Current Pharmaceutical Analysis - Volume 16, Issue 7, 2020

This article describes and reviews the steps involved in risk assessment of the twenty-four (24) potential elemental impurities in pharmaceutical drug products, as per the permitted daily exposure limits. Screening and estimation of prescribed elemental impurities in pharmaceutical drug substances, inactive excipients and drug products by inductively coupled plasma mass spectrometry or inductively coupled plasma optical emission spectrometry and their controls involved are also reviewed, as referred in the general chapters <232> & <233> of the United States Pharmacopoeia, Q3D guideline for elemental impurities as per international conference on harmonization and q3d elemental impurities: guidance for industry as per U. S., Food and Drug Administration USFDA.

Background: Thiamphenicol belongs to the amphenicol class of antibiotic and possesses a broad-spectrum antimicrobial activity. An alternative microbiological assay for quantification of thiamphenicol in pharmaceutical formulations has not yet been reported in the literature. Objective: This study aimed to develop and validate an agar diffusion method for quantification of thiamphenicol in soft capsules. Methods: The assay was based on the inhibitory effect of thiamphenicol on the following: a strain of Kocuria rhizophila ATCC 9341, used as the test microorganism, Antibiotic 1culture medium, phosphate buffer pH 6, 0, inoculum at a concentration of 1%, as well as standard and sample solutions at the concentrations of 20.0, 40.0 and 80.0 μg mL-1. Results: The method validation yielded good results for the parameters of linearity, precision, accuracy, robustness and selectivity. The experimental statistic results were analyzed using analysis of variance (ANOVA). The method was found to be linear (r2 = 0.9992) in the range of 20-80 μg mL-1, precise (inter-assay R.S.D = 0.09%), accurate (R.S.D. = 4.65%), specific, and robust. Conclusion: The results demonstrated the validity of the proposed bioassay, which allows for reliable quantification of thiamphenicol in a pharmaceutical sample. An alternative methodology for thiamphenicol determination in routine quality control has been reported herein.

Background: Teriflunomide is an immunosuppressive agent. Immunosuppressive agents are drugs that inhibit or prevent activity of immune system. Teriflunomide was investigated as a medication for multiple sclerosis (MS). Various studies have reported the HPLC, UPLC, LC/MS methods for the estimation of teriflunomide. However, till date stability indicating HPTLC analysis method has not been reported for the estimation of teriflunomide in bulk and tablet dosage form. Objective: Objective of the present work was to develop and validate stability indicating high performance thin layer chromatography method for the determination of teriflunomide in bulk and tablet dosage form. Methods: Chromatography was performed on aluminium plates coated with silica gel 60F254 using toluene, methanol and acetic acid (7.5: 2.5: 0.05 v/v/v) as mobile phase. Densitometric analysis was performed at 254 nm. The method was validated with different parameters such as linearity, precision, accuracy, specificity, robustness, limit of detection (LOD) and limit of quantitation (LOQ). The RF value of teriflunomide was 0.56 ± 0.03. The method is sensitive (limit of quantification 17.83 ng/band), precise (RSD ≤ 1.34%), accurate (drug recovery 98.49–99.53 %), and linear over the range 100–600 ng/band (r2 0.998). Results: Degradation products were found in stress conditions did not interfere with the detection of teriflunomide; therefore, the proposed technique can be considered stability-indicating. Teriflunomide did not degrade under alkaline hydrolysis, thermal and photolytic conditions but showed degradation under acid hydrolysis and oxidation with about 14.5 and 13.8 % decompositions respectively. Conclusion: The developed method was satisfactorily applied for the analysis of pharmaceutical preparations and proved to be specific and accurate for quality control of the cited drugs in their tablet dosage form.

Background: Sertraline Hydrochloride, an oral anti-depressant, has two chiral centers and its cis enantiomers and trans diasteromers are defined as related substances by United State Pharmacopoeia and British Pharmacopoeia. Introduction: A selective, stereospecific and economical high performance liquid chromatographic (HPLC) method was developed for the determination of sertraline enantiomeric forms. The HPLC-UV method was developed and optimized in the terms of system suitability parameters. Methods: The elution was made using a mixture of -cyclodextrin (-CD) and hydroxypropyl - cyclodextrin (HP -CD). Analysis was performed on a Zorbax SB C-18 column (250 x 4.6mm), 5μ with the mobile phase consisting of 170mM KH2PO4 containing -CD and HP -CD (pH: 3.0 with dil. H3PO4) and acetonitrile (75:25, v/v). Flow rate was kept at 1.0mL/min and the detection was carried out by UV at 220nm. Enantio-separation for sertraline was carried out using two different CDs (β-CD and HP β- CD) at different concentrations in the mobile phase. Results: Complete resolution of all the four isomers was achieved using 9mM β-CD and 15mM HP β- CD in the mobile phase. The development was optimized using central composite quadric model, where concentration of -CD and HP -CD were varied and resolution between trans diastereomeric impurities was calculated as a response. Conclusion: Resolution between any pair of isomers was found to be more than 2. Method development and optimization leading to the best resolution between the isomers of sertraline is described in detail.

