INTRODUCTION:

Dissolution of drugs from tablet dosage forms is a key parameter in assessing the quality of product and uniformity at the formulation stage and throughout the product's shelf-life (M.A. E1-Massik et al., 1996).The in vitro dissolution profiles have also been used in a way to characterize the in vivo behavior of drugs with little success and is a requirement for regulatory approval for product marketing (Suraj D. Jadhav, 2011, Fernandes et al., 2006). The pharmaceutical industry and the regulatory agencies focus on the evaluation of the release kinetics from dosage forms, and this study is generally performed on official or nonofficial dissolution devices (Yalc¸ın O8 zkan, 2000, Jashnani et al., 1993). The United States Pharmacopoeia (USP) contained dissolution methods for zidovudine tablet (900 ml, distilled water at 50 rpm for 30 min) and for combination of lamivudine and zidovudine tablet (900 ml.0.1N HCl at 75 rpm for 30 minute).

The tolerance limit (Q) is not less than 85 % of the labeled amount of zidovudine and lamivudine dissolved (Pharmacopeia, 2007).According to FDA, validation is a process that generates sufficient data so as to assume that the process undertakes and will produce that which it is expected to. The EEC Guide to Good Manufacturing Practice for Medicinal Products 111/2244/87-EN. Rev. 3., 1989 consider that all analysis methods used in quality control must be validated (Manuel C6rdoba Borrego a and Rodriguez, 1995).

Lamivudine and zidovudine (Figure 1 and figure 2) are synthetic nucleoside analogues with activity against human immunodeficiency virus (HIV) and form one of the first line regimens in HIV treatment as fixed dose combination. The chemical name of Lamivudine is 2(1H)-Pyrimidinone, 4-amino-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-, (2R-cis) and the Zidovudine is 3'-Azido-3'deoxythymidine. Lamivudine is soluble in water, sparingly soluble in methanol, slightly soluble in ethanol (96%) and zidovudine is sparingly soluble in water, soluble in anhydrous ethanol (Pharmacopoeia, 2007).Both drugs are classified as class 1 drug in BCS classification system.

Figure 1. Structure of Lamivudine (a) and Zidovudine (b).

The aim of our study was to develop a prospective validation method of a dissolution test containing distilled water as a dissolution medium for zidovudine-lamivudine film coated tablets that contain mg of zidovudine and mg of lamivudine and liquid chromatographic method for the quantification of the drug from the dissolution test, as well as to evaluate the dissolution profiles for tablets.

MATERIALS AND METHODS:

Chemicals:

Lamivudine (99.69 %) and Zidovudine (99.58%) were obtained from GlaxoSmithKline, India. Dipotassium hydrogen phosphates (AR Grade), acetonitrile (HPLC Grade) were purchased from E. Merck (India) Ltd. Worli, Mumbai, India. The 0.45 µm nylon filters were purchased from Advanced Micro Devices Pvt. Ltd. Chandigarh, India. Double distilled water was used throughout the experiment. In house film coated tablets containing Lamivudine 150 mg and Zidovudine 300 mg per tablet, were used for the study.

Instrumentation:

The dissolution test was performed in Electro lab TDT 08L, in accordance with United States Pharmacopoeia (USP) general methods (Pharmacopeia, 2007). Analysis was performed on a chromatographic system of Waters, Alliance System, UV/VIS Detector (Waters 2487), System Software (Waters Empower). Chromatographic separation was achieved on Hypersil SS C18 (250 mm × 4.6 mm, 5µm) analytical column.

Chromatographic condition:

An HPLC method with UV detection, developed in our laboratory was selected due to its ability to separate Lamivudine and Zidovudine from the tablets excipients. The reverse-phase HPLC procedure utilized a bondpack C18; (4.6 x 250 mm I.D, 5 µm particle size, Thermo) column maintained at ambient temperature. The mobile phase was pumped through the column with flow rate of 1.5 mL/ min. Injection volume 10 µL was used in all experiments. The optimum wavelength selected was 270 nm, which represents the wavelength of maximum response for both Lamivudine and Zidovudine.

