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Journal of Chromatography A, 1218 (2011) 9384– 9389

Contents lists available at SciVerse ScienceDirect

Journal of Chromatography A

j our na l ho me p ag e: www.elsev ier .com/ locate /chroma

dentification of �6-monounsaturated fatty acids in human hairnd nail samples by gas-chromatography–mass-spectrometrysing ionic-liquid coated capillary column

rédéric Destaillats ∗, Marjorie Guitard, Cristina Cruz-Hernandezestlé Research Center, Lausanne, Switzerland

r t i c l e i n f o

rticle history:eceived 25 September 2011eceived in revised form 20 October 2011ccepted 21 October 2011vailable online 15 November 2011

eywords:as-chromatographyair

onic liquidonounsaturated fatty acidailebum

a b s t r a c t

Lipids found in human sebum contain specific fatty acids such as sapienic (cis-6 16:1), cis-8 18:1 andsebaleic (cis-5, cis-8 18:2) acids. These fatty acids belong to the n-10 series and the initial step involvedin their synthesis is the desaturation of palmitic acid by the �6-desaturase to form sapienic acid. Theoccurrence in human hair and nail of sapienic (cis-6 16:1), cis-8 18:1 and sebaleic (cis-5, cis-8 18:2) acidshas not been reported to our knowledge nor has the formation of �6-monounsaturated fatty acids fromother saturated fatty acids such as stearic acid. The pre-requisite for such identification is the ability toseparate cis-6 from cis-8 monounsaturated fatty acid derivative (i.e. cis-6 18:1 from cis-8 18:1 methylesters) by gas-chromatography (GC) and such separation is not achievable using cyanoalkyl based highlypolar capillary columns. In the present study, we used the 100 m SLB-IL 111 ionic liquid based cap-illary column recently commercialized by Supelco (Bellefonte, PA). The identification was performedby gas-chromatography–mass-spectrometry (GC–MS) with electronic impact (EI) ionization using 4,4-dimethyloxazoline (DMOX) derivatives. Baseline separation between critical cis-6 18:1 and cis-8 18:1isomers was obtained allowing unambiguous identification based on MS fragmentation and pure stan-

dards. In sebum, hair and nail samples, sapienic, cis-8 18:1 and sebaleic acids were found and moreimportantly, petroselinic acid was identified in these human tissues for the first time. In addition, weidentified in hair and nail lipids cis-6 14:1, cis-6 15:1, iso-cis-6 16:1, aiso-cis-6 17:1 and cis-6 17:1 as theirDMOX derivatives based on molecular ion as well as diagnostic ion fragments at m/z 167, 180 and 194.Possible biosynthesis scenario is postulated to explain the occurrence of these �6-monounsaturatedfatty acids in human sebum, hair and nail lipids.

© 2011 Elsevier B.V. All rights reserved.

. Introduction

In human, fatty acids from the n-10 series have been reportedo occur in sebum [1,2] as well as lipid lung surfactants [3]. The

ain n-10 fatty acids found in these samples are sapienic (cis-66:1), cis-8 18:1 and sebaleic (cis-5, cis-8 18:2) acids. The initialtep of the formation of this series involved the desaturation ofalmitic acid by the �6-desaturase to form sapienic acid [1]. Thisiosynthetic pathway seems to be specific to these tissues whenxposed to the environment but their biological significance has

ot been extensively investigated to date [2].

The occurrence of sapienic (cis-6 16:1), cis-8 18:1 and sebaleiccis-5, cis-8 18:2) acids has not been reported to our knowledge in

∗ Corresponding author at: Nestlé Research Center, Vers-chez-les-Blanc, P.O. Box4, CH-1000 Lausanne 26, Switzerland. Tel.: +41 21 785 8937; fax: +41 21 785 8553.

E-mail address: frederic.destaillats@rdls.nestle.com (F. Destaillats).

