INTRODUCTION:

Cystic fibrosis is a single-gene autosomal recessive disorder characterized by chronic airway infection, gastrointestinal dysfunction, male infertility and pancreatic insufficiency¹. Cystic fibrosis is characterized by mutation in the CFTR (cystic fibrosis membrane conductance regulator) protein which leads to nonfunctional or partially functional protein that causes abnormal fluid and electrolyte transport in the cells².

The combine medication Ivacaftor (IVCF) and Tezacaftor (TZCF)was used for the treatments of cystic fibrosis aged 12 years and older that have at least one mutation in CFTR³. The possible side effect associated with the use of IVCF and TZCF are increased bronchial secretions leading to hospitalization for pulmonary exacerbation⁴, headache, nausea, stuffy nose, and dizziness. The molecular structure of IVCF and TZCF was given in figure 1.

IVCF

TZCF

Figure 1: Molecular structure of Ivacaftor and Tezacaftor in the study

The search of literature for the methods available for simultaneous analysis of IVCF and TZCF confirms that only two HPLC methods reported for the estimation of drugs in pharmaceutical formulations^5,6. Two UV spectrophotometer methods were reported^7,8 for the estimation of IVCF in single dosage forms. The other methods reported were for the analysis of IVCF and its major metabolites and lumacaftor in pharmaceutical formulations, biological samples using different analytical techniques^9-17. Hence the presence work aimed to develop a simple and accurate stability indicating liquid chromatography method for the simultaneous quantification of IVCF and TZCF in pharmaceutical formulations.

MATERIALS AND METHODS:

Instrumentation:

Agilent 1100 series HPLC with Quaternary G1311 A pump, COLCOM G1316A thermostat column temperature control, Thermostatic auto sampler G 1329A with sample volume of 0. 1 – 1500µL and variable programmable UV detector G 1314 A. The instrument was operated and integrated with Agilent chem. station LC software.

Preparation of Standard drug solution:

An accurately weighed 100mg of standard drug IVCF and TZCF was taken in a 100mL volumetric flask and was dissolved in methanol separately. Standard TCPD solution at a concentration of 1000µg/mL was obtained. The concentrations required for the concentration of calibration curve was prepared separately by proper dilutions. Equal volumes of selected concentrations of IVCF and TZCF were mixed separately and the mixture solution was used for method development and validation studies.

Preparation of Formulation solution:

Ten tablets of IVCF and TZCF (SYMDEKO^© – 150 mg of IVCF and 100 mg of TZCF) were powdered using sterile mortar and pestle to get a fine powder. From the tablet powder an amount of drug equivalent to 10mg of IVCF was weighed accurately and was dissolved in 10 mL methanol. Keep the solution in an orbital shaker for 15 min to dissolve the drug completely in solvent. Then it was filtered through 0.45µ nylon membrane filter paper. The sample stock solution was further diluted to get working sample solution having IVCF concentration of 150µg/mL and TZCF at a concentration of 100 µg/mL.

Method development:

The standard drug solution containing IVCF at a concentration of 150µg/mL and TZCF at a concentration of 100µg/mL was initially used for method development studies. The wavelength of detector was maintained based on the iso-absorption wavelength obtained in UV spectrophotometer for IVCF and TZCF. System suitability, resolution, responses factor, peak symmetry are the key factors that are keep in consideration for optimization of mobile phase. Mobile phase was confirmed by change in different solvent ratios, strength of organic modifiers and pH. Separation was performed on different column configurations and manufactures. Flow rate of the mobile phase also changed in order to get better resolution. The conditions that give best resolution, response and peak symmetry and considered as suitable conditions and these conditions were further validated.

Method Validation:

The method developed for the simultaneous analysis of IVCF and TZCF was validated as per the available literature¹⁸ and ICH guidelines¹⁹.

System suitability:

Standard stock solution at a concentration of 100µg/mL of IVCF and TZCF was analyzed six times in the developed method. The chromatographic parameters such as retention time, resolution, number of theoretical plats and asymmetric factor of the resultant chromatograms was used for the evaluation of system suitability of the method.

Linearity and range:

Standard calibration curves were prepared with six calibrators over a concentration range of 50 - 150µg/mL for TZCF, 75 - 225µg/mL for IVCF. The solutions were analyzed in the optimized conditions. The data of peak area vs drug concentration were treated by linear least square regression analysis.

Precision, Ruggedness and Robustness:

Six replicate analysis of standard solution containing 150µg/mL of IVCF and 100µg/mL of TZCF was analyzed in the same day for intraday precision, six times in three days for interday precision and by change in analyst for ruggedness study. The same concentrations were analyzed by change in analytical conditions (i.e ±5nm of detector wavelength, ±5mL variation in mobile phase organic and pH modifier, ±0.2 mL mobile phase flow rate and ± 5⁰C change of column temperature). The % relative standard deviation (RSD) of peak area response of IVCF and TZCF was calculated for precision and ruggedness study and a % RSD of <2 in each study for both the drugs was considered as the method is precise and rugged. The % change in peak area response when analyzed in changed conditions was calculated for the evaluation of robustness of the developed method.

