1. INTRODUCTION:

The Combination antiretroviral therapy (cART) has evolved considerably over the past two decades leading to better control of human immunodeficiency virus (HIV), preservation of the immune system and decreased incidence of opportunistic infections, malignancies and deaths¹. The human immunodeficiency virus (HIV) is a retrovirus that infects the cells of the immune system and is characterized by a decline in CD4? cell count and immune function, which can result in life-threatening opportunistic infections (OI), HIV-related cancer and acquired immunodeficiency syndrome (AIDS). HIV represents a global pandemic affecting an estimated 34 million individuals and causing 1.7 million deaths through AIDS or HIV-related illness per year².

The most used antiretroviral therapy against HIV virus is based in the use and combination of two or three drugs from at least two different families. This combination is called “Highly Active Antiretroviral Therapy” (HAART)³. There are two drug combination for assessing the safety and efficacy of switching from stable antiretroviral regimens composed of two nucleosidenucleotide reverse transcriptase inhibitors (NtRTIs) plus a protease inhibitor (PI), INSTI or NNRTI to a combination of dolutegravir (DOL) plus rilpivirine (RIL)⁴. Dolutegravir sodium chemically, (4R,12aS)-9-{[(2,4-difluorophenyl)methyl]carbamoyl}-4-methyl-6,8-dioxo-3,4,6,8,12,12a-Hexahydro-2H-pyrido[1',2':4,5]pyrazino[2,1-b][1,3]oxazin-7-olate⁵. [Figure 1] Dolutegravir is a novel integrase stand transfer inhibitor active against Human Immunodeficiency Virus. Dolutegravir sodium is a white to light yellow powder and is slightly soluble in water⁶.

Figure 1: Structure of Dolutegravir

Chemically Rilpivirine is 4-[[4[(E)-2-cyanoethenyl]-2,6-dimethylphenyl]-2-pyrimidinyl]amino]benzonitrile.the chemical structure shown in figure 2⁷. A molecular formula of C22H18N6. It is mainly used to treat HIV-1 infection in combination with other antiretroviral agents⁸.

Figure 2: Structure of Rilpivirine

From Literature survey, various methods UV, HPLC⁹, HPTLC and LC-MS methods were reported for the estimation of these drugs either individually or combined with other drugs¹⁰. The objective of present investigation deals with development of simple, accurate, precise and more economical uv spectrophotometric method for estimation of dolutegravir and rilpivirine in synthetic mixture by simultaneous equation method and their validation as per ICH guidelines¹¹.

1.1. Simultaneous equation method (Vierodt’s Method):

If a sample contains two drugs with reasonably dissimilar λmax, each of which exhibits absorbance at the λmax of other, then it is possible to determine the drugs by simultaneous equation method (Vierodt’s Method) ^[12]. The information required is (a) The aborptivities of X at and λ1 and λ2 are ax1 and ax2 respectively (b) The aborptivities of Y at and λ1 and λ2 are ay1 and ay2 respectively. (c) The absorbances of the diluted sample at λ1 and λ2 are A1 and A2 respectively. Let Cx and Cy be the concentrations of X and Y respectively in the diluted sample. Two equations are constructed based upon the fact that at λ1 and λ2, the absorbance of the mixture is the sum of the individual absorbance of X and Y¹³.

At λ1 A1 = a X1 b Cx + aY1b Cy ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐-- (1)

At λ2 A2 = a X2 b Cx + aY2 b Cy‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐- (2)

For measurements in 1cm cell, b = 1, therefore,

^Cx ^{= A}2^ay1 ^{– A}1^ay2^/ax2^ay1 ^{– a}x1^ay2

^Cy ^{= A}1^ax2 ^{– A}2^ax1^/ax2^ay1 ^{– a}x1^ay2

Using the above two equations the concentration of component X and component Y in the sample mixture can be determined¹⁴.

Figure 3: Overlain spectra of component X, Y and Mixture containing X and Y

2. MATERIALS AND METHODS:

2.1. Instrument:

Instrument used was an UV‐Visible double beam spectrophotometer, make: Shimadzu (model UV‐1800) with a pair of 1 cm matched quartz cells. With spectral bandwidth of 1 nm, wavelength accuracy of ± 0.3nm and 1.0cm matched quartz cells was use.

2.2 Chemicals and reagents:

Pharmaceutical grade DOL and RIL was obtained as a gift by Cipla Pharmaceuticals, Mumbai. India. Methanol were purchased from MERK laboratory Mumbai India. All other chemicals and reagents used were for analytical grade.

