Sensitive HPLC Method Research Articles

Stability Indicating RP-HPLC Method for Quantitative Estimation of Lenalidomide and Its Impurities in Solid Dosage Form

The main aim and objectives of the research are to develop an effective, sensitive, economical, and simple reverse-phase HPLC method developed for determining and quantifying Lenalidomide impurities in Lenalidomide solid dosage formulations. The lack of research work and no compendial methods available for estimating this drug influenced the current research investigation to give a simple, sensitive, rapid, precise, accurate and robust gradient high-performance liquid chromatographic method for the determination and quantification of Lenalidomide and its impurities. Samples are analyzed using reverse phase (RP-HPLC) using stationary phase an Inertsil ODS-3V (150 x 4.6 mm, 3μm), and the mobile phase consists of two channels A and B. channel-A: pH 3.0 phosphate buffer and Channel-B: Acetonitrile: water (90:10 v/v) in the proportion of gradient elution. The flow rate is 1.0 mL/min. The column temperature was maintained at 40°C, and the sample cooler temperature was maintained at 5°C, injection volume of 20 μL, and wavelength of 210 nm. The developed HPLC method was validated concerning specificity, and the chromatograms were recorded for blank, placebo, standard, sample, and spiked sample solutions of Lenalidomide and its related substances. Specificity studies reveal that the peaks are well separated from each other. For precision, the results were found to be within acceptable limits. The limit of detection (LOD) and limit of quantitation (LOQ) for impurity-A 0.1124 μg/mL and 0.0371μg/mL, Impurity-B 0.2247μg/mL and 0.0742μg/mL, respectively. The linearity results for Lenalidomide and all the impurities in the specified concentration range are satisfactory, with a correlation coefficient greater than 0.99. The accuracy studies were shown as % recovery for Lenalidomide and its impurities at the specification level; the results obtained were within limits. Solution stability parameter was established; standard, sample, and spiked sample solutions are stable up to 48 hrs on a bench top at the refrigerator. Filter validation parameters were established, and the filtered spiked sample solutions are compatible with both 0.45 μm PVDF & 0.45 μm Nylon filters.

Analysis of Acetylated Hyaluronic Acid in Moisturizing Lotion and Milk Lotion by HPLC with Fluorescence Detection.

We developed a simple and sensitive analytical HPLC method for the determination of acetylated hyaluronic acid (AcHA) in moisturizing and milk lotions. AcHA with different molecular weights was separated as a single peak using a C4 column and detected through post-column derivatization using 2-cyanoacetamide. The limits of detection and quantification were 60 and 200 ng, respectively. We found that AcHA in water was successfully extracted into a strong anion exchange (SAX) spin column with a recovery rate of AcHA was 63.8 ± 1.8%. Although the supernatant from acetone precipitation of lotions could pass through the spin column, the recovery rate (%) and accuracy of AcHA were affected by the viscous properties of cosmetics and acidic and acetone-soluble ingredients. Upon conducting analytical methods in this study, the concentration of AcHA in nine lotions was found to have ranged from 7.50 to 83.3 µg/mL. These values are comparable to the concentration range of AcHA in emulsions that have been previously evaluated for their superior effects. We believe that the analytical and extraction method is useful for the qualitative analysis of AcHA in moisturizing and milk lotions.

Validation and standardization of Gallic acid and Ellagic acid in Quercus infectoria, Terminalia chebula and Pistacia integerrima.

