Sulfamethoxazole and trimethoprim combined trace detection method and application of sulfamethoxazole and trimethoprim combined trace detection method in drug monitoring after treatment of hematopoietic stem cell transplantation patient

Abstract

The invention belongs to the field of analysis and detection, and particularly relates to a sulfamethoxazole and trimethoprim combined trace detection method and application to drug monitoring after hematopoietic stem cell transplantation patient treatment. The detection method comprises the following steps: (1) pre-treating a sample: treating the sample by adopting a releasing agent containing an internal standard substance which comprises an isotope labeled compound; (2) carrying out qualitative and quantitative analysis by adopting liquid chromatography-tandem mass spectrometry, wherein a mobile phase A is an aqueous solution of formic acid; b is methanol. According to the invention, a micro method for detecting the blood concentration of sulfamethoxazole / trimethoprim in a hematopoietic stem cell transplanted child patient by a liquid chromatography-tandem mass spectrometry technology is established; combined determination of sulfamethoxazole and trimethoprim is achieved through a mass spectrum electrospray positive ion multi-reaction monitoring mode, selectivity, residue, accuracy, precision, stability and matrix effect are comprehensively evaluated, and the method is verified completely, stable and reliable.

Description

Technical Field

[0001] The invention belongs to the field of analysis and detection, and specifically relates to a method for detecting a trace amount of sulfamethoxazole combined with trimethoprim and its application in drug monitoring after treatment of hematopoietic stem cell transplantation patients. Background Art

[0002] The immune system of children after hematopoietic stem cell transplantation (HSCT) is suppressed for a period of time, especially during the immune reconstitution period after transplantation, which makes them extremely susceptible to various pathogens, including Pneumocystis jirovecii, which leads to Pneumocystis jirovecii pneumonia (PCP). PCP is a serious lung infection with a high mortality rate for immunocompromised patients. The 2016 ECIL guidelines point out that early intravenous administration of antimicrobial drugs is very important in patients with PCP in hematological malignancies, with high-dose trimethoprim-sulfamethoxazole being the first choice. According to the 2017 guidelines for the prevention of Pneumocystis jirovecii pneumonia in children with solid tumors, pediatric patients receiving allogeneic hematopoietic stem cell transplantation and leukemia treatment need to prevent PCP. Preventing PCP can not only reduce the risk of death in children, but also reduce the high medical costs incurred by treating PCP.

[0003] Trimethoprim-sulfamethoxazole (TMP-SMZ) is a fixed-dose combination of sulfamethoxazole (SMZ) and trimethoprim (TMP). The synergistic mechanism of TMP-SMZ is to use DHPS and DHFR as two important targets to block folic acid and protein synthesis, thereby playing a bactericidal role. The combined use of sulfamethoxazole and trimethoprim expands the antibacterial spectrum, including a wide range of antibacterial activity against Gram-positive and Gram-negative bacteria, anaerobic bacteria, parasites, etc. Clinically, trimethoprim-sulfamethoxazole can be used to treat infections caused by a variety of pathogens, such as urinary tract infections, prostatitis, acute exacerbation of chronic bronchitis, skin and soft tissue infections caused by methicillin-resistant Staphylococcus aureus, etc.

[0004] Although trimethoprim-sulfamethoxazole is widely used in clinical practice, its blood concentration varies greatly between individuals. Long-term exposure to high blood concentrations may lead to toxic reactions, and too low blood concentrations may lead to prevention failure or even induce drug resistance. Therefore, it is particularly important to conduct therapeutic drug monitoring (TDM) for hematopoietic stem cell transplantation patients using trimethoprim-sulfamethoxazole. Children are very different from adult patients due to their unique physiological and pathological characteristics.

[0005] Sulfamethoxazole is well absorbed after oral administration, and about 90% of the administered dose can be absorbed, but the absorption rate is slow. After absorption, the drug is widely distributed in body tissues and various extracellular fluids, including pleural fluid, peritoneal fluid, synovial fluid, aqueous humor, saliva, sweat, urine, bile, etc. Sulfamethoxazole can enter the cerebrospinal fluid through the blood-cerebrospinal fluid barrier, and can also enter breast milk and pass through the placental barrier. Sulfamethoxazole is acetylated in the liver, with an acetylation rate of about 20% to 40% in the blood, and is bound to glucuronic acid. It is mainly excreted through glomerular filtration, and some free drugs can be reabsorbed through the renal tubules. Within 24 hours after administration, 20% to 40% of the administered dose is excreted in urine in its original form. The excretion of sulfamethoxazole is related to the urine pH value, and the excretion amount increases in alkaline urine. The protein binding rate of sulfamethoxazole is about 60% to 70%, and the protein binding rate may be reduced in patients with severe renal impairment. The elimination half-life of sulfamethoxazole is 6 to 12 hours in patients with normal renal function, and can be extended to 20 to 50 hours in patients with impaired renal function.

