Liquid chromatography–tandem mass spectrometric assay for diclofenac and three primary metabolites in mouse plasma
Introduction
Diclofenac (DF, Fig. 1) is an important analgesic and anti-inflammatory drug, widely used in the treatment of post-operative pain, rheumatoid arthritis, and chronic pain associated with cancer. When given orally, absorption is rapid and complete in rat, dog, rhesus monkey, and man [1], [2]. Extensive first pass metabolism (Fig. 1) combined with low enterohepatic circulation reduces oral bioavailability of DF in humans to 50–60% of the administered dose [3], [4]. In rats and dogs, however, significant enterohepatic circulation due to biliary excretion of DF and its acyl-glucuronide (DF-G, Fig. 1) has been reported [1], [5]. Biliary excretion of DF-G in rats is critically dependent on multidrug resistance protein (Mrp) 2, an efflux pump located at the canalicular membrane of hepatocytes [6]. To further evaluate the roles of Mrp2 and other ATP-binding cassette multidrug transporters, disposition of DF and its primary metabolites was studied at our laboratory in recently generated knockout mouse models [7], [8]. To support this study, a sensitive bioanalytical assay for simultaneous quantification of DF and its three principal metabolites (Fig. 1) in small volumes of mouse plasma samples (ca. 25 μl) is required.
Chromatographic methods capable of quantifying diclofenac and one or more hydroxy-metabolites in human plasma [9], [10], [11] or urine [12], [13], [14], [15], [16] and in rat plasma [17] have been reported using LC-UV [9], [14], [15], [16], [17], GC-electron capture detection [10], [12], [13] and LC-electrochemical detection [11]. Lower limits of quantification (LLQs) in the range 5–25 ng/ml can be obtained using these techniques using 200–2000 μl of urine or plasma [9], [10], [11], [13], [14], [17]. For sample pre-treatment liquid–liquid extraction [9], [10], [12], [13], [14], [15], [16], [17], mostly using an ethereal solvent [9], [10], [12], [14], [15], [17], is often used. Solid-phase extraction (SPE) was also reported as a suitable option [11]. No validated LC/MS(/MS) method has been published for diclofenac and these metabolites so far.
On the other hand, quantification of the acyl-glucuronide of diclofenac, the main metabolite of this drug, seems to be an almost unexplored field. Two concise method descriptions for the quantification of DF-G in microsomal incubation mixtures using LC/MS/MS [18], [19] and one using LC-UV [6] could be found. The LC/MS/MS method of King et al. [19] only quantified DF-G (LLQ = 10 ng/ml) after SPE while the method of Kumar et al. [18] was capable of quantifying DF, 4′-hydroxy-DF (4′-H-DF, Fig. 1) and 4′-H-DF-acyl-glucuronide at, depending on the analyte, 6 ng/ml or lower, using protein precipitation. Seitz et al. [6] did not report the sensitivity of their LC-UV assay for DF and DF-G using protein precipitation.
Therefore, a chromatographic assay for the simultaneous quantification of DF, 4′-H-DF, 5-hydroxy-diclofenac (5-H-DF, Fig. 1) and DF-G, with a simple pre-treatment procedure using electrospray-MS/MS as detection technique for 10 μl sample volumes of mouse plasma was developed and validated. The final aim was to support in vivo pharmacokinetic studies in mice, using small plasma samples obtained from the tail vein.
Section snippets
Animals
Mice were housed and handled according to institutional guidelines complying with Dutch legislation. Animals used in this study were male wild-type mice of a FVB genetic background, between 9 and 15 weeks of age. Animals were kept in a temperature-controlled environment with a 12 h light/12 h dark cycle and received a standard diet (AM-II, Hope Farms, Woerden, The Netherlands) and acidified water (to pH 2.4–2.5 using hydrochloric acid to suppress bacterial growth) ad libitum.
