Concurrent Determination of Daclatasvir and Sofosbuvir in Pure Binary Mixture and Their Combined Film Coated Tablets by a Simple Stability Indicating RP-HPLC Method

Ramreddy Godela^1,2*, Sowjanya G¹

¹GITAM Institute of Pharmacy, GITAM Deemed to be University, Visakhapatnam, India, 530045.

²Bhaskar Pharmacy College, Hyderabad, Rangareddy, India, 500075.

*Corresponding Author E-mail: ramreddy.godela@yahoo.com

ABSTRACT:

A trouble-free, simple, specific and highly sensitive stability indicating phase HPLC method was developed for concurrent assessment of Daclatasvir  and Sofosbuvir in pure and in their combined tablet formulation. An effectual separation was accomplished by using XDB Phenyl (250 x 4.6mm, 5µ,100 A⁰) column, mobile phase composition of Acetonitrile: buffer(0.1%v/v Trifluoroaceticacid in water) (50:50 v/v) and isocratic elution at a flow rate of 1ml/min and detection wavelength of 275nm. The extreme stress conditions like hydrolysis with acid and base, peroxide oxidation, thermal decomposition were used as per ICH specifications to assess the stability of the analytes in bulk and dosage forms. The retention times of Daclatasvir and Sofosbuvir were found at 2.8 and 3.7min respectively. The proposed method has linear response in the concentration ranges from 12 to 36µg/ml and 80 to 240 µg/ml for Daclatasvir and Sofosbuvir respectively. The detection and quantification limits calculated as 2.5μg/ml and 7.8μg/ml for DCL, 5.2μg/ml and 15.8μg/ml SOF respectively. All the method validation parameters were met the acceptance limits of Q2 specifications of ICH procedures. The degradation products produced by forced degradation studies were have good resolution from Daclatasir and Sofosbuvir peaks, which represents the methods stability. The proposed RP-HPLC method was highly sensitive, precise, stability indicating and economical. That’s why the method has the capacity to employ in the pharmaceutical manufacturing of Daclatasvir and Sofosbuvir and routine analysis in quality control department.

KEYWORDS: Daclatasvir, Sofosbuvir, Stability indicating, Phenyl column, Isocratic elution.

INTRODUCTION:

The multiple drug combination therapy is an effective, competent and great progression in the treatment of the chronic disease such as chronic hepatitis-C and human immune virus infections. The two drug combination of Daclatasvir  (DCL) and Sofosbuvir (SOF) is proficient in the treatment of chronic hepatitis C which causes inflammation of the liver leads to  hepatoma or hepatocellular carcinoma^1-3. Chemically DCL is methyl N-[(2S)-1-[(2S)-2-[5-[4-[4-[2-[(2S)-1-[(2S)-2-(methoxycarbonylamino)-3-methylbutanoyl]pyrrolidin-2-yl]-1H-imidazol-5-yl]phenyl]phenyl]-1H-imidazol-2-yl]pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-yl]carbamate ⁴.Which inhibits NS5A, a non-structural protein mainly involved in replication of viral genome ^5,6.

SOF, chemically propan-2-yl (2S)-2-[[[(2R,3R,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyloxolan-2-yl]methoxy-phenoxyphosphoryl]amino]propanoate^7,8. It is a prodrug and analog of uridine nucleotide. 5^’ triphosphate nucleotide is an active moiety of SOF competitively inhibits the NS5B polymerase leads to ceases the viral RNA replication ^9-12. The chemical structures of DCL and SOF were mentioned in Figure 1.

A proficient analytical method is basic need for a drug substance to estimate alone or in combine with other drug substances concurrently. The broad literature investigation disclosed that a small number of analytical methods like UV, RP-HPLC methods were reported to estimate DCL and SOF alone and in combined tablet forms^{7, 13-18}. Besides RP-HPLC methods, bio analytical liquid chromatographic methods and LC-MS methods were described in literature for estimation of DCL and SOF^16,19–21. Along with above cited method few methods were reported with SOF in combination with other antiviral agents^22-27.  Till date a competent stability indicating RP-HPLC method was not on hand for the concurrent estimation of DCL and SOF in blended powder and combined tablet. In this regards, research attempts have been done to develop a competent, highly sensitive and economical RP-HPLC method for determination of the assay of DCL and SOF and for the assessment of the stability of DCL and SOF in blended powder and combined tablets concurrently. After the development of method, validation was done as denoted in Q2 guidelines of ICH.

