A New Validated Rp-Hplc Method For The Determination Of Lumacaftor And Ivacaftor In Its Bulk And Pharmaceutical Dosage Forms

Introduction

Ivacaftor is Cystic fibrosis is caused by any one of several defects in a protein, cystic fibrosis transmembrane conductance regulator, which regulates fluid flow within cells and affects the components of sweat, digestive fluids, and mucus. The defect, which is caused by a mutation in the individual’s DNA, can be in any of several locations along the protein, each of which interferes with a different function of the protein. One mutation, G551D, lets the CFTR protein reach the epithelial cell surface, but doesn’t let it transport chloride through the ion channel. Ivacaftor is a potentiator of the CFTR protein. The CFTR protein is a chloride channel present at the surface of epithelial cells in multiple organs. Ivacaftor facilitates increased chloride transport by potentiating the channel-open probability (or gating) of the G551D-CFTR protein.

Lumacaftor I Orkambi is a combination of lumacaftor and ivacaftor, both of which are oral cystic fibrosis transmembrane conductance regulator (CFTR) modulators. The CFTR protein is a chloride channel present at the surface of epithelial cells in multiple. Ivacaftor is currently approved for use in combination with Lumacaftor (as the combination product Orkambi) for the treatment of chronic cysticfibrosis ^1-4

Literature review reveals that there few HPLC^7-13and HPTLC^14-15 methods are available for the determination of Lumacaftor and Ivacaftor in different dosage forms.

For Lumacaftor and Ivacaftor there are several HPLC^17-21methods available in combined dosage forms.

The structures of Lumacaftorand Ivacaftor were shown in figures 1 and 2.

Figure 1: Structure of Lumacaftor. Click here to View figure

 

Figure 2: Structure of Ivacaftor. Click here to View figure

 

Materials and Methods

Instrumentation

The chromatography was performed on a Waters 2695 HPLC system, equipped with an auto sampler, UV detector and Empower 2 software. The analysis was carried out at 254 nm with an inertsil ODS (4.6 x 250mm, 5mm) dimensions at ambient temperature(25⁰c).

Chemicals and reagents

Ivacaftor and Lumacaftor were supplied  fromMylon laboratories, Hyderabad. Orthophosphoric acid (OPA) (Merck),Methanol(MERCK HPLC grade) Acetonitrile (Molychem, HPLC grade) and Water for HPLC (LICHROSOLV (MERCK). were employed in the present work.

Preparation of solutions

Preparation of buffer

1ml of Orthophosphoric acidin1000 ml of HPLC water. The P^His adjusted to 3.0with TEA. The final solution is filtered through 0.45 m membrane filter and sonicate it for 10 mins.

Preparation of mobile phase

Accurately measured 600 ml (60%) of  P^H=3.0 buffer and 300 ml(30%) of Acetonitrile  and 100ml(10%) of  Methanol. mixed and degassed in an ultrasonic water bath for 10 minutes and then filtered through 0.45 µmmembrane filter under vacuum filtration. Figure4 represents the Chromatograms of mobile phase(blank solution).

Diluent Preparation

The Mobile phase was used as the diluent.

Preparation of standard stock solution

20 mg of Lumacaftor and 12.5 mg of Ivacaftorwere accurately weighed and transferred into a 10 ml clean dry volumetric flask.Add about 7 mL of Diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent.

Further, 1.5 ml of the above prepared stock solution is pipetted into a 10ml volumetric flask and dilute up to the mark with diluent.

Preparation of Sample Solution

Accurately weighthe samples of 10 tablets.It is crushed in mortor and pestle.Transfer equivalent to 20 mg ofLumacaftor and 12.5 mg Ivacaftor sample into a 10 mL clean dry volumetric flask.Add about 7 mL of Diluent and sonicate it up to 30 mins to dissolve it completely and make volume up to the mark with the same solvent.Then, it is Filtered through 0.44 micron Injection filter.

Further, pipette 1.5 ml of Lumacaftor and Ivacaftor from the above sample solution into a 10ml volumetric flask and dilute up to the mark with diluent. The standard solutions wereprepared on daily basis from which  stock solutions were prepared.

Procedure

20 μL of the standard, stock and sample solution are injected into the chromatographic system. The areas are measured for Lumacaftor and Ivacaftor peaks are calculated.The %Assay by using the standard formula.

