INTRODUCTION:

High-performance liquid chromatography (HPLC) is the fastest growing analytical technique for analysis of drugs. Its simplicity, high specificity, and wide range of sensitivity make it ideal for the analysis of many drugs in both dosage forms and biological fluids. High performance liquid Chromatography (HPLC) is the term used to describe liquid chromatography in which the liquid mobile phase is forced through the column at high speed as a result, the analysis time is reduced by 1-2 orders of the magnitude relative to classical column chromatography and the use of much smaller particles of the adsorbent or support becomes possibly increasing the column efficiency substantially.

The importance of chromatography is increasing rapidly in pharmaceutical analysis for the exact differentiation, selective identification, quantitative determination of structurally closely related compounds. Another important field of application of chromatographic methods is the purity testing of final products and the intermediates. The reasons for the popularity of the method is its sensitivity, its ready adaptability to accurate quantitative determinations, its suitability for separating non-volatile species or thermally fragile ones and its wide spread applicability to substances that are of prime interest to the industry. [1-6]

Cobicistat is chemically as 1,3-thiazol-5-ylmethyl N-[(2R,5R)-5-[[(2S)-2-[[methyl [(2propa2-yl-1, 3-thiazol-4-yl) methyl] carbamoyl] amino]-4 morpholin-4-yl butanoyl] amino]-1, 6-diphenylhexan-2-yl] Carbamate, is a mechanism-based inhibitor of cytochrome P450 3A (CYP3A) isoforms. Inhibition of CYP3A-mediated metabolism by Cobicistat increases the systemic exposure of CYP3A substrates Atazanavir and Darunavir and therefore enables increased anti-viral activity at a lower dosage. Cobicistat does not have any anti-HIV activity on its own.[7]

Elvitegravir is chemically as: 6-(3-Chloro-2-fluorobenzyl)-1-[(2S)-1-hydroxy 3methylbutan-2yl] 7-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, an HIV-1 integrase strand transfer inhibitor (INSTI).Integrase is an HIV-1 encoded enzyme that is required for viral replication inhibition of integrase prevents the integration of HIV-1 DNA into host genomic DNA, blocking the Formation of the HIV-1 provirus and propagation of the viral infection. Elvitegravir does not inhibit human topoisomerases. [8]

In the present work, attempts were made to develop analytical method of simultaneous estimation of Cobicistat and Elvitegravir pharmaceutical formulations by RP – HPLC method.

Figure1: (A) Chemical structure of Cobicistat

Figure2: (B) Chemical structure of Elvitegravir

MATERIALS AND METHODS:

Instruments:

Shimadzu HPLC, Model: LC 2010, Photo diode array detector (PDA), with an automated sample injector. The output signal was monitored and integrated using LC solutions Ver 1.23 software synchronized to shimadzu class VP series work station. AGILENT ZORBAX C18 column (150 X 4.6 mm id, ODS 2.5μm) column was used for separations. List of instruments are listed in Table No.1 and chemicals are listed in Table no.2.

Experimental work:

Optimized chromatographic conditions:

Mobile phase: Methanol: OPA (75:25 % V/V)

Column: Agilent Zorbax column

Wavelength: 265 nm

Flow rate: 1 mL/min

Column temperature: Ambient

Sample temperature: Ambient

Injection volume: 20μL

Run time: 6 min

Figure: 3: Optimized Chromatogram of Cobicistat and Elvitegravir

RESULTS:

By injecting the standard mix solution which contains Cobicistat and Elvitegravir, retention times were found to be at 2.11 min and 3.93 min respectively. Peaks are selectively separated with required resolution and tailing factors of peaks are within the limits. Impurities are well separated.

PREPARATION OF SOLUTIONS: [10-11]

Preparation of mobile phase:

1 mL of Ortho Phosphoric acid was added to 1000 mL of water. Mixed well filtered and degassed through 0.45μm membrane filter. Methanol (HPLC grade) of 650 mL was taken and 350 mL of the above prepared OPA buffer was added to it and finally this solution was filtered through 0.45μm membrane filter followed by degassing.

Preparation of standard stock solution:

About 200.8 mg of Elvitegravir and 10.1 mg of Cobicistat were accurately weighed into a 250mL volumetric flask, to this 10 mL of diluent (mobile phase) was added, sonicated and the volume was made up with the diluent and then filtered through 0.45μm membrane filter.

