Development and Validation of UV Spectroscopic by Q-absorption Ratio, RP-HPLC Method for Simultaneous Estimation of Atazanvir and Ritonavir in bulk and Pharmaceutical Dosage Form

 

K. Vijaya Sri*, S. Deepthi, M. Madhuri, P. V. Aishwarya

 

ABSTRACT:

The main objectives of presented work were developed and validate the UV-spectrophotometric by Q-absorption ratio and RP-HPLC method for the simultaneous estimation of Atazanavir and Ritonavir as per ICH guidelines in bulk and pharmaceutical dosage form. In UV-spectrophotometric, RP-HPLC method was developed by using Acetonitrile used as a solvent and Acetonitrile/ acetate buffer in the ratio of 60:40(%v/v) used as a mobile phase. The RP-HPLC method was developed in Eclipse C₁₈ column (100 mm × 4.6 mm, 3.5 μm particle size). In UV-spectrophotometric method, the λ_max of Atazanavir and Ritonavir was 250 and 239.4nm (iso absorption point) and for HPLC –UV detection at 250 nm were found to be respectively. It was proved that linearity in the concentration range of 5–35 μg/ml for Atazanavir, 10-60 µg/ml for Ritonavir and for HPLC the linearity range between 3-150µg/ml for Atazanavir and Ritonavir 1-50 µg/ml with a correlation coefficient value of 0.999. The % RSD for intraday precision and interday precision was less than 2% for UV-spectrophotometric and RP-HPLC method. A new, simple, accurate UV-spectrophotometric and RP-HPLC method were developed for the simultaneous estimation of Atazanavir and Ritonavir in the bulk and in the pharmaceutical dosage form.

 

 

 

INTRODUCTION:

Atazanavir is chemically (3S, 8S, 9S, 12S)-3, 12-bis(1, 1-dimethylethyl)-8-hydroxy-4, 11- dioxo-9-(phenylmethyl)-6-[[4-(2-pyridinyl)phenyl]methyl]-2, 5, 6, 10, 13 Pentaazatetradecanedioic acid dimethyl ester, sulfate (1:1) is a selective inhibitor of the HIV-1 aspartic protease enzyme that cleaves viral Gag and Gag-Pol polyproteins, preventing the formation of infectious virons^[1-3].

 

Its molecular formula is C₃₈H₅₂N_6O7•H₂SO₄, which corresponds to a molecular weight of 802.9 (sulfuric acid salt) shown in Fig.1.

 

Fig 1 Structure of Atazanavir

 

Fig 2 Structure of Ritonavir

 

Ritonavir is chemically 1, 3-thiazol-5-ylmethyl N-[(2S, 3S, 5S)-3-hydroxy-5-[(2S)-3-methyl-2-{[methyl({[2-(propan-2-yl)-1, 3-thiazol-4yl]methyl})carbamoyl] amino} butanamido]-1, 6-diphenylhexan-2-yl]carbamate is an antiretroviral (anti-HIV) drug. Its molecular formula is C₃₇H₄₈N₆O5S₂, and its molecular weight is 720.95. Fig.2

 

Literature survey reveals that there are several UV^4-8, HPLC, LC-MS, HPTLC methods available for estimation of Atazanavir and Ritonavir in combination and the other methods reported were meant for the determination of the two drugs singly or in combination with other drugs in pharmaceutical dosage form and biological dosage form^9-24. The proposed method was validated according to ICH guidelines²⁵. Basing on the above literature study it made essential to develop a new suitable UV- Q absorption ratio and RP-HPLC method for routine analysis of the above said drugs in combined formulations, and in this accord attempts were made by the authors to developed new, simple, precise and accurate RP-HPLC method for the simultaneous assay of the titled drugs and extended it for their determination in formulations.

 

MATERIALS AND METHODS:

Chemicals:

Atazanavir and Ritonavir was gifted from Hetero labs, Hyderabad. Acetonitrile was purchased from Merck Chemical Company. Acetic acid purchased from SDFCL chemicals. Whatman no 5 filter paper was obtained from Modern Science lab, (Nasik, India). All the glassware used were class A grade.

 

Instrumentation:

Double beam UV-Visible Spectrophotometer (Shimadzu-1800) connected to a computer loaded with Shimadzu UV Probe 2.33 software was used for all the spectrophotometric measurements in all proposed spectrophotometric methods.

