The determination of 2beta-(3-hydroxypropoxy)-1alpha,25-dihydroxy vitamin D3 (ED-71) in human serum by high-performance liquid chromatography-electrospray tandem mass spectrometry.
A liquid chromatography-electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method for the determination of 2beta-(3-hydroxypropoxy)-1alpha,25-dihydroxy vitamin D3 (ED-71) in human serum has been developed. ED-71 in human serum was extracted using two solid-phase extraction steps on Bond Elut C18 and NH2 cartridge. The separation of ED-71 and preED-71 isomer was attained by LC using 2 mmol/L ammonium acetate-methanol (15:85, v/v) as a mobile phase on a Symmetry C18 column (5 microm, 150 mm x 2.1mm i.d.). ESI-MS/MS analysis was operated using selected reaction monitoring (SRM) in positive ion mode. The method achieved a lower limit of quantitation of 25 pg/mL. The calibration curve (25-3200 pg/mL) gave acceptable linearity (r>0.9964). Intra-assay precision ranged from 2.3 to 9.7%. Inter-assay precision ranged from 1.0 to 3.4%. The accuracy was within 90.8-107.0%. This highly sensitive and reproducible method is able to determine only biologically active ED-71 by separating it from preED-71, which is considered to be applicable for the determination of serum samples from pharmacokinetic studies in human.
Murao
Naoaki
N
Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Company Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan. muraonoa@chugai-pharm.co.jp
Ohishi
Norihisa
N
Nabuchi
Yoshiaki
Y
Ishigai
Masaki
M
Kawanishi
Takehiko
T
Aso
Yoshinori
Y
D002117:Calcitriol / Q000031:analogs & derivatives* / Q000097:blood; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013696:Temperature; D013997:Time Factors; D014807:Vitamin D / Q000031:analogs & derivatives
D014807:Vitamin D; D002117:Calcitriol; C547512:eldecalcitol