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 RESEARCH ARTICLE
Year : 2013  |  Volume : 45  |  Issue : 2  |  Page : 159-167

Rapid resolution liquid chromatography method development and validation for simultaneous determination of homocysteine, vitamins B 6 , B 9 , and B 12 in human serum


Human Genome Centre, School of Medical Sciences, University Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia

Correspondence Address:
Siew Hua Gan
Human Genome Centre, School of Medical Sciences, University Sains Malaysia, 16150 Kubang Kerian, Kelantan
Malaysia
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Source of Support: Research University Grant (1001/PPSP/815094), Universiti Sains Malaysia, Conflict of Interest: None


DOI: 10.4103/0253-7613.108303

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Aim: Hyperhomocysteinemia and vitamins B 6 , B 9 , and B 12 deficiencies usually result in various neurological, vascular, ocular, renal, and pulmonary abnormalities. However, to date, there are no simultaneous detection methods available for determining homocysteine, vitamins B 6 , B 9 , and B 12 levels in various biological fluids. In this study, we aim to develop a new validated simultaneous detection method for all four compounds to save both cost and time of analysis. Materials and Methods: The mobile phase consisted of a mixture of methanol and 1-heptanesulfonic acid sodium salt (33:67) with 0.05% triethylamine. The pH of the entire mixture was adjusted to 2.3 and the flow rate was 0.5 mL/min. Separation was achieved using a C-18 column (5 μm; 150 mm × 4.6 mm) maintained at 28°C in a column oven and the detection was conducted at 210 nm. Results: The method was linear between 50 and 1600 ng/mL for all of the drugs. The limits of detection for homocysteine, vitamins B 6 , B 9 , and B 12 were 5, 5, 10, and 10 ng/mL, respectively, while the limits of quantification were 10, 10, 25, and 25 ng/L, respectively. The developed method achieved good precision and accuracy and complies with the Food and Drug Administration (FDA) requirements. Conclusion: The developed and validated method is suitable to be used for the routine analysis of homocysteine, vitamins B 6 , B 9 , and B 12 simultaneously in human serum.






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