Elsevier

Microchemical Journal

Volume 75, Issue 3, December 2003, Pages 179-187
Microchemical Journal

Solvent and salting effects on sample preparation for the determination of fenofibric acid in human plasma by HPLC-DAD

https://doi.org/10.1016/S0026-265X(03)00093-6Get rights and content

Abstract

The solvent and salting effects induced on the sample preparation procedure applied to plasma samples containing fenofibric acid and 4-chlorophenyl-4′-hydroxyphenyl methanone (internal standard) are evaluated. Sodium chloride addition during a deproteinization step using both methanol and phosphoric acid influences the recovery of the analytes as well as the selectivity of the process. The chromatographic method allows high sample volume injection (500 μl) with the focusing of both analytes in the stationary phase. The synthesized high porosity octadecylsilica material allows a fast elution gradient at 4 ml/min flow-rate and a complete analysis within 7 min. UV-detection is made at 295 nm and quantitation limit in the 20 ng/ml concentration level can be achieved. The method can be successfully applied for bioequivalence studies on fenofibrate, administrated as prodrug (fenofibric acid represents its main active metabolite) in pharmaceutical formulations. The main parameters used in studying the retention behavior of the internal standard and FEFA were also estimated.

Introduction

Fenofibrate is a known prodrug used in the hyperlipidemia treatment. After absorption, it is rapidly and completely metabolized, mainly to its major active metabolite, fenofibric acid (FEFA) by plasma and tissue esterases [1], [2], [3]. No unchanged fenofibrate can be detected in the plasma after an oral dose administration [4], [5], [6]. Therefore, FEFA in plasma samples after fenofibrate administration. After oral administration of a single dose of 100 or 200 mg of fenofibrate, the maximum concentration in plasma is obtained after 5–8 h and is situated within a large interval (0.5 to 10 μg/ml). Analysis of FEFA in plasma samples during bioequivalence studies is a rather difficult task, due to the high variability on the adsorption and metabolism of fenofibrate. The molecular structure of fenofibrate, fenofibric acid and internal standard are given in Fig. 1.

A few methods for the determination of FEFA in plasma samples have been reported in literature; all of them are based on high-performance liquid chromatography (HPLC). Three approaches have been emphasized for plasma sample preparation: (1) solid-phase extraction [7]; (2) liquid–liquid extraction [8]; and (3) plasma deproteinisation [9], [10]. Extraction–based methods use acid addition to avoid the dissociation of the metabolite and subsequently to induce a low solubility in the aqueous phase. Internal standards (naproxen or sulindac) have been recommended for sample preparation methods involving solid-phase or liquid–liquid extraction (liquid extractants are mainly n-hexane or ethyl acetate). Such techniques are characterized by high recoveries of the analytes, high sensitivity (due to concentration of the samples), but sometimes requires tedious stages inducing also significant errors.

Plasma deproteinization by means of acid addition (perchloric acid, trichloroacetic acid), or by means of organic solvent addition (methanol, acetonitrile, acetone) has been reported in the literature [9], [10]. Such procedures are fast, easy to achieve, but suffer from a lack of sensitivity. Acid induced deproteinization generates low matrix interferences, but poor recoveries due to the low intrinsic solubility of FEFA as well as the adsorption phenomena on precipitated proteins.

Deproteinization by means of the addition of an organic solvent is characterized by higher recoveries. However, the matrix interferences and poor sensitivity are the main problems related to the technique.

Certainly, the sensitivity of the method should not be considered as a major concern for FEFA determination in plasma samples, but the large variability of the concentration values imposes a lower quantitation limit in the tenths of ppb range. The proposed deproteinization procedure is based on the addition of both acid and organic solvent. Simultaneous salt addition during the deproteinization stage induces advantages in terms of recovery and selectivity. In order to compensate the loss on sensitivity, large volume injection can be applied, with the focusing of analytes in the stationary phase. A fast gradient elution at a flow rate of 4 ml/min is affordable, due to the use of a synthesized stationary phase (with more than 80% porosity).

Section snippets

Experimental

The HPLC experiments were performed using an Agilent 1100 series liquid chromatograph consisting of a quaternary pump, solvent degasser, autosampler, column oven and diode-array detector (DAD). Chromatographic data were acquired by means of the Chemstation software (Agilent Technologies).

A Chromolith Performance RP-18e column (Merck), 100×4.6 mm, protected by a guard column (cartridge RP-18e, 10–4.6 mm) was used. The stationary phase consists in a synthesized chemically modified rod having

Results and discussions

Although the present method is not based on an extraction procedure, we still preferred to use an internal standard such that to eliminate the random errors due to the possible adsorption phenomenon on plasma precipitates. For this purpose, 4-chlorophenyl-4′-hydroxyphenyl methanone was chosen as internal standard (I.S.) due to the following reasons:

  • it is not a metabolite of the initial analyte, and it does not result from any degradation process from fenofibrate or FEFA;

  • it has quite a similar

Conclusions

The analytical method (sample preparation and chromatographic analysis) can be used for the determination of fenofibric acid in plasma samples. The sample preparation procedure is simple and based on the salting effects of sodium chloride added to the samples upon the solubilities of the fenofibric acid, internal standard and plasma pattern in final supernatant solution. The composition of supernatant solution allows high volume injection without focusing phenomenon and, consequently, reaching

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