Specific Detection of Anabasine, Nicotine, and Nicotine Metabolites in Urine by Liquid Chromatography Tandem Mass Spectrometry

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1 Clinical Chemistry / DETECTION OF TOBACCO ALKALOIDS IN URINE Specific Detection of Anabasine, Nicotine, and Nicotine Metabolites in Urine by Liquid Chromatography Tandem Mass Spectrometry Andrew N. Hoofnagle, MD, PhD, Thomas J. Laha, MT(ASCP), Petrie M. Rainey, MD, PhD, and Sayed M.H. Sadrzadeh, PhD Key Words: Anabasine; Nicotine; Cotinine; Nornicotine; Trans-3'-hydroxycotinine; Liquid chromatography mass spectrometry; Urine; Direct injection; Interference Abstract The sensitive and specific detection of nicotine, its metabolites, and the tobacco alkaloid anabasine is useful in evaluating the success of smoking cessation treatments and detecting tobacco use, passive exposure, and nontobacco nicotine exposure in potential transplant recipients, insurance clients, and elective surgical patients. Rapid sample preparation and extended high-performance liquid chromatographic separation of tobacco alkaloids and metabolites was interfaced with tandem mass spectrometry. By using deuterated internal standards and appropriate confirmatory ion mass transitions, direct injection of centrifugally clarified urine was possible. The method had excellent precision, limit of quantitation, and linearity. The rigorous separation method revealed an interferent of nicotine that had coeluted with anabasine in more rapid chromatography and that may result in tobacco use misclassification. The method provides more specific detection of tobacco exposure and illustrates the potential of centrifugal clarification for sample preparation in the detection of multiple analytes in urine. Nicotine is a plant alkaloid found in certain vegetables (eg, potatoes, green peppers, and tomatoes) at very low concentrations and in tobacco products (eg, cigars, cigarettes, chewing and dipping tobacco, and snuff) at very high concentrations. 1 Approximately 1 to 2 mg of the nicotine present in a cigarette is absorbed by the buccal and respiratory mucosa of smokers. 2 Once circulating in the bloodstream, nicotine acts as a central and peripheral nervous system stimulant. In addition, nicotine is highly addictive and is associated with shortterm adverse health effects, including elevated blood pressure, heart rate, and blood glucose levels. 3 Besides increased cancer rates, long-term tobacco use is associated with increased incidence of atherosclerotic arterial disease, chronic obstructive pulmonary disease, hypertension, and low birth weight of infants born to mothers who smoke. 2-5 For patients undergoing surgery, tobacco and nicotine use have important implications. Several studies have shown delayed healing times and increased infection and thrombosis rates in nicotine users after surgical procedures. Tobacco use in patients receiving organ transplants is associated with elevated rates of graft loss, mortality, and new cancer. For these reasons, assays for nicotine and its metabolites in the urine of patients scheduled for elective surgery or organ transplantation can help in planning surgery and establishing transplant eligibility. 6-8 Nicotine replacement therapies (ie, chewing gum, medicated patch, and inhalants) are highly purified nicotine preparations designed to reduce the desire for tobacco products. Smoking cessation programs use these therapies to replace the physiologic need for nicotine from tobacco products while using other modalities to reduce the psychological desire to use tobacco. 9 The success of these programs can be accurately evaluated only if tobacco and nontobacco nicotine users are 880 Am J Clin Pathol 2006;126: Downloaded 880 from

2 Clinical Chemistry / ORIGINAL ARTICLE categorized appropriately. Distinguishing the 2 groups requires the detection of tobacco alkaloids distinct from nicotine and its metabolites. One such alkaloid that has been well studied is anabasine. 