Determination of polysulphides in blood by gas chromatography and gas chromatography-mass spectrometry.

A sensitive and simple method to determine polysulphides in human blood, using an extractive alkylation technique and gas chromatography, has been devised. Polysulphides were alkylated with pentafluorobenzyl bromide, and then converted into bis(pentafluorobenzyl)disulphide by desulphuration with potassium cyanide. The disulphide was analysed qualitatively by mass fragmentography and quantitatively by gas chromatography with electron-capture detection. The lower limit of detection was 0.005 mumol/ml. Field testing in a suicide case confirmed the validity of the method.     

Determination of sulphonamides by electron-capture gas chromatography. Preparation and properties of perfluoroacyl and pentafluorobenzyl derivatives.

The derivatization of benzenesulphonamide, N-ethylbenzenesulphonamide and N-phenylbenzenesulphonamide with trifluoroacetic and heptafluorobutyric anhydride and pentafluorobenzyl bromide has been studied. A rapid quantitative acylation is obtained in benzene in the presence of trimethylamine. Pentafluorobenzylation is performed by the extractive alkylation technique using tetrabutylammonium as counter ion and methylene chloride as solvent. Less than 20 min are required for a quantitative derivatization. The derivatized sulphonamides have a hydrophobic character, making them very suitable for gas chromatography. Trifluoroacetylation and heptafluorobenzylation decreases it. The derivatives have a high electron-capture detector response (minimum detectable quantity, 1-2 X 10-minus 16 moles/sec). A standard curve is given for the determination of N-phenylbenzenesulphonamide as trifluoroacetyl derivative in the range 1.8-90 ng/ml.     

Simplex optimization of extractive alkylation procedures for organic acids in aqueous samples

Simplex optimization procedures may be used to optimize the yield from an extractive alkylation procedure. Three variables from the derivatization reaction (pH, methylating agent concentration, and phase transfer agent concentration) were chosen for optimization because they interacted with each other and could not be optimized by conventional means. The yield for methylation of adipic acid was doubled with relatively little effort. This procedure may be easily applied to other systems.     

Chemical derivatization of amino acids for in situ analysis of Martian samples by gas chromatography

Three different methods of derivatization are tested in order to select and optimize one for the in situ analysis of amino acids in Martian samples. The silylation procedure can easily be automated with a high yield and a linear response in a large range of concentrations. The alkylation method is simple and easily automated, but irreproducible data are obtained for the reaction in the GC liner at quite a high temperature (300 degrees C). Moreover by-products of the reaction interfere in the GC chromatograms and mass spectrometry detection is needed for product identification. The chloroformate derivatization has several advantages such as one-step reaction and short time analysis. The main problem of this procedure is the shaking step which difficult to develop in space application.     

Quantitative Determination of Catechol Estrogens by Mass Spectrometry-a Model Study with 2-Hydroxy-Estradiol

Abstract

An approach to the determination of catechol estrogens is described. The study involves the di-O-ethylation of a catechol estrogen by extractive alkylation and quantitative determination using selected ion monitoring mass spectrometry. The effects of antioxidant and base concentrations on reaction yield are discussed. It is shown that the derivatization is both reproducible and quantitative when nanogram amounts of analyte are derivatized.     

Simplified methods for preparation of microbial fatty acids for analysis by gas chromatography with electron-capture detection

Analysis of bacterial metabolites and constituents by gas chromatography (GC) with frequency-pulsed electron-capture detection (FPECD) has been suggested as an approach to rapid identification of infection in man. In such methods conversion of analytes to electrophoric derivatives is obligatory. Present methods for analysis of microbial carboxylic acids by GC-FPECD use trichloroethylation. In order to simplify and possibly automate such sample preparation procedures, we investigated pentafluorobenzylation via extractive alkylation and via solid-supported reagents. The sensitivity in terms of minimum amounts of bacteria detectable were determined for the trichloroethyl and pentafluorobenzyl derivatives and results from solid-supported reagents were compared to extractive alkylation.     

Determination of diclofenac in plasma and urine by capillary gas chromatography-mass spectrometry with possible simultaneous determination of deuterium-labelled diclofenac.

