Ultra-high performance liquid chromatography tandem mass-spectrometry for simple and simultaneous quantification of...

Cannabis is used widely in the United States, both recreationally and for medical purposes. Current methods for analysis of cannabinoids in human biological specimens rely on complex extraction process and lengthy analysis time. We established a rapid and simple assay for quantification of Δ⁹-tetrahydrocannabinol (THC), cannabidiol (CBD), 11-hydroxy Δ⁹-tetrahydrocannabinol (11-OH THC) and 11-nor-9-carboxy-Δ⁹-tetrahydrocannbinol (THCCOOH) in human plasma by U-HPLC-MS/MS using_{Δ9-tetrahydrocannabinol-D3 (THC-D3)} as the internal standard. Chromatographic separation was achieved on an Acquity BEH C18 column using a gradient comprising of water (0.1% formic acid) and methanol (0.1% formic acid) over a 6 min run-time. Analytes from 200μL plasma were extracted using acetonitrile (containing 1% formic acid and THC-D₃). Mass spectrometry was performed in positive ionization mode, and total ion chromatogram was used for quantification of analytes. The assay was validated according to guidelines set forth by Food and Drug Administration of the United States. An eight-point calibration curve was fitted with quadratic regression (r²>0.99) from 1.56 to 100ngmL^-1 and a lower limit of quantification (LLOQ) of 1.56ngmL^-1 was achieved. Accuracy and precision calculated from six calibration curves was between 85-115% while the mean extraction recovery was >90% for all the analytes. Several plasma phospholipids eluted after the analytes thus did not interfere with the assay. Bench-top, freeze-thaw, auto-sampler and short-term stability ranged from 92.7 to 106.8% of nominal values. Application of the method was evaluated by quantification of analytes in human plasma from six subjects.