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Development and validation for the simultaneous estimation of lamivudine, tenofovir disproxil and dolutegravir in drug product by RP-HPLC
Abstract
The aim of the method was to develop and validate a rapid, sensitive and accurate method for simultaneous estimation of Lamivudine, Tenofovir DF and Dolutegravir in drug product by liquid chromatography. The chromatographic separation was achieved on column (Luna C8 150*4.6mm) at ambient temperature. The separation was achieved employing a mobile phase consists of 0.1%v/v TFA in water and Acetonitrile with simple gradient programme. The flow rate was 1.0ml/ minute and ultra violet detector at 260nm. The average retention time for Lamivudine, Tenofovir DF and Dolutegravir found to be 2.023 min, 5.330 min and 7.673. The proposed method was validated for selectivity, precision, linearity and accuracy. All validation parameters were within the acceptable range. The assay methods were found to be linear from 75.0 – 225.0µg/ml for Lamivudine, 75.0 – 225.0µg/ml of Tenofovir DF and 12.5 – 37.50µg/ml of Dolutegravir.
... They came to the conclusion from this experiment that the newly created approach for simultaneously estimating the TENOFOVIR Disoproxil Fumarate discovered towards straightforward, accurate, exact, high resolution, with time-efficient which made this technique extra palatable and economical. 16 Yogesh Pawar et. al. in 2017: Tenofovir Disoproxil Fumarate RP-HPLC method was created by means of column C-18 (4.6 x 250 mm) as immobile phase, 0.1% OPA in Methanol: Water (85:15 v/v) as mobile phase. ...
Many questions have been raised regarding the management of acquired immunodeficiency syndrome (AIDS) which is caused by a retrovirus called as HIV, (human immunodeficiency virus) is what causes AIDS. Infection caused by HIV is particularly the world's most serious health and development challenges. Although there is no known complete cure for HIV, several drugs can help you stay healthy by lowering the amount of HIV in your body. When treating HIV infection, antiretroviral therapy is used, and a variety of medications are available from this category. Tenofovir and its salt versions, both by themselves and in combination with emtricitabine, are the most often utilized medications. HIV levels should be lowered so that your immune system can function more effectively. This article offers a summary and evaluation of several analytical techniques used on the antiretroviral medication tenofovir over the previous five years. It covers forced degradation, HPLC and RP-HPLC, HPTLC, UPLC and RP-UPLC, LC-MS.
... Extensive literature survey was carried out which revealed that there is no work carried out especially on Forced degradation method of the Lamivudine and Dolutegravir in tablet dosage form using UPLC method. The specific aim of the research was to develop a UPLC method and performing forced degradation studies of Lamivudine and Dolutegravir in bulk and formulated dosage form and to validate the proposed methods in accordance with ICH guidelines for the intended analytical application [5][6][7][8][9][10][11][12] ...
A simple, fast, precise, specific, and accurate reversed phase Ultra Performance Liquid Chromatographic (UPLC) method was developed and validated for the forced degradation studies of the Lamivudine and Dolutegravir in tablet dosage form. Chromatogram was run HSS C18 (2.6 x 50mm, 1.6µm). Mobile phases containing 70% 0.01N disodium hydrogen phosphate: 30% Methanol). The elution of analytes was achieved with a flow rate at 0.3 mL/min. Potassium dihydrogen phosphate was used as a buffer in this experiment. Temperature was maintained at 30°C. Optimized wavelength selected at 260nm. The detector response was linear in the concentration range of 25-150μg/mL respectively. Retention time of Lamivudine and Dolutegravir were found to be 1.408min and 1.739min. %RSD of the Lamivudine and Dolutegravir were and found to be 0.8 and 0.8. The % recovery was obtained as 100.39% and 100.37% for Lamivudine and Dolutegravir. LOD, LOQ values obtained from regression equations of Lamivudine and Dolutegravir were 0.41, 1.25 and 0.09, 0.26. Regression equation of Lamivudine was y = 24270x + 12218 and for Dolutegravir was y = 34783x + 1060. Since retention times and run times were reduced, the method established was easy and premium and it could be used in periodic quality control tests in industries.
