A novel method development and validation for determination of 2,4,6-Trinitrotoluene and its metabolites on LC-MS/MS.

LC-MS/MS has recently emerged as the best practice for simultaneous analysis of 2, 4, 6 Trinitrotoluene (TNT) and its metabolites. We have developed and validated an LC-MS/MS method for simultaneous quantification of 2, 4, 6 Trinitrotoluene (TNT) and its metabolites 4-ADNT, 2-ADNT, 2,4-DNT, and 2,6-DNT in urine samples. These four metabolites were acid hydrolyzed using 1 mL of urine followed by extraction using n-Hexane and ethyl acetate as an extracting solvent. Separation was achieved by centrifugation, and the supernatant was dried under nitrogen, reconstituted with water and acetonitrile, and then filtered. Chromatographic separation was achieved on Agilent Poroshel 120 EC-C18 column (2.1 mm × 75 mm × 2.7 µm) utilizing two mobile phases 0.1% formic acid in water and 0.1% formic acid in acetonitrile in gradient flow. The validated AMR of TNT and its metabolites was 7.8-1000 ng/mL. The method showed an excellent correlation (>0.99) for TNT and its metabolites. Accuracy and within/between day precision of TNT and its metabolites were within ±15%. The integrity of diluted samples was maintained for each dilution factor. The method was found stable after storage and freeze-thaw cycle. The presented method can be used for TNT screening in occupationally exposed ordnance factory workers.

Determination of crizotinib in human and mouse plasma by liquid chromatography electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

An LC-ESI-MS/MS method using high-throughput solid-phase extraction (SPE) was developed and validated to measure crizotinib in human and mouse plasma to support ongoing clinical and preclinical pharmacokinetic studies. Chromatographic separation of mouse or human plasma extracts was performed on a Supelco Discovery c18 column (50 mm × 2.1mm, 5.0 µ) with gradient elution using a combination of acidified aqueous and methanol (MeOH) mobile phases. The mass-to-charge transition monitored for detection and quantitation of crizotinib was m/z 450.2>260.2 while the stable label internal standard (ISTD) was monitored at m/z 457.2>267.3. The validation studies demonstrated that the assay is both precise and accurate with %CV<9% and accuracies within 8% of nominal target concentration across all concentrations tested for both the human and mouse plasma matrices. Sample volumes required for analysis were 50 and 25 µL for human plasma and mouse plasma, respectively. Calibration curves were linear over a range of 5-5,000 ng/mL for human plasma and 2-2,000 ng/mL for mouse plasma. The use of a 96-well plate format enabled rapid extraction as well as compatibility with automated workflows. The method was successfully applied to analyze crizotinib concentrations in plasma samples collected from children enrolled on a phase I pediatric study investigating the use of crizotinib for treatment of pediatric brain tumors.