Kande et al Journal of Drug Delivery & Therapeutics.

2019; 9(3):740-744

Available online on 22.05.2019 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research
© 2011-18, publisher and licensee JDDT, This is an Open Access article which permits unrestricted
non-commercial use, provided the original work is properly cited

Open Access Review Article

Simultaneous estimation of atazanavir and ritonavir in combined Tablet
dosage form: A Review
*Akash Shankar Kanade, Vishwas Bhagat, Rajkumar V. Shete
Rajgad Dnyanpeeth’s College of Pharmacy, Bhor, Pune, Maharashtra 412206, India

ABSTRACT
In present study describe the development of new method and parameter which will be considered during method development and validation.
Article give brief idea for a simple, sensitive, precise and accurate High-performance thin-layer chromatographic method for simultaneous
determination of Ritonavir and Atazanavir in their combined tablet dosage form has been developed, validated and used for determination of
the compounds in commercial pharmaceutical products. Chromatographic separation will be achieved on different column like C8, C18, etc.
used as the stationary phase and different mobile phase.
Keywords: High-performance thin-layer chromatography, Ritonavir, Atazanavir

Article Info: Received 08 April 2019; Review Completed 15 May 2019; Accepted 20 May 2019; Available online 22 May 2019
Cite this article as:
Kanade AS, Bhagat V, Shete RV, Simultaneous estimation of atazanavir and ritonavir in combined Tablet dosage form: A
Review, Journal of Drug Delivery and Therapeutics. 2019; 9(3):740-744 http://dx.doi.org/10.22270/jddt.v9i3.2725

*Address for Correspondence:

Akash Shankar Kanade, Rajgad Dnyanpeeth’s College of Pharmacy, Bhor, Pune, Maharashtra 412206, India

1. INTRODUCTION Ritonavir is a Antiretroviral drug 1,3-thiazol-5-ylmethyl N-

[(2S,3S,5S)-3-hydroxy-5-[(2S)-3-methyl-2{[methyl({[2-
Atazanavir Sulphate Methyl is a Antiretroviral drug N- [(1S)- (propan-2-yl)-1,3-tiazole-4-
1-{ [(2S,3S) - 3 - hydroxy-4- [(2S)-2- [(methoxycarbonyl) yl]methyl})carbamoyl]amino}butane mido]-1,6-diphenyl
amino] -3, 3-dimethyl-N'-{[4-(pyridin-2-yl)phenyl]methyl} hexan-2-yl] carbamate. Ritonavir inhibits the HIV viral
butanehydrazido]-1- phenylbutan-2-yl] carbamoyl}-2, 2 - protease enzyme. This prevents cleavage of the gag-pol
dimethyl propyl] carbamate sulphate is a azapeptide HIV-1 polyprotein and, therefore, improper viral assembly results.
protease inhibitor. The compound selectively inhibits the This subsequently results in noninfectious, immature viral
virus-specific processing of viral Gag and Gag-Pol particles. 2
polyproteins in HIV-1 infected cells, thus preventing
formation of mature virions. 1

Figure 1: chemical structure of Atazanavir Sulphate Figure 2: chemical structure of Ritonavir

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Kande et al Journal of Drug Delivery & Therapeutics. 2019; 9(3):740-744

