1. Introduction Institute for Pheromone Research and Department of Chemistry, Indiana University, 800 E. Kirkwood Ave., Bloomington, IN 47405, USA 1 Ludwig-Boltzman-Institute for Urban Ethology, Department of Anthropology, Althanstrasse 14, A-1090 Vienna, Austria 2 Centre for Chemometrics, School of Chemistry, University of Bristol, Cantocks Close, Bristol BS8 ITS, UK 3 Konrad Lorenz Institute for Ethology, Austrian Academy of Sciences, Savoyenstr. 1a, A-1160 Vienna, Austria GC- MS GC-AED (Atomic Emission Detection) Genetic factors and biochemical individuality of human skin volatiles studied through GC-MS and chemometric methodologies H. A. Soini, M. V. Novotny, D. Wiesler, I. Klouckova, E. Oberzaucher 1, K. Grammer 1, S. J. Dixon 2, F. Gong 2, Y. Xu 2, R.G. Brereton 2, and D.J. Penn 3 Sampling and Analysis References Acknowledgments Conclusions Agilent Quadrupole GC-MS 6890N-5973i  2 identical systems - Column: DB- 5MS (20 m x 0.18 mm, 0.18 μm film, J&W Scientific), helium flow 0.6 ml/min ml/min (9 psi head-pressure) -Temperature program: 50 °C (1 min), 5 °C /min to 160 °C, then 3 °C/min min to 200 °C (hold 10 min) - Transfer line temperature: 280 °C - Quadrupole and ion source temperatures 150 °C and 230 °C, respectively. - Spectra acquisition: Positive EI, 70 eV, scans 35-350 msu (4.51 scans/s) Gerstel TDSA –CIS sample introduction -TDSA (splitless mode): 20 °C (0.5 min), then 60 °C/min to 250 °C (3 min) -CIS-4 (solvent vent mode): -80 °C, 12 °C /s to 280 °C (10 min) Agilent GC-AED, 6890 – G2350A with Gerstel TDSA-CIS - Column: DB- 5MS (30 m x 0.25 mm, 0.25 μm film, J&W Scientific) - Helium 1.2 ml/min, constant flow mode, 14 psi head-pressure - Temperature program: 50 °C (2 min), 3 °C/min to 200 °C (12 min) - Detection at emission lines of 194 nm (carbon), 181 nm (sulfur) and 174 nm (nitrogen) Sample introduction (TDSA-CIS): -Similar to GC-MS sample introduction, except CIS initial temperature -60 °C Results Chemometric methods Stir bar roller device designed and constructed at Indiana University, Department of Chemistry, Mechanical Instrument Services by Mr. Gary Fleener Stir bar: 10 mm, 0.5 mm PDMS film (Twister , Gerstel GmbH) Quantitative comparisons of VOC profiles  Addition of embedded internal standards into the stir bar before sample collection – SBSE aqueous extraction - 13 C- labelled benzyl alcohol (50 ng) - 7- tridecanone (8 ng)  Storage under refrigeration - Internal standards stable for 20 days - Sample VOCs stable for 14 days  Sampling location can be different from the analysis location 8,9 Subjects: 197 adults - males and females within 16 families Samples: repeatedly - collected axillary samples during 10 weeks period  985 samples analyzed Reproducibility of compound measurement, one individual (RSD %, n=4) Nonanal 9.0 % Decanal 6.1 % Geranylacetone 6.7 % 1-Dodecanol 18.1 % 1-Hexadecanol 11.0 % Internal standard reproducibility by GC-MS Short term reproducibility n=22, over 5 days RSD = 8.1 % (7-tridecanone) RSD = 11.2 % 13 C-benzyl alcohol Long term reproducibility n=958, over 3 months RSD = 14.3 % (7-tridecanone) RSD = 14.7 % ( 13 benzyl alcohol) Pilot study – 5 individuals Quantitative and stable GC-MS method for a skin sample set of 965  chemometric data evaluation with novel methods  Gender differences and gender specific compounds  Family differences and family marker compounds  Individual marker compounds Gender differences GC-AED sulfur line GC-MS TIC Male Female Family differences -Novel GC-MS peak alignment methods were developed to locate constant compounds among 4987 peaks 6,7  373 constant compounds were found (appearance at least 4 times among 5 repeats) - peaks were square-rooted and normalized 1)Exploratory methods (PCA analysis, univariate t-statistics) were used for finding biological marker compounds 2)The second set of predictive and class modeling methods were used for predicting the origin of the unknowns  12 compounds strongly related to gender  70 related to family and or HLA  45 individual marker compounds Precision of the method Male richer in sulfur compounds Among 373 constant compounds, identifications for 160 compounds - linear and branched hydrocarbons, acids, esters, alcohols, ketones and aldehydes Additional sampling application: collection of finger prints from the glass surface GC-MS TICs of individuals A, B, C 1. Jacob,S.; McClintock, M.K. Horm.Behav. 