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Clinical Chemistry 45: 2164-2172, 1999;
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(Clinical Chemistry. 1999;45:2164-2172.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Analysis of Nicotine, 3-Hydroxycotinine, Cotinine, and Caffeine in Urine of Passive Smokers by HPLC-Tandem Mass Spectrometry

Tapani Tuomia,1, Tom Johnsson1 and Kari Reijula1

1 Finnish Institute of Occupational Health (FIOH), Indoor Air & Environment Program of the FIOH, Uusimaa Regional Institute, Arinatie 3A, 00370 Helsinki, Finland.
a Author for correspondence. Fax 358-9-5061087; e-mail tapani.tuomi{at}occuphealth.fi


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Background: A method is described for the simultaneous analysis of nicotine and two of its major metabolites, cotinine and 3-hydroxycotinine, as well as for caffeine from urine samples. The method was developed to assess exposure of restaurant and hotel workers to environmental tobacco smoke.

Methods: The method includes sample pretreatment and reversed-phase HPLC separation with tandem mass spectrometric identification and quantification using electrospray ionization on a quadrupole ion trap mass analyzer. Sample pretreatment followed standard protocols, including addition of base before liquid-liquid partitioning against dichloromethane on a solid matrix, evaporation of the organic solvent using gaseous nitrogen, and transferring to HPLC vials using HPLC buffer. HPLC separation was run on-line with the electrospray ionization-tandem mass spectrometric detection.

Results: The detection limits of the procedure were in the 1 µg/L range, except for nicotine (10 µg/L of urine). Still lower detection limits can be achieved with larger sample volumes. Recoveries of the sample treatment varied from 99% (cotinine) to 78% (3-hydroxycotinine).

Conclusions: The method described is straightforward and not labor-intensive and, therefore, permits a high throughput of samples with excellent prospects for automation. The applicability of the method was demonstrated in a small-scale study on restaurant employees.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
In far too many occupations, environmental tobacco smoke (ETS),1 classified by the US Environmental Protection Agency as a class A carcinogen (1), is the greatest single preventable occupational health risk (2)(3)(4)(5). In fact, in the US, it has been estimated that ~50–75% of the nonsmoking population is exposed to ETS either at home or at work (1), making it the major source of indoor pollution. ETS is, however, preventable in the sense that measures can be taken to inhibit the spread of tobacco smoke in indoor environments. This includes the restriction of smoking in premises as well as ventilation and construction of solutions that prevent exposure of nonsmokers to ETS. Both approaches have been used in Finland, where the tobacco act of 1994, restricting smoking in the workplace, was recently updated to include restaurants as well (6). Before, during, and after the adaptation of the new legislation, it has been of interest to evaluate the impact of ETS in occupational environments (7). This is particularly urgent inasmuch as it concerns heavily exposed groups, such as hotel and restaurant workers.

Dispite a high affinity to surfaces and textiles, with resulting off-gassing from furnishings, nicotine frequently is considered the best marker of ETS in indoor air, because it is specific to tobacco (8)(9)(10). Approximately 95% of tobacco-derived nicotine is present in the gas phase in concentrations inherent to indoor air, i.e., in concentrations below or slightly above 30 µg/m3 (5)(11)(12). Hence, nicotine uptake is the best biological marker for ETS exposure (13). Nicotine, however, is rapidly metabolized, with a half-life of 2 h, to yield more stable metabolites, such as nicotine-1'-N-oxide, nornicotine, and cotinine (14)(15)(16)(17). Cotinine, with a half-life of ~20 h (17), is by far the best documented and most frequently utilized marker (13), although in the urine of both active and passive smokers, 3-hydroxycotinine (3-OH-cotinine) is the predominant nicotine metabolite, corresponding to ~40% of the total nicotine excretion (18)(19)(20). 3-OH-cotinine has been suggested to have a shorter serum half-life than cotinine (18)(21); however, it is beneficial to measure 3-OH-cotinine instead of cotinine, or 3-OH-cotinine alongside cotinine, particularly when monitoring passive ETS exposure using urine samples (22)(23).

