A method and kit for rapid on-site detection of blood drug concentration by direct ionization mass spectrometry

Abstract

This invention discloses a rapid on-site detection method for blood drug concentration using direct ionization mass spectrometry, comprising: acquiring a serum sample to be tested and performing precipitation pretreatment to obtain a supernatant; selecting a suitable ion source from nano-electrospray ionization source and probe electrospray ionization source according to the type of drug to be tested; ionizing the supernatant through the selected ion source and then sending it to a direct ionization mass spectrometer for secondary mass spectrometry detection; calculating the blood drug concentration according to a pre-established standard curve; wherein the total time from the start of pretreatment to obtaining the detection result does not exceed 7 minutes. This invention uses a nano-electrospray ionization source for carbamazepine and valproic acid, and a probe electrospray ionization source for linezolid and voriconazole, and is equipped with a precipitant kit containing a specific internal standard concentration. This invention requires no complex pretreatment, is simple to operate, rapid in detection, and highly sensitive, making it suitable for on-site, real-time detection in clinical settings and for poisoning treatment, with sensitivity meeting the needs of clinical therapeutic drug monitoring.

Description

Technical Field

[0001] This invention relates to the field of biological sample drug detection and mass spectrometry analysis technology, specifically to a rapid blood drug concentration detection method and kit based on direct ionization mass spectrometry technology. Background Technology

[0002] Therapeutic Drug Monitoring (TDM) is a pharmacokinetic-guided process that uses modern analytical techniques to detect the concentrations of drugs and their metabolites in a patient's blood. It is used to assess efficacy, predict toxicity, and optimize individualized dosing regimens, making it a crucial tool for achieving precision medicine in clinical practice. TDM is necessary in clinical settings where drugs exhibit significant individual variability in blood concentrations, have narrow safety margins, high toxicity, or nonlinear pharmacokinetic characteristics, as well as in patients with comorbid organ diseases, those using combined medications, those with poor adherence, those suspected of drug poisoning, those with poor efficacy, or those whose toxic symptoms are difficult to identify. Furthermore, TDM can provide relevant medical and legal evidence for clinical drug use, meeting the complex clinical monitoring needs.

[0003] Multiple authoritative guidelines, both domestic and international, have clearly defined the TDM monitoring levels and therapeutic concentration ranges for commonly used clinical drugs: The consensus guidelines of the Association for Neuropsychopharmacology and Pharmacopsychiatry (AGNP) list carbamazepine TDM as a first-level recommendation, with a therapeutic reference concentration range of 4-10 μg / mL. -1 The Chinese Expert Consensus on the Clinical Application of Psychiatric Therapeutic Drug Monitoring (2022 Edition) strongly recommends valproic acid, with a therapeutic reference concentration range of 50-100 μg / mL. -1 The expert consensus on linezolid therapeutic drug monitoring and personalized dosing lists linezolid therapeutic drug monitoring (TDM) as a Level 1 recommendation, with a therapeutic reference concentration range of 2-8 μg / mL. -1 The "Guideline for Personalized Voriconazole Use" published by the Therapeutic Drug Monitoring Research Committee of the Chinese Pharmacological Society recommends a therapeutic window of 0.5-5 μg / mL for voriconazole trough concentration. -1 All of the above-mentioned drugs require precise blood drug concentration monitoring, which places high clinical demands on the speed, accuracy, and convenience of the detection methods.

