This Application Note describes a method for analysis of vitamin D2 and D3 using the Varian 320-MS Triple Quadrupole LC/MS/MS. LC/MS/MS is a very sensitive and accurate tool for vitamin D screening, and gives clear chromatographic peaks that could easily be applied to quantitative analysis in blood plasma or serum.
The document details a method for separating and analyzing active forms of vitamin D (Vitamin D2 and Vitamin D3) in plasma or serum. The analysis utilizes the Varian 320-MS Triple Quadrupole mass spectrometer equipped with an APCI source, coupled with Varian Prostar 430 AutoSampler and binary solvent delivery modules.
Specific HPLC and MS parameters are provided for the procedure. The method involves injecting a standard solution at 10 ppm concentration to generate MS/MS data. Results indicate that while isocratic flow was used in this specific demonstration, the system allows for rapid analysis of multiple transitions.
| Ionization Mode | APCI (positive) |
|---|---|
| Nebulizing Gas Pressure | 55 psi |
| Vaporizer Gas Temperature | 100 °C |
| Needle Voltage | 5000 V |
| Corona Current | 2.00 pA |
| Shield Voltage | 600 V |
| Column Type | Polaris C18-A 3 um, 100 x 2.0 mm ID |
| Injection Volume | 25 pL |
| Vitamin D2 Retention Time | 4.01 min. |
| Vitamin D3 Retention Time | 4.27 min. |
The analysis uses APCI (positive) ionization mode.
The document specifies an injection volume of 25 pL.