High-sensitivity liquid chromatography detection method for drug metabolites

Abstract

The invention discloses a high-sensitivity liquid chromatography detection method for drug metabolites, and relates to the technical field of drug analysis. Aiming at the problems of low sensitivity, serious matrix interference and complicated operation of the existing method, the method realizes efficient detection by improving sample pretreatment and optimizing chromatography-mass spectrometry conditions: removing more than 99% of matrix impurities from a biological sample by using an acetonitrile-methanol mixed precipitator (with the volume ratio of 7: 3); a core-shell C18 chromatographic column and a gradient elution program are adopted to realize high-efficiency separation of metabolites, and specific detection is performed in combination with a tandem mass spectrum multi-reaction monitoring mode. According to the method, the detection limit is as low as 0.0008-0.01 ng / mL, compared with a traditional method, the sensitivity is improved by 50-100 times, the metabolite recovery rate reaches 90-98%, the quantitative accuracy RSD is smaller than or equal to 5%, the pretreatment time consumption is smaller than or equal to 15 min, and complex derivatization steps are not needed. The method is suitable for accurate quantification of low-concentration drug metabolites in biological samples such as plasma and urine, can be widely applied to drug research and development, pharmacokinetic research and clinical treatment drug monitoring, and has remarkable practical value.

Description

Technical Field

[0001] This invention relates to the field of drug analysis and detection technology, specifically to a highly sensitive liquid chromatography method for detecting drug metabolites, which is suitable for the accurate quantitative detection of low-concentration drug metabolites in drug development, clinical therapeutic drug monitoring, and pharmacokinetic studies. Background Technology

[0002] Metabolites produced after a drug enters the body directly reflect the drug's absorption, distribution, metabolism, and excretion processes, serving as crucial evidence for assessing drug efficacy and safety and adjusting dosing regimens. In the innovative drug development stage, qualitative and quantitative analysis of metabolites can clarify drug metabolic pathways and reveal the toxic risks of metabolites, providing core data for screening drug candidate compounds. In clinical treatment, accurate detection of low-concentration metabolites is of great significance for individualized dosing regimens and early warning of adverse drug reactions, especially suitable for monitoring medication use in special populations such as patients with impaired liver or kidney function and elderly patients.

[0003] Currently, liquid chromatography is widely used for the detection of drug metabolites due to its high separation efficiency and ease of operation; however, existing methods have significant drawbacks:

[0004] 1) Severe interference from the sample matrix: Proteins, lipids, and endogenous small molecules (such as amino acids, hormones, fatty acids, etc.) in biological samples (plasma, urine, tissue homogenate, etc.) are easily co-eluted with metabolites, resulting in overlapping chromatographic peaks and peak shape distortion, which significantly affects the accuracy of detection. In some cases, it is even impossible to distinguish between target metabolites and interfering substances.

[0005] 2) Insufficient sensitivity: low concentrations of metabolites (below ng / mL) have weak signal responses in traditional liquid chromatography-UV detectors or fluorescence detectors, and the detection limit is usually only 0.5 to 1 ng / mL, which is difficult to meet the quantitative requirements of early generation and late elimination of metabolites in pharmacokinetic studies.

[0006] 3) The pretreatment process is complex. Traditional methods often use solid phase extraction (SPE) or derivatization technology. Solid phase extraction requires multiple steps such as activation, equilibration, sample loading, and elution, which takes up to 30 to 60 minutes. In addition, the extraction column is expensive. Derivatization reaction has problems such as reagent contamination, harsh reaction conditions (such as high temperature and specific pH value), and loss of metabolites.

[0007] 4) Narrow linear range and poor reproducibility. Due to the limitations of column separation efficiency and detector specificity, the linear range of existing methods can usually only cover 1 to 3 orders of magnitude, and the inter-day precision RSD of batch sample detection often exceeds 10%, which affects the reliability of data.

[0008] These problems severely limit the application of liquid chromatography in low-concentration metabolite detection scenarios, especially failing to meet the needs of high-throughput screening in innovative drug development and rapid and accurate quantification in clinical monitoring. There is an urgent need to develop a detection method that is highly sensitive, resistant to interference, easy to operate, and has good reproducibility. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention provides a highly sensitive liquid chromatography method for the detection of drug metabolites, enabling accurate quantification of low-concentration metabolites (below ng / mL level). It also simplifies the pretreatment process, reduces matrix interference, broadens the linear range, and improves detection reproducibility, thus meeting the diverse needs of drug development, pharmacokinetic studies, and clinical drug monitoring.

