Stroke is the second most life-threatening disease annually; as 15 million people suffer worldwide due to stroke as per the American Stroke Organization. According to the World Health Organization, Ischemic stroke is a major cause of death over hemorrhagic stroke as it happens to lack blood flow to the brain1. Treatment often used for stroke is thrombolytic agents, antiplatelet agents, and blood thinners whereas the tissue plasminogen activator is the majorly administering thrombolytic agent2,3. Anticoagulant therapy is the most promising therapy for the management of ischemic stroke as there is recurrent rate of stroke is also higher. CPD is an approved antiplatelet agent that prevent platelets from clinging and forming a clot3. CPD belongs to the thienopyridine class of drugs that hinder the antiplatelet P2Y12 adenosine 5-phosphate receptor and are used to restrain blood clots. By blocking the glycoprotein IIb/IIIa pathway block this receptor results in the inhibition of platelet aggregation4. The major challenge to treat Cerebral stroke is the blood-brain barrier5. To overcome that challenge clopidogrel can be potentially administered via nasal route to avoid the BBB6 and so, it is necessary to estimate drug concentration in NSF for in vitro estimation7. The literature survey uncovered the method for estimation of CPD in NSF in previous data access through various information sources which gives an idea to develop and validate the method for analysis of CPD in NSF using the HPLC method.

Figure1: Structure of Clopidogrel Bisulphate

A QbD is well defined as "A systemic approach to the method development that begins with predefined objectives and emphasizes product⁸ and process understanding and process control, based on sound science and quality risk management ⁹”. QbD approach towards the development of the method helps to increase the cost-effectiveness and robustness of the method which leads to an emphasis on the reproduction of the method. The aim of present investigation is developed and validate HPLC method for CPD in NSF by using QbD approach ¹⁰.

Material

CPD was obtained as a gratis sample from Dr Reddy’s Laboratory, Buddi, Himachal Pradesh, India. All other Chemicals used were of analytical grade. Purified water was of Mili-Q 3 U.V. Acetonitrile was of HPLC grade and other chemicals were of analytical grade.

Method

Instrumentation

The instruments immersed for the method development were SHIMADZU/DGU/20/A5R with SHIMADZU 1800 UV-Spectrophotometer and Hypersil C-18 Column having length 4.6× 250 mm. Data analysis was done by the software Chemstation version 10.04.

Working solution preparation

10 mg of accurately weighed quantity of CPD was transferred to a 10 ml volumetric flask containing acetonitrile and NSF in the ratio 65:35 and dissolved into it. The standard working solution having distinct concentrations was prepared by using a standard working solution with a mobile phase.

Preparation of NSF.

In 1 Liter of purified water, accurately weighed quantities of sodium chloride, calcium chloride, and potassium chloride were evanesced to produce NSF. Triethanol amine was used to adapt pH 6.8 ¹¹.

Selection of maximum wavelength

The drug solution in a simulated nasal fluid having a concentration of ten µg/ml was scanned in the range of 200 to 400 nm using UV Spectrophotometer. The maximum wavelength obtained was 242 nm in NSF.

Chromatographic condition

The column Hypersil C-18 was introduced for method development with acetonitrile: NSF (55:45), having PH of NSF 6.8, as a mobile phase having flow rate of 1 ml/min having loop volume 20µl at ambient temperature with detection wavelength was 242 nm. A satisfactory peak with separation and symmetry for the pure drug was obtained with the mentioned Chromatographic conditions. CCD was put in an application to develop the design HPLC method using the parameters ratio of acetonitrile: NSF and PH of NSF.

Figure 2: Maximum wavelength of CPD in NSF using UV- Spectrophotometer

Method optimization

Resolution of the highest peak, separation of the peak of CPD, and optimization were earned with parameters like mobile phase solvents, composition of solvent, and pH of NSF.

Development of HPLC method by QbD approach

Quality Target Product Profile (QTPP) Parameter Selection

The effect of QTPP parameters can be greatly recognized by QTPP. Retention time, area of peak, and the theoretical plate were QTPP parameters identified for the proposed HPLC method.

Determination of Critical Quality Attributes (CQA).

The QTPP parameters were directly affected by CQA as the Ratio of acetonitrile and NSF pH of NSF are the critical quality attributes for the proposed method of HPLC.

Design of Experiment

The optimization of the proposed method of HPLC was carried out by applying CCD and independent parameters and dependent parameters were chosen for the Design. The various interactions and quadratic effects of the Ratio of acetonitrile and pH of NSF affect the retention time, area, and theoretical plates of the chromatogram. The 2 factors, the Ratio of acetonitrile and pH at 3 different levels were designed by CCD given by the Software design of the experiment (version 13). The quadratic or second-order equation is best befitted for the response surface.