Preparation of sample stock solution:

Twenty tablets (STRIBILD) were taken (each tablet containing 150mg of Cobicistat and 150mg of Elvitegravir) into a mortar and finely powdered with a pestle. An equivalent weight of powder containing 150mg of Cobicistat and 150mg of Elvitegravir were accurately weighed and transferred to 10 mL volumetric flask and diluted with the Methanol and shaken mechanically for 10 minutes and then centrifuged. The clear supernatant liquid was taken and sonicated in ultrasonic bath for 5 minutes. The solution was filtered through 0.45μm membrane filter and then final volume was made up with Methanol. From this stock solution 1 mL was taken into a 10mL volumetric flask and made up the volume with diluents.

Method Validation: [9]

Linearity:

The calibration curves were constructed with six concentrations. The linearity was evaluated by linear regression analysis, which was calculated by the least square regression method.

Preparation of solution-1:

From the stock solutions 0.050ml of Cobicistat and 0.035 ml of Elvitegravir were taken and diluted up to 2 ml with 1.915ml of diluent to get a concentration of 2.52μg/ml Cobicistat and 2.45μg/ml of Elvitegravir respectively.

Preparation of solution-2:

From the stock solutions 0.100ml of Cobicistat and 0.070 ml of Elvitegravir were taken and diluted up to 2 ml with 1.830ml of diluent to get a concentration of 5.04μg/ml Cobicistat and 4.91μg/ml of Elvitegravir respectively.

Preparation of solution-3:

From the stock solutions 0.200ml of Cobicistat and 0.140ml of Elvitegravir were taken and diluted up to 2 ml with 1.830ml of diluent to get a concentration of 10.09μg/ml Cobicistat and 9.82μg/ml of Elvitegravir respectively.

Preparation of solution-4:

From the stock solutions 0.300ml of Cobicistat and 0.210ml of Elvitegravir were taken and diluted up to 2 ml with 1.490 ml of diluent to get a concentration of 15.13μg/ml Cobicistat and 14.73μg/ml of Elvitegravir respectively.

Preparation of solution-5:

From the stock solutions 0.400ml of Cobicistat and 0.280ml of Elvitegravir were taken and diluted up to 2 ml with 1.320ml of diluent to get a concentration of 20.18μg/ml Cobicistat and 19.64μg/ml of Elvitegravir respectively.

Preparation of solution-6:

From the stock solutions 0.500ml of Cobicistat and 0.350ml of Elvitegravir were taken and diluted up to 2 ml with 1.150ml of diluent to get a concentration of 25.22μg/ml Cobicistat and 24.55μg/ml of Elvitegravir respectively. The results are listed in the Table-5

Accuracy and precision:

Preparation of LQC sample:

From stock solutions 0.125ml of Cobicistat and 0.085 ml of Elvitegravir were taken into a 2ml volumetric flask and made up the volume with 1.790ml of mobile phase to get a concentrations of 6.31μg/mL of Cobicistat and 5.96μg/ml of Elvitegravir respectively.

Preparation of MQC sample:

From stock solutions 0.25ml of Cobicistat and 0.175ml of Elvitegravir were taken into a 2ml volumetric flask and made up the volume with 1.525ml of mobile phase to get a concentration of 12.61μg/ml of Cobicistat and 12.27μg/ml of Elvitegravir respectively.

Preparation of HQC sample:

From stock solutions 0.375ml of Cobicistat and 0.255ml of Elvitegravir were taken into a 2ml volumetric flask and made up the volume with 1.370ml of mobile phase to get a concentration of 18.92μg/ml of Cobicistat and 17.89μg/ml of Elvitegravir respectively.

Accuracy of the assay method was determined for both intra-day and inter-day variations using the triplicate analysis of QC samples. Precision of the assay was determined by repeatability (intra-day) and intermediate precision (inter-day). Repeatability refers to the use of the analytical procedure within a laboratory over a short period of time that was evaluated by assaying the QC samples during same day. Intermediate precision was assessed by comparing assays on different days (3days).

The results are listed in the Table-6,7

Specificity:

The specificity of the method is performed by separately injecting the blank, Elvitegravir sample, Cobicistat sample and sample containing both Elvitegravir and Cobicistat. The interference observed (if any) at the retention times of each analyte in all the chromatograms is evaluated.

Over lay chromatogram for the above discussed specificity solutions were recorded as shown in the Figure -6

Robustness:

The MQC sample solution prepared was analysed under different chromatographic conditions stated below.

a) Change in flow rate:

Flow rate – 0.9 mL/min

Flow rate – 1.1 mL/min

Change in mobile phase:

Methanol: OPA (80:20 % v/v)

Methanol: OPA (70:30% v/v)

The results are listed in the Table

Ruggedness:

The Ruggedness was determined by using the data obtained by the analysis performed by two different analysts. Each analyst prepared 5 MQC samples of the same batch and the results obtained.

