Capillary gas chromatography/mass spectrometry with chemical ionization and negative ion detection for confirmation of identity of patulin in apple juice

Gas chromatography/mass spectrometry (GC/MS) with negative ion chemical ionization permits detection of underivatized patulin in apple juice extracts while minimizing co-extractive responses. The technique has been used with a variety of capillary columns in quadrupole, ion trap, and magnetic sector GC/MS instruments to confirm presumptive findings of patulin in apple juice at concentrations ranging from 68 to 3700 micrograms/L. The demonstrated ability to use any of these 3 mass spectrometers and several capillary columns to confirm the identity of patulin are significant strengths of the technique.     

Identification of chlorinated fatty acids in fish by gas chromatography/mass spectrometry with negative ion chemical ionization of pentafluorobenzyl esters

This paper reports the development of a technique for identifying and confirming chlorinated fatty acids previously detected in fish by gas chromatography (GC) with halogen-specific detection (XSD). Fatty acid methyl esters (FAMEs) including chlorinated FAMEs within fractions of reversed-phase high-performance liquid chromatography of transesterified fish extracts were derivatized to pentafluorobenzyl esters, which were subjected to GC/mass spectrometry (MS) with negative ion chemical ionization (NICI). Pentafluorobenzyl esters displayed reasonably good GC characteristics, a very high ionization efficiency and a low degree of fragmentation. Chloride ion chromatograms extracted at m/z 35 and 37 from full scans were utilized for locating traces of chlorinated unknowns in the GC elution profile so that their mass spectra could be readily displayed. Significant ions displayed in the mass spectrum scanned in a narrow range of retention time where a chlorinated unknown was located were evaluated using ion chromatograms extracted at the m/z of these ions. The chromatographic peaks of those ions derived from the analyte were expected to center at that specific retention time, whereas those originating from matrix compounds were not. The isotopic patterns of chlorinated ions were also examined against their theoretical relative abundances. Using this approach, three metabolism-related dichloro fatty acids previously identified by GC/XSD in filet extracts of white sucker sampled downstream from a bleached kraft pulp mill were confirmed: dichlorooctadecanoic, dichlorohexadecanoic and dichlorotetradecanoic acids. In addition, an isomer of dichlorotetradecanoic acid was found in a reference fish sample. As sample preparation is critical in this application, improved conditions for hydrolysis and pentafluorobenzyl esterification are also discussed.     

Quantitative analysis of memantine in human plasma by gas chromatography/negative ion chemical ionization/mass spectrometry

A sensitive and specific method for the determination of memantine in human plasma is presented. Memantine was extracted from plasma and derivatized to the pentafluorobenzoyl derivative in a one-step procedure avoiding any sample concentration steps. Amantadine was used as an internal standard. The compounds were measured by gas chromatography/negative ion chemical ionization mass spectrometry without any further processing. Using this detection mode, the fragment ions at m/z 353 and 325 were obtained at high relative abundance. Calibration graphs were linear over the range 0.117-30 ng ml(-1). At the limit of quantification (LOQ), the inter-assay precision was 2.00% and the intra-assay variability was 3.22%. The accuracy at the LOQ showed deviations of -1.42% (intra-assay) and -2.47% (inter-assay). The method is rugged, rapid and robust and was applied to the batch determination of memantine during pharmacokinetic profiling of the drug.     

Determination of misoprostol free acid in human breast milk and serum by gas chromatography/negative ion chemical ionization tandem mass spectrometry

To study an expected transition of misoprostol from human blood into breast milk, a novel method for the determination of its active metabolite misoprostol acid (MPA) was developed. MPA was determined in serum and breast milk samples by an isotope dilution assay using gas chromatography/negative ion chemical ionization tandem mass spectrometry (GC/NICI-MS/MS). After addition of (15S)-15-methylprostaglandin E(2) (15-methyl-PGE(2)) as an internal standard, MPA was extracted from both matrices using a reversed-phase cartridge. The prostanoids were derivatized with O-2,3,4,5,6-pentafluorobenzylhydroxylamine hydrochloride (PFBHA) and 2,3,4,5,6-pentafluorobenzyl bromide (PFBB) to the pentafluorobenzyl oxime (PFBO)-pentafluorobenzyl ester (PFB) derivatives. The sample was subjected to thin-layer chromatography with ethyl acetate-hexane (1 : 1 (v/v)) as the developing solvent. The corresponding zone was extracted. After derivatization to the trimethylsilyl ether, MPA was determined by GC/NICI-MS/MS using the [molecule (M) - pentafluorobenzyl (PFB)](-) ([P](-)) ions as precursor in the negative ion chemical ionization mode. The product ions used for quantification were [P - 2TMSOH - C(6)F(5)CH(2)OH](-) (MPA) and [P - 2TMSOH - C(6)F(5)CH(2)OH - CO(2)](-)(15-methyl-PGE(2)), respectively. The limit of quantification for MPA was approximately 1 pg ml(-1) in breast milk and serum samples. The correlation coefficients of the calibration curves for MPA were r > 0.997 in the 0.5-2000 pg ml(-1) range for both tested matrices.     

