fluorescence detection in the analysis of phenols in mainstream cigarette smoke

Journal of Chromatography A, 1141 (2007) 90–97

Gas chromatography/mass spectrometry versus liquid chromatography/fluorescence detection in the analysis of phenols in mainstream cigarette smoke Serban C. Moldoveanu ∗ , Melissa Kiser R.J. Reynolds Tobacco Co., 950 Reynolds Boulevard, Winston-Salem, NC 27105, USA Received 19 October 2006; received in revised form 28 November 2006; accepted 29 November 2006 Available online 19 December 2006

Abstract A new gas chromatographic/mass spectrometric (GC/MS) technique for the analysis of hydroxybenzenes (phenols) in mainstream cigarette smoke has been developed. The technique allows the measurement of 24 individual compounds, and the sum of a few other alkyl-dihydroxybenzenes. A critical evaluation is done for the new technique and for an established high-performance liquid chromatographic (HPLC) technique reported in the literature for the analysis of hydroxybenzenes in cigarette smoke, which uses fluorescence detection. Compared with the HPLC procedure, the new technique has similar accuracy, precision, and robustness. However, the GC/MS procedure allows for a larger number of phenols to be analyzed simultaneously, and eliminates any potential interference that may appear in the HPLC method. Using the GC/MS analysis, it was found that besides the main phenols typically measured in mainstream cigarette smoke such as phenol, catechol, hydroquinone, and cresols, many other phenols that are present at lower levels can be quantitated in mainstream cigarette smoke. © 2006 Elsevier B.V. All rights reserved. Keywords: Phenols; Hydroxybenzenes; HPLC; GC/MS; Mainstream cigarette smoke

1. Introduction Hydroxybenzenes (phenols) are present in cigarette smoke and contribute to cigarette sensory properties [1]. Also, some phenols are considered toxic compounds [2] and their presence in smoke has been related to environmental and health issues. Thus, the analysis of phenols in smoke has been the subject of several studies, some reported in peer reviewed literature [3–10]. Outside the tobacco industry, numerous published reports describe phenol analysis, which has been commonly performed on samples such as water [11–18], food [19–21], plant materials (fruits, vegetables) [22], pharmaceuticals [23,24], etc. Phenols can be analyzed by both high-performance liquid chromatographic (HPLC) and gas chromatography (GC). The HPLC analysis can be done with UV detection [25,26] or fluorescence detection [3,23,27]. Several advantages of fluorescence versus UV detection are described in the literature, the sensi-

∗

Corresponding author. Tel.: +1 336 741 7948; fax: +1 336 728 9112. E-mail address: [email protected] (S.C. Moldoveanu).

0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.11.100

tivity and selectivity of fluorescence detection being in general better than that of UV detection [23]. The analysis of phenols by GC or gas chromatography/mass spectrometry (GC/MS) is usually done after derivatization [7,10,24,28,29]. Other analytical techniques for phenols determination are also reported such as micellar electrokinetic chromatography [30]. The phenols typically analyzed in cigarette smoke by the HPLC method include 1,4-dihydroxybenzene (hydroquinone), 1,2-dihydroxybenzene (catechol), 1,3-dihydroxybenzene (resorcinol), hydroxybenzene (phenol), 4-methyl + 3-methyl-hydroxybenzene (p- + mcresol), and 2-methylhydroxybenzene (o-cresol). Although 2-methoxyphenol (guaiacol) is not usually analyzed by HPLC this compound can be easily measured by this technique. Besides the six (seven, if 2-methoxyphenol is added) phenols typically analyzed by HPLC/fluorescence, a number of other phenols are present in cigarette smoke [31]. They are not listed as biologically active agents [2] and their levels are relatively low. Nevertheless, their presence in smoke may contribute to the properties of smoke, and a full chemical characterization of cigarette smoke should include them. However, the analysis of these additional phenols by HPLC/fluorescence raises a selec-