Due to its medicinal properties, Pistacia integerrima is in high demand and is extensively used as a key ingredient in various formulations. However, its popularity has led to IUCN threatened category list. In Ayurvedic texts, such as Bhaishajaya Ratnavali, Quercus infectoria is recommended as a substitute for P. integerrima in different formulations. Additionally, Yogratnakar highlights that Terminalia chebula shares similar therapeutic properties with P. integerrima. The objective of the current study was to gather scientific data on metabolite profiling and markers-based comparative analysis of Q. infectoria, T. chebula and P. integerrima. In present study, hydro-alcoholic and aqueous extracts of all three plants were prepared and standardized for the comparative evaluation of secondary metabolites. Thin-layer chromatography was carried out for the comparative fingerprinting of the extracts using chloroform: methanol: glacial acetic acid: water (60: 8: 32: 10, v/v/v/v) as a solvent system. A fast, sensitive, selective, and robust HPLC method was developed to determine gallic acid and ellagic acid from both extracts of all three plants. The method was validated for precision, robustness, accuracy, limit of detection and quantitation as per the International Conference on Harmonization guidelines. The TLC analysis revealed the presence of several metabolites, and the pattern of metabolites in the plants exhibited a certain degree of similarity. A highly precise and reliable quantification technique was created for gallic acid and ellagic acid, operating within the linear concentration range of 81.18-288.22 µg/mL and 3.83-13.66 µg/mL, respectively. The correlation coefficients for gallic acid and ellagic acid were 0.999 and 0.996, indicating strong relationships. The gallic acid content in all three plants ranged from 3.74% to 10.16% w/w, while the ellagic acid content ranged from 0.10% to 1.24% w/w. This pioneering scientific approach highlights the phytochemical similarities between Q. infectoria, T. chebula and P. integerrima.

RP UFLC method for estimation of valsartan chemometricaly

A simple, sensitive, rapid, precise and economical reverse phase HPLC method has been developed for the estimation of valsartan from pharmaceutical dosage forms. The method was carried out using Chromatographic conditions were established by employing a C18 analytical phenomenal (Kinetex) column (5micron -C18, 250x4.6mm) with a mobile phase of phosphate buffer (PH 3.5) and acetonitrile in a 60:40 ratio. The sample was injected at a volume of 20 microliters, and the mobile phase was degassed using a Sonica ultrasonic sonicator before being pumped into the HPLC system. The flow rate was set at 1 ml per minute, and the wave length 273 nm was chosen for detection. The column temperature was kept at 25oC respectively. The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantitation and solution stability. The proposed method can be used for the estimation of these drugs in dosage forms.

Development of a sensitive and quantitative HPLC-FLD method for the determination of obestatin in human plasma.

Obestatin is a gastrointestinal system peptide. The quantification of this peptide is conventionally performed using immunological techniques. In this study, a selective and sensitive HPLC method coupled with fluorescence detection for the quantitation of obestatin in human plasma was developed and validated. The separation was obtained on a C18 (4.6 × 100 mm, 3.5-μm particles) column using a mobile phase composed of acetonitrile and water, both including 0.1% trifluoroacetic acid. The developed method was found to be linear in the concentration range of 20 to 1000 ng/mL, with a coefficient of determination of 0.9982. The precision results were less than 10%, and the accuracy results were between 92% and 107%. The detection and quantification limit values were obtained as 2.8 and 9.4ng/mL, respectively. Analyte solutions were found stable for 24 h at room temperature, three freeze-thaw cycles, and 2weeks at -20°C. The developed method was successfully used for the quantification of obestatin in human plasma samples. In conclusion, the developed method is sensitive and specific for measuring the plasma concentrations of obestatin.

Quality by design-steered development and validation of analytical and bioanalytical methods for raloxifene: Application of Monte Carlo simulations and variance inflation factor.

A sensitive, rapid, reproducible, and economical HPLC method is reported for the quantification of raloxifene hydrochloride employing Quality by Design (QbD) principles. Factor screening studies, employing Taguchi design, indicated buffer volume percentage and isocratic flow rate as the critical method parameters (CMPs), which significantly influence the chosen critical analytical attributes, that is, tailing factor and theoretical plate number. Method conditions were subsequently optimized using face-centered cubic design with magnitude of variance inflation factor for assessing multicollinearity among CMPs. Method operable design region (MODR) was earmarked and liquid chromatographic separation optimized using 0.05 M citrate buffer, acetonitrile, and methanol (57:40:3 v/v/v) as ggmobile phase at 0.9 mL min-1 flow rate, λmax of 280 nm, and column temperature of 40°C. Validation of the developed analytical method was accomplished as per International Council on Harmonization (ICH) guidelines confirming high levels of linearity, precision, accuracy, robustness, and sensitivity. Application of Monte Carlo simulations enabled the attainment of best plausible chromatographic resolution and corroboration of the demarcated MODR. Establishment and validation of the bioanalytical method using rat plasma samples, along with forced degradation and stability studies, corroborated the aptness of developed HPLC methods for drug quantification in the biological fluids, as well as in bulk and marketed dosage forms.

