Methane Negative Chemical Ionization Research Articles

Using an internal body residue approach to assess acute pesticide toxicity in juvenile Chinook salmon (Oncorhynchus tshawytscha)

The use of internal body residues has the potential to improve toxicological assessments of hydrophobic pesticides. The acute toxicity of three classes of pesticides were assessed in juvenile Chinook salmon using internal body residues. Chinook salmon were exposed to two current-use pesticides bifenthrin and fipronil, and 4,4′- dichlorodiphenyldichloroethylene (DDE), which is a degradation product of the legacy pesticide dichlorodiphenyltrichloroethane (DDT). After 96-h of aqueous exposure to each pesticide, the pesticide content in whole-body Chinook salmon homogenates was measured using gas chromatography/mass spectrometry with methane negative chemical ionization. The wet-weight (ww) normalized lethal residue at 50% mortality (LR50) was lowest for bifenthrin (0.654 nmol/g ww), followed by fipronil (7.17 nmol/g ww) and the sum of fipronil and its sulfone, sulfide, and desulfinyl degradation products (8.72 nmol/g ww). No lethality was observed for DDE, even at the highest body residue (>116 nmol/g ww). LR50 estimates were also normalized to dry weight and lipid content and compared to field-caught fish to assess risk. The use of a risk quotient approach indicated that bifenthrin imparts the highest risk of acute toxicity in juvenile Chinook salmon among the three pesticides tested. In comparison to external dose metrics, the use of internal body residues has the potential to improve risk assessment by providing a more direct link between pesticide concentration at the receptor site and toxicological effects.

Quantitation of Diethylene Glycol and Its Metabolites by Gas Chromatography Mass Spectrometry or Ion Chromatography Mass Spectrometry in Rat and Human Biological Samples

The misuse of the commonly used chemical diethylene glycol (DEG) has lead to many poisonings worldwide. Methods were developed for analysis of DEG and its potential metabolites; ethylene glycol, glycolic acid, oxalic acid, diglycolic acid and hydroxyethoxy acetic acid in human urine, serum and cerebrospinal fluid samples, collected following a DEG-associated poisoning in the Republic of Panama during 2006. In addition, methods were developed for rat blood, urine, kidney and liver tissue to support toxicokinetic analysis during the conduct of DEG acute toxicity studies in the rat. Sample analysis was conducted using two techniques; ion chromatography with suppressed conductivity and negative ion electrospray ionization with MS detection or with gas chromatography using electron impact ionization or methane negative chemical ionization with MS detection. Stable-isotope-labeled analogs of each analyte were employed as quantitative internal standards in the assays.

Determination of midazolam and 1-hydroxymidazolam from plasma by gas chromatography coupled to methane negative chemical ionization mass spectrometry after sublingual administration of midazolam

A sensitive and selective gas chromatographic mass spectrometric method for the determination of midazolam and its biologically active metabolite, 1-hydroxymidazolam, in rabbit plasma has been developed and validated. Sample preparation includes mixed-mode solid-phase extraction and derivatization with silylating reagents. Midazolam-d4 was used as an internal standard for the determination of parent drug and its active metabolite. The instrumentation consisted of a capillary column gas chromatography and a single quadrupole mass spectrometer with a negative chemical ionization. The method was found to be valid in terms of selectivity, linearity, precision, accuracy, and recovery over the concentration range of 2–200 ng/ml and 1–100 ng/ml for midazolam and 1-hydroxymidazolam, respectively. For both analytes, the lower limit of quantification was 2 ng/ml. Midazolam was stable in stock solutions stored three months at −20 °C and in human plasma stored for three months at −80 °C. In addition, no degradation of midazolam was found after three freeze–thaw cycles, in short-term stability at room temperature for 24 h, or in post-preparative stability in the autosampler. The validity of the method was further tested by performing a pharmacokinetic study of sublingual administration of midazolam in rabbits. The method will be used in studies related to a formulation development of novel midazolam formulations for use in paediatric anaesthesia.

