Chromatography-negative Chemical Ionization Mass Spectrometry Research Articles

Solid-phase extraction versus solid-phase microextraction for the determination of chlorinated paraffins in water using gas chromatography–negative chemical ionisation mass spectrometry

Solid-phase extraction (SPE) and solid-phase microextraction (SPME) were evaluated for the analysis of short-chain chlorinated paraffins (SCCPs) in water samples using gas chromatography coupled to negative chemical ionisation mass spectrometry (GC-NCI-MS). For SPE optimisation, four commercially available SPE cartridges were tested and several SPE parameters, such as the elution solvent, elution volume and breakthrough volume were studied. The best results were obtained with Varian Bond Elut-C 18. In order to achieve a high selectivity in the determination of SCCPs, GC-NCI-MS was used. Quality parameters of the optimised SPE and SPME procedures were determined, and the best results were obtained for the SPE/GC-NCI-MS method with LODs of 5 and 20 ng l −1 for tap and river water, respectively. This method was successfully applied to the analysis of SCCPs in river water samples at concentrations below the μg l −1 level.

Determination of polychlorinated biphenyl congeners in human milk by gas chromatography–negative chemical ionization mass spectrometry after sample clean-up by solid-phase extraction

This paper describes a simple and efficient procedure for measuring 25 congeners of polychlorinated biphenyls in human milk. The limit of quantitation was 0.1 ng/ml for five less chlorinated congeners (PCB 70, 74, 87, 99,101), and 0.01 ng/ml for the remaining 20 congeners (PCB 77, 105, 118, 126, 128, 138, 151, 153, 156, 169, 170, 180, 183, 187, 191, 194, 205, 206, 208 and 209). Solid phase extraction technology was applied to extract the analytes from the matrix and to remove lipids. Three columns were used sequentially, and they were a Bond Elut C 18, a Sep-Pak Plus NH2 and a Bond Elut PCB cartridge. The instrumental method was gas chromatography–mass spectrometry with negative chemical ionization, and selected ion monitoring mode was used.

Determination of organophosphorus pesticide residues in human tissues by capillary gas chromatography–negative chemical ionization mass spectrometry analysis

We describe an analytical method that allows the determination of organophosphorus pesticides (OPs) in different human tissues. It involves an extraction procedure with ethanol–ethyl acetate, followed by gel permeation chromatography clean-up step and analysis by capillary gas chromatography–negative chemical ionization mass spectrometry in the selected ion monitoring mode. The method was tested for 37 OPs and the recoveries obtained vary between 60 and 106% with standard deviations ranging between ±2 and ±10. These values are independent of the analyzed tissue. Peak area repeatability as RSD for some OPs was ≤4.8% while a good linear relationship in the range 1.0–500 pg μl −1 with r 2≥0.9878 was obtained. The limit of detection for the 37 OPs falls between 0.01 and 0.09 ng g −1 with an RSD≤9.5%. The analytical set up in this paper has been used to analyze different samples of human tissues (liver, healthy kidney, cancer kidney and adipose tissue) of 24 patients. The number of the identified OPs in the tissue samples is different (max. 20) according to the sample while their concentration ranges between the limit of detection and 28.0 ng g −1. The highest concentrations have been determined in liver samples without any pathology (0.4–28.0 ng g −1) while the lowest concentrations have been determined in healthy kidney samples (0.01–1.50 ng g −1). In the cancer kidney samples OP concentrations vary between 0.03 and 4.6 ng g −1: these concentrations are more elevated than those determined in healthy kidney samples. The comparison between the concentration of OPs determined in the healthy part, when possible, and those determined in the cancer part of the same kidney sample are very interesting: in fact, in the latter the OP concentration is generally 1–2-times higher than that in the former, an index of lower enzymatic activity in the cancer tissue.

Analysis of estrogens in river water and effluents using solid-phase extraction and gas chromatography–negative chemical ionisation mass spectrometry of the pentafluorobenzoyl derivatives

A procedure was developed for the analysis of estrogens in environmental water and effluents. Samples were extracted by passing through polymer-impregnated solid-phase extraction discs or C 18 cartridges, followed by gas chromatography–negative chemical ionisation mass spectrometry of the pentafluorobenzoyl derivatives. The derivatives were stable and gave diagnostic negative molecular ions as the base peak for each of the major estrogens studied. The absolute recovery of estrogens spiked into clean groundwater using the disc procedure was 84–116% at the 10 ng l −1 level (calculation not based on use of internal standards). Using doubly deuterated estradiol as internal standard added prior to extraction, the % relative standard deviation of estrogen extraction and analysis in spiked groundwater at the 10 ng l −1 level was 2.6–9.8%. Detection limits were 0.2 ng l −1 or below for the major estrogens, based on a 2.5 litre sample. The most abundant estrogen was estrone, with concentrations over the range 6.4–29 ng l −1 in effluents, and 0.2 to 17 ng l −1 in water from the River Thames.