Background and Objectives: Kang Fu Xin liquid (KFX) is an official preparation made from the ethanol extract product from P. Americana. The present quality control method cannot control the quality of the preparation well. The aim of the present study is to establish a convenient HPLC method for multicomponents determination combined with fingerprint analysis for quality control of KFX. Methods: An HPLC-DAD method with gradient elution and detective wavelength switching program was developed to establish HPLC fingerprints of KFX, and 38 batches of KFX were compared and evaluated by similarity analysis (SA), hierarchical clustering analysis (HCA), and principal component analysis (PCA). Meanwhile, six nucleosides and three amino acids, including uracil, hypoxanthine, uric acid, adenosine, xanthine, inosine, tyrosine, phenylalanine and tryptophan in KFX were determined based on the HPLC fingerprints. Result: An HPLC method assisted with gradient elution and wavelength switching program was established and validated for multicomponents determination combined with fingerprint analysis of KFX. The results demonstrated that the similarity values of the KFX samples were more than 0.845. PCA indicated that peaks 4 (hypoxanthine), 7 (xanthine), 9 (tyrosine), 11, 13 and 17 might be the characteristic contributed components. The nine constituents in KFX, uracil, hypoxanthine, uric acid, adenosine, xanthine, inosine, tyrosine, phenylalanine and tryptophan, showed good regression (R2 > 0.9997) within test ranges and the recoveries of the method for all analytes were in the range from 96.74 to 104.24%. The limits of detections and quantifications for nine constituents in DAD were less than 0.22 and 0.43 μg•mL-1, respectively. Conclusion: The qualitative analysis of chemical fingerprints and the quantitative analysis of multiple indicators provide a powerful and rational way to control the KFX quality for pharmaceutical companies.

Background: Metoprolol is a selective β1-adrenergic receptor antagonist (β-blockers). It is widely used for the treatment of hypertension and other related diseases. Metoprolol can be used as a doping agent in sports, thus has been included in the list of forbidden drugs. In Iran, therapeutic drug monitoring (TDM) of β-blockers is an applied procedure in some cases. A therapeutic regimen could be easily managed by the determination of drug levels in biological fluids which is a relatively costly process and requires highly skilled technical staff. Using a simple and low-cost analytical procedure may help to use TDM in routine clinical practice. Methods: A real biological sample was prepared and its pH was adjusted to 3-4, then metoprolol was quickly extracted using magnetic iron oxide nanoparticles (MIONPs) modified by sodium dodecyl sulfate (SDS) and determined by applying spectrofluorimetry at 340 ± 3 nm after excitation at 283 ± 3 nm. Results: The extraction and determination conditions including, the amount of MIONPs and SDS, pH of the solution, standing time, desorption solvent type and volume were investigated and adjusted. Calibration curves were linear over the concentration range of 6–100 ng/mL for plasma and 5–100 ng/mL for water, urine and exhaled breath condensate samples, respectively. Intra and inter-day precision values for determination of metoprolol in different samples were less than 5.6 % and 6 %, respectively, and accuracy (as a relative error) was better than 5 %. Moreover, standard addition recovery tests were carried out, and the analytical recoveries ranged from 86 % to 113 %. The limits of detection (LOD) and limits of quantification (LOQ) of metoprolol were found to be in the range of 2.1-3.4 ng/mL and 6.3- 10.2 ng/mL, respectively. Conclusion: The developed method was successfully applied to biological samples taken from a volunteer who was given an oral tablet of 50 mg metoprolol.

