Development and validation of a high-performance liquid chromatography-mass spectrometric assay for the determination of 17alpha-hydroxyprogesterone caproate (17-OHPC) in human plasma.
A sensitive and specific method for the determination of 17alpha-hydroxyprogesterone caproate (17-OHPC) in human plasma using high-performance liquid chromatography and mass spectrometry has been developed and validated. Plasma samples were processed by a solid phase extraction (SPE) procedure using Oasis HLB extraction cartridge prior to chromatography. Medroxyprogesterone acetate (MPA) was used as the internal standard. Chromatography was performed using Waters C18 Symmetry analytical column, 3.5 microm, 2.1 mm x 10 mm, using a gradient elusion with a mobile phase consisting of acetonitrile [A] and 5% acetonitrile in water [B], with 0.1% formic acid being added to both [A] and [B], at a flow rate 0.2 ml/min. The retention times of 17-OHPC and MPA were 8.1 and 5.0 min, respectively, with a total run time of 15 min. Analysis was performed on Thermo Electron Finnigan TSQ Quantum Ultra mass spectrometer in a selected reaction-monitoring (SRM), positive mode using electron spray ionization (ESI) as an interface. Positive ions were measured using extracted ion chromatogram mode. The extracted ions following SRM transitions monitored were m/z 429.2-->313.13 and 429.2-->271.1, for 17-OHPC and m/z 385.1-->276 for MPA. The extraction recoveries at concentrations of 5, 10 and 50 ng/ml were 97.1, 92.6 and 88.7%, respectively. The assay was linear over the range 0.5-50 ng/ml for 17-OHPC. The analysis of standard samples for 17-OHPC 0.5, 1, 2.5, 5, 10, 25 and 50 ng/ml demonstrated a relative standard deviation of 16.7, 12.4, 13.7, 1.4, 5.2, 3.7 and 5.3%, respectively (n=6). This method is simple, adaptable to routine application, and allows easy and accurate measurement of 17-OHPC in human plasma.
Zhang
Shimin
S
Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA 15261, USA.
Mada
Sripal Reddy
SR
Mattison
Don
D
Caritis
Steve
S
Venkataramanan
Raman
R
D019326:17-alpha-Hydroxyprogesterone / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D011247:Pregnancy; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization
D019326:17-alpha-Hydroxyprogesterone