A high performance liquid chromatography-tandem mass spectrometry method for detecting isoniazid concentration in human cerebrospinal fluid

Abstract

The application discloses a high performance liquid chromatography-tandem mass spectrometry method for detecting the concentration of isoniazid in human cerebrospinal fluid, and belongs to the technical field of analytical chemistry and clinical therapeutic drug monitoring. The method comprises the following steps: short column and rapid gradient elution are adopted to realize chromatographic separation within 3.5 minutes; isoniazid and a deuterium internal standard thereof are specifically detected in a multiple reaction monitoring mode by optimizing mass spectrometry parameters; and sample pretreatment is performed by using one-step acetonitrile precipitation. Verification shows that the lower limit of quantification of the method reaches 5 ng / mL, the linear range is 5-4000 ng / mL, and the precision, accuracy and stability all meet the standards. The application has the advantages of ultrahigh sensitivity, rapid analysis, simple operation and strong anti-interference capability, and provides a precise and reliable drug concentration monitoring tool for individualized treatment of patients with tubercular meningitis.

Description

Technical Field

[0001] This invention relates to a high-performance liquid chromatography-tandem mass spectrometry method for detecting isoniazid concentration in human cerebrospinal fluid, at the intersection of analytical chemistry and clinical medicine. Background Technology

[0002] Tuberculous meningitis (TBM) is a non-suppurative inflammation of the meninges caused by Mycobacterium tuberculosis (MTB), accounting for 5%-10% of extrapulmonary tuberculosis cases and only 1% of all tuberculosis cases. However, its mortality rate can reach 27%, and its prognosis is poor, making it the most deadly type of tuberculosis, posing a serious threat to global public health and imposing a heavy economic burden. In recent years, factors such as population mobility, increased MTB drug resistance, and rising HIV incidence have led to a gradual increase in the prevalence of TBM. Isoniazid is a first-line drug for the treatment of TBM. Although it has good blood-brain barrier permeability, there are significant inter-individual differences. After intravenous or oral administration, the concentration of isoniazid in cerebrospinal fluid (CSF) varies considerably, thus affecting the treatment efficacy of TBM. Therefore, monitoring the concentration of isoniazid in CSF and developing individualized dosing regimens is crucial. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) is widely used for therapeutic drug monitoring (TDM) due to its advantages such as high sensitivity, excellent specificity, simple method development, and low detection cost.

[0003] Currently, the main specimen type used for LC-MS / MS detection of isoniazid is blood, with very few literature reports on its use in human CSF specimens, and the following issues exist: Insufficient sensitivity: The lower limit of quantitation (LLOQ) of existing methods is high (e.g., above 58.6 ng / mL), which is difficult to meet the monitoring needs of patients receiving low-dose treatment.

[0004] Low analytical efficiency: Some methods have long elution times (4.5 minutes) and large injection volumes, resulting in low throughput.

[0005] The pretreatment is complicated: protein precipitation in CSF samples is incomplete, and traditional liquid-liquid extraction is time-consuming and cumbersome.

[0006] Matrix interference: Existing technologies mostly use artificial CSF or plasma as the matrix, which differs from the matrix effect of human CSF and may affect the accuracy of detection.

[0007] In summary, there is an urgent clinical need for an LC-MS / MS detection method that offers higher sensitivity, faster analysis speed, simpler pretreatment, and thorough validation based on real human CSF matrix, in order to achieve accurate and efficient monitoring of isoniazid concentration in the CSF of TBM patients. This invention is therefore proposed. Summary of the Invention

[0008] The purpose of this invention is to provide a high-performance liquid chromatography-tandem mass spectrometry method for detecting isoniazid concentration in human cerebrospinal fluid, which solves the problems of insufficient sensitivity, low analytical efficiency, complex pretreatment, and poor accuracy due to matrix differences in the prior art. It can achieve ultra-high sensitivity, rapid analysis, simple pretreatment, and reliable verification based on real matrix, providing a precise monitoring tool for individualized treatment of tuberculous meningitis.