[0004] The mainstream method for detecting clinical blood drug concentrations is liquid chromatography-mass spectrometry (LC-MS), which generally suffers from cumbersome pretreatment steps, long detection time, expensive and sophisticated equipment, and high operational expertise requirements. It relies solely on specialized laboratories for testing, failing to achieve rapid, point-of-care detection and real-time dynamic monitoring, thus hindering the clinical implementation of precision medicine and failing to meet the needs of rapid medication dispensing and risk assessment. Compared to traditional detection techniques, direct ionization mass spectrometry (DIMS) offers significant speed advantages, requiring no complex sample pretreatment and enabling rapid ionization analysis under normal pressure. It provides a technological foundation for rapid in-situ detection of biological samples. However, its application in quantitative blood drug concentration detection still faces key technological shortcomings. Existing research is largely limited to qualitative analysis of simple systems, failing to address core issues such as interference from complex serum matrices and poor accuracy in quantifying trace amounts of drugs. A suitable on-site detection system for clinical serum samples is lacking, thus preventing truly rapid and accurate on-site quantification of blood drug concentrations and failing to meet the clinical needs of total drug detection (TDM). Summary of the Invention

[0005] The purpose of this invention is to provide a rapid, on-site detection method and kit for direct ionization mass spectrometry of blood drug concentration. This invention features the ability to rapidly and accurately quantify drug concentrations in serum.

[0006] The technical solution of this invention: a rapid on-site detection method for blood drug concentration by direct ionization mass spectrometry, characterized by comprising the following steps: Obtain the serum sample to be tested; The serum sample was subjected to precipitation pretreatment to obtain a supernatant; Depending on the type of drug to be tested, a suitable ion source is selected from multiple ion sources, including at least nanoelectrospray ionization sources and probe electrospray ionization sources. The supernatant is ionized by the appropriate ion source and then sent to a direct ionization mass spectrometer for secondary mass spectrometry detection. The blood drug concentration of the serum sample to be tested was calculated based on the pre-established standard curve; The total time from the start of the pretreatment to obtaining the blood drug concentration in the detection method shall not exceed 7 minutes.

[0007] The aforementioned method for rapid on-site detection of drug concentration in serum via direct ionization mass spectrometry is characterized by selecting an appropriate ion source based on the type of drug to be tested, and is characterized by: When the drug to be tested is carbamazepine or valproic acid, the nano-electrospray ionization source is selected; When the test drug is linezolid or voriconazole, the probe electrospray ionization source is selected.

[0008] The aforementioned method for rapid on-site detection of serum drug concentration by direct ionization mass spectrometry is characterized in that the direct ionization mass spectrometer is a small direct ionization mass spectrometer.

[0009] The aforementioned method for rapid on-site detection of serum drug concentration by direct ionization mass spectrometry is characterized in that the pretreatment for precipitation includes: adding the serum sample to a precipitant containing an internal standard, vortexing to mix evenly, centrifuging for no more than 5 minutes, and taking the supernatant.

[0010] The aforementioned method for rapid on-site detection of serum drug concentration by direct ionization mass spectrometry is characterized in that the volume ratio of the carbamazepine sample to the formic acid-methanol mixed solution with internal standard is 1:39, wherein the volume ratio of formic acid to methanol is 1:249. The volume ratio of the valproic acid sample to the acetonitrile-methanol mixed solution with internal standard was 1:19, wherein the volume ratio of acetonitrile to methanol was 1:4. The precipitant for linezolid is a formic acid-methanol mixed solution containing an internal standard, with a volume ratio of formic acid to methanol of 1:319. The volume ratio of serum sample to precipitant is 1:4. After centrifugation, the supernatant is taken and diluted with methanol at a ratio of 1:9. The precipitant for voriconazole is a mixed solution of formic acid and ethyl acetate with an internal standard of 1:249 (volume ratio of formic acid to ethyl acetate). The volume ratio of the clear sample to the precipitant is 1:4. After centrifugation, the supernatant is diluted with 0.4% formic acid and ethyl acetate at a ratio of 1:9.

[0011] The aforementioned method for rapid on-site detection of serum drug concentration by direct ionization mass spectrometry is characterized in that: the internal standard concentration in the carbamazepine precipitant is 0.1 μg·mL. -1 The internal standard concentration in the valproic acid precipitant is 2.63 μg·mL. -1 The internal standard concentration in the linezolid precipitant is 1 μg·mL. -1 The internal standard concentration of the voriconazole precipitant is 0.5 μg·mL. -1 .

