Bioanalysis of captopril: two sensitive high-performance liquid chromatographic methods with pre- or postcolumn fluorescent labeling

https://doi.org/10.1016/S0378-4347(97)00002-9Get rights and content

Abstract

This study describes the development and comparison of two HPLC methods for the analysis of the antihypertensive drug captopril. The first method is based on a precolumn derivatization of captopril with the fluorescent label monobromobimane (MBB). The second method is based on a postcolumn reaction with the fluorescent reagent o-phthaldialdehyde (OPA). Since the disulfide metabolites of captopril can be reconverted to the active drug in vivo, the bioanalysis of captopril should involve both the determination of its free thiol form (free captopril) and the total amount of free thiol and reducible disulfides (total captopril). For total captopril analysis, disulfides were reduced with tributylphosphine (TBP) prior to protein precipitation. Since the reducing agent interfered with the MBB derivatization reaction, this method was not suitable for total captopril analysis. Both methods were validated for the bioanalysis of free captopril in human plasma. After removal of plasma proteins, samples were analyzed without an additional extraction procedure. The limit of quantitation in plasma was 12.5 ng/ml for the MBB method (limit of detection 30 pg) and 25 ng/ml for the OPA method (limit of detection 50 pg). The OPA method was also validated for total captopril analysis in human plasma and urine. The limit of quantitation was 25 ng/ml in plasma and 250 ng/ml in urine (limit of detection 50 pg). We conclude that for the analysis of free captopril the precolumn MBB method is superior to the OPA method since only the derivatization reaction has to be carried out immediately. The postcolumn OPA method is especially suitable for the analysis of total captopril since reducing reagents and high concentrations of endogenous thiols do not interfere with the derivatization reaction.

Introduction

The angiotensin converting enzyme (ACE) inhibitor captopril (1-[(2S)-3-mercapto-2-methylpropionyl]-l-proline, Fig. 1), is a widely prescribed antihypertensive drug. This article describes the development and comparison of two HPLC methods with fluorescent derivatization of captopril. The first method is based on precolumn derivatization with monobromobimane (MBB) (Fig. 2). MBB has been used as fluorescent label in the analysis of several important biological thiols and thiol drugs 1, 2, 3, 4, 5. The other method is based on postcolumn derivatization of captopril with o-phthaldialdehyde (OPA). The fluorescent label OPA is commonly applied for the derivatization of primary amines and amino acids. However, when an amine is added as a reagent, OPA can also be used for the selective derivatization of thiols 5, 6, 7, 8. The reaction of captopril with OPA is depicted in Fig. 3.

Like other thiols, captopril undergoes rapid oxidation to disulfide metabolites both in vitro and in vivo [9]. Intracellularly, disulfide metabolites are reduced to the free thiol and as such they can act as a reservoir for free captopril [10]. The kinetics of captopril can be interpreted correctly only when both the free thiol (free captopril) and the total amount of reducible disulfide metabolites (total captopril) are determined.

Several methods have already been reported for the quantitative determination of free and total captopril in biological fluids, including gas chromatography–mass spectrometry (GC–MS) 11, 12, radioimmunoassay (RIA) [13]and high-performance liquid chromatography (HPLC) 14, 15, 16, 17, 18, 19. The effective reduction of captopril disulfides with tributylphosphine (TBP) has been reported by several authors 13, 14, 15, 16. Some of the reported methods require very sophisticated and expensive equipment and other methods are very laborious. A major drawback of the existing methods is that relatively large sample volumes are required. Small sample volumes can be of importance when analyzing for captopril in small children 17, 20and in laboratory-animal studies. Both of the methods presented in this paper are sensitive, easy to perform and require only 100 μl of biological sample per assay. The methods were validated for the analysis of either free or total captopril in human plasma and for the analysis of total captopril in urine. In order to test their applicability in clinical and pharmacokinetic studies, the methods were used in a pharmacokinetic experiment in a human volunteer and in an in vivo rat experiment.

Section snippets

Materials

Captopril and MBB were purchased from Sigma (St. Louis, MO, USA). TBP (purum) and OPA (purum) were obtained from Fluka (Buchs, Switzerland). Glycine (analytical grade) was obtained from Merck (Darmstadt, Germany). All solvents were of HPLC quality (Labscan, Dublin, Ireland). All other chemicals were of analytical-reagent grade. Water was purified with a Milli-Q water system (Millipore, Bedford, MA, USA). Captopril tablets (Capoten) were obtained from Bristol-Myers Squibb (Woerden, Netherlands).

Instrumental

Derivatization procedures

The quantitative analysis of thiols is complicated by the reactive nature of this functional group. Most of the existing analytical methods for thiols are based on a precolumn derivatization reaction, protecting the thiol group from further oxidation [9]. We used MBB as precolumn derivatization reagent because adduct formation of thiols with MBB at room temperature is usually complete within a few minutes 1, 2and MBB adducts are stable at −20°C 2, 4, 5, 9. The addition of 10 μl of 10% MBB in

Conclusion

Both HPLC methods are sensitive, easy to perform and require only a small volume of biological sample. The MBB method and the OPA method can be applied for the analysis of free captopril, although the MBB method is superior to the OPA method for this type of analysis with regard to sensitivity and sample stability. The OPA method proves especially suitable for total captopril analysis.

Since both methods can be operated on the same chromatograph with only a small modification of the

Acknowledgements

This study was financially supported by the Dutch Organization for Scientific Research (NWO) Grant No. 902-21-151. GIDS is part of the research school Groningen Utrecht Institute for Drug Exploration (GUIDE).

References (24)

  • R.C. Fahey, G.L. Newton, R. Dorian and E.M. Kosower, Anal. Biochem., 111 (1981)...
  • I.A. Cotgreave and P. Moldeus, J. Biochem. Biophys. Methods, 13 (1986)...
  • L. Slordal, A. Andersen, L. Dajani and D.J. Warren, Pharmacol. Toxicol., 73 (1993)...
  • D.J. Warren and L. Slordal, Ther. Drug Monit., 15 (1993)...
  • C.C. Yan and R.J. Huxtable, J. Chromatogr. B, 672 (1995)...
  • H. Nakamura and Z. Tamura, Anal. Chem., 54 (1982)...
  • B. Gabard and H. Mascher, Biopharm. Drug Dispos., 12 (1991)...
  • C. Parmentier, P. Leroy, A. Visvikis and A. Nicolas, LC·GC Int., 9 (1996)...
  • D. Perret and S.R. Rudge, J. Pharm. Biomed. Anal., 3 (1985)...
  • O.H. Drummer, L. Routley and N. Christophidis, Biochem. Pharmacol., 36 (1987)...
  • O.H. Drummer, B. Jarrott and W.J. Louis, J. Chromatogr., 305 (1984)...
  • H.J. Leis, M. Leis, W. Welz and E. Malle, J. Chromatogr., 529 (1990)...
  • Cited by (0)

    View full text