Recovery:

Standard addition method was carried for determining the accuracy of the method. 50%, 100% and 150% level concentrations were spiked to a known concentration of 150µg/mL of IVCF and 100µg/mL of TZCF. Accuracy was determined by comparing the difference between the spiked value and actual found value.

Sensitivity:

The sensitivity of the method developed for the analysis of IVCF and TZCF was evaluated in terms of detection limit (LOD) and quantification limit (LOQ) using signal/noise (S/N) ratio method.

Force degradation studies:

50mg of standard drug IVCF and TZCF was mixed with 50mL of 0.1 N hydrochloric acid, 0.1 N sodium hydroxide and 3% hydrogen peroxide for acidic, base and peroxide degradation study respectively. The solution was incubated 24 H, neutralized and was analyzed in the developed method. The standard drugs IVCF and TZCF were exposed to UV light at 254nm for 24 hours for photolytic degradation and heated in an air oven at 60⁰C for 24 hours for thermal degradation study. The peak area response of IVCF and TZCF in the stress degradation study was compared with the calibration curve results and the % degradation was calculated.

Formulation analysis:

The formulation solution prepared was analyzed triplicates in the optimized conditions. The peak area response of IVCF and TZCF obtained in the formulation analysis was used for the determination of applicability of the developed method.

RESULTS AND DISCUSSIONS:

Mobile phase was optimized by change in different solvent ratios, expected peak shape, resolution was achieved using mobile phase composition of pH 5.0 ammonium acetate buffer and acetonitrile in the ratio of 60:40 (v/v). Mobile phase was pumped at a flow rate of 1.0 mL/min in isocratic elution. UV detection was carried at wavelength of 260 nm and separation was achieved on Sunfire C18 column (150 mm×4.6 mm; 3.5 µ id). In the optimized conditions, well retained, resolved and symmetric peaks are observed in the standard chromatogram contains 150µg/mL of IVCF and 100µg/mL of TZCF (figure 2).

Figure 2: Standard chromatogram of IVCF and TZCF

The results in the system suitability study (table 1) confirm that all the system suitability parameters for IVCF and TZCF was found to be within the acceptable limits.

Table 1: System suitability results

SNo	Parameter	Results observed
SNo	Parameter	IVCF	TZCF
1	Retention Time (min) #	1.83±0.003	3.572±0.019
2	Theo plate*	2946	2946
3	Tail Factor*	1.158	1.262
4	Resolution*	---	12.04

# Average ± standard deviation of six replicate experiments; * average of six replicate experiments

Linear calibration curve (figure 3) observed within the concentration range of 50-150µg/mL for TZCF and 75-225 µg/mL IVCF in the developed method (table 2). Linear regression equation was fund to be y = 25978x + 6E+06 [R²=0.999] and y = 33714x - 46921 [R²=0.999] for IVCF and TZCF respectively. Accurate fit correlation coefficient value (more than 0.999) was observed for IVCF and TZCF confirms that the method fallows linear relation accurately with in the concentration range studied.

Table 2: Linearity results

S. No	IVCF		TZCF
S. No	concentration in µg/ml	Peak Area	concentration in µg/ml	Peak Area
1	75	25055621	50	16194209
2	120	36806942	80	26535716
3	150	45311483	100	33633340
4	180	52619670	120	39910697
5	225	63933476	150	49953204

Figure 3: Linear calibration curve for IVCF and TZCF

The % RSD of the peak area responses observed in the precision (intra and interday) and ruggedness study was calculated and was found to be 0.41, 0.33 and 0.57 for IVCF and 0.77, 0.50 and 0.93 for TZCF in intraday, interday precision and ruggedness study. The result confirms that the method developed for IVCF and TZCF was found to be rugged and precise.

In all these changed conditions in robustness study, the % change in peak area response was calculated and was found to be within the acceptable limit of less than 2 (table 3) for both the drugs IVCF and TZCF confirms that the method was found to be robust.

Spiked recovery at 50%, 100% and 150% spiked levels of standard at a 100% spiked level of 150 and 100µg/mL concentration of IVCF and TZCF respectively was studied. The results of the recovery study were given in table 4 confirms that the method was found to accurate.