Dissolution condition:

Dissolution testing was performed in compliance with USP 29 [23] testing apparatus II rotating at 50 rpm and 900 ml of the distilled water (method I) and 0.1M HCl (method II). Dissolution apparatus was set up by programming temperature, rotation and run time at 37°C, 50 rpm and 30 minutes respectively. Degassed the dissolution medium and placed 900 ml of dissolution medium in each of the six vessels of the dissolution apparatus, equilibrated the dissolution medium to 37°C±0.5°C.Placed one tablet in each of the six vessels, and allowed the tablet to sink to the bottom of the vessel before starting rotation of the paddle. The 900 ml glass dissolution vessels were covered to minimize evaporation. Samples were collected using a glass hypodermic syringe equipped with a stainless steel needle at each sample time interval (1,2,3,4,5,6,8,10,15,20,25 and 30 min), an exact volume of sample was withdrawn from each flask, and immediately replaced with an identical volume of fresh medium. Aliquots were filtered using a 0.45 µ millipore membrane filtrate and assayed by HPLC.

Validation:

To demonstrate weather the method was optimum for dissolution testing. Method was validated through the analysis of Specificity, Linearity, Precision, Accuracy and Robustness Study (Samanta C. Mourãoa, 2010, Ju´ lia Menegola, 2007, ICH, 1996).

Specificity:

It was evaluated by preparing a placebo sample and test samples of the reference formulation of tablets in their usual concentration. The placebo sample was transferred to vessels with 900 ml of two different dissolution media deaerated ( 0.1 M HCl and distilled water ) and stirred at 37°C±0.5°C for 50 rpm and 30 minutes using paddle (USP apparatus 2). Aliquots of this solution were filtered with quantitative filter and 0.45 µ millipore membrane filter and analyzed by HPLC.

Linearity:

To study the linearity range of each component, the serial dilutions of standard stock were made in the range of 8.5-26.11 µg/ml of lamivudine and 17.30-51.91 µg/ml of Zidovudine (i.e. 50–150% of test concentration).Each solution was prepared in triplicate. A graph was plotted as concentration of drugs versus peak area response. The linearity was evaluated by linear regression analysis which was calculated by the least square regression method and analysis for variance (ANOVA).

Precision:

Repeatability and intermediate precision were used to assess the precision of the method. Repeatability was evaluated through relative standard deviation (RSD) from the recovery data at 100% level [3] at intra-vessel and the intermediate precision through the RSD inter-vessel., The dissolution test was done for 30 min using 900 ml of dissolution medium 0.1 M HCl, apparatus II rotating at 50 rpm. Aliquots of 10.0 ml were filtered with quantitative filter and then with 0.45 µ millipore membrane filter and analyzed by HPLC method. The recovery data were performed, in triplicate.

Accuracy:

Accuracy study was carried out by performing recovery studies at three levels (50%, 100% and 120% of test concentration) in triplicate. The drug was dissolved in ethanol and spiked to the dissolution media-containing placebo. The dissolution test was done for 30 min using 900 ml of dissolution medium 0.1 M HCl, apparatus II rotating at 50 rpm. Aliquots of 10.0 ml were filtered with quantitative filter and then with 0.45 µ millipore membrane filter and analyzed by HPLC method. The drug recovered was calculated and compared with the drug added to the dissolution media. The data given in the table represents the percentage recovery at three different levels. Each concentration was prepared in duplicate and each one was injected in triplicate.

Robustness:

The robustness of an analytical procedure gives its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. Robustness of the proposed dissolution method was studied by analyzing aliquot of a homogenous test sample by deliberately changing the parameters of the method. An experimental design was used to determine how the influence of filter type affects the dissolution profile of lamivudine and zidovudine from the tablets.

Stability:

The stability of the standard solution and test solution was evaluated by analyzing them at different time intervals up to 24 hrs keeping them at room temperature in solution form. The samples were analyzed against the fresh standard and the results were compared with the initial value.

Dissolution Comparison:

The dissolution profiles were compared through the similarity factor (f2), dissolution efficiency (DE) and dissolution parameters (k and t50). The f2 factor is a logarithmic transformation of the sum squared error of differences between the test and reference dissolution method over all time points. This factor was calculated as follow:

where and are the average the percent dissolved compound from the preparation at the ith time point and n is the total number of time points. This factor is 100 when the test and reference methods are identical and approaches zero as the profiles become increasingly dissimilar. According to the SUPAC-MR guidance document, two dissolution profiles are considered similar if the f2 value is between 50 and 100 (Ju´ lia Menegola, 2007, N.H. Anderson and Sardaro, 1998, FDA, September 1997).(FDA, September 1997, Michael D. Likar, 2005, S. I. Guseva, 2010)

Dissolution efficiency (DE) was also determined. DE is defined as the area under the dissolution vs time curve at time t, expressed as a percentage of the area of the rectangle that would correspond to 100% dissolution at time t.