Application of a Quality by Design-based approach in development and validation of an RP-HPLC method for simultaneous estimation of methotrexate and mangiferin in dual drug-loaded liposomes.

The present study delineates the development of a novel rugged and sensitive stability-indicating risk-based HPLC method for the concurrent estimation of methotrexate and mangiferin in dual drug-loaded nanopharmaceuticals based on an analytical QbD approach. Preliminary screening trials along with systemic risk analysis were performed, endeavouring to explicate the critical method attributes, namely pH, percentage orthophosphoric acid content and percentage methanol content, that influence critical quality attributes. Box-Behnken design was utilized for the optimization of the tailing factor as response for methotrexate and mangiferin in short run time. The chromatographic conditions were optimized by performing 17 experimental runs acquired from Design-Expert software. The chromatographic conditions after the analysis of an optimized zone within the confines of the design space were chosen as mobile phase water-methanol adjusted to pH3.0 with 0.05% orthophosphoric acid (65:35, v/v) and flow rate 1.0ml/min using a C18 analytical column at an isosbestic wavelength of 265 nm. Furthermore, the validation of the optimized method was done in accordance with International Conference on Harmonization guidelines and were reckoned to be in the prescribed limits. The developed RP-HPLC method has a high degree of practical utility for synchronous detection of methotrexate and mangiferin in pharmaceutical nano-dosage forms such as protein-based-nanoparticles, nanocrystals, polymeric nanoparticles and metallic nanoparticles in in vivo and in vitro studies.

A simple and sensitive HPLC method for simultaneous quantification of macrocyclic spermidine alkaloids in root, stem and leaf of Tripterygium wilfordii

AbstractThere has been a lively interest on macrocyclic polyamine alkaloids due to their remarkable pharmacological activities such as anti-tumor, anti-inflammatory, anti-Alzheimer's disease and anti-parasitic. Tripterygium wilfordii is a widely used traditional Chinese medicine, which is abundant in alkaloids including macrocyclic polyamine alkaloids. However, there are rarely studies on macrocyclic spermidine alkaloids of T. wilfordii so far. In this article, we use three known macrocyclic spermidine alkaloids celafurine, celabenzine and celacinnine, and successfully develop a simple and sensitive HPLC method for simultaneous quantification of macrocyclic spermidine alkaloids in root, stem and leaf of T. wilfordii.

Application of QbD-based approach to the development and validation of an RP-HPLC method for simultaneous estimation of pregabalin and naringin in dual-drug loaded liposomes.

The current work delineates the development of a novel, rugged and sensitive stability-indicating risk-based HPLC method based on an analytical quality-by-design (QbD) approach for the concurrent estimation of naringin and pregabalin in dual-drug-loaded nanopharmaceuticals. Preliminary screening trials were conducted, along with systemic risk analysis, in order to identify the critical method attributes, namely injection volume, pH and acetonitrile content, that influence critical quality attributes. The Box-Behnken design was used to optimize the tailing factor as a response to pregabalin and naringin in a short run time. The chromatographic conditions were improved by running 17 experimental runs generated by design expert software. After analysing the optimized zone within the confines of the design space, the following chromatographic conditions were chosen: mobile phase water-acetonitrile adjusted to pH6.9 with phosphate buffer (80:20, %v/v), at flow rate of 1.0ml/min using a C18 analytical column at an isobestic wavelength of 212 nm. Furthermore, the optimized method was validated in accordance with International Conference on Harmonization guidelines and was found to be within the prescribed limits. The developed RP-HPLC method has a high degree of practical utility in in vivo and in vitro studies for the synchronous detection of pregabalin and naringin in pharmaceutical nanodosage forms such as protein-based nanoparticles, nanocrystals, polymeric nanoparticles and metallic nanoparticles.

