Palavan Chinnaiah /J Compr Phar 2015;2(3):71-83

Journal of Comprehensive Pharmacy

Research Article Available Online at: www.jcponline.in ISSN NO: 2349-5669

NEW VALIDATED RP-HPLC METHOD FOR IDENTIFICATION AND

QUANTITATION OF PROCESS AND DEGRADATION RELATED
IMPURITIES IN THE COMBINED DOSAGE TABLETS OF
ATAZANAVIR AND RITONAVIR
Palavan Chinnaiah *, a, Appala R Lanka b, Srinivasu Pamidi c, Palapatla PR Govada d, Venkata LNSR Jillella e
a
Research Scholar, Andhra University College of Pharmaceutical Sciences, Visakhapatnam-530003, A.P, India.
b
Assistant General Manager, Analytical Development, Hetero Labs Limited, Unit-III, Jeedimetla, Hyderabad-500055,
Telangana, India.
c
General Manager, Analytical Development, Hetero Labs Limited,Unit-III, Jeedimetla, Hyderabad-500055, Telangana, India.
d
Director, Hetero Labs Limited, Unit-III, Jeedimetla, Hyderabad-500055, Telangana, India.
e
Principal, Srinivasarao College of Pharmacy, P. M. Palem, Visakhapatnam-530041, A.P, India.

ARTICLE INFO ABSTRACT

Aim: The objective of the present study is to develop an accurate and precise high performance
Article history: liquid chromatographic method for the simultaneous determination of eight process and degradation
related impurities in the combined dosage tablets of atazanavir and ritonavir.
Received 27 April 2015
Method: Separation of the analytes was achieved on a SunFire C18 column (250 × 4.6 mm; 5µm) by
Accepted 21 May 2015 gradient programming of mobile phase-A (phosphate buffer of pH 4.0) and mobile phase-B (mixture
Available online 25 June 2015 of acetonitrile and tetrahydrofuran in the ratio of 80:20 v/v) at a flow rate of 1.5 mL/min. The
*Corresponding author: analytes in the eluate were monitored at 250 nm.
Results: By applying the proposed method, the relative retention times of ritonavir impurity-E,
Palavan Chinnaiah ritonavir impurity-F, atazanavir related compound 01, atazanavir, ritonavir impurity-L, ritonavir,
Email: atazanavir related compound 02, ritonavir impurity-O, atazanavir related compound 03, and ritonavir
[email protected] impurity-T were found to be 0.33, 0.44, 0.85, 1.00, 0.94, 1.00, 1.34, 1.19, 1.50 and 1.72 respectively.
The relative response factor values for atazanavir related compound 01, atazanavir related compound
Tel.:+91-9000450662. 02, atazanavir related compound 03, ritonavir impurity-E, ritonavir impurity-F, ritonavir impurity-L,
ritonavir impurity-O, and ritonavir impurity-T were found to be 0.95, 1.03, 0.97, 0.72, 0.73, 0.66,
0.83, and 1.02 respectively. The proposed method was validated for other parameters like accuracy,
precision, LOD, LOQ, forced degradation studies, robustness, filter variability and solution stability.
Conclusion: The proposed HPLC method is sensitive, accurate, precise, robust and stability
indicating. Thus the method can be used for identification and quantitation of the process-related and
degradation impurities of atazaunavir and ritonavir in tablet dosage forms.

KEY WORDS
Atazanavir, Ritonavir, Impurities, HPLC, Gradient elution.

INTRODUCTION belongs to the class of protease inhibitors [1-4]. HIV-1

protease is an enzyme required for the proteolytic
Atazanavir (methyl N-[(1S)-1-{[(2S,3S)-3-hydroxy-4- cleavage of the viral polyprotein precursors into the
[(2S)-2-[ (methoxycarbonyl) amino]-3,3-dimethyl-N'- individual functional proteins found in infectious HIV-
{[4-(pyridin-2-yl)phenyl]methyl} butanehydrazido]-1- 1. Both atazanavir and ritonavir binds to the protease
phenylbutan-2-yl]carbamoyl}-2,2dimethylpropyl] active site and inhibits the activity of the enzyme. This
carbamate)and ritonavir (1,3-thiazol-5-ylmethyl N- inhibition prevents cleavage of the viral polyproteins
[(2S,3S,5S)-3-hydroxy-5-[(2S)-3-methyl2{[methyl({[2- resulting in the formation of immature non-infectious
(propan-2-yl)-1,3-thiazol-4-yl]methyl})carbamoyl] viral particles. Protease inhibitors are almost always
amino}butanamido]-1,6-diphenylhexan-2yl]carbamate)