10 Previous assays for nicotine and/or metabolites in urine have used many different technologies, including chromatographic techniques interfaced with mass spectrometry, thinlayer chromatography, and several immunologically based detection systems Each of these methods has its advantages. However, the chromatographic assays can have the capability of being more specific, especially if interfaced with mass spectrometry (MS) or tandem mass spectrometry (MS/MS). In addition, when interfaced with MS, chromatographic techniques are able to detect and quantitate anabasine, enabling them to distinguish tobacco users from nontobacco nicotine users. Electrospray ionization mass spectrometry requires samples that are relatively free of salts and other contaminating ion-suppressive compounds. Published sample preparation steps range from syringe filtration to solid phase extraction (SPE) However, to adequately resolve anabasine from an unrelated isobaric compound present in the urine of tobacco and nicotine replacement users, we found that more rigorous chromatography was required. Because urine is relatively protein-free and because prolonged chromatography removes many ion-suppressive compounds in-line, we thought it might be possible to use a simple and rapid specimen preparation. In evaluating rapid preparation steps, we demonstrated that centrifugal clarification (removal of particulate matter by simple centrifugation) was sufficient. Accordingly, this method provided very specific detection and quantitation of anabasine in urine with limited sample preparation. Materials and Methods Reagents Potassium hydroxide and chromatography-grade methanol were obtained from Fisher (Houston, TX), glacial acetic acid from J.T. Baker (Phillipsburg, NJ), dibasic potassium phosphate from Mallinckrodt (Phillipsburg, NJ), Oasis HLB SPE columns from Waters (Milford, MA), 0.2-µm nylon and cellulose acetate microcentrifuge filters from Alltech (Deerfield, IL), urine Multistix 10 SG from Bayer (Tarrytown, NY), and ammonium acetate from ICN Biomedicals (Aurora, OH). Nicotine, cotinine, nornicotine, anabasine, and cotinine-d 3 were from Sigma-Aldrich (St Louis, MO); trans-3'-hydroxycotinine, trans-3'-hydroxycotinine-d 3, nornicotine-d 4, anabasine-d 4, and isonicotine were from Toronto Research Chemicals (North York, Canada); and nicotine-d 4 was from Cerilliant (Round Rock, TX). Calibrator and control samples were made up in certified drug-free urine from UTAK Laboratories (Valencia, CA). Preparation of Samples Each urine, control, and calibrator specimen was spiked with 0.1 volume of an internal standard solution containing 210 ng/ml of each deuterated analog in methanol. SPE was performed as described previously. 16 Centrifugal filtration was performed using nylon or cellulose acetate membrane filters spinning at 3,000g for 5 minutes in a swinging bucket rotor, with the filtrate used for further analysis. Alternatively, for centrifugal clarification, samples were spun at 13,000g for 5 minutes in a fixed angle rotor, with the supernatant used for analysis. The extracts, filtrates or supernatants, respectively, were used directly in the analysis described in the following text. High-Performance Liquid Chromatography Separation Separation of analytes was performed using Waters Alliance 2795 high-performance liquid chromatography (HPLC) and a mm Restek (Bellefonte, PA) pentafluorophenyl-propyl column with guard column at 35 C. Mobile phase A consisted of 2 mmol/l of ammonium acetate/10 mmol/l of acetic acid in water and mobile phase B of 2 mmol/l of ammonium acetate/10 mmol/l of acetic acid in 99.5% methanol. The column was first equilibrated with 1 ml/min of 95% A/5% B. Then, a 30-µL sample was injected, and after 2 minutes, the solvent was changed in a single step to 30% A/70% B at a flow rate of 600 µl/min. The eluate without analytes was diverted to waste for an additional 1.5 minutes, after which the flow was decreased to 400 µl/min and the eluate was directed to the mass spectrometer for the next 2.5 minutes to quantitate trans-3'-hydroxycotinine and cotinine Figure 1. Eluate was then diverted to waste for 1 minute at 600 µl/min before quantitating nicotine, nornicotine, and anabasine during the next 5 minutes at 400 µl/min (total run time, 12 minutes). The column was then reequilibrated for 2 minutes at initial conditions before the next injection (total cycle time, 14 minutes). Mass Spectrometry Ions were introduced into a Waters/Micromass Quattro micro API mass spectrometer via electrospray ionization in positive ion mode. The capillary voltage was 0.75 kv; extractor voltage, 2.00 V; RF Lens voltage, 0.2 V; source temperature, 130 C; desolvation temperature, 400 C; cone gas flow, 50 L/h; and desolvation gas flow, 800 L/h. Confirmatory Ion Selection and Analyte Quantification Multiple reaction monitoring data were collected for several significant daughter ions present in the MS/MS spectrum for each analyte. The SD of the ratio of each combination of quantifying and confirming ion was determined for 53 replicates at Downloaded from Am J Clin Pathol 2006;126:

