Quantification of 25-hydroxyvitamin D3 in Rat Serum Using Derivatization to Enhance LC-MS/MS Sensitivity

By Dr. Feng Li June 1, 2013

Circulating 25-hydroxyvitamin D3 (25-OHVD3) is widely accepted as the most useful biomarker for evaluating vitamin D status and diagnosing certain diseases. 

Determination of 25-OHVD3 is important during some drug development since the increasing concern of these drugs potentially affecting vitamin D absorption. 

Detecting 25-OHVD3 in biological matrix using LC-MS/MS has been challenged due to its poor ionization efficiency and lack of a predominant daughter ion, which causes a low sensitivity.  Isomeric metabolites of vitamin D and other sterol-like endogenous interferences pose an additional challenge with chromatographic separation.

In this study, a sensitive and robust LC-MS/MS method was developed and validated for the determination of 25-OHVD3 in rat serum.

LC-MS/MS Bioanalysis Study Alliance Pharma

Sample Preparation                                                                  

The samples were prepared in 96-well format by liquid-liquid extraction with MTBE, followed by a 30-minute derivatization with picolinic acid. 25-OHVD3-d6 was employed as internal standard.

LC-MS/MS Analysis

Column: ACE C4, 100 X 4.6 mm, 3 µm particle size

Mobile Phase A: 0.1% formic acid in water

Mobile Phase B: 0.1% formic acid in acetonitrile

Gradient: 0-0.2 min, 75%B; 0.2-3.0min, 75-85%B; 3.05-5.50min, 95%B; 5.55-6.50min, 75%B

Flow rate: 1.5 mL/min

Detector: Sciex API 4000, ESI+

MRM transition: m/z 506.6 à 383.3 for 25-OHVD3

                              m/z 512.4 à 389.3 for 25-OHVD3-d6

Bioanalysis Results

A nitrogen-containing moiety was introduced to the 25-OHVD3 molecule via a derivatization reaction with picolinic acid to increase ionization efficiency (Figure 1).

Derivatization also ensured the formation of predominant product ions that can be used in SRM detections for both 25-OHVD3 and 25-OHVD3-d6 (internal standard) (Figure 2).

Due to high endogenous levels of 25-OHVD3 in blank serum, calibration standards were prepared in a surrogate matrix (5% BSA). Precision and accuracy was evaluated by spiking known concentrations of analyte in pre-quantified “blank” matrix.

Figure 3. Representative chromatograms of 25-OHVD3 and 25-OHVD3-d6 for LLOQ (0.5 ng/mL ) and in surrogate matrix and a rat serum   sample showing endogenous 25-OHVD3.

This method was fully validated with a quantitation limit of 0.5 ng/mL and required only 50 mL of rat serum.  The assay showed excellent linearity (R2>0.998) using a calibration range of 0.5 – 250 ng/mL (Figure 4).


The derivatization reaction with picolinic acid increased ionization efficiency of the 25-hydroxyvitamin D3 molecule and ensured the formation of predominant product ions, which in turn enhanced the LC-MS/MS sensitivity.

The method was validated as linear, accurate, precise and reproducible. It can be used to determine the concentration of 25-hydroxyvitamin D3 in rat serum as low as 0.5 ng/mL using only 50 mL of sample. 


Yinghe Li, Yifan Shi, Meng Fang, and Pam Rogers 


Posted in: lc-ms/ms