Simultaneous Quantification of Vitamin D-2 , Vitamin D-3 , and their 25-Hydroxy Metabolites in Human Plasma by High Performance Liquid Chromatography

Vitamin D plays an important role in protecting from bone and potentially other metabolic diseases in humans [1-2]. Vitamin D exists in two forms: vitamin D-3 (cholecalciferol) synthesized from 7-dehydrochlesterol in the skin by the action of UVB radiation and vitamin D-2 (ergocalciferol) derived from plant/yeast by irradiation of ergosterol. Vitamin D made in the skin or supplied in the diet is metabolized in the liver to 25-hyroxyvitamin D, which is the major circulating form of vitamin D and best indicates vitamin D status. 25-hydroxyvitamin D is further metabolized in the kidney to 1, 25 dihydroxyvitamin D the active metabolite [3,4].


Introduction
Vitamin D plays an important role in protecting from bone and potentially other metabolic diseases in humans [1][2]. Vitamin D exists in two forms: vitamin D-3 (cholecalciferol) synthesized from 7-dehydrochlesterol in the skin by the action of UVB radiation and vitamin D-2 (ergocalciferol) derived from plant/yeast by irradiation of ergosterol. Vitamin D made in the skin or supplied in the diet is metabolized in the liver to 25-hyroxyvitamin D, which is the major circulating form of vitamin D and best indicates vitamin D status. 25-hydroxyvitamin D is further metabolized in the kidney to 1, 25 dihydroxyvitamin D the active metabolite [3,4].
Deficiency of vitamin D is common with inadequate sun exposure. Recent studies reported that 30-50% of children and adults in many countries had 25 hydroxyvitamin D levels below 20 ng/mL [5].

Apparatus
Chromatography was performed on Waters Alliance HPLC 2695 (Waters Associates Inc, Milford, MA, USA) consisting of quaternary pump, auto sampler, column thermostat, and photodiode array detector set at 265 nm. A reversed-phase Zorbax C18 (4.6 x 250 mm, 5-μm) column protected by a guard pak pre-column C18 (4.6 x 20 mm, 5µm) insert was used for separation. The data were collected with a Pentium IV computer using Empower Chromatography Manager Software.

Chemicals and reagents
All chemicals were of analytical grade unless stated otherwise. Vitamin D-2 and vitamin D-3 were purchased from Acros Organics, New Jersey, USA, whereas 25(OH) VD-2 and 25(OH) VD-3 were purchased from Sigma-Aldrich, USA. Benzopyrene (100 µg/mL solution in dichloromethane) was purchased from Ultra scientific, Kingstown, RI, USA. 2-Proponol, methanol, hexane, and acetonitrile were purchased from Fisher Scientific, Fairlawn, NJ, USA. Water for HPLC was prepared by reverse osmosis and further purified by using synergy water purification system (Millipore, Bedford, MA, USA). Drug-free human plasma was obtained from the blood bank of King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia, after approval of the institution Research Ethics Committee.

Chromatographic conditions
The mobile phase consisted of solvent-A: acetonitrile, methanol, and water (35:50:15, v/v) and solvent-B: actonitrile: methanol: water (pH=3.0 adjusted with phosphoric acid) (10:80:10, v/v) and was used in a gradient elution mode. The gradient started with solvent-A at a flow rate of 1.2 ml/min for 19 minutes and switched immediately to solvent B at a flow rate of 1.5 ml/min for 25 minutes.

Standard and control solutions
VD-2, VD-3, 25(OH) VD-2, and 25(OH) VD-3 stock solutions were prepared in methanol (100 μg/mL) and stored at -20ºC. Working solutions were prepared in human plasma (100 ng/mL). Eight calibration standards were prepared in human plasma in the range of 5-100 ng/mL. Benzopyrene (IS) working solution was prepared in methanol (0.25 µg/ml). Three quality control samples were prepared: 15(low), 50 (middle), and 90 (high) ng/mL. The solutions were vortexed for one minute, and then 1.5 ml aliquots were transferred into 7 mL glass culture tubes and stored at 4°C until used.

Sample preparation
100 µL of the IS working solution was added to 1.5 ml of plasma samples, calibration standards, or quality control samples in a 7 mL culture tubes and vortexed. A mixture of methanol and 2-proponol (9:1, v/v) was added. After further vortexing, the samples were extracted twice with 3 mL hexane and centrifuged at 4000 rpm (8°C) for 10 min., and the clear supernatant layer was transferred to a clean culture tube and dried under gentle steam of nitrogen at 40°C. The residue was reconstituted in 150 µL methanol containing water 4.0%. 100 µL of the clear solution was injected into the HPLC system.