A specific and sensitive method for the determination of diclofenac at concentrations down to ca. 1 ng/ml, the limit of detection being 100 pg/ml, in human plasma and urine by gas chromatography-mass spectrometry with 2H4-labelled diclofenac as internal standard is described. The method is also suitable for the simultaneous assay of these two compounds when both are present in samples of human plasma or urine. In this case, 5-chlorodiclofenac is used as internal standard. After toluene extraction from plasma or without extraction for urine, the method involves the formation of a dimethylindolinone derivative by extractive alkylation. The technique was applied to determine low plasma concentrations and urinary excretion of labelled and unlabelled diclofenac after percutaneous applications of Voltaren Emulgel to humans applied simultaneously under occlusive dressing as deuterated diclofenac sodium, and without occlusive dressing as unlabelled diclofenac sodium.     

Determination of clenbuterol in pharmaceutical preparations by reaction with o-phthalaldehyde using a flow-injection fluorimetric procedure

A new procedure for the determination of clenbuterol is proposed using flow-injection and fluorimetric detection. The method is based on the derivatization reaction of the primary amine group with o-phthalaldehyde in the presence of 2-mercaptoethanol. The calibration graph based on peak area was linear in the range 0.2-5 mug ml(-1) and the detection limit was 0.06 mug ml(-1). The method was validated using a reference spectrophotometric procedure and was applied to the determination of the drug in commercial pharmaceutical preparations.     

High-performance liquid chromatographic determination of netilmicin in guinea-pig and human serum by fluorodinitrobenzene derivatization with spectrophotometric detection

A high-performance liquid chromatographic procedure for netilmicin determination in guinea-pig and human serum using pre-column derivatization with 1-fluoro-2,4-dinitrobenzene and UV detection is described. Linearity was established over the range 0.5-40 micrograms/ml using only 50 microliters of serum. Accuracy and precision were good, with a mean coefficient of variation less than 5% and a mean relative error less than 4%. This procedure correlates well with an enzyme multiplied immunoassay technique and has a sensitivity similar to those of published fluorescence derivatization methods.     

Spectrophotometric determination of tellurium in trace quantities by use of an ion-association complex

The formation of an ion-association complex by the interaction of iodotellurate(IV) with cetyltrimethylammonium bromide is used as the basis of an extractive procedure to determine tellurium in the range 2.5-12.5 mug in a final aqueous phase volume of 20 ml. The method is simple, reliable and sensitive. Selectivity is achieved by separation of tellurium on aluminium hydroxide as collector.     

Determination of piperazine in working atmosphere and in human urine using derivatization and capillary gas chromatography with nitrogen- and mass-selective detection

A reliable routine method is presented for the determination of piperazine down to the sub-ppm level in aqueous solutions and in urine. The method includes a two-phase derivatization procedure with ethyl- or isobutyl chloroformate as the reagent, followed by a capillary gas chromatographic determination using nitrogen- or mass selective detection. The addition of ammonia ensured a quantitative recovery. Detection limits for piperazine in urine were ca. 20 ng/ml using nitrogen-selective and ca. 1 ng/ml with mass-selective detection. The calibration plots were linear in the investigated range, 100-10,000 ng/ml with nitrogen-selective and 30-3000 ng/ml with mass-selective detection. The precision was ca. 6% at a concentration of 300 ng/ml. Acid anhydrides were investigated as alternative reagents in the two-phase derivatization procedure, and heptafluorobutyric acid anhydride in aqueous solutions gave approximately 100% recovery. However, in urine the recoveries of the investigated acid anhydride derivatives were unsatisfactory.     

Analysis of baclofen by capillary electrophoresis with laser-induced fluorescence detection

A new analytical method for baclofen (4-amino-3-p-chlorophenylbutyric acid) based on capillary electrophoretic separation and laser-induced fluorescence detection has been developed. Naphthalene-2,3-dicarboxaldehyde was used for precolumn derivatization of the non-fluorescent drug. Optimal separation and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH 9.5) and a He-Cd laser (excitation at 442 nm, emission at 500 nm). Linearity (r > or = 0.99) over three orders of magnitude was generally obtained and the concentration limit of detection was in the nanomolar level. Coupled with a simple cleanup procedure, the method was successfully applied to the analysis of baclofen in human plasma. Recovery of spiked baclofen in plasma was 98%. The relative standard deviation values on peak size and migration time were 7.9% and 0.4%, respectively. The limit of detection of baclofen in plasma was 10 ng/ml.     