Figure No: 1. Chemical structures of DCL and SOF

MATERIALS AND METHODS:

API form of DCL and SOF were procured from Fortune Pharma, Hyderabad. All HPLC grade and analytical grade solvents were gained from local distributor of Merck India limited. The method was done by using WATERS HPLC (2695model, PDA detector and Empower-2 software). In addition 1mg sensitive balance (SCALETEC-SAB224CL)), a digital pH meter (SMIS-PH-7000), water (Milli-Q) were used, prepared

Chromatographic method conditions:

An adequate and effective separation of the both DCL and SOF were achieved with XDB-Phenyl (250 x 4.6mm, 5µ) column, using a mobile phase composition of ACN: Buffer (0.1% Trifluoro acetic acid in water) (50:50 v/v) at a flow rate of 1ml/min and maximum absorption wavelength of 275nm. Ambient temperature was used in both injection port and column. Water and ACN in 50:50 ratio used as diluent.

Preparation of standard solution:

24mg of DCL and 160mg of SOF API powders were weighed and transferred into 100 ml volumetric flask, volume made with diluent to 100mL. 1ml of above solution was diluted 10 ml to obtain concentration of 24µg/ml, 160µg/ml for DCL and SOF respectively represented as 100% level concentrations.

Preparation of sample solution:

The tablet (Hepcinat Plus) powder equivalent to 24 mg of DCL and 160mg of SOF was weighed and dissolved with diluents to 100ml. 1ml of above solution was further diluted to 10 ml obtain concentration of 24µg/ml, 160µg/ml for DCL and SOF respectively. Sample solution was filtered by using 0.4 µm Nylon filter before injecting.

Method validation:

Validation is printed evidence which furnishes reasonable and high degree level assurance about method or process. Analytical methods are validated in accordance with Q2 specification of the ICH guidelines.

System suitability Test:

The system suitability of the method was performed by injecting 100% level solution in 6 subsequent successive injections and further evaluation of the parameters such as theoretical plates (N), percentage relative standard deviation (%RSD) and tailing factors (T) was done.

Linearity:

The linearity of an analytical method represents the proportional relation between the experiential results and the mentioned concentrations. It was performed for the concentrations ranges from 12µg/ml to 36µg/ml and 80µg/ml to 240µg/ml of DCL and SOF respectively. A liner plot was drawn between concentration and peak area   to calculate the regression coefficient (R²).

Precision:

Close proximity between the experimental values of the homogeneous sample on more than one consecutive samplings called as precision. In common it has to be performed within the same day (intraday) and repeated in three successive days. The % RSD values of observed peak areas, and retention time (RT) were calculated.

Accuracy:

To a large extent the % recovery method can be followed to find out the accuracy of the analytical procedures. In which specified amount of sample solution is spiked in to standard solution at 50, 100, and 150% levels of standard concentration. The % mean recovery added amount was computed at each % level.

Specificity:

It is the capacity of an analytical method to determine the intended drug substance in the existence of other compounds without any interference. Around10µl of of blank solution, standard, placebo and standard spiked with placebo solutions were separately injected into HPLC. In depth observation was done to check the interference of other peaks with the intended analyte peak.

Sensitivity:

The LOD and LOQ were determined by using standard deviation method.

LOD=3σ/S

               LOQ = 10 σ/S

Where, σ is the standard deviation of the intercept

S is the slope of the linear curve

Robustness:

Robustness of the method was evaluated by intentionally altering the optimized method conditions vaguely. The parameters like flow rate (± 0.1 ml/min), mobile phase ratio (± 1ml), and detection wavelength (± 2nm) were altered vaguely and intentionally.

Forced degradation studies:

In the forced degradation (FD) studies consciously drug product or substance was exposed to more stress than accelerated stability conditions. The FD studies provide the chemical stability of the drug and to produce a stable formulation with appropriate storage conditions. ICH regulatory guidelines are significantly mentioned some degradation conditions like acid hydrolysis and base hydrolysis, oxidative degradation, photo and thermal degradation etc. To perform acid, base hydrolysis and oxidative degradation separate thee 10ml portions of standard stock solution was mixed with 2ml of 0.1N HCl, 2ml of 0.1N NaOH and 2ml ml of 3% hydrogen peroxide respectively, reflux the produced solutions at 70 C for 2hr and kept those aside for 24hr. Further dilutions have been done to all the three solutions to obtain a concentration o about 24µg/ml of DCL and 160µg/ml of SOF respectively. Thermal degradation was done by keeping the standard stock solution in a hot air oven at 80°C/75% RH for 24hr. 1ml of the above exposed solution further diluted to get a concentration of 24µg/ml and 160µg/ml for DCL and SOF respectively. The degradation solutions have been introduced into HPLC system and calculate the percentage degradation of DCL and SOF. As stated by regulatory guidelines upto 20% degradation of the drug substance can be advised as the most effectual and suitable for the validation of stability- indicating HPLC method.

