Comparison of the limulus amebocyte lysate test and gas chromatography-mass spectrometry for measuring lipopolysaccharides (endotoxins) in airborne dust from poultry-processing industries

The lipopolysaccharide (endotoxin) content in airborne dust samples from three different poultry slaughterhouses was determined with both the chromogenic Limulus amebocyte lysate assay and gas chromatography-mass spectrometry analysis of lipopolysaccharide-derived 3-hydroxy fatty acids. Gram-negative cell walls were also measured by using two-dimensional gas chromatography/electron-capture analysis of diaminopimelic acid originating from the peptidoglycan. The correlation between the results of the Limulus assay and those of gas chromatography-mass spectrometry for determination of the lipopolysaccharide content in the dust samples was poor, whereas a good correlation was obtained between lipopolysaccharide and diaminopimelic acid concentrations with the gas chromatographic methods. The results suggest that it is predominantly cell-wall-dissociated lipopolysaccharides that are measured with the Limulus assay, whereas the gas chromatographic methods allow determination of total concentrations of lipopolysaccharide, including Limulus-inactive lipopolysaccharide, gram-negative cells, and cellular debris.     

Quantification of ergosterol and 3-hydroxy fatty acids in settled house dust by gas chromatography-mass spectrometry: comparison with fungal culture and determination of endotoxin by a Limulus amebocyte lysate assay.

Ergosterol and 3-hydroxy fatty acids, chemical markers for fungal biomass and the endotoxin of gram-negative bacteria, respectively, may be useful in studies of health effects of organic dusts, including domestic house dust. This paper reports a method for the combined determination of ergosterol and 3-hydroxy fatty acids in a single dust sample and a comparison of these chemical biomarkers determined by gas chromatography-mass spectrometry with results from fungal culture and Limulus assay. Analyses of replicate house dust samples resulted in correlations of 0.91 (ergosterol in six replicates; P < 0.01) and 0.94 (3-hydroxy fatty acids in nine replicates; P < 0.001). The amounts of ergosterol (range, 2 to 16.5 ng/mg of dust) correlated with those of total culturable fungi (range, 6 to 1,400 CFU/mg of dust) in 17 samples, (r = 0.65; P < 0.005). The amounts of endotoxin (range, 11 to 243 endotoxin units/mg of dust) measured with a modified chromogenic Limulus assay correlated with those of lipopolysaccharide (LPS) determined from 3-hydroxy fatty acid analysis of 15 samples. The correlation coefficient depended on the chain lengths of 3-hydroxy acids used to compute the LPS content. The correlation was high (r = 0.88 +/- 0.01; P < 0.001) when fatty acid chains of 10 to 14 carbon atoms were included; the correlation was much lower when hydroxy acids of 16- or 18-carbon chains were included. In conclusion, the results of the described extraction and analysis procedure for ergosterol and 3-hydroxy fatty acids are reproducible, and the results can be correlated with fungal culture and endotoxin activity of organic dust samples.     

Sodium-Dependent Growth of Azotobacter chroococcum

Selected ion-monitoring gas chromatography-mass spectrometry was used for detection of beta-hydroxy fatty acids as an independent assay for the presence or absence of endotoxin in materials claimed to induce nonspecific activation of Limulus amoebocyte lysate. To this end, suspensions of gram-negative and -positive bacteria, one fungal species, cerebrospinal fluid, and hollow-fiber hemodialyzer rinses were assayed for endotoxin by gas chromatography-mass spectrometry and the Limulus amoebocyte lysate assay. Good qualitative agreement was shown for both methods when suspensions of test organisms were assayed. Two false-negative results were obtained by gas chromatography-mass spectrometry assays of cerebrospinal fluid and were shown to be a result of insufficient endotoxin in the cerebrospinal fluid specimens for detection by gas chromatography-mass spectrometry. Hemodialyzer rinses were Limulus assay positive; however, no beta-hydroxy fatty acids were detected by gas chromatography-mass spectrometry. These data were compared with data obtained from USP rabbit pyrogen tests of the rinse materials (nonpyrogenic) and chemical characterization of the Limulus assay-reactive rinses, which showed the rinses to be cellulosic in nature. It is suggested that beta-hydroxy fatty acids, as assayed by selected ion-monitoring gas chromatography-mass spectrometry, be used as chemical marker molecules for the presence or absence of endotoxin in materials reported to cause nonspecific activation of Limulus amoebocyte lysate.     