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tivity problem, since the separation of some of these phenols may be difficult by HPLC. Also, their positive identification without a mass spectrum can be questionable. For this reason, a GC/MS technique for phenol analysis in cigarette mainstream smoke has been developed and is described in this study. The application of the HPLC/fluorescence analysis and of GC/MS analysis on the same samples allows a good comparison of advantages and disadvantages of both techniques. 2. Experimental 2.1. Smoke collection Smoke collection was done with a Borgwaldt RM20 CSR smoking machine, tuned for conditions similar to those recommended by US Federal Trade Commission (FTC) [32]. That consisted of 35 mL puff volume, each puff taken every 2 s, with 60 s puff interval, the cigarettes being smoked to 3 mm distance to the filter overwrap. Other smoking regimes [33–36] can also be utilized depending on the purpose of the analysis. Five different cigarettes were used in the study. This included two Kentucky references cigarettes indicated as 1R5F and 2R4F (University of Kentucky, Kentucky Tobacco Research & Development Center, KY, USA) and three commercial cigarettes. The description of these cigarettes is given in Table 1. Smoke from five cigarettes was collected in each run on a 92 mm Cambridge pad. The results previously reported in the literature [3] indicate that phenols are effectively collected from mainstream smoke by this procedure. A chromatographic standard consisting of 100 ␮L solution containing 250 ␮g/mL of 4-chlorophenol was added to the pad. The pad was extracted on a mechanical shaker for 30 min with 25 mL water containing 1% acetic acid and 0.1% ascorbic acid (both from Aldrich/Sigma, Milwaukee, WI, USA). The solution extract was used as is, after filtration through 0.45 ␮m pore size polyvinilydene fluoride (PVDF) filters, for the HPLC analysis. The solutions can be kept in a freezer for at least 1 week without affecting the phenols content. For the GC/MS analysis a sample preparation using solid-phase extraction (SPE) was necessary before the chromatographic step. 2.2. HPLC/fluorescence detection analytical procedure The analysis by HPLC/fluorescence followed a procedure described in the literature [3]. One difference in the present work was the use of a fixed amount of extracting solution (25 mL)

Fig. 1. HPLC/fluorescence chromatogram obtained for the main phenols in mainstream smoke of a 2R4F Kentucky reference cigarette.

instead of adjusting this volume to obtain a solution containing approximately 1 mg/mL of total particulate matter (TPM). The ascorbic acid in the present method was added to prevent any potential oxidation of the phenols. Also, a chromatographic standard (further described) was used in the present work. The instrumentation used for the HPLC analysis consisted of two Waters 510 pumps, a 717plus Waters autosampler and a 474 Waters scanning fluorescence detector (Waters, Milford, MA, USA). The separation was achieved on a Beckman Ultrasphere ODS column 15 cm × 4.6 mm I.D., 5 ␮m particle size (Beckman Coulter, Fullerton, CA, USA). The separation took place under gradient conditions using two solutions, one being water with 1% acetic acid (sol. A) and the other acetonitrile with 1% acetic acid (sol. B). The acetic acid is necessary for improving the stability of phenols fluorescence [3]. The initial solution contained 4% sol. B (and it is not pure water) in order to avoid the outgasing commonly seen when mixing pure water and acetonitrile. The flow rate was 1.4 mL/min. The composition of the mobile phase was changed (linear) to reach 31% sol. B at 10.5 min and 100% sol. B at 15.5 min. Sol. B was flown through the column for another 4.5 min and then the conditions were restored to the initial mobile phase composition. Multiple injections were done every 27 min. The fluorescence was measured initially at 304 nm λex and 338 nm λem . The conditions were changed to 274 nm λex and 298 nm λem after 3.5 min, and to 232 nm λex and 310 nm λem after 13.2 min. The data acquisition was performed for 20 min. The injection volume was 8 ␮L. A typical chromatogram obtained in these conditions for the mainstream smoke of the 2R4F Kentucky reference cigarette is shown in Fig. 1. For the quantitation of phenols, calibration curves were generated using a series of four standards. The standards

Table 1 Description of Kentucky reference and commercial cigarettes Descriptor

1R5F Kentucky ref.

