Serum samples from patients are precipitated with zinc sulfate solution and an organic solvent with the addition of a deuterated internal standard. Then a centrifugation follows. Due to the high quantity of samples 96-deep-well collecting plates (e.g., REF 738650.5) are used instead of 1.5 mL standard vials. The sample pretreatment is directly carried out in the cavities of the plate.

The enrichment of analyts as well as the removal of interfering substances are received by online solid phase extraction (online-SPE) with C8 ec extraction columns. For a faster working process two SPE extraction columns (1) + (2) are used with changing steps (s. column switching). Lipophilic substances like vitamin D3 are retained, but polar compounds like e.g., salts, proteins, amino acids, carbohydrates and water soluble vitamins can pass. After sample loading onto the SPE extraction column the analyts are eluated by changing the flow direction and an increase of the organic part of eluent. The eluent flow is diverted onto the analytical HPLC column (3) for the chromatographic separation. The analytes are ionized with the MS electrospray method and detected by tandem mass spectrography.

Eluent A: 2 mL formic acid is filled up with water (LCMS Optigrade) to 1 L volume Eluent B: 2 mL formic acid is filled up with 700 mL methanol (Optigrade) and 300 mL acetonitrle (Optigrade)

Injection volume: 5 µL / 20 µL

Gradient and flow rate:

Online-SPE (extraction) 0 to 1.1 min: A 98 % 0 to 1.1 min: flow 0.8 mL/min 1.1 to 4 min: flow 0.4 mL/min 2.7 to 3 min: A 100 % 3 to 4 min: A 2%

HPLC (analysis) 0 to 1.2 min: A 30 % 1.25 to 2.65 min: A 20 % 2.7 to 3 min: A 10 % 3.1 min: A 30 % 0 to 4 min: flow 0.4 mL/min

Oven temperature: 40 °C Rack temperature: 15 °C