(mebendazole) was performed on an RP-18 column using acetonitrile-O.25N sodium acetate buffer (3:7, v/v), pH 5.0, as the mobile phase and using detection at ...
Journal of Analytical Toxicology, Vol. 18, March/April 1994
Determinationof AlbendazoleMetabolitesin Plasma by HPLC M a r i a E s t h e r C. Valois
Faculty of Pharmacy, Federal University of Maranh&o Osvaldo Massaiti Takayanagui
Faculty of Medicine of Ribeir~o Preto, University of S~o Paulo P i e r i n a S u e l i B o n a t o * , Vera Ldcia L a n c h o t e , and D e r m e v a l C a r v a l h o
Faculty of Pharmaceutical Sciences of Ribeir~o Preto, University of S&o Paulo, 14040-903, Ribeir&o Preto, SP, Brazil I Abstract Albendazole is an antihelminthic agent belonging to the benzimidazole class and has been used successfully in the treatment of neurocysticercosis. We report here a method for the determination of the two major albendazole metabolites in plasma, albendazole sulfone and albendazole sulfoxide. The method consists of drug extraction from 500 pL of plasma previously acidified with chloroform-isopropanol (9:1, v/v) and extract purification with n-hexane immediately before chromatographic analysis. Separation of drugs and of the internal standard (mebendazole) was performed on an RP-18 column using acetonitrile-O.25N sodium acetate buffer (3:7, v/v), pH 5.0, as the mobile phase and using detection at 290 nm.
detect it in plasma in an unchanged form after oral administration (10,13-15). This low bioavailability explains the fact that most analytical methods can measure ASOX, a drug with effective activity against neurocysticercosis, and ASON, which has no pharmacological activity (11). In the present report, we propose a simple method that requires a single extraction step and is of sufficient sensitivity for the analysis of albendazole metabolites to be used in therapeutic control and pharmacokinetic studies.
Experimental Reagents
Introduction
Neurocysticercosis is a serious public health problem in several countries in Latin America, Asia, and Africa, especially in developing countries in which precarious sanitary and socioeconomic conditions combine to perpetuate its dissemination. In our university hospital, neurocysticercosis is responsible for 7.3% of all admissions and for 2.7% of all outpatient clinic attendance in the discipline of neurology (1,2). Therapy for neurocysticercosis, formerly restricted to palliative measures, advanced with the advent of albendazole (3-5). Although it is considered the medication of choice for active parenchymal brain cysticercosis (6-8), it is necessary to establish a better dosage regimen as well as appropriate methods for the analysis of this drug and its metabolites in biological fluids (9). High-performance liquid chromatography (HPLC) is the technique of choice for this type of study (10-12) because it has the sensitivity and specificity necessary for the analysis of albendazole and its metabolites. Albendazole is practically insoluble in water and, therefore, barely absorbed in the gastrointestinal tract. Furthermore, it is rapidly metabolized to albendazole sulfoxide (ASOX) and then to albendazole sulfone (ASON) (Figure 1); thus, it is difficult to *Author to whomcorrespondenceshouldbe addressed.
86
The reagents used were analytical grade and chromatographic grade (methanol and acetonitrile, Merck). Standard ASOX (Robert Young & Co. Ltd., Glasgow, Scotland, U.K.) solutions were prepared in methanol at concentrations of 0.50, 1.20, 5.00, and 10.00 ~g/mL, and ASON (Robert Young & Co Ltd.) solutions were prepared at concentrations of 0.10, 0.24, 1.00, and 2.00 lag/mL. Mebendazole (Johnson & Johnson, Sao Paulo, Brazil) was used as the internal standard at the concentration of 0.50 gg/mL methanol. These solutions were stable for at least three months when stored at -20~ Equipment
We used a Marian liquid chromatographic system model 5000 equipped with a Rheodyne injector model 7125 (20-gL loop), a Varian ultraviolet absorption detector model UV-100, and a Varian integrator model 4290. Chromatographic separations were obtained on a 125- x 4-mm Lichrocart RP- 18 column (Merck) with 5-grn particles, protected by a precolumn of the same material (Lichrocart RP-18, 4 x 4 mm). Acetonitrile-0.25N sodium acetate buffer (3:7, v/v), pH 5.0, was used as the mobile phase, with a flow rate of 1.5 mL/min and detection at 290 nm. Sample preparation
The drugs were extracted with 2 mL chloroform-isopropanol (9:1, v/v) from 500-gL plasma aliquots to which 25 gL of the internal standard solution (mebendazole) and 200 IlL of 0.01M He1 (containing sodium metabisulfite at 4 g/L) were added. After
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Journal of AnalyticalToxicology,Vol. 18, March/April 1994 shaking for 20 min in a mechanical shaker (220 • 10 cycles/min) and later centrifuging at 1800 x g for 5 rain, the organic phases were transferred to conical tubes and evaporated dry under an air flow at room temperature. The residues were reconstituted with 100 laL n-hexane and 100 pL of the mobile phase. After shaking in a mixer for 20 s and centrifuging at I800 x g for 5 rain, 20/aL of the lower phase was chromatographed. The calibration curve was constructed in a similar manner, using 500-I.tL aliquots of blank plasma from healthy individuals who had received no medication for 72 h before collection and 100 laL of each standard drug solution added at the concentrations specified previously. Evaluation of the method Relative recovery was obtained from blank plasma samples enriched with ASOX and ASON at the same concentrations used to obtain the calibration curve. After extraction and chromatographic analysis, the relative recoveries were determined on the basis of a calibration curve that had not been submitted to the extraction procedure. For the determination of absolute recovery, the internal standard was added to a fixed volume (1 mL) of the organic phases obtained during the extraction step. The linearity of the method was evaluated by analyzing blank plasma samples, with ASOX and ASON added at the concentrations of 0.10-50.00 lag/mL and 0.02-10.00 lag/mL, respectively. The concentration ranges were considered to be linear when the coefficients of variation of the K values obtained (standard area/[intemal standard area • standard concentration]) were lower than 10%.