... 7 From the literature survey, methods for simultaneous analysis of LMN have been reported in combination with TNF or DLG by High Performance Liquid Chromatography (HPLC). [8][9][10][11][12] Kavya et al. 13 Mallikarjuna Rao et al. 14 Kalpana et al. 15 and Mastanamma et al. 16 have reported the HPLC methods for simultaneous determination of DLG, LMN and TNF in bulk and tablet dosage form. All these methods, however, have a longer chromatographic run time of greater than 10 min. ...
A simple, selective and rapid ultra performance liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous estimation of dolutegravir, lamivudine and tenofovir in bulk and tablet dosage form. Chromatographic separation was attained on Acquity Ethylene Bridged Hybrid (BEH) C18 column (50 × 2.1 mm, 3.5 µm), using a mixture of acetonitrile and 0.1% formic acid in water (60:40, v/v) as a mobile phase at a flow rate of 0.12 mL/min. The total run time of analysis was 3.5 min. The analytes were detected using tandem mass spectrometry, operating in positive ionization and multiple reaction monitoring modes. The method's linearity was determined to be in the range of 10–150 ng/mL with r ² > 0.99. The proposed method was validated as per the International Council for Harmonization (ICH) guidelines, and the results were found well within the acceptance limits. The method was successfully applied for the simultaneous quantification of all the three analytes in the combined tablet dosage form.
... A few analytical methods were developed for simultaneous analysis of LMV with other antiviral agents and TDF with other antiviral agents [10][11][12][13][14][15][16]. Certain analytical methods were described to estimate TDF, LMV with other antiretroviral agents [17][18][19][20][21][22][23][24][25][26]. An immense exploration of literature exposed that only two HPLC methods were recently reported for simultaneous analysis of DOR, LMV and TDF in a tablet dosage form [27,28]. ...
Background
To establish a simple, sensitive, accurate, precise, efficient, economical RP-UPLC method for simultaneous estimation of Doravirine, Lamivudine and Tenofovir disoproxil fumarate in bulk and their combined pharmaceutical formulations. Optimization of Chromatographic separation was achieved on analytical column HSS C18 (100 × 2.1 mm, 1.8 μ) maintained at temperature 30 °C and mobile phase consisting of 0.01 N Potassium dihydrogen orthophosphate buffer (pH-4.8) and acetonitrile in the ratio 60:40 v/v and at a flow rate 0.3 mL/min in isocratic mode. The injection volume was set as 1 µl detection wavelength is 260 nm. The proposed method validation was done as per International Council on Harmonization Q2 (R1) guidelines.
Results
Doravirine, Lamivudine and Tenofovir disoproxil fumarate were eluted at retention times of 1.2, 1.5, and 1.8 min respectively. The proposed method was identified an excellent linearity over concentration range of 12.5–75.0 µg/mL for Doravirine and 37.5–225.0 µg/mL for Lamivudine and 37.5–225.0 µg/mL for Tenofovir disoproxil fumarate. The percentage relative standard deviation for intra-day and inter-day precision of the present method was less than 2% for Doravirine, Lamivudine and Tenofovir disoproxil fumarate. Accuracy of the present method was evaluated by recovery studies which were in the range of 99.62–99.88% for Doravirine and 98.78–99.44% for Lamivudine and 99.67–100.52% for Tenofovir disoproxil fumarate. The limit of detection and limit of quantification were found to be 0.249 µg/mL and 0.756 µg/mL for Doravirine and 0.24 µg/mL and 0.727 µg/mL for Lamivudine and 0.797 µg/mL and 2.966 µg/mL for Tenofovir disoproxil fumarate. Forced degradation studies were carried out under various stress conditions like acid, base, peroxide, thermal, photo and neutral conditions.
Conclusions
The present method makes sure about no degraded impurity peak interference at the retention time of analyte peak hence can be applied for quality control investigation of Doravirine, Lamivudine and Tenofovir disoproxil fumarate in bulk and pharmaceutical formulations.