2.3. Preparation of Stock Solutions:

Accurately weighted 10mg of Dolutegravir and Rilpivirine was transferred to 100ml volumetric flask, dissolved separately in Methanol: Water (8:2) that give final concentration of 100µg/ml both the drugs.

2.4. Preparation of working solution:

Appropriate volume 1ml and 0.25ml of standard stock solution of Dolutegravir and Rilpivirine was transferred into 10ml volumetric flask, diluted to mark with Methanol: Distilled Water (8:2) to give 10μg/ml and 2.5 μg/ml concentration of each drugs. The resulting solution was scanned in UV range (200nm – 400nm). In spectrum Dolutegravir and Rilpivirine showed absorbance maximum at 259.20 nm and 305.80 nm respectively.

2.5. Method: Spectrophotometric Method for Estimation of Dolutegravir and Rilpivirine HCl in Synthetic Mixture by Simultaneous Equation Method

2.5.1. Selection of analytical wavelength:

Appropriate dilutions were prepared for drugs from the standard stock solutions and scanned in the spectrum mode from 400nm to 200nm and Dolutegravir and Rilpivirine showed absorbance maxima at 259.20 nm and 305.80 nm respectively.

2.6. Preparation of Calibration curve:

From the standard stock solution of Dolutegravir and Rilpivirine, appropriate aliquot was pipetted out into 10 ml volumetric flask and dilution was made with Methanol:Water (8:2) to obtain concentration 100µg/ml. Appropriate aliquots were pipette out into 10ml volumetric flasks and dilutions were made with Methanol:Water (8:2) to obtain working standard solutions of concentrations 4, 8, 12, 16, 20, 24µg/ml. Absorbance’s for these solutions were measured at 259.20nm (Table 1) and a calibration curve of absorbance against concentration was plotted figure 4. Similarly, a series of standard solutions of concentration 1, 2, 3, 4, 5, 6, 7, 8µg/ml were prepared for and their absorbance’s were measured at 305.80nm (Table 1). A standard calibration curve of absorbance against concentration was plotted. Both drugs followed the beer lamberts law in the range of 4-24µg/ml and 1-8µg/ml for DOL and RIL respectively. In Table. 2. summarises the optical characteristics of both the drugs.

Figure 4: Overlain Spectrum of Dolutegravir and Rilpivirine

Table 1: Standard Calibration Table DOL and RIL

Concentration in µg/ ml	Abs. of DOL at 259.20nm	Concentration in µg/ ml	Abs. of RIL at 305.80nm
0	00	0	00
4	0.220	1	0.099
8 10	0.346 0.402	2	0.202
12	0.511	3	0.398
16	0.943	4	0.539
20	0.925	5	0.671
24	1.604	6	0.819
		7	0.983
		8	1.120

Figure 5: Calibration Curve of Dolutegravir λmax 259.20nm.

Figure 6: Calibration Curve of Rilpivirine λmax 305.80 nm.

Table 2: Optical characteristics and Statistical data of the Regression equation in Simultaneous equation methods.

Parameter	Dolutegravir	Rilpivirine
λ max (nm)	259.20 nm	305.80 nm
Beer’s law limits (μg / ml)	4-24 μg/ml	1-8 µg/ml
Regression equation (Y*)	Y = 0.041x + 0.013	Y = 0.148x - 0.062
Slope (b)	0.041	0.148
Intercept (a)	0.013	0.062
Correlation coefficient (r2)	0.999	0.998

2.7. Determination of Absorptivity Values of drugs at Selected Wavelengths:

Aliquot portions of DOL from stock solution were transferred to 10ml volumetric flasks; volume was adjusted to mark to obtain the concentration of 10µg/ml. Similarly, Aliquat portions of RIL from stock solution were transferred to three 10ml volumetric flasks; volume was adjusted to mark to obtain the concentration of 2.5µg/ml. Absorbance of these solutions were recorded at two wavelength 259.20nm and 305.80nm. Results of Absorptivity values of drugs are shown in Table No.3 for DOL and RIL respectively.

Table 3: Absorptivity Measurement.