Method development and selection of wavelength

UV spectrum of 10 µg/ml Lumacaftorand 10 µg/ml Ivacaftor in diluents (mobile phase composition) are recorded by scanning in the range of 200nm to 400nm. The UV Spectrum obtain for From the UV spectrum LumacaftorandIvacaftoris shownin thefigure.1.Form the UV spectrum , the wavelength is selected as 254 nm. At this wavelength both the drugs show good absorbance.

Figure 3: UV Spectra of Lumacaftor and Ivacaftor for Selection of Wavelength. Click here to View figure

 

Construction of calibration curve

Aliquots of different concentrations of standard solution were prepared and their chromatograms were recorded at the optimized chromatographic conditions. The mean peak areas at different concentration levels were calculated from the chromatograms. Then the linearity plot was constructed using the mean peak areas at their respective concentrations. (Figures 8 &9)

Method of Validation

The developed method was validated for linearity, accuracy, precision, and limit of detection, limit of quantitation, robustness and system suitability parameters as described in ICH guidelines.

Linearity

From the stock solution, 100, 200, 300, 400, 500μg/ml solutions for Lumacaftor and 62.5, 125, 187.5,250, 312.5μg/ml solutions for Ivacaftor were made and their chromatograms were recorded. From the recorded chromatograms, their respective mean peak areas were calculated and the linearity plot was constructed using the mean peak areas at their respective concentrations. The correlation coefficient was found to be 0.999. The linearity data of Lumacaftor and Ivacaftorare shown in the Tables 1 & 2.The calibration plots, are given in the figures 4&5.

Table 1: Showing assay results.

S. No	Name of compound	Amount taken(mg)	%purity
1	Lumacaftor	200mg	100.39
2	Ivacaftor	125mg	100.17

 

 

 

Table 2: Linearity results for Lumacaftor.

 

Figure 4: Showing calibration graph for Lumacaftor. Click here to View figure

 

Table 3: Linearity results for Ivacaftor.

S. No	Linearity Level	Concentration(mm/ml)	Area
1	I	62.5	71267
2	II	125	99725
3	III	187.5	127369
4	IV	250	155275
5	V	312.5	179461
Correlation Coefficient			0.999

 

Figure 5: Showing calibration graph for Ivacaftor. Click here to View figure

 

Results and Discussion

The present investigation reported by the authors are to develop a new validated method for the simultaneous estimation of Lumacaftor and Ivacaftor by RP-HPLC method. Mobile phase contains the mixture of 60% PH 3 Buffer(1ml OPA in 1000ml water) and 30% of Acetonitrile and 10% of Methanol. It is used as diluent in the present study. An intersil ODS column of 5μ (4.6X250mm) is employed for the simultaneous determination of Lumacaftor and Ivacaftor by RP-HPLC method. A flow rate of 1ml for minute is used in this method. UV detection wavelength at 254mm and temperature of 25^oC were maintained. Two sharp peaks were obsorbed at 3.101mts and 4.025 mts for Ivacaftor and Lumacaftor respectively. The representative chromatograms of blank solution, Lumacaftor and Ivacaftor shown in this figure.4 Chromatograms of assay of sample injection and standard of sample injection are shown in the figures5 & 6 and assay results of purity in the table 1. The % purity of Lumacaftor and Ivacaftor were found to be 100.39 and 100.17 respectively.

Figure 6: Chromatogram showing blank Solution (mobile phase). Click here to View figure

 

Linearity

Figures 7a to 7e represent the chromatograms showing different linearity levels with different concentrations of Lumacaftor and Ivacaftor and results of given in the tables 2 & 3. Both Lumacaftor and Ivacaftor obey Beer Lamberts Law in the range of concentrations of100µg /ml to 500 µg /ml and 62.5µg /ml to 312.5µg/ml respectively with regression equations Y= 332.76 X + 31993(correlation and coefficient) R²= 0.999 for Lumacaftor and Y= 27194 X + 45038 , R²= 0.999. forIvacaftor.

Precision

This validated method is more precise and the percentage of relative standardization (%RSD) and intermediate precision / Ruggedness were found to be 0.8 and 0.7 for Lumacaftor  and 0.8 and 0.6 for Ivacaftor . The results are given the in the tables 6 and 7.