Determination of <Emphasis Type="SmallCaps">D</Emphasis>- and <Emphasis Type="SmallCaps">L</Emphasis>-amino acids produced by cyanobacteria using gas chromatography on Chirasil-Val after derivatization with pentafluoropropyl chloroformate

A rapid and simple method was developed for the determination of free amino acids (AAs) released from cyanobacteria. The procedure involves trapping of AAs from the centrifuged cyanobacterial culture fluid on a cation-exchange resin, their release together with the resin by direct treatment with the reaction medium, followed by immediate derivatization with a corresponding chloroformate. The extractive alkylation transfers the analytes into an organic phase, an aliquot of which is subjected to GC analysis. Identification and quantification of AAs was performed by GC/MS and GC/FID, respectively, using propyl chloroformate (PCF) as the derivatization reagent. For chiral analysis, the cyanobacteria extracts were treated with 2,2,3,3,3-pentafluoropropyl chloroformate (PFPCF) to create more volatile analytes. Separation of the AA enantiomers was accomplished on a Chirasil-Val capillary column and the D/L enantiomeric ratios were determined. AAs of cyanobacteria are considered to be important for the assessment of energy flow in an aquatic food web, nutrition value of cyanobacteria in a food web and for cell–cell communication within cyanobacteria. The highest levels of AAs were found in the summer period at the beginning of the season (July). In the September and October samples, the amount of AAs was lower, the number of D-AAs decreased and the D/L ratio was higher than in the July sample. Based on the obtained results it can be assumed that young populations excrete AAs in higher concentrations and a different composition compared to actively growing populations. Figure PFPCF derivatization scheme     

Mass fragmentographic determination of diphenylhydantoin and its main metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin, in human plasma.

A method is described for the mass fragmentographic determination of diphenylhydantoin and its main metabolite, 5-(-4-hydroxyphenyl)-5-phenylhydantoin (4-OH-DPH), in human plasma as their dimethyl and trimethyl derivatives, respectively. The derivatives are formed by using the recently described extractive alkylation technique. Pentadeuterated 4-OH-DPH is used as the internal standard. Following acidic hydrolysis of the plasma sample, conjugated 4-OH-DPH and, indirectly, the dihydrodiol metabolite, 5-(3,4-dihydroxy-1,5-cyclohexadien-1-yl)-5-phenylhydantoin, are measured. Using 100-mul plasma samples, the lower limit of detection is about 10 ng/ml 00.03 nmole/ml).     

Screening and confirmation of thyreostatics in urine by gas chromatography with nitrogen-phosphorus detection and gas chromatography-mass spectrometry after sample clean-up with a mercurated affinity column

Methods are described for the screening and confirmation of residues of the thyreostatics thiouracil, methylthiouracil and propylthiouracil in urine samples of cattle at levels down to 25 micrograms/l. After a selective preconcentration of the thiol-containing thyreostatics on a mercurated affinity column, the analytes are derivatized by extractive alkylation and analysed by gas chromatography with nitrogen-phosphorus or mass spectrometric detection.     

Determination of Cyanide in Blood for Forensic Toxicology Purposes—A Novel Nci Gc-Ms/Ms Technique

One of the recently evolving methods for cyanide determination in body fluids is GC-MS, following extractive alkylation with pentafluorobenzyl bromide or pentafluorobenzyl p-toluenesulfonate. The aim of this study was to improve previous GC methods by utilizing a triple quadrupole mass spectrometer, which could enhance selectivity and sensitivity allowing for the reliable confirmation of cyanide exposure in toxicological studies. Another purpose of this study was to facilitate a case investigation including a determination of cyanide in blood and to use the obtained data to confirm the ingestion of a substance, found together with a human corpse at the forensic scene. The blood samples were prepared following extractive alkylation with a phase transfer catalyst tetrabutylammonium sulfate and the PFB-Br derivatization agent. Optimal parameters for detection, including ionization type and multiple reaction monitoring (MRM) transitions had been investigated and then selected. The validation parameters for the above method were as follows—linear regression R² = 0.9997 in the range of 0.1 µg/mL to 10 µg/mL; LOD = 24 ng/mL; LOQ = 80 ng/mL and an average recovery of extraction of 98%. Our study demonstrates the first attempt of cyanide determination in blood with gas chromatography-tandem mass spectrometry. The established method could be applied in forensic studies due to MS/MS confirmation of organic cyanide derivative and low matrix interferences owning to utilizing negative chemical ionization.     