021-9673/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2011.10.095

human hair and nail lipids. In addition, it can be hypothesized thatthe formation of �6-monounsaturated fatty acids may occur fromother saturated fatty acids such as stearic acid. The pre-requisitefor such identification is the ability to separate cis-6 from cis-8monounsaturated fatty acid derivative such as petroselinic (cis-618:1) from cis-8 18:1 acid methyl esters by gas-chromatography(GC). Such separation cannot be achieved using cyanoalkyl basedhighly polar capillary columns.

Delmonte et al. [4] reported significant improvement of criticalseparation of geometric and positional isomers using the recentlyreleased 100 m SLB-1L 111 ionic-liquid coated capillary column(Supelco, Bellefonte, PA). The authors evaluated the performanceof this column to separate 14:1, 16:1 18:1, 20:1 and 18:3 isomersand compared the results obtained with SP-2560 (Supelco, Belle-

fonte, PA) and CP-Sil 88 (Varian, Middelburg, The Netherlands).From our perspective, the more significant progress achieved byDelmonte et al. [4] is the baseline separation of petroselinic (cis-618:1) from cis-7/cis-8 18:1 acid methyl esters. This improvement

F. Destaillats et al. / J. Chromatogr. A 1218 (2011) 9384– 9389 9385

9

Hair

Nails

Sebum

Standards

1

2 3

4

5

6

9

1. Palmitic acid (16:0)2. Sapienic acid (cis-6 16:1)3. Stearic ac id (18:0)4. Petroseli nic acid (cis-6 18:1)5. cis-8 Ocatedecenoic acid (cis-8 18 :1)6. Oleic ac id (cis-9 18:1)7. Unidentified8. cis-Vaccenic acid (cis-11 18:1)9. Sebaleic acid (cis-5,cis-8 18:2)

2

2

24 5

3

3

24.518.5 Retention Time (min)

9

78

F owingc vacceS s-8 18

asi

2

2

SS1(

2

hahTohtnafitmicoMa

ig. 1. Fatty acid methyl ester (FAME) profile of hair, nails and sebum samples shomprising petroselinic acid (cis-6 18:1), cis-8 18:1, oleic acid (cis-9 18:1) and cis-LB-IL 111 (Supelco). Standard FAME of cis-6 16:1, cis-6 18:1, cis-8 18:1 and cis-5,ci

llows conducting a thorough examination of the occurrence ofpecific �6-monounsaturated fatty acids such as petroselinic acidn human sebum, hair and nail lipids.

. Materials and methods

.1. Chemicals and reagents

2-Amino-2-methyl-1-propanol was obtained fromigma–Aldrich (Saint Louis, USA) and methanol/HCl (3 N) fromupelco (Bellefonte, PA). Pure methyl cis-6 16:1, cis-6 18:1, cis-8-8:1 and cis-5, cis-8 18:2 acid esters were obtained from LipidoxSweden).

.2. Sampling procedure

Hairs, nails and a superficial skin sample were collected fromuman volunteers (N = 6) following the procedure described here-fter. Hair sample collection: approximately 20 hairs including theair follicles were collected from females scalp using tweezers.he hairs were further cut with a clean razor blade in order tobtain approximately 1 cm length from the hair follicle. The 1 cmairs including hair follicles were further pooled in a 15 mL falconube before derivatization. Nail sample collection: 1–2 mL of fingerails were cut with a scissor from the ten fingers of the subjectnd pooled in a 15 mL falcon tube before derivatization. Super-cial skin sample: superficial skin samples were collected usingape stripping methodology. Tape stripping is a commonly used

ethod to investigate the stratum corneum (SC) physiology. The SCs the superficial layer of the epidermis which consists of corneo-

ytes embedded in lipid bilayers. Present within these lipids are thenes produced in the sebum. Briefly, regular adhesive tape (ScotchagicTM Tape), 2 cm wide and 5 cm long was used. The tape was

pplied to the skin on the human volunteer’s right cheek, rubbed

the occurrence of a complex pattern (left panel) of cis-octadecenoic acid isomersnic (cis-11 18:1) acid. Analysis performed on ionic liquid coated capillary column:2 were obtained from Lipidox (Sweden).

lightly to assure adhesion and then pulled off with one fluent anddecisive movement. This was repeated 10 times on the same spot.And the 10 strips were pooled in a 50 mL falcon tube to be processedfor extraction.