[0012] The aforementioned method for rapid on-site detection of serum drug concentration by direct ionization mass spectrometry is characterized in that the pre-established standard curve is obtained by: using filtered horse serum as a blank matrix, adding drug standards of different concentrations, and plotting the curve after the same pretreatment and mass spectrometry detection as the sample to be tested; wherein the concentration range of the carbamazepine standard curve is 2-20 μg·mL. -1 Valproic acid is 20-200 μg·mL -1 Linezolid is 1-20 μg·mL -1 Voriconazole is 0.4-10 μg·mL -1 .

[0013] The aforementioned method for rapid on-site detection of serum drug concentration by direct ionization mass spectrometry is characterized in that, when using the nano-electrospray ionization source, the mass spectrometry conditions are: positive / negative ion mode, ion source voltage 5000 V, isolation voltage amplitude 3 V, fragmentation voltage 0.1-1 V, and injection volume 10 μL; when using the probe electrospray ionization source, the mass spectrometry conditions are: positive ion mode, ion source voltage 2000 V, isolation voltage amplitude 3.6 V, fragmentation voltage 1 V, and the injection volume is limited to ensure that the injection solution does not touch the metal inside the injection needle.

[0014] The aforementioned direct ionization mass spectrometry method for rapid on-site detection of drug concentration in serum is characterized in that, in the secondary mass spectrometry detection: the precursor ion of carbamazepine is m / z 237.3, and the qualitative and quantitative daughter ions are m / z 192.2 and m / z 194.2, respectively; the precursor ion of valproic acid is m / z 143, and the qualitative and quantitative daughter ions are m / z 143; the precursor ion of linezolid is m / z 338, and the qualitative and quantitative daughter ions are m / z 195 and m / z 296, respectively; the precursor ion of voriconazole is m / z 349.2, and the qualitative and quantitative daughter ions are m / z 224.1 and m / z 280.9, respectively.

[0015] A rapid on-site detection kit for blood drug concentration using the method described above, characterized in that it comprises: a precipitant, wherein the precipitant is an organic solvent mixture containing an internal standard; and an instruction manual containing rules for selecting an ion source, wherein the rules are: a nano-electrospray ionization source is used for detecting carbamazepine or valproic acid, and a probe electrospray ionization source is used for detecting linezolid or voriconazole.

[0016] Compared with existing technologies, this invention optimizes and improves upon the aforementioned technical bottlenecks, and possesses the following outstanding innovations and beneficial effects: Overcoming matrix interference and achieving precise quantification: This invention optimizes the detection parameters and pretreatment methods of direct ionization mass spectrometry, effectively suppressing complex matrix interference in serum, and solving the technical deficiency of traditional direct ionization mass spectrometry that can only qualitatively identify but cannot accurately quantify trace amounts of blood drugs, thus significantly improving detection sensitivity and accuracy.

[0017] Enables rapid, on-site bedside testing with high timeliness: This invention eliminates the need for complex pretreatment processes such as purification and chromatographic separation. Relying on atmospheric pressure direct ionization technology, it greatly simplifies the detection process and shortens the detection time, freeing it from the limitations of large instruments and laboratories. It can achieve real-time on-site clinical testing and meet the needs of real-time TDM monitoring.

[0018] Good versatility and wide clinical applicability: This invention can stably detect the concentration of a variety of commonly used TDM drugs such as carbamazepine, valproic acid, linezolid, and voriconazole, which fully conforms to the therapeutic concentration monitoring range of authoritative guidelines and is applicable to a wide range of clinical scenarios.

[0019] Easy to operate, low cost, and easy to promote: The testing process is simple, the equipment dependence is low, and no high-end professional personnel are required to operate it, which greatly reduces the time and economic cost of clinical TDM and is suitable for routine application in hospitals at all levels and primary medical institutions. Attached Figure Description

[0020] Figure 1 The concentration of carbamazepine in the serum sample was 12 μg·mL. -1 MS 2 picture.

[0021] Figure 2 The concentration of valproic acid in the serum sample was 120 μg·mL. -1 MS 2 picture.

