Hikoto Ohta

Determination of Aconitum alkaloids in blood and urine samples. I. High-performance liquid chromatographic separation, solid-phase extraction and mass spectrometric confirmation

Determination of four toxic Aconitum alkaloids, aconitine, mesaconitine, hypaconitine and jesaconitine, in blood and urine samples has been established using high-performance liquid chromatography (HPLC) combined with ultraviolet absorbance detection, solid-phase extraction and mass spectrometry (MS). These alkaloids were hydrolyzed rapidly in alkaline solution (half lives (t1/2)<one day), were stable in solutions of acetonitrile, tetrahydrofuran and diluted hydrochloric acid (t1/2>five months) and were unstable in solutions of methanol and ethanol (t1/2<one month). These alkaloids were separated on an octadecylsilica column with isocratic elution using a solvent mixture of tetrahydrofuran and 0.2% trifluoroacetic acid (14:86, v/v), which was found to be the optimal solvent of the elution systems examined. Calibration curves with UV detection were linear on injection of amounts ranging from 2.5 to 500 ng, and the limit of detection was 1 ng (S/N=3). These four alkaloids in aqueous solution were recovered almost totally by solid-phase extraction using the styrene polymer resin, Sep-Pak Plus PS-1, and were eluted using a mixture of acetonitrile and hydrochloric acid. These Aconitum alkaloids were confirmed by HPLC coupled with fast atom bombardment MS, giving their protonated molecular ions as base peaks. These alkaloids were detected by HPLC with UV detection from blood samples spiked with more than 50 ng ml(-1) of alkaloids, but were not detectable from urine samples spiked with 5 microg ml(-1) of alkaloids because of severe sample interference.

Determination of blood cyanide by headspace gas chromatography with nitrogen-phosphorus detection and using a megabore capillary column

Abstract A method for the determination of cyanide in blood using head-space capillary gas chromatography (HS-GC) with nitrogen-phosphorus detection (NPD) was developed. The separation efficiency and sensitivity for hydrogen cyanide (HCN) were improved by using a GS-Q megabore capillary column and by injection of 500 μl of HS gas with a splitting ratio of 5. No interference or column deterioration was observed. Optimum HS conditions were found to be incubation of 50°C for 30 min in the presence of 10% phosphoric acid as an acidifying reagent. The distribution coefficient ( k ) was 75.7 and 158.9 for HCN acetonitrile (CH 3 CN; candidate for internal standard), respectively. HCN showed a different vaporization behaviour to CH 3 CN with respect to temperature and matrix effect. The salting-out effect was relatively small for HCN and CH 3 CN. An increase in the blood content in the liquid phase resulted in a significant increase in k for HCN. The calibration graph was linear over a wide concentration range of cyanide, and the limit of determination was 1 ng ml −1 (R.S.D. 10%). Because a considerable amount of CH 3 CN was detected in control blood, the direct calibration method could be used instead of the internal standard method using CH 3 CN. The detectable amount of cyanide was significantly decreased during the preincubation of cyanide and blood at 2°C. Thiocyanate in blood showed a positive interference. Contents determined by this HS-GC method agreed closely with those obtained by the microdiffusion-spectrophotometric method for blood samples from fire victims.

Determination of Aconitum alkaloids in blood and urine samples. II. Capillary liquid chromatographic-frit fast atom bombardment mass spectrometric analysis.

Determination of fourteen alkaloids, toxic Aconitum alkaloids, aconitine, mesaconitine, jesaconitine, hypaconitine and deoxyaconitine, and their hydrolysis products, benzoylaconines and aconines, have been established using capillary liquid chromatography (LC) fast atom bombardment mass spectrometry (FAB-MS) with a frit interface. Protonated molecular ions were observed as base peaks in the FAB-MS for these fourteen alkaloids. All the alkaloids were simultaneously quantified with linear gradient LC elution by solvent mixture of acetonitrile and 0.3% trifluoroacetic acid using selected ion monitoring of the protonated molecular ions. The calibration curves of these alkaloids were linear in injection amounts ranging from 5 to 500 pg, and their detection limits were 1 pg per injection (S/N=3). Solid-phase extraction using Sep-Pak Plus PS-1 was also investigated to clean-up and concentrate alkaloids in blood and urine samples, and showed satisfactory recoveries. This capillary LC-frit-FAB-MS method enables determination of low levels of Aconitum alkaloids in blood and urine samples, coupled with solid-phase extraction.