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] A highly sensitive liquid chromatography method for the detection of drug metabolites includes three steps: sample pretreatment, chromatographic separation, and mass spectrometry detection, as detailed below:

[0012] (1) Sample pretreatment:

[0013] Take 50–200 μL of biological sample, selected from one or more of plasma, urine, and tissue homogenate, transfer it to a centrifuge tube, add a protein precipitant mixture of acetonitrile and methanol at a volume ratio of 1:3 to 1:5 (7:3 volume ratio of acetonitrile to methanol); place the centrifuge tube on a vortex mixer and vortex at 3000–4000 r / min for 1–2 min, then place it in a high-speed refrigerated centrifuge and centrifuge at 4℃ and 12000 r / min for 5–8 min. After centrifugation, collect the supernatant and dilute it with 0.1–0.2 times the volume of ultrapure water. The diluted sample should be tested within 24 h under refrigeration at 4℃.

[0014] (2) Chromatographic separation:

[0015] A core-shell C18 column with dimensions of 100 mm × 2.1 mm and a particle size of 1.7 μm was used. The column temperature was controlled at 30–35 °C with a temperature fluctuation of ≤ ±0.5 °C. The mobile phase consisted of ultrapure water containing 0.05–0.1% formic acid as phase A and acetonitrile containing 0.05–0.1% formic acid as phase B. A gradient elution program was used: 0–2 min, phase B ratio 20–30%; 2–8 min, phase B ratio 30–80%; 8–10 min, phase B ratio 80%; 10–10.1 min, phase B ratio decreasing from 80% to 20%; 10.1–15 min, phase B ratio 20%. The flow rate was set at 0.25–0.3 mL / min, and the injection volume was 5–10 μL. The injection needle was rinsed three times with sample solution before injection to avoid cross-contamination.

[0016] (3) Mass spectrometry detection:

[0017] A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) system was used with an electrospray ionization (ESI) source. Positive or negative ion mode was selected based on the polarity of the drug metabolites, with positive ion mode preferred for amine metabolites and negative ion mode preferred for carboxylic acid metabolites. Multiple reaction monitoring (MRM) was employed for specific detection. The optimized ion source temperature was 300–350 °C, the spray voltage was 3500–4000 V, the sheath gas pressure was 40–50 psi, and the auxiliary gas pressure was 8–12 psi. The collision energy was adjusted according to the structural characteristics of the drug metabolites: 15–25 eV for carboxylic acid metabolites, 20–30 eV for amine metabolites, and 18–28 eV for alcohol metabolites, to obtain the maximum fragment ion response signal.

[0018] Furthermore, in step 1, the volume ratio of the protein precipitant to the biological sample is 1:4.

[0019] Furthermore, in step 2, the chromatographic column was a Waters XBridge BEH C18 core-shell column, the column temperature was 32℃, phase A was ultrapure water containing 0.08% formic acid, phase B was acetonitrile containing 0.08% formic acid, the flow rate was 0.28 mL / min, the injection volume was 8 μL, and the gradient elution program was as follows: 0–2 min, phase B ratio was 25%; 2–8 min, phase B ratio linearly increased from 25% to 75%; 8–10 min, phase B ratio was 75%; 10–10.1 min, phase B ratio decreased from 75% to 25%; 10.1–15 min, phase B ratio was 25%.

[0020] Furthermore, the drug metabolite is a small molecule metabolite with a molecular weight of 100-500 Da, selected from one or more of alcohol, carboxylic acid, amine, and ester metabolites.

[0021] Furthermore, the dwell time of the multiple reaction monitoring mode in step 3 is 80–120 ms.

[0022] Furthermore, in the biological sample pretreatment process, the vortex oscillation time is 1.5 min, the high-speed refrigerated centrifugation time is 6 min, and the dilution volume ratio of the supernatant to ultrapure water is 1:0.17.

[0023] Beneficial effects of this invention:

[0024] (1) Significantly improved sensitivity: Through the high separation efficiency of the core-shell chromatographic column, gradient elution optimization and the specific MRM mode of the MS / MS detector, the method detection limit is as low as 0.005-0.01 ng / mL and the quantitation limit is 0.02-0.03 ng / mL, which is 50-100 times more sensitive than the traditional liquid chromatography method. It can effectively detect low concentrations of metabolites in the early and late stages of drug metabolism and in special populations (those with liver and kidney dysfunction).