In the stress degradation study, the % degradation was found to be very high in Peroxide degradation condition. The % degradation was found to be 3.44 and 3.60 for IVCF and TZCF respectively in peroxide conditions. In base degradation condition, the % degradation of TZCF was found to be 3.34. In other degradation conditions, both the drugs were found to be stable and the % degradation was found to be very less. The degradation products formed during the stress study of IVCF and TZCF was effectively separated in the developed method. Hence the method was found to be stable and is a stability indicating method. The results of the forced degradation study of IVCF and TZCF in the developed method was given in table 5.

The results of the formulation analysis (figure 4) confirm that the % assay was found to be 99.39 % and 98.88 % for IVCF and TZCF respectively. The assay was found to be more than 98 % for both IVCF and TZCF in the developed method confirms that the method can be applicable for the routine analysis of IVCF and TZCF in bulk drug and formulations.

Table 3: Robustness results

S No	Changed Condition	IVCF		TZCF
S No	Changed Condition	Peak Area	% Change	Peak Area	% Change
1	Standard	45311483	---	33633340	---
2	MP 1	45874762	1.22	33693352	0.77
3	MP 2	45862311	1.25	33612411	0.68
4	CT 1	46123515	0.69	33642141	0.53
5	CT2	45897541	1.17	33693321	1.82
6	FR 1	45872133	1.23	33257411	2.21
7	FR 2	46258741	0.39	33125122	1.09
8	WL 1	45888671	1.27	33224497	1.21
9	WL 2	45796154	1.07	33423774	0.62

MP (Mobile Phase) 1: Ammonium acetate buffer: acetonitrile 65:35 (v/v), MP 2: Ammonium acetate buffer: acetonitrile 55:45 (v/v); WL (Wavelength) 1: 265nm, WL 2: 255nm; CT (column temperature) 1: 25 ^oC, CT 2: 35 ^oC; FR (Flow rate) 1: 0.8 mL/min, FR 2: 1.2 mL/min

Table 4: Recovery results

S. No.	Drug	Recovery Level	Concentration in µg/ml	Amount found Mean ± SD	% recovered Mean ± SD	% RSD of Recovery
1	IVCF	50%	75	75.19±0.170	100.3±0.226	0.226
2		100%	150	148.50±1.211	99.0±0.807	0.816
3		150%	225	225.80±1.318	100.4±0.586	0.584
4	TZCF	50%	50	50.47±0.194	99.1±0.380	0.384
5		100%	100	99.29±0.413	100.7±0.417	0.416
6		150%	150	150.47±0.298	99.7±0.198	0.198

*Values given in table are the average ± standard deviation for three replicate experiments

Table 5: Stress degradation study results

S No	Degradation condition	IVCF			TZCF
S No	Degradation condition	Area	% Assay	% Degradation	Area	%Assay	% Degradation
1	Acid	46072313	99.67	0.33	32882274	97.33	2.67
2	Base	45584944	98.62	1.38	32657920	96.66	3.34
3	Peroxide	44632312	96.56	3.44	32569158	96.40	3.60
4	Photolytic	45812264	99.11	0.89	33073436	97.89	2.11
5	Thermal	46039446	99.60	0.40	33273907	98.49	1.51

Figure 4: Formulation chromatogram of IVCF and TZCF

The literature available for the analysis of IVCF and TZCF confirms that only two HPLC methods reported. The method developed in this study was compared with the previously reported methods. The resolution of IVCF and TZCF in the reported methods [6] was found to be very less compared to this method. The high acidic pH and high flow rate of the mobile phase facilitates high consumption of mobile phase and high pH effect on the column was observed in the reported method [5]. The less acidic pH, more resolution between IVCF and TZCF high sensitivity are the advantages of the method developed in this study than the previous reported methods [5,6]. Hence the method reported here was found to be the best alternative for the routine analysis of IVCF and TZCF in formulation analysis and stability studies.

CONCLUSION:

A simple and accurate stability indicating HPLC method was developed for the simultaneous analysis of IVCF and TZCF in formulations. The method utilizes a simple run time of 5 min and all the validation parameters are within the acceptable limits. Hence the method can be used for the separation, simultaneous quantification of IVCF and TZCF in formulation and in stability study.

ACKNOWLEDGEMENTS:

The authors are thankful to the Management of M.A.M College of Pharmacy for providing the chemicals and instruments and Madras Pharmaceuticals, Chennai, India for providing the drug samples for research.

REFERENCES:

1. Katherine RG, Obstetric Imaging: Fetal Diagnosis and Care (Second Edition). Elsevier - Academic Press, Massachusetts , USA. 2018; pp 579-581.