3 Hoofnagle et al / DETECTION OF TOBACCO ALKALOIDS IN URINE Relative Intensity 1 2 Nicotine Nornicotine Anabasine Trans-3'-hydroxycotinine Cotinine Time (min) Figure 1 Chromatogram and qualitative ion suppression analysis. Pictured in black are filled-in traces of ion intensity for each analyte transition. Thin black traces represent the signal detected for each ion transition during the ion suppression analysis described in the Materials and Methods section. Arrow 1 indicates where the flow rate was increased to 600 µl/min and the eluate was diverted away from the mass spectrometer. Arrow 2 indicates when the eluate was directed back to the spectrometer for quantitation of the remaining analytes with a flow rate of 400 µl/min. different analyte concentrations. Confirming ions were selected based on signal/noise, precision of quantifying/confirming ion ratio, and, in the case of anabasine, the ability to avoid interference with isobaric compounds. The transitions tested for each analyte were (precursor ion/fragment ion m/z) as follows: nicotine, 163/80, 163/84, 163/106, 163/130, and 163/132; cotinine, 177/80, 177/98, and 177/146; trans-3'-hydroxycotinine, 193/80, 193/86, 193/106, 193/134, and 193/149; nornicotine, 149/65, 149/70, 149/80, 149/93, and 149/130; and anabasine, 163/80, 163/84, 163/94, 163/120, 163/134, and 163/146. The final method used with the following transitions for each analyte in order of elution (analyte quantifying ion mass transition, confirmatory ion mass transition): trans-3'-hydroxycotinine, 193/80, 193/86; cotinine, 177/80, 177/98; nicotine, 163/132, 163/106; nornicotine, 149/80, 149/130; and anabasine, 163/146, 163/134. For deuterated internal standards, transitions were as follows: trans-3'-hydroxycotinine-d 3, 196/80; cotinine-d 3, 180/80; nicotine-d 4, 167/136; nornicotine-d 4, 153/84; and anabasine-d 4, 167/84. Characterization of the Method Calibrators were made in drug-free human urine at 3 levels for nicotine, cotinine, trans-3'-hydroxycotinine, nornicotine, and anabasine, respectively: low, 2, 2, 10, 2, and 2 ng/ml; medium, 250, 375, 625, 50, and 50 ng/ml; and high, 1,000, 1,500, 2,500, 200, and 200 ng/ml. The analytic measurement range for each analyte was determined by analyzing 6 spiked concentrations of each analyte in drug-free urine. The middle 4 concentrations were equally spaced between the highest and lowest concentrations tested, which are listed in Table 1. Precision for each analyte was determined using 20 aliquots each of low- and high-concentration control specimen analyzed on the same day (intra-assay) or 31 aliquots each of low and high concentration during 7 days (interassay). Low- and high-concentration control specimens contained 25 and 800 ng/ml of nicotine, 37.5 and 1,200 ng/ml of cotinine, 62.5 and 2,000 ng/ml of trans-3'-hydroxycotinine, and 5 and 20 ng/ml of nornicotine and anabasine, respectively. Analytic recovery was assessed for SPE and filtration methods by comparing analytes in water directly injected onto the HPLC column vs processed as described in the preceding text. Low and high concentrations of analytes in water were the same as for precision studies. The limit of quantitation was established using four 2-fold serial dilutions of the lowest calibrator in drug-free urine. Each dilution was analyzed 10 times with the centrifugal clarification method, and the data were extrapolated to an imprecision of coefficient of variation percentage of 20%. Table 1 Method Characteristics of Liquid Chromatography Tandem Mass Spectrometry With Centrifugal Clarification Sample Preparation * Intra-assay Interassay Linearity (ng/ml) Imprecision (CV%) Imprecision (CV%) Limit of Quantitation Analyte Low High Low High Low High (ng/ml) Nicotine 2 4, Cotinine 2 6, Trans-3'-hydroxycotinine 10 10, Nornicotine Anabasine CV, coefficient of variation. * Linearity, imprecision, and sensitivity were determined as described in the Materials and Methods section. 882 Am J Clin Pathol 2006;126: Downloaded 882 from

