[Determination of monofluoroacetic acid in plasma and urine by stable isotope dilution ion chromatography-triple quadrupole mass spectrometry].

A method was developed for the determination of monofluoroacetic acid (MFA) in plasma and urine by ion chromatography-triple quadrupole mass spectrometry (IC-MS/MS). A plasma sample was extracted with 3% (v/v) perchloric acid aqueous solution, and the extract was centrifuged to remove the protein and lipids. A urine sample was acidulated with 3% (v/v) perchloric acid aqueous solution. The target analyte was extracted with methyl tert-butyl ether (MTBE) at a pH between 0.5 and 1.0. After the MTBE was removed by blowing with nitrogen, the MFA in the residues was dissolved into 0.1% (v/v) ammonia solution. The separation of MFA was carried out on a Dionex Ionpac AS 19 analytical column (250 mm×2 mm, 7.5 µm) with gradient elution using KOH solution electrolytically generated from an on-line eluent generation cartridge. Before the eluent flow entered the mass spectrometer, an in-line suppressor was used to remove potassium ions. The MFA was detected with a negative electrospray ionization source in the multiple reaction monitoring (MRM) mode, and quantified with the stable isotope internal standard method. The correlation coefficient of the linear calibration curve of MFA was greater than 0.999 at the corresponding ranges of 0.1-1000 µg/L. The average recoveries were 96.2%-120% of MFA in plasma and urine samples with relative standard deviations of 1.1%-13.1% (n=6). The limits of detection of MFA in plasma and urine samples were 0.03 µg/L and 0.1 µg/L, respectively. The method is simple, sensitive and accurate, and can be applied for the determination of MFA in plasma and urine samples.

Analysis of monofluoroacetic acid in urine by liquid chromatography-triple quadrupole mass spectrometry and preparation of the positive sample by the bioconversion from monofluoroacetamide to monofluoroacetic acid in vitro.

Whether as a rodenticide or as a natural product, monofluoroacetic acid (FAcOH) may cause poisoning to humans or animals for its high acute toxicity. Urine is one of the most typical specimens for forensic diagnosis when poisoning case about FAcOH happens. The positive sample containing FAcOH plays a key role for the development of an accurate and reliable analytical method. The bioconversion from monofluoroacetamide (FAcNH2) to FAcOH in urine in vitro was studied for the preparation of positive urine sample containing FAcOH without standard spiking or animal experiment. The average bioconversion rates were 0%, 18.6% and 41.3% when incubated the FAcNH2 spiked urine in vitro for 21days at -20°C, room temperature (RT) and 37°C, respectively. Afterwards, a fast and sensitive analytical method was developed for determination of FAcOH in urine. Samples were diluted with water containing formic acid and cleaned with polymeric anion exchange (PAX) cartridge. The acid eluate was neutralized with ammonium hydroxide and directly measured by hydrophilic interaction liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) using basic mobile phase condition. The limit of detection and limit of quantification of FAcOH in urine were 2 and 5ngmL(-1), respectively. The linear range was 5-1000ngmL(-1) with a correlation coefficient of r=0.9993 in urine calibrated with internal standard. The recoveries at four spiking levels (5, 10, 50 and 500ngmL(-1) in urine) were 87.2%-107% with relative standard deviations ranged between 4.3%-8.8%.

Quantification of monofluoroacetate and monochloroacetate in human urine by isotope dilution liquid chromatography tandem mass spectrometry.

The rodenticide monofluoroacetate (MFA) and monochloroacetate (MCA), a chemical intermediate from several chemical syntheses, have been identified as potential agents of chemical terrorism due to their high toxicity. In preparation for response to poisonings and mass exposures, we have developed a quantification method using isotopic dilution to determine MFA and MCA in urine from 50 to 5000 ng/mL. Both analytes were extracted from urine using solid-phase extraction; extraction recoveries were 62% (MFA) and 76% (MCA). The extracts were then separated with isocratic high-performance liquid chromatography and identified using electrospray ionization tandem mass spectrometry, with detection limits of 0.9 and 7.0 ng/mL for MFA and MCA, respectively. Selectivity was established for both analytes with unique chromatographic retention times which were correlated with isotopically labeled internal standards and the use of two mass spectral transitions for each compound. The intra-day variability was less than 5% for both analytes and the inter-day variability was 7% for MFA and 6% for MCA.

Simultaneous determination of two acute poisoning rodenticides tetramine and fluoroacetamide with a coupled column in poisoning cases.

A coupled column system was developed for the simultaneous determination of both rodenticides fluoroacetamide and tetramine in this paper by gas chromatography/mass spectrometry (GC/MS). A short length of strong polar column (1.5 m of Innowax) was coupled to the top of a 30 m of DB-5 ms with a quartz capillary column connector. Peak width at half height (W(h)) was used to evaluate the band broadening of the coupled column system. The length of the short couple column and oven temperature program were discussed according to W(h). The precisions of the coupled column were analyzed with peak area and retention time. Good linear correlations were found for both rodenticides. Typical samples were discussed for each rodenticide and some poisoning cases were presented.