[0006] After oral administration, trimethoprim is completely absorbed, and about 90% of the administered dose can be absorbed. After absorption, the drug is widely distributed in tissues and body fluids, and the concentrations in the kidneys, liver, spleen, lungs, muscles, bronchial secretions, saliva, vaginal secretions, prostate tissues and prostatic fluids all exceed the blood concentration. Trimethoprim can cross the blood-cerebrospinal fluid barrier. When there is no inflammation in the meninges, the cerebrospinal fluid drug concentration is 30% to 50% of the blood concentration, and can reach 50% to 100% when it is inflammatory. TMP can also cross the blood-placental barrier, and the drug concentration in the fetal circulation is close to the maternal blood concentration. The protein binding rate of trimethoprim is 30% to 46%. The elimination half-life of trimethoprim is about 8 to 10 hours, and the half-life can be extended to 20 to 50 hours in anuric patients. Trimethoprim is mainly excreted in the urine through glomerular filtration and tubular secretion. About 50% to 60% of the dose can be excreted within 24 hours, of which 80% to 90% is excreted in the original form of the drug, and the rest is excreted in the form of metabolites. The average urine drug concentration is 90 to 100 mg / L, and the peak concentration in urine is about 200 mg / L. The excretion of trimethoprim in urine increases in acidic urine, and decreases in alkaline urine.

[0007] It can be seen that the blood concentration of sulfamethoxazole and trimethoprim in vivo is affected by multiple factors such as oral bioavailability, liver and kidney function, and protein binding rate. Children are in the growth and development period, and their liver and kidney function also show dynamic changes. Graft-versus-host disease is a common complication of children with hematopoietic stem cell transplantation. Gastrointestinal rejection often causes vomiting, diarrhea, gastrointestinal inflammation, etc., which seriously affects the bioavailability of the drug. The rejection of the liver and kidneys successively affects the metabolism and excretion of the drug. At the same time, a large part of children are in infancy during the transplantation period, and it is not easy to cooperate with the medication. Many factors lead to prominent individual differences in the use of sulfamethoxazole in children, and concentration determination is very necessary. In addition, due to the small weight of children, blood collection is very difficult. Therefore, it is urgent to develop a micro-determination method suitable for children.

[0008] TDM is an important means to ensure the efficacy and safety of drugs. In clinical practice, immunological methods are often used for TDM because of their simple operation and rapid detection. However, for small molecule drugs such as trimethoprim (TMP) and sulfamethoxazole (SMZ), it is difficult to prepare high-affinity antibodies due to their poor immunogenicity, which limits the application of immunological methods in the detection of TMP and SMZ. Therefore, there are currently few immunological methods and kits for these two drugs, which makes other detection technologies become mainstream. For example, liquid chromatography (HPLC) is another commonly used method for detecting TMP and SMZ. Since the levels of these two drugs at therapeutic concentrations are high, usually at the level of tens or even hundreds of micrograms per milliliter, the HPLC method can meet the detection requirements in terms of sensitivity. This method usually uses a UV detector, but because the absorption of ultraviolet light is not specific, it is easily interfered by other substances in the sample, which may bring certain risks in clinical practice and affect the accuracy of the results. Summary of the invention

[0009] In view of the problems existing in the prior art, the present invention provides a method for detecting a trace amount of sulfamethoxazole combined with trimethoprim and its application in drug monitoring after treatment of hematopoietic stem cell transplantation patients. The present invention establishes a method for detecting the blood concentration of sulfamethoxazole / trimethoprim in children with hematopoietic stem cell transplantation by liquid chromatography-tandem mass spectrometry; the combined determination of sulfamethoxazole and trimethoprim is achieved by mass spectrometry electrospray positive ion multiple reaction monitoring mode, and a comprehensive evaluation of selectivity, residue, accuracy, precision, stability and matrix effect is performed, and the method is fully verified, stable and reliable.

[0010] The technical solution of this application is as follows:

[0011] The first object of the present invention is to protect a method for detecting a trace amount of sulfamethoxazole combined with trimethoprim, comprising the following steps:

[0012] (1) Sample pretreatment: using a release agent containing an internal standard, which includes an isotope-labeled compound;

[0013] (2) Liquid chromatography-tandem mass spectrometry was used for qualitative and quantitative analysis: mobile phase A was an aqueous solution of formic acid; mobile phase B was methanol.

