Use of quadrupole GC-MS and ion trap GC-MS-MS for determining 3-hydroxy fatty acids in settled house dust: relation to endotoxin activity Anita Saraf,a Ju-Hyeong Park,b Donald K. Miltonb and Lennart Larsson*a aDepartment of Infectious Diseases and Medical Microbiology, Section of Bacteriology, University of Lund, S-223 62 Lund, Sweden. E-mail: Lennart.Larsson@mmb.lu.se bDepartment of Environmental Health, Harvard School of Public Health, Boston, MA 02115, USA Received 18th November 1998, Accepted 8th February 1999 Gas chromatography-mass spectrometry (GC-MS) using a quadrupole instrument and GC-tandem MS (GC-MSMS) using an ion trap instrument were applied to determine 3-hydroxy fatty acids (3-OH FAs) with 10–18 carbon chain lengths, specific components of the endotoxin ( lipopolysaccharide, LPS) of Gram-negative bacteria, in 30 house dust samples. The two methods provided similar detection sensitivity for methyl ester/trimethylsilyl derivatives of the 3-OH FAs and allowed these acids to be distinguished from co-eluting 2-OH FA derivatives.The correlation coeYcients between endotoxin activity (Limulus test) and the combined amounts of 3-OH C10, 3-OH C12, and 3-OH C14 were 0.60 and 0.61 when using GC-MS and GC-MS-MS, respectively. The superior selectivity of GC-MS-MS was illustrated in analyses of sub-milligram amounts of dust, where the chromatograms achieved by GC-MS were diYcult to interpret due to a high background and several closely eluting compounds. GC-MS-MS is therefore preferable to GC-MS for determining 3-OH FAs in minute (sub-milligram) amounts of dust.degrees of correlation have been found between the results of Introduction 3-OH FA determinations and Limulus assays.18–21 One finding Endotoxin is the name given to a class of biological molecules has been that LPS associated with 3-OH C16 and 3-OH C18 that have certain characteristic toxic eVects. Endotoxins are has lower potency than when associated with 3-OH C10, 3- lipopolysaccharides (LPS) present in the outer membrane of OH C12, and 3-OH C14.18,22 Gram-negative bacteria, occurring abundantly in domestic Gas chromatography-mass spectrometry (GC-MS) is the house dust and in several agricultural and occupational preferred analytical technique for determining 3-OH FAs in environments.Exposure to high concentrations of airborne chemically complex environments.The GC-MS method preendotoxin is associated with bronchial inflammation, acute dominantly used includes analysis of these acids as methyl airflow obstruction and inhalation fever.1–7 Endotoxin has ester (Me)/trimethylsilyl (TMS) derivatives by using a quadrualso been associated with work-related illness in non- pole type of instrument.Selected ion monitoring (SIM) is agricultural environments.8–10 Endotoxin in house dust has used in order to achieve high detection sensitivity; it is inevibeen associated with increased asthma severity.11 Inhaled table, however, that by applying SIM the detection specificity endotoxin is a potent proinflammatory substance that triggers will be lowered in comparison with full scan analysis.We have a cascade of cellular inflammatory responses primarily through previously shown that Me/TMS derivatives of 3-OH FAs can activating pulmonary macrophages.12 be distinguished from the corresponding 2-OH FA derivatives Environmental endotoxin is usually measured by diVerent by focusing at a fragment specific for the former (m/z 175).23 versions of the Limulus amebocyte lysate (LAL) assay.However, whether this fragment is specific enough to dis- Measurement of clotting enzyme activity by means of a tinguish 3-OH FAs also from other co-eluting compounds that synthetic chromogenic substrate and the formation of a gel may be present in dust samples is not known. clot