Journal of Chemical Ecology, Vol. 21, No. 7, 1995

QUANTITATIVE

HIGH-RESOLUTION

CHROMATOGRAPHIC STEREOISOMERIC VOLATILE

DETERMINATION

COMPOSITION

COMPOUNDS

RANGE

GAS OF

OF CHIRAL

IN THE PICOGRAM

BY EC-DETECTION

ANN-BRITT WASSGREN and GUNNAR BERGSTROM* Department of Chemical Ecology GOteborg University Reutersgatan 2 C, S-413 20 GOteborg, Sweden (Received February 14, 1995; accepted March 10, 1995)

Abstraet--A gas chromatographic method using electron capture detection for determination of the stereocbemical composition of chiralolatile compounds, e.g., pheromones, at the picogram level is described. Substances of interest are analyzed as pentafluorobenzoate derivatives on fused silica capillary columns coated with CP Si1-88. This method was developed primarily for the quantitative analyses of stereoisomerism of the pheromone precursor, diprionol, in female sawflies, Neodiprion sertifer. It was possible to separate the stereoisomers showing (2S, 3S, 7R) and (2S, 3S, 7S) configurations. Resolution of the (2S, 3S, 7S)- and (2S, 3R, 7R)-diastereomers was high enough to allow the quantification of these stereoisomers. A quantitative analysis of the diprionol production in individual N. ser~ifer females yielded 7.59.7 ng/female. Key Words--Chiral separation, HRGC, stereoisomers, diastereomers, enantiomers, electron capture detection, pentafluorobenzoates, pheromones, volatile compounds.

INTRODUCTION

Methods for identifying substances in biological material can be greatly facilitated by the conversion of a functional group to a suitable derivative according to the mode of determination. For example, detection of various phenols and carboxylic acids as pentafluorobenzoyl derivatives has been satisfactory using a tritium foil ECD, and analyses at trace concentrations have been successfully *To whom correspondence should be addressed.

987 0098-0331/95/0700-0987507.50/0 © 1995 Plenum PublishingCorporalion

988

WASSGREN AND BERGSTROM

performed by Kawahara (1968). Methods for determining the composition and concentration of carboxylic acids and phenols in water using derivatives have been described by Fogelqvist et al. (1980). Esterification with halogenated alcohols has been used for the identification of pesticides at nanogram levels (Satter and Paasivirta, 1979). The determination of trace amounts of different estrogens, in the form of heptafluorobutyric esters, has made it possible to measure picogram amounts by using 63Ni ECD, according to Kern and Brander (1979). Selective analysis of enantiomers of chiral compounds in biological material presents an interesting problem. In the oldest and most commonly used GC procedures for enantiomeric separations, an optically pure chiral reagent is employed to convert enantiomers into their diastereomeric derivatives, which are then separated on a nonchiral stationary phase. These methods were described by, among others, St/illberg-Stenhagen (1970). Today it is customary to perform determinations of enantiomeric compositions by chiral GC analysis on modified cyclodextrin phases, and in some cases it is possible to derivatize the compounds to trifluoroacetyl (TFA) derivatives (Krnig et al., 1990). Separations of underivatized enantiomers and diastereomers on chiral metal chelate dissolved in an achiral stationary phase is described by Schurig and Weber (1984). In the present report, pentafluorobenzoate was chosen as a derivatizing agent. It introduces an electron capturing moiety into the molecule, which provides good response to a 63Ni detector and is suitable for gas chromatographic separation at the picogram level, as exemplified by the analysis of diprionol (2S, 3S, 7S)-3,7-dimethyl-2-pentadecanol, the pheromone precursor in Neodiprion sert(fer (Geoffr.) (Hymenoptera: Diprionidae) females. Since some of the stereoisomers of diprionol occur in picogram amounts only, their determination requires sensitive methods. The gas chromatographic separation of diprionol has been performed earlier using two different methods, which we have described in Hrgberg et al. (1990). In both methods, the substances were derivatized, either to isopropyl carbamates or to benzoates. The isopropyl carbamates were separated on a chiraI column CP-XE-60-(S)-valine-(S)-2-phenylethylamide (K6nig, 1982; Krnig et al., 1982) and detected by N/P detection (Wassgren and Bergstrrm, 1984; Bergstrrm, et al., 1992), while the benzoates were separated on a fused silica column coated with Sil-88 and detected by a flame ionization detector. Previously, we separated the diastereomers showing (2S, 3S, 7R)-configuration from the (2S, 3S, 7S) pair in synthetic diprionol as benzoates on a Sil-88 column, but problems arose when identifying small amounts in biological materials, i.e., in the picogram range (Hrgberg et al., 1990). The objective of this work was to develop a convenient, sensitive method for the identification and quantification of the enantiomeric composition of diprionol from natural material in the picogram range and the quantification of the production of diprionol in single females. We think that this method may be of general interest in connection with separations of small amounts of many different chiraI natural compounds.