[0014] According to a specific embodiment of the present application, the sample in step (1) includes a calibration sample and / or a quality control sample;

[0015] The sample pretreatment process includes: adding a release agent containing an internal standard to the standard curve sample and / or the quality control sample, vortexing, centrifuging, taking the supernatant, and diluting with methanol.

[0016] According to a specific embodiment of the present application, the internal standard in step (1) includes trimethoprim-D9 and sulfamethoxazole-D4.

[0017] According to the specific implementation of the present application, the specific conditions of step (2) include:

[0018] Chromatographic parameters: chromatographic column is ProChrom C18, column temperature is 45°C; sample temperature is 10°C; injection volume is 1 μL; flow rate is 0.3 mL / min; mobile phase A is an aqueous solution containing 0.1% formic acid; B is methanol.

[0019] According to the specific implementation of the present application, the specific conditions of step (2) include:

[0020] Mass spectrometry parameters: ionization mode adopted electrospray positive ion mode (ESI+), capillary voltage 3.0 kV; desolvation temperature 500°C; desolvation gas flow rate 800 L / h; cone backflush gas flow rate 150 L / h; ion source temperature 150°C.

[0021] According to the specific implementation of the present application, the optimal ion pair is selected: trimethoprim 291.1 / 230.0, sulfamethoxazole 254.1 / 155.9.

[0022] The second purpose of the present application is to protect the application of the above-mentioned sulfamethoxazole combined with trimethoprim trace detection method in drug monitoring after treatment of hematopoietic stem cell transplantation patients.

[0023] The third purpose of the present application is to protect a drug monitoring method for patients after hematopoietic stem cell transplantation treatment, characterized in that the content of sulfamethoxazole and trimethoprim in the blood is detected by using the method described in any one of claims 1 to 7.

[0024] The fourth purpose of the present application is to protect a drug monitoring method for children after hematopoietic stem cell transplantation treatment, using the method described in any one of claims 1 to 6 to detect the content of sulfamethoxazole and trimethoprim in the blood.

[0025] According to a specific embodiment of the present application, the amount of blood used is greater than or equal to 50 μL. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the mass spectrum of trimethoprim;

[0027] Figure 2 is the mass spectrum of sulfamethoxazole;

[0028] Figure 3 Mass spectra of trimethoprim daughter ions and collision energy optimization; from bottom to top, the cone voltages are 5, 10, 15, 20, 25, 30, 35, and 40 eV, respectively;

[0029] Figure 4 The mass spectrum of the sulfamethoxazole daughter ion and collision energy optimization; from bottom to top, the cone voltages are 5, 10, 15, 20, 25, 30, 35, and 40 eV respectively;

[0030] Figure 5 Optimize the chromatogram for trimethoprim product ion response;

[0031] Figure 6 Optimize the chromatogram for sulfamethoxazole product ion response;

[0032] Figure 7 The standard curve of sulfamethoxazole obtained in Example 4;

[0033] Figure 8 This is the trimethoprim standard curve obtained in Example 4.

[0034] The beneficial effects of this application are:

[0035] (1) The present invention conducts qualitative analysis of standard substances through characteristic spectrum and mass spectrum, and uses isotope internal standard to correct the error in the operation process, thereby improving the accuracy and reliability of the analysis; by optimizing the sample pretreatment method and chromatographic mass spectrometry conditions, the simultaneous determination of sulfamethoxazole and trimethoprim is achieved, which improves the detection efficiency and can monitor the residues of the two drugs at the same time. Trimethoprim and sulfamethoxazole of appropriate concentration are prepared for mixed work, methanol and acetonitrile are used as strong elution solvents, and water with a certain amount of formic acid and ammonium acetate added is used as weak elution solvent, and the starting gradient of the strong elution solvent is continuously optimized, so that trimethoprim and sulfamethoxazole avoid chromatographic problems such as front edge, tailing, and low signal-to-noise ratio, avoid dead time and impurity interference when washing the chromatographic column, and peak at the appropriate time, and finally the optimized mobile phase is A: 0.1% formic acid / water, B: methanol.