forms the basis of the chromogenic and gel clot LAL Tandem MS (GC-MS-MS), using ion-trap technology, has assay.13,14 However, the toxicity of endotoxin from diVerent been applied to achieve improved detection selectivity.In this Gram-negative bacteria can vary substantially both quantitat- technique, the ions formed initially are subjected to further ively and qualitatively,15,16 and the LAL gel assay may not fragmentations, and the fragment (daughter) ions are monireflect all these diVerences.tored. However, a disadvantage of this approach may be the Determination of 3-hydroxylated fatty acids (3-OH FAs), unwanted side-reactions and other events that may occur in of 10–18 carbon chain lengths, has been applied to measure an ion trap upon introduction of samples containing relatively LPS in environmental samples. This method is reproducible large amounts of compounds that co-elute with the analytes.and excludes the risk of interference from other biologically In the present study, results from analyses of Me/TMS active components, such as b-1–3 glucan, that can be encoun- derivatives of 3-OH FAs in settled house dust samples by tered in the LAL assays. The analysis also provides some using quadrupole GC-MS were compared with those obtained information about the origin of the endotoxin, because the using ion trap GC-MS-MS.The aims were (1) to evaluate the relative distribution of the individual 3-OH FAs varies between specificity of the m/z 175 fragment for determining LPS in diVerent species of Gram-negative bacteria.17 However, this such samples, (2) to assess the performance of ion trap GCmethod measures the total mass of LPS, irrespective of the MS-MS for measuring 3-OH FAs in samples containing coeluting compounds, including 2-OH FAs, and (3) to correlate bioactivity, and sensitivity is lower than in LAL assays.Various J. Environ. Monit., 1999, 1, 163–168 163the results of the GC-MS and GC-MS-MS analyses of 3-OH endotoxin units with reference to EC5 reference standard endotoxin (US Pharmacopeia, 1 ng EC5=10 endotoxin FAs with Limulus activity.units, EU). Experimental Preparation of samples for GC-MS and GC-MS-MS Chemicals and glassware Dust samples were heated in 2 ml of 4 M methanolic HCl Pentafluoropropionic anhydride (PFPA) and pyridine were (10 ml of acetyl chloride added to 22 ml of methanol ) for 18 h purchased from Fluka Chemie (Buchs, Switzerland), triethyl- at 100 °C and cooled to room temperature, after which amine was obtained from Sigma (St.Louis, MO, USA), and deuterated 3-OH C14 methyl ester was added as an internal N,O-bis-(trimethylsilyl ) trifluoroacetamide (BSTFA) was from standard. A volume of 1 ml of water was added, and the Acros (Geel, Belgium). The 2- and 3-OH FA methyl esters with preparations were extracted with 2×1.5 ml of heptane, dried chains of 10, 12, 14, 16, and 18 carbons were purchased from using a stream of nitrogen, dissolved in 1 ml of dichloro- Larodan Lipids (Malmo� , Sweden), and deuterated 3-OH-C14 methane–heptane (1+1, v/v), and applied to a silica gel was a gift from Dr.David White. Solvents were of analytical- column (1 ml, 100 mg Si; Varian, Harbor City, CA, USA) reagent grade and used without further purification.Glass that had been preconditioned with 1 ml of diethyl ether and test-tubes were equipped with PTFE-lined screw caps. 1 ml of dichloromethane–heptane (1+1, v/v). The column was washed twice with 1 ml of dichloromethane–heptane (1+1, Dust samples v/v), whereafter hydroxylated fatty acid methyl esters were eluted with two 1 ml portions of diethyl ether.The eluates House dust samples were collected with a Eureka Mightywere then dried under a stream of nitrogen, dissolved in 1 ml Mite vacuum cleaner modified to collect dust in cellulose