Where y is the percentage of dissolved product. DE is then the area under the dissolution curve between time points t1 and t2 expressed as a percentage of the curve at maximum dissolution, y100, over the same time period. In the current study t1 = 0 and t2 =30 min. The integral of the numerator, i.e. the area under the curve was calculated by the trapezoidal method. The area under the curve is the sum of all the trapeziums defined by:

Where ti is the ith time point, yi is the percentage of dissolved product at time ti. If the difference and the 95% confidence interval of difference are within appropriate limits (9 -10% for example),indicated that the reference and test dissolution profiles are equivalent (N.H. Anderson and Sardaro, 1998, Samanta C. Mourãoa, 2010).

Dissolution profile were treated with first order kinetics and processed by linear regression. Dissolution rate constant (k) was calculated by slopes of the respective curves and extrapolation on each curve gives value of t50 (time required to 50 % drug release) (Manuel C6rdoba Borrego a and Rodriguez, 1995, Dr. Ritesh Patel, 2010, S. I. Guseva, 2010).

Statistical Analysis:

The pharmacokinetic data of zidovudine and lamivudine were statistically analyzed using GraphPad Instat software package. K and t50 were evaluated by F variance ratio test, followed by student t test (p<0.05).

Result and discussion:

The discriminatory power of the dissolution method depends on the method’s ability to detect changes in the drug product. The physicochemical property and solubility are important properties to be considered when selecting dissolution medium. K.prakash reported that lamivudine and zidovudine were soluble in 0.1N HCl and in distilled water. In present study we selected US FDA recommended 0.1 N HCl as a reference medium and distilled water as a test dissolution medium.

Method validation:

The HPLC was used for quantitative and qualitative determination of the dissolution samples. The HPLC was validated according to current ICH and FDA guidelines. Method was validated through the analysis of specificity, linearity, precision, accuracy and robustness study. The specificity of the method was evaluated by injecting blank solution, placebo solution, standard solution and test solution. Fig.2 shows the specificity of the HPLC method which illustrates the complete separation of Lamivudine and Zidovudine in presence of tablet excipients. There were no interferences at the retention time of Lamivudine and Zidovudine in the chromatogram of the placebo solution. These results showed the specificity of the method.

Figure 2. Chromatogram of blank (A),placebo (B),standard (C) and test (D) dissolution sample.

The linearity of the HPLC detector response for Lamivudine and Zidovudine was determined by analyzing series of different concentrations of Zidovudine (17.30-51.9µg/ml) and lamivudine(8.5-26.11µg/ml). The plots of area under the curve (AUC) of the peak responses of the compound against their corresponding concentrations (fig 3), they exhibit linear responses with r> 0.999 for both compounds. Different linearity study parameter such as a correlation coefficient, concentration ranges are summarized in Table 1.

Table 1. Different linearity study parameters.

Parameter	Zidovudine	Lamivudine
Linearity range(µg/ml)	17.30-51.91	8.5-26.11
slope	14271.38	18474.28
intercept	9567.068	7041.58
Correlation coefficient (r)	0.9994	0.9996

The system and repeatability of the method were evaluated by replicate intra-vessel and inter-vessel dissolution respectively. The %RSD was ≤ 1.88 for zidovudine and ≤ 1.46 for lamivudine. This reproducibility was considered acceptable and hence method is precise.

Accuracy of the dissolution method was calculated by recovery studies at three concentrations of 50%, 100%, and 150% levels by standard addition method (Table 2). The mean percentage recoveries obtained for zidovudine was 99.83%, while 100.55% for lamivudine. The %RSD was ≤0.76 for zidovudine and ≤ 1.05 for lamivudine.

