Derivatization of Ethinylestradiol with Dansyl Chloride To Enhance Electrospray Ionization:  Application in Trace Analysis of Ethinylestradiol in Rhesus Monkey Plasma

Abstract

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.

Analysis of steroidal estrogens as pyridine-3-sulfonyl derivatives by liquid chromatography electrospray tandem mass spectrometry

“…The product ion spectrum of the dansyl derivative of E2 showed the dominant ion of m/z 171 (Fig. 2D), as has been previously observed and assigned to an ion representing the 5-(dimethylamino)naphthyl moiety, formed by homolytic cleavage of the C-S bond between the naphthalene and sulfonyl groups [14,23].…”

“…A major obstacle in steroid analysis by LC-MS and LC-MS/MS had been that many steroids are weakly ionizable in the electrospray ionization (ESI) and atmospheric pressure chemical ionization techniques of LC-MS and LC-MS/MS, leading to low inherent sensitivity. Over the years chemical derivatization techniques have been developed to enhance the ionization of various analytes in LC-MS [11][12][13][14]. The deficiency of poor ionization of steroids in LC-MS has now been largely overcome; a number of derivatization procedures that involve either the introduction of a group carrying a formal charge [15][16][17][18] or the attachment of a group with high proton affinity [14,[19][20][21][22] are now used to enhance ionization efficiency for positive ion detection in steroid analysis by LC-MS and LC-MS/MS.…”

“…For the analysis of steroidal estrogens by LC-ESI-MS/MS, pre-column derivatization by reaction with dansyl chloride at the C-3 hydroxyl groups of these compounds is by far the most popular technique, and is now widely used in both biomedical [14,[23][24][25][26][27][28][29] and environmental [30][31][32] applications. There are, however, aspects of the analysis of estrogens as dansyl derivatives that are not desirable.…”

“…There are, however, aspects of the analysis of estrogens as dansyl derivatives that are not desirable. With dansyl derivatization and LC-ESI-MS/MS of steroidal estrogens, the vast majority of the ion current after collision-induced dissociation (CID) of the [M+H] + ion is carried by a single ion derived from the protonated 5-(dimethylamino)-naphthyl moiety [14,[23][24][25][26], and very little of it by estrogen-derived ions, thereby making MS/MS and third stage MS (MS 3 ) analyses structurally uninformative. The identification of unknown estrogens and their metabolites is therefore not significantly aided by the formation and analysis of dansyl derivatives.…”

Derivatization of bisphenol A and its analogues with pyridine‐3‐sulfonyl chloride: multivariate optimization and fragmentation patterns by liquid chromatography/Orbitrap mass spectrometry

“…Derivatization reagents used for LC/ESI‐MS should enhance the ESI response by attaching a readily ionizable group to the original molecule 17, 18. Nevertheless, most of them provide only reagent‐specific ions upon MS/MS fragmentation of the corresponding [M+H] + ions, which usually results in numerous co‐eluting interferences when analyzing complex samples 28…”