... These determinations require highly sophisticated instruments and methods like Mass Spectroscopy, Gas chromatography, HPTLC 14 , HPLC [15][16][17][18]etc. RPHPLC 11,[19][20][21] method is sensitive, accurate, precise and desirable for routine estimation of drugs in formulations. Thereby it is advantageous than volumetric methods. ...
Requirement for a sophisticated analytical method using HPLC and HPTLC is in high demand to meet the needs of a small scale industry for analysis of drugs that are relatively expensive. Hence a simple method was proposed in the routine determination of Mafenide acetate in pharmaceutical formulations and bulk dosage forms that can be less expensive. An analytical method was developed for the estimation of Mafenide acetate drug substance by liquid chromatography. The chromatographic separation was achieved on phenyl column (Eclipse XDB-Phenyl 250*4.6, 5um) at ambient temperature. The separation was achieved employing a mobile phase consisting of 0.1 %v/v Trifluoroacetic acid in water: Methanol (10:90). The flow rate was 1.0 ml/ minute and ultraviolet detector at 245nm. The average retention time for Mafenide acetate was 3.3 minutes. The proposed method was validated for selectivity, precision, linearity and accuracy. All validation parameters were checked and are found within the acceptable range. The assay methods were found to be linear ranging from 50-150 µg/ml for Mafenide acetate. The parameters considered for the procedure are related limit, selectivity, linearity, range, accuracy and precision are defined. Thesample solution leads to unequivocal, absolute identification of the analyte peak of interest apart from all other matrix components. The objective of our work is to form a basis for production procedure and control, which are designed to assure that the drug products have the identity, Quality, and purity. The results obtained could be treated as simple, sensitive and reproducible for determination of Mafenide acetate in pharmaceutical formulations.
... A wide variety of analytical methods were disclosed in the literature for determination of LMV as single moiety and LMV in combination with other antiretroviral agents [12][13][14][15][16][17][18][19]. Similarly, several methods were described in literature to assess TDF alone and in combination with other antiretroviral agents including Emtricitabine, Efavirenz, Dolutegravir and LMV [20][21][22][23][24][25][26][27]. Only Two HPLC methods have been recently reported for the tablet consisting of three drugs including DRV, LMV and TDF [28,29]. ...
Objectives: The main aim of the study was to develop an economical, insightful,accurate and simple RP-HPLC-DAD method with high precision and good sensitivity for concurrent determination of Tenofovir disoproxil fumarate, Doravirine and Lamivudine in blended bulk form and their combined tablet form. Material and methods: A method with Ascentis C18 (150 x 4.6mm, 5μm) column, mobile phase ratio of 0.1% ortho phosphoric acid and Acetonitrile in 70:30 (v/v), 1ml/min flow rate and detection wavelength of 260nm was highly proficient in effective separation of all three drugs. The developed method was validated in accordance with ICH specifications.
Results: The retention times of Doravirine, Lamivudine and Tenofovir disoproxil fumarate observed were 2.4, 2.9, and 3.6 min respectively. The linear responses were observed for Doravirine, Lamivudine and Tenofovir disoproxil fumarate in the range of 12.5-75 μg/ml, 75-225μg/ml and 75-225μg/ml respectively. The limit of detection and quantification values were calculated to be 0.36μg/ml and 0.11μg/ml for Lamivudine, 0.55μg/ml and 1.66 μg/ml for Tenofovir disoproxil fumarate and 0.03 μg/ml and 0.09μg/ml for Doravirine. The %RSD values of the intraday and inter-day precision were calculated in the range of 0.134-1.749. The mean percentage recovery of all three analytes was in the range of 98.85% - 100.18%. The statistical results of the validation parameters ensured that the method was accurate, specific, and precise with good sensitivity. Investigation of analytes under different stressed conditions ensures the stability of analytes reflecting the stability indication of the method. The developed method has high proficiency in separation of Tenofovir disoproxil fumarate, Doravirine and Lamivudine. The degradation products generated due to stress conditions also separated with good resolution.
