Validation of Pepsin-Assisted Extraction and Immunoaffinity-HPLC/DAD Analysis for Vitamin B12 in Seafood

Vitamin B₁₂ exists in free and bound forms in foods. Use of proteolytic enzymes is recommended for vitamin B₁₂ extraction from protein-rich foods, but information applicable to seafood is very limited. In this study, pepsin-assisted extraction (PE) coupled with immunoaffinity-HPLC/DAD (I/H/D) was validated and applied for determination of total vitamin B₁₂ contents (sum of free and bound) in four types of seafood with different matrices (littleneck clam, blue crab, mackerel, and squid). PE-I/H/D showed good specificity and linearity (R² =0.9992). Limits of detection and quantification of PE-I/H/D were about twice as high (0.015 and 0.106 ㎍/100 g, respectively) as those of non-PE-I /H/D. Both PE- and non-PE-I/H/D showed good accuracy based on higher than 95% recoveries from the certified reference material, BCR-487. Precision of PE-I/H/D was acceptable based on AOAC guidelines, although it decreased upon application of PE (coefficient variations of repeatability and reproducibility: 9.4 and 13.6% for PE-I/H/D and 6.0 and 10.5% for non-PE-I/H/D, respectively). Free and total vitamin B12 contents of the four types of seafood were analyzed by using the validated PE-I/H/D. The total vitamin B₁₂ contents of littleneck clam (55.16 ㎍/100 g) and mackerel (5.60 ㎍/100 g) were significantly higher than corresponding free vitamin B₁₂ contents (46.91 and 4.64 ㎍/100 g, respectively), indicating that 18~20% of total vitamin B₁₂ exists in bound form in littleneck crab and mackerel. On the other hand, there was no significant difference between free and total vitamin B12 contents in blue crab and squid within the ranges of 6.09~6.87 and 2.39~2.45 ㎍/100 g, respectively. The results of this study suggest that PE-I/H/D assay would be effective to release vitamin B₁₂ from protein-rich seafood tissues and to produce acceptable selectivity, sensitivity, accuracy, and precision of vitamin B₁₂ analysis, indicating PE-I/H/D is applicable for the determination of vitamin B12 at low levels in seafood.