Four years of LC-MS/MS method for quantification of 25-hydroxyvitamin D (D2+D3) for clinical practice

Many methods for routine total plasma/serum 25-hydroxyvitamin D (25OHD) measurements are available from automated immunoassays to the most specific LC-MS/MS techniques. These last ones are nowadays numerous, still perfectible but more powerful than immunoassays in specific illnesses. We presented a robust method for simple quantification of 25-hydroxyvitamin D(2) (25OHD(2)) and 25-hydroxyvitamin D(3) (25OHD(3)) in human plasma by LC-APCI-MS/MS. This method is reliable and easy to perform for clinical measurements as we report a 4-year of clinical laboratory use. A brief off-line sample pretreatment (protein precipitation with addition of the internal standard) was realized then the supernatant was loaded into 96-well plates and analyzed by an online SPE-LC/MS/MS method on an APCI mode. 25OHD(2) and 25OHD(3) were both measured. The chromatographic system was thought and optimized for providing a dedicated line for this measurement on a shared instrument. The linearity was tested up to 380 nmol/L for both 25OHD(2) and 25OHD(3). The limit of quantification (LOQ) was 7 and 8 nmol/L for 25OHD3 and 25OHD(2), respectively. Routine imprecision and bias were found in agreement with recommended limits for routine testing, CV≤10% and bias≤5%. Since July 2010, our participation to DEQAS was successfully validated. This simple robust online SPE-LC/MS/MS method is suitable for routine measurement of 25OHD(2) and 25OHD(3) in human adult plasma. The assay operates for 4 years and has performed more than 40,000 patient samples on a shared instrument.