Determination of isotopic enrichments of [1-13C]homocysteine, [1-13C]methionine and [2H3-methyl-1-13C]methionine in human plasma by gas chromatography–negative chemical ionization mass spectrometry

We describe a reliable method for the simultaneous determination of isotopic enrichments of [1-13C]homocysteine, [1-13C]methionine and [2H3-methyl-1-13C]methionine in human plasma. Accurate [1-13C]homocysteine calibration standards were prepared by chemical conversion via thiolactonisation of [1-13C]methionine standards. Based upon anion-exchange chromatography, (di)acetyl-3,5-bis–trifluoromethylbenzyl derivatives, preparation of accurate calibration curves and gas chromatography–negative chemical ionization mass spectrometry, isotopic enrichments in human plasma could be determined with TTR (%) <±0.2% (N=3) for [1-13C]homocysteine (enrichment range 0–8%), [1-13C]methionine (enrichment range 0–3%) and [2H3-methyl-1-13C]methionine (enrichment range 0–12%). The method was applied in a [2H3-methyl-1-13C]methionine tracer infusion study in a biological model.

Cortisol Production Rates in Subjects with Suspected Cushing’s Syndrome: Assessment by Stable Isotope Dilution Methodology and Comparison to Other Diagnostic Methods1

It can be difficult to establish the diagnosis of Cushing's Syndrome (CS) in patients with mild or nonspecific clinical and biochemical findings, because available diagnostic tests have limited predictive values. We hypothesized that measurement of 24-h cortisol production rates (CPRs) might be a more sensitive indicator of CS in such patients. We measured CPRs in 28 patients with suspected CS (but equivocal biochemical findings) and in 22 healthy control subjects, by infusing tracer amounts of deuterated cortisol, with simultaneous measurements of 24-h urine free cortisol (UFC) levels; and we frequently sampled serum cortisol levels. CPRs were calculated from the ratio of isotopic enrichment to isotopic dilution of cortisol measured by gas chromatography-negative ion chemical ionization mass spectrometry. Nine of the patients proved to have CS by surgery (CS-Yes), whereas 19 patients were determined not to have CS by biochemical testing (CS-No). Mean 24-h UFCs, nocturnal serum cortisol levels, and CPRs were higher in CS-Yes, compared with CS-No and normal subjects. However, one CS-Yes patient had a normal 24-h UFC, two had normal nocturnal serum cortisol levels, and two had normal 24-h CPRs. There was extensive overlap in all of the biochemical parameters between the CS-Yes and the CS-No groups. Thus, measurement of CPR does not seem to offer any diagnostic advantage over available tests for the diagnosis of CS. Patients with proven CS can have normal UFC levels, normal CPRs, or normal nocturnal cortisol levels, whereas patients not thought to have CS may have elevated levels of any one or more these parameters.

Analysis of a residual diamine in a pharmaceutical polymer using solid phase extraction with analysis by gas chromatography mass spectrometry

A method was developed for the analysis of 4,4′-methylenebiscyclohexylamine (DMDA) and 4,4′-methylenedicyclohexylisocyanate (DMDI) in a pharmaceutical polymer. The DMDA was extracted from the polymer with either buffer (0.1 M potassium phosphate pH 3.1) and the extract was passed through a SCX solid phase extraction cartridge. It was eluted from the cartridge with methanolic ammonia and then converted to its heptafluorobutyramide (HFB) derivative prior to analysis by gas chromatography–negative chemical ionisation mass spectrometry (GC–MS) in the negative ion chemical ionisation (NICI) mode. It was not possible to directly measure DMDI and it was thus analysed by selecting extraction conditions such that it would decompose to DMDA. The quantification of the residues in the polymer was based on the method of standard additions since this gave a better indication of the recovery from the complex matrix.

