Extractive determination study of Etravirine by using Tpooo as an Analytical reagent in Pure and Pharmaceutical dosage forms

Murali Dadi¹*, Indra Sen Singh¹, Purnachandra Rao G²

¹Department of Chemistry, Copperbelt University, Jambo Drive, Kitwe, Zambia.

²Department of Chemistry, NRI Institute of Technology, Pothavarpadu, Agiripalli, Andhra Pradesh, India.

*Corresponding Author E-mail: murali.dadi@gmail.com

ABSTRACT:

This study developed a sensitive and straightforward extractive spectroscopic method to estimate Etravirine (ETR) using TPooo as an analytical reagent in pure and pharmaceutical dosage forms. This method was achieved based on the extractable chloroform complex formed with Tropeoline ooo (TPooo) in an acidic media. Following Beer’s law, the extractable complex showed the absorbance maximum at 485nm at the concentration ranges between 12.5-75 µg/ml with the molar absorptivity 2.195 x 10³ L/mole /cm and the Sandell’s sensitivity 0.1549 µg cm^-2. The result of Etravirine estimation for the present method has been validated statistically by recovery studies, and the developed method was simple, sensitive, accurate, and precise. It was validated following International Conference on Harmonization (ICH) guidelines and also successfully applied for the estimation of Etravirine in tablet dosage forms.

KEYWORDS: Etravirine, Extractive Spectrometry, TPooo, tablet dosage forms.

INTRODUCTION:

Etravirine (ETR), a non-nucleoside analog, is a diarylpyrimidine second-generation non-nucleoside reverse transcriptase inhibitor¹. ETR is active against HIV that confers resistance to the commonly prescribed first-generation non-nucleoside reverse transcriptase inhibitors. ETR binds directly to reverse transcriptase and hinders the RNA-dependent and DNA-dependent DNA polymerase activities. The result is the blockage of the growth of HIV². In combination with other antiretrovirals, ETR is designated for treating HIV type 1 infection in patients who are resistant to non-nucleoside reverse transcriptase inhibitor and other antiretroviral agents^3,4,5. Various methods are reported in the literature for ETR determination in biological and pharmaceutical samples. They include UV spectrophotometry^6,7,8, UPLC^9,10,11, LC-MS^12-16, HPLC-MS^17,18, HPTLC¹⁹, HPLC^20,21 and Pharmacokinetic studies²².

The UV spectrophotometry method^6,7,8 is less selective as it involves absorbance measurements at a shorter wavelength where the interference from the excipients is more. The reported UPLC, LC-MS, HPTLC, and HPLC methods involve time-consuming procedures, sophisticated and expensive instruments that are mostly not available in pharmaceutical quality control laboratories. Furthermore, the LC-MS methods were applied for the determination of ETR in biological fluids.

To the best of our knowledge, few visible spectrophotometric methods are found in the literature to quantify ETR. Also, no UV spectrophotometric method has been reported by using TPooo. Hence the need arises to develop specific sensitive, precise, accurate, and flexible visible spectrophotometric methods, which prompted the author to choose ETR for the investigation based on various chemical reactions by exploiting various functional groups from the structure. The present study describes the estimation of ETR through an extractable ion-pair complexation of ETR with Tpooo.

MATERIALS AND METHODS:

Instrumentation:

The present investigation used an ELICO (Hyderabad, India) double beam model SL 244 digital spectrophotometer with 1cm matched quartz cells. Samples were weighed by using a Schimadzu electronic weighing balance (Kyoto, Japan) TW223L model.

Materials and reagents:

This study used analytical grade chemicals. ETR was obtained as a gift sample from Arabindo Laboratories Pvt. Ltd, Hyderabad, and used as received. The Tropeoline ooo (TPooo) was procured from Merk specialties Pvt. Ltd, Mumbai, India. Hydrochroric acid and chloroform were procured from Sd-fine Chemicals, Mumbai, India. All the solutions were prepared using double distilled water.

Preparation of stock and standard working solutions:

A stock solution of ETR was prepared by dissolving 100 mg of drug in 20mL of methanol in a 100mL volumetric flask and then made up to the mark with distilled water (1.0mg/mL). The stock solution was diluted stepwise with the distilled water to obtain standard working solutions of 250μg/mL concentration for this method.

The other solutions were freshly prepared for this study, as described below.

The Tropeolineooo (TPooo) solution of 0.2% (w/v) concentration was prepared by dissolving 200mg of TPooo (Merk specialties Pvt. Ltd, Mumbai, India) in 100mL of distilled water.