[0036] (2) The present invention performs quantitative evaluation on the matrix effect, fully eliminates the interference of the matrix effect, selects the internal standard method for quantification, and thus improves the accuracy of the quantitative analysis; sets a quantitative linear range suitable for clinical use, which provides a standardized operating procedure for clinical application and ensures the consistency and comparability of the results; optimizes the dilution multiple suitable for clinical samples, which helps to improve the flexibility and adaptability of sample processing, especially when processing samples with large concentration differences, it can ensure the accuracy of the test results.

[0037] (3) The present invention uses a small amount of plasma sample (50 μL) to measure the concentration of sulfamethoxazole and trimethoprim. This method reduces the demand for patient blood. Especially for pediatric patients, this minimally invasive sampling technology has significant clinical advantages. DETAILED DESCRIPTION

[0038] Material

[0039] Trimethoprim (catalog number 100031, batch number 202207, purity 99.8%) and sulfamethoxazole (catalog number 100025, batch number 201906, purity 99.8%) were purchased from China Food and Drug Inspection Institute; trimethoprim-D9 (batch number MD-7586D9, batch number G0094317, purity 99.7%, isotopic abundance 98%) was purchased from Manhage (Shanghai) Biotechnology Co., Ltd.; sulfamethoxazole-D4 (catalog number CMS-Y91002312, batch number APS466, purity 99%, isotopic abundance 98%) was purchased from Shanghai Pufen Biotechnology Co., Ltd.

[0040] Chromatographic grade methanol and acetonitrile were purchased from Merck (Germany), and ultrapure water was prepared using a Cascada I ultrapure water system (Avidity, USA).

[0041] Example 1

[0042] Optimization of mass spectrometry conditions

[0043] 1. Screening of parent ions

[0044] The single standard working solution of trimethoprim (100 ng / mL) was used as the sample, and the mass spectrometer MS Scan function was used in the ESI+ ionization mode to scan in the range of 50-350 m / z. The scanning time was 0.3 s and the cone voltage was 20 V. The results are shown in Figure 2. Figure 1 As shown, the mass-to-charge ratio of 291.0 is the parent ion of trimethoprim [M+H]+, which is consistent with the theoretical parent ion of trimethoprim [M+H]+ 291.1. No other ionized forms such as [M+Na]+ or [M+K]+ were found. Therefore, 291.1 was selected as the parent ion of trimethoprim.

[0045] The single standard working solution of sulfamethoxazole (100 ng / mL) was used as the sample, and the mass spectrometer MS Scan function was used in the ESI+ ionization mode to scan in the range of 50-350 m / z. The scanning time was 0.3 s and the cone voltage was 20 V. The results are shown in Figure 2. Figure 2 As shown, the mass-to-charge ratio of 254.0 is the parent ion of sulfamethoxazole [M+H]+, which is consistent with the theoretical parent ion of trimethoprim [M+H]+ 254.1. No other ionized forms such as [M+Na]+ or [M+K]+ were found. Therefore, 254.1 was selected as the parent ion of trimethoprim.

[0046] Select 291.1 as the parent ion of trimethoprim and 254.1 as the parent ion of sulfamethoxazole.

[0047] 2. Screening of product ions and optimization of collision energy

[0048] Using the mixed standard working solution (50ng / mL) as the sample, the mass spectrometer Daughters Scan function was used in the ESI+ ionization mode, with 291.1 and 254.1 as the parent ions, respectively, and scanning was performed in the range of 50-350m / z. The scanning time was 0.30s, the cone voltage was 20V, and the collision energy was gradually increased in the range of 5-40eV with a step size of 5eV to screen the daughter ions of trimethoprim and sulfamethoxazole and optimize their collision energy. The results are shown in Figure 3 , Figure 4 As shown, 275.0 (25), 261.0 (25), 230.0 (25), and 123.0 (30) are daughter ions available for detecting trimethoprim, and 155.9 (15), 108.0 (25), and 92.0 (25) are daughter ions available for detecting sulfamethoxazole. The numbers in brackets are the optimal collision energies.

[0049] Then, the mixed standard working solution (50 ng / mL) was used as the sample, and the above parent ion, daughter ion and collision energy conditions were used to form the MRM method for injection and detection. The results are shown in Figure 5 , Figure 6 As shown, when the injection concentration is the same, the ion channels described in Table 1 are selected for subsequent work by comprehensively considering the response value and the product ion size.

[0050] Table 1 Trimethoprim, sulfamethoxazole daughter ions and collision energies

[0051]

[0052] Note: The ions marked with * are quantification products.