Components.	Absorptivity at
Components.	259.20nm	305.80nm
DOL	189	462
RIL	140	596

2.8. Application of Proposed Method for Simultaneous Estimation of Drug in Laboratory Solution:

In order to see the feasibility of proposed method for estimation of Dolutegravir and Rilpivirine in marketed pharmaceutical formulations, the method was first tried for estimation of drugs in standard laboratory solution. Accurately about 10mg of pure drug (DOL) and 10mg of another pure drug (RIL) was weighed and transferred in the 100ml volumetric flask Dissolve in Methanol: Distilled Water (8:2) to give stock solution of concentration (100µg/ml). Appropriate aliquot portion (10 and 2.5mg/mL) was transferred to 10mL volumetric flask and diluted with Methanol: distilled water (8:2) to obtain the concentration of 10/2.5µg/mL respectively. Absorbance was measured in the range 259.20nm and 305.80nm against solvent as blank. Concentrations of drugs were determined by using simultaneous equation 3 and 4 and results are reported in Table 4.

Where,

C_Xand C_y = Concentration of DOL and RIL respectively

A₁and A₂= Absorbance at 259.20 nm and 305.80 nm

a_x1 = 189 a_x2 = 462

a_y1 = 140 a_y2= 596 CX=

CY=

% Drug found was calculated using following formula:

% Estimation =×100

Table 4: Analysis of DOL and RIL in Synthetic Mixture

Sr. No.	Absorbance at		%Estimation
Sr. No.	259.20nm	305.80nm	Dolutegravir	Rilpivirine
1	0.612	0.537	100.48	99.16
2	0.613	0.538	100.63	99.33
3	0.610	0.537	99.73	100.14
		Mean	100.28	99.54
		SD	0.482	0.524
		%RSD	0.480	0.527

2.9. Validation of Proposed method:

The Proposed method was validated as per the ICH guidelines. To the pre-analysed sample solutions (20 µg/ml of DOL and 1 µg/ml of RIL), a known amount of standard solutions of the pure drugs (DOL and RIL) were added i.e. 16, 20, and 24 µg/ml of DOL and 4.5, 5 and 5.5 µg/ml of RIL (standard stock solution) was added, and total conc. of above dilution is measured by using equation I and II. The results are reported in Table 5.

Where, A= Total amount of drug estimated

B= Amount of drug found on preanalysed bases

C= Amount of pure drug added

Table 5: Results of Recovery Studies

Sr. No.	Conc. In µg/ml		Amount Added (µg/ml)		Absorbance At		Amount Recovered		% Recovery
Sr. No.	DOL	Ril	DOL	RIL	259.20nm	305.80nm	DOL	RIL	DOL	RIL
1	10	2.5	18	4.5	1.095	0.973	17.84	4.54	100.8	99.11
2	10	2.5	20	5.0	1.206	1.050	19.89	5.09	100.5	99.86
3	10	2.5	22	5.5	2.406	0.299	22.15	5.69	99.32	99.27
								Mean	100.20	99.41
								SD	0.7823	0.3950
								%RSD	0.7807	0.3973

Table 6: Results of Accuracy Studies

Sr. No.	% of	Conc. In µg/ml	Amount Added (µg/ml)		% Recovery
Sr. No.	Drug	DOL RIL	DOL	RIL	DOL	RIL
1	80	10 2.5	18	100.8	99.11	99.50
2	100	10 2.5	20	100.5	99.86	99.86
3	120	10 2.5	22	99.32	99.27	99.29
				Mean	100.20	99.41
				SD	0.7823	0.3950
				% RSD	0.7807	0.3973

3. Validation Parameters:

3.1. Accuracy:

Accuracy of an analytical method is the closeness of the test results obtained by that of the true value. It was ascertained on the basis of recovery studies performed at different levels of concentrations. (Table-6).

3.2. Precision:

Precision of an analytical method is the degree of agreement among individual test results. It is expressed as ± S.D. or %RSD of series of measurements. Precision of the method was verified by using stock solutions in the ratio of 1:4 containing 10µg/ml DOL and 2.5µg/ml of RIL. System repeatability was done by repeating the assay three times of three replicate dilutions of the same concentration after every two hours on the same day for intraday precision. Interday precision was carried out by performing the assay of tree sample sets after 24 hours and 48 hours, results are reported in Table 7 and 8.

Table7: Results of Precision Studies (Intra-day)

Sr.No.	Absorbance at		%Estimation*
Sr.No.	259.20nm	305.80nm	Dolutegravir	Rilpivirine
Obser.1	0.612	0.537	100.48	99.16
Obser.2	0.613	0.539	100.52	99.71
Obser.3	0.614	0.541	100.56	100.26
		Mean	100.52	99.71
		SD	0.400	0.550
		%RSD	0.397	0.551

*Results are mean of three replicates

Table 8: Results of Precision Studies (Inter-day)

Sr. No.	Absorbance at		%Estimation*
Sr. No.	259.20nm	305.80 nm	Dolutegravir	Rilpivirine
Day 1	0.615	0.542	100.70	100.47
Day 2	0.614	0.539	100.78	99.57
Day 3	0.613	0.541	100.56	100.26
		Mean	100.68	100.10
		SD	0.1113	0.6586
		%RSD	0.1101	0.6579

*Results are mean of three replicates

3.3. Ruggedness:

Ruggedness of the proposed method is determined by analysis of aliquots from homogenous slot by two analyst using same operational and environmental conditions, the results are reported in Table 9.