Chemicals
DF was obtained from
Method development
Because of the high selectivity and sensitivity of the MS/MS detection, the simple pre-treatment procedure for a small sample volume could be used. Initially, an isocratic chromatographic system was developed using a mixture of 65% (v/v) methanol and 35% (v/v) of 0.05% (v/v) formic acid in water. The increased resolution of a 10 cm column was used to reduce the observed ion suppression by endogenous compounds using a 5 cm column and the 50 °C column temperature was used to reduce back pressure,
Conclusions
The first validated LC/MS/MS assay for the simultaneous quantitative analysis of diclofenac and three primary metabolites, DF-G, 4′-H-DF and 5-H-DF in plasma has been reported now. The assay uses a simple sample pre-treatment and meets common criteria for precision, accuracy and recovery. The sensitivity is in the same range as previous bioanalytical assays for only diclofenac and its hydroxy-metabolites but uses no more than 5% of the sample volume [9], [10], [11], [13], [14], [17]. Sufficient
References (25)
- et al.
J. Chromatogr.
(1986) - et al.
J. Chromatogr.
(1991) - et al.
J. Chromatogr.
(1981) - et al.
J. Chromatogr.
(1980) - et al.
J. Chromatogr.
(1985) - et al.
J. Chromatogr. B: Anal. Technol. Biomed. Life Sci.
(2003) - et al.
J. Chromatogr. B: Anal. Technol. Biomed. Life Sci.
(2006) - et al.
J. Chromatogr.
(1993) - et al.
J. Pharmacokinet. Biopharm.
(1991) - et al.
Clin. Pharmacokinet.
(1997)
Eur. J. Clin. Pharmacol.
Eur. J. Clin. Pharmacol.
Cited by (62)
Mass spectrometry imaging of diclofenac and its metabolites in tissues using nanospray desorption electrospray ionization
2022, Analytica Chimica ActaCitation Excerpt :In a QWBA study that was performed to determine the distribution of [14C] diclofenac in rats [48], the highest concentration of radio-labelled drug related material after 1 h was found in the bile, followed by the esophagus, kidney, and liver which coincided with the typical metabolism route [48]. Concentrations of diclofenac and its metabolites in mouse, rabbit, and human plasma [46,59,60] along with mouse and human urine [43,49,61] have been previously determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). With LC analysis, the isomeric 4′- and 5-hydroxy metabolites have been separated [46,49].
Development and validation of an LC-MS/MS assay for the quantification of cintirorgon (LYC-55716) in mouse plasma and tissue homogenates
2022, Journal of Pharmaceutical and Biomedical AnalysisCitation Excerpt :For this reason, a simple and fast 96-well format was used for the protein precipitation step which also enables working with low sample volumes. Because of the lack of a commercially available stable isotopically-labeled IS, several compounds (e.g. monensin, salinomycin, and diclofenac) were tested as IS alternatives [10,11]. The retention time of monensin was most similar to that of cintirorgon, with a difference of only 0.2 min, and was thus selected for further use as IS (Fig. 1).
Application of ultra-sensitive GC-QqQ-MS/MS (MRM) method for the determination of diclofenac in whole blood samples without derivatization
2021, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life SciencesCitation Excerpt :Diclofenac poisoning is a major cause of population decline in some vulture species that feed on the meat of animals treated with diclofenac prior to death [7]. Determination of diclofenac in biological samples was performed by the use of high-performance thin-layer liquid chromatography [8] and high-performance liquid chromatography with electrochemical detection [9], fluorimetric detection [10], UV detection [11,12,13] and coupled with tandem mass spectrometry [14]. Among gas chromatographic methods, detection of diclofenac was achieved by the use of electron-capture detector [15] flame ionization detector [16,17,18], and mass spectrometry [19–33].
Characterization of the catabolic pathway of diclofenac in Raoultella sp. KDF8
2019, International Biodeterioration and BiodegradationImmobilized copper ions on MWCNTS-Chitosan thin film: Enhanced amperometric sensor for electrochemical determination of diclofenac sodium in aqueous solution
2017, International Journal of Hydrogen EnergyCitation Excerpt :That is why they expose advantageous catalytic activity for many chemical reactions [45,46]. Multiple attempts have been established to determine DS that include thin layer chromatography (TLC) [47], spectrophotometry [48,49], high performance liquid chromatography (HPLC) [50,51], gas chromatography [52,53], spectrofluorimetry [54], liquid chromatography (LC) [55], capillary zone electrophoresis [56], voltammetry [57,58] from the biological fluids. Spectrophotometry requires extensive preparations of samples by extraction process or chemical reaction.
Bioconversion of non-steroidal anti-inflammatory drugs diclofenac and naproxen by chloroperoxidase
2017, Biochemical Engineering Journal