Cotinine monitoring, particularly from passive smokers, is associated with several inherent difficulties. The cotinine concentration seldom exceeds 50 µg/L in urine and commonly varies from <1 µg/L to 10 µg/L in urine (2)(13)(24). For active smokers, the concentrations are rarely <100 µg/L, with mean values in most studies amounting to several hundred nanograms per milliliter of urine or milligram of creatinine (25)(26)(27)(28). In addition, HPLC methods that rely on nonspecific detection, such as the traditionally used ultraviolet (UV) detection at 260 nm, tend to have a high interference of caffeine because it is difficult to separate caffeine from cotinine by means of HPLC (29)(30)(31). In nations in which per capita coffee or tea consumption is high, such as Finland, this is of particular concern because the caffeine concentrations for most nonsmokers are several orders of magnitude higher than the cotinine concentrations.

Consequently, to facilitate accurate and reliable measurements of passive smoking, a limit of quantification (LOQ) for cotinine in urine approaching 1 µg/L must be met, and caffeine interference must be accounted for. To this end, methods using compound-specific detection, such as mass spectrometry, are preferable to less specific methods. Recently, Bernert et al. (32) and Bentley et al. (33) have published HPLC-atmospheric pressure chemical ionization tandem mass spectrometry (MS/MS)-based methods, with a LOQ of 0.05 µg/L from serum and saliva samples, respectively. In addition, Pacifici et al. (34), McManus et al. (35), and Rustemeier et al. (36) have reported HPLC-MS methodology for the same purpose (Table 1 ).


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  		Table 1. LOD for cotinine analysis, using various selective detectors with accompanying chromatographic separation.1

In forthcoming studies, we will assess the exposure of restaurant and hotel employees to ETS. This requires the simultaneous analysis of nicotine, cotinine, and 3-OH-cotinine at concentrations relevant to passive smoking. For this aim, a new method had to be developed because no published methods were suited to the equipment at hand. This report describes the adaptation of the method of Pichini et al. (30) to electrospray ionization (ESI)-MS/MS detection of cotinine, as well as the introduction of this methodology for the investigation of passive smoking in restaurant environments. Further reliability was sought by including nicotine, 3-OH-cotinine, and caffeine to the list of measured compounds.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
chemicals
Native cotinine [(-)-cotinine, 98%] and caffeine (98%; 1.0 g/L in methanol ± 5%) were purchased from Sigma. 1'-N-Ethylnorcotinine was provided by Dr. Georg B. Neurath (Contract Research, Hamburg, Germany). Nicotine (98%) was obtained from BDH Chemicals. All solvents were of either HPLC or proanalysis grade. Water was of MilliQ (Millipore) quality.

subjects and sampling
Urine samples for monitoring of passive smoking were collected from three restaurants where smoking was allowed, in Jyväskylä, in central Finland, during 1 consequent working week. One working week constituted 5 subsequent days, with a routine day lasting 8 h. Two nonsmokers were recruited from each restaurant. The subjects donated samples twice each day, once before the work shift and once after the shift. The restaurant workers were asked to record the length of each work shift, as well as to crudely estimate the number of customers present during the shift and the number of cigarettes smoked per customer.

Urine samples were stored at -20 °C for a maximum of 2 weeks before delivery. Frozen samples were shipped by express mail to the laboratory. Delivery lasted ~4 h and allowed for melting of samples. On arrival, samples were kept at 5 °C for a maximum of 2 days before analysis.

sample preparation
A 1.5-mL aliquot of urine, with 15 µL of 1'-N-ethylnorcotinine (100 mg/L) as internal standard, was thoroughly mixed with 1.4 mL of 0.5 mol/L sodium hydroxide. Samples were then transferred to prepacked Extrelut®-3 glass columns (Merck KGaA). The columns had been washed with 15 mL of dichloromethane and left to dry overnight 1 day before analyses.

