J Chem Ecol (2010) 36:10351042 DOI 10.

1007/s10886-010-9846-7

Analysis of Volatile Organic Compounds in Human Saliva by a Static Sorptive Extraction Method and Gas Chromatography-Mass Spectrometry
Helena A. Soini & Iveta Klouckova & Donald Wiesler & Elisabeth Oberzaucher & Karl Grammer & Sarah J. Dixon & Yun Xu & Richard G. Brereton & Dustin J. Penn & Milos V. Novotny

Received: 3 March 2010 / Revised: 27 May 2010 / Accepted: 16 August 2010 / Published online: 31 August 2010 # Springer Science+Business Media, LLC 2010

Abstract Human saliva not only helps control oral health (with anti-microbial proteins), but it may also play a role in chemical communication. As is the case with other mammalian species, human saliva contains peptides, proteins, and numerous volatile organic compounds (VOCs). A highthroughput analytical method is described for profiling a large number of saliva samples to screen the profiles of VOCs. Saliva samples were collected in a non-stimulated fashion. The method utilized static stir bar extraction followed by gas chromatography-mass spectrometry (GC-MS). The method provided excellent reproducibility for a wide range of salivary compounds, including alcohols, aldehydes, ketones, carboxylic acids, esters, amines, amides, lactones, and hydrocarbons. Furthermore, substantial overlap of salivary VOCs and the
H. A. Soini : I. Klouckova : D. Wiesler : M. V. Novotny (*) Institute for Pheromone Research and Department of Chemistry, Indiana University, 800 E Kirkwood Ave., Bloomington, IN 47405, USA e-mail: novotny@indiana.edu E. Oberzaucher : K. Grammer Department for Anthropology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria S. J. Dixon : Y. Xu : R. G. Brereton Centre for Chemometrics, School of Chemistry, University of Bristol, Cantocks Close, Bristol BS8 1TS, UK D. J. Penn Konrad Lorenz Institute for Ethology, Austrian Academy of Sciences, Savoyenstr. 1a, 1160 Vienna, Austria

previously reported skin VOCs in the same subject group was found in this study by using pattern recognition analyses. Sensitivity, precision, and reproducibility of the method suggest that this technique has potential in physiological, metabolomic, pharmacokinetic, forensic, and toxicological studies of small organic compounds where a large number of human saliva samples are involved. Key Words Chemical communication . Gas chromatography-mass spectrometry (GC-MS) . Human saliva . Stir bar extraction . Volatile organic compounds

Introduction Human saliva is a complex secretion produced by glands in the oral cavity (parotid, submandibular, and sublingual), minor salivary glands in the soft mucosa tissues, and the von Ebner gland in the tongue (Marcotte and Lavoie, 1998; Dodds et al., 2005). Functions of the saliva include regulating oral microbial ecology, affecting condition of the oral soft tissue and hard tooth enamel, and involvement in the food intake and pre-digestion processes (Black, 1977; Dodds et al., 2005). Human saliva also may play a role in chemical communication in humans, as it does in other mammals, such as rodents (Block et al., 1981; Ferkin and Johnston, 1995; Taha et al., 2009); boar (Marchese et al., 1998); elephants (Rasmussen et al., 1997); and other primates (Laidre, 2009). R.A. Fisher (1915) pointed out that bad breath may be sexually unattractive precisely because it indicates a variety of diseases. Indeed, physicians have long used breath odor, which may relate to the saliva headspace, to diagnose a variety of diseases (Penn and Potts, 1998; Gaspar et al., 2009; Preti et al., 2009).