Conclusion: The current method is a stability-indicating assay method consisting of appropriate specificity, accuracy, precision and sensitivity. The developed method has a good potential to be adopted by the pharmaceutical industrial sector.
... The broad literature search disclosed that a small number of analytical methods like UV and RP-HPLC methods were at hand for determination of LAM. TDF and EVZ individually and in combined dosage form [3,5,[12][13][14][15][16]. In addition to those methods, few RP-HPLC methods were described for determination of LAM, TDF with dolutegravir, and other antiviral agents [17][18][19][20][21]. ...
Background
An easy, defined, rapid, and accurate reverse phase high-performance liquid chromatography method was developed and subsequently validated for the concurrent estimation of lamivudine, efavirenz, and tenofovir disoproxil fumarate in their pure blend and combined tablet formulation. An efficient and appropriate separation of the three analytes was attained with Zorbax eclipse XDB-Phenyl column, with a mobile phase of methanol: buffer (0.1% v/v formic acid in water) (73:27 v/v) at a flow rate of 1mL/min and isocratic elution by using 260nm as detection wavelength. Equal ratio of acetonitrile and water was used as diluent.
Results
The retention times of lamivudine, tenofovir disoproxil fumarate, and efavirenz were found at 2.6, 4.4, and 5.9 min respectively. The linear response for lamivudine, tenofovir disoproxil fumarate, and efavirenz was in the range of 15.0–45.0μg/mL, 15.0–45.0μg/mL, and 20.0–60.0 μg/mL respectively. The method validation was done in accordance to ICH guidelines and all validation parameters in compliance with ICH standards. The degradants produced by stress testing were well resolved from the peaks of active analytes, which stipulates the stability-indicating property of the method.
Conclusion
The method has the ability to separate lamivudine, efavirenz, and tenofovir disoproxil fumarate concurrently in blended powder and their combined tablet. All degradants produced by application of stress conditions were separated with high resolution and determined with good sensitivity that ensures the stability-indicating property of the method. Thus, the projected method has high probability to adopt in the pharmaceutical industrial sector.
In the pharmaceutical industry, effective risk management and control strategies for potential genotoxic impurities are of paramount importance. The current study utilized GC–MS to evaluate a precise, linear, and accurate analytical method for quantifying ethylenediamine present in tripelennamine hydrochloride using phthalaldehyde as a derivatizing agent. When phthalaldehyde is sonicated for 10 min at room temperature, it reacts with ethylenediamine to form (1z,5z)‐3,4‐dihydrobenzo[f][1,4]diazocine. This approach minimizes matrix interference issues and resolves sample preparation difficulties encountered during ethylenediamine identification in GC–MS. In this method, helium serves as the carrier gas, while methanol acts as the diluent. The stationary phase consists of a DB‐5MS column (30 m × 0.25 mm × 0.25 μm) with a flow rate of 1.5 mL/min. The retention time of (1z,5z)‐3,4‐dihydrobenzo[f][1,4]diazocine was determined to be 6.215 min. The method validation demonstrated limits of detection and quantification for (1z,5z)‐3,4‐dihydrobenzo[f][1,4]diazocine at 0.4 and 1.0 ppm, respectively, with a linearity range spanning from 1 to 30 ppm concentration with respect to the specification level. System suitability, precision, linearity, and accuracy of the current method were assessed in accordance with guidelines, yielding results deemed suitable for the intended use.