After sample application, two different extraction conditions were compared. The first conditions were according to Pichini et al. (30), and included elution of lipophilic substances, including the nonionized forms of caffeine, cotinine, 3-OH-cotinine, nicotine, and 1'-N-ethylnorcotinine, with 15 mL of dichloromethane. The dichloromethane phase eluting from the column was retained. In the second extraction procedure, the modifications suggested by Zuccaro et al. (31) were included. This included extraction of the Extrelut-3 column with 15 mL of 900 mL/L dichloromethane-100 mL/L isopropyl alcohol and the addition of 300 µL of methanolic HCl (25 mmol/L) to the organic phase after extraction. Regardless of the extraction procedure used, the organic phase eluting from the column was stored in 15 mL Extrelut coned glass vials (Merck) and evaporated to dryness under pressurized nitrogen before transfer to HPLC vials, using 150 µL of HPLC buffer (aqueous acetate-methanol-acetonitrile; 736:245:20, by volume). The acetate solution contained 8.73 mmol/L NH4CH3CO2, 27.2 µmol/L NaCH3CO2 · 3 H2O, and 1.625 mL/L glacial acetic acid at pH 4.3.

hplc conditions
The analytes were introduced to the MS detector by injecting 20 µL of sample through a HPLC system consisting of an Alliance 2690 separations module (Waters) connected to a µBondapak C18 2 x 300 mm column (particle size, 10 µm; Waters) operated at 30 °C (column oven model 7981; Jones Chromatography). Samples were separated isocratically, using a methanol-acetonitrile-aqueous buffer solvent system (see above) at flow rate 0.5 mL/min. Runtime was 15 min.

esi-collision-induced dissociation ms/ms
Mass spectral analysis was performed on a Finnigan LCQ (Finnigan) fitted with an ESI probe. The operating conditions were optimized in the working flow range using caffeine and were as follows: The ESI probe was operated in the positive-ion mode and set at a voltage of 4.3 kV. Pressurized nitrogen (6.90 kPa) was used as auxiliary and sheath gas with a flow rate of 20 L/min and 45 L/min, respectively. Helium was used for collision-induced dissociation at a pressure of 2.75 kPa. Capillary temperature was set to 225 °C and capillary voltage to 3.0 V with a tube lens offset of 60 V. The system includes two octapole ion guides with an interoctapole lens in between. The first octapole DC offset potential was -2.93 V, and the second was -5.49 V, with the interoctapole lens voltage set at -14.82 V and the octapole RF amplitude at 400. The electron multiplier voltage was set to -800 V. Maximum inject time was 200.03 ms and total microscans set to three. It should be pointed out, however, that these operating conditions are optimal, or nearly optimal, for the specific instrument used in this study only and that optimization should be done individually for other similar instruments.

method yield, linear range, and error limits
The error limits of the method were measured by performing a double-sided Student t-tests with 95% confidence intervals on the deviation from the mean of 36 calibrators of nine different concentrations prepared in distilled water. The calibrators all contained the same concentration of internal standard (Table 2 ). The calibrators were subjected to the same treatment as the samples. The overall recoveries of the compounds were expressed as the mean recovery of the calibrators. The term "recovery" in this report means the amount of substance obtained in the last quantification step in relation to the amount of substance added to the material before extraction, and is expressed as a percentage.


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  		Table 2. Recovery of extraction procedure and linearity, accuracy, and sensitivity of the detection methods.

precision and accuracy
Separate dilutions of calibrators were prepared in urine to assay compound-specific precision and accuracy of the method. Six replicate analyses at three different points spanning the concentration range of interest were analyzed to yield precision expressed as the coefficient of variation (CV) and accuracy (percentage of expected value) at these points (Table 3 ). For further assurance, six replicates of the same blank urine was analyzed (Table 3 ).


