Novel Method Development, Validation and Forced Degradation Studies for the Concurrent Determination of Lamivudine, Tenofovir Disoproxil Fumarate and Doravirine in Active Pharmaceutical Ingredient and Formulation using RP-UPLC

Introduction

Lamivudine^1-3 (Fig. 1a), chemically known as 4-Amino-1-1[(2R, 5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2-dihydropyrimidin-2-one. It is a white or almost white powder, soluble in water, sparingly soluble in methanol and practically insoluble in acetone. It has a molecular formula of C₈H₁₁N₃O₃S, molecular weight 229.3g/mol and a pKa value of 14.29. Lamivudine acts as an antiretroviral drug as it inhibits nucleoside reverse transcriptase enzyme and hence used for the treatment of HIV/ AIDS and chronic hepatitis B. Tenofovir disoproxil fumarate^4-6 (Fig. 1b), chemically known as 9-[(R)-2-[[bis[[(isopropoxycarbonyl) oxy] methoxy] phosphinyl] methoxy]propyl]adenine fumarate (1:1). It has a molecular weight of 635.52g/mol and a molecular formula of C₁₉H₃₀N₅O₁₀P.C₄H₄O₄. It is a white to off-white crystalline powder, soluble in water and aqueous solvents. It belongs to a category of antiretroviral drug which acts by inhibiting the activity of HIV-I reverse transcriptase and HBV reverse transcriptase enzymes. Doravirine^7,8 (Fig. 1c), chemically known as 3-chloro-5-[[1-[(4,5-dihydro-4-methyl-5-oxo-1H-1,2,4-triazol-3-yl)methyl]-1,2-dihydro-2-oxo-4-(trifluoromethyl)-3-pyridinyl]oxy] benzonitrile. It has a molecular formula of C₁₇H₁₁ClF₃N₅O₃ with a molecular weight of 425.75g/mol practically insoluble in water. It belongs to a category of antiviral agent which acts by inhibiting the HIV-1 non-nucleoside reverse transcriptase inhibitor (NNRTI).

Figure 1a: Structure of lamivudine. Click here to View figure

Figure 1b: Structure of tenofovir disoproxil fumarate Click here to View figure

Figure 1c: Structure of doravirine Click here to View figure

According to the review of the literature, numerous analytical techniques have been established for estimating lamivudine, tenofovir, and doravirine separately. But only few methods such as RP-HPLC^9-14, UPLC^15-17 were developed for the concurrent determination of lamivudine, doravirine and tenofovir in multidrug combination. The major goal of the proposed research was to simultaneously design and validate a new UPLC method for stability indication that could be used to estimate lamivudine, tenofovir, and doravirine in pharmaceutical dose form.

Material and Methods

Chemicals and reagents

Working standards for lamivudine, tenofovir, and doravirine were obtained as free samples from spectrum labs in Hyderabad. Delstrigo pills were acquired from a nearby drugstore. The analytical reagent (AR) grade chemicals and reagents utilised for the method development and validation were all purchased from Merck in Mumbai, India. Each and every solvent utilised in the procedure was of the AR grade and was obtained from Merck in Mumbai, India.

Instruments and chromatographic conditions

The acquity UPLC system operated using empower 2 software was used as chromatographic instrument for the development of the novel method. HSS C18(100mm×2.1mm,1.8µ)column, acetonitrile and 0.1% ortho phosphoric acid (65:35) mobile phase, 0.3ml/min flow rate, 260nm detection wavelength and 30°C column temperature were used as optimized chromatographic conditions for the simultaneous estimation of drugs. Other instruments used during the process of the method were weighing balance (Sartorius), ultrasonic bath (Phoenix), and pH meter (Lab India). Glassware such as volumetric flasks, pipettes, burettes and beakers made of borosil was used.  

Experimental work

Preparation of 0.1%OPA buffer

0.1% OPA buffer was created for 1000 ml by diluting 1ml of concentrated ortho phosphoric acid in 1 litre of distilled water.

Preparing mobile phase

The mobilephase was created by combining 350ml of acetonitrile with 650ml of 0.1% OPA buffer.

Diluent preparation

Equal parts of distilled water and methanol were combined and used as a diluent to create the solutions (Both sample and standard) for measurements.

Preparation of Standard and sample solutions

An accurate amounts of 75 mg of lamivudine, 75 mg of tenofovir & 25 mg doravirine are taken and transferred to a 50 ml volumetric flask which was cleaned and dried previously. The standard medicines were dissolved using sonication and the addition of 25ml of diluent. Later, using the diluent to obtain the stock solution, the volume was raised to the required level. The standard solution, which contains lamivudine (150 mg/ml), tenofovir (150mg/ml), & doravirine (50mg/ml) was created by diluting 1 ml stock solution in to 10 ml solution by using a diluent.   

Twenty Delstrigo tablets were weighed on average. In a mortar and pestle, the tablets are fine grounded into powder. Accurately Weighed, dissolved in 50ml of diluent was a quantity equal to 75mg of lamivudine. The solution underwent filtering and sonication. The sample solution was created by diluting 1ml of the filtrate with diluent to a volume of 10ml.

