Analytical Method Development and Validation for Simultaneous Estimation of Reltegravir and Lamivudine in Bulk and Pharmaceutical Formulation

Patil Jyoti, Nadare Archana,

doi:10.5281/zenodo.19993747

Research Paper | Open Access
Volume 04 | Issue 05 | Article Id IJPS/260401255

Analytical Method Development and Validation for Simultaneous Estimation of Reltegravir and Lamivudine in Bulk and Pharmaceutical Formulation
Patil Jyoti* Nadare Archana
¹ Pravara Rural Education Society’s Institute of Pharmacy, Loni, Ahmednagar
2 Padm. Dr . D. Y. Patil College of Pharmacy, Akurdi, Pune

Abstract

Analytical chemistry finds application in many applied areas. Especially in pharmaceutical industry quality of drug in manufactured formulations like tablets, solution, suspension, emulsion etc must be carefully controlled. Slight variation in purity of drug because of other component can affect therapeutic value of drug so, there is need to develop newer and better method for pharmaceutical analysis. Multicomponent formulations have gained lot of importance in pharmaceuticals due to greater patient acceptability, increased potency , decreased side effect. Lamivudine nucleoside reverse transcriptase inhibitor & Raltegravir HIV integrase inhibitor , both used in management & treatment AID (HIV ).there are various combination available in market such as Lamivudine-Reltegravir , Lamivudine – Tenofovir, Lamivudine – Ziduvudine, Reltegravir Potassium – Maraviroc. Lamivudine potassium & Raltegravir recent combination available In market. Literature survey revel that few UV & HPLC method have been reported to determine Lamivudine potassium & Raltegravir as single component in combination or with other drug. Rapid & easy analytical method are needed due to increasing number of multicomponent formulations. . The aim of this research is to develop and validate simple precise, accurate sensitive, rapid & economic Ultra violet spectroscopic & Reveres phase High Performance Liquid Chromatography method for estimation of Reltegravir & Lamivudine in bulk & pharmaceutical formulation. Multi component analysis method based on composite absorption spectrum & Q analysis also known as absorption method is ratio of absorbance at any wavelength is constant independent of concentration or path length

Keywords

Ultra violet spectroscopic analysis, spectrophotometry, Q analysis method, multicomponent method

Introduction

Introduction to Analytical Chemistry:

Analytical chemistry may be defined as the science and the art of determining components of the material in terms of the element or the compounds contained. Analytical chemistry seeks ever improved means of measuring of chemical composition of natural and artificial materials. The techniques of the science are used to identify the substances which may be present in a material and to determine the exact amounts of the identified substances. Analytical chemist work to improve the reliability of existing technique to meet the demands for better chemical measurement, which arise constantly in our society, they adapt proven methodologies to new kind of material or to answer question about their composition and their reactivity mechanisms.

In industry, analytical chemistry provide the mean of testing raw material and for assuring the quality of finished products whose chemical composition is critical. Analytical techniques play an important role in assuring and maintaining the quality of substance which are critical components of Q.A. /Q.C. The reliability, utility, accuracy, interception and specificity of measurement is the responsibility of an analytical chemist. In general terms, pharmaceutical analysis comprises of those procedures which are necessary to determine the identity, strength, quality and purity of the drugs and chemicals.¹

The discipline of analytical chemistry consists of

Qualitative Analysis: Qualitative analysis deals with the identification of elements, ions or compounds present in sample.²
Quantitative Analysis: Quantitative analysis deals with the identification of much amounts of one or more constituents are presents in the sample.²

Various instrumental methods:

Spectroscopic methods
Thermal methods
Electrochemical methods
Chromatographic methods
Hyphenated Techniques

Introduction to methods for quantitative analysis by UV-visible spectroscopy.

Quantitative Spectrophotometric assay of medicinal substances. Preparing a solution in a transparent solvent and measuring its absorbance at a suitable wavelength may quickly carry out the assay of an absorbing substance. The wavelength normally selected is a wavelength of maximum absorption (λmax) where small error is setting the wavelength scale has little effect on measured absorbance. Ideally the concentration should be adjusted to give an absorbance of approximately 0.9, around which the accuracy and precision of the measurement are optimal. The concentration of absorbing substances is then calculated from the measured absorbance using one of three following principal procedures,

Use of standard absorptivity value
Use of calibration graph
Single or double point standardization

MATERIALS:

For developing a simple, accurate, precise method the following material were used.

