INTRODUCTION:

Emtricitabine, an HIV medication, has the chemical structure “4-amino-5-fluoro-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl] pyrimidin-2-one”². and functions as a nucleoside reverse transcriptase inhibitor (NRTI) in the treatment of adult Human immune virus infections. Chemical structure of emtricitabine is given in Figure 1². The drug inhibits reverse transcriptase enzyme to prevent the multiplication of HIV RNA into viral DNA. Emtricitabine works by acting as a competitor for the substrate deoxycytidine 5'-triphosphate to insert as such viral DNA strands leading to inhibition of DNA chain synthesis. The incorporation of emtricitabine within the DNA synthesis pathway separates HIV-1 reverse transcriptase from performing its activity. The antiviral effect of emtricitabine on reverse transcriptase leads to lower HIV replication in the body alongside improved immune resistance against infection which makes it essential for managing HIV infection³.

The antiviral medication Efavirenz comes under the chemical name “(4S)-6-chloro-4-(2-cyclopropylethynyl)-4-(trifluoromethyl)-2,4-dihydro-1H-3,1-benzoxazin-2-one”⁴. which has its structure shown in Figure 2⁴. Efavirenz exists as a key component of antiretroviral therapy used to treat HIV infection. Patients exposed to HIV transmission situations commonly receive Efavirenz combined with other antiviral medications in extended post-exposure prophylaxis to prevent HIV infection⁵.

Previous literature studies showed various analytical methods estimated emtricitabine and efavirenz individually or when combined with other dosage forms but no RP -UPLC method exists for the assessment of these drugs in fixed dosage (tablet) formulations^6, According to literature review, planned work aims to develop an easy-to-use sensitive and reproducible Ultra Performance Liquid Chromatographic method with superior resolution speed and sensitivity by utilizing appropriate columns as an implementation strategy^8,9. UPLC ¹⁰operates under extreme pressure levels (up to 1500psi) to perform the simultaneous detection of emtricitabine and efavirenz that exists within tablet dosage forms¹⁰.

MATERIALS AND METHODS:¹¹

Chemicals¹¹ and Reagents:¹¹

Standard drugs of Emtricitabine and Efavirenz were obtained as gifted samples from a reputed pharmaceutical industry. Teevir tablets manufactured by Mylan labs was obtained from local pharmacy. Ortho phosphoric acid H₃PO₄¹¹, potassium dihydrogen phosphate, UPLC grades methanol all were obtained from Merck.

Instrumentation:

UPLC system with PDA detector controlled by LC software with Grace Smart C18 (4.6 X 50mm,1.7µm) column. Wave length was scanned by UV-VIS spectrophotometer¹² Lab India 3000⁺. All the ingredients weighing is done using semi micro balance model Aarson SS. pH meter manufactured by ELICO was used. Sonicator manufactured by IKON LABS was used. Constant water bath manufactured by Thermo lab GMP were used for the method development¹³.

Wave length selection and chromatographic conditions:

10µg/ml solutions of Emtricitabine (EM) and Efavirenz (EF) were prepared. The resulting solutions were analyzed using double beam UV-Visible spectrometer within range of 200-400nm¹⁴. Both the drugs show maximum absorbance at 260nm. Therefore, all subsequent measurements were conducted at this wavelength.

Preparation of Mobile phase:

In 1000ml volumetric flask, 2.5grams of potassium dihydrogen ortho phosphate (KH₂PO₄)¹⁵ was solvated with UPLC grade water and pH was made to 3 using ortho phosphoric acid. The above prepared buffer and UPLC grade methanol were degassed in an ultrasonicator for 5 mins, followed by filtration through a 0.45µm membrane filter¹⁵.

Preparation of Standard solution:

A precise amount of 10 milligrams of Emtricitabine and 20 milligrams of Efavirenz¹⁶ were solubilized in 10mL of diluent by ultrasonication to obtain a concentration of 1000µg/ml¹⁶ (Stock solution). Suitable aliquots of above solutions were diluted to attain the concentration levels to 30µg/ml, 60µg/ml of Emtricitabine & Efavirenz respectively¹⁶.

Preparation of sample solutions:

Weigh approximately 10 tablets and grind them to powder in a mortar¹⁷. Transfer the powder equivalent to 10 milligrams emtricitabine and 20 milligrams efavirenz to another clean and dry 10mL flask, and diluted with the solvent upto the mark to obtain a concentration of 1000µg/ml¹⁷. 10 millilitres volumetric flask was filled with 0.3millilitres of the above solution, diluted upto the mark with the solvent resulting in a concentrations matching that of the standard solution. Before injecting into the UPLC system, the solution was filtered through a 0.45µm filter¹⁷.

Preparation of placebo:

10mL volumetric flasks were filled with precisely weighed amounts of inactive components, equivalent to the amount in ten tablets and diluted with the solvent upto the marked of 10mL and the precipitate was allowed to settle. The solution was then divided into aliquots for further dilutions. 0.1mL of the previously prepared solution was transferred to a 10millilitres volumetric flask and the diluent was added upto the mark. The solution was filtered through a 0.45µm membrane filter before injecting into the UPLC system¹⁷.

Method validation:

This RP-UPLC analytical method was validated following the ICH guidelines. The validation process includes specificity, linearity, accuracy, precision¹⁸, robustness, and ruggedness, Limit of detection and Limit of quantification¹⁸.

Optimization of chromatographic conditions:

RP-UPLC method operated in an isocratic mode through a “Grace Smart C18 column” (4.6 x 50mm, 1.7 µm) to optimize chromatographic operations. The liquid phase consist of methanol and phosphate buffer of pH 3.0 which was mixed in a 65:35 volume ratio (v/v) and operated in the column at 0.3mL/minute with 260nm¹⁴ absorption maxima. The separation quality of Emtricitabine and Efavirenz showed satisfactory results since peak asymmetry stayed within the acceptable range while impurity sources had no adverse effects. Results complied with defined acceptance standards indicating complete optimization of the method. The chromatogram obtained during optimization is available in Figure 3 while the drug percentage purities were computed from the recorded chromatographic information. The method developed through optimization capabilities demonstrated stability in addition to accuracy and precision standards for assessing drugs simultaneously in pharmaceutical products¹⁴.

RESULTS AND DISCUSSION:

System suitability:

Emtricitabine (EM) and Efavirenz (EF) of working standard solutions were injected 6 times and system suitability results were calculated. The results obtained are shown in¹⁹ Table 1.

Specificity:

Specificity of the method ensures that the measured signal comes from the substance of interest¹⁹, and not affected by excipient and/or degradation products and/or impurities¹⁹. The above parameter was demonstrated without the effect of diluents used in blank¹⁹. Specificity shown in figure 5 and 6

Linearity:

Standard solutions of various concentrations were injected separately, and the chromatograms were recorded¹⁹ for each concentration. The peak areas which correspond to injected drug concentration were measured, and a calibration curve was plotted with concentration Vs peak area¹⁹. The linearity performance parameters are detailed below, and the results are summarized in Table 2 and Figures 7 and 8. The linearity data revealed that the method was linear within the concentration ranges of 10-50µg/mL for Emtricitabine and 20-100µg/mL for Efavirenz. The correlation coefficient for the calibration curves was found to be 0.999¹⁹.

Precision: (Intraday and Interday)

The standard solution of the drug was injected in replicates, and the peak area was recorded for each individual injections in UPLC. The %RSD for these replicate injections was found to be within the acceptable limits¹⁹ which shows that the method meets the criteria for repeatability and precision. The %RSD should not exceed 2%. The experimental findings are presented in Table 3.

