Derivatization of ethinylestradiol with dansyl chloride to enhance electrospray ionization: Application in trace analysis of ethinylestradiol in rhesus monkey plasma

被引:172
作者
Anari, MR [1 ]
Bakhtiar, R [1 ]
Zhu, B [1 ]
Huskey, S [1 ]
Franklin, RB [1 ]
Evans, DC [1 ]
机构
[1] Merck Res Labs, Preclin Drug Metab, Rahway, NJ 07065 USA
关键词
D O I
10.1021/ac025712h
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
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 spectrometxy 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-muL plasma sample and 10-muL 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.
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收藏
页码:4136 / 4144
页数:9
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