Quantification of xylitol in foods by an indirect competitive immunoassay

Sugar alcohols are widely used as food additives and drug excipients. d-Xylitol (INS 967), an important five-carbon sugar alcohol, is a natural constituent of many fruits and vegetables. The critical reagent for an immunoassay of haptens is the requirement of hapten-specific antibodies. Here, affinity-purified xylitol-specific antibodies generated earlier [Sreenath, K.; Venkatesh, Y. P. Reductively aminated D-xylose-albumin conjugate as the immunogen for generation of IgG and IgE antibodies specific to D-xylitol, a haptenic allergen. Bioconjugate Chem. 2007, 18, 1995-2003] have been utilized for developing an indirect competitive ELISA for xylitol. With xylitol-BSA conjugate as the coating antigen, a working range of 5-400 ng of xylitol could be determined in the immunoassay; the limit of detection was 1 ng of xylitol. Onion (Allium cepa) and strawberry (Fragaria nilgerrensis) were selected as the food sources containing D-xylitol. The amount of D-xylitol was found to be 12.6 and 44 mg/100 g fresh weight of onion and strawberry, respectively, and the results are in good agreement with the reported values by HPLC and GC. The recovery analyses showed that added amounts of D-xylitol were recovered fairly accurately with recoveries in the range of 89.2 to 94.9% in the case of onion, and 88.4 to 95.9% in the case of strawberry. The indirect competitive ELISA for xylitol quantification is a simple method using a 3 kDa ultrafiltrate of whole food extract, and does not require extensive sample preparation and derivatization as in the case of GC and HPLC analyses. This is the first immunoassay developed for the sugar alcohol, xylitol.