Rapid resolution liquid chromatography method development and validation for simultaneous determination of homocysteine, vitamins B6, B9, and B12 in human serum

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 mu m; 150 mm x 4.6 mm) maintained at 28 degrees 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.