STEREOISOMERIC COMPOSITION OF VOLATILES

989

METHODS AND MATERIALS

Biological Material. Females ofN. sertifer were collected in 1991 in V~irmland, Sweden, from two populations. Chemical Materials. All synthetic references were synthesized in high stereochemical purity (see H6gberg et al., 1990). The substances are the following: (2S, 3S, 7S)-3,7-dimethyl-2-tridecanol, (C~5-SSS), (2S, 3S, 7S)-3,7-dimethyl2-tetradecanol, (Ct6-SSS), (2S, 3S, 7S)-3,7-dimethyl-2-pentadecanol, (C~7-SSS), (2S, 3R, 7R)-3,7-dimethyl-2-pentadecanol, (C~7-SRR), (2S, 3S, 7R)-3,7dimethyl-2-pentadecanol, (C57-SSR), and (2S, 3S, 7S)-3,7-dimethyl-2-hexadecanol, (C~8-SSS). The solvents were of high analytical quality, containing no halogenated compounds. Ethyl acetate was obtained from American Burdick & Jackson, Fluka AG, Switzerland; pentane was distilled in our laboratory, and the water was Millipore-distilted. Other solvents and reagents, such as pyridine, methanol, pentafluorobenzoyl chloride, and isopropyl isocyanate were of Fluka analytical reagent grade. Chromabond polypropylene columns containing silica gel (SiOH) (Skandinaviska GeneTec AB), 1 ml volume, were used for sample preparation. Preparation of Extracts. Single female N. sertifer were extracted in about 100 p.l ethyl acetate at room temperature for 72 hr. The sample was transferred to a test tube and 50 ng of (2S, 3S, 7S)-3,7-dimethyl-2-hexadecanol was added as internal standard. In addition, two standards were made with 10 and 20 ng, respectively, of (2S, 3S, 7S)-3,7-dimethyl-2-pentadecanol, with 50 ng internal standard in each. Two blank samples containing only pentane were treated in a similar manner. The solvent was concentrated to about 20 t.tl by evaporation under a slow stream of nitrogen; the residue was then immediately dissolved in 50 #1 pentane for subsequent liquid chromatography. Liquid Chromatography. The cleanup was performed with solid-phase extraction on a Chromabond SiOH column (1 ml). The chromatography was initiated by wetting the adsorbent material with the eluent, in this case pentane; the material was then placed on the column and a gradient elution was applied (Wassgren et al., 1992) by increasing the polarity with 1-9% ethyl acetate in pentane, with 150 p.1 elution volume in each fraction. Fractions 7 and 8 were mixed in a small conical glass tube with a stopper before evaporation to dryness. Preparation of Pentafluorobenzoates. The sample was dissolved in 3 p.1 of a mixture consisting of 2/~1 pentafluorobenzoyl chloride in 20/zl pyridine, and the closed reaction tube was kept in a water bath at 60-70°C for 2 min. The solvent and excess reagent were then evaporated to dryness with water suction for 0.5-1 hr. The yellow residue was dissolved with 3 #1 methanol, and 3 /~1 Millipore-redistilled H20 was added. The pentafluorobenzoate was extracted twice with pentane (2 x 25 ~1). The organic phase was washed twice with MiUipore-redistilled H20, dried with Na2SO4, and concentrated to about 20 p,l for GC analyses.

