Effect of creatinine and specific gravity normalization techniques on xenobiotic biomarkers in smokers' spot and 24-h urines.

Renal excretion mechanisms are xenobiotic-specific; therefore, accurate exposure assessment requires an understanding of relationships of xenobiotic biomarker concentration and excretion rate to urine flow, specific gravity and creatinine concentration. Twenty-four-hour urine collection for xenobiotic exposure assessment is considered the "gold standard" procedure. Random spot-urine collection is convenient and minimizes subject compliance concerns but requires that normalization techniques be employed to account for diuresis and diurnal variation in xenobiotic biomarker excretion. This paper examines and makes recommendations concerning normalization techniques and conditions under which spot-urine results most accurately reflect 24-h urine results. Specific gravity, creatinine, and xenobiotic biomarkers were determined in smokers' spot and 24-h urines. Normalization techniques were applied, variance-component analyses were performed to estimate variability, spot urines were pooled mathematically to simulate 24-h urines and analyses of variance were performed to evaluate spot urines' ability to reflect 24-h urine concentrations. For each xenobiotic biomarker concentration, log-linear relationships were observed with urine flow, specific gravity, and creatinine. For most xenobiotic biomarker excretion rates, log-linear relationships were observed with urine flow; creatinine, however, was unaffected by urine flow. The conventional creatinine ratio-normalization technique demonstrated greater variability (within-day, between-day and between-subject) than other normalization techniques. Comparisons of simulated 24-h urines to spot urines suggest that spot-urine collection be performed only between 2 p.m. and 2 a.m. and that the modified specific-gravity-adjusted-creatinine ratio-normalization technique and the creatinine-regression normalization technique yield the best agreement between spot- and simulated 24-h urine results.

Validation and application of a method for the determination of nicotine and five major metabolites in smokers' urine by solid-phase extraction and liquid chromatography-tandem mass spectrometry.

An SPE-LC-MS/MS method was developed, validated and applied to the determination of nicotine and five major metabolites in human urine: cotinine, trans-3'-hydroxycotinine, nicotine-N-glucuronide, cotinine-N-glucuronide and trans-3'-hydroxycotinine-O-glucuronide. A 500 microL urine sample was pH-adjusted with phosphate buffer (1.5 mL) containing nicotine-methyl-d3, cotinine-methyl-d3 and trans-3'-hydroxycotinine-methyl-d3 internal standards. For the unconjugated metabolites, an aliquot (800 microL) of the buffered solution was applied to a 30 mg Oasis HLB-SPE column, rinsed with 2% NH4OH/H2O (3.0 mL) and H2O (3.0 mL) and eluted with methanol (500 microL). The eluate was analyzed isocratically (100% methanol) by LC-MS/MS on a diol column (50 x 2.1 mm). For the total metabolites, a beta-glucuronidase/buffer preparation (100 microL) was added to the remaining buffered solution and incubated at 37 degrees C (20 h). An aliquot (800 microL) of the enzymatically treated buffered solution was extracted and analyzed in the same manner. The conjugated metabolites were determined indirectly by subtraction. The quantitation range of the method (ng/mL) was 14-10,320 for nicotine, 15-9800 for cotinine and 32-19,220 for trans-3'-hydroxycotinine. The validated method was used to observe diurnal variations from a smoker's spot urine samples, elimination half-lives from a smoker's 24 h urine samples and metabolite distribution profiles in the spot and 24 h urine samples.