Vitamin D is an essential nutrient, and there is a growing appreciation of its clinical significance. In addition, there has recently been a lot of discussion as to the best method of measuring this analyte in serum.
To develop a robust and rapid LC-MS/MS assay to detect 25-hydroxyvitamin D3 and D2 in serum. Additionally, to determine the levels of 25-hydroxyvitamin D in patients served by San Francisco General Hospital.
We used a 3200 QTrap® LC-MS/MS from Applied Biosystems coupled with an Agilent 1200 LC and a Luna C18, 2.0 x 50mm, 5µm column from Phenomenex maintained at 60oC. Gradient elution with 1% formic acid in water and 0.5mM ammonium acetate, 0.1% formic acid in methanol at 0.4 mL/minute had a run-time of 5 minutes. Liquid-liquid extraction was performed with n-heptane on 250 µL of serum. The MS/MS operates with an atmospheric pressure chemical ionization (APCI) source using multiple reaction monitoring and an enhanced ion scan in an information-dependent acquisition mode (MRM-IDA-EPI). We used one MRM transition for each of the 2 analytes and for the deuterated internal standard.
For both analytes, extraction recovery was around 70% and ion suppression was <30%. Calibrators ranged from 0 to 95 ng/mL for D3 and 0 to 49 ng/mL for D2 with concentrations verified using the National Institute of Standards and Technology Standard Reference Material. The assay was linear between these concentrations. Quality controls were at 15 and 35 ng/mL. Within and between day precision studies yielded CVs of <10% and <12% for both D3 and D2 respectively. During a study period of 3 months, 40% of our patients were deficient in total Vitamin D (<20 ng/mL), 35% were insufficient (20-29 ng/mL), and 25% were sufficient (>30 ng/mL). The highest recorded value was 59 ng/mL. Our investigations are ongoing.
We have developed a robust and rapid LC-MS/MS assay to detect 25-hydroxyvitamin D3 and D2. Additionally, the patients served by San Francisco General Hospital have significantly low serum vitamin D levels that may be reflective of the status of the patients served by this county hospital.