High-performance liquid chromatographic assay for acetaminophen glucuronide in human liver microsomes

doi:10.1016/S0378-4347(00)00566-1

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 753, Issue 2, 5 April 2001, Pages 303-308

https://doi.org/10.1016/S0378-4347(00)00566-1 Get rights and content

Abstract

A rapid and specific high-performance liquid chromatographic assay was developed for the determination of acetaminophen glucuronide formed by human liver microsomes. In addition, incubation conditions were systematically evaluated. Conditions that yielded the optimal rate of acetaminophen glucuronide formation over various concentrations of acetaminophen (0.15–30 mM) consisted of the following: 0.1 M potassium phosphate buffer, 1 mM magnesium chloride, 30 μg/mg alamethicin, 4 mM uridine 5′-diphosphoglucuronic acid at a pH of 7.1. Alamethicin produced higher and more consistent APAPG formation rates compared to Brij-58. Adding saccharolactone to the incubation medium reduced the velocity of the reaction. Acetaminophen glucuronide, acetaminophen, and the internal standard (paraxanthine), were analyzed on a C₁₈ column with UV detection at 250 nm. The mean correlation coefficient (r²) of the standard curves for acetaminophen glucuronide was >0.99 over the range of 0.1–25 nmol. The intra- and inter-day coefficients of variation were <4%. This method is suitable for in vitro studies using acetaminophen glucuronide formation as an index reaction for UGT activity.

Introduction

Glucuronidation is a major drug biotransformation reaction in the body. This reaction catalyzes the transfer of glucuronic acid from uridine 5′-diphosphoglucuronic acid (UDPGA) to a multitude of endogenous and exogenous lipophilic aglycon substrates through the activity of UDP-glucuronosyltransferases (UGTs). UGTs are glycoproteins that are mainly localized within the endoplasmic reticulum (ER) [1]. Acetaminophen (N-acetyl-P-aminophenol, 4-acetamidophenol, APAP) is a widely used analgesic drug that undergoes significant glucuronidation (Fig. 1). UGT1A6 is the major UGT isoenzyme catalyzing APAP glucuronide (APAPG) formation in vivo; UGT1A9 also glucuronidates APAP but with much lower affinity [2], [3]. Few assay reports have been published to quantify APAPG formation in liver microsomes [4], [5]. Pacifici et al. utilized tritium-labeled acetaminophen and determined APAPG formation as radioactivity remaining in the aqueous portion after an ethyl acetate-extraction [4]. Miners et al. isolated APAPG using solid-phase extraction with subsequent evaporation and reconstitution in mobile phase; analysis was by HPLC with UV detection [5]. Although these techniques were suitable for APAPG determination, a non-radioactive assay that does not require sample extraction was desired.

Interest in phase II metabolic pathways, notably glucuronidation, has recently increased due to their role in drug disposition and disease pathogenesis [6], [7], [8]. Indeed, APAP glucuronidation in human liver microsomes shows marked variability between individuals [4], [5]. Thus, a thorough evaluation of methods that are commonly utilized to measure glucuronidation activity in vitro is needed. In this report, we describe a simple, sensitive, and specific HPLC assay for the determination of APAPG using human liver microsomes. We also evaluated the effect of several incubation conditions on APAP glucuronidation in an attempt to optimize the rate of glucuronide formation under physiologic conditions.

Section snippets

Reagent and chemicals

APAP, APAPG, alamethicin, Brij-58 (polyoxyethylene [20]-cetyl ether), UDPGA, magnesium chloride, Trizma hydrochloride (Tris [hydroxymethyl] aminomethane hydrochloride), saccharolactone (d-saccharic acid 1,4-lactone), paraxanthine (1,7-dimethylxanthine) were purchased from Sigma (St. Louis, MO, USA). Potassium phosphate, ethanol, and methanol were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Acetic acid was purchased from Aldrich Chemical Company, Inc. (Milwaukee, WI, USA). All utilized

Chromatographic separation

Representative chromatograms of microsomal incubations are shown in Fig. 2. APAP, APAPG, and the internal standard (paraxanthine) were separated within 10 min of the chromatographic run. The retention times for APAPG, APAP and paraxanthine were approximately 3.1, 4.5 and 9.3 min, respectively. APAPG was stable in processed microsomal incubates for at least 24 h at room temperature.

Calibration, precision and accuracy

Standard curves for APAPG were linear over the range of 0.1–25 nmol. The mean correlation coefficient (r²) for the

Discussion

APAP glucuronidation represents about 55% of the drug’s primary biotransformation in the body. APAP has been proposed as an in vivo probe of UGT activity in part due to its safety at therapeutic doses [15]. This paper describes an HPLC assay for measuring APAP glucuronidation in human liver microsomes. The method described provides rapid, sensitive and specific detection of APAPG with a total run time of 10 min.

An additional objective was to evaluate different incubation conditions on the rate

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The phase II metabolic pathway(s) have notably increased in interest, due to their important role in drug disposition (Alkharfy and Frye, 2001).
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For the first time, electrocatalytic oxidation and selective determination of methadone (Mtd), as a long-acting opioid, in the presence of acetaminophen (Ac) has been investigated at a glassy carbon electrode modified with functionalized multi-walled carbon nanotubes. This simple and sensitive electrochemical sensor was fabricated through the drop-casting of functionalized multi-walled carbon nanotubes (fMWCNT) on the surface of a glassy carbon electrode (GCE). The electrocatalytic oxidations of Ac and Mtd are both individually and simultaneously investigated at the surface of the fMWCNT modified glassy carbon electrode (fMWCNT/MGCE) through using cyclic and differential pulse voltammetric studies. The fMWCNT/MGCE offered a considerable enhancement in the anodic peak current of Ac and Mtd associated with separating their overlapping voltammetric responses with potential difference of 290 mV. The catalytic peak currents obtained from differential pulse voltammetry of Ac and Mtd increased linearly with their concentration at the ranges of 0.45–90.0 μM and 0.5–100.0 μM, respectively, and the detection limits for Ac and Mtd were sequentially 0.35 μM and 0.28 μM. Furthermore, this electrochemical sensor was successfully implemented for the quantitative determination of Ac and Mtd in human urine, saliva and pharmaceutical samples using standard addition method and the obtained results were found to be satisfactory.
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High-performance liquid chromatographic assay for acetaminophen glucuronide in human liver microsomes

Abstract

Introduction

Section snippets

Reagent and chemicals

Chromatographic separation

Calibration, precision and accuracy

Discussion

Biochem. Pharmacol.

Biochem. Pharmacol.

Biochem. Pharmacol.

Biochem. Pharmacol.

Clin. Chim. Acta

J. Biol. Chem.

Gastroenterology

Arch. Biochem. Biophys.

Biophys. J.

Drug Metab. Rev.

Clin. Pharmacokinet.