High-temperature gas chromatography-mass spectrometry for skin surface lipids profiling
2010; Elsevier BV; Volume: 52; Issue: 1 Linguagem: Inglês
10.1194/jlr.d008094
ISSN1539-7262
AutoresRime Michael‐Jubeli, Jean Bleton, Arlette Baillet‐Guffroy,
Tópico(s)Dermatology and Skin Diseases
ResumoSkin surface lipids (SSLs) arising from both sebaceous glands and skin removal form a complex lipid mixture composed of free fatty acids and neutral lipids. High-temperature gas chromatography coupled with electron impact or chemical ionization mass spectrometry was used to achieve a simple analytical protocol, without prior separation in classes and without prior cleavage of lipid molecules, in order to obtain simultaneously i) a qualitative characterization of the individual SSLs and ii) a quantitative evaluation of lipid classes. The method was first optimized with SSLs collected from the forehead of a volunteer. More than 200 compounds were identified in the same run. These compounds have been classified in five lipid classes: free fatty acids, hydrocarbons, waxes, sterols, and glycerides. The advantage to this method was it provided structural information on intact compounds, which is new for cholesteryl esters and glycerides, and to obtain detailed fingerprints of the major SSLs. These fingerprints were used to compare the SSL compositions from different body areas. The squalene/cholesterol ratio was used to determine the balance between sebaceous secretion and skin removal. This method could be of general interest in fields where complex lipid mixtures are involved. Skin surface lipids (SSLs) arising from both sebaceous glands and skin removal form a complex lipid mixture composed of free fatty acids and neutral lipids. High-temperature gas chromatography coupled with electron impact or chemical ionization mass spectrometry was used to achieve a simple analytical protocol, without prior separation in classes and without prior cleavage of lipid molecules, in order to obtain simultaneously i) a qualitative characterization of the individual SSLs and ii) a quantitative evaluation of lipid classes. The method was first optimized with SSLs collected from the forehead of a volunteer. More than 200 compounds were identified in the same run. These compounds have been classified in five lipid classes: free fatty acids, hydrocarbons, waxes, sterols, and glycerides. The advantage to this method was it provided structural information on intact compounds, which is new for cholesteryl esters and glycerides, and to obtain detailed fingerprints of the major SSLs. These fingerprints were used to compare the SSL compositions from different body areas. The squalene/cholesterol ratio was used to determine the balance between sebaceous secretion and skin removal. This method could be of general interest in fields where complex lipid mixtures are involved. Even if the barrier property of skin is localized in the stratum corneum (SC), skin surface lipids (SSLs) present in hydrolipidic film have a close relationship with SC lipids. Thus, SSLs participate in this barrier function (1Fluhr J.W. Mao-Qiang M. Brown B.E. Wertz P.W. Crumrine D. Sundberg J.P. Feingold K.R. Elias P.M. Glycerol regulates stratum corneum hydration in sebaceous gland deficient (Asebia) mice.J. Invest. Dermatol. 2003; 120: 728-737Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 2Mavon A. Zahouani H. Redoules D. Agache P. Gall Y. Humbert P. Sebum and stratum corneum lipids increase human skin surface free energy as determined from contact angle measurements: a study on two anatomical sites.Colloids Surf. B Biointerfaces. 1997; 8: 147-155Crossref Scopus (88) Google Scholar, 3Thiele J.J. Weber S.U. Packer L. Sebaceous gland secretion is a major physiologic route of vitamin E delivery to skin.J. Invest. Dermatol. 1999; 113: 1006-1010Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 4Pilgram G.S.K. van der Meulen J. Gooris G.S. Koerten H.K. Bouwstra J.A. The influence of two azones and sebaceous lipids on the lateral organization of lipids isolated from human stratum corneum.Biochim. Biophys. Acta. 2001; 1511: 244-254Crossref PubMed Scopus (68) Google Scholar, 5Man M.Q. Xin S.J. Song S.P. Cho S.Y. Zhang X.J. Tu C.X. Feingold K.R. Elias P.M. Variation of skin surface pH, sebum content and stratum corneum hydration with age and gender in a large chinese population.Skin Pharmacol. Physiol. 