Ultra-Sensitive Method for the Determination of 1,25 di-OH Vitamin D2 and 1,25 di-OH Vitamin D3 in Serum Using Supported Liquid Extraction Prior to LC-MS/MS

Introduction

This application note describes a protocol for the extraction of 1,25 di-OH Vitamin D2 and 1,25 di-OH Vitamin D3 metabolites from serum using supported liquid extraction prior to LC-MS/MS detection. The method described in this application achieves highly reproducible recoveries for 1,25 diOH Vitamins D2 and D3. A calibration range between 5 and 500 pg/mL is demonstrated using a starting volume of 0.25 mL of serum. Sensitivity is maximized through the use of a simple PTAD derivatization and formation of a methylamine complex.

Figure 1. Analyte structures

ISOLUTE^® SLE+ Supported Liquid Extraction plates and columns offer an efficient alternative to traditional liquid-liquid extraction (LLE) for bioanalytical sample preparation, providing high analyte recoveries, no emulsion formation, and significantly reduced sample preparation.

Analytes

1,25 diOH Vitamin D2 and 1,25 diOH Vitamin D3 with deuterated 1,25 diOH Vitamin D3 as internal standard (all purchased from Sigma Aldrich Chemical Co., Poole, UK).

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