The 0.1M HCl (v/v) hydrochloric acid was prepared by diluting 8.6ml of concentrated HCl (Sd-fine Chemicals, Mumbai, India) to 1000 mL with distilled water.

General Assay Procedure:

The aliquots of (0.5–3.0 mL) standard ETR solution (250 μg/ml) were transferred into a series of 125mL separating funnels. The volume in each separating funnel was adjusted to 3.0mL with distilled water. The volumes of 6.0mL of 0.1 M HCl and 2.0mL of 0.2% TPooo were added to each separating funnel, which leads to the formation of an ion association complex between the ETR and TPooo. The funnels were shaken vigorously with 10 mL of chloroform for 20 minutes and then allowed for a clear separation of the two phases. The separated organic phase was transferred to a 10 mL volumetric flask. The extract was diluted to the mark with chloroform and mixed well. The absorbance of the colored ion-pair complex was measured at 485 nm against a similar reagent blank. The amount of ETR was deduced from the calibration curve.

Assay of Etr in Tablet Dosage Form:

Intelence tablets (Janssen Pharmaceuticals, Inc., Titusville, NJ) labeled to contain 100mg/200 mg ETR per tablet were used in the present investigation. Twenty tablets were weighed and finely powdered. An accurately weighed quantity of the powder equivalent to 100mg of ETR was transferred into a 100mL calibrated flask and dissolved in about 20ml of methanol. The contents of the flask were swirled, sonicated for 10 min, and filtered through Whatmann No. 1 filter paper. The filtrate was completed to volume with distilled water. This solution was diluted quantitatively with distilled water to obtain suitable concentrations for the analysis by the proposed spectrophotometric method. The content of ETR in the tablets was calculated from the corresponding calibration curve or corresponding regression equation.

Optimization of experimental conditions:

The experimental variables were optimized to achieve maximum sensitivity and adherence to Beer’s law. The optimization was achieved by carrying out control experiments by measuring the absorbance at 485 nm for several solutions. The control experiments involved the variation in one and fixing the other parameters in each case, such as the concentration of TPooo, HCl, and organic solvent used for extraction.

RESULTS AND DISCUSSION:

Optimization of experimental conditions:

The experimental variables in the proposed spectrophotometric method, which was found to affect the color intensity and stability of the resulting colored complex, were optimized following procedure 2.6, as described earlier.

The results are incorporated in (Table 1).

Determination of absorption maxima (λ_max):

To determine the analytic wavelength, the absorption spectra of the colored chromogen were scanned in the wavelength region of 400-700 nm against a corresponding reagent blank. The results are graphically represented (Figure 1).

Figure 1: Absorption spectrum of ETR-TPooo

Method Validation:

The proposed method was validated for linearity, sensitivity, selectivity, stability of the colored species, accuracy, precision, robustness, and recovery according to the current ICH guidelines (ICH, 2005)²³.

Linearity and sensitivity:

Under the optimum conditions, a linear relation was obtained between absorbance and concentration of ETR in the ranges given in (Table 2). The calibration graph (Figure 2) in each instance is described by the equation: Y = mX + c, (where Y = absorbance, c = intercept, m = slope and X = concentration of ETR in μg/ml). The regression coefficient, intercept, and slope for the calibration data are summarized in (Table 2). The values specify a good correlation between absorbance values and concentration of ETR in the proposed method.

Sensitivity parameters, such as apparent molar absorptivity and Sandell’s sensitivity values, the limits of detection (LOD), and limits of quantification (LOQ) are calculated as per the current International Conference on Harmonization guidelines (ICH, 2005) and compiled in (Table 2). The values indicated that the proposed method has adequate sensitivity for the analysis of ETR.

Table 2: Optical characteristics, precision and accuracy of the proposed methods for the proposed spectrophotometric method

Parameter	Values
Linearity range (μg/ml)	12.5-75
Molar Absorbtivity (L/mole/cm)	2.195 x 10³
Sandell’s sensitivity (µg cm^-2)	0.1549
Optimum photometric range(μg/ml)	10.0-100.0
Regression equation (Y=mx+c)^*	Y = 0.0062x + 0.0030
Regression coefficient (R²)	0.9975
Slope (m)	0.0062
Intercept (c)	0.0030
LOD (µg/ml)	0.760
LOQ (µg/ml)	2.303
Stability of colored species (hr)	1.10

*Y = Absorbance; x = Concentration of drug in μg/ml

Figure 2: Beer’s law plot of ETR-TPooo

Stability of colored species:

The stability of the colored species formed in the proposed method was also studied. The stability determination was done by measuring the solution's absorbance at their corresponding optimum wavelength at regular intervals of time. The results are presented in (Table 2). In the proposed method, the colored species formed was stable for at least more than 1 hr. The considerable stability helped in proceeding with a large number of samples and their comfortable measurements.