2009; 22: 190-199Crossref PubMed Scopus (149) Google Scholar). SSLs are formed from a complex mixture of free fatty acids (FFAs) and neutral lipids arising from both sebaceous secretion and skin removal (6Greene R.S. Downing D.T. Pochi P.E. Strauss J.S. Anatomical variation in the amount and composition of human skin surface lipid.J. Invest. Dermatol. 1970; 54: 240-247Abstract Full Text PDF PubMed Scopus (212) Google Scholar). Freshly liberated sebum contains predominantly triglycerides, wax esters, and squalene (7Vantrou M. Venencie P.Y. Chaumeil J.C. Les lipides cutanés de surface chez l'homme: origine, synthése, régulation.Ann. Dermatol. Venereol. 1987; 114: 1115-1129PubMed Google Scholar, 8Downing D.T. Wertz P.W. Stewart M.E. The role of sebum and epidermal lipids in the cosmetic properties of skin.Int. J. Cosmet. Sci. 1986; 8: 115-123Crossref PubMed Scopus (23) Google Scholar, 9Saint-Léger D. Lévêque J-L. Les methodes d’évaluation quantitative des lipides de surface chez l'homme. Présentation d'une nouvelle procédure.Int. J. Cosmet. 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Several lipid classes are then present in SSLs, and the complexity of the mixture is emphasized by the structural microheterogeneity within each class. Hydrolipidic film represents an important modulator of cutaneous barrier functions (1Fluhr J.W. Mao-Qiang M. Brown B.E. Wertz P.W. Crumrine D. Sundberg J.P. Feingold K.R. Elias P.M. Glycerol regulates stratum corneum hydration in sebaceous gland deficient (Asebia) mice.J. Invest. Dermatol. 2003; 120: 728-737Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 2Mavon A. Zahouani H. Redoules D. Agache P. Gall Y. Humbert P. Sebum and stratum corneum lipids increase human skin surface free energy as determined from contact angle measurements: a study on two anatomical sites.Colloids Surf. B Biointerfaces. 1997; 8: 147-155Crossref Scopus (88) Google Scholar, 3Thiele J.J. Weber S.U. Packer L. Sebaceous gland secretion is a major physiologic route of vitamin E delivery to skin.J. Invest. Dermatol. 1999; 113: 1006-1010Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 4Pilgram G.S.K. van der Meulen J. Gooris G.S. Koerten H.K. Bouwstra J.A. The influence of two azones and sebaceous lipids on the lateral organization of lipids isolated from human stratum corneum.Biochim. Biophys. Acta. 2001; 1511: 244-254Crossref PubMed Scopus (68) Google Scholar, 5Man M.Q. Xin S.J. Song S.P. Cho S.Y. Zhang X.J. Tu C.X. Feingold K.R. Elias P.M. Variation of skin surface pH, sebum content and stratum corneum hydration with age and gender in a large chinese population.Skin Pharmacol. Physiol. 2009; 22: 190-199Crossref PubMed Scopus (149) Google Scholar), particularly in SC hydration. Moreover, sebum transports antioxidants to the skin surface (e.g., vitamin E), preventing aging (3Thiele J.J. Weber S.U. Packer L. Sebaceous gland secretion is a major physiologic route of vitamin E delivery to skin.J. Invest. 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Maintaining the stability of the amounts and composition of SSL is of major importance to preserving skin barrier properties. Moreover, information provided by SSL analyses, such as fine profiling, squalene/cholesterol ratio, and intact glyceride patterns, contribute to the wide knowledge of physiological and pathological evolution of hydrolipidic film. Our study falls within the framework of lipidomics, in which global SSLs profiles are determined in the entire sample, keeping the structural integrity of the compounds. Our purpose was to develop a simple analytical protocol using a noninvasive sampling method without time-consuming sample preparation steps that would provide a qualitative characterization of individual SSL compounds and a quantitative evaluation of different lipid classes. Previously, SSLs were studied mostly after being separated into lipid classes, using thin-layer chromatography. 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Individual compounds were identified using additional techniques such as HPLC/atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) (30Vrkoslav V. Urbanová K. Cvacka J. Analysis of wax ester molecular species by high performance liquid chromatography/atmospheric pressure chemical ionisation mass spectrometry.J. Chromatogr. A. 2010; 1217: 4184-4194Crossref PubMed Scopus (39) Google Scholar), GC-FID (24Nazzaro-Porro M. Passi S. Boniforti L. Belsito F. Effects of aging on fatty acids in skin surface lipids.J. Invest. Dermatol. 