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Abstract BACKGROUND
Determination of the effectiveness of patient smoking cessation programs often requires careful monitoring of both nicotine and its primary metabolite, cotinine. Accurate measurement of blood nicotine and cotinine concentrations are important in the qualification of transplant candidates and evaluating their exposure to tobacco smoke post-transplantation, but the levels of these compounds are much lower in serum or plasma than urine. Significant opportunities for improvement in the existing method employed in our laboratory were also identified in both the sample preparation procedure as well as in mitigation of carryover. A method was thus needed that could quantitate nicotine and cotinine both at low levels (2 ng/mL) as well as much higher levels (50 ng/mL and 1000 ng/mL for nicotine and cotinine, respectively) to ascertain the degree of potential exposure. Our aim was to develop and validate a method for more efficiently quantitating nicotine and cotinine in serum over an expanded analytical measurement range with a reduced sample volume while streamlining sample preparation.
METHODS
Nicotine, cotinine, and their deuterated internal standards were extracted by protein precipitation. Briefly, 100 µl of samples, controls, or calibrators were transferred to a 96-well plate and mixed with 200 µl of protein precipitation solution (aqueous 0.2 M ZnSO4 in methanol, 3:7 v/v) containing the internal standards (nicotine-d3 and cotinine-d3 at 25 ng/mL). The plate was vortexed vigorously for one minute followed by centrifugation for five minutes, after which the supernatant was ready for injection without further manipulation. This procedure eliminated both an extended incubation step as well as the addition of caustic potassium hydroxide solution utilized in the predecessor assay. Each sample was analyzed on a Transcend II LX-2 LC coupled to a TSQ Quantis™ triple quadrupole mass spectrometer (Thermo Scientific). Online sample cleanup was performed using a turbulent flow method on a TurboFlow™ Cyclone MCX-2 mixed-cation exchange column (0.5 × 50 mm; Thermo Scientific). The compounds were separated chromatographically on a porous graphitic carbon Hypercarb™ analytical column (3.0 × 50 mm, 5 µm particle size, Thermo Scientific) at ambient temperature with an injection volume of 20 µl. Multiple reaction monitoring (MRM) was used to monitor quantifier and qualifier transitions for nicotine, nicotine-d3, cotinine, and cotinine-d3. An extended column washing step was implemented after each injection to mitigate carryover. The total analytical runtime was 6.0 minutes. Ion suppression/enhancement, matrix effects, reportable range, analytical sensitivity, carryover, intra- and inter-assay precision, and method comparison were assessed and validated.
RESULTS
The chosen alternative matrix (SeraCon™ II CD Hormone Depleted Double-Stripped Negative Diluent, SeraCare) was found to be suitable for calibrator use (maximum bias observed -6.7% for nicotine, -6.9% for cotinine when mixed with patient samples). No ion suppression or enhancement near the retention time of either analyte was observed. No interferences were found for hemolyzed, lipemic, or icteric samples, other nicotine metabolites or related compounds (nornicotine, trans-3'-hydroxycotinine, anabasine, cotinine-N-oxide), or commercial interference mixtures (Cerilliant Interference Mixtures 2-7, 40+ potential interferents). Reportable range was validated from 2 ng/mL to 50 ng/mL for nicotine, and from 1.6 ng/mL to 1000 ng/mL for cotinine. For nicotine, the mean recovery at the LLOQ was 96.5%; for cotinine, the mean recovery at the LLOQ was 103.5%. To evaluate the analytical sensitivity of the method, the limit of detection (LoD), limit of blank (LoB), and lower limit of quantitation (LLOQ) were determined for each analyte. For nicotine, the LoD was 0.34 ng/mL, the LoB was 0.26 ng/mL, and the LLOQ was 2.08 ng/mL (2.55% CV). For cotinine, the LoD was 0.41 ng/mL, the LoB was 0.32 ng/mL, and the LLOQ was 2.08 ng/mL (2.87% CV). Acceptable carryover limits (<1 ng/mL) were found for nicotine at 100 ng/mL and cotinine at 1500 ng/mL. For nicotine, the intra-day precision CV ranged from 1.3% to 1.8% and the inter-day precision CV ranged from 1.4% to 3.0%. For cotinine, the intra-day precision CV ranged from 0.8% to 1.5% and the inter-day precision CV ranged from 1.6% to 1.9%. Comparison with an independent LC-MS/MS method showed an average bias of -3.6% for nicotine and -7.4% for cotinine. Deming regression analysis of the results indicated a slope of 1.049 and intercept of -1.0 (R=0.9915) for nicotine, and a slope of 0.915 and intercept of 2.8 (R= 0.9985) for cotinine.
CONCLUSION
The described LC-MS/MS method for quantitation of nicotine and cotinine in serum was developed to minimize sample preparation time while retaining accuracy and precision. The sample preparation employs a streamlined one-step protein precipitation step intended to increase the efficiency of workflow in our clinical laboratory, and the higher carryover limits target a significant reduction in the number of patient sample analyses that must be repeated.
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= Discovery stage. (24.37%, 2023)
= Translation stage. (39.50%, 2023)
= Clinically available. (36.13%, 2023)