[0022] Figure 3 The concentration of linezolid in the serum sample was 7.5 μg·mL. -1 MS 2 picture.

[0023] Figure 4 The concentration of voriconazole in the serum sample was 10 μg·mL. -1 MS 2 picture.

[0024] Figure 5 Standard curve of carbamazepine.

[0025] Figure 6 Carbamazepine MS in blank samples 2 picture.

[0026] Figure 7 Carbamazepine MS in actual samples 2 picture. Detailed Implementation

[0027] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0028] The instrument used in this embodiment is the CRAIV-120 miniaturized direct ionization mass spectrometer from Ningbo Huayi Ningchuang Intelligent Technology Co., Ltd. The instrument dimensions are 43 cm (length) x 28 cm (width) x 37 cm (height), and the total weight is less than 22 kg. This miniaturized mass spectrometer can analyze and detect various compounds in both positive and negative ion modes. Instrument control and data acquisition utilize the CRAIV-120 software from Ningbo Huayi Ningchuang Intelligent Technology Co., Ltd. The nano-electrospray ionization source is an open-type electrospray ionization source with a nano-coating modified on the discharge tip.

[0029] Materials used in the examples: Carbamazepine, CAS No. 298-46-4, molecular formula C 15 H 12 N₂O, molecular weight 236.269. Carbamazepine internal standard: Carbamazepine- d 10 CAS No. 132183-78-9, Molecular Formula C 15 D 10 H2N2O, molecular weight 246.33.

[0030] Valproic acid, CAS number 99-66-1, molecular formula C8H 16 O2, molecular weight 144.211. Valproic acid internal standard: valproic acid- d 6 CAS No. 87745-18-4, Molecular Formula C8H 10 D6O2, molecular weight 150.25.

[0031] Linezolid, CAS No. 165800-03-3, molecular formula C 16 H 20 FN3O4, molecular weight 337.35. Linezolid internal standard: Linezolid - d 3 CAS No. 1127120-38-0, Molecular Formula C 16 H 17 D3FN3O4, molecular weight 340.36.

[0032] Voriconazole, CAS No. 137234-62-9, molecular formula C 16 H 14 F3N5O, molecular weight 349.3. Voriconazole internal standard: Voriconazole- d 3 CAS No. 1217661-14-7, Molecular Formula C 16H 11 D3F3N5O, molecular weight 352.33.

[0033] Standard serum sample: filtered horse serum. Serum sample to be tested: serum collected from volunteers to obtain the sample to be tested.

[0034] The direct ionization mass spectrometry method for rapid on-site detection of drug concentration in serum, as described in this embodiment, includes the following steps: 1. Preparation of standard serum samples: Add different amounts of standard substance (the standard substance is dissolved in methanol with a concentration of 1000 μg·mL) to the blank sample. -1 A series of standard serum samples were obtained by using carbamazepine, valproic acid, linezolid, and voriconazole solutions.

[0035] 2. Sample processing: Carbamazepine, take 25 μL, concentration is 2 μg·mL -1 5 μg·mL -1 8 μg·mL -1 12 μg·mL -1 15 μg·mL -1 20 μg·mL -1 The standard serum sample was added to 975 μL of a mixed solution containing formic acid and methanol containing internal standard, vortexed for 15 s, centrifuged at 12000 rpm for 5 min, and the supernatant was taken as the injection sample.

[0036] Valproic acid, take 25 μL, concentration is 20 μg·mL -1 50 μg·mL -1 80 μg·mL -1 120 μg·mL -1 150 μg·mL -1 200 μg·mL -1 The standard serum sample was added to 475 μL of methanol-acetonitrile mixed solution containing internal standard, vortexed for 15 s, centrifuged at 12000 rpm for 5 min, and the supernatant was taken as the injection sample.