1979; 73: 112-117Abstract Full Text PDF PubMed Scopus (96) Google Scholar, 29Yamamoto A. Serizawa S. Ito M. Sato Y. Effect of aging on sebaceous gland activity and on the fatty acid composition of wax esters.J. Invest. Dermatol. 1987; 89: 507-512Crossref PubMed Scopus (71) Google Scholar, 32Robyn N.S. Anna B. George A.V. Neil J.M. The effect of a low glycemic load diet on acne vulgaris and the fatty acid composition of skin surface triglycerides.J. 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The columns available today that are stable at high temperature are suitable for successful separations of compounds with high molecular weights (33Myher J.J. Kuksis A. General strategies in chromatographic analysis of lipids.J. Chromatogr. B Biomed. Sci. Appl. 1995; 671: 3-33Crossref PubMed Scopus (0) Google Scholar, 34Kuksis A. Myher J.J. Geher K. Quantitation of plasma lipids by gas-liquid chromatography on high temperature polarizable capillary columns.J. Lipid Res. 1993; 34: 1029-1038Abstract Full Text PDF PubMed Google Scholar, 35Moldovan Z. Jover E. Bayona J.M. Systematic characterisation of long-chain aliphatic esters of wool wax by gas chromatography-electron impact ionisation mass spectrometry.J. Chromatogr. A. 2002; 952: 193-204Crossref PubMed Scopus (30) Google Scholar, 36Jover E. Moldovan Z. Bayona J.M. Complete characterisation of lanolin steryl esters by sub-ambient pressure gas chromatography-mass spectrometry in the electron impact and chemical ionisation modes.J. 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Both electron impact (EI) and chemical ionization (CI) procedures were necessary to obtain structural information and molecular masses. The protocol was optimized to preserve the integrity of thermolabile compounds and to provide good separation between the lipid classes and between individual compounds. Each compound was identified by its retention time, molecular mass, and fragmentation pattern and then assigned to the proper class. Thus, informative fingerprints of SSLs were obtained. To illustrate this method, the differences among SSL compositions from different body areas have been studied. The peak area normalization with the response factor approach was applied (40Miller J.M. Chromatography: concepts and contrasts. John Wiley and Sons, Inc., Hoboken, NJ2005: 300-301Google Scholar). Relative amounts of lipid classes were determined, and the squalene/cholesterol ratio was assessed to describe the balance between sebum secretion and skin removal (41Mélissopoulos A. Levacher C. La peau: structure et physiologie. Tec & Doc Lavoisier, Paris, France1998: 71-73Google Scholar). To sum up, this method gives structural information and detailed lipid profiles by using only one analytical protocol. It could be of a great interest for establishing the proof for medical treatment efficacy in diseases such as acne, atopic dermatitis, seborrhea, or psoriasis. More generally, this method can be extended to numerous other applications in medical, pharmaceutical, cosmetic, and alimentary fields where complex lipid mixtures are involved. Diethyl ether, isooctane, palmitic acid, monostearin (also glyceryl monostearate), squalene, cholesterol, palmityl palmitate, dipalmitin, cholesteryl palmitate, tripalmitin, pyridine, and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) were obtained from Sigma-Aldrich (St. Louis, MO). Lipid-free absorbent papers were purchased from Rizla UK Ltd. (Pontypridd, South Wales, UK). Medical tapes, i.e., elastic tubular nettings for the application of gauzes and medications (Surgifix), were purchased from Fra Production Spa (Cisterna d'Asti, Italy). After cleaning the body sampling areas with water and soap, 12 h before sampling, the volunteer did not apply any cosmetic or pharmaceutical product to these areas until the end of sampling. Skin surface lipids were collected from six areas (forehead, back, thorax, forearm, thigh, and calf) of a female volunteer, 26 years old, with healthy skin, living in France for more than 6 months before the date of sampling. Human samples were obtained following review and approval from an institutional review board, and informed consent was obtained from the volunteer. Two lipid-free absorbent papers were