990

WASSGREN AND BERGSTROM

GC-ECD. Gas chromatography was performed with a Chrompack model CP-9000 gas chromatograph equipped with an electron capture detector, model 902A, which contains a foil of radioactive 63Ni, a splitless injector, and a HP3396A integrator. The gas chromatograph was fitted with a 1.5-m x 0.32mm-ID methyl deactivated silica retention gap, connected with a Quick-Seal column connector to fused silica columns coated with Sil-88 (Chrompack, the Netherlands) with the following dimensions: (a) 30 m × 0.15 mm ID, df = 0.24/~m; CO) 25 m x 0.25 mm ID, df = 0.22 t~m; (c) 50 m × 0.25 mm ID, df = 0.22 #m. A setup of oxygen trap, molecular sieve, and charcoal filter was placed before the gas inlet. Separations of pentafluorobenzoates of the synthetic enantiomeric mixture of diprionol and the corresponding chiral homologs were carried out by tandem GC. The deactivated retention gap was fitted with a QuickSeal column connector to the first fused silica capillary column Sil-88 (30 m x 0.15 mm ID). The second fused silica column was also coated with Sil-88 (25 m × 0.25 mm ID). Nitrogen was used as carrier gas at a pressure of 145 kPa, as well as make-up gas and purge gas at flows of 40 ml/min and 35 mI/min, respectively. The oven temperature were 250 ° and 275°C, respectively. The ECD was working in range 0, and attenuation 0.1. Individual analyses of N. sertifer samples were also performed on Sil-88 columns, namely a 50-m × 0.25-mm column coupled to a 25-m x 0.25-mm column in series. The carrier gas pressure was 90 kPa, and the oven temperature 150°C. RESULTS AND DISCUSSION

It has always been a special problem to isolate and identify biologically active components in extracts of whole insects. Such extracts, which are complex mixtures of volatile and nonvolatile compounds, usually require cleanup with solid sorbent extraction. This is a rapid isolation technique for compounds such as the alcohols present in pine sawflies. The recovery in this case, when we used the extract of an individual female, was 90%. In earlier studies we presented the quantification of the amount of diprionol in extracts containing 20 females by using a flame ionization detector. This detector is not sufficiently sensitive to detect diprionol from single individuals or for determining possible homologs. We have therefore improved the method by using a selective electron capture detector (ECD), which is used for very sensitive detection of components with high electron affinity, especially halogenated hydrocarbons. The alcohols were transformed to pentafluorobenzoates with pentafluorobenzoyl chloride, which gives high sensitivity on ECD. The derivatizing procedure is quantitative, and it is therefore possible to perform the whole procedure in vitro; in this manner the loss o f material is exceedingly small; recovery is 80% after the derivatization procedure. The reproducibility