Table 9: Results of Different analyst studies

Sr. No.	Absorbance at		%Estimation*
Sr. No.	259.20 nm	305.80nm	Dolutegravir	Rilpivirine
Analyst 1	0.615	0.542	99.94	99.44
Analyst 2	0.614	0.539	99.33	99.83
		Mean	99.63	99.63
		SD	0.4313	0.2757
		%RSD	0.4329	0.2767

* Results are mean of three replicates

3.4. Robustness:

It expresses the precision within laboratories, Variation like different solvent. Robustness of the methods was assessed by carrying out assay 3 times with different solvent by using same equipment. The results of the same are presented in Table 10.

Table 10: Results of Different solvent studies

Solvent Methanol: Water	Absorbance at		%Estimation*
Solvent Methanol: Water	259.20 nm	305.80 nm	Dolutegravir	Rilpivirine
9:1	0.610	0.540	99.62	100.4
7:3	0.609	0.539	99.47	100.2
		Mean	99.54	100.3
		SD	0.1060	0.1414
		%RSD	0.1064	0.1409

4. RESULT AND DISCUSSION:

A new drug combination containing Dolutegravir 50 mg and Rilpivirine HCl 25mg was selected for Analytical method development as no any method was reported for its simultaneous estimation. Pure samples of Dolutegravir sodium and Rilpivirine HCl were procured as gift sample from industries.

4.1. Method: UV-Spectrophotometric Method for Estimation of Dolutegravir and Rilpivirine HCl in Synthetic Mixture by Simultaneous Equation Method:

As both drugs are soluble in 8:2(Methanol: Water) solution estimation was carried out by dissolving in solvent and diluting with same solvent. DOL and RIL showed absorbance maxima at 259.20nm and 305.8nm, respectively. Since there λmax differ by more than 15 nm and at λmax of one drug other drug absorbs minimum. Both these drugs, obeyed linearity in the concentration range 4 - 24µg/ml and 1- 8µg/ml, respectively. Absorbance of both drugs was found to additive at both wavelengths. The proposed method was applied for synthetic mixture and % estimation of DOL and RIL was found to be 100.28 and 99.54, respectively. The amount of drugs estimated by proposed method was in good agreement with the estimation. Accuracy of the method was checked by recovery studies at different levels. The % recovery for DOL and RIL was found to be in the range 99.32 – 100.20 and 99.11 – 99.86, respectively; the %RSD values less than 2 indicative of accuracy of the method. The method was found to be precise as indicated by the inter-day, intra-day and repeatability analysis; showing %RSD less than 2. The result did not show any statistical difference between operators suggesting that method developed was rugged. The summary of validation parameters is reported in Table 11

Table 11: Summary of Validation Parameter DOL and RIL

Parameters	DOL	RIL
Linearity range [µg/ml]	4 – 24	1– 8
Regression equation [Y = mX +C]	Y = 0.041x + 0.013	Y = 0.148x - 0.062
Recovery [% RSD, n = 3]	0.7807	0.3973
Precision [% RSD]
Intra-day [n = 3]	0.397	0.551
Inter-day [n = 3]	0.1101	0.6579
Ruggedness [% RSD]
Analyst 1 [n = 3]	0.4329	0.2767
Analyst 2 [n =3]	0.4329	0.2767
Robustness [% RSD]
9:1 MeOH:H2O[n = 3]	0.1064	0.1409
7:1 MeOH:H2O [n = 3]	0.4329	0.2767

5. CONCLUSION:

The UV- Spectrophotometric ‘Simultaneous equation methods have been developed for determination of DOL and RIL. From the statistical results, it can be concluded that ‘simultaneous equation’ is more accurate, precise and simple as least calculations are involved. Statistical analysis proves that the developed methods can be used for routine analysis of said drugs in synthetic mixture.

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Received on 09.04.2022 Modified on 10.05.2022

Asian J. Pharm. Ana. 2022; 12(3):181-186.

DOI: 10.52711/2231-5675.2022.00031