J. High Resol. Chromatogr. VOL. 21, FEBRUARY 1998 133

Determination of Petroselinic Acid in Umbelliferae Seed Oils byAutomated Gas ChromatographyBirgit Reiter *, Marion Lechner, Eberhard Lorbeer

Institute of Organic Chemistry, Wa¨hringer Str. 38, A-1090 Vienna, Austria

Ms received: August 8, 1997; accepted: November 5, 1997

Key Words:GC; analysis of vegetable oil; petroselinic acid; butyl esters

1 Introduction

Petroselinic acid (C18:1 (6c)) and its cleavage products lauricand adipic acid are important oleochemical raw materials: petro-selinic acid is used for the synthesis of fine chemicals, lauricacid is useful in the production of surfactants, emulsifiers, deter-gents, and soaps, and adipic acid is utilized as raw material forsofteners, polyamides, and nylon. Petroselinic acid is one of themajor fatty acids (seeTable 1) in Umbelliferae seeds, whichinclude species like fennel, coriander, celery, parsley,etc., andthere it amounts up to 80% [1–3].

Alongside petroselinic acid, two other positional isomers of octa-decenoic acid occur in Umbelliferae seeds: oleic (C18:1 (9c))andcis-vaccenic acid (C18:1 (11c)). With regard to the technicaluse of petroselinic acid the determination of these different iso-mers is necessary to perform a quick screening of Umbelliferaeseeds. Therefore, an automated gas chromatographic methodusing a commercial capillary column and a quick derivatizationprocedure is important. Different possibilities for the gaschromatographic determination of petroselinic acid have alreadybeen proposed. Normally, fatty acid patterns of vegetable oils aredetermined and quantified as fatty acid methyl esters by gaschromatography [4]. However, the separation of these isomers asmethyl esters is insufficient for quantitative analysis [5, 6].Uccianiet al. [7, 8] formed trimethylsilyl ester derivatives of pet-roselinic and oleic acid and analyzed them on a 100 m Carbowax20 M capillary column but baseline separation was not possible.With isopropyl esters [9] of the positional isomers, analyzed on a50 m CP-Sil 88 fused-silica capillary column, one obtained near-baseline separation, but the derivatization procedure is time-con-suming. The resolution of the named fatty acids as methylthio-derivates [10] on a 25 m FFAP capillary column is insufficientfor quantification. The phenylethyl ester [11] of petroselinic andoleic acid allow baseline separation, but the derivatization proce-dure is laborious.

All methods mentioned above are either very time-consumingand complex for an automated procedure, or separation is in-sufficient for quantitative analysis.

Thies[12] used fatty acid butyl ester for the analysis of petroseli-nic acid in seed oils of coriander and analyzed them under iso-thermal conditions with a 60 m DB-23 capillary column. Not

even a near-baseline separation was reached. It is assumed thattemperature programmed analysis and optimized gas chromato-graphic conditions could improve separation efficiency. Thesebutyl esters were also examined with regard to their use in auto-mated analysis.

2 Material and Methods

2.1 Reagents and Samples

Methyl esters of petroselinic, oleic,cis-vaccenic, palmitic, lino-leic, and margaric acid as internal standard were purchased fromSigma, Deisenhofen, Germany, Natert-butylate from Fluka,Buchs, Switzerland.n-Hexane andn-butanol were used in p.a.quality. Fennel and caraway seeds were purchased from the Bun-desamt und Forschungszentrum fu¨r Landwirtschaft, Vienna, Aus-tria. Oil was obtained by Soxhlet extraction.

2.2 Preparation of Fatty Acid Butyl Ester

500ll 0.1 M Na tert-butylate in butanol was added to 2 mg ofvegetable oil. After 10 min at room temperature 500ll 5%NaHSO4 was added. 300ll of the butanol phase were transferredinto a vial (Reacti-Vial, Pierce, Oud-Bejierland, The Nether-lands).