Accuracy and Precision:

Pure ETR solution at three different concentration levels (12.5, 37.5 and 75µg/ml) were prepared and analyzed in five replicates during the same day (intra-day precision) to determine the accuracy and precision of the proposed method. The same exercise was repeated on three consecutive days (inter-day precision) by the proposed method. The results are presented in Table 3.

The percentage recovery of intra- and inter-day analysis was in the range of 99.90-100.16%, 99.62-100.12%, 99.60-100.18%, 99.40-100.0%, 99.04-100.12%, and 99.40-100.25% for the present method. These values indicate that the accuracy of the method is satisfactory.

Percentage relative standard deviation for intra- and inter-day analysis was in the range of 0.061-0.190%, 0.093-0.725%, 0.079-0.547%, 0.069-0.518%, 0.108-1.247% and 0.207-1.310% for this method. The low values were indicating the repeatability of the proposed methods in the routine analysis of ETR.

Robustness:

The experimental variables were altered deliberately, and the effect of these changes on the performance of the proposed method was studied to evaluate the robustness of the proposed spectrophotometric method. The robustness of the method was studied at two different concentration levels (12.5 and 75µg/ml).

The volume change of 0.1M HCl solution from 5.8mL to 6.2mL at a constant volume of 0.2% TPooo reagent and volume change of 0.2% TPooo from 1.8mL to 2.2 mL at a constant volume of 0.1M HCl was used to test the robustness of the method. The results of this study are presented in (Table 4). From the % RSD values presented in Table 7 (0.020-0.126%), it can be concluded that the proposed methods are robust.

Table 4. Robustness of the proposed spectrophotometric method

Parameter	Concentration of ETR (μg/ml)		SD	Recovery (%)	RSD (%)
Parameter	Taken	Found*	SD	Recovery (%)	RSD (%)
Volume of 0.1M HCl (6.0 ± 0.2 ml)	12.5	12.48	0.0141	99.84	0.113
Volume of 0.1M HCl (6.0 ± 0.2 ml)	75	74.98	0.0150	99.97	0.020
Volume of 0.2% TPooo (2.0 ± 0.2 ml)	12.5	12.52	0.0158	100.16	0.126
Volume of 0.2% TPooo (2.0 ± 0.2 ml)	75	74.97	0.0187	99.96	0.024

* Average of three determinations

Analysis of Tablets Containing Etravirine:

The proposed method was successfully applied to the estimation of ETR in commercially available tablets (Intelence tablets, Janssen Pharmaceuticals, Inc., Titusville, NJ, labeled to contain 100 mg/200 mg ETR per tablet). The results are compiled in (Table 8). The excellent percent recovery value with a low relative standard deviation (<1%) value confirms the suitability of the proposed method for the routine analysis of ETR in tablet dosage forms.

The results obtained were statistically compared with the labeled claim values by applying the Student’s t-test for accuracy and F-test for precision at 95% confidence level. As can be seen from (Table 5), the calculated t- and F- values at the 0.05 confidence level did not exceed the tabulated values of 1.833 and 5.19, respectively. These findings indicate that the proposed method possesses sufficient accuracy and precision

Table 3: Evaluation of intra-day and inter-day precision and accuracy of the proposed spectrophotometric method

Intra-day precision and accuracy
Concentration of ETR (μg/ml)		SD	RSD(%)	Recovery (%)	Confidence limit
Taken	Found*	SD	RSD(%)	Recovery (%)	0.05	0.01
12.5	12.488	0.02126	0.170	99.90	0.01776	0.02630
37.5	37.503	0.02315	0.061	100.00	0.01935	0.02863
75	75.07	0.08258	0.110	100.09	0.06905	0.10215
Inter-day precision and accuracy
Concentration of ETR (μg/ml)		SD	RSD(%)	Recovery (%)	Confidence limit
Taken	Found*	SD	RSD(%)	Recovery (%)	0.05	0.01
12.5	12.490	0.02383	0.190	99.92	0.01992	0.02947
37.5	37.498	0.02932	0.078	99.99	0.02451	0.03627
75	75.125	0.12094	0.160	100.16	0.10112	0.14961

* Average of five determinations

Table 5. Assay of ETR in tablets by the proposed spectrophotometric method

Labeled claim (mg)

Found *

RSD

% Recovery

t-Value **

F-Value ***

100

99.50

0.02640

0.2653

99.50

0.3629

1.2975

200

0.06029

0.0301

99.97

0.1254

1.5027

*Average of six determinations

** tabulated t value = 1.833

*** tabulated F value = 5.19

Chemistry of The Colored Products:

The result obtained in the present method was based on extractive spectrophotometry. The ETR exhibits basic character essentially due to the presence of an amino group. Under the acidic condition, the amino group of ETR was protonated to form ammonium derivative of Etravirine. The sulphonic group of TPooo²⁴disassociates to form a negatively charged sulphonate derivative of TPooo. The positively charged ETR derivative and anionic TPooo thus generated form a yellow colored ion association complex that is extractable with chloroform from the aqueous phase. The ion association complex exhibits maximum absorption at 485 nm. Scheme for the developed yellow colored complex shown below figure 3.