2.3. Preparation of fatty acid methyl esters (FAME)

Hair and nail samples (>20 mg) were placed in a mortar andcrushed into very small pieces under very cold conditions (liquidnitrogen-dry ice). Pulverized samples were then transferred into10 mL screw cap test tubes with methanol (2 mL), methanol/HCl(2 mL, 3 N) and hexane (1 mL). After vigorous shaking, the methy-lation was performed at 100 ◦C for 60 min and shaken vigorouslyevery 20 min. After cooling-down to room temperature, water(2 mL) was added and tubes were centrifuged at 1200 × g for 5 min.If necessary, sample was further concentrated before GC analysis.Sebum samples were extracted from tape by immersion and homo-geneization in hexane (1 mL) and methanol (2 mL) for 1 min. Afterremoving the tape from the tube, methanol/HCl (2 mL, 3 N) wasadded and methylation was conducted as described for hair andnail samples.

2.4. Preparation of 4,4-dimethyloxazoline (DMOX) derivative forGC–MS analysis

DMOX were prepared as previously described with slight mod-ification [5]. Briefly, FAME were dried over nitrogen and mixedwith 2-amino-2-methyl-1-propanol (0.5 mL) and heated at 190 ◦Covernight. After cooling down to room temperature, hexane (2 mL)

and water (2 mL) were added and the tube was vigorously shakenand centrifuged at 1000 rpm for 2 min. The organic phase wasrecovered and hexane evaporated using nitrogen. Sample wasdiluted in fresh hexane and analyzed by GC–MS.

9386 F. Destaillats et al. / J. Chromatogr

Fig. 2. EI mass-spectra of cis-6 18:1 (A), cis-6 16:1 (B) and cis-6 14:1 (C) and acidsas their 4,4-dimethyloxazoline (DMOX) derivatives in hair sample.

. A 1218 (2011) 9384– 9389

2.5. Gas-chromatography

Analysis of FAME were performed on a 7890 gas-chromatograph(Agilent Technologies, Santa Clara, CA), equipped with a fused-silica SLB-IL 111 capillary column (100 m, 0.25 mm i.d., 0.2 �mfilm thickness; Supelco, Bellefonte, PA) split–splitless injector (5:1,250 ◦C) and flame-ionization detector (300 ◦C). Oven temperatureprogramming was 60 ◦C isothermal for 5 min, increased to 165 ◦C at15 ◦C/min, isothermal for 1 min at this temperature, then increasedat 2 ◦C/min to 195 ◦C, isothermal for 1 min and then increased at5 ◦C/min to 215 ◦C and held isothermal for 8 min. Hydrogen wasused as a carrier gas in constant flow mode at 1.5 mL/min.

2.6. Gas-chromatography–mass-spectrometry (GC–MS)

DMOX samples were analyzed on a 6890 Series II gas-chromatograph (Agilent Technologies, Santa Clara, CA) attached toa 5973N quadrupole mass selective detector (Agilent Technologies,Santa Clara, CA) equipped with electron ionization (EI) ion source.The instrument was operated in positive ion mode using the stan-dard 70 eV electron energy. The GC injector was operated in splitmode (split ratio 25:1) at 250 ◦C, the GC–MS interface was main-tained at 250 ◦C. Helium was used as carrier gas under constantflow rate of 1 mL/min. Oven temperature programming was 60 ◦Cisothermal for 5 min, increased to 165 ◦C at 15 ◦C/min, isothermalfor 1 min at this temperature, then increased at 2 ◦C/min to 195 ◦C,isothermal for 1 min and then increased at 5 ◦C/min to 215 ◦C andheld isothermal for 8 min. Electron ionization mass spectra wererecorded in the m/z 50–400 range.

