Zak K. Shihabi

Department of Pathology,Wake Forest UniversitySchool of Medicine,Winston-Salem, NC, USA

Fenofibrate and fenofibric acid analysis by capillaryelectrophoresis

A capillary electrophoresis method has been developed to measure fenofibrate incapsules based on micellar electrokinetic capillary chromatography with detectionat 280 nm using a borate buffer containing sodium dodecyl sulfate (SDS). However,the metabolite of this drug (fenofibric acid) in serum and whole blood was analyzedby capillary zone electrophoresis (CZE) in a borate-carbonate buffer using aceto-nitrile stacking. The analysis is rapid, , 7 min with no interferences. Incubation offenofibrate in whole blood caused hydrolysis of the ester bond with the release offenofibric acid.

Keywords: Estrase / Fenofibrate / Fenofibric acid / Lipoprotein / StackingDOI 10.1002/elps.200305849

1 Introduction

Fenofibrate (Tricor) is a fibric acid derivative approved forthe treatment of hyperlipidemia. It decreases the serumlevel of cholesterol, and triglycerides and increases thelevel of high-density lipoprotein (HDL) [1–3]. Fenofibrate,like other fibrates, limits the availability of fatty acids fortriglyceride synthesis within the liver. Also it increaseslipoprotein lipase activity, stimulates reverse cholesteroltransport, and suppresses activity of 3-hydroxy-3-meth-ylglutaryl coenzyme A (HMG-CoA) reductase within theliver. It has also other desirable effects such as inhibitingangiogenesis, which is involved in plaque progressionand instability. Also it inhibits endothelial cell proliferation.Fenofibrate has a beneficial effect on fibrinogen and uricacid levels [4]. Elevated plasma fibrinogen is an independ-ent risk factor for cardiovascular disease. Fenofibrate hasbeen shown to decrease plasma fibrinogen levels by 15%and to decrease uric acid levels by 13% [4, 5].

Fenofibrate is highly protein bound (,99%). After absorp-tion, fenofibrate is rapidly and completely metabolized,essentially to its major active metabolite, fenofibric acidby plasma and tissue esterases [6]. The metabolite is pri-marily conjugated with glucuronic acid and excreted inthe urine with a half-life of approximately 20 h. Interest-ingly, a high correlation between plasma fenofibric acid

levels and its effect on b-lipoprotein changes was foundand it is thought to reflect the effect of fenofibrate on thecatabolism of plasma low-density lipoprotein (LDL) by theLDL-receptor [7].

Several HPLC methods for fenofibric acid analysis havebeen described [8–11]. These methods require high con-centration of organic solvent (,50%) for elution in addi-tion to sample extraction. Capillary electrophoresis is anattractive alternative method, which has several advan-tages over the HPLC; mainly lower cost and more flexibil-ity. Here, we show that CE can be used for rapid analysisof both fenofibrate and fenofibric acid. Fenofibrate in cap-sules can be measured by MEKC; while fenofibric acid inblood can be determined by CZE based on stacking bytransient pseudo-isotachophoresis [12, 13].

2 Materials and methods

2.1 Instrument

A Model 2000 CE (Beckman Instruments, Fullerton, CA,USA) equipped with a capillary of 25 cm650 mm (ID) wasset at 12 kV for MEKC and 5.5 kV for CZE with detectionat 280 nm. The sample was injected hydrodynamically (atlow pressure, 3447 Pa) for 15 s for MEKC or for 30 s forCZE.

2.2 Buffers

MEKC for fenofibrate: 100 mmol/L borate (Sigma, St.Louis, MO, USA) buffer, pH 8.0 containing 2% sodiumdodecyl sulfate (SDS) (Sigma) was used for the MEKC.CZE for fenofibric acid: 7 g boric acid, 7 g sodium carbon-

Correspondence: Prof. Z. K. Shihabi, Department of Pathology,Wake Forest University School of Medicine, Winston-Salem, NC27157, USAE-mail: zshihabi@wfubmc.eduFax: 1336-716-9944

Abbreviations: HDL, high-density lipoprotein; HMG-CoA, 3-hy-droxy-3-methylglutaryl coenzyme A; LDL, low-density lipopro-tein

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Electrophoresis 2004, 25, 1648–1651 Fenofibrate analysis 1649

ate (Mallinckrodt, St. Louis, MO, USA) and 5 g/L polyeth-ylene glycol 6000 (Fisher Scientific, Fair Lawn, NJ, USA)were used for the CZE analysis [14].