Background: Clofazimine has antibacterial and leprostatic properties, which has its use in Multidrug Therapy (MDT) of leprosy. As per the FDA guidance for industry, each NDA and ANDA must include the analytical procedures necessary to ensure the identity, strength, quality, purity, and potency of the drug substance and drug product. However, it was noticed that no stability indicating method is available in the literature for the estimation of degradation impurities of Clofazimine. Objective: Objective of the proposed work is to develop and validate a rapid, specific, linear, robust, accurate and sensitive Ultra High-Performance Liquid Chromatography (UHPLC) method with LC-MS compatible mobile phase for the quantification of degradation impurities of Clofazimine in a pharmaceutical dosage form (topical gel 0.5% w/w). Method: Ultra High-Performance Liquid Chromatography equipped with PDA and Tunable UV (TUV) detector at a wavelength of 284 nm, stationary phase with a fused core particle technology, LC-MS compatible mobile phase was employed in this study. Gradient elution was employed for ensuring the selectivity of degradation impurities and clofazimine. This method was validated in accordance with ICH Q2 guidelines. This is the first reported Ultra High-Performance Liquid Chromatography method for estimation of degradation impurities of clofazimine. Results: The method showed good linearity over the range of 0.25 -1.5μg/ml of clofazimine. All the validation parameters were within the acceptance criteria. The product is found to degrade in the acid and peroxide degradation condition. The major degradant impurities are eluted at relative retention times of 0.35, 0.89 and 0.95. The developed method successfully separated the degradation products of clofazimine and able to quantitate accurately in its formulation. Conclusion: To date, there is no UHPLC method for determination of degradation impurities of clofazimine. in pharmaceutical dosage forms. Being a specific, linear, accurate and robust method, this would help in determining the chemical stability of drug product during the product development as well as in the shelf life of the drug product.

Introduction: Polyvinyl alcohol (PVA), a polymer, is in demand due to its usage in different applications such as pharmaceutical, biomedical and textile, paper, food industries. Methods: A new sensitive reversed phased high-pressure liquid chromatography (RP-HPLC) method with refractive index detector (RID) was developed for determination of PVA in an ophthalmic solution containing dexpanthenol and PVA as active substances and it was validated according to The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline. Results: Chromatographic separation was achieved on a Chiral-AGP (150 mm × 4.0 mm, 5 μm) column kept at 30°C with an isocratic flow at a flow rate of 1.0 ml/min. The detector temperature was 30°C, the retention time of PVA was around 1.0 min and the total run time was 5 minutes. Conclusion: The proposed method showed linearity, accuracy, precision, specificity, robustness, solution stability, and system suitability results within the acceptance criteria.

Background: Abuse of drugs is associated with several medical, forensic, toxicology and social challenges. “Drugs of abuse” testing is therefore an important issue. Objective: We propose a simple CE-based method for the quantification of amphetamine, codeine and morphine after direct injection of Exhaled Breath Condensate (EBC) by the aid of simple stacking mode and an off-line pre-concentration method. Methods: Using graphene oxide adsorbents, amphetamine, codeine and morphine were extracted from EBC in order to eliminate the proteins and other interferences. In addition to off-line method, an online stacking mode was applied to improve the analytical signal obtained from the instrument. Results: The validation parameters were experimented on the developed method based on the FDA guideline over concentration ranges of 12.5-100, 30-500 and 10-1250 ng/mL associated with amphetamine, codeine and morphine, respectively. Small volumes (around 100 μL) of EBC were collected using a lab-made setup and successfully analyzed using the proposed method where precisions and accuracies (within day and between days) were in accordance with the guideline (recommended less than 15 % for biological samples). The recovery tests were used to evaluate the matrix effect and data (94 to 105 %) showed that the proposed method can be applied in different EBC matrix samplings of subjects. Conclusion: The proposed method is superior for simultaneous determination of amphetamine, codeine and morphine over chromatographic analyses because it is fast and consumes fewer chemicals, with no derivatization step.

Background: Among the existing antifungal drugs, Amphotericin B is the first drug in the treatment of systemic fungal infections. However, its large adverse reactions limit the clinical application and Liposome Amphotericin B resolves the problem. Objective: In the present study, a rapid, simple, sensitive and efficient method based on LCMS/ MS for determination of liposomal Amphotericin B in rat plasma and tissue samples using natamycin as the internal standard has been developed and validated. Methods: The analytical samples contain the plasma and various tissues disposed of by protein precipitation and determination of liposomal Amphotericin B by an LC-MS/MS. Chromatographic separation was achieved on a Poroshell 120 EC-C18 column (4.6 mm x 50 mm, 2.7 μm) with 10 mmol/L ammonium acetate in water-acetonitrile by gradient elution at a flow rate of 0.7 mL/min. The MS analysis was conducted in positive electrospray ionization with Multiple Reaction Monitoring (MRM). Results: The calibration curves of plasma and tissues showed good linear range from 50 to 10000 ng/mL. The analytical samples containing plasma and tissues were stable under different storage conditions and temperature. Conclusion: The developed LC-MS/MS method has been successfully applied to the studies of toxicokinetics and tissue distribution after intravenous injection of liposomal Amphotericin B to rats.