2R4F Kentucky ref.

16.2 mg ‘tar’

10.6 mg ‘tar’

5.0 mg ‘tar’

FTC ‘tar’ (mg/cig) Cigarette length (mm) Filter length (mm) Filter ventilation (%) Blend type Nicotine (mg/cig) CO (mg/cig)

1.7 84 32 72 American 0.16 3

8.9 84 27 28 American 0.75 12.0

16.2 83 21 23 American 1.31 13.9

10.6 83 27 32 American 0.92 10.7

5.0 83 27 54 American 0.5 7.4

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Table 2 GC/MS operating parameters Parameter

Description

Parameter

Description

GC column Column dimensions Film thickness Initial oven temperature Initial time Oven ramp rate Oven final first ramp Final time first ramp Oven ramp rate Oven final temperature Final time Total run time Inlet temperature Inlet mode

BPX-5 Two 30 m columns connected, 0.25 mm i.d. 0.25 ␮m 125 ◦ C 7.0 min 4 ◦ C/min 220 ◦ C 0 min 25 ◦ C/min 320 ◦ C 0 min 34.75 min 280 ◦ C Split

Injection volume Split ratio Split flow Carrier gas Flow mode Flow rate Nominal initial pressure GC outlet MSD transfer line Ion source temperature Quadrupole temp. MSD EM offset MSD solvent delay MSD acquisition mode

1.0 ␮L 25:1 27.4 mL/min Helium Constant flow 1.1 mL/min 24.73 psi MSD 280 ◦ C 230 ◦ C 150 ◦ C 250 V 6.0 min TIC or SIM

were made in a two-step dilution from an initial stock solution, which was obtained by accurately weighing 600 ± 50 mg hydroquinone, 600 ± 50 mg catechol, 600 ± 50 mg resorcinol, 150 ± 20 mg phenol, 150 ± 20 mg o-cresol, 150 ± 20 mg m-cresol, 150 ± 20 mg p-cresol, and 150 ± 20 mg guaiacol, followed by dissolving the phenols in 1% aqueous acetic acid to a volume of 1000 mL (all chemicals from Aldrich/Sigma, Milwaukee, WI, USA). The initial stock solution was first diluted ten times with 1% aqueous acetic acid to obtain a secondary stock solution. From this secondary stock solution the calibration standards were obtained by taking 1, 5, 10, and 15 mL into separate 100 mL flasks and diluting farther with 1% aqueous acetic acid. Calibration curves representing quantity versus peak areas were generated for each compound and were used for the measurement of phenols. These curves were linear and the R2 values for the dependence were all above 0.999. The chromatographic standard, 4-chlorophenol, was used only to verify the constancy of the chromatographic response. 2.3. Sample preparation for the GC/MS analytical procedure A number of procedures for sample preparation in phenols analysis are described in the literature [10,22,37]. In this study, an aliquot of the smoke extract to be used for the GC/MS analysis was processed by SPE. For this purpose, 2 mL of the extract obtained with 25 mL 1% aqueous acetic acid from the particulate phase smoke (the same solution used for the HPLC analysis) was processed. Larger volumes (4 mL, 8 mL or even higher) can also be used (about 20 mL solution can be recovered from the pad extract). The smoke extract was passed through a Strata X SPE cartridge of 200 mg, 3 mL format (Phenomenex, Torrance, CA, USA). A vacuum manifold was typically used for this procedure. The cartridge containing the sample was then washed three times with 2–3 mL 1% aqueous acetic acid. The phenols are retained from the smoke extract and the washing does not remove the phenols from the cartridge. However, the attempt to wash the cartridge with water containing 10% acetone or 10% methanol lead to the elution of some of the dihydroxybenzenes.