QbD green analytical procedure for Novel study of a genotoxic and carcinogenic compound trace determination in physiological solution compatibility

Regulatory bodies more focusses on the safety and efficacy of the pharmaceuticals, through this work we want to express the necessary control of 5-Hydroxymethyl furfural (5-HMF) impurity level in intra-venous (IV) injections. Genotoxic and carcinogenic compound 5-HMF determination in IV injections was determined by using the simple, sensitive, green, and eco-friendly HPLC method. Box-Behnken Design (BBD) was utilized to optimize the final method conditions prior to method validation, resulting in 15 design of experiments (DOE) using critical method parameters (CMPS) and critical assumptions. The identification and quantification were done with Phenomenex prodigy C8 (150 × 4.6) mm, 5 μm column with a flow rate of 0.6 mL/min. The peak detection was done at 284 nm and with a total run time of 6 min. The mobile phase consists of 0.1 M potassium dihydrogen phosphate (PDP) pH 2.5 buffer and acetonitrile (ACN) in the ratio of 80:20 (v/v) used for the isocratic elution. The method was validated as per the current regulatory guidelines. The accuracy results from LOQ to 150% level were attained between 95.1% and 103.4%. The 5-HMF response is linear from LOQ to 150% with a correlation coefficient (r) > 0.999. The %coefficient of variation (CV) from method precision and intermediate precision (n = 6) results were found to be 1.67% and 3.45%, respectively. Trace analysis of 5-HMF was done in selected IV injections Pancuronium Bromide (PMB), Meropenem (MPM), Doripenem (DPM), and Cefotaxime (CTM) injections at different time points. The method greenness was evaluated by utilizing the Green analytical procedure index (GAPI), Analytical GREEness (AGREE), National environmental procedure index (NEMI), and analytical eco-scale and concluded that the method is green.

DEVELOPMENT AND VALIDATION OF A NEW RP HPLC ANALYTICAL METHOD FOR THE DETERMINATION OF ETODOLAC SUCCINIC ACID CO-CRYSTALS IN SPIKED RABBIT PLASMA

Objective: The aim of the study was to develop and validate a novel and sensitive HPLC method for the determination of Etodolac content in Etodolac succinic acid co-crystals in spiked rabbit plasma.
 Methods: Chromatographic separation was achieved on an Eclipse C18 column (4.6 mm,100 mm, 3.5 μm spherical particles) using acetonitrile: methanol: acetic acid (100%) (50:49:1) as the mobile phase at a flow rate of 0.8 ml/min and monitored at 278 nm. Tinidazole was used as the internal standard. The run time was 6 min. The method was validated to fulfill International Conference on Harmonisation (ICH) guidelines, which included specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision and robustness.
 Results: The calibration curve was linear over the concentration range from 2.5 to 15 μg/ml, and the lower limit of detection was 0.3700 μg/ml. and lower limit of quantification was 1.121μg/ml for determination in spiked rabbit plasma. The accuracy and precision of the method were within the acceptable limit of±2% at the lower limit of quantification.
 Conclusion: A simple, sensitive, rapid and reproducible RP-HPLC method was developed with short runtime and less flow rate. Statistical analysis of the method proved that this method is suitable for the estimation of Etodolac in Co-crystalin plasma. Hence this method can be employed in the routine assay of the Etodolac Succinic acid co-crystals.

Insights of different analytical approaches for estimation of budesonide as COVID-19 replication inhibitor in its novel combinations: green assessment with AGREE and GAPI approaches

Simple, direct, rapid, and sensitive HPLC and spectrophotometric methods were established for simultaneous estimation of a novel combination of budesonide and azelastine (BUD/AZL) in their laboratory-prepared mixture and dosage form according to the medicinally recommended ratio 1:4.28. Budesonide is an important inhalation corticosteroid that plays a vital role in the inhibition of COVID-19 replication and cytokine production. The first chromatographic method was created for the simultaneous estimation of BUD epimers in the presence of AZL with excellent efficiency in a relatively short chromatographic run (< 9 min). The separation of BUD epimers with AZL was carried out on a C18 column using acetonitrile: phosphate buffer of pH 3.5 adjusted by 0.2 M orthophosphoric acid (40:60, v/v) as a mobile phase, UV detection at 230 nm and a flow rate of regulated at 2 mL/min. Besides, three spectrophotometric methods were applied for the simultaneous determination of the provided mixture adopting zero order, first order derivative, and ratio first derivative approaches. The Zero-order spectrophotometry was used for the determination of AZL in presence of BUD, where BUD shows no absorbance at 290 nm. The first derivative amplitude at 265 nm (1D265) (zero-crossing of AZL) and the ratio of first derivative amplitudes at 270 nm (1DD270) using 10.0 µg mL−1 AZL as divisor was chosen for the simultaneous determination of BUD in the presence of AZL in the binary mixture. The proposed methods were found to be rectilinear in the concentration range of (0.4–40.0 µg mL−1) and (0.05–40.0 µg mL−1) for BUD and AZL, respectively in the HPLC method. Whereas the concentration range for AZL in the zero-order method was (1.0–35.0 µg mL−1) and for BUD in the first derivative and ratio derivative method was (6.0–20.0 µg mL−1). Validation of the suggested approaches according to the ICH criteria was performed. Furthermore, to ensure the proposed approaches' greenness, The AGREE and GAPI metrics were utilized, and the afforded results revealed an excellent greenness of the proposed approaches.

