Validation of a UV-spectrophotometric analytical method for determination of LPSF / AC 04 from inclusion complex and liposomes

1Universidade Federal de Pernambuco, UFPE, Laboratório de Imunopatologia Keizo-Asami, LIKA, Recife, PE, Brazil, 2Universidade Estadual da Paraíba, UEPB, Laboratório de Síntese e Vetorização de Moléculas, João Pessoa, PB, Brazil, 3Centro Acadêmico de Vitória, CAV, Recife, PE, Brazil, 4Universidade Federal de Pernambuco, UFPE, Laboratório de Planejamento e Síntese de Fármacos, Departamento de Antibióticos, Recife, PE, Brazil

The rational design of drugs is one of the most useful approaches for the introduction of new drugs in therapy and widely used by researchers in the medicinal chemistry field.In this context, LPSF/AC04 is an acridine-based derivative, part of a series of new anticancer agents synthesized for the purpose of developing more effective and less toxic anticancer drugs.This molecule, with potential antitumor activity, was synthesized at the Laboratory of Rational Design and Synthesis of Drugs (LPSF) of the Federal University of Pernambuco, Brazil.LPSF/AC04 has shown antitumor activity with tumor inhibition of more than 85% in a murine sarcoma 180 model after 8 days of treatment with 100 mg/kg i.p/day (De Lima, Lins, Pitta, 2007).In addition, a recent study has reported preliminary pharmacokinetics of LPSF/AC04 with a half-life of 66 h and accumulation in different tissues (Pigatto et al., 2011).However, heterocyclic acridine derivatives such as LPSF/AC04 are characterized by low solubility in aqueous solutions, limiting both clinical trials and its therapeutic use.
In order to minimize this problem, incorporation of the drug into controlled release systems is required.Liposomes are widely used in pharmaceutical applications, such as for drug delivery vehicles of several therapeutic agents, given their versatility and clinical efficacy (Torchilin, 2006).In addition, cyclodextrins are used to improve the solubility of poorly hydrophilic drugs.Cyclodextrins (CyDs) have a relatively nonpolar cylindrical cavity that can bind, and thereby solubilize, a wide range of hydrophobic molecules (Loftsson, Hreinsdóttir, Másson, 2005;Loftsson, Duchêne, 2007).The main purpose of inclusion complex-loaded liposomes is to combine the advantages of cyclodextrins as drug solubility enhancement agents with those of liposomes as drug-targeting agents.Some authors have cited the use of cyclodextrins in the formation of inclusion complexes with hydrophobic drugs, including acridine derivatives, to enhance their solubility (Schuette et al., 1991;Correia et al., 2002;Loftsson, Hreinsdóttir, Másson, 2005;Loftsson, Duchêne, 2007;Mishur et al., 2011;Al Omari et al., 2011).
The quality control of pure drugs and dosage forms are carried out using official and validated methods.Despite the clear advantages of using the HPLC chromatographic technique, the method has several limitations such as the high cost of instrumentation and operation, relatively long analysis times and the need for experience in handling the equipment and in processing samples (Siqueira-Moura et al., 2008).Spectrophotometry is a highly convenient analytical technique that is widely used in laboratories for quality control given its simplicity, low cost and wide availability (Darwish et al., 2009).
The main objective of this work was to develop and validate a simple, precise, accurate and economical analytical method for quantifying LPSF/AC04 by UVspectrophotometry. Subsequently, the resultant method can be applied to determine pure drug and dosage forms, such as liposomes and inclusion complexes containing LPSF/AC04.

Analytical method validation
The validation of the UV spectrophotometric analytical method was carried out based on parameters including linearity, limits of detection and quantification, specificity, precision, accuracy and robustness (ANVISA, 2003;ICH, 1995A;ICH, 1996B).All assays were performed at 25 °C except for the robustness assay, where samples were also stored at 4 °C and 37 °C before analysis.

Specificity
The specificity of the method was evaluated by comparing the UV spectra of blank samples (HP-β-CD and unloaded liposomes) against LPSF/AC04 standard solution.The analysis of LPSF/AC04-loaded liposomes and LPSF/AC04:HP-β-CD inclusion complex scanning was also performed from 225 to 340 nm and checked for changes in absorbance at the respective wavelengths.

Linearity
The linearity of the proposed method was verified by preparing three different standard solutions of LPSF/AC04 (0.3, 0.5, 1.0, 1.5 and 2.0 mg.mL -1 ), analyzed in triplicate, to plot nine derived analytical curves.The linearity of the analytical curve was evaluated by linear regression analysis using the least squares method and analysis of variance (ANOVA).