Table 2. Result of recovery analysis of lamivudine and zidovudine

Compound	Wt. Spiked(mg)	Wt.recovered (mg)	% Recovery	% RSD
Zidovudine	166.39	167.15	100.46	0.53
	303.56	304.65	100.36	0.76
	449.43	443.50	98.66	0.29
Lamivudine	75.38	75.75	100.5	0.456
	150.77	150.17	100.06	0.285
	226.15	228.63	101.1	1.05

R.S.D.: Relative standard deviation. Wt: weight

Robustness of the method was evaluated as shown in table 3 and included filter type. Vinyl filter (0.45µ) and disc filter (0.45µ) were evaluated. The result demonstrated that filter type had no significant effect on quantitative analysis of dissolution sample.

The combined standard solution of lamivudine and zidovudine and dissolution sample were stored, unprotected from light and assayed after 3,6,9,12,15,24 hr.% variance within this period(table 4) ≤±0.76% and ≤±4.26 for test and standard zidovudine sample respectively, while ≤±3.74 and ≤±1.30 for standard and test lamivudine sample.

Dissolution comparison:

Analysis of the data in Fig. 4 showed that zidovudine and lamivudine release in both method are within tolerance limit Q (not less than 85 % within 30 min) (Pharmacopeia, 2007).Zidovudine drug release was 98.22±0.89 and 99.73±0.35 for method I and II respectively, while lamivudine drug release was 96.78±1.62 and 95.36±1.28 for method I and II respectively. Dissolution comparison between both method performed by analyzing dissolution efficiency, similarity factor (f2), t50 and k value.

Dissolution efficiency data are presented in table 5.It can be seen that DE value for zidovudine was 88.30±0.12 and 88.30±0.12 for method I and method II respectively, while 89.72±0.018 and 89.24±0.017 was DE value of lamivudine for method I and method II respectively.DE value for both methods was not found to be significantly different.

To compare dissolution profile of test method (method II) with reference method (method I) and to determine their equivalence, similarity factor (f2) was calculated. Similarity factor (f2) reflect the difference between two curves over all time points. The curves can be considered similar if the f 2 values are from 50 to 100.The similarity factor was 78.088 and 79.59 for zidovudine and lamivudine respectively (Table 5).It can be concluded that method II was equivalent to method I.

The mean K (rate constant) value and mean t50 for different experimental design are shown in table 5. Table 5 and 6 gives the corresponding first-order K (min-) and t50 values resulting from the adjustment of each profile with a regression curve. K and t50 were evaluated by( F) variance ratio test, followed by student t test(p<0.05).

Data analysis of t50 and k:

Data analysis of dissolution rate constant:

The statistic parameters n, number of data; K, mean value; S, standard deviation; and S², variance were calculated from the individual measurement obtained with each method for both compounds: (Manuel C6rdoba Borrego a and Rodriguez, 1995)

For Zidovudine:

nI= 6 nII=6

KI=0.3028 KII=0.2917

SI=0.00673 SII=0.00475

SI2=0.272×10-3 SII2=0.475×10-3

The experimental value for variance ratio (Fexp) is compared with the tabulated value (Ftab):

Ftab (DFI = 5,DFII=5,p=0.05) = 5.05

As values indicated Fexp < Ftab ,means method I and method II have a similar level of precision in regards to the dissolution rate constant with a probability of 95%,further compare ttab value with texp value was obtained as follow:

S² , is combined variance is obtain from:

Applying the value for S² to texp gives: texp = 1.346, ttab (DF = 5 + 5 = 10, p = 0,05)= 2.228.

Table 4. Stability study of zidovudine and lamivudine sample.

Time interval (hr)	% Variance
	Zidovudine		Lamivudine
	Standard	Test	Standard	Test
0 hour	--	--	--	--
3 hour	-0.07	0.51	0.00	0.61
6 hour	-0.91	0.76	-1.15	0.90
9 hour	-1.56	0.76	-1.51	1.30
12 hour	-2.40	0.45	-2.62	1.05
15 hour	-3.83	-0.23	-3.54	0.93
24 hour	-4.26	-0.11	-3.74	1.27

Table 5. Dissolution parameter comparison for method I and method II.

Parameter	Zidovudine		Lamivudine
	Method I	Method II	Method I	Method II
DE (%)	88.30±0.12	89.17±0.09	89.72±0.018	89.24±0.017
t50	1.63±0.057	1.46±0.046	1.91±0.17	2.064±0.085
k	0.305±0.006	0.297±0.004	0.42±0.057	0.38±0.028
f2	78.088		79.59

Table 6. Resulting values from six tablets in each method for zidovudine.