[0037] Linezolid, take 100 μL of the solution, the concentration is 1 μg·mL -1 2.5 μg·mL -1 5 μg·mL -1 7.5 μg·mL -1 10 μg·mL -1 20 μg·mL -1The standard serum sample was added to 400 μL of a mixed solution containing formic acid and methanol containing internal standard, vortexed for 15 s, centrifuged at 12000 rpm for 5 min, and 100 μL of the supernatant was diluted with 900 μL of methanol as the injection sample.

[0038] Voriconazole, 100 μL, concentration 0.4 μg·mL -1 1 μg·mL -1 2.5 μg·mL -1 5 μg·mL -1 10 μg·mL -1 The standard serum sample was added to 400 μL of a mixed solution containing internal standard formic acid-ethyl acetate, vortexed for 15 s, centrifuged at 12000 rpm for 5 min, and 100 μL of the supernatant was diluted with 900 μL of 0.4% formic acid-ethyl acetate as the injection sample.

[0039] 3. Instrument testing: Carbamazepine and valproic acid were ionized using a nano-electrospray ionization source (NS). A 10 μL liquid sample was loaded onto the tip of a triangular nanosheet. The parameters for miniaturized direct ionization mass spectrometry were set, and the triangular tip was placed close to the mass spectrometer inlet. After ionization, the sample was analyzed by mass spectrometry.

[0040] Carbamazepine was analyzed qualitatively and quantitatively using positive ion mode and two-stage scanning. In positive ion mode, a relatively stable precursor ion (m / z 237.3) was formed. This precursor ion then fragmented into stable qualitative and quantitative daughter ions (m / z 192.28 and m / z 194.2) through collision-induced fragmentation. The MS analysis of carbamazepine... 2 As shown in the picture Figure 1 As shown.

[0041] Valproic acid was analyzed qualitatively and quantitatively using negative ion mode and two-stage scanning. In negative ion mode, a relatively stable precursor ion (m / z 143) was formed. This precursor ion exhibited unique characteristics and did not fragment upon collision induction; both the qualitative and quantitative product ions were m / z 143. (MS of valproic acid...) 2 As shown in the picture Figure 2 As shown.

[0042] Linezolid and voriconazole were treated with PDESI as the ion source. The sample was fed through a glass needle tip, and an appropriate amount of sample was drawn (ensuring the solution did not touch the metal inside the needle). The parameters for miniaturized direct ionization mass spectrometry were set, and the glass needle tip was placed close to the mass spectrometer inlet. After ionization, the sample was analyzed by mass spectrometry.

[0043] Linezolid was analyzed qualitatively and quantitatively using positive ion mode and a two-stage scanning method. In positive ion mode, a relatively stable precursor ion (m / z 338) was formed. This precursor ion then fragmented into stable qualitative and quantitative daughter ions (m / z 195 and m / z 296) through collision-induced fragmentation. The MS analysis of linezolid... 2 As shown in the picture Figure 3 As shown.

[0044] Voriconazole was analyzed qualitatively and quantitatively using positive ion mode and two-stage scanning. In positive ion mode, a relatively stable precursor ion (m / z 349.2) was formed. This precursor ion then fragmented into stable qualitative daughter ions (m / z 224.1) and quantitative daughter ions (m / z 280.9) through collision-induced fragmentation. The MS analysis of voriconazole... 2 As shown in the picture Figure 4 As shown.

[0045] Example 1

[0046] The purpose of this embodiment is to evaluate the methodology to verify the feasibility and effectiveness of direct ionization mass spectrometry in detecting serum drug concentrations.

[0047] 1. Construction of standard curve: After processing the standard serum samples of a series of concentrations according to step 2, they were loaded and analyzed according to the detection conditions in step 3. A target drug standard curve containing the matrix was constructed, with the ratio of the target drug response to the internal standard as the ordinate and the target drug concentration as the abscissa. The standard curve is shown below. Figure 5 As shown (using carbamazepine as an example).

