*Corresponding Author:
Prasanthi Chengalva
Department of Pharmaceutical Analysis, Annamacharya College of Pharmacy, Rajampet 516126, India
E-mail: [email protected]
Date of Received 27 April 2020
Date of Revision 16 September 2020
Date of Acceptance 23 December 2020
Indian J Pharm Sci 2020;82(6):1006-1014  

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Abstract

A simple and rapid stability indicating reverse phase ultra-performance liquid chromatographic method has been established and validated for the simultaneous quantification of lamivudine, tenofovir disoproxil fumarate, doravirine and efavirenz in bulk and pharmaceutical formulations. The chromatographic separation was performed on Acquity Ethylene Bridged Hybrid Phenyl (50 mm×2.1 mm, 1.7 μm) column. The isocratic elution system of water and acetonitrile in the ratio 50:50 v/v pumped at a flow rate 0.4 ml/min in isocratic mode. The injection volume set was 1 μl and the detection wavelength was 238 nm. The column temperature was set at 30º. The retention times of lamivudine, tenofovir disoproxil fumarate, doravirine and efavirenz were found to be 1.012 min, 1.233 min, 1.428 min and 1.666 min respectively with a total run time of 3 min. The proposed method was validated according to International Council on Harmonisation Q2 (R1) guidelines. The percentage recoveries were found to be in the range of 99.56 -100.40 %. The relative standard deviation values obtained during precision studies were found to be less than 2. Linearity between concentration and response was found within the specified concentration range and the correlation coefficient was found to be 0.999 for all the drugs. Degradation studies were carried out under various stress conditions such as acid, base, oxidation, heat and light and found no interference of degraded impurity peaks at the retention time of analyte peaks. Hence, the proposed ultra-performance liquid chromatographic method can be utilized in the routine quality investigation of lamivudine, tenofovir disoproxil fumarate, doravirine and efavirenz either individually or simultaneously in bulk and co-formulated formulations).

Keywords

Lamivudine, tenofovir disoproxil fumarate, doravirine, efavirenz, reverse phase ultraperformance liquid chromatographic method, method development and validation

Antiretroviral drugs are used for the treatment of retroviral infection, primarily human immune virus (HIV). Combinational therapy of antiretroviral drugs is preferred for highly active treatment. Antiretroviral drug molecules such as lamivudine, tenofovir disoproxil fumarate, doravirine and efavirenz were selected for the study, as these are the drugs among most commonly prescribed for HIV therapy. Lamivudine (fig. 1A) is chemically 4-amino-1-[(2R,5S)-2-(hydroxymethyl)- 1,3-oxathiolan-5-yl]pyrimidin-2-one. It is formulated as tablets and oral solution. Tenofovir disoproxil fumarate (fig. 1B) is {[(2R)-1-(6-aminopurin-9-yl)propan-2- yl]oxymethyl-(propan-2-yloxycarbonyloxymethoxy) phosphoryl}oxymethylpropan-2-yl carbonate;(E)-but-2-enedioic acid. Doravirine (fig. 1C) is 3-chloro-5- ({1-[(4-methyl-5-oxo-4,5-dihydro-1H-1,2,4- triazol-3-yl)methyl]-2-oxo-4-(trifluoromethyl)-1,2- dihydropyridin-3-yl}oxy)benzonitrile. Efavirenz (fig. 1D) is (4S)-6-chloro-4-(2-cyclopropylethynyl)-4- (trifluoromethyl)-2,4-dihydro-1H-3,1-benzoxazin- 2-one. These are the drugs that belong to the class of reverse transcriptase inhibitors. They are available as single drug formulations with varied strengths and also as co-formulated dosage forms with other HIV drugs used in the effective management of retro viral infections and Hepatitis B. A combination of lamivudine, tenofovir disoproxil fumarate and efavirenz tablets are available in the market with different trade names such as Trioday, Vonaday and Telura by various manufacturers. Lamivudine and tenofovir disoproxil fumarate with doravirine combinational tablets are available with the trade name Delstrigo. An extensive literature survey revealed analytical methods for the estimation of specified drugs individually[1,2] and also in combination with other drugs[3-5]. An ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was reported for the impurities characterization of doravirine[6]. Few liquid chromatographic methods were reported for the simultaneous estimation of efavirenz along with impurities[7,8]. There were simultaneous Reverse-phase high performance liquid chromatography (RP-HPLC) methods proposed for efavirenz in combination with lamivudine and tenofovir disoproxil fumarate[9-12], but in those methods efavirenz has longer retention time (RT) and the run time of the studies was high leading to longer analysis time. In addition to these, there was no ultra-performance liquid chromatography (UPLC) method yet reported for the simultaneous estimation of the proposed lamivudine, tenofovir disoproxil fumarate, doravirine and efavirenz. Henceforth, there is a scope for the development of a simple and fast method that can analyze all the four drugs either in single or combination formulations as well as in bulk. The present investigation depicts the development and validation of accurate and isocratic Reverse-phase ultra-performance liquid chromatography (RP-UPLC) method with excellent resolution for the assay of lamivudine, tenofovir disoproxil fumarate, doravirine and efavirenz. Therefore, this method can be used for the routine quality control of bulk and marketed formulations of selected drugs, utilizing a simple mobile phase in short time.