Table 1-List of Materials

Sr. No.	Material	Source
1	Reltegravir Potassium	Mylan Laboratory, Aurangabad
2	Lamivudine	Mylan Laboratory, Aurangabad
3	Acetonitrile HPLC Grade	Changshu Yanhun Chemicals

EXPERIMENTAL METHOD

Simultaneous Estimation of Reltegravie Potassium and Lamivudine by Uv-Spectrophotometric Method.

Method of estimation

Method A: Q-Analysis Method.
Method B: Multicomponent Method.

RESULT

U.V-VISIBLE SPECTROSCOPIC METHODS OF LAM AND REL

Selection of analytical wavelength for simultaneous estimation of drug.

Here λ max 269 nm for LAM and 330 nm for REL were selected. Fig. 1 & Fig.2 represent the spectra of LAM and REL.

Figure 1- U. V. Spectra of Lamivudine

Figure 2U. V. Spectra of Reltegravir Potassium

Calibration curve of LAM and REL

The standard calibration data and Calibration curves for LAM and REL are given in Table 2 & Table 3 and Fig. 3 and Fig 4 respectively.

Table 2 Standard Calibration data for LAM.

Sr. No.	Concentration (µg/ml)	Absorbance ( nm)
1	5	0.053
2	10	0.106
3	15	0.159
4	20	0.21
5	25	0.242
6	30	0.318
7	35	0.371
8	40	0.426
9	45	0.477
10	50	0.531

Figure 3-Standard calibration curve of LAM.

Table 3Standard Calibration data of REL.

Sr. No.	Concentration (µg/ml)	Absorbance ( nm)
1	10	0.088
2	20	0.178
3	30	0.294
4	40	0.392
5	50	0.491
6	60	0.588
7	70	0.686
8	80	0.784
9	90	0.882
10	100	0.981

Figure 4 Standard calibration curve of REL.

ESTIMATION OF LAM AND REL BY Q-ANALYSIS METHOD

Selection of the iso-absorptive point.

Isobestic point is selected at 247.0 nm, the overlain spectra of both drugs in Fig. 5.

Figure 5 Overlain Spectra of LAM and REL for Q-Analysis

Analysis data of Mixed Standards by Q-Analysis Method.

The results obtained were accurate and reproducible shown in table 4.

Table 4: Analysis of Mixed Standards by Q-Analysis Method.

Sr. No.	Conc. (µg/ml)		% of drugs found*		% R.S.D.*
Sr. No.	LAM	REL	LAM	REL	LAM	REL
1	5	10	99.49	99.88	0.132	0.1089
2	10	20	99.53	99.91	0.055	0.0731
3	15	30	99.53	99.91	0.050	0.0889
4	20	40	99.66	99.90	0.037	0.0861
5	25	50	99.58	99.87	0.085	0.0565

*Average of six readings

From Table 4 it was studied that % R.S.D was found to be less than 2.

Analysis data of Tablet formulations by Q- Analysis Method.

Analysis of Tablet Formulations and its statistical validation by Q- Analysis method is given in Table 5. and Table 6.

Table 5 Analysis of Tablet formulations by Q-Analysis method.

Sr. No.	Amount Present (mg)		Amount Found (mg)		% Amount
Sr. No.	LAM	REL	LAM	REL	LAM	REL
1	150	300	149.70	296.52	99.80	98.84
2	150	300	149.37	299.64	99.58	99.88
3	150	300	149.50	300.60	99.67	100.2
4	150	300	150.10	294.75	100.1	98.25
5	150	300	146.73	299.22	97.82	99.74
6	150	300	147.45	293.85	98.30	97.95

Statistical validation of Tablet Formulation by Q- Analysis Method

Table 6 Statistical validation of tablet formulation

Conc.( mg)		% drug found*		S.D.		% R.S.D*
LAM	REL	LAM	REL	LAM	REL	LAM	REL
150	300	98.71	99.31	0.9464	0.9545	0.9587	0.9611

*Average of six determinations

From Table 6 it was found that the S.D and %R.S.D were less than 2 by Q- Analysis Method.