[0048] Quantitative analysis using a standard curve revealed that the limit of detection (LOD) for carbamazepine using the miniaturized direct ionization mass spectrometer was 1 μg·mL⁻¹. -1 The limit of quantitation (LOQ) is 2 μg / mL. -1 The limit of detection (LOD) for valproic acid is 10 μg·mL. -1 The limit of quantitation (LOQ) is 20 μg / mL. -1 .

[0049] Table 1. Linearity, Limit of Detection, and Limit of Quantification of Carbamazepine and Valproic Acid

[0050] Quantitative analysis using a standard curve revealed that the limit of detection (LOD) for linezolid using a miniaturized direct ionization mass spectrometer was 0.5 μg·mL⁻¹. -1The limit of quantitation (LOQ) is 1 μg·mL. -1 The limit of detection (LOD) for voriconazole is 0.2 μg·mL⁻¹. -1 The limit of quantitation (LOQ) is 0.4 μg·mL. -1 .

[0051] Table 2 Linearity, Limit of Detection, and Limit of Quantification of Linezolid and Voriconazole

[0052] The results showed that the POCT detection method for carbamazepine established using the CRAIV-120 miniaturized direct ionization mass spectrometer has high sensitivity and can meet the clinical needs for POCT detection of carbamazepine, valproic acid, linezolid, and voriconazole.

[0053] 2. Precision, accuracy, and recovery rate: Following the standard curve series solution preparation steps, carbamazepine was prepared at low, medium, and high concentrations of 4 μg·mL⁻¹. -1 10 μg·mL -1 16 μg·mL -1 Quality control samples; valproic acid was prepared at low, medium, and high concentrations of 40 μg·mL⁻¹. -1 62.5 μg·mL -1 125 μg·mL -1 Quality control samples were used. Six sample analyses were performed at each concentration over three consecutive days. Method precision was obtained from the intra-day and inter-day RSD (%), and accuracy was obtained from the ratio of measured to theoretical values. Six sample analyses were performed at each concentration, and the responses were recorded. Spiked samples of the same determination concentration were prepared using standard substances and pretreated blank matrix, and the corresponding responses were obtained. These responses were compared with those of the quality control samples to obtain the sample extraction recovery rate. The analytical results are shown in Table 3.

[0054] Table 3. Precision, accuracy, and recovery of carbamazepine and valproic acid.

[0055] Experimental results showed that the intra-day and inter-day precision of carbamazepine were within 0.75%–1.71% and 1.44%–2.87%, respectively, with an accuracy range of -0.64%–-2.87%, both meeting the standard of <±15%, indicating that the method has good precision and accuracy. The extraction recoveries of carbamazepine at three concentrations were 98.69%, 93.62%, and 93.91%, respectively, all meeting the requirements for rapid clinical detection of carbamazepine. The intra-day and inter-day precision of valproic acid were within 1.15%–4.43% and 3.37%–6.62%, respectively, with an accuracy range of -2.72%–5.18%, both meeting the standard of <±15%, indicating that the method has good precision and accuracy. The extraction recoveries of valproic acid at three concentrations were 100.72%, 93.09%, and 92.94%, respectively, all of which meet the requirements for rapid clinical detection of valproic acid, indicating that the method is accurate and reliable.

[0056] Following the standard curve series solution preparation steps, linezolid was prepared at low, medium, and high concentrations of 2 μg·mL⁻¹. -1 8 μg·mL -1 15 μg·mL -1 Quality control samples were prepared; three concentrations of voriconazole were prepared at low, medium, and high levels of 0.4 μg·mL⁻¹, 2 μg·mL⁻¹, and 8 μg·mL⁻¹, respectively. Each concentration was analyzed three times over three consecutive days. Method precision was obtained from the intra-day and inter-day RSD (%), and accuracy was obtained from the ratio of measured to theoretical values. The response was recorded for each concentration. The analytical results are shown in Table 4.

[0057] Table 4. Precision, accuracy, and recovery of linezolid and voriconazole.