Sodium-Dependent Growth of Azotobacter chroococcum

Selected ion-monitoring gas chromatography-mass spectrometry was used for detection of beta-hydroxy fatty acids as an independent assay for the presence or absence of endotoxin in materials claimed to induce nonspecific activation of Limulus amoebocyte lysate. To this end, suspensions of gram-negative and -positive bacteria, one fungal species, cerebrospinal fluid, and hollow-fiber hemodialyzer rinses were assayed for endotoxin by gas chromatography-mass spectrometry and the Limulus amoebocyte lysate assay. Good qualitative agreement was shown for both methods when suspensions of test organisms were assayed. Two false-negative results were obtained by gas chromatography-mass spectrometry assays of cerebrospinal fluid and were shown to be a result of insufficient endotoxin in the cerebrospinal fluid specimens for detection by gas chromatography-mass spectrometry. Hemodialyzer rinses were Limulus assay positive; however, no beta-hydroxy fatty acids were detected by gas chromatography-mass spectrometry. These data were compared with data obtained from USP rabbit pyrogen tests of the rinse materials (nonpyrogenic) and chemical characterization of the Limulus assay-reactive rinses, which showed the rinses to be cellulosic in nature. It is suggested that beta-hydroxy fatty acids, as assayed by selected ion-monitoring gas chromatography-mass spectrometry, be used as chemical marker molecules for the presence or absence of endotoxin in materials reported to cause nonspecific activation of Limulus amoebocyte lysate.     

Use of gas chromatography-mass spectrometry versus the limulus amebocyte lysate test for the determination of airborne endotoxin in confined swine buildings

Airborne endotoxin (lipopolysaccharide, LPS) in filter samples collected in three different swine confinement buildings was determined by using the Limulus amebocyte lysate test and by applying gas chromatography-mass spectrometry (GC-MS) to analyze 3-hydroxy fatty acids (3-OH-FAs). The amounts of LPS as shown by GC-MS were 30–50 times larger than the amounts detected by the Limulus test. GC-MS revealed that 21% of the LPS collected on cellulose acetate filters and 26% on polycarbonate filters remained on the filters after buffer extraction. Better correlation with the Limulus test and the 3-OH-FA measurements was achieved when considering the sum of 3-OH C12:0 and 3-OH C14:0 rather than the sum of all of the detected 3-OH-FAs (i.e. those with 12–18-carbon chains), indicating that the bioactivity of the LPS was dependent upon the relative distribution of the 3-OH-FAs. Linear regression analysis between air concentrations of dust and endotoxin/LPS gaveR ²values that varied from 0.407 to 0.739. The air concentrations of LPS were lower in the uninsulated swine house (a green house with an alternative housing system) than in the two insulated buildings (conventional housing systems; one designed as a climate chamber, i.e. climatic parameters could be controlled), whereas the opposite was found for the concentrations of LPS in airborne dust from the three buildings. The numbers of viable bacteria and fungi were highest in the uninsulated swine house. Moreover the relative distribution of 3-OH-FAs in that building differed from the distribution in the insulated buildings, which reflects differences in the microflora, probably mainly due to differences between the housing systems and the design of the buildings.     

Characterization of lipopolysaccharides present in settled house dust

The 3-hydroxy fatty acids (3-OHFAs) in lipopolysaccharides (LPS) play an important role in determining endotoxin activity, and childhood exposure to endotoxin has recently been associated with reduced risk of atopic diseases. To characterize the 3-OHFAs in house dust (HD), we used gas chromatography-mass spectrometry to assay 190 HD samples. Dust from beds, bedroom floors, family rooms, and kitchen floors was collected as part of a birth cohort study of childhood asthma (study 1) and a longitudinal study of home allergen and endotoxin (study 2). We also measured endotoxin activity with a Limulus assay and computed specific activity (endotoxin activity per nanomole of LPS). Longer-chain (C(16:0) and C(18:0)) 3-OHFAs were predominant in HD compared with short-chain (C(10:0), C(12:0), and C(14:0)) acids. Endotoxin activity was positively correlated with short-chain 3-OHFAs in both studies. In study 2, 3-OH C(16:0) was negatively correlated and 3-OH C(18:0) was not correlated with endotoxin activity, consistent with previous findings that the Limulus assay responds preferentially to LPS containing short-chain 3-OHFAs. Kitchen dust contained the highest concentrations of 3-OH C(10:0), the highest endotoxin activities, and the highest specific activities (P < 0.03). Bed dust contained the largest amounts of long-chain 3-OHFAs, the highest concentrations of LPS, and the lowest specific activities. Apartments had significantly different types of LPS (P = 0.03) compared with single-family homes in study 2. These data suggest that the Limulus assay may underestimate exposure to certain types of LPS. Because nontoxic LPS may have immune modulating effects, analysis of 3-OHFAs may be useful in epidemiologic studies.     