Development and validation of an RP-HPLC method for determination of Metformin and Evogliptin in bulk and pharmaceutical dosage forms

Introduction and Aim: In this study, a new sensitive and rapid HPLC method was developed to determine Metformin hydrochloride and Evogliptin tartrate in pharmaceutical dosage forms. Materials and Methods: Chromatographic separation of Metformin and Evogliptin was achieved on Waters Alliance-e2695 by using Waters XTerra RP-18 150X4.6 mm, 3.5µ column, and the mobile phase containing Acetonitrile: KH2PO4: Methanol in the ratio of 50:40:10% v/v. Results: The flow rate was 1.0 ml/min; detection was carried out by absorption at 228 nm using a photodiode array detector at ambient temperature. Conclusion: This method was a simple, economical, suitable, precise, accurate and robust method for quantitative analysis of Metformin and Evogliptin study of its stability.

Development of a sensitive HPLC method with fluorescence detection for the determination of motilin in human plasma.

Background: Motilin is a peptide-structured gastrointestinal system hormone. In this study, a sensitive HPLC-fluorescence detection method was developed and validated for the quantification of motilin in human plasma. Materials & methods: Optimization processes were carried out with the experimental design methodology. Analyses were performed on a C8 column (4.6×150mm, 3.5μm particles) using water and acetonitrile containing trifluoroacetic acid as the mobile phase. Results & conclusion: The method was linear from 2 to 200ng/mlof motilin. The assay variability was less than 5%. The limit of quantification was found to be 1.84ng/ml. The applicability of the developed method was successfully demonstrated by quantifying the levels of motilin in human plasma samples.

LC-MS-Compatible Chromatographic Method for Quantification of Potential Organic Impurities of Elagolix Sodium in Tablet Dosage Form with Identification of Major Degradation Products.

Elagolix is a gonadotropin-releasing hormone (GnRH) modulator and used for pain relief from endometriosis. The present research was performed to develop and validate a simple, novel, fast, sensitive, and cost-effective LC-MS-compatible chromatographic method for quantification of all prominent organic impurities of elagolix sodium in tablet formulation with identification of major degradation products. The optimum separation of the organic impurities of elagolix sodium was achieved on an ACE C18-PFP (250 mm × 4.6 mm, 5 µm) column by employing pH 5.6 acetate buffer-acetonitrile (95 + 5, by volume) as mobile phase A, and acetonitrile-methanol (90 + 10, by volume) as mobile phase B. UV detection of the drug and impurities was carried out at 210 nm. A forced degradation study was carried out by employing acid, alkali, oxidative, thermal, and photolytic stress conditions on elagolix sodium drug substance and its drug product. The major degradation products observed during the stress study were identified by using mass spectrometry. Elagolix sodium and its prominent organic impurities were resolved in the developed method through a gradient elution program of 46 min at a flow rate of 1.3 mL/min. Significant degradation was observed during alkali hydrolysis and oxidative stress conditions with a mass balance of more than 97.0%. The method was validated in line with present International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2(R1) guidelines. The forced degradation study suggested that the developed method is specific and stability-indicating and can be used for related substance analysis of elagolix drug substance and its dosage forms. This is the first research paper which describes a simple and sensitive (LOD 0.08 µg/mL) HPLC method for quantification of all probable impurities of elagolix in tablet dosage forms. The noticeable feature of the developed method is resolution of impurities of similar structures in a short time using routine solvents which are easily available in the QC laboratory.

Application of QbD based approach in development and validation of RP-HPLC method for simultaneous estimation of methotrexate and baicalin in dual-drug-loaded liposomes.