Limits of detection and quantification
Limits of detection (LOD) and quantification (LOQ) were estimated according to ICH guidelines (ICH, 1995A; ICH, 1995B).Limit of detection was calculated by: LOD = 3.3(σ/S), and limit of quantification was calculated by: LOQ = 10(σ/S), where σ is the standard deviation of the response of the blank and S is the slope of the analytical curve.

Accuracy
The accuracy of the method was determined by the recovery of a known amount of LPSF/AC04 added to samples of unloaded liposomes and HP-β-CD.Briefly, to determine the accuracy of the proposed method, different levels of drug concentrations were used: lower concentration (0.5 mg.mL -1 ), intermediate concentration (1.0 mg.mL -1 ) and higher concentration (1.5 mg.mL -1 ).A known aliquot of LPSF/AC04 stock solution was transferred to a 10mL volumetric flask containing unloaded liposomes or an accurately weighed amount of HP-β-CD equivalent to the quantity in the LPSF/AC04:HP-β-CD inclusion complex (Section 2.6.1) and filled to volume with methanol.A 1:10 dilution in methanol of the sample was then performed.
The samples were prepared in triplicate and analyzed by the proposed method.The relative standard deviation and LPSF/AC04 recovery percentage were employed to evaluate the accuracy of the method by applying equation 1.The paired t-test at a 95% level of significance was performed to compare the mean absorbance of the samples.Eq. 1

Precision
Inter-day, intra-day and inter-analyst variations were studied to determine repeatability and intermediate precision of the proposed analytical method.Intermediate precision was determined by analyzing three different levels of LPSF/AC04 concentrations at 0.5, 1.0, 1.5 mg.mL -1 .Different solutions were prepared in triplicate by two different analysts at two different times during one day and analyzed for intra-day variations.The same procedure was followed for two different days to study inter-day and inter-analyst variations (ANVISA, 2003).The percentage relative standard deviation (%RSD) of the predicted concentrations from the regression equation was taken as the measure of precision.The paired t-test at a 95% level of significance was performed to compare the mean absorbance of the samples.

Robustness
The robustness of the method was evaluated by changing the solvent suppliers and the temperature of the LPSF/AC04 samples (1.5 mg.mL 1 ) at 4, 25 and 37 °C.The samples were previously prepared and transferred to sealed tubes and refrigerated at 4 °C or maintained at different temperatures (25 and 37 °C) for 24 h prior to analysis.Assays were performed three times under the same conditions (ANVISA, 2003;Ulu, Elmali, 2010).

Preparation of LPSF/AC04:HP-b-CD inclusion complex
LPSF/AC-04:HP-β-CD complex was prepared using freeze-drying.First, stoichiometric amounts of HP-β-CD and LPSF/AC04 were dissolved in purified water and the mixture stirred for 72 h at 25 °C, then frozen in liquid nitrogen and finally lyophilized at 4x10 -6 Bars for 72 h.

Preparation of LPSF/AC04 and LPSF/AC04:HP-b-CDloaded liposomes
LPSF/AC04 and LPSF/AC04:HP-β-CD-loaded liposomes were prepared using the thin lipid film method (Mendonça et al., 2012).Drug entrapment efficiency was determined using ultrafiltration by the ultracentrifugation technique (Ultrafree ® units, Millipore, USA).Samples of LPSF/AC04 and LPSF/AC04:HP-β-CD-loaded liposomes (400 µL) were placed into the filtration unit and submitted to ultracentrifugation at 8,792g for 1 h.The samples were analyzed at 250 nm for determination of LPSF/AC04 content using a LPSF/AC04 analytical curve with concentrations ranging from 0.3 to 2.0 mg.mL -1 .All samples were prepared in triplicate.Data are expressed as percentage of total content of LPSF/AC04 entrapped in liposomes.

Analytical method validation
The absorption spectra of the LPSF/AC04 in methanol were recorded at concentrations ranging from 0.3 to 2.0 μg.mL -1 (Figure 3).LPSF/AC04 was found to exhibit a maximum absorption peak (l max ) at 250 nm with a molar absorptivity (ε) of 7.60×10 4 L.mol −1 .cm−1 .In addition, no interference from the solvent in LPSF/AC04 shoulder peaks near l max was verified.Methanol can thus be considered a suitable solvent for validating the proposed method, since it showed no interference in the analysis, thus supporting the reproducibility of the results.