[0058] Experimental results show that the intra-day and inter-day precision of linezolid are within 1.49%~4.93% and 4.04%~6.07%, respectively, and the accuracy range is -0.86%~7.95%, both meeting the standard of <±15%. The intra-day and inter-day precision of voriconazole are within 2.89%~5.68% and 6.89%~10.42%, respectively, and the accuracy range is -0.88%~3.3%, both meeting the standard of <±15%. This indicates that the method has good precision and accuracy.

[0059] Example 2

[0060] The application of the detection method of this invention is the detection of carbamazepine in the serum of volunteers who have taken the medication.

[0061] The standard working curve is created in the same way as in Example 1.

[0062] Sample processing and measurement were the same as in Example 1.

[0063] The detection capabilities of the four drugs in spiked samples are shown in Table 5. Both the detection capability and accuracy requirements meet the quantitative requirements. Taking carbamazepine as an example, the MS of carbamazepine in blank and spiked samples... 2 The figures are as follows: Figure 6 and 7 As shown, this demonstrates that the method exhibits good selectivity.

[0064] Table 5. Detection results of carbamazepine, valproic acid, linezolid, and voriconazole spiked samples.

[0065] The above results demonstrate that the method of the present invention can effectively distinguish between positive and negative samples, and can accurately quantify the four drugs in actual clinical serum samples. Moreover, the detection results are consistent with clinical expectations, verifying the practicality and reliability of the method of the present invention in clinical therapeutic drug monitoring.

[0066] Example 3

[0067] This embodiment is used to verify that the total time from the start of pretreatment to obtaining the detection result of the method of the present invention does not exceed 7 minutes. The timing starts from the start time of pretreatment (i.e., when the precipitant is added to the serum sample) and continues until the mass spectrum is generated and the quantitative result is exported: (1) Vortex mixing: sampling and fixing the vortex takes 30 seconds; (2) Centrifugation: fixing takes 5 minutes (during centrifugation, the miniaturized mass spectrometer is simultaneously turned on for preheating, which takes about 1 minute and runs in parallel with the centrifugation operation without adding extra time); (3) Sampling and injection detection: after centrifugation, the supernatant is taken for injection, and the mass spectrometry data acquisition and processing takes about 20 seconds. In summary, the total time is approximately: 30 seconds + 5 minutes + 20 seconds = 5 minutes and 50 seconds, which meets the requirement of not exceeding 7 minutes.

[0068] In comparison, the study "Simultaneous Determination of 12 Antiepileptic Drugs in Serum by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry" mentions the detection of 12 antiepileptic drugs, including valproic acid and carbamazepine. Excluding the time for preparing liquid chromatography and mass spectrometry conditions, the pretreatment and detection process takes no less than 25 minutes. In contrast, direct ionization takes no more than 7 minutes from sampling to obtaining the result, and eliminates the need for complex steps such as solid-phase extraction, nitrogen drying, and chromatographic separation. This significantly improves detection efficiency, reduces operational difficulty, and is suitable for on-site clinical testing scenarios.

[0069] Table 6 Method Comparison

[0070] The embodiments described above are only preferred embodiments of the present invention and do not limit the scope of the present invention. All modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A rapid on-site detection method for blood drug concentration using direct ionization mass spectrometry, characterized in that, Includes the following steps: Obtain the serum sample to be tested; The serum sample was subjected to precipitation pretreatment to obtain a supernatant; Depending on the type of drug to be tested, a suitable ion source is selected from multiple ion sources, including at least nanoelectrospray ionization sources and probe electrospray ionization sources. The supernatant is ionized by the appropriate ion source and then sent to a direct ionization mass spectrometer for secondary mass spectrometry detection. The blood drug concentration of the serum sample to be tested was calculated based on the pre-established standard curve; The total time from the start of the pretreatment to obtaining the blood drug concentration in the detection method shall not exceed 7 minutes.

2. The method according to claim 1, characterized in that, The selected suitable ion source includes: When the drug to be tested is carbamazepine or valproic acid, the nano-electrospray ionization source is selected; When the test drug is linezolid or voriconazole, the probe electrospray ionization source is selected.

