Simultaneous quantitative determination of the major phase I and II metabolites of ibuprofen in biological fluids by high-performance liquid chromatography on dynamically modified silica

doi:10.1016/S0378-4347(97)00239-9

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 696, Issue 2, 29 August 1997, Pages 235-241

https://doi.org/10.1016/S0378-4347(97)00239-9 Get rights and content

Abstract

Ibuprofen has previously, after ingestion by man, been demonstrated to yield four major phase I metabolites, which are excreted in the urine partly as glucuronic acid conjugates. However, in previous investigations the quantitative determinations of the conjugates were performed by indirect methods. The purpose of the present investigation was to develop a high-performance liquid chromatographic (HPLC) system for the simultaneous determination of the major phase I and II metabolites of ibuprofen in biological fluids. The separation was performed using bare silica dynamically modified with N-cetyl-N,N,N-trimethylammonium hydroxide ions contained in the mobile phase. The separation of the metabolites of ibuprofen is greatly improved with this system compared to other published reversed-phase HPLC systems intended for the same purpose. The method developed makes it possible to simultaneously determine the intact glucuronic acid conjugates of ibuprofen as well as its phase I metabolites in human urine. In a study involving four healthy volunteers, a total recovery in urine of the dose given was found to be 58–86% within 8 h. This may be compared to an average of 67% earlier reported in the literature.

References (23)

S.S. Adams et al.
Toxicol. Appl. Pharmacol.
(1969)
M. Spraul et al.
J. Pharm. Biomed. Anal.
(1992)
A.C. Rudy et al.
J. Chromatogr.
(1990)
B. Chai et al.
J. Chromatogr.
(1988)
A. Shah et al.
J. Chromatogr.
(1986)
G. Geisslinger et al.
J. Chromatogr.
(1989)
G.F. Lockwood et al.
J. Chromatogr.
(1982)
U.G. Thomsen et al.
J. Chromatogr.
(1995)
S.H. Hansen
J. Chromatogr.
(1989)
J. Tjørnelund et al.
J. Chromatogr.
(1991)

S.H. Hansen et al.

J. Chromatogr.

(1983)