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  		Table 3. Replicate analyses of supplemented urine for estimation of precision and accuracy.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
sample pretreatment
The recovery of measured compounds in the basic sample pretreatment procedure varied from 99% for cotinine to a less acceptable 34% and 30% for 3-OH-cotinine and nicotine, respectively (Table 2Up ). Modifying the solid-phase extraction procedure as suggested by Zuccaro et al. (37) raised the recoveries of nicotine and 3-OH-cotinine to >75% (Table 2Up ). The method was modified from the method of Pichini et al. (30), which included both cotinine and 3-OH-cotinine. It seems that this methodology will yield a better recovery than traditional liquid-liquid extraction for 3-OH-cotinine but not for cotinine (30)(32). In addition, the present method is easy to automate because it allows for a high throughput of samples, with few and not very labor-intensive sample treatment steps. In our laboratory, this meant that 24 samples could be treated by one person, in 1 working day, still allowing time to set up and carry out HPLC-MS/MS analyses of the previous day’s samples.

chromatography and detection
Chromatographic conditions were adapted to the ESI process, while at the same time ensuring reasonable chromatographic separation between all compounds within a short runtime. Positive-ion mode ESI was clearly more effective than negative ionization of these alkaloids. Protonated molecules accounted for >60% of ionized species when an acidic ammonium acetate buffer was included in the eluent at the low concentration of 10 mmol/L to not impair the ESI.

With the given conditions, the five detected compounds eluted within 8 min. Nicotine and 3-OH-cotinine coeluted to some extent (resolution, 0.80 in UV and 0.52 in MS/MS). All other peaks had resolutions above 1.0, even in MS/MS, where a certain amount of additional band broadening was brought about by the characteristics of the ionization and detection as well as the additional sample line (Fig. 1 ). With the MS detector, peak interference had no bearing because the MS/MS detection enabled separation of coeluting peaks as well as reduction of background influence (Fig. 1 ).



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  		Figure 1. HPLC-UV and HPLC-MS/MS chromatograms of a calibrator containing 20 mg/L each of nicotine, 3-OH-cotinine, cotinine, caffeine, and 1-N'-ethylnorcotinine.

Clearly, the limits of detection (LODs) of the UV detector were insufficient for monitoring passive smoking with the present sample volumes and methodology (Table 2B). MS/MS, however, allowed a LOD of 1 µg/L and a LOQ of 2 µg/L for cotinine. This is sufficient for monitoring passive smoking in premises where the tobacco load is relatively high (2)(13)(24). Previously, a LOQ as low as 0.05 µg/L for cotinine had been achieved by Bernert et al. (32) with serum samples and Bentley et al. (33) with saliva samples. Other HPLC-MS procedures have detection limits similar to the present method. Apart from HPLC-MS or HPLC-MS/MS, monitoring of cotinine from passive smokers can be achieved using gas chromatography-MS (Table 1Up ). The LODs for 3-OH-cotinine and nicotine were 2 and 10 µg/L, respectively (Table 2C). This means that concentrations in the urine of passive smokers are, for the most part, too low to facilitate analysis of nicotine with the present methodology (38). 3-OH-cotinine, on the other hand, can be monitored from the urine of passive smokers with the present methodology because the concentrations of 3-OH-cotinine in urine are higher than the corresponding cotinine concentrations (18)(19)(20)(22)(23).

esi-ms/ms spectra
Similar molecular structures yielded similarities in the ESI-MS/MS fragmentation patterns of nicotine and its analogs (Table 4 ). Consequently, all spectra included the pyridinium ion at m/z 80.1. This ion results from the loss of the 1-methylpyrrolidine group in the case of nicotine, and in the case of cotinine, hydroxycotinine, and N'-ethylnorcotinine, from the loss of the 2-pyrrolidinone group, with accompanying substituents. The counter ion, i.e., the protonated pyrrolodine or pyrrolidinone groups, resulting from loss of the pyridyl group was present in the spectra of nicotine (m/z 84), N-ethylnorcotinine (m/z 112), and cotinine (m/z 98). Another common fragment was the indole ion at m/z 118. This ion, however, was not included in the spectrum of nicotine because nicotine, contrary to the other tobacco alkaloids, lacks the carbonyl oxygen at position 2, which would facilitate the formation of the proposed ion (Table 4 ).