2.3 Procedure

Fenofibrate: capsule contents (or tablets, investigational)were weighed and dissolved in methanol followed by10 times dilution in 2% SDS prepared in 20 mmol/Lborate buffer, pH 8.0. Fenofibric acid: 100 mL serum orwhole blood was vortex-mixed for 15 s with 200 mL ofacetonitrile (containing 500 mg/L iohexol). The mixturewas centrifuged at 15 0006g for 20 s and the supernatantwas injected on the capillary for 30 s.

2.4 Standards

Stock standard of 2 g/L fenofibrate in methanol (Sigma).Stock standard of fenofibric acid: 200 mg fenofibrate in5 mL of 5 mol/L NaOH was heated at 957C for 4 h. Bothstandards were appropriately diluted before analysis.

3 Results and discussion

3.1 Fenofibrate

Because fenofibrate is a neutral compound (an ester) it issuited for analysis by MEKC while its metabolite being anacid is more suited for analysis by CZE. Fenofibrate is not awater-soluble compound, and thus has to be dissolved inorganic solvents such as acetone or methanol. Injection ofdirect organic solvents onto the capillary is not recom-mended in MEKC. Thus, the sample is diluted in an aque-ous buffer containing SDS to decrease the concentrationof the organic solvent. Figure 1 shows the analysis of feno-fibrate by MEKC. Several compounds can be added di-rectly to the dilution buffer and used as internal standardssuch as caffeine, Cl-theophylline, acetoacetanalide, and4-Cl-phenol. As the SDS concentration in the electropho-resis buffer is increased from 0.3 to 2% the fenofibratepeak becomes sharper and taller (higher plate number);however, the migration time is increased from 3 to 4.5 min.

Figure 2 shows the effect of pH on peak height and migra-tion. As the pH is increased, the migration time increaseswithout too much effect on peak height. We selectedpH 8.0 for routine work. The test was linear between 0.25and 4 mg/mL with 0.02 mg/mL as the minimum detectionlevel (three times the baseline noise). The RSD for peakheight was 4.9%, n = 11. The method was applied to anal-ysis of seven capsules, some which were placebo,Table 1. The expected and analyzed values are very close.

Figure 1. Analysis of 200 mg/L fenofibrate (F) by MEKC,several compounds can be added as internal standards.Peak 1, caffeine; 2, acetoacetanalide; 3, Cl-theophylline;4, 4-Cl-phenol.

Figure 2. Effect of pH on the peak height and migrationtime of fenofibrate.

3.2 Fenofibric acid

Fenofibric acid is the active metabolite of fenofibrate pres-ent in serum (or blood). Drugs in serum are more difficultto analyze than those present in tablets or capsules be-cause of the presence of many interfering compounds.

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CE

and

CE

C

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Table 1. Analysis of seven investigational capsules by CE(mg/capsule; n = 2)

Capsule Expected Found 6 SD

A 200 201 3.5B 200 198 8.4C 200 194 7.1D 54 59 4.3E 0 0F 0 0G 0 0

Since this metabolite is an acid it was more suited to anal-ysis by CZE. The CZE has the advantage of ability to con-centrate compounds present in low concentration easilyand directly on the capillary by acetonitrile stacking(pseudotransient isotachophoresis) as described earlier[12, 13]. This mechanism is different from that of the tran-sient isotachophoresis [15, 16]. The acetonitrile performsthe function of a terminating ion in eliciting the neededhigh field strength without being one [12]. The borate buf-fer was chosen because it is a general buffer used forseparating proteins as well as other compounds in ourlab [14]. Fenofibric acid compound migrates in theborate-carbonate buffer at about 6.5 min while the inter-nal standard migrates ahead at about 5.5 min (Fig. 3).Since the level of this compound in serum is relativelylow (5–30 mg/L), sample concentration and protein elim-ination are important for successful analysis.

