1. Computer-assisted HPLC method development and validation for the routine analysis of 25-Hydroxyvitamins D 2 & D 3 “A comparative-assay study” Mohamed A. Abu el Maaty (1), Rasha S. Hanafi (2), Mohamed Z. Gad (1) (1) Department of Biochemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo, Egypt (2) Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo, Egypt Introduction Objectives Materials & Methods Results Conclusions Bibliography The vitamin D deficiency pandemic has been attracting substantial attention in the past couple of years. The mere irrefutable fact that approximately 1 billion people worldwide aren’t getting enough of the “sunshine” vitamin and thus are deficient is enough to drive medical professionals to take a stand [1]. Recent advances in vitamin D research have yielded an array of techniques for the assessment of this vitamin’s status in humans by quantifying 25- Hydroxyvitamin D [25(OH)D], the agreed upon biomarker [1] however, such advances have been rendered clinically unappreciated due to the numerous reports of abundant variability among them [2-4]. While chromatographic techniques have been identified as the “Gold Standard” for this task, not all could elute both forms of the vitamin which is a necessity for an overall clinical assessment of vitamin D status [5-7]. 1. Holick, M.F., Vitamin D deficiency. N Engl J Med, 2007. 357(3): p. 266-81. 2. Barake, M., et al., 25-hydroxyvitamin D assay variations and impact on clinical decision making. J Clin Endocrinol Metab, 2012. 97(3): p. 835-43. 3. Heijboer, A.C., et al., Accuracy of 6 routine 25-hydroxyvitamin D assays: influence of vitamin D binding protein concentration. Clin Chem, 2012. 58(3): p. 543-8. 4. Lai, J.K., et al., Variability in vitamin D assays impairs clinical assessment of vitamin D status. Intern Med J, 2012. 42(1): p. 43-50. 5. Wang, Z., et al., Simultaneous measurement of plasma vitamin D(3) metabolites, including 4beta,25-dihydroxyvitamin D(3), using liquid chromatography-tandem mass spectrometry. Anal Biochem, 2011. 418(1): p. 126-33. 6. Brunetto, M.R., et al., HPLC determination of Vitamin D(3) and its metabolite in human plasma with on-line sample cleanup. Talanta, 2004. 64(5): p. 1364-70. 7. Kand'ar, R. and P. Zakova, Determination of 25-hydroxyvitamin D3 in human plasma using HPLC with UV detection based on SPE sample preparation. J Sep Sci, 2009. 32(17): p. 2953-7. 1.To use HPLC method development software, DryLab ®, to develop a method that elutes both forms of the vitamin along with their mono- hydroxyl metabolites, in a commercially appealing run-time to provide enough incentive for analysts to adopt the method and utilize it for routine work. 2.To use this method, after validating it, as well as a commercially available ELISA kit, to assess the vitamin levels of recruited individuals and to identify possible variability between the assays. Figure 1: Method development chromatograms. Conditions from top to bottom: t G = 50 minutes T = 30°C ; t G = 50 minutes T = 60°C ; t G = 100 minutes T = 30°C ; t G = 100 minutes T = 60°C. Figure 2: The two dimensional resolution map of T versus tG generated by DryLab ®. While some sort of separation could be obtained by employing any condition provided here, the best resolution is achieved by utilizing the conditions offered by the red area of the map, as opposed to the blue areas which offer the worst resolutions. Colors in between however, offer a variety of resolution options. Figure 3: An experimental chromatogram of a standard mixture of the four analytes of interest (bottom) versus the predicted one proposed by DryLab ® (top). Peak comparisons were made based on retention factors. Error in prediction ranged from 0.2% to 11.9%. Figure 4: Chromatograms of both spiked (top) and unspiked (bottom) plasma samples. Vitamins D 2 and D 3 are naturally undetectable as indicated in the bottom chromatogram. The described method permits accurate and efficient quantitation of both forms of the vitamin’s metabolite independently and in a timely fashion making it’s extrapolation to service-providing laboratories possible. Advantages of this method include the ease of it’s application as well as the utilization of HPLC as opposed to LC-MS, which is usually only present in high-end research laboratories and requires completely versed operators to use it. Vitamin D analysis remains a challenge whose difficulty is increasing exponentially in spite of the efforts made world-wide to optimize and standardize it. Figure 5: Deming regression comparing total 25(OH)D levels, in ng/mL, of 46 samples analyzed by both assays. Statistical analysis rendered the two assays incomparable. Slope: -6.4 y-intercept: 147.7 r = - 0.22 Method validation: The described method was validated by determining the limits of detection and quantitation (2 and 5 ng/mL respectively for either metabolite), linearity was tested for up to 60 ng/mL for each metabolite, extraction yield for OH-D 2 and OH-D 3 was 53.5 ± 3.2 and 54.9 ± 3.4 % respectively, Intra- and Inter-day precision (% coefficient of variation) and accuracy (% bias) for OH-D 2 was 1.0-3.1% and -1.6-5.2% whereas for OH-D 3 1.1-3.8% and 0.8-4.1%. Consumables: Cholecalciferol Ergocalciferol 25-Hydroxycholecalciferol 25-Hydroxyergocalciferol Methanol (HPLC grade) Acetonitrile (HPLC grade) Sigma-Aldrich Chemie GmbH, Steinheim (Germany) Equipment & Software: HPLC- Thermo Finnigan Spectrasystem ® : 1) Pump: Spectrasystem P2000 2) Detector: Spectrasystem UV3000 3) Autosampler: Spectrasystem AS3000 4) Acquisition software: Chromquest 4.2 data system 5) Column: BDS Hypersil C18 (150 x 4.6 mm – 5µm) HPLC method development software: DryLab ® 2000 and PeakMatch ® [Molnár Institute for Applied Chromatography, Berlin, Germany] Figure 6: Mean 25(OH)D concentrations of both female (n=36) and male (n=10) participants of mean ages (SD) 54.7 (3.2) and 25.8 (2.1) years respectively. Chromatographic conditions: Mobile phase A: 0.02M phosphate buffer pH 2.6 Mobile phase B: methanol : acetonitrile (1:1) Gradient: 78-95% B t G : 25 minutes Temperature: 57°C