Monitoring of bacterial sugars and hydroxy Fatty acids in dust from air conditioners by gas chromatography-mass spectrometry

Bacterial levels in dust collected from hospital air-conditioning filters were examined by chemical analysis (without prior culture). The dust was analyzed by gas chromatography-mass spectrometry after hydrolysis and derivatization. l-Glycero-d-mannoheptose and hydroxy fatty acids (3-OH 12:0 and 3-OH 14:0) (primarily found in lipopolysaccharide) and muramic acid (a chemical marker for bacterial peptidoglycan) were present at higher levels in dust collected from filters primarily contacting outdoor (as opposed to indoor) air. The ratio of l-glycero-d-mannoheptose to muramic acid in dust (compared with those of a group of gram-positive and gram-negative bacteria) suggested that both dust types contained appreciable numbers of gram-negative bacteria. There is potential for the chemical assessment of the microbial content of airborne dust.     

Airborne environmental endotoxin: a cross-validation of sampling and analysis techniques.

A standard method for measurement of airborne environmental endotoxin was developed and field tested in a fiberglass insulation-manufacturing facility. This method involved sampling with a capillary-pore membrane filter, extraction in buffer using a sonication bath, and analysis by the kinetic-Limulus assay with resistant-parallel-line estimation (KLARE). Cross-validation of the extraction and assay method was performed by comparison with methanolysis of samples followed by 3-hydroxy fatty acid (3-OHFA) analysis by gas chromatography-mass spectrometry. Direct methanolysis of filter samples and methanolysis of buffer extracts of the filters yielded similar 3-OHFA content (P = 0.72); the average difference was 2.1%. Analysis of buffer extracts for endotoxin content by the KLARE method and by gas chromatography-mass spectrometry for 3-OHFA content produced similar results (P = 0.23); the average difference was 0.88%. The source of endotoxin was gram-negative bacteria growing in recycled washwater used to clean the insulation-manufacturing equipment. The endotoxin and bacteria become airborne during spray cleaning operations. The types of 3-OHFAs in bacteria cultured from the washwater, present in the washwater and in the air, were similar. Virtually all of the bacteria cultured from air and water were gram negative composed mostly of two species, Deleya aesta and Acinetobacter johnsonii. Airborne countable bacteria correlated well with endotoxin (r2 = 0.64). Replicate sampling showed that results with the standard sampling, extraction, and Limulus assay by the KLARE method were highly reproducible (95% confidence interval for endotoxin measurement +/- 0.28 log10). These results demonstrate the accuracy, precision, and sensitivity of the standard procedure proposed for airborne environmental endotoxin.     

Determination of MK-287, a new platelet-activating factor antagonist, in plasma and serum by gas chromatography chemical ionization mass spectrometry

MK-287 is a novel platelet-activating factor antagonist. A sensitive and specific gas chromatographic/mass spectrometric assay has been developed for the determination of the drug in serum and plasma. The assay utilizes an extraction with methyl-t-butyl ether and subsequent trimethylsilylation of the hydroxyl function. The gas chromatographic/mass spectrometric determinations are carried out with temperature-programmed capillary gas chromatography and ammonia negative chemical ionization mass spectrometry. The method has sufficient sensitivity, precision, accuracy and selectivity for the analysis of drug concentrations in clinical samples.     

Determination of 4-(4-chlorophenyl)-4-hydroxypiperidine, a metabolite of haloperidol, by gas chromatography with electron-capture detection

An electron-capture gas chromatographic procedure was developed for the analysis of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), a metabolite of haloperidol. The assay involved basic extraction of this metabolite from the biological samples, followed by back-extraction with HCl. After basification of the acid phase, extractive derivatization with pentafluorobenzoyl chloride in toluene was conducted. The pentafluorobenzoyl derivative was quantified on a gas chromatograph equipped with a fused-silica capillary column, an electron-capture detector and a printer-integrator. N-(3-Trifluoromethylphenyl)piperazine was carried through the procedure as an internal standard and calibration curves were determined for each assay run. The procedure was demonstrated to be linear and reproducible and was utilized to detect and quantify CPHP in urine, plasma, brain and liver samples from rats treated with haloperidol. The structure of the derivatized metabolite was confirmed by gas chromatography-mass spectrometry.     