Effect of cation-exchange pretreatment of aqueous soil extracts on the gas chromatographic-mass spectrometric determination of nerve agent hydrolysis products after tert.-butyldimethylsilylation

The efficiency of pretreatment of aqueous soil extracts using a cation-exchange resin has been investigated by gas chromatographic-mass spectrometric (GC-MS) determination of nerve agent hydrolysis products after tert.-butyldimethylsilyl (TBDMS) derivatization. An aqueous solution containing methylphosphonic acid (MPA) and its monoalkyl esters, ethyl methylphosphonic acid, isopropyl methylphosphonic acid and pinacolyl methylphosphonic acid, was dried, and these phosphonic acids were derivatized with N-methyl-N-(tert.-butyldimethylsilyl)trifluoro-acetamide and analyzed by GC-MS. The yields of TBDMS derivatives were significantly decreased by the addition of calcium and magnesium ions to an aqueous solution (approximately 0.5 mM) before derivatization. The extent of lowered yields was related to the hydrophilicity of phosphonic acids. MPA and its monoalkyl esters were spiked into soil samples (sand, alluvial soil and volcanic ash soil), extracted with distilled water, dried, silylated and applied to GC-MS. The yields of TBDMS derivatives of monoalkyl esters from soil samples were low (3-42%) and MPA derivative was scarcely detected (yield: < 0.7%). By desalting the aqueous soil extract by passage through a strong cation-exchange resin, the yields of TBDMS derivatives of monoalkyl esters were significantly improved (12-69%) and MPA derivative was detected (yield: 2-36%). The extent of improved yields was related to the concentrations of divalent metal cations in aqueous soil extracts. In combination with desalting by the cation-exchange resin, GC-MS after TBDMS derivatization enables detection of nerve agent hydrolysis products in soils at sub-ppm (0.2 microgram/g) concentrations.

Microspectrophotometric discrimination of single fibres dyed by indigo and its derivatives using ultraviolet-visible transmittance spectra

Single fibres dyed by indigo and its seven derivatives were measured by microspectrophotometric examination of transmittance spectra in the ultraviolet-visible region (240-760 nm). Glycerin was the most suitable embedding reagent for this measurement. Small intra-sample variation of the transmittance intensity in single woollen fibres was observed, but relatively large inter-sample variation was observed among the fibres obtained from different areas in the same textile. There were no differences in the hmax, hmin and the position of shoulders observed in their spectra. This method was applied to the discrimination of single cotton fibres dyed by vat dyes, such as indigo and seven indigo derivatives which had similar structures. On the basis of the ultraviolet-visible transmittance spectra, these sample fibres could be distinguished from one another. It is a non-destructive method compared with chromatographic dyestuff extraction methods; furthermore, utilization of the ultraviolet region gave spectra providing useful information for evaluating fibre evidence.

Simultaneous determination of neonicotinoid insecticides in human serum and urine using diatomaceous earth-assisted extraction and liquid chromatography-tandem mass spectrometry.

A rapid and sensitive analytical method was developed for simultaneous determination of eight neonicotinoid insecticides (acetamiprid, clothianidin, dinotefuran, flonicamid, imidacloprid, nitenpyram, thiacloprid and thiamethoxam) and three specific metabolites of acetamiprid (N-desmethylacetamiprid, 5-(N-acetyl-N-methylaminomethyl)-2-chloropyridine and 5-(N-acetylaminomethyl)-2-chloropyridine) in human serum and urine. A diatomaceous earth-assisted extraction using Extrelut NT3 column with chloroform/2-propanol (3:1, v/v) as eluent was selected for the single step cleanup procedure for all the target compounds. Qualitative and quantitative analyses were conducted by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring mode. The limits of detection and the limits of quantification of eleven compounds were in the ranges of 0.1-0.2ng/mL and 0.5-10ng/mL for serum, 0.1-1ng/mL and 1-10ng/mL for urine, respectively. The extraction recoveries were between 80.9% and 101.8% for serum samples, 91.9% and 106% for urine samples. The intra-day RSDs and the inter-day RSDs were less than 11.5% and 13.2% for serum, less than 8.3% and 8.8% for urine. The proposed procedure will be suitable for forensic investigations of human poisoning cases with neonicotinoid insecticides. This is the first report of simultaneous determination of eight neonicotinoids in serum and urine samples.

Thin layer chromatography/fluorescence detection of 3,4-methylenedioxy-methamphetamine and related compounds.

