Rapid method for the determination of total 5-methyltetrahydrofolate in blood by liquid chromatography with fluorescence detection.
A liquid chromatographic method is described for the determination of total 5-methyltetrahydrofolate (5-MTHF) in whole-blood samples. The method was applied to a survey of whole-blood total 5-MTHF levels of women at child-bearing age. To determine whole-blood total 5-MTHF content, a whole-blood sample was frozen and thawed to break red blood cells and the 5-MTHF polyglutamates were released and hydrolyzed into 5-MTHF monoglutamate by endogenous polyglutamates hydrolase in the plasma. In brief, an aliquot of 0.1 ml whole-blood sample was mixed with 0.3 ml 57 mmol/l ascorbic acid and incubated at 37 degrees C for 60 min, then diluted with 0.6 ml buffer solution (0.2 mol/l potassium phosphate dibasic and 30 mmol/l mercaptoethanol, pH 8.5). After the sample was heated at 100 degrees C for 10 min and centrifuged, the supernatant was analyzed by reversed-phase liquid chromatography with fluorescence detection. The recoveries from spiked samples were from 95 to 105% with within-day and day-to-day relative standard deviations less than 6.5%. The detection limit was estimated to be 30 nmol/l based on three times the noise level (peak to peak). Application of the method to a survey of whole-blood total 5-MTHF levels of women at child-bearing age showed that the method was reliable and suitable for the determination of blood total 5-MTHF.
Lu
Wenhong
W
The Central Laboratory, Shantou University Medical College, Guangdong, China. whluo@stu.edu.cn
Li
Hui
H
Zhang
Yuan
Y
Ang
Catharina Y W
CY
D002853:Chromatography, Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013763:Tetrahydrofolates / Q000097:blood*
D013763:Tetrahydrofolates; C005984:5-methyltetrahydrofolate