INTRODUCTION:

Atazanavir sulphate (ATV) is a 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 penta aza tetra decane dioic acid dimethyl ester, sulphate (1:1) (figure1). ATV is an antiretroviral agent for the treatment of HIV infection and consequently it is clinically useful in the treatment of AIDS¹. Atazanavir sulfate, azapeptide inhibitor of HIV-1 protease, is indicated in combination with other antiretroviral agents for the treatment of HIV-1 infection². Atazanavir sulfate is a white to pale yellow powder. It is slightly soluble in water. The drug was approved by the United States Food and Drug Administration (USFDA) in June 2003. Structure–activity studies with a series of azadipeptides designed to mimic the transition state of the peptide-cleavage reaction catalyzed by HIV-1 protease identified lead compounds that had either potent antiviral activity against mutant HIV-1 strains or good oral bioavailability, but not both.

Atazanavir is given with food by mouth as the sulfate and the usual adult dose is 400 mg once daily³. The drug is well absorbed when administered orally with food (bioavailability 68%). The drug is highly bound to plasma proteins (86%) and is metabolized by CYP3A isozyme. It is a moderate inhibitor of CYP3A. Literature survey revealed few spectrophotometric methods^4-6, RP-HPLC methods^7-13 and HPTLC methods¹⁴ available for estimation of Atazanavir sulfate in bulk and pharmaceutical formulation. Out of these analytical methods HPLC^15-24 is most widely used technique in quantitative analysis of drugs. In spite of various HPLC methods available for the estimation of atazanavir sulfate, still there is a need of a economical and rapid HPLC method for its estimation in dosage forms. The present study, a new RP-HPLC method was developed for estimation of atazanavir sulfate in pharmaceutical formulation, which shown high reproducibility, sensitivity and economical. The developed method was validated as per ICH guidelines²⁵.

Fig.1: Structure of atazanavir sulfate

MATERIALS AND METHODS:

Instrumentation:

Chromatographic separation was performed on a Cyberlab HPLC system equipped with a Flowrosil C18 column (250 × 4.6mm, with 5μm particle), single pumps, degasser, variable wave length detector and Rheodyne injector with 20μl loop volume. ‘LC solution’ software was used to collect and process the data. Ultra sonicator (Citizen ultra sonicator) was used for sonicating the drug and sample solution. Digital weighing balance (SHIMADZU AUX 220) used for weighing.

Chemicals and Reagents:

Atazanavir sulfate pure drug (purity 99.1%) was obtained as gifted sample from Hetro Drugs Ltd. Marketed formulation Atazor capsules (label claim 300 mg) was procured from local market. HPLC grade water, Acetonitrile was from MERCK India Ltd. HPLC grade methanol was from standard reagent Pvt Ltd Hyderabad. Analytical grade glacial acetic acid was from SD Fine chemicals Mumbai, India. Nylon membrane filters 0.2 µm and 0.45µm were from PALL life sciences Mumbai, India.

Preparation of mobile phase:

Mobile phase was prepared by mixing 800mL of HPLC grade acetonitrile with 200mL 1% glacial acetic acid (2 ml glacial acetic acid diluted to 200ml with HPLC grade water). The mobile phase was sonicated for 60 min and filtered through the 0.22μm membrane filter.

Preparation of standard stock solutions:

The standard stock solutions of 100μg/mL of the drug were prepared by dissolving 50mg of pure drug in the mobile phase in a 50mL volumetric flask and the volume was made up to the mark. Resulting solutions were further diluted with mobile phase to obtain a final concentration of 100μg/mL and stored under refrigeration. Aliquots of standard stock solutions were put in a 10mL volumetric flask and diluted up to the mark with mobile phase. In such a way, the final concentrations of the drug were in the range of 5–30 μg/Ml

Preparation of sample solution:

To determine the content of atazanavir sulfate in capsule dosage form (Label claim: 300mg/capsule) ten capsules were accurately weighed and powder was collected. The powder weight equivalent to 10 mg of atazanavir sulfate was taken and dissolved in 100ml of mobile phase. The resulting solution (2ml) was transferred to a 10ml volumetric flask and diluted up to the mark with mobile phase. The final solution was filtered through 0.45μ membrane filter using injection filter. A 20µL of the filtrate was injected into chromatographic system. The peak area of the atazanavir sulfate was determined and concentration was found using linear regression equation obtained from calibration curve.