 

Reverse phase - High performance liquid chromatography (Agilent) equipped with VW detector. The software used is EZ Chrome and the column employed is Eclipse C₁₈ (100 mm × 4.6 mm, 3.5 μm particle size).

 

UV METHOD:

Selection of solvent:

Acetonitrile was found to be a common solvent for these two drugs. Because of the high solubility of Atazanavir and Ritonavir in Acetonitrile. So it was selected as solvent.

 

SELECTION OF WAVELENGTH:

Working standard solutions of 30μg/ml of Atazanavir and 10μg/ml of Ritonavir were prepared by appropriate dilution of standard stock solutions. which the wavelengths of 250nm and 239.4nm were selected for Atazanavir and Ritonavir respectively for further studies.

 

PREPARATION OF STANDARD STOCK SOLUTIONS:

Standard stock solution of Atazanavir and Ritonavir was prepared separately by dissolving 30 mg of Atazanavir and 10mg Ritonavir in Acetonitrile and made the volume up to 10 ml with same solvent. (3000μg/ml) and (1000μg/ml ) Stock A solution. From the above stock solutions 1ml of the each aliquots were pipette out in a 10 ml volumetric flask separately and the volume was made up to the mark with same solvent to obtain the final concentration of 300μg/ml of Atazanavir and 100μg/ml of Ritonavir (stock B solution).

 

PREPARATION OF SAMPLE SOLUTION:

20 tablets were taken and their average weight was determined they were crushed to fine powder. The powder equivalent to 30mg of Atazanavir and 10 mg of Ritonavir was taken in 100ml volumetric flask and sonicated for about 25 min. Dissolved in 100ml Acetonitrile solvent with vigorous shaking for 5-10 minutes The solution was then filtered through Whatman filter paper No 41. The filtrate contains 300 μg/ml of Atazanavir and 100 μg/ml Ritonavir. The amount of Atazanavir and Ritonavir were calculated using Q absorption ratio method.

 

Cx= {(QM-Qy)/ (Qx-Qy)}* (A1/ax1)

Cy= {(QM-Qx)/ (Qy-Qx)}* (A1/ay1)

QM = A2/A1; Qx= ax2/ ax1, Qy = ay2/ay1.

A₁ = absorbance of formulation at 239.4 nm.

A₂ = absorbance of formulation at 250nm.

ax1 = absorptivity of Ritonavir at 239.4 nm.

ax2 = absorptivity of Ritonavir at 250 nm.

ay1 = absorptivity of Atazanavir at 239.4nm.

ay2 = absorptivity of Atazanavir at 250 nm.

_Cx = concentration of Ritonavir

_Cy = concentration of Atazanavir

 

HPLC METHOD:

Selection of solvent for RP-HPLC:

With the knowledge of properties of the selected drugs Eclipse C₁₈- 100mm x 2.5mm column was chosen as stationary phase and mobile phase with different compositions such as Acetonitrile and acetate buffer were used.

Table: 1 Trials done for the selection of mobile phase:

Trial	Mobile Phase Composition	Injection volume	Flow ml/min	Defect
1	Methanol: 0.2M phosphate buffer-(50:50) v/v	20	1.0	No peak was observed. Not satisfactory
2	Methanol: acetonitrile-(50:50) v/v	20	1.0	Single peak was observed Not satisfactory
3	Acetonitrile: water: methanol (20:30:50) v/v	20	1.0	Single peak was observed.Not satisfactory
4	Methanol: water (70:30) v/v	20	1.2	Single peak was observed. Not satisfactory
5	Methanol: water containing 0.1% formic acid.	20	1.2	Single peak was observed. Not satisfactory
6	Acetonitrile:0.1M acetate buffer (60:40) v/v	20	1.0	Sharp peaks were observed Satisfactory

 

SELECTION OF CHROMATOGRAPHIC METHOD FOR RP-HPLC:

Proper selection of the method depends on the nature of the sample (ionic or ionisable or neutral molecules), its molecular weight, pka value and stability. The selected drugs in the present study is polar and so reverse phase or ion exchange chromatography can be used. The reversed phase HPLC was selected for the initial separation because of its simplicity and suitability. Table 1.

 

From the literature survey and with the knowledge of properties of the selected drug, Eclipse C₁₈- 100mm x 2.5mm column was chosen as stationary phase and mobile phase with different compositions such as Acetonitrile and acetate buffer were used.