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used in combination with at least two other anti-HIV pyridinyl)phenyl]methyl]-2,5,6,10,13-

drugs. pentaazatetradecanedioic acid di methyl ester.
(Atazanavir related compound-03)
4. Thiazol-5-ylmethyl(2S,3S,5S)-3-hydroxy-5-[2-(3-
{[2-(2-hydroxypropan-2-yl)thiazol-4-yl]methyl}-
3-methylureido)acetamido]-1,6-diphenylhexan-2-
ylcarbamate. (Ritonavir impurity-E)
5. Thiazol-5-ylmethyl(2S,3S,5S)-3-hydroxy-5-[(S)-
4-isopropyl-2,5-dioxoimidazolidin-1-yl]-1,6-
diphenylhexan-2-ylcarbamate.(Ritonavir impurity-
F)
6. (4S,5S)-4-benzyl-5-[(2S)-2-[[(2S)-3-methyl-2-
[[methyl[[2-(1-methylethyl)thiazol-4-yl]methyl]
carbamoyl]amino]butanoyl]amino]-3-
Fig. 1: Chemical Structure of atazanavir phenylpropyl]oxazolidin-2-one.(Ritonavir
impurity- L)
7. Thiazol-5-ylmethyl[(1S,2R,4S)-1-benzyl-2-
hydroxy-4-[[(2S)-3-methyl-2-[[methyl[[2-
(1methylethyl)thiazol-4-yl]methyl]
carbamoyl]amino]butanoyl]amino]-5-
phenylpentyl] carbamate. (Ritonavir impurity- O)
8. (2S)-N-[(1S,2S,4S)-1-benzyl-2-hydroxy-4-[[(2S)-
3-methyl-2-[[methyl[[2-(1-methylethyl)thiazol-
4yl] methyl]carbamoyl]amino]butanoyl]amino]-5-
phenylpentyl]-3-methyl-2-[[methyl[[2-(1-
methylethyl)thiazol-4-yl]methyl]carbamoyl]
amino] butanamide. (Ritonavir impurity- T)

Fig. 2: Chemical Structure of ritonavir

MATERIALS AND METHODS

Sreenivasa Rao et al reported an RP-HPLC method for Drugs and chemicals

the separation of potential impurities of atazanavir Reference standard samples of atazanavir sulfate
sulfate [5]. Method for the separation and estimation of (purity 99.8%), ritonavir (purity 99.4%), atazanavir
impurities from the bulk drug of ritonavir and ritonavir related compound-01 (purity 93.9%), atazanavir related
tablets was recommended by USP 37 [6, 7]. No HPLC compound-02 (purity 95.2%), atazanavir related
method was reported for simultaneous determination of compound-03 (purity 96.0%), ritonavir impurity-E
impurities in combined dosage forms containing (purity 93.9%), ritonavir impurity-F (purity 89.9%),
atazanavir and ritonavir. ritonavir impurity-L (purity 95.9%), ritonavir impurity-
The present investigation by the author describes an O (purity 95.4%), ritonavir impurity-T (purity 96.8%),
accurate and precise RP-HPLC method for the and in-house tablets of atazanavir and ritonavir (each
simultaneous determination of the following process tablet containing 300 mg of atazanavir and 100 mg of
and degradation related impurities in combined dosage ritonavir) were obtained from Hetero Labs Ltd.
tablets of atazanavir and ritonavir. (Hyderabad, India). AR grade potassium dihydrogen
phosphate, barium chloride dihydrate and sodium
1. (3R,8S,9S,12R)-3,12-Bis(1,1-dimethylethyl)-8- hydroxide were purchased from Finar Chemicals
hydroxy-4,11-dioxo-9-(phenylmethyl)-6-[[4-(2 Limited. GR grade orthophosphoric acid, hydrochloric
pyridinyl)phenyl]methyl]-2,5,6,10,13- acid and hydrogen peroxide were purchased from
pentaazatetradecanedioic acid di methyl ester. Merck Limited. HPLC grade acetonitrile and
(Atazanavir related compound-01) tetrahydrofuran were purchased from Merck Limited.
2. (3S,8R,9S,12S)-3,12-Bis(1,1-dimethylethyl)-8- HPLC grade water was prepared by using Millipore
hydroxy-4,11-dioxo-9-(phenylmethyl)-6-[[4-(2- Milli-Q system.
pyridinyl)phenyl]methyl]-2,5,6,10,13-
pentaazatetradecanedioic acid di methyl ester. Instrumentation
(Atazanavir related compound-02) The chromatographic system consisted of a Waters
3. (3R,8R,9S,12R)-3,12-Bis(1,1-dimethylethyl)-8- Alliance liquid chromatograph (model 2695) fitted
hydroxy-4,11-dioxo-9-(phenylmethyl)-6-[[4-(2- with a diode array detector (model 2996) and an auto