Tablet	Method I		Method II
	t50	k	t50	k
I	1.677	0.2856	1.5563	0.2902
II	1.8547	0.2971	1.4232	0.3017
III	1.6728	0.2879	1.5448	0.2879
IV	1.593	0.3017	1.5164	0.2948
V	1.5645	0.3178	1.5007	0.3040
VI	1.4232	0.3270	1.2551	0.2717

As values indicated texp < ttab, means there is no significant difference between the mean K values of either method, with a probability of 95%.

For lamivudine:

nI= 6 nII=6

KI=0.4229 KII=0.3818

SI=0.05746 SII=0.02853

SI²=3.301×10^-3 SII²=0.814×10^-3

The experimental value for variance ratio (Fexp) was compared with the tabulated value(Ftab): Fexp=0.2466 and Ftab (DFI = 5,DFII=5,p=0.05) = 5.05. As values indicated Fexp < Ftab ,means method I and method II have a similar level of precision in regards to the dissolution rate constant

with a probability of 95%,further compare ttab value with texp value was obtained as follow: Texp=0.1569 and Ttab(DF = 5 + 5 = 10, p = 0,05)= 2.228, as values indicated Texp < Ttab ,means there is no significant difference between the mean K values of either method, with a probability of 95%.

Data analysis of t50:

The statistical parameters as earlier are calculated: n, number of data; t50 mean value, etc.

For Zidovudine:

nI= 6 nII=6

t50I=1.6294 t50II=1.4660

SI=0.0574 SII=0.0463

SI²=0.0198 SII²=0.0128

Table 7. Resulting values from six tablets in each method for lamivudine.

Tablet	Method I		Method II
	t50	k	t50	k
I	1.96	0.3731	1.93	0.4007
II	2.12	0.3616	2.071	0.3569
III	1.71	0.4813	2.00	0.4145
IV	1.96	0.3984	2.15	0.3431
V	1.69	0.5020	2.09	0.3730
VI	2.05	0.4214	2.15	0.4030

The experimental value for variance ratio (Fexp) was compared with the tabulated value(Ftab): Fexp=0.646and Ftab (DFI = 5,DFII=5,p=0.05) = 5.05. As values indicated Fexp < Ftab ,means method I and method II have a similar level of precision in regards to the dissolution rate constant with a probability of 95%,further compare Ttab value with Texp value was obtained as follow: Texp=0.0688 and Ttab(DF = 5 + 5 = 10, p = 0,05)= 2.228, as values indicated Texp < Ttab ,means there is no significant difference between the mean K values of either method, with a probability of 95%.

For lamevudine:

nI= 6 nII=6

t50I=1.9143 t50II=2.0641

SI=0.1774 SII=0.0850

SI²=31.499×10^-3 SII²=7.294×10^-3

The experimental value for variance ratio (Fexp) was compared with the tabulated value(Ftab): Fexp=0.218 and Ftab (DFI = 5,DFII=5,p=0.05) = 5.05. As values indicated Fexp < Ftab ,means method I and method II have a similar level of precision in regards to the dissolution rate constant with a probability of 95%,further compare ttab value with texp value was obtained as follow: Texp=1.863 and Ttab(DF = 5 + 5 = 10, p = 0,05)= 2.228, as values indicated Texp < Ttab ,means there is no significant difference between the mean K values of either method, with a probability of 95%.

CONCLUSION:

The proposed HPLC methods is simple, rapid, accurate, precise, sensitive and easy to be applied to the simultaneous determination of dissolution rate studies of lamivudine and zidovudine in tablet dosage forms. The study have demonstrated that the results obtained with the test method used here to measure release profile present no statistically significant difference with compare to those obtained with the official method described in the USP for combination zidovudine and lamivudine tablets.

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Received on 05.09.2011 Modified on 25.09.2011

Asian J. Research Chem. 4(11): Nov., 2011; Page 1793-1799

Factor	Type	Component	Mean Area		% Variation
			Standard	Test	Standard	Test
	Unfiltered	Zidovudine	485963	465462	--	--
	Vinyl filter		494195	472505	1.69	1.51
Filter Type	Disc filter		488467	463532	0.52	0.42
	Unfiltered	Lamivudine	314207	298097	--	--
	Vinyl filter		319846	301161	1.80	1.03
	Disc filter		314811	296928	0.19	0.39