After washing (with 1% aqueous acetic acid), the cartridge was dried prior to phenols elution. For this purpose, ambient air was allowed to pass through the cartridge using vacuum in the manifold, for 1 h. Traces of water remaining in the sorbent after drying do not interfere with the analysis, but larger quantities disturb the derivatization process that follows. The dried cartridge was then eluted with 1 mL dimethylformamide (DMF). Only 0.5 mL of this DMF solution was taken from the eluate, placed into a GC vial, and 100 ␮L of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) was added (Pierce, Rockfort, IL, USA). After capping the vials, the solution was heated at 78 ◦ C for 30 min to obtain the trimethylsilyl (TMS) derivatives of phenols that are further analyzed by GC/MS. 2.4. GC/MS analytical procedure The GC/MS analysis was performed on a 6890 GC/5973 MS instrument (Agilent, Wilmington, DE, USA), in either total ion mode (generating a total ion chromatogram, TIC) or in selected ion monitoring mode (SIM), with the parameters for the instrument given in Table 2. SIM parameters were set to allow for the detection of individual phenols. The use of the above conditions for a set of standards generated in SIM mode produced the chromatogram shown in Fig. 2. The identification of the peaks from this chromatogram is given in Table 3, which also lists

Fig. 2. SIM chromatogram for a series of standards of phenols processed through the SPE. Peak identification based on retention times is given in Table 3 (4chlorophenol used as an internal standard is not present in this standard mixture).

S.C. Moldoveanu, M. Kiser / J. Chromatogr. A 1141 (2007) 90–97 Table 3 List of standards of phenols, their retention times, and m/z values used for the detection/quantitation No.

Compound

Retention time (min)

m/z

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Phenol o-Cresol m-Cresol p-Cresol 2-Ethylphenol 2,5-Dimethylphenol 3,5-Dimethylphenol 2,4-Dimethylphenol 2-Methoxyphenol 4-Ethylphenol (I.S.) 4-Chlorophenol 2,6-Dimethylphenol 2,3-Dimethylphenol 3,4-Dimethylphenol 3-Methoxyphenol 4-Methoxyphenol Catechol (1,2-dihydroxybenzene) Resorcinol (1,3-dihydroxybenzene) 4-Methylcatechol Hydroquinone (1,4-dihydroxybenzene) 3-Methylcatechol 3-Methylresorcinol 2-Methylresorcinol + methylhydroquinone 4-Ethylresorcinol 2,5-Dimethylresorcinol

6.88 8.57 8.76 9.08 10.28 10.70 11.07 11.20 11.28 11.59 11.71 11.79 12.02 12.32 13.17 13.47 13.88 16.05 16.27 16.73 16.71 18.19 18.66 19.90 20.18

166 180 180 180 194 194 194 194 196 194 185 194 194 194 196 196 254 254 268 254 268 268 268 282 282

the ions (m/z) used for the measurement of TMS derivatives. The standards solution was processed through the SPE similar to a smoke extract (2 mL added on the cartridge). The individual concentrations in the standards solution varied between 2.0 and 2.5 ␮g/mL. The SIM chromatogram for an extract of smoke from a 2R4F cigarette generated the chromatogram shown in Fig. 3. In addition to the dimethyl- and/or ethyl-dihydroxybenzenes (C2-dihydroxybenzenes) for which standards were available, several other peaks in the chromatogram shown in Fig. 3, eluting between 20.5 and 22.5 min were identified based only on their spectrum as C2- or C3-dihydroxybenzenes (C3 indicating any alkyl with three carbon atoms). Because of the similarity of the spectra of these compounds, the exact position of the substitu-

Fig. 3. SIM chromatogram of the phenols from the smoke of a 2R4F cigarette. Peak identification based on retention times given in Table 3.

