Water-soluble vitamins stability by robust liquid chromatography-mass spectrometry

Introduction: The measurement of water-soluble vitamins is essential to diagnose and monitor various vitamin deficiencies. Establishing stability limits for these vitamins is crucial to ensure accurate laboratory testing. This study aimed to assess the stability of commonly measured water-soluble vitamins under different storage conditions to improve the accuracy of water-soluble vitamin measurement. Methods: The stabilities of thiamine, riboflavin, nicotinamide, pantothenic acid, pyridoxic acid and pyridoxal, biotin, 5-methyltetrahydrofolic acid (5-MTHF), and ascorbic acid were measured using liquid chromatography-tandem mass spectrometry. We investigated three pre-analytical factors: the effect of different temperatures and time durations on serum stability, variation between serum and plasma samples, and the impact of transferring samples to an ice bath before serum separation. We evaluated stability based on differences from the baseline. Results: Thiamine, pyridoxal, and ascorbic acid in serum exhibited instability at room temperature and 2–8℃. Riboflavin and 5-MTHF in serum were only stable for up to 48 and 72 h at 2–8℃. However, when stored at -20℃, all water-soluble vitamins remained stable for up to 72 h, whereas at -80℃, stability was maintained for up to 7 days. All vitamins in whole blood, except nicotinamide, were stable for up to 2–4 h when stored in an ice bath. Conclusions: Water-soluble vitamins, such as thiamine, riboflavin, pyridoxal, and ascorbic acid, are unstable at room temperature and 2–8℃. All vitamins were stable for up to 7 days and stored at -80℃. The ice bath improved the stability of whole blood samples before centrifugation. Thus, laboratories should ensure appropriate storage conditions to maintain pre-analytical quality for vitamin measurements.