Calculation
In order to correct for endogenous 25-hydroxyvitamin D-3 level in "blank" plasma, we used the difference in peak area ratios between each consecutive concentration as the response (rather than the peak area ratio). The difference in peak ratio was plotted against the concentration. For the other three compounds the peak area ratios was plotted against concentration. Bias (%) was calculated as the difference between measured and nominal concentration divided by nominal level times 100, whereas coefficient of variation (100%) was calculated as standard deviation divided by mean concentration times 100.

Method validation
The method was validated according to US FDA guidelines for industrial bio analytical method validation [14]. As shown in Table 1, the calibration curves for the determination of VD-2, VD-3, 25(OH) VD-2, and 25(OH) VD-3 levels in human plasma were linear over the range 5-100 ng/mL. The linearity of this method was statistically confirmed. For each calibration standard, the concentrations were back-calculated from the equation of the linear regression. Precision and bias were determined in five replicates of three concentrations (15, 50 and 90 ng/mL). The results are presented in Table 2. The largest coefficient of variation and bias of the assays were 9.7% and 13.1%, respectively. The analytical recoveries at three different levels (1.5, 50 and 90 ng/mL) were determined by comparing peak areas of VD-2, VD-3, 25(OH) VD-2, 25(OH) VD-3, and the IS prepared in methanol    and to those prepared in plasma. Mean recoveries were 97%, 91%, 80%, and 81% for 25(OH) VD-3, 25(OH) VD-2, VD-2, VD-3, respectively, and 65% for the IS. The limit of detection was 2 ng/mL for 25(OH) VD-3, and 25(OH) VD-2, and 3 ng/mL for VD-2 and VD-3, whereas the limit of quantification was 5 ng/mL for all components. Table 3 summarizes the results of stability studies of the two forms of vitamin D and their metabolites.

Stability
Freeze and thaw stability: Stability of VD-2, VD-3, 25(OH) VD-2, and 25(OH)VD-3 was determined over three freeze and thaw cycles. Fifteen aliquots of each of two QC samples (15 ng/mL and 90 ng/mL) for the four compounds were stored at -20ºC. After 24 hours, all aliquots were left to thaw unassisted at room temperature. When completely thawed, 5 aliquots of each QC sample were analyzed. The other aliquots were returned to -20ºC and kept for 24 hours. The same procedure was repeated three times. The concentrations in freeze-thaw samples were compared with the concentration of freshly prepared and analyzed samples.
Stability in processed samples: Fifteen aliquots of each of two QC samples (15 ng/mL and 90 ng/mL) for the four compounds were processed. Five aliquots of each QC sample were analyzed immediately.
The other aliquots were analyzed after being stored at room temperature for 24 hours or at -20ºC for 48 hours.
Stability in unprocessed samples: Thirty aliquots of each of two QC samples (15 ng/mL and 90 ng/mL) for the four compounds were prepared. Five aliquots of each QC sample were analyzed immediately. Five aliquots of each QC sample were allowed to stand on the benchtop for 24 hours at room temperature before extraction. Five aliquots of each QC sample were stored at -20ºC for 1, 3, 5, or 10 weeks before analysis.

Application to volunteer samples
Plasma samples from volunteers in a clinical trial were processed according to the method and subjected to HPLC analysis. Figure 3 depicts a chromatogram of sample collected from a representative volunteer. Over 200 plasma samples were analyzed by this method without any significant loss of resolution. No change in column efficiency or increase in backpressure was observed.

Discussion
The aim of the study was to develop a reliable method for simultaneous analysis of the two forms of vitamin D and their metabolites in human plasma. The method developed is well suited for routine application in the clinical laboratory because of a simple extraction procedure and easy to handle readily available LC system. We used benzopyrene as IS, despite the fact that it does not have a similar structure to the analytes because it is well separated from endogenous components and potential interfering plasma substances and is characterized by consistent recovery under applied conditions. In regard to stability, the results showed that VD-2, VD-3, 25(OH)VD-2, and 25(OH)VD-3 are stable at least for 10 weeks at -20°C (94-103%) and after three cycles of freeze at-20°C and thaw at RT (95-106%).
Since the level of 25(OH) VD-2 and 25(OH)VD-3 in human plasma is considered an important parameter in determining health status, many manufacturers developed commercial kits. However, for mass-scale analysis, such kits may not be affordable. The described assay offers simultaneously determine the levels of the two forms of vitamin D and their 25-hydroxy metabolites in human plasma/serum simultaneously in an affordable way.

Conclusions
The described HPLC method is simple, precise, and accurate for the simultaneous measurement of vitamin D-2, vitamin D-3, and their 25-hydroxy metabolites in human plasma. The assay was applied to monitor stability of these compounds under various condition encountered in the clinical laboratory. Further, the assay was successfully applied to determine the levels of these compounds in plasma samples from volunteers for clinical study.