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sampler using Empower2 data handling system. A Preparation of formulation sample solution
SunFire C18 column (250 × 4.6 mm; 5µm) was used
Ten tablets (Each tablet contains 300 mg of atazanavir
for the separation of the analytes. Solubility of all the
and 100mg of ritonavir) were crushed and ground to a
compounds was enhanced by sonication on an
fine powder. The tablet powder equivalent to 100 mg
ultrasonicator. All the weighings in the experiments
of ritonavir was accurately weighed and transferred
were done with Sartorius balances (model CPA225D
into a 100 mL volumetric flask. About 60 mL of
and model ME36S). Bandelin Sonorex was used for
diluent was added into it and sonicated for 30 minutes
ultrasonication. PVDF and nylon membrane filters
with occasional shaking. The contents were made up to
were purchased from Merck Millipore.
volume with the diluent, mixed well and filtered
Preparation of the buffer (pH 4.0; Mobile phase-A) through a 0.45 µm membrane filter (The first few
mLof the filtrate was discarded). This solution was
2.72 g of potassium dihydrogen phosphate was
used as the formulation sample solution (3000 µg/mL
weighed and dissolved in a beaker containing 1000 mL
of atazanavir and 1000 µg/mL of ritonavir).
of water (0.02M potassium dihydrogen phosphate
solution). The pH of the solution was adjusted to 4.0 Preparation of individual standard solutions of
with 10% orthophosphoric acid and was filtered related compounds and impurities
through a 0.45 µ membrane filter followed by
About 3 mg of each of atazanavir related compound 01,
sonication. This solution was used as mobile phase-A.
atazanavir related compound 02, atazanavir related
Preparation of mobile phase - B compound 03, ritonavir impurity-E and ritonavir
impurity-L and 2 mg of each of ritonavir impurity-O
A mixture of acetonitrile and tetrahydrofuran in the
and ritonavir impurity-T were weighed separately and
ratio of 80:20 v/v was used as mobile phase-B.
transferred into four separate 10 mL volumetric flasks.
Preparation of the diluent About 13 mg of ritonavir impurity-F was weighed and
transferred into a 20 mL volumetric flask. 5.0 mL of
Mobile phase - A and acetonitrile were mixed in the
the diluent was added into each of the above
ratio of 50:50 v/v and was used as the diluent for
volumetric flasks and sonicated for 10 min. The
preparation of various drug solutions.
volumes were made up with the diluent and mixed
Preparation of mixed working standard solution of well. These solutions were used as stock solutions of
the drugs impurities.
About 69 mg of atazanavir sulfate and 40 mg of Using the above stock solutions, dilutions containing 6
ritonavir were weighed and transferred into a 100 mL µg/mL each of atazanavir related compound 01,
volumetric flask. 60 mL of the diluent was added and
atazanavir related compound 02 and atazanavir related
sonicated to dissolve. The contents were made up to
compound 03, 30 µg/mL of ritonavir impurity-F, 3
volume with the diluent and mixed. This solution was
µg/mL each of ritonavir impurity-E and ritonavir
filtered through a 0.45 µm membrane filter (The first
impurity-L and 2 µg/mL each of ritonavir impurity-O
few mL the filtrate was discarded). 5.0 mL of the and ritonavir impurity-T were prepared. These
above solution was transferred into a 200 mL
solutions were used as individual working standard
volumetric flask and diluted to volume with the diluent
solutions of related compounds and impurities (100%
to make a mixed working standard solution containing concentration level).
17.25 µg/mL of atazanavirsulfate (15.14 µg/mL of
atazanavir) and 10 µg/mL of ritonavir. Preparation of the resolution solution
Preparation of placebo solution 100 mg of ritonavir was accurately weighed and
transferred in to a 100 mL volumetric flask. About 60
Ten typical placebo tablets were crushed and finely
mL of diluent was added into it and sonicated for 30
powdered. From this, a quantity equivalent to the minutes with occasional shaking. 1.0 mL of stock
weight of a tablet was a transferred into a 100 mL solution of ritonavir impurity-L was transferred in to
volumetric flask containing 60 mL of diluent. The the flask and the contents were made up to volume
contents were mixed well and sonicated for 30 minutes
with the diluent, mixed well and filtered through a 0.45
with occasional shaking (The temperature of water-
µm membrane filter (The first few mL of the filtrate
bath of thesonicator was maintained at 20-25°C). The was discarded). This solution was used as the
volume of the mixture was made up to the volume with resolution solution.
the diluent and mixed. A portion of this mixture was
filtered through a 0.45 µm membrane filter (The first Optimization of the chromatographic conditions
few mLof the filtrate was discarded). This placebo
Mobile phase-A and mobile phase-B were pumped
solution was later used for the testing the interference
through the column in gradient proportions at a flow
of the excipients used in tablets.

