Liquid chromatography–tandem mass spectrometric assay for diclofenac and three primary metabolites in mouse plasma

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Abstract

The first liquid chromatography–tandem mass spectrometric assay for the simultaneous determination of diclofenac, 4′-hydroxy-diclofenac, 5-hydroxy-diclofenac and diclofenac-acyl-glucuronide in mouse plasma, using a simple sample pre-treatment procedure, was developed and validated. Analytes in plasma were stabilized using acetic acid and ascorbic acid. After addition of the internal standard D4-diclofenac to a 10 μl sample volume and protein precipitation with acetonitrile, the supernatant was supplemented with an equal volume of water and injected into the chromatographic system. A polar embedded reversed-phase column with gradient elution using formic acid and ammonium acetate in water–methanol were used. The eluate was totally transfered into an electrospray interface with positive ionization and the analytes were quantified using triple quadrupole mass spectrometry. The assay was validated in the ranges 10–5000 ng/ml for 4′-hydroxy-diclofenac and 20–10,000 ng/ml for the other analytes, the lowest levels of these ranges (10 or 20 ng/ml) being the lower limits of quantification. Within day precisions were ≤10%, between day precisions ≤13% and accuracies were between 90 and 108%. The analytes were chemically stable under all relevant conditions. The assay was successfully applied in a pharmacokinetic study with diclofenac in mice.

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

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