[0009] The high-performance liquid chromatography-tandem mass spectrometry method for detecting isoniazid concentration in human cerebrospinal fluid provided by this invention includes the following steps: S1. Sample pretreatment: Add internal standard solution and protein precipitant to human cerebrospinal fluid sample, mix, centrifuge and take the supernatant; S2. Chromatographic separation: The supernatant obtained in step S1 is injected into a high-performance liquid chromatography system, and a reversed-phase C18 column is used for gradient elution with acetonitrile and an aqueous solution containing 10 mM ammonium acetate as the mobile phase. S3. Mass spectrometry detection: Isoniazid and its internal standard are monitored by multiple reaction monitoring in electrospray ionization positive ion mode; the monitoring ion pair of isoniazid is m / z 138.0→121.0; the internal standard is deuterated isoniazid, and its monitoring ion pair is m / z 142.0→125.0. S4. Quantitative analysis: Based on the peak area ratio of isoniazid to the internal standard, combined with the standard curve, the concentration of isoniazid in human cerebrospinal fluid was obtained.

[0010] In step S1, the internal standard is D4-isocyanate; the protein precipitant is acetonitrile. The volume ratio of the cerebrospinal fluid sample, the internal standard solution, and the acetonitrile is 20:1:60; Preferably, the acetonitrile precipitation step is as follows: Sampling and addition of internal standard: Accurately measure 200 μL of human cerebrospinal fluid (CSF) sample and place it in a centrifuge tube. Add 10 μL of deuterated internal standard (D4-isoniacinamide) working solution (concentration of 0.50 μg / mL), and then vortex mix for 30 seconds to ensure that the internal standard and sample are fully homogenized.

[0011] Protein precipitation: Add 600 μL of acetonitrile to the above mixture (precipitant to sample volume ratio is 3:1). After capping, vortex vigorously for 3 minutes to allow the protein to fully denature and precipitate.

[0012] Centrifugation: Place the centrifuge tubes in a centrifuge pre-cooled to 4°C and centrifuge for 10 minutes at a relative centrifugal force of 16,200 × g.

[0013] Supernatant injection: After centrifugation, the sample will separate into a clear supernatant layer and a dense protein precipitate layer. Carefully aspirate a certain amount of the supernatant and transfer it directly to the injection vial of the LC-MS / MS instrument for chromatographic-mass spectrometric analysis.

[0014] In step S2, the specifications of the reversed-phase C18 column are: column length 50 mm, inner diameter 4.6 mm, and packing particle size 3.5 μm. Compared to a conventional 150 mm column, the shorter 50 mm column length significantly shortens the retention path of the analyte within the column, which is a key hardware foundation for achieving an ultra-fast analysis cycle of 3.5 minutes. The smaller the packing particles, the higher the theoretical plate number and the higher the column efficiency. This ensures sufficient separation efficiency within an extremely short column length, effectively separating the target peak from matrix interference peaks and avoiding quantification inaccuracies caused by co-elution. The standard inner diameter (4.6 mm) is fully compatible with conventional liquid chromatography systems. Combined with an ultra-low injection volume of only 5 μL, this ensures that the sample enters the mass spectrometer in a high-concentration, narrow-band form, greatly improving ionization efficiency and is one of the important conditions for achieving an ultra-low limit of quantitation (LLOQ) of 5 ng / mL.

[0015] In step S2, the gradient elution procedure is as follows: 0~1.0 minutes: The volume ratio of acetonitrile to 10 mM ammonium acetate aqueous solution is 10:90; 1.0~1.2 minutes: Acetonitrile ratio increases linearly from 10% to 90%; 1.2~2.0 minutes: Maintain the volume ratio of acetonitrile to 10 mM ammonium acetate aqueous solution at 90:10; 2.0~2.5 minutes: Acetonitrile ratio decreases linearly from 90% to 10%; 2.5~3.5 minutes: Maintain the volume ratio of acetonitrile to 10 mM ammonium acetate aqueous solution at 10:90.

[0016] The gradient elution procedure described in this invention enables ultra-fast analysis and increases throughput. The entire elution-rebalancing cycle takes only 3.5 minutes, which is significantly shorter than existing technologies (typically ≥4.5 minutes). This directly solves the problem of "low analytical efficiency" in the prior art and meets the clinical demand for large-volume, rapid sample output.

[0017] The gradient elution program of this invention produces sharp peak shapes, ensuring high sensitivity: a steep elution gradient (1.0-1.2 minutes) causes the target compound to be "pushed" out of the column in a very short time, resulting in narrow peak widths and sharp peak shapes (e.g., Figure 2(As shown). The synergistic low injection volume (5 μL) and sharp peak shape mean higher peak concentration, which greatly enhances the response signal of the mass spectrometer detector. This is the key chromatographic basis for achieving an ultra-low limit of quantitation (LLOQ) of 5 ng / mL.