The repeatability of the method was evaluated by analyzing blank plasma samples enriched with ASOX at concentrations of 0.50 and 1.50 lag/mL and with ASON at concentrations of 0.10 and 0.30 lag/mE The intra-assay precision was evaluated by analyzing 10 samples enriched with albendazole metabolites, and the interassay precision was evaluated by analyzing the samples in duplicate over 5 consecutive days. The quantitation limit, defined as the lowest plasma concentration of the drugs that could be quantitated with an error of less than 10%, was determined by analyzing plasma samples (n =5) enriched with ASOX at concentrations of 0.06, 0.04, and 0.02 lag/mL andwith ASON at a concentration of 0.01 pg/mL. In order to determine possible interferents, we first chromatographed several drugs at the maximal concentrations in the therapeutic range under the conditions previously established for chromatographic analysis. Solutions of these drugs were prepared in methanol, and, after solvent evaporation, the residues were reconstituted in an equal volume of the mobile phase, and 20 taL of the resulting solutions were chromatographed. When the retention time found was similar to the retention time of ASOX, ASON, or the internal standard, the drug was added to blank plasma, submitted to the extraction procedure, and rechromatographed.
Results Figure 2 shows the chromatograms obtained in the analysis of blank plasma, of plasma enriched with ASOX, ASON, and the
A CH,-CH2- CH2/ s H
B 0
II
Ch~-Ch~-C~/ $~)---NH-C'O- OCh~ C 0
I!
g [2~ " f L-N H
D 0
FNH- CO-OCH= Figure 1. Structural formulas for (A) albendazole, (B) alhendazole sulfoxide, (C) albendazolesulfone, and (D) the internal standard, mebendazole.
p 2
4
6 rain
Figure 2. Chromatographic separation of albendazole metabolites: (A) blank plasma; (B) blank plasma containing (1) 1.00 pg/mL of AS0X, (2) 0.20 pg/mL of ASON,and (3) mebendazole;and (C) plasma from a patient under treatment with albendazole.
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Joumalof AnalyticalToxicology,Vol. 18, March/April1994
internal standard, and of plasma from a patient submitted to treatment with albendazole. Relative recovery was determined within the concentration range of 0.10-2.00 pg/mL and 0.02-0.40 lag/mL for ASOX and
ASON, respectively. The values obtained for ASOX (105.9%) and ASON (112.0%) were independent of concentration in the range analyzed. Using the same concentration range, absolute recoveries of
Table I. Determination of the Repeatability of the Method for Analysis of Albendazole Metabolites in Plasma Intra-assay precision
Interassay precision
Concentration added (pg/mL)
Mean concentration determined (pg/mL)
n*
ASOX
0.50 1.50
0.42 1.47
ASON
0.10 0.30
0.09 0.30
Drug
CVt (%)
Concentration added (pg/mL)
Mean concentration determined (pg/mL)
n*
CVt(%)
10 10
2.9 1.7
0.50 1.50
0.42 1.52
5 5
7.0 3.5
10 10
3.4 1.6
0.10 0.30
0.09 0.31
5 5
6.4 3.5
* n = n u m b e r of determinations. t C V = coefficient of variation.
Table II. Retention Times of the Drugs Studied as Possible Interferents Drug Salicylicacid Paracetamol Caffeine ASOX Valproicacid ASON Ephedrine Phenobarbital Carbamazepine Mebendazole Nitrazepam Clonazepam Chlordiazepoxide N-Desmethyldiazepam Haloperidol Clobazam Diazepam Amitriptyline Atropine Carbamazepine-10,11-dihydroxide Carbamazepine-10,11-epoxide Codeine Chlorimipramine Chlorpromazine Desipramine Phenylbutazone Phenylephrine Phenytoin Imipramine Levomepromazine Lidocaine Nortriptyline Primiclone Thioridazine Triazolam 9 t R = retention time.
t N D = not detected over a period of 30 rain.
88
y = 3.978x + 0.025
10 9"
Concentration (pg/mL)
tn* (min)
200.0 25.0 20.0 1.0 100.0 0.2 0.25 40.0 8.0 0.5 1.0 0.1 10.0 1.0 1.0 0.5 1.0 1.0 0.2 2.0 2.0 0.2 1.0 0.2 1.0 100.0 0.5 20.0 1.0 0.2 5.0 1.0 20.0 2.0 0.025
0.73 0.83 0.86 1.16 1.39 1.93 2.21 2.26 4.28 5.58 5.70 6.80 6.90 10.00 10.40 11.20 22.00 NDt ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
A
7. O3
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8" 5"