2000, 37, 57-78. 2. Stern, K.; McClintock, M.K. Nature 1998, 392, 177-179. 3.Sato, K.; Kang, W.H.; Saga, K.; Sato, K.T. J. Am. Acad. Dermatol. 1989, 20, 537-563. 4. Jacob,S. ; McClintock, M.K. Nature Genet. 2002, 30, 175-179. 5.Gower, D.B.; Holland, K.T.; Mallet, A.I.; Rennie, P.J.; Watkins, W.J. J.Steroid.Biochem.Molec.Biol, 2003, 48, 409-418. 6.Brereton, R.G. Chemometrics: Data Analysis for the Laboratory and Chemical Plant (Wiley, Chichester, UK, 2003) 7.Dixon, S.J.; Brereton, R.G.; Soini, H.A.; Novotny, M.V.; Penn, D.J., submitted 2006 8. Soini, H.A.; Bruce, K.E.; Klouckova, I.; Brereton, R.G.; Penn, D.J.; Novotny, M.V., submitted 2006 9. Penn, D.J.; Oberzaucher, E.; Grammer, K.; Fischer, G., Soini, H.A.; Wiesler, D.; Novotny, M.V.; Dixon, S.J.; Xu, Y.; Brereton, R.G, submitted 2006 This work was sponsored jointly by Lilly Chemistry Alumni Chair (Indiana University) and ARO contract DAAD19-03-1-0215 (  ). Opinions, interpretations, conclusions, and recommendations are those of authors and are not necessarily endorsed by the United States Government. Human skin surface contains four different types of glands that excrete compounds from polar and non-polar lipids and peptides to small volatile compounds (VOCs). Gland distribution differs within the different skin regions. Underarm (axillary) area contains all four types of glands: sebaceous, eccrine (sweat), apocrine and apoeccrine glands. Human body odors can reflect physiological state and mood of individuals. 1,2 Metabolic (disease) disorders have been reported altering body odors as well. 3 Body odors may also have their genetic attributes 4 (e.g., MHC related odors) while resident microflora may contribute to their occurrence. 5 The studies of human skin VOCs have been limited by the lack of quantitative analytical techniques for comparing a large number of individuals. Also, the previously reported studies are tedious and not suitable for analyzing a large number of samples. We have developed a high-throughput and highly quantitative technique applicable for profiling VOCs from human skin. The method allows us to monitor precisely about 400 compounds by gas chromatography-mass spectrometry (GC-MS). Repeatedly collected underarm VOC samples of nearly 200 subjects were analyzed. The sampling approach facilitated sample collection at the geographically different location (Europe) from the analysis location (USA). Advanced chemometric exploratory and predicting methods were employed for evaluation of the large data set of the VOC profiles. 6,7 Additionally, the analytical method was demonstrated as useful for collection of biomaterials associated with human latent finger- prints from a non-biological surface. Latent fingerprints: - Sweat gland contents from fingers - Gland contents from face, etc. through touching - Environmental components  The novel rolling stir bar sampling method for collecting human skin VOCs provided highly quantitative and reproduciple results for a large data set with high analytical through-put.  Samples could be collected in a remote site and analyzed later (within 10 days) in another location.  Using newly developed chemometric methods for the data evaluation, 1) distinct individual marker compounds, 2) compounds defining families, and 3) gender-unique compounds were found in the VOC profiles  The method may have implications for research in human chemosensory communication and large metabolomic studies on human skin 8,9 Family H Family B Family A GC-MS TICs PCA of the family A – effect of individual marker compounds “Approved for Public Release, Distribution Unlimited” ()()