Development and Validation of a Simple and Rapid Capillary Zone Electrophoresis Method for Determination of NNRTI Nevirapine in Pharmaceutical Formulations

Um método simples e rápido por eletroforese capilar foi desenvolvido e validado para a quantificação do inibidor não nucleosídeo da transcritase reversa (NNRTI) nevirapina, em medicamentos. A análise foi otimizada utilizando tampão fosfato de sódio 10 mmol L, pH 2,5, voltagem aplicada de +25 kV, injeção hidrodinâmica a 0,5 psi por 5 s e detecção UV em 200 nm. Diazepam (50,0 μg mL) foi usado como padrão interno. Sob estas condições, nevirapina foi analisada em aproximadamente menos de 2,5 min. A curva analítica apresentou um coeficiente de correlação de 0,9994. Os limites de detecção e quantificação foram 1,4 μg mL e 4,3 μg mL, respectivamente. Precisões intra e inter-dia expressas como desvio padrão relativo foram 1,4% e 1,3%, respectivamente e a recuperação média foi de 100,81%. O fármaco foi submetido a testes de hidrólises (ácida, básica e neutra) e a estresse oxidativo. Não foi observada interferência por parte dos produtos de degradação, nem dos excipients na análise da nevirapina. Este método mostrou ser rápido, simples, preciso, exato e econômico para a determinação de nevirapina em produtos farmacêuticos e é apropriado para o controle de qualidade em análise de rotina uma vez que a eletroforese capilar oferece benefícios em termos de desenvolvimento rápido dos métodos e custos muito reduzidos de operação.


Introduction
Nevirapine (11-cyclopropyl-4-methyl-5,11-dihydro-6H-dipyrido[3,2-b:2',3'-e][1,4]diazepin-6-one) 1,2 is a non-nucleoside reverse transcriptase inhibitor (NNRTI) that stops HIV from multiplying by preventing the reverse transcriptase enzyme from working. This drug is usually included in the generic fixed-dose combinations and has shown efficacy in advanced HIV disease. 3 Several analytical methods have been developed for analyzing nevirapine and other antiretroviral drugs including derivative UV spectrophotometry 4 and high performance thin layer chromatography (HPTLC). 5 High performance liquid chromatography (HPLC) has been widely used for determination of antiretrovirals in both biological fluids and pharmaceutical formulations, [6][7][8][9][10][11] hyphenated HPLC-MS [12][13][14][15][16][17] systems have also been introduced for the determination of antiretrovirals in plasma. Many of these methods require the use of expensive and hazardous chemicals which make the process complex, time consuming and costly. Capillary electrophoresis has become an active research and development area of considerable interest in analytical chemistry and has been widely adopted for the analysis of drugs and pharmaceuticals 18 due to its simplicity, wide application, small sample volume requirement, short analysis time, high resolution and low running cost with eco-friendly solvents as compared to HPLC.
Several CE methods were also available in the recent literature for the determination and separation of antiretroviral drugs in serum samples using solid phase extraction procedures. [19][20][21][22] Hyphenated CE-MS systems were also used. 23 A few CE methods were reported for the determination and validation of antiretrovirals drugs in bulk drugs and pharmaceutical formulations. 24, 25 Sekar and Azhaguvel, 25 separated and determined lamivudine and stavudine with the nevirapine (NNRTI) in pharmaceutical formulations by micellar electrokinetic capillary chromatographic (MEKC) method, all antiretroviral drugs were separated within 14 min using 10 mmol L -1 sodium tetraborate (pH 9.8), 100 mmol L -1 SDS and 15% 2-propanol as an organic modifier. The development of a simple CZE method when compared with MEKC method can supply a very useful alternative for routine analysis of pharmaceutical formulations.
Thus, as part of our ongoing anti-HIV research and considering that nevirapine is currently part of the AIDS treatment, there is a need to develop specific methodologies for its quality control. The aim of this study was to develop and validate a rapid, simple, inexpensive and environmental friendly capillary zone electrophoresis method, which is much faster than the existing MEKC or even CZE methods, for the determination of nevirapine in drug substance and formulated products suitable for routine quality control analysis and stability tests.