Apolipoprotein C-III isoforms: kinetics and relative implication in lipid metabolism

Apolipoprotein C-III (apoC-III) production rate (PR) is strongly correlated with plasma triglyceride (TG) levels. ApoC-III exists in three different isoforms, according to the sialylation degree of the protein. We investigated the kinetics and respective role of each apoC-III isoform in modulating intravascular lipid/lipoprotein metabolism. ApoC-III kinetics were measured in a sample of 18 healthy men [mean age (+/-SD) 42.1 +/- 9.5 years, body mass index 29.8 +/- 4.6 kg/m2] using a primed-constant infusion of l-(5,5,5-D3) leucine for 12 h. Mono-sialylated and di-sialylated apoC-III (apo-CIII1 and apoC-III2) exhibited similar PRs (means +/- SD, 1.22 +/- 0.49 mg/kg/day vs. 1.15 +/- 0.59 mg/kg/day, respectively) and similar fractional catabolic rates (FCRs) (0.51 +/- 0.13 pool/day vs. 0.61 +/- 0.24 pool/day, respectively). Nonsialylated apoC-III (apoC-III0) had an 80% lower PR (0.25 +/- 0.12 mg/kg/day) and a 60% lower FCR (0.21 +/- 0.07 pool/day) (P < 0.0001 for comparison with CIII1 and CIII2 isoforms). The PRs of apoC-III1 and apoC-III2 were more strongly correlated with plasma TG levels (r > 0.8, P < 0.0001) than was apoC-III0 PR (r = 0.54, P < 0.05). Finally, the PR of apoC-III2 was strongly correlated with the proportion of LDL <255 A (r = 0.72, P = 0.002). These results suggest that all apoC-III isoforms, especially the predominant CIII1 and CIII2 isoforms, contribute to hypertriglyceridemia and that apoC-III2 may play a significant role in the expression of the small, dense LDL phenotype.

MUCOSAL AND HEPATIC PROTEIN SYNTHESIS MEASURED SIMULTANEOUSLY WITH INTRAGASTRIC AND INTRAVENOUS STABLE ISOTOPIC TRACERS IN FED PIGLETS

The pool of amino acids used for intestinal mucosal and hepatic protein synthesis can derive from the diet, from intracellular proteolysis and from arterial amino acids. The relative contributions of these sources to mucosal and hepatic protein were quantified in piglets (N=4; 7.4 kg body weight), previously implanted with gastric (IG), portal (PORT), jugular (IV) and arterial (ART) catheters. After an overnight fast, the animals were fed hourly meals of a milk-based diet and were infused, for 6 h, with U13C-algal protein (IG) and 2H5-phenylalanine(PHE; IV). The tracer:tracee ratios of [U13C] and [2H5]PHE in ART and PORT blood, as well as in VLDL apolipoproteinB-100 (APOB), fibrinogen, hepatic and mucosal protein were measured by methane negative chemical ionization selected ion monitoring mass spectrometry. The table shows the mean ratios (± SD) of the isotopic enrichments of IV tracer to that of the IG tracer in ART, PORT and APOB-100 over the last h, and in plasma fibrinogen and hepatic protein at the end of the infusion. The ratio of the IV:IG tracers was (P<0.01) lower in PORT than in ART PHE. The tracer:tracee ratio of the IG tracer in APOB (4.98 ± 1.69 mol%) was higher (P<0.025) than in hepatic free PHE (1.92 ± 0.85). The IV:IG tracer ratio of APOB-100, fibrinogen and hepatic protein were all lower (P<0.05) than that of ART PHE, but the IV:IG ratio was higher in APOB than in fibrinogen and liver constitutive protein. The ratio of the two tracers in mucosal protein (0.27 ± 0.02) was 30% (P=0.05 by paired t-test) higher than their ratio in mucosal free PHE (0.21 ± 0.06). We conclude (i) that PHE for mucosal and hepatic protein synthesis derives from the extracellular input rather than from amino acids released by intracellular proteolysis and (ii) that ART PHE may be a preferential source of mucosal and PORT PHE a preferential source of hepatic protein synthesis.

Determination of trifluoroacetylated glycosides by gas chromatography coupled to methane negative chemical ionization mass spectrometry

Gas chromatography coupled to methane chemical ionization mass spectrometry was used for qualitative determination of trifluoroacetylated plant glycosides. The mass spectra obtained exhibited characteristic fragment ions of the sugar moiety and molecular or pseudo-molecular ions. The pattern of these chemical ionization spectra was influenced by source temperature. This method was applied to the structural determination of glycosides from purple passion fruit and muscat wine (Muscat of Frontignan) extracts obtained by adsorption on Amberlite XAD-2.

