| Research Article |
Open Access |
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| Quantitative Estimation of Lopinavir and Ritonavir in Tablets by RP-HPLC
Method |
| Jagadeeswaran M1*, Gopal N2, Pavan kumar K1 and Siva kumar T1 |
| 1Nandha College of Pharmacy and Research Institute, Department of Pharmaceutical Analysis, Erode -638 052, Tamil Nadu, India |
| 2Balaji Institute of Pharmaceutical Sciences, Department of Pharmaceutical Chemistry, Narsampet, Warangal-506 331, Andhra Pradesh, India |
| *Corresponding author: |
Jagadeeswaran M
Assistant Professor, Department of
Pharmaceutical Analysis
Nandha College of Pharmacy and Research Institute
Perundurai
Road, Erode-638 052, TamilNadu, India
E-mail: jagantgode@rediffmail.com |
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| Received February 15, 2012; Accepted July 10, 2012; Published July 14, 2012 |
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Citation: Jagadeeswaran M, Gopal N, Pavan kumar K, Siva kumar T (2012)
Quantitative Estimation of Lopinavir and Ritonavir in Tablets by RP-HPLC Method.
Pharmaceut Anal Acta 3:160.
doi:10.4172/2153-2435.1000160 |
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| Copyright: © 2012 Jagadeeswaran M, et al. This is an open-access article
distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided
the original author and source are credited. |
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| Abstract |
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| A reversed phase high-performance liquid chromatographic method was developed and validated for the
quantitative determination of two antiviral drugs viz. lopinavir and ritonavir. Chromatography was carried out by
gradient technique on a reversed-phase C18 Column, Phenomenex (250 x 4.6 mm, 5 μ) with mobile phase mixture
of Buffer: Acetonitrile (45:55 v/v) was used as a mobile phase and the pH was adjusted into 4.5 by using with
O-phosphoric acid, at a flow rate of 1.2 ml/min. The UV range was detected at 240 nm for lopinavir and ritonavir
respectively. The different analytical performance parameters such as linearity, precision, accuracy, specificity,
limit of detection (LOD) and limit of quantification (LOQ) were determined according to International Conference
on Harmonization ICH Q2B guidelines. The linearity of the calibration curves for each analyte in the desired
concentration range is good (r2 >0.9). The recovery of the method was between 102.1% and 100.1% for lopinavir
and ritonavir respectively. Hence the proposed method is highly sensitive, precise and accurate and it successfully
applied for the reliable quantification of API content in the commercial formulations of lopinavir and ritonavir. |
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| Keywords |
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| Lopinavir; Ritonavir; UV spectrophotometry; RP-HPLC |
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| Introduction |
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| One of the deadliest and unmanageable chronic health catastrophes
is HIV/AIDS. It requires lifelong treatment with potent life saving
essential drugs that include nucleoside reverse transcriptase inhibitors,
non nucleoside reverse transcriptase inhibitors and protease inhibitors.
Amongst these lopinavir and ritonavir drug combination is a protease
inhibitor used as a second line regimen to treat patients with HIV [1]. |
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| Lopinavir (the active ingredient) (Figure 1A) is chemically
designated as [1S-[1R*,(R*) 3R*, 4R*]]-N-[4-[[(2,6-dimethylphenoxy)
acetyl]amino]-3 -hydroxy-5-phenyl-1-(phenylmethyl) pentyl]
tetrahydro-alpha-(1-methylethyl)-2-oxo-1 (2H)-pyrimidineacetamide.