IJPS-disoproxil

Figure 1:Structure of analytes. Structures of A. lamivudine, B. tenofovir disoproxil fumarate, C. doravirine and D. efavirenz

Materials and Methods

Combinational tablets containing 300 mg of lamivudine, 300 mg of tenofovir disoproxil fumarate, 100 mg of doravirine (formulation A) and the reference standards were procured from Spectrum Pharma Research Solutions, Hyderabad. Trioday tablets (Cipla, formulation B) were purchased from the local market. Acetonitrile and HPLC grade water of analytical grade from Merck Ltd., Mumbai. The diluent used was water and acetonitrile in the ratio of 50:50 v/v.

Chromatographic conditions:

The instrument used was Acquity UPLC H-Class system of Waters, Milford, USA. It consists of binary solvent manager, auto sampler and Ultraviolet (UV) detector. The output signal was monitored and processed using Empower software. Chromatographic separation was performed on Acquity Ethylene Bridged Hybrid (BEH) Phenyl column (50 mm×2.1 mm, 1.7 μm) with UV detection at 238 nm. The mobile phase consisting of water and acetonitrile in the ratio 50:50 v/v was pumped at a flow rate 0.4 ml/min in isocratic mode with an injection volume of 1 μl and the column temperature was set at 30º.

Preparation of standard solution:

Accurately 7.5 mg of lamivudine, 7.5 mg of tenofovir disoproxil fumarate, 2.5 mg of doravirine and 15 mg of efavirenz were weighed and transferred into a clean and dry 25 ml volumetric flask. To this, 15 ml of diluent was added, sonicated for 10 min and made up to the final volume with diluent. This was considered as the primary stock solution. From the primary stock, 1 ml was taken into a 10 ml volumetric flask and made up to the volume with diluent. This was considered as working standard solution with final concentrations of 30 μg/ml of lamivudine, 30 μg/ml of tenofovir disoproxil fumarate, 10 μg/ml of doravirine and 60 μg/ml of efavirenz.

Sample solution preparation (Formulation A):

Combinational tablets containing 300 mg of lamivudine, 300 mg of tenofovir disoproxil fumarate, 100 mg of doravirine were taken for the sample solution preparation. Twenty tablets were grinded into fine powder in a mortar. From this, the weight of powder equivalent to 7.5 mg of lamivudine, 7.5 mg of tenofovir disoproxil fumarate, 2.5 mg of doravirine was taken and transferred into 25 ml clean dry volumetric flask, 15 ml of diluent was added, sonicated for 15 min, filtered and made up to the final volume with diluent. From the above solution, 1 ml was taken into a 10 ml volumetric flask and made up to the volume with diluent.

Sample solution preparation (Formulation B):

Trioday tablets were taken for the sample solution preparation. Twenty tablets were grinded into fine powder in a mortar. From this, the weight of powder equivalent to 7.5 mg of lamivudine, 7.5 mg of tenofovir disoproxil fumarate, 15 mg of efavirenz was taken and transferred into 25 ml clean dry volumetric flask, 15 ml of diluent was added, sonicated for 15 min, filtered and made up to the final volume with diluent. From the above solution, 1 ml was taken into a 10 ml volumetric flask and made up to the volume with diluent.