Dolutegravir sodium, Lamivudine and Tenofovir disoproxil fumarate are anti-viral drugs. A new stability indicating UHPLC method has been proposed for the simultaneous determination of Dolutegravir sodium, Lamivudine and Tenofovir disoproxil fumarate tablets on gradient mode with 1.0mL/min flow rate and a diluent consisting of a mixture of phosphate buffer (pH 3.0) and acetonitrile (62: 38) was used for the study. Shimadzu NexeraX2 Model UPLC system with (PDA detector) Zorbax SB-C18 column (100mm × 4.60mm, 3.5μ) was employed for the present study. Beer-Lambert’s law was obeyed over the concentration range 5-900, 2-700 and 2-150µg/mL with linear regression equationsy = 1724.x-11307 (R² = 0.999), y = 1737.x-1009 (R² = 0.999) and y = 3538.x+587.5 (R² = 0.999) for Lamivudine, Tenofovir disoproxil fumarate and Dolutegravir sodium and the method was validated as per ICH guidelines. The LOQ values were found to be 4.4926, 1.8342 and 1.7963 µg/mL and that of LOD values as 1.4811. 0.6024 and 0.5903µg/mL forD Lamivudine, Tenofovir disoproxil fumarate and Dolutegravir sodium respectively. The combination of Dolutegravir sodium, Lamivudine and Tenofovir disoproxil fumarate was exposed to various stress conditions and the stability of the proposed method was carried out at UV detection 260nm. The proposed UHPLC method is simple, precise, accurate, robust and used for the routine analysis of tablet dosage forms.
Objective: The purpose of this research was to develop and validate a stability-indicating RP – HPLC technique for simultaneous quantification of Emtricitabine (EMT), Tenofovir Alafenamide Fumarate (TEN), and Dolutegravir Sodium (DOL) in bulk and in their combined formulation.
Material and Methods: The developed approach was done on Exterra C18 column (150 × 4.6 mm, 5 μm) and Methanol and Buffer (comprising 0.1 (v/v) of Triethylamine and o-phosphoric acid in water, pH 2.6) as mobile phase in the proportion of 75:25 (v/v), eluted at 1 mL/min. The analytes were quantified using DAD detector at 265 nm.
Results: The approach was validated in accordance with the ICH guidelines. Linearity, precision, accuracy, specificity, Limit of Detection (LOD), Limit of Quantitation (LOQ), and robustness were used to validate the proposed method. Linear response was found in the range of 500 – 1500 µg/mL for EMT, 62.5 - 187.5 µg/mL for TEN and 125 – 375 µg/mL for DOL. The LOD values of EMT, TEN and DOL were found 91.78 μg/mL, 10.47 μg/mL and 19.28 μg/mL correspondingly. The LOQ values of EMT, TEN and DOL were found and 278.11 μg/mL, 31.74 μg/mL and 58.42 μg/mL correspondingly. The assay outcomes for all drugs were observed between 99.11 – 100.84%. To access the method's stability indicating capabilities, the drugs were exposed to various environmental (acid, alkaline, neutral, oxidative, photolytic and thermal) conditions.
Conclusion: The established approach was considered to be accurate, linear, precise, specific, robust and it can be utilized to analyse the drugs mentioned in its tablet.
New RP-HPLC method have been developed for simultaneous analysis of lamivudine and dolutegravir in pharmaceutical dosage forms and applied to stability studies of drugs. The title analytes were eluted rapidly with phosphate buffer (pH 5.0) and acetonitrile (60:40 v/v) on Std discovery C18 (150 x 4.6 mm, 5 µ) column. The detection was carried out using PDA detector at 260 nm. The solutions were chromatographed at a constant flow rate of 1 mL/min. Lamivudine and Dolutegravir were eluted at 2.37 min and 2.97 min respectively with good resolution. Method was validated as ICH guidelines. The linearity range of lamivudine and dolutegravir were found to be of 18.75 - 112.5 µg/mL and 3.125 - 18.75 µg/mL, respectively. The % RSD values (< 2) in precision studies indicates the reproducibility of method. The percentage recoveries were 100.17 % and 100.36 % respectively for lamivudine and dolutegravir, found to be within the limits. The proposed validated method was fruitfully applied for assay of formulation and stability studies of drugs under various stress conditions.
In the analysis of the pharmaceutical agents new sophisticated chromatographic methods have been utilized for the quality control purpose. In the current scenario ample amount of new drugs and newer pharmaceutical formulations are available intended in the cure of diseases. Diseases like HIV, AIDs , Hepatitis, and other viral diseases requires newer drugs and their combinations. As a result of this there is a need for analyse the drugs for quality control purposes. Here the api-drugs Lamivudine LAM, tenofovir TEN, Doravirine DOR, has been analysed by the RP-HPLC method in the tablet dosage-forms. This method is developed for the analysis, of these three drugs in combined forms for rapid analysis with very less amount of analytes drugs utilized for analysis purposes. The concentration range for the linearity selected was 7.5 to 45 µg/ml for Lamivudine LAM & Tenofovir TEN, where as for Doravirine DOR it is 2.5 to 15 µg/ml. Wavelength selected for estimation was 269nm and chromatographic column used was Acclaim 120 C-18 column ( 250 mm x 4.6 mm, 5 µm id ).