Specificity
The method described was specific for the determination of LPSF/AC04 in inclusion complex and liposomes.A well-defined peak of LPSF/AC04 was observed at 250 nm.In addition, the absorption spectrum of blank samples (HP-β-CD without LPSF/AC04) and unloaded liposomes showed no peak at the specific wavelength of LPSF/AC04 (Figure 3).The absorption peak of LPSF/AC04 at 250 nm was unchanged in the presence of the constituents of the liposomal formulation and HP-β-CD, thereby demonstrating the specificity of the method.Mean absorbance (± SD) a 0.3 0.056 ± 0.002 0.5 0.095 ± 0.004 1.0 0.186 ± 0.007 1.5 0.271 ± 0.004 2.0 0.365 ± 0.007 a Values represent the mean of three analytical curves.

Linearity
Absorbance at l max = 250 nm and concentrations of LPSF/AC04 ranging from 0.3 to 2.0 µg.mL -1 presented a linear relationship (Table I), and the regression analysis data are summarized in Table II.The analytical curves were fitted by least squares treatment to give the following mean regression equation: Absorbance = 0.18068 x [LPSF/ AC04, µg.mL −1 ] + 0.00348 (r 2 = 0.9995, n=9) (Figure 4).

Limits of detection and quantification
The limit of detection (LOD) and the limit of quantification (LOQ) of LPSF/AC04 were 0.047 µg.mL -1 and 0.143 µg.mL -1 , respectively.These results showed that the method was sensitive even at low concentrations of LPSF/AC04.1.0 and 1.5 mg.mL -1 ) spiked in unloaded liposomes and HP-β-CD.LPSF/AC04 recoveries ranged from 101.06 ± 2.09% to 100.71 ± 1.35% for HP-β-CD (Table III) and 99.36 ± 0.67% to 101.51 ± 1.91% (Table IV) for liposome.The mean LPSF/AC04 recovery values were close to 100%, and their low standard deviation values evidence the high accuracy of the analytical method.These results reveal that small changes in the drug concentration in the solutions can be accurately determined by the proposed analytical method.

Precision
Repeatability (RSD%) ranged from 4.20 to 4.34 (%) for three levels of LPSF/AC04 concentrations (Table V).The results of repeatability (intra-day precision) of the method indicated the precision under the same operating conditions over a short period of time.Inter-day precision expresses within-laboratory variations on different days by different analysts (ANVISA, 2003).In the precision study, RSD% values were less than 5% in all cases and    were well within the acceptable range, indicating that the method has good repeatability and inter-day precision (ANVISA, 2003).

Robustness
The analytical method proved to be robust (Table VI), since no statistically significant differences (Student´s t-test) were found when samples were subjected to temperature variations and diluted in solvents from different manufacturers.The satisfactory recovery of LPSF/AC-04 from liposomes (>99%) stored at different temperatures confirmed that the molar absorptivity of LPSF/AC04 is not dependent on temperature.

Application of the method: determination of LPSF/AC04 in inclusion complex and liposomes
The encapsulation efficiencies of LPSF/AC04 and LPSF/AC04:HP-β-CD in liposomes were 99.02 ± 1.56% and 93.57± 0.37%, respectively.In the literature, the quantification of hydrophobic molecules in inclusion complexes and liposomes have been performed successfully using the proposed spectrophotometry method (Siqueira-Moura et al., 2008;Cavalcanti et al., 2012;Lapenda et al., 2012).
The high encapsulation efficiency of LPSF/AC04 and LPSF/AC04:HP-β-CD from liposomes indicated the effectiveness of the proposed method in the quantification of LPSF/AC04.

CONCLUSION
The proposed spectrophotometric analytical method for determination of LPSF/AC04 proved simple, rapid, accurate, precise and low-cost.The method was applied to quantify the LPSF/AC04 in inclusion complex as well in liposomes and can therefore be used for routine analysis.

TABLE I -
Experimental results of validation parameters

TABLE III -
Recovery of LPSF/AC04 in HP-β-CD matrix to evaluate the accuracy of the UV method Values represent the mean of nine measurements.b t cal is the calculated value and t crit is the theoretical value based on the paired t-test at the level of significance of p = 0.05.

TABLE IV -
Recovery of LPSF/AC04 in liposomes to assess the accuracy of the proposed method Values represent the mean of nine measurements.b t cal is the calculated value and t crit is the theoretical value based on the paired t-test at the level of significance of p = 0.05.

TABLE V -
Precision of proposed analytical method a t cal is the calculated value; b t tab is the theoretical value based on the paired t-test at the level of significance of p = 0.05 (n=6).

TABLE VI -
Robustness of the UV method using different solvent suppliers and temperatures of LPSF/AC04 samples t cal is the calculated value.The theoretical value t crit = 4.30 is based on the paired t-test at the level of significance of p = 0.05 (n=3). a