Extraction of Vitamin D Metabolites from Human Serum Using Supported Liquid Extraction

July 2, 2012

The Application Notebook

Volume 0, Issue 0

Biotage Application Note

This application note describes a simple and effective procedure for the extraction of 25-OH vitamin D2 and 25OH vitamin D3 from human serum, using ISOLUTE SLE+ supported liquid extraction plates, with analysis by LC–MS–MS. The procedure incorporates an integral protein binding disruption step which eliminates the need for any offline protein disruption. Supported liquid extraction is a simple, high throughput alternative to traditional liquidliquid extraction for bioanalytical fluid samples, providing high analyte recovery, clean extracts and reduced sample preparation time.

Extraction Conditions

Sample pre-treatment: Dilute human serum (150 µL) with water:isopropanol (50:50, v/v, 150 uL). Cap and shake for 1 min.

Sample load: Load pre-treated serum (300 µL in total) onto the ISOLUTE SLE+ extraction plate (part number 8200400-P01). Pulse vacuum to initiate flow, then leave sample to absorb for 5 min.

Analyte elution: Apply heptane (750 µL) and wait 5 min for the solvent to absorb. Apply a second aliquot of heptanes (750 µL), allow to soak for a further 5 min, then apply a final pulse of vacuum.

Post extraction: Evaporate to dryness at room temperature and re-constitute ininjection solvent (mobile phase A: mobile phase B, 70:30, v/v, 100 µL,). Cap and vortex gently for 60 s.

Note: analytes are light sensitive and so amber glassware is recommended.

Analytical Conditions

Instrument: Waters Acquity UPLC with 20 µL injection loop interfaced to Premier XE triple quadrupole mass spectrometer equipped with an electro spray interface for mass analysis.

Column: Restek Pinnacle DB Biphenyl (1.9 µ, 50 + 2.1 mm i.d.)

Mobile phase: Isocratic 20%A: 80% B at 0.4 mL/min. Totalrun time 2.0 min.

A: 2 mM Ammonium Formate (aq) with 0.1% formic acid

B: 2 mM Ammonium Formate (99% MeOH 1% aq) with 0.1% formic acid

Injection volume: 15 µL (partial loop with overfill)

Sample temp: 10 °C

Column temp: 40 °C

Desolvation temp: 450 °C

Source temp: 120 °C

For full MRM conditions please see AN757 at www.biotage.com/applications


Recoveries of > 90% were achieved for all analytes, with relative standard deviation (RSD) < 10%. Limit of quantifications (LOQs) were 3.75 ng/mL for 25-hydroxyvitamin D2 and 1 ng/mL for 25-hydroxyvitamin D3. Excellent linearity (> 0.99) for both analytes was demonstrated (see Figure 1). DEQAS samples extracted using this method showed excellent correlation with nominal values.

Figure 1: Calibration curve for 25-OH Vitamin D3 from serum


Supported liquid extraction using ISOLUTE SLE+ plates provides a simple, robust sample preparation method for extraction of Vitamin D metabolites from serum. The integral sample pre-treatment step is sufficient to eliminate analyte-protein binding, without any offline steps being required, leading to accurate determination of the analytes in serum samples.

Biotage AB

Kungsgatan 76, SE-753, 18, Uppsala, Sweden

Tel. +46 18 56 59 00 fax +46 18 59 19 22



(1) For complete details of this application, see Application Note AN757, available from www.biotage.com/applications