ESTIMATION OF LAM AND REL BY MULTICOMPONENT METHOD

Spectra of both the drugs by multi component Method

Figure 6 Overlain Multicomponent Spectra of LAM and REL

Analysis data of Mixed Standards by Multicomponent method of analysis

The results of Multicomponent analysis are given in Table 7.

Table 7: Analysis data of mixed standards by Multicomponent Method of analysis

Sr. No.	Conc. (µg/ml)		% of drug found		%RSD*
Sr. No.	LAM	REL	LAM	REL	LAM	REL
1	5	10	99.89	99.87	0.052	0.087
2	10	20	99.85	99.91	0.069	0.041
3	15	30	99.88	99.90	0.063	0.046
4	20	40	99.86	99.89	0.079	0.038
5	25	50	99.88	99.89	0.070	0.041

*Average of Six Readings.

3. Analysis data of Tablet Formulation by Multicomponent Method of analysis

The results of Tablet Formulation and its statistical validation are given in Table 8. and Table 8.9.

Table 8 Analysis data of Tablet Formulation by Multicomponent method of analysis

Sr. No.	Amount Present (mg)		Amount Found (mg)		%Amount Found
Sr. No.	LAM	REL	LAM	REL	LAM	REL
1	150	300	149.73	299.94	99.82	99.98
2	150	300	149.74	299.88	99.83	99.96
3	150	300	149.67	299.70	99.78	99.90
4	150	300	149.92	299.82	99.95	99.94
5	150	300	149.80	299.88	99.87	99.96
6	150	300	149.77	299.82	99.85	99.94

Table 9 Statistical validation of Tablet Formulation by Multicomponent method of analysis

Conc. (mg)		% of drug found*		S.D.		%R.S.D.*
LAM	REL	LAM	REL	LAM	REL	LAM	REL
150	300	99.85	99.94	0.0576	0.0384	0.0576	0.0384

*Average of Six determinations

From Table 9. it was found that the S.D and %R.S.D were less than 2 by Multicomponent method of analysis.

VALIDATION OF DEVELOPED UV-SPECTROPHOTOMETRIC METHODS

Precision.

Intermediate precision (Intra-day and Inter-day precision).

The S.D. and % R.S.D. were calculated and are presented in Table 10.

Table 10 Statistical validation for Intermediate Precision

Method	Drug	Intra-day Precision*			Inter-day Precision*
Method	Drug	% Mean	S.D	%R.S.D.	% Mean	S.D.	%R.S.D.
A	LAM	99.90	0.05921	0.05926	99.93	0.06324	0.06328
A	REL	99.87	0.05752	0.05759	99.90	0.05683	0.05688
B	LAM	99.86	0.03653	0.03658	99.91	0.06462	0.06467
B	REL	99.90	0.04633	0.04637	99.89	0.05357	0.05362

*Average of six determinations.

From Table 10.it was found that the S.D and %R.S.D were less than 2 by all two Methods. Hence, the methods are precise.

Limit of Detection (LOD) and Limit of Quantitation (LOQ).

The results of the same are shown in Table11.

Table 11 Limit of Detection (LOD) and Limit of Quantitation (LOQ)

Method	Limit of Detection (LOD)*		Limit Of Quantitation (LOQ)*
Method	LAM	REL	LAM	REL
A	0.2548	0.6254	0.4584	0.7589
B	0.2814	0.6598	0.4825	0.7963

*Average of six determinations.

From Table 11. The LOD and LOQ by the two methods were found to be in the range as per ICH guidelines.

Robustness Studies.

The S.D. and % R.S.D. were calculated and are presented in table 8.12.

Table 12Robustness Study

Method	Parameters	% Mean		S.D.*		%R.S.D.*
Method	Parameters	LAM	REL	LAM	REL	LAM	REL
A	Instrument	99.91	99.91	0.04593	0.06153	0.03659	0.06158
A	Analyst	99.90	99.89	0.03898	0.05316	0.03901	0.05321
B	Instrument	99.88	99.91	0.05741	0.06583	0.05747	0.06588
B	Analyst	99.90	99.92	0.06377	0.05692	0.06383	0.05696

*Average of six determinations.

