Derivatization of ethinylestradiol with dansyl chloride to enhance electrospray ionization: application in trace analysis of ethinylestradiol in rhesus monkey plasma.
The extensive metabolism and administration of low doses of ethinylestradiol (EE) in preclinical animal species necessitates a sensitive analytical method to quantify the drug at low picogram-per-milliliter concentrations in biological matrixes. A highly sensitive and accurate method based on the derivatization of EE with dansyl chloride coupled with liquid chromatography/tandem mass spectrometry is described. The dansyl derivatization of EE introduced a basic secondary nitrogen into the molecule that was readily ionized in commonly used acidic HPLC mobile phases. The derivative showed an intense protonated molecular ion at m/z 530 under positive turbo ion spray ionization. The collision-induced dissociation of this ion formed a distinctive product at m/z 171, corresponding to the protonated 5-(dimethylamino)naphthalene moiety. The selected reaction monitoring, based on the m/z 530 --> 171 transition, was highly specific for EE, since no background signal was observed from blank plasma obtained from rhesus monkeys. The limit of detection, at a signal-to-noise ratio of 5, was 0.2 fg/mL EE spiked into blank plasma. This allowed for a lower limit of quantitation of 5 pg/mL using a 50-microL plasma sample and 10-microL injection of dansylated derivative into the CTC-PAL Leap autosampler coupled to a Sciex API 4000 mass spectrometer. Using fast-gradient liquid chromatography, the analyte peak eluted at 1.6 min. The validation results showed high accuracy (% bias < 4) and precision (% CV < 7.5) at broad linear dynamic ranges (0.005-20 ng/mL), using deuterated EE as internal standard. Therefore, the facile dansyl derivatization coupled with tandem mass spectral analysis allowed the development of a highly sensitive and specific method for quantitation of trace levels of EE in the plasma of rhesus monkeys dosed orally and intravenously with EE.
Anari
M Reza
MR
Preclinical Drug Metabolism, Merck Research Laboratories, Rahway, New Jersey 07065, USA. reza_anari@merck.com
Bakhtiar
Ray
R
Zhu
Bing
B
Huskey
Su
S
Franklin
Ronald B
RB
Evans
David C
DC
D000818:Animals; D003619:Dansyl Compounds / Q000737:chemistry; D045166:Estradiol Congeners / Q000097:blood*; D004997:Ethinyl Estradiol / Q000097:blood*; D005260:Female; D008253:Macaca mulatta; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods* / Q000592:standards
D003619:Dansyl Compounds; D045166:Estradiol Congeners; D004997:Ethinyl Estradiol; C029038:dansyl chloride