Simultaneous Quantification of Sofosbuvir & Ledipasvir in Bulk and Tablet Dosage by Validated RP-HPLC Method

The chemical name is Isopropyl (2S)-2-[[[(2R, 3R, 4R, 5R) -5-(2, 4-dioxopyrimidin-1-yl) -4-fluoro-3- hydroxy-4-methylfuran-2-l] methoxy phenoxyphosphoryl] amino] propionate. It have a molecular  formula  of  C22H29FN3O9P  and  a molecular weight of 529.453gm/mol. Generously soluble in methanol, dimethyl form amide (DMF) and a little soluble in water. It has the structural formula shown in [Figure No.1].

Ledipasvir

The  chemical  name  is  (2S)-1-[(6S)-6-[5-(9,  9 difluoro-7-{2-[(1R, 3S, 4S)-2-[(2S)-2- {[hydroxyl (methoxy)        methylidene]  amino}-3- methylbutanoyl]-2-azabicyclo [2.2.1] heptan-3-yl]- 1H-1, 3-benzodiazol-6-yl}-9H-fluoren-2-yl)-1H- imidazole-2-yl]-5-azaspiro[2.4] heptan -5-yl] -2-{[hydroxyl methoxy) methylidene] amino} -3- methylbutan-1-one. It has a molecular formula of C49H54F2N8O6 and molecular weight of 889.00gm/mol. Liberally soluble in methanol, dimethylformamide (DMF) and to some extent soluble in water. It has the structural formula shown in [Figure No.2].

The literature survey reveals that there are a very few HPLC3-7 methods available for the determination individual Sofosbuvir and Ledipasvir in bulk and dosage forms. There were few analytical methods reported for simultaneous estimation Sofosbuvir and Ledipasvir in bulk and their combined dosage forms8-11. Hence an author made an attempt to develop specific, sensitive, accurate and precise RP-HPLC method for simultaneous estimation of these drugs with isocratic elution mode.

MATERIAL AND METHODS

Instrumentation

HPLC experiment is carry out on a Waters Alliance 2690 separation module, with waters 2996 photodiode array detector using Auto sampler. Data gathering and processing was prepared using EMPOWER PDA 2 software. The analytical column used for the separation was Inertsil- C18 ODS, 250mm 4.6mm, 5mm Column, Other equipments used were sonicator (FAST CLEAN), Electronic Balance (SARTORIOUS).

Preparation of solutions Mobile phase

Mix Degassed Methanol and Water in the ratio of 60:40v/v. Filter through 0.45m. Membrane filter paper.

Diluents

Mobile phase is used as diluents.

Preparation of stock solution

Reference solution

The solution was prepared by dissolving 20.0mg of accurately weighed Ledipasvir and 25.0mg Sofosbuvir in Mobile phase, in two 100.0mL volumetric flasks separately and sonicate for 20min. From the above solutions take 10.0mL from each solution into a 50.0mL volumetric flask and then makeup with mobile phase and sonicate for 10min.

Preparation of working standard solution:

The stock solutions equivalent to 20ppm to 80ppm with respect to both drugs were prepared in combination of Ledipasvir and Sofosbuvir above, sonicated and filtered through 0.45µ membrane.

Chromatographic conditions

HPLC experiment is carry out on a Waters Alliance 2690  separation  module,  with  waters  2996 photodiode array detector using Auto sampler. Data gathering and processing was prepared using EMPOWER PDA 2 software. The analytical column used for the separation was Inertsil-C18, 250mm, 4.6mm, 5mm Column, BDS Column. Other equipments used were sonicator (FAST CLEAN), Electronic balance (SARTORIOUS).

Selection of wavelength

Absorbance maxima of Sofosbuvir and Ledipasvir were detected at 227nm (λ1) and 264nm (λ2), respectively. Both the spectra’s were overlapped at 254nm. Both the drugs showed linearity with absorbance in the range 20-80μg/ml and 20- 80μg/ml respectively, when measured at 227nm and 264nm. Calibration curves were plotted from the absorbance values at these wavelengths.