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Acute ecotoxicological effects on daphnids and green algae caused by the ozonation of ibuprofen
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Due to its ubiquitous presence in wastewaters, wastewater treatment plant effluents and even surface waters, the removal of the pharmaceutical ibuprofen from water is of special interest. Ozonation is widely applied for the treatment of micropollutants in wastewater treatment plants and is already known to also degrade ibuprofen. However, the formation of a wide range of transformation products during such oxidation steps might affect the aquatic environment. This study focuses on the acute ecotoxicological impact of the ibuprofen ozonation products on the two model organisms Daphnia magna and Desmodesmus subspicatus. For the identification of possibly ecotoxic products, a new workflow combining ecotoxicological testing, analytical methods and toxicity prediction was applied. Examination at different pH conditions with increasing ozone doses can point to respective products for further systematic examination.
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Citation Excerpt :
Moreover, Batt et al. (2008) reported the presence of 2′-OH-IBU in WWTP effluents in USA. 2′-OH-IBU is the main hydroxyl derivative that is produced via metabolism, while 1′-OH-IBU and 3′-OH-IBU were also found in urine but at very small concentrations (Kepp et al., 1997); however, there is scarce data for their presence in wastewaters. This is in agreement with Pérez and Barceló (2007) who pointed out that the three isomers of OH-IBU could not be differentiated unequivocally based on their mass spectra alone, but the most likely metabolite was believed to be 2′-OH-IBU.
Pharmaceuticals and personal care products (PPCPs) along with illicit drugs (IDs) are newly recognized classes of environmental pollutants and are receiving considerable attention because of their environmental impacts: frequent occurrence, persistence and risk to aquatic life and humans. However, relatively little information is often available with regard to their possible biotic and abiotic transformation products (TPs). This lack of knowledge has resulted in a substantial amount of ongoing effort to develop methods and approaches that would assess their occurrence, degradability potential elimination mechanisms and efficiencies in sewage treatment plants as well as environmental and human health risks. In this article, an extensive literature survey was performed in order to present the current stage of knowledge and progress made in the occurrence of TPs of PPCPs and IDs in raw and treated wastewaters. Apart from the TPs resulting from structural transformations of the parent compound in the aquatic environment or in technological treatment facilities (e.g. sewage and drinking water treatment plants), free metabolites and drug conjugates formed during human metabolism have also been included in this review as they are also released into the aquatic environment through wastewaters. Their concentration levels were reported in influents and effluents of WWTPs, hospital effluents and their removals in the treatment plants were discussed. Finally, information on the toxicity of TPs has been compiled when available.
Ultrasound-assisted emulsification microextraction combined with ultra-high performance liquid chromatography-tandem mass spectrometry for the analysis of ibuprofen and its metabolites in human urine
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IBU was isolated from biological fluids by deproteination [10,23], liquid–liquid extraction [8,11,14,21,25,26], solid-phase extraction (SPE) [8,12,18,22] solid-phase microextraction (SPME) [13,17], hollow fiber-based liquid phase microextraction (HF-LPME) [15,16] and hollow-fiber liquid membrane-protected solid-phase microextraction (HFLM-SPME) [6]. Three papers have described methods for the simultaneous determination of IBU and its metabolites (only selected two metabolites) [12,14,17]. Recently, much attention is being paid to the development of miniaturized, more efficient and environmentally friendly extraction techniques which could greatly reduce the consumption of organic solvents.
In this study, a fast, simple and efficient method based on ultrasound-assisted emulsification-microextraction (USAEME) coupled with ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) was successfully developed for the determination of ibuprofen (IBU) and its four metabolites (1-hydroxyibuprofen (1-HIBU), 2-hydroxyibuprofen (2-HIBU), 3-hydroxyibuprofen (3-HIBU), carboxyibuprofen (CIBU)) in human urine. For this purpose, the influence of the different parameters affecting the USAEME procedure was evaluated in order to optimize the efficiency of the process. The optimum conditions were found to be: 100 μL of 1-octanol as extraction solvent, 2 mL of urine sample, 15% (w/v) NaCl to control the ionic strength, ultrasonication for 10 min; and centrifugation for 5 min at 6500 rpm. After sample preparation, chromatographic separation was achieved on a Zorbax Rapid Resolution High Definition (RRHD) SB-C18 column using the mobile phase consisting of 0.1% formic acid in water and acetonitrile in an elution gradient. Detection was performed in a triple quadrupole tandem mass spectrometer using the multiple reaction monitoring (MRM) mode and negative ionization.
