A rapid LC-MS-MS method for the determination of nicotine and cotinine in serum and saliva samples from smokers: validation and comparison with a radioimmunoassay method.

The development and validation of a rapid liquid chromatography (LC)-tandem mass spectrometry (MS-MS) method for determination of nicotine and cotinine in smokers' serum is described. The method is based on solid-phase extraction in a 96-well plate format and requires only 100 microL of serum. Using normal-phase chromatography, both analytes elute in less than 1 min, which permits high sample throughput applications. The calibrated range is 2-100 ng/mL nicotine and 20-1,000 ng/mL cotinine. For known samples, recovery is 95-116% for nicotine and 93-94% for cotinine. The method is extended to rat serum and human saliva (cotinine only) using partial validation techniques. When compared with an existing radioimmunoassay method in our laboratory, the LC-MS-MS method gives improved accuracy, precision, and sample throughput.

Determination of nicotine N-1-glucuronide, a quaternary N-glucuronide conjugate, in human biological samples.

[Methyl-d3]-N-1-beta-D-glucopyranosyl-(+/-)-nicotinium inner salt ((+/-)-[methyl-d3]nicotine N-1-glucuronide) was synthesized from (+/-)-[methyl-d3]nicotine via reaction with methyl-2,3,4-tri-O-acetyl-1-bromodeoxy-alpha-D-glucopyranouronate, followed by deprotection with 1 M aqueous NaOH and purification by preparative TLC. Nicotine N-glucuronide was identified and determined directly in smokers' urine. A solid phase extraction method was used to partially isolate the material from urine. Subsequent determination was by thermospray-LC/MS using the synthetic d3-labeled nicotine N-glucuronide as internal standard. The identified urinary component had the same retention time as a synthetic standard and gave the same mass spectrum. The thermospray mass spectrum was characterized from the protonated molecular ion (m/z 339) and the protonated aglycone ion (m/z 163). Quantitative results from this direct method were compared with those from an indirect method, which calculated the nicotine glucuronide in the biological sample from the amount of nicotine released following treatment of the sample with the deconjugating enzyme, beta-glucuronidase. On average, the concentration of nicotine N-glucuronide determined by the direct method was 34% greater than that determined by the indirect method. Concentrations of nicotine N-glucuronide in urine ranged from 2.2 to 7.6 nmol/ml with a limit of detection of 1.3 nmol/ml.

A further study of FTC yield and nicotine absorption in smokers.

The relationship between nicotine yield as determined by the FTC method and nicotine absorption was examined in 72 smokers in a more rigorous repetition of a previous study of 33 smokers. For this study, 113 smokers evenly distributed across four FTC "tar" yield ranges were recruited, only 72 demonstrated reasonable compliance with the study criteria with regard to sample collections and cigarette brand style consistency. Subjects recorded the number of cigarettes smoked daily and collected a 24-h urine sample and a saliva sample on 3 consecutive days. Nicotine absorption was determined by monitoring urinary excretion of nicotine and its metabolites. In addition, saliva samples were monitored for cotinine using radioimmunoassay (RIA). The correlation of the relationship for nicotine absorbed per cigarette was positive and significant (r = 0.31, P = 0.008) but weaker than in the previous study. Only smokers in the highest yield range showed any statistical difference from smokers in the lower ranges. Our results suggest that FTC nicotine yield is weakly related to nicotine absorption and that smoker-controlled factors exert a great influence on the amount of nicotine absorbed by smokers. Compensation is substantial but incomplete for the minority (by market share) of smokers at the low end of the yield scale. It is uncertain how well any alternative set of machine parameters would predict nicotine absorption for the majority of smokers, even if it were more predictive for the small number of smokers at the lower yield part of the range.

Long-term mechanical circulatory support system reliability recommendation: American Society for Artificial Internal Organs and Society of Thoracic Surgeons: long-term mechanical circulatory support system reliability recommendation.

Jointly developed by members of the American Society for Artificial Internal Organs and the Society of Thoracic Surgeons along with staff from the Food and Drug Administration, the National Heart, Lung and Blood Institute and other experts, this recommendation describes the reliability considerations and goals for Investigational Device Exemption and Premarket Approval submissions for long-term, mechanical circulatory support systems. The recommendation includes a definition of system failure, a discussion of an appropriate reliability model, a suggested in vitro reliability test plan, reliability considerations for animal implantation tests, in vitro and animal in vivo performance goals, the qualification of design changes during the Investigational Device Exemption clinical trial, the development of a Failure Modes Effects and Criticality Analysis, and the reliability information for surgeons and patient candidates. The document will be periodically reviewed to assess its timeliness and appropriateness within five years.