Screening for diuretics in human urine by gas chromatography-mass spectrometry with derivatisation by direct extractive alkylation

A rapid, sensitive and reliable gas chromatographic-mass spectrometric (GC-MS) screening procedure for diuretics in human urine has been developed. The procedure uses derivatisation by extractive methylation directly from the urine. The suitability of a number of phase transfer reagents and solvents were studied for the detection of sixteen diuretics. The results obtained indicate that the screening procedure employing tetrahexylammonium hydrogensulphate at pH 12 with methyl iodide in toluene at room temperature was the most effective. This method gives selectivity and sensitivities down to 0.03-0.1 microgram/ml and provides a substrate suitable for GC-MS confirmation without further manipulation. The application of the method is demonstrated by the screening of urine for bumetanide, ethacrynic acid, acetazolamide, chlorothiazide and hydrochlorothiazide.     

Determination of 4-alkylphenols by novel derivatization and gas chromatography-mass spectrometry

A simple and sensitive method for the determination of alkylphenols in water samples has been developed using gas chromatography-mass spectrometry. Alkylphenols were determined after the extractive derivatization with pentafluoropyridine. The derivatization of alkylphenols efficiently proceeded to give the corresponding 4-tetrafluoropyridyl derivatives under the biphasic reaction system. The derivatization conditions including the phase-transfer catalyst, the amount of pentafluoropyridine, the reaction time, the concentration of NaOH and organic solvent were optimized. On the mass spectra of these derivatives, intense specific ion peaks were observed: m/z 256 for 4-n-alkylphenols and m/z 284 for 4-tert.-alkylphenols. Calibration curves were linear in the range of 20-1000 ng/l (200-10,000 ng/l for nonylphenol), and the detection limits varied between 6.93 and 15.7 ng/l (85.2 ng/l for nonylphenol). The average recoveries of the alkylphenols in a fortified river water sample (100 ng/l except for nonylphenol: 1000 ng/l) ranged from 91.1 to 112%. The relative standard deviations were found to be between 5.6 and 16%. This method was successfully applied to the determination of alkylphenols in river water.     

Determination of hydrazine by liquid chromatography with preliminary derivatization with naphthalene-2,3-dialdehyde

A method is proposed for the quantification of hydrazine by reversed-phase chromatography after its derivatization with naphthalene-2,3-dialdehyde. The conditions of derivatization and the chromatography separation on a Zorbax Eclipse XDB-C8 column in the gradient mode are optimized. The derivatization and chromatography analysis take 1 and 16 min, respectively. If fluorimetry detection (λ_ex = 273 nm, λ_em = 500 nm) is used and the injection volume is 100 μL, the detection limit is 0.05 μg/L. The procedure is applicable to the quantification of hydrazine in natural waters and soil extracts. A simple and rapid procedure is elaborated for the determination of 0.1–50 μg/L hydrazine in natural waters, RSD = 12% (n = 3).     

Bioanalysis of the neuropeptide des-enkephalin-gamma-endorphin by high-performance liquid chromatography with on-line sample pretreatment using gel permeation and solid-phase isolation.

A bioanalytical method is described that allows the determination of a number of beta-endorphin-related peptides. The method is based on the application of fluorescence detection after high-performance liquid chromatography followed by post-column derivatization with o-phthaldialdehyde. Concentrations exceeding 10-25 ng/ml could be determined by using conventional fluorescence detection, whereas lower concentrations demand the use of laser-induced fluorescence detection. The sample pretreatment includes the use of on-line gel permeation, on-line solid-phase isolation and heart cutting of a peak from reversed-phase gradient elution. The sample pretreatment procedure does not discriminate between the dodecapeptide des-enkaphalin-gamma-endorphin (DE gamma E) and its metabolites in order to obtain similar recoveries for all components. The final chromatographic phase system is based on ion-pair formation, which permits the separation of DE gamma E from its metabolites and degradation products. The optimized procedure allows the determination of these peptides in plasma at concentration levels down to about 1 ng/ml, demanding a sample volume of 1 ml.