DIETARY SUPPLEMENTS Determination of Pantothenic Acid in Foods by Optical Biosensor Immunoassay SIMON A. HAUGHEY ,ANTHONY A. O’KANE, and G. ANDREW BAXTER Xenosense Ltd, Unit 18, The Innovation Centre, Northern Ireland Science Park, Queen’s Rd, Queen’s Island, Belfast, N. Ireland, BT3 9DT ANDRAS KALMAN and MARIE-JOSÉ TRISCONI Nestlé Research Centre, PO Box 44, Lausanne 26, Switzerland HARVEY E. INDYK and GAYLENE A. WATENE Fonterra Waitoa, Cnr No. 1 Rd & SH26, Waitoa, New Zealand An optical biosensor inhibition immunoassay was developed using a specific pantothenic acid-binding protein for the quantitation of free pantothenic acid (vitamin B 5 ) in foodstuffs. Samples were prepared by a simple extraction procedure in buffer, and vitamin content was estimated against authentic calibrants in the same buffer. Performance parameters included a working range of 10–5000 ng/mL, a limit of detection of 4.4 ng/mL, precision relative standard deviation of 5.4–7.1% over a range of concentrations, and recoveries >95% in the matrixes tested. A wide range of foodstuffs, including National Institute of Standards and Technology reference samples, were tested in 3 independent laboratories and the results were compared with microbiological assay and liquid chromatography/mass spectrometry (LC/MS) methods. The results indicate that the biosensor technique is appropriate for the estimation of pantothenic acid in a wide range of foodstuffs. V itamins are routinely added as supplements to various health and nutritional foodstuffs. The European Union (EU) has recently published a draft proposal on the addition of vitamins, minerals, and certain other substances to foodstuff (1), which relates to the voluntary addition of these substances and specifies the purposes of fortification. Food supplements are governed by EU Directive (2002/46/EC; 2), which requires that maximum levels for vitamins and minerals intake are set. Because the concentration of vitamins in foods is usually established after processing, it is mandatory that compliance analysis is robust and accurate. Pantothenic acid (vitamin B 5 ) is a member of the B-group of water-soluble vitamins, and because of its high level of abundance in most foods, human deficiency of this vitamin is restricted to the severely malnourished. As an integral component of the critical enzymes coenzyme A (CoA) and acyl-carrier protein (ACP), it performs multiple roles within intermediate cellular metabolism. It is a viscous, hygroscopic liquid that is unstable to extremes of pH and heat. Pantothenic acid exists in foods largely in its free form, although is also present at lower levels in its protein-bound forms. It is usually added as a supplement to food in its more stable and biologically active D-enantiomeric calcium pantothenate form. Related compounds include pantethine, panthenol, 4-phosphopantothenic acid, CoA, and ACP. Methods for the determination of pantothenic acid in foods have been reviewed (3). The conventional and most widely used method to determine pantothenic acid levels is a microbiological assay (MBA) using Lactobacillus plantarum (4), which has been approved by AOAC INTERNATIONAL (5) as an official method for the determination of pantothenic acid in pharmaceutical formulations and infant formula. MBA methods require maintenance of sterile conditions, may suffer from matrix interference in complex foods and poor precision, and may require analysis time of 2–3 days. Enzyme-linked immunosorbent assays (ELISAs) have been developed and used to test foodstuffs (6–9) but are not commercially available; a radioimmunoassay (RIA) method has been developed for plasma analysis (10). These alternatives to MBA are sensitive and less labor-intensive, but require pantothenic acid to be released in its free form by enzymatic digestion. Liquid chromatography (LC; 11, 12) using the inherently poor spectral characteristics of the vitamin and gas chromatography (GC; 13) have shown relatively low specificity and sensitivity and have generally been restricted to supplemented foods. A more recent LC technique for total pantothenic acid using dual ion exchange solid-phase extraction cleanup and post-column derivatization with sensitive fluorescence detection has also been reported (14). A sensitive stable isotope dilution assay (SIDA) method has been developed (15, 16) using gas chromatography/mass spectrometry (GC/MS) for application to a range of foodstuffs and blood plasma. This approach has been further developed and simplified to eliminate derivatization requirements with 1008 HAUGHEY ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 4, 2005 Received November 9, 2004. Accepted by AP December 10, 2004. Corresponding author's e-mail: simon.haughey@xenosense.co.uk. Downloaded from https://academic.oup.com/jaoac/article-abstract/88/4/1008/5657487 by guest on 25 July 2020