Chromatographic analysis of lipoic acid and related compounds

The analysis of lipoic acid and related compounds, such as its reduced form dihydrolipoic acid, its amide form lipoamide and other analogues, in biological and food samples is important in biochemistry, nutritional and clinical chemistry. This review summarizes the chromatographic methods for the determination of lipoic acid and related compounds, and their applications to various samples such as bacteria, tissues, drugs and food. Gas chromatographic methods with flame ionization detection and flame photometric detection are commonly used for the quantification of lipoic acid present as its protein-bound form, after acid or base hydrolysis of these samples. High-performance liquid chromatographic methods with ultraviolet, fluorescence and electrochemical detection are mainly used for the determination of free lipoic acid and related compounds, such as dihydrolipoic acid, lipoamide and other analogues. Moreover, gas chromatography–mass spectrometry and capillary electrophoresis methods are also developed.     

Determination of muramic acid in organic dust by gas chromatography-mass spectrometry

A method is described for the quantitation of muramic acid, a marker of bacterial peptidoglycan, in organic dust. House dust samples were hydrolysed in hydrochloric acid and then extracted with hexane to remove hydrophobic compounds. The aqueous phase was evaporated, heated in a silylation reagent to form trimethylsilyl derivatives, and analysed by gas chromatography-mass spectrometry. The muramic acid derivative gave two peaks upon injection into the gas chromatograph-mass spectrometer. Injection of 10 pg of the derivative gave a signal-to-noise ratio of 17 for the dominating peak when using selected ion monitoring in the electron impact mode, and a linear calibration curve was achieved upon analysis of samples containing 5–1500 ng of muramic acid. In a house dust sample, 40 ng of muramic acid was found per mg of dust; the coefficient of variation was 8.2% (n = 6, 1.2 mg of dust analysed). The described method is rapid and simple to apply, and should therefore become widely used for measuring peptidoglycan in many types of environmental samples, including organic dust.     

Diagnostic methods for the determination of iduronic acid in oligosaccharides

A high-performance liquid chromatography (HPLC) method with pulsed amperometric detection (PAD) was used for the determination of the acid hydrolysis products of L-iduronic acid containing oligosaccharides isolated from biological sources. This HPLC-PAD method was compared with gas chromatographic (GLC) methods. Since acid hydrolysis of oligosaccharides can produce a number of products, several uronic acid derivatives were prepared by chemical synthesis. These well characterized standards in conjunction with mass spectrometry allowed for the identification of most of the products of methanolysis or hydrolysis of glycosamino-glycans, which included chondroitin sulfates A and B (dermatan sulfate), heparin, and hyaluronic acid. (4 M) HCl in methanol 100 degrees C for 24 h was found to be optimum for GLC and 1 M aqueous HCl for 4 h at 100 degrees C for HPLC-PAD. All of the monosaccharides, hexosamines, and uronic acids could be separately identified in a single chromatographic step using either technique. Good resolution, high sensitivity (low microgram samples) and rapid analysis makes these methods particularly useful for the determination of small amounts of glycosaminoglycans and other glycoconjugates found in samples isolated from biological sources. These two techniques are specifically designed to allow the qualitative determination of the carbohydrate content and composition of samples whose carbohydrate composition and content is completely unknown.     

Simultaneous radioenzymatic determination of plasma and tissue adrenaline,noradrenaline and dopamine within the femtomole range

A radiometric-enzymatic assay for measuring simultaneously femtomole quantities of adrenaline, noradrenaline and dopamine has been developed. The three catecholamines are first converted to their O-methylated analogues by catechol-O-methyltransferase in the presence of S-adenosyl-methionine-³H and thereafter extracted following addition of sodium tetraphenylborate. This extraction, together with an improved quick chromatographic separation and the oxidation of the adrenaline and noradrenaline derivatives to vanillin, yields an extremely high sensitivity and specificity of the method.The present assay allows the determination of adrenaline, noradrenaline and dopamine in tissue samples with a protein content of 100 μg or less and in plasma volumes of 20 – 100 μl. The amine content of 40 – 50 samples can be determined in two days by one person.Due to the high sensitivity achieved, this method promises to be a valid alternative to the gas chromatography-mass spectrometry technique.     