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  		Table 4.

For quantification purposes, the combined abundance of the three major fragments, or in the case of nicotine the two major fragments, were used (Table 4Up ; Fig. 1Up ). In other words, in the first MS stage the protonated molecular ions were selected as base peaks, i.e., they were trapped in the ion trap. In the second MS stage, the entrapped ions were fragmented by collision with helium and full-scan spectra were acquired. From each acquired compound-specific spectrum, the combined intensities of the two or three major peaks were used to quantify peaks. It follows from this that the identity of each peak was verified by the presence of the spectra shown in Table 4Up . This approach gave a better correlation with concentration than the abundance of a single peak or the use of single reaction monitoring, although single reaction monitoring would have yielded a lower LOQ by compromising selectivity.

precision and accuracy of method
The accuracy over the whole calibration concentration interval using different methods of detection and sample preparation is expressed as the standard error of the estimate (Sy|x; Table 2Up ). Clearly, the modifications suggested by Zuccaro et al. (37) improved the accuracy of the method for nicotine without having any negative effect on the accuracies for the other compounds. This was, therefore, the method of choice. When this method was used, the precision and accuracy of six replicate analyses at three different concentrations of each compound added to urine was calculated (Table 3Up ). The precision (CV) and accuracy (percentage of expected value) of all compounds within the concentration range of interest were excellent, considering the properties of the ion trap, and supported the compound-specific LOQs and LODs (Tables 2C and 3). The blank urine used in quality control contained caffeine in excess of 1000 µg/L, which exceeded the highest concentration used for calibration (Table 2C). Judging from the accuracy and precision of the measurements, however, the supplemented urine samples were within the linear range of the method (Table 3Up ). Nevertheless, studies aimed at measuring caffeine in urine of coffee- or tea-consuming subjects would benefit from a 100-fold dilution of urine samples before sample preparation.

application of the method for monitoring the ets and caffeine loads of restaurant workers
The three workplaces examined differed in their customer types and frequencies as well as their ventilation properties. Therefore, the ETS concentrations at different sites were certain to be different, as were the resulting mean cotinine and hydroxycotinine values (Fig. 2 ). The nicotine concentrations in the indoor air were not measured in this study, although this would certainly have contributed valuable information, because the emphasis was on methodology pertaining to cotinine and hydroxycotinine in subjects in restaurant environments.



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  		Figure 2. Cotinine (A) and 3-OH-cotinine (B) load during working week in two nonsmoking workers from restaurants A (A/1 and A/2), B (B/1 and B/2), and C (C/1 and C/3).

The cotinine concentrations in all subjects remained well below the 50 µg/L cutoff value that can be used to distinguish active smokers from nonsmokers exposed passively to ETS (13). There was a clear correlation between cotinine and hydroxycotinine values (Pearson correlation coefficient r = 0.86). Surprisingly, measured hydroxycotinine values were in some cases up to 10-fold higher than the corresponding cotinine values, suggesting background interference. However, as mentioned previously, there are indications that the hydroxycotinine concentration in urine significantly exceeds the cotinine concentration in both active and passive smokers. In fact, the differences can be as high as 10-fold (36). This is somewhat contradictory if one is to assume that cotinine is the sole source of hydroxycotinine, unless the terminal half-life of cotinine is shorter than that of its oxidation product, 3-OH-cotinine. Nicotine values were in most cases below the LOD.