of pentane–dichloromethane (1+1, v/v), and divided into two extraction thimbles (19×90 mm; Whatman, Maidstone, Kent, portions.The hydroxy acid esters in one portion were subjected UK) for an ongoing study of home endotoxin exposure and to TMS derivatization (using BSTFA) as previously childhood asthma. Separate clean rigid tubes were used described,23 and those in the other portion were subjected to between the sampling surface and the thimble for each sample pentafluoropropionyl (PFP) derivatization as follows.Extracts to prevent cross-contamination. Thimbles were placed iairwere evaporated using a stream of nitrogen and dissolved in tight plastic bags immediately after collection. No later than 30 ml of triethylamine-containing (1% v/v) acetonitrile. the day after collection the recovered dust was weighed, sifted Thereafter, 20 ml of PFPA were added, and the mixture was through a 500 mm mesh sieve, and the fine dust reweighed and heated at 37 °C for 15 min.After cooling to room temperature, aliquoted for various analyses. Of a total of 143 dust samples 750 ml of hexane and 1 ml of 0.1 M phosphate buVer (pH 7.0) that were subjected to GC-MS analysis for 3-OH FAs, a subwere added, and the mixture was vortexed and centrifuged set of 30 samples was taken for the comparison with GC-MS- (approximately 1000g).The hexane phase was then applied to MS in this study. The sub-set was taken by randomly choosing a disposable silica gel column that had previously been rinsed 15 samples from the lowest quintile and another 15 samples with 3×1 ml of dichloromethane, and the Me/PFP derivatives from the highest quintile of house dust endotoxin activity (EU were eluted using 3×1 ml of the same solvent.After evapor- per mg dust). ation, the product was redissolved in heptane and used for Endotoxin assays analysis. Furthermore, five aliquots ranging from 0.15 to 1.0 mg of The endotoxic biological activity of the dust samples was one of the 30 dust samples were prepared for 3-OH FA measured by the kinetic Limulus assay with the resistantanalysis.parallel-line estimation (KLARE) method, described pre- Apart from the TMS and PFP derivatives, several other viously.24,25 Limulus amebocyte lysate (LAL) was obtained derivatives of 2- and 3-OH fatty acid methyl ester (FAME) from BioWhittaker (Walkersville, MD, USA), reference stan- standards, prepared essentially according to previously dard endotoxin from the US Pharmacopeia (Rockville, MD, described methods, were analyzed.These included methoxy,26 USA), and control standard endotoxin from Associates of pentafluorobenzoyl,27 tert-butyldimethylsilyl,28 heptafluoro- Cape Cod (Woods Hole, MA, USA). All glassware was heated butyryl (prepared in the same way as the PFP derivatives in to 270 °C for 30 min prior to use. Control and reference the present study), acetate, propionate and butyrate derivastandards and field samples were serially diluted for endotoxin tives.29 FAMEs with underivatized hydroxy groups were also analysis in a standard buVer (of 0.01% triethylamine – 0.05 M studied.potassium phosphate). A 25 mg portion of sifted dust was placed in glass tubes with 5 ml of buVer and bath-sonicated GC-MS and GC-MS-MS for 1 h with vortexing at 15 min intervals.An initial 1+24 dilution of dust extracts with suspended particulate was made A VG Trio-1 S quadrupole mass spectrometer (VG, Manchester, UK) connected to a Hewlett-Packard Model before the start of the serial dilutions used in the assay. Duplicate 50 ml aliquots of five serial dilutions of dust extracts 5890 gas chromatograph was used.The fused-silica capillary column (CP-Sil 5CB-MS, 0.25 mm film thickness, and a control standard endotoxin were placed in an endotoxinfree 96-well, flat-bottomed polystyrene microplate (Associates 30 m×0.25 mm id; Chrompack, Middelburg, The Netherlands) was temperature programmed, at 20 °Cmin-1, of Cape Cod); 50 ml of LAL were added and the microplate was agitated.The absorbance of each well was recorded at from 90 to 280 °C. The injector was kept at 280 °C, the interface at 290 °C, and the ion source at 200 °C. Injections 405 nm every 30 s for 120 min during incubation at 37 °C. The response parameter for the LAL reaction was the maximum were made in the splitless mode using a Hewlett-Packard Model 7673 autosampler; helium, at a flow rate of 1 ml min-1, rate of absorbance change (Vmax).The log potency and its variance were computed using resistant regression and Tukey’s was used as a carrier gas. This instrument was used to analyze TMS-derivatized samples only, operating in SIM mode with jackknife. Interference was detected by analysis of covariance and a standard algorithm was used to eliminate such eVects electron impact (EI ) ionization; the ionization energy was 70 eV.during an iterative data analysis. Results were considered valid if the final data analysis found no statistically significant An ion trap GC-MS-MS instrument, Model Saturn 2000 (Varian, Palo Alto, CA, USA), equipped with the same type diVerence between the dose-response slopes for the control standard and environmental sample.Results are reported in of fused-silica capillary column as in the quadrupole instru- 164 J. Environ. Monit., 1999, 1, 163–168ment (see above), was used. Samples were injected in the splitless mode using a Varian Model 8200 CX autosampler. Column temperature was programmed from 90 to 280 °C at a rate of 20 °Cmin-1; the temperature of the injector was 280 °C and that of the transfer line (between GC and MS) 280 °C.The ion trap was held at 180 °C for the TMS derivatives and at 200 °C for all other derivatives studied. The derivatized acid standards were all analyzed by GC-MS on this instrument; in addition, TMS- and PFP-derivatized standards and dust samples were also subjected to GC-MS-MS analysis. Conditions for formation of daughter ions from suitable parent ions selected from the mass spectra of TMS- and PFPderivatized 3-OH FAMEs were optimized by repeatedly injecting 1000–5000 pg of each marker derivative and studying the influence of diVerent fragmentation conditions [voltage, time, resonance/non-resonance mode, digital/analogue conversion values (DAC), and excitation storage level ].PFP derivatives were analyzed in selected ejection chemical ionization (SECI) mode, using isobutane as the reagent gas; the selected parent ions were fragmented in resonance mode.TMS derivatives were analyzed by using EI; the parent ion m/z 175 was fragmented (non-resonance mode). A 1ml aliquot (total volume 100 ml ) of a prepared dust sample was injected for each analysis. The results of the analyses of the TMS-derivatized dust samples by GC-MS-MS were compared with the results obtained when the same preparations were analyzed by quadrupole GC-MS.Statistical analyses Because the samples analyzed were taken from the lowest and highest quintiles, the distribution of endotoxin and concentrations were not Gaussian, even after log transformation. Therefore, the Spearman rank correlations were computed to examine associations between endotoxin level and LPS content of dust samples. Results Standard solutions PFP derivatives of the 2- and 3-OH FAMEs were well separated chromatographically.The EI spectra of the 3-OH FA derivatives showed excessive fragmentation. The SECI spectra were dominated by ions of m/z [M-163] ( loss of CF3–CF2–COO) which were therefore used for fragmentation in the MS-MS analyses, resulting in fragments of m/z [M-163-32] (further loss of CH3OH; Fig. 1). Fragmentation Fig. 1 Mass spectra