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tion on the benzene ring was not possible (no standards were obtained for these compounds). The quantitation of phenols by the GC/MS technique was also done with calibration curves. The calibration curves for hydroquinone, catechol, resorcinol, phenol, p-cresol, m-cresol, o-cresol, and guaiacol were generated with the same series of four standards used for the calibration of HPLC method. Curves representing quantity versus peak areas normalized by the area of the internal standard (4-chlorophenol) were generated. The curves were linear and the R2 values for the dependence were all above 0.993. For the other phenols only three point calibrations were generated (phenols from Aldrich/Sigma, Milwaukee, WI, USA). 3. Results and discussion Several aspects of the two analytical techniques for phenols analysis, the HPLC and the GC/MS, were investigated with the purpose of their comparison. These aspects included selectivity, accuracy, precision, limit of detection and of quantitation, recovery, and robustness. A comparison of advantages and disadvantages of the two techniques was possible following the description of these parameters. 3.1. Methods selectivity The selectivity of the HPLC procedure was difficult to verify, since no sample was available containing the same matrix but without phenols. However, potential interferences from the matrix are not likely for the phenols found in relatively high levels in smoke. The extraction with water of smoke condensate eliminates many compounds that are not water soluble, and the fluorescence of phenols is selective. The separation of pcresol from m-cresol was not achieved, and the results for the two compounds were given as their sum. Some changes in the elution gradient that modified the retention times of the analytes, as well as the use of a longer column (Ultrasphere ODS 5 ␮m, 4.6 mm × 25 cm) did not change peak areas for the analytes for samples generated from a 2R4F Kentucky reference cigarette as well as those from the analyzed commercial cigarettes. This basically indicated that the measured peaks are pure. Selectivity was enhanced for the GC/MS procedure compared to the HPLC. The SPE processing of the sample provided besides concentration and solvent change, a cleanup step. The availability of the mass spectra for identification and the use of selected ions for quantitation also increased the method selectivity. This allowed the separation of cresols (m- and p-cresol were not separated using the HPLC procedure) and the peaks of other hydroxybenzenes that were not analyzed by HPLC were very well separated in the GC/MS method (see Fig. 2). Only 2-methylresorcinol and methylhydroquinone had identical retention times and very similar mass spectra and were not separated in the conditions described in this study. A potential interference in the measurement of hydroquinone for both GC/MS and HPLC procedures may occur if p-benzoquinone is present. This substance is reduced to hydroquinone in the presence of ascorbic acid. In order to prove that

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Table 4 Comparison for the results of phenols from 2R4F cigarette obtained by the HPLC procedure using solution for pad extraction with and without 0.1% of ascorbic acid Compound

Average ␮g/cig. HPLC with ascorbic acid

RSD %

Average ␮g/cig. HPLC without ascorbic acid

RSD %

Difference %

Phenol o-Cresol m + p-Cresol Catechol Hydroquinone Resorcinol Guaiacol

9.21 2.34 6.95 35.60 28.80 0.84 1.61

2.3 6.1 4.6 1.9 4.9 10.6 4.0

9.19 2.14 6.77 35.81 28.71 0.79 1.52

3.5 2.8 4.7 3.8 3.8 8.9 4.5

0.22 8.93 2.62 −0.59 0.31 6.13 5.75

Table 5 Comparison with literature data for the results obtained using the HPLC and the GC/MS procedures for the phenols from 2R4F and from 1R5F cigarettes 2R4F

1R5F

Compound

Average ␮g/cig. HPLC

RSD % HPLC

Average ␮g/cig. GC/MS

RSD % GC/MS

Ref. [38] ␮g/cig.

Average ␮g/cig. HPLC

RSD % HPLC

Average ␮g/cig. GC/MS

RSD % GC/MS

Ref. [10] ␮g/cig.