Long-term mechanical circulatory support system reliability recommendation: American Society for Artificial Internal Organs and The Society of Thoracic Surgeons: long-term mechanical circulatory support system reliability recommendation.

Jointly developed by members of the American Society for Artificial Internal Organs and the Society of Thoracic Surgeons along with staff from the Food and Drug Administration, the National Heart, Lung and Blood Institute and other experts, this recommendation describes the reliability considerations and goals for Investigational Device Exemption and Premarket Approval submissions for long-term, mechanical circulatory support systems. The recommendation includes a definition of system failure, a discussion of an appropriate reliability model, a suggested in vitro reliability test plan, reliability considerations for animal implantation tests, in vitro and animal in vivo performance goals, the qualification of design changes during the Investigational Device Exemption clinical trial, the development of a Failure Modes Effects and Criticality Analysis, and the reliability information for surgeons and patient candidates. The document will be periodically reviewed to assess its timeliness and appropriateness within five years.

Comparison of measured and FTC-predicted nicotine uptake in smokers.

Cigarette smokers have a wide variety of "tar" and nicotine yields to choose from in the current market, ranging from 0.5 mg "tar" and less than 0.05 mg nicotine to 27 mg "tar" and 1.8 mg nicotine by the Federal Trade Commission (FTC) method. To understand better the relationship between FTC nicotine yields and actual nicotine uptake in smokers, we have studied nicotine uptake in 33 smokers of self-selected products representing four "tar" groupings: 1 mg "tar" (1MG), ultra-low "tar" (ULT), full-flavor low "tar" (FFLT), and full flavor (FF) cigarettes. These cigarette categories had mean FTC nicotine yields of 0.14, 0.49, 0.67, and 1.13 mg/cigarette, respectively. The subjects smoked their usual brand of cigarette ad libitum and provided a 24-h urine sample for total nicotine uptake analysis over a period during which the number of cigarettes smoked was recorded. Nicotine uptake was determined by monitoring urinary nicotine and its metabolites, including the glucuronide conjugates. Daily nicotine uptake was 9.1 +/- 7.3 mg (range 1-21 mg) for 1MG, 19.2 +/- 10.0 mg (range 4-42 mg) for ULT, 21.8 +/- 9.4 mg (range 13-38 mg) for FFLT, and 37.1 +/- 14.4 mg (range 21-60 mg) for FF smokers. On a per cigarette basis, yields were 0.23 +/- 0.11, 0.56 +/- 0.23, 0.60 +/- 0.18, and 1.19 +/- 0.43 mg nicotine, respectively. Although individual variability was fairly large (CVs of 0.39-0.80), means for the different groups showed that lower FTC yield smokers not only absorb less nicotine per 24-h period, but also per cigarette smoked. These data suggest that nicotine uptake is a function of individual smoking behavior within product design limits. We conclude from these data that, while FTC yield cannot precisely predict nicotine uptake for an individual smoker, it is useful in predicting and comparing actual nicotine uptake by smokers who select cigarettes with a particular FTC yield.

Direct determination of cotinine-N-glucuronide in urine using thermospray liquid chromatography/mass spectrometry.

A thermospray liquid chromatographic/mass spectrometric method has been developed for direct determination of cotinine-N-glucuronide in the urine of smokers. Quantification was performed using methyl-d3-cotinine-N-glucuronide as internal standard and monitoring the protonated aglycons. Using a simple preparation, urine samples from four smokers were analyzed and the results compared favorably with those from a previously reported method that quantifies aglycon release following beta-glucuronidase treatment. Amounts of cotinine-N-glucuronide found in urine from smokers ranged from less than 0.7 to 21 nmol ml-1, indicating wide inter-individual variability in the metabolic production of this metabolite. Cotinine-N-glucuronide was found to be the second most abundant urinary nicotine metabolite. A similar method was developed for trans-3'-hydroxycotinine-N-glucuronide but this compound was not detected in smokers' urine.

Determination of 3-quinuclidinyl benzilate (QNB) and its major metabolites in urine by isotope dilution gas chromatography/mass spectrometry.

In response to the scheduled destruction of U.S. military stockpiles of the hallucinogenic agent 3-quinuclidinyl benzilate (QNB), a specific confirmatory test for human exposure to QNB was developed. The amount of the parent compound in the urine as well as the two major metabolites, 3-quinuclidinol (Q) and benzilic acid (BA), was determined because the relationship between QNB dose and levels of QNB and its metabolites in human urine is not known. QNB was determined in urine samples spiked at a target level of 0.5 ng/mL, and the metabolites BA and Q were determined at a target level of 5 ng/mL. The method uses solid-phase extraction to isolate each analyte from the urine and isotope dilution gas chromatography/mass spectrometry for quantitation. Each analyte is converted to its trimethylsilyl derivative for analysis. The analytical method was tested on eight different urine samples spiked with known amounts of the analytes near the target levels, at 10 times the target levels, and blank (unspiked) urine samples. The variabilities in the method are for the most part evenly distributed between three imprecision categories: GC/MS measurement, sample preparation, and the urine samples. The total imprecision (1 standard deviation) of a single measurement is about 15% of the value for each analyte.