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rate of 1.5 mL/min. The gradient time program was set RESULTS AND DISCUSSION
as T/%B: 0/40, 40/40, 60/70, 65/70, 67/40, and 75/40.
The injection volume was 20µL and the column was The described method has been extensively validated
kept at 40°C. The detector wavelength was set at 250 was according to ICH guideline Q2 (R1) for
nm. Prior to injection of the drug solution, the column specificity, linearity, accuracy, precision, LOD, LOQ,
was equilibrated with the initial composition of the and robustness [8]. Solution stability studies and forced
mobile phase for 30 minutes. degradation studies were also performed.

Typical chromatograms obtained from the analysis of Specificity

the blank solution, mixed working standard solution, Individual reference solutions of atazanavir sulfate,
placebo sample solution, formulation sample solution, ritonavir and impurities at standard working
formulation sample solution spiked with impurities, concentration level, mixed standard solution,
and resolution solution are shown in the Fig. 3, 4, 5, 6, formulation sample solution and formulation sample
7, and 8, respectively. solution spiked with known impurities at standard

Fig. 3: Representative chromatogram of the blank solution.

Fig. 4: Representative chromatogram obtained from the analysis of mixed working standard solution.

Fig. 5: Representative chromatogram of the placebo sample solution.

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Fig. 6: Representative chromatogram obtained from the analysis of the formulation sample solution.

Fig. 7: Representative chromatogram obtained from the analysis of the formulation sample solution
spiked with the impurities.

Fig. 8: Representative chromatogram obtained from the analysis of the resolution solution.

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Table No 1: Retention times of the peaks

Component Retention time (min)

Mixed standard solution Formulation sample solution
Atazanavir 25.408 25.417
Ritonavir 30.517 30.835
Individual Reference Solutions
Ritonavir impurity-E 10.004
Ritonavir impurity-F 13.484
Atazanavir related compound 01 21.021
Atazanavir 25.063
Ritonavir impurity-L 29.100
Ritonavir 30.049
Atazanavir related compound 02 33.365
Ritonavir impurity-O 36.649
Atazanavir related compound 03 37.261
Ritonavir impurity-T 53.489
Spiked test solution
Ritonavir impurity-E 10.138
Ritonavir impurity-F 13.755
Atazanavir related compound 01 21.680
Atazanavir 25.484
Ritonavir impurity-L 29.166
Ritonavir 30.926
Atazanavir related compound 02 34.220
Ritonavir impurity-O 36.825
Atazanavir related compound 03 38.131
Ritonavir impurity-T 53.152

working concentration levels were analyzed in six System suitability

replicates by HPLC. The retention times obtained for
For system suitability, six replicates of the mixed
the drugs and impurities for the mixed working
standard solution were injected and the parameters like
standard solution, formulation sample solution and
peak area, number of theoretical plates and tailing
formulation sample solution spiked with known
factor of the peaks were calculated. These results are
impurities were compared with those of the respective
shown in the Table 2. Resolution between ritonavir
reference compounds.
impurity-L and ritonavir is also an integral part of
The blank (diluent) and placebo solutions were injected system suitability studies. This was determined by
into the chromatographic system. No interfering peaks analyzing the resolution solution (Fig. 8). Resolution
were observed at the retention times of the analytes and between ritonavir impurity-L and ritonavir was found
the known impurities due to the presence of excipients. to be 1.6.