Phenol o-Cresol m + p-Cresol Catechol Hydroquinone Resorcinol Guaiacol

9.21 2.34 6.95 35.60 28.80 0.84 1.61

2.3 6.1 4.6 1.9 4.9 10.6 4.0

7.49 2.45 6.22 35.88 32.74 1.27 1.42

4.5 2.6 2.7 1.8 5.7 7.9 3.2

7.32 1.89 5.84 37.90 32.40 0.91 Not available

1.18 0.47 0.74 6.42 6.58 0.24 0.37

8.6 13.1 4.6 8.2 8.2 23.5 13.6

0.76 0.35 0.62 6.90 6.30 0.51 0.20

3.5 0.8 2.4 3.8 3.4 4.5 6.1

0.9 0.2 0.3 7.4 4.9 0.6 Not available

the addition of 0.1% ascorbic acid in the extraction solution of phenols does not affect the results regarding hydroquinone (or other phenols), the analysis for the 2R4F cigarette was done by the same procedure as previously described without the addition of ascorbic acid in the Cambridge pad extracting solution [3]. The results are shown for triplicate samples with each chromatographic runs performed twice (total of six data points) in Table 4 for the HPLC method only. The agreement between the data is very good. Further comparison of the accuracy of the results obtained with the present procedure for 2R4F and 1R5F Kentucky reference cigarettes with the literature data also proved to be very good. This indicates that the equilibrium quinone/hydroquinone is strongly displaced toward hydroquinone in mainstream cigarette smoke. 3.2. Methods accuracy The results obtained for 2R4F and 1R5F Kentucky reference cigarettes were compared with the data from the literature [10,35] ([3] shows data only for 1R4F Kentucky reference cigarette). Only results for seven common phenols in cigarette smoke were available for comparison. The results are given in Table 5. For this comparison, triplicates of each sample were smoked and processed, followed by chromatographic runs performed twice for each sample (total of six data points). The results from Table 5 show good agreement between the HPLC results, the GC/MS results, and also the results published in literature ([38] provides data from a collaborative study between six laboratories). Larger discrepancies were noticed only in the results for resorcinol, which is present in cigarette smoke at lower levels compared to the other phenols shown in Table 5.

This indicates that both the HPLC and the GC/MS procedure have good accuracy. 3.3. Precision, limit of detection and of quantitation The RSD values given in Table 5 show that both the HPLC and GC/MS procedures have good precision. For the HPLC analysis of the 1R5F cigarette the RSD values were higher than those obtained by the GC/MS procedure. The fluorescence signal for the 1R5F samples was relatively low compared to the noise, which may explain the increased RSD values. However, a lower volume of solution used to extract the Cambridge pads can increase the concentration of phenols in the analyzed solution and, therefore can possibly improve the repeatability of the technique. Calculated as 3 × SD (where SD is the standard deviation for a sample with low levels of phenols), the limit of detection (LOD) obtained from Table 5 is given in Table 6. The limits of detection given in Table 6 can be considerably improved if certain changes are performed in the sample processing. For the Table 6 Limit of detection calculated as 3 × SD, where SD is the standard deviation for a low sample Compound

LOD ␮g/cig. HPLC

LOD ␮g/cig. GC/MS

Phenol o-Cresol m + p-Cresol Catechol Hydroquinone Resorcinol Guaiacol

0.30 0.18 0.10 1.58 1.62 0.17 0.15

0.08 0.01 0.04 0.79 0.64 0.07 0.04

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Table 7 Other phenols detected in the mainstream cigarette smoke of 2R4F and 1R5F cigarettes No.

Compound

Average 2R4F ␮g/cig.

RSD % 2R4F

Average 1R5F ␮g/cig.

RSD % 1R5F

1 2 3 4 5 6 7 8 9 11 12 13 14 15 15 17 18 19 20 21

m-Cresol p-Cresol 2-Ethylphenol 2,5-Dimethylphenol 3,5-Dimethylphenol 2,4-Dimethylphenol 4-Ethylphenol 2,6-Dimethylphenol 2,3-Dimethylphenol 3,4-Dimethylphenol 3-Methoxyphenol 4-Methoxyphenol 4-Methylcatechol 3-Methylcatechol 3-Methylresorcinol 2-Methylresorcinol + methylhydroquinone 4-Ethylresorcinol 2,5-Dimethylresorcinol Other C2-dihydroxybenzenes Sum of C3-dihydroxybenzenes

1.76 4.46 0.57 0.57 0.62 1.09 1.43 0.58 1.33 0.50 0.28 0.46 4.32 4.52 0.83 3.91 0.40 1.26 3.97 0.63

