A novel high-performance liquid chromatography with diode array detector method for the simultaneous quantification of the enzyme-reactivating oximes obidoxime, pralidoxime, and HI-6 in human plasma.

Oximes such as pralidoxime (2-PAM), obidoxime (Obi), and HI-6 are the only currently available therapeutic agents to reactivate inhibited acetylcholinesterase (AChE) in case of intoxications with organophosphorus (OP) compounds. However, each oxime has characteristic agent-dependent reactivating efficacy, and therefore the combined administration of complementary oximes might be a promising approach to improve therapy. Accordingly, a new high-performance liquid chromatography method with diode-array detection (HPLC-DAD) was developed and validated allowing for simultaneous or single quantification of 2-PAM, Obi, and HI-6 in human plasma. Plasma was precipitated using 5% w/v aqueous zinc sulfate solution and subsequently acetonitrile yielding high recoveries of 94.2%-101.0%. An Atlantis T3 column (150 × 2.1mm I.D., 3 µm) was used for chromatographic separation with a total run time of 15 min. Quantification was possible without interferences within a linear range from 0.12 to 120 µg/mL for all oximes. Excellent intra-day (accuracy 91.7%-98.6%, precision 0.5%-4.4%) and inter-day characteristics (accuracy 89.4%-97.4%, precision 0.4%-2.2%) as well as good ruggedness were found. Oximes in processed samples were stable for at least 12 h in the autosampler at 15°C as well as in human plasma for at least four freeze-thaw cycles. Finally, the method was applied to plasma samples of a clinical case of pesticide poisoning.

A quantitative NMR protocol for the simultaneous analysis of atropine and obidoxime in parenteral injection devices.

A rapid selective and accurate quantitative (1)H NMR method was developed for the simultaneous analysis of obidoxime chloride and atropine sulfate, the active components in parenteral injection devices (PID) used for the emergency treatment of poisoning by toxic organophosphates. The spectra were acquired in 90% H(2)O-10% D(2)O using sodium 3-(trimethylsilyl)-1-propane sulfonate hydrate as the internal standard. Both synthetic mixtures and dosage forms were assayed. The results were compared with those obtained from a reported HPLC method.

Simple high-performance liquid chromatographic method for determination of atropine and obidoxime in a parenteral injection device.

Atropine and obidoxime in a parenteral injection device are determined by simple HPLC method simultaneously without any pretreatment at 228 nm. The relative standard deviations (R.S.D.) were below 1.6% for the compounds. The correlation coefficient was greater than 0.999 for both compounds in the calibration range. The recoveries at 5 mg/L concentration averaged as 95% for atropine and 102% for obidoxime. The uncertainty of the measurements for atropine and obidoxime was 2.8% and 2.4%, respectively.

Determination of atropine and obidoxime in automatic injection devices used as antidotes against nerve agent intoxication.

A capillary gas-liquid chromatographic (GLC) and an ion-pair high performance liquid chromatographic (HPLC) method were developed for the assay of atropine sulphate and obidoxime chloride from a parenteral solution in commercial automatic injection devices. The injectors are aimed for the emergency treatment of poisoning by nerve agents. The two-step GLC method consists of extraction of atropine as a free base prior to GLC analysis using scopolamine as an internal standard. Obidoxime is determined directly in a diluted sample solution by reversed-phase HPLC using sodium 1-heptanesulphonate as a counter ion in the mobile phase. The relative standard deviation (R.S.D.) was 1.81% for the GLC procedure with injectors containing only atropine and 2.37% for the GLC of atropine in atropine-obidoxime injectors. The R.S.D. for the HPLC procedure of obidoxime in atropine-obidoxime injectors was 0.82%. The corresponding R.S.D.s for the sampling of atropine-obidoxime injectors were 0.36% and 0.27%. The coefficient of determination (r2) was 1.000 for both methods. The recoveries at the target concentration averaged 101.0% and 98.7% with a standard error of the mean of 0.30 for both methods. The retention times for atropine and obidoxime were 6.27 and 4.29 min, respectively.

Ion-exclusion chromatography of compounds of biological interest.

The principle of ion exclusion was examined as a method for the separation of small ionic compounds. The systems employed consisted of very porous column packings, substituted with fixed charges, which were eluted by buffer solutions of low ionic strength. DEAE-Sephadex A-50 was principally employed, and it was shown that there was a linear relationship between the net charge on a cation and its partition coefficient into the gel phase. A similar relationship existed in the chromatography of amino acids on various columns bearing fixed negative charges. It was concluded that this was an efficient form of chromatography, which gave results directly related to the ionic charge of the sample being examined. The charge characteristics of biologically active compound could be determined by this method.