 

Optimized conditions:

The following optimized parameters were used as a final method in estimation of Ritonavir in bulk and pharmaceutical formulations. The instrument used was Agilent model 1220 and Eclipse C₁₈- 100mm x 2.5mm column, flow rate was 1ml/min and ambient column temperature, the capacity of injection sample volume is 20ml by using mobile phase of Acetonitrile: Acetate buffer (60:40 v/v) with runtime of 5mins.

 

PREPARATION OF STANDARD STOCK SOLUTIONS:

Standard stock solution of 300 mg of Atazanavir and 100mg Ritonavir dissolved in Acetonitrile and made the volume up to 100 ml with same solvent. (3000μg/ml) and (1000μg/ml) Stock 1 solution. From the above stock solutions 1ml of the each aliquots were pipetted out in a 10 ml volumetric flask separately and the volume was made up to the mark with same solvent to obtain the final concentration of 300μg/ml of Atazanavir and 100μg/ml of Ritonavir (stock B solution).

 

PREPARATION OF SAMPLE SOLUTION:

20 Tablets were taken and their average weight was determined they were crushed to fine powder. The powder equivalent to 300mg of Atazanavir and 100mg of Ritonavir was taken in 100ml volumetric flask and sonicated for about 25 min. Dissolved in 100ml Acetonitrile solvent with vigorous shaking for 5-10 minutes. The solution was then filtered through What man filter paper No 41 µm. The filtrate contains 300 μg/ml of Atazanavir and 100 μg/ml Ritonavir. Determine the amount of % Atazanavir and Ritonavir according to the following formula.

 

                   AT x WS × Sample D.F x Average Weight

Assay % = –––––––––––––––––––––––––––––––––––––––× PR

                         AR x Standard D.F x WT× LA

 

Where,

WS = weight of standard; WT = weight of sample

AT = Absorbance of Atazanavir and Ritonavir in the test solution

AR = Absorbance of Atazanavir and Ritonavir in the standard solution

Std. D.F = Standard dilution factor

Sample D.F = Sample dilution factor

PR = Purity of working standard [%]

LA = Labeled amount of Atazanavir and Ritonavir.

 

METHOD VALIDATION:

As per the International Conference on Harmonization (ICH) guidelines, the method validation parameters like linearity, precision, accuracy, limit of detection, limit of quantitation, specificity and robustness were experimentally determined and the method was validated.

 

Specificity:

Specificity for an assay ensures that the signal measured comes from the substance of interest, and that there is no interference from excipient and/or degradation products and/or impurities. Specificity of the method was done by comparing the chromatogram of drug with the chromatogram of blank (mobile phase).

 

Linearity for UV and HPLC:

The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample Five levels are required to allow detection of curvature in the plotted data.

 

The calibration curve was obtained at six concentrations levels of Atazanavir and Ritonavir solutions (5-35μg/ml of Atazanavir and 10-60μg/ml of Ritonavir for UV and 3-150 µg/ml for HPLC Method). Absorbances of the Atazanavir and Ritonavir solutions were measured at 250nm and 239.4nm for UV and peak area at 250nm for HPLC. The linearity was evaluated by the least square regression method with triplicate determinations at each concentration level.

 

Precision for UV and HPLC:

Precision studies were carried out to ascertain the reproducibility of the proposed method.

1. Intraday precision: (Intermediate precision) 2. Inter day precision 3. Repeatability

 

Repeatability:

Repeatability for UV expresses the precision under same operating conditions over a short interval of time. Repeatability of a method can be determined by multiple replicate preparations of the same sample. % RSD was calculated the % RSD should be < 2.0 %.

 

Repeatability for HPLC was determined by preparing six replicates of both Atazanavir (15 µg/ml) and for Ritonavir (5 µg/ml) separately inject equal volumes (20 µl) of each solution. Record the chromatograms and measure the peak response of both Atazanavir and Ritonavir.

 

Intraday precision (Intermediate precision):

Intraday precision for UV was found by carrying out the analysis of the standard drugs at three different concentrations in the linearity range of drugs for three times on the same day. Each concentration was applied in triplicate and % RSD was calculated.

 

Intraday precision study for HPLC was carried out by preparing drug solution of only one concentration (15 µg/ml of Atazanavir) and (5 µg/ml) of Ritonavir analyzing it at three different times in a day. Record the chromatograms and measure the peak response of both drugs.