For automated injection butanol was removed in a stream ofnitrogen, and butyl esters were taken up into 3 mln-hexane. Thesolution was transferred into a septum vial. 1ll of this solutionwas injected into the gas chromatograph.

2.3 Instrumentation

A Carlo Erba HRGC 5300 gas chromatograph equipped with aflame ionization detector (FID) and anon-columninjector (CarloErba, Milano, Italy) was used. In addition, for automated mea-surements, a GC 8000 gas chromatograph (Fisons Instruments,Milano, Italy) was used, equipped with a FID,on-columninjec-tor, an AS 800 autosampler (Fisons Instruments, Milano, Italy),and an uncoated fused-silica precolumn deactivated withDPTMDS (diphenyltetramethyldisilazane), (2 m6 0.53 mm i.d.,BGB Analytik, Rothenfluh, Switzerland), which was combinedwith the analytical column. Hydrogen was used as carrier gas.

Determination of fatty acid butyl esters, dissolved in butanol,was carried out on a fused-silica capillary column, coated with50%-cyanopropyl methylpolysiloxane (DB-23, 30 m60.32 mm i.d., 0.25lm film; J & W Scientific, Folsom, CA,USA), under the following conditions: 1208C increased at 208/

Table 1.Content of major fatty acids in Umbelliferae seed oils.

Petroselinic acid 35–78%Oleic acid 5–24%Linoleic acid 8–33%

Determinationof Petroselinic Acid in UmbelliferaeSeedOils

134 VOL. 21,FEBRUARY 1998 J.High Resol.Chromatogr.

min up to 1408C andincreasedat 38/min up to 2308C. Theflowratewasheldat 2.6ml/min.

Automateddeterminationof fatty acid butyl esters,dissolvedinn-hexane,wascarriedout on a DB-23underthefollowing condi-tions:detectortemperature 2408C. 708C increasedat 208/min upto 1108C and increasedat 38/min up to 2008C. The flow ratewasheldat 2.6ml/min.

A personalcomputerand the softwareprogramChromcardforWindows(FisonsInstruments,Milano, Italy) wereusedfor dataacquisitionandevaluation.

3 Resultsand Discussion

3.1 Analysisof Butyl Esters

Unlike Thies[12], who performedextractionandtransesterifica-tion of the lipids in onestep,we preparedfatty acid butyl estersdirectly from the extractedoil. The derivatizationprocedureisfastandhighly suitablefor simultaneoussamplepreparation.

Fattyacidbutyl estersof fennelandcarawayseedoils werealsoanalyzedon differentcapillary columns:30 m DB-5, 25m DB-225,30 m DB-210,30 m DB-1701,and30m DB-23. Accordingto theliterature[12], thebestresultsfor thedeterminationof pet-roselinic acid are forthcomingwith a DB-23 fused-silicacapil-lary column.Temperatureprogrammedanalysisgaveimprovedseparationefficiency on using a 30 m DB-23. Near-baselineseparationwasachieved.Figure 1 andFigure 2 showchromato-gramsof fatty acidsof fennelandcarawayseedoils, separatedasbutyl estersona DB-23capillarycolumn.

Subsequently, fatty acidbutyl estersweresubjectedto automatedanalysis,but the useof a precolumn,which is necessaryfor on-columninjectionwith anautosampler, hasa detrimentaleffect onpeak resolution. After a few injections, strong peak tailing

decreasedthe separation efficiency. Better separation wasobtainedfor 2–3 injectionsafterinstallationof anewprecolumn,thenthepeakshapedeterioratedagain(Figure 3).

A modifiedderivatizationusingn-hexaneinsteadof n-butanolassolventavoidedthedecreaseof separationefficiency.