3. Results and discussion

Hair, nail and sebum samples were obtained from human vol-unteers following the sampling procedure described in Section 2.The preparation of FAME from these samples was carried-out usingdirect methylation protocol conducted using acid catalyst at hightemperature as described by various matrices [6]. Direct prepara-tion of FAME has the advantage of working with small samples,saving the time required for extraction and in some cases avoidingthe loss of labile compounds during the extraction step. In addi-tion, the use of an acid at 100 ◦C allows for the disruption of thematrix thereby enhancing the recovery of lipids from the sample.Preliminary analysis of FAME prepared from hair samples usingstandard capillary column (100 m, CP-Sil 88 obtained from Var-ian, data not shown) confirmed the occurrence of a peak elutingjust before methyl oleate with the same retention time as cis-818:1 acid. The occurrence of this fatty acid in hair sample has beenhypothesized based on indirect analyzes for the first time in 1947 byWeitkamp et al. [7] and is formed by the elongation of sapienic acid(cis-6 16:1). Similar sample was then analyzed using the 100 m SLB-IL 111 ionic liquid coated capillary column recently commercializedfollowing the promising results published by Delmonte et al. [4].

Results of the analysis using the SLB-IL 111 show two peakseluting before methyl oleate instead of the single one observedwith the CP-Sil 88 column (see Fig. 1). Similar results were obtainedfor the sebum and nail samples. Co-chromatography experimentswere performed using pure standard of petroselinic (cis-6 18:1)and cis-8 18:1 acids. Results indicate that the two peaks elutingbefore methyl oleate have a similar retention to cis-6 18:1 andcis-8 18:1 acid isomers. These data confirmed the superior reso-

lution power of the SLB-IL 111 column [4], but more importantlyindicate that hair lipids might contain petroselinic acid. GC anal-yses also revealed the putative occurrence of sapienic (cis-6 16:1)and sebaleic (cis-5, cis-8 18:2) acids. These fatty acids have been

F. Destaillats et al. / J. Chromatogr. A 1218 (2011) 9384– 9389 9387

(B) as

rk

aitcd

Fig. 3. EI mass-spectra of cis-8 18:1 (A) and cis-5,cis-8 18:2 acids

eported in sebum [1,2], but not in hair and nail samples to ournowledge.

Confirmatory experiments were conducted by GC–MS usingzo-derivatives suitable for structural analysis by electron-impact

onization [8–10]. DMOX derivatives are particularly interesting inhe present context since their fragmentation under EI yield to spe-ific ion fragments that allow allocating the position of ethylenicouble bonds and their retention time are very similar to methyl

their 4,4-dimethyloxazoline (DMOX) derivatives in hair sample.

ester homologues [8,9]. In addition, spectra of pure cis-6 16:1, cis-6 18:1, cis-8 18:1 and cis-5, cis-8 18:2 standard DMOX derivativeshave been already published and can be found on the Lipid Libraryrepository [9]. DMOX were prepared from FAME samples by the

direct method developed by Fay and Richli [5] and these sampleswere analyzed in full scan mode with a mass range of 50–400 amu.DMOX of pure cis-6 16:1, cis-6 18:1, cis-8 18:1 and cis-5, cis-8 18:2acid were prepared using similar procedure from methyl esters. A

9388 F. Destaillats et al. / J. Chromatogr. A 1218 (2011) 9384– 9389

F dimett -6 17:

tD

rdcopdat(id[

FT

ig. 4. Total ion chromatogram (upper panel) and extracted chromatogram of 4,4-he occurrence of cis-6 14:1, cis-6 15:1, iso-cis-6 16:1, cis-6 16:1, aiso-cis-6 17:1, cis

otal ion chromatogram displaying the fatty acid identified as theirMOX derivatives is provided in Fig. 4.