Background: Nardostachys jatamansi DC. is indigenous to the Himalayan regions of India and Tibetan plateau of China, and widely used as drugs for treating various neurological disorders, insomnia, hysteria and depressive illness. However, beyond the huge efforts to the organic components in N. jatamansi, the elemental contents have not been investigated, which are important for the quality control and biosafety evaluation of N. jatamansi. Objective: In this study, we quantified the element concentrations in N. jatamansi. Methods: Twenty-five N. jatamansi samples were collected in Sichuan, Tibet, Qinghai and Gansu provinces. The samples were digested and subjected to inductively coupled plasma optical emission spectrometer (ICP-OES) measurements. Results: The results indicated that Al, B, Ba, Ca, Cd, Co, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, Pd, Si, Sr, Ti and Zn were detected in N. jatamansi samples. The chemometrics approaches indicated that N. jatamansi samples from Sichuan and Tibet shared similar elemental patterns. The altitude did not statistically influence the elemental patterns of N. jatamansi samples, while the K, P, Ba and Cd showed meaningful decreases. The high Ba contents in all samples suggested the potential toxicity of heavy metal to patients. Conclusion: The inorganic element quantification of N. jatamansi is useful in the genuine regional drug identification, quality control and biosafety evaluations.

Background: The main aim of the study was to develop an automated static headspace gas chromatography (SHS-GC) method for determination of dichloromethane (DCM) in ampicillin sodium by using a capillary column. Methods: SHS-GC also known as gas chromatography-headspace is the technique of choice due to its high sensitivity, excellent separation abilities, low limit of detection and simplicity of the instrumentation used for the technique. The headspace sampling method has more appropriate sensitivity than the direct injection method because it can clearly separate volatile analytes from the sample matrix and effectively concentrate them. Therefore, this method results in less complex sample preparation, decreased instrument contamination, and increased capillary column life. Results: The developed SHS-GC method showed symmetrical peak shape reasonable retention time for DCM. A linear relationship was obtained over the range of 2-240 μg mL-1 with a correlation coefficient (r2) of 0.993. The recovery, system precision and robustness of the method were within the acceptable values. The Limit of Detection (LOD) and Limit of Quantitation (LOQ) were 0.5 μg mL-1 and 2 μg mL-1 respectively. Conclusions: The results obtained in this study demonstrate that SHS-GC method is selective, precise, linear, accurate and robust for determination of dichloromethane in ampicillin sodium and its formulation (injection).

Introduction: Loratadine (LOR) (Fig. 1), an active H1 receptor antagonist, is often used in the treatment of allergic disorders such as seasonal allergies and skin rash [1]. LOR was clinically approved for symptomatic relief of nasal and non-nasal symptoms of allergic rhinitis in children ≥2years. Materials and Methods: An adapted method of liquid chromatography-mass spectrometry (LC/MS/MS) was developed and validated to measure the concentrations of loratadine (LOR) and its active metabolite descarboethoxyloratadine (DCL) from pediatric plasma. After being mixed with the internal standard (IS, propranolol) and precipitated with methanol, samples were centrifuged and 20 μL of the supernatants were injected into the HPLC system. Separation was carried out on a reversed-phase C18 gradient column using a mobile phase consisting of water (containing 0.1 % formic acid) and acetonitrile. The flow rate was 0.5 mL/min and the running time was 5.0 min for each sample. Results and Conclusion: Quantitation of LOR, DCL and IS was performed using MRM mode and the transitions were: 383.1 → 337.1 for LOR, 311.1 → 259.0 for DCL and 260.2 → 116.0 for propranolol, respectively. The method was validated according to FDA guidelines, precisions and accuracies met the requirements in all cases. Calibration curves were 0.2–50.0 ng/mL for both LOR and DCL. This method was then applied for a pilot study examining the pharmacokinetics and therapeutic drug monitoring of LOR in children.

