Evaluation of High Performance Liquid Chromatography and Liquid Chromatography-Tandem Mass Spectrometry Methods for 25 (OH) D3 Assay.

BACKGROUND

This study was designed to compare the performances of HPLC (High Performance Liquid Chromatography) and LC-MS/MS (Liquid Chromatography-Tandem Mass Spectrometry) methods in 25 (OH) D3 testing.

METHODS

This study is comprised of 306 randomly chosen plasma samples from the subjects who applied for routine measurement of 25 (OH) D3. Plasma 25 (OH) D3 levels were quantified using HPLC and LC-MS/MS. The LC-MS/MS method was used as the reference method. The linearity, precision, carry-over, limit of blank, limit of detection (LoD), and comparison studies were done for method validation. Accuracy was tested using external quality assurance samples.

RESULTS

Coefficients of variation for both methods were at around 10.0%. The HPLC and LC-MS/MS assays were linear over the working range from 5.0 to 100 ng/mL (r > 0.99). The HPLC assay showed a higher LoD compared to LC-MS/MS (5.1 vs. 1.6 ng/mL, respectively). Results from external quality assurance samples were within ? 1 SD range for both methods. The comparison study revealed good correlation between HPLC and LC-MS/MS methods (y = 1.054x - 1.981 with a small mean bias (-0.953) (r = 0.9752)), when all samples were included, regardless of their 25 (OH) D3 levels. However, the correlation was poor for samples with 25 (OH) D3 concentrations lower than 10 ng/mL.

CONCLUSIONS

Both methods have acceptable performance characteristics for use in clinical diagnostic applications. A good comparability was obtained between HPLC and LC-MS/MS methods. However, LoD of HPLC assay was higher and there was a poor correlation between the two systems for samples with 25 (OH) D3 concentrations below 10 ng/mL, showing that LC-MS/MS system is more successful in measuring samples with low 25 (OH) D3 concentration.