[0053] Example 2

[0054] A method for detecting a trace amount of sulfamethoxazole combined with trimethoprim comprises the following steps:

[0055] (1) Prepare standard solution;

[0056] Dissolve trimethoprim and sulfamethoxazole in methanol and prepare 1 mg / mL and 10 mg / mL stock solutions, respectively. Then pipette 90 μL of trimethoprim stock solution, 900 μL of sulfamethoxazole stock solution and 10 μL of methanol into the same EP tube, mix evenly, and prepare a mixed working solution of trimethoprim and sulfamethoxazole to a concentration of 0.9 and 9 mg / mL, respectively. Mix the mixed working solution with 50% methanol aqueous solution in proportion and dilute it into a mixed working solution of each gradient concentration. Take 50 μL of the mixed working solution and mix it with 950 μL of plasma to complete the preparation of calibration products and quality control products for each concentration gradient. See Table 1 for details.

[0057] The isotope-labeled compounds trimethoprim-D9 and sulfamethoxazole-D4 were dissolved in methanol to prepare 1 mg / mL and 10 mg / mL stock solutions, respectively. Subsequently, 1.5 μL of trimethoprim-D9 and sulfamethoxazole-D4 were taken in a 15 mL centrifuge tube, and 10 mL of acetonitrile was added to complete the preparation of the sample release agent containing the internal standard, wherein the concentrations of trimethoprim-D9 and sulfamethoxazole-D4 were 0.15 μg / L and 1.5 μg / mL, respectively.

[0058] Table 1: Preparation and concentration of standard and quality control samples

[0059]

[0060] (2) Sample pretreatment;

[0061] Accurately pipette 50μL of sample (or standard curve sample, quality control sample, patient plasma) into an EP tube and dilute with 450μL ultrapure water. Take 50μL of diluted sample, add 100μL of sample release agent containing internal standard, vortex for 5 seconds, let stand for 5 minutes, and centrifuge at 12000g centrifugal force and 10℃ for 10 minutes. Accurately pipette 1μL of supernatant, dilute with 999μL 50% methanol, and then test.

[0062] (3) Qualitative and quantitative analysis using liquid chromatography-tandem mass spectrometry

[0063] Chromatographic parameters: chromatographic column is ProChrom C18 (2.1mm×100mm, 3μm), column temperature is 45℃; sample temperature is 10℃; injection volume is 1μL; flow rate is 0.3mL / min; mobile phase A is an aqueous solution containing 0.1% formic acid; B is methanol. Mass spectrometry parameters: ionization mode adopts electrospray positive ion mode (ESI+), capillary voltage is 3.0kV; desolvation temperature is 500℃; desolvation gas flow rate is 800L / h; cone backflush gas flow rate is 150L / h; ion source temperature is 150℃.

[0064] Table 2: Gradient elution conditions

[0065] time Mobile phase A ratio Mobile phase B ratio Starting 70％ 30％ 2min 0％ 100％ 2.4min 0％ 100％ 2.5min 70％ 30％ 4.5min 70％ 30％

[0066] Component ion pair parameters:

[0067] Table 3: Multiple reaction monitoring parameters

[0068]

[0069] Note: The ions marked with * are quantitative product ions.

[0070] Example 3

[0071] Sensitivity

[0072] After treatment with blank solvent methanol, blank plasma, and plasma with sulfamethoxazole / trimethoprim and internal standard working solution, the assay was performed under the conditions of Example 2. The concentration levels were all LLOQ (sulfamethoxazole 3 μg / mL, trimethoprim 0.3 μg / mL); the sensitivity results are shown in Table 2, with an accuracy of 85-115% and RSD within 15%, from which it can be seen that the sensitivity meets the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0073] Table 2 Sensitivity investigation and analysis

[0074]

[0075] Example 4

[0076] Standard curve

[0077] A series of standard working solutions were prepared, with the concentration of sulfamethoxazole / trimethoprim in plasma as the horizontal coordinate X, and the peak area ratio of sulfamethoxazole / trimethoprim to internal standard in the chromatogram as the vertical coordinate Y. The weighted least squares method was used for regression calculation (weight W=1 / CC), and the equation of the sulfamethoxazole standard curve was obtained as follows: y=0.015651*x+0.00457479, R2=0.999746; the equation of the trimethoprim standard curve was obtained as follows: y=0.927434*x+0.0106652, R2=0.999700. This indicates that the plasma concentration of sulfamethoxazole / trimethoprim has a good linear relationship in the range of 3-300μg / mL / 0.3-30μg / mL. The results are shown in Figure 7 , Figure 8 .