4 Clinical Chemistry / ORIGINAL ARTICLE Ion suppression was characterized in 2 ways. In the first, a mixture of analytes at 10 ng/ml in water was injected with a T fitting directly into the eluate from a drug-free urine specimen, which had been previously extracted with SPE, centrifugally filtered, or centrifugally clarified. In the second, an analyte mixture was diluted into 10 urine specimens ranging in specific gravity ( ) and protein concentration (0-4+) as determined by urine dipstick. The peak area and final quantitation of each analyte were determined after centrifugal clarification or SPE. Concentrations of analytes for the spiked urine specimens were 250 ng/ml of nicotine, 375 ng/ml of cotinine, 625 ng/ml of trans-3'-hydroxycotinine, and 50 ng/ml of nornicotine and anabasine. Method comparison with a reference laboratory used 29 cotinine-positive specimens and modified Deming regression with Analyse-it Clinical Laboratory, version 1.68 (Analyse-It Software, Leeds, England). Results nonsmoking volunteer 4 hours and 96 hours after chewing nicotine gum, indicating that it is an alkaloid found in tobacco and a metabolite of nicotine. Given the similar but distinct retention times and daughter ion fragmentation patterns of this compound, we hypothesized the compound was nicotine glucuronide. However, nicotine glucuronide eluted between 6 and 7 minutes in our chromatographic run, a period when the eluate is diverted to waste (data not shown). We then hypothesized that the interferent might be isonicotine (1-methyl-3'-(3-pyridyl)pyrrolidine), a ring shifted analog of nicotine. Although isonicotine had the same retention time as the interferent, the daughter spectrum was distinct (data not shown). Therefore, the identity of the isobaric compound remains uncertain. Comparison of SPE and Filtration We hypothesized that the extensive HPLC purification needed to separate nicotine, the isobaric interferent, and anabasine may eliminate the need for SPE described in published methods. 16,18 For this reason, we compared results from samples of analytes in water prepared with centrifugal filtration Development of a New Liquid Chromatographic Technique Previous work has demonstrated the importance of adequate separation of anabasine and nicotine. 16,18,19 The analytes have identical mass, and, although the fragment ion patterns are distinct for each, with very high levels of nicotine, anabasine cannot be quantitated owing to noise when nicotine coelutes. For this reason, we compared several liquid chromatographic techniques. Of the stationary phases tested, including C18, cyano, intrinsically base-deactivated (polar groups embedded within alkyl side chains), and phenyl groups, the combined hydrophilic-lipophilic pentafluorophenyl-propyl column separated nicotine and anabasine most completely (Figure 1). By using this column, a step gradient of 70% methanol at 400 µl/min and a diverting valve with increased flow rates between analysis of cotinine and nicotine, the total run took 14 minutes, including reequilibration. Detection of an Isobaric Interferent As we were determining the optimal column for this separation, we noticed another ion with m/z of 163 eluting separately from nicotine and anabasine in urine from smokers. Indeed, as the separation between nicotine and anabasine improved, the isobaric compound was resolved more completely and eluted as the second of three peaks with m/z of 163 Figure 2 (upper panel). The daughter ion spectrum of peak II was very similar to nicotine (Figure 2). To further characterize the peak, we extracted tobacco and nicotine gum with methanol and analyzed the extract diluted into cotinine-negative urine. The compound was present in tobacco but not in nicotine gum. It was also present in the urine of a healthy Figure 2 Daughter fragment ions of nicotine, anabasine, and an interferent. The uppermost trace is the total ion current detected for the parent mass 163 as daughter ion spectra were obtained throughout chromatographic elution from the high-performance liquid chromatography column of a tobacco extract spiked into drug-negative urine. Three major peaks are detected and labeled I, II, and III. The corresponding daughter fragmentation patterns for each peak are illustrated below the total ion current trace. Peak I corresponds to nicotine and peak III to anabasine; peak II is an isobaric interferent. Downloaded from Am J Clin Pathol 2006;126:

5 Hoofnagle et al / DETECTION OF TOBACCO ALKALOIDS IN URINE and SPE. Interestingly, nornicotine and cotinine seemed to become partially adsorbed to the nylon and cellulose acetate membranes to give similar extraction efficiencies to SPE (mean absolute recovery range for the 5 analytes, 56%-106%). The imprecision (coefficient of variation percentage) was also similar between the 2 methods (mean interassay imprecision range, 4%-24%). Because the 2 methods both seemed to suffer from analyte loss during sample preparation, we moved to centrifugal clarification as another possible preparation method. Method Characteristics The performance characteristics of the final method using centrifugal clarification and liquid chromatography (LC)- MS/MS are outlined in Table 1. Analytic measurement ranges encompassed entire clinically relevant ranges, which include concentrations observed in unexposed, passively exposed, actively tobacco-exposed and nontobacco nicotine exposed, and tobacco users abstaining for up to 2 weeks. 2,3,18,20 The imprecision, interassay and intra-assay, was less than 8% for all analytes and less than 5% for nicotine and cotinine. Limits of quantitation for each analyte compared very well with published methods ,21-23 Ion Suppression Ion suppression was evaluated in 2 ways. First, a solution containing constant concentrations of each analyte was infused into the eluate from the HPLC separation of a negative urine specimen (Figure 1). Qualitatively, each of the analytes eluted at times reasonably free of ion suppression, which is detected as decreased signal from the infused analyte. Second, we quantitatively determined the effect of ion suppression on results generated with our method by analyzing 10 drugscreen negative urine specimens with a range of specific gravity values and protein concentrations, each spiked with known amounts of analytes Table 2. Trans-3'-hydroxycotinine elutes early and closest to the void volume, where the majority of the small molecule contaminants are expected to elute. For the other analytes, ion suppression caused at most 25% variability in the final quantitation. These mean relative recoveries were similar to urine specimens prepared with off-line SPE, where recoveries ranged from 84% to 151% for nicotine and cotinine. Further analysis of the data demonstrated that when the peak area of the internal standard was substantially decreased owing to ion suppression, imprecision in the final result was significantly higher. We determined that for all analytes, up to 90% suppression of the internal standard peak area could be tolerated before imprecision exceeded 25%. Method Comparison With Reference Laboratory We next compared our new method with the results from a reference laboratory that used a previously published LC- MS/MS method. 18 For the most part, the comparative method displayed a positive bias compared with our method Table 3. The large intercept for nicotine points to the increased Table 2 Recovery of Analytes From 10 Drug-Free Urine Specimens * Sample No. Trans-3'-hydroxycotinine Cotinine Nicotine Nornicotine Anabasine * Mean recoveries of two concentrations run in duplicate. Data are given as percentages. Table 3 Method Comparison With That of Another Reference Laboratory Analyte N Range (ng/ml) Slope Intercept Pearson Correlation (r) Nicotine , Cotinine , Trans-3'-hydroxycotinine , Nornicotine Anabasine Anabasine with interferent * * Values derived by summing anabasine and isobaric interferent signal as described in the text. 884 Am J Clin Pathol 2006;126: Downloaded 884 from

6 Clinical Chemistry / ORIGINAL ARTICLE variability between the 2 methods at higher concentrations of this analyte. In addition, the 2 methods correlated poorly for trans-3'-hydroxycotinine and anabasine. Similar to previously published methods, trans-3'-hydroxycotinine elutes close to the void volume, and increased ion suppression likely explains the large variations between our results and the results of the comparative method. Characterization of Interference by the Nicotine/Anabasine Isobaric Compound The differences seen for anabasine were more difficult to explain. Of 29 cotinine-positive specimens, 7 were negative for anabasine by our method but positive at more than 2 ng/ml by the comparative method. Suspecting that the nicotine/anabasine isobaric compound might be responsible for the discrepant anabasine levels seen in the comparative method, we recalculated a correlation coefficient between the reference method laboratory result and the sum of the anabasine concentration and 13% of the concentration of the interferent using the anabasine calibration curve and the 163/80 transition in our method; the Pearson correlation coefficient improved from 0.72 to 0.88, and the slope of the Deming regression rose from 0.60 to 1.07 (Table 3). Although not conclusive, the aggregate of this evidence is suggestive of isobaric interference with anabasine in the comparative method. Confirmatory Ion The specificity of LC-MS/MS is derived