[0018] The gradient elution program described in this invention effectively purifies the chromatographic column, ensuring method robustness. A strong elution step (1.2-2.0 minutes, 90% acetonitrile) effectively elutes highly retained impurities such as phospholipids and nonpolar metabolites that may be present in cerebrospinal fluid, preventing their accumulation on the column, protecting the column, and maintaining long-term stable column efficiency and back pressure. This makes it suitable for continuous analysis of large numbers of clinical samples. Excellent separation specificity is achieved; the initial weak elution conditions (10% acetonitrile, 0-1.0 minutes) allow most highly polar matrix interferences to be washed away before the target analyte. Subsequently, the target analyte achieves baseline separation from the remaining interferences under an optimized gradient (e.g., ...). Figure 2 As shown in Figure C, this effectively reduces matrix inhibition or enhancement effects during mass spectrometry detection, ensuring the accuracy of quantification.

[0019] In step S2, the flow rate for chromatographic separation was 0.5 mL / min, the injection volume was 5 μL, and the total analysis time was 3.5 minutes.

[0020] In step S3, the parameters for mass spectrometry detection are: curtain gas pressure 40 psi, collision gas medium, ion source gas 1 pressure 20 psi, ion source gas 2 pressure 20 psi, ionization voltage 5500 V, and ion source temperature 500℃.

[0021] In step S4, the linear range of the standard curve is 5 ng / mL to 4000 ng / mL; The lower limit of the method of this invention is 5 ng / mL, which is direct proof of the ultra-high sensitivity of the method of this invention. It is far lower than the common methods reported in the literature (usually >50 ng / mL), marking a significant methodological breakthrough.

[0022] The lower limit of the method of this invention is 5 ng / mL, which can cover low-concentration samples. This lower limit ensures that the drug concentration can be accurately measured in the following key situations: patients receiving low-dose treatment (such as children, patients with hepatic or renal insufficiency); patients with highly individual differences in blood-brain barrier permeability, whose CSF drug concentration may be extremely low; and samples in the trough concentration period after administration. Accurate monitoring of this low concentration is crucial for assessing efficacy and avoiding subtherapeutic concentrations.

[0023] The method of this invention has an upper limit of 4000 ng / mL, covering peak therapeutic concentrations and high-dose regimens: this upper limit is significantly higher than the expected peak concentration in CSF after routine oral or intravenous administration (typically in the range of hundreds of ng / mL to low μg / mL). It reliably measures: high-concentration samples after high-intensity administration such as intrathecal injection; abnormally high-concentration samples due to individual metabolic differences, avoiding the need for dilution and retesting due to concentration exceeding the range, thus improving work efficiency; and its future application scalability: it reserves space for research on higher-dose treatment regimens or special cases, enhancing the method's versatility and prospective application.

[0024] The present invention further provides a kit for carrying out the method, comprising: Deuterated isoniazid internal standard solution; A series of isoniazid standard solutions of various concentrations used to establish a standard curve.

[0025] This invention provides an LC-MS / MS method specifically for the detection of isoniazid concentration in human cerebrospinal fluid through systematic optimization, with the following outstanding advantages: Extremely high sensitivity: with a quantitation limit of 5 ng / mL, it can accurately detect trace amounts of drugs and meet the needs of low-concentration monitoring.

[0026] Fast analysis speed: The total running time is only 3.5 minutes, and combined with simple pretreatment, it greatly improves the detection throughput.

[0027] Simple and stable operation: It adopts a one-step acetonitrile precipitation method, which is simple and fast, with high and stable recovery rate.

[0028] The results are accurate and reliable: the use of real cerebrospinal fluid matrix and matched deuterated internal standards for verification effectively overcomes matrix interference.

[0029] Wide range of applications: The linear range is 5–4000 ng / mL, covering all therapeutic concentrations, requiring no dilution and making it widely applicable.

[0030] This invention provides a precise, efficient, and reliable concentration monitoring tool for individualized treatment of tuberculous meningitis. Attached Figure Description

[0031] Figure 1 The representative ion spectra of the isoniazid precursor ion (138.0 m / z) and fragment ions are shown in Figure A and Figure B, respectively.