Table No 2: System suitability parameters of the proposed method

S.No. Peak Area Number of theoretical plates Tailing factor

Atazanavir Ritonavir Atazanavir Ritonavir Atazanavir Ritonavir
1 249090 64330 15550 18081 1.1 1.1
2 249007 64341 15354 17927 1.1 1.0
3 249064 65270 15205 17315 1.1 1.0
4 250170 65884 15120 16401 1.1 1.1
5 247684 66047 15617 15520 1.1 1.1
6 251934 64573 15516 15159 1.1 1.1
Mean 249492 65074 - - - -
SD 1433.31 772.41 - - - -
%RSD 0.574 1.187 - - - -

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Linearity and range concentrations were constructed for individual

Six linearity study solutions (calibration) were compounds. The results are tabulated below.
prepared by using reference standards of atazanavir Limit of detection (LOD) and Limit of quantitation
sulfate, ritonavir, atazanavir related compound 01, (LOQ)
atazanavir related compound 02, atazanavir related
compound 03, ritonavir ritonavir impurity-E, ritonavir The LOD and LOQ values of atazanavir, ritonavir and
impurity-F, ritonavir impurity-L, ritonavir impurity-O their impurities were estimated by preparing their
and ritonavir impurity-T at different concentration solutions at lower concentrations and injecting the
levels ranging from LOQ to 150% of standard working solutions into the chromatographic system and
concentration levels. LOQ level and highest level were calculating the S/N ratio (signal/noise). LOD and LOQ
analyzed in six replicates and other levels in duplicate. are the concentrations where S/N ratio is 3.3 and 10
From these chromatograms, the mean peak areas were respectively. The LOD and LOQ values are shown the
calculated and linearity plots of mean peak areas over Table 4.

450000 160000
400000 140000
Mean peak area

Mean peak area

350000 120000
300000
100000
250000
80000
200000
y = 17364x + 653.2 60000
150000 y = 16548x + 105.0
100000 R² = 0.999 40000 R² = 0.999
50000 20000
0 0
0 5 10 15 20 25 0 2 4 6 8 10
Concentration of atazanavir related
Concentration of atazanavir (µg/mL)
compound 01(µg/mL)
(a) (b)

180000 180000
160000 160000
Mean peak area
Mean peak area

140000 140000
120000 120000
100000 100000
80000 80000
60000 y = 17808x - 290.5 60000 y = 16886x - 467.1
40000 R² = 1 40000 R² = 1
20000
20000
0
0
0 2 4 6 8 10
0 2 4 6 8 10
Concentration of atazanavir related Concentration of atazanavir related
compound 02(µg/mL) compound 03(µg/mL)
(c) (d)

Fig. 9: Linearity plots of atazanavir and its impurities

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120000 25000
105000 22500
Mean peak area

20000

Mean peak area

90000
17500
75000 15000
60000 12500
45000 10000 y = 4992.x - 93.18
y = 6900.x + 387.6
7500 R² = 0.999
30000 R² = 0.999
5000
15000 2500
0 0
0 5 10 15 20 0 1 2 3 4 5

Concentration of ritonavir (µg/mL) Concentration of ritonavir impurity-E

(µg/mL)
(a) (b)

250000 22500
225000 20000
Mean peak area
Mean peak area

200000 17500
175000 15000
150000
12500
125000
10000
100000
y = 5010.x + 544.7 7500
75000 y = 4577.x - 41.53
50000 R² = 0.999 5000 R² = 0.999
25000 2500
0 0
0 10 20 30 40 50 0 1 2 3 4 5
Concentration of ritonavir impurity-F Concentration of ritonavir impurity-L
(µg/mL) (µg/mL)

(c) (d)

21000 24500

18000 21000
Mean peak area
Mean peak area

15000 17500
12000 14000
9000 y = 5728.x - 99.32 10500 y = 7061.x - 86.83
R² = 0.999 R² = 0.999
6000 7000
3000 3500
0
0
0 1 2 3 4
0 1 2 3 4
Concentration of ritonavir impurity-O Concentration of ritonavir impurity-T
(µg/mL) (µg/mL)
(e) (f)

Fig. 10: Linearity plots of ritonavir and its impurities

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Table No 3: Linearity data of the proposed method

