Analysis of Forced Degradation Products in Rilpivirine using RP-HPLC and Peak Purity Evaluation

Introduction

Rilpivirine¹, (RLP) an aminopyrimidine which is a pyrimidine-2,4-diamine in that position of amino groups 2and 4 were interchangedby 4-cyanophenyl and 4-[(E)-2-cyanovinyl]-2,6-dimethylphenyl groups in proper place to class of NNRTIs². This should be utilized to manage HIV-1 infections individually or in combination with  dolutegravir and cabotegravir. The remarkable potency and reduced risk of resistance, outstands RLP among other NNRTIs³. This Character arises from its adaptability within its binding site and its internal conformational flexibility.Its approval for various treatment regimens, including the pioneering Juluca, showcases its impact on HIV-1 management. The flexibility in dosing intervals further underscores its evolution in improving treatment options.  The substance profile of drug is crucial task to the regulatory circumstances. Because of unknown foreign substances, unwanted solvents, at very less levels, may vary the consequence of efficiency drug along with side effects. Accordingly, profile of foreign substance of drug should be performedwith using the method for stability indicative.The literature review reveals that few methods were published for the analysis of RLP individually and in with other combinations using HPLC and LCMS^4,5. But, no method is published till now for the impurity profiling of RLP by RP-HPLC with peak purity assessment. Separate a mixture of compounds to diagnose and compute into separateconstituentswith the help ofHPLC^6,7.  Different authors used various methods for the forced degradations studies and the results obtained are in the limit for the mobile phase, injection of the sample and wavelength^8-9.  The present work focussed to develop HPLC method to separate foreign substances and their degradation products^10-12by taking everything in the mind limit ofspecification along with short run time and validated this process as per the rules and regulations given by ICH¹³.

Table 1: Structures of RLP and its Impurities. Click here to View Table

Materials and Methods

Rilpivirine and related foreign substances are procured from  clearsynth laboratory. Acetonitrile and methanol of HPLC standard are purchased from Rankem India Pvt. Ltd. Other chemicals used for the preparation of buffer were of analytical grade, pure milli-Q water is used from Millipore purification structure.

Instrumentation and chromatographic conditions

WATERS  HPLC Model: 2695 furnished by 2996  photo diode array detector was  utilized to  developmentalong with validation of the method, byusing sample injector which is a automated. Agilent Zorbax Eclipse XDB C18 column (150 x 2.1mm, 1.8µm) was used for the separation. 0.3%v/v solution of perchloric acid in water adjusted to pH 3.0 with 5% sodium hydroxide solution utilized as a Eluent A and eluent B asAcetonitrile. Analysis wasperformedby gradient mode.  The rate of flow is 0.55mL/min.   3µL volume is injected into the system. Temperature of the column is maintained at 55^oC.   40minutes is the runtime.   The gradient program is represented in Table 1. At a wavelength of 220nm the data is identified. Final output signal is observed and also integrated with the help of Empower 2 software.

Table 2: Gradient Programme to separateforeign substancesin RLP

Time (Min)	% Eluent A	% Eluent B
0	68	32
7.0	60	40
21.9	20	80
22.0	68	32

 

Preparation of solutions

Diluent is prepared by mixing both water as well as acetonitrile in the proportions of 70v/v and 30v/v

Preparation of sample solution

RLP HCl powder (about 20 mg) was weighed and treated as indicated and diluted to 50 mL with diluents(400 µg/mL). The obtained solutions were injected for Impurity profile. For assay determination and Peak purity each treated solution was diluted 5/20 with diluents(100µg/mL)

Results And Discussion

This HPLC validation processis performed out to determine foreign substances in RLP as per the rules and regulations given by ICH for to demonstrating that this proposed process is a stability indicative topredetermined usage. Also forced degradation studies were done along peak purity assessment.

Figure 1: Chromatogram of RLP(Standard and sample). Click here to View Figure

Figure 2: Peak purity plot  for  unstressed sample of RLP. Click here to View Figure

Table 3: Specificity experimental data

Name of the Sample	Retention time in Min.	Relative retention time
RLP amide B	9.298	0.54
RLP Amide A	10.181	0.59
Z-RLP	16.260	0.94
RLP.HCl	17.319	1
Blank	–	–

 

Figure 3: Spiked sample chromatogram. Click here to View Figure

By this chromatogram , authors are observed that there is no interference because of diluent blank in retention times of RLP and its related foreign substances. All these foreign substanceswere apportioned by very good resolution.