Confirmation of Chloramphenicol Residues in Bovine Milk by Gas Chromatography/Mass Spectrometry

Abstract A gas chromatographic/mass spectrometric method was developed to confirm the presence of chloramphenicol (CAP) at 0.5 ng/mL in bovine milk. meta-Nitrochloramphenicol (20 ng) is added to 10 mL milk as a surrogate standard. The milk is mixed with ethyl acetate and then loaded onto a diatomaceous earth-filled, solid-phase extraction (SPE) column; CAP is eluted with additional ethyl acetate. A solution of 4% NaCI in water is added to the eluant, and ethyl acetate is removed by evaporation under a flow of N2. The sample is defatted with hexane and then loaded onto a C18-SPE column. The column is washed with water, and CAP is eluted with methanol. The methanol is evaporated under a flow of N2, and the trimethylsilyl derivative is prepared with Sylon HTP (hexamethyldisilazane–trimethylchlorosilane–pyridine, 3 + 1 + 9). The excess reagent is evaporated under N2, and the residue is taken up in cyclohexane-hexane (60 + 40). CAP is separated on a 30 m × 0.25 mm id methylsilicone column having a film thickness of 0.25 pm and analyzed by methane negative chemical ionization with selected ion monitoring. The ions with m/z 304, 322, 358, 360, 466, and 468 are monitored. Confirmation is based on the presence of 5 ions with relative ion abundances within 10% of that obtained by using standards.

Isotopic determination of organic keto acid pentafluorobenzyl esters in biological fluids by negative chemical ionization gas chromatography/mass spectrometry

A rapid, single-step procedure for the extraction and derivatization of organic alpha-keto acids from microliter quantities of human plasma has been developed. The keto acids were analyzed as the pentafluorobenzyl (PFB) ester by methane negative chemical ionization gas chromatography/mass spectrometry. The PFB esters possess excellent chromatographic properties and required no further derivatization to block the keto group. They fragment to produce intense carboxylate anions, often as the sole ion in the spectrum, and offer detection limits below 1 pmol. This derivative is suitable for isotopic analysis of organic keto acids because it does not introduce any additional isotopic complexity into the target molecule. Normal human plasma 4-methyl-2-oxopentanoic acid levels were 34.9 +/- 5.3 mumol.L-1 and could be determined with 1.1% precision by isotope dilution GC/MS. We have used this procedure to study leucine and 4-methyl-2-oxopentanoic acid metabolism by using stable isotopically labeled tracers in a variety of normal and abnormal conditions.

Analysis of (dichloromethylene) bisphosphonate in urine by capillary gas chromatography—mass spectrometry

An anion exchange extraction method of bisphosphonates from urine is described. More than 90% of the (dichloromethylene) bisphosphonate (Cl 2MBP, clodronate) was recovered from urine. The extracted bisphosphonates were trimethyl-silylated and analysed with capillary gas chromatography—mass spectrometry (GC/MS). The mass spectrometric techniques used were electron ionization (EI), ammonia chemical ionization (CI), ammonia CI tandem mass spectrometry and methane negative chemical ionization (NCI). The limit of detection of Cl 2MBP was 25 pg/injection in the NCI/selective ion recording (SIR)-mode. At 100 ng ml −1 of Cl 2MBP the precision of the whole assay method was 17.9% ( N = 6). The NCI/SIR technique offers a sensitive and highly selective method for the quantitation of Cl 2MBP in urine.

Quantification of dideoxycytidine in human plasma by gas chromatography/mass spectrometry.

A gas chromatographic/mass spectrometric procedure has been developed for the quantification in plasma of dideoxycytidine (DDC), a candidate anti-AIDS drug. The assay uses an extraction with ethyl acetate containing 10% methanol followed by three derivatization steps: (i) reaction with t-butyl dimethyl chlorosilane to silylate the 5'-hydroxyl group; (ii) pentafluorobenzoylation of the amino group with pentafluorobenzoyl chloride; (iii) methylation of the ring nitrogen adjacent to the amino group with diazomethane. The resulting derivative is quantified using stable isotope dilution, selective ion monitoring and methane negative chemical ionization mass spectrometry. Plasma concentrations of DDC were measured over a range of 2-200 ng ml-1 using 1 ml plasma for extraction.

Direct coupling of open-tubular liquid chromatography with mass spectrometry.

The design and performance of a probe that allows direct liquid introduction of the total effluent of an open-tubular liquid chromatography (OTLC) column into a mass spectrometer are described. No modification of the mass spectrometer source is required, and the probe is introduced through the direct insertion probe inlet. Because flow rates are less than 0.1 ..mu..L/min, this system can be used under both conventional electron impact and chemical ionization mass spectrometric conditions. The probe has been used with glass OTLC columns (0.5-mm o.d., 16-..mu..m i.d.) and fused silica columns (0.35-mm o.d., and 5- or 10-..mu..m i.d.). Tapering and heating the end of the column allow for the detection of compounds of low volatility while maintaining good peak shape and minimizing band broadening. A separation of five pesticides is shown, for which detection limits in the low picogram levels in the full-scan mode were obtained by using methane negative chemical ionization (MNCI). The analysis of two isomeric triazine herbicides by using OTLC with electron impact ionization mass spectrometry is also shown.