Preparation of forced degradation samples:

As a part of forced degradation studies, the sample of acid degradation was prepared by taking 1 ml of standard stock solution and 1 ml of 1 Normal (1 N) hydrochloric acid into a 10 ml volumetric flask and refluxed for 30 min at 60º. After refluxing, the resultant solution was neutralized by adding 1 N sodium hydroxide solution and made up to the volume with diluent. The base degradation sample was prepared by taking 1 ml of standard stock and 1 ml of 1 N sodium hydroxide into a 10 ml volumetric flask and refluxed for 30 min at 60º. After refluxing, the resultant solution was neutralized by adding 1 N hydrochloric acid solution and made up to the volume with diluent. During oxidative degradation, 1 ml of standard stock and 1 ml of 10 % hydrogen peroxide was added into a 10 ml volumetric flask. The resultant solution was kept undisturbed for 30 min at 60º. During sample preparation of thermal degradation, the standard stock solution was kept in the oven at 60º for 6 h. From the resultant stock solution, 1 ml was taken into 10 ml volumetric flask and made up to the volume with diluent. As a part of the photo stability study, 10 ml of stock solution was kept under UV light for 3 d. From the resultant stock solution, 1 ml was taken into a 10 ml volumetric flask and made up to the volume with diluent[13].

Method development:

Varied chromatographic conditions were tried to get sharp peaks with good resolution and for rapid elution. Different combinations of solvents in different ratios as mobile phase were examined and found that the suitable mobile phase for the separation was water and acetonitrile in the ratio of 50:50 v/v. It was pumped at a flow rate of 0.4 ml/min in an isocratic mode found quite well for the detection as well as simultaneous estimation of four drugs. Acquity BEH Phenyl column (50 mm×2.1 mm, 1.7 μm) with UV detection at 238 nm was considered best for the separation. The injection volume of 1 μl and a column temperature of 30º were optimized for the study. Standard solutions were prepared and injected six times into the chromatographic system. The evaluated system suitability parameters were tabulated in Table 1. The resolution between two peaks must be greater than 2. The number of theoretical plates must be more than 2000 and the tailing factor must be less than 2. From the results, it was found that all the parameters were in compliance with the acceptance limits. Hence, these chromatographic conditions were optimized for the estimation of lamivudine, tenofovir disoproxil fumarate, doravirine and efavirenz. A typical UPLC chromatogram from standard preparation was shown in fig. 2.

IJPS-hromatogram

Figure 2: Chromatogram of standard preparation

Analytes RT (min)* Resolution* Theoretical plates* Tailing Factor*
Lamivudine 1.013±0.002 - 3968.7±251.6 1.11±0.06
Tenofovir disoproxil fumarate 1.236±0.002 3.45±0.10 5977.8±341.4 1.20±0.03
Doravirine 1.430±0.002 3.00±0.09 7854.0±739.2 1.22±0.06
Efavirenz 1.671±0.003 3.62±0.12 9716.7±556.7 1.27±0.01

Table 1: System Suitability

Results Discussion

The developed reverse phase UPLC method was validated for parameters like specificity, accuracy, linearity, precision, Limit of detection (LOD), limit of quantitation (LOQ) and robustness according to ICH guidelines[14].

The specificity of the developed method was performed to analyze the interference components which were expected to be present include solvent, excipients, degradants and impurities with the analyte peaks during determination of analytes. To establish specificity, sample, blank and the impurities generated by forced degradation were injected into the chromatographic system and observed for any interfering peaks at the RT of analytes. The degradation was carried out intentionally by exposing the samples into various stress conditions. The sample chromatograms were shown in fig. 3. After the injection of sample, blank and degradation samples into the chromatographic system, no interfering peaks were observed at the RT of analytes. Hence, the method was found to be specific for the estimation of drugs.

IJPS-sample

Figure 3: Chromatograms of sample preparations. Chromatograms of A. Formulation A and B. Formulation B

The precision of the method expresses the closeness of agreement between a series of measurements. It was examined using pure samples. The working standard solutions were prepared and injected six times into the system. The results were tabulated in Table 2. The mean and standard deviation (SD) of peak areas were considered; percentage relative standard deviation (RSD) for peak areas was calculated and reported. RSD values were found within the limits, inferring good repeatability of the developed method.