991

STEREOISOMERIC COMPOSITION OF VOLATILES

and accuracy of this approach were determined in an experimental series of standards consisting of diprionol, 10 and 20 ng, respectively, with 50 ng internal standard in each. The pentafluorobenzoyl derivatives of diastereomericdiprionols were separated on commercially available high-polar fused-silica capillary columns, CP Sil-88. The stationary phase unfortunately is not chemically bonded, since the relatively active cyanate groups (CN-) do not allow cross-linking without vinyl groups. It is therefore necessary to inject a reference mixture at regular intervals, to check retention time, and carry out coinjection of the material with pure, known stereoisomers. This ensures that the stereoisomers elute at the same time. The uncoated retention gap avoids problems with solvent condensation that occur with splitless injection. One of the main problems we encountered was the occurrence of residual water after extraction, because water both distorts the chromatography and severely reduces the sensitivity of the ECD; anhydrous sodium sulfate can be used to remove residual water. It was also possible to use this gas chromatographic method for the quantification of low concentrations of diprionol its stereoisomers and occurring homologs in both natural and synthetic material (Figure 1). The detection limit is below 1 pg of diprionol homologs and diastereomers. Furthermore, Figure 1 shows the separation of a mixture of known stereochemical composition of isomeric diprionols and homologs. The erythro-isomers of the C~5- and C~6-homologs which have the (S, S, S)-configuration have the same retention time as the (R, R, R)-isomers (Hrgberg et al., 1990). The erythro-isomers (S, S, S) and (R, R, R) were separated as N-isopropylcarbamates. The separations of (2S, 3S, 7R)-diprionol from (2S, 3S, 7S)-diprionol by a 1.5-min retention difference and the resolution between (2S, 3S, 7S)-diprionol and (2S, 3R, 7R)-diprionot are sufficient to identify and (2S,3S. TS)-3,7-Dimethyl-2-pentadecanol (C=v-SSS) OH

.\

..\. c s

H CH3

H CH3

C~

l C~,-S~

FIG. 1. Enantiomer separation of diprionols as pentafluorobenzoate derivatives, detected by ECD. Columns: fused silica capillary columns, 30 m x 0.15 mm and 25 m × 0.25 mm coated with CP-Sil-88; d/= 0.22/zm, coupled in series. Oven temperature, 150°C; inlet temperature, 145 kPa nitrogen. CIs-SSS (2 pg); CI6-SSS(10 pg); C~7-SSS(200 pg); Ct7-SSR (4 pg) and CIT-SRR(2 pg).

992

WASSGREN AND BERGSTRIDM

Ni(II)-or Mn(II)-CHELATE i

¢, ~- or-~ - CYCLODEXTRIN

CLEAN UP OF NATURAL MATERIAL

i_~.

DERIVATIZED

F--PENTAFLUOROBENZOATES

I

I

SCHEME 1. Summary. of procedures used in chiral analyses of sawfly and antlion volatiles. quantify them. With this derivatization method we can determine the configuration at the 7-position for erythro isomers of diprionols. Scheme 1 shows, in summary, the procedure we have used for the analysis of chiral volatiles from sawflies and antlions. We refer here especially to H6gberg et al. (1990) and Bergstr6m et al. (1992), respectively, The analytical results of the quantification of diprionol from single individuals of N. sertifer are presented in Figure 2A, which shows a GC-ECD chromatogram of natural material. Two females from the same population were analyzed, and we found 7.5 and 8.2 ng per individual, respectively. A female from another population produced 9.7 ng diprionol. The second chromatogram presents a standard with 10 ng diprionol (Figure 2B). For each quantification, a standard curve was plotted using the peak area of the diprionol per internal standard. This standard curve for diprionol, i.e., the curve obtained by plotting the ratio between the gas chromatographic peak area of the substance being analyzed and the internal standard against the amount of substance determined, was linear. The blank sample, which was prepared equivalently to the other samples, contained extraneous peaks from the reagents and solvents used (Figure 2C).

STEREOISOMERICCOMPOSITIONOF VOLATILES

993

1~ 21~1IS A

a~ 2T

2~ i l l~ IS

B

2l' I

C

FIG. 2. Quantitative GC-ECD analysis of diprionol in an individual N. sertifer female. Column: fused silica capillary columns 50 m x 0.25 mm coupled to 25 m × 0.25 mm coated with Sil-88; d; = 0.22 #m, Oven temperature, 150°C; carrier gas, nitrogen (90 kPa); injector temperature, 250°C; EC-detector temperature, 275°C. (A) Derivatized LC fractions 7 and 8 from one female: C~7-SSS (a), internal standard C~8-SSS (IS), attenuation: 2]" ~ and 2T°. (B) Standard mixture; 10 ng C~7-SSS (a), CIs-SSS internal standard (IS), attenuation: 2T° and 21"-2. (C) A blank chromatogram with the disturbed peaks from the reagent, attenuation: 21"0.