[0078] Example 5

[0079] Accuracy and Precision

[0080] Quality control samples with low (L), medium (M), and high (H) concentrations (7.5, 120, and 225 μg / mL, 0.75, 12, and 22.5 μg / mL, respectively) were prepared, with 7 samples of each concentration. The samples were measured on the same day under specific analytical conditions to investigate the accuracy and intra-day precision. Three consecutive batches were measured by the same method to investigate the inter-day precision. The results are as follows. The accuracy of the quality control of the three concentrations of sulfamethoxazole / trimethoprim L, M, and H was between 85% and 115%, and the intra-day precision and inter-day precision were controlled within 3%. The results are shown in Table 3, from which it can be seen that the accuracy and precision meet the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0081] Table 3 Accuracy and precision analysis

[0082]

[0083]

[0084]

[0085] Example 6 Stability

[0086] 6.1 Freeze-thaw stability

[0087] Two concentrations of sulfamethoxazole / trimethoprim reference plasma, low (L) and high (H), were prepared, three portions for each concentration, placed at -80°C for 24 hours and then thawed naturally at room temperature. After complete thawing, they were refrozen under the same conditions for 24 hours. After three freeze-thaw cycles, the samples were processed and measured. The concentration results of the samples after freeze-thaw are shown in Table 4.

[0088] Table 4 Freeze-thaw stability test results

[0089]

[0090] The drug content in plasma did not change much after three freeze-thaw cycles at -80°C, indicating that sulfamethoxazole / trimethoprim had good freeze-thaw stability in plasma samples.

[0091] 6.2 Stability at room temperature

[0092] Quality control samples of sulfamethoxazole (7 μg / mL, 225 μg / mL) / trimethoprim (0.7 μg / mL, 22.5 μg / mL) at two concentrations (L) and H) were prepared, three times each, and placed at room temperature for 4 hours. After being treated according to the conditions of Example 2 (2), the samples were measured under the analytical conditions of Example 2 (3). The results of the sample concentration measurement after being placed at room temperature are as follows (see Table 5).

[0093] After being placed at room temperature for 4 hours, the drug content in plasma does not change much. After collection, clinical samples can be placed at room temperature and measured within 4 hours.

[0094] Table 5 Stability test results at room temperature

[0095]

[0096] 6.3 Stability of samples after treatment

[0097] Quality control samples of sulfamethoxazole (7 μg / mL, 225 μg / mL) / trimethoprim (0.7 μg / mL, 22.5 μg / mL) at low (L) and high (H) concentrations were prepared, treated according to the conditions of Example 2 (2), and placed in an automatic sampler (4° C.) for 19 hours.

[0098] The determination was carried out under the analytical conditions of Example 2, and the results of the sample concentration determination were as follows (see Table 6). After the treated sample was placed in the automatic sampler for 19 hours, the drug content in the sample did not change much. After the clinical sample was processed, it was placed in the automatic sampler and the determination was completed within 19 hours.

[0099] Table 6 Stability test results of treated samples

[0100]

[0101] Clinical sample measurement of implementation effect

[0102] 7.1 Standard curve

[0103] Prepare the standard curve sample and quality control sample according to the conditions of Example 2(1).

[0104] 7.2 Sample processing and determination

[0105] Take 50 μL of the standard curve sample, quality control sample, and clinical patient plasma, treat them according to the conditions of Example 2(2), and measure them according to the analytical conditions of Example 2(3).

[0106] 7.3 Sample measurement results and clinical application

[0107] (1) Evidence-based data show that:

[0108] 1) For common pathogens: the effective peak concentration of sulfamethoxazole is 30-60 μg / mL, and the effective peak concentration of trimethoprim is 1.5-2.5 μg / mL;

[0109] 2) For Pneumocystis jiroveci: the effective peak concentration of sulfamethoxazole is 100-200 μg / mL, and the effective peak concentration of trimethoprim is 5-10 μg / mL;

[0110] 3) For Nocardia: The effective peak concentration of sulfamethoxazole is 100-150 μg / mL.

[0111] 4) When the sulfamethoxazole concentration is >200 μg / mL, adverse reactions should be closely monitored and intervention should be made as appropriate.