If the restaurant sites and monitoring days are treated as one uniform group, then the postshift cotinine values could not be deemed significantly higher than the preshift values. Weighing the postshift values against the preshift values yielded P <0.40 (Fig. 2Up ). For 3-OH-cotinine and nicotine, the statistical difference was even less significant. There was, however, in all sites, a significant accumulation of both cotinine and hydroxycotinine during the working week. This is perhaps best illustrated graphically (Fig. 2Up ). The accumulation of nicotine metabolites could also be established by averaging the postshift cotinine and hydroxycotinine values from the first and second days and comparing these values with the mean values from the end of the last 2 working days. The mean postshift values at the end of the last 2 working days were higher with a statistical significance of 0.95 (P <0.05). It seems, therefore, that the daily uptake of nicotine exceeded excretion and that during days off, when the nicotine source was eliminated for 2 whole days, the cotinine and hydroxycotinine concentrations decreased to what they were at the start of the first shift.

Caffeine concentrations were in most cases at least 10-fold higher than either cotinine or hydroxycotinine (data not shown). Clearly, with HPLC conditions similar to those in the present study and with cotinine concentrations as low as those associated with passive smoking, it is wise to distinguish cotinine from caffeine. This will allow elimination of caffeine interference attributable to possible coelution of these two compounds. The same logic will also prescribe distinguishing nicotine from 3-OH-cotinine because these might coelute to some extent as well.

In conclusion, quadrupole ion trap MS/MS with positive ESI is well suited for the simultaneous detection and quantification of nicotine, cotinine, hydroxycotinine, and caffeine, with N-ethylnorcotinine serving as internal standard. Because the sample treatment steps in the method described are few and are not labor-intensive, it allows for a high throughput of samples with good prospects of automation. The method was applied successfully to investigate exposure to ETS in restaurant environments. Exposure to ETS can be measured from several sources, including blood, urine, and saliva. The advantages with using urine in the investigations included lower viscosity and ease of handling when compared with saliva and, when compared with blood, a relatively nonintrusive sample collection/donation method without an occupational health risk. In addition, samples could be collected more frequently than would have been the case with blood samples. Frequent sampling as well as an extended sample collection period is particularly important when estimating occupational exposure to ETS. Samples should be collected over a certain time period, e.g., a work week or a similar time frame for the following reasons: (a) there is a delay between initial exposure and excretion of nicotine metabolites to urine; (b) there are individual differences in metabolic rates; (c) there seems to be a possibility for build up of cotinine and 3-OH-cotinine during a working week; (d) differences in the emission of ETS in relation to time and space can thus better be accounted for; (e) if the results are not weighed against creatinine or the density of urine, additional variations will arise; and (f) in this way exposure to ETS during time off can be accounted for. The ion trap, particularly when used as a MS/MS device as in the present study, is qualitatively reliable. However, the accuracy of the quantitative analysis was limited by the characteristics of the ion trap, which is a semiquantitative rather than a precise quantitative instrument. Furthermore, using the method to monitor passive smoking in occupational environments other than bars and restaurants would in most cases require larger sample volumes to achieve lower LODs. For example, using 15 mL of urine instead of the 1.5 mL used in the present scheme would in all probability yield a 10-fold lower LOD for all compounds.


   Acknowledgments
 
We thank the Finnish Ministry of Social Affairs and Health for financial support. Hilkka Martinkauppi is gratefully acknowledged for accurately and efficiently carrying out the sample pretreatments, and Markku Raivio is gratefully acknowledged for conducting the field study in the restaurants.


   Footnotes
 
1 Nonstandard abbreviations: ETS, environmental tobacco smoke; 3-OH-cotinine, 3-hydroxycotinine; UV, ultraviolet; LOQ, limit of quantification; MS/MS, tandem mass spectrometry; ESI, electrospray ionization; and LOD, limit of detection.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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