before the addition of ammonium acetate, while maintaining its stability. This improves the detection sensitivity of tacrolimus because tacrolimus is primarily detected as [M+Na] during electrospray ionization. + With [M+NH4] + Two adduct ion forms exist ( Figure 1 (As shown in a). Although [M+Na + The peak signal response is high, but its signal is easily affected by background fluctuations of exogenous sodium ions in the pipeline and environment, leading to decreased signal stability and analytical reproducibility. Adding non-volatile sodium salts to enhance sodium adduct ion formation may cause ion source contamination and increase instrument maintenance costs. Therefore, [the following option is chosen]. m / z 821.6 ([M+NH4]) + ) is the parent ion, and further based on its secondary mass spectrometry fragmentation characteristics (see Figure 2 ),choose m / z 821.6→ m / z 768.4 as a quantitative ion pair, m / z 821.6→ m / z 786.4 was used as a qualitative ion pair to establish the MRM channel for tacrolimus. Internal standard tacrolimus- 13 C, d2 (choose) m / z 824.6→771.4 was used as the quantitative ion pair. The optimized MRM parameters of tacrolimus and its internal standard are shown in Table 1.

[0037] Table 1. Tacrolimus and its internal standard tacrolimus- 13Optimal MRM parameters for C,d2

[0038] a Quantitative ions b Qualitative ions In acoustic-excited mass spectrometry (AES), the type of carrier liquid with an open interface significantly affects the ionization efficiency and signal response of tacrolimus. Therefore, the carrier liquid type was screened and optimized. The results are as follows: Figure 3 As shown, among the four carrier solutions investigated, the methanol solution containing 0.1% formic acid yielded the highest tacrolimus signal response with a well-defined peak shape. In contrast, the signal intensity was significantly reduced in the methanol solution containing 2 mM ammonium acetate, the methanol solution containing 2 mM ammonium fluoride, and the acetonitrile:water solution (7:3, v / v). Based on these results, the methanol solution containing 0.1% formic acid was selected as the carrier solution for the acoustic excitation mass spectrometry detection of tacrolimus in this invention.

[0039] The injection volume for acoustic-excited mass spectrometry was then optimized. For example... Figure 4 As shown, within the injection volume range of 2.5-200 nL, the response intensity of tacrolimus increases with increasing injection volume, exhibiting a good linear trend. Considering both signal response and sample consumption, 60 nL was selected as the single injection volume for subsequent detection.

[0040] Example 2

[0041] A high-throughput quantitative detection method for tacrolimus in blood based on acoustic excitation mass spectrometry includes the following steps: (1) Establishment of working curve and investigation of limit of quantitation (LOQ) and limit of detection (LOD) A series of tacrolimus standard solutions of different concentrations (concentration gradients of 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL) were prepared. 10 μL of each solution was dried under nitrogen, reconstituted with 10 μL of blank whole blood, and then an extraction solution containing an internal standard (acetonitrile:water = 1:1.08, v / v) was added. The internal standard concentration was 0.5 ng / mL. After mixing, the samples were stored at -20°C. o Freeze in a C refrigerator for 10 min; take 20 μL of the supernatant, add an equal volume of ammonium acetate aqueous solution (4 mM), vortex for 30 s, and then add to a 384-well plate for ultrasonic-excited mass spectrometry detection. Linear regression fitting of the tacrolimus concentration to the peak height ratio of the tacrolimus standard to the internal standard was performed, and the LOQ and LOD of the established method were calculated using 10-fold and 3-fold signal-to-noise ratios (Table 2).

[0042] Table 2. Linear range, LOD and LOQ of tacrolimus

[0043] (2) Testing of quality control (QC) samples Commercial quality control (QC) samples were used to validate the performance of this method and evaluate its accuracy. The QC samples used contained tacrolimus concentrations of 2.29, 6.47, 13.9, and 30.4 ng / mL, covering multiple levels within the operating curve range of this method. The QC samples were processed according to the sample pretreatment procedure described in Example 1 and detected using acoustic excitation mass spectrometry. Figure 5 The peak height ratio of tacrolimus to internal standard was obtained and substituted into the established working curve for quantitative calculation, thereby obtaining the concentration of tacrolimus in each QC sample (Table 3).

[0044] Table 3. Comparison of standard values ​​and measured results of tacrolimus in quality control samples

[0045] The results showed that the method established in this invention for the quantitative analysis of tacrolimus exhibited good linearity in the range of 0.5-50 ng / mL, with a coefficient of determination ( ). R 2 The concentration was 0.9969. The LOD and LOQ were 0.15 ng / mL and 0.50 ng / mL, respectively, indicating that the method has high sensitivity (see Table 2). Quality control samples were pretreated and detected using the same procedure as in Example 1. The calculated concentrations and values ​​all fell within the reference range of the given standard values ​​for the quality control samples, indicating that the method has good accuracy (see Table 3). Therefore, this method is suitable for high-throughput quantitative detection of tacrolimus concentrations in blood samples from organ transplant patients.