of the methyl ester/PFP derivative of 3-OH C16 was achieved by using the resonance mode at an excitation obtained in the EI (upper) and in the SECI (middle) mode, and a storage level of 48 m/z for 20 ms and voltages of 0.24 V (3- SECI-MS-MS spectrum of the same compound after fragmentation of m/z 269 (lower).OH C10), 0.25 V (3-OH C12), 0.26 V (3-OH C14), 0.22 V (3-OH C16) and 0.23 V (3-OH C18). The heptafluorobutyryl derivatives also separated chromatographically and the SECI spectra were identical with those of the PFP derivatives. The FAMEs. However, a considerable peak tailing was observed, leading to poor sensitivity.The tert-butyldimethylsilyl deriva- detection sensitivity for both derivatives was comparable; however, the PFP derivatives had shorter retention times, thus tives of the corresponding 2- and 3-OH FAMEs showed a partial chromatographic separation that decreased with they were used in subsequent studies. Mass spectra of the 3-OH FAME TMS derivatives were increasing carbon chain length.The spectra of the 2- and 3- OH FA derivatives were virtually identical and dominated by dominated by ions of m/z 175 (cleavage of the C3–C4 linkage) and m/z [M-15] ions ( loss of CH3). The 2-OH FA derivatives m/z [M-57] {loss of –C(CH3)3}. The 3-OH FAME/methoxy derivatives produced dominant m/z [M-15] ions ( loss of also produced m/z [M-15] ions, but not m/z 175; as reported earlier,23 there was no chromatographic separation of the CH3), whereas the 2-OH FA derivatives gave [M-59] ions (cleavage of C1–C2 linkage); however, the yields were low corresponding 2- and 3-OH FAs.Fragmentation of m/z 175 ions for 50 ms at 18 V and an excitation storage level of 48 which resulted in poor detection sensitivity. None of the other derivatives of the 2- and 3-OH FAs of m/z (non-resonance mode) resulted in two distinctive fragment ions, m/z 131 and m/z 73.30 identical chain lengths was separated chromatographically.The Me/pentafluorobenzoyl derivatives of both 2- and 3-OH Methyl esters of the 2- and 3-OH FAs (with non-derivatized hydroxy groups) were well separated chromatographically; FAs gave identical spectra, dominated by ions of m/z 195 (pentafluorobenzoyl group).The spectra of 2- and 3-OH ions of m/z 103 (fragmentation between C3 and C4) were found for all of the 3-OH FAMEs but not for the 2-OH FAME acetate, propionate and butyrate derivatives were J. Environ. Monit., 1999, 1, 163–168 165dominated by m/z [M-59] ( loss of CH3COO), [M-73] ( loss instrument. After the preparations had been diluted 10-fold with heptane and re-analyzed using the same GC-MS-MS of CH3CH2COO), and [M-87] ( loss of CH3CH2CH2COO), respectively.conditions, a marked increase in the relative amounts of 3- OH C16 and 3-OH C18 was noticed (Fig. 3). The fragment ion spectra of the 3-OH FA derivatives were dominated by m/z Dust samples 131. However, when the amounts injected were small ( less PFP derivatives.All dust samples studied contained detect- than approximately 50 pg) the spectra were dominated by m/z able amounts of both 2- and 3-OH FAs of 10, 12, 14, 16, and 175 provided that background subtraction was not applied 18 carbon chain lengths. As mentioned, chromatographic (Fig. 4). These results demonstrate that only part of the m/z separation was achieved for all of the acids when analyzed as 175 ions originate from 3-OH FAs, and that SIM may not be Me/PFP derivatives (Fig. 2). Preparations were analyzed both specific enough for determining 3-OH FAs, e.g., in small in the SECI-MS mode and by using SECI-MS-MS; as expected, amounts of dust. SECI-MS-MS gave lower background noise than SECI-MS (data not shown). In the MS-MS experiments, ions of m/z Detection sensitivity.Both quadrupole GC-MS and ion trap [M-195] were used for quantification. The