Optimization and validation of a high-performance liquid chromatographic method with UV detection for the determination of ketoconazole in canine plasma

doi:10.1016/j.jchromb.2006.03.010

Journal of Chromatography B

Volume 839, Issues 1–2, 24 July 2006, Pages 62-67

https://doi.org/10.1016/j.jchromb.2006.03.010 Get rights and content

Abstract

An isocratic high-performance liquid chromatographic method with detection at 240 nm was developed, optimized and validated for the determination of ketoconazole in canine plasma. 9-Acetylanthracene was used as internal standard. A Hypersil BDS RP-C₁₈ column (250 mm × 4.6 mm, 5 μm particle size), was equilibrated with a mobile phase composed of methanol, water and diethylamine 74:26:0.1 (v/v/v). Its flow rate was 1 ml/min. The elution time for ketoconazole and 9-acetylanthracene was approximately 9 and 8 min, respectively. Calibration curves of ketoconazole in plasma were linear in the concentration range of 0.015–10 μg/ml. Limits of detection and quantification in plasma were 5 and 15 ng/ml, respectively. Recovery was greater than 95%. Intra- and inter-day relative standard deviation for ketoconazole in plasma was less than 3.1 and 4.7%, respectively. This method was applied to the determination of ketoconazole plasma levels after administration of a commercially available tablet to dogs.

Introduction

Ketoconazole, cis-1-Acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl] piperazine, shown in Fig. 1A, is a synthetic imidazole type of oral broad-spectrum antifungal agent. It is effective in the treatment of superficial and systematic infections [1], [2] and has been widely used in immunocompromized patients and advanced prostatic carcinoma [3]. Compared with other similar drugs, it has a broad-spectrum activity and few unwanted side effects.

Several analytical methods have been developed for the determination of ketoconazole in biological samples. Among them microbiological assays [4], [5] lack specificity because they are based on measurement of antifungal activity, which may not only be due to ketoconazole but to other bioactive plasma components or metabolites as well. HPLC methods with UV [6], [7], [8], [9], [10], [11], [12], fluorescence [13], [14], electrochemical [15] and MS–MS [16] detection have been reported in plasma samples. Chen et al. [16] have summarized the limitations of all the above HPLC methods. More specifically, main drawbacks of the existing HPLC methods with UV detections are either high detection limits (>50 ng/ml) [8], [9], [10], [11], [12], or complicated and time consuming sample preparation procedures including liquid-liquid extraction, evaporation and reconstitution in order to lower LODs [6], [7], [8], [12] or long total time elution time (>20 min) with the internal standard eluted after ketoconazole [7], [8], [9], [10] or lack of internal standard [6], [11], [12].

The aim of the present work was to develop and validate a simple, fast and reliable isocratic RP-HPLC method with UV detection for the determination of ketoconazole in plasma samples. The important features and novelty of the proposed method include simple sample treatment with acetonitrile precipitation, centrifugation and direct injection of the clear supernatant to the HPLC system; short elution time (less than 10 min) with internal standard eluted prior to ketoconazole; short analysis time (less than 20 min); low limit of detection 5 ng/ml; good precision (less than 5%) and high recovery (greater than 95%). Confirmation of the applicability of the developed method to pharmacokinetic studies of ketoconazole was also performed in dogs after single administration of a commercially available tablet (Fungoral^®).

Section snippets

Instrumentation

The chromatographic system consisted of a Spectra System P1000 pump, a Spectra System UV 2000 absorbance detector extended to the visible region and an autosampler AS 3000. The above system was controlled by a Specta System Controller SN 4000 and a software package Chromquest (Thermoquest Inc., San Jose, USA). A Hettich centrifuge Universal 32R (Tuttlingen, Germany) was utilized to centrifuge plasma samples.

Chemicals and reagents

Methanol (MeOH) and acetonitrile (ACN) of HPLC grade was purchased from E. Merck

Mobile phase

In the search of a proper and simple mobile phase, trying to avoid use of phosphate buffers, several solvent mixtures containing methanol (or acetonitrile) and water were examined. Triethylamine or diethylamine was necessary for minimizing peak asymmetry even though a BDS (Base Deactivated Silica)-C₁₈ column was used. The finally accepted mobile phase was methanol-water-diethylamine 74:26:01 (v/v/v), where peaks of the drug and IS were clearly separated and not interfered with plasma

Acknowledgments

The authors are grateful for the partial support of the General Secretariat of Research and Technology (PABET 2000), Ministry of Development, Greece and Galenica S.A. (Greece).

References (22)

S. Bajad et al.
J. Chromatogr. A
(2002)
P. de Bruijn et al.
J. Chromatogr. B
(2001)
C.M. Riley et al.
J. Chromatogr.
(1986)
N.R. Badcock
J. Chromatogr.
(1984)
K.B. Alton
J. Chromatogr.
(1980)
K.H. Yuen et al.
J. Chromatogr. B
(1998)
S.F. Swezey et al.
J. Chromatogr.
(1982)
D.W. Hoffman et al.
Anal. Biochem.
(1988)
Y.L. Chen et al.
J. Chromatogr. B
(2002)
J.G. Baxter et al.
J. Pharm. Sci.
(1986)

F.C. Odds et al.

J. Antimicrob. Chemother.

(1980)

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^☆: This paper was presented at the 4th International Symposium on Separations in the BioSciences (SBS ’05), Utrecht, The Netherlands, 18–21 September 2005.

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Optimization and validation of a high-performance liquid chromatographic method with UV detection for the determination of ketoconazole in canine plasma☆

Abstract

Introduction

Section snippets

Instrumentation

Chemicals and reagents

Mobile phase

Acknowledgments

J. Chromatogr. A

J. Chromatogr. B

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr. B

J. Chromatogr.

Anal. Biochem.

J. Chromatogr. B

J. Pharm. Sci.

J. Antimicrob. Chemother.