Reagents and chemicals
All the reagents used in the method development and validation were of analytical grade, the solvents of chromatographic purity and the water was purified by deionization (Milli-Q, Millipore, Bedford, MA, USA). The electrolyte buffer phosphate at 10 mmol L -1 concentration and pH 2.52 was prepared by dissolution of equimolar amounts of sodium dihydrogenphosphate dehydrate (Merck, São Paulo, Brazil) and o-phosphoric acid (Sigma, Milwaukee, WI, USA). Nevirapine ( Figure 1A) and diazepam ( Figure 1B) standards were kindly donated by Farmanguinhos (Rio de Janeiro, Brazil) and used without further purification. Commercial samples of nevirapine tablets containing 200.0 mg of nevirapine were obtained from three local laboratories (São Paulo, Brazil) named laboratory A, B and C.

Equipment and capillary conditioning
The experiments were performed using two CE systems: model P/ACE 5510 (Beckman Coulter Instruments, Fullerton, CA, USA), equipped with a variable UV-Vis, software for data acquisition and treatment (Beckman P/ ACE System Gold Software) and model HP 3D CE (Agilent Technologies, Palo Alto, CA, USA), equipped with a diode array detector, software for data acquisition and treatment (HP ChemStation, rev A.08.03). Samples were introduced onto the capillary via hydrodynamic injection (0.5 psi for 5 s, Beckman instrument, 1 psi = 6894.76 Pa; 50 mBar for 5 s, Agilent instrument, 1 mBar = 100 Pa). The instruments were operated under positive polarity, with a constant voltage of +25 kV. Detection wavelength was set at 200 nm and the capillary temperature was 25 ºC.  For capillary conditionating an uncoated fused-silica capillaries (Polymicro Technologies, Phoenix, AZ, USA) with an inner diameter of 75 µm and total length of 27.0 cm (19.4 cm to the detector) for Beckman instrument and 31.5 cm (23.0 cm to the detector) for Agilent instrument were used. New capillaries were conditioned by flushes with 1 mol L -1 NaOH (30 min), followed by deionized water (30 min) and electrolyte (10 min). At the beginning of the day, the capillary was conditioned with 1 mol L -1 of NaOH for 5 min, followed by deionized water for 5 min and electrolyte buffer for 10 min. In between runs, the capillary was rinsed with the electrolyte solution for 1 min. At the end of the day, a final 5 min wash with 1 mol L -1 NaOH and water was performed.

Preparation of standard solutions
Separate standard stock solutions of nevirapine (200.0 µg mL -1 ) and diazepam (500.0 µg mL -1 ), used as internal standard (IS), were prepared in acetonitrile and methanol, respectively. Working standard solutions were prepared fresh daily by diluting appropriately the stock solutions with deionized water.

Sample preparation
Twenty tablets of each sample (laboratories A, B and C) were finely powdered. Amounts corresponding to 20.0 mg of nevirapine of each sample were weighed, transferred into separate 100 mL volumetric amber flasks; 50 mL of acetonitrile was added to each flask for dissolution. The volume was completed with deionized water. The solutions were sonicated for 10 min and filtered using a 0.45 µm filter (Millipore), rejecting the first 5 mL. Aliquots of 5 mL of these solutions and 1.0 mL of standard stock solution of diazepam (500.0 µg mL -1 ) were transferred into 10 mL volumetric amber flasks and volumes were completed with deionized water. Final concentrations were 100.0 µg mL -1 and 50.0 µg mL -1 for nevirapine and diazepam, respectively. A standard solution was prepared at the same concentration of the sample, following the procedure described above. The samples and the standards solutions were sonicated for 10 min prior to introduction onto the capillary column.

Stress testing
For stress degradation studies the drug substance, nevirapine, was subjected to neutral, acid and alkaline hydrolysis and chemical oxidation. Individually 10.0 mg mL -1 of the drug were diluted with 5 mL of water, 1 mol L -1 HCl and 1 mol L -1 NaOH solutions in 10 mL volumetric flasks, the volumes were completed with acetonitrile. To analyze chemical oxidation, 10.0 mg mL -1 of the drug was diluted with 5 mL 3% H 2 O 2 solution in a 10 mL volumetric flask. These mixtures were heated at 80 ºC for 2 h. After completion of the degradation processes, the acid and alkaline solutions were neutralized. After stressing time adequate dilutions were made from each solution. The final concentration of nevirapine was 100.0 µg mL -1 .