Background: In some cases, lifestyle changes are not enough to keep type 2 diabetes under control, so there are several medications that may help. Metformin can lower your blood sugar levels, Glimepiride makes more insulin, whereas Empagliflozin prevents the kidneys from reabsorbing sugar into the blood and sending it out in the urine. Methods: Mean centering, double divisor, ratio spectra-zero crossing, and successive derivative were applied for the estimation of metformin, empagliflozin, and glimepiride respectively, in their prepared laboratory mixtures and in pharmaceutical tablets, without prior chemical separation. The absorption spectra of the mentioned drugs were recorded in the range of 200-400nm. Results: These methods were linear over concentration ranges of 1.0-10, 2.5-30, and 1.0-10 μgmL-1 of metformin, empagliflozin, and glimepiride respectively. Mean centering for metformin was measured at 234 and 248 nm, while empagliflozin and glimepiride had amplitude values at 276 and 262 nm, respectively. The derivative of double divisor was measured at 234, 278, and 288 nm for metformin, empagliflozin and glimepiride, respectively. The ratio of spectra-zero crossing was quantified at amplitude values of the analytical signal at 234 and 274 nm for metformin and empagliflozin, respectively, whereas glimepiride was determined at 242 and 286 nm. The successive ratio of metformin, empagliflozin, and glimepiride was determined at 284, 242, and 266 nm, respectively. Conclusion: The methods are validated according to the ICH guidelines where accuracy, precision and repeatability are found to be within the acceptable limit. The methods were studied and optimized. Upon validation linearity, precision, accuracy, LOD, LOQ and selectivity were proved to be operative for the analysis of specified drugs in pharmaceutical dosage configuration. Statistical illustration was done between the suggested methods with the reported methods with consideration to accuracy and precision. No significant difference was found by student’s t-test, F-test and one-way ANOVA.

Background: Esculin (ESCN) is used in the pharmaceutical industry with intravenous effect, stimulant and anti-inflammatory capillaries, like vitamin P. It is a significant component of many anti-inflammatory remedies such as esqusan, esflazid and anavenol [14]. It is also found in numerous other remedies available in the market such as proctosone, anustat, and ariproct. Objective: To determine experimental conditions, to elucidate retention behavior of esculin in HILIC mode. Moreover, to suggest new ways to separate and determinate esculin in ointments. Methods: Two hydrophilic columns were obtained by attaching molecules of sulfobetaine to polystyrene- divinylbenzene particles were studied for chromatographic separation of esculin. The various lengths of the chain are used as an investigative instrument for esculin retention conduct. The change of ACN percentage, buffer concentrations and pH of mobile phase in order to study the retention conduct of esculin. Results: A mixed mode hydrophilic interaction and ion exchange is the separation mechanism of esculin. A calibration graph was created for two columns. The concentration range was 8-1200 ng.ml-1, LOD 2.33 and 1.40, RSD% 0.31-1.02, , LOQ 7.07 and 4.25 ng.ml-1, Erel% 0.83 ± 0.68 and 0.545 ± 0.45, Recovery% 100.86 ± 0.68 and 1054 ± 0.45. Conclusion: The findings of the current study introduced new ZIC-HILIC methods for the separation and quantitation of esculin.

Background: This study reports an easy method of a veterinary drug investigation in raw milk, based on QuECHERS extraction followed by RP-HPLC-UV analysis. Use of this benchtop system was motivated by its availability and moderate cost relatively to other sophisticated methods such as LC-MS which are more efficient. Methods: This developed method has been optimized and then after validation according to EU legislation, it demonstrated good linearity with R²>0.997, acceptable peak resolution within a short time (<9.5 min) and good recovery of the analyzed drugs (OXY, ALZ and IVR, respectively 87.08, 99.02 and 92.01 %). Additionally, we applied the method to the analysis of cow milk, collected in Nouakchott, capital of Mauritania. Results: The obtained results indicated a mixed level of drug use according to targeted molecules. In 42% of sampled farms, the anti-parasitics IVR and ALZ were detected whereas the antibiotic OXY was detected in 50%. Conclusion: This investigation shows that 17% of the sampled farms exceeded European standards for IVR drug.