Method validation^18,19

System suitability

Into the UPLC system the prepared standard solution is injected five replicate times and the parameters for system suitability i.e tailing factor, plate count, % relative standard deviation (RSD), resolution were evaluated.

Linearity

A concentration range of 37.5 µg/ml to 225µg/ml, 37.5µg/ml to 225µg/ml and 12.5µg/ml to 75µg/ml are accurately prepared from stock solution for lamivudine, tenofovir and doravirine respectively, and injected in the UPLC system. The measured peak areas plotted vs the concentration of the drug solution and correlation coefficients were determined.

Accuracy

Three levels of drug solutions are prepared 50%, 100% and 150% using spiking method; and the peak areas were measured in triplicates for these levels. 

Precision

Repeatability

For the estimation of repeatability of the method, the prepared sample solution is injected into the UPLC six times, the respective peak areas were noted. From the measurements, % RSD was evaluated.

Intermediate Precision

For determining the intermediate precision, the % RSD was calculated when the sample solutions were injected into the UPLC six times in two consecutive days.

Specificity

The interference of the placebo peaks with the reference peaks was evaluated to determine the method’s specificity.

Limit of detection & limit of quantification

Utilizing standard deviation & slope values from linearity results, the limits of detection (LOD) & quantification (LOQ) were accurately calculated.

Robustness

The method robustness is assessed by slightly modifying the final optimized chromatographic conditions such as ± 0.1ml/min flow rate,  ± 5% organic phase in a mobile phase composition and ±5°C column temperature; later the %RSD was calculated. 

Solution stability

In order to study the effect of diluent on the standard drugs, the prepared standard solution was made to stand for 24 hours under normal conditions. The solution was assessed after 24 hours to observe for any degradation of the drugs.

A study on forced degradation

The chosen standard solution of drug is implicated to a variety of stressed conditions during forced degradation studies^20–22, including photolysis (at 105°C, 6 hours), thermo lytic conditions (keeping the solution under UV light continuously for 7 days), peroxide conditions (Hydrogen peroxide 20% continuously 30 minutes at a temperature of 60 °C), acid conditions (2Normal HCl continuously for 30 minutes at a temperature of  60 °C), and base conditions (2Normal Sodium hydroxide continuously for 30 minutes at a temperature of 60˚C)

Results and Discussion

Initially for the method development, various stationary phases and mobile phase compositions were tried. Based on trial and error method, HSS C18 (100×2.1 mm,1.8 µ) column and 0.1% OPA buffer & ACN (65:35) were selected as stationary phase and mobile phase respectively, as the peak shape and resolution were observed good. Flow rates were selected as 0.3ml/minute and isocratic mode of elution was used. The detection wavelength for the analysis was selected depends on the overlay UV spectrum of the three drugs as 260nm and the figure was shown in Fig. 2.

Figure 2: UV overlay Spectrum for lamivudine, tenofovir and doravirine Click here to View figure

To assess the system appropriateness characteristics, the produced std. solution is injected to the UPLC system. All the results are given in Table 1. Figs. 3a and 3b, respectively, show the chromatograms of the std, sample & blank solutions. Plate count, tailing factor, resolution, and percent RSD—all system suitability parameters—were discovered to be within acceptable bounds. Lamivudine’s retention time was determined to be 0.48 minutes, tenofovir’s to be 1.08 minutes, and doravirine’s to be 1.59 minutes.

Table 1: Validation parameter results

Parameters	Lamivudine	Tenofovir	Doravirine
Specificity	Specific	Specific	Specific
Linearity, Regression equation	y = 7832.3x + 4142	y = 9031.6x + 12149	y = 7212x + 3474.3
Correlation coefficient	0.9998	0.9992	0.9997
Accuracy Level I (50%)	99.15%	100.88%	99.99%
(recovery)Level II (100%)	99.76%	100.67%	100.24%
n=3 Level III (150%)	100.16%	100.08%	100.78%
Precision, Repeatability (%RSD)	1.0	0.5	0.8
Intermediate precision Day 1	1.1	0.6	0.4
(% RSD), n=6    Day 2	0.2	0.8	1.2
LOD (µg/ml)	1.05	0.50	0.46
LOQ (µg/ml)	3.19	1.52	1.40
Robustness	Robust	Robust	Robust
Solution Stability	Stable	Stable	Stable
System suitability, Plate  count Tailing   Resolution	2917 1.66 –	3930 1.76 5.8	3527 1.16 3.6

Figure 3a: UPLC Chromatogram for Std. solution Click here to View figure

Figure 3b: UPLC Chromatogram of sample solution. Click here to View figure

In the concentration ranges of 37.5g/ml to 225g/ml for lamivudine, 37.5g/ml to 225g/ml for tenofovir & 12.5g/ml to 75g/ml for doravirine, it was discovered that the procedure was linear and adhered to Beer’s law. In all three linearity plots, the correlation coefficient was discovered to be within the acceptable range, or 0.999. The plots were depicted in Figs. 4a, 4b, and 4c, and Table 2 showed the linearity results.

