LC Evaluation of Intestinal Transport of Praziquantel
2009, 69, S213–S217
Priscila O. Cinto1, Ana Luiza Ribeiro Souza1, Andre´a C. Lima1, Marco V. Chaud2, Maria Palmira D. Gremia ˜o1,& 1
2
Departamento de Fa´rmacos e Medicamentos, Faculdade de Cieˆncias Farmaceˆuticas, Universidade Estadual Paulista, Rodovia Araraquara-Jau´, km. 01, Araraquara, SP CEP 14801-902, Brazil; E-Mail:
[email protected];
[email protected] Universidade de Sorocaba, Curso de Farma´cia, UNISO, Rod. Raposo Tavares, 92,5, Sorocaba, SP CEP 18023-000, Brazil
Received: 9 September 2008 / Revised: 19 December 2008 / Accepted: 20 January 2009 Online publication: 1 March 2009
Introduction
Abstract Praziquantel (PZQ) is a highly lipophilic drug with low aqueous solubility. Despite this, it is well absorbed from the gastrointestinal tract. In this study, a simple LC method was developed and validated, in order to monitor the concentration of PZQ in TC-199 buffer in vitro, in the rat everted gut sac absorption model. PZQ was analyzed by a reversed-phase LC method with an isocratic mobile phase containing acetonitrile and water in the proportions 45:55. The flow-rate was 1 mL min-1 and PZQ was determined by measuring absorbance at 215 nm, at 25 °C. The method was found to be specific, as none of the components of TC-199 or intestinal sac artefacts interfered with the drug peak. Recovery was within acceptable statistical limits. The limit of detection was 0.54 lg mL-1 and the limit of quantitation was 1.63 lg mL-1. The calibration curve was found to be linear in the concentration range of 10–90 lg mL-1 PZQ. The proposed method was found to be rapid and selective and hence can be applied in the monitoring of the absorption of PZQ in in vitro everted gut sac absorption studies.
Keywords Column liquid chromatography Intestinal permeation assay Praziquantel
Praziquantel (PZQ), 2-cyclohexylcarbonyl[1–3,6,7,11b]hexahydro-4H-pyrazino[2,1-a]isoquinolin-4-one, is a synthetic heterocyclic antihelminthic agent, broadly effective against flatworms, including trematodes, human and veterinary cestodes, and displays cysticidal effects. PZQ is active against all major human schistosome parasites and has become, at present, the drug of choice for the treatment and morbidity control of schistosomiasis, a disease that affects about 200 million people worldwide, leading to the loss of 1.53 million disability-adjusted life years [1–4]. The low efficacy of orally administered PZQ is considered to be the result of its pharmacokinetic pathway and hydrophobic character [5, 6]. PZQ is a candidate for incorporation into liposomes, given its low solubility in water and very short half-life [7, 8]. By this means, it is possible to reduce the rate of metabolism and increase its bioavailability, thus increasing
Separation Analysis Applied to Pharmaceutical Sciences in Brazil.
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its efficiency. Compounds with potent activity in vitro may fail to generate good activity in vivo owing to their poor water-solubility, poor permeability and/or poor stability. In fact, drug dissolution and permeability across the intestinal mucosa is one of the most critical factors in defining drug bioavailability and biological activity [9, 10]. A number of in vivo, in situ, and in vitro experimental methods have been developed to determine intestinal drug permeability, such as artificial membranes, cultured cells, isolated tissues, and organ perfusion. However, these test systems always represent a compromise between high throughput with low predictive potential and low throughput with high predictive potential. [11]. Several in vitro experimental models have been used early in the development of drug candidates or delivery systems, to determine the intestinal absorptive potential of a drug. Such in vitro experimental models used to study absorption have advantages and disadvantages. Among the various test systems, the everted rat gut sac model is commonly used to investigate transport mechanism, classify permeability and predict in vivo absorption of drugs in humans for the reduced labor and experimental costs compared to in vivo animal studies [12, 13]. In recent years, this technique has been used to evaluate the permeation of drugs incorporated in drug delivery systems [10, 14, 15]. The aim of this study was to develop a simple isocratic reversed-phase LC method to measure the absorption of PZQ in vitro in an everted rat gut sac absorption model.
and filtered through a 0.45 lm membrane, were used throughout the study.
Chromatographic System and Instrumentation A Varian LC system was used for the analysis of PZQ, consisting of a ProStar/ Dynamax 210/215 solvent delivery module, ProStar 330 UV–Vis PDA spectrophotometric detector (set at 215 nm), and a Rheodine VS 7125 injection valve with a 100 lL loop. The analytical column employed was RP-18 (Varian, Chromsep) (250 mm 9 4.6 mm i.d. 5 lm particle size). The mobile phase was comprised of acetonitrile–water (45:55, v/v). All separations were performed isocratically at a flow-rate of 1.0 mL min-1 and column condition was maintained at 25 °C. A sonicator (ultrasonic liquid processor, heat systems) was employed to prepare small unilamellar vesicles (SUV).