Effect of applied and endogenous indol-3-yl-acetic acid on maize root growth

The level of endogenous Indol-3-yl-acetic acid (IAA) measured by gas chromatography-mass spectrometry in the elongating zone of intact primary roots of Zea mays showed a good linear correlation with the growth rate of these roots. When they were treated with IAA, their relative elongation decreased; this indicates a supraoptimal content of endogenous IAA. However, the growth of some of the relatively rapidly extending roots was enhanced by such treatment. Interactions between endogenous and applied IAA in the control of root growth are discussed.Abbreviations GC-MS gas chromatography-mass spectrometry - IAA Indol-3-yl-acetic acid     

Galactonate determination in urine by stable isotope dilution gas chromatography-mass spectrometry

A stable isotope dilution assay was developed for the sensitive determination of D-galactonic acid. D-[U-13C(6)]galactono-1,4-lactone was prepared as internal standard. Unlabelled and U-13C-labelled D-galactonic acid species were converted to the N-(1-butyl)galactonamide pentaacetate derivatives and assessed by gas chromatography-mass spectrometry (GC-MS). Positive chemical ionisation and monitoring of the [MH-60](+)-ions in the galactonate chromatographic peak at m/z 402 and m/z 408 were used for quantification. The procedure was applied to study the variability of D-galactonate excretion in healthy subjects and galactosemic patients and to monitor the D-galactonate-D-galactitol ratio in human urine.     

Chromatographic separation of 2,3-benzodiazepines

A review of chromatographic methods for the determination of 2,3-benzodiazepines (2,3-BZs) is presented. The determinations are performed to investigate the presence of potential impurities in drug substances and to study their pharmacokinetic profile in biological samples, either in animals or in humans. Several methods dealt with a pretreatment of samples, i.e., liquid–liquid extraction by using a variety of solvents, solid-phase extraction, direct injection of specimens into the chromatographic apparatus. Different chromatographic techniques have been used. High-performance liquid chromatography allows optimal sensitivity and specificity by using ultraviolet or diode array detection methods. Gas chromatography-mass spectrometry and gas chromatography with nitrogen-phosphorous or electron-capture detectors have been also reported. Suitable methods for the separation of enantiomers of 2,3-BZs have been described. Thin-layer chromatography has been shown to be capable to isolate analytes from biological samples as urine or faeces. The reported chromatographic techniques are currently applied to define the metabolic pathways of 2,3-BZs in experimental and clinical studies.     

Isolation of 9-hydroxy-δ-tetradecalactone from lipid A of Pseudomonas diminuta and Pseudomonas vesicularis

Abstract A lipid component was isolated from the fatty acid fraction of acid hydrolysates of lipid A derived from Pseudomonas diminuta JCM 2788 and Pseudomonas vesicularis JCM 1477 lipopolysaccharide. By structural analysis of the lipid and its trimethylsilyl and acetyl derivatives by thin-layer chromatography, gas chromatography-mass spectrometry, mass spectrometry, infrared spectrometry and ¹³C-NMR, it was identified as 9-hydroxy-δ-tetradecalactone.     

Determination of glyphosate, glyphosate metabolites, and glufosinate in human serum by gas chromatography-mass spectrometry

This paper describes an assay for the determination of glyphosate (GLYP), glyphosate metabolites [(aminomethyl) phosphonic acid] (AMPA), and glufosinate (GLUF) in human serum. After protein precipitation using acetonitrile and solid-phase extraction, serum samples were derivatized and analyzed by gas chromatography-mass spectrometry (GC-MS). The assay was linear over a concentration range of 3-100.0 microg/ml for GLYP, AMPA, and GLUF. The overall recoveries for the three compounds were >73%. The intra- and inter-day variations were <15%. Precision and accuracy were 6.4-10.6% and 88.2-103.7%, respectively. The validated method was applied to quantify the GLYP and AMPA content in the serum of a GLYP-poisoned patient. In conclusion, the method was successfully applied for the determination of GLYP and its metabolite AMPA in serum obtained from patient of GLYP-poisoning.     

人的粪便、血浆、唾液、呼出气体中短链脂肪酸的分析

目的建立一种顶空气相色谱-串联质谱法(HS-GC/MS)快速检测人的粪便、血浆、唾液、呼出气体中短链脂肪酸(SCFAs)的方法,初步探索人的粪便、血浆、唾液、呼出气体中短链脂肪酸的相关性。方法样品无需处理直接封存于顶空进样瓶中,顶空进样;采用DB-FFAP毛细管柱(30 m×0.25 mm×0.25μm)分离;全扫描模式检测。结果人的粪便、血浆、唾液、呼出气体中均含有短链脂肪酸。在人的粪便、唾液样本中均检测到8个短链脂肪酸(乙酸、丙酸、异丁酸、丁酸、异戊酸、戊酸、异己酸、己酸);血浆、呼出气体样本中均检测到7个短链脂肪酸(未检测到异己酸)。结论初步推测人的粪便、血浆、唾液、呼出气体中的短链脂肪酸具有一定的相关性。本方法简单、快速、灵敏,可用于人的生物样品中短链脂肪酸的快速检测。