Determination of Dydrogesterone in Human Plasma by Tandem Mass Spectrometry: Application to Therapeutic Drug Monitoring of Dydrogesterone in Gynecological Disorders M. E. Abdel-Hamid 1,& , L. H. Sharaf 1 , S. B. Kombian 2 , F. M. E. Diejomaoh 3 1 Department of Pharmaceutical Chemistry, Kuwait University, Safat, Kuwait; E-Mail: Abdel-Hamid@hsc.edu.kw 2 Department of Applied Therapeutics, Faculty of Pharmacy, Kuwait University, Safat, Kuwait 3 Department of Obstetrics and Gynecology, Faculty of Medicine, Kuwait University, Safat, Kuwait Received: 24 June 2006 / Revised: 12 July 2006 / Accepted: 12 July 2006 Online publication: 23 August 2006 Abstract A sensitive and specific tandem mass spectrometric (MS–MS) method was developed and vali- dated for the determination of dydrogesterone (Duphaston Ò ), an orally active synthetic proges- togen, in human plasma. Multiple reaction monitoring (MRM) scans at m/z 313.1 > 105.5 (dydrogesterone) and m/z 393 > 147 (dexamethasone, internal standard) were selected to determine dydrogesterone by the internal standard method. Linear correlations (r: 0.99 ± 0.05) of the calibration curves were established over the concentration range 10–60 ng mL )1 with a lower limit of quantification (LLQ) of 10 ng mL )1 (RSD% 14.9 and %DEVs )10.5 to +15.6). Solid- phase extraction (SPE) technique was used for extraction of dydrogesterone and internal standard from patient plasma samples using Oasis Ò Max C18 cartridges. Ion suppression studies indicated negligible effects of plasma matrix on the mass ions detection of dydrogesterone and IS, when measured in MRM mode. Validation data showed that RSD% values were <22.0%, whereas %DEV values were in the range of )20.2 to +13.3 for intra- and inter-day precision and accuracy, respectively. Analytical recoveries of dydrogesterone from supplemented plasma samples with the drug were in the range of 100.7–112%, indicating the efficiency of the SPE for separation of dydrogesterone from human plasma. Stability studies conducted at )20 °C, showed that dydrogesterone was stable in plasma as indicated from the measured degradation kinetic parameters. The developed method was applied for monitoring plasma levels of dydrogesterone in 25 patients treated with Duphaston Ò tablets at a dose of 10 mg three times daily. Mean plasma concentration of 16.1 ± 3.5 ng mL )1 of dydrogesterone were measured at the steady state. The data suggest the utility of tandem mass method in therapeutic drug monitoring of plasma levels of dydrogesterone in gynecological disorders treated with Duphaston Ò tablets. Keywords Solid-phase extraction Tandem mass spectrometry Clinical application Dydrogesterone Introduction Dydrogesterone (Fig. 1) is an orally active synthetic progestin hormone with almost no androgenic or estrogenic properties [1]. Dydrogesterone is a pro- gestogen structurally related to proges- terone. Dydrogesterone reduces uterine contractility and increases the expansile property of the uterus. Dydrogesterone does not inhibit or interfere with ovu- lation. It is uniquely non-thermogenic and does not alter basal body temper- ature. Dydrogesterone is clinically indi- cated in conditions associated with progesterone deficiency including men- orrhagia [2], premenstrual syndrome [3], functional uterine bleeding due to hor- monal imbalance, endometriosis [4], and as adjunct to estrogen replacement therapy [5]. Dydrogesterone is widely indicated to improve women infertility and to treat recurrent spontaneous miscarriage [6]. Adverse drug reactions such as anomalies of genito-urinary tract of newborn were reported, when treatment with dydrogesterone is con- tinued during pregnancy period [7]. As for progestogens, the pharmacokinetic profiles of orally administered dydro- gesterone dosage forms showed low therapeutic plasma concentration at the steady state, first-pass hepatic metabo- lism, short elimination half-life and high protein binding [8]. Monitoring of plasma levels of dydrogesterone is important for dose adjustment to achieve optimal therapeutic effects and to avoid adverse drug reactions. Devel- opment of analytical techniques for monitoring dydrogesterone in blood is highly demanded. To our knowledge, no article has been reported on the analysis of dydrogesterone in biological matrices. Recently, tandem mass spec- trometry has been extensively used in our laboratories for analysis of drugs DOI: 10.1365/s10337-006-0035-3 0009-5893/06/09 Ó 2006 Friedr. Vieweg & Sohn/GWV Fachverlage GmbH 2006, 64, 287–292 Original Chromatographia 2006, 64, September (No. 5/6) 287