Ethanol analysis from biological samples by dual rail robotic autosampler

Detection, identification, and quantitation of ethanol and other low molecular weight volatile compounds in liquid matrices by headspace gas chromatography-flame ionization detection (HS-GC-FID) and headspace gas chromatography-mass spectrometry (HS-GC-MS) are becoming commonly used practices in forensic laboratories. Although it is one of the most frequently utilized procedures, sample preparation is usually done manually. Implementing the use of a dual-rail, programmable autosampler can minimize many of the manual steps in sample preparation. The autosampler is configured so that one rail is used for sample preparation and the other rail is used as a traditional autosampler for sample introduction into the gas chromatograph inlet. The sample preparation rail draws up and sequentially adds a saturated sodium chloride solution and internal standard (0.08%, w/v acetonitrile) to a headspace vial containing a biological sample, a calibrator, or a control. Then, the analytical rail moves the sample to the agitator for incubation, followed by sampling of the headspace for analysis. Using DB-624 capillary columns, the method was validated on a GC-FID and confirmed with a GC-MS. The analytes (ethanol, acetonitrile) and possible interferences (acetaldehyde, methanol, pentane, diethyl ether, acetone, isopropanol, methylene chloride, n-propanol, and isovaleraldehyde) were baseline resolved for both the GC-FID and GC-MS methods. This method demonstrated acceptable linearity from 0 to 1500 mg/dL. The lower limit of quantitation (LOQ) was determined to be 17 mg/dL and the limit of detection was 5 mg/dL.     

Proposal of an analytical method for the study of the oxidation products of membrane lipids

A method of lipid sample preparation for GLC and GC-MS analysis is presented which would seem particularly suitable for studying the chemical composition of the oxidation products of membrane lipids. The method requires small amounts of lipid, is quite rapid and avoids the formation of oxidation artifacts during the different analytical steps. Due to the small quantities of lipid material used it is possible that the method is not rigorously quantitative. Transmethylation of lipids is carried out with methanolic NaBH4 in the presence of NaOH. The reaction is complete in 20 min both on neutral and on polar lipids. In the course of the transmethylation the hydroperoxidic groups are reduced to the corresponding hydroxy groups and can be located through the GC-MS spectra of the corresponding TMS derivatives. The epoxidic rings that may be present are not hydrolyzed. They are located by opening the ring with BF3/MeOH and by GC-MS analysis of the corresponding methoxy-hydroxy derivatives.     

Analysis of a novel antiinflammatory agent, 1-(7-tert.-butyl-2,3-dihydro-3,3-dimethylbenzo[b]furan-5-yl)-4-cyclopropylbutan-1-one (PGV-20229), in plasma matrices by stable-isotope-dilution gas chromatography-mass spectrometry

A sensitive and selective GC-MS method was developed for the determination of low levels of a novel antiinflammatory agent, 1-(7-tert.-butyl-2,3-dihydro-3,3-dimethylbenzo[b]furan-5-yl)-4-cyclopropylbutan-1-one (I), in small volumes of animal plasma. The method involved the addition of ¹³C₆-labeled-I to plasma samples, followed by a simple liquid-liquid extraction with hexane to isolate the analytes from matrix components. The levels of I in the sample extracts were determined by isotope-dilution GC-MS analysis using selected-ion monitoring. The method was linear over three orders of magnitude, with a limit of quantitation of 1.8 ng/ml I, using plasma sample volumes of 0.1 ml. The method was utilized to determine the pharmacokinetic parameters of I in rats and dogs, following intravenous administration.     

Acetic acid improves the sensitivity of theophylline analysis by gas chromatography-mass spectrometry

In the analysis of theophylline by gas chromatography-mass spectrometry (GC-MS), we found that the addition of acetic acid to the solvent (ethyl acetate) decreased the adsorption of theophylline to the glass wool packed into the inlet liner. The addition of acetic acid to ethyl acetate improved the sensitivity for theophylline (optimum concentration of 3%). This simple and sensitive method without derivatization can be applied to the quantification of theophylline in serum samples in clinical and toxicological practice.     

Simple and sensitive determination of free and total morphine in human liver and kidney using gas chromatography-mass spectrometry

We developed a reliable, simple and sensitive method to determine free and total morphine in human liver and kidney, using gas chromatography-mass spectrometry (GC-MS). Free morphine or total morphine obtained by acid hydrolysis from 0.2g tissue sample was extracted using an Extrelut NT column with an internal standard, dihydrocodeine, followed by trimethylsilylation. The derivatized extract was submitted to GC-MS analysis of EI-SIM mode. The calibration curves of morphine in both liver and kidney samples were linear in the concentration range from 0.005 to 5 microg/g. The lower limits of detection of morphine were 0.005 microg/g. This method proved successful when we determined free and total morphine in liver and kidney obtained from an autopsied man who was mis-ingested morphine compound in the hospital, which resulted in the cause of death being morphine intoxication.     

Affinity chromatography of bull seminal proteins on mannan-Sepharose

4-Hydroxynon-2-enal (4-HNE) is one of the major aldehydic products of lipid peroxidation (LPO) and is involved in a number of pathophysiological processes. Since LPO products are useful indicators for oxidative stress in vivo, a number of detection methods for LPO products in biological tissues were developed. However, none of these methods is presently used in clinical settings. In order to introduce LPO products as biomarkers in clinical studies a suitable GC-MS method for 4-HNE detection was adapted to meet clinical requirements. As one result, the minimal sample volume could be decreased to 50 microl of plasma so that the method might even be suitable for pediatric purposes. The best internal standard (I.S.) for 4-HNE detection by GC-MS 9,9,9-D(3)-4-hydroxynon-2-enal was introduced by van Kuijk et al. [Anal. Biochem., 224 (1995) 420]. However, because of its limited availability, benzaldehyde-ring-d(5), 4-hydroxybenzaldehyde, and 2,5-dihydroxybenzaldehyde were tested to find an alternative. Out of these three, 4-hydroxybenzaldehyde was shown to serve best as I.S. To examine the applicability of the adapted method, tests on the stability of 4-HNE in samples during storage were carried out. It was shown that plasma samples need to be stored at -80 degrees C or less to avoid greater loss of 4-HNE. Samples with 4-HNE concentrations close to the physiological level were shown to be stable over 22 months at -80 degrees C. The introduction of a new and easily available I.S., reduction of the sample volume, and information about sample stability provided by this study facilitate 4-HNE determination in most clinical settings.     

A microbead array chemical sensor using capillary-based sample introduction: toward the development of an "electronic tongue"

The development of a micromachined fluidic structure for the introduction of liquid samples into a chip-based sensor array composed of individually addressable polymeric microbeads is presented. The micromachined structure consists of micromachined storage cavities combined with a covering glass layer that confines the microbeads and fluidic channels. In our sensor array transduction occurs via optical (colorimetric and fluorescence) changes to receptors and indicator molecules that are covalently attached to termination sites on the polymeric microbeads. Spectral data are acquired for each of the individual microbeads using a charged-coupled device (CCD) allowing for the near-real-time analysis of liquid sample. Hence the micromachined fluidic structure must allow for both optical access to the microbeads and fluid flow through the micromachined cavities that serve as the microreactors/analysis chambers. One of the key parts of the structure is a passive fluid introduction system driven only by capillary force. This simple means of fluid introduction realizes a compact device. The capillary flow on the inlet channel has been studied, and the responses of the microbeads (alizarin complexone) to a liquid sample have been characterized. The test results show that this system is useful in a micro-total-analysis-system (mu-TAS) and biomedical applications.     

Screening and semi-quantitative analysis of post mortem blood for basic drugs using gas chromatography/ion trap mass spectrometry

The study presented here shows that GC-MS with ion trap detection can be used for screening post mortem blood. The method described was used to simultaneously screen for unknowns, identify basic drugs present and semi-quantitate 14 drugs commonly encountered in coroner's toxicology (i.e. was used to determine whether the drugs were present in sub-therapeutic, therapeutic or greater than therapeutic amounts). The equipment used included a Varian Saturn 2000 GC-MS operating in full scan mode, a CP-3800 GC, a CP-8400 autosampler and Saturn GC-MS workstation Version 5.5 software. Post mortem blood samples were extracted using a standard liquid-liquid procedure; diethylether followed by back extraction into 0.1 M HCl. Standard curves for the 14 drugs which were semi-quantitated (amitriptyline, citalopram, clozapine, cocaine, cyclizine, diazepam, dihydrocodeine, dothiepin, methadone, mirtazapine, procyclidine, sertraline, tramadol, venlafaxine) were prepared covering the concentration range 0-1.0 ug/mL. The procedure is in routine use for coroners toxicology; semi-quantitation has been used (i) to speed-up the through put of cases where drugs are an incidental finding and (ii) for cases where the amount of sample submitted for analysis was too small to allow for screening, identification and quantitation on separate sample volumes.     

Rapid quantification of major reaction products formed during thermochemical pretreatment of lignocellulosic biomass using GC-MS

Accurate quantification of reaction products formed during thermochemical pretreatment of lignocellulosic biomass would lead to a better understanding of plant cell wall deconstruction for production of cellulosic biofuels and biochemicals. However, quantification of some process byproducts, most notably acetamide, acetic acid and furfural, present several analytical challenges using conventional liquid chromatography methods. Therefore, we have developed a high-throughput gas chromatography based mass spectrometric (GC-MS) method in order to quantify relevant compounds without requiring time-consuming sample derivatization prior to analysis. Solvent extracts of untreated, ammonia fiber expansion (AFEX) treated and dilute-acid treated corn stover were analyzed by this method. Biomass samples were extracted with acetone using an automated solvent extractor, serially diluted and directly analyzed using the proposed GC-MS method. Acetone was the only solvent amongst water, methanol and acetonitrile that did not contain detectable background levels of the target compounds or facilitate a buildup of plant-derived residues in the GC injector, which decreased analytical reproducibility. Quantitative results were based on the method of standard addition and external standard calibration curves.     

Gas chromatography-mass spectrometric analysis of hexanal and heptanal in human blood by headspace single-drop microextraction with droplet derivatization

In this work, we developed a new approach to the analysis of the lung cancer biomarkers, hexanal and heptanal in human blood that was based on headspace single-drop microextraction (HS-SDME) with droplet derivatization, followed by gas chromatography-mass spectrometry (GC-MS). Aldehydes in blood were headspace extracted, concentrated, and derivatized by a suspended microdrop solvent containing the derivatization agent O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride. The aldehyde oximes formed in the microdrop solvent were analyzed by GC-MS. The optimal HS-SDME with droplet derivatization parameters extraction solvent of decane, sample temperature of 40 degrees C, extraction time of 6 min, stirring rate of 1100 rpm, and solvent volume of 2.0 microL were obtained and used for analysis of hexanal and heptanal in blood. The method reproducibility, linearity, recovery, and detection limit were studied and the obtained results demonstrated the method feasibility. Finally, the proposed method was applied to the quantification of hexanal and heptanal in cancer blood and normal blood. Due to sample extraction, concentration, and derivatization being performed in a single step, the method provided a simple, rapid, low-cost, and efficient approach to analysis of aldehydes in blood samples.     

Predictive metabolite profiling applying hierarchical multivariate curve resolution to GC-MS data--a potential tool for multi-parametric diagnosis

A method for predictive metabolite profiling based on resolution of GC-MS data followed by multivariate data analysis is presented and applied to three different biofluid data sets (rat urine, aspen leaf extracts, and human blood plasma). Hierarchical multivariate curve resolution (H-MCR) was used to simultaneously resolve the GC-MS data into pure profiles, describing the relative metabolite concentrations between samples, for multivariate analysis. Here, we present an extension of the H-MCR method allowing treatment of independent samples according to processing parameters estimated from a set of training samples. Predictions or inclusion of the new samples, based on their metabolite profiles, into an existing model could then be carried out, which is a requirement for a working application within, e.g., clinical diagnosis. Apart from allowing treatment and prediction of independent samples the proposed method also reduces the time for the curve resolution process since only a subset of representative samples have to be processed while the remaining samples can be treated according to the obtained processing parameters. The time required for resolving the 30 training samples in the rat urine example was approximately 13 h, while the treatment of the 30 test samples according to the training parameters required only approximately 30 s per sample (approximately 15 min in total). In addition, the presented results show that the suggested approach works for describing metabolic changes in different biofluids, indicating that this is a general approach for high-throughput predictive metabolite profiling, which could have important applications in areas such as plant functional genomics, drug toxicity, treatment efficacy and early disease diagnosis.     

A rapid gas chromatography-mass spectrometry method for quantification of alkylresorcinols in human plasma

Alkylresorcinols (ARs) are phenolic lipids that among foods are found almost exclusively in whole grain and bran products based on wheat and rye. They have been suggested to be used as selective biomarkers of whole grain wheat and rye intake and, thus, may serve as an alternative/complement to commonly used dietary assessment methods in epidemiological studies. For such investigations where analysis of large numbers of valuable samples is required, rapid, sensitive, and repeatable methods are essential. In this article, we describe a rapid and sensitive gas chromatography-mass spectrometry (GC-MS) method for quantification of AR homologues C17:0, C19:0, C21:0, C23:0, and C25:0 in human plasma. The method uses Oasis MAX solid phase extraction cartridges for sample cleanup. A plasma sample of 0.2 ml could be used without preincubation with water. Samples in the range of 7 to 8750 nmol total AR/L were successfully analyzed with the method described, and the average total AR recovery within the reported range was 92 ± 12%. The within- and between-day precision values of total AR concentration in a quality control sample, determined as the coefficients of variation, were on average 7 and 10%, respectively. Approximately 30 to 50 samples could be analyzed within 1 day. The improved GC-MS method presented can be used for rapid analysis of ARs in relatively small sample volumes.     

Ultrasonic-assisted derivatization reaction of amino acids prior to their determination in urine by using single-drop microextraction in conjunction with gas chromatography

A derivatization-extraction method that avoids tedious preconcentration steps is established in order to determine amino acids accurately at nanogram levels. The method involves conversion of the analytes of concern to N(O,S)-ethoxycarbonyl amino acid ethyl esters and subsequent extraction by single-drop microextraction (SDME) followed by GC analysis. The reaction proceeds smoothly and rapidly under ultrasonication which removes the bubbles from the bulk solution. Precision is acceptable and 12 non-hydrolyzed amino acids can be determined in urine in this manner. As long as the extraction conditions are consistently applied, quantitative analysis can be performed accurately. The limits of detection were satisfactory in the range 0.010-0.025 microg/ml for GC-FID and 0.26-68 ng/ml for GC-MS(SIM) with 1 ml sample volume.     

Identification and quantification of methyl jasmonate in leaf volatiles of Arabidopsis thaliana using solid-phase microextraction in combination with gas chromatography and mass spectrometry

Static headspace sampling with solid-phase microextraction has been used in combination with GC-FID and GC-MS for the specific enrichment, identification and quantification of volatile methyl jasmonate secreted by wounded leaves of Arabidopsis thaliana. The microsample method of analysis was found to be precise, accurate, sensitive and rapid. The detection limit of the procedure is 1.5 ppb (approximately 1.3 ng) per injection, which is of adequate sensitivity to detect the natural baseline levels of methyl jasmonate (approximately 10-100 ng/g) present in plant tissues. The method can be applied to most plants, requires a minimum of sample material, and shows the additional advantage that it is suitable for automation and could thus be used for high-throughput screening.     

Rapid and sensitive method for prenatal diagnosis of propionic acidemia using stable isotope dilution gas chromatography-mass spectrometry and urease pretreatment

Propionic acidemia is one of the most frequent inborn errors of metabolism caused by a deficiency of propionyl-CoA carboxylase. Methylcitric acid, a key indicator of this disorder, is increased in amniotic fluid when a fetus is affected. Therefore, the direct chemical analysis of cell-free amniotic fluid for methylcitric acid, using stable isotope dilution gas chromatography-mass spectrometry, was carried out for the prenatal diagnosis of propionic acidemia. We developed a simple, highly sensitive, and accurate method for quantitation of this polar methylcitric acid in amniotic fluids by applying a simplified urease pretreatment which we devised earlier for urine. As the recovery of methylcitric acid from amniotic fluid was as high as 91% with a coefficient of variation lower than 3% in this procedure, only 0.02 ml of sample was required for the analysis of the affected fetus. This new procedure takes 1 h for sample pretreatment, including derivatization, and 15 min for GC-MS measurement and provides final results within 1.5 h.     

Improved detection of polyunsaturated fatty acids as phenacyl esters using liquid chromatography-ion trap mass spectrometry

The fatty acid composition of Shewanella pealeana was determined by the analysis of fatty acid methyl esters via gas chromatography-mass spectrometry (GC-MS) and fatty acid 2-oxo-phenylethyl esters via high-performance liquid chromatography-mass spectrometry (LC-MS) combined with ultra violet (UV) detection. There was good agreement between the percentage composition of components determined by GC-MS and LC-UV analyses. However, LC-MS analysis using Atmospheric Pressure Chemical Ionization (APCI) demonstrated dramatically enhanced detection of unsaturated fatty acid 2-oxo-phenylethyl esters. The degree of enhancement was proportional to the degree of unsaturation. Tests with a pure polyunsaturated fatty acid (PUFA) standard gave an absolute detection limit in full scan mode of 200 pg. In samples, the selectivity of MS over UV gave a significantly lower detection limit due to lack of chemical interferences. In 'Selected Reaction Monitoring' (SRM) mode, the detection limit was 5 pg. This was essentially independent of whether the sample is a standard or complex mixture of fatty acids. Tandem mass spectrometry was used to support structural information and to enhance the ability to target specific fatty acids. Several PUFAs which were not evident from GC-MS analysis were detected and identified by APCI LC-MS, including some rare or novel PUFAs from S. pealeana and a menhaden oil standard. Detailed analysis of bacterial fatty acid composition by either GC-MS or APCI LC-MS is highly preferable to analysis systems based solely on retention time identification.     

A micro-technique for preparing homogenates of biological samples

A method is described for preparing homogenates of insects, mites, nematodes, or fungal spores for electrophoretic analysis which overcomes the disadvantages of existing methods. It is also applicable to the homogenization of microsamples of animal tissue. The method involves grinding the samples in glass tubes, closed at one end, prepared from melting-point capillaries. A range of plungers is fabricated from the stainless-steel plungers from broken microsyringes, or from stainless-steel wire. The plunger is operated by hand to homogenize a sample in 2-20 microliter of buffer in a tube. The techniques for handling the samples before and after homogenization and for labeling and centrifuging them are described. Compared with existing methods, the procedure minimizes sample loss and risk of cross-contamination and eliminates the possibility of overheating the sample during homogenization.     

气相色谱-质谱联用技术及其在代谢组学中的应用

代谢组学是以高通量、高灵敏度、高分辨率的现代仪器分析方法为手段,对细胞、体液、组织中所有代谢物进行无偏向的定性与定量分析的一门学科。气相色谱-质谱联用技术具有较高的检测灵敏度和鉴定准确度,通过标准谱图库的比对可对代谢物进行快速的鉴定,因此被广泛应用于生物样品的代谢产物的检测中。文中对近年来气相色谱-质谱联用技术的发展以及在代谢组学研究中取得的成果进行了综述。首先介绍了气相色谱-质谱联用技术的分类和常用的样品衍生化方法;继而从样品预处理、定性与定量分析、数据分析三方面介绍了气相色谱-质谱联用技术分析代谢物的方法,并系统地对该技术在微生物、植物、疾病诊断领域的应用实例进行了评述;最后提出了当前气相色谱-质谱联用技术在代谢组学研究中存在的问题并对后续的研究进行了展望。     

Accurate mass measurement using multiple sprayer nano-electrospray mass spectrometry combined with nano-scale high-performance liquid chromatography on a magnetic sector instrument

A new technique for accurate mass measurement utilizing multiple sprayer nano-electrospray ionization mass spectrometry (nano-ESI-MS) combined with nano-scale high-performance liquid chromatography (nano-HPLC) on a magnetic sector instrument is described. Both metal-coated glass capillaries and fused-silica capillaries were used as nano-ESI sprayers. A metal-coated glass capillary was used for the introduction of the Ref. compound solution, and a metal-coated fused-silica capillary was used for connection to the nano-HPLC column. By shifting each sprayer's position relative to the sampling orifice, spectra were obtained of both the sample components as eluted from the column and reference compounds. Several standard compounds were examined and satisfactory accurate masses were obtained. Problems arising from differences in ionization efficiency between the sample and reference compounds were not observed.     

Gas chromatography-mass spectrometry method for determination of phenylalanine and tyrosine in neonatal blood spots

In this paper we developed a simple, rapid and sensitive method for the quantitative analysis of phenylalanine (Phe) and tyrosine (Tyr) in dried blood spots of newborns by gas chromatography-mass spectrometry (GC-MS). Phe and Tyr in blood samples were reacted with N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide at 120 degrees C for 30 min and their corresponding single derivatives were obtained. Phe and Tyr were determined by measurement of their derivatives by GC-MS in the selected ion monitoring mode. Contents of Phe and Tyr in blood spots were calculated by external standard method. The ratio of Phe to Tyr was used for neonatal screening for phenylketonuria. The present method only took a few minutes to perform and required minimal sample preparation. In addition it provided low detection limits of 1.2 micromol l(-1) for Phe and 1.6 micromol l(-1)for Tyr.