methods reported for the simultaneous estimation of absorbed techniques and skills from all other four branches
Atazanavir and Ritonavir gives out information that there are so by the 1950s, analytical chemistry was finally accepted as
few separate methods reported for the quantitative a branch of chemistry in its own right. 4
estimation of Atazanavir sulfate in bulk, pharmaceutical
The ability to provide timely, accurate, and reliable data is
dosage forms and in plasma by HPLC, likewise a very few
methods have been reported for the quantitative estimation central to the discovery, development, and manufacture of
of Ritonavir by HPLC but till date no method has been pharmaceuticals. Analytical data are used to screen potential
drug candidates, aid in the development of drug synthesis,
reported for the simultaneous quantitative estimation of
support formulation studies, monitor the stability of bulk
Atazanavir and Ritonavir by HPLC [3-8]. There is just one
spectrophotometric method reported for the simultaneous pharmaceuticals and formulated products, and test final
estimation of Atazanavir sulfate and Ritonavir in tablets. The products for release. The quality of analytical data is a key
factor in the success of a drug development program. 5
present developed method was used determine the
Atazanavir and Ritonavir present in the formulation and 3. INTRODUCTION TO HPLC
method validated according to the ICH guidelines. 3
HPLC originally referred to the fact that high pressure was
2. INTRODUCTION TO DRUG ANALYSIS needed to generate the flow required for liquid
chromatography in packed columns. In the beginning,
Analytical chemistry is a branch of chemistry that
determines the nature and identity of a substance and its instrument components only had the capability of generating
pressures of 500psi (35 bar). This was called High Pressure
composition. In the early twentieth century there were only
four accepted branches of chemistry namely, organic Liquid Chromatography (HPLC). 6
chemistry, inorganic chemistry, physical chemistry and 3.1 Instrumentation
biochemistry. Its importance grew, and in the process,

Figure 3: Schematic Diagram of HPLC Instrument

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Kande et al Journal of Drug Delivery & Therapeutics. 2019; 9(3):740-744

4. INTRODUCTION TO METHOD DEVELOPMENT & required systematic method development involving accurate
VALIDATION modeling of the retention behavior of the analyte.

Method development and optimization in liquid 4.1 Critical steps for HPLC method development 7
chromatography is an attractive field of research for
academia and industry. Complex mixtures or samples

Information on a sample, define separation goals

Need for special HPLC procedure, Sample pretreatment, etc

Choose Detector and detector settings

Choose LC Method, preliminary runs, estimate best separation conditions

Optimize separation conditions

Check for problems or requirements for special procedure

Recover purified material, qualitative Calibration, Quantitative Method

Validate Method for released routine laboratory

Table 1: Preferred experimental conditions for the initial HPLC run

Separation variable Initial condition
Column
Dimensions (length, diameter) 150 × 4.6mm
Particle size 5μm(3.5μm alternatively)
Stationary phase C8 or C18

Mobile phase
Solvent A and B Buffer- Acetonitrile
% of strong solvent 80-100%
Buffer (compound, pH, and conc.) 25mM Potassium phosphate
pH range 2.0 < 3.0
Additives (e.g. Ion pair reagent, amine modifier) Do not use initially

Flow rate 1.5-2.0 ml/min.

Temperature 350-450 C.
Sample size
Volume < 25μl
Weight < 100μg

4.2 Analytical Method Validation Parameters various Performance parameters, which are addressed in a
validation exercise, are grouped as follows. 8
Before performing validation of analytical method it is
necessary to understand the validation parameters. The

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5.4.2 Determination of Reproducibility: Reproducibility

means the precision of the procedure when it is carried
out under different conditions, usually in different
laboratories on separate, putatively identical samples
taken from the same homogeneous batch of material. 13
5.5 Limit of Detection and Limit of Quantization
5.5.1 Limit of Detection: The limit of detection is the
parameter of limit tests. It is the lowest level of analyte
that can be detected, but not necessarily determined in
a quantitative fashion, using a specific method under
the required experimental conditions. The limit test
thus merely substantiates that the analyte
concentration is above or below a certain level. 14
5.5.2 Limit of Quantitation: Limit of quantitation is a
parameter of quantitative assays for low levels of
compounds in sample matrices such as impurities in
bulk drugs and degradation products in finished
pharmaceuticals. The limit of quantitation is the lowest
concentration of analyte in a sample that may be
Figure 4: Method Validation Parameters as per USP determined with acceptable accuracy and precision
and ICH when the required procedure is applied. 15
5. PARAMETER FOR METHOD VALIDATION 5.6 Robustness and Ruggedness
5.1 Selectivity and Specificity: 5.6.1 Robustness: The robustness of an analytical method is
a measure of its capacity to remain unaffected by small
The selectivity of an analytical method is its ability to but deliberate variation in method parameters and
measure accurately and specifically the analyte of interest in provides an indication of its reliability during normal
the presence of components that may be expected to be usage. The determination of robustness requires that
present in the sample matrix. methods characteristic are assessed when one or more
5.2 Linearity and Range: operating parameter varied. 16