Assay:

The percentage purities of DCL and SOF the in commercial tablet were estimated by injecting successive injections of standard and sample solution.

RESULTS AND DISCUSSION:

The method development was started with solubility studies of the both DCL and SOF. It has been found that DCL was freely soluble in water, ACN and methanol on other hand SOF was freely soluble in water, ACN but slightly soluble in methanol. Based on the solubility of DCL and SOF, water and ACN in 50:50 ratios selected as diluent.

Figure No: 2. Chromatogram of the optimized method

Table 1. Results of system suitability parameters of standard solution

	DCL				SOF
Injection	RT	Peak area	Plate count	Tailing factor	RT	Peak area	Plate count	tailing factor
1	2.805	515911	8379	1.12	3.732	951078	12545	1.08
2	2.811	515450	8515	1.09	3.736	956132	12584	1.09
3	2.805	519435	8412	1.12	3.734	952200	12368	1.08
4	2.806	514412	8429	1.12	3.733	953459	12400	1.08
5	2.806	514972	8456	1.12	3.733	958399	12393	1.08
6	2.811	515450	8515	1.09	3.736	956132	12584	1.09
MEAN	2.807	515938.3	8451	1.11	3.734	954566.7	12479	1.083
SD	0.0028	1787.05	55.5	0.015	0.001	2777.99	102.336	0.005
%RSD	0.1	0.34	0.65	1.39	0.04	0.29	0.82	0.47

SD, Standard Deviation; %RSD, Relative Standard Deviation

Method optimization:

Method optimization was done with trial and error method. After numerous trials a method with XDB-Phenyl (250 x 4.6mm, 5µ) column, using a mobile phase ratio of ACN: Buffer (0.1% Trifluoro acetic acid in water) (50:50 v/v) at a flow rate of 1ml/min and absorption wavelength of 275nm preferred as optimized conditions. The chromatogram of the optimized method shown in Figure 2.

Method validation:

System suitability:

The experimental data attained by injecting standard solution in six consecutive injections satisfying the acceptance limits of parameters such as % RSD (≤ 2), tailing factor (≤ 2), and number of plate (> 2000). The results were illustrated in table-1.

Linearity:

The method has considerable linearity for DCL and SOF were in the range of 12 to 36 µg/ml 80 to 240 µg/ml determined by linearity curve plotted between concentration and peak area (Figure 3). The statistically obtained R² values for both the drugs were 0.999, which were satisfied the acceptance limit.

Figure No: 3. Linearity curve of DCL and SOF

Table 2. Results of percentage recovery

	DCL
% Level	Amount added (µg/ml)	Amount recovered (µg/ml)	% recovery	% mean recovery
50	12	12.024	100.2	99.7
	12	11.988	99.9
	12	11.88	99
100	24	23.784	99.1	99.1
	24	23.736	98.9
	24	23.856	99.4
150	36	35.928	99.8	99.7
	36	35.856	99.6
	36	35.892	99.7
SOF
50	80	79.68	99.6	99.6
	80	79.2	99
	80	80.16	100.2
100	160	159.2	99.5	99.6
	160	159.68	99.8
	160	159.52	99.7
150	240	239.28	99.7	99.4
	240	239.04	99.6
	240	237.6	99

The percentage recovery at each percentage level within the acceptable limit

Precision:

The %RSD values of the peak responses thus attained by injecting standard solution in five successive replicates were ≤ 2. The %RSD of the peak areas of the SOF and DCL found to be 0.04 and 0.03 respectively for intra-day and 0.08 and 0.04 for inter day precision respectively.

Sensitivity:

The LOD and LOQ concentrations were calculated as 2.5μg/ml and 7.8μg/ml for DCL, 5.2μg/ml and 15.8μg/ml SOF respectively.