From Table 12 it is studied that there are no specific changes in the observations for both the drugs. The S.D and R.S.D were found to be less than 2. Hence all the two methods are Robust.

Recovery studies.

The Result of Recovery Studies of tablet given in table 13 to table 17

Table 13 Recovery Studies for Tablet formulation by Q-Analysis Method

Level of % Recovery	Amount Present (mg/tablet)		Amount of Standard added (mg)		Total amount recovered (mg)		% Recovery*
Level of % Recovery	LAM	REL	LAM	REL	LAM	REL	LAM	REL
80%	150	300	120	240	119.85	239.59	99.87	99.83
80%	150	300	120	240	119.82	239.66	99.85	99.86
80%	150	300	120	240	119.89	239.64	99.86	99.89
100%	150	300	150	300	149.79	299.94	99.86	99.96
100%	150	300	150	300	149.77	299.94	99.85	99.98
100%	150	300	150	300	149.82	299.52	99.88	99.84
120%	150	300	180	360	179.94	359.92	99.97	99.98
120%	150	300	180	360	179.85	359.82	99.92	99.95
120%	150	300	180	360	179.91	359.78	99.95	99.94

Table 14Statistical validation of Recovery Studies by Q- Analysis Method

Level of % Recovery	% Recovery		Mean % Recovery		S.D.*		%R.S.D.*
	LAM	REL	LAM	REL	LAM	REL	LAM	REL
80%	99.87	99.83	99.86	99.84	0.010	0.01527	0.010	0.01522
	99.85	99.86
	99.86	99.85
100%	99.86	99.98	99.86	99.93	0.01527	0.08082	0.01529	0.08087
	99.85	99.98
	99.88	99.84
120%	99.97	99.98	99.94	99.95	0.02516	0.02081	0.02517	0.02082
	99.92	99.95
	99.95	99.94

*Average of three determinants

Table 15 Recovery Studies for Tablet formulation by Multicomponent Method

Level of % Recovery	Amount Present (mg/tablet)		Amount of Standard added (mg)		Total amount recovered (mg)		% Recovery
Level of % Recovery	LAM	REL	LAM	REL	LAM	REL	LAM	REL
80%	150	300	120	240	119.78	239.66	99.82	99.86
80%	150	300	120	240	119.95	239.85	99.96	99.90
80%	150	300	120	240	119.85	239.66	99.88	99.84
100%	150	300	150	300	149.80	299.85	99.87	99.95
100%	150	300	150	300	149.89	299.91	99.93	99.97
100%	150	300	150	300	149.77	299.58	99.85	99.86
120%	150	300	180	360	179.73	359.85	99.85	99.96
120%	150	300	180	360	179.78	359.74	99.88	99.93
120%	150	300	180	360	179.96	359.71	99.98	99.92

Table 16 Statistical validation of Recovery Studies for Tablet formulation Multicomponent Method

Level of % Recovery	% Recovery		Mean %Recovery		S.D.*		%R.S.D.*
	LAM	REL	LAM	REL	LAM	REL	LAM	REL
80%	99.82	99.86	99.88	99.86	0.07211	0.04618	0.07219	0.04653
	99.96	99.90
	99.88	99.84
100%	99.87	99.95	99.88	99.92	0.04163	0.05859	0.04168	0.05863
	99.93	99.97
	99.85	99.86
120%	99.85	99.96	99.90	99.92	0.06806	0.04582	0.06812	0.04585
	99.88	99.93
	99.98	99.92

*Average of three determinants

Table 17 Statistical study of Recovery studies.

Method	Level of % Recovery	%Mean		S.D.		%R.S.D.
Method	Level of % Recovery	LAM	REL	LAM	REL	LAM	REL
A	80%	99.86	99.84	0.010	0.01527	0.010	0.01522
	100%	99.86	99.93	0.01527	0.08082	0.01529	0.08087
	120%	99.94	99.95	0.02516	0.02081	0.02517	0.02082
B	80%	99.88	99.88	0.07211	0.04618	0.07219	0.04653
	100%	99.88	99.92	0.04163	0.05859	0.04168	0.05863
	120%	99.90	99.92	0.06806	0.04582	0.06812	0.04585

*Average of three determination

From the Table 8.17 it is confirmed that the drug was recovered by adding the standard amount of LAM and REL in the tablet formulation. The S.D and % R.S.D were found to be less than 2. The results were found be accurate and satisfactory by all the two methods.