The results are shown in Table-8

System suitability:

The system suitability was assessed by six replicate analysis of drugs at concentrations of ranging from 100.90μg/mL for Cobicistat and 20039.84μg/mL for Elvitegravir. By taking 0.4 mL of Cobicistat and 0.4mL of Elvitegravir from stock solutions in 10mL volumetric flak and made up the volume with mobile phase. The acceptance criteria were ± 2% for percent coefficient variation (CV %) for peak area and retention time for both Cobicistat and Elvitegravir respectively.

The results are listed in the Table-3.

Limit of detection (LOD):

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.

Several approaches for determining the detection limit are possible, depending on whether the procedure is a non-instrumental or instrumental.

1. Based on Signal-to-Noise:

This approach can only be applied to analytical procedures which exhibit baseline noise. Determination of the signal-to-noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected. A signal- to-noise ratio 3:1 is generally considered acceptable for estimating the detection limit.

2. Based on the Standard Deviation of the Response and the Slope:

The detection limit (DL) may be ex pressed as:

LOD= 3.3 σ / S

Where,

σ = standard deviation of intercepts of calibration curves

S = mean of slopes of the calibration curves

The slope S may be estimated from the calibration curve of the analyte.

Limit of quantitation (LOQ):

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.

The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/or degradation products. Several approaches for determining the quantitation limit are possible, depending on whether the procedure is a non-instrumental or instrumental. Determination of the signal-to-noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and by establishing the minimum concentration at which the analyte can be reliably quantified

1. A typical signal-to-noise ratio is 10:1.

2. Based on the Standard Deviation of the Response and the Slope

The quantitation limit (QL) may be expressed as:

LOQ = 10 σ / S

Where,

σ = standard deviation of intercepts of calibration curves

S = mean of slopes of the calibration curves

The slope S may be estimated from the calibration curve of the analyte.

The results are listed in the Table -10

Procedure for assay:

This combination is not official in any pharmacopoeia. Therefore, a general method for the assay of tablets is followed as per the procedure mentioned under Tablets of General Procedures in The Indian Pharmacopoeia 2010.

Accordingly, 20 tablets of marketed formulation (STRIBILD) containing 150mg of Cobicistat and 150 mg of Elvetigravir were individually weighed and taken into a mortar. The average weight of each tablet is then calculated. The tablets were crushed into a fine powder. Accurately weighed quantity of one tablet equivalent to weight was transferred to a 1000 ml volumetric flask containing mobile phase. This mixture is then sonicated for 10 minutes and then made up to the volume with mobile phase. This is named as Solutions 1.

5 ml of the solution -1 is then taken and diluted to 10 ml volumetric flask with mobile phase and this solution is named as Solution 2. Solution -2 is injected and concentration is calculated. The results are listed in the Table-4

Stability:

The stability of drug solutions was determined QC samples for short term stability by keeping at room temperature for 6hrs and then analyzed. The long-term stability was determined by storing at 4°c for 30 days. Auto sampler stability was determined by storing the sample for 24hrs in the auto sampler. The results are listed in the Table-11

7.2.10 Degradation studies:

Forced degradation or accelerated degradation is a process whereby the natural degradation rate of a product or material is increased by the application of additional stress.

Forced degradation or stress test is undertaken to demonstrate specificity when developing stability- indicating methods, particularly when little information is available about potential degradation products. These studies also provide information about the degradation pathways and degradation products that could form during storage. Forced degradation studies may help facilitate pharmaceutical development as well in areas such as formulation development, manufacturing, and packaging, in which knowledge of chemical behaviour can be used improve a drug product.

a) Acid degradation:

From stock solutions 1 mL of Cobicistat and1 mL of Elvitegravir were taken separately into a 10mL volumetric flask, sonicated with 5mL of diluent with intermediate shaking for 15 min. To both the flasks 1mL of 0.1 N HCl was added and these solutions were placed in water bath at 60°C for 1 hr. Then the solution was allowed to cool at room temperature, and the sample solutions were neutralized with 1mL of 0.1N NaOH. The volume was made up to the mark with diluent and the resulting solutions were filtered. 10μL of the solutions were injected and chromatograms were recorded for the Cobicistat and Elvitegravir separately.

b) Alkali degradation:

From stock solutions 1 mL of Cobicistat and 1 mL of Elvitegravir were taken separately into a 10mL volumetric flask, sonicated with 5mL of diluent with intermediate shaking for 15 min. To both the flasks 1mL of 0.1 N NaOH was added and these solutions were placed in water bath at 60°C for 1 hr. Then the solutions were allowed to cool at room temperature, and the sample solutions were neutralized with 1mL of 0.1N HCl. The volume was made up to the mark with diluent and the resulting solutions were filtered. 10μL of the solutions were injected and chromatograms were recorded for the Cobicistat and Elvitegravir separately.