Acetonitrile stacking as described earlier can accomplishboth steps conveniently at the same time. The capillary isfilled to 13% of its volume with sample without band broad-ening. Detection at 280 nm resulted in cleaned electropher-ograms (Fig. 3). However, this is not the case if the wave-length is set at 214 nm. Fenofibrate, the parent compound,migrates here with the neutral compounds. The calibrationcurve was linear between 3 and 40 mg/L (r = 0.998) with0.5 mg/L as the low detection limit (three times baselinenoise). This linearity covers the expected therapeutic levelof this drug in serum of 5–30 mg/L [7]. The apparent recov-ery of 25 mg/L of fenofibric acid in serum relative to that in1% NaCl was 86%. The apparent recovery is performed in1% NaCl rather than water because this concentration isvery close to that of the serum. Under these conditions,this concentration favors stackingandaffects themigrationclose to that of the serum [12, 13]. However, in order toavoid corrections for the difference in peak shape [17] orrecovery, the standards were added directly to a serumpool free of this drug. The RSD was 3.8% (n = 8). Iohexolcan be added as internal standard (Fig. 3). It is importantto note that there is a relationship between fenofibric acidserum level and the decrease in the lipids fractions [7].Thus, monitoring the serum level of this drug is useful.

Figure 3. Fenofibric acid (F) analysis in pooled serum. Theserum was deproteinated with two volumes of acetonitrile.(A) Pooled serum spiked with 18 mg/L; (B) serum beforespiking and (C) aqueous standard of 25 mg/L fenofibricacid (of1%NaCl).Sample volume, 13%of thecapillary vol-ume; separation at 5.5 kV. I, iohexol, internal standard.

Esterases are nonspecific enzymes present in most tis-sues including the serum and the white cells. They hydro-lyze a wide range of esters. Incubation of fenofibrate,which is an ester, in whole blood at 377C for 3 h caused ahydrolysis of the ester bond with the release of fenofibricacid presumably through the action of serum esterases(Fig. 4). This figure illustrates that whole blood can bealso deproteinated and stacked with acetonitrile similarto serum. The resulting electropherograms in this assayare also free from interference. Seventeen different bloodsamples were measured for the production of fenofibricacid from fenofibrate with a mean of 11 mg/L/3 h with arange of 2–22 mg. This shows that different blood sam-ples have widely different activities towards this drug.This in vitro hydrolysis brings the attention to the impor-tance of proper storage and rapid analysis of the sample

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Figure 4. Enzymatic production of fenofibric acid (F) inblood. Fenofibrate was added to whole blood at a con-centration of 200 mg/L; incubated at 377C for: (A) 0 h and(B) 3 h, and deproteinated with two volumes of acetoni-trile. Sample size, 13% of the capillary volume; separationat 6.5 kV. I, iohexol.

to avoid false-elevated values. Furthermore, investigatingthe use of fenofibrate as substrate for the blood esterasesmay yield clinical diagnostic significance about leakage ofthis enzyme from the injured tissues.

4 Concluding remarks

Fenofibrate and fenofibric acid have not been analyzed byCE before. This technique is well suited for analysis ofthese compounds both in capsules and in serum. MEKCis well suited for the analysis of the neutral drug fenofi-brate while CZE is more suited for the analysis of fenofi-

bric acid. The RSD for fenofibrate and fenofibric acid was4.9 and 3.8%. The LOD for both compounds is adequatefor practical analysis both in capsules and in serum. Theadvantage of the CE for fenofibrate analysis is the elimi-nation of the need for expensive organic solvents and col-umns and elimination of the need for sample preparationsoften used in the HPLC methods. The analysis by CE isalso simple and rapid.

Received December 8, 2003

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