Robustness:

Deliberate changes in mobile phase ratio, flow rate and absorption wavelength of the optimized method to little extent could not influence the system suitability parameters results which were attained inside the acceptance limit only (Table 3), those results indicates the methods robustness.

Forced degradation:

In majority of the stability-indicating analytical methods, up to 20% degradation in the drug substance is considered by scientists. The percentage degradation of drug substance was assessed by comparing the peak areas produced by pure standard solution and stressed standard solution.

Table 3. Results of robustness of standard solution

Parameter		DCL				SOF
		RT	Peak area	Plate count	Tailing factor	RT	Peak area	Plate count	Tailing factor
Mobile phase ratio (Buffer: ACN)	49:51	2.807	510589	8536	1.12	3.735	950886	12481	1.08
	51: 49	2.805	505388	8443	1.11	3.733	950290	12332	1.08
Flow rate (±0.1ml)	0.9	3.10	520621	8321	1.09	4.6	935649	12385	1.11
	1.1	2.349	492310	7169	1.08	3.12	935649	10723	1.09
Maximum wavelength	273	2.806	514635	8429	1.12	3.733	953459	12400	1.08
	277	2.806	515071	8456	1.12	3.733	958399	12393	1.08

%RSD, Relative Standard Deviation Time; slight change in method parameter could not influence the USP plate count and tailing factor

The attained results were shown in the Table 4. Acid hydrolysis chromatograms mentioned in Figure 4. Both the analytes were highly stable at mention thermal degradation conditions

Table 4. Results of forced degradation studies

Type of degradation	% Degradation
	DCL	SOF
Acid hydrolysis	9.8	15.6
Base hydrolysis	16.4	51.2
Oxidation	7.0	2
Thermal degradation	0.6	1.2

Percentage degradation was less than 20% in most of the different stress conditions

Assay:

The percentage purity of the DCL and SOF in the film coated tablets form were in the limit of 100%±15 (Table 5).

In the past several RP-HPLC were developed for DCL and SOF in combined tablet form. But single RP-HPLC method with simple mobile phase and fewer elution times was not existed. Therefore attempts have been made to develop a method with simple mobile phase and less RT. The RT described for DCL and SOF were less than the earlier mentioned method which is advised as economical, as it lowers the elution time and consumption of mobile phase. Hence sample analysis time was lowered and more number of samples can be tested in the stipulated time.

Figure No: 4. Chromatograms different forced degradation studies

Table  5. Results of % assay of the tablet dosage form

Drug	Peak name	Retention time	Peak Area	USP Tailing	USP Plate count	%Assay
DCL	Standard	2.807	511279	1.12	8579	99.6
	Test	2.807	505209	1.11	8612
SOF	Standard	3.736	954657	1.08	12651	99.5
	Test	3.732	952165	1.08	12157
Average weight of tablet-620g, Label claim Daclatasir- 60 mg, Sofosbuvir-400mg

CONCLUSION:

An uncomplicated, unambiguous, perceptive, accurate, sensitive, precise and isocratic RP HPLC method was developed to estimate DCL and SOF simultaneously in bulk blended powder and their combined tablets. Application extreme and deliberate forced degradation conditions to the analytes represent the stability of the method.  The proposed method was effectively separate DCL, SOF and their degradants with excellent resolution and high sensitivity. The currently established method has god stability-indicating, considerable specificity and sensitivity. Therefore, the proposed method has substantial implementation in the analytical research division in the pharmaceutical industrial field.

ACKNOWLEDGMENT:

The authors are thankful to Fortune pharma, Hyderabad for providing lab facilities and chemicals. The authors are also thankful to department of Pharmacy, GITAM University, Vizag, Andhra Pradesh for encouragement.

AUTHORS’ CONTRIBUTIONS:

All the authors contributed equally in design and frame of the work, acquisition and interpretation of data and manuscript preparation, all authors have read the prepared manuscript and approved for the publication.

CONFLICT OF INTEREST:

No conflict of interest from all the authors.

LIST OF ABBREVIATIONS:

DCL: Daclatasir 

SOF:  Sofosbuvir

RT: Retention Time

LOD: Limit of Detection

LOQ: Limit of Quamtification

ICH: International Conference on Harmonisation of Technical Requirements For Registration of Pharmaceuticals for Human Use

SD : Standard Deviation

RSD: Relative Standard Deviation

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Received on 30.08.2020            Modified on 13.10.2020

Accepted on 07.12.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(11):5913-5918.

DOI: 10.52711/0974-360X.2021.01028