[0025] (2) Strong anti-interference ability: The modified protein precipitant can remove more than 99% of the sample matrix protein. Combined with the high efficiency separation of the core-shell chromatographic column and the specific monitoring of the MRM mode, the matrix effect factor is controlled between 0.9 and 1.1, and the quantitative accuracy RSD ≤ 5%, which greatly reduces the interference of endogenous impurities.

[0026] (3) Simple and efficient operation: The pretreatment process only takes less than 15 minutes, without the need for complex solid phase extraction or derivatization steps. The efficiency of batch sample detection is increased by 2 to 3 times, and the metabolite recovery rate reaches 90 to 98%, meeting the needs of high-throughput detection.

[0027] (4) Wide applicability and good reproducibility: Chromatographic and detection parameters can be adjusted for drug metabolites of different polarities with molecular weights of 100–500 Da (such as small molecule alcohols, carboxylic acids, amines, esters, etc.), with a linear range covering 0.02–10 ng / mL (R0). 2 ≥0.999), intraday RSD≤3.5%, interday RSD≤4.5%, suitable for detection of various biological sample types;

[0028] (5) Cost controllable: The pretreatment only uses conventional reagents such as acetonitrile and methanol, without the need for expensive extraction columns or derivatization reagents. The detection cost is reduced by 40-60% compared with traditional methods, which is convenient for promotion and application. Attached Figure Description

[0029] Figure 1 This is a schematic flowchart of the drug metabolite detection method of the present invention;

[0030] Figure 2 This is the MRM chromatogram of the target metabolite (salicylic acid) of this invention. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0032] The highly sensitive liquid chromatography detection method for drug metabolites proposed in this invention includes the following steps:

[0033] (1) Sample pretreatment:

[0034] Take biological samples (plasma, urine, tissue homogenate), accurately transfer 50–200 μL into centrifuge tubes, and add a modified protein precipitant (acetonitrile-methanol mixture, volume ratio 7:3) at a ratio of 1:3 to 1:5. This ratio has been verified through extensive experiments to balance protein precipitation efficiency and metabolite solubility. Acetonitrile, as a highly polar organic solvent, can rapidly disrupt the hydrogen bond network of proteins to achieve denaturation and precipitation, while methanol can improve the solubility of lipid-soluble metabolites, avoiding the precipitation loss of some metabolites caused by acetonitrile precipitation alone. Then place the centrifuge tubes on a vortex mixer and vortex at 3000–4000 rpm for 1–2 min (vortexing time less than 1 minute). If the protein precipitation is too short (less than 2 minutes, protein precipitation will be insufficient; if it is longer than 2 minutes, metabolite degradation is likely to occur). Then, place the sample in a high-speed refrigerated centrifuge and centrifuge at 12000 r / min for 5-8 minutes. During centrifugation, the temperature should be controlled at 4℃ to further reduce the loss of metabolite volatilization. After centrifugation, use a pipette to accurately aspirate the supernatant (avoiding the aspiration of bottom precipitate) and dilute it with 0.1-0.2 times the volume of ultrapure water. The dilution step can reduce the proportion of organic phase in the supernatant, avoid high concentration of organic phase entering the chromatographic column and causing damage to the stationary phase, and improve the ionization efficiency of subsequent mass spectrometry detection. The diluted sample should be detected within 24 hours under refrigeration at 4℃ to prevent metabolite degradation.

[0035] (2) Optimization of chromatographic conditions:

[0036] Chromatographic column: A core-shell C18 column (100mm × 2.1mm, particle size 1.7μm) is used. The core-shell structure of this column consists of a solid silica core and a porous silica shell. Compared with traditional fully porous columns, the mass transfer resistance is significantly reduced, and the column efficiency is improved by more than 30%. The small particle size of 1.7μm further enhances the separation ability, effectively separating metabolites from trace interfering substances in the matrix. The column temperature is strictly controlled at 30-35℃ and is kept constant by a column oven. The temperature fluctuation is ≤±0.5℃ to ensure the reproducibility of metabolite retention time.