maintained on the defined area for 30 min, using medical tape, and then removed with tweezers and introduced into a closed vial. This step was repeated four times. The collected lipids were extracted from papers twice with 40 ml of diethyl ether. The solution was concentrated in a rotary evaporator at 30°C, then transferred into a 2-ml vial, and dried under a gentle stream of nitrogen. The dry extract was stored at −20°C until further use. The trimethylsilylation reagent consisted of BSTFA/pyridine, 50:50 (v/v). The dry extract was trimethylsilylated at room temperature for 30 min with 100 µl of reagent. The excess reagent was then removed using rotary evaporation at 30°C, and the dried residue was dissolved in 500 µl of isooctane. For forearm, thigh, and calf samples, the solution was directly injected (1 μl, on-column). For forehead, back, and thorax samples, the solution was diluted in isooctane (1:5; v/v) before injection. A Thermo Scientific (Austin, TX) gas chromatography unit (Trace GC Ultra) equipped with an on-column injector was coupled to a quadrupole DSQII mass spectrometer via a high-temperature interface. The separation was achieved using a 30 m × 0.32 mm ZB-5HT capillary column (Phenomenex, Torrance, CA) coated with 0.1 µm of 5% diphenyl/95% dimethylpolysiloxane, connected to a 5-m 0.32-mm HT-deactivated tubing guard column. Helium was used as a carrier gas at a constant flow of 2 ml/min. The injector and transfer line temperatures were set to 80°C and 350°C, respectively. The oven temperature was programmed from 80°C to 240°C at 5°C/min; 240°C to 320°C at 2.5°C/min; and 320°C to 350°C at 1°C/min. EI mass spectra were recorded in the total ion current (TIC) monitoring mode. The operating conditions for EI-MS were source temperature at 250°C, ionizing energy at 70 eV, and scan range from m/z 45 to 1,000 with a period of 1 s. For CI-MS, ionizing energy was 120 eV, and ammonia was used as the reagent gas at a constant flow of 1ml/min. The SSL derivatization step was used to improve the detection of compounds having acid and/or alcohol functions. We checked to make sure that with the BSTFA/pyridine (50:50; v/v) mixture, a 30-min reaction at room temperature led to results similar to those obtained at 80°C. Thus, we chose cold silylation in order to prevent any thermodegradation of the samples (33Myher J.J. Kuksis A. General strategies in chromatographic analysis of lipids.J. Chromatogr. B Biomed. Sci. Appl. 1995; 671: 3-33Crossref PubMed Scopus (0) Google Scholar). On-column injection was used to avoid discrimination between analytes of very different volatility and to keep the structural integrity of thermolabile compounds like steryl esters. A high-temperature stable capillary column with apolar stationary phase was chosen because it is chemically inert toward silylated compounds (42Donike M. Die temperaturprogrammierte Analyse von Fettsäuretrimethylsilylestern: Ein kritischer Qualitätstest für gas-chromatographische Trennsäulen.Chromatographia. 1973; 6: 190-195Crossref Scopus (54) Google Scholar). At the beginning of the study, rapid oven temperature programming was tested, from 80°C to 390°C at 30°C/min and an 8-min isothermal step at 390°C. That program led to a lot of coelutions, very high background noise at the end of the chromatogram, and a partial degradation of steryl esters. To remedy these problems, an optimized oven temperature program was achieved. A method with three temperature gradients was adopted: from 80°C to 240°C at 5°C/min to elute FFAs; then to 320°C at 2.5°C/min to elute wax esters and diglycerides; and finally to 350°C at 1°C/min to elute steryl esters and triglycerides (Fig. 1). Thus, selectivity was improved and coelutions and background noise were decreased, which provided mass spectra of better quality. Samples were collected from the forehead of a female volunteer by using absorbent papers. This noninvasive method allows collecting exclusively SSLs. In the SSL sample studied, a compound corresponding to a given chromatographic peak was identified through its retention time and MS data (molecular mass and fragmentation pattern) and assigned to the proper class regardless of isomers and unsaturation positions in order to establish the profiles of all SSLs. In addition to the molecular [M]+ ion, the [M-CH3]+ ion, due to the loss of a methyl from the trimethylsilyl (TMS) group, was used for identification. Under our chromatographic conditions, major FFA (trimethylsilyl esters) were contained between decanoic acid (C10:0), and tetracosenoic acid (C24:1) (Table 1). These acids have been partly described in a study by Green et al. (28Green S.C. Stewart M.E. Downing D.T. Variation in sebum fatty acid composition among adult humans.J. Invest. Dermatol. 