| Objective:1.To establish the qualitative and quantitate method of Ion chromatography(IC)for detection of the thiocyanate in biological samples;2.To establish a liquid-liquid extraction,LC-MS/MS method of 2-aminothiazole-4-carboxylic acid(ATCA)in biological samples;3.To obtain the normal reference range of the thiocyanate and2-aminothiazoline-4-carboxylic acid in Taiyuan area healthy human;Method:1.VolunteersAfter signing an informed consent,124 volunteers(44 male and 75 female)from Taiyuan were recruited to join the experiment,who was 18 to 27,no medicine within past two weeks,no chronic illness.2.Specimen CollectingVolunteers were fasted for twelve hours and then was taken from the elbow vein.The blood was added to the blood collection tube containing no anticoagulant and anticoagulant heparin and EDTA.After coagulation,(5000r/min,5min),the supernatant was collected and stored at-80?for determination of thiocyanate and2-aminothiazoline-4-carboxylic acid.Six poisoning case specimen were collected from Shanxi Medical University Forensic Center.3.Extraction and analysisIon chromatography was used to measure the concentrations after precipitated by acetonitrile and extracted by deionized water.The qualitative analysis was based on retention time and add sample experiment.The quantitative analysis was based on external standard method and calibration curve.0.1mL internal standards 2-aminothiazoline-4-carboxylic acid-13C,15N2(ATCA-13C,15N2)was added to biological samples and extracted by water and determined by LC-MS/MS.The qualitative analysis was based on retention time and MRM ions.The quantitative analysis was based on internal standard method and working curve.4.Data analysisSPSS20.0 statistical software was used for variance analysis and rank and inspection.There was significant statistical differences between groups when P<0.05.Results:1.The calibration curves for thiocyanate was linear ranging from 125ng/m L~5000ng/mL and had a coefficient of determination(R~2)>0.9999.The limit detection and the limit quantification were found at 20ng/ml and 40.7ng/m L for thiocyanate,respectively.The intraday precision and interday precision were less than 10%.The normal range of 45 healthy male volunteers in serum levels of thiocyanate from64.1ng/mL to 943.4ng/mL,the mean value was 374.8±209.6ng/ml.The normal range of 75 healthy female volunteers in serum levels of thiocyanate from 109.7ng/m L to807.5ng/mL,the mean value was 395.8±145.9ng/mL.There was no statistical difference between male and female.The normal range of 45 healthy male volunteers in plasma levels of thiocyanate from 53.5ng/mL to 681.6ng/m L,the mean value was261.6±146.7ng/ml.The normal range of 78 healthy female volunteers in plasma levels of thiocyanate from 62.4ng/m L to 557.3ng/m L,the mean value was273.7±114.9ng/m L.There was no statistical difference between male and female.The normal range of 43 healthy male volunteers in whole blood levels of thiocyanate from40.7ng/mL to 548.9ng/mL,the mean value was 208.0±131.4ng/ml.The normal range of 79 healthy female volunteers in whole blood levels of thiocyanate from172.5ng/mL to 539.8ng/m L,the mean value was 222.7±105.3ng/m L.There was no statistical difference between male and female.2.The calibration curves for 2-aminothiazoline-4-carboxylic acid was linear ranging from 12.5ng/mL~1000ng/m L and had a coefficient of determination(R~2)>0.9999.The limit detection and the limit quantification were found at 1ng/m L and 5ng/m L for 2-aminothiazoline-4-carboxylic acid respectively.The intraday precision and interday precision were less than 10%.The normal range of 44 healthy male volunteers in serum levels of 2-aminothiazoline-4-carboxylic acid from101.6ng/mL to 385.1ng/m L,the mean value was 238.4±82.51ng/ml.The normal rangeof76healthyfemalevolunteersinserumlevelsof2-aminothiazoline-4-carboxylic acid from 86.8ng/mL to 381.0ng/mL,the mean value was 215.3±86.36ng/m L.There was no statistical difference between male and female.The normal range of 45 healthy male volunteers in plasma levels of2-aminothiazoline-4-carboxylic acid from 81.4ng/mL to 440.0ng/mL,the mean value was 236.1±85.36ng/ml.The normal range of 79 healthy female volunteers in plasma levels of 2-aminothiazoline-4-carboxylic acid from 81.6ng/m L to 387.7ng/mL,the mean value was 200.1±96.26ng/m L.There was no statistical difference between male and female.The normal range of 44 healthy male volunteers in whole blood levels of2-aminothiazoline-4-carboxylic acid from 22.6ng/mL to 163.1ng/mL,the mean value was 73.7±35.38ng/ml.The normal range of 79 healthy female volunteers in whole blood levels of 2-aminothiazoline-4-carboxylic acid from 25.5ng/mL to 177.0ng/mL,the mean value was 77.1±38.16ng/m L.There was no statistical difference between male and female.3.The range of 6 poisoning samples in whole blood levels of thiocyanate from1166ng/mL to 11966ng/m L,the mean value was 4872±4198ng/m L.The range of 6poisoning samples in whole blood levels of 2-aminothiazoline-4-carboxylic acid from318ng/mL to 3396ng/m L,the mean value was 1026±1169ng/mLConclusion:1.The method which analyzed thiocyanate in serum,plasma,blood by ion chromatography conductivity have high extraction recovery,specificity,high sensitivity and accuracy.The statistical results shows that the serum level of thiocyanate is higher than plasma level,the plasma level of thiocyanate is higher than blood level.The reference range of serum level of thiocyanate is from 64.1ng/mL to943.4ng/mL and the 95%reference value limit is 775.2ng/m L;plasma level of thiocyanate is from 53.5ng/mL to 681.6ng/mL and 95%reference value limit is477.6ng/mL;whole blood level of thiocyanate is from 41.7ng/m L to548.9ng/mL and95%reference value limit is 406.0ng/m L.2.The LC-MS/MS method can be used to detect 2-aminothiazoline-4-carboxylic acid.The qualitative analysis is based on retention time and ratio of ions.The quantitative analysis is based on internal standard method and working curve.This method have high extraction recovery,specificity,high sensitivity and accuracy,strong specificity,high recovery of high specificity and can meet the requirements of qualitative and quantitative analysis of normal population and poisoning cases.Results suggest that the serum and plasma level of 2-aminothiazoline-4-carboxylic acid have no statistical difference,but are significantly higher than whole blood level of 2-aminothiazoline-4-carboxylic acid.The reference range of serum level of2-aminothiazoline-4-carboxylic acid is from 86.84ng/mL to 385.1ng/m L and the 95%reference value limit is 366.32ng/mL;plasma level of 2-aminothiazoline-4-carboxylic acid is from 81.43ng/m L to 440.0ng/m L and 95%reference value limit is375.73ng/mL;whole blood level of 2-aminothiazoline-4-carboxylic acid is from22.64ng/mL to 177.0ng/m L and 95%reference value limit is136.7ng/mL.3.This study have established methods to detect two metabolites of cyanide in serum,plasma,whole blood and can meet can meet the requirements of qualitative and quantitative analysis of normal population and poisoning cases.We make the reference range so as to provide a certain basis for forensic medicine identification. |
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