Determination of urine melamine by validated isotopic ultra-performance liquid chromatography/tandem mass spectrometry.

Little is known about melamine (MEL) analysis in children's urine. In this study, an isotopic ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method was developed and systematically validated for the analysis of MEL in urine. The method is easily performed and comprises acidification, solid-phase extraction (SPE) and UPLC/MS/MS analysis. (13)C(3)N(3)((15)NH(2))(3) was used as the internal standard (IS) for calibration. Transition ions m/z 127 > 85 of MEL and m/z 133 > 89 of the IS were used for quantification and m/z 127 > 68 of MEL was used for quantitative confirmation. Recovery and precision were assessed to guarantee the applicability of the method. The limit of quantification (LOQ) was 0.01 microg/mL while the calculated method detection limit was 0.006 microg/mL. The mean recoveries ranged from 96-99%. The method was then applied to analyze urine samples from children who had potentially consumed MEL-tainted dairy products during screening in Taiwan. Ten nephrolithiasis cases and 20 age- and gender-matched controls were selected for this study. Three out of the 10 nephrolithiasis cases had elevated levels of MEL. Comparatively, twenty age- and gender-matched non-nephrolithiasis controls consuming Taiwan brand milk powder all showed MEL levels lower than the detection limit except for two children with background levels of 0.02 microg/mL. The background level in these children urine samples was established by UPLC/MS/MS analysis. Positive results of urine MEL tests might be associated with nephrolithiasis in these candidates. Measurement of urine MEL concentration can be helpful in confirming MEL-related nephrolithiasis, but its clinical application needs further clarification.

Effect of chenodeoxycholic acid on the toxicity of 5alpha-cyprinol sulfate in rats.

Attempts are made to elucidate the effect of bile acid chenodeoxycholic acid on the toxicity of bile alcohol 5alpha-cyprinol in rats. Twenty-four male Wistar rats were divided into four groups and treated orally at 3-days periodic treatment with each 160 mg/kg of 5alpha-cyprinol sulfate/chenodeoxycholic acid (9:1), 5alpha-cyprinol sulfate and chenodeoxycholic acid for 19 days. After treated with 5alpha-cyprinol sulfate/chenodeoxycholic acid (9:1), 5alpha-cyprinol sulfate and chenodeoxycholic acid, the relative ratios of liver and kidney weight to body weight, the concentrations of red blood cells (RBC), hemoglobin and hematocrit in the blood, the levels of aspartate transferase (AST), alanine transferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN) and creatinine in the plasma, and the levels of BUN and creatinine in the urine of rats were significantly increased, but body weight of rats and the levels of Na(+), K(+), Ca(++) in the urine of rats were significantly decreased, especially for both groups of 5alpha-cyprinol sulfate/chenodeoxycholic acid (9:1) and 5alpha-cyprinol sulfate. The pathological examination of liver and kidney also showed the cell enlargement and lesion in cell integrity in these treated groups, especially for both groups with 5alpha-cyprinol sulfate/chenodeoxycholic acid (9:1) and 5alpha-cyprinol sulfate. The toxicity of 5alpha-cyprinol sulfate/chenodeoxycholic acid (9:1) and 5alpha-cyprinol sulfate was similar to each other, and the toxic effect of chenodeoxycholic acid was less.

Identification of tetrodotoxin and fish species in a dried dressed fish fillet implicated in food poisoning.

There were five victims of neurotoxic food poisoning from a dried dressed fish fillet in Changhua County, Taiwan, in February 2000. The toxicity of the dried dressed fish fillets was 243 mouse units per g according to a tetrodotoxin bioassay. The partially purified toxin was identified as tetrodotoxin and anhydrotetrodotoxin. The sequence of the 376-nucleotide region in the cytochrome b gene of the mitochondrial DNA exhibited the same genotype as that of the toxic puffer fish Lagocephalus lunaris. The same single restriction site for Hinfl was found in the polymerase chain reaction (PCR) products from the dried dressed fish fillet and the muscle of L. lunaris, yielding two DNA fragments of 170 and 206 bp. However, no restriction site for Hinfl was found in the PCR products from other toxic puffer fishes, including Takifugu niphobles, Takifugu oblongus, and Takifugu rubripes. Therefore, the species of the dried dressed fish fillet was identified as L. lunaris and its causative agent was identified as tetrodotoxin.

Short-term toxicity of grass carp bile powder, 5alpha-cyprinol and 5alpha-cyprinol sulfate in rats.

We compared the short-term toxicity of toxic components of grass carp bile juice (GCBJ) in rats. Twenty-four male Wistar rats were divided into four groups and treated orally every 3 days with 40 mg each of freeze-dried GCBJ powder, 5alpha-cyprinol and 5alpha-cyprinol sulfate for 19 days. After treatment, the relative ratio of liver and kidney weight to body weight, the concentrations of RBC, hemoglobin and hematocrit in the blood, the levels of aspartate amino transferase, alanine amino transferase, alkaline phosphatase, blood urea nitrogen and creatinine in the plasma, and the levels of urinary urea nitrogen and creatinine in the urine were significantly increased. Body weight of rats and the levels of Na+, K+, Ca2+ in the urine were significantly decreased, especially for groups treated with GCBJ powder and 5alpha-cyprinol sulfate. Pathological examination of liver and kidney also showed cell enlargement and lesions, especially for groups treated with GCBJ powder and 5alpha-cyprinol sulfate. The grass carp bile juice exhibited significant toxicity, and the short-term toxicity of 5alpha-cyprinol sulfate and GCBJ powder was similar to each other. Renal but not hepatic failure induced by grass carp bile juice could be prevented by pentoxifylline.