Development and comparison of three liquid chromatography-atmospheric pressure chemical ionization/mass spectrometry methods for determining vitamin D metabolites in human serum

摘要

Liquid chromatographic methods with atmospheric pressure chemical ionization mass spectrometry were developed for the determination of the vitamin D metabolites 25-hydroxyvitamin D _2 (25(OH)D _2), 25-hydroxyvitamin D _3 (25(OH)D _3), and 3-epi-25-hydroxyvitamin-D _3 (3-epi-25(OH)D _3) in the four Levels of SRM 972, Vitamin D in Human Serum. One method utilized a C18 column, which separates 25(OH)D _2 and 25(OH)D _3, and one method utilized a CN column that also resolves the diastereomers 25(OH)D _3 and 3-epi-25(OH)D _3. Both methods utilized stable isotope labeled internal standards for quantitation of 25(OH)D _2 and 25(OH)D _3. These methods were subsequently used to evaluate SRM 909c Human Serum, and 25(OH)D _3 was the only vitamin D metabolite detected in this material. However, SRM 909c samples contained matrix peaks that interfered with the determination of the [~2H _6]-25(OH)D _3 peak area. The chromatographic conditions for the C18 column were modified to remove this interference, but conditions that separated the matrix peaks from [~2H _6]-25(OH)D _3 on the CN column could not be identified. The alternate internal standard [~2H _3]-25(OH)D _3 did not suffer from matrix interferences and was used for quantitation of 25(OH)D _3 in SRM 909c. During the evaluation of SRM 909c samples, a third method was developed using a pentafluorophenylpropyl column that also separates the diastereomers 25(OH)D _3 and 3-epi-25(OH)D _3. The 25(OH)D _3 was measured in SRM 909c using all three methods, and the results were compared.