Analysis of Cannabinoids and Their Metabolites in Human Urine

Biologically monitoring marijuana exposure from active and passive use requires both a wide linear range and sensitive detection. We have developed and validated a multifunctional method using ultrahigh performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS) for analysis of urinary Δ9-tetrahydrocannabinol (THC), cannabidiol and cannabinol, and two major metabolites of THC, 11-nor-9-carboxy-THC and 11-hydroxy-THC, in active users and particularly in people exposed to secondhand marijuana smoke (SHMS). The method used positive electrospray ionization (ESI) mode to reach the sensitivity needed to detect trace SHMS exposure with limits of detection (LOD) ranging from 0.002 to 0.008 nanograms per milliliter (ng/mL) and 0.005 to 0.017 ng/mL for “free” (unconjugated forms) and “total” (unconjugated plus conjugated forms) measurements, respectively. These LODs were approximately 10–100 times more sensitive than those reported in the literature. To reduce or avoid time-consuming repetitive sample preparation and analysis, the method simultaneously monitored multiple reaction monitoring transitions in negative ESI mode to quantify high analyte levels typically found in the urine of active marijuana users (linear dynamic range of 12.5–800 ng/mL). The validation results indicated this method was accurate (average inter/intra-day bias, <10%), precise (inter/intra-day imprecision, <10%), and fast (6 min run time). In addition, sample preparation throughput was greatly improved using an automation liquid-handling system, meeting the needs for potential large-scale population studies

Validation of a LC-MS/MS Method for Quantifying Urinary Nicotine, Six Nicotine Metabolites and the Minor Tobacco Alkaloids—Anatabine and Anabasine—in Smokers' Urine

<div><p>Tobacco use is a major contributor to premature morbidity and mortality. The measurement of nicotine and its metabolites in urine is a valuable tool for evaluating nicotine exposure and for nicotine metabolic profiling—i.e., metabolite ratios. In addition, the minor tobacco alkaloids—anabasine and anatabine—can be useful for monitoring compliance in smoking cessation programs that use nicotine replacement therapy. Because of an increasing demand for the measurement of urinary nicotine metabolites, we developed a rapid, low-cost method that uses isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) for simultaneously quantifying nicotine, six nicotine metabolites, and two minor tobacco alkaloids in smokers' urine. This method enzymatically hydrolyzes conjugated nicotine (primarily glucuronides) and its metabolites. We then use acetone pretreatment to precipitate matrix components (endogenous proteins, salts, phospholipids, and exogenous enzyme) that may interfere with LC-MS/MS analysis. Subsequently, analytes (nicotine, cotinine, hydroxycotinine, norcotinine, nornicotine, cotinine N-oxide, nicotine 1′-N-oxide, anatabine, and anabasine) are chromatographically resolved within a cycle time of 13.5 minutes. The optimized assay produces linear responses across the analyte concentrations typically found in urine collected from daily smokers. Because matrix ion suppression may influence accuracy, we include a discussion of conventions employed in this procedure to minimize matrix interferences. Simplicity, low cost, low maintenance combined with high mean metabolite recovery (76–99%), specificity, accuracy (0–10% bias) and reproducibility (2–9% C.V.) make this method ideal for large high through-put studies.</p></div

The molar percent each analyte contributed to the combined concentrations of nicotine and 6 nicotine metabolites in this study (n = 94).

<p>The molar percent each analyte contributed to the combined concentrations of nicotine and 6 nicotine metabolites in this study (n  =  94).</p

Comparison of standard curves generated by urine or enzyme standards to water standard curve.

<p>Comparison of standard curves generated by urine or enzyme standards to water standard curve.</p

Limits of Detection (LOD) for “free” and ”total” analytes.

<p>Limits of Detection (LOD) for “free” and ”total” analytes.</p

HPLC Gradient Elution Table.

<p>HPLC Gradient Elution Table.</p

Standard Concentrations and Reportable Ranges (sample volume 200 µL urine–1∶5 dilution factor).

<p>Standard Concentrations and Reportable Ranges (sample volume 200 µL urine–1∶5 dilution factor).</p

Accuracy and Precision from the Repetitive Analysis of Five Fortified Urine Pools.

<p>Accuracy and Precision from the Repetitive Analysis of Five Fortified Urine Pools.</p

Representative chromatograms.

<p>(A) Standards Analysis (cotinine, 200 ng/mL); (B) Smoker Urine Sample (“free” cotinine, 14.1 ng/mL); (C) Smoker Urine Sample (“free” cotinine, 2767 ng/mL); (D) Smoker Urine Sample (“total” cotinine, 4195 ng/mL). Abbreviations: Cotinine-oxide (COX); Nicotine-oxide NOX); Hydroxycotine (HCT); Norcotinine (NCT); Cotinine (COT); Nornicotine (NNC); Anatabine (ANT); Anabasine (ANB); Nicotine (NIC). The fourth letter “T” in the abbreviations in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101816#pone-0101816-g002" target="_blank">Figure 2(D)</a> represents the “total” concentrations for measured analytes.</p