 

Inter day precision:

Inter day precision for UV was found out by carrying out the analysis of the standard drugs at three different concentrations in the linearity range of drugs for next day and % RSD was calculated.

 

Interday precision study for HPLC was carried out by preparing drug solution of only one concentration (15 µg/ml of Atazanavir) and (5 µg/ml Ritonavir) by analyzing it at three different days to determine interday precision. Record the chromatograms and measure the peak response of both drugs.

 

Accuracy for UV and HPLC:

The accuracy of measurement is defined as the closeness of the measured value to the true value. In a method with high accuracy, a sample (whose “true value” is known) is analyzed and the measured value should ideally be identical to the true value. Typically, accuracy is represented and determined by recovery studies. Accuracy of the proposed method was determined using recovery studies by standard addition method. The recovery studies were carried out by adding known amounts (50, 100 and 150%) of the pure drug to the pre-analyzed formulation. The solutions were prepared in triplicates and the % recovery was calculated.

 

Limit of Detection and Limit of Quantitation for UV and HPLC:

The limit of quantification (LOQ) and limit of detection (LOD) were based on the residual standard deviation of the response and the slope of the constructed calibration curve (n=3), as described in International Conference on Harmonization guidelines Q2 (R1).

                        σ                    σ

LOD =3.3 × –– LOQ=10× –––

                       S                     S

Where,

σ = the standard deviation of the response and

S = slope of the calibration curve

 

Ruggedness Studies for UV:

Ruggedness studies for UV were performed by preparing three replicates of 25μg/ml of Atazanavir, 40μg/ml of Ritonavir analyzing by different analyst and different at similar operational and environmental conditions and the results are reported as %RSD.

 

Robustness for HPLC:

Robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. It is carried by changing the flow rate of mobile phase from 0.8to 1.2 mL/min and also changing in wavelength of both Atazanavir and Ritonavir and the %RSD is determined.

 

In this study, experimental conditions were deliberately altered one factor after the other. The effect of change in flow rate and wavelength on the retention time, peak area and peak asymmetry were studied.

 

a.       Flow rate variation:

Standard and check standard solutions were prepared as per the test method and injected into HPLC system with flow rates of 0.8 mL/minute and 1.2 mL/minute.

b.       The wave length was varied 248 to252nm.

Standard solution of 15µg/ml of Atazanavir and for Ritonavir 5µg/ml were analysed at different wavelength 248 to 250nm.

 

Analysis of fixed dose combination tablets for UV:

For the analysis, six replicates of each batch were assayed. 20 Tablets were finely powdered and a portion of the powder, equivalent to about 300 mg of Atazanavir and 100 mg of Ritonavir was accurately weighed and transferred into a 100 ml volumetric flask followed by the addition of 100 ml of Acetonitrile. The solution was sonicated for 30 min and diluted with Acetonitrile to volume. 3ml of sample stock solution was taken in a 10ml volumetric flask and made the volume with Acetonitrile(30ml) The above solution was analyzed at 259 and 239.4 wavelengths and values of the absorbance were substituted in “respective equations” to obtain the content of Atazanavir and Ritonavir. The % recovery and mean recovery should be between 98.0% - 102.0%.

 

Analysis of fixed dose combination tablets for HPLC:

For the analysis, six replicates of each batch were assayed. 20 Tablets were finely powdered and a portion of the powder, equivalent to about 300 mg of Atazanavir and 100 mg of Ritonavir was accurately weighed and transferred into a 100 ml volumetric flask followed by the addition of 100 ml of Acetonitrile. The solution was sonicated for 30 min and diluted with mobile phase to volume. 20μl of the sample solution i.e. 30 μg/mL of Atazanavir and 10 μg/mL and was injected in to the chromatographic system and the areas of sample solution was measured. The amount of Atazanavir and Ritonavir in bulk dosage form was determined by fitting the responses into the regression equations of the calibration curve and the results obtained were comparable with the corresponding label claim to obtain % recoveries.

 

RESULTS AND DISSCUSSION:

UV-SPECTROSCOPIC METHOD:

Selection of wavelength:

The standard stock solution of 30μg/ml of Atazanavir sulfate and 10μg/ml of Ritonavir was scanned from 200-400nm and the absorption spectra’s were recorded at 250nm and 239.4nm wavelength in UV spectrophotometer which is presented in Fig 3.