c) Photolytic degradation:

From stock solutions 1 mL of Cobicistat and 1 mL of Elvitegravir were taken separately into a 10mL volumetric flask, sonicated with 5mL of diluent with intermediate shaking for 15 min and made up to the volume with diluent. The samples were exposed to UV light about 6 hrs. 10μL of the solutions were injected and chromatograms were recorded for Cobicistat and Elvitegravir separately.

d) Oxidation:

From stock solutions 1 mL of Cobicistat and 1 mL of Elvitegravir were taken separately into a 10mL volumetric flask, sonicated with 5mL of diluent with intermediate shaking for 15 min. To both the flasks 1mL of 1 % H2O2 was added and these solutions were placed in water bath at 60°C for 1 hr. Then the solutions were allowed to cool at room temperature. The volume was made up to the mark with diluent and the resulting solutions were filtered. 10μL of the solutions were injected and chromatograms were recorded for the Cobicistat and Elvitegravir separately.

The results are listed in the Table-12

Table .1: List of Instruments

S.NO	Equipment’s	Company
1	HPLC	Shimadzu class
2	Electronic Balance	ASCOSET
3	Ultra-Sonicator	ENERTECH
4	Heating Mantle	BIO TECHNICS INDIA
5	Thermal oven	NARANG
6	pH Meter	ADWA
7	Filter Paper 0.45 microns	MILLI PORE

Table No.2 List of chemicals and reagents used

S. No.	Chemicals/standards and reagents	Grade	Make
1	Methanol	HPLC	Merck
2	Milli Q Water	HPLC	Merck
3	Ortho Phosphoric Acid	AR	Merck
4	Hydrochloric Acid	AR	Merck
5	Sodium hydroxide	AR	Merck
6	Hydrogen Peroxide	AR	Merck
7	Cobicistat	NA	Hetero
8	Elvitegravir	NA	Hetero

Table3: system suitability

System suitability Parameters	Cobicistat	Elvitegravir
% RSD for six replicates injections of standard	0.81	0.58
Tailing factor	1.43	1.63
Theoretical plates	3794	10726
Resolution	-	14.20

System suitability:

Fig.4 Chromatogram for system suitability

ASSAY OF STRIBILD:

Results of Cobicistat and Elvitegravir in marketed product:

Fig.5 Chromatogram for Assay of marketed sample

Table4: Assay results of marketed samples

Marketed formulation	Drug	% Assay
STRIBILD	Cobicistat- 150 mg	99.62
STRIBILD	Elvitegravir- 150mg	100.198

Method validation:

Linearity:

Six different concentrations were prepared ranging from 2.52-25.22 µg/mL of Cobicistat and 2.45-24.55 µg/mL of Elvitegravir and chromatograms were recorded for same.

Linearity graph of Elvitegravir

Linearity graph of Cobicistat

Table5: Linearity table

Parameters	Cobicistat	Elvitegravir
Slope	31110.2481	120544.3938
Intercept	-20949.3008	-1008295619
Correlation coefficient	0.9998	0.9996

Accuracy and Precision of Cobicistat:

Table 6: Accuracy and Precision of Cobicistat:

	Nominal Concentration (µg/mL)
	6.31	12.61	18.92
DAY 1
MEAN (n=6)	101.43	100.97	99.99
SD	0.76	0.47	1.17
% CV	0.75	0.47	1.17
DAY 2
MEAN (n=6)	101.40	100.93	99.96
SD	0.74	0.45	1.15
% CV	0.73	0.46	1.15
DAY 3
MEAN (n=6)	100.43	100.95	99.98
SD	0.75	0.46	1.16
% CV	0.75	0.45	1.16

Accuracy and Precision of Elvitegravir:

Table 7: Accuracy and Precision of Elvitegravir:

	Nominal Concentration (µg/mL)
	5.96	12.27	17.89
DAY 1
MEAN (n=6)	102.26	98.32	98.52
SD	0.07	0.32	0.04
% CV	0.07	0.32	0.04
DAY 2
MEAN (n=6)	102.23	98.30	98.50
SD	0.05	0.30	0.02
% CV	0.05	0.30	0.02
DAY 3
MEAN (n=6)	102.25	98.31	98.51
SD	0.06	0.31	0.03
% CV	0.06	0.31	0.03

Specificity:

The specificity of the analytical method was indicated in fig 6 where the retention time of Cobicistat does not interfere with the retention time of the Elvitegravir.