The present study delineates the development of a novel, rugged and sensitive stability-indicating risk-based HPLC method for the concurrent estimation of methotrexate (MTX) and baicalin (BCL) in dual-drug-loaded-nanopharmaceuticals based on an analytical quality-by-design approach. Preliminary screening trials along with systemic risk analysis were performed, endeavouring to explicate the critical method attributes, namely pH, percentage of orthophosphoric acid and percentage of acetonitrile, that influence the critical quality attributes. Box-Behnken design was utilized for the optimization of the tailing factor as the response for MTX and BCL in a short run time. The chromatographic conditions were optimized by performing 17 experimental runs using design expert software. The chromatographic conditions were selected after the analysis of the optimized zone within the confines of the design space: water:acetonitrile adjusted to a pH of 3.0 with 0.05% orthophosphoric acid (60:40, %v/v) was the mobile phase, the flow rate was 1.0ml/min and an analytical C18 column was used at an isobestic wavelength of 282 nm. Furthermore, the optimized method was validated in accordance with the International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines and was found to be within the prescribed limits. Therefore, the developed reversed-phase-high-performance liquid chromatography method has a high degree of practical utility for synchronous detection of MTX and BCL in pharmaceutical nano-dosage forms such as protein-based nanoparticles, nanocrystals, polymeric nanoparticles and metallic nanoparticles in in vivo and in vitro studies.

A DoE-based fast and synchronous quantification of rhein, aceclofenac, and diclofenac by RP-HPLC-DAD method in spiked Wistar rat plasma: application to pharmacokinetic studies

A fixed-dose combination of diacerein and aceclofenac (ACF) is commonly used in the treatment of osteoarthritis for their disease-modifying and analgesic effects, respectively. We introduce a simple, fast, sensitive, and green HPLC method for synchronous quantification of rhein (RH) (a primary metabolite of diacerein), ACF, and diclofenac (DL) (an active metabolite of ACF) in rat plasma using fenofibric acid as an internal standard (IS). The chromatographic conditions were optimized using Fractional Factorial and Box-Behnken design. Drugs were extracted by acetonitrile-methanol-based simple protein precipitation technique. The separation was performed on C18 column (Phenomenex, 250 × 4.6 mm), using acetonitrile: phosphate buffer pH 3.5 (60:40, v/v) at a 1 mL/min flow rate in a 10 min run time and detected at 264 nm. As per the USFDA guidelines, the established method was validated. RH, ACF, and DL showed good linearity in the 25–3000, 50–6000, and 50–6000 ng/mL concentration ranges respectively. The precision and accuracy results were found to be well within the acceptable range, with average recovery ranging between 92.27 and 96.10%. The developed method was found to be stable and successfully used to determine the pharmacokinetic parameters in Wistar rats after oral administration of Dycerin A tablet.

Method Development and Validation of a Novel Anti-Depressant Bupropion by RP-HPLC

Bupropion is an atypical antidepressant and smoking cessation aid. The drug is a non-tricyclic antidepressant and differs from most commonly prescribed antidepressants such as SSRIs, as its primary pharmacological action is thought to benorepinephrine-dopamine reuptake inhibition. A new sensitive and rapid HPLC method was developed for the determination of buproprion in bulk and pharmaceutical dosage forms; it was validated according to ICH and FDA guidelines. The HPLC analysis was performed on the Alliance Waters e2695 Separations Module system equipped with a Waters X – Bridge C-18 5µm, 4.6 X 150 mm column 5, with a mixture of Acetonitrile: Ammonium bicarbonate (5mM) pH-9 adjusted with 1% Ammonium hydroxide (50:50, %v/v). Flow rate was 1 ml/min quantification was done by PDA detector at 254nm. The proposed method showed linearity in the concentration range of 50 to 250 ppm for Bupropion. The linear regression equation of Bupropion was found to be y = 6E+06x + 91344 and correlation coefficient value was found to be 0.997 indicating a high degree of linearity for the drug. The proposed method was stable except the basic condition during analysis. The stability of the analyte solution was determined at interval of 1st, 2nd, 3nd, 4th and 5th day.&#x0D; The low values of %recovery and %C.V. showed that the method is precise within the acceptance limit of 5% (according to ICH guidelines).The limit of detection (LOD) of Bupropion was 0.5 ppm and limit of quantification (LOQ) was 2.0 ppm respectively. The proposed method showed excellent linearity, accuracy, precision, specificity, robustness, LOD, LOQ, and system suitability results within the acceptance criteria.