Table 2: Linearity results

S. No.	Lamivudine			Tenofovir		Doravirine
	concentration (µg/ml)	peakarea		concentration (µg/ml)	peakarea	concentration (µg/ml)	peakarea
1 2 3 4 5 6	37.5 75.0 112.5 150.0 187.5 225.0	293918 604734 868835 1201390 1469115 1758968		37.5 75.0 112.5 150.0 187.5 225.0	342605 720084 997205 1414172 1695828 2027558	12.5 25.0 37.5 50.0 62.5 75.0	93273 184223 276192 371973 452428 539380
Correlation coefficient	0.9998		0.9992			0.9997

Figure 4a: Linearity plot of lamivudine Click here to View figure

Figure 4b: Linearity plot of tenofovir disoproxil fumarate. Click here to View figure

Figure 4c: Linearity plot of doravirine Click here to View figure

The above method is said to be Accurate as the % recovery values for lamivudine are 99.15% – 100.16%, for tenofovir it was 100.08% – 100.88% and for doravirine it was 99.99% – 100.78%, which were within the limits. For the determination of precision, % RSD was calculated in two ways, repeatability and intermediate precision. The % RSD for repeatability was 1.0, 0.5 and 0.8 for lamivudine, tenofovir and doravirine respectively. The % RSD for day 1 was 1.1 and for day 2, it was found to be 0.2 for lamivudine. The % RSD for day 1 was found to be 0.6 and for day 2, it was found to be 0.8 for tenofovir. The % RSD for day 1 was found to be 1.2 and for day 2, it was found to be 0.4 for doravirine. The above values indicate that the method was precisely developed.

Since the placebo peak did not interfere with the typical drug peaks, the approach was proven to be precise. In Fig. 5, the placebo chromatogram was displayed.

Figure 5: UPLC chromatogram for placebo solution. Click here to View figure

The linearity results were used to calculate the LOD & LOQ values. LOD & LOQ data for lamivudine were found to be 1.05 and 3.19, respectively. Tenofovir’s LOD & LOQ were discovered as 0.50 and 1.52, respectively. For doravirine, the LOD was determined as 0.46 and the LOQ as 1.40.

The robustness of the method is detected by slightly changing the finally selected chromatographic conditions and the method was found to be robust as % RSD values were observed within the acceptance criteria. The robustness results were summarized in Table3.

Table 3: Robustness results

Parameter	Lamivudine	Tenofovir	Doravirine
Optimized   condition	1.1	0.6	1.2
Less flow rate	1.8	0.8	1.6
More flow rate	0.9	1.7	1.3
Decrease mobile  phase (organic)	1.5	0.8	1.0
Increase mobile phase (organic)	0.4	0.4	0.5
Low column temperature	0.2	1.0	0.5
More column temperature	0.2	0.3	0.3

 

After 24 hours standing of standard drug in solution form, the drugs were found to be stable as there was no deviation in results.

The drug solution was exposing to several stressed conditions, like acidic, alkaline, peroxide, thermal, and photolytic, to assess the analytical method stability. Since the net deterioration was found to be within acceptable bounds, the medication solution was determined to be stable. Degradation chromatograms were displayed in Fig. 6 together with the final results of the forced degradation research, which are provided in Table 4.

Table 4: Forced degradation study results

Drug		Forced degradation condition
		Acid	Alkaline	Peroxide	Thermal	Photolytic
Lamivudine	% Assay % Degradation	93.89 6.11	80.89 19.11	95.37 4.63	97.26 2.74	98.70 1.30
Tenofovir	% Assay % Degradation	93.77 6.23	95.67 4.33	95.36 4.68	97.06 2.94	98.01 1.99
Doravirine	% Assay % Degradation	94.41 5.59	95.57 4.43	95.58 4.42	97.27 2.73	98.17 1.83

 

Figure 6a: Chromatogram for Acid degradation Click here to View figure

Figure 6b: Chromatogram for Basic degradation Click here to View figure

Figure 6c: Chromatogram for Oxidation stress degradation Click here to View figure

Figure 6d: Chromatogram for Photolytic degradation. Click here to View figure

Figure 6e: Chromatogram for Thermal degradation Click here to View figure

Conclusion

Novel technique is developed for concurrent determination of the compounds lamivudine, tenofovir and doravirine in active pharmaceutical ingredient & formulation by UPLC. According to ICH requirements, the devised method was validated, and it was discovered to be specific, accurate, linear, sensitive, and highly robust. The stressed degradation studies was also conducted to determine the stability of the analytical method. The degradation studies revealed that the approach remained stable even when subjected to stressful situations. For routine quality control analysis of simultaneous estimation of lamivudine, tenofovir, & doravirine in combination dosage forms, the developed UPLC method can be employed.

Acknowledgement

The Hetero Labs Pvt. Ltd. in Hyderabad and the Spectrum labs in Hyderabad, India, who provided the resources needed to conduct this research are both gratefully acknowledged by the authors.

Conflict of Interest

We have no conflicts of interest to disclose

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