Identification of cyclopropene triacylglycerols by high-temperature gas chromatography and high-temperature gas chromatography-negative chemical ionization mass spectrometry

The seed oil of Choriosa speciosa, which is known to be rich in cyclopropene triacylglycerols, has been investigated by high-temperature gas chromatography using glass capillary columns coated with the stationary phase SOP-50 (50% diphenyl–50% dimethylpolysiloxane, methoxy terminated). Cyclopropene rings were characterized after a mild catalyzed hydrogenation procedure. The partial π-character of the resulting cyclopropane ring supports the separation of individual triacylglycerol species on the polarizable stationary phase. Characterization of the separated compounds was performed by negative chemical ionization–mass spectrometry with ammonia as reagent gas. ©1999 John Wiley & Sons, Inc. J Micro Sep 11: 223–229, 1999

Determination of bromate at parts-per-trillion levels by gas chromatography–mass spectrometry with negative chemical ionization

The ozonation of bromide-containing source waters produces bromate as a class 2B carcinogenic disinfection by-product. The present work describes the determination of bromate by gas chromatography–negative chemical ionization mass spectrometry (GC–NCIMS) following a bromate reaction/extraction procedure. The method detection limit for bromate based on 3.14 σ n−1 of seven replicate injections was 22 ng/l (parts-per-trillion). The overall GC–NCIMS analysis time was less than 5 min. In chlorinated waters, an interference is observed. Therefore, the current application is for unchlorinated waters, such as those produced in ozone contactors within water treatment plants, prior to the addition of chlorine. The bromate levels determined in these ozone contactor waters agreed within experimental error to the levels determined by an ion chromatographic technique. These water samples were fortified and recoveries ranged between 96% and 114% with relative standard deviation between 4% and 14%.

Determination of S-1 (combined drug of tegafur, 5-chloro-2,4-dihydroxypyridine and potassium oxonate) and 5-fluorouracil in human plasma and urine using high-performance liquid chromatography and gas chromatography-negative ion chemical ionization mass spectrometry.

A high-performance liquid chromatography (HPLC) and gas chromatography-negative ion chemical ionization mass spectrometry (GC-NICI-MS) method was developed for the analysis of the combined antitumor drug S-1 (tegafur, 5-chloro-2,4-dihydroxypyridine and potassium oxonate) and active metabolite 5-fluorouracil in human plasma and urine. Tegafur was fractionated from biological fluids by extraction with dichloromethane and analyzed by HPLC. 5-Fluorouracil and 5-chloro-2,4-dihydroxypyridine were extracted with ethyl acetate from the residual layer after extraction of tegafur, and converted to pentafluorobenzyl (PFB) derivatives. Potassium oxonate was cleaned up with an anion-exchange column (Bond Elut NH2). The extracted potassium oxonate was degraded to 5-azauracil and converted to PFB derivatives. The PFB derivatives were analyzed by GC-NICI-MS. A stable isotope was employed as the internal standard in the GC-NICI-MS analysis. The limits of quantitation of tegafur, 5-fluorouracil, 5-chloro-2,4-dihydroxypyridine and potassium oxonate in plasma were 10, 1, 2 and 1 ng/ml, respectively. The reproducibility of the analytical method according to the statistical coefficients is approximately 10%. The accuracy of the method is good; that is, the relative error is < 10%. The methods were applied to pharmacokinetic studies of S-1 in patients.

Characterization of a novel diclofenac metabolite in human urine by capillary gas chromatography-negative chemical ionization mass spectrometry.

A sensitive analytical method was developed to characterize diclofenac metabolites in small amounts of body fluids. Desalted and lyophilized urine samples were extracted with supercritical carbon dioxide directly or after acidic hydrolysis. The extracts were derivatized with N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamide. The derivatives were separated by capillary gas chromatography and identified by negative chemical ionization mass spectrometry. Full mass spectra were obtained at a level of 1.10(-9) g/ml. With direct extraction, the metabolites could be analysed in one step as open-chained acids and as (cyclic) oxindoles. By acidic hydrolysis the conjugates were transformed to the oxindoles. With both methods, a new main metabolite, [2-[2,6-dichloro-4-hydroxy-3-methoxyphenyl)amino]phenyl]acetic acid, was identified The mechanism of its formation is discussed.

Determination of Ro 19-6327 (Lazabemide) in human plasma and urine by gas chromatography-negative chemical ionization mass spectrometry.