The proposed method showed satisfactory linearity over a wide concentration range (correlation coefficients over 0.9994). The lower limit of quantification (LLOQ) was 0.0005 ng/mL for IBU and its metabolites. The intra- and inter-day precisions were in the range of 2.19–10.8% and the accuracies were between −5.93% and 6.29%. The mean recovery of analytes ranged from 90.7 to 104%. As a result, this method has been successfully applied for the sensitive determination of IBU and its metabolites in human urine samples.
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Citation Excerpt :
Both compounds were found to form fragment ions due to loss of CO2 (loss of 43.9898 mass units) and loss of C3H8 (loss of 44.0626 mass units) from the deprotonated compounds. The known human phase I metabolites carboxy-IBF and 1-OH-IBF were not found in the trout bile samples (Brooks and Gilbert, 1974; Kepp et al., 1997; Tan et al., 1997, 2002). The ion peak at tR 16.0 min gave a m/z of 381.1556 (peak 4), which corresponds to the mass of acyl glucuronide of IBF, while the m/z 397.1512 ± 0.0001 recorded for the peaks eluting at tR 10.0–11.9 min (peaks marked with 5) and at peak at tR 1.6 min (peak 6) corresponded to the mass of the acyl glucuronide of hydroxylated IBF or ether glucuronide of hydroxylated IBF.
The biotransformation of the anti-inflammatory drug ibuprofen (IBF) was studied by exposing rainbow trout (Oncorhynchus mykiss) to IBF via intraperitoneal (i.p.) injection, and via water at four (0.17, 1.9, 13 and 145 μg L⁻¹) exposure levels for 4 d. Following exposure, the bile was collected and analyzed by LC–MS/MS methods. The identification of the formed metabolites in i.p. injected fish bile was based on the exact mass determinations by a time-of-flight mass analyzer (Q–TOF–MS) and on the studies of fragments and fragmentation patterns of precursor ions by ion trap mass analyzer (IT-MS). In addition to unmetabolized IBF, several phase I and phase II metabolites were found in the bile. The main metabolites were acyl glucuronides and taurine conjugates of IBF and of hydroxylated IBFs. The bioconcentration factors (BCF_bile), defined as the ratio of the sum of IBF and its metabolites in fish bile to the concentration of IBF in water, was determined following enzymatic deconjugation and was found to range from 14 000 to 49 000. The highest BCF_bile was found at the lowest exposure concentration (0.17 μg L⁻¹). The results show that rainbow trout has a high capacity for biotransformation of IBF, and the exposure of fish to sub μg L⁻¹ concentrations of IBF can be determined by the analyses of the biliary metabolites of the compound.
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A complex system of different enzymes is responsible for Ibu metabolism, including several CYPs, dehydrogenases (Chang et al., 2008; McGinnity et al., 2000) and UDP glucuronosyl transferases (UGTs) (Spraul et al., 1993). The main phase-I metabolites found in human urine are 2-hydroxy-Ibu (2-OH-Ibu), 3-carboxy-Ibu, and 3-OH-Ibu (Kepp et al., 1997). CYP2C9 is a highly polymorphic enzyme that is responsible for the metabolism of numerous clinically important therapeutic drugs, but not of procarcinogens (Johansson and Ingelman-Sundberg, 2011).
The anti-inflammatory drug ibuprofen (Ibu) is metabolized in the human liver to a number of metabolites including 1-hydroxyibuprofen (1-OH-Ibu), 2-OH-Ibu, and 3-OH-Ibu, respectively. The only human CYP known to produce relevant amounts of 3-OH-Ibu is CYP2C9 and as genetic polymorphisms of CYP2C9 influence the metabolization of numerous drugs, the availability of reference standards for CYP2C9-specific metabolites is of considerable interest. The aim of this study was to develop a biological production process for 3-OH-Ibu and to affirm its NMR characteristics. The recombinant fission yeast strain CAD68 coexpressing human CYP2C9 and CPR was used for the whole-cell biotransformation of Ibu to 3-OH-Ibu in 1 L batch-scale for 75 h. The average space-time yield for the bioproduction of 3-OH-Ibu (125 ± 34 μmol/L d) considerably exceeded that of 2-OH-Ibu (44 ± 10 μmol/L d). Accordingly, average biotransformation activities normalized to dry biomass weight were 5.0 ± 0.8 μmol/g d (3-OH-Ibu) and 1.9 ± 0.7 μmol/g d (2-OH-Ibu). The metabolite was prepurified on preparative TLC-plates, isolated by HPLC fractionation, and characterized by LC–MS and NMR. As expected, differential fragmentation patterns of 2-OH-Ibu and 3-OH-Ibu were detected in ESI-LC–MS analysis. 44 mg of 3-OH-Ibu was efficiently purified from four 1 L batch cultures and its structure was clearly confirmed by one- and two-dimensional NMR.

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Regular paperSimultaneous quantitative determination of the major phase I and II metabolites of ibuprofen in biological fluids by high-performance liquid chromatography on dynamically modified silica

Abstract

Toxicol. Appl. Pharmacol.

J. Pharm. Biomed. Anal.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

Regular paper
Simultaneous quantitative determination of the major phase I and II metabolites of ibuprofen in biological fluids by high-performance liquid chromatography on dynamically modified silica