The linearity of an analytical method is its ability to elicit test 5.6.2 Ruggedness: The ruggedness of an analytical method
results that are directly (or by a well defined mathematical is the degree of reproducibility of test results obtained
transformation) proportional to the analyte concentration in by the analysis of the same samples under a variety of
samples within a given range. 9 normal test conditions such as different laboratories,
different analysts, using operational and environmental
5.3 Accuracy conditions that may differ but are still within the
The accuracy of an analytical method may be defined as the specified parameters of the assay. 17
closeness of the test results obtained by the method to the 5.7 Stability of Analytical Solution:
true value. It is the measure of the exactness of the analytical
method developed. 10 Stability of the sample, standard and reagents is required for
a reasonable time to generate reproducible and reliable
5.4 Precision results. 18
The precision of an analytical method is the degree of 5.8 System Suitability:
agreement among individual test results when the method is
applied repeatedly to multiple samplings of homogenous System suitability is the checking of a system to ensure
samples. This is usually expressed as the standard deviation system performance before or during the analysis of
or the relative standard deviation (coefficient of variation). 11 unknowns. Parameters such as plate count, tailing factors,
resolution and reproducibility (% RSD, retention time and
5.4.1 Determination of Repeatability: Repeatability can be area for six repetitions) are determined and compared
defined as the precision of the procedure when against the specifications set for the method. 19
repeated by same analyst under the same operating
conditions like same reagents, equipments, settings and
laboratory over a short interval of time. 12

Table 2: System Suitability Parameters and Recommendations20

Parameters Recommendation

Capacity Factor (k”) The peak should be well-resolved from other peaks and the void volume, generally K” > 2
Repeatability RSD ≤ 1% N ≥ 5 is desirable
Relative retention Not essential as the resolution stated
Resolution (Rs) Rs of > 2 between the peak of interest and the closest eluting potential interferent (impurity,
excipients, degradation products, internal standard, etc.)
Tailing factor (T) T≤2
Theoretical plates (N) In general should be > 2000

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Table 3: Characteristics to be validated in HPLC 21

Characteristics Acceptance Criteria
Accuracy / trueness Recovery 98-102% (individual)
Precision RSD < 2%
Repeatability RSD < 2%
Intermediate Precision RSD < 2%
Specificity/ Selectivity No interference
Detection Limit S/N > 2 or 3
Quantitation Limit S/N > 10
Linearity Correlation coefficient r2 > 0.999
Range 80-120%
Stability >24 h or > 12 h

5.9 Development of Stability indicating HPLC Method 22

Figure 6: Overview of the Method Development Process

6. CONCLUSION 11. Gurdeep R. Chatwal, Sham K. Anand, “Instrumental Method of
Chemical Analysis,” 5st Edition, PP- 2.567.
The results of the validation study indicate that the analytical 12. Lippncott Williams and Wilkins, “Remington the Science and
method developed for the determination of assay is found to Practice of pharmacy,” Vol. - I, 21st Edition, PP- 667
be accurate and precise. The percentage RSD for all 13. Gurdeep R. Chatwal, Sham K. Anand, “Instrumental Method of
parameters was found to be less than two, which indicates Chemical Analysis,” 5st Edition, PP- 2.624.
14. Gurdeep R. Chatwal, Sham K. Anand, “Instrumental Method of
the validity of the method and assay results obtained bythis
Chemical Analysis,” 5st Edition, PP- 2.570.
method are in fair agreement. The method is both repeatable 15. Ravi Shankar, “Instrumental Method of Analysis”, 2 nd Edition,
and rugged. PP- 18-60.
16. Ravi Shankar, “Instrumental Method of Analysis”, I nd Edition,
7. REFERENCES PP- 17-36.
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2. British Pharmacopoeia, British pharmacopoeia commission, 18. International Journal of Pharmacy and Technology,” Vol. – II,
London, UK. 2001; 1: 305. Issue June 2010, PP- 429-439.
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Analysis. 2011; 55(1):31-47. human plasma by liquid chromatography– tandem mass
4. Arianna Loregia Journal of Pharmaceutical and Biomedical spectrometry method: application to a bioequivalence study of
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