Fig:6 Specificity

Robustness:

Table8: Robustness Results

Drug Name	Para meters	Variation	Rt	Tailing factor	Plate count
Cobi cistat	Flow rate	0.9 mL/min	2.39	1.45	4021.00
	Flow rate	1.1mL/min	1.92	1.46	3710.00
	Mobile phase	80% organic phase	1.87	2.09	3523.00
Elvi tegravir	Mobile phase	70% organic phase	2.83	1.69	6472.50
	Flow rate	0.9 mL/min	4.40	1.61	11355.33
	Flow rate	1.1mL/min	3.50	1.61	10186.00
	Mobile phase	80% organic phase	2.84	1.71	9399.00
	Mobile phase	70% organic phase	6.01	1.63	11579.50

Ruggedness:

Table9: Ruggedness Results

ID	Drug name	Rt	Tailing factor	Theoretical plates	Reso lution
Analyst 1	Cobicistat	2.13	1.41	3919.33	0.00
Analyst 1	Elvitegravir	3.86	1.62	10820.00	7.97
Analyst 2	Cobicistat	2.15	1.72	5968.00	0.00
Analyst 2	Elvitegravir	3.85	1.65	10645.67	7.84

LOD and LOQ

Table10: Results LOD and LOQ

Drug name	Para meter	Peak area	Tailing factor	Theoretical plates
Cobicistat	LOD	24428.67	1.69	6359.67
Cobicistat	LOQ	44682.67	1.73	6091.33
Elvitegravir	LOD	49082.00	1.64	11108.33
Elvitegravir	LOQ	101903.00	1.63	10916.00

Stability studies:

Table11: Results stability studies

S. No.	Sample ID	Drug Sample	RT	Peak Area	Theore tical Plates	Tailing Factor
1.	Fresh Sample	Cobi cisat	2.15	386160	3794	1.46
1.	Fresh Sample	Elvi tegravir	3.92	1791413	10507	1.62
2.	Stability Sample	Cobi cisat	2.14	504262	4118	1.34
2.	Stability Sample	Elvi tegravir	3.90	1714870	10599	1.61

Forced degradation studies:

Table 12: Results of Forced Degradation Studies:

S.NO.	Degradation Studies	Cobicistat % Stability	Elvitegravir % Stability
1.	Oxidation	96.58	97.95
2.	Photolytic	94.92	98.47
3.	Acid	-	98.90
4.	Alkaline	94.05	-

Summary:

The summary of the analytical method is tabulated in below

Table: 13: Summary

Columm		AZILENT ZORBAX C18 column (150 X 4.6 mm id, ODS 2.5μm)
Mobile phase		Methanol and OPA (Ortho phosphoric Acid) Buffer in the ratio of 75:25
Injection volume		Injection volume of 20 μl
flow rate		1 ml/min.
Detector wave length		265 nm.
Cobicistat	Retention time	2.11
	Linearity	2.52-25.22 μg/mL
	Slope	31110.2481
	Intercept	20949.3008
	correlation coefficient	0.9998
Elvitegravir	Retention time	3.93
	Linearity	2.45-24.55 μg/mL.
	Slope	20544.3938,
	Intercept	-100829.5619
	correlation coefficient	0.9996
System suitability
Cobicistat	%RSD for six replicates injections of standard	0.81
	Tailing factor	1.43
	Theoretical plates	3794
	Resolution	-
Elvitegravir	%RSD for six replicates injections of standard	0.58
	Tailing factor	1.63
	Theoretical plates	10726
	Resolution	14.20

CONCLUSION:

The RP-HPLC method development was done for Cobicistat and Elvitegravir using Mobile phase A and B containing Methanol and Buffer (Ortho phosphoric Acid) in the ratio of 75:25 and detection was performed at 265nm with a retention time of 2.11 min and 3.93 min. The method was validated for all validation parameters as per ICH guidelines. The linearity values of given method with respect to r2 value is 0.999, found was within acceptable limits. The % RSD for Intra and Inter day precision was < 2%. The accuracy of method was validated by recovery studies and was found to be significant and under specification limits, within acceptable range 98-102%.

ACKNOWLEDGEMENT:

The author thankful to Dr. SVUM Prasad, Program Director School of Pharmacy, JNTUK for provide all the facilities and supports for accomplishment and completion of this research work.

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Received on 08.04.2017 Accepted on 12.07.2017

Asian J. Pharm. Ana. 2017; 7(3): 151-158.

DOI: 10.5958/2231-5675.2017.00024.2