Method validation
The proposed CZE method was validated based on the recommendations of the International Conference on Harmonization 26 and United States Pharmacopeia 2 guidelines.

Linearity
Aliquots of 4.0, 4.5, 5.0, 5.5, and 6.0 mL from the standard stock solution of nevirapine (200.0 µg mL -1 ) and aliquots of 1.0 mL from standard stock solution of diazepam (500.0 µg mL -1 ) were transferred into separate 10 mL volumetric amber flasks. The volumes were completed with deionized water. Concentration range from 80.0 to 120.0 µg mL -1 of nevirapine, and 50.0 µg mL -1 of diazepam were obtained. The solutions were sonicated for 10 min, and filtered using a 0.45 µm filter (Millipore) prior injection.

Precision
The repeatability (intra-day precision) was determined preparing samples solutions of nevirapine at three concentration levels (80.0, 100.0 and 120.0 µg mL -1 ) of the analytical curve with a constant amount of diazepam (50.0 µg mL -1 ) (IS). Three determinations of each concentration level were performed.
The intermediate precision (inter-day precision) was evaluated as described in repeatability, but it was performed in three different days. Precision was expressed by the relative standard deviation (RSD%) for peak area ratio (PAR) of nevirapine/IS.

Accuracy (recovery test)
To determine the accuracy of the CZE method, recovery experiments were performed according to procedures endorsed by AOAC International. 27 Nevirapine standard stock solution was added to commercial sample solutions and analyzed by the proposed method, according to the procedure depicted in Table 1.

Robustness
To determine the robustness of the method statistical comparison of assay results was performed by two different

