The Scientific World Journal Volume 2012, Article ID 359721, 8 pages doi:10.1100/2012/359721 The cientificWorldJOURNAL Research Article A Novel HPLC Method for the Concurrent Analysis and Quantitation of Seven Water-Soluble Vitamins in Biological Fluids (Plasma and Urine): A Validation Study and Application Margherita Grotzkyj Giorgi, 1 Kevin Howland, 2 Colin Martin, 3 and Adrian B. Bonner 1 1 Centre for Health Services Studies, University of Kent, Canterbury, Kent CT2 7NF, UK 2 Department of Biosciences, University of Kent, Canterbury, Kent CT2 7PD, UK 3 School of Health, Nursing and Midwifery, University of the West of Scotland, Ayr Campus, Ayr KA8 0SX, Scotland, UK Correspondence should be addressed to Margherita Grotzkyj Giorgi, mg209@kent.ac.uk Received 10 October 2011; Accepted 30 November 2011 Academic Editors: D. Agbaba and M. Mahmoud Copyright © 2012 Margherita Grotzkyj Giorgi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. An HPLC method was developed and validated for the concurrent detection and quantitation of seven water-soluble vitamins (C, B 1 ,B 2 ,B 5 ,B 6 ,B 9 ,B 12 ) in biological matrices (plasma and urine). Separation was achieved at 30 ◦ C on a reversed-phase C18- A column using combined isocratic and linear gradient elution with a mobile phase consisting of 0.01% TFA aqueous and 100% methanol. Total run time was 35 minutes. Detection was performed with diode array set at 280 nm. Each vitamin was quantitatively determined at its maximum wavelength. Spectral comparison was used for peak identification in real samples (24 plasma and urine samples from abstinent alcohol-dependent males). Interday and intraday precision were <4% and <7%, respectively, for all vitamins. Recovery percentages ranged from 93% to 100%. 1. Introduction Water-soluble vitamins include B group vitamins (B 1 ,B 2 , B 3 ,B 5 ,B 6 ,B 9 ,B 12 ) and ascorbic acid (vitamin C). Vitamins are micronutrients that are essential to life, and many of them play an important role in regulation of brain functioning (e.g., vitamin B 1 deficiency causes biochemical brain lesions that causes Wernicke’s encephalopathy, an acute neuropsychiatric condition often observed in chronic alcohol abusers [3]). The reference methods for vitamins analysis in bio- logical fluids are often based on time-consuming micro- biological assays that may lack specificity [4]. In addition, vitamin extraction involves pretreatment through complex chemical reactions followed by individual methods for the determination of each vitamin. During the last decades, there has been an increasing interest for the simultaneous determination of vitamins. Thus, various analytical methods have been developed over recent years [5–9]. A number of recent studies have focused on validation of analytical methodologies for multivitamins analysis but the vast major- ity of them applied their methods to analysis of food matrices, drinks, polyvitaminated premixes, and vitamins supplements [10, 11]. On the other hand, only a relatively small number of experimental studies focused on validation of analytical methodologies for multivitamins analysis in biological samples (blood and urine) and with limited results in terms of lengthy sample preparation steps and method’s robustness and reproducibility [8]. Because of this lack of a robust and validated analytical test for multivitamin analysis in biological samples in routine clinical assessment and in those investigations where a timely and robust analytical method is needed, the aim of this study was to develop and validate a novel HPLC methodology for rapid detection and quantitation of seven water-soluble vitamins (B 1 ,B 2 , B 5 ,B 6 ,B 9 ,B 12 , C) in biological fluids (plasma and urine). The validated method was then applied to quantify water- soluble vitamins in plasma and urine samples obtained from 24 abstinent alcohol-dependent males.