Ritonavir (Figure 1B) is chemically designated as 10-Hydroxy-2-
methyl-5-(1-methylethyl)-1-[2-(1-methylethyl)-4-thiazolyl]-3,6-
dioxo-8,11-bis(phenylmethyl)-2,4,7,12-tetraazatridecan -13-oic acid,
5-thiazolylmethyl ester, [5S-(5R*,8R*,10R*,11R*)]. |
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Figure 1: Chemical structures of lopinavir (A) and ritonavir (B). |
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| Literature survey revealed several analytical methods for
the determination of ritoavir and lopinavir in tablets, capsules,
and syrups which employ techniques such as high-performance
liquid chromatography (HPLC) [2-4], Ultra performance liquid
chromatography (UPLC) [5], and high performance thin layer
chromatography (HPTLC) [6]. In biological fluids, the active principles
as well as their metabolites have been quantitatively determined by
HPLC with UV detection, LC/MS/MS [7,8], Spectroscopic method
[9], Micellar electrokinetic chromatography method [10] and Tandem
mass spectrometry [11]. |
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| The proposed method was optimized and validated in accordance
with International Conference on Hormonization (ICH) guidelines
[12]. The aim of present work is to develop a simple, rapid, precise,
accurate and selective reversed phase chromatographic method and to
estimate the lopinavir and ritoavir in bulk and its solid dosage forms. |
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| Materials and Methods |
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| Chemicals |
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| The bulk drugs of lopinavir and ritonavir were obtained as gift
samples from Abbott Laboratories Ltd, Guwahati, India. HPLC
grade acetonitrile and ammonia were obtained from Sigma Aldrich
(Switzerland). Combination tablets of Lopinavir 200 mg and Ritonavir
50 mg from abbott were purchased from local market. Milli-Q-Water
was used in all experiments. All the solutions for analysis were prepared
and analyzed freshly. |
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| Instrumentation and analytical conditions |
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| Chromatography was performed using a shimadzu LC-10ATvp
series, (Kyoto, Japan) equipped with SPD-10A UV-Vis detector. Data
acquisition and processing was performed using chemistry station
software (LC solution). The methods were conducted using a gradient reverse phase technique. The analytical conditions (mobile phase
composition, flow rate and analytical wavelengths) for the two drugs
have been summarized in Table 1. The mobile phases were prepared
freshly, filtered through 0.45 μ membrane filter (Millipore, USA) and
sonicated (Branson sonicator 3210, Germany) for 10 min before use in
order to deaerate. |
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Table 1: The optimized chromatographic conditions for analysis of lopinavir and
ritonavir. |
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| Preparation of standard and quality control solutions |
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| Primary stock solutions of lopinavir (100 mg) and ritonavir (100
mg) were prepared in ultra pure water and further diluted with water
to obtain working standards in the concentration range of 40–200 μg/
ml and 10-50 μg/ml for lopinavir and ritonavir respectively. Quality
control (QC) samples were run with each batch of working standards in
order to calculate the validation parameters. QC samples were prepared
in ultra pure water spiked with analytes at different concentrations
following the same procedure as for calibration standards, using a
different primary stock. The samples were analyzed with reagent
blanks. All the solutions were prepared in triplicates. |
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| Results and Discussion |
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| RP-HPLC method |
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| A RP-HPLC method was developed for two anti-retroviral drugs,
which can be conveniently employed for routine quality control in
pharmaceutical dosage forms. The chromatographic conditions were
optimized in order to provide a good performance of the assay. The
mobile phase for each drug was selected based on its polarity. Different
ratios of Buffer: ACN combinations were tried for lopinavir and ritonavir
and the fixed mobile phase are listed in Table 1. The optimization
of flow rate is critical since the extent of longitudinal broadening is
inversely related to flow rate of mobile phase. In either case of high
or low flow rates, an ideal Gaussian curve of the peak is not obtained
as the peak symmetry parameters are affected, i.e. asymmetry factor
deviates from unity (Figure 2-4). The retention times of ritonavir and
lopinavir were 4.323 and 5.650 min, respectively. The chromatograms
have been shown in Figure 5. The methods were specific as none of the
excipients interfered with the analytes of interest. Hence, the methods
were suitably employed for assaying the commercial anti-retroviral
individual formulations. |
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Figure 2: Overlay spectrum of lopinavir and ritonavir. |
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Figure 3: Peak purity curve of ritonavir. |
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Figure 4: Peak purity curve of lopinavir. |