Standard Solution Praziquantel was accurately weighed and dissolved in absolute ethanol to prepare a primary stock solution at a concentration of 1.0 mg mL-1. This solution was diluted appropriately with TC-199 buffer to obtain working standards in the concentration range of 10–90 lg mL-1. The TC-199 buffer used in all preparations was freshly prepared and filtered through a 0.22 lm acetate membrane filter; its composition was: 145 mM NaCl; 4.56 mM KCl; 1.25 mM CaCl22H2O; 5 mM NaHPO4 and 10 mM glucose.
Experimental
Method Validation
Materials
The analytical method presented in this article was validated by confirming linearity over the tested concentration range and assessing specificity, precision, recovery and detection and quantitation limits [16–18].
Soya phosphatidylcholine (PC) Epikuron 200 (Lucas Meyer, Hamburg, Germany), praziquantel (PZQ) (Reiza-Kern, Brusque, Brazil), pentobarbital sodium (Abbott, Sa˜o Paulo, Brazil), analytical grade chloroform (Merck – Darmstadt, Germany), LC grade acetonitrile (J. T. Baker, Mexico City, Mexico) and purified water, obtained by reverse osmosis (Millipore)
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Assay Specificity
Specificity, according to USP [19], is the ability to measure accurately and spe-
cifically the analyte of interest in the presence of the other components that might be expected to be present in the sample matrix. To examine the specificity of the proposed LC method, samples from inside the intestinal sac (without PZQ) were analyzed to determine possible interferences and were used as blank and as blank plus analyte (with added to PZQ).
Linearity
The linearity of an analytical procedure is its ability (within a given range) to produce test results which are directly proportional to the concentration of analyte in the sample. The linearity of the method was studied by injecting PZQ at nine concentrations in the range 10– 90 lg mL-1. Each concentration was prepared in triplicate. The mean peak areas versus concentration data was treated by least-squares linear regression analysis. The relative standard deviation (RSD) for the slope and Y-intercept of the calibration curve was calculated. In linearity studies, the acceptance criterion is a correlation coefficient of at least 0.998, otherwise the calibration curve is rejected.
LOD and LOQ
The limit of detection (LOD) of an analytical method is the lowest analyte concentration in a sample that can be detected by the method of analysis. It is expressed as a concentration at a specified signal-to-noise ratio, usually two or three to one. The limit of quantitation (LOQ) is defined as the lowest concentration of the analyte in a sample that can be determined with acceptable precision and accuracy under the stated operational conditions of the method. The signal-to-noise ratio of 10:1 can be taken as LOQ of the method [20]. The LOD and LOQ were determined using Eqs. (1) and (2), respectively [17]. LOD ¼ 3:3r=S LOQ ¼ 10r=S
ð1Þ ð2Þ
where r is the standard deviation of the Y-intercept and S is the slope of the standard curve.
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Fig. 1. LC chromatograms of a sample of TC-199 buffer; b sample taken from inside the everted gut sac after incubation in TC-199 buffer (blank); c 75 lg mL-1 PZQ added to blank, and d sample from inside the everted rat gut sac after incubation with 250 lg mL-1 PZQ in TC-199 buffer. Absorbance at 215 nm
Intra- and Inter-Day Precision
Precision is the parameter that expresses the closeness of agreement (or degree of scatter) among a series of measurements obtained from multiple analysis of the same homogeneous sample under the prescribed conditions. To determine intra- and inter-day precision of the method, replicate (n = 3) sets of calibration samples were analyzed within the same day and between different days. Peak areas were determined and precision was expressed as relative standard deviation (RSD) of each curve [16].
racy of the proposed method, recovery studies were carried out by the standard addition technique in which quality control samples were prepared by spiking TC-199 with known amounts of the drug to obtain three different concentration levels (30, 50 and 70 lg mL-1) within the calibration-curve range. Each level of the drug was determined in triplicate (n = 3) by the LC assay. The amount of PZQ recovered was calculated in relation to the added amount (recovery percent).
Intestinal Permeation Assay Accuracy (Recovery Method)
Accuracy of an analytical method is its exactness or the closeness of the agreement between the correct value (which is accepted either as a conventional true result of a validated procedure or known amount of reference material) and the value found [17]. To confirm the accuFull Short Communication
All the animal studies were done after approval of the protocol by the Institutional Animal Ethical Care Committee (IAEC) of State of Sa˜o Paulo University (Protocol Number: 22/2007). In vitro absorption studies were performed with segments of everted rat small intestine.
Male adult rats (210–250 g) were kept in a fasting state for 8 h, anesthetized with sodium pentobarbital and the small intestine was immediately dissected, flushed with TC-199 buffer at 10 °C and placed in a beaker containing cold and oxygenated (O2:CO2—95:5) TC-199. The intestine was then gently everted over a glass rod (*2.5 mm in diameter) with its end protected by a fine fabric (mini brush), one of the ends was tied with suture wire and the intestinal segment filled with TC-199. The other end was then closed so that the length of the intestinal sac was 6 cm. The sac was incubated in oxygenated TC-199 buffer containing PZQ at various concentrations (50–1,000 lg mL-1) for 90 min, with gentle agitation. After this time, the gut sac was removed and PZQ concentrations inside the gut were determined by LC after filtration of the medium through cellulose acetate membrane (0.22 lm).