[0032] Figure 2MRM chromatograms of blank cerebrospinal fluid (Figure A), MRM chromatograms of solutions containing 5 ng / ml isoniazid and 200 ng / ml internal standard (Figure B), and MRM chromatograms of clinical samples (containing 6.74 ng / ml isoniazid) collected after intrathecal injection of 100 mg isoniazid (Figure C). Detailed Implementation

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0034] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0035] This invention provides a liquid chromatography-tandem mass spectrometry (LC-MS / MS) method for detecting isoniazid concentration in human cerebrospinal fluid, comprising the following steps: Sample pretreatment: Take human cerebrospinal fluid samples, add deuterated internal standard (D4-isoniacinamide) solution and acetonitrile for protein precipitation, centrifuge and take the supernatant as the test solution.

[0036] Chromatographic separation: The test solution is injected into a high-performance liquid chromatography system, and a reversed-phase C18 column of a specific specification (preferably 50 mm × 4.6 mm, 3.5 μm) is used. Optimized gradient elution is performed with acetonitrile and an aqueous solution containing 10 mM ammonium acetate as the mobile phase, resulting in a short total analysis time.

[0037] Mass spectrometry detection: Isoniazid (monitoring ion pair m / z 138.0→121.0) and its internal standard (monitoring ion pair m / z 142.0→125.0) were specifically detected using electrospray ionization positive ion mode and multiple reaction monitoring (MRM) scanning.

[0038] Quantitative analysis: The concentration is calculated based on the ratio of the peak areas of the target analyte to the internal standard, combined with the standard curve.

[0039] The method of this invention is characterized by high sensitivity, rapid analysis, simple operation, and high accuracy and reliability, providing an effective tool for achieving precise individualized treatment.

[0040] Example 1: Establishment of the detection method 1. Solution preparation Accurately weigh an appropriate amount of isoniazid standard, dissolve and dilute it with 50% methanol aqueous solution to prepare a 1 mg / mL stock solution, and store at -20℃. Before use, serially dilute with 50% methanol aqueous solution to prepare a standard working solution of the required concentration.

[0041] Accurately weigh an appropriate amount of D4-isoniazid internal standard and prepare a 1 mg / mL internal standard stock solution using the same method. Dilute with 50% methanol aqueous solution to prepare a 0.50 μg / mL internal standard working solution.

[0042] 2. Sample pretreatment Take 200 μL of human cerebrospinal fluid sample and place it in a 1.5 mL plastic centrifuge tube. Accurately add 10 μL of 0.50 μg / mL D4-isoniazid internal standard working solution and mix on a vortex mixer for 30 seconds. Then add 600 μL of acetonitrile and vortex vigorously for 3 minutes. Place the centrifuge tube in a pre-chilled centrifuge at 4°C and centrifuge at 16,200×g for 10 minutes. After centrifugation, transfer the supernatant to the inner liner of an LC-MS / MS vial for analysis.

[0043] 3. Chromatographic conditions Chromatography system: Agilent 1290 Infinity II liquid chromatography system.

[0044] Chromatographic column: Agilent Eclipse Plus C18 column (50 mm × 4.6 mm, 3.5 μm).

[0045] Mobile phase: Phase A is an aqueous solution containing 10 mM ammonium acetate; Phase B is acetonitrile.

[0046] Gradient elution program: 0-1.0 min, 10% B; 1.0-1.2 min, 10% B → 90% B; 1.2-2.0 min, 90% B; 2.0-2.5 min, 90% B → 10% B; 2.5-3.5 min, 10% B.

[0047] Flow rate: 0.5 mL / min; column temperature: 40℃; injection volume: 5 μL.

[0048] 4. Mass spectrometry conditions Mass spectrometry system: AB Sciex QTRAP 5500+ mass spectrometer, equipped with an electrospray ionization source.

[0049] Ionization mode: Positive ion electrospray ionization.

[0050] Scanning mode: Multiple response monitoring.

[0051] Ion source parameters: curtain gas 40 psi, collision gas medium, ion source gases 1 and 2 are both 20 psi, ion spray voltage 5500 V, ion source temperature 500℃.

[0052] MRM parameters: Isoniazid, parent ion m / z 138.0, daughter ion m / z 121.0; D4-Isoniazid (internal standard), parent ion m / z 142.0, daughter ion m / z 125.0.