System suitability 

The results for Lumacaftorand Ivacaftor are given in the tables 473. It was performed to ensure that complete testing system was suitable for the intended application. The USP tailing factor for Lumacaftorand Ivacaftor were 1.58 and 1.15 which is <2 and the USP plate found were 2693.56 and 2310.88 which is >2000 the results for actual flow of 1.0ml/min is considered from assay standard. Tablets for all shows system suitability results with change in the organic composition in the mobile phase for Lumacaftorand Ivacaftorchromatograms of and Lumacaftorand Ivacaftorare show in the figures 12 and 13.

Accuracy 

The accuracy study was performed for 50%, 100% and 150 % for Lumacaftorand Ivacaftor. Each level was injected in triplicate into chromatographic system. The area of each level was used for calculation of % recovery. These results were given in the tables 4 & 5. The Mean % of recovery is 100.53  forLumacaftor and 100.13 for Ivacaftor.(NLT 98% and NMT 102%)

Figure 7: Chromatogram showing assay of sample injection. Click here to View figure

 

Figure 8: Chromatogram showing standard of sample injection –Linearity. Click here to View figure

 

Linearity 

Figure 9: a,b Level 1,2 Chromatograms showing Linearity of Lumacaftor and Ivacaftor. Click here to View figure

 

Figure 9: c,d. Level 3,4Chromatograms showing Linearity of Lumacaftor and Ivacaftor. Click here to View figure

 

Figure 9: e. Level 5 Chromatograms showing Linearity of Lumacaftor and Ivacaftor. Click here to View figure

 

Table 4: Linearity results for Ivacaftor.

S. No	Linearity Level	Concentration(mm/ml)	Area
1	I	62.5	71267
2	II	125	99725
3	III	187.5	127369
4	IV	250	155275
5	V	312.5	179461
Correlation Coefficient			0.999

 

Accuracy 

The accuracy study was performed for 50%, 100% and 150 % for Lumacaftor and Ivacaftor. Each level was injected in triplicate into chromatographic system. The area of each level was used for calculation of % recovery.

The accuracy results for Lumacaftor

Table 5: Showing accuracy results for Lumacaftor.

 

Table 6: Showing accuracy results for Ivacaftor.

 

Precision

Table 7: Showing% RSD results for Lumacaftor and Ivacaftor.

Injection	Area for Lumacaftor	Area for Ivacaftor
Injection-1	141368	128876
Injection-2	140717	127224
Injection-3	142655	129055
Injection-4	143939	128739
Injection-5	143013	126699
Injection-6	142282	129220
Average	14239	128302.2
Standard Deviation	1156.8	1064.1
%RSD	0.8	0.8

Intermediate precision/Ruggedness

Table 8: Showing results for intermediate precision of Lumacaftor and Ivacaftor.

Injection	Area for Lumacaftor	Area for Ivacaftor
Injection-1	139453	122535
Injection-2	137162	121224
Injection-3	139458	122915
Injection-4	138377	123391
Injection-5	138482	123108
Injection-6	139771	122959
Average	138783.8	122688.7
Standard Deviation	976.1	769.7
%RSD	0.7	0.6

 

Figure10: Chromatogram showing less flow rate. Click here to View figure

 

Figure11: Chromatogram showing more flow rate. Click here to View figure

 

Table 9: System suitability results for Lumacaftor.

S. No	Flow Rate (ml/min)	System Suitability Results
		USP Plate Count	USP Tailing
1	0.9	2910	1.16
2	1.0	2310.88	1.58
3	1.1	2245.12	1.13

 

Table 10: System suitability results forIvacaftor.

S. No	Flow Rate (ml/min)	System Suitability Results
		USP Plate Count	USP Tailing	USP Resolution
1	0.9	3425.70	1.19	3.62
2	1.0	2693.11	1.16	3.43
3	1.1	2675.84	1.17	3.35

 

Figure 12: Chromatogram showing less organic composition in the mobile phase.Click here to View figure

 

Figure 13: Chromatogram showing more organic composition in the mobile phase. Click here to View figure

 

Table 11: Showing system suitability results for Lumacaftor.

S. No	Change in Organic Composition in the Mobile Phase	System Suitability Results
		USP Plate Count	USP Tailing
1	10% less	2425.70	1.21
2	*Actual	2310.88	1.58
3	10% more	2705.45	1.12

 

Table 12: Showing system suitability results for Ivacaftor.

S. No	Change in Organic Composition in the Mobile Phase	System Suitability Results
		USP Plate Count	USP Tailing	USP Resolution
1	10% less	2910.66	1.16	3.61
2	*Actual	2693.11	1.16	3.43
3	10% more	2248.50	1.12	2.96

 

Detection limit

As per ICH guidelience S/N Ratio value shall be 3 for LOD solution.