METHOD DEVELOPMENT

To saturate the column, mobile phase was pump for about 20min thus to get the base line corrected. Standard calibration lines were constructed for each drug. A series of aliquots were prepared from the above stock solutions using diluents to get the concentrations 20- 80µg/ml for Ledipasvir, 20- 80µg/ml for Sofosbuvir. Inject each concentration 6 times in to the chromato-graphic system. Every time peak area and retention time were recorded separately for both the drugs. Calibration curves are constructing as by taking average peak area on Y- axis and concentration on X-axis individually for both drugs. Regression equations were calculated from the calibration curves, these regression equations is used to calculate drug substance in formulation.

Estimation of Sofosbuvir and Ledipasvir in tablet dosage forms

20 tablets were taken and their average weight was determined, they were crushed to fine powder. The powder equivalent to 20mg Sofosbuvir and 5mg Ledipasvir was weighed and dissolved in 20ml of mobile phase with the aid of ultra-sonication for 20min. The content was diluted to 50 with mobile phase to furnish a stock test solution. The stock solution is filtered through a 0.5µm nylon syringe filter and 10ml of filtrate was diluted in to a 50ml volumetric flask to give a test solution containing 80µg/ml Sofosbuvir and 20µg/ml Ledipasvir. This solution was estimated by above developed method. The assay procedure was repeated 6 times (n=6) the drug content was estimated using above calculated regression equation.

MEHOD VALIDATION

The described method has been validated for linearity, accuracy, limit of detection, precision, and robustness, as per the ICH guidelines.

Linearity

The linearity of the method was determined by preparing six different concentrations of both Ledipasvir and Sofosbuvir in the concentration range of 20- 80µg/ml and 20-80 µg/ml. Each solution was prepared in triplicate. The calibration curves were obtained by plotting peak area versus concentration. Linearity was check over the same concentration range on three successive days and the results obtained.

Accuracy, as recovery

The accuracy of the method was determined at three different concentration levels 50%, 100%, and 150% by spike known quantities of the drug analyte and % of recovery were calculated.

Precision

Method precision (repeatability)

The method precision is determined by inject six working standard solutions and six sample injections. The areas of all the injections were taken and standard deviation, % relative standard deviation (RSD), % assay were calculated.

Intermediate Precision

The intermediate precision was determined by inject six working standard solutions and six sample injections on different days by different operator or by different instruments. The areas of all the injections were taken and standard deviation, % relative standard deviation (RSD), % assay was calculated.

LOD and LOQ

LOD

It is lowest amount of analyte in a sample that can be detected but not necessarily quantified as an exact value under the stated experimental conclusions.  The  detection  limit  is  usually expressed as the concentration of analyte. The standard deviation and response of the slope and the results obtained.

LOQ

The Quantitation limit of an analytical procedure is the lowest amount of an analyte of a sample which can be quantitatively determined with suitable precision and accuracy. The standard deviation and response of the slope and the results obtained.

System suitability parameters

For assessing system suitability, six replicates of working standards samples of Ledipasvir and Sofosbuvir were injected and studied the parameters like plate number(N), tailing factor (K),re-solution, relative retention time and peak asymmetry of samples.

Robustness

The robustness of the assay method was established by introducing small changes in the chromatographic condition which included wavelength (225nm - 229nm) flow rate (1.0 and 1.2mL/min) and organic phase (+5% to -5%).

Specificity and selectivity

Specificity is the degree to which the procedure applies to a single analyte and is checked in each analysis by examining blank matrix samples for any interfering peaks. The specificity of the method was evaluated with regard to interference due to presence of any other placebos. Two different samples were injected and studied with respective placebos. The HPLC chromatograms recorded for the drug matrix (mixture of the drug and placebos) showed almost no interfering peaks with in retention time ranges. The obtained figures shows that the selected drugs were cleanly separated. Thus, the HPLC method proposed in this study was selective.

