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Table 1.

Urine samples were treated by β-glucuronidase ( Escherichia coli K12) at 45°C for 120 min. to minimize the THCCOOH-glucuronide fraction. Hundred microlitre of pre-treated urinary samples, serum sample standards and quality controls were prepared by adding 50 μl 0.1 M phosphate buffer solution (pH 6), 50 μl internal standard (50 ng/ml, d9-THCCOOH and d3-THC) and 1 ml diethyl ether/n-hexane/ethyl acetate (1:1:1) as extractants. Samples were analysed by liquid chromatography–tandem mass spectrometry (LCMSMS) using a Waters Acquity UPLC system with an autosampler and a binary solvent delivery system (Waters, Milford, MA, USA) interfaced to Waters Micromass® Quattro Premier™ XE benchtop tandem quadrupole mass spectrometer (Waters, Manchester, UK). Chromatography was performed on a 2.1 × 50 mm Waters Acquity BEH C8 1.7 mm column. The mobile phase consisted of 68% methanol in 0.34 g/l aqueous ammonium acetate with an isocratic flow rate of 0.4 ml/min. For quantitative analysis of THCCOOH and THC, the following MRM transitions were used: m/z 343→299 (quantification ion), 345→327 (qualifier ion) and m/z 315→193 (quantification ion), 315→259 (qualifier ion), respectively. MRM transition m/z 352→308, 354→336 and m/z 318→196, 318→262 were used for the internal standards d9-THCCOOH and d3-THC, respectively. The linear dynamic range ( r 2  > 0.99) for THCCOOH and THC was 3–5000 ng/ml and 0.6–240 ng/ml, respectively. LOD for THC and THCCOOH were 0.2 ng/ml and 0.17 ng/ml, respectively. Between-day coefficients of variation (CVs) for THCCOOH calculated from quality control samples were <6% at 30 ng/ml and <4% at 3000 ng/ml. For THC, the CV was <11% at 2.5 ng/ml and <6% at 250 ng/ml.

Serum and urinary cannabinoid levels in six regular cannabis users, before and after 24 hr of food deprivation.

Specimen collection.

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Urine creatinine was analysed photometrically after complex formation with picric acid in an alkaline solution by a routine method (Jaffé's method) on a Cobas Integra 400 + multianalyser (Roche Diagnostics, Basel, Switzerland). The LOQ was 0.11 mg/ml, and the CV was <10%.