[0112] (2) Measurement results and clinical application:

[0113] Patient 1: male, 63 years old, had a fever for 5 days after more than 1 year of kidney transplantation. After admission, the patient completed relevant tests and auxiliary examinations. Chest CT showed lung infection. Bronchoscopy + alveolar lavage was performed on December 4, 2024. The NGS results indicated Pneumocystis jiroveci infection. The dosage of immunosuppressive drugs was adjusted, and symptomatic supportive treatment such as caspofungin and hormones were given. Oxygen support treatment, the patient later developed hemoptysis, heparin was discontinued, the patient was advised to control the total intake to 2000ml, furosemide was given, and the changes in urine volume were closely monitored. The patient's urine volume decreased, 2024-12- 13 was given femoral vein catheterization + CRRT, during which the blood oxygen saturation was maintained at 83-92 mmHg. The patient complained of shortness of breath and difficulty maintaining high-flow oxygen inhalation, so he was intubated and continued to be given oral intubation and ventilator-assisted breathing. ECG monitoring, blood pressure, and blood oxygen saturation monitoring were given. Imipenem and cilastatin sodium were used for antibacterial treatment, resuscitating sulfamethoxazole and caspofungin were used for antifungal treatment, expectorant, intermittent nicardipine pump for antihypertensive treatment, diuresis, enoxaparin for anticoagulation, acid suppression and gastric protection, enteral and parenteral nutrition support, blood transfusion, bronchoscopy + alveolar lavage, etc. The patient's condition improved after treatment. , the oral endotracheal tube was removed on 2024-12-26. After extubation, the patient's oxygen saturation decreased. The parameters of high-flow humidified oxygen therapy were adjusted, but the patient's oxygenation could not be maintained. On 2024-12-29, the endotracheal tube was intubated again and the patient was connected to a ventilator for assisted breathing. On 2024-12-30, a minimally invasive bedside tracheotomy under the guidance of a fiberoptic bronchoscope was performed and the patient was connected to a ventilator for assisted breathing. The patient is now in a continuous offline state, and the oxygenation index has improved compared with before. Diagnosis: 1. Severe pneumonia Pneumocystis jiroveci infection 2. Acute respiratory failure 3. Arrhythmia, atrial fibrillation, ventricular premature beats 4. Hypertension 5. Diabetes 6. Chronic bronchitis 7. emphysema 8. bilateral pleural effusion 9. post-renal transplantation 10. severe anemia 11. hypoproteinemia 12. nutritional risk. The peak concentration of sulfamethoxazole was 63.648 μg / mL, and the peak concentration of trimethoprim was 3.377 μg / mL. For Pneumocystis jirovecii, the effective peak concentration of sulfamethoxazole should be 100-200 μg / mL, and the effective peak concentration of trimethoprim should be 5-10 μg / mL. The patient's current concentration is low. Considering the normal renal function, it is recommended to increase the dosage from 2 tablets tid to 3 tablets tid.

[0114] Patient 2: Male, 40 years old, was admitted to the hospital for fever for 1 day after more than 3 months of kidney transplantation. The patient's general condition is good, diet and appetite are good, no nausea, vomiting, no acid reflux, heartburn, chest tightness, chest pain and other discomfort symptoms, and sleep is good at night. Physical examination: The breath sounds of both lungs are clear, and no dry or wet rales are heard. There is no bulge or depression in the precordial area, no enlargement of the heart border, regular rhythm, and no pathological murmurs are heard in the auscultation area of ​​each valve. The size of the transplanted kidney area is normal, the texture is good, and the blood flow murmur is clear. Laboratory test results: Procalcitonin test (2025-01-1308:57) Procalcitonin: 0.092ng / mL↑(0--0.05); Blood cell analysis (five-category classification) (2025-01-1309:32) Neutrophil percentage: 0.902↑(0.40--0.75), Lymphocyte percentage: 0.055↓(0.2--0.5), Eosinophil percentage: 0.000↓(0.004--0.080), Lymphocyte count: 0.31×10^9 / L↓(1.1--3.2), Eosinophil count: 0.00×10^9 / L↓(0.02--0.52), hemoglobin: 100.0g / L↓(130--175), hematocrit: 0.323↓(0.4--0.5), mean corpuscular volume: 72.40fL↓(82--100), mean hemoglobin content: 22.40pg↓(27--34), mean hemoglobin concentration: 310g / L↓(316--354), red blood cell width-CV value: 19.00%↑(11.5--14.5); glucose determination (enzymatic method)^electrolytes^renal function^liver function (2025-01-1312:21) total bile Bilirubin: 2.30umol / L↓(5.0--24.0), indirect bilirubin: 0.60umol / L↓(2.8--23.8), total protein: 53.50g / L↓(65--85), albumin (bromocresol green method): 28.10g / L↓(40--55), albumin-to-globulin ratio: 1.11RATIO↓(1.2--2.4), cholinesterase: 4812.0U / L↓(5320--12920), superoxide dismutase: 105.00U / mL↓(129--216), urea: 21.90mmol / L↑(2.8 6--8.20), creatinine (enzymatic method): 250.00umol / L↑(59--104), uric acid: 189.0umol / L↓(202.3--416.5), cystatin C: 2.69mg / L↑(0.57--1.30), β2 microglobulin: 4.11mg / L↑(1.0--3.0), retinol binding protein: 72.80mg / L↑(25.0--70.0), glucose: 10.32mmol / L↑(3.89--6.11), complement C1q: 15.00mg / dL↓(15.7--23.7); Blood cell analysis (five categories) (2025-01-15 09:34) Neutrophil percentage: 0.907↑(0.40--0.75), lymphocyte percentage: 0.059↓(0.2--0.5), eosinophil percentage: 0.000↓(0.004--0.080), lymphocyte count: 0.37×10^9 / L↓(1.1--3.2), eosinophil count: 0.00×10^9 / L↓(0.02--0.52), hemoglobin: 98.0g / L↓(130--175), hematocrit: 0.333↓(0.4--0.5), mean corpuscular volume: 75.70fL↓(82--100), mean hemoglobin content: 22.30pg↓(27--34), mean hemoglobin concentration: 294g / L↓(316--354), red blood cell width-CV value: 19.60%↑(11.5--14.5); the peak concentration of sulfamethoxazole was 79.079μg / mL, and the peak concentration of trimethoprim was 3.420μg / mL. The patient is currently infected with Pneumocystis jirovecii. For Pneumocystis jirovecii, the effective peak concentration of sulfamethoxazole should be 100-200 μg / mL, and the effective peak concentration of trimethoprim should be 5-10 μg / mL. The patient's current concentration is low, so it is recommended to increase the dosage from 3 tablets tid to 4 tablets tid.