3. The method according to claim 1, characterized in that, The direct ionization mass spectrometer is a small direct ionization mass spectrometer.

4. The method according to claim 1, characterized in that, The pretreatment for precipitation includes: adding the serum sample to a precipitant containing an internal standard, vortexing to mix evenly, centrifuging for no more than 5 minutes, and taking the supernatant.

5. The method according to claim 4, characterized in that: When the test drug is carbamazepine, the precipitant is a formic acid-methanol mixed solution containing an internal standard, and the volume ratio of serum sample to precipitant is 1:39, wherein the volume ratio of formic acid to methanol is 1:

249. When the test drug is valproic acid, the precipitant is an acetonitrile-methanol mixed solution containing an internal standard, and the volume ratio of serum sample to precipitant is 1:19, wherein the volume ratio of acetonitrile to methanol is 1:

4. When the test drug is linezolid, the precipitant is a formic acid-methanol mixed solution containing internal standard formic acid with a volume ratio of formic acid to methanol of 1:319, the volume ratio of serum sample to precipitant is 1:4, and after centrifugation, the supernatant is taken and diluted with methanol at a ratio of 1:

9. When the test drug is voriconazole, the precipitant is a mixed solution of formic acid and ethyl acetate with an internal standard of 1:249, and the volume ratio of serum sample to precipitant is 1:

4. After centrifugation, the supernatant is diluted with 0.4% formic acid and ethyl acetate at a ratio of 1:

9.

6. The method according to claim 5, characterized in that, The precipitant contains carbamazepine with an internal standard concentration of 0.1 μg·mL⁻¹. -1 The internal standard concentration in the precipitant for valproic acid was 2.63 μg·mL. -1 The internal standard concentration in the precipitant of linezolid is 1 μg·mL. -1 The internal standard concentration in the voriconazole precipitant is 0.5 μg·mL. -1 .

7. The method according to claim 1, characterized in that, The pre-established standard curve was obtained as follows: using filtered horse serum as a blank matrix, different concentrations of drug standards were added, and the curve was plotted after the same pretreatment and mass spectrometry detection as the test sample; wherein the concentration range of carbamazepine in the standard curve was 2-20 μg·mL. -1 Valproic acid is 20-200 μg·mL -1 Linezolid is 1-20 μg·mL -1 Voriconazole is 0.4-10 μg·mL -1 .

8. The method according to claim 2, characterized in that: When using the aforementioned nano-electrospray ionization source, the mass spectrometry conditions are: positive / negative ion mode, ion source voltage 5000 V, isolation voltage amplitude 3 V, fragmentation voltage 0.1-1 V, and injection volume 10 μL; when using the aforementioned probe electrospray ionization source, the mass spectrometry conditions are: positive ion mode, ion source voltage 2000 V, isolation voltage amplitude 3.6 V, fragmentation voltage 1 V, and the injection volume is limited to ensure that the injection solution does not touch the metal inside the injection needle.

9. The method according to claims 1-2, characterized in that, In the secondary mass spectrometry detection: the precursor ion of carbamazepine is m / z 237.3, and the qualitative and quantitative daughter ions are m / z 192.2 and m / z 194.2, respectively; the precursor ion of valproic acid is m / z 143, and the qualitative and quantitative daughter ions are m / z 143; the precursor ion of linezolid is m / z 338, and the qualitative and quantitative daughter ions are m / z 195 and m / z 296, respectively; the precursor ion of voriconazole is m / z 349.2, and the qualitative and quantitative daughter ions are m / z 224.1 and m / z 280.9, respectively.

10. A rapid on-site detection kit for blood drug concentration using the method of any one of claims 1-9, characterized in that, include: A precipitant, wherein the precipitant is a mixed solution of organic solvents containing an internal standard; The instruction manual contains rules for selecting the ion source, which states that a nano-electrospray ionization source should be used for detecting carbamazepine or valproic acid, and a probe electrospray ionization source should be used for detecting linezolid or voriconazole.

Images