Determination of 4-chloroindoleacetic acid methyl ester in Vicieae species by gas chromatography-mass spectrometry

The natural chlorinated auxin, 4-chloroindoleacetic acid methyl ester, was identified in immature seeds of Lathyrus sativus L., Lathyrus maritimus (L.) Bigel and Lathyrus odoratus L. by thin layer chromatography and gas chromatography-mass spectrometry. In immature seeds of Vicia sativa L. and Lens culinaris Medik. the hormone was identified by selected ion monitoring. The hormone was determined quantitatively using pentadeuterated 4-chloroindoleacetic acid methyl ester as internal standard. Contents varied from 1 mg/kg fresh weight in Lathyrus sativus to 0.02 mg/kg in Lens culinaris. Lathyrus maritimus also contained indoleacetic acid methyl ester (0.3 mg/kg) besides the chlorinated analogue.     

Analysis of trimethyl carboxyphosphate by gas chromatography-mass spectrometry

Analysis of trimethyl carboxyphosphate samples by gas chromatography-mass spectrometry, using typical conditions resulted in significant decomposition of the analyte. Optimization of injection conditions, including conditioning of the injection port liner, produced a dramatic increase in observed peak areas and afforded an effective method for detection of trimethyl carboxyphosphate at the <1μg mL⁻¹ level.     

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.     

Pyrolysis gas chromatography-mass spectrometry of mycobacterial mycolic acid methyl esters and its application to the identification of Mycobacterium leprae

Pyrolysis gas chromatography-mass spectrometry of methyl mycolates from 32 species of mycobacteria, including Mycobacterium leprae, was carried out. The mycobacteria could be classified into four groups in respect of the fatty acid ester patterns detected within the range C20 to C26. The applicability of this pyrolysis-gas chromatographic method for identifying M. leprae is discussed.     

Selected ion monitoring in quantitative gas-liquid chromatographic-mass spectrometric detection of fatty acid methyl esters from environmental samples

To calculate selected ion monitoring (SIM) gas-liquid chromatography (GLC)-mass spectrometry (MS) results of phospholipid fatty acids (PLFAs) from environmental samples, coefficients were calculated for each fatty acid by dividing the sum of ion intensities in SCAN with that of ions followed in SIM. The SIM chromatogram areas were multiplied with the coefficients, and then processed as in SCAN. The results were compared to those obtained using calibration curves and SCAN. The calibration curve and coefficient based results had the greatest errors of 7.8 and 6.7%, respectively, outside standard deviations of SCAN percentages. The PLFA contents calculated using calibration curves and coefficients were 104.9+/-7.3% and 101.5+/-8.6%, respectively, of SCAN values. SIM increased sensitivity approximately 10-fold from SCAN, and the smallest detectable injected amount was approximately 50 ng (0.18 nmol) for 20 fatty acids, corresponding to 4 x 10(6) cells.     

Determination of dihydroetorphine in biological fluids by gas chromatography-mass spectrometry using selected-ion monitoring

A method for the determination of dihydroetorphine hydrochloride, a powerful anaesthetic and analgesic drug, in biological fluids by GC-MS with selected-ion monitoring using etorphine as internal standard was established. Dihydroetorphine was extracted from human blood and urine with dichloromethane and then derivatized with N-heptafluorobutyrylimidazole after concentration to dryness. A dihydroetorphine monoheptafluorobutyl derivative was formed which showed good behavior on GC-MS with electronic-impact ionization. The main fragment, m/z 522, which is the base peak, was selected as the ion for quantitation and the corresponding ion, m/z 520, was selected for monitoring the internal standard, etorphine. The recoveries and coefficients of variation of the whole procedure were determined with five controlled dihydroetorphine-free urine and plasma samples spiked with different concentrations of dihydroetorphine. The concentration of dihydroetorphine for quantitation was in the range 1–20 ng/ml for urine and 2.5–250 ng/ml for plasma. The correlation coefficients of the standard curves are sufficient to determine the dihydroetorphine. The accuracy for quantitation of dihydroetorphine in urine and plasma is less than 10.6%.     

Determination of urinary trans,trans-muconic acid by gas chromatography-mass spectrometry

A sensitive and specific method for the determination of trans,trans-muconic acid (t,t-MA) in urine is described. After clean-up on an anion-exchange cartridge, t,t-MA was derivatized with BF₃-methanol to the dimethyl ester and analyzed by gas chromatography-mass spectrometry (GC-MS), with 2-bromohexanoic acid as an internal standard. The limit of detection was 0.01 mg/l, the coefficient of variation for duplicate analysis in a series of urine samples (n = 50) was 2.6% and the recovery rate ranged from 93.3 to 106.3%. The between-day and within-day precision for the analysis were 7.4 and 14.6%, respectively. The method was applied to the determination of t,t-MA in urine samples from smokers and non-smokers. The mean concentration of t,t-MA in urine of 10 smokers was 0.09 ± 0.04 mg/g creatinine and was significantly (p = 0.012) higher than that found in urine of 10 non-smokers (0.05 ± 0.02 mg/g creatinine). In contrast to the results obtained with the commonly used high-performance liquid chromatographic ultraviolet detection (HPLC-UV) methods, no interference between t,t-MA and other urinary compounds was found. This GC-MS method is both specific and sensitive for biomonitoring of low environmental benzene exposure.     

Determination of ajulemic acid and its glucuronide in human plasma by gas chromatography-mass spectrometry

A method using gas chromatography-mass spectrometry (GC-MS) and solid-phase extraction (SPE) was developed for the determination of ajulemic acid (AJA), a non-psychoactive synthetic cannabinoid with interesting therapeutic potential, in human plasma. When using two calibration graphs, the assay linearity ranged from 10 to 750 ng/ml, and 750 to 3000 ng/ml AJA. The intra- and inter-day precision (R.S.D., %), assessed across the linear ranges of the assay, was between 1.5 and 7.0, and 3.6 and 7.9, respectively. The limit of quantitation (LOQ) was 10 ng/ml. The amount of AJA glucuronide was determined by calculating the difference in the AJA concentration before ("free AJA") and after enzymatic hydrolysis ("total AJA"). The present method was used within a clinical study on 21 patients suffering from neuropathic pain with hyperalgesia and allodynia. For example, plasma levels of 599.4+/-37.2 ng/ml (mean+/-R.S.D., n=9) AJA were obtained for samples taken 2 h after the administration of an oral dose of 20 mg AJA. The mean AJA glucuronide concentration at 2h was 63.8+/-127.9 ng/ml.     

Rapid determination of para-phenylenediamine by gas chromatography-mass spectrometry with selected ion monitoring in henna-containing cosmetic products

A rapid method for the determination of para-phenylenediamine (PPD) in cosmetic products, such as henna tattoos has been developed and evaluated. This analytical procedure involved extracting a 10mg test portion of cosmetic product in 10 mL of ethyl acetate, followed by determination by gas chromatography-mass spectrometry in the selected ion monitoring mode (GC/MS-SIM). 1,4-Phenylenediamine-2,3,5,6-d(4) was selected as an internal standard that was added at the beginning of the extraction procedure and used to correct for recovery and matrix effects. The linearity ranged from 1.0 to 1275 μg/mL with a coefficient of determination (r(2)) greater than 0.999. LOQ and LOD were 1.0 and 0.10 μg/mL, respectively. The recovery in a tattoo product containing PPD was 94% and that for a tattoo product containing no PPD reached 105%. Extraction efficiency of 98% was obtained. This method has been successfully applied to henna temporary tattoo and other henna-related cosmetic products for the determination and quantitation of PPD.     

Bile acid metabolism in isolated rat hepatocytes: studied by gas-liquid chromatography-mass spectrometry-selected ion monitoring

Bile acid contents in isolated rat hepatocytes were determined by gas-liquid chromatography-mass spectrometry-selected ion monitoring with the use of deuterium-labeled internal standards. This allowed us first to monitor the actual amounts of not only major but also minor bile acid components present with sufficient sensitivity and specificity and to follow the changes of individual bile acids in cultured rat hepatocytes simultaneously. In freshly isolated rat hepatocytes, cholic and beta-muricholic acids were the major components, comprising 35 and 46% of the total bile acids, respectively. These two bile acids were found to be most actively synthesized during the first 2 h of incubation and continued to increase thereafter for up to 6 h (the end of the period studied). In contrast, chenodeoxycholic and alpha-muricholic acids, which are the precursors of beta-muricholic acid, showed slight increases only in the first hour of incubation and decreased thereafter. These results suggested that the conversion to beta-muricholic acid from chenodeoxycholic acid via alpha-muricholic acid occurred rapidly in cultured rat hepatocytes. The secondary bile acids such as deoxycholic, hyodeoxycholic, and 3 alpha, 12 beta-dihydroxy-5 beta-cholanoic acids declined steadily from the start of incubation, which supported the findings that further hydroxylation of these dihydroxy bile acids occurs in rat liver.     

Determination of albuterol in plasma after aerosol inhalation by gas chromatography-mass spectrometry with selected-ion monitoring

Albuterol is a β₂-adrenergic agonist commonly used as a bronchdilator for the treatment of patients with asthma. We have developed an assay to determine plasma levels as low as 50 pg/ml of albuterol by gas chromatography-mass spectrometry (GC-MS). This assay utilizes isotopically labeled albuterol ([¹³C]albuterol) as an internal standard. In this assay albuterol and the internal standard are recovered from 1 ml of plasma using solid-phase extraction. The samples are then derivatized to trimethylsilyl ethers using N,O-bis(trimethylsilyl)trifluoro-acetamide with 1% trimethylchlorosilane. The samples are then analyzed by GC-MS with selected-ion monitoring (SIM) for the ions m/z 369.15 and 370.15. The method has been validated for a concentration range of 50–10000 pg/ml in plasma.     

Simultaneous determination of urinary androgen glucuronides by high temperature gas chromatography-mass spectrometry with selected ion monitoring

An efficient procedure is described for the simultaneous determination of 9 androgen glucuronides including androsterone, etiocholanolone, 11-ketoandrosterone, 11-ketoetiocholanolone, 11beta-hydroxyandrosterone, 11beta-hydroxyetiocholanolone, and dehydroepiandrosterone (DHEA) in 3-glucuronide form and dihydrotestosterone (DHT) and testosterone in 17-glucuronide form from urine specimens. The method involves solid-phase extraction of the urinary steroids using Serdolit PAD-1 resin, with subsequent conversion to methyl ester-trimethylsilyl (Me-TMS) ether derivatives for the direct analysis by gas chromatography-mass spectrometry (GC-MS) using high temperature MXT-1 (Silcosteel-treated stainless steel) capillary column. Upon split injection of Me-TMS steroids at 330 degrees C into the MXT-1 capillary column initially maintained at 300 degrees C then programmed to 322 degrees C at 2 degrees C/min, each androgen glucuronide was well separated in excellent peak shape. The characteristic ions at m/z 217 constituting the base peaks in the electron-impact (20 eV) mass spectra for most steroids permitted their sensitive detection by GC-MS with selected-ion monitoring (SIM), whereas base peak ion at m/z 271 was used for the SIM of dehydroepiandrosterone-3-glucuronide. The detection limits for SIM of most of the steroids were 15 pg except for the 3-glucuronides of 11-ketoandrosterone and 11-ketoetiocholanolone, which could be detected down to 20 pg. The SIM responses were linear with correlation coefficients varying from 0.981 to 0.993 in the concentration range of 20 to 3000 ng/ml for the androgens studied. When applied to urine samples, the present method allowed rapid screening for the 7 androgens in their glucuro-conjugated forms simultaneously with good overall precision and accuracy within the normal concentration ranges of 15.1 to 3124.6 ng/ml.     

p-Toluenesulfonic acid/methanol: Mild reagent for the preparation of bile acid methyl esters

An improved method for the preparation of bile acid methyl esters is described. This is achieved by the addition of catalytic amounts of p-toluenesulfonic acid in a solution of bile acid in methanol. Advantages of this procedure over conventional methods include (1) use of a mild solid acid catalyst which prevents the formation of undesirable byproducts, (2) isolation of a solid product of high purity and (3) utilization of a relatively safe reagent in comparison to other methods involving diazomethane, hydrochloric acid or sulfuric acid.     

Determination of 2-butoxyethanol and butoxyacetic acid in rat and human blood by gas chromatography-mass spectrometry

A sensitive and selective gas chromatographic-negative-ion chemical ionization mass spectrometric method was developed to simultaneously quantitate 2-butoxyethanol (BE) and butoxyacetic acid (BAA) in rat and human blood at low ng/g levels as pentafluorobenzoyl and pentafluorobenzyl derivatives, respectively. Analysis of ¹³C-labeled analogs of BE and BAA were found to improve the limits of quantitation to below 2 ng/g. Deuterium-labeled BE and BAA were used as internal standards. Calibration curves were generally linear over three orders of magnitude, with limits of quantitation of 16–18 ng/g for both BE and BAA, and 1.5 and 0.4 ng/g for [¹³C₂]BE and [¹³C₂]BAA, respectively, in human blood. Linearity in rat blood was similar, with limits of quantitation of 22 ng/g for BE and 5 ng/g for BAA. This method was developed for the support of mammalian metabolism studies and human biomonitoring studies involving exposure to BE or [¹³C₂]BE.     

Determination of corticosterone in rat and mouse plasma by gas chromatography-selected ion monitoring mass spectrometry

A simple, highly sensitive and specific method based on gas-chromatography-selected ion monitoring (SIM) mass spectrometry has been developed for the quantitation of corticosterone in rat and mouse plasma. After extraction of the plasma with ethyl acetate, the residue was trimethy-silylated with pentafluorobenzyl hydroxylamine-trimethylsilyl (PFBO-TMS). Detection of the derivatives was accomplished by a quadruple mass spectrometer in the selected ion monitoring mode (m/z of 316, 648, 663 and 678). The detection limit of the assay was 0.1 pg on column. The results show that in the plasma of non-stressed animals, only minor amounts of corticosterone were found; whereas in the plasma of stressed animals, it was dramatically increased. The method developed here can be used to examine corticosterone levels as a marker of stress in rats and mice and may also be used for estimation of the effect of stress-release medications.     

Determination of neuraminidase-susceptible and total N-acetylneuraminic acid in cells by selected ion monitoring.

N-acetylneuraminic acid (NANA) is released from glycoproteins by neuraminidase or acid hydrolysis and quantified by monitoring the protonated molecular ions of fully silylated NANA ($\text{m}\text{e}\text{= 814}$) and neuraminic acid β-methylglycoside (internal standard, $\text{m}\text{e}\text{= 714}$) in a gas chromatograph—mass spectrometer system using isobutane ionization. Detection limit is 200 pg (0.6 pmol, underivatized weight) of NANA injected. In biological samples 5 ng (15 pmol) of NANA can be detected in 50 μl of hydrolysate. Only 1 to 50 μl of hydrolysate is needed, sample preparation is simple, NANA is positively identified in every analysis, 2-deoxy carbohydrates and other sialic acids do not interfere, only free NANA is determined, and the internal standard increases reliability. The NANA content of neuraminidase-treated human leukemic cells was on the order of $\text{0.3μ}\text{mol}\text{10}{}^9$ cells. NANA was quantified using as few as 5 × 10⁴ cells, in contrast to the conventional colorimetric (thiobarbituric) technique which requires 2.5 × 10⁷ cells.     

Determination of cyanide and thiocyanate in blood by gas chromatography and gas chromatography-mass spectrometry

We devised a sensitive and simple method for determining cyanide And its major metabolite, thiocyanate, in blood using an extractive alkylation technique. Pentafluorobenzyl bromide was used as the alkylating agent, and tetradecyldimethylbenzylammonium chloride was used as the phase-transfer catalyst. The derivatives obtained were analyzed qualitatively by gas chromatography-mass spectrometry and quantitatively by gas chromatography with an electron-capture detection. The detection limits of cyanide and thiocyanate were 0.01 and 0.003 μmol/ml, respectively, while the gross recovery of both compounds was 80%. The calibration curve was linear over the concentration range from 0.02 to 1.0 μmol/ml for cyanide and from 0.01 to 1.0 μmol/ml for thiocyanate. The accuracy and precision of the method were evaluated, and the coefficients of variation were found to be within 10%. Using this method, the blood levels of two victims who had died from cyanide poisoning were determined.     

Determination of copper in urine and serum by gas chromatography-mass spectrometry

A stable isotope dilution gas chromatography-mass spectrometry method using enriched 65Cu as an internal standard is described for the determination of Cu in urine and serum. Chelating agents N,N'-ethylenebis-(trifluoroacetylacetoneimine) [H2(enTFA2)] and lithium bis(trifluoroethyl)dithiocarbamate [Li(FDEDTC)] were used and evaluated for memory effect. H2(enTFA2) did not show any appreciable memory effect, whereas Li(FDEDTC) was found to have a strong memory effect. Overall precision of 1.6% was obtained for determining Cu isotope ratios at a 10-ng level using H2(enTFA2). Cu concentrations in the National Institute of Standards and Technology (NIST) reference materials, freeze-dried urine SRM 2670, and human serum SRM 909 determined using the H2(enTFA2) chelating agent were in good agreement with the NIST-certified values. Isotope ratios determined by gas chromatography-mass spectrometry on samples with altered isotopic composition were in good agreement with the inductively coupled plasma-mass spectrometry data.