“…In light of the low ionization and fragment efficiency of the EE core structure, we sought to introduce a highly ionizable group into the molecule using a facile aqueous phase derivatization reaction. , Our simulation of the ionization efficiency for a number of feasible derivatives indicated that the addition of a dansyl moiety should enhance the ionization of EE significantly (Table ). The derivatization of phenolic groups with dansyl chloride is a rapid, aqueous-phase, chemical reaction that has been used widely for the fluorometric detection of estrogens. ,, The introduction of a dansyl functional group bearing a basic nitrogen shifted the predicted p K a of the molecule to 3.3 ± 0.4 (p K a of the conjugated acid form of the basic amine), which as we postulated, should significantly enhance ionization under acidic conditions (for example, mobile phases containing 0.1% formic acid, pH 3) (Table ). 1 Schematic representation for the aqueous-phase chemical derivatization of ethinylestradiol by dansyl chloride. 2 Reconstructed ion chromatogram and product ion scan of the isolated 3-dansyl-ethinylestradiol.…”
Section: Resultsmentioning
confidence: 96%
“…A number of other classical assays based on the fluorometric or chemiluminescence detection of dansyl derivatized estrogens have been reported. − The fluorescence is not related to EE per se, but rather originates from the dansyl moiety. Therefore, other endogenous estrogens and their metabolites that are coderivatized, are detected simultaneously .…”
mentioning
confidence: 99%
“…A number of other classical assays based on the fluorometric or chemiluminescence detection of dansyl derivatized estrogens have been reported. − The fluorescence is not related to EE per se, but rather originates from the dansyl moiety. Therefore, other endogenous estrogens and their metabolites that are coderivatized, are detected simultaneously . Even if all of the interfering analytes are separated by using a longer chromatographic separation, the nanogram detection limit reported for EE is higher than the peak plasma concentration of the drug in humans and preclinical species…”
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.
“…In light of the low ionization and fragment efficiency of the EE core structure, we sought to introduce a highly ionizable group into the molecule using a facile aqueous phase derivatization reaction. , Our simulation of the ionization efficiency for a number of feasible derivatives indicated that the addition of a dansyl moiety should enhance the ionization of EE significantly (Table ). The derivatization of phenolic groups with dansyl chloride is a rapid, aqueous-phase, chemical reaction that has been used widely for the fluorometric detection of estrogens. ,, The introduction of a dansyl functional group bearing a basic nitrogen shifted the predicted p K a of the molecule to 3.3 ± 0.4 (p K a of the conjugated acid form of the basic amine), which as we postulated, should significantly enhance ionization under acidic conditions (for example, mobile phases containing 0.1% formic acid, pH 3) (Table ). 1 Schematic representation for the aqueous-phase chemical derivatization of ethinylestradiol by dansyl chloride. 2 Reconstructed ion chromatogram and product ion scan of the isolated 3-dansyl-ethinylestradiol.…”
Section: Resultsmentioning
confidence: 96%
“…A number of other classical assays based on the fluorometric or chemiluminescence detection of dansyl derivatized estrogens have been reported. − The fluorescence is not related to EE per se, but rather originates from the dansyl moiety. Therefore, other endogenous estrogens and their metabolites that are coderivatized, are detected simultaneously .…”
mentioning
confidence: 99%
“…A number of other classical assays based on the fluorometric or chemiluminescence detection of dansyl derivatized estrogens have been reported. − The fluorescence is not related to EE per se, but rather originates from the dansyl moiety. Therefore, other endogenous estrogens and their metabolites that are coderivatized, are detected simultaneously . Even if all of the interfering analytes are separated by using a longer chromatographic separation, the nanogram detection limit reported for EE is higher than the peak plasma concentration of the drug in humans and preclinical species…”
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.
“…20 It has been proven that dansylation of estrogens is a simple, rapid, aqueous phase chemical reaction producing high yields of approximately 92%. 19,21 Supernatants were pre concentrated by Hydrophilic Lipophilic Balance (HLB) polymeric SPE cartridges and subsequently matrix components were reduced by a couple of cleanup steps including an in-test tube liquid-liquid extraction and QuEChERS dSPE cleanup. The HLB polymeric SPE cartridges were utilized for aqueous sample extractions because of its advantages over traditional silica-based reversed phase (RP) SPE cartridges such as C-8 and C-18.…”
“…The estrogenic components have been analyzed using such methods as argentimetry (2), colorimetry (1, 3, 5), fluorometry (2), UV spectrophotometry (6), GLC (6), TLC with fluorescent detection (7), and high-performance liquid chromatography (HPLC) (8)(9)(10).…”
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