Claims

1. A method for detecting trace amounts of sulfamethoxazole combined with trimethoprim, characterized in that: The following steps are involved: (1) Sample pretreatment: using a release agent containing an internal standard, which includes an isotope-labeled compound; (2) Liquid chromatography-tandem mass spectrometry was used for qualitative and quantitative analysis: mobile phase A was an aqueous solution of formic acid; mobile phase B was methanol.

2. The method for detecting trace amounts of sulfamethoxazole combined with trimethoprim according to claim 1, characterized in that: The samples in step (1) include standard samples and / or quality control samples; The sample pretreatment process includes: adding a release agent containing an internal standard to the standard curve sample and / or the quality control sample, vortexing, centrifuging, taking the supernatant, and diluting with methanol.

3. The method for detecting trace amounts of sulfamethoxazole combined with trimethoprim according to claim 1 or 2, characterized in that: The internal standard substances in step (1) include trimethoprim-D9 and sulfamethoxazole-D4.

4. The method for detecting trace amounts of sulfamethoxazole combined with trimethoprim according to any one of claims 1 to 3, characterized in that: The specific conditions of step (2) include: Chromatographic parameters: chromatographic column is ProChrom C18, column temperature is 45°C; sample temperature is 10°C; injection volume is 1 μL; flow rate is 0.3 mL / min; mobile phase A is an aqueous solution containing 0.1% formic acid; B is methanol.

5. The method for detecting trace amounts of sulfamethoxazole combined with trimethoprim according to any one of claims 1 to 3, characterized in that: The specific conditions of step (2) include: Mass spectrometry parameters: ionization mode adopted electrospray positive ion mode (ESI+), capillary voltage 3.0 kV; desolvation temperature 500°C; desolvation gas flow rate 800 L / h; cone backflush gas flow rate 150 L / h; ion source temperature 150°C.

6. The method for detecting trace amounts of sulfamethoxazole combined with trimethoprim according to claim 5, characterized in that: The best ion pairs were selected: trimethoprim 291.1 / 230.0, sulfamethoxazole 254.1 / 155.

9.

7. Use of the sulfamethoxazole combined with trimethoprim trace detection method according to any one of claims 1 to 6 in drug monitoring after treatment of hematopoietic stem cell transplantation patients.

8. A method for drug monitoring after treatment of hematopoietic stem cell transplantation patients, characterized in that: The method according to any one of claims 1 to 7 is used to detect the content of sulfamethoxazole and trimethoprim in blood.

9. A drug monitoring method for children with hematopoietic stem cell transplantation after treatment, characterized in that: The method according to any one of claims 1 to 6 is used to detect the content of sulfamethoxazole and trimethoprim in blood.

10. The drug monitoring method according to claim 9, characterized in that: The blood volume is greater than or equal to 50 μL.

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