 

 

Fig 3 Overly spectra of Atazanavir and Ritonavir in Acetonitrile

 

Table : 2 Linearity studies of Atazanavir sulfate and Ritonavir for UV Method

 

Linearity:

The linearity was found in the concentration range of Atazanavir sulfate(5-35μg/ml) and Ritonavir (10-60μg/ml) for the developed UV spectroscopy method. The X-axis is concentration and the Y-axis is absorbance. The correlation coefficient was found to be 0.9991 and the regression equation was found to be Y=0.3076x + 0.0025 for Atazanavir and Ritonavir and the results are shown in Table 2 and Fig 4-7.

 

 

Fig 4 Calibration curve of Atazanavir Fig 5 Overlay spectra's of Atazanavir

 

Fig 5 Overlay spectra's of Atazanavir

 

 

Fig 6 Calibration curve of Ritonavir Fig 7 Overlay spectra’ of Ritonavir

 

Precision:

Repeatability studies for UV were carried out by taking test concentration and repeating it six times.Interday and intraday precision were done by taking three concentrations and repeating it three times and the values for both system precision and method precision in terms of %RSD was found to Atazanavir 0.532 and for Ritonavir 0.905.The results show that the %RSD Values obtained are represented in Table 3.

 

Fig 7 Overlay spectra’ of Ritonavir

 

The values for intraday is 10, 15 and 20µg/ml for Atazanavir and 10, 20, and 30 µg/ml for Ritonavir in the linearity range for three times on the same day and the values are represented. The values for inter day is10, 15 and 20µg/ml for Atazanavir and 10, 20, and 30 µg/ml for Ritonavir in the linearity range for three times on the same day and the values are represented in the Table 4.

 

Accuracy:

Recovery studies for UV were carried out at 50%, 100% and 150% by adding known amount of standard drug solution i.e. 15, 30 and 45 µg/ml to the standard concentration of 30 µg/ml for Atazanavir and 5, 10 and15 µg/ml to the standard concentration of 10 µg/ml for Ritonavir. The solutions were prepared in triplicates and the % recovery was calculated. % recovery was found to between 99.1 to 99.6 (Atazanavir) 98.5 to 99.1 (Ritonavir). Results are shown in the Table 5.

 

Limit of Detection and Limit of Quantization:

The LOD and LOQ for UV were calculated by using the slope and SD of response (intercept). The mean slope value and the SD of response were obtained from the calibration curve. The LOD and LOQ calculations were done and reported in the results shown Table 6.

 

 

 

 

Table: 3 Repeatability results for Atazanavir and Ritonavir for UV Method

 

 

Table: 4 Intermediate precision studies of Atazanavir and Ritonavir for UV

 

 

Table: 5 Accuracy results for Atazanavir (250n.m) and Ritonavir (239.4n.m) for UV

 

 

Table: 6 Limit of detection and limit of quantification for UV

Parameter	Atazanavir at 250nm	Ritonavir at 239.4
Limit of detection	1.514	2.48
Limit of quantification	4.588	7.53

 

 

 

Ruggedness Studies:

The results for UV indicate that the % RSD values for different analysts and different instruments was found to be in the range of 0.61-0.8 and 0.49-0.61. A method is said to be robust if the %RSD values is <2% and reported in the results shown in the Table 7.

 

 

 

 

Table :7 Ruggedness results for Atazanavir (250nm) and Ritonavir (239.4nm) for UV

Different Analysts
Parameter	Conc (µg/ml)	Absorbance of Analyst Iand Instrument 1(shimadzu)	mean absorbance ± SD(n=3)	%RSD	Absorbance of Analyst II	mean absorbance ± SD(n=3)	% RSD
Atazanavir	15	0.359	0.357±0.002	0.741	0.358	0.356±0.002	0.584
	15	0.358			0.357
	15	0.354			0.354
Ritonavir	20	0.285	0.285±0.002	0.728	0.288	0.287±0.001	0.348
	20	0.288			0.287
	20	0.284
Different Instruments
Atazanavir	15	0.359	0.357±0.002	0.741	0.359	0.356±0.002	0.706
	15	0.358			0.356
	15	0.354			0.354
Ritonavir	20	0.285	0.285±0.002	0.728	0.285	0.286±0.001	0.533
	20	0.288			0.288
	20	0.284			0.286

 

 

Table: 8 Assay studies of Atazanavir and Ritonavir for UV

Drug name	Formulation	Label claim	Amount found	Mean % purity± S.D(n=3)	% RSD	% Purity
Atazanavir	SYNTHAVIN (Tablets300+100mg)	300mg	299.5	99.86±0.305	0.305	99.86%
Ritonavir		100mg	99.3	99.79±0.730	0.732	99.79%

 

 

 

Analysis of fixed dose combination tablets:

The results show that the %purity was found to be 99.86% and reported in the results shown in Table 8.

