Quantitative determination of duloxetine and its metabolite in rat plasma by HPLC‐MS/MS

Abstract

The major metabolite of duloxetine is a glucuronide conjugate of 4-hydroxy duloxetine (4-HD). However, interestingly, there have been no reports determining concentrations of 4-HD and no fully validated method has been established for measuring duloxetine and 4-HD in rat plasma. We developed a method for the simultaneous quantification of duloxetine and its metabolite in rat plasma using high-performance liquid chromatography tandem mass spectrometry. Duloxetine and 4-HD were analyzed on a reverse-phase C 18 analytical column after protein precipitation of the plasma sample with methanol, using carbamazepine as an internal standard. The isocratic mobile phase of 5 mM ammonium acetate-methanol (4:6, v/v) was eluted at 0.4 mL/min. Quantification was performed on a triple-quadrupole mass spectrometer using electrospray ionization, and the ion transition monitored in selective reaction monitoring mode. The coefficient of variation for assay precision was <18.0%, and the accuracy was 84.0-118.0%. This method was successfully used to measure the concentrations of duloxetine and its metabolite in plasma following the oral administration of a single 40 mg/kg dose in rats. Copyright © 2013 John Wiley & Sons, Ltd.Keywords: duloxetine; 4-hydroxy duloxetine (4-HD); rat plasma; LC/MS/MS Duloxetine hydrochloride, chemically known as (+)-N-methyl-3 (1-naphthalenyloxy)-2-thiophenepropanamine hydrochloride, is a potent inhibitor of both serotonin and norepinephrine reuptake that binds to 5-hydroxytryptamine (5-HT) and norepinephrine (NE) transport sites with comparable affinities. It is used clinically for the treatment of major depressive disorders, pain owing to diabetic peripheral neuropathy and incontinence (Ma et al., 2007). The major metabolite of duloxetine is a glucuronide conjugate of 4-hydroxy duloxetine (Lantz et al., 2003). Estimations of duloxetine in biological matrices using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) have been reported (Zhao et al., 2009Radhe et al., 2012, and HPLC has been used to quantify 4-hydroxy duloxetine-b-D-glucuronide in human plasma (Satonin et al., 2007). However, there have been no reports determining concentrations of 4-hydroxy duloxetine (4-HD) and no fully validated method has been established for measuring duloxetine and 4-hydroxy duloxetine in rat plasma.Here, we present a simple, sensitive HPLC-MS/MS method for the simultaneous determination of duloxetine and its metabolite in rat plasma. This method was successfully used to characterize the pharmacokinetics of duloxetine and 4-HD following the oral administration of duloxetine in rats.Duloxetine and 4-HD were purchased from Waterstone Technology (Carmel, IN, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). Carbamazepine, used as an internal standard (IS), was purchased from Sigma Chemical (St Louis, MO, USA). HPLC-grade methanol was obtained from Merck (Darmstadt, Germany). Duloxetine and 4-HD were dissolved in methanol at 1 mg/mL. These standard solutions were diluted serially ...

Inhibition of Cytochrome P450 by Propolis in Human Liver Microsomes

“…The incubations were performed at 37°C for 60 min in a shaking water bath and terminated by adding 200 μL of cold acetonitrile containing 50 nM cabamazepine as an internal standard after a 60 min incubation period, and agitating with a vortex mixer before centrifugation. The samples for each enzyme assay were centrifuged at 2,500 × g for 20 min at 4°C, and the supernatants of the individual reaction samples were analyzed by LC-MS/MS ( 14 ). Compounds were separated on an Atlantis C18 column (50 × 2.1 mm i.d., 3 μm; Waters) with an isocratic mobile phase consisting of methanol and 5 mM ammonium acetate (6 : 4, v/v).…”

Common Cholinergic, Noradrenergic, and Serotonergic Drugs Do Not Block VNS-Mediated Plasticity

“…Doses were selected based on reported ranges that produced intended pharmacological effects but were below levels found to cause analogous unwanted effects observed in clinical use. All drugs have plasma half-lives that fall within the timing of behavioral training ( Jaillon, 1980 ; Chae et al, 2013 ; Tian et al, 2019 ). Groups received motor training paired with VNS and daily injections of oxybutynin chloride (10 mg/kg, s.c., Fisher Scientific, Hampton, NH, United States – 18604931), prazosin hydrochloride (5 mg/kg, s.c., Fisher Scientific, Hampton, NH, United States – AAJ61712MD), duloxetine (20 mg/kg, s.c., Fisher Scientific, Hampton, NH, United States – D42231G), or saline (s.c.).…”

Application of an Inter-Species Extrapolation Method for the Prediction of Drug Interactions between Propolis and Duloxetine in Humans

“…DLX and 4-HD were extracted from the plasma by a liquid-liquid extraction method, followed by analysis with a validated HPLC-MS/MS method as described in our previous report [56]. In detail, DLX and 4-HD were ionized in the positive electrospray ionization mode and detected through multiple reaction monitoring transitions at 298.00→44.00 and 314.13→154.10 m/z, respectively.…”