3.2 Calibration

Calibrationof the responsefactor wascarriedout with margaricacid ME. Standardsolutionscontainingknown amountsof thereferencesubstances(petroselinicacid ME, oleic acid ME, cis-vaccenicacid ME, and linoleic acid ME) and the internal stan-dardmargaricacidME weretransformedto their butyl estersandanalyzedthree times eachby automatedGC in order to deter-mine the ratio Ac/Ast (peakareaof component(Ac)/peakareaofinternalstandard(Ast)). The following calibrationfunctionshavebeenobtained:

Cc/Cst = (1.21l 0.003)6 Ac/Ast + (–0.0067)for oleic acid;Cc/Cst

= (1.07 l 0.012)6 Ac/Ast for petroselinicacid; Cc/Cst = (1.02 l0.002)6 Ac/Ast for cis-vaccenic,palmitic, andstearicacid(thesethreefatty acidshaveaboutthe sameconcentrationin the seedoil, so the sameresponsefactor was used); Cc/Cst = (1.14 l0.006) for linoleic acid (Cc, concentrationof component(mg/ml)); Cst, concentrationof internalstandard(mg/ml)). Goodline-arity of the calibration functionswas observedwithin the con-centrationrangeof interest.Table2 showscalibrationfunctionsof petroselinicacid, oleic acid, linoleic acid and cis-vaccenic,stearicandpalmitic acid.

3.3 Quantitativeanalysis

The concentrationsof petroselinic,oleic, and cis-vaccenicacidweredeterminedin carawayandfennelseedoil usingmargaric

Figure 1. Chromatogramof fatty acidsof fenneloil, separatedasbutyl esters.

Determinationof PetroselinicAcid in UmbelliferaeSeedOils

J.High Resol.Chromatogr. VOL. 21,FEBRUARY 1998 135

acidME asinternalstandard.Concentrationswerecalculatedonthebasisof following equations:

Cc = Cst 6 [Rf 6 (Ac/Ast) + coeff.]; (Cc, concentration of fattyacid as butyl esterin the sample(mg/ml); Cst, concentrationoftheinternalstandard(mg/ml); Rf, responsefactorof fatty acid).

Cfa = (Cc/Ct) 6 100; (Cfa, concentrationof fatty acid (%); Ct,total of all Cc (mg/ml)). Table 3 showstheconcentrationsof pet-roselinic acid, oleic acid, and cis-vaccenicacid in carawayandfennelseedoils.

4 Conclusion

Petroselinicacid, oleic acid and cis-vaccenicacid can be sepa-ratedasbutyl esterwith a 30m DB-23 fused-silicacapillarycol-umn within 32 min. The methodis suitablefor routine labora-tories,becausederivatizationprocedureis fastandapplicableforsimultaneoussamplepreparation. Changingsolventafter deriva-tization – n-hexaneinsteadof n-butanol – allows the useof anautosampler.

If an economicaluseof petroselinicacid is considered,a quickscreeningof differentUmbelliferaeseedoils canbeperformedina shorttime.

AcknowledgmentsTheauthorsthankG. Benesch,N. Wimmer, andM. Zillinger for practicalsupport.This work was financially supportedby the Austrian FederalMinistry of AgricultureandForestry(ProjectNr. L 896/94).

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Figure 2. Chromatogramof fatty acidsof carawayseedoil, separatedasbutyl esters.

Figure 3. Deteriorationof peak separationby using a precolumnforautomaticinjection.

Table2. Calibration functionsfor petroselinicacid, oleic acid and cis-vaccenicacid.

Rf coeff. SE r2

Petroselinicacid 1.07 – 0.012 0.996Oleic acid 1.21 –0.0067 0.003 0.994cis-Vaccenic,palmitic, stearicacid 1.02 – 0.002 0.997Linoleic acid 1.14 – 0.006 0.996

Abbreviations:Rf, responsefactor;coeff., coefficient; SE,standarderror;r2, mean-squareerror.

Table3. Concentrationof petroselinicacid, oleic acid andcis-vaccenicacidin caraway(Carumcarvi) andfennel(Foeniculumvulgare) seedoil.

C18:1(6c) C18:1(9c) C18:1(11c)

Carumcarvi 42.4% 15.2% 0.6%Foeniculumvulgare 73.7% 4.8% 0.3%

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136 VOL. 21,FEBRUARY 1998 J.High Resol.Chromatogr.

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