Solid-phase microextraction and chiral HPLC analysis of ibuprofen in urine

https://doi.org/10.1016/j.jchromb.2005.01.010Get rights and content

Abstract

A simple and rapid solid-phase microextraction method was developed for the enantioselective analysis of ibuprofen in urine. The sampling was made with a polydimethylsiloxane-divinylbenzene coated fiber immersed in the liquid sample. After desorptioning from the fiber, ibuprofen enantiomers were analyzed by HPLC using a Chiralpak AD-RH column and UV detection. The mobile phase was made of methanol–pH 3.0 phosphoric acid solution (75:25, v/v), at a flow rate of 0.45 mL/min. The mean recoveries of SPME were 19.8 and 19.1% for (−)-R-ibuprofen and (+)-(S)-ibuprofen, respectively. The method was linear at the range of 0.25–25 μg/mL. Within-day and between-day assay precision and accuracy were below 15% for both ibuprofen enantiomers at concentrations of 0.75, 7.5 and 20 μg/mL. The method was tested with urine quality control samples and human urine fractions after administration of 200 mg rac-ibuprofen.

Introduction

(R,S)-ibuprofen [(±)-(R,S)-2-(4-isobutylphenyl) propionic acid], a non-steroidal anti-inflammatory drug (NSAID) widely used for the treatment of pain and inflammation in rheumatic disease and other musculoskeletal disorders, is marketed, with the exception of Austria and Switzerland [1], as a racemate [2], [3], [4], [5], [6]. However, its anti-inflammatory action is mainly associated with the (+)-(S)-enantiomer [7], [8], [9].

Ibuprofen undergoes stereoselective metabolism, resulting in stereoselective pharmacokinetics parameters, with higher plasma and urinary concentrations for the (+)-(S)-isomer [2], [3]. In addition, the disposition of the enantiomers of ibuprofen is particularly complex because (−)-(R)-ibuprofen undergoes biotransformation with inversion of configuration at the chiral center to yield (+)-(S)-enantiomer of the drug [10]. As a consequence, enantioselective methods are required for the analysis of ibuprofen in biological samples to evaluate the contributions of these stereoselective processes [11], [12].

Several HPLC methods have been developed for the analysis of ibuprofen enantiomers in biological human samples [13], [14], [15], [16], [17], [18], [19], [20], [21], almost of them based on the use of chiral stationary phases [13], [14], [17], [18], [20]. In addition, these methods were developed using common methods of extraction, mainly liquid–liquid extraction (LLE) [13], [14], [16], [17]. This traditional extraction technique has some disadvantage, such as the use of toxic and expensive solvents and being tedious and time consuming. These drawbacks can be avoided by the use of solid-phase microextraction (SPME), introduced by Arthur and Pawliszyn in 1990 [22]. This technique enables simultaneous extraction and pre-concentration of analytes from gaseous, aqueous, and solid matrices. SPME is based on the equilibrium of the analytes between the sample matrix and an organic polymeric phase usually coated on a fused-silica fiber; the amount of the analyte absorbed/adsorbed by the fiber is proportional to the initial concentration. It is also possible to obtain good extraction and reliable analysis under non-equilibrium conditions [23], if the extraction conditions are held constant. Selection of the fiber coating is mainly based on polarity of the analyte. Non-polar analytes have relatively high affinity for apolar phases, whereas polar fibers are the first choice for the extraction of polar analytes.

Most SPME methods developed until now are used in combination with gas chromatography (GC) with the fiber placed in the hot injector of the equipment, where the analytes are thermally desorbed. SPME and high-performance liquid chromatography (HPLC) were first coupled in 1995 [24] but in numerous applications, such as the analysis of drugs in biological samples, they have not been fully explored.

For SPME-HPLC coupling, the extraction procedure is similar to that used for GC analysis. The main difference between SPME-GC and SPME-HPLC is the second step, the desorption procedure. In HPLC analysis, an organic solvent or the mobile phase is used to desorb the analytes from the fiber. The desorption can be performed in a desorption chamber (on-line SPME-HPLC), or in a separate vial filled with the desorption solvent (off-line SPME-HPLC) [25], [26].

In this paper, we describe for the first time the development, validation and application of an SPME-HPLC method to the analysis of ibuprofen enantiomers in human urine. Three kinds of fiber coatings were compared: carbowax–templated resin (CW–TPR), polydimethylsiloxane–divinylbenzene (PDMS–DVB) and polyacrylate (PA). The extraction was carried out by direct immersion (SPME-DI) of the fiber into urine samples and off-line desorption was performed. The developed and validated method was applied to determine ibuprofen enantiomers in urine samples collected from a healthy volunteer after a single oral administration of 200 mg of rac-ibuprofen.