Results and Discussion
Prior to method development, a few structural characteristics of the compound structure were taken into account. The basic character of nevirapine ( Figure 1A) makes it a suitable molecule for CE analysis. The overall charge of the molecule and consequently its mobility can be modified by controlling the electrolyte buffer pH. At low pH, the amine groups are readily protonated and the molecule bears a positive charge. At this low pH condition, only the electrophoretic mobility contributes for the net transport of the analyte towards the detector, since a negligible electroosmotic flow is observed. Thus, a good choice for the electrolyte is a phosphate buffer, which presents adequate buffering capacity around pH 2.5. An internal standard (diazepam, Figure 1B) was selected. Internal standards are often used to minimize injection volume fluctuations, dilution errors and errors during sample treatment. The proper choice of an internal standard can substantially improve precision of peak area determinations. The choice of detection wavelength was based on the inspection of the UV spectra of nevirapine and diazepam in the optimal electrolyte (not shown). In order to assure the method maximum sensitivity and accuracy, 200 nm was selected. Figure 2 illustrates the separation of diazepam and nevirapine standards under selected optimal conditions, confirming that diazepam performs well as internal standard. Migration times were approximately 2.0 and 2.6 min, respectively.
Sekar and Azhaguvel, 25 using micellar electrokinetic chromatography (MEKC) method separated nevirapine from lamivudine and stavudine (tablets) in approximately 12 min. The migration time of nevirapine is approximately five times higher when compared with the proposed CZE method. Furthermore, the CZE method did not generate hazardous organic solvents in contrast to MEKC method above described which uses 2-propanol.
In the HPLC methods described by Sarkar 9 and Kapoor 10 the authors identified nevirapine in about 5 and 14.5 min, respectively in pharmaceutical tablet form. Rebiere 11 separated nevirapine from the others antiretrovirals drugs in approximately 2.7 min but nevirapine was not well separated from fosamprenavir drug.
The applicability of proposed method to commercial sample tablets containing nevirapine is demonstrated by Figure 3B.
Before a method is routinely used, it must be validated. The validation process provides that the method is acceptable for its purpose. In the present work the CZE method was validated by determining its performance characteristics regarding specificity, linearity, limits of detection and quantification, precision, accuracy, robustness and system suitability. 2,26 All validation parameters were determined using Beckman instrument except for specificity (Agilent instrument) and robustness, which was assessed during sample analyses in both Beckman and Agilent instruments.
System suitability tests are used to verify that the resolution and reproducibility of the chromatographic system are adequate for the analysis to be done. 2 According  to the FDA 28 theoretical plate number must be higher than 2000, tailing factor ≤ 2, and resolution higher than 2. Table 2 shows the results obtained for nevirapine (100.0 µg mL -1 and diazepam (50.0 µg mL -1 ) standard solutions. All parameters are in accordance with FDA recommendations.
Since sample placebo was not available, the method specificity was attested by inspection of the spectral purity of the nevirapine peak in the sample electropherogram (Agilent instrument, not shown). A purity index superior to 98.8% was obtained, confirming the method specificity for nevirapine. Furthermore, was demonstrated noninterference between nevirapine and excipients from the sample ( Figure 3B).
To establish the method linearity, diluted fortified standard solutions were injected in triplicate and average peak area ratios (nevirapine/diazepam) were plotted against the corresponding nevirapine concentrations. Linearity was assessed by least-squares regression analysis. 29 Coefficients of correlation (0.99 or greater) and intercept close to the origin should be obtained. 30 As shown by the statistical data organized in Table 3, the method exhibited excellent linearity (r > 0.999) over the concentration range 80.0-120.0 µg mL -1 . Limits of detection and quantification were 1.4 µg mL -1 and 4.3 µg mL -1 , respectively. The criterion used to determine the limit of detection (LOD) and quantification (LOQ) was based on the residual standard deviation (SE) of the regression line of the analytical curve and its slope (S) ( Table 3), according to LOD = 3.3 SE/S and LOQ = 10 SE/S. 31 The intra-and inter-day precisions of the proposed analytical method were determined at concentrations in the low, middle and high linearity range with the constant amount of IS. The RSD % values for the peak area ratios are given in Table 4. Averages values of 1.4% and 1.3% RSD for intra-and inter-day precision were obtained indicating a good agreement among individual test results. The criterion for precision demands a RSD smaller than 2%. 18 The method accuracy was evaluated as the percentage recovery of a known amount of standard added to samples as endorsed by ICH 26 and AOAC International 27 protocols. Table 5 shows that recoveries ranged from 99.64 to 102.46% for three concentration level. Mean recoveries should be within 100 ± 2% at each concentration level over the range 80-120% of the target concentration. 1   Samples of nevirapine tablets from three different laboratories were assayed against a reference standard solution of nevirapine to establish the assay. Both solutions were prepared at 100.0 mg mL -1 of nevirapine. The results in Table 6 demonstrated that the proposed method is suitable for determination of nevirapine in tablets within tolerance limits (90-110% label claim).
The robustness of the proposed method was evaluated by statistical comparison of assay results generated by two different CE instruments (Table 6). Significance tests were performed in the results ( Table 7). As observed, calculated F values are smaller than the critical F value, attesting that there is no statistical difference in the assay results, at a confidence level of P = 95%. 32 Furthermore, no significant changes were observed in the instrumental responses when the analytical parameters were deliberately modified. When the voltage was changed from 22 kV to 28 kV, the values of RSD% were 1.52% and 1.53%, respectively for PAR of nevirapine/IS and when the temperature was changed from 22 ºC to 28 ºC, the RSDs% were 1.07% and 1.17%, respectively for PAR of nevirapine/IS. These results obtained were similar to those of the initial conditions (25 kV and 25 ºC). Thus, the proposed method can be considered robust and reliable.
CZE studies of drug nevirapine obtained on stress testing suggested the following degradation behavior. Degradation was not observed when nevirapine was subjected to neutral hydrolysis (water at 80 ºC for 2 h) ( Figure 4B). Significant degradation was observed when the drug was subjected to alkaline hydrolysis (1 mol L -1 NaOH at 80 ºC for 2 h), leading to the formation of unknown degradation products in approximately 1.2 (major peak) and 4.6 min ( Figure 4C). The same was observed under oxidative conditions (3% H 2 O 2 at 80 ºC for 2 h) leading to the formation of unknown degradation peaks in approximately 1.4, 1.6, and 3.7 min, the major product was seen at around 1.6 min ( Figure 4E). Under acidic conditions (1 mol L -1 HCl at 80 ºC for 2 h) nevirapine was almost completely degraded ( Figure 4D). The proposed CZE method was appropriate for quantitative determination of nevirapine in the presence of its degradation products.