The results demonstrate that c o m p l e x a t i o n gas chromatography can be used as an effective method for stereochemical analyses o f molecules that contain suitable functional groups for derivatization. This method may prove generally useful for determination o f stereochemical composition.

Acknowledgments--We thank the Swedish National Science Research Council (NFR) for financial support and Dr. Heidi Dobson for linguistic assistance. This work represents part of a more extensive collaboration on sex pheromones of pine sawflies with colleagues at the Universities of Lund and Sundsvall, in Sweden.

994

WASSGREN AND BERGSTROM REFERENCES

BERGSTROM,G., WASSGREN,A.-B., HOGBERG, H.-E., HEDENSTROM, E., HEFETZ, A., SIMON, D., O~LSSON, T., and LOFQVIST,J. 1992. Species-species, two-component, volatile signals in two sympatric ant-lion species: Synclysis baetica and Acanthaclisis occitanica (Neuroptera, Myrmeleontidae). J. Chem. Ecol. 18:1177-1188, FOGELQVIST,E., JOSEFSSON,B., and Roos, C. 1980. Determination of carboxylic acids and phenols in water by extractive alkylation using pentafluorobenzylation, glass capillary GC and electron capture detection. HRC CC 3:568-574. HOGBERG, H.-E., HEDENSTROM, E., WASSGREN, A.-B., HJALMARSSON, M., BERGSTROM, G., L6FQVlST, J., and NOr~IN, T~ 1990. Synthesis and gas chromatographic separation of the eight stereoisomers of diprionol and their acetates, components of the sex pheromone of pine sawflies. Tetrahedron 46:3007-3018. KAWAHARA,F.K. 1968. Microdetermination of derivatives of phenols and mercaptans by means of electron capture gas chromatography. Anal. Chem. 40: 1009-1010. KERN, H., and BRANDER,B. 1979. Precision of an automated all-glass capillary gas chromatography system with an electron capture detector for the trace analysis of estrogens. HRC CC 2:312318. KONIG, W.A. 1982. Separation of enantiomers by capillary gas chromatography with chiral stationary phases. HRC CC 5:588-595. KONIG, W.A., FRANCKE,W., and BENECKE, 1. 1982. Gas chromatographic enantiomer separation of chiral alcohols. J. Chromatogr. 239:227-231. KON1G~W.A., KREBBER, R., EVERS, P., and BRUHN, G. 1990. Stereochemical analysis of constituents of essential oils and flavor compounds by enantioselective capillary gas chromatography. HRC CC 13:328-332. SATTAR, M.A., and PAASlVtRTA,J. 1979. Simultaneous determination of 4-chloro-2-methylphenoxyacetic acid and its metabolites in soil by gas chromatography. Anal. Chem. 51:598-602. SCHUaIG, V., and WEBER, R. 1984. Use of glass and fused-silica open tubular columns for the separation of structural, configurational and optical isomers by selective complexation gas chromatography. J. Chrornatogr. 289:321-332. STALLBERG-STENHAGEN, S. 1970. The absolute configuration of terrestrot. Acta Chem. Scand. 24:358-360. WASSGREN, A.-B., and BERGSTROM,G. 1984. A simple effluent splitter for capillary gas chromatography. HRC CC 7:154-155. WASSGREN, A.-B., ANDERBRANT,O., LOFQVIST, J., HANSSON, B., BERGSTROM,G., HEDENSTROM, E., and HOGBERG, H.-E. 1992. Pheromone related compounds in pupal and adult female pine sawflies, Neodiprion sertifer, of different age and in different parts of the body. J. Insect Physiol. 38:885-893.