amounts of the 3- GC-MS-MS revealed the presence of all of the studied 3-OH OH FAs compared with the corresponding 2-OH FAs were FAs (from C10 to C18) in the five (0.15–1.0 mg) aliquots of lower for C16 and C18 than for C10, C12 or C14. Thus, the relative amounts (mean values) of 3-OH FAs to the sum of 2- and 3-OH FAs were 0.86, 0.95, 0.75, 0.24, and 0.44 for C10, C12, C14, C16, and C18, respectively (Table 1A).TMS derivatives. Ions of m/z 175, m/z 178 (internal standard) and m/z [M-15] were monitored in the quadrupole GC-MS analysis. The 3-OH FAs were quantified by dividing the area of the m/z 175 ions by the area of the m/z 178 ions for each sample (m/z 175 is considered to be specific for 3-OH FAs, whereas m/z [M-15] is assumed to represent the sum of 2-OH and 3-OH FAs).The relative amounts (mean values) of the area of the m/z 175 ion to that of the [M-15] ion were 0.73, 1.0, 0.8, 0.23, and 0.56 for C10, C12, C14, C16, and C18, respectively (Table 1B). The same preparations were also run on the ion trap instrument in the EI-MS-MS mode, fragmenting the m/z 175 ion; m/z 131 was used for quantification.Relatively small amounts of both 3-OH C16 and 3-OH C18 were detected as compared with the results obtained with the quadrupole Fig. 2 SECI-MS-MS analysis of methyl ester/PFP-derivatized 2- and 3-OH FAs, with chains of 10–18 carbon atoms (blackened), present in a house dust sample. The amount injected corresponded to 100 mg of dust and 2.4 ng of internal standard (I.S).Table 1 Ratios (mean) of 3-OH FAs to sum of 2- and 3-OH FAs (methyl ester/PFP derivatives) (A) and ratios (mean) of the intensity of the m/z 175 to the m/z [M-15] ions (methyl ester/TMS derivatives of hydroxy acids) (B) determined for the set of 30 dust samples Fig. 3 Methyl ester/TMS-derivatized hydroxy FAs of 10–18 carbon atom chain lengths in a house dust sample analyzed by quadrupole A: B: GC-MS in the EI-SIM mode, monitoring m/z 175, m/z 178 (internal Acid Mean±s Mean±s standard), and m/z [M-15].The amount injected corresponded to 50 mg of dust and 1.2 ng of internal standard (upper three tracings). C10 0.86±0.10 0.73±0.15 Results are also shown for the same preparation before (middle two C12 0.95±0.03 1.00±0.05 tracings) and after a 10-fold dilution ( lower two tracings), analyzed C14 0.75±0.17 0.80±0.26 by ion-trap EI-MS-MS, with fragmentation of m/z 175 monitoring C16 0.24±0.11 0.23±0.09 m/z 131; fragment m/z 134 was monitored for the internal standard C18 0.44±0.24 0.56±0.21 (I.S ). 166 J. Environ. Monit., 1999, 1, 163–168Fig. 5 Methyl ester/TMS-derivatized 3-OH FAs of 10–18 carbon atom chain lengths in a house dust sample analyzed by ion-trap EI-MS-MS Fig. 4 Fragment ion spectrum (using m/z 175) of the methyl ester/ (fragmentation of m/z 175, monitoring m/z 131; upper) and by TMS derivative of 3-OH C16 in a standard solution (upper) and in a quadrupole GC-MS in the EI-SIM mode (lower). The amount injected dust sample ( lower); background subtraction was not applied.corresponded to 1.35 mg of dust. the dust samples that were analyzed separately as Me/TMS derivatives. However, the superior selectivity of MS-MS specificity be achieved. In the first studies published on this resulted in chromatograms that were easier to interpret than subject,19,20 where GC-MS of Me/pentafluorobenzoyl derivathose obtained by quadrupole analysis (Fig. 5). The sensitivit- tives was used in the CI-negative ion mode, the presence of ies for the Me/PFP derivatives were considerably lower (data substantial amounts of 2-OH FAs of 10–18 carbon chain not shown). lengths in organic dust samples was overlooked; thus, the reported amounts of 3-OH FAs were overestimated since Correlation between GC-MS/GC-MS-MS and Limulus results chromatographic