RESULTS AND DISCUSSION

Optimized chromatographic conditions

Most of all reported HPLC methods till date use C- 8 or C-18 columns. Most of these uses Complex mobile phase compositions. Hence, attempts were directed towards development of a Simple and better method on commonly used Inertsil- C18, BDS column with good resolution. Different logical Modifications were tried to get good separation among the drugs and the degraded products. These changes included change in mobile phase composition in isocratic elution as well as gradient modes on different HPLC columns. The optimized chromatographic conditions Figure No.3. The best peak shape and maximum separation was achieved with mobile phase composition of Methanol and Water (60:40), peak symmetry and reproducibility were obtained on inertsil-C18, 250mm 4.6mm, 5mm Column. The optimum wave-length for detecting the analyte was found to be 254nm, a flow rate of 1.0ml/min yielded optimum separation and peak symmetry. Chromatogram of Sofosbuvir and Ledipasvir (Figure No.3) and optimized chromatographic condition is shown in (Table No.1).

Accuracy and Precision

Accuracy as recovery was evaluated by spiking previously analyzed test solution with additional Placebo at three different concentration levels (Table No.4a, b). Recovery of previously analyzed test solution drug concentration added was found to be 99.9% for Sofosbuvir and 100.06% for Ledipasvir with the value of RSD less than 1% indicating that the proposed method is accurate for the simultaneous estimation of both drugs from their combination drug products in presence of their degradation products. The low RSD values indicate the repeatability and reproducibility of the Method (Table No.5a, b).

Linearity, LOD and LOQ

The calibration plot was linear over the concentration range investigated (20-80µg/ml) for Ledipasvir, (20-80µg/ml) for Sofosbuvir respectively. Average correlation coefficient R2¼ 0.999 for both the drugs with %RSD values 2.0 across the concentration ranges studied, was obtained from regression analysis. The LOD that produced the requisite precision and accuracy was found to be 0.32mg/ml Sofosbuvir and 0.33mg/ml Ledipasvir drug. The resultant %RSD values were 1.00% (Table No.3a, b, c). The LOQ for Sofosbuvir and Ledipasvir were found to be 0.98mg/ml and 1.01mg/ml respectively. The Regression results indicate that method was linear in the concentration range studied and can be used for detection and quantification of Sofosbuvir and Ledipasvir in very wide concentration range for Sofosbuvir and Ledipasvir respectively. (Figure No.4a, b).

Specficity and selectivity

Specificity is checked in each analysis by examining blank and placebo samples for any interfering peaks. The specificity of the method was evaluated with regard to interference due to presence of any other excipient.

Robustness

The elution order and resolution for all components were not significantly affected. RSD of peak areas were found to be well within the limit of 2.0%.

System suitability

Parameters calculated for system suitability was a number of theoretical plates, tailing factor, resolution, retention time, and area. Results are shown in (Table No.6).

Assay of the tablet dosage form

(Sofosbuvir 400mg/tab and Ledipasvir 90mg/tab) The proposed validated method was successfully applied to determine Sofosbuvir and Ledipasvir in tablet dosage form. The result obtained for Sofosbuvir and Ledipasvir were comparable with corresponding labeled amounts (Table No.2).

Table No.1: Optimized chromatographic conditions

Table No.2: Results for marketed Formulation analysis

Table No.3a: Linearity of Ledipasvir and Sofosbuvir

Table No.3b: Statistical Analysis

Table No.3c: LOQ AND LOD

Table No.4a: Accuracy for Sofosbuvir

Table No.4b: Accuracy for Ledipasvir

Table No.5a: Method Precision of Sofosbuvir

Table No.5b: Method Precision of Ledispavir

Table No.6: System Suitability Parameters

Figure No.1: Chemical structure of Sofosbuvir

Figure No.2: Chemical structure of Ledipasvir

Figure No.3: A typical chromatogram of Sofosbivir and Ledipasvir

Figure No.4: UV Spectrum of Sofosbivir and Ledipasvir

Figure No.4a: Linearity plot for Ledispavir

Figure No.4b: Linearity plot for Sofosbuvir