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Silylation can be omitted in gas-liquid chromatographic/mass spectrometric analysis of ergosterol.

Ergosterol (5,7,22-ergostatrien-3b-ol) is a cell membrane sterol compound characteristic of fungi, and absent or a minor compound in other living organisms, including plants, animals, and bacteria. Ergosterol extracted from soil largely results from living cellular material, due to a quite rapid mineralisation in soil. Therefore, ergosterol can be used as a chemical marker for measuring fungal biomass, and coefficients have been calculated for the conversion of ergosterol content into biomass. Ergosterol has been used to analyse fungal communities in a wide range of environments including leaves, soil, house dust, building material, indoor air and activated sludge. In the analysis of ergosterol, lipidbound fatty acids are commonly hydrolysed and ergosterol released, e.g. by the direct saponification in alcoholic KOH. Ergosterol is then extracted with a solvent such as hexane. For analysis by gas-liquid chromatography/mass spectrometry (GC/MS), the hydroxyl group of ergosterol is then converted, for example, into a trimethylsilyl (TMS) derivative. The conversion of ergosterol into its TMS derivative gives a sharper GC peak, whereas without derivatisation ergosterol may be adsorbed onto the column in the free form, resulting in peak tailing. We present data on ergosterol GC/MS analysis, which support the possibility of omitting the resource-consuming derivatisation procedure. Leaves collected in September 2002 from five silver birches (Betula pendula) in Helsinki (Finland) were dried at 22 28C for 14 days. Approximately 1 g of leaves in separate nylon litter bags (10 15 cm, pore size 1mm) was decomposed in soil at the depth of 5 cm for 8 months. The leaves were lyophilised, pulverised in liquid nitrogen, and 75.3–90.0mg (dry weight) were weighed in duplicate for the ergosterol analysis. Leaves were supplemented with 10% KOH in 3mL of methanol, heated at 808C for 90min, and cooled. The internal standard, 7-dehydrocholesterol, and 1mL of water were added, and samples were extracted twice with 2mL of hexane. After evaporation under a stream of nitrogen at 21 28C, the solid residue was dissolved in 1mL of dichloromethane/hexane (1:1, v/v). The sample was applied to a silica solid-phase extraction (SPE) column (100mg of silica, Varian, Palo Alto, CA, USA) which has been preconditioned with 1mL of diethyl ether and 1mL of dichloromethane/hexane (1:1, v/v), and washed twice with 1mL of dichloromethane/hexane. The sterols were eluted twice with 1mL of diethyl ether, and the solvent was evaporated under a stream of nitrogen. Hexane (0.5mL) was added or, alternatively, the trimethylsilyl (TMS) derivative of the alcohol group in ergosterol was prepared by adding 55mL of N,Obis(trimethylsilyl)trifluoroacetamide/ pyridine/trimethylchlorosilane (5:5:1, v/v/v) and heating at 708C for 15min. Pyridine was evaporated, and 0.5mL of hexane was added. The sample was washed twice with 0.5mL of distilled water, and dried with anhydrous Na2SO4. 17 The amount of egosterol at the end of the sample preparation was calculated using an external calibration curve, and in leaves using an internal standard. The ratio of ergosterol after sample preparation to that in leaves, that is the ergosterol recovery, was without silylation 69.0 10.4% and with silylation 51.7 8.4% (average standard deviation, n1⁄4 8). Methanol, KOH and Na2SO4 were from Merck (Darmstadt, Germany), hexane and dichloromethane from Mallinckrodt (Deventer, The Netherlands), diethyl ether from Riedel de-Häen (Seelze, Germany), silylation reagents from Pierce (Rockford, IL, USA), and 7dehydrocholesterol from Sigma (St. Louis, MO, USA). The sterols were analysed with a model GC-17A gas chromatograph terscience.wiley.com) DOI: 10.1002/rcm.3893 (Shimadzu, Kyoto, Japan) equipped with a model GCMC-QP5000 massselective detector and an model AOC17, long automatic sampler. The gasliquid chromatography conditions were as follows: Zebron ZB-5 capillary column (30m 0.25mm 0.25mm) coated with 5% phenyl–95% dimethylpolysiloxane (Phenomonex, Torrance, CA, USA); carrier gas, helium at a flow rate of 1mLmin ; injection of 1mL, splitless; injector temperature, 3108C; and detector interface temperature, 3008C. The oven temperature was programmed to hold at 1708C for 1min, and then increased by 208C min 1 to 290, 300, 310, or 3208C. The electron ionisation (EI) mass spectra were recorded at an electron energy of 70 eV and emission current of 75mA. The ion source temperature was 2158C. In the GC/MS analysis using selected ion monitoring (SIM) and a final oven and column temperature of 2908C, peaks were produced in positions where ergosterol and 7-dehydrocholesterol and their TMS derivatives were assumed to elute according to the retention times of standards (Fig. 1). In leaf samples without TMS derivatisation, two additional unknown peaks observed could be plant sterols. Saraf et al. could not analyse accurately the TMS derivative of egosterol from 4 of 31 house dust preparations to quantify fungi, due to an unknown compound with the same retention time and the same ion in SIM as the ergosterol TMS derivative. The interference of an unknown substance co-eluting with ergosterol or 7-dehydrocholesterol was not observed in this study. The TMS derivative of ergosterol was detected from ions atm/z 337 [M–131]þ, 363 [M– 105]þ and 468 [M]þ, and the TMS derivative of 7-dehydrocholesterol from ions at m/z 325 [M–131]þ, 351 [M–105]þ and 456 [M]þ. The ions in the mass spectra of the TMS-derivatised ergosterol at m/z 337 [M-131]þ, and the TMSderivatised 7-dehydrocholesterol at 325 [M–131]þ, result from the loss of trimethylsilanol and the C1-C3 fragment. The ions in the TMS-derivatised ergosterol at m/z 363 [M–105]þ, and the TMS-derivatised 7-dehydrocholesterol at m/z 351 [M–105]þ, are due to the loss of trimethylsilanol and a methyl group. The direct analysis of ergos-