from the combination of the retention time, the parent ion mass, and the daughter ion mass for an analyte. With consistent elution and fragmentation conditions from run to run, ratios of quantitative ions to confirmatory ions can also be characteristic for a given molecule. To assess the effects of ion suppression and variable analyte concentrations on these ratios, we analyzed 20 replicates of 2 control specimens and 13 replicates of the low standard. The target ratios and the SD of the ratios for each analyte are shown in Table 4. In addition, because the daughter ion spectra are similar for nicotine and the isobaric interferent (Figure 2), it might be expected that the ratio would be very similar as well. However, when we determined the ratio for the isobaric interferent using the nicotine-specific transitions (132/106), it was significantly different at 0.65 ± 0.03 (mean ± SD) from that for nicotine at 1.24 ± 0.04 (P <.0001). The ratio observed for the isobaric compound using the anabasine-specific transitions (146/134) was also significantly different from that for anabasine, 0.45 ± 0.25 vs 2.71 ± 0.62 (P <.0001). Based on the variance data, we chose a cutoff of ±2.5 times the SD for each ratio as positive identification of each analyte. Discussion We have developed a highly specific method to measure nicotine and its metabolites, as well as the tobacco alkaloid anabasine, in urine. This method requires only centrifugal clarification of the specimen before analysis by LC-MS/MS. It is linear over 3 to 4 orders of magnitude and has good precision, an excellent limit of quantitation, and typical interference by ion suppression. It is important to note that it avoids potential contamination of the anabasine quantitation by an isobaric compound. Improvement of Specificity With Better LC and Qualifying Ions Distinguishing tobacco from nontobacco nicotine exposure in research and evaluation of patients for transplantation eligibility or for insurance premium adjustment relies on specific detection of anabasine and other tobacco alkaloids. 10,24 Anabasine is a plant alkaloid absorbed during tobacco use that is not present in nicotine replacement therapies. Nornicotine, because it represents less than 1% of the metabolites of a nicotine load, will be only slightly elevated in people exposed to nicotine replacement therapies and can be used with anabasine to assess tobacco exposure. By extending the chromatographic Table 4 Ratio of Quantifying Ion to Confirmatory Ion for Each Analyte * Quantifying Confirmatory Empirically Transition Transition Mean SD (CV%) Acceptable Range Nicotine 163/ / (6.4) Cotinine 177/80 177/ (4.4) Trans-3'-hydroxycotinine 193/80 193/ (8.6) Nornicotine 149/80 149/ (11.6) Anabasine 163/ / (22.9) Interferent 163/ / (4.6) Interferent 163/ / (55.6) CV, coefficient of variation. * Mean and SD for nicotine, cotinine, trans-3'-hydroxycotinine, nornicotine, and anabasine were determined from 53 ratios for each analyte. Mean and SD for the isobaric interferent were determined from 15 ratios. Ratio determined using transitions for nicotine. Ratio determined using transitions for anabasine. Downloaded from Am J Clin Pathol 2006;126:

7 Hoofnagle et al / DETECTION OF TOBACCO ALKALOIDS IN URINE run, we were able to achieve more reliable identification of anabasine. More specifically, we have documented that for at least 23% of our clinical specimens, the comparative method classified the patient as a tobacco user when we could find no evidence of this. Although this does not interfere with recognizing patients using nicotine, it does affect research into smoking-cessation therapies. In fact, this type of interference could cause tobacco replacement therapies to seem less effective than they actually might be. Confirmatory, or qualifying, ion ratios are used to confirm the identity of mass fragments in MS; often an arbitrary cutoff of ±20% to 25% is used to rule out interference. By examining the ratios of each of our analytes at different concentrations, we demonstrated nonoverlapping ratio ranges for 3 isobaric molecules nicotine, anabasine, and the interfering analyte eluting at 9.8 minutes (Table 4). The data presented herein indicate that arbitrary ratios may be overly general for use with analytes that have isomeric interference but that the development of method specific ranges of acceptable ratios is possible. More Complete Chromatographic Separation Adequately Removes Suppressive Ions A potential complication that must be evaluated in