Friday, April 17, 2009
Instrumentation
Table 1 - Optimized instrumental conditions and working parameters
A Star 3800 CX gas chromatograph (Varian, Mississauga, Ontario, Canada) equipped with a splitless/split injector and a Saturn 3 ion trap MS detector using helium carrier gas was used for analyses. The GC-MS was equipped with a Combi PAL autosampler (CTC Analytics, Zwingen, Switzerland) fitted with a fiber heater, sample tray, and sample heater/agitator. PAL Cycle Composer software (version 1.4.1, CTC) running on the spectrometer controlled all extraction conditions and timings for SPME headspace sampling, heating, agitating, and injection control in the sampling method. The GC injector port (constant, 230 °C) contained an SPME glass insert and was connected to a fused capillary 5%−diphenyl−95%−dimethyl polysiloxane J&W DB-5MS column: 30 m × 0.25 mm i.d. with a 0.25-μm phase (Restek, Brockville, Ontario, Canada). Optimized GC-MS parameters were: helium carrier flow rate of 1.6 mL/min, and samples injected with closed split at 5 psi (see also Table 1); the transfer line and ion trap were held at 260 °C and 220 °C, respectively. The electron multiplier was operating at 2000 V, and the ion trap detection (ITD) mass setting was set to cover the range 40−480 m/z.
A 9012 HPLC system with 9050 UV-VIS diode array detector under software control using ChromStar 5.31 software (Varian) with Varian LC-18 column in a thermostated box (Supelco), with a 10-μm injection loop, and a dual pump methanol and water solvent system, were used to determine ergosterol from extracted and standard samples. The UV detector was set at a wavelength of 282 nm and 233 nm to monitor for ergosterol and methyl benzoate, respectively, for calibration and linearity determinations.
Standards for analysis and optimization
A set of methyl benzoate in 95% water:5% methanol (v/v) standards were prepared (serial dilution) to cover a 1–1000 ppb (ng mL–1) range so that each sample had a final volume of 25 mL.
For GC-MS analysis, all experimental parameters and optimization were under automation control and run on 25-mL sample volumes in 30-mL vials containing a magnetic stirrer bar and sealed with screw-top PTFE-coated septum caps. To prevent carryover, the SPME fiber was reconditioned for 1 min at 220 °C between runs. The extraction conditions for methyl benzoate (10 ppb) in water were optimized: extraction time (5–30 min intervals), spinning rate (500–1200 rpm), extraction temperature (25–70 °C in 5° intervals), pH (2–9) by acid/base adjustment, and with/without the presence of salt.
For HPLC analysis, ergosterol standards were prepared (serial dilution) to cover the range 5–1000 ppb (ng mL–1). During analyses, ergosterol samples were wrapped in metal foil whenever possible to avoid photodecomposition. Otherwise, samples were stored in freezers at –20 °C. Optimized conditions included temperature (25, 30, and 35 °C), different solvent combinations, mobile phase flow rate (0.8–1.6 mL min–1), and pH (2–9).
Surrogate building material samples
Pieces of wallboard and ceiling tile samples (without glue layer) unaffected by mold were collected from a lecture room at Memorial University of Newfoundland (Corner Brook, Canada). A sample blank was prepared by stirring 1 g of dried material (16 hr at 40 °C) in 25 mL of water. Four wallboard and three ceiling tile samples (without the glue layer) with visible fungal growth were collected from the same test room.
For methyl benzoate analysis, each 1 g of dried sample was cut and transferred to a 30-mL vial containing 25 mL water. Analysis was carried out under optimized conditions and compared to sample blanks.
The ergosterol content of the fungal biomass from similar building samples was determined by an extraction method similarly outlined by Grant and West.7 The moldy sample (1 g dry weight) with methanol (80 mL) was sonicated at 5 °C (10 min, ultrasonic bath) then kept at 0 °C for 30 min. The suspension was filtered and the residue washed with 25 mL of methanol. The combined filtrate was rotary evaporated (30–35 °C) to about 20 mL and was combined with ethanol (8 mL), potassium hydroxide (0.2 g), and PTFE boiling chips. This mixture was saponified under reflux (30 min) and cooled to room temperature. After addition of distilled water (10 mL), the ergosterol was extracted with hexanes. The combined extracts were dried (magnesium sulfate), filtered, and evaporated (25 °C) to about 1 mL and then sealed under nitrogen and stored at –20 °C. Immediately prior to assay, the ergosterol was dissolved in methanol (2.0 mL) and filtered through a 0.2-μm Teflon filter (Pall Corp./Gelman Laboratory, East Hills, NY). HPLC with 282-nm wavelength detection was used for analysis.