Component Linearity range Regression equation and Relative response

(μg/mL) coefficient factor
Atazanavir 0.340-22.676 y = 17364x + 653.2 (R² = 0.999) -
Atazanavir related compound-01 0.318-9.082 y = 16548x + 105.0 (R² = 0.999) 0.95
Atazanavir related compound-02 0.458-9.153 y = 17808x - 290.5 (R² = 1) 1.03
Atazanavir related compound-03 0.497-9.037 y = 16886x - 467.1 (R² = 1) 0.97
Ritonavir 0.663-15.077 y = 6900.x + 387.6 (R² = 0.999) -
Ritonavir impurity-E 0.599-4.495 y = 4992.x - 93.18 (R² = 0.999) 0.72
Ritonavir impurity-F 0.675-45.023 y = 5010.x + 544.7 (R² = 0.999) 0.73
Ritonavir impurity-L 0.752-4.510 y = 4577.x - 41.53 (R² = 0.999) 0.66
Ritonavir impurity-O 0.606-3.029 y = 5728.x - 99.32 (R² = 0.999) 0.83
Ritonavir impurity-T 0.516-3.094 y = 7061.x - 86.83 (R² = 0.999) 1.02

Table No 4: Limits of detection and quantitation

S.No. Compound name LOD (µg/mL) LOQ (µg/mL)

1 Ritonavir impurity-E 0.198 0.599
2 Ritonavir impurity-F 0.225 0.675
3 Atazanavir related compound-01 0.106 0.318
4 Atazanavir 0.112 0.340
5 Ritonavir impurity-L 0.248 0.752
6 Ritonavir 0.221 0.663
7 Atazanavir related compound-02 0.153 0.458
8 Ritonavir impurity-O 0.200 0.606
9 Atazanavir related compound-03 0.166 0.497
10 Ritonavir impurity-T 0.170 0.516

Accuracy very precise. The results of repeatability and

intermediate precision studies are shown in the Table 6
Accuracy was performed by spiking the impurities to
and Table 7.
the placebo solution at 50%, 100% and 150% of
working concentration level in triplicate at each level. Forced degradation studies
These solutions were injected into the chromatographic
Ten tablets were crushed and grinded to a fine powder.
system and the percent recovery was calculated.
This powdered tablet was then subjected to various
Accuracy at LOQ was also performed similarly by
stress conditions like acid (1M HCl, 80°C, 2 hr), base
spiking the known impurities to the placebo in
(0.25M NaOH, 80°C, 1 hr), peroxide (3% H2O2, 2 hr),
triplicate and analyzing these solutions. The percent
photo degradation (254 nm, 168 hr), thermal
recoveries of impurities at all the levels were between
degradation (90°C, 2 hr) and humidity induced
the limits of 85.0 and 115.0. Hence the method is very
degradation (90% relative humidity). These stressed
accurate.
samples were analyzed and was found that the samples
Precision were stable to the stress induced by peroxide,
photolysis and humidity. The purity angles of
System precision was studied by preparing working
atazanavir and ritonavir peaks were less than their
standard solution and analyzing them in six replicates.
purity thresholds in all the stress-induced samples.
Peak areas of atazanavir and ritonavir were measured
and their percent relative standard deviations were Robustness
found to be 0.57 and 1.19 respectively. Repeatability
The formulation sample solution spiked with impurities
and intermediate precision was studied by preparing
and mixed standard solution were prepared and
formulation sample solution and formulation sample
analyzed in three and six replicates respectively, after
solution spiked with known impurities at specification
deliberately changing the chromatographic parameters
level and analyzed in six replicates. A very small %
(one at a time) like flow rate of the mobile phase,
RSD value of recoveries describes that the method is
temperature of the column and pH of the buffer.

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Table No 5: Results obtained from the recovery studies

Compound name Mean percent recovery at different levels

LOQ level 50% level 100% level 150% level
Atazanavir related compound 01 100.94 99.34 100.50 99.90
Atazanavir related compound 02 93.06 99.67 99.52 99.52
Atazanavir related compound 03 99.02 100.69 100.17 100.43
Ritonavir impurity-E 100.31 99.65 100.24 100.48
Ritonavir impurity-F 100.74 100.45 100.42 100.39
Ritonavir impurity-L 100.14 100.69 100.13 100.22
Ritonavir impurity-O 99.69 100.06 99.83 100.37
Ritonavir impurity-T 100.03 100.46 100.22 100.32

