A validated high‐performance liquid chromatographic method for the determination of moclobemide and its two metabolites in human plasma and application to pharmacokinetic studies

Abstract

A rapid and sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) with ultraviolet detection has been developed for the determination of moclobemide and its metabolites, p-chloro-N-(-2-morpholinoethyl)benzamide N'-oxide (Ro 12-5637) and p-chloro-N-[2-(3-oxomorpholino)ethyl]-benzamide (Ro 12-8095), in human plasma. The assay was performed after single liquid-liquid extraction with dichloromethane at alkaline pH using phenacetin as the internal standard. Chromatographic separation was performed on a C(18) column using a mixture of acetonitrile and water (25:75, v/v), adjusted to pH 2.7 with ortho-phosphoric acid, as mobile phase. Spectrophotometric detection was performed at 239 nm. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The quantification limit for moclobemide and Ro 12-8095 was 10 ng/mL, and for Ro 12-5637 was 30 ng/mL. Linearity of the method was confirmed for the range 20-2500 ng/mL for moclobemide (r = 0.9998), 20-1750 ng/mL for Ro 12-8095 (r = 0.9996) and 30-350 ng/mL for Ro 12-5637 (r = 0.9991). Moreover, within-day and between-day precisions and accuracies of the method were established. The described method was successfully applied in pharmacokinetic studies of parent drug and its two metabolites after a single oral administration of 150 mg of moclobemide to 20 healthy volunteers.

Modern chromatographic and electrophoretic measurements of antidepressants and their metabolites in biofluids

“…In a few reports, the use of HPLC with UV detection for the assay of MOC and its metabolites in biofluids such as plasma (Misztal et al ., 2002; Rakic et al ., 2007; Plenis et al ., 2007; Ignjatovic et al ., 2009) and urine samples (Hoskins et al ., 2001b) was also reported. In these methods, the chromatographic separations were performed with C 6 (Hoskins et al ., 2001b) C 8 (Misztal et al ., 2002) and C 18 (Rakic et al ., 2007; Plenis et al ., 2007) analytical columns, respectively. For the improvement of the peak shape of the analytes, addition of triethylamine to mobile phase was the most frequently used.…”

“…In these studies, the recoveries of VEN and ODV were above 76% whereas for SER and DMS they were 83.2 and 76.1%, respectively. In the literature, there is only one report where one‐step LLE with dichloromethane in alkaline pH (1 m NaOH) was applied before HPLC determination of plasma concentations of MOC and its two metabolites: Ro 12‐5637 and Ro 12‐8095 (Plenis et al ., 2007). The obtained recovery was between 87.02 and 93.08%.…”

Strategies in quantitative LC‐MS/MS analysis of unstable small molecules in biological matrices

“…Although some N‐ oxides, e.g. Ro12‐5637 (Plenis et al ., 2007), nicotine‐ N‐ oxide and cotinine‐ N‐ oxide (Miller et al ., 2010), etc., are stable, many compounds in this category are not only thermally labile, but also unstable in solutions and/or biological matrices during sample extraction, especially under strong acidic or basic conditions. Light exposure may further accelerate the decomposition of these molecules (Dell, 2004).…”

Validated UPLC‐MS/MS method for determination of moclobemide in human brain cell supernatant and its application to bidirectional transport study

“…Several of analytical methods have been applied for the determination alone or with other antidepressants of moclobemide and in some cases also their metabolites in biological fluids, such as plasma, whole blood and urine samples (Geschke et al ., ; Rakic et al ., ; Titier et al ., ). For determination of moclobemide in these physiological fluids, gas chromatography with nitrogen‐selective detection (Maguire et al ., ) and high‐performance liquid chromatography, with either ultraviolet detection (Geschke et al ., ; Misztal et al ., ; Amini et al ., ; Malfara et al ., ; Plenis et al ., ; Rakic et al ., ) or electrospray ionization‐mass spectrometry (ESI‐MS; Hoskins et al ., ; Choong et al ., ) are widely used. However, these methods present some disadvantages such as poor reproducibility, low sensitivity (Geschke et al ., ; Misztal et al ., ; Amini et al ., ) and requirement for complex sample processing (Geschke et al ., ; Hoskins et al ., ; Rakic et al ., ), thus they only can provide limited assistance for bidirectional transport assay involving determination of moclobemide in cell supernatant.…”