Igor Rodin (Presenter)
Lomonosov Moscow State University
Authorship: Timur Baygildiev, Igor Rodin, Andrey Stavrianidi, Arcady Braun, Igor Rybalchenko, Irina Anan’eva, Oleg Shpigun
Chemistry Department, Lomonosov Moscow State University, 119991 Moscow, Russia
| Short Abstract Very simple, rapid and direct approach for the determination of the most stable nerve agent biomarker – methylphosphonic acid in urine samples by anion-exchange liquid chromatography tandem mass spectrometry was firstly proposed and developed. Chromatographic separation was performed using anion-exchange column. Application of anion-exchange chromatography allowed completely overcome matrix effect influence of urine substances. Tandem mass spectrometric detection has provided reliable and very sensitive determination of methylphosphonic acid in urine. Achieved limit of detection in urine 4 ng ml-1 were low. Precision of the method was good, within run and between run repeatability were lower than 12%. |
Long Abstract
Introduction
Nowadays there is a threat of using chemical weapons during local armed conflicts and terrorist attacks. One of the most dangerous toxic chemicals that have been used as chemical weapons are nerve agents. The most well-known nerve agents are sarin, soman, VX. These substances are not stable and in environment or living organisms undergo rapid hydrolysis to the alkyl methylphosphonic acids, which further finally hydrolysis to the methylphosphonic acid (MPA). MPA is the most stable and specific marker of nerve agents. Determination of MPA is a direct proof of using nerve agents.
Biomedical sample analysis is very important because the exposure of workers involved in demilitarization activities may be monitored in addition to the analyses of biomedical samples of people treated by CW agents in terrorist attacks or in local armed conflicts. MPA is very polar substance and its determination by chromatographic tools is very challenging. Besides, low sub ppm concentrations of MPA likely to be encountered in biological matrices and their great complexity makes determination of MPA in biological matrices even more complicated.
MPA exist in urine in anionic form (pKa1=2.12) so application of ion-exchange chromatography may be useful for separation MPA from another urine matrices components.
Methods
Urine samples were defrosted at a room temperature, mixed and centrifuged. After separation of supernatant from the residue, supernatant was diluted with deionized water and 20 µL of resulted solution were injected into LC-MS/MS system.
Chromatographic separation was performed on an anion-exchange Seporus A-UNI column (BiASep, Russia) with quaternary ammonium functional groups with a mobile phase consisting of formic acid in water at a flow rate of 0.95 mL min-1.
Tandem mass spectrometric conditions were as follows: Negative electro spray ionization mode (ESI). MRM measurements were conducted using the precursor ion and two product ions of MPA and were as follows: 95→79 (quantitative) and 95→63 (qualitative).
Results
The goal was to develop an approach that allows to determine MPA by anion-exchange liquid chromatography with tandem mass spectrometric detection using ordinary LC-MS/MS system. Most of mobile phases that have been using in IC are not compatible with mass spectrometric detection. So our idea was to use anion-exchange column compatible with wide range of eluents and to try most suitable for mass spectrometry mobile phases that have ionic nature and may be used as eluents for ion-exchange chromatography. Formic acid solution was chosen as the best mobile phase. MPA peak has appropriate retention factor (k’=4.1) which allows to separate MPA from matrix components of urine.
It was found that calibration curve was linear over a concentration range of 10-500 ng mL-1 with three injections at each concentration level. Calibration curve showed excellent linearity with correlation coefficient of 0.9999 and residuals lower than 10%. LOD and LOQ in spiked blank urine were 4 ng mL-1 and 10 ng mL-1 respectively.
It was discovered that proposed sample preparation technique in combination with separation on anion-exchange column and tandem mass spectrometric detection completely remove influence of urine components. Within-run and between-run repeatability were good at three concentrations and did not exceed 8 and 12 % respectively (Table 1).
Conclusions
The developed approach demonstrates application of anion-exchange chromatography with tandem mass spectrometric detection for determination of MPA in urine samples. Very simple sample pretreatment, anion-exchange separation and MS/MS detection allows completely overcome matrix effect and provide rapid, non laborious, reproducible and reliable determination of MPA in urine with the lowest LOD (4 ng mL-1) among existing direct methods. Our results show that this approach may become an effective alternative to the existing GC methods that require time-consuming and laborious derivatization.
References & Acknowledgements:
This work was supported by Russian Foundation for Basic Research (Grant No. 16-33-00804 and grant No. 14-03-00125)
| Description | Y/N | Source |
| Grants | yes | Russian Foundation for Basic Research |
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