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Saliva consists of macromolecular compounds and small organic substances, including volatile organic compounds (VOCs), but the composition and functions of VOCs are not as well defined as some macromolecules. Saliva includes antibacterial, anti-viral, and antifungal proteins, such as lysozyme, lactoferrin, peroxidases, carbonic anhydrase, cystatins (cysteine proteinase inhibitors), and secretory immunoglobulin A. Antimicrobial peptides, histidinerich histatins, and -defensins, also are found in saliva from inflammation sites (Marcotte and Lavoie, 1998; Dodds et al., 2005). The von Ebner gland protein (VEGh), a member of the lipocalin family, represents one of the several saliva components that also are present ubiquitously in other body secretions (e.g., tears and semen). Lysozyme, also found in saliva, is present in other body fluids such as blood and sweat (Nieuw Amerongen and Veerman, 2002). Digestionaiding salivary enzymes, such as -amylase and a humanspecific, hydrophobic lipase, participate in the first step of food digestion in the oral cavity (Marcotte and Lavoie, 1998; Isenman et al., 1999). Saliva also contains glycosylated (mucin-type) proteins that are responsible for the viscous gel-like properties of saliva (Wu et al., 1994). The small organic molecules previously described in saliva include hormones, amino acids, peptides (Marcotte and Lavoie, 1998), and nitric oxide (Benjamin et al., 1994; Palmerini et al., 2003). More than 300 bacterial species have been found in the oral cavity, which also contribute to saliva chemical composition through secretion of their metabolic byproducts (Scannapieco, 1994; Marcotte and Lavoie, 1998). Analysis of the sulfur-containing volatile compounds in breath has been reported in the investigation of sources for malodors in the oral cavity, and anaerobic bacterial activity contribute particularly to malodorous breath (Ochiai et al., 2001; Rodrguez et al., 2002; van den Velde et al., 2007). Small organic compounds of environmental origin may be transported into the saliva via the digestive tract, through the lungs (Kostelc et al., 1981; Amorim and Cardeal, 2007), or via transdermal absorption through the skin into the blood stream, followed by filtration into saliva (Jimbo, 1983; Jiang et al., 1996; Cross et al., 1997; Chatelain et al., 2003). Several papers report comparative measurements in plasma and saliva for single organic compounds such as steroid hormones or their metabolites at relatively high concentrations (Hill et al., 2001; Jnsson et al., 2003; Contrerars et al., 2004; Kumar et al., 2005; Higarshi et al., 2007). Additionally, drug metabolite measurements in pharmacokinetic (Matin et al., 1974; Guo et al., 2007) and forensic studies (Lo Muzio et al., 2005; Gognard et al., 2006) have been reported in saliva. Samyn et al. (2007) recently reviewed various analytical procedures when using oral fluids in drug abuse investigations. However, there are

relatively few reports on the endogenous volatile compounds in saliva. Lochner et al. (1986) report finding 39 compounds after solvent extraction and derivatization followed by gas chromatography-mass spectrometry (GCMS). This approach is relatively tedious and, therefore, not suitable for a large number of analyses. Sastry et al. (1980) have reviewed different analytical methods for measuring VOCs in the salivary headspace by gas chromatography. However, these approaches are not applicable for a large number of analyses. Ligor (2009) recently reviewed preconcentration methods used in breath analysis, including solid-phase microextraction (SPME) methods. In a previous study, we described the VOC profiles found on human skin (Soini et al., 2006; Penn et al., 2007), and in the present work we examined the VOCs in saliva samples from the same large group of subjects living in the Austrian Alps (Penn et al., 2007). As a prelude to systematic studies of a human population, it was necessary to develop a reliable analytical procedure for the large number of samples. We describe here an efficient and precise quantitative method for screening the native volatile compounds present at low levels in human saliva. We used a novel static stir bar sorptive extraction step followed by chemical analysis by GC-MS. The data from a large number of volatile compound profiles in human saliva and skin were evaluated by using a previously described chemometric pattern recognition technique (Dixon et al., 2006, 2007; Penn et al., 2007; Xu et al., 2007). The analytical method may have further potential in physiological, metabolic, pharmacokinetic, environmental, and forensic applications that require high analytical throughput and high sensitivity in analyzing various volatile compound levels in human saliva.

