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technique(s)

HPLC: suitable

test parameters

column: Ascentis Express  F5, 15 cm x 2.1 mm I.D., 2.7 μm particles (53571-U)
mobile phase: [A] 5 mM ammonium formate water; [B] methanol; (25:75, A:B)
flow rate: 0.4 mL/min
pressure: 5178 psi (357 bar)
column temp.: 40 °C
detector: ESI(+), 100-1000 m/z
injection: 2 μL, each compound 300 ng/mL in 40:60 (water:methanol)

suitability

application for HPLC

application(s)

clinical
vitamins, nutraceuticals, and natural products

Analysis Note

The metabolic role and health implications of the various vitamin D isoforms are of current clinical interest. Therefore, it is important to have an analytical method that will resolve all of the known isoforms with necessary sensitivity and specificity.This application shows that a combination of Ascentis Express F5 with MS detection provided rapid resolution of seven such compounds. The highest grade LC-MS solvents were used to supply low background interference and low particulate contaminants for robust, trouble-free operation. Cerilliant CRMs provided reliable identification and quantification.

Legal Information

Ascentis is a registered trademark of Merck KGaA, Darmstadt, Germany

Analyte

Description
    1α,25-Dihydroxyvitamin D3 solution, 5 μg/mL in ethanol, ampule of 1 mL, certified reference material, Cerilliant®
    1α,25-Dihydroxyvitamin D2 solution, 5 μg/mL in ethanol, ampule of 1 mL, certified reference material, Cerilliant®
    25-Hydroxyvitamin D3 solution, 100 μg/mL in ethanol, ampule of 1 mL, certified reference material, Cerilliant®
    3-epi-25-Hydroxyvitamin D3, 98% (CP)
    3-epi-25-Hydroxyvitamin D2 solution, 100 μg/mL in ethanol, 98% (CP)
    1α-Hydroxyvitamin D3, ≥97.0% (HPLC)

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