Enantioselective trace analysis of amphetamine in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry

A method for the quantitative enantioselective analysis of amphetamine in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry (GC/NICI-MS) is presented. Five-fold deuterated analogues of both enantiomers were used as internal standard. Plasma sample preparation was performed by a rapid liquid-liquid extraction using n-hexane. Derivatization with (S)-(-)-N-(heptafluorobutyryl)prolyl chloride was accomplished directly in the n-hexane extract to avoid loss of amphetamine during sample concentration. The method was validated in the expected concentration range of 0.006 for a pharmacokinetic study. Calibration curves were linear within a range 0.006-50 ng/mL plasma. Precision and accuracy were acceptable over the entire calibration range. Baseline separation of the enantiomers was easily achieved on a 15-m nonchiral apolar column. The method is simple and robust, and has been applied to the batch analysis of amphetamine enantiomers.     

Pulsed-valve chemical ionization for gas chromatography/fourier-transform mass spectrometry

The pressure requirements for chemical ionization g.c./F.t.m.s. which restrict mass resolution and accuracy are overcome through use of a pulsed valve that provides momentary reagent gas pressures. For alternate electron impact (EI)/chemical ionization (c.i.) g.c./F.t.m.s., similar resolution for both e.i. and c.i. data is demonstrated. The efficiency of chemical ionization with the pulsed valve is similar to static high pressure c.i. measurements of several model compounds. Results from the analysis of peppermint oil and a fuel additive illustrate the potential information available from a single g.c./F.t.m.s. experiment.     

Determination of 4,4'-methylenebis(2-chloroaniline) in urine using capillary gas chromatography and negative ion chemical ionization mass spectrometry.

A highly sensitive and specific gas chromatographic-mass spectrometric (GC-MS) assay for the determination of 4,4'-methylenebis(2-chloroaniline) (MBOCA) in urine is reported. It is based on the solvent extraction of the hydrolysed MBOCA conjugates, together with deuterium-labelled benzidine-d8 added as an internal standard, and a two-phase derivatization procedure involving use of pentafluoropropionic anhydride in the presence of ammonia as the phase-transfer catalyst. The reaction is complete within 2 min at room temperature. The pentafluoropropyl derivatives are determined by use of capillary column GC-MS with selected-ion monitoring in the negative ion chemical ionization mode. The lower limit of detection for MBOCA was 1 microgram dm-3 and the calibration graph showed linearity between 10 and 250 micrograms dm-3. The recovery of the analyte added to pooled urine was above 86%. Thirty urine specimens from workers employed in a polyurethane-producing plant were analysed for MBOCA by this method.     

Confirmation of heptachlor epoxide and octachlor epoxide in milk via fused silica gas chromatography/negative ion chemical ionization mass spectrometry

A method for the mass spectral confirmation of heptachlor epoxide and octachlor epoxide in milk is described. The method includes the use of a 30 m × 0.25 mm DB-1701 fused silica gas chromatography column combined with negative ion chemical ionization mass spectrometry. By using this technique, confirmation of these compounds in milk can be performed at a level of 0.1 parts-per-million in milk fat.     

Determination of nitrosamines in water by gas chromatography/chemical ionization/selective ion trapping mass spectrometry

A gas chromatography/mass spectrometry (GC/MS) method for determination of nine N-nitrosamines (NAs) in water is described. Two ionization modes, electron impact (EI) and chemical ionization (CI) with methanol, as well as different ion analysis techniques, i.e. full scan, selected ion storage (SIS) and tandem mass spectrometry (MS/MS) were tested. Chemical ionization followed by SIS resulted the mass spectrometric method of choice, with detection limits in the range of 1-2ng/L. Solid Phase Extraction (SPE) with coconut charcoal cartridges was applied to extract NAs from real samples, according EPA Method 521. Drinking water samples were collected from seven surface- and two groundwater treatment plants. Three surface water treatment plants were sampled before and after addition of O(3)/ClO(2) to observe the effect of disinfection on NAs' formation. N-nitrosodiethylamine (NDEA), n-nitrosodipropylamine (NDPA), n-nitrosomorpholine (NMOR) and n-nitrosodibutylamine (NDBA) were found up to concentrations exceeding three times the risk level of 10ng/L set by the California Department of Public Health. Because dermal adsorption has been recently indicated as a new contamination route of exposure to NAs for people who practice swimming activity, water samples from five swimming pools in the Bologna (Italy) area were collected. N-nitrosopyrrolidine (NPYR) was detected in all samples at concentrations larger than 50ng/L, likely as a disinfection by-product from the amino acid precursor proline, a main constituent of skin collagen.     