Methods and Materials Sample Collection Saliva samples were collected in a nonstimulated fashion from 175 healthy volunteers within a geographically distinct area in a village in the Austrian Alps (Penn et al., 2007). Subjects did not have any dietary restrictions. Their food intake was documented in a questionnaire, and they were instructed to rinse their mouths with water before saliva collection, without brushing their teeth or using any mouthwashes. After 5 min of the water rinse, about 3 ml of saliva per person were accumulated in a plastic container by spitting. Samples were frozen at 20C until analyses. Reagents and Materials All analytical standard compounds were purchased from Sigma-Aldrich/ Fluka Chemical Company (Milwaukee, WI, USA) or TCI America (Portland, OR, USA). Stir bars (Twister, 10 mm, 0.5 mm film

J Chem Ecol (2010) 36:10351042

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thickness, 24-l polydimethylsiloxane (PDMS) volume) used for sample collection were provided by Gerstel GmbH (Mlheim an der Ruhr, Germany). The stir bars were conditioned prior to and between individual runs in the TC2 tube conditioner (Gerstel GmbH ) at 300C under a purified stream of helium. Saliva samples (0.5 ml) were placed in 10-ml headspace vials (Gerstel) with screw caps equipped with high purity silicon/PFTE septa (Gerstel). Samples were diluted with 2.0 ml of high-purity water (OmniSolv, EM Science, Gibbstown, NJ, USA). As an internal standard, 8 ng 7-tridecanone (Aldrich) were added in 5 l of methanol (Baker Analyzed, Mallinckrodt Baker, Inc., Phillisburg, NJ, USA) to each vial, followed by the addition of a preconditioned stir bar into the liquid phase. The vials were closed and were subsequently incubated at 40C for 3 h in an oven. The sampling bars were not stirred, but rather equilibrated in the vials in a static mode. After sampling, stir bars were rinsed with high-purity water (OmniSolv), dried with paper tissue, and placed in thermal desorption autosampler device (TDSA) tubes for the analysis. After the analysis, TDSA tubes were washed with 50% OmniSolv acetonitrile (EMD Chemicals, Darmstadt, Germany) in water containing 0.1% trifluoroacetic acid (99%, Aldrich Chemical Company, Milwaukee, WI, USA), followed by a rinse with high-purity OmniSolv water and acetone (EMD Chemicals), and dried in the oven at 85C for 60 min. Analytical Instruments The GC equipment consisted of an Agilent 6890N gas chromatograph connected to the 5973i MSD mass selective detector (Agilent Technologies, Inc., Wilmington, DE, USA). Positive electron ionization (EI) mode at 70 eV was used with the scanning rate of 4.51 scans/sec over the mass range of 35350 amu. The MSD transfer line temperature was set at 280C. The ion source and quadrupole temperatures were set at 230C and 150C, respectively. Samples were thermally desorbed in a TDSA automated system (Gerstel), followed by injection into the column with a cooled injection assembly, CIS-4. The TDSA was operated in splitless mode. The TDSA temperature program for desorption was 20C (hold for 0.5 min), then a 60C/min ramp to 250C (final hold of 3 min). Temperature of the transfer line was set at 280C. The CIS4 was cooled with liquid nitrogen to 80C. After desorption and cryotrapping, CIS-4 was heated at 12C/ sec to 280C, with the hold time of 10 min. The CIS-4 inlet was operated in the solvent vent mode, a vent pressure of 14 psi, a vent flow of 50 ml/min, and a purge flow of 50 ml/min. Compounds in the samples were separated on a DB-5MS [20 m 0.18 mm, i.d., 0.18 m film thickness] capillary column from Agilent (J&W Scientific, Folsom, CA, USA). The temperature program in the GC operation was 50C for 1 min, then increased to 160C at a rate of 5