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Figure 5: Typical chromatogram showing the elution of ritonavir and lopinavir
at their respective retention times. |
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| Linearity |
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| Calibration curves were obtained from the peak area and
concentration of the drug were subjected to regression analysis and
correlation coefficients. Table 2 represents the mean RP-HPLC area
responses for ritonavir and lopinavir at different concentrations. As
shown, the responses for the drug was strictly linear (r2 > 0.999) in the
concentration range of 10-50 μg/ml for ritonavir and 40-200 μg/ml for
lopinavir respectively. The slope and intercept for lopinavir was found
to be 21201 and 10566 where as for ritonavir was found to be 15278 and
26980 respectively. |
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Table 2: Linearity of the developed RP-HPLC method. |
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| Accuracy and precision |
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| Accuracy and precision were determined by elaboration of three
standard calibration curves, two from the same day (intra-day) and
third one from a different day (inter-day). The intra-day and inter-day
precisions (% RSD) at different concentration levels were found to be
less than 2% (Table 3). Moreover the % RSD (less variation) showed
good precision of the developed HPLC methods. |
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Table 3: Precision data for the analysis of lopinavir and ritonavir. |
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| The respective RP-HPLC area responses from the accuracy
determination study are shown in Table 4. Recovery experiment was
carried out by applying the standard addition method. Drug assay
was performed in triplicate by spiking with equivalent amount of
raw material into each volumetric flask for each spike level to get the concentrations of lopinavir and ritonavir equivalent to 80%, 100%, and
120% of the standard concentrations of lopinavir and ritonavir. The
average percentage recovery of both the drugs was found to be within
the limits and it is highly accurate |
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Table 4: Analysis of formulations and recovery studies by RP-HPLC. |
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| LOQ and LOD |
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| The LOD and LOQ were determined from the calculated standard
deviations of each calibration standard and it was found to be 0.013
μg/ml and 0.465 μg/ml for lopinavir and ritonavir respectively. The
calculated LOQ and LOD concentrations confirmed that the method
is sensitive. |
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| Specificity |
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| The developed method is specific as none of the excipients interfered
with the analytes of interest. Hence, this method is suitably employed
for assaying the commercial anti-retroviral individual formulations. |
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| Conclusion |
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| The proposed RP-HPLC is simple, reliable and selective. It also
provides satisfactory accuracy and precision with lower limits of
detection and quantification. Moreover the shorter duration of analysis
for lopinavir and ritonavir make these reported methods suitable for
routine quantitative analysis in pharmaceutical dosage forms. The
recoveries achieved are good by both the methods. |
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| Acknowledgements |
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| The authors are thankful to Abbott Laboratories Ltd., Guwahati, India for
procuring the gift samples and also thankful to the management, Nandha College
of Pharmacy and Research Institute, Erode, Tamilnadu, India. |
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| References |
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- Sean CS Martindale: The complete drug reference, (34th edn). The Pharmaceutical Press.
- Usami Y, Oki T, Nakai M, Sagisaka M, Kaneda T (2003) A simple HPLC method for simultaneous determination of lopinavir, ritonavir and efavirenz. Chem Pharm Bull (Tokyo) 51: 715-718.
- Donato EM, Dias CL, Rossi RC, Valente RS, Froelich PE et al. (2006) LC method for studies on the stability of lopinavir and ritonavir in soft gelatin capsules. Chromatogr 63: 437-443
- Phechkrajang CM, Thin EE, Sratthaphut L, Nacapricha D, Wilairat P (2009) Quantitative Determination of Lopinavir and Ritonavir in Syrup Preparation by Liquid Chromatography. Mahi Univ J Pharm Sci 36: 1-12.
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- Vaishali P Nagulwar, Kishore PB (2010) Simultaneous estimation of ritonavir and lopinavir by absorption ratio (Q-analysis) UV spectrophotometric method in combined tablet dosage form. Der Pharmacia Lettre 2: 196-200.
- Carvalho AZ, El-Attug MN, Zayed SE, Hove EV, Duppen JV et al. (2010) Micellar electrokinetic chromatography method development for determination of impurities in ritonavir. J Pharm Biomed Anal 53: 1210-1216.
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- Anonymous, ICH Guidelines (1994). Validation of Analytical Procedures: Text and Methodology Q2 (R1).
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