Determination of vitamin D levels in human biological specimens has gained a high relevance over the last decades, essentially because low levels have been associated with several biological disorders. In fact, vitamin D deficiency has become a worldwide health concern covering all ages and genders. The storage of biofluids has to be considered for determination of vitamin D and metabolites in order to fully preserve matrices status. This study attempts to evaluate lyophilization of serum and plasma as a pre-processing step for sample storage prior to quantitative analysis of vitamin D-3 and its main hydroxylated metabolites-25(OH)D-3, 24,25(OH)(2)D-3 and 1,25 (OH)(2)D-3. The protocol including sample lyophilization was characterized in terms of analytical features and compared to the same method, based on SPE-LC-MS/MS, without lyophilization. Sensitivity, precision and accuracy were not affected when we operated with lyophilized serum and plasma and results provided by a set of twenty-four serum samples from DEQAS (Vitamin D External Quality Assessment Scheme) were in agreement with reported concentrations for 25(OH)D-3 and 1,25(OH)(2)D-3. A stability study programmed for 9 months allowed ensuring that the concentration of vitamin D-3 and metabolites in lyophilized serum and plasma stored at room temperature was not affected during this period. This research has demonstrated that the quantitation of target metabolites is not under the influence of lyophilization. Therefore, including lyophilization prior to analysis could reduce shipment and storage costs, avoid delays of sample processing, and increase the stability of the target analytes due to an effective quenching process.

Tetanus toxoids (TT) commercially available for use in horses and livestock are commonly used to vaccinate elephants and rhinoceros that are in human care. Although recommendations for booster intervals have changed in human and horse protocols to reduce the risks associated with hyper-immunity (i.e. B-cell anergy and hypersensitivity reactions) these have generally not been adopted in zoo protocols. Additionally, there is no evidence to demonstrate commercial TT immunogenicity in rhinoceros. In this study, a preliminary analysis of rhinoceros antibody responses to TT was conducted, in addition to an exploration of the impact of various booster frequencies on antibody responses in elephant. Retrospective analysis of archived serum samples was conducted for 9 Asian elephants (Elephas maximus), 7 southern black (Diceros bicornis minor), one southern white (Ceratotherium simum simum), and two greater one-horned (Rhinoceros unicornis) rhinoceros. Pre-vaccination (baseline) samples and those following priming vaccination (rhinoceros only), annual and non-annual boosters were targeted. A commercially available competitive ELISA kit was used to quantify serum anti-TT antibodies. Average baseline and post-vaccination anti-tetanus antibody concentrations were greater in elephant (92 mg/L +/- 42, n = 3, N = 3; 125 +/- 76, n = 82, N = 9) than in rhinoceros (47 mg/L +/- 39, n = 8, N = 8; 44 mg/L +/- 37, n = 16, N = 7). Rhinoceros antibody concentrations did not differ markedly following vaccinations from their naturally acquired high pre-vaccination concentrations. Eight elephants demonstrated antibody maintenance for 3-5 years without a tetanus booster. Additionally, although five out of nine elephants developed local reactions consistent with delayed type IV hypersensitivity following some boosters, there was no association between high antibody concentrations and increased incidence of adverse reactions. In addition, no decrease in antibody concentrations was detected as a result of annual vaccination in elephants, though this does not entirely rule out potential for B-cell anergy.