CONCLUSION:

In the present investigation, the author reported an extractive spectrophotometric method for quantifying ETR in pure drugs and tablets by using TPooo as an analytical reagent. The method is based on the characteristic properties of different functional groups, such as a primary amine in this case.

The methodology which author followed in the present investigation meet all the criteria of the demands of analytical chemists namely simplicity, accuracy, reliability and cost-effective analysis. The developed method was validated by testing their linearity, sensitivity, accuracy, precision, selectivity, and robustness. The statistical data from the validation studies demonstrated the methods to be accurate, precise, selective, sensitive, linear, and robust. The application of the proposed method was checked by analyzing ETR in commercially available tablets that revealed good recovery with low relative standard deviation values ensuring the suitability for routine quality control and drug analysis of ETR in tablet dosage forms.

ACKNOWLEDGEMENTS:

The authors thank the authorities of the Copperbelt University and NRI Institute of Technology for providing funding, reagents, and facilities to carry out the present study. The authors gratefully thank Arabindo Labs Pvt.Ltd., Hyderabad, India, for providing a gift sample of the drug.

CONFLICT OF INTEREST:

The authors declared no conflicts.

REFERENCES:

1. Schiller DS, Youssef BM. Etravirine: a second-generation non-nucleoside reverse transcriptase inhibitor (NNRTI) active against NNRTI-resistant strains of HIV. 2009; 31 (4): 692-704.

2. García FG, Estévez MA, Suay VG. Chemical characteristics, mechanism of action and antiviral activity of etravirine. Enfermedades Infecciosas Y Microbiologia Clinica. 2009; 27 (Suppl 2): 2-5.

3. Ciaffi L, Cavassini M, Genne D, Delhumeau C, Spycher ER, Hill A, et al., Switch to Etravirine for HIV-positive patients receiving statin treatment: a prospective study. European Journal of Clinical Investigation. 2015; 45 (7): 720-730.

4. Casado JL, Bañón S, Rodriguez MA, Moreno A, Moreno S. Efficacy and pharmacokinetics of the combination of Etravirine plus raltegravir as novel dual antiretroviral maintenance regimen in HIV-infected patients. Antiviral Research. 2015; 113: 103-106.

5. Vingerhoets J, Calvez V, Flandre P, Marcelin AG, Ceccherini-Silberstein F. Efficacy of Etravirine combined with darunavir or other ritonavir-boosted protease inhibitors in HIV-1- infected patients: an observational study using pooled European cohort data. HIV Medicine. 2015; 16 (5): 297-306.

6. Reddaiah CV, Devi PR, Mukkanti K, Rao KS. Estimation of Etravirine by UV-Visible spectroscopic method in tablet dosage form and it’s in vitro dissolution assessment. International Journal of Pharmacy Research and Development. 2012; 4 (3): 287-295.

7. Murali D, Venkatrao S.V, Rambabu C. Spectrophotometric Determination of Etravirine in Bulk and Pharmaceutical formulations. American Journal of Analytical Chemistry. 2014; 5: 77-82.

8. Barath M., Chandan R.S., Maruthi R., Paramakrishnan. Analytical method development and validation of Etravirine by UV Spectroscopy. Research Journal of Pharmcy and Techology. 2020; 13(10): 4707-4710.

9. Aleem A, Krishnamurthy G, Bhojyanaik HS, Ramesh S. Development and validation of stability indicating ultra performance liquid chromatographic method for Etravirine. International Journal of Pharmacy and Pharmaceutical Sciences. 2012; 4 (1): 255-261.

10. Reddy CM, Reddy KH. A novel validated stability indicative UPLC method for Etravirine for the determination of process related and degradation impurities. American Journal of Analytical Chemistry. 2012; 3 (12): 840-848.

11. Djerada Z, Feliu C, Tournois C, Vautier D, Binet L, Robinet A, et al. Validation of a fast method for quantitative analysis of elvitegravir, raltegravir, maraviroc, Etravirine, tenofovir, boceprevir and 10 other antiretroviral agents in human plasma samples with a new UPLC-MS/MS technology. Journal of Pharmaceutical and Biomedical Analysis, 2013; 86: 100-111.