Headspace solid-phase microextraction with 1-pyrenyldiazomethane on-fibre derivatisation for analysis of fluoroacetic acid in biological samples.

A new and in part automated headspace solid-phase microextraction method for quantitative determination of the highly toxic rodenticide fluoroacetic acid (FAA) in serum and other biological samples has been developed. FAA and deuterated acetic acid (internal standard) were extracted from acidified samples by a StableFlex divinylbenzene-Carboxen on polydimethylsiloxane fibre. The acids were derivatised on the fibre in-situ with 1-pyrenyldiazomethane and detected using gas chromatography-mass spectrometry with electron impact ionisation and selected ion monitoring. The calibration curve for FAA in serum was linear over the range from 0.02 to 5 microg/ml, with limits of detection and quantification of 0.02 and 0.07 microg/ml, respectively. The method was also tested with spiked whole blood, urine, stomach contents and kidney samples. It was sufficiently reliable, reproducible and sensitive for use in routine forensic toxicology applications.

Cardiotoxicity of high-dose continuous infusion fluorouracil: a prospective clinical study.

PURPOSE: A prospective clinical study was performed to determine the incidence of high-dose continuous intravenous infusion fluorouracil (5FU-CIV) cardiotoxicity. PATIENTS AND METHODS: Three hundred sixty-seven patients who were given first-cycle high-dose 5FU-CIV were monitored for cardiac function by clinical examination, ECG, and laboratory tests. 5FU-CIV was administered during a 96- or 120-hour period at doses that ranged from 600 to 1,000 mg/m2/d. Associated drugs included cisplatin (56%), mitomycin (12.5%), folinic acid (leucovorin) (7%), and others (14%). Thirty-nine patients (10.5%) received 5FU as a single agent. RESULTS: 5FU-induced cardiac events occurred in 28 patients (7.6%; 95% confidence interval, 4.9% to 10.3%). Nine of them had a history of cardiac disease. Primary tumors included head and neck (n = 13), gastrointestinal (n = 6), breast (n = 3), and others (n = 6). The mean onset time of cardiac symptoms was 3 days (range, 2 to 5). Inaugural symptoms included angina pectoris (n = 18), hypotension (n = 6), hypertension (n = 5), malaise (n = 4), dyspnea (n = 2), arrhythmia (n = 1), or sudden death (n = 1). At 5FU discontinuation, six patients' cardiac symptoms returned to baseline, but 21 patients experienced unstable angina (n = 8), hypotension/cardiovascular collapse (n = 11), pulmonary edema (n = 1), or sudden death (n = 4). The lethality rate was 2.2% (five sudden deaths plus three irreversible collapses). ECG showed repolarization changes (ST segment deviation; T-wave inversion) in 65% and/or diffuse microvoltage in 22% of the patients who presented with cardiac events. Echocardiography showed partial or global hypokinesia in nine of the 16 patients who were examined, and one case of prolonged akinesia. Cardiac enzymes rarely showed an increase (n = 2). In severe but reversible cases, clinical, ECG, and echographic parameters returned to baseline status within 48 hours after the drug discontinuation. A fluorine 19 nuclear magnetic resonance (19F NMR) analysis of urine was performed on 14 patients; six had cardiac symptoms and eight did not. Fluoroacetate (FAC), a known cardiotoxic compound, was detected in all cases. CONCLUSION: In our study, the incidence of high-dose 5FU-CVI cardiotoxicity was 7.6%. The hypothesis of a toxic cardiomyopathic process requires further confirmation.

Nephrotoxicity of mercapturic acids of three structurally related 2,2-difluoroethylenes in the rat. Indications for different bioactivation mechanisms.

