A simplified liquid chromatography-mass spectrometry assay for artesunate and dihydroartemisinin, its metabolite, in human plasma.

Artesunate (AS) is a potent antimalarial that is used worldwide for the treatment of malaria. A simple method with a total run time of 12 min was developed and validated for the quantification of AS and dihydroartemisinin (DHA), its active metabolite, in human (heparinized) plasma based on one-step protein precipitation in acetonitrile using artemisinin (ARN) as an internal standard, followed by liquid chromatography with a single quadrupole mass spectrometry system connected to a C18 column. Peak area ratio responses were fitted to the 2nd-order curve type, polynomial equation with weighting (1/concentration) over a quantification range between 3.20/5.33-3,000/5,000 nM (1.23/1.52-1153/1422 ng/mL) of AS/DHA showing linearity with very good correlation (r2>0.999). Single ion recordings of 5 µL injections of plasma extracts allowed for limits of detection of 1.02 nM (0.39 ng/mL) for AS and 0.44 nM (0.13 ng/mL) for DHA. The inter-assay and intra-assay accuracy and precision of the method was very good with an inaccuracy of ±12.4% and coefficients of variation of ≤10.7% at all tested concentrations. The recovery of the analytes from plasma was ≥95%. Other commonly used antimalarials including mefloquine, quinine, and chloroquine, did not interfere with the analysis. Post-preparative tests over 24 h in an autosampler (10 °C) showed that the DHA response was only 2.1% of AS from auto-hydrolysis, and ß-DHA was the major, stable epimer that was used for quantification of DHA. In contrast, α-DHA increased steadily up to 600%. Artesunate and DHA in plasma were stable through three freeze/thaw cycles for up to 6 h at room temperature and up to one year at -80 °C.

A sensitive HPLC-ESI-MS-MS method for the determination of cotinine in urine.

A simple, sensitive, selective, and reproducible method based on high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry was developed for the determination of nicotine and its major metabolite cotinine in human urine. The internal standard (acetaminophen) was separated from cotinine on a Hypersil Gold C(18) column with retention times of 9.3 and 13.0 min, respectively. The mobile phase consisted of a mixture of 10 mM acetate buffer (pH 5.4) and methanol (45:55, v/v), running through the column at a flow rate of 0.6 mL/min. The chromatographic analysis was operated at 25 degrees C. Sample preparation was prepared by liquid-liquid extraction with a mixture of methyl-t-butyl ether in dichloromethane (1:1, v/v). The precision of the method based on within-day repeatability and reproducibility (day-to-day variation) was below 15% (% coefficient of variation). Good accuracy was observed for both the intra-day or inter-day assays. Limit of quantification was accepted as 0.02 ng using 100 mL samples. The mean recoveries for cotinine and the internal standard were greater than 90%. The method has been applied to the investigation of a 2-h urinary excretion of cotinine in 154 healthy non-smoking Thai volunteers (aged 18-45 years) following the administration of a half-piece (2 mg) of nicotine gum.

The influence of iron stores on cadmium body burden in a Thai population.

Cadmium is a toxin of increasing public health concern due to its presence in most human foodstuffs and in cigarette smoke. Exposure to cadmium leads to tissue bioaccumulation and, in particular, has nephrotoxic effects. The aim of the present study was to investigate the association between cadmium body burden and iron stores in a Thai population. A total of 182 healthy adult Thai subjects of both genders (89 males, 93 females) aged between 18 and 57 years and weighing 40-95 kg were included in this study. The total amounts of cadmium excreted in urine over 2 h (microg/g creatinine) were used as an index of long-term cadmium exposure. Quantitation of cadmium was performed using electrothermal (graphite furnace) atomic absorption spectrometry. The urinary cadmium excreted displayed a normal frequency distribution. The average urinary cadmium level did not exceed the WHO maximum tolerable internal dose for the non-exposed population (2 microg/g creatinine). Body iron stores reflected by serum ferritin levels did not show any correlation with cadmium burden in both males and females, although a relatively stronger influence of body iron store status on cadmium burden was shown in females. When the levels of serum ferritin were stratified into five levels (<20, 20-100, 101-200, 201-300, and >300 microg/l), a significant difference in total cadmium body burden was observed between females and males only in the group with a low level of serum ferritin of <20 microg/l. The cadmium body burden in females was about twice that in males in this group.

Cadmium exposure in Thai populations from central, northern and northeastern Thailand and the effects of food consumption on cadmium levels.

The aim of the present study was to investigate the association between cadmium body burden and the areas of exposure in Thailand, as well as blood pressure levels, the types and frequencies of foods, and alcohol consumption. A total of 182 healthy adult Thai subjects of both genders (89 males, 93 females) ages 18 to 57 years old weighing 40-95 kg were included in this study. Participants were residents from three main areas of Thailand: Pathum Thani Province (central Thailand; n=50), Khon Kaen Province (northeastern Thailand; n=43) and Mae Sot District, Tak Province (northern Thailand; n=89). The total amount of cadmium excreted in urine over 2 hours (microg/g creatinine) was used as an indicator of long-term cadmium exposure. Quantitation of cadmium was performed using electrothermal (graphite furnace) atomic absorption spectrometry (GFAAS). The urinary cadmium excreted displayed a normal frequency of distribution. Significantly higher mean cadmium levels were observed in subjects residing in Mae Sot, Tak Province (0.63 +/- 1.41 microg/g creatinine) and Khon Kaen (0.51 +/- 0.76 microg/g creatinine) compared to Pathum Thani Province (0.23 +/- 0.35 microg/g creatinine). The proportion of subjects with elevated blood pressure was significantly higher in the group exposed to higher (n=39) as opposed to lower (n=5) levels of cadmium. There were no significant differences in the mean total amounts of cadmium excreted in the 2-hour urine samples from subjects who consumed different types of meat and offal, or from those who consumed them at different frequencies.

The influence of CYP2A6 polymorphisms and cadmium on nicotine metabolism in Thai population.

We investigated the influence of genetic, cadmium exposure and smoking status, on cytochrome P450-mediated nicotine metabolism (CYP2A6) in 182 Thai subjects after receiving 2mg of nicotine gum chewing for 30min. The urinary excretion of cotinine was normally distributed over a 2h period (logarithmically transformed). Individuals with urinary cotinine levels in the ranges of 0.01-0.21, and 0.52-94.99µg/2h were categorized as poor metabolizes (PMs: 6.5%), and extensive metabolizers (EMs: 93.5%), respectively. The majority of EMs (45%) carried homozygous wild-type genotypes (CYP2A6*1A/*1A, CYP2A6*1A/*1B and CYP2A6*1B/*1B), whereas only 1% of PMs carried these genotypes. Markedly higher frequencies of EMs were also observed in all heterozygous defective genotypes including the null genotype (*4C/*4C; 1 subject). A weak but significant positive correlation was observed between total amounts of urinary cadmium excretion and total cotinine excretion over 2h. Our study shows generally good agreement between CYP2A6 genotypes and phenotypes. Smokers accumulated about 3-4-fold higher mean total amounts of 2-h urinary cadmium excretion (127.5±218.2ng/2h) than that of non-smokers (40.5±78.4ng/2h). Among the smokers (n=16), homologous wild-type genotype *1/*1 was significantly the predominant genotype (6/16) compared with other defective allele including *4C/*4C. In addition, 2h urinary excretion of cotinine in smokers of all genotypes was significantly higher than non-smokers. The proportion of smokers who smoked more than 5 cigarettes/day was significantly higher in EMs in all CYP2A6 genotypes (n=14) than in PMs (n=0).