the development of any LC-MS/MS technique is ion suppression. It results from the inhibition of analyte ionization at the electrospray source due to other polyatomic ions and inorganic ions. The effects of ion suppression can be compensated with deuterated analogs that elute simultaneously. However, severe ion suppression will result in imprecise quantitation, even with a deuterated analog. Because sample cleanup is minimal with centrifugal clarification, ion suppression could present a significant difficulty. We investigated this potential problem by analyzing 10 different drug-free urine specimens with a variety of protein and salt concentrations that had been spiked with the analytes of interest. For all of the analytes except trans-3'-hydroxycotinine, variability was less than 25% owing to ion suppression, similar to the variability observed when SPE was used. Fortunately, because extended chromatographic separation was necessary for accurate identification of analytes, SPE was not required for the removal of suppressive ions. Overall Benefits of Clarification vs SPE Because extraction and derivatization have been necessary for quantitative gas chromatography MS methods, LC- MS/MS seems to be an ideal replacement to achieve the rapid, sensitive detection of small-molecule analytes in urine. Traditional liquid-liquid extraction methods require derivatization for adequate recovery of many analytes of interest. As recently demonstrated by Nordgren and Beck 25 and Nordgren and colleagues, 26 use of unextracted urine specimens may be possible for general toxicologic urine drug screens. This would save time and money and would provide a more sensitive method of detection for more compounds than current methods. Our new method for the detection of nicotine, its metabolites, and anabasine in unextracted urine provides more evidence that this may be an achievable goal. SPE may be necessary to separate analytes from highly proteinaceous matrices, such as serum. Urine represents a much simpler matrix and, as we have shown, is amenable to simple clarification and subsequent LC-MS/MS. This can substantially reduce the time and cost of sample preparation. It is important to note that centrifugal clarification also avoids analyte loss during extraction. In 1 SPE-based method, analytic recovery of nicotine and its metabolites from urine was less than 85% for every analyte and as low as 21% for trans-3'- hydroxycotinine. 18 Because this step was entirely removed in our new method, sensitivity was increased, up to 5-fold for trans-3'-hydroxycotinine vs the SPE method. Insights Into Developing Clinical LC-MS/MS Assays From our experiences with isobaric interference in anabasine quantitation, we include a word of caution. When developing HPLC-mass spectrometric techniques for use in the clinical laboratory, we think it is prudent to rule out interferents with identical mass. For example, as demonstrated herein, one may start with a rigorous liquid chromatographic separation and evaluate patient specimens for interferents with identical mass in analyte-positive patients. Simply demonstrating that small-molecule pharmaceuticals and a few of their metabolites do not interfere with the analysis of laboratory-derived calibrators may not be sufficient. Conclusions LC-MS/MS is an appealing alternative to the immunologically based assays requiring expensive reagents that may vary in performance and occasionally be in limited supply. In addition, sample preparation may be more straightforward than in gas chromatography MS techniques. As we describe, the detection of small-molecule analytes in urine by direct injection onto an LC-MS/MS platform is possible and has acceptable sensitivity, linearity, and imprecision and remarkable specificity. It is important to note that when isomeric interference is present and extended HPLC separation of analytes is needed, the speed of sample preparation that centrifugal clarification provides can compensate for increased chromatographic duration. Our LC-MS/MS technique offers improved specificity for the detection of anabasine, the bestcharacterized marker for tobacco exposure. From the Department of Laboratory Medicine, University of Washington Medical Center, Seattle. 886 Am J Clin Pathol 2006;126: Downloaded 886 from