1984; 83: 114-117Abstract Full Text PDF PubMed Scopus (50) Google Scholar) from C13 to C18. Due to the branching of chains or unsaturation positions, several FFA with the same total number of carbon atoms, the so-called carbon number, were eluted at different retention times. Hexadecenoic acid (C16:1) was the most abundant acid detected in the sample. According to different authors (31Marzouki Z.M.H. Taha A.M. Gomaa K.S. Fatty acid profiles of sebaceous triglycerides by capillary gas chromatography with mass-selective detection.J. Chromatogr. B Biomed. Sci. Appl. 1988; 425: 11-24Crossref Scopus (15) Google Scholar, 43Drake D.R. Brogden K.A. Dawson D.V. Wertz P.W. Thematic review series: skin lipids. Antimicrobial lipids at the skin surface.J. Lipid Res. 2008; 49: 4-11Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar), we could identify it as sapienic acid (C16:1Δ6).TABLE 1Lipid classes identified in trimethylsilylated SSL samples analyzed by HTGC-MSFree Fatty AcidsFree Fatty Acids (continued)RTHydrocarbonsMonoglyceridesRTRCORTRCO29.29SqualeneRTRCO8.05C10:018.89C16:031.62Squalene25.81C14:011.32C12:019C16:033.14Epoxysqualene28.26C16:012.06C12:019.13C16:128.71C16:012.14C13:019.28C16:1RTFree sterols31C18:112.9C13:019.4C16:035.91Cholesterol31.45C18:013.09C13:019.53C16:038.22Lanosterol13.32C13:019.67C16:038.943 hydroxy lanosta-8,24-dieneDiglycerides13.46C13:019.57C16:0RTR'COR’’CO14.05C13:019.85C16:046.83C15:0C16:014.34C13:020.21C17:1Cholesteryl esters47.67C14:0C18:014.84C14:020.34C17:0RTR1CO48.17C15:0C16:115.27C14:020.53C17:061.48C14:148.69C15:0C16:115.49C14:120.87C17:161.72C14:149.20C15:0C16:015.99C14:021.17C18:162.01C14:049.92C16:0C16:116.65C15:121.39C17:063.17C15:149.92C16:0C16:016.74C15:021.91C18:163.48C15:150.05C16:0C16:116.93C15:022.14C18:263.82C15:050.37C16:0C16:017.17C15:022.43C18:264.57C16:150.61C16:0C16:117.32C15:022.53C18:164.99C16:051.05C16:0C16:017.17C15:022.8C18:065.31C16:153.51C18:1C16:017.32C15:023.06C18:065.71C16:054.61C18:0C16:017.86C15:029.05C20:166.46C17:157.45C18:0C18:018.11C15:030.45C21:166.81C17:158.10C18:0C18:018.46C16:131.8C22:166.84C17:018.58C16:032.85C23:167.20C17:018.69C16:034.79C24:168.59C18:1Several trimethylsilylated FFA with the same total carbon number were eluted at different retention times (RT, min); this could due to the branching of chains or unsaturation positions. RCO, R1CO, R′CO and R″CO, acid moieties. Open table in a new tab Several trimethylsilylated FFA with the same total carbon number were eluted at different retention times (RT, min); this could due to the branching of chains or unsaturation positions. RCO, R1CO, R′CO and R″CO, acid moieties. Other fatty acids were detected in trace amounts: C7:0 (3.44 min), C8:0 (4.78 min), C9:0 (6.45 min), C11:0 (10.38 min), and C12:1 (12.26 min). Their amounts were too low to significantly affect the total sum of fatty acids. This result was consistent with the findings of Vantrou et al. (7Vantrou M. Venencie P.Y. Chaumeil J.C. Les lipides cutanés de surface chez l'homme: origine, synthése, régulation.Ann. Dermatol. Venereol. 1987; 114: 1115-1129PubMed Google Scholar) and James et al. (25James A.T. Wheatley V.R. Studies of sebum. 6. The determination of the component fatty acids of human forearm sebum by gas–liquid chromatography.Biochem. J. 1965; 63: 269-273Crossref Scopus (35) Google Scholar). Three main hydrocarbons were identified using the [M]+ and [M-CH3]+ ions (Table 1). The squalene, which is the marker of the sebaceous function (44Strauss J.S. Pochi P.E. Downing D.T. The sebaceous glands: twenty-five years of progress.J. Invest. Dermatol. 1976; 67: 90-97Abstract Full Text PDF PubMed Scopus (35) Google Scholar, 45Pappas A. Epidermal surface lipids.Dermatoendocrinol. 2009; 1: 72-76Crossref PubMed Google Scholar), was clearly the most abundant compound in the sample studied. In addition, epoxy-squalene was also detected. It could be an intermediate in the cholesterol synthesis from squalene (46Nikkari T. Comparative
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