Development and validation of amitriptyline and its metabolite in human plasma by ultra performance liquid chromatography–tandem mass spectrometry and its application to a bioequivalence study

Abstract

A method based on ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) in combination with solid-phase extraction for sample pretreatment has been developed for the simultaneous analysis of amitriptyline and its main metabolite in human plasma. The extraction of the analytes from plasma samples was carried out by means of a selective SPE procedure using hydrophilic-lipophilic balance cartridges. The assay involves a simple solid-phase extraction (SPE) procedure of 0.2 mL of human plasma and analysis was performed on a triple-quadrupole tandem mass spectrometer by multiple reactions monitoring (MRM) mode via electrospray ionization (ESI). The standard calibration curve was linear over the ranges 0.370-95.539 ng/mL for amitriptyline and 0.365-94.374 ng/mL for nortriptyline, expressed by the linear correlation coefficient r², which was better than 0.995 for both. The intra- and inter-day precision and accuracy of the quality control samples were within 10.0%. The recovery was 85.3, 88.4 and 80.7% for amitriptyline, nortriptyline and doxepin respectively. Total run time was 1.2 min only for each sample, which makes it possible to analyze more than 400 samples per day. The method was highly reproducible and gave peaks with excellent chromatography properties.

Simultaneous quantification of selective serotonin reuptake inhibitors and metabolites in human plasma by liquid chromatography–electrospray mass spectrometry for therapeutic drug monitoring

“…Method selectivity can be improved with MS/MS, which use is increasing in bioanalytical chemistry. Several methods have recently been published for plasma quantification of SSRI drugs using MS/MS [22][23][24][25][26][27][28][29][30][31]. However, a compromise has to be found between method complexity and ease-of-use.…”

“…Recently, ultra high performance liquid chromatography (UHPLC)-MS/MS methods have also been proposed [30,31]. Sample preparation was performed mainly by liquid-liquid extraction [8,9,[11][12][13]16,17,22,23], solid-phase extraction (SPE) [10,14,18,19,24,30], protein precipitation [25,26,31] or on-line SPE using a column-switching system [15,20,21,[27][28][29]. The main drawback of almost all of these methods is that multiple analytes were quantified using the same IS [8][9][10][11][12][13][14][15][16][18][19][20][22][23][24][25][26][27][28][29].…”

“…This approach could decrease the analytical precision, as it is difficult to have a single IS with physico-chemical properties and ionization behavior similar to several analytes. Furthermore, in many publications, a drug was chosen as IS [8,11,[13][14][15][17][18][19][21][22][23]25,30], which could lead to an important interference if the patient had taken this compound. Although stable isotope-labeled IS were used in some methods [10,16,24,[27][28][29]31], no method proposed the quantification of all SSRI with a stable isotope-labeled IS for each matching analyte.…”

“…Methods using gas chromatography (GC) have been proposed [8][9][10], however, separation was mainly performed by HPLC coupled with ultraviolet (UV) [11][12][13][14][15], MS [16][17][18][19][20][21] or MS/MS detection [22][23][24][25][26][27][28][29]. Recently, ultra high performance liquid chromatography (UHPLC)-MS/MS methods have also been proposed [30,31]. Sample preparation was performed mainly by liquid-liquid extraction [8,9,[11][12][13]16,17,22,23], solid-phase extraction (SPE) [10,14,18,19,24,30], protein precipitation [25,26,31] or on-line SPE using a column-switching system [15,20,21,[27][28][29].…”

Quantitative Determination of Amitriptyline and Its Metabolite in Rat Plasma by Liquid Chromatography-tandem Mass Spectrometry

“…A recent study by described a liquid-liquid extraction (LLE) and solid-phase extraction (SPE) method improved sensitivity. 7,8 These reported methods have still higher quantification limits than our developed method.…”