Degradation studies

Preliminary SIM studies showed that Methanol as dilution solvent is participating in the degradation chemistry of RLPHCl  under acidic and basic stress conditions. Therefore and due to RLP HCl poor solubility in organic solvents and water, DMSO was used as co solvent.  These samples  ofRLP are stressed by using acid, base, oxidation, heat as well as humidity. Samples those are degraded are analysedwith a photo diodie array detector. Peak purity of RLP as well as  its related impurities are recognized the forced degradation circumstanceswhich are denoted in Table 3 and the obtained results were specified in Table 4

Table 4: Forced degradation conditions of RLP

Stressed condition	Solvent	Temp (0C)	Exposed time in hours
Acid	0.5MHCl	70	48
Base	0.5M NaOH	RT	8.5
Oxidation	30%H2O2	RT	6
Photolytic Solution	Diluent	765W/m2 at 35°C	1
Powder	–	765W/m2, 35°C	6.9
Heat solution	Diluent	70	46
Powder	–	120	48
Humidity80%	Diluent	–	–

 

Impurity Profile by HPLC – Acid Degradation

The following degradation products were formed during degradation.

Figure 4: Degradation with  (A). Acid.(B.)Base. Click here to View Figure

Figure 5: Exposed toirradiation of xenon arc lamp ( Z-RLP as degradant)A. Solution B. Powder. Click here to View Figure

Figure 6: RLP HCl solution under heat treatment degrades to minor degradation product at RRT 1.33A. Solution B. Powder.No degradation products (equal or higher than 0.02% w/w) were observed. Click here to View Figure

Figure 7: Degraded under 80% humidity: No degradation products (equal or higher than 0.02% w/w) were observed. Click here to View Figure

Table 5: Summary of Assay and Peak Purity of degraded RLP HCl on various conditions

Stress Action	Conditions		Time interval	% Assay	Purity Angle	Purity Thresh-hold	Match Angle	Match Thresh-hold
Unstressed	NA	NA	NA	NA	0.044	0.083	0.00	0.087
Oxidation	1 mL H2O2 30%	RT	6hrs	98.4	0.045	0.085	0.057	0.088
Acid	1 mL HCL 0.5M	70ºC	48hrs	99.6	0.039	0.095	0.062	0.103
Base	1 mL NaOH 0.5M	RT	8.5hrs	90.2	0.069	0.225	0.076	0.147
Sun cabinet	Sun cabinet of solution	765W/m2 35ºC	1hrs	93.5	0.039	0.085	0.064	0.091
	Sun cabinet of powder	765W/m2 35ºC	6.9hrs	99.6	0.040	0.084	0.059	0.089
Heat	Heating of solution	70ºC	46hrs	100.2	0.043	0.096	0.055	0.103
	Heating of powder	120ºC	48hrs	101.9	0.044	0.065	0.051	0.091
Humidity	Exposure to humidity	RT	24hrs	99.4	0.069	0.225	0.076	0.147

 

The forced degradation tests demonstrate that the peak of RLP HCl is spectroscopically pure in all stressed conditions, all degradation products are separated from the main peak and do not interfere with main substance. RLP HCl powder was found to be stable under heat treatment.RLP HCl solution was found to be stable under oxidation treatment.RLP HCl solution was found to be stable under acidic treatment without heat.Acidic degradation combined with heat: The main degradation products are RLP Amide A and RLP Amide B. Basic degradation: Main products of degradation were RLP Amide A as well as RLP Amide B.Exposure of RLP HCl solution to heat: RLPHCl solution main degradation product is peak at RRT 1.33.Exposure of RLP HCl solution to irradiation of xenon arc lamp: Main degradation product is Z-RLP.RLP HCl powder under irradiation of Xenon arc lamp 765W/m², 35°C was decomposed to Z-RLP and some unknown impurities: RRT 1.49 and RRT 1.51.Exposure of RLP HCl powder to humidity no degradation product formed.

Conclusion

By theexperimentalvalues it was finalized that, this newly developed method to  simultaneous estimation of related substances in RLP is identified as simple, more precise and highly accurate along with the high resolution. This present approach provides cost effective and should be furnishedto routine analysis in pharma industry. The outcomes of this meticulous study unveiled the susceptibilities of RLP to a spectrum of stress factors, in the generation of impurity profile RLP-Amide A, RLP-Amide Band Z-RLP with peak purities. The forced degradation tests demonstrate that the peak of RLPHCl is spectroscopically pure in all stressed conditions. All degradation products are separated from the main peak and do not interfere with main substance. This exploration not only augments the comprehension of RLP’s stability profile but also underscores the pivotal role of analytical techniques in upholding the safety and efficacy benchmarks of pharmaceutical formulations.

Acknowledgement

We sincerely thank Department of chemistry, KRU, Machilipatnam, for providing Ph.D registration and facilities.

Conflicts of Interest

There are no conflicts of interest among the authors.

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