| High-dose methotrexate(MTX) combined with calcium folinate is widely used to treat malignant lymphoma, osteosarcoma, acute lymphoblastic leukemia and other malignant tumors. Methotrexate is of narrow therapeutic range, exhibits obvious individual difference and has certain cytotoxic effect to rapidly dividing normal cells, which leads to adverse reactions. Therapeutic drug monitoring of blood drug concentration could help achieving maximum efficacy and reduce the adverse reactions in the application of high-dose methotrexate. At present, fluorescence polarization immunoassay and high performance liquid chromatography are prevalently used in clinical practice. However, the former can't distinguish between parent drug and its metabolite, the price of kit in a complete set of equipments is high. Cross reaction may affect the accuracy of analysis results as well. The analysis time is long and sample preparation is complex by high performance liquid chromatography detection, which can't satisfy timeliness in clinical practice. In order to, the study aims to establish a rapid, simple and accurate method for therapeutic drug monitoring of methotrexate to guide clinical individualized medication, define the safety and therapeutic window, improve curative effect and reduce adverse reactions.For therapeutic drug monitoring, this research applied the UHPLC-MS/MS method for simultaneous quantification of methotrexate and its major metabolite 7-hydroxy methotrexate(7-OH-MTX) in human serum. Protein precipitation was used for sample pretreatment with D3-methotrexate set as the internal standard. Separation was carried out on an Agilent ZORBAX C18 column(2.1×100 mm, 3.5 ?m) using a gradient elution with methanol and 0.2% formic acid aqueous solution at a flow rate of 0.3 mL/min, and the column temperature was maintained at 35 °C. Detection was achieved on Agilent 6460 tandem mass spectrometer coupled with ESI in positive mode monitoring the following mass transitions: m/z 455.1?308.1 for MTX, 471.0?324.1 for 7-OH-MTX and 458.2?311.1 for D3-methotrexate. Methotrexate and 7-hydroxy methotrexate showed good linearity in concentrations ranging from 5 to 10000 ng/mL(r>0.99). The intra-day and inter-day precision(RSD/%) of methotrexate and 7-hydroxy-methotrexate were <15%. The extraction recovery of methotrexate and 7-hydroxy-methotrexate were >90%. The results indicated that the established method was of high specificity and sensitivity for simple pre-treatment and rapid determination. This method was applicable for therapeutic drug concentration monitoring to guide individualized medication.The additives in blood vessels could affect determination results of blood drug concentration and were one of the important factors. This study examined the interferences of four different additives in vacuum blood collection tube on analysis of methotrexate and 7-hydroxy methotrexate in human blood. The additives included coagulant, EDTA-K2, EDTA-K3 and lithium heparin in vacuum blood collection tubes. Samples prepared from lab blank plasma with addiction of neat standards of methotrexate and 7-hydroxy methotrexate were the control group, and those from coagulant, EDTA-K2, EDTA-K3 and lithium heparin vacuum blood collection tubes were test groups. The deviation of methotrexate analysis results were-8.52%~-11.05%,-1.78%~-5.71%,-2.73%~-13.20% and-2.92%~-5.74% among four vacuum blood collection tubes, respectively. The deviation of 7-hydroxy methotrexate analysis results were 0.34%~-3.29%,-38.17%~-55.46%,-67.09%~-77.29% and-7.20%~-17.00% among four vacuum blood collection tubes, respectively. The results showed that four additives in vacuum blood collection tube had less effects on determination results of methotrexate(<15%). But EDTA-K2 and EDTA-K3 in vacuum blood collection tube had greater effects on quantification results of 7-hydroxy methotrexate(>35%). In