[0037] Mobile phase: Phase A is ultrapure water containing 0.05–0.1% formic acid, and Phase B is acetonitrile containing 0.05–0.1% formic acid. The addition of formic acid adjusts the pH of the mobile phase to 2.5–3.5, promoting the protonation of metabolites (especially those containing amino and carboxyl groups), improving the response signal of subsequent mass spectrometry detection, and the 0.05–0.1% concentration range avoids excessive acidification that could lead to hydrolysis of the stationary phase. An optimized gradient elution program is used: 0–2 min, Phase B 20–30%, with a low proportion of B in this stage. Phase B can quickly elute polar impurities in the sample, avoiding interference with subsequent metabolite separation; 2–8 min, Phase B 30–80%, linear gradient elution can achieve efficient separation based on the polarity difference between metabolites and interfering substances; 8–10 min, Phase B 80%, maintain a high proportion of Phase B to flush the column and remove residual non-polar impurities; 10–10.1 min, Phase B 80–20%, quickly return to the initial ratio; 10.1–15 min, Phase B 20%, equilibrate the column and prepare for the next injection;

[0038] Flow rate: 0.25-0.3 mL / min. Too low a flow rate will result in prolonged analysis time and peak broadening, while too high a flow rate will increase column pressure (core-shell columns have a pressure range of 0-6000 psi and are compatible with this flow rate range). Injection volume: 5-10 μL. Precise control is achieved through an autosampler. The injection needle should be rinsed with sample solution three times before injection to avoid cross-contamination.

[0039] (3) Detection conditions:

[0040] A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) system was used with an electrospray ionization (ESI) source. The system allows selection of either positive or negative ion mode based on the metabolite polarity (positive ion mode is preferred for amine metabolites, and negative ion mode is preferred for carboxylic acid metabolites). Multiple reaction monitoring (MRM) mode was employed, which selectively monitors specific precursor and daughter ions of metabolites, effectively eliminating interference from other substances in the matrix and improving detection specificity. Key detection parameters were optimized: the ion source temperature was controlled between 300 and 300 °C. 50℃ is a temperature range that balances solvent evaporation efficiency and metabolite stability, preventing thermal decomposition of metabolites due to excessively high temperatures. The spray voltage is 3500–4000V to ensure sufficient ionization of metabolites; too low a voltage results in insufficient ionization efficiency, while too high a voltage can lead to ion source contamination. The collision energy is adjusted according to the structural characteristics of the metabolites: 15–25 eV for carboxylic acid metabolites, 20–30 eV for amine metabolites, and 18–28 eV for alcohol metabolites. Optimizing the collision energy yields the maximum fragment ion response, further enhancing detection sensitivity. Specific implementation examples:

[0042] Taking the detection of salicylic acid, the main metabolite of aspirin, as an example, the implementation process of the present invention is described in detail. This embodiment is only used to explain the present invention and does not constitute a limitation on the scope of protection of the present invention.

[0043] 1. Experimental materials and instruments:

[0044] Reagents: Salicylic acid standard (purity ≥99.5%, Sigma-Aldrich); acetonitrile, methanol, and formic acid (all chromatographic grade, Thermo Fisher); ultrapure water (prepared using a Milli-Q ultrapure water system, resistivity ≥18.2 MΩ·cm); blank human plasma (provided by volunteers with no history of aspirin use, heparin anticoagulated);

[0045] Instruments: High-performance liquid chromatography-tandem mass spectrometry system (Agilent 1290HPLC coupled with Agilent 6470MS / MS); high-speed refrigerated centrifuge (Eppendorf 5810R); vortex mixer (Vortex-Genie 2); ultrapure water system (Millipore Synergy); analytical balance (Mettler XS205DU, accuracy 0.01mg);

[0046] Preparation of standard curve: Accurately weigh 10.0 mg of salicylic acid standard, dissolve it in methanol and dilute to 10 mL to prepare a standard stock solution with a concentration of 1.0 mg / mL. Store at -20℃ protected from light for 3 months. Before use, take out the stock solution and dilute it stepwise with methanol to prepare a series of standard working solutions with concentrations of 0.01, 0.02, 0.1, 0.5, 1.0, 5.0, and 10.0 ng / mL. At the same time, weigh p-hydroxybenzoic acid (internal standard, purity ≥99.0%) and prepare an internal standard working solution with a concentration of 0.1 μg / mL using methanol. Take 90 μL of blank human plasma, add 10 μL of the series of standard working solutions and 10 μL of the internal standard working solution, and vortex for 30 s to prepare matrix-matched standard curve samples with concentrations of 0.001, 0.002, 0.01, 0.05, 0.1, 0.5, and 1.0 ng / mL to eliminate the influence of matrix effect on quantification.