Pharmaceutical medicines play an important role in human life that helps to cure different diseases. For the chemical and pharmaceutical analysis of the drug effective quality control and pharmacodynamic and pharmacokinetic studies are needed. Several methods have been developed and validated for its pharmaceutical and biological materials since it was introduced as an important antiretroviral agent. The literature survey reveals that only four RP HPLC method and one HPTLC was developed for the simultaneous estimation of Dolutegravir, Lamivudine and Tenofovir disoproxil fumarate in tablet dosage form. These three drugs are used as antiretroviral medicines which are used for HIV or AIDS prevention and treatment. The goal of this review is to define and establish a simple, precise and selective method for estimating the dosage of Lamivudine, Tenofovir Disoproxil fumarate and Dolutegravir in biological and pharmaceutical dosage form using the HPLC, HPTLC, UPLC, UV Visible spectroscopy, LC/MS, Infrared spectroscopy, NMR spectroscopy, Microbiological assay, Electrochemical studies and Capillary electrophoresis. UV-detector HPLC is commonly used in pharmaceuticals and LC-MS are widely used for biological materials with mass and tandem mass spectrometer detector systems. Various parameters such as device suitability, process accuracy, precision, linearity, detection limit will validate the UV Visible spectroscopy and RP-HPLC technique.
A simple, rapid, and economical method has been developed for the simultaneous estimation of the latest FDA approved antiviral drug combination, Dolutegravir, Lamivudine, and tenofovir disoproxil fumarate in tablet dosage form using Shimadzu LC-20 AT HPLC with a Phenomenex Luna column compartment., the method was developed using HPLC graded methanol with o-phosphoric acid as a mobile phase and successfully validated the developed method as per the ICH guidelines. The method was found to be linear, accurate, precise, robust, and rugged. The limit of detection and the limit of quantification was found to be 2.6μg/ml and 8.18μg/ml for Dolutegravir, 14.63 μg/ml and 44.35 μg/ml for Lamivudine and 16.43 μg/ml and 49.81 μg/ml for tenofovir disoproxil fumarate respectively. The retention time was found to be 3.0, 2.3 and 2.7 min for Dolutegravir, Lamivudine and tenofovir disoproxil fumarate respectively. All of assessed parameters complied with the acceptance criteria hence indicated the usefulness of the RP-HPLC method for the determination of assay and in-vitro dissolution study for tablet dosage form which contains lamivudine, tenofovir disoproxil fumarate, and dolutegravir active substances. Hence the method can be applied for routine quality control of the drugs.
In this paper, for the first time, the study of electrochemical investigation and voltammetric determination of HIV integrase inhibitor dolutegravir was described. The effect of pretreatment (anodically or cathodically) of boron-doped diamond electrode was investigated in detail on the stripping voltammetric performance for this compound. Dolutegravir presented one or two oxidation peaks within the pH range 2.0–12.0 by using both pretreated electrodes. Employing square-wave stripping mode on the surface of cathodically pretreated boron-doped diamond electrode (after 60 s accumulation at +0.3 V) in Britton-Robinson buffer at pH 10.0, there was a linear dependence of the peak current at +0.79 V (vs. Ag/AgCl, 3 M NaCl) and dolutegravir concentration in the range of 1.0–50.0 μg mL⁻¹ (2.38 × 10⁻⁶–1.19 × 10⁻⁴ M), with a detection limit of 0.26 μg mL⁻¹ (6.20 × 10⁻⁷ M). The applicability of the developed approach for the quantification of dolutegravir was tested in the commercial tablet formulations and model human urine samples.
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