[0046] (3) Quantitative detection of tacrolimus in actual samples The whole blood of organ transplant patients was processed using the sample pretreatment method in Example 1. Combined with acoustic excitation mass spectrometry detection, the peak height ratio of tacrolimus to internal standard was obtained. Substituting this ratio into the working curve, the concentration of tacrolimus in the actual sample can be calculated.

[0047] The above embodiments, in conjunction with the accompanying drawings, are only used to illustrate preferred embodiments of the present invention and are not intended to limit the present invention. Modifications, equivalent substitutions, or improvements made by those skilled in the art without departing from the spirit of the present invention should be included within the scope of protection defined by the claims.

Claims

1. A high-throughput quantitative detection method for tacrolimus in blood based on acoustic excitation mass spectrometry, characterized in that, Includes the following steps: (1) Take a blood sample, add internal standard solution, mix well to obtain a mixture; (2) The mixture obtained in step (1) is subjected to phase separation treatment to obtain an extract containing tacrolimus; (3) Add volatile ammonium salt aqueous solution to the extract, mix well to obtain the test solution; (4) Perform acoustic excitation mass spectrometry detection on the solution to be tested.

2. The high-throughput quantitative detection method for tacrolimus in blood based on acoustic excitation mass spectrometry as described in claim 1, characterized in that, In step (1), the blood sample is whole blood, plasma or serum.

3. The high-throughput quantitative detection method for tacrolimus in blood based on acoustic excitation mass spectrometry as described in claim 1, characterized in that, In step (1), tacrolimus- 13 C,d2 is used as an internal standard, and the solvent of the internal standard solution is a mixture of acetonitrile and water in a volume ratio of 1:0.6-1.

5.

4. The high-throughput quantitative detection method for tacrolimus in blood based on acoustic excitation mass spectrometry as described in claim 1, characterized in that, In step (1), the volume ratio of blood to internal standard solution is 1:1-1000, and the concentration of internal standard is 0.1-10 ng / mL.

5. The high-throughput quantitative detection method for tacrolimus in blood based on acoustic excitation mass spectrometry as described in claim 1, characterized in that, In step (2), the phase separation process involves freezing the supernatant and taking the supernatant liquid. The freezing temperature is 0 ~ -80 ℃ and the freezing time is 1-30 min.

6. The high-throughput quantitative detection method for tacrolimus in blood based on acoustic excitation mass spectrometry as described in claim 1, characterized in that, In step (3), the concentration of the volatile ammonium salt aqueous solution is 0.1-10 mM, and the volume ratio of the volatile ammonium salt aqueous solution to the extract is 1:0.5-1.

7. The high-throughput quantitative detection method for tacrolimus in blood based on acoustic excitation mass spectrometry as described in claim 1, characterized in that, In step (3), the volatile ammonium salt includes at least one of ammonium formate, ammonium acetate and ammonium fluoride.

8. The high-throughput quantitative detection method for tacrolimus in blood based on acoustic excitation mass spectrometry as described in claim 1, characterized in that, In step (4), nano-level droplets are injected using an acoustic droplet jetting method, and the single injection volume of the acoustic droplet jetting is 2.5-1000 nL.

9. The high-throughput quantitative detection method for tacrolimus in blood based on acoustic excitation mass spectrometry as described in claim 1, characterized in that, In step (4), the carrier liquid for acoustic excitation mass spectrometry detection is selected from any of the following: acetonitrile: aqueous solution (7:3, v / v), methanol solution containing 0.1% formic acid, methanol solution containing 2 mM ammonium acetate, or methanol solution containing 2 mM ammonium fluoride.

10. The high-throughput quantitative detection method for tacrolimus in blood based on acoustic excitation mass spectrometry as described in claim 1, characterized in that, In step (4), the detection conditions for the acoustic excitation mass spectrometry are as follows: electrospray positive ion mode is used for acquisition, the ion spray voltage is 5500 V, the spray gas is 90 psi, the auxiliary heating gas is 45 psi, the curtain gas is 20 psi, the collision gas is 9 psi, and the ion source temperature is 600 ℃; the residence time of a single MRM channel is 15 ms, the pause time is 3 ms, and the total scan time is 0.054 s; the carrier liquid flow rate is 360 μL / min, and the excitation interval is 2500 ms.

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