Background: Clarithromycin is widely used for infections of helicobacter pylori. Clarithromycin belongs to polymorphic drug. Crystalline state changes of clarithromycin in sustained release tablets were found. Objective: The aim of this study was to find the influential factor of the crystal transition of clarithromycin in preparation process of sustained-release tablets and to investigate the possible interactions between the clarithromycin and pharmaceutical excipients. Methods and Results: The crystal transition of active pharmaceuticals ingredients from form II to form I in portion in clarithromycin sustained release tablets were confirmed by x-ray powder diffraction. The techniques including differential scanning calorimetry and infrared spectroscopy, x-ray powder diffraction were used for assessing the compatibility between clarithromycin and several excipients as magnesium stearate, lactose, sodium carboxymethyl cellulose, polyvinyl-pyrrolidone K-30 and microcrystalline cellulose. All of these methods showed compatibilities between clarithromycin and the selected excipients. Alcohol prescription simulation was also done, which showed incompatibility between clarithromycin and concentration alcohol. Conclusion: It was confirmed that the reason for the incompatibility of clarithromycin with high concentration of alcohol was crystal transition.

Introduction: In this study, we used UPLC-MS/MS to detect shanzhiside methylester in rat plasma, and investigated its pharmacokinetics in rats. Materials and Methods: Diazepam was utilized as an internal standard (IS), and acetonitrile precipitation method was used to process the plasma samples. Chromatographic separation was achieved using a UPLC BEH C18 column using mobile phase of methanol-0.1 % formic acid with gradient elution. Electrospray ionization (ESI) tandem mass spectrometry in multiple reaction monitoring (MRM) mode with positive ionization was applied. Results: The results indicated that within the range of 5-4000 ng/mL, linearity of shanzhiside methylester in rat plasma was acceptable (r>0.995), and the lower limit of quantification (LLOQ) was 5 ng/mL. Intra-day and inter-day precision RSD of shanzhiside methylester in rat plasma were lower than 14%. Accuracy range was between 87.3 % and 109.1 %, and matrix effect was between 99.2% and 106.3%. Conclusion: The method was successfully applied in the pharmacokinetics of shanzhiside methylester in rats after intravenous administration.

Background: Sequential injection chromatography (SIC) with monolithic column has been proposed with potential benefits for separation and quantification. Objective: To utilize SIC to develop a new assay method for the separation and quantification of some phenothiazines (promethazine, chlorpromazine and perphenazine) in human urine and synthetic pharmaceutical formulations. Methods: The 32 full-factorial design was adopted to study the effect of mobile phase composition on separation efficiency, retention time, peak height and baseline. The separation was conducted on a C18 monolithic column (100 x 4.6 mm) using a mobile phase composition of phosphate: acetonitrile:methanol (60:28:12) at pH 4.0. The detection was carried out using a miniaturized fiber optic spectrometer at 250 nm. Results: Satisfactory analytical features, including number of theoretical plates (1809-6232), peak symmetry (1.0-1.3), recovery (95.5-99.1% in pharmaceutical formulations and 91.6-94.7% in urine), intra-day precision (0.36-1.60% for pharmaceutical formulation and 2.96-3.67 for urine), inter-day precision (1.47-2.28% for pharmaceutical formulation), limits of detection (0.23-0.88 μg/ml) and limits of quantification (0.77-2.90 μg/ml), were obtained. Conclusion: The remarkable advantages of the proposed SIC method are the inexpensiveness in terms of instrumentation and reagent consumption.

Background: The present study was focused on the development of HPLC method for purity testing of sofosbuvir by the Design of Experiments and determination of the activation energy of hydrolytic degradation reactions of sofosbuvir using HPLC based on the kinetics of sofosbuvir degradation. Methods: Following four factors for the Design of Experiments were selected, stationary phase, an organic modifier of the mobile phase, column temperature and pH of the mobile phase. These factors were examined in two or three level experimental design using Modde 11.0 (Umetrics) software. The chromatographic parameters like resolution, USP tailing and discrimination factor were calculated and analysed by partial least squares. The chromatography was performed based on Design of Experiments results with the mobile phase containing ammonium phosphate buffer pH 2.5 and methanol as an organic modifier. Separation was achieved using gradient elution on XBridge BEH C8 at 50 °C and a flow rate of 0.8 mL/min. UV detection was performed at 220 nm. The activation energy of hydrolytic degradation reactions of sofosbuvir was evaluated using two different calculation methods. The first method is based on the slope of dependence of natural logarithm of the rate constant on inverted thermodynamic temperature and the second approach is the isoconversional method. Results and Conclusion: Calculated activation energies were 77.9 ± 1.1 kJ/mol for the first method and 79.5 ± 3.2 kJ/mol for the isoconversional method. The results can be considered to be identical, therefore both calculation methods are suitable for the determination of the activation energy of degradation reactions.