Determination of Genotoxic Impurities N-nitrosamine's in Efavirenz Drug Substance Using Rp-Hplc Technique

Aim: The main purpose of the research is to develop a effective, sensitive, economical and simple reverse phase HPLC method for the determination of genotoxic N-Nitrosamine's impurities in Efavirenz drug substance.&#x0D; Study Design: HPLC based quantification studies&#x0D; Place and Duration of Study: Department of Chemistry, Acharya Nagarjuna University, Guntur, Andhra Pradesh between April 2022 and August 2022.&#x0D; Methodology: Determination of genotoxic N-Nitrosamine's in Efavirenz drug substance. N-Nitrosamine's impurities were determined by RP-HPLC method using Zorbax SB C18 (150x4.6mm, 3.5µ) column as stationary phase. Column temperature maintained 25°C, injection volume 20 µL, flow rate 1.0 mL/min and sample cooler temperature 5°C and run time was 15 minutes. The mixture of 0.1% formic acid buffer and methanol in the ratio of 50:950 (v/v) was used as mobile phase. wavelength 240 nm. respectively.&#x0D; Results: There is no interference of blank at N-Nitrosamine's impurities peaks. The elution order and the retention times of impurities and Efavirenz obtained from individual standard preparations and mixed standard preparations are comparable.&#x0D; The limit of detection (LOD) and limit of quantitation (LOQ) for NDMA 0.03 &amp; 0.09 µg/mL, NDEA 0.02 &amp; 0.06 µg/mL and NDIPA 0.03 &amp; 0.09 µg/mL respectively.&#x0D; The linearity results for N-Nitrosamine's impurities in the specified concentration range are found satisfactory, with a correlation coefficient greater than 0.99.Calibration curve was plotted and correlation co-efficient for impurities found to be 1.000, 1.000 and 1.000 respectively.&#x0D; The accuracy studies were shown as %recovery for N-Nitrosamine's impurities at specification level. The limit of % recovered shown is in the range of 80 and 120% and the results obtained were found to be within the limits. Hence the method was found to be accurate.&#x0D; The technique has been validated in accordance with ICH rules, and all validation parameters meet the acceptance criteria of the ICH Q2 specification.&#x0D; Conclusion: According to ICH guidelines, an RP-HPLC method that can assess genotoxic Nitrosoamines in Efavirenz at trace level concentration has been established. The method's specificity, precision, linearity, and accuracy ensured its efficacy. As a result, the technique is appropriate for the goals it was designed to achieve and can be successfully used for release testing of efavirenz into the market.

Development and Validation of HPLC-DAD Method for the Determination of Favipiravir and Studying the Impact of Vitamin C on the Pharmacokinetics of COVID-19 Antiviral Drug Favipiravir

A novel, sensitive, and low-cost HPLC method for the rapid determination of favipiravir (FVR) in rat plasma was developed and validated, and the effect of vitamin C on FVR pharmacokinetic parameters was investigated. FVR and oxcarbazepine (IS) were separated using a mobile phase of 50% acetonitrile and 50% water (with 0.25% trifluoroacetic acid) at 1.0 mL/min flow rate and detected at λmax 289 nm. The intra- and interday values for FVR in plasma were less than 15%, with low, medium, and high QC levels for the relative recovery rate, according to ICH guidelines. Cmax values in the control and experimental groups were 558 ± 124.42 and 979.13 ± 138.10 ng/mL, respectively; t1/2 values were 7.15 ± 1.60 and 9.09 ± 1.14 h, AUC(0-t) values were 5697.70 ± 536.58 and 7381.62 ± 1577.58 ng.h/mL, and AUC(0-∞) values were 5697.70 ± 536.58 and 8192.36 ± 1721.67, respectively. According to the results, the experimental group’s Cmax of FVR was 75.17% higher than the control group’s, the Vz/F was lower, and the t1/2 was 1.86 h longer. The technique developed for determining FVR in plasma was useful for FVR pharmacokinetics and food–drug interaction investigations.