Accuracy:

The accuracy of the developed UPLC method was¹⁹ evaluated through recovery studies conducted at 3 different concentration levels: 50%, 100%, and 150% ¹⁹of the target analyte concentration. This involved spiking known amounts of standard drug solutions into a matrix or placebo to simulate the actual sample environment.

Preparation of Solution:

50% Solution: A standard solution corresponding to 50% of the¹⁹ target concentration was spiked into the matrix.

100% Solution: A standard solution was spiked¹⁹ to 100% of the target concentration.

150% Solution: A standard solution corresponding to 150% of the target concentration was spiked.

The % recovery for all three levels—50%, 100%, and 150%—was within the acceptance criteria, exemplifying the accuracy of the method. The accuracy results are presented in Table 4.

Robustness:

The robustness of the UPLC method for concurrent determination of emtricitabine and efavirenz was²⁰ evaluated by making small, deliberate changes in analytical parameters to evaluate its reliability and consistency. The robustness of the developed UPLC method for emtricitabine and²⁰efavirenz was evaluated by varying key parameters such as liquid phase composition (±2%), and flow rate (±0.2mL/min)²⁰.

Results demonstrated that, the evaluation of the above results²⁰ showed that the developed method remained robust even with change in the flow rate although it proved to be most robust only in low rate flow condition²⁰. The results were shown in table 5.

Additionally, an investigation was done to assess the effects of varying the liquid phase composition²⁰. The composition was altered from 30% to 70%, and standard solutions of emtricitabine (30µg/mL) and efavirenz (60 µg/mL) were prepared and analyzed using different liquid phase ratios, along with the original composition²⁰. The prepared standard solutions were injected into the UPLC system, and the retention time values were recorded, as shown in Table 6.

Limit Of Detection (LOD):

LOD was calculated based on slope of calibration curve, for the sample injected in triplicates and the standard deviation²⁰was measured. LOD for Emtricitabine& Efavirenz were determined to be 0.004 and 0.03µg/ml respectively.

Limit of Quantification (LOQ):

By injecting the sample in triplicates, the LOQ was evaluated, and the standard deviation along with slope of calibration curve were computed. Emtricitabine and Efavirenz were found to have limits of quantification of 0.012 and 0.09 µg/ml²⁰ respectively.

Fig 1 & 2: Structure of Emtricitabine & Efavirenz

Figure 3: Standard chromatogram of Emtricitabine and Efavirenz

Figure 4: Assay chromatogram of EM and EF

Figure 5: Chromatogram of Placebo Interference

Fig 6 : Chromatogram of Blank Interference.

Figure 7: Calibration curve of Emtricitabine.

Figure 8: Calibration curve of Efavirenz

Figure 9: LOD Chromatogram for Emtricitabine & Efavirenz:

Figure 10: LOQ Chromatogram for Emtricitabine & Efavirenz

Table 1: Chromatogram values for System suitability

Parameters	Emtricitabine(EM)	Efavirenz(EF)
Peak area (% RSD)	0.322	0.8206
Retention time	0.494	2.237
Tailing factor	1.34	0.928
Plate count (% RSD)	0.135	1.23
Resolution	-	1.11

Table 2: Linearity results:

Emtricitabine		Efavirenz
Concentration	10-50µg/ml	Concentration	20-100 µg/ml
Correlation coefficient	0.9988	Correlation coefficient	0.9978

Table 3: Results for Intra-day Precision:

Sample no	Emtricitabine		Efavirenz
Sample no	Peak area	Rt	Peak area	Rt
1	12472562	0.431	9548542	2.324
2	12485792	0.432	9376152	2.314
3	12432763	0.432	9506942	2.326
4	12436576	0.433	9402542	2.317
5	12498763	0.437	9229572	2.334
Mean	12465291.20	0.43	9412750.00	2.32
Std. dev	29479.46419	0.002345	124809.2511	0.007874
%RSD	0.236492383	0.541618	1.325959482	0.338959

