A Rapid APCI-LC-MS/MS Method for the Measurement of 25OH-Vitamin D

J Duffy, C Webster

Department of Clinical Biochemistry and Immunology, Heartlands Hospital, Bordesley Green East, Birmingham, UK.

Introduction:  Vitamin D status is commonly assessed by measuring the concentration of 25OH vitamin D in serum.  LC-MS/MS has recently become the method of choice for its measurement.

The method described here is a gradient LC-MS/MS method which is both quick and precise and has excellent sensitivity.

Method:  Serum samples were initially precipitated with methanol containing deuterated vitamin D3 (internal standard).  The samples were then submitted to a single hexane extraction and evaporated to dryness.  Samples were reconstituted in 50:50 Methanol:water and 60uL injected onto a Luna C8(2) Mercury Cartridge 20x2mm (Phenomenex).  HPLC was performed using a gradient elution starting with 70% B and increasing to 95% B over 0.5 minutes.  Mobile phase A was 0.1% formic acid in water, mobile phase B was 0.2% formic acid in methanol and the flow rate was 0.8ml/min.  Sample analysis was in positive mode on an API 3200 Q Trap(r) tandem mass spectrometer (Applied Biosystems, Warrington, UK) using an APCI ion source and transitions m/z 413.2>355.4, m/z 401.3>365.4 and m/z 407.4>371.4 for vitamin D2, D3 and deuterated D3 respectively.

Results:  Run time was 2.5 minutes in total with D2 eluting at 1.62 minutes and D3 at 1.60 minutes.  The method was linear up to 309nmol/L for D2 (r=0.9977) and 314nmol/L for D3 (r=0.9976).  Intra-assay variation at 5nmol/L was 25 % for D2 and 21% for D3.  Inter-assay variation was 10.2% and 11.0% for D2 and 4% and 11.2% for D3 at 15.6nmol/L and 43.9nmol/L respectively.

Conclusion:  The method described above is a rapid and sensitive gradient method for quantitation of 25 OH Vitamin D using APCI ionisation to increase sensitivity and decrease background noise.