Adduct ion formation by metal complexes under methane negative chemical ionization conditions

AbstractNegative chemical ionization mass spectrometry is used as a probe to examine reactions between hydrocarbon radicals and metal complexes in the gas phase. The methane negative chemical ionization mass spectra of 27 complexes of cobalt(II), nickel(II) and copper(II) in the presence of O4, O2N2 and N4 donor atom sets are characterized by two dominant series of adduct ions of the form [M + CnH2n]− and [M + CnH2n+1]− at m/z values above the molecular ion, [M]−. Insertion of the CH radical into the ligand followed by radical/radical recombination and electron capture is proposed as the major mechanism leading to the formation of [M + CnH2n]− adduct ions. A second pathway involves ligand substitution by CnH2n+1 radicals concomitant with H elimination and electron capture. Oxidative addition at the metal followed by ionization is suggested as the principal pathway for the formation of [M + CnH2n+1]− adduct ions.

Determination of an antisecretory trimethyl prostaglandin E2 analog in human plasma by combined capillary column gas chromatography-negative chemical ionization mass spectrometry.

A method is described for measuring a trimethyl prostaglandin E2 analog, TM-PGE2, in human plasma. Trideuterated and monofluorinated analogs of TM-PGE2 and added to plasma as internal standard and carrier, respectively. The plasma is adjusted to pH 3.0 and is extracted with a mixture of benzene-dichloromethane (9:1). The residue, following removal of the extracting solvent, is reacted consecutively with pentafluorobenzyl bromide and bistrimethylsilyltrifluoroacetamide. The excess derivatizing reagents are removed by evaporation, and an aliquot of the reconstituted residue is analyzed by capillary column gas chromatography using methane as the carrier gas. A quadrupole mass spectrometer is set to monitor in the gas chromatographic effluent the (M - C7H2F5)-fragment ion of TM-PGE2 (m/e 449) and trideuterated TM-PGE2 (m/e 452) generated by methane negative chemical ionization. Quantitation of unknowns is based on a comparison of the m/e 449 to m/e 452 ion ratio in each unknown to that obtained from the analysis of control plasma spiked with known amounts of TM-PGE2 and fixed amounts of internal standard and carrier. The sensitivity limit of the assay is approximately 100 pg ml-1, which is equivalent to 1 pg injected. The assay was used to measure the concentration of TM-PGE2 in the plasma of two subjects following a single 10 micrograms kg-1 oral dose of the drug.

Methane Negative Chemical Ionization Analysis of 1,3-Dihydro-5-Phenyl-1,4-Benzodiazepin-2-Ones

The methane negative chemical ionization (NCI) mass spectra of the medically important 1,3-dihydro-5-phenyl-1,4-benzodiazepin-2-ones generally consisted solely of M- and (M-H)- ions. Attempts to find the location of the H lost in the generation of the (M-H)- ion were unsuccessful, although many possibilities were eliminated. A Hammett correlation analysis of the relative sensitivities of a series of 7-substituted benzodiazepines suggested that the initial ionization takes place at the 4,5-imine bond. For certain benzodiazepines, the (M-H)- ion generated by methane NCI was 20 times more intense than the MH+ ion generated by methane positive chemical ionization (PCI). By using NCI, a sensitive and simple GC-MS assay for nordiazepam was developed that can quantitate this important metabolite of many of the clinically used benzodiazepines in the blood and brain of rats.

Methane negative chemical ionization analysis of 1,3-dihydro-5-phenyl-1,4-benzodiazepin-2-ones.

The methane negative chemical ionization (NCI) mass spectra of the medically important 1,3-dihydro-5-phenyl-1,4-benzodiazepin-2-ones generally consisted solely of M- and (M-H)- ions. Attempts to find the location of the H lost in the generation of the (M-H)- ion were unsuccessful, although many possibilities were eliminated. A Hammett correlation analysis of the relative sensitivities of a series of 7-substituted benzodiazepines suggested that the initial ionization takes place at the 4,5-imine bond. For certain benzodiazepines, the (M-H)- ion generated by methane NCI was 20 times more intense than the MH+ ion generated by methane positive chemical ionization (PCI). By using NCI, a sensitive and simple GC-MS assay for nordiazepam was developed that can quantitate this important metabolite of many of the clinically used benzodiazepines in the blood and brain of rats.