Development of free 25-hydroxyvitamin D3 assay method using liquid chromatography-tandem mass spectrometry

Abstract

The free hormone hypothesis has triggered controversies regarding the measurement of free vitamin D metabolites, such as free 25-hydroxyvitamin D (25(OH)D), as a suitable indicator for total vitamin D for clinical use. This issue can be addressed by developing a precise and accurate method for free 25(OH)D measurement. In the present study, a novel assay method for free 25(OH)D3 based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Sample preparation first involved ultrafiltration to remove vitamin D-binding protein-bound and albumin-bound 25(OH)D, followed by extraction with a column, derivatization, evaporation, dissolution, and injection into the LC-MS/MS system. The coefficient of variation of repeatability and reproducibility obtained were 3.8-4.5% and 4.8-5.9%, respectively. Satisfactory linearity (r=0.999) was obtained up to 80 pg/ml. The lower quantification limit was 0.97 pg/ml and the S/N ratio on the peak of 1.0 pg/ml sample was 24.8 (which is more than the acceptable value of 10). The recovery rate was between 84.5 and 92.4% with a negligible matrix effect (94.5-104.9%). Levels of free 25(OH)D3, but not total 25(OH)D3, in the serum of the patients with chronic kidney disease (CKD) and hepatic cirrhosis (HC) were substantially lower than those in healthy subjects. The correlation coefficient between total and free 25(OH)D3 was 0.738 in all samples, while the linear regression equations were different between the patients with CKD and HC. In conclusion, LC-MS/MS assay for free 25(OH)D3 might be useful to evaluate high-throughput methods, including ELISA.

Keywords: free 25(OH)D3; liquid chromatography-tandem mass spectrometry (LC-MS/MS); ultrafiltration; vitamin D metabolites; vitamin D-binding protein (DBP).

Figure 1. Validation of LC-MS/MS assay for measuring free 25(OH)D₃ concentration

(A) Calibration curves were generated using four standards corresponding to 1.1, 10.975, 21.95, and 43.9 pg/ml of free 25(OH)D₃ based on the ratio of the peak area of each standard to that of the corresponding IS. (B) The linearity was evaluated according to the CLSI guideline using four samples prepared by adequately diluting the JeoQuant™ calibrator. (C) The LLoQ was determined as the lowest concentration with less than 20% CV according to the repeated measurement (n=10, duplicate for 5 days) of five samples prepared by serially diluting the JeoQuant™ calibrator. The inside figure indicates the peak obtained by LC-MS/MS for 1 pg/ml of free 25(OH)D₃ (S/N ratio: 24.8).

Figure 2. Distribution of total and free 25(OH)D₃ levels in the patients with CKD and HC and PRG women

Box plots indicate the distribution of (A) total and (B) free 25(OH)D₃ levels in serum obtained from the patients with CKD (n=15) and HC (n=15), as well as PRG (n=15). Mann–Whitney U-test was used for data analysis between NOR (n=15) and test individuals; *P<0.05.

Figure 3. Correlation between total and free 25(OH)D₃ levels

The correlation between total and free 25(OH)D₃ levels were evaluated for subjects from all groups (NOR, CKD, HC, and PRG). The linear regression equation and the correlation of coefficient of each group are indicated under the correlation diagram.

Figure 4. Correlation between calculated free 25(OH)D and LC-MS/MS-measured total and free 25(OH)D₃ levels

Free 25(OH)D levels calculated using the formula described by Bikle et al. [19] were compared with free and total 25(OH)D₃ measured by LC-MS/MS for a part of subjects (n=34) whose DBP and Alb levels in serum were known. Correlations were evaluated between (A) free and total 25(OH)D₃ levels, (B) calculated 25(OH)D and total 25(OH)D₃ levels, and (C) calculated 25(OH)D and 25(OH)D₃ levels.