[0053] Figure 1The characteristic ion information of the target compound (isoniacinamide) and its internal standard (D4-isoniacinamide) monitored in this invention in mass spectrometry is shown: Figure 1 Figure A shows the mass spectrum of isoniazid, which displays the precursor ion (parent ion) formed by the isoniazid molecule in electrospray ionization in positive ion mode. Its mass-to-charge ratio (m / z) is 138.0, corresponding to the protonated molecule of isoniazid [M+H]. + The precursor ion further fragments in the collision chamber, producing a main characteristic fragment ion (daughter ion) with an m / z of 121.0, presumably formed by the loss of a molecule of ammonia (NH3) from the parent ion. Therefore, this invention ultimately selects the pair of characteristic ions with m / z 138.0→121.0 as the monitoring channel for the qualitative and quantitative analysis of isoniazid.

[0054] Figure 1 Figure B shows the mass spectrum of the deuterated internal standard D4-isoniacinamide. The figure shows that its precursor ion has an m / z of 142.0, which is due to the substitution of four hydrogen atoms by deuterium atoms, resulting in a 4-unit increase in molecular weight. Its main characteristic fragment ion has an m / z of 125.0, corresponding to a fragment that has lost one molecule of ND3 or NH3. Therefore, this invention ultimately selected m / z 142.0→125.0 as the monitoring channel for the internal standard.

[0055] Figure 1 The specificity and rationality of the selected monitoring ion pair in this invention have been verified. Using a deuterated internal standard (D4-isoniacinamide) ensures that its chemical properties and chromatographic behavior are highly consistent with the target analyte (isoniacinamide), but they are distinguishable in mass spectrometry due to their different mass numbers. This lays a crucial foundation for subsequent accurate multiple reaction monitoring (MRM) and quantification using the internal standard method to correct for pretreatment losses and matrix effects during mass spectrometry ionization. The clear and single ion peaks in the figure also indicate that, under optimized mass spectrometry conditions, the ionization and fragmentation processes of the target analyte and the internal standard are stable, efficient, and with minimal interference.

[0056] Example 2: Methodological Validation—Standard Curve, Linear Range, and Lower Limit of Quantitation 1. Preparation of standard curve Take the mixed blank human cerebrospinal fluid, add appropriate amounts of isoniazid standard working solution and internal standard working solution, and prepare a series of standard curve samples with isoniazid concentrations of 5.0, 10.0, 50.0, 100.0, 500.0, 1000.0, 2000.0, and 4000.0 ng / mL, and the internal standard concentration of each is 4000 ng / mL. Prepare five replicates for each concentration point. Perform pretreatment and analysis according to the method in Example 1.

[0057] 2. Standard Curve Plotting and Linear Range The peak area ratio of isoniazid to internal standard was plotted on the ordinate (y), and the concentration of isoniazid on the abscissa (x). A weighted least squares method (with a weighting coefficient of 1 / x) was used. 2 Linear regression was performed. The standard curve equation was obtained as y = 0.00511x + 0.00438, with a correlation coefficient (r) of 0.9966. The calibration curves from three runs are shown in Table 1. The results indicate that isoniazid exhibits good linearity in the concentration range of 5.0–4000.0 ng / mL.

[0058] Table 1. Calibration curves of isoniazid in three runs

[0059] 3. Determination of the lower limit of quantitation Six independent analyses were performed on samples with a concentration of 5.0 ng / mL (LLOQ). The average signal-to-noise ratio (S / N) at this concentration was 13.31 (≥10), with an accuracy (RE) ranging from 1.6% to 15.2% and a precision (RSD) of 5.29% (all less than 20%). This demonstrates that the limit of quantitation for the method of this invention is 5.0 ng / mL, which meets the requirements for trace detection.

[0060] Example 3: Methodology Validation—Precision and Accuracy 1. Experimental Design Isoniazid standard was added to blank human cerebrospinal fluid to prepare quality control samples at three concentrations: low (15.0 ng / mL), medium (800.0 ng / mL), and high (3200.0 ng / mL). Six samples at each concentration level were analyzed on the same day for three consecutive days to examine intra-day and inter-day precision and accuracy.