12. Abobo CV, Wu L, John J, Joseph MK, Bates TR, Liang D. LC-MS/MS determination of Etravirine in rat plasma and its application in pharmacokinetic studies. Journal of Chromatography B Analytical Technologies in the Biomedical and Life Sciences. 2010; 878 (30): 3181-3186.

13. Fayet A, Beguin A, Zanolari B, Cruchon S, Guignard N, Telenti A et al. A LC-tandem MS assay for the simultaneous measurement of new antiretroviral agents: Raltegravir, maraviroc, darunavir and Etravirine. Journal of Chromatography B. 2009; 877 (11-12): 1057-1069.

14. Heine R, Rosing H, Gorp ECM, Mulder JW, Beijnen JH, Huitema ADR. Quantification of Etravirine (TMC125) in plasma, dried blood spots and peripheral blood mononuclear cell lysate by liquid chromatography tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis. 2009; 49 (2): 393-400.

15. Rezk NZ, White NR, Jennings SH, Kashuba ADM. A novel LC-ESI-MS method for the simultaneous determination of Etravirine, darunavir and ritonavir in human blood plasma. Talanta. 2009; 79 (5):1372-1378.

16. Anna KB, Yonghou J, Dale W, Kim A.W. Simultaneous measurement of Etravirine, maraviroc and raltegravir in pigtail macaque plasma, vaginal secretions and vaginal tissue using a LC–MS/MS assay. Journal of Chromatography B Analytical Technologies in the Biomedical and Life Sciences. 2016; 1025: 110-118.

17. Else L, Watson V, Tjia J, Hughes A, Siccardi M, Khoo S, et al.Validation of a rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for the simultaneous determination of existing and new antiretroviral compounds. Journal of Chromatography B. 2010; 878 (19): 1455-1465.

18. D’Avolio A, Simiele M, Siccardi M, Baietto L, Sciandra M, Bonora S et al., HPLC-MS method for the quantification of nine anti-HIV drugs from dry plasma spot on glass filter and their long term stability in different conditions. Journal of Pharmaceutical and Biomedical Analysis. 2010; 52 (5): 774-780.

19. Raja AP, Venkateshwar RJ. HPTLC method development and validation for determination of Etravirine in bulk and tablet dosage form. International Journal of Pharmacy and Biological Sciences. 2013; 3(3): 515-522.

20. Murali D, Rambabu C. Development and validation of stability indicating RP-HPLC method for the quantification of Etravirine in tablet dosage form. Der Pharmacia Lettre. 2015; 7 (12): 216-226.

21. Somsubhra Ghosh, Sailaja A and Ravikumar BVV. Analytical method development and validation of Etravirine in its bulk dosage form by using reverse phase high performance liquid chromatography method as per international conference on harmonization guidelines. Asian Journal of Pharmaceutical and Clinical Research, 2015. 8(2): 147-150.

22. Monika S G, Thomas N K, Goedele D S, Hilde V, Marie P B, Monika P et al. A pharmacokinetic study of Etravirine (TMC125) co-administered with ranitidine and omeprazole in HIV–negative volunteers. British Journal Clinical Pharmacology. 2008; 66 (4): 508-516.

23. ICH Validation of analytical procedures; Text and methodology; Q2 (R1), International Conference on Harmonization, 2005.

24. Raghu Babu K, Chandra Sekhar N, Aruna Kumari V, Jagannadha Rao. Novel spectrophotometric methods for the determination clopidogrel bisulphate in bulk and pharmaceutical formulations by cobalt thiocyanate and Tpooo. Der Pharmacia Lettre, 2015; 7(3): 241-246.

Received on 01.12.2020 Modified on 19.04.2021

Research J. Pharm.and Tech 2022; 15(3):1145-1150.

DOI: 10.52711/0974-360X.2022.00192

Parameter	Investigation conditions	Optimized condition	Remarks
λ_max (nm)	450 – 550	485	-
Volume of 0.1M HCl (mL)	1-10	6.0	Variation of the volume of HCl above the 6.0 mL and below 6.0 mL resulted in low absorbance values.
Volume of 0.2% TPooo (mL)	1-5	2.0	2.0 mL of dye solution was sufficient for covering a broad range of Beer’s law limit
Choice of organic solvent for extraction	Chloroform, Benzene, Carbon Tetrachloride and Butanol	Chloroform	Chloroform was preferred for its slightly higher efficiency on color intensity, selective extraction of ion-pair complex from the aqueous phase.