Abstract:  A rapid and sensitive method for the detection of six methylenedioxylatedphenethylamines, 3,4‐methylenedioxymethamphetamine (MDMA); 3,4‐methylenedioxyamphetamine; 3,4‐methylenedioxyethylamphetamine; N‐methyl‐1‐(3,4‐methylenedioxyphenyl)‐2‐butamine; N‐methyl‐1‐(3,4‐methylenedioxyphenyl)‐3‐butamine; and 3,4‐methylenedioxydimethylamphetamine, by thin‐layer chromatography with fluorescence detection is proposed. These compounds form fluorophores on the developing plate following spraying with a reagent consisting of sodium hypochlorite, potassium hexacyanoferrate (III), and sodium hydroxide, and heating for 3 min at 100°C. Blue fluorescent spots were observed under ultraviolet light in a wavelength range of 250–400 nm. The detection limits for MDMA and the above related compounds were 50 ng. The proposed method was effectively applied to the detection of MDMA in urine samples.

Effects of oximes on mitochondrial oxidase activity

Oximes, including 2-pyridinealdoxime methiodide (2-PAM), are reactivators of acetylcholinesterase (AChE) inhibited by organophosphate poisoning. Unfortunately, their clinical use has been limited by their toxicity. To investigate the mechanism of this toxicity, the effects of oximes on the enzymes choline oxidase (ChOD) and cytochrome c oxidase (CyCOD) of the respiratory chain in mitochondria were examined. The oximes 2-PAM, obidoxime, and diacetylmonoxime significantly (P<0.01) inhibited ChOD activity, and the extent of inhibition correlated with the ability to reactivate inhibited AChE. When ChOD activity in mitochondrial extracts was tested, 2-PAM inhibited the activity by 75%, obidoxime and diacetylmonoxime did not significantly inhibit it, and 4-[(hydroxy-imino)methyl]-1-decylpyridinium bromide (4-PAD), which has greater toxicity, increased the amount of product generated in the assay to approximately 200% of normal levels. Similarly, 2-PAM inhibited the activity of CyCOD in mitochondrial extracts whereas obidoxime and diacetylmonoxime did not. One explanation for these findings is that, in addition to their inhibition of mitochondrial oxidases, the oximes may produce excessive reactive oxygen species such as H(2)O(2) in the mitochondrial fraction, which may account for some of their toxicity. This is a preliminary report related to the toxicities of oximes that may participate in the inactivation of mitochondrial oxidase enzymes. This hypothesis should be further investigated by in vivo study, including kinetic determination and free radical work.

Liquid chromatography-tandem mass spectrometry method for determination of the pyridinium aldoxime 4-PAO in brain, liver, lung, and kidney.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was validated and applied to the in vitro determination of 4-[(hydroxyimino)methyl]-1-octylpyridinium cation (4-PAO), which can penetrate the blood-brain barrier and reactivate acetylcholinesterase (AChE) inhibited by alkylphosphonate in the brain, liver, lung, and kidney. The limit of detection (LOD) was 0.235 microg cation/g wet weight, and the quantification range and linearity of the calibration curve extended over a range of 0.470-941 microg cation/g wet weight. For the proof of applicability, when 4-PAO was administrated intravenously via the rat tail vein at 10% LD(50), we were able to quantify the 4-PAO concentration in the tissues: brain 7.60+/-1.32 microg cation/g wet weight (mean+/-SD, n=5), liver 26.8+/-2.82 microg cation/g, lung 76.4+/-24.9mugcation/g, and kidney 638+/-266 microg cation/g. In addition, the methods for 4-[(hydroxyimino)methyl]-1-decylpyridinium bromide (4-PAD) and 4-[(hydroxyimino)methyl]-1-(2-phenylethyl) pyridinium bromide (4-PAPE) were partly validated referring to the findings of the 4-PAO full validation. Thus, the LC-MS/MS method described in this study can be useful for quantification of pyridinium aldoxime methiodide (PAM)-type oximes in biological samples.

Lipophilicity indices derived from the liquid chromatographic behavior observed under bimodal retention conditions (reversed phase/hydrophilic interaction): application to a representative set of pyridinium oximes.

The liquid chromatographic behavior observed under bimodal retention conditions (reversed phase and hydrophilic interaction) offers a new basis for the determination of some derived lipophilicity indices. The experiments were carried out on a representative group (30 compounds) of pyridinium oximes, therapeutically tested in acetylcholinesterase reactivation, covering a large range of lipophilic character. The chromatographic behavior was observed on a mixed mode acting stationary phase, resulting from covalent functionalization of high purity spherical silica with long chain alkyl groups terminated by a polar environment created through the vicinal diol substitution at the lasting carbon atoms (Acclaim Mixed Mode HILIC 1 column). Elution was achieved by combining different proportions of 5 mM ammonium formiate solutions in water and acetonitrile. The derived lipophilicity indices were compared with logP values resulting from different computational algorithms. The correlations between experimental and computed data sets are significant. To obtain a better insight on the transition from reversed phase to hydrophilic interaction retention mechanisms, the variation of the thermodynamic parameters determined through the van׳t Hoff approach was also discussed.