order to ensure accuracy of quantification results of clinical samples, we selected the coagulant vacuum blood tube for blood specimen collection.This study collected 44 h, 68 h and 92 h blood samples from patients with malignant lymphoma and 2 h, 4 h, 6 h, 12 h, 24 h, 30 h, 44 h, 54 h, 63 h, 72 h and 87 h blood samples from patients with osteosarcoma. The study used UHPLC-MS/MS method for therapeutic drug monitoring. At present, there were 11 patients with malignant lymphoma and 4 patients with osteosarcoma, there were 88 blood samples and 29 times for therapeutic drug monitoring. To analyze blood drug concentration of patients with lymphoma, methotrexate C44h>1 ?M was 31.82%, C68h>0.1 ?M was 38.09%. The discrepancy of methotrexate C44 h, C68 h and C92 h of different individual was 3.31 ?M, 0.63 ?M and 0.26 ?M, respectively.And the discrepancy of 7-hydroxy methotrexate of different individual C44 h, C68 h and C92 h was 11.29 ?M, 3.32 ?M and 1.62 ?M, respectively. The discrepancy of methotrexate C44 h and C68 h of the same individual was 0.02~2.76 ?M and 0.29~0.58 ?M. And the discrepancy of 7-hydroxy methotrexate C44 h and C68 h of the same individual was 0.04~8.56 ?M and 0.76~2.79 ?M. SPSS 18.0 statistical software was applied for bivariate correlation analysis through single sample Kolmogorov Smirnov test and independent samples t-test analysis. Regression equation was obtained as follows: MTX C68h=0.172×MTX C44h-0.018(r=0.938, P=0.000), MTX C92h=0.087×MTX C44h-0.030(r=0.904, P=0.035), MTX C92h=0.447×MTX C68h+0.003(r=0.961, P=0.009), 7-OH-MTX C68h=0.245×7-OH-MTX C44h+0.031(r=0.626, P=0.004), 7-OH-MTX C92h=0.290×7-OH-MTX C68h+0.056(r=0.959, P=0.041), 7-OH-MTX C44h=2.565×MTX C44h+1.976(r=0.764, P=0.000). The difference of MTX C44 h, 7-OH-MTX C44 h, MTX C68 h and 7-OH-MTX C68 h at between MTX?2.5 g/m2 and MTX >2.5 g/m2 group and between CF?30 mg and CF>30 mg group was not statistically significant(p>0.05). The difference of MTX C68 h and 7-OH-MTX C44 h at between MTX C44h?1 ?M and MTX C44h>1 ?M was statistically significant(p<0.05) and the difference of 7-OH-MTX C68 h at between MTX C44h?1 ?M and MTX C44h>1 ?M and between MTX C68h?0.1 ?M and MTX C68h>0.1 ?M was not statistically significant(P>0.05). The incidence rate of mucositis was 19.18%, the incidence rate of renal toxicity was 9.09%, the incidence rate of toxicity of blood system was 19.18% and the incidence rate of liver toxicity was 9.09% from patients with lymphoma. The pharmacokinetic parameters were analyzed to 2 patients with osteosarcoma, residence time of MTX was 7.76 and 6.67 h, elimination half-life of MTX was 0.114 and 0.109 h, clearance of MTX was 2.18 and 5.76 L/h/m2. Residence time of 7-OH-MTX was 21.81 and 18.59 h, elimination half-life of 7-OH-MTX was 15.44 and 10.03 h, vivo clearance of 7-OH-MTX was 20.22 and 16.90 L/h/m2. The incidence rate of mucositis was 25% and the incidence rate of hepatotoxicity was 50%. The results showed that methotrexate and 7-hydroxy methotrexate exhibited large individual differences, drug concentration at different blood collection points had certain correlation, treatment protocols had no statistical significance for blood drug concentration, when methotrexate was detected of C44h>1 ?M or C68h>0.1 ?M, rescued scheme should be adjusted to reduce adverse reactions, adverse reactions could be reduced through therapeutic drug monitoring.In summary, when patients with malignant lymphoma and osteosarcoma choosed high-dose methotrexate chemotherapy regimens, the established UHPLC-MS/MS method could be used for therapeutic drug monitoring, provided instructions for individualized clinical medication and reduced adverse reactions. However, this study collected relatively small number of samples, so the conclusions need to validation by collecting more samples. |
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