Elsevier

Journal of Chromatography B

Volume 780, Issue 2, 25 November 2002, Pages 341-348
Journal of Chromatography B

Validation of a liquid chromatographic method for the determination of ibuprofen in human plasma

https://doi.org/10.1016/S1570-0232(02)00543-3Get rights and content

Abstract

A simple, rapid method of determining the ibuprofen concentration in small volumes of human plasma (50 μl) by HPLC was developed. The sample was prepared for injection using a solid-phase extraction method, with naproxen as the internal standard. A 96-well extraction plate was used, easing sample preparation and allowing the simultaneous extraction of multiple plasma samples directly into the HPLC injection vials. Samples were stable at room temperature for at least 48 h prior to injection. The HPLC method used an ultraviolet detector with a 5-min run time and measured concentrations across the range typically seen with the clinical use of this drug. The calibration curve was linear across the concentration range of 0.78–100 μg/ml with a limit of quantitation (LOQ) of 1.56 μg/ml. The coefficient of variation for intra-day and inter-day precision was 6% or less with accuracies within 2% of the nominal values for low (4.5 μg/ml), medium (40 μg/ml) and high (85 μg/ml) ibuprofen concentrations. For ibuprofen concentrations at the LOQ, the intra-day and inter-day precision and accuracy were within 10 and 15%, respectively. Recovery was 87% or greater for ibuprofen. This method was used to analyze plasma samples for unknown ibuprofen concentrations in bioequivalence and limited food effect studies of different formulations of ibuprofen. Thus, this method has been fully validated and used in the analysis of unknown plasma samples for ibuprofen.

Introduction

Ibuprofen, a propionic acid derivative, was the first non-steroidal antiinflammatory drug to be commonly used for the treatment of pain and inflammation [1]. Because this is a commonly used drug, there are frequently studies in which ibuprofen concentrations must be determined.

We report a method for measuring ibuprofen concentrations in human plasma using a simple solid-phase extraction technique and a rapid HPLC method with naproxen used as the internal standard. The advantages of this method over frequently reported methods include the ease of sample preparation, the small amount of plasma sample required (50 μl), stability of the prepared sample prior to injection for at least 48 h at room temperature, and an HPLC analysis using an ultraviolet detector with a run time of only 5 min. Limitations to other reported methods include solvent evaporation in the extraction process [7], [8], [9], requirement of larger plasma volumes [6], [7], [8], [9] or no validation in human plasma [10].

Also, this method was developed using a 96-well extraction plate further easing sample preparation by reducing the number of tube–tube sample transfers and allowing the simultaneous extraction of multiple plasma samples directly into the HPLC injection vials.

This method has been validated according to the criteria established by the Journal of Chromatography B [2] and the United States Center for Drug Evaluation and Research [3]. Included with this report, as proof of applicability, are the results of the analysis of human plasma samples for ibuprofen performed for bioequivalence studies supporting abbreviated new drug applications of new formulations of ibuprofen to the United States Food and Drug Administration (US FDA).

Section snippets

Chemicals and reagents

Ibuprofen and the internal standard, naproxen, where both from United States Pharmacopeia primary reference standards (Ibuprofen: lot 1, catalog number 335508; Naproxen: lot 1, catalog number 457301). Methanol and acetonitrile were both HPLC grade and were obtained from Fisher (Houston, TX, USA). Phosphoric acid was purchased from Sigma (St Louis, MO, USA). Water used in this study was obtained using a Millipore water purification system (Bedford, MA, USA). Drug-free human plasma was obtained

Assay validation

Standards of ibuprofen and naproxen were injected onto the HPLC column and found to elute at 3.1 and 1.7 min, respectively (Figure 1). Plasma obtained from six different individuals was tested for interference and showed no interfering peaks at these retention times. Plasma spiked with caffeine, aspirin and acetaminophen did not have any interfering peaks with ibuprofen and naproxen (Figure 1).

The concentration–response relationship for the calibration curve was described by a simple

Discussion and conclusions

Reports are available in the medical literature which describe methods for the chromatographic analysis of ibuprofen which have utility in different settings [6], [7], [8], [9], [10]. However, there are also some limitations to these methods, including solvent evaporation in the extraction process [7], [8], [9], larger plasma volumes required than used in the method described in this report [6], [7], [8], [9] or no validation in human plasma [10]. While there is increasing interest in the

Acknowledgements

This research was supported in whole by BASF Corporation (Shreveport, LA, USA). The investigators would like to thank Phil Burns, Lynn Massad and Ranga Velagaleti at BASF for their technical assistance.

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