 

HPLC METHOD:

Selection of mobile phase and flow rate:

Initially various mobile phase compositions were tried, to separate title ingredients. Mobile phase composition and flow rate selection was based on peak parameters (height, tailing, theoretical plates, capacity or symmetry factor) and run time. As we have performed 6 different ratios i.e, from Fig 8.a-f Out of 6 trials made in the lab, the 6^th trial was selected for further studies because when compared to other trials 6^th trial was found less in retention time, with good peak symmetry, and plate count within acceptable limits. The system with Acetonitrile: Buffer (60:40) of pH 4.5 with 1ml/min flow rate was found to be quite robust. The optimum wavelength for detection was 250nm at which better detector response for both Atazanavir and Ritonavir were obtained. The retention time was found to be 2.497 and for Ritonavir 2.953min and the total runtime for this method was 5 min and indicates that the developed method is quite fast and economical. The standard chromatogram is shown in Fig 9.

 

 

Fig 8. Different mobile phases of Standard chromatogram of Atazanavir and Ritonavir

a. Methanol: 0.2M phosphate buffer-(50:50)v/v b. Methanol: acetonitrile-(50:50)v/v c. Acetonitrile: water: methanol (20:30:50)v/v d. Methanol: water (70:30)v/v e. Methanol: water containing 0.1% formic acid f. Acetonitrile:0.1M acetate buffer (60:40)v/

 

 

Fig 9 Standard chromatogram of Atazanavir and Ritonavir

 

 

Fig 10 Chromatogram of specificity

 

Specificity:

The analyte was assessed in the presence of the components and it was found that there was no interaction with the analyte. Hence the method is said to be specific and shown in Fig 10.

 

Linearity:

The linearity was found in the concentration range of (3-150μg/ml) for the developed HPLC method. Each of these drug solutions (20μL) was injected into the chromatographic system for three times. The X-axis is concentration and the Y-axis is area under curve.

 

 

Fig 11 Calibration curve for Atazanavir

 

The peak area and retention time were recorded and the mean values of peak areas were plotted against concentrations. The correlation coefficient was found to be 0.999 and the regression equation was found to be Y = 6E+06x + 15314 for Atazanavir and Ritonavir and the results are shown in Fig 11, 12.

 

Fig 12 Calibration curve for Ritonavir

 

Precision:

Repeatability studies for HPLC were carried out by taking test concentration and repeating it six times. 15µg/ml for Atazanavir and 5 µg/ml Chromatographs were recorded and area of each chromatograph was measured and the values are represented in the Table 9. Interday and intraday precision were done by taking three concentrations and repeating it three times and the values for both system precision and method precision in terms of %RSD were found to be <2.0% The results show that the %RSD value for repeatability, intraday and interday in Table 10.

 

 

 

Table: 9 Repeatability results for Atazanavir and Ritonavir for HPLC

Atazanavir				Ritonavir
Concentration (µg/ml)	Peak Area (250nm)	Area mean± S.D (n=6)	%RSD	Concentration (µg/ml)	Peak Area (250nm)	Area mean± S.D (n=6)	% RSD
15	3733331	38267±74582	1.94	5	942366	969834±14889	1.53
	3733331				968326
	3826036				971020
	3817327				983574
	3962625				971254
	3962824				967452

 

Table: 10 Intermediate Precision For Atazanavir and Ritonavir for HPLC

Conc. (µg/ml)	Atazanavir sulfate				Conc. (µg/ml)	Ritonavir
	Intraday		Interday			Intraday		Interday
	Peak Area (mean± S. D) n=3	% RSD	Peak Area (mean± S.D) n=3	% RSD		Peak Area (mean± S. D) n=3	% RSD	Peak Area (mean± S.D) n=3	% RSD
15	4065118±142828	0.351	4074727±288965	0.709	5	971989±155501	1.59	1050069±16206	1.54