Chromatographic conditions:

The chromatographic system used for method development and validation includes the LC-P100 pump, variable wavelength programmable LC-UV100 UV detector and SCL20A system controller at CYBERLAB HPLC. A Rheodyne injector 7725i equipped with a 20 μL loop was used and the data was recorded and evaluated using LC solution software version 5.0. Separation was performed at Flowrosil C18 (250 × 4.6 mm i.d., 5µm) at the ambient temperature. A mixture of 1% glacial acetic acid and acetonitrile in a 20: 80 v / v ratio was found to be the ideal mobile phase for the ideal chromatographic analysis of atazanavir sulfate. The solvent mixture was filtered through a 0.22μ membrane filter and sonicated before use. It is pumped through the column at a flow rate of 1.0mL/min. The injection volume is maintained in the column at 20µL and room temperature. The column was balanced by pumping the mobile phase through the column for at least 20 min before injecting the drug solution. The detection was monitored at 249nm. Run time is set to 10 minutes. Optimized chromatographic conditions are shown in Table 1.

Table 1: Optimized chromatographic conditions

Parameters	Conditions
Stationary Phase (Column)	C₁₈ (250 × 4.6 mm i.d.,5µ)
Mobile Phase	Acetonitrile: 1 % glacial acetic acid (80:20,v/v)
Flow rate(ml/min)	1.0 mL/min
Run time(min)	10 min
Column temperature (°C)	Ambient
Volume of injection loop(μL)	20
Detection wavelength(nm)	249 nm
Retention time(min)	5.027

Fig. 2: Chromatogram of standard solution of atazanavir sulfate

Method Validation:

The developed method was validated as per ICH guidelines by evaluating linearity, accuracy, precision, robustness, ruggedness, detection limit, quantification limit and stability. Coefficients of variation and relative errors of less than 2 % were considered acceptable.

System Suitability Test:

Before performing validation experiments, system suitability test (SST) has to be applied to indicate that HPLC system and method are capable of providing data with admissible quality. SST was performed by investigating capacity factor, tailing factor, theoretical plates number, and also relative standard deviation (RSD) of the peak areas.

Linearity:

A stock solution of Atazanavir sulfate of 1000μg/mL was prepared with mobile phase. From it, various working standard solutions were prepared in the range of 5 to 50μg/ml and injected into HPLC. It was shown that the selected drug had linearity in the range of 5–30 μg/mL. The calibration plot (peak area of Atazanavir sulfate versus Atazanavir sulfate concentration) was generated by replicate analysis (n=6) at all concentration levels and the linear relationship was evaluated using the least square method within Microsoft Excel® program.

Accuracy:

The accuracy of the method was carried out using one set of different standard addition methods at different concentration levels, 50%, 100% and 150%, and then comparing the difference between the spiked value (theoretical value) and actual found value.

Precision:

The precision of the method was ascertained from the peak area obtained by actual determination of six replicates of a fixed amount of the drug (20μg/mL). The precision of the assay was also determined in terms of intra- and inter-day variation in the peak areas of a set of drug solutions on three different days. The intra- and inter-day variation in the peak area of the drug solution was calculated in terms of relative standard deviation (RSD).

Robustness:

Robustness of the proposed method for Atazanavir sulfate was carried out by the slight variation in flow rate, analytical wavelength and mobile phase ratio. The percentage recovery and RSD were noted for Atazanavir sulfate.

Ruggedness:

The test solutions were prepared as per test method and injected under variable conditions. Ruggedness of the method was studied by different analysts.

Specificity:

The specificity of the proposed method was determined against blank and placebo applications. Here mobile phase was used as blank and excipients like starch, lactose, magnesium stearate were used as placebo.

RESULTS AND DISCUSSION:

Method validation:

System Suitability Test:

After setting the optimum conditions, system suitability parameters for the developed method were determined and compared with recommended limits. To determine the parameters, the study was performed with standard solution of 20µg/ml concentration and the results were acquired from six injections. According to the results, all of the system suitability parameters were within the recommended limits and the method was found to be suitable for the analysis.

Linearity and sensitivity:

Linearity study was performed with calibration standards with 5, 10, 15, 20, 25, and 30µg/ml concentrations. The standards were injected in triplicate. Calibration curves were obtained by plotting the peak areas against the given concentrations. The calibration curve was evaluated by the determination coefficient. The determination coefficient (R²) of the calibration curves was 0.999. Therefore, the calibration curve for Atazanavir sulfate was found to be linear within the range of 5–30µg/ml concentrations as shown in Fig.3. The regression equations were calculated from the calibration graphs. The sensitivity of the analytical method was evaluated by determining the limits of detection (LOD) and quantitation (LOQ). The values of LOD and LOQ are given in Table 2. The low values of LOD and LOQ indicates the sensitivity of method.

Table 2: Spectral and statistical data for determination of Istradefyllin by proposed RP-HPLC method.

Parameter	Result
Detection wavelength (nm)	249
Linearity range (µg/ml)	5-30
Coefficient of determination (r²)	0.999
Regression equation (Y^a)	Y= 16744x – 4639.7
Slope (m)	16744
Intercept (c)	-4639.7
Limit of detection, LOD (µg/ml)	0.04
Limit of quantitation, LOQ (µg/ml)	0.12

^aY = mx + c, where x is the concentration (µg/ml).