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Table 1. Intra-day and inter-day precision of quality control samples of praziquantel (n = 3)
Intra-day
Inter-day
Theoretical concentration (lg mL-1)
Concentration found (lg mL-1)
Precision as RSD
30.0 50.0 70.0 30.0 50.0 70.0
30.68 51.89 68.12 30.13 51.42 71.12
5.2 2.9 3.5 3.8 2.1 2.3
Table 2. Percent recovery of PZQ from TC-199 buffer Concentration (lg mL-1)
PZQ% recovery
RSD
30 50 70
101.50 108.73 102.29
2.27 1.23 4.30
trations of this drug in in vitro rat gut sac absorption experiments performed with TC-199. Good linearity was observed over the concentration range 10–90 lg mL-1. The mean (±RSD) values of slope and Y-intercept were 1,826,714.496 (±1.48) and 10,510,680.11 (±2.46), respectively. The correlation coefficient (r) was found to be 0.9986, indicating a functional linear relationship between the concentration of analyte and area under the peak. LOD and LOQ
The limit of detection of praziquantel was approximately 0.54 lg mL-1 and the limit of quantitation was set at 1.63 lg mL-1. This undoubtedly indicated the excellent sensitivity of the analytical method. Intra- and Inter-Day Precision
The intra- and inter-day precision of the method for praziquantel was evaluated by analyzing three replicates of 30, 50 and 70 lg mL-1 quality control samples. Intra-day precision ranged from 2.9 to 5.2%, while the inter-day precision ranged from 2.1 to 3.8% as shown in Table 1. These values were considered satisfactory, given the complexity of the matrix, and these results demonstrated that the method had excellent intra- and inter-day precision. Accuracy (Recovery Method) Fig. 2. Quantity of PZQ absorbed across the gut membrane in sacs incubated in various concentrations of PZQ in TC-199
Results and Discussion Method Validation Tests Specificity
The mean retention time (tR) of PZQ was found to be 10 min. The drug peak did not overlap with peaks of any of the components of TC-199, the medium that will be used in the in vitro absorption studies. Representative chromatograms of samples of TC-199 buffer, TC-199 from inside the intestinal sac without
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PZQ (blank), blank with PZQ added and the sample from inside the sac incubated with PZQ are shown in Fig. 1. The method is highly specific and detects the peak of the drug in the presence of the gut sac artefacts, as is clear from chromatograms in Fig. 1. Linearity
The calibration curve of PZQ was constructed in TC-199 buffer. This was used as the solvent because this method is intended to be used to monitor concen-
The mean absolute recovery values of the present LC method throughout the linear range are presented in Table 2. The results show that the method is obviously accurate, ensuring reliable results, since appropriate recovery values reported by Brazilian Legislation (2003) [16], were 80–120%.
In Vitro Everted Gut Sac Absorption - Standard Studies The ability to predict drug absorption through the gastrointestinal barrier is a key issue in the selection of new drug candidates for oral delivery [21]. Several
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in vitro models can be used early in drug development to determine the intestinal absorptive potential of a drug. The everted gut sac model is a relatively simple and very reproducible technique. Barthe et al. and Binks et al. [9, 22, 23], demonstrated the viability of this technique, using a complex biological tissue culture medium for the incubation (TC199). This incubation medium is very important in this assay, because it maintained the integrity of the everted intestinal fragment and the viability of the cells. Compared to other in vitro models, the everted gut sac model is simple, quick, very reproducible and inexpensive. On the other hand, the technique had disadvantages such as cell heterogeneity and the fact that the luminal and basolateral membranes were studied together. Being an in vitro animal experimental model, the absorption results obtained may not reflect the real in vivo absorption profile in humans. The measurements may also be affected by contrary flux and paracellular transport. The everted gut sac model was used to carry out intestinal permeation analysis in order to verify the permeation efficiency of PZQ through rat intestinal membrane. The drug concentration in samples obtained from inside the gut sac was analyzed by LC. The amount of drug that permeated through the mucosal membrane of the sac is plotted in Fig. 2 against the external concentration of PZQ.
Conclusions The proposed LC method is a simple isocratic reversed-phase method which was developed for the determination of PZQ concentrations in vitro in an
Full Short Communication
everted rat intestinal sac absorption model. The data generated by this method was analyzed to determine drug flux through the rat intestine. The LC method is capable of detecting low concentrations of PZQ in in vitro samples (0.54 lg mL-1). The peak area versus concentration curve was linear over the concentration range of 10–90 lg mL-1 (r = 0.9986). There was no interference between the analyte peak and intestinal sac artefacts or components of TC-199. The method is accurate and precise, as is evident from recovery values.
Acknowledgments Financial support by Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´ vel Superior (CAPES) and Conselho Nacional de Desenvolvimento Cientı´ fico e Tecnolo´gico (CNPq) is gratefully acknowledged.
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