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

• A routine LC–MS/MS method for plasma 25 hydroxyvitamin D (D2 + D3) determination is proposed.
• On a shared LC–MS/MS with a special design allowing dedicated lines for three groups of users.
• Long-term use (4 years) with high throughput (at least 100 samples per set) two times per week.
• Matrix effects and stability data on real samples were performed.
• Low cost but high efficiency for clinical diagnosis have been particularly studied.

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 D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) 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. 25OHD2 and 25OHD3 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 25OHD2 and 25OHD3. The limit of quantification (LOQ) was 7 and 8 nmol/L for 25OHD3 and 25OHD2, 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 25OHD2 and 25OHD3 in human adult plasma. The assay operates for 4 years and has performed more than 40,000 patient samples on a shared instrument.