Validation of a new HPLC method for determination of midazolam and its metabolites: Application to determine its pharmacokinetics in human and measure hepatic CYP3A activity in rabbits

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Abstract

Midazolam (MDZ) is a commonly used benzodiazepine in clinical practice. In addition, its metabolic oxidation is used as a surrogate marker for Cytochrome P450 (CYP) 3A enzyme activity as well. Thus, a new simpler method to measure MDZ and its metabolites is welcomed. Herein we report a new and simple HPLC method with ultraviolet detection for the simultaneous determination of midazolam and its hydroxyl metabolites using lorazepam as an internal standard. A liquid–liquid extraction was used to extract the compounds from rabbit hepatic microsomes and human plasma. The separation was performed on a Zorbax Eclipse XDB C18 column using a mobile phase composed of 0.05 M Na2PO4 (pH 4.5) and acetonitrile mixture (67:33) pumped at 1.2 mL/min. The calibration curves showed good linearity with correlation coefficient higher than 0.999 for all analytes in the range 10–500 ng/mL. Accuracy in the measurement of quality control (QC) samples was in the range 95–106% of the nominal values. The intra-day and inter-day precisions in the measurement of QC samples were less than 11% coefficient of variation. Although less sensitive than GC–MS, the proposed method was adequately sensitive to measure midazolam hydroxylase activity as a marker for CYP3A activity, and was applied to measure midazolam pharmacokinetics in human plasma.

Introduction

Midazolam (MDZ) is a benzodiazepine with a rapid onset and short duration of action [1], [2]. Due to its favorable pharmacokinetic properties, MDZ is commonly used in clinical practice to induce anaesthesia and to treat status epilepticus and induce sedation in patients in intensive care units [1], [3]. Clearance of MDZ has been used as a marker for the activity of Cytochrome P450 (CYP) 3A, the most abundant class of CYP enzymes in the liver [4], [5]. Midazolam is rapidly and extensively metabolized by CYP3A isoforms to 1-hydroxy (1-OH MDZ) and 4-hydroxy (4-OH MDZ) midazolam with the ratio of these metabolites indicative of the CYP3A isoform [5], [6]. Although 4-OH MDZ is pharmacologically inactive, 1-OH MDZ is 80% as potent as its parent drug [2].

A significant correlation appears to exist between plasma concentration of MDZ and its active metabolite and the degree of sedation [3]. A routine drug monitoring for MDZ has been recommended in patients with neurologic damage and in patients who have renal failure [3]. In addition, measurement of midazolam and its hydroxyl metabolites in body fluids and tissue is commonly used as a surrogate marker for CYP3A activity. Accordingly, analytical methods should be available to measure MDZ and its metabolites to allow for therapeutic drug monitoring at clinical settings, and to measure CYP3A activity for drug metabolism studies. An ideal analytical method should be sensitive, accurate and precise. The method should also be specific to avoid interference from endogenous chemicals and drugs that may be co-administered with MDZ. A short analysis time is crucial in clinical practice, routine drug analysis, and when large number of samples is processed per day.

Several analytical methods have been published for the determination of MDZ with or without its metabolites in different biological fluids. These techniques utilized high performance liquid chromatography (HPLC) with UV detection [7], [8], [9], [10], [11], [12], [13], HPLC–mass spectrometry (MS) [1], [14], [15], [16], [17], [18], [19], gas chromatography (GC)–MS [20], [21]. Although methods utilizing MS detectors are the most sensitive, and usually require small sample volume, these detectors are not always available in clinical settings and traditional research laboratories. Most of the HPLC–UV methods suffer from various limitations, including inadequate sensitivity [8], [9], [10], [12], long run times [10], [12], and use of expensive solid phase extraction cartridges [9], [10], [21].

Herein, we present a HPLC–UV method for the simultaneous assay of MDZ and its hydroxyl metabolites in human plasma and rabbit hepatic microsomes. The method offers the advantage of simplicity, specificity, sensitivity, and low sample volume to perform pharmacokinetic studies of midazolam and measure CYP3A activity. The simplicity of our proposed method is facilitated by using a single-step liquid extraction procedure rather using the expensive and tedious solid phase extraction cartridges. Endogenous compounds in human plasma and rabbit hepatic microsomes did not show any interference with the analytes of interest during the run time. The method's sensitivity was good enough to monitor MDZ level in human plasma for 6 h after oral administration and capture its pharmacokinetic parameters. Lower sample volume (200 μL) compared to other existing methods [8], [11], [22], [23] provides an advantage when plasma volume or hepatic microsomal fraction is very low or limited. As well, we report an application of the method to determine MDZ pharmacokinetics in human after a single oral administration, and to measure CYP3A activity in hepatic microsomes from rabbits.

Section snippets

Chemicals and reagents

Midazolam (MDZ), 1-hydroxymidazolam (1-OH MDZ), 4-hydroxymidazolam (4-OH MDZ) and all chemicals used for microsomal preparation, determination of microsomal protein content, and enzyme assays were purchased from Sigma–Aldrich Canada Ltd. (Oakville, ON, Canada). Lorazepam (LOR, 4 mg/mL) was purchased from Sandoz Canada Inc. HPLC grade acetonitrile and methanol were purchased from EMD chemicals (Darmstadt, Germany). Euthanol (Pentobarbital Sodium) was purchased from Bimeda-MTC Animal Health Inc.

Preparation of hepatic microsomes

Hepatic microsomes were prepared as described previously [24], [25]. Briefly, 0.5 g of liver was homogenized in 2 mL homogenization buffer (50 mM Tris buffer, 150 mM KCl, 0.1 mM dithiothreitol, 1 mM EDTA, 20% glycerol, and 0.1 mM phenylmethyl sulfonylfluoride). The homogenate was centrifuged at 9000 × g for 30 min in a Beckman L8-55 Ultracentrifuge (Palo Alto, CA, USA). The supernatant was carefully transferred to clean ultracentrifuge tubes and centrifuged at 100,000 × g for 30 min. The pellet was washed

Results and discussion

Midazolam and its hydroxyl metabolites (4-OH MDZ and 1-OH MDZ) are basic compounds with a tertiary amine group in their structure. These analytes were rendered non-polar in the biological samples (plasma or hepatic microsomes) through using a strong alkaline solution (2.5N NaOH), then extracted with a non-polar organic solvent (Toluene). After injection into a reversed phase column, MDZ and its metabolites were eluted from the column by using an acidic (pH 4.5) mobile phase. Lorazepam is

Study limitation

The method was applied to measure CYP3A activity in rabbit hepatic microsomes. Although rabbit is not commonly used as a model for this kind of study, we use it in our lab as a model for hypercholesterolemic atherosclerosis. An ongoing project is studying the correlation between hypercholesterolemia and activity of different CYP enzymes, e.g. CYP3A. Accordingly having a validated HPLC assay to measure CYP enzymes in rabbit hepatic microsomes is critical to accomplish our studies.

Conclusion

In this study, we describe a simple HPLC-UV method for the simultaneous determination of MDZ and its metabolites for their drug therapy monitoring and as a surrogate marker for CYP3A enzyme activity as well. This new method offers the advantages of simplicity, precision, adequate sensitivity, and low sample volume. We successfully applied the method to determine CYP3A activity in rabbit hepatic microsomes and to study the pharmacokinetics of MDZ in healthy human subjects.

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