8 Clinical Chemistry / ORIGINAL ARTICLE Address reprint requests to Dr Hoofnagle: Dept of Laboratory Medicine, University of Washington Medical Center, Campus Box , 1959 NE Pacific, Seattle, WA References 1. Doolittle DJ, Winegar R, Lee CK, et al. The genotoxic potential of nicotine and its major metabolites. Mutat Res. 1995;344: Bergen AW, Caporaso N. Cigarette smoking. J Natl Cancer Inst. 1999;91: Yildiz D. Nicotine, its metabolism and an overview of its biological effects. Toxicon. 2004;43: Centers for Disease Control and Prevention. Annual smoking: attributable mortality, years of potential life lost, and economic costs: United States, MMWR Morb Mortal Wkly Rep. 2002;51: Lackmann GM, Salzberger U, Tollner U, et al. Metabolites of a tobacco-specific carcinogen in urine from newborns. J Natl Cancer Inst. 1999;91: Wing KJ, Fisher CG, O Connell JX, et al. Stopping nicotine exposure before surgery: the effect on spinal fusion in a rabbit model. Spine. 2000;25: Ehlers SL, Rodrigue JR, Widows MR, et al. Tobacco use before and after liver transplantation: a single center survey and implications for clinical practice and research. Liver Transpl. 2004;10: Kaufman T, Eichenlaub EH, Levin M, et al. Tobacco smoking: impairment of experimental flap survival. Ann Plast Surg. 1984;13: Henningfield JE, Fant RV, Buchhalter AR, et al. Pharmacotherapy for nicotine dependence. CA Cancer J Clin. 2005;55: Jacob P, Yu L, Shulgin AT, et al. Minor tobacco alkaloids as biomarkers for tobacco use: comparison of users of cigarettes, smokeless tobacco, cigars, and pipes. Am J Public Health. 1999;89: Dhar P. Measuring tobacco smoke exposure: quantifying nicotine/cotinine concentration in biological samples by colorimetry, chromatography and immunoassay methods. J Pharm Biomed Anal. 2004;35: Gariti P, Rosenthal DI, Lindell K, et al. Validating a dipstick method for detecting recent smoking. Cancer Epidemiol Biomarkers Prev. 2002;11: Ziegler UE, Kauczok J, Dietz UA, et al. Clinical correlation between the consumption of nicotine and cotinine concentrations in urine and serum by competitive enzymelinked immunosorbent assay. Pharmacology. 2004;72: Ghosheh OA, Browne D, Rogers T, et al. A simple high performance liquid chromatographic method for the quantification of total cotinine, total 3'-hydroxycotinine and caffeine in the plasma of smokers. J Pharm Biomed Anal. 2000;23: Jung BH, Chung BC, Chung SJ, et al. Simultaneous GC-MS determination of nicotine and cotinine in plasma for the pharmacokinetic characterization of nicotine in rats. J Pharm Biomed Anal. 1999;20: Xu X, Iba MM, Weisel CP. Simultaneous and sensitive measurement of anabasine, nicotine, and nicotine metabolites in human urine by liquid chromatography tandem mass spectrometry. Clin Chem. 2004;50: Tuomi T, Johnsson T, Reijula K. Analysis of nicotine, 3- hydroxycotinine, cotinine, and caffeine in urine of passive smokers by HPLC tandem mass spectrometry. Clin Chem. 1999;45: Moyer TP, Charlson JR, Enger RJ, et al. Simultaneous analysis of nicotine, nicotine metabolites, and tobacco alkaloids in serum or urine by tandem mass spectrometry, with clinically relevant metabolic profiles. Clin Chem. 2002;48: Meger M, Meger-Kossien I, Schuler-Metz A, et al. Simultaneous determination of nicotine and eight nicotine metabolites in urine of smokers using liquid chromatography tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2002;778: Hukkanen J, Jacob P, Benowitz NL. Metabolism and disposition kinetics of nicotine. Pharmacol Rev. 2005;57: Cognard E, Staub C. Determination of nicotine and its major metabolite cotinine in plasma or serum by gas chromatography mass spectrometry using ion-trap detection. Clin Chem Lab Med. 2003;41: Bernert JT, Turner WE, Pirkle JL, et al. Development and validation of sensitive method for determination of serum cotinine in smokers and nonsmokers by liquid chromatography atmospheric pressure ionization tandem mass spectrometry. Clin Chem. 1997;43: Bentley MC, Abrar M, Kelk M, et al. Validation of an assay for the determination of cotinine and 3-hydroxycotinine in human saliva using automated solid-phase extraction and liquid chromatography with tandem mass spectrometric detection. J Chromatogr B. 1999;723: Jacob P, Hatsukami D, Severson H, et al. Anabasine and anatabine as biomarkers for tobacco use during nicotine replacement therapy. Cancer Epidemiol Biomarkers Prev. 2002;11: Nordgren HK, Beck O. Multicomponent screening for drugs of abuse: direct analysis of urine by LC-MS-MS. Ther Drug Monit. 2004;26: Nordgren HK, Holmgren P, Liljeberg P, et al. Application of direct urine LC-MS-MS analysis for screening of novel substances in drug abusers. J Anal Toxicol. 2005;29: Downloaded from Am J Clin Pathol 2006;126: