Quantification of clotiazepam in human plasma by gas chromatography-mass spectrometry

An analytical procedure was developed and validated for the quantification of clotiazepam in human plasma. After subjecting plasma samples to solid-phase extraction, the extract was evaporated and the residue re-constituted. An aliquot of the mixture was injected onto a gas chromatography-mass spectrometry system. The detector response was linear for clotiazepam concentrations in the range of 5-200 ng/ml. Intra- and inter-day precision for the assay over the concentration range was below 13.1 and 13.5%, and the accuracy ranged between 99.0-107.9% and 92.4-101.3%, respectively. The drug was found to be stable under various processing conditions used. The method is applicable to human pharmacokinetic studies of clotiazepam.     

Quantification of urinary 5-aminolevulinic acid by gas chromatography-mass spectrometry

In this report, we describe a method for the specific quantification of urinary 5-aminolevulinic acid. It is based on gas chromatographic mass spectrometric measurement of the trimethylsilyl ester of the ethylacetoacetate pyrrole derivative of 5-aminolevulinic acid. Selected ion monitoring (SIM) was used for quantification using 3-hydroxymyristic acid as an internal standard. The detection limit of this method was 0.1 nmol/ml. This method was applied to the determination of urinary 5-aminolevulinic acid from a tyrosinemia type I patient and normal subjects, and 21.4 mmol/mol creatinine and 0.54+/- 0.49 mmol/mol creatinine (n = 7), respectively, were detected. Less than 0.2 ml urine was sufficient for the determination of 5-aminolevulinic acid in healthy subjects.     

Quantification of glyceollins in non-elicited seedlings of Glycine max by gas chromatography-mass spectrometry

A sensitive gas chromatography-mass spectrometry method is described for detection and quantification of small amounts of phytoalexins, especially glyceollins. This method allowed identification of canescacarpin (glyceollin V) besides appreciable amounts of glyceollin isomers I–III and glyceofuran in unchallenged parts of soybean seedlings. Based on this quantification method the glyceollin content of soybean roots, either untreated, wounded or incubated with a glucan elicitor from Phytophthora sojae, was compared. The proportions of glyceollin isomers in the mixture of compounds were determined in different soybean tissues. Single-ion monitoring revealed the presence of four additional glyceollin isomers with benzofuranoid structure.     

Prostacyclin metabolites,in urine of adults and neonates,studied by gas chromatography-mass spectrometry and radioimmunoassay

Endogenous levels of two metabolites of prostacyclin, 6-keto-prostaglandin F_1α (spontaneous hydrolysis product) and 6,15-diketo-13,14-dihydroprostaglandin F_1α (enzymatic degradation product) were measured in urine of adults and neonates by gas chromatography-mass spectrometry, using deuterated internal standards. The method comprised extraction of prostanoids by reverse-phase cartridges and purification by silicic acid column chromatography and reverse- and straight-phase high-performance liquid chromatographies. Exogenous 6-keto-prostaglandin F_1α and 6,keto-13,14-dihydroprostaglandin F_1α added to urine were recovered quantitatively by gas chromatography-mass spectrometry. Endogenous levels of 6-keto-prostaglandin F_1α in urine of adults were 0.11 ± 0.05 (S.D.) ng/ml (n = 12), whereas in urine of neonates the levels were much higher: 1.41±0.36 (S.D.) ng/ml (n = 5) on the 3rd day of life declining to 0.51 ± 0.21 (S.D.) ng/ml (n = 5) on the 5th day. 6-keto-prostaglandin F_1α was also estimated in both age groups by radioimmunoassay. Urinary levels of 6,15-diketo-13,14-dihydroprostaglandin F_1α in neonates on the 3rd day of life were 2.12 ± 0.70 (S.D.) ng/ml (n = 4) and declined until the 5th day. In adult urine this metabolite was below the limit of detection (0.20 ng/ml).     

Quantification of indol-3-yl acetic acid in pea and maize seedlings by gas chromatography-mass spectrometry

A procedure is described for the identification and quantification of IAA in plant tissues by GC/MS analysis of the N-heptafluorobutyryl ethyl ester of IAA using [²H₅]IAA as an internal standard. The detection limit is ca 3 pmol IAA/tissue sample. By using this method, IAA levels of 30–90 pmol/g fr. wt were obtained for dark-grown Pisum sativum epicotyls and 71–199 pmol/g fr. wt for dark-grown Zea mays seedlings. When either methanol or ethanol was used as extraction solvent, some esterification of IAA during sample preparation was observed. No evidence for the natural occurrence of methyl or ethyl esters of IAA in Pisum sativum seedlings was found.     

Plasma levels of norethisterone measured by radioimmunoassay and gas chromatography-mass fragmentography

Assays designed to quantitate the plasma levels of norethisterone (NET) are compared. Results from radioimmunoassay were compared with those from gas chromatography-mass fragmentography which were established in 5 subjects after a 1-mg oral dose of the steroid. In general, the levels of steroid measured by radioimmunoassay were usually higher than those measured by gas chromatography-mass fragmentography; there was however a correlation coefficient of .96 (P .001). For concentrations of the steroid above 500 pg/ml, the mean overestimate by radioimmunoassay was less than 30%.     

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.     

Analytical methods for thromboxane B2 measurement and validation of radioimmunoassay by gas liquid chromatography-mass spectrometry

A sensitive and specific radioimmunoassay using a ¹²⁵I tracer of high specific radioactivity was developed for thromboxane B₂ and was applied to the determination of the biosynthesis of this compound in some systems (i.e. human washed platelets, human platelet rich plasma (PRP) and rat spleen homogenates). The assays were also evaluated by mass spectrometry; levels measured by these two analytical methods were very similar. the results obtained for washed human platelets with a thin layer radiochromatographic method were in good agreement with the two preceeding methods.     

Mercury determination in blood by gas chromatography-mass spectrometry

A stable isotope dilution gas chromatography-mass spectrometry method using¹⁹⁶Hg as an internal standard is described for determining Hg in blood. In this method, the blood samples are not subjected to any digestion to avoid the loss of Hg. A solution of 0.6M HCl is used to free Hg present in blood from proteins. The pH of the solution is adjusted to 9 using borate buffer and Hg chelated using lithiumbis(trifluoroethyl)dithiocarbamate. All isotope ratio measurements are made using an organic mass spectrometer. Overall precision values for the five major Hg isotopes relative to²⁰²Hg are 1.6–2.3% when 10 ng samples of chelated Hg are analyzed. No appreciable memory or carryover effect is observed when two synthetic mixtures differing in¹⁹⁶Hg/²⁰²Hg ratios by a factor of 30 are sequentially analyzed. The method is validated by determining Hg in blood samples using isotope dilution GC-MS.     

Quantification of histamine in blood plasma and cell culture supernatants: a validated one-step gas chromatography-mass spectrometry method

A novel one-step ethylchloroformate (ECF) derivatization of histamine in biological liquid matrices that allows the sensitive quantification by gas chromatography and mass spectroscopic detection (GC-MS) from small volumes of blood plasma or cell culture supernatants within 15 min is described. After addition of ECF/chloroform directly to the crude sample, histamine has been found to be quantitatively derivatized within seconds. Following centrifugation, the organic phase is transferred to a fresh vial, dried by addition of anhydrous sodium sulfate, and subjected to GC-MS analysis. The reliability of the results is verified by use of two different ion pairs for detection. The method is validated according to DIN 38402. Linearity is given from 0.0054 to 13 microg/ml and the limit of detection is 2 ng/ml (10 pg absolute, at a signal to noise ratio of 3:1). The limit of quantification, as calculated at a confidence level of 95%, is 15.6 ng/ml. Practical application is exemplified by the determination of the histamine content in blood plasma of birch pollen-sensitized mice and in the culture supernatant of rat basophil leukemia cells after Ca(2+) ionophore-mediated degranulation.     

Quantification of 31 volatile organic compounds in whole blood using solid-phase microextraction and gas chromatography-mass spectrometry

The prevalence of exposure to volatile organic compounds (VOCs) has raised concern about possible health effects resulting from chronic human exposure. To support studies exploring the relation between VOC exposure and health effects, we developed an automated analytical method using solid-phase microextraction (SPME), capillary gas chromatography (GC), and quadrupole mass spectrometry (MS). This method quantifies trace levels (low parts per trillion) of 14 halogenated alkanes, 5 halogenated alkenes, 10 aromatic compounds, and 2 other VOCs in human blood. Detection limits for the SPME-GC-MS method range from 0.005 to 0.12 microg/L, with linear calibration curves spanning three orders of magnitude. The improved throughput of this method will enable us to expand biomonitoring efforts to assess nonoccupational VOC exposure in large epidemiological studies.     

Measurement of 8-hydroxy-2'-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry

Measurement of 8-hydroxy-2'-deoxyguanosine (8-OH-dGuo) in DNA by high-performance liquid chromatography/mass spectrometry (LC/MS) was studied. A methodology was developed for separation by LC of 8-OH-dGuo from intact and modified nucleosides in DNA hydrolyzed by a combination of four enzymes: DNase I, phosphodiesterases I and II and alkaline phosphatase. The atmospheric pressure ionization-electrospray process was used for mass spectral measurements. A stable isotope-labeled analog of 8-OH-dGuo was used as an internal standard for quantification by isotope-dilution MS (IDMS). Results showed that LC/IDMS with selected ion-monitoring (SIM) is well suited for identification and quantification of 8-OH-dGuo in DNA at background levels and in damaged DNA. The sensitivity level of LC/IDMS-SIM was found to be comparable to that reported previously using LC-tandem MS (LC/MS/MS). It was found that approximately five lesions per 10(6) DNA bases can be detected using amounts of DNA as low as 2 microgram. The results also suggest that this lesion may be quantified in DNA at levels of one lesion per 10(6) DNA bases, or even lower, when more DNA is used. Up to 50 microgram of DNA per injection were used without adversely affecting the measurements. Gas chromatography/isotope-dilution MS with selected-ion monitoring (GC/IDMS-SIM) was also used to measure this compound in DNA following its removal from DNA by acidic hydrolysis or by hydrolysis with Escherichia coli Fpg protein. The background levels obtained by LC/IDMS-SIM and GC/IDMS-SIM were almost identical. Calf thymus DNA and DNA isolated from cultured HeLa cells were used for this purpose. This indicates that these two techniques can provide similar results in terms of the measurement of 8-OH-dGuo in DNA. In addition, DNA in buffered aqueous solution was damaged by ionizing radiation at different radiation doses and analyzed by LC/IDMS-SIM and GC/IDMS-SIM. Again, similar results were obtained by the two techniques. The sensitivity of GC/MS-SIM for 7,8-dihydro-8-oxoguanine was also examined and found to be much greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS for 8-OH-dGuo. Taken together, the results unequivocally show that LC/IDMS-SIM is well suited for sensitive and accurate measurement of 8-OH-dGuo in DNA and that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results.     

Quantitation of lipid peroxidation products by gas chromatography-mass spectrometry

A method for the quantitation of lipid peroxidation products in total hepatic lipid has been developed. Lipid extracts are reduced and subsequently transmethylated with sodium methoxide. The hydroxy fatty acid methyl esters are isolated by silicic acid chromatography and derivatized to their trimethylsilyl ethers prior to analysis by gas chromatography-mass spectrometry. Three isomers, 11-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE), are quantitated using selected ion monitoring techniques relative to the internal standard, methyl 15-hydroxyarachidate. In mice treated with carbon tetrachloride (2 ml/kg), the HETE levels in total hepatic lipid were 20-fold greater than those found in control animals. HETE levels were also elevated (5- to 10-fold) in hepatic lipid from rats treated with the same dose of carbon tetrachloride. Studies on subcellular fractions with this methodology show that these lipid peroxidation products are 5- to 6-fold higher in hepatic plasma membrane vesicles isolated from rats treated with carbon tetrachloride when compared with those isolated from control animals.     

Quantification of cyanamide in young seedlings of Vicia species, Lens culinaris, and Robinia pseudo-acacia by gas chromatography-mass spectrometry

We quantified the cyanamide content of young leaves of nine Vicia species, Lens culinaris, and Robinia pseudo-acacia using a modified analytical procedure that made it possible to measure the cyanamide content of a single leaf. Recent molecular phylogenetic analysis suggests that cyanamide is present in V. benghalensis, which is placed in a monophyletic group with cyanamide-biosynthesizing plants, V. villosa and V. cracca; this suggestion was verified.     

Acetylcholine turnover estimation in brain by gas chromatography-mass spectrometry

A gas chromatograph/quadrupole mass spectrometer system has been employed to estimate the turnover of acetylcholine in mouse brain $\text{in vivo}$ following a pulse intravenous injection of choline, using discrete deuterium labelled variants of choline and acetylcholine as tracer and internal standards. There appear to be two functional pools with turnover rates of 1.4 and 7.9 nmol gm^?1min^?1.     

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.