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

Jun 1, 2013 9:13:00 AM / by Dr. Feng Li

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 


Topics: lc-ms/ms

Dr. Feng Li

Written by Dr. Feng Li

Feng (Frank) Li, Ph.D., is President of Alliance Pharma; he obtained his Ph.D. degree in Bioanalytical Chemistry jointly from Concordia University and the National Institute of Scientific Research (Canadian Doping Control Center) in Montreal, Canada. Subsequently, Dr. Li did his post-doctoral fellowship at the Biomedical Mass Spectrometry Facility at the Mayo Clinic in Rochester, Minnesota. Furthermore, Dr. Li has an M.Sc. degree in Natural Product Chemistry and a B.S. in Pharmacy. He has held leadership roles in the Department of Drug Discovery Metabolism at Phoenix International Life Sciences, Inc., a major CRO at the time in Montreal, Canada, which was later acquired by MDS Pharma Services; in the Drug Analysis group in the Department of Drug Metabolism and Pharmacokinetics (DMPK) at GlaxoSmithKline; and in the Drug Metabolism group in the Department of Drug Safety and Disposition at Cephalon, Inc. Dr. Li has extensive DMPK experience in discovery and developmental phases of drug development. With more than 20 years in the pharmaceutical biotechnology, and CRO industry, he is well versed in bioanalytical techniques for both qualitative (drug metabolite identification) and quantitative (PK/TK) drug analysis and has published numerous articles in the area of drug metabolite identification and quantitation.