Analysis of EI mass-spectra of pure standards confirmed theesults obtained by co-chromatography with pure standard andemonstrate the evidence of cis-6 16:1, cis-6 18:1, cis-8 18:1 andis-5, cis-8 18:2 in hair, sebum and nail samples. EI mass-spectrumf cis-6 16:1 and cis-6 18:1 DMOX derivative in hair sample isrovided in Fig. 2. DMOX derivative of petroselinic acid (Fig. 2A)isplayed molecular ion at m/z 335 and a diagnostic ion fragmentt m/z 167 as reported in the literature [8–10]. Similar fragmenta-ion characteristics are observed for sapienic acid DMOX derivative

Fig. 2B) that displayed molecular ion at m/z 307 and a diagnosticon fragment at m/z 167. The diagnostic ion fragment at m/z 167 isue to the charge remote fragmentation between carbons 6 and 78–10].

14:0myris�c acid

cis-6 14:1

16:0palmi�c acid

cis-8 18:1

sebaleic acid

Elongase

cis-5,cis-8 18 :2

cis-6 16:1sapienic acid

Δ5-desaturase

Iso-16:0Iso-methyl pentadecanoic a

Iso-cis-6 16:1

15:0pentadecanoic acid

cis-6 15 :1

Δ6-desaturas

cis-8 16 :1

Elongase

cis-8 17:1

Elongase

ig. 5. Proposed biosynthetic pathways involved in the formation of �6-monounsaturathe n-10 series biosynthesis pathway stating from palmitic acid has been adapted from P

hyloxazoline (DMOX) derivatives (ions fragment at m/z 167, lower panel) showing1 and cis-6 16:1 acids in hair sample.

Analysis of the full scan data by filtering signal for the ion frag-ment at m/z 167 allowed the identification of cis-6 14:1, cis-6 15:1,iso-cis-6 16:1, aiso-cis-6 17:1 and cis-6 17:1 as their DMOX deriva-tives (Fig. 4). EI mass-spectrum of cis-6 14:1 is provided in Fig. 2Cand displayed molecular ion at m/z 279 and ion fragment at m/z167. It is important to note that a similar feature of the EI spec-tra of the DMOX derivatives of cis-6 14:1, cis-6 16:1 and cis-6 18:1acids is the high abundance of the ion fragment at m/z 126. Forother DMOX derivatives, the typical base peak is at m/z 113 [10].The extraction of ion fragment at m/z 167 (Fig. 4) provided a seriesof mass-spectrum having similar features comprising diagnostic

ion fragments at m/z 180 and 194. Chain length and the numberof unsaturations of the FA were deduced according to molecularions. The eludication of the structures of the two branched chain�6-monounsaturated fatty acids was tentatively done based the

17:0heptadecanoic acid

cis-6 17:1

18:0stearic acid

petroselinic acidcis-6 18:1

cidAiso-17:0

aiso-methyl hexadecano ic acid

aiso-cis-6 17:1

e

ed fatty acids in hair, nails and sebum by �6-desaturation of saturated fatty acids.icardo et al. [1].

atogr.

pa

aDic9uEdail

h1ioesifotToiosddsmssf

F. Destaillats et al. / J. Chrom

attern of ion fragments in the high mass range (i.e. M+−15, M+−29nd M+−43) according to Christie [9].

MS analyzes also confirmed the occurrence of cis-8 18:1 in hairnd nail samples eluting just before oleic acid DMOX derivative.MOX derivative of cis-8 18:1 acid (Fig. 3A) displayed molecular

on at m/z 335 and diagnostic ion fragments at m/z 182, 194 and 208onfirming the location of the double bond between carbons 8 and

[8–10]. Similarly, cis-8 16:1 and cis-8 17:1 acids were identifiedsing identical diagnostic ion fragments in hair sample (Fig. 4). TheI mass-spectra of sebaleic acid, also found in the analyzed sampleisplayed molecular ion at m/z 333 and diagnostic ion fragmentst m/z 180, 192 and 206 for the �8 double bond and a character-stic ion fragment at m/z 153 for the �5 double as reported in theiterature [8–10].