Table 4: Results for Inter-day Precision:

Sample no	Emtricitabine		Efavirenz
Sample no	Peak area	Rt	Peak area	Rt
1	12314042	0.429	9149222	2.299
2	12331962	0.43	9093352	2.302
3	12310082	0.431	9132662	2.304
4	12385752	0.434	9094182	2.312
5	12324072	0.435	9114962	2.339
Mean	12333182.00	0.43	9116876.00	2.31
Std.dev	30610.59865	0.002588	24328.58977	0.01627
%RSD	0.248197089	0.599452	0.266852261	0.703946

Table 5: Accuracy data of Emtricitabine and Efavirenz:

Sample	Concentration (%)	Amount Recovered	Recovery (%)	% RSD
Emtricitabine	50	8382805± 2686	100.00± 1.0	1.0
	100	12506696± 2222	99.31± 0.4	0.4
	150	16385136± 2836	99.89± 0.2	0.2
Efavirenz	50	6345805± 2582	101.00± 0.6	0.6
	100	8995372± 2379	98.81± 0.9	0.9
	150	11893562± 2836	99.76± 0.5	0.5

Table 6: Robustness results on flow rate

Emtricitabine	Flow rate	0.2ml/min	0.3ml/min	0.4ml/min
	% RSD for Rt	0.435	0.5994	1.01
	USP Theoretical plate	2511	2490	2484
	USP tailing factor	1.12	1.12	1.23
Efavirenz	% RSD for Rt	1.96	0.703	0.21
	USP Theoreticalplate	2651	2870	2984
	USP tailing factor	1.15	1.12	1.12

Table 7: Robustness results on mobile phase variation:

Emtricitabine	% Mobile phase	75%	65%	55%
	% RSD for Rt	0.692	0.531	0.341
	USP Theoretical plate	2510	2490	2492
	USP tailing factor	1.54	1.12	1.12
Efavirenz	% Mobile phase	75%	65%	55%
	% RSD for Rt	0.262	0.703	0.269
	USP Theoretical plate	2454	2870	2030
	USP tailing factor	1.2	1.12	1.14

CONCLUSION:

The UPLC method provides efficient analysis of various categories of samples in a less time, offering excellent stability, accuracy, and precision, while delivering valuable information. Emtricitabine and Efavirenz were determined to be freely soluble in water and alcohol. Methanol and potassium dihydrogen orthophosphate (pH 3) were selected as the diluents, and run time was about 5 minutes. The method has been validated for various parameters, including system suitability, linearity, sensitivity, accuracy, specificity, robustness, LOD, and LOQ. The system suitability parameters were within acceptable limits, confirming the system’s reliability for performing the assay. The advanced method demonstrated linearity within different dilution ranges of 10-50µg/ml for Emtricitabine and 20-100µg/ml for Efavirenz. Recovery values for both Emtricitabine and Efavirenz were found to be between 98% and 102%. Notably, the method showed no interference from excipients or the mobile phase. Robustness and ruggedness were confirmed by the minimal variation in results due to variations in flow rate, liquid phase composition, and across different analysts. This method also exhibited good correlation with assay results of pharmaceutical formulations, establishing it as a reliable approach. Therefore, the advanced method is suitable for regular analysis of Emtricitabine and Efavirenz in bulk drugs and pharmaceutical formulations.

CONFLICT OF INTEREST:

There is no disagreement consecrated the authors regarding this research.

ACKNOWLEDGMENT:

The authors would like to thank the Management and Principal of Aditya College of Pharmacy Surampalem for his continuous support and encouragement to authors since carrying out the research work.

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Received on 12.03.2025 Revised on 05.04.2025

Accepted on 25.04.2025 Published on 12.07.2025

Available online from July 21, 2025

Asian Journal of Pharmaceutical Analysis. 2025; 15(3):191-196.

DOI: 10.52711/2231-5675.2025.00030

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