The Qualitative And Quantitative Methods Of RDX And HMX By LC-MS/MS And Its Application In Rats

Objective:To study the mass spectrometric behavior of RDX and HMX by the High-throughput platform of liquid chromatography tandem mass spectrometry, and develop a rapid and efficient liquid chromatography-electrospray quadrupole linear ion trap mass spectrometry (LC-MS/MS) method suitable for the simultaneous determination of1,3,5-Trinitroperhydro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in rat plasma. The detection method will be tested by the rat plasma after intravenous administration of two nitramine compounds mixed solution, in order to verifing the accuracy of the quantitative analysis methods and providing a clinically useful tool for the monitoring and early warning of intoxication. At the same time, the pharmacokinetics and histopathological of rats after intravenous administration of two nitramine compounds mixed solution will be investigated.Methods:The standard solutions of analytes were infused into the mass spectrometer using a syringe pump. Both of the concentrations of RDX and HMX were10μg·ml/-1. The mass spectrums of the Q1full scan and secondary fragment ion scan were obtained. Looking for regularities of the nitramines the fragmentation. Then the mass spectrometry parameters, chromatographic conditions, and pre-treatment methods were groped and optimized. A rapid and efficient liquid chromatography-electrospray quadrupole linear ion trap mass spectrometry (LC-MS/MS) method suitable for the simultaneous determination of RDX and HMX in plasma was developed. Then the selectivity, linearity and dynamic range, method precision and accuracy, recovery, matrix effects and stability studies of the estabilished LC-MS/MS method were validated. Rats were given a single dose of nitramine compounds intravenously and plasma samples were collected. The concentrations of RDX and HMX in plasma were detected by the established LC-MS/MS method. Pharmacokinetics parameters were determined from the plasma concentration-time data with the DAS2.0software. The ALT, ALP and AST in plasma were also detected, and the rats pathologies after intravenous ing RDX and HMX were investigated after been sacrificed.Results:Both of RDX and HMX were adducted with the ions in the mobile phase. These adduct ions were [M+CL]-and [M+CH2COO]-, their second fragment ions were characterized NO2-、CH2NNO2-ions. At the same time, RDX and HMX were adducted with fragment ions from themselves, respectively. RDX were adducted with NO2-, CH2NNO-and NO3-ions, and HMX were adducted with C3H3N3-ion. The C3H3N3-ion was characterized and can be applied for the structure determination of HMX. A rapid and efficient LC-MS/MS method suitable for the simultaneous determination of RDX and HMX were established. RDX, HMX and internal standard mycophenolic acid were extracted with protein precipitation by acetonitrile, and then eluted for8.0min on a reverse phase C18analytical column with water/acetonitrile mixture as mobile phase. The detections of nitramines were carried out by multiple reaction monitoring (MRM) on3200QtrapTM mass spectrometry. The electrospray ionization (ESI) source was applied and operated in negative ion mode. RDX was detected at m/z284.1�'61.7, HMX at m/z331.0�'108.8and mycophenolic acid (IS) at m/z319.2�'m/z191.1. While the Calibration plots in solvent were adapted as calibration standard, the absolute concentration of unknowns was quantified by the ratio of the calculated concentration to that of the recovery coefficient. The linear calibration curves were obtained at the concentration range of5-200ng·mL-1for RDX and HMX. The extraction recoveries of RDX and HMX were60.04±4.18%and79.57±3.35%, respectively. The precision and accuracy meet the requirements. The analytes in plasma revealed perfect stability when assessed severally at-20℃for60days, after three freeze-thaw cycles, and after extracting in the autosampler at room temperature for0h,2h and4h, for2times successively. After intravenouse administration of nitramines, the plasma samples were collected. The concentrations of rat plasma samples were quantified by the LC-MS/MS analytical method. The concentrations of RDX were141.72ng·ml-1at5min down to14.69ng·ml-1at8h after intravenouse. The concentrations of HMX were30.39ng·ml-1at5min down to1.24ng·ml-1at8h after intravenouse. The established LC-MS/MS method was applied to rat plasma successfully. After intravenouse administration of nitramines, t1/2values of RDX and HMX were respectively (324.68±188.91) min and (134.88±100.01) min, reaching Cmax of (127.50±28.39) ng·mL-1of RDX and (26.78±7.82) ng·mL-1of HMX, and their AUC0-twere (24164.75±5401.43) ng·min·mL-1and (3787.55±2587.03) ng·min·mL-1, respectively. After intravenouse administration of nitramines, the liver kidney tissue showed no pathological changes.Conclusion:Both of RDX and HMX form intricate adduct ions with the impurities in the mobile phase. Their mass spectrometric behavior has some similarities, but HMX has a secondary fragment ions [C3H3N3]-which is characterized and can be applied for the structure determination of HMX. The present LC-MS/MS method is miniaturized, effective, portable and rapid, which can be easily used for simultaneous quantification study of RDX and HMX in rat plasma. RDX and HMX are absorbed rapidly after intravenous administration, but only a very small part of them were distributed in the blood. There was difference on the eliminate rate of the two nitramines. Flatulence was observed of the rats after intravenous administration, but no pathological changes were found.