Sapienic, cis-8 18:1 and sebaleic acids are usually found inuman sebum [1,2]. In hair lipids, we only found a report from947 by Weitkamp et al. who reported the occurrence of cis-8 18:1

n hair free fatty acids [7]. Longmuir et al. reported the occurrencef sapienic and cis-8 18:1 acids in lipid lung surfactants [3]. How-ver, in the present study, we show the fatty acids from the n-10eries are found in human hair, nail and sebum samples confirm-ng the assumption of Longmuir et al. that these fatty acids are onlyound in tissues exposed to the environment [3]. The Formationf sapienic acid, the initial precursor of the n-10 series, occurs byhe �6-desaturation of palmitic acid as reported in sebum [1,2].his pathway is specific to human and has not been reported toccur in other animals [1]. The elongation of sapienic acid resultsn the formation of cis-8 18:1 acid which can be further metab-lized by �5-desaturation to form sebaleic acid [1]. A syntheticcheme of this biosynthetic pathway is provided in Fig. 5. Theata obtained in the present study provide evidences that �6-esaturase is active in hair and nail as was previously known forebaceous gland [1]. Therefore, it can be hypothesized that the for-

ation of the series of �6-monounsaturated fatty acids in hair,

ebum and nail lipids happens by �6-desaturation of the corre-ponding saturated fatty acids in a similar manner to the processor sapienic acid (Fig. 5).

[

[

A 1218 (2011) 9384– 9389 9389

4. Conclusion

The identification of the occurrence of petroselinic acid inhuman skin, hair and nails was not achievable without the newionic liquid coated SLB-IL 111 column. We confirmed in the presentstudy that this column can be used to obtain baseline resolutionbetween petroselinic acid and cis-8 18:1 acid methyl esters. Similarseparation can be obtained with DMOX derivatives and this columncan be efficiently used for structure elucidation experiments by EIGC–MS. Although we provide evidences that fatty acids from then-10 series are found in hair, nail and sebum, the results obtainedin this study show that petroselinic acid is not specifically found inplants (i.e. Umbelliferae, Apiaceae and Geraniaceae) as it is currentlyreported in the literature [11]. The experiments described in thepresent study demonstrate that petroselinic acid is also endoge-nously produced in human skin, nail and hair. We report for thefirst time the occurrence of a series of �6-monounsaturated fattyacids in samples of human origin comprising, in addition to sapienicand petroselinic acids, cis-6 14:1, cis-6 15:1, iso-cis-6 16:1, aiso-cis-617:1 and cis-6 17:1 acids. The biological relevance of the presence ofpetroselinic acid in these tissues of common origin is still unknownand calls for further investigations.

References

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[2] Z.M.H. Marzouki, J. Chromatogr. 425 (1988) 11.[3] K.J. Longmuir, J.L.C. Resele-Tiden, M.E. Rossi, J. Lipid Res. 29 (1988) 1065.[4] P. Delmonte, F.K. Ali-Reza, J.K.G. Kramer, M.M. Mossoba, L. Sidisky, J.I. Rader, J.

Chromatogr. A 1218 (2011) 545.[5] L. Fay, U. Richli, J. Chromatogr. 541 (1991) 89.[6] F. Destaillats, P.-A. Golay, F. Giuffrida, B. Hug, F. Dionisi, Lipid Technol. 16 (2004)

183.[7] A.W. Weitkamp, A.M. Smiljanic, S. Rothman, Am. J. Chem. Soc. 69 (1947) 1936.[8] W.W. Christie, G.W. Robertson, W.C. McRoberts, J.T.G. Hamilton, Eur. J. Lipid

Sci. Technol. 102 (2000) 23.[9] http://lipidlibrary.aocs.org.10] J.Y. Zhang, Q.T. Yu, B.N. Liu, Z.H. Huang, Biomed. Environ. Mass Spectrom. 15

(1988) 33.11] N. Tsevegsuren, K. Aitzetmuller, K. Vosmann, Lipids 39 (2004) 571.