Method validation

As per ICH guidelines parameters like linearity, accuracy, sensitivity, and stability are the parameters of validation of the developed method of HPLC for active pharmaceutical ingredients, CPD ¹².

Linearity

Linear regression analysis is used to check the linearity of the proposed method using different concentrations having a range of 2 to 10 µg/ml. The various dilutions like 2,4,6,8 and 10 µg/ml from stock solution were prepared using mobile phase. The graph was plotted as concentration versus absorbance and the linearity was calculated.

Accuracy 

The accuracy was determined by preparing variants of analyte separately like 50 %, 100 %, and 150% and the chromatogram was obtained for each.

Precision

The area of chromatograms was determined by HPLC by giving six injections of solutions. Relative deviation from the standard was calculated.

Robustness

A parameter like flow rate was used to check the changes. The changes were observed and reported¹³.

LOD and LOQ

The lowest limit of drug concentration can be accurately verified and distinguished by using a limit of detection and a limit of quantification. The formula used to calculate LOD and LOQ is

“LOD =¼ 3:3 σ/S

LOQ =¼ 10 σ/S

Where, σ = Standard Deviation

               S = Slope” ⁹

Assay

Accurately weighed and powdered marketed tablets of CPD were taken for the assay, out of that powder equivalent to 75 mg CPD was transferred to 100 ml of volumetric flask. By dissolving the weighed amount of powder into a small quantity of mobile phase, volume was made with the mobile phase up to 100 ml. The resultant solution was filtered through the Whatman filler paper (0.42µ) and dilution was prepared by using mobile phase as dilution media from 2 to 10 µl.

Degradation study

Force degradation study focused on a stability profiling of active pharmaceutical ingredient in pharmaceutical industry. Force degradation was studied using factors such as photodegradation, acid degradation, and thermal degradation. By heating a 10 ml volumetric flask holding a mobile phase containing 10 mg of medication to 80ºC in an oven, thermal degradation was examined. For the acid degradation investigation, a mobile phase containing 10 mg of medication was introduced to a 10 ml volumetric flask along with 2 ml of hydrochloric acid. Using UV light, a volumetric flask holding 10 ml of the drug in the mobile phase was utilized for the photodegradation investigation. The HPLC method was used to analyse the sample¹⁴.

Design by experiment

Initially different ratios like 50:50,80:20 and, 70:30 of acetonitrile to NSF were tried and the chromatogram was observed for the retention time. The best of the retention time given by the ratio were further selected for study and levels of the independent parameters were decided for the Experimental design. Improvement of peak shape and peak symmetry was tested by the adjustment of different PH and out of that the best-suited PH for the levels of independent parameters were selected for the Design of Experiment.

Table 1: Values of independent variables

Table 2: Design by design of experiments software

The optimized system suitability best satisfied the chromatographic conditions, as the ratio of acetonitrile to NSF as a mobile phase obtained by the design, was 65:35 and PH was 6.8 with the desirability of design 0.918.

Table 3: Solution for optimised batch

Figure 3: 3D Response surface graph for independent variables

Fig 4: Chromatogram of CPD in NSF

Linearity

The linearity assessment was done by obtaining a calibration curve by plotting concentration against area. The regression equation having coefficient was found to be y = 1171.8x-24.667 & 0.9971.

Figure 5: Linearity curve in acetonitrile: NSF (65:35)

Accuracy

The accuracy was calculated at three levels with different concentrations of the drug. % Recovery was calculated using peak area obtained in a chromatogram and it was found to be 99.80 %, 99.59%, and 99.73% for 5mg,10 mg, and 15 mg of drug respectively.

Table 4: Accuracy of optimized batch in acetonitrile: NSF (65:35) all value expressed in Mean ± S.D. where n=3

Precision

The precision of the developed method was determined by intermediate (Interday) and repeatability (Intraday) of evaluation. The % recovery for the intermediate evaluation was obtained at 98.34%, 98.98% and,99.73% for 5mg, 10mg and, 15 mg of drug concentration respectively. Whereas, repeated evaluation gives 99.82%, 99.36% and, 99.73% of % recovery for 5mg, 10mg and, 15 mg of drug concentration respectively.

Table 5: Intraday precision in acetonitrile: NSF (65:35) all value expressed in Mean ± S.D. where n=3

Table 6: Interday precision of optimized batch in acetonitrile: NSF (65:35), all value expressed in Mean ± S.D. where n=3

Robustness

Table 7: Robustness of optimized batch in acetonitrile: NSF (65:35), all value expressed in Mean ± S.D. where n=3

Limit of Detection and Limit of quantification

The limit of detection and limit of quantification of the optimized batch given by the quality by design of the software was detected at 0.47µg/ml and 0.77 µg/ml.   