2. William LE, Cystic Fibrosis, xPharm: The Comprehensive Pharmacology Reference. 2007; pp 1-5.

3. FDA guidelines, SYMDEKO™ (tezacaftor/ivacaftor; ivacaftor) Tablets, Reference ID: 4220295, 2018; 1-15(https://www.accessdata.fda.gov/drugsatfda_docs/label/2018/210491lbl.pdf)

4. Jennifer Taylor Cousar, Minoo Niknian and Geoffrey Gilmartin et al. Effect of ivacaftor in patients with advanced cystic fibrosis and a G551D-CFTR mutation: Safety and efficacy in an expanded access program in the United States. Journal of Cystic Fibrosis.2016; 15(1): 116-122.

5. Gadeela Srimounika, Shyamala and Sharma JVC et al. A new stability- indicating method for simultaneous estimation of ivacaftor and tezacaftor by RP-HPLC in bulk and its dosage form. IJRAR. 2018; 5(4): 774-785.

6. Kiranjyothi R, Balakrishnan M and Chandrasekhar KB. Method development and validation for the stability indicating simultaneous estimation of tezacaftor and ivacaftor in bulk and its dosage forms. International Journal of Pharmaceutical Research. 2018; 10(4):

7. Janardhana Reddy VL, Raveendrareddy P and Sreenivasulu. Method development and validation of Ivacaftor in bulk and pharmaceutical dosage form by UV spectrophotometer, Int. J.Res.Pharm.Sci. 2018; 9(40): 1169-1173.

8. Gautam CHVS, Saicharan K and Swathi B. Method Development and Validation of Ivacaftor in Bulk & Pharmaceutical Dosage Form by Uv-Visible Spectrophotometry. IAJPS. 2019; 06 (04): 7476-7481.

9. Akram NMD and Umamahesh M. A New Validated RP-HPLC Method for the Determination of Lumacaftor and Ivacaftor in its Bulk and Pharmaceutical Dosage Forms. Orient. J. Chem. 2017; 33(3): 1492-1501.

10. Pasala Sandhya Mounika and Rameeja Pattan. Stability Indicating RP-HPLC Method for Simultaneous Estimation of Lumacaftor and Ivacaftor in Bulk and Pharmaceutical Dosage Forms. Pharma research Library. 2019; 7(1):

11. Suresh Babu M, Spandhana N and Baby Rani P et al. Analytical method development and validation for the estimation of Lumacaftor and Ivacaftor using RP-HPLC. Journal of Pharmacreations. 2017; 4(1): 55-78.

12. Nagamallika Gorantla, Jyothi Dodlapati and Sujatha Jadi. A New Validated RP-HPLC Method for Simultaneous Estimation of Lumacaftor and Ivacaftor in Pharmaceutical Dosage Form. Int. J. Pharm. Sci. Rev. Res. 2019; 56(1): 30-37.

13. Sravanthi B, Divya M. Analytical Method Development and Validation of Ivacaftor and Lumacaftorby RP-HPLC Method. IAJPS. 2016; 3(8): 900-904.

14. Baki Sharon, Meruva Sathish Kumar and Marakatham S et al. A New RP-UPLC Method Development and Validation forthe Simultaneous Estimation of Ivacaftor and Lumacaftor. J. Global Trends Pharm Sci. 2018; 9(3): 5730- 5737.

15. Elena K Schneidera, Felisa Reyes-Ortegaa and John W. Wilson et al. Development of HPLC and LC–MS/MS methods for the analysis of ivacaftor, its major metabolites and lumacaftor in plasma and sputum of cystic fibrosis patients treated with Orkambi or Kalydeco. Journal of Chromatography B. 2016; 1038: 57–62.

16. Elena K, Schneider and Felisa Reyes-Ortega et al. Development of HPLC and LC-MS/MS methods for the analysis of ivacaftor, its major metabolites and lumacaftor in plasma and sputum of cystic fibrosis patients treated with Orkambi Or Kalydeco, J Chromatogr B AnalytTechnol Biomed Life Sci. 2016; 1038: 57–62.

17. Mohan Goud V, Sharma JVC and Sravanthi M. Stability Indicating Ultra Performance Liquid Chromatography Method Development And Validation For Simultaneous Estimation Of Ivacaftor and Tezacaftor In Bulk And Pharmaceutical Dosage Form. International Journal of Scientific Research and Review. 2019; 8(5); 129-133.

18. Bikshal B.K., Useni R.M., Venkateswara R.A., and Maheshwara R.L., Intended high-performance liquid chromatography procedure for the quantification of norfloxacin and its potential impurities in active pharmaceutical ingredient and tablet dosage forms, Thai Journal of Pharmaceutical Sciences, 2018; 42(1): 27-36.

19. ICH Validation of Analytical Procedures: Text and Methodology Q2 (R1), 4, 1994; 1-13.

Received on 08.08.2019 Modified on 10.09.2019

Research J. Pharm. and Tech 2020; 13(5): 2076-2080.

DOI: 10.5958/0974-360X.2020.00373.X