C/min, followed by the second ramp at a rate of 3C/min to 200C (hold time: 10 min). The carrier gas head pressure was 14 psi (flow rate, 0.7 ml/min at constant flow mode and under retention time-locking conditions). Data Analysis Saliva and skin volatile compound data were combined and evaluated with a newly developed chemometric pattern recognition method as described in Dixon et al. (2006); Penn et al. (2007); and Xu et al. (2007). Method Development and Optimization All volunteers for the initial optimization study provided informed consent to participate in this study (05-9661), as approved by the Indiana University Bloomington Institutional Review Board. Saliva samples from healthy donors (by their own recognition) first were pooled and 0.5 ml aliquots were used for method development. The highly viscous sample matrix appeared to retain small organic volatile compounds, effectively competing with the PDMS material used in sorptive extraction. We learned this when stir bar extraction with stirring in the aqueous phase resulted in low recoveries and a high variation in the peak areas (4050% relative standard deviation, RSD). Similar results were obtained by using a conventional headspace approach (Soini et al., 2005) with the stir bars. When the saliva samples and stir bars were set in the extraction vials at elevated temperatures for 1 hr without stirring (35 and 40C), the recoveries increased, but peak area variability still exceeded 20% (RSD). At 40C, extraction times of 1, 2, 3, and 4 hr revealed that peak areas reached the maximum plateau with a 3-h extraction time (data not shown), whereas the reproducibility of peak areas was less than 5% (RSD, N=4) for most of the compounds.

Results Reproducibility Chromatographic analysis of seven selected salivary components in a pooled sample showed relatively good within-day reproducibility (Table 1). An internal standard was not added to the aliquots of pooled saliva. The long-term reproducibility of the method is reflected in the consistent variability (RSD=14.7%) in integrated peak area of the internal standard during an analysis of 175 samples (Fig. 1). Characterization of Volatile Organic Compounds (VOCs) in Saliva In our previous report of the axillary skin VOCs for the same subject group, we found 373 repeatedly appearing compounds on the skin among about 5,000 VOCs for the 196 subjects (Penn et al., 2007). The chemometric data evaluation of the 175 salivary VOC profiles [consisting typically of

1038 Table 1 Within-day reproducibilitiy of the peak areas (x 106) of selected salivary compounds in four aliquots of a pooled saliva sample Compound 4-methylbenzaldehydeb borneola geraniol 1-dodecanol 1,12-dodecanediolb caffeine hexadecanoic acid Retention time (min) 9.2 11.63 13.76 19.66 26.32 28.30 31.92

J Chem Ecol (2010) 36:10351042 Average peak area counts (N=4) 22.97 4.18 1.54 35.26 2.99 3.06 11.82 SDa 0.43 0.05 0.03 0.84 0.10 0.17 1.68 RDSa (%) 1.87 1.20 1.95 2.38 3.34 5.55 14.21

SD standard deviation; RSD relative standard deviation Tentative identification

100300 peaks (Dixon et al., 2006; Penn et al., 2007)], revealed that 166 of the 373 compounds from the constant axillary sweat compound group (i.e., 44.5%) also were found in saliva. We identified or tentatively identified 90 saliva VOCs (Table 2) among the 166 compounds that were found in common to saliva and skin (Penn et al., 2007). Thus, among the compounds in common, 76 remain unidentified. There was a substantial profile variability of total ion chromatograms (TIC) among the individual saliva samples (Fig. 2). All lactones, and amides (100%) and nearly all carboxylic acids (89%) and aldehydes (83%) found in the group of constant compounds on skin also were present in saliva. Smaller overlap was found for ketones (60%), ethers (50%), hydrocarbons (47%), esters (44%), alcohols and phenols (42%), and amines (12%). No androgen steroids were found in saliva, possibly due to the low detection limits.