2.26 2.68 2.40 1.44 2.28 1.16 0.98 11.35 1.16 11.96 9.44 12.23 4.78 3.42 11.06 2.11 6.87 7.33 8.10 4.33

0.15 0.47 0.17 0.14 0.16 0.26 0.28 0.23 0.14 0.15 0.13 0.22 0.88 0.93 0.30 0.65 0.17 0.33 0.49 0.27

0.49 2.41 1.10 4.15 11.90 3.35 3.56 14.72 3.19 7.10 4.90 2.52 3.48 4.04 2.00 5.96 6.85 7.12 9.10 5.97

GC/MS technique for example, the volume of the solution of particulate phase smoke extract added to the SPE cartridge can be increased from 2 mL to a higher volume, without resulting in any breakthrough of the analytes. However, for the analysis of phenols in mainstream cigarette smoke this increase was not considered necessary. The values for the limit of quantitation (LOQ) can be evaluated as 3 × LOD.

during the drying of the SPE cartridge. The presence of water in the DMF eluate, noticeable by strong heating of the sample when the reagent BSTFA is added for derivatization, leads to lower peaks in the chromatogram and incorrect quantitation (slight heating of the samples still occurs even with dry samples). However, after 1 h of drying the SPE cartridge with ambient air, the potential problem of hydrolyzing the TMS derivatives was eliminated.

3.4. Recovery 3.6. Comparison of HPLC and GC/MS procedures The recovery of the HPLC procedure has been previously reported [3]. The efficiency of phenols extraction from the cigarette smoke particulate matter (TPM) collected on a Cambridge pad using a 1% acetic acid aqueous solution was thoroughly evaluated in this previously reported HPLC technique [3]. For this reason, the dissimilarity between the extraction of phenols from TPM or from the clean pad was not expected to generate considerable differences. The recovery for the GC/MS was performed by adding on a Cambridge pad 100 ␮L solution of a mixture of standards containing between 500 and 625 ␮g/mL of individual phenols. The pad was further processed by the procedure previously described, and the results compared to the initial amount added. Recoveries between 93 and 105% were obtained for all the phenols analyzed. This indicated that no recovery problems are encountered during pad extraction and the SPE step. 3.5. Robustness Robustness refers to the quality of an analytical procedure to not be influenced by small experimental modifications during its performance. Both HPLC and the GC/MS techniques described in this study were simple and no difficult steps were encountered. Some attention is required in the GC/MS procedure

Both HPLC and GC/MS procedures give good results for the analysis of the major phenols from cigarette mainstream smoke. The HPLC procedure is simpler since after the pad extraction, the solutions are directly subject to the chromatographic process. A slightly lower precision noticed during the analysis of the 1R5F cigarette by the HPLC method is not critical, and can be improved by using smaller extraction volumes of the Cambridge pad. The advantage of the GC/MS technique consists mainly in the extension of the list of analytes and in the separation of m- and p-cresols. Also, the detection based on the ions characteristic for each phenol further eliminates the chances for interferences. The results for the level of other phenols present in smoke of the 2R4F and 1R5F Kentucky reference cigarettes as obtained by the GC/MS technique are given in Table 7. As seen from Table 7, only the C1-dihydroxybenzenes and to a lower extent C2-dihydroxybenzenes are present in smoke of the two Kentucky reference cigarettes at appreciable levels compared to those of the major phenols. Most other phenols in smoke besides phenol, catechol, hydroquinone, and cresols are present at lower levels, however their total sum accounts for about 25% of total phenols in smoke. The same conclusion was obtained from the analysis of smoke from other cigarettes. As an example, the analysis of phenols from the mainstream smoke of three

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Table 8 Comparison of phenol levels in three common commercial cigarettes Cigarette No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Compound