No. of injections Lamivudine Tenofovir disoproxil fumarate Doravirine Efavirenz
RT (min) Peak area RT (min) Peak area RT (min) Peak area RT (min) Peak area
1 1.009 318566 1.233 301462 1.427 101594 1.666 642993
2 1.012 312155 1.235 299482 1.428 103694 1.670 645839
3 1.012 316678 1.236 300968 1.430 102241 1.671 642575
4 1.013 313755 1.236 298624 1.431 101216 1.672 644333
5 1.014 315265 1.238 298743 1.432 102155 1.674 645871
6 1.015 314625 1.238 300474 1.433 101927 1.675 642637
Mean 315174 299959 102138 644041
SD 2245.2 1185.8 851.2 1541.8
RSD (%) 0.7 0.4 0.8 0.2

Table 2: Results Of Precision Studies Results Of Precision Studies

The linearity was developed between concentration of standard solutions and their responses. The test solutions were prepared from standard solution at six concentration levels from 25 % to 150 % of assay concentration. The obtained peak area versus concentration was treated by the least squares linear regression analysis. The calibration plots were shown in fig. 4. The results were tabulated in Table 3, which have shown excellent linearity between peak areas and concentration within the specified concentration range.

IJPS-tenofovir

Figure 4: Calibration plots of analytes. Calibration plots of A. lamivudine, B. tenofovir disoproxil fumarate, C. doravirine and D. efavirenz

Percentage level Lamivudine Tenofovir disoproxil fumarate Doravirine Efavirenz
Concentration
(µg/ml)
Peak area Concentration
(µg/ml)
Peak
area
Concentration
(µg/ml)
Peak
area
Concentration
(µg/ml)
Peak
area
25 7.5 84266 7.5 75499 2.5 25201 15 164858
50 15 154822 15 150629 5 50845 30 328328
75 22.5 223455 22.5 226368 7.5 74935 45 488277
100 30 311473 30 308325 10 99965 60 650628
125 37.5 384776 37.5 377326 12.5 124983 75 818475
150 45 469557 45 450843 15 148844 90 968192
Correlation coefficient (r2) 0.999 0.999 0.999 0.999

Table 3: Results Of Linearity

The correlation coefficients were found to be 0.999 for all the four drugs, which met the acceptance criteria and hence the method was said to be linear within the specified concentration range.

The accuracy, also called as trueness, was determined by carrying out recovery studies where known quantities of the drug substance have been added to drug product. The developed analytical procedure has been applied so as to determine the added amount of drug substance. The percent recoveries were calculated and reported. The results of accuracy using formulation A and B were presented in Table 4 and Table 5. The results were found under the acceptance criteria which must be in the range of 98-102 %. Hence, the developed method was found to be accurate for the estimation of the mentioned drugs.

Analyte Accuracy level (%) *Amount spiked (µg/ml) *Amount found (µg/ml) #Mean percentage recovery
Lamivudine 50
100
150
15
30
45
15.06
30.21
45.04
100.40
Tenofovir disoproxil fumarate 50
100
150
15
30
45
14.93
30.26
45.07
100.19
Doravirine 50
100
150
5
10
15
5.03
10.00
15.02
100.23

Table 4: Results Of Accuracy (Formulation A)

Analyte Accuracy level (%) *Amount spiked (µg/ml) *Amount found (µg/ml) #Mean percentage recovery
Lamivudine 50
100
150
15
30
45
14.85
30.18
45.06
99.91
Tenofovir disoproxil fumarate 50
100
150
15
30
45
14.97
30.01
45.08
100.01
Efavirenz 50
100
150
30
60
90
30.16
59.35
89.28
99.56

Table 5: Results Of Accuracy (Formulation B)

The LOD and LOQ specify the lowest amount of analytes that can be detected and necessarily quantified respectively. Several methods were available for determining these limits. Among which, formula method has been applied to determine the limits. The values were calculated using the formulae. LOD=3.3 σ/S and LOQ=10 σ/S, Where, σ is the SD of the y-intercept and S is the slope of the calibration curves. The detection and quantitation limits were presented in Table 6. The itemized results showed the sensitivity of the developed method.