Quantitative analysis of estrogens in human urine using gas chromatography/negative chemical ionisation mass spectrometry

A sensitive procedure for the determination of estrogens in urine has been developed, using enzymatic hydrolysis and ether extraction followed by capillary gas chromatography with negative chemical ionisation mass spectrometry (GC/NCI-MS) of the pentafluorobenzoyl derivatives. These derivatives were superior for GC/NCI-MS to trifluoroacetyl, heptafluorobutyl, pentadecafluorooctanoyl and perfluorotolyl derivatives, giving characteristic negative molecular ions as the base peak for each of the naturally occurring estrogens estrone, estradiol and estriol, and for the synthetic estrogen ethynylestradiol used in contraceptive pills. The method was also suitable for determination of some metabolites of estrone and estradiol. The specificity and sensitivity of the GC/NCI-MS method simplifies sample preparation. Recovery of spiked samples was in the range 84-101% for the natural estrogens based on additions to increase the concentration by 5 and 1 microg L(-1). The repeatability of the method was 1-8% for all estrogens.     

Determination of anabolic steroids by gas chromatography/negative-ion chemical ionization mass spectrometry and gas chromatography/negative-ion chemical ionization tandem mass spectrometry with heptafluorobutyric anhydride derivatization

A gas chromatography/mass spectrometry (GC/MS) method is described which uses negative ion chemical ionization (NCI) and tandem mass spectrometry (MS/MS) for the determination of eight anabolic steroids in human urine. Eight anabolic steroids were derivatized by heptafluorobutyric anhydride (HFBA), and were determined using GC/NCI-MS and GC/NCI-MS/MS. The linear correlation coefficients for calibration in NCI-MS/MS were in the range 0.9880-0.9988. This method of derivatization with HFBA for use with GC/NCI was useful in determinations of 19-norandrosterone, boldenone, 19-noretiocholanolone, 2-methylandrosterone, nandrolone, 1-methyleneandrosterone, 1-methylandrosterone, 4-dihydroboldenone and mesterolone. The detection limits of this procedure were 5-20 ppb at a signal-to-noise (S/N) ratio of 3.     

Gas chromatography/negative ion chemical ionization mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry quantitative profiling of N-acetylcysteine conjugates of valproic acid in urine: application in drug metabolism studies in humans

We report a GC/NICI-MS assay and a LC/ESI-MS/MS assay for the analysis of N-acetylcysteine (NAC) conjugates of (E)-2,4-diene VPA (NAC I and NAC II) identified in humans. The assay also includes the analysis of the NAC conjugate of 4,5-epoxy VPA (NAC III), an identified metabolite in rats treated with 4-ene VPA for its use in metabolic studies in animals. The highly sensitive GC/MS assay was designed to monitor selectively the diagnostic and most abundant [M - 181](-) fragment anion of the di-PFB derivatives of NAC I, NAC II, and NAC IV, the internal standard (IS) and the PFB derivative of NAC III. The higher selectivity of LC/MS/MS methodology was the basis for an assay which could identify and quantitate the underivatized conjugates simultaneously using MRM of the diagnostic ions m/z 130 and 123 arising from the CID of their protonated molecular ions [MH](+). The GC/MS assay employed liquid-liquid extraction whereas the LC/MS/MS assay used a solid-phase extraction procedure. Linearity ranges of the calibration curves were 0.10-5.0microg ml(-1) by GC/MS and 0.10-1.0microg ml(-1) by LC/MS/MS for NAC I, NAC II and NAC III (r(2) = 0.999 or better). Both assays were validated for NAC I and NAC II and provided good inter- and intra-assay precision and accuracy for NAC I and NAC II. The LOQ by LC/MS/MS was 0.1microg ml(-1), representing 1 ng of NAC I and NAC II. The same LOQ (0.1microg ml(-1)) was observed by GC/MS and was equivalent to 100 pg of each metabolite. NAC III was detected at concentrations as low as 0.01 microg ml(-1) by both methods. The total urinary excretion of the NAC conjugates in four patients on VPA therapy was determined to be 0.004-0.088% of a VPA dose by GC/MS and 0.004-0. 109% of a VPA dose by LC/MS/MS.     

