1H NMR Spectroscopy and GC-MS Analysis of  -Chioralose. Application to Two Poisoning Cases

Abstract

Alpha-chloralose, a compound widely used as a rodenticide and in the control of bird pests, is readily available. Two cases of intentional poisoning are reported. Both patients became comatose and presented hypersialorrhea and myoclonal crises in the legs. They were discharged from hospital after several days. As clinical signs of alpha-chloralose poisoning lack specificity, anamnesis might be difficult, particularly in the case of delayed diagnosis. Toxicological analysis is therefore critical, and this article reports the investigation of serum and urine samples by gas chromatography-mass spectrometry (GC-MS) in the electron-impact mode, and by 1H nuclear magnetic resonance (1H NMR) spectroscopy. Non-hydrolyzed urinary samples and those hydrolyzed by beta-glucuronidase were taken into consideration. After acetylation, GC-MS analysis was based on characteristic mass-to-charge ratio values of 272 for alpha-chloralose and 206 for beta-hydroxyethyltheophylline, which was used as internal standard. Characterization of alpha-chloralose species by 1H NMR spectroscopy was performed taking two parameters into account: chemical shift and coupling-constant values. Without any pretreatment, 1H NMR spectroscopy revealed the presence of free (5.50 and 6.15 ppm) and conjugated forms of alpha-chloralose by characteristic resonances of H1 and chloral-type protons, respectively. Quantitative analysis was performed by relative integration of peak areas. Serum alpha-chloralose showed concentrations below the quantitation limit of both methods. In urine samples, the free chemical species rapidly decreased. GC-MS analysis revealed the predominence of conjugation after a beta-glucuronidase hydrolysis step. 1H NMR analysis directly showed that on admission of the first patient, average urinary concentrations were 1.73 mmol/L (535 mg/L) for the free form and 13.72 and 6.25 mmol/L for the two conjugated forms. A later enzymatic treatment confirmed the total concentration of alpha-chloralose chemical species. Analysis of alpha-chloralose in urine by either GC-MS or 1H NMR spectroscopy methods proved to be comparable.

Distribution of Chloralose in a Fatal Intoxication

“…Glucuronide conjugate exceeded the free parent drug by 7.3-fold. This finding confirms that chloralose is excreted primarily as the glucuronide metabolite, as previously reported (9,16). Other analytical findings indicate the recent intake of lorazepam, chlorpromazine and mesterolone, consistent with scene investigation.…”

Toxicological analysis of acute pesticides poisoning among Moroccan population

“…For the alphachloralose cases, chromatographic analysis helped to identify the cause of the poisoning. This ascertainment is found in other studies [15][16][17]. Nevertheless, the negative results is related to the kinetics of the compounds.…”

Determination of chloralose residues in animal tissues by liquid chromatography-electrospray ionisation tandem mass spectrometry

“…Diethyl ether and diethyl ether/chloroform mixtures have been used to extract residues from animal tissues or physiological fluids [6][7][8][9], however, they have the disadvantage of co-extracting a significant proportion of lipid material. In-house development of the GC-ECD method had demonstrated that acetonitrile was a suitable extractant.…”

“…Illegal baits are most frequently prepared either using the carcasses of rabbits, hares, game birds or pigeons to target predatory or scavenging animals; or using eggs, grain or bread to target other animals. Chloralose is rapidly absorbed from the gastrointestinal tract and is mainly excreted in urine, partly as the parent compound and partly as glucuronides [6,7]. Investigation of a fatal human poisoning demonstrated the presence of chloralose residues in a wide range of tissues and body fluids [8].…”

“…Early analytical methods for the determination of chloralose usually involved indirect determination or colorimetry [10][11][12][13][14]. Subsequently, analyses by gas chromatography (GC) or gas chromatography-mass spectrometry (GC-MS) following derivatisation of chloralose or the indirect determination as chloral [6,7,[15][16][17] were reported. A selective GC procedure, utilising electron capture detection of the trimethylsilyl (TMS)-derivative for the determinative stage, was applied for residues in animal tissues [9,18].…”