[0047] 2. Sample pretreatment optimization:

[0048] Take 100 μL of the matrix-matched standard curve sample or clinical plasma sample and transfer it to a 1.5 mL centrifuge tube. Accurately add 400 μL of acetonitrile-methanol mixed precipitant (volume ratio 7:3), ensuring that the volume ratio of precipitant to sample is 1:4 (verified by orthogonal experiments, this ratio achieves a protein removal rate of 99.2% and the highest salicylic acid recovery rate). Place the centrifuge tube on a vortex mixer and vortex at 3500 r / min for 1.5 min to ensure thorough mixing of the sample and precipitant. Then place it in a high-speed refrigerated centrifuge, set the temperature to 4℃, and the speed to 12000 r / min for 6 min. After centrifugation, slowly aspirate 180 μL of supernatant using a 200 μL pipette (avoid touching the protein precipitate at the bottom of the tube) and transfer it to a sample vial. Add 30 μL of ultrapure water (0.17 times the volume) to dilute and vortex for 30 s. At this point, the proportion of organic phase in the sample drops to 82%, which protects the stationary phase of the chromatographic column and improves the mass spectrometry ionization efficiency. All processed samples were placed in an autosampler at 4°C and the test was completed within 12 hours.

[0049] 3. Optimization of chromatographic and mass spectrometric parameters:

[0050] Chromatographic system: A Waters XBridge BEH C18 core-shell column (100 mm × 2.1 mm, 1.7 μm) was used. The column temperature was precisely controlled at 32 °C using a column oven, with temperature fluctuations ≤ ±0.3 °C to ensure reproducibility of retention times. The mobile phase A was ultrapure water containing 0.08% formic acid (the amount of formic acid added was optimized to improve the protonation efficiency of salicylic acid by 40%), and the mobile phase B was acetonitrile containing 0.08% formic acid. The flow rate was set to 0.28 mL / min, and the injection volume was 8 μL. The gradient elution program was optimized as follows: 0–2 min, phase B 25% (eluting polar impurities); 2–8 min, phase B linearly increases from 25% to 75% (achieving baseline separation of salicylic acid and internal standard); 8–10 min, phase B is maintained at 75% (rinsing residual impurities in the column); 10–10.1 min, phase B rapidly decreases to 25%; 10.1–15 min, phase B 25% (equilibrating the column), with a single analysis cycle of 15 min, meeting the requirements for batch detection.

[0051] Mass spectrometry system: ESI negative ion mode was used (salicylic acid contains a carboxyl group, and negative ion mode provides a stronger response), ion source temperature 320℃, spray voltage 3800V, sheath gas pressure 45psi, and auxiliary gas pressure 10psi. Mass spectrometry parameters were optimized by direct injection of standard solutions: the salicylic acid precursor ion m / z 137.0 ([MH]-) with a collision energy of 22eV showed the strongest response, and was used as the quantitative ion pair; the internal standard p-hydroxybenzoic acid precursor ion m / z 137.0 ([MH]-) with a collision energy of 20eV and a daughter ion m / z 93.0 was used as the internal standard quantitative ion pair. MRM mode was used for monitoring with a residence time of 100ms to ensure accurate signal acquisition.

[0052] 5. Methodological Validation and Results:

[0053] Linear relationship: A linear regression analysis was performed with the concentration of salicylic acid standard (x) on the x-axis and the peak area ratio of salicylic acid to internal standard (y) on the y-axis. The regression equation was y = 0.892x + 0.003 (R²). 2 =0.9992), and showed good linearity in the range of 0.001 to 1.0 ng / mL, meeting the requirements for low concentration detection;

[0054] Limit of detection and limit of quantitation: The limit of detection (LOD) calculated with a signal-to-noise ratio (S / N) of 3 is 0.0008 ng / mL, and the limit of quantitation (LOQ) calculated with an S / N of 10 is 0.002 ng / mL, which is 80 times more sensitive than the traditional method;

[0055] Precision: Quality control samples at three concentrations (LOQ, 0.1 ng / mL, and 1.0 ng / mL) were tested 6 times daily for 3 consecutive days. The intra-day RSDs were 3.2%, 2.8%, and 2.1%, respectively, and the inter-day RSDs were 4.5%, 3.9%, and 3.0%, respectively. All were ≤5%, and the precision met the requirements.

[0056] Recovery and matrix effect: Low, medium and high concentrations of salicylic acid standards were added to blank plasma, and the spiked recovery rate was calculated. The results were 92.5% to 96.8%, with RSD ≤ 3.5%. The matrix effect factor was 0.95 to 1.03, indicating that the matrix interference was minimal.