Analysis of 7-methylguanine using isotope dilution and gas chromatography/electron-capture negative chemical ionization mass spectrometry

This paper presents results obtained for in vivo endogenous and exogenous 7-methylguanine (7-MG) analyzed using a method incorporating gas chromatography with electron-capture negative chemical ionization mass spectrometry and isotope dilution (GC/EC-ID-MS). 13C4-Labeled 7-MG was synthesized to serve as an internal standard to improve accuracy of quantitation, and was used to analyze 7-MG in livers of control mice and dacarbazine-treated mice. The results confirm that 7-MG in tissue DNA can be measured using this GC/EC-ID-MS method with excellent sensitivity and specificity. Administration of 0, 30, and 60 mg/kg dacarbazine to mice led to dose-dependent increases in the formation of 7-MG. The results indicate that this method could be applied to the analysis of endogenous and exogenous 7-MG in human tissues for future molecular epidemiology studies on potential health effects caused by methylating agents.     

Analysis of triacetone triperoxide by gas chromatography/mass spectrometry and gas chromatography/tandem mass spectrometry by electron and chemical ionization

The explosive triacetone triperoxide (TATP) has been analyzed by gas chromatography/mass spectrometry (GC/MS) and sub-nanogram detection limits are reported by ammonia positive ion chemical ionization (PICI), electron ionization (EI) and methane negative ion chemical ionization (NICI). Analysis by methane PICI and ammonia NICI gave detection limits in the low nanogram range. Analyses were carried out on (linear) quadrupole and ion trap instruments. Analysis of TATP by PICI using ammonia reagent gas is the preferred analytical method, producing low limits of detection as well as an abundant (greater than 60% of base peak) diagnostic adduct ion at m/z 240 corresponding to [TATP + NH4]+. Isolation of the [TATP + NH4]+ ion with subsequent collision-induced dissociation (CID) produces extremely low abundance product ions at m/z values greater than 60, and the m/z 223 ion corresponding to [TATP + H]+ was not observed. Density functional theory (DFT) calculations at the B88LYP/DVZP level indicate that dissociation of the complex to form NH4+ and TATP occurs at energies lower than peroxide bond dissociation, while protonation of TATP leads to cleavage of the ring structure. These results provide a method for pico-gram detection levels of TATP using commercial instrumentation commonly available in forensic laboratories. As a point of comparison, a detection limit of 15 ng was obtained by flame ionization detection.     

Studies on steroids. CCXXVIII Trace analysis of bile acids by gas chromatography-mass spectrometry with negative ion chemical ionization detection

A suitable derivatization method for the trace analysis of bile acids by gas chromatography (GC) in combination with negative ion chemical ionization (NICI) mass spectrometry is described. Of various derivatives for the carboxyl group, the pentafluorobenzyl (PFB) ester provided the highest value of the ratio of the negative to the positive ion current. A characteristic carboxylate anion [M - 181]- was produced as the most abundant ion by the loss of the PFB group in NICI. The PFB esters were further derivatized to the dimethylethylsilyl (DMES) ethers, whereby lithocholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid and cholic acid were distinctly separated by GC on a cross-linked methyl silicone fused-silica capillary column. The detection limit for the PFB-DMES derivatives of dihydroxylated bile acids was 2 fg when the fragment ion was monitored at m/z 563 in the NICI mode using isobutane as a reagent gas.     

On-line derivatization gas chromatography with furan chemical ionization tandem mass spectrometry for screening of amphetamines in urine

A simple alternative method with minimal sample pretreatment is investigated for screening of amphetamines in small volume (using only 20 microL) of urine sample. The method is sensitive and selective. The method uses gas chromatography (GC) direct sample introduction (DSI) for on-line derivatization (acylation) of amphetamines to improve sensitivity. Furan as chemical ionization (CI) reagent in conjunction with tandem mass spectrometry (MS/MS) is used to improve selectivity. Low background with sharp protonated molecular ion peaks of analytes is the evidence of improvement in sensitivity and selectivity. Blank urine samples spiked with known amounts of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine and 3,4-methylenedioxyethylamphetamine is analyzed. Selected ion monitoring of the characteristic product ions (m/z 119+136+150+163) using furan CI-MS/MS in positive ion mode is used for quantification. Limits of detection (LOD) between 0.4 and 1.0 ng mL(-1) and limits of quantitation (LOQ) between 1.0 and 2.0 ng mL(-1) are established. Linear response over the range of 1-1000 ng mL(-1) (r(2)>0.997) is observed for all analytes, except for methamphetamine (2.0-1000 ng mL(-1)). Good accuracy between 86 and 113% and precision ranging from 4 to 18% is obtained. The method is also tested on real samples of urine from suspected drug abusers. This method could be used for screening and determination of amphetamines in urine samples, however needs additional work for full validation.