Assay

The chromatogram of CPD obtained with conditions of the optimized batch by QbD Design gives a retention time of 4.491 when the assay is performed from the tablet. For the label claim of CPD, % the drug concentration by assay was estimated at 99.351% which indicates the ability of the developed method to carry out estimation in the presence of another component also.

Degradation Study

The effect of various stress conditions like degradation by acid and alkali, thermal degradation, and Photodegradation was considered on the stability of CPD by using NSF. The degradation of the drug in the presence of acid and alkali was 5.235% &28.013% respectively,15.346% degradation was seen thermally, and photodegradation in the presence of UV light was estimated at 8.236%. The observations in stress conditions state that CPD is unstable in stressed conditions as the changes in the tailing of peaks were observed but without disturbing the parent peak.

Table 8: Degradation study

Figure 6: Acid degradation of CPD

Figure 7: Alkali degradation of CPD

Figure 8: Photodegradation of CPD

Figure 9: Thermal degradation of CPD

The chromatographic method development for recognition and quantification of active pharmaceutical ingredient highly approachable towards quality control parameters. The analytical QbD approached HPLC method plays a crucial role in quality control which provide the optimised batch in development of method which is required towards accuracy of the method. Development of HPLC method in NSF is required for nasal administration of drugs as NSF mimics the nasal mucosa some extent which is important to evaluate the release profile of drug which administered by the nasal route of administration.¹⁵ As Previously QbD based HPLC method development for CPD was not reported and CPD is USFDA approved drug for some neurodegenerative diseases for which nasal administration of CPD is novel route of administration and for the nasal route administration of CPD development of method for HPLC is necessary tool for estimation of drug in nasal formulations¹⁶.QbD based or design or the approach towards the HPLC method development gives an idea to develop method with improvement of method and reduce the chances of failure of development of method. The method of HPLC was developed by using the QbD approach and validated with distinct parameters. QbD approach towards the development of HPLC method critically analyse the corelation between the process and the material attributes which get affect the quality attribute as it provides the absolute results. The parameters considered for analytical target product profiling were retention time, area, and theoretical plates which were affected by two parameters namely ratio of acetonitrile to NSF and PH of NSF ¹¹The critical parameters which affect the critical parameters were analysed by risk assessment study ¹⁷Design of experiment version 13.0 used to apply CCD design with two factors having three different levels. Hypersil C-18 column was used to optimize the HPLC method which gives the retention time 4.216, area 2694.866, and 14214.5 theoretical plates with 0.918 desirability for a ratio of acetonitrile: NSF was 65:35 and PH of NSF was 6.8. The linearity was checked by using different concentrations like 2 to 10 µg/ml giving the regression coefficient 0.9971 with regression equation y = 1171.8x-24.667. The method was precise as intraday and interday precision was below 2 percent when measured in terms of %RSD. Below 2 percent of %, RSD shows the robustness of the optimized batch of method development with %, recovery of 99.351%. The forced degradation was shown maximum in the presence of alkali than the acid, photo, and thermal degradation. So, the method was developed as per ICH conditions.

The analytical HPLC method by using QbD was developed to estimate CPD in NSF and validated by using different parameters. The emerging method was found ideal for selectivity and specificity in NSF with expressed chromatographic conditions. The solution is given by the Design of the experiment cast about components of the HPLC method like the ratio of mobile phase and the PH of NSF. CPD HPLC method development in NSF was extended by using QbD, to check the most suitable performing system with the final design space which has important variations in several components like a combination of different two components such as the ratio of mobile phase and the PH of NSF with three different levels. The CCD is used to study their interrelation and optimized at three different levels. The validation parameters of the developed method were found within an acceptance limit. The validated method was seen to be linear, accurate, precise, robust, specific, and stable CPD was found in NSF as no degradation was observed during the development and validation of the method.

Acknowledgment

We would like to express our sincere gratitude towards Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur for their valuable support and to provide research laboratory to carry out this project.

Funding

This research is funded by Mahatma Jyotiba Phule Research and Training Institute (MAHAJYOTI), Nagpur.

Author Contribution

Bhagyashree Kokate: Idea, manuscript drafting, Swati Bodhankar: Interpretation of data and statistics, Rishabh Agade: Experimentation and Drafting, Veena Belgamwar: supervision and finalization.

Conflict of Interest

All authors do not have any conflict of interest.

List of abbreviations

QbD-Quality by Design, HPLC-High pressure liquid chromatography, CPD- Clopidogrel Bisulphate, NSF- Nasal simulated Fluid, CCD- Central composite Design, UV-ultra violet, QTPP-Quality target product profile, CQA-Critical quality attributes, LOD-limit of detection, LOQ-Limit of quantification. RSD-Relative Standard Deviation.

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