Purpose: Vitamin D metabolites, 25-OH Vitamin D2/D3 are challenging bio-markers for many reasons. The separation of compounds, labor intensive sample prep, and matrix effects are often challenges of the assay. In this study, we explore the effectiveness of an automated on-line solid phase extraction method coupled with LC-MS/MS analysis in human serum. The assay is associated with a shorter Kinetex C18, 2.6 µm, 30x3.0 mm analytical column and a Strata RP extraction column 50x0.5 mm, which reduces the total run time to less than 6 minutes including the system equilibration. The assay shows the accuracy and precision including LLOQ (n=6) of the resulting method are assed for a linear dynamic range from 2-100 ng/mL.
Methods: Sample Pretreatment
Dilute 150 µL of human serum with 200 µL of Precipitating Reagent*
Add 10 µL of 25-OH Vitamin-D3-2H6 (1µg/mL), mix for one minute, then centrifuge at 14,000 RPM for 10 minutes
Transfer 200 µL supernatant to autosampler injection vial
*Precipitating Reagent prepared as (5:2:1) MeOH/Acetonitrile/ZnSO4
Cohesive System run in TX Mode (LX-2) with Sciex 4000 QTRAP, APCI, Positive ion mode
Strata RP, 50x0.5mm, Part No: 00B-S326-AF
Kinetex C18, 2.6µm, 30x3.0mm, Part No: 00A-4462-Y0
Injection Volume: 40 µL
Gradient LC method with
Mobile Phase A: 0.1% formic acid in water
Mobile Phase B: 0.1% formic acid in methanol
Needle Wash 1: 50:50 MeOH: Water
Needle Wash 2: 0.1% formic acid in water
Results: The on-line extraction is supplemented with an off-line protein precipitation. The precipitating reagent of Methanol:Acetonitrile:ZnSO4 (5:2:1) has been optimized for both efficient protein removal and acceptable analyte recovery. The online extraction recovery of the assay for 25-OH vitamin D2 (n=6) is 95.6% with CV% at 3.09%, and 25-OH vitamin D3 (n=6) is 93.3%, while the relative standard deviation is 2.10%, respectively.
This assay was subsequently evaluated using four different level QCs at n=6, including LLOQ for each sample set. The accuracy and precision of 25-OH vitamin D2 analyte presented at 100 - 104% with CV% at 3.42-8.80% across all four level QCs, and 91.7- 106% with CV% at 3.11-9.78% for 25-OH vitamin D3. The results demonstrated the assay meets a GLP/GCP environment requests.
The linear dynamic range of this method was also tested with seven calibrators (n=2) from 2-100 ng/mL with linearity (R2) volume is greater than 0.9995 for both 25-OH vitamin D2/D3. Double charcoal-stripped serum was used for calibration standards and QCs preparation during the assay evaluation. The endogenous level of compounds in the blank matrix was checked, indicating that there is no detectable levels of 25-OH Vitamin D2/D3, respectively.
Conclusion: This assay has combined on-line solid phase extraction and bio-analytical LC-MS/MS method with a total run time of less than six minutes. The speed of this method associated with its accuracy and precision make it ideal for the high throughput research and production environment.