[0061] 2. Results Intraday precision (RSD) was 9.15% (low), 5.48% (medium), and 6.72% (high); interday precision was 2.16% (low), 12.78% (medium), and 0.95% (high).

[0062] Intraday accuracy (RE) ranges from 5.48% to 9.15%; interday accuracy ranges from 0.95% to 12.78%.

[0063] All results met the requirements of the guidelines for validation of analytical methods for biological samples (such as FDA guidelines) (RE within ±15%, LLOQ within ±20%; RSD ≤15%, LLOQ ≤20%).

[0064] Table 2. Intra-day and inter-day precision (RSD) and accuracy (RE) of isoniazid (n=6)

[0065] Example 4: Method Validation—Recovery and Matrix Effect 1. Extraction recovery rate The quality control samples (n=6) of low, medium, and high concentrations were processed according to the method in Example 1, and the peak areas (A) were measured. A separate pure standard solution of the same concentration (the standard was reconstituted with the supernatant after extraction using a blank matrix without prior extraction) was prepared and injected directly, and the peak area (B) was measured.

[0066] Recovery rate = (A / B) × 100%.

[0067] Results: As shown in Table 3, the average extraction recoveries of isoniazid at the three concentration levels were 100%, 93.16%, and 104.74%, respectively; the recovery rate of the internal standard was 125.09%. The recoveries were stable and reproducible.

[0068] 2. Matrix effect Three pure standard solutions of low, medium, and high concentrations (dissolved in the mobile phase) were prepared and directly injected to measure the peak area (C). Six blank human cerebrospinal fluid matrices from different sources were processed according to the method in Example 1, and the same concentration of standard was reconstituted with the supernatant and injected to measure the peak area (D).

[0069] Matrix effect = (D / C) × 100%.

[0070] Results: As shown in Table 3, the matrix effect of isoniazid ranged from 97.43% to 104.74% (RSD < 10%), while the matrix effect of the internal standard was 40.55 ± 5.33% (RSD = 13.14%). This indicates that under the conditions of the method of this invention, the human cerebrospinal fluid matrix has no significant inhibitory or enhancing effect on the ionization efficiency of isoniazid and the internal standard.

[0071] Table 3. Matrix effect and recovery rate of isoniazid (n=6)

[0072] Example 5: Methodological Validation—Stability Study 1. Examination conditions Stability was assessed by placing two quality control samples (n=3) at low and high concentrations under the following conditions: (1) Place at room temperature for 4 hours; (2) Underwent three cycles of freezing at -80℃ and thawing at room temperature; (3) Store frozen at -80℃ for 36 days.

[0073] 2. Results Under the above conditions, the samples were reanalyzed. Compared with the measured values ​​of freshly prepared samples, the RE values ​​of isoniazid concentration were all within ±12%, as shown in Table 4. This demonstrates that under the sample processing and analysis conditions established by the method of this invention, isoniazid in human cerebrospinal fluid has good stability and can ensure the accuracy of the detection results.

[0074] Table 4. Stability of isoniazid in cerebrospinal fluid (n=6)

[0075] Example 6: Clinical Sample Testing Application The LC-MS / MS method established in Example 1 was used to test a cerebrospinal fluid sample from a patient clinically diagnosed with tuberculous meningitis who received intrathecal isoniazid treatment.

[0076] Take a 200 μL sample of the patient's cerebrospinal fluid.

[0077] Perform the pretreatment according to the steps in Example 1.

[0078] The analysis was performed under the chromatographic and mass spectrometric conditions described in Example 1.

[0079] The peak area ratio of isoniazid to internal standard was substituted into the standard curve equation established in Example 2 for calculation.

[0080] The results showed that the concentration of isoniazid in the patient's cerebrospinal fluid was 6.74 ng / mL. Its MRM chromatogram is shown below. Figure 2 As shown in Figure C, the peaks of isoniazid and internal standard are sharp and symmetrical, and completely separated from the adjacent matrix interference peaks, proving the feasibility and reliability of the method of the present invention in complex real clinical samples.

[0081] in, Figure 2 Figure A shows the MRM chromatogram of a blank human cerebrospinal fluid sample. The results show that no obvious chromatographic peaks appeared near the retention times of isoniazid (approximately 1.2 minutes) and its internal standard (approximately 1.2 minutes). This indicates that the blank matrix did not interfere with the detection of the target analyte, demonstrating the excellent specificity of the method of this invention.