A sensitive and specific analytical method was developed for determination of Ro 19-6327 (Lazabemide) in human plasma and urine samples to provide pharmacokinetic data from clinical trials. The new method employs a simple liquid-liquid extraction to isolate the drug from biological samples. The extract is reacted to form the trifluoroacetyl derivative of Ro 19-6327 and then analyzed by gas chromatography-negative chemical ionization mass spectrometry (GC-NCIMS). The lower limit of quantitation of the assay is 0.05 ng/ml for plasma and 5.0 ng/ml for urine, based on 1-ml aliquots. No interferences from anticoagulants, collection devices, or endogenous constituents of plasma and urine were observed. Recovery (64.3%), inter-assay precision (< 8% R.S.D.), and accuracy (> 85%) of the method were considered acceptable. The assay proved reliable enough to be automated for unattended sample analysis of approximately 50 samples daily. In an additional series of tests, Ro 19-6327 was shown to be stable under conditions that might be encountered during the analysis of samples from clinical trials.

Quantitative measurement of a new synthetic hetrazepine derivative, BN50730, in human plasma and urine by combined liquid chromatography-negative chemical ionization mass spectrometry using a particle beam interface.

A new simple and sensitive assay has been developed for the quantitative measurement of BN50730 at the picomole level in human plasma and urine. The drug and the internal standard (BN50765) were measured by combined liquid chromatography-negative chemical ionization mass spectrometry with methane as the reagent gas. A simple solid-liquid extraction procedure was used to isolate BN50730 from complex biological matrices. Mild operating conditions were required to assay the parent drug with a particle beam interface from Hewlett-Packard. The mass spectrometer was tuned to monitor the intense ion m/z 333, which was generated in the ion source by a dissociative capture process. This assay was performed with 1 ml of plasma or 0.1 ml of urine, and the quantification limit of the method was statistically calculated as 1 ng ml-1. The very low relative standard deviation and mean percentage of error calculated during the different within-day or between-day repeatability assays clearly demonstrate the ruggedness of the technique for the routine determination of BN50730 in the biological fluids. Some preliminary results on the pharmacokinetics of the drug are presented to illustrate the applicability of this new powerful LC-MS method.

Is arterial pressure a determinant of renal prostaglandin release?

The effect of changes in renal perfusion pressure (RPP) on renal prostaglandin (PG) release was investigated in conscious dogs (n = 10). PGE2, PGF2 alpha, and 6-keto-PGF1 alpha levels in renal venous and aortic plasma were measured in response to controlled reductions of RPP by an inflatable cuff implanted around the renal artery. PG plasma concentrations were determined by gas chromatography-negative ion chemical ionization mass spectrometry. At control RPP, PGE2 and PGF2 alpha concentrations in renal venous plasma were severalfold higher than in aortic plasma (PGE2, 54.2 +/- 13.4 vs. 9.3 +/- 2.7 pg/ml, P < 0.01; PGF2 alpha, 40.3 +/- 10.4 vs. 10.4 +/- 3.4 pg/ml, P < 0.01), whereas only a small secretion rate was found for 6-keto-PGF1 alpha (renal vein, 24.3 +/- 2.6 pg/ml; aorta, 17.4 +/- 3.4 pg/ml; P < 0.01). Concentrations of all three PGs in renal venous or aortic plasma did not change in response to reductions of RPP within the normal range of renal blood flow (RBF) autoregulation. The mean difference between PG release at a RPP of 70 mmHg and control was for PGE2 -7.9 pg/ml with a 95% confidence interval (CI) from -20.7 to + 5.0 pg/ml; for PGF2 alpha it was -13.7 pg/ml (95% CI -29.4 to +2.0 pg/ml); and for 6-keto-PGF1 alpha it was -0.9 pg/ml (95% CI -6.0 to +4.1 pg/ml). Reductions of RPP below the lower limit of RBF autoregulation (< 66 mmHg) had no effect on 6-keto-PGF1 alpha secretion rate but decreased secretion rates of PGE2 and PGF2 alpha (n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Quantification of acitretin in human plasma by microbore liquid chromatography—negative chemical ionization mass spectrometry

A highly sensitive liquid chromatographic—mass spectrometric procedure has been developed to quantitate plasma concentrations of acitretin, a dermatologic agent used to treat severe psoriasis. The assay utilizes the combination of normal-phase microbore high-performance liquid chromatography, negative chemical ionization mass spectrometry, selective ion monitoring and stable isotope dilution. The method has been used to measure acitretin and its metabolite, 13- cis-acitretin, over a range of 1–20 ng/ml in human plasma. The inter-assay precision was 5.3% for acitretin and 3.9% for 13- cis-acitretin, while the intra-assay precisions for acitretin and 13- cis-acitretin were 10.8 and 12.7%, respectively. Reproducibility of the assay for acitretin and 13- cis-acitretin, which was determined by the relative standard deviation of multiple analyses of the same quality assurance sample, was 5.9 and 8.1%, respectively.