Discussion Individual variability observed among the salivary VOC profiles likely is due to several variables, including personal habits such as diet, environment, and genetics. Interestingly,
Fig. 1 Peak areas for the internal standard 7-tridecanone measured at m/z 113 (base peak) by sample number analyzed over a 17-d period (relative standard deviation = 14.7%, N=175)

almost half of the repeatedly appearing 373 VOCs on the skin found in the previous study (Penn et al., 2007) also were found in saliva. This suggests that these compounds could be endogenous. Because of the many possible entry routes for VOCs into the salivary flow, elucidation of their origin and route of entry is likely to be a difficult and complex task. For example, small organic compounds, such as pharmaceuticals, can move from blood to saliva via passive transcellular diffusion, ultrafiltration, or active transport mechanisms. Lipophilicity and molecular weight of the compounds of interest are among properties that influence the blood-saliva transfer rate (Hld et al., 1995). Targeted metabolic experiments containing a particular compound could verify some of the hypothesized excretion routes into saliva through the body and clarify which compounds are exogenous (externally transferred) or endogenous (internally transferred) in origin. Salivary VOC composition could be an excellent indicator of environmental and occupational chemical exposure through transdermal absorption, inhalation through lungs, or via food intake. Environmental exposure to phthalates has been well documented in different body fluids. Multifunctional phthalate compounds can originate from soaps, shampoos, cosmetics, plastics, paints, and pesticide formulations. Their metabolites have been found

J Chem Ecol (2010) 36:10351042 Table 2 Compounds identified in saliva that have also been found on skin in a previous study of the same subject group (Penn et al., 2007) Alcohols and phenols Rt (min) 12.47 12.90 13.82 16.48 27.75 Aldehydes Rt (min) 12.74 13.93 14.38 15.46 18.10 20.28 20.61 23.00 23.58 26.04 Ketones Rt (min) 8.98 14.99 17.47 17.69 18.27 19.66 20.22 20.54 22.52 23.09 24.92 28.70 Carboxylic acids Rt (min) 12.24 14.76 21.90 23.18 24.20 25.74 26.80 28.36 28.65 29.31 31.70 31.96 34.37 34.94 Compound octanoic acid nonanoic acid dodecanoic acid 9-methyldodecanoic acida tridecanoic acid 10-methyltridecanoic acida myristic acid (tetradecanoic acid) a methyltetradecanoic acida a methyltetradecanoic acida pentadecanoic acid 9-hexadecenoic acida palmitic acid (hexadecanoic acid) 9-heptadecenoic acida heptadecanoic acid Table 2 (continued) 37.31 38.03 Esters Rt (min) 11.40 16.37 17.16 21.24 21.95 22.21 23.30 23.73 24.15 24.46 27.53 28.23 30.93 32.40 32.99 33.70 37.35 43.35 Lactones Rt (min) 12.27 16.70 18.60 Amines Rt (min) 15.19 Amides Rt (min) 17.18 18.04 21.98 Hydrocarbons Rt (min) 12.26 12.50 15.15 17.55 17.77 18.22 19.72 19.96 20.36 20.38 21.77 22.56 oleic acid stearic acid (octadecanoic acid)

1039

Compound -terpineola 2-phenoxyethanola geraniol eugenol a hexadecadienola Compound decanal p-anisaldehydea geranial undecanal dodecanal liliala tridecanal tetradecanal pentylcinnamaldehydea E-2-hexylcinnamaldehydea Compound acetophenone 2-undecanone jasmonea 2-dodecanone -iononea -iononea 2-tridecanone Z--ironea 2-tetradecanone benzophenonea 2-pentadecanone 2-acetyl-3,5,5,6,8,8-hexa-methyl-5,6,7, 8-tetrahydronaphthalene a