Table: 11 Accuracy studies of Atazanavir and Ritonavir for HPLC

 

 

Fig 13 Typical chromatogram of Accuracy at 50%

 

Fig 14 Typical chromatogram Accuracy at 100%

 

Fig 15 Typical chromatogram Accuracy at 150%

 

 

Accuracy:

The recovery studies were carried out by adding known amounts (50, 100 and 150%) i.e. 15, 30 and 45µg/ml to the standard concentration of 30 µg/ml for Atazanavir and 5, 10 and15 µg/ml to the standard concentration of 10 µg/ml for Ritonavir. The solutions were prepared in triplicates and the % recovery was calculated. % recovery was found to between 99.1 to 99.6 (Atazanavir) 98.5 to 99.1 (Ritonavir). Results are shown in the Table 11 and Fig 13-15.

 

Limit of Detection and Limit of Quantization:

The limit of quantification (LOQ) and limit of detection (LOD) were based on the residual standard deviation of the response and the slope of the constructed calibration curve (n=3), as described in International Conference on Harmonization guidelines Q2 (R1).the results are shown in Table 12.

 

 

 

 

Ruggedness Studies:

Standard solution of Atazanavir and Ritonavir for HPLC was analysed by analyst 1 and analyst 2 at similar operational and environmental conditions using developed method was found to be in the range of 0.61-0.88 and 0.49-0.61 and reported the results in Table 13.

 

Table: 12 Determination of LOD and LOQ results for Atazanavir and Ritonavir at 250nm for HPLC

Parameter	Atazanavir at 250nm	Ritonavir at 239.4
Limit of detection	1.717	0.646
Limit of quantification	2.15	0.90

 

Robustness:

Robustness studies were carried out by changing one factor at a time to study the effect. Variation of wavelength by 2nm (248nm, 267nm and 252nm) and mobile phase flow rate by 0.2ml (0.8ml, 1ml and 1.2ml/min) has no significant effect on the retention time and chromatographic response of the 20 µg/ml solution, indicating that the method was robust and the results are displayed in Table 14.

 

System suitability testing (SST):

System suitability tests are an integral part of chromatographic method. They were used to verify that the reproducibility of the chromatographic system is adequate for the analysis. It is defined as tests to measure that the method can generate result of acceptable accuracy and precision. Table 15.

 

Table: 13 Ruggedness results for different instruments for HPLC

 

 

 

Table: 14 Robustness of Atazanavir and Ritonavir for HPLC

 

Table: 15 System suitability test for HPLC

 

 

 

Table:16 Assay results of Atazanavir and Ritonavir for HPLC

 

 

Fig 16 Assay chromatogram of Atazanavir and Ritonavir

 

Table: 17 Summary of UV-spectrophotometric and HPLC validation parameters

 

Analysis of fixed dose combination tablets:

The results show that the %purity was found to be 98.45% and was reported and the results shown in Table 16 and Fig 16.

 

 

CONCLUSIONS:

Optimum conditions, optical characteristics and statistical data of the regression equation in uv and hplc method. The optical characteristics such as Beer’s law limits molar absorptivity, LOD and LOQ in each method were calculate and the results are displayed in Table 17. Also the regression equation like slope (b), intercept (a) and correlation coefficient (R²) using the method of least squares were calculated. The results show the methods are reasonably simple, precise, accurate UV-spectrophotometric and RP-HPLC method were developed for the simultaneous estimation of Atazanavir and Ritonavir in the bulk and in the pharmaceutical dosage form.

 

ACKNOWLEDGEMENT:

The authors are grateful to Malla reddy College of Pharmacy for providing necessary research facilities to carry out the research work and Hetero drugs, Hyderabad, India for providing the gift sample of the drug.

 

REFERENCE:

1.      Gilman A G, Goodman L S. The pharmacologic basis of therapeutics. 10th ed. New York: McGraw-Hill; 1999.

2.      Indian Pharmacopoeia. Indian Pharmacopoeial Commission, Ghaziabad, 2007, p. 697-701, 1057-1070.

3.      United States Pharmacopoeia, The United States Pharmacopoeia official compendia of standards, 30th edn. USP Convention, Rockville, MD, 2007, p. 3143.

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Received on 26.05.2019                 Accepted on 21.06.2019

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Asian J. Pharm. Ana. 2019; 9(3):138-150.