DISCUSSION

The UV Spectrometric methods were based on the development of calibration curve for Lamivudine and Reltegravir potassium separately was plotted and the isobestic point of the both drugs was found to be at 247.0 nm in Q Analysis (Method A). Further the solutions were scanned at their isobestic point 247.0 nm and at λ_maxof Lamivudine 269.0 nm. The analysis of the formulation was done. The % RSD values for the analysis were found to be less than 2. The drug recovered by this method was 99.88% for LAM and 99.90% for REL from the tablet formulation.
Method B was Multicomponent Method, calculations done by the inbuilt software between two selected wavelengths from the spectrum of the standard drug using the plot of Absorbance against the Wavelength. The drug recovered by this method was 99.88 % for LAM and 99.89% for REL from the tablet formulation.
Results obtained with proposed methods confirm the suitability of these methods for pharmaceutical dosage forms. The other active ingredients and excipients did not interfere in the estimation of the dosage form which was analyzed by the methods. The accuracy of methods was confirmed by recovery studies, by adding a known amount of pre analyzed pure drug to the pharmaceutical formulation. The drug recovered by this method was 99.88% for LAM and 99.90% for REL from the tablet formulation.
A comparative study of all the two methods is given in the table 18

Table 18 Comparative Study of U.V. Spectrophotometric Methods

Sr. No.	Parameters	Method A		Method B
Sr. No.	Parameters	LAM	REL	LAM	REL
1	λ_maxin nm	247.0nm	269.0nm	269.0nm	330.0nm
2	LOD	0.2548	0.6254	0.4584	0.7589
3	LOQ	0.2814	0.6598	0.4823	0.7963
4	%Recovery	99.88	99.90	99.88	99.89
5	Precision (RSD) intraday	0.05926	0.05759	0.06328	0.05688
6	Precision (RSD) inter-day	0.03658	0.03658	0.06467	0.06467

The method developed for the quantitative simultaneous estimation of Lamivudine and Reltegravir Potassium in combined tablet dosage form was simple, rapid, reproducible, accurate and precise.

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        Lakshmana Rao A., and Raghuram M.S., Validated Reverse Phase HPLC Method for Determination of Raltegravir in Pharmaceutical Preparations, Int. J. Res. Pharm. Chem. 2(1), 2012, 217-221.
        
        Rambabu K., Balmurti K. K., and Rao S., New RP - HPLC Method Development and validation for Analysis of Antiviral Drug Raltegravir, Int. J. Res. in Pharm. Biomed. Sci., 2 (1), 2011, 132-135.
        
        Indian Pharmacopoeia, Government of India Ministry Healthcare, 18thed., Published by Indian Pharmacopoeia Commission Gaziabad, Vol-II, 2014, 2054-2064.
        
        Sean C. S., Martindale, The Complete Drug Reference, 36th ed., published by Pharmaceutical Press, Vol-I, 902.
        
        www.drugbank.ca/drug/db06817, 2010.
        
        www.drugbank.ca/drug/db00109, 2005.

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Lakshmana Rao A., and Raghuram M.S., Validated Reverse Phase HPLC Method for Determination of Raltegravir in Pharmaceutical Preparations, Int. J. Res. Pharm. Chem. 2(1), 2012, 217-221.
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Patil Jyoti

Corresponding author

Pravara Rural Education Society’s Institute of Pharmacy, Loni, Ahmednagar

Nadare Archana

Co-author

Padm. Dr . D. Y. Patil College of Pharmacy, Akurdi, Pune

Patil Jyoti, Nadare Archana, Analytical Method Development and Validation for Simultaneous Estimation of Reltegravir and Lamivudine in Bulk and Pharmaceutical Formulation, Int. J. of Pharm. Sci., 2026, Vol 4, Issue 5, 293-305. https://doi.org/10.5281/zenodo.19993747

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Analytical Method Development and Validation for Simultaneous Estimation of Reltegravir and Lamivudine in Bulk and Pharmaceutical Formulation

Abstract

Keywords

Introduction

Reference

Patil Jyoti

Nadare Archana

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