Evidence for urinary excretion of glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine in smokers.

Urinary nicotine metabolic output was profiled for 11 smokers who smoked their regular cigarette brands ad libitum. Thermospray liquid chromatography/mass spectrometry was used to monitor nicotine and eight metabolites, including glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine that were determined indirectly using enzyme hydrolysis. These results were used to estimate an average, steady-state concentration in a 24-hr urine sample during ad libitum smoking and to assess interindividual variability in the excretion of these metabolites. The variability in absolute amount among the nine analytes ranged from 35 to 70% for these smokers. The glucuronide conjugates constituted an average of 29% of all urinary metabolites monitored in this study. trans-3'-Hydroxycotinine in the free form constitutes the largest single metabolite in smokers' urine, with an average of 35% of the total. The sums of nicotine metabolites determined here are very close to the Federal Trade Commission yields of nicotine for the total number of cigarettes smoked by these subjects during the urine collection interval. These results indicate that a large proportion of the nicotine absorbed while smoking can be accounted for as urinary metabolites of nicotine, including glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine.

Characterization of the glucuronide conjugate of cotinine: a previously unidentified major metabolite of nicotine in smokers' urine.

Recent studies in our laboratories have confirmed that a major unidentified metabolite of nicotine in smokers' urine was susceptible to enzymatic degradation by beta-glucuronidase to afford (S)-(-)-cotinine. In order to establish the identity of this metabolite, the quaternary ammonium conjugate, viz., (S)-(-)-cotinine N-glucuronide, was synthesized. Reaction of methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucopyranuronate with (S)-(-)-cotinine at 60 degrees C for 3 days affords the fully protected conjugate as the bromide salt. Deprotection was accomplished in 1 M NaOH overnight at 25 degrees C. The deprotected inner salt was isolated by Dowex-50W cation-exchange chromatography. Electrospray mass spectra of the inner salt revealed the presence of ions with m/z 353 (M + H)+, 375 (M + Na)+, and 391 (M + K)+ as well as ions resulting from loss of water and cleavage of the glycosidic bond. Proton and carbon nuclear magnetic resonance spectra established that the position of glucuronidation was the pyridyl nitrogen. The magnitude of the coupling between H1" and H2" of the sugar ring (8.71 Hz) and nuclear Overhauser enhancements were consistent with the beta-isomer of the glucuronide conjugate. The synthetic (S)-(-)-cotinine N-glucuronide was susceptible to enzymatic hydrolysis by beta-glucuronidase to afford (S)-(-)-cotinine. Application of a cation-exchange high-performance liquid chromatographic method enabled the collection of a fraction containing (S)-(-)-cotinine N-glucuronide from a smoker's urine. The electrospray mass spectrum of this fraction contained ions consistent with the presence of (S)-(-)-cotinine N-glucuronide. The concentrated fraction was subjected to enzymatic hydrolysis by beta-glucuronidase to afford (S)-(-)-cotinine.(ABSTRACT TRUNCATED AT 250 WORDS)

Isotope dilution gas chromatography-mass spectrometry in the determination of benzene, toluene, styrene and acrylonitrile in mainstream cigarette smoke.

A cryogenic trapping method with isotope dilution gas chromatography-mass spectrometry analysis has been developed for the determination of benzene, toluene, styrene and acrylonitrile in mainstream vapor phase cigarette smoke. The method is simple, direct, and quantitative. Vapor phase samples are collected cryogenically in a series of four traps following removal of the particulate phase with a Cambridge filter pad. For all four analytes, 75-85% of the total amounts recovered were found in the initial trap and less than 1% in the final trap. Assessment of instrumental precision by multiple injections of a sample gave relative standard deviations of less than 2%. Linear calibration for all analytes over the analysis range gave an r2 value greater than 0.99 with average relative standard deviations at the mean ranging from 1.4 to 8.2%. The cigarettes analyzed include a reference cigarette (Kentucky 1R4F), a commercial ultra-low "tar" mentholated cigarette, and two cigarettes that heat but do not burn tobacco. The values determined for the four analytes in the 1R4F samples are comparable to reported values of similar cigarettes. The cigarettes which heat rather than burn tobacco yield less of all four analytes compared to the other cigarettes in the study.