separation was not achieved.In later investigations, Me/TMS-derivatized samples were used and analyzed Endotoxin levels observed in the 30 dust samples ranged from in the EI mode, and it was found that 2- and 3-OH FAs could 24 to 516 EU mg-1 house dust and generally no evidence of be distinguished even though they co-eluted from the GC interference was shown during Vmax data analysis to estimate column; only the latter produced m/z 175 ions, whereas both log potency of endotoxin.The correlation between results of 2- and 3-OH FA derivatives produced m/z [M-15].23 In the the Limulus assays and the 3-OH FA analyses (using Me/TMS present study, we showed that Me/PFP derivatives of 2-and derivatives) depended upon the individual acid or the combi- 3-OH FAs separate chromatographically and that 2-OH FAs, nations of acids chosen to calculate the number of moles of mainly those with 16 and 18 carbon chain lengths, are ubiqui- LPS (moles of acids divided by four). Correlation was best tous in house dust.We also found that the discrepancies when 3-OH FAs of 10, 12 or 14 carbon chain lengths were between the intensities of the m/z 175 and m/z [M-15] ions considered, either individually or in diVerent combinations.In seen when analyzing Me/TMS derivatives were primarily due the quadrupole GC-MS analyses, correlation coeYcients of to co-elution of the 2- and 3-OH FA derivatives, since the 0.59, 0.57, and 0.61 were obtained for 3-OH C10, 3-OH C12, ratios of the intensities of these ions and the ratios of the 3- and 3-OH C14, respectively.A correlation coeYcient of 0.60 OH FAs to the sum of 2- and 3-OH FAs were in general ( p=0.0006) was obtained when all three of these acids were agreement (Table 1). taken into consideration. With 3-OH C16 and C18 the corre- Compared with GC-MS analysis in the SIM mode, GC- lation coeYcients were 0.22 and 0.38, respectively. In the ion MS-MS provides superior analytical specificity, and ion trap trap GC-MS-MS analyses of the 10-fold diluted samples use instruments are particularly suitable for the latter type of of 3-OH C10 gave a correlation coeYcient of 0.65, with 3-OH analysis.We have previously reported details concerning detec- C12 it was 0.56 and with 3-OH C14 it was 0.65.The correlation tion limit,30 reproducibility,18 and quantification18 of the 3- coeYcient was 0.61 (p=0.0004) when a combination of these OH FAME/TMS derivatives.However, it is also generally three acids was used. With 3-OH C16 and C18 the correlation accepted that the sample capacity of ion traps is limited. coeYcients were 0.27 and 0.44, respectively. Introduction of a large amount of a component into a trap results in a shorter ionization time (which is regulated auto- Discussion matically).In our analyses of undiluted dust preparations, the ionization time was approximately 2000–3000 ms for the For a successful application of 3-OH FA analysis to determine LPS in environmental samples, it is essential that high detection C10–C14 3-OH FAs but only about one-third as long for the J. Environ. Monit., 1999, 1, 163–168 1675 R.Rylander, P. Haglind andM. Lundholm, Am. Rev. Respir. Dis., C16 and C18 3-OH FAs, probably due to the simultaneous 1985, 131, 209. elution of appreciable amounts of C16 and C18 2-OH FA 6 D. A. Schwartz, P. S. Thorne, S. J. Yagla, L. F. Burmeister, S. A. derivatives. The results of these analyses showed that the Olenchock, J. L. Watt and T. J. Quinn, Am.J. Respir. Crit. Care relative amounts of the C16 and C18 3-OH FAs compared with Med., 1995, 152, 603. the C10–C14 3-OH FAs were considerably smaller than the 7 A. Thelin, O� . Tegler and R. Rylander, Eur. J. Respir. Dis., 1984, 65, 266. results obtained with the quadrupole instrument. However, 8 D. K. Flaherty, F. H. Deck, J. Cooper, K. Bishop, P. A. after a 10-fold dilution and re-analysis, ionization times were Winzenburger, L.R. Smith, L. Bynum and W. B. Witmer, Infect. substantially longer, and these diVerences in the relative peak Immun., 1984, 43, 206. areas were not observed (Fig. 3). 