Robust HPLC Research Articles

Establishment and Validation of a Robust Reversed-Phase HPLC Method for the Determination of Calotropis gigantea in Bulk Material and Marketed Product

Establishment and Validation of a Robust Reversed-Phase HPLC Method for the Determination of Calotropis gigantea in Bulk Material and Marketed Product

Incompatibility of Y-site administered drugs: the case of acyclovir and ciprofloxacin

Abstract Objectives Multiple drugs administration is a common practice in hospitals and clinics. This is the case when multiple pathologies are treated. In this context, frequently, antivirals and antibiotics are co-administrated either simultaneously or sequentially ignoring potential incompatibilities. In this study, we focused on an antiviral (Acyclovir) and an antibiotic (Ciprofloxacin) compatibility/incompatibility when they are co-administrated. Methods In this study, we focused on the effect of drugs ratio, pH and delay by implementing robust HPLC methods in order to determine the main factors leading to potential modification of concentration of one of the two drugs in the mixture. Results The results showed that acyclovir is quantitatively recovered in all conditions related to pH, concentration and time (up to 24 hours). However, ciprofloxacin is strongly altered depending on the acidic and basic conditions, the quantity of ciprofloxacin used and time after mixing the two drugs. The most valuable piece of information is that the ratio of acyclovir/ciprofloxacin is crucial. Conclusions The volume ratio of acyclovir/ciprofloxacin (prepared at 2mg/mL each) should be calculated and managed to provide at least two fold quantity of acyclovir versus ciprofloxacin or at least 9 fold quantity of ciprofloxacin versus acyclovir in order to maintain the administrated doses and prevent physicochemical interactions.

Development and Validation of a Simple HPLC–UV‐Based Bioanalytical Method for Estimation of Acalabrutinib in Rat Plasma and Its Application in Evaluation of Drug‐Loaded Nanocrystal Formulation

ABSTRACTA robust and reproducible HPLC–UV‐based bioanalytical method for acalabrutinib (ACP) was developed and validated in rat plasma using prednisone as internal standard. The samples were prepared by single‐step protein precipitation method using methanol with 1% v/v formic acid. A polar C18 stationary phase was used with a combination of 10 mM ammonium acetate (pH 3.5) and methanol (60:40 ratio), flow rate 1.1 mL/min, and 50 µL injection volume. ACP and internal standard were retained at ∼5.96 and 8.29 min, respectively. The method was validated according to the United States Food and Drug Administration and ICH M10 bioanalytical method validation guidelines. It was linear in the calibration range (0.08–5 µg/mL) with as 0.9989; accurate (% bias ≤ 0.50 ± 0.67), and precise (%RSD ≤ 0.986) for any quality control standard. Further, the method was applied in estimating ACP in rat plasma samples collected during in vivo pharmacokinetic study of the bulk drug as well as a nanocrystal (NC) suspension formulation. ACP‐NCs were formulated by bottom‐up solvent–antisolvent precipitation with mean particle size 266.32 ± 29.90 nm, 0.25 ± 0.05 PDI, with 92.7% ± 2.3% yield. The NC formulation improved both in vitro dissolution and in vivo absorption, supporting a further development of ACP‐loaded nanodrug delivery systems.

Determination of 3-acetyl-11-keto-β-boswellic acid in analytical and biological samples using streamlined and robust RP-HPLC method.

AKBA (3-acetyl-11-keto-β-boswellic acid) is a phytoconstituent derived from Boswellia serrata extract and utilized in the management of rheumatoid arthritis. Drug delivery approaches showed interest in delivering AKBA with advanced nanotechnology. There is a need for a simple, sensitive, and robust HPLC method that can determine AKBA in complex nanoformulation and in vitro and ex vivo samples. In the proposed work, the RP-HPLC method was developed using a mobile phase comprising a mixture of acetonitrile : milli Q (90 : 10) at detection λmax 250 nm. The method exhibited a linearity of 250 to 20 000 ng mL-1 with a high correlation coefficient of 1. The limit of detection and limit of quantification for the analytes were found to be 41.32 ng mL-1 and 125.21 ng mL-1, respectively. In the accuracy study, the % recovery of AKBA was found to be 98% to 102%, and the precision study showed less than 2% relative standard deviation. The developed method was found to be robust under chromatographic conditions with changes in pH and mobile phase mixture ratio. The method was also explored for forced degradation study, and the results showed the successful separation of degradation products from the AKBA. Further, the RP-HPLC method was applied for the quantification of AKBA in topical nanoformulations and different matrices, such as skin matrices and adhesive tapes. The method was able to measure entrapment efficiency (93.13 ± 1.94%), drug loading (25.83 ± 0.54%), drug assay in a gel matrix (96.99 ± 3.89%), drug amount in stratum corneum (7.90 ± 0.62 μg cm-2), and drug amount in viable skin layers (33.94 ± 0.21 μg cm-2) with high-speed reproducibility. The developed method can be utilized for the routine analysis of AKBA in conventional and complex formulations in academia and industry.

Chemical Relationship among Genetically Authenticated Medicinal Species of Genus Angelica.

The genus Angelica comprises various species utilized for diverse medicinal purposes, with differences attributed to the varying levels or types of inherent chemical components in each species. This study employed DNA barcode analysis and HPLC analysis to genetically authenticate and chemically classify eight medicinal Angelica species (n = 106) as well as two non-medicinal species (n = 14) that have been misused. Nucleotide sequence analysis of the nuclear internal transcribed spacer (ITS) