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.
Preclinical Drug Metabolism, Merck Research Laboratories, Rahway, New Jersey 07065, USA. reza_anari@merck.com
M Reza
MR
Anari
Ray
R
Bakhtiar
Bing
B
Zhu
Su
S
Huskey
Ronald B
RB
Franklin
David C
DC
Evans
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