Parameter Lamivudine Tenofovir  disoproxil fumarate Doravirine Efavirenz
LOD (µg/ml) 0.15 0.10 0.04 0.32
LOQ (µg/ml) 0.44 0.29 0.11 0.97

Table 6: Results Of Lod And LOQ

Robustness of the analytical method indicates the reliability of the method upon normal usage. It was examined by intentionally altering the chromatographic conditions so as to prove the capability of the method remained unaffected by those variations in chromatographic parameters. The parameters such as flow rate, column temperature, and mobile phase composition were altered to prove the robustness of the developed method. A deviation of ±0.1 ml/min in the flow rate, ±5º in the column temperature and 5 % variation in mobile phase ratio were tried individually. A standard solution at test concentration with the specified changes in the operational conditions was injected into the chromatographic system for six times.

The results of robustness were presented in Table 7. The results infer that all the parameters were found to be within the limits even after slight variations in the chromatographic conditions. Hence, the method was found to be robust.

Parameter Lamivudine Tenofovir  disoproxil fumarate Doravirine Efavirenz
Tailing* Plate count* Tailing* Plate count* Tailing* Plate count* Tailing* Plate count*
Low flow rate
(0.3 ml/min)
1.16 3430.7 1.20 6147.3 1.21 8193.2 1.25 10002.2
High flow rate
(0.5 ml/min)
1.23 4014.0 1.17 5833.5 1.22 7280.7 1.26 8786.7
Low column temperature (25º) 1.16 3684.8 1.17 5451.5 1.22 7491.8 1.26 9392.0
High column temperature (35º) 1.21 3586.5 1.24 5530.7 1.18 7577.5 1.29 8995.1
Low organic phase (55:45) 1.19 3995.5 1.21 5553.2 1.18 8271.2 1.27 10152.8
High organic phase (45:55) 1.22 3623.3 1.18 5864.0 1.22 7468.2 1.28 8765.0

Table 7: Results Of Robustness

During the forced degradation study, the standard and degraded samples were injected into the system with a run time of 10 min and the results were tabulated in Table 8. The percentage of drug degraded in the solution was calculated. The significant degradation was found in the presence of acid and base. Drugs were slightly degraded upon oxidation, under light and heat. The degradation products produced as a result of stress studies did not interfere with the RT of analytes. Therefore, the assay was considered as stability-indicating for the simultaneous estimation of lamivudine, tenofovir disoproxil fumarate, doravirine and efavirenz.

  Stress conditions Lamivudine Tenofovir  disoproxil fumarate Doravirine Efavirenz
Percentage Degraded
Acid 17.37 13.54 9.55 8.31
Base 12.76 15.01 3.34 6.82
Peroxide 3.81 6.93 8.52 8.51
Thermal 2.71 1.72 2.27 3.12
Photolysis 5.01 2.09 2.37 1.41

Table 8: Forced Degradation Studies

The applicability of the proposed method was confirmed by assaying formulations A and B. The standard and sample solutions were prepared and injected into the chromatographic system. The amount of drug present in the formulation was calculated and the results were tabulated in Table 9 and Table 10. The results were in conformity with the label claim and hence the developed method can be effectively applied for quality analysis of formulations.

S. No. Lamivudine Tenofovir disoproxil
fumarate
Doravirine
Percentage Assay
1 99.40 99.96 99.58
2 99.28 100.25 101.33
3 99.14 99.54 100.00
4 99.63 99.43 100.47
5 99.65 100.24 99.52
6 100.09 99.54 101.29
Mean 99.53 99.83 100.36

Table 9: Assay Results Of Formulation A

S. No. Lamivudine Tenofovir  disoproxil fumarate Efavirenz
Percentage Assay
1 99.26 99.92 100.28
2 100.32 99.20 100.07
3 99.28 100.70 99.12
4 99.23 100.29 99.10
5 100.25 100.29 98.97
6 100.30 99.98 99.99
Mean 99.77 100.06 99.59

Table 10: Assay Results Of Formulation B

The proposed stability indicating UPLC method was found to be simple, accurate, precise, robust, quick and economic. All the four drugs were eluted within 3 min with good resolution utilizing a simple mobile phase, thereby drastically lessening the analysis time. This method can be used for the assay of drugs in bulk as well as in their dosage forms either individually or in combinations. Thus, the developed method can be employed for routine assay of bulk and tablets containing lamivudine, tenofovir disoproxil fumarate, doravirine and efavirenz in quality control divisions.

Acknowledgements:

The author expresses sincere thanks to Spectrum Pharma Research Solutions, Hyderabad for providing necessary facilities to carry out the research work.

Conflict of Interests:

The authors declared no conflict of interest.

References