Section snippets

Drugs and reagents

Rac-Ibuprofen (99.9%) was kindly supplied by Knoll Pharmaceuticals (Nottingham, England). Commercial rac-ibuprofen formulation (Advil, Whitehall) was obtained in a local drug store. (+)-(S)-ibuprofen (99%) was obtained from Sigma-Aldrich (St. Louis, MD, USA). Trifluoroacetic acid was supplied from Fluka (Buchs, Switzerland). Sodium chloride and sodium dihydrogen phosphate were obtained from Merck (Darmstadt, Germany). Sodium hydroxide was obtained from Nuclear (São Paulo, SP, Brazil) and

Optimization of the extraction procedure

These experiments were performed using 0.5 mL drug-free urine samples spiked with 1.25 μg/mL of each ibuprofen enantiomer. Before immersion of the fiber for extraction, urine samples were submitted to alcaline hydrolysis as detailed in 2.6. Because ibuprofen is a weak acid (pKa = 3.8), the pH of the solutions must be adjusted to 2.5– 3, to keep it mainly in the undissociated form, that will be extracted by the fiber. The sample pH was adjusted by neutralizing the NaOH previously added during the

Conclusion

This paper describes for the first time the use of SPME-DI for the determination of ibuprofen enantiomers in human urine sample. The method is simple, highly sensitive and solvent-free. A single fiber was able to perform more than 100 extractions, showing that the desorption mode employed is reproductible. The validated method allows the determination of ibuprofen in the 0.25–25 μg/mL range with a quantification limit of 0.25 μg/mL for both enantiomers.

Acknowledgments

The authors are grateful to Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for financial support and for granting a research fellowship.

References (36)

  • G.T. Tucker

    Lancet

    (2000)
  • J. Caldwell et al.

    Biochem. Pharmacol.

    (1988)
  • K.M. Williams

    Pharm. Ther.

    (1990)
  • P.S. Bonato et al.

    J. Chromatogr. B

    (2003)
  • R. Bauza et al.

    Anal. Chim. Acta

    (2001)
  • C.H. Lemko et al.

    J. Chromatogr. Biomed. Appl.

    (1993)
  • K.J. Pettersson et al.

    J. Chromatogr. Biomed. Appl.

    (1991)
  • G. Geisslinger et al.

    J. Chromatogr. Biomed. Appl.

    (1989)
  • D.A. Nicollgriffith et al.

    J. Chromatogr. Biomed. Appl.

    (1988)
  • S. Ulrich

    J. Chromatogr. A

    (2000)
  • P. Popp et al.

    J. Chromatogr. A

    (2000)
  • M.N. Sarrión et al.

    J. Chromatogr. A

    (2002)
  • H. Lord et al.

    J. Chromatogr. A

    (2000)
  • M. Moeder et al.

    J. Chromatogr. A

    (2000)
  • H.-H. Lin et al.

    J. Chromatogr. A

    (2003)
  • S.-D. Huang et al.

    Talanta

    (2004)
  • F. Monteil-Rivera et al.

    J. Chromatogr. A

    (2004)
  • R. Causon

    J. Chromatogr. B

    (1997)
  • Cited by (76)

    • Spectrophotometric nanodrop system for quantification of trace concentrations of ibuprofen in water samples using silver-functionalized magnetic nanoparticles

      2022, Microchemical Journal
      Citation Excerpt :

      In recent years, various methods have been developed for the analysis of trace concentrations of IBU in different matrices. High resolution liquid chromatography has been one of the most used techniques for this purpose, although almost always coupled to mass detector, making the analysis technique not accessible to most laboratories [3,8,24,28]. Additionally, when this coupling is not performed, the detection limits reached are high, not being able to quantify the IBU traces found in drinking or wastewater, even in seawater [2,19,26].

    • Exploiting the capsule phase microextraction features in bioanalysis: Extraction of ibuprofen from urine samples

      2022, Microchemical Journal
      Citation Excerpt :

      The proposed CPME-UHPLC protocol was compared to other microextraction techniques followed by chromatographic determination of ibuprofen that were published in the literature in the last decade (Table 3). Until now, various microextraction techniques including solid-phase microextraction (SPME) [36], dispersive liquid–liquid microextraction (DLLME) [37], microextraction by packed sorbents (MEPS) [38,39] and fabric phase sorptive extraction (FPSE) [27] have been used for the extraction of IBU from biological matrices. The research group of Locatelli developed a FPSE method coupled to HPLC-PDA for the determination of IBU among other NSAIDs in human saliva.

    • Simultaneous enantioseparation of nonsteroidal anti-inflammatory drugs by a one-dimensional liquid chromatography technique using a dynamically coated chiral porous silicon pillar array column

      2020, Journal of Chromatography A
      Citation Excerpt :

      While the (S)-naproxen enantiomer binds to the cyclooxygenase enzyme to decrease prostaglandin production at the site of injury for decreasing pain, (R)-naproxen isomer does not inhibit inflammation properties and is furthermore toxic to the liver [23]. Also, (S)-ibuprofen is the only active form and (R)‑ibuprofen even causes side effects [24]. The (R) and (S)-ketoprofen enantiomers produce different therapeutic actions.

    View all citing articles on Scopus
    View full text