9 D. K. Milton, J. Amsel, C. E. Reed, P. L. Enright, L. R. Brown, The fragmentation conditions used in GC-MS-MS were G. L. Aughenbaugh and P.R. Morey, Am. J.Ind. Med., 1995, adjusted so that most of the m/z 175 fragment formed ions of 28, 469. 10 D. K. Milton, D. Wypij, D. Kriebel, M.Walters, S. K. Hammond m/z 131 (used for quantification) and m/z 73. However, when and J. Evans, Am. J. Ind. Med., 1996, 29, 3. analyzing small amounts of dust samples, ions of m/z 175 11 O. Michel, J. Kips, J. Duchateau, F. Vertongen, L. Robert, H. dominated over m/z 131 provided that background subtraction Collet, R.Pauwels and R. Sergysels, Am. J. Respir. Crit. Care was not applied (Fig. 4), indicating that an appreciable pro- Med., 1996, 154, 1641. portion of the former ion represents compound(s) other than 12 R. Rylander and L. Beijer, Am. Rev. Respir. Dis., 1987, 135, 83. 3-OH FAs. The diVerence in specificity between quadrupole 13 S.Iwanaga, T. Morita, T. Harada, S. Nakamura, M. Niwa, K. Takada, T. Kimura and S. Sakakibara, Haemostasis, 1978, 7, 183. GC-MS and ion trap GC-MS-MS was illustrated by the 14 J. Levin and F. B. Bang, Thromb. Diath. Haemorrh., 1968, 19, analysis of a 0.15 mg dust sample; the identities of the 186. extraneous peaks seen in the quadrupole GC-MS trace are not 15 I. Helander, M.Salkinoja-Salonen and R. Rylander, Infect. known (Fig. 5). Immun., 1980, 29, 859. The correlation between 3-OH FA composition and endo- 16 M. W. Baseler, B. Fogelmark and R. Burrell, Infect. Immun.,1983, toxin activity (Limulus test) was similar regardless of whether 40, 133. 17 S. G. Wilkinson, in Microbial Lipids, ed. C. Ratledge and S. G. GC-MS-MS or GC-MS was applied; however, several milli- Wilkinson, Academic Press, London, 1988, vol. 1, pp. 299–488. grams of dust samples were used in this comparison. Our 18 A. Saraf, L. Larsson, H. Burge and D. Milton, Appl. Environ. results show that GC-MS-MS is the preferred method when Microbiol., 1997, 63, 2554. only sub-milligram amounts of dust are available for analysis, 19 A. Sonesson, L. Larsson, A. Fox, G.Westerdahl and G. Odham, which may be the case, e.g., when studying airborne particles J. Chromatogr. B, 1988, 431, 1. in various indoor environments. 20 A. Sonesson, L. Larsson, A. Schu� tz, L. Hagmar and T. Hallberg, Appl. Environ. Microbiol., 1990, 56, 1271. 21 M. Walters, D. Milton, L. Larsson and T. Ford, Appl. Environ. Acknowledgements Microbiol., 1994, 60, 996. 22 L. Ma°rtensson, W. Gradowska and L. Larsson, Aerobiologia, Financial support from the Swedish Council for Building 1997, 13, 99. Research, Swedish Society for Medical Research, The Royal 23 Z. Mielniczuk, E. Mielniczuk and L. Larsson, J. Microbiol. Physiographic Society in Lund, Medical Faculty at the Methods, 1993, 17, 91. 24 D. K. Milton, H. A. Feldman, D. S. Neuberg, R. J. Bruckner and University of Lund, The Crafoord Foundation and the US I. A. Greaves, Environ. Res., 1992, 57, 212. National Institute of Environmental Health Sciences grant 25 D. K. Milton, D. K. Johnson and J. H. Park, Am. Ind. Hyg. Assoc. number ES07036 is greatly acknowledged. J., 1997, 58, 861. 26 W. Gradowska and L. Larsson, J. Microbiol. Methods, 1994, 20, 55. References 27 Z. Mielniczuk, S. Alugupalli, E. Mielniczuk and L. Larsson, J. Chromatogr., 1992, 623, 115. 1 R. M. Castellan, S. A. Olenchock, K. B. Kinsley and J. L. 28 B. O. Axelsson, A. Saraf and L. Larsson, J. Chromatogr. B, 1995, Hankinson, N. Engl. J. 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