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Abstract INTRODUCTION
Cigarette smoking is the leading preventable cause of death in the United States. Nicotine, present in tobacco products, is an addicting substance that causes individuals to continue use of tobacco despite concerted efforts to quit. Nicotine is metabolized to its major metabolites of cotinine, and nornicotine. Nornicotine is pharmacologically active and may accumulate to a greater degree in the brain. The presence of anabasine is indicative of active use of a tobacco product. As a result, tobacco users in abstinence programs are routinely monitored by laboratory tests that measure nicotine, its metabolites, and anabasine.
Measurements of nicotine and metabolites in urine are prone to quantifier/qualifier mismatch, poor chromatography of anabasine and nornicotine, and increasing interferences with nicotine. Therefore, we present an improved method to monitor nicotine, cotinine, nornicotine, and anabasine in urine.
METHODS
Solid phase extraction was applied to clean up the urine samples. 50 mcL of calibrator, QC, blank and patient samples were pipetted into 96-well plate, followed by 50 mcL of deuterium labeled internal standard mixtures (200 ng/mL each), diluted with 500 mcL of 0.1% formic acid in water, followed by vortexing the plate. The strong cation Bond Elut PlexaPCX 30mg plate (Agilent) was conditioned with 0.1N HCL in methanol followed by 0.1N HCL in water. Samples were transferred to the SPE plate, pushed through with positive pressure, washed with 0.1N HCL in water and 0.1N HCL in methanol, and eluted twice with 5% of ammonium hydroxide in methanol. The final eluent was diluted with the ion-pair reagent, 1-octanesulfonic acid, at a concentration of 1.25g/L. The analytes were injected and analyzed on the HPLC-MS/MS (Thermo Scientific TLX-4 coupled to Sciex triple quadrupole 6500), separated over 10 minutes using a 3 x 50 mm, 2.6 um PS C-18 column (Kinetex) with the mobile phase A (0.1% formic acid in water and 2mM of ammonium formate), mobile phase B (0.1% formic acid in ACN). The method was validated with patient samples over 20 runs, with a maximum of two runs per day. All necessary studies, including accuracy, precision, linearity, specificity, sensitivity, LOD, interferences and ion suppression studies were performed.
RESULTS
Compared to our current dilute and shoot method, the new SPE method provided significantly reduced quantifier/qualifier discrepancies and eliminated interferences. The analytical measuring range was 5-1,200 ng/mL for nicotine/cotinine, and 2-120 ng/mL for nornicotine/anabasine No significant carryover was seen following a sample twice the upper limit of quantitation. To assess the improvements from the new method, 119 samples that previously exhibited interferences or quantifier/qualifier discrepancies were tested. With the new method, the quantifier and qualifier match were within 15% for nicotine, 7% for cotinine, 11% for nornicotine. Anabasine samples results matched within 20% with the exception of 4 samples which matched <35%. For intra-day and inter-day imprecision, the %CVs were less than 6.8% and 7.1% for all analytes. Accuracy included a minimum of 63 patient samples of each analyte that was cross validated with our current LC-MS/MS method. The results for each analyte met the CSLI validation criteria. No interferences were observed among 92 common prescriptive drugs, as well as vitamins and hormones. No hemolysis, bilirubin/icterus, and bilirubin/icterus conjugate interferences were present. Minimal ion suppression/enhancement was seen for anabasine. Some degree of ion suppression/enhancement was observed in the analytes of nicotine, cotinine and nornicotine. However, the internal standard was able to compensate for the ion suppression/enhancement.
DISCUSSION
An accurate and robust method was established for nicotine and its metabolites in urine. Our new SPE extraction, polar analytical column, and ion-pairing reagent achieved the goal of reducing quantifier/qualifier mismatches and interferences. The specificity, precision, accuracy, and quantifier-qualifier match were all improved through enhanced chromatographic performance and eliminating the potential interferences. As a result, the improved method significantly reduces the need to repeat samples; therefore, improving the turn-around time, increasing throughput, and dramatically reducing the workload for the laboratory.
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= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