The biotransformation and the hepato- and nephrotoxicity of the mercapturic acids (N-acetyl-1-cysteine S-conjugates) of three structurally related 2,2-difluoroethylenes were investigated in vivo in the rat. All mercapturic acids appeared to cause nephrotoxicity, without any measureable effect on the liver. The mercapturic acid of tetrafluoroethylene (TFE-NAC) appeared to be the most potent nephrotoxin, causing toxicity upon an i.p. dose of 50 mumol/kg. The mercapturic acids of 1,1-dichloro-2,2-difluoroethylene (DCDFE-NAC) and 1,1-dibromo-2,2-difluoroethylene (DBDFE-NAC) were nephrotoxic at slightly higher doses, i.e. at 75 and 100 mumol/kg, respectively. In the urine of TFE-NAC-treated rats significant amounts of difluoroacetic acid (DFAA) could be detected. With increasing doses, the relative amount of DFAA in urine increased progressively (5-18% of dose). In urine of rats treated with DCDFE-NAC and DBDFE-NAC, however, the corresponding dihaloacetic acids, dichloroacetic acid and dibromoacetic acid, could not be detected. Formation of DFAA and pyruvate could also be observed during in vitro metabolism of the cysteine conjugate of tetrafluoroethylene (TFE-CYS) by rat renal cytosol. Inhibition by aminooxyacetic acid (AOA) pointed to a beta-lyase dependency for the DFAA-formation. Next to DFAA and pyruvate, also formation of hydrogen sulfide and thiosulfate could be detected. These results suggest that TFE-CYS is bioactivated to a significant extent to difluorothionacyl fluoride, which most likely is subsequently hydrolysed to difluorothio(no)acetic acid and difluoroacetic acid. According to formation of pyruvate, the cysteine conjugates derived from DCDFE-NAC and DBDFE-NAC also were efficiently metabolized by rat renal beta-lyase. However, the formation of corresponding dihaloacetic acids, dichloroacetic acid and dibromoacetic acid, could not be detected in vitro at all. Only very small amounts of hydrogen sulfide and thiosulfate were detected. These results suggest that bioactivation of the latter two conjugates to a dichloro- or dibromothionoacyl fluoride represents only a minor route. Because of better leaving group abilities of chloride and bromide compared to fluoride, rearrangement of the initially formed ethanethiol to a thiirane might be favoured. Based on the present in vivo and in vitro data, it is concluded that the nephrotoxicity of the structurally related mercapturic acids of 2,2-difluoroethylenes is dependent on halogen substitution and presumably the result of at least two different mechanisms of bioactivation.

Analytical isotachophoresis utilizing computer simulation. II. Assessment of optimum separation conditions for urinary trifluoroacetic acid metabolized from anaesthetic halothane.

Experimentally determined optimum separation conditions for a metabolite of anaesthetic halothane in urine, trifluoroacetic acid, were assessed by means of computer simulation of the isotachophoretic steady-state. The simulation confirmed that urinary acids and trifluoroacetic acid can be separated in the limited pH range of 3.5-3.7 buffered by beta-alanine, as far as the pH dependence of effective mobility is utilized. The separated fraction of the trifluoroacetic acid zone was identified by mass spectrometry. The simulated coefficient of the calibration curve agreed well with the observed value.

Mechanism of defluorination of enflurane. Identification of an organic metabolite in rat and man.

Difluoromethoxydifluoroacetic acid (CHF2OCF2CO2H) has been identified as a metabolite of enflurane (CHF2OCF2CHCIF) in rat liver microsomes in vitro and in human urine by gas chromatography mass spectrometry. The formation of the metabolite in rat liver microsomes was dependent upon the presence of NADPH and O2, and was inhibited when SKF 525-A or CO/O2 (8:2, v/v) were present in the reaction mixture. When the C-H bonds of the CHCIF group of enflurane or of the CHCI group of isoflurane (CHF2OCHCICF3) were replaced with a C-CI bond, virtually no fluoride ion was produced from either derivative in rat liver microsomes. These results indicate that cytochrome P-450 catalyzes the oxidative dehalogenation of CHF2OCF2CHCIF at its CHCIF group to form CHF2OCF2CO2H and chloride and fluoride ions. In contrast, the CHF2 group does not appear to be appreciably susceptible to metabolic oxidative dehalogenation. These results can be used for the more rational design of new inhalation anesthetics that would not be appreciably metabolized to the potential kidney toxin, F-.

Quantitative analysis of trifluoroacetate in the urine and blood by isotachophoresis.

Trifluoroacetate (TFA), the major metabolite of halothane, was assayed by a newly developed isotachophoretic technique. This technique has several advantages over the presently used methods of analysis. It requires no special preparation of urine or blood samples. The sample volume is small (5--100 microliters) and the analysis time is short (30--90 min per sample). In addition, the method provides an analysis that is both qualitative and quantitative over a wide range of concentrations (from 2 nanomoles in 200 microliters to 200 nanomoles in 5 microliters). In this study, the assay was performed using HCl (0.001 M) in 1 per cent Triton X-100, titrated with beta-alanine to a pH value of 3.6--3.9 as the leading electrolyte and n-caproic acid (0.01 M) as the terminal electrolyte (50--100 muA migration current). Using this technique, daily urinary TFA excretion of seven patients was measured during halothane anesthesia and for 14 days postoperatively. The TFA values were highest on the second postoperative day (317--1,259 mg). The mean values of the urinary TFA excreted during the entire study (2,501 +/- 493 mg, mean +/- SEM) were much higher than those reported previously. The isotachophoretic technique provides a sensitive assay for future research into the biotransformation of halothane.