[0082] Figure 2 Figure B shows the MRM chromatogram of the spiked sample, where the isoniazid concentration is 5 ng / mL (i.e., the lower limit of quantitation of the method of this invention), and the internal standard concentration is 200 ng / mL. As can be seen in the figure, sharp, symmetrical, and clearly identifiable peaks of isoniazid and the internal standard appear at approximately 1.2 minutes, with a signal-to-noise ratio much greater than 10. This figure directly demonstrates that the method of this invention still possesses reliable detection capability at the LLOQ level, i.e., ultra-high sensitivity.

Claims

1. A high-performance liquid chromatography-tandem mass spectrometry method for detecting isoniazid concentration in human cerebrospinal fluid, comprising the following steps: S1. Sample pretreatment: Add internal standard solution and protein precipitant to human cerebrospinal fluid sample, mix, centrifuge and take the supernatant; S2. Chromatographic separation: The supernatant obtained in step S1 is injected into a high-performance liquid chromatography system, and a reversed-phase C18 column is used for gradient elution with acetonitrile and an aqueous solution containing 10 mM ammonium acetate as the mobile phase. S3. Mass spectrometry detection: Isoniazid and its internal standard are monitored by multiple reaction monitoring in electrospray ionization positive ion mode; the monitoring ion pair of isoniazid is m / z 138.0→121.0; the internal standard is deuterated isoniazid, and its monitoring ion pair is m / z 142.0→125.

0. S4. Quantitative analysis: Based on the peak area ratio of isoniazid to the internal standard, combined with the standard curve, the concentration of isoniazid in human cerebrospinal fluid was obtained.

2. The method according to claim 1, characterized in that: In step S1, the internal standard is D4-isocyanate; the protein precipitant is acetonitrile. The volume ratio of the cerebrospinal fluid sample, the internal standard solution, and the acetonitrile is 20:1:

60.

3. The method according to claim 1 or 2, characterized in that: In step S2, the specifications of the reversed-phase C18 chromatographic column are: column length 50 mm, inner diameter 4.6 mm, and packing particle size 3.5 μm.

4. The method according to any one of claims 1-3, characterized in that: In step S2, the gradient elution procedure is as follows: 0~1.0 minutes: The volume ratio of acetonitrile to 10 mM ammonium acetate aqueous solution is 10:90; 1.0~1.2 minutes: Acetonitrile ratio increases linearly from 10% to 90%; 1.2~2.0 minutes: Maintain the volume ratio of acetonitrile to 10 mM ammonium acetate aqueous solution at 90:10; 2.0~2.5 minutes: Acetonitrile ratio decreases linearly from 90% to 10%; 2.5~3.5 minutes: Maintain the volume ratio of acetonitrile to 10 mM ammonium acetate aqueous solution at 10:

90.

5. The method according to any one of claims 1-4, characterized in that: In step S2, the flow rate for chromatographic separation was 0.5 mL / min, the injection volume was 5 μL, and the total analysis time was 3.5 minutes.

6. The method according to any one of claims 1-5, characterized in that: In step S3, the parameters for mass spectrometry detection are: curtain gas pressure 40 psi, collision gas medium, ion source gas 1 pressure 20 psi, ion source gas 2 pressure 20 psi, ionization voltage 5500 V, and ion source temperature 500℃.

7. The method according to any one of claims 1-6, characterized in that: In step S4, the linear range of the standard curve is 5 ng / mL to 4000 ng / mL.

8. A kit for carrying out the method according to any one of claims 1-7, comprising: Deuterated isoniazid internal standard solution; A series of isoniazid standard solutions of various concentrations used to establish a standard curve.

9. The use of the method according to any one of claims 1-7 in the preparation of a diagnostic and therapeutic kit for personalized drug administration in tuberculous meningitis, characterized in that: The concentration of isoniazid in human cerebrospinal fluid is detected to guide the adjustment of medication dosage for patients with tuberculous meningitis.

10. A system for individualized drug delivery to patients with tuberculous meningitis, comprising: The data acquisition module is used to acquire isoniazid concentration data in the cerebrospinal fluid of a patient as determined by the method according to any one of claims 1-7; The dosing regimen adjustment module is used to generate a suggestion to adjust the isoniazid dosage based on the comparison results between the isoniazid concentration data and the target therapeutic concentration.

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