Simultaneous determination of amitraz, chlordimeform, formetanate and their main metabolites in human urine by high performance liquid chromatography-tandem mass spectrometry

- Simultaneous determination of amitraz, chlordimeform, formetanate and their main metabolites in urine was achieved.
- A fast and practical strategy was established and validated for forensic and clinical analysis with satisfying sensitivity and accuracy.
- HPLC-MS/MS coupled with solid-support liquid-liquid (SLE) technique was applied.
- Low matrix effects were obtained in detection.

A rapid, simple and reliable high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for simultaneous determination of amitraz, chlordimeform, formetanate and their main metabolites, N-(2,4-dimethylphenyl)-N-methyl-formamidine (DMPF), 2,4-dimethylformamidine (DMF), 2,4-dimethylaniline (DMA), 4-chloro-2-methylaniline and 3-hydroxyacetanilide in human urine. The urine samples were mixed with buffer solutions (pH 8) and subsequently cleaned up by solid supported liquid/liquid extraction (SLE). The target analytes were efficiently separated with a Waters Atlantis T3 column (150 mm × 4.6 mm, 5 μm), ionized with electrospray ion source in positive mode, and quantitatively determined by tandem mass spectrometry in the multiple reaction monitoring (MRM) mode. In order to minimize matrix effects, the matrix-matched calibration curves of eight analytes were adopted with correlation coefficients (R2) above 0.99. The method were further validated by determining the limits of detection (LODs, 0.3-0.6 ng/mL), the limits of quantitation (LOQs, 1.0-2.0 ng/mL) and recoveries (89.1%-108.4%) with intra-day and inter-day relative standard deviation (RSD, <11%). The established method was applied and demonstrated in a real case by assaying a urine sample from a female poisoned by formetanate. The achieved results proved this method to be rapid, sensitive and accurate for simultaneous quantitation of eight analytes in human urine for intended forensic cases of human poisoning.