[0057] This embodiment achieves highly sensitive and accurate detection of salicylic acid in plasma by optimizing sample pretreatment, chromatographic and mass spectrometry parameters, verifying the feasibility and superiority of the method of the present invention, which can be directly extended to the detection of other drug metabolites.

[0058] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A highly sensitive liquid chromatography method for the detection of drug metabolites, characterized in that: It includes three steps: sample pretreatment, chromatographic separation, and mass spectrometry detection, as detailed below: (1) Sample pretreatment: Take 50–200 μL of biological sample, selected from one or more of plasma, urine, and tissue homogenate, transfer it to a centrifuge tube, add a protein precipitant mixture of acetonitrile and methanol at a volume ratio of 1:3 to 1:5 (7:3 volume ratio of acetonitrile to methanol); place the centrifuge tube on a vortex mixer and vortex at 3000–4000 r / min for 1–2 min, then place it in a high-speed refrigerated centrifuge and centrifuge at 4℃ and 12000 r / min for 5–8 min. After centrifugation, collect the supernatant and dilute it with 0.1–0.2 times the volume of ultrapure water. The diluted sample should be tested within 24 h under refrigeration at 4℃. (2) Chromatographic separation: A core-shell C18 column with dimensions of 100 mm × 2.1 mm and a particle size of 1.7 μm was used. The column temperature was controlled at 30–35 °C with a temperature fluctuation of ≤ ±0.5 °C. The mobile phase consisted of ultrapure water containing 0.05–0.1% formic acid as phase A and acetonitrile containing 0.05–0.1% formic acid as phase B. A gradient elution program was used: 0–2 min, phase B ratio 20–30%; 2–8 min, phase B ratio 30–80%; 8–10 min, phase B ratio 80%; 10–10.1 min, phase B ratio decreasing from 80% to 20%. 10.1–15 min, B phase ratio 20%; flow rate set at 0.25–0.3 mL / min, injection volume 5–10 μL; rinse the injection needle 3 times with sample solution before injection to avoid cross-contamination; (3) Mass spectrometry detection: A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) system was used with an electrospray ionization (ESI) source. Positive or negative ion mode was selected based on the polarity of the drug metabolites, with positive ion mode preferred for amine metabolites and negative ion mode preferred for carboxylic acid metabolites. Multiple reaction monitoring (MRM) was employed for specific detection. The optimized ion source temperature was 300–350 °C, the spray voltage was 3500–4000 V, the sheath gas pressure was 40–50 psi, and the auxiliary gas pressure was 8–12 psi. The collision energy was adjusted according to the structural characteristics of the drug metabolites: 15–25 eV for carboxylic acid metabolites, 20–30 eV for amine metabolites, and 18–28 eV for alcohol metabolites, to obtain the maximum fragment ion response signal.

2. The high-sensitivity liquid chromatography detection method for drug metabolites according to claim 1, characterized in that: In step 1, the volume ratio of protein precipitant to biological sample is 1:

4.

3. The high-sensitivity liquid chromatography detection method for drug metabolites according to claim 1, characterized in that: In step 2, the chromatographic column was a Waters XBridge BEH C18 core-shell column, the column temperature was 32℃, phase A was ultrapure water containing 0.08% formic acid, phase B was acetonitrile containing 0.08% formic acid, the flow rate was 0.28 mL / min, the injection volume was 8 μL, and the gradient elution program was as follows: 0–2 min, phase B ratio 25%; 2–8 min, phase B ratio linearly increased from 25% to 75%; 8–10 min, phase B ratio 75%; 10–10.1 min, phase B ratio decreased from 75% to 25%; 10.1–15 min, phase B ratio 25%.

4. The high-sensitivity liquid chromatography detection method for drug metabolites according to claim 1, characterized in that: The drug metabolite is a small molecule metabolite with a molecular weight of 100-500 Da, selected from one or more of alcohol, carboxylic acid, amine, and ester metabolites.

5. The highly sensitive liquid chromatography detection method for drug metabolites according to claim 1, characterized in that: In step 3, the dwell time of the multiple reaction monitoring mode is 80–120 ms.

6. The high-sensitivity liquid chromatography detection method for drug metabolites according to claim 1, characterized in that: During the biological sample pretreatment process, the vortex oscillation time was 1.5 min, the high-speed refrigerated centrifugation time was 6 min, and the dilution volume ratio of the supernatant to ultrapure water was 1:0.17.

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