Preparation of 4,4′-diaminodiphenylmethane-(2H4) for use as internal standard in the quantification of 4,4′-diaminodiphenylmethane

A simple method has been developed for the synthesis of 4,4′-diaminodiphenylmethane-(2H4) for analytical purposes in gas chromatography-negative ion chemical ionization mass spectrometry (GC-NICIMS). It consists in a proton-deuterium exchange reaction of the diamine using deutero-hydrochloric acid in deuterated methanol. The reaction was conducted at 60°C. Deuteration of the diamine in the ortho positions of the aromatic rings was observed after eight days in these conditions, using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy.

Modulation of prostanoid formation by various polyunsaturated fatty acids during platelet-endothelial cell interactions

Previous studies have reported that polyunsaturated fatty acids (PUFAs) of nutritional interest may influence arachidonic acid (20:4n-6) metabolism in both platelets and endothelium, when tested separately. In the present study, platelets (PL) and cultured endothelial cells (EC) were first pre-enriched with eight different PUFAs for a two hour incubation in the presence of free fatty acid albumin pre-coated with each acid. EC, PL or both cell populations in combination, were then stimulated by thrombin (0.1 U/ml) for five minutes. Prostanoids were extracted, purified by thin-layer chromatography, and TxB 2, 6-keto-PGF 1α and PGE 2 were quantitated by radioimmunoassays. Prostanoids or dihomoprostanoids formed from cyclooxygenase substrates other than 20:4n-6 were measured by gas chromatography-negative chemical ionisation mass-spectrometry (GC-MS). When co-incubated with EC, PL produced less TxB 2 (−15 and −85% in the absence and presence of thrombin, respectively). In contrast, 6-keto-PGF 1α increased by 189 (basal conditions) and 358% (thrombin stimulation) when PL were added to EC, in agreement with PGH 2 transfers from PL to EC. PGE 2, produced by both cell populations, reached amounts which roughly represent the sum of those measured in PL and EC alone, except when cells were pre-enriched with linoleic (18:2n-6) and the n-3 family fatty acids (18:3-, 20:5- and 22:6n-3). 6-keto-PGF 1α was markedly inhibited by adrenic acid (22:4n-6), while this acid was converted into dihomo-6-keto-PGF 1α the stable metabolite of dihomoprostacyclin. 22:4n-6 also inhibited TxB 2 formation and was converted into dihomo-TxA 2. Finally, we found that 20:5n-3 was oxygenated by both cell populations into TxB 3 and Δ17-6keto-PGF 1 α but at a much lower level than TxB 2 and 6-keto-PGF 1α from endogenous 20:4n-6.

Gas chromatography/negative chemical ionization mass spectrometry of intact glucuronides

Gas chromatography-negative chemical ionization mass spectrometry (GC–NCIMS) has been applied to the analysis of O- and S-glucuronides. The glucuronides were derivatized with pentafluorobenzyl (PFB) bromide and Tri-Sil, and analyzed by GC–NCIMS. All glucuronides gave a large [M—PFB]− ion indicative of the molecular weight of the compound. This methodology has been successfully applied to the identification of a glucuronide of unknown structure.

Pharmacokinetics of Rioprostil in Human Plasma as Assayed by Combined Capillary Gas Chromatography-Negative Ion Chemical Ionization Mass Spectrometry

To investigate the pharmacokinetics of rioprostil in man an assay is developed to analyse levels of rioprostil in plasma. After extraction and purification by solid-phase cartridges rioprostil is measured by negative ion chemical ionization mass spectrometry using a deuterated internal standard. The method is validated by a recovery experiment. Levels of rioprostil in the plasma of four volunteers following a single oral dose of 600 micrograms rioprostil are found as always below 100 pg/ml. The data presented here suggest that rioprostil is transformed rapidly in man to its more polar metabolites.

Harman in alcoholic beverages: pharmacological and toxicological implications.

Harman, a beta-carboline compound, was identified and quantitated in beer and wine samples using a combination of high performance liquid chromatography with fluorescence detection and gas chromatography-negative chemical ionization mass spectrometry. The concentration of harman in beer (7.3-140.0 ng/ml) was greater than in wine (0.8-10.5 ng/ml) and was not related to alcohol content. The pharmacological and toxicological implications of harman in alcoholic beverages are discussed.