Table No 6: Repeatability and intermediate precision data of formulation sample solution

Compound name %RSD of recoveries

Repeatability studies Intermediate precision studies
Atazanavir related compound-01 NA NA
Atazanavir related compound-02 NA NA
Atazanavir related compound-03 NA NA
Ritonavir impurity-E NA NA
Ritonavir impurity-F 1.01 0.88
Ritonavir impurity-L NA NA
Ritonavir impurity-O NA NA
Ritonavir impurity-T 2.04 2.23
MSUI 1.16 1.29
Total impurities 0.93 0.70

Note: 1. NA: Not applicable

2. MSUI: Maximum single unspecified impurity

Table No 7: Repeatability and intermediate precision data of formulation sample solution spiked with impurities

Compound name %RSD of recoveries

Repeatability studies Intermediate precision studies
Atazanavir related compound 01 0.74 0.40
Atazanavir related compound 02 0.58 0.44
Atazanavir related compound 03 0.82 0.40
Ritonavir impurity-E 0.96 0.53
Ritonavir impurity-F 0.38 0.40
Ritonavir impurity-L 0.72 0.47
Ritonavir impurity-O 0.82 0.52
Ritonavir impurity-T 1.20 0.60
MSUI 2.23 1.66
Total impurities 0.22 0.31

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Fig. 11: Representative chromatogram of the formulation sample subjected to acid hydrolysis.

Fig. 12: Representative chromatogram of the formulation sample subjected to base hydrolysis.

Fig. 13: Representative chromatogram of the formulation sample subjected to oxidation.

Fig. 14: Representative chromatogram of the formulation sample subjected to thermal.

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Fig. 15: Representative chromatogram of the formulation sample subjected to photo degradation.

Fig. 16: Representative chromatogram of the formulation sample subjected to humidity degradation.

Table No 8: Summary of results obtained after analyzing mixed standard solution (n=6)

Variation in Peak area %RSD Minimum theoretical Maximum

chromatographic plates tailing factor
condition Atazanavir Ritonavir Atazanavir Ritonavir Atazanavir Ritonavir
Unchanged condition 0.57 1.19 15120 15159 1.10 1.10
Flow rate 1.35 0.32 1.38 16045 15953 1.08 1.09
(1.5 mL/min) mL/min
1.65 0.92 1.79 14442 15013 1.06 1.06
mL/min
Column oven 35°C 0.87 2.17 14501 14209 1.08 1.12
temperature 45°C 0.37 0.43 16289 16629 1.04 1.03
(40°C)
Change in pH 3.8 0.63 1.37 15672 15278 1.05 1.05
of buffer 4.2 0.56 0.90 15848 15764 1.05 1.06
(4.0)

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 Palavan Chinnaiah /J Compr Phar 2015;2(3):71-83

Table No 9: Summary of results obtained after analyzing formulation sample solution spiked with known
impurities (n=3)

Chromatographic %RSD values of recoveries

condition Atazanavir Atazanavir Atazanavir Ritonavir Ritonavir Ritonavir Ritonavir Ritonavir
related related related impurity- impurity- impurity- impurity- impurity-
compound compound compound E F L O T
01 02 03
Flow rate 1.35 1.72 3.09 2.27 2.05 3.09 2.11 1.65 2.43
(1.5 mL/min) mL/min
1.65 1.19 2.18 1.68 1.98 1.99 2.02 2.22 1.58
mL/min
Column 35°C 1.58 1.97 2.22 2.33 4.13 1.64 1.39 2.56
temperature 45°C 2.55 2.63 2.76 1.69 3.45 2.27 2.71 3.02
(40°C)
pH of the 3.8 2.25 2.53 2.86 2.21 2.44 2.88 1.54 1.62
buffer 4.2 1.23 1.25 2.07 1.87 2.27 1.45 1.32 2.75
(4.0)

The system suitability parameters obtained after

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Cite this article as: Chinnaiah P, Lanka AR, Pamidi S, Govada PPR , Jillella VLNSR. NEW VALIDATED RP-
HPLC METHOD FOR IDENTIFICATION AND QUANTITATION OF PROCESS AND DEGRADATION OF
PROCESS AND DEGRADATION RELATED IMPURITIES IN THE COMBINED DOSAGE TABLETS OF
ATAZANAVIR AND RITONAVIR. J Compr Phar 2015;2(3):71-83.

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