As per ICH guidelience S/N Ratio value shall be 10 for LOQ solution.

Figure14: Chromatogram showing LOD. Click here to View figure

 

Figure 15: Chromatogram showing LOQ. Click here to View figure

 

Conclusion

The proposed HPLC method was found to be simple, precise, accurate and sensitive for the simultaneous estimation of Lumacaftor and Ivacaftorin pharmaceutical dosage forms. The results are accordance with ICH guidelines. Hence, this method can easily and conveniently adopt for routine quality control analysis of Lumacaftor and Ivacaftor   in pure and its pharmaceutical dosage forms.

Acknowledgment

Authors are thankful to the Pharma Train Lab, Kukatpally, for providing instrumental and analytical support. We extended our thanks to the Principal and Management for their timely help.

References

1. Alter, M.J.;  Hepatology. 1997, 26, 62-65
   CrossRef
2. Benhamou, Y.; DeMartinio, V.; Bochet,  M.;  Colombet, G.; Thibault, V.; Liou, A.; Katlama, C. Hepatology, 34, 283-287
   CrossRef
3. “Elbasvir Hydrochloride”. The American Society of Health-System Pharmacists. Retrieved Jan 2016.
4. Hirst, J.;  Farmer, A. J.; Ali, R.; Roberts, N. W.; Stevens,  R. J. 2012, 35(2), 446-54
5.  Lilian, B. A. R.; Marilia,  B. G. Elbasvir: an old but still the best treatment for type 2 diabetes, 2013
6. Arayne, M. S.; Najma S.; Hashim, Z.  Pak. J. Pharm. Sci., 2006, 19(3), 231-235.
7. Teng, X .W.;  Foe, K.; Brown, K.;   Cutler, D. J.;  Davies; J. Pharm. Biomedi. Anal.  2001, 26, 313-19
   CrossRef
8.  Shaikh, S.;  Muneera, M.S,; Thusleem, O.A  Orient. J. Chem., 2013, 29(2), 579-587.
9.  Kondaguli, A.V.  Jour. of Chromatographic Sci., 2009, 47(2), 178-183
   CrossRef
10.  Ravi, R. Y.; Manoj, D. R., Deepali M. G.; Ganesh S. Holkar.  Intern. Jour. of Theo. & Appl.Sci. 2012, 4(2), 145-156
11.  Kabeer, A. S.; Ashish, T. P. Jour. of Trace Analysis in Food and  Drugs, 2013, 1, 14-21
12.  Yadav, R.R.; Rokade, M.D.;  Salunke, S.A.; Gangrade,  R.M.; . Holkar, G.S. V.N. Biological Forum – An Int. Jour. 2009, 4(21), 1128(-21071
13. Hiral, N. D.; Ashlesha, G. M.;  Chaitanya, B.;  Killambi, P. k.; Inter. Jour. of App. Sci. and Eng. 2011, 9, 177-185
14.  Ravi, G.; Padmakar, A.; Sudesh, B.; Biological Forum – An International Jour.2013, 5(1),  33-41
15.  Heidurnezhad,  Z,; Heydari, F.; Bahranian, E. Orient. J. Chem.,  2013, 29(1), 69-74
16. Rajesh, M. K.; Santosh, G. S.; Shrawan, S.; E-Jour. of Chem., 2011, 8, 340-346
    CrossRef
17. Soleymani, R.; Dehgharian, A.; Ebtesam, S., Orient J. Chem., 2012, 28(4),  1291-1304
18. Abdollahpour, A.;  Forouhi, M.;  Shamsipur, M.; Yamini, Y. J. Iran. Chem. Soc., 2010, 7, 516-520
    CrossRef
19. Holkar, G.S.;  Rokade, M.D.;  Yadav, R.R.; Daphal, V.N.; Int. Jour.of Theo. & App. Sci.,  2012, 4(2), 56-65
20. Gopalakrishnan, S.; Chitra, T.A.;  Aruna, A.; Chenthilnathan, A. Der. Pharma. Chemica,  2002, 4, 1003-1015
21. Geetha, M.; Venkat, R.; ShakilL, S.; Sripal, R. P. Orient. J. Chem. 2013, 29, 579-587.
    CrossRef

---

This work is licensed under a Creative Commons Attribution 4.0 International License.