Accuracy:

To study the reliability, the suitability, and the accuracy of the method, recovery experiments were carried out. Known quantities of the pure drug were added to the preanalyzed sample to make samples at the levels of 50 %, 100%, and 150%, and were assayed by the proposed method. Accuracy was calculated as the percentage of recovery. The recovery and relative standard deviation for each of the analytes are given Table 3. From the recovery studies it is evidence that the method is highly accurate and can give excellent results.

Fig. 3: Calibration curve of Atazanavir sulfate

Table 3: Accuracy results

% spike Level	Sample	Amount Added (Std)	Amount Found (µg/ml)	% Recovery	Statistical Parameters
50	20	10	9.91	99.1	Mean = 99.1 SD= 0.458 % RSD=0.462
	20	10	9.96	99.6
	20	10	9.87	98.7
100	20	20	19.90	99.5	Mean = 98.9 SD= 0.655 % RSD=0.663
	20	20	19.64	98.2
	20	20	19.80	99.0
150	20	30	29.73	99.1	Mean = 98.9 SD= 0.529 % RSD=0.535
	20	30	29.49	98.3
	20	30	29.79	99.3

Precision:

The precision was demonstrated at three levels: repeatability, intermediate precision, and reproducibility (between laboratories’ precision). Each level of precision was investigated by 3 sequential replicate of injections of three concentrations of 10, 20 and 30µg/mL. The precision was expressed as relative standard deviation (RSD) or coefficient of variation (CV). The results of three levels of precision are shown in Table 4. The developed method was found to be precise as the RSD values for repeatability, intermediate precision and reproducibility studies were < 2%, respectively as recommended by ICH guidelines (ICH Q2 (R1), 2005).

Table 4: Precision results

Precision

Results

Concentration (µg/mL)

% RSD of Peak area

% RSD of Retention Time

Repeatability

0.89

1.21

1.11

0.02

0.08

0.12

Intermediate precision

1.42

0.75

0.67

0.08

0.06

Reproducibility

1.64

0.78

0.85

0.11

0.17

0.09

Robustness and Ruggedness:

Robustness of the method was studied by deliberate variations of the analytical parameters such as flow rate (1.0±0.1mL/min), mobile phase composition (±5% organic phase) and analytical wavelength (±2nm). The result shown that have the negligible effect on retention time, recoveries and peak area of Atazanavir sulfate indicating the developed method is robustness. Ruggedness of the method was carried out by different analysts. There is no variation in peak areas and retention time of Atazanavir sulfate from studies carried out by two analysts as indicated by % RSD < 2 gives the method ruggedness.

Mobile phase stability:

The stability of the mobile phase was evaluated, so the mobile phase was stored at 4–8 °C for 1 week. The aged mobile phase was compared using a freshly prepared one. The mobile phase was stable up to 1 week at 4–8 °C.

Specificity:

Specificity is the ability to unequivocally assess the analyte in the presence of components that may be expected to be present. Typically, these might include impurities, degradants or matrix. Specificity of an analytical method is its ability to accurately and specifically measure the analyte of interest without interference from blank or placebo. The peak purity of Atazanavir sulfate was assessed by comparing the retention times of standard Atazanavir sulfate and the sample, and good correlation was obtained between the retention time of the standard and sample. Placebo and blank were injected and there were no peaks. There is no interference of blank and placebo on drug peaks hence, the method is specific.

Sample Analysis:

The developed and validated method was applied for analysis of capsule formulation contains Atazanavir sulfate. The sample was analyzed in triplicate. Analysis results were evaluated using a calibration curve. The amount of Atazanavir sulfate in the samples was calculated from calibration curve equation and recovery and RSD values were determined. The recoveries were in good agreement with the label claims. The chromatogram obtained was clear as shown in Fig. 4. It was concluded that the method can be applied successfully for the analysis of Atazanavir sulfate in tablet dosage form.

Fig.4: Chromatogram of sample Atazanavir sulfate

CONCLUSION:

The proposed method for the estimation of Atazanavir sulfate was validated as per the ICH guidelines and it is simple, specific and economical. Furthermore, this simple and rapid RP-HPLC method can also be used successfully for the determination of Atazanavir sulfate in pharmaceutical formulations without any interference from the excipient.

ACKNOWLEDGEMENT:

The authors thankful to the management of Srikrupa Institute of Pharmaceutical Sciences for providing the instrumental and chemical facilities.

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Received on 21.10.2020 Modified on 06.03.2021

Research J. Pharm. and Tech. 2022; 15(7):2928-2932.

DOI: 10.52711/0974-360X.2022.00488