Phenol o-Cresol m-Cresol p-Cresol 2-Ethylphenol 2,5-Dimethylphenol 3,5-Dimethylphenol 2,4-Dimethylphenol 2-Methoxyphenol 4-Ethylphenol 2,6-Dimethylphenol 2,3-Dimethylphenol 3,4-Dimethylphenol 3-Methoxyphenol 4-Methoxyphenol Catechol Resorcinol 4-Methylcatechol Hydroquinone 3-Methylcatechol 3-Methylresorcinol 2-Methylresoecinol + methylhydroquinone 4-Ethylresoecinol 2,5-Dimethylresorcinol C2-Dihydroxyphenol C3-Dihydroxyphenol Total phenols

5.0 mg ‘tar’

10.6 mg ‘tar’

16.2 mg ‘tar’

Average ␮g/cig.

RSD %

Average ␮g/cig.

RSD %

Average ␮g/cig.

RSD %

4.86 1.23 0.93 2.12 0.31 0.29 0.36 0.56 0.65 0.77 0.30 0.54 0.29 0.15 0.25 20.00 0.72 2.43 19.78 2.37 0.46 1.83 0.21 0.63 1.29 0.34 63.69

2.85 0.03 1.91 2.69 1.60 2.63 9.59 2.68 4.05 3.32 7.55 6.29 3.10 6.42 11.88 4.61 9.86 4.52 6.36 3.74 7.94 7.89 5.23 7.09 10.94 12.96

9.29 2.62 1.97 5.02 0.70 0.67 0.82 1.28 1.43 1.67 0.73 0.84 0.60 0.36 0.63 45.20 1.81 6.19 43.00 5.58 1.16 3.57 0.53 1.49 2.93 0.57 140.68

5.30 4.14 2.25 3.25 3.97 2.98 2.04 2.54 2.31 1.78 9.33 11.34 5.95 9.53 5.36 2.48 8.48 6.08 6.41 1.75 7.57 13.11 7.51 9.84 11.10 4.61

16.07 3.70 2.95 6.97 0.75 0.79 1.00 1.47 2.40 2.10 0.68 1.62 0.73 0.33 0.53 62.35 1.80 7.59 56.44 6.50 1.19 5.70 0.47 1.73 4.65 0.81 190.29

4.26 4.96 4.44 4.69 2.85 3.72 3.40 2.61 2.48 3.67 2.43 1.32 4.24 11.61 3.36 1.58 1.01 2.13 1.81 2.92 0.58 2.32 2.22 3.28 2.76 1.99

common commercial cigarettes of different ‘tar’ levels was performed. The cigarettes included a 5.0 mg FTC ‘tar’ cigarette, (where ‘tar’ is defined as the weight of total mainstream smoke condensate (or wet particulate matter) minus the weight of nicotine and water) a 10.6 mg ‘tar’ and a 16.2 mg ‘tar’ cigarette, with the description given in Table 1. The results for the phenols analysis are given in Table 8. As it can be calculated from Table 8 the proportion of different phenols relative to the total amount of phenols in smoke is not very different for different cigarettes, and phenol, catechol, hydroquinone, and cresols account for about 75% of total phenols in smoke. 4. Conclusions This study presents a new GC/MS analytical technique for phenol analysis, which allows the measurement of 24 individual compounds and of the sum of certain C2-dihydroxybenzenes and of C3-dihydroxybenzenes. The main characteristics of the GC/MS analytical procedure are compared to those of a HPLC technique with fluorescence detection. Both techniques provide very reliable results for the analysis of phenols. Both methods are equally accurate for the analysis of 6–7 major phenolic compounds from mainstream cigarette smoke. Using the GC/MS procedure it was found that besides the phenols typically analyzed in cigarette smoke some other phenols are

present, accounting together for about 25% of total phenols in mainstream smoke. However, the GC/MS method requires two additional steps compared to the HPLC method (the SPE and the derivatization). Also, the GC/MS equipment is typically more expensive than the HPLC one. For routine laboratories the HPLC technique is probably recommended, while the GC/MS is necessary only when a detailed analysis of phenols in mainstream cigarette smoke is desired.

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