Compound benzyl acetatea -terpineyl acetatea geranyl acetate -trichloromethylbenzyl acetatea pentyl salicylatea isooctanediol dibutyratea isopropyl dodecanoatea methyl-cis-dihydrojasmonatea 3Z-1-hexenyl salicylatea 1-hexyl salicylatea 2-ethylhexyl salicylatea isopropyl hexadecanoatea methyl hexadecanoate (methyl palmitate)a hexyl dodecanoatea ethyl hexadecanoate (ethyl palmitate) isopropyl hexadecanoatea 2-ethyl-hexyl-4-methoxycinnamatea 1-octyl-4-methoxycinnamatea Compound -heptanolactonea -nonanolactone coumarin Compound an aliphatic aminea Compound methyl N,N-diethylthiocarbamatea a hydroxyl acetanilidea n-propylbenzamidea Compound 1-dodecene dodecane tridecane 1-tetradecene tetradecane -caryophyllene trans-muurola-4(14),5-dienea a methyl biphenyla -farnesene pentadecane 4-methylpentadecanea hexadecane

1040 Table 2 (continued) 23.18 25.04 27.48 30.22 32.93 35.79 38.79 Other (ethers) Rt (min) 17.65 24.43 29.26
a

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6-phenylundecanea heptadecane octadecane nonadecane eicosane (C-20 linear hydrocarbon)a heneicosane (C-21 linear hydrocarbon)a docosane (C-22 linear hydrocarbon)a Compound diphenyl ethera dioctylethera bis(benzyloxy)methanea

Tentative identification

in human urine (Silva et al., 2004a), breast milk (Calafat et al., 2004), amniotic fluid (Silva et al., 2004b), serum (Kato et al., 2003), and saliva (Silva et al., 2005). In general, salivary sampling is a less invasive alternative compared to serum collection for many biomedical applications (Cook, 2002; Grschl and Rauh, 2006; Chiappin et al., 2007). Certain limitations may pertain in sampling for sufficient volumes of saliva from infants, geriatric subjects, or subjects with certain physiological states that
Fig. 2 Representative GC-MS total ion chromatograms of salivary VOC profiles from two subjects from different families: a: a female, and b: a male. Analytical conditions are described in the text. Numbers and names for selected compounds are a: 1=acetophenone, 2=eugenol, 3=-caryophyllene, 4=hexadecane, 5=methyldihydro-cis-jasmonate, 6=hexadecanoic acid, 7=ethyl hexadecanoate, 8=oleic acid, 9=docosane and b: 1=acetophenone, 2=-heptanolactone, 3=decanal, 4=nonanoic acid, 5=nicotine, 6=tetradecane, 7=dodecanoic acid, 8=tetradecanoic acid, 9=caffeine, 10=methylhexadecanoate, 11=9-hexadecenoic acid, 12=hexadecanoic acid, 13=oleic acid, 14=octadecanoic acid. For a and b, IS=the internal standard (7-tridecanone)

cause reduced saliva production (Granger et al., 2007). Saliva collection methods also may influence sample composition and induce interference to the analytical methods. Stimulated (e.g., by chewing gum) vs. nonstimulated saliva flow, or use of certain devices such as cotton balls or swabs placed in mouth can influence the final composition of the saliva (Grschl and Rauh, 2006; Schipper et al., 2007). In this study, a non-stimulated saliva collection approach was chosen because stimulating agents and polymer-containing collection devices could introduce further contaminants in the analysis of the innate salivary VOC analysis. In summary, the static stir bar extraction method for analyzing small organic compounds in saliva provided a sensitive and precise analytical approach for a large variety of compounds with different functional groups. The longterm reproducibility of the method allowed the analysis of a large number of samples in a high-throughput fashion. In this study, there was a large number of volatile compounds identified in saliva from a group of 175 subjects that were in common with components in previously analyzed sweat samples from the same subject group. This suggests that saliva presents a systemic body fluid that is potentially suitable for monitoring small organic compounds in physiological, metabolomic, pharmacokinetic, forensic, and toxicological studies.

J Chem Ecol (2010) 36:10351042 Acknowledgments This work was sponsored jointly by Lilly Chemistry Alumni Chair (Indiana University), and the Indiana METACyt Initiative, funded in part through a major grant from the Lilly Endowment to Indiana University, and ARO Contract DAAD1903-1-0215. Approved for Public Release, Distribution Unlimited.

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