Quantitative determination of antalarmin, a novel corticotropin-releasing hormone receptor-1 antagonist, in canine plasma by HPLC-MS.
A simple and rapid method was developed for the quantitation of antalarmin from plasma using high performance liquid chromatography coupled with electrospray ionization mass spectrometry (ESI/MS). Separation of antalarmin from interfering compounds was achieved using reversed phase chromatography on a C-8 micro-column with an isocratic mobile phase comprised of 80% acetonitrile, 20% water, and 5 mM triethylamine. Detection by ESI/MS was accomplished in positive ion mode using single ion monitoring of the protonated molecular ions of antalarmin and its 13C2-isotopimer. The area ratio of the integrated peaks of interest in the extracted ion chromatogram was used for quantitation. The lower limit of detection was 1 picogram (pg) and the quantitation showed a linear response up to 4 nanograms loaded on column. To achieve acceptable accuracy at or around the limit of quantitation of 20 pg, a 1/x weighting was applied to the calibration data. Accuracy and precision variation for intra and inter-day validation were below the acceptable limit (15%) for pharmacokinetic studies.
Fox
Stephen D
SD
Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., National Cancer Institute at Frederick, P.O. Box B, Frederick, MD 21702-1201, USA.
Roman
John M
JM
Cizza
Giovanni
G
Veenstra
Timothy D
TD
Issaq
Haleem J
HJ
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D013058:Mass Spectrometry; D015394:Molecular Structure; D011743:Pyrimidines / Q000097:blood* / Q000737:chemistry; D011758:Pyrroles / Q000097:blood* / Q000737:chemistry; D018019:Receptors, Corticotropin-Releasing Hormone / Q000037:antagonists & inhibitors*; D000099052:CRF Receptor, Type 1
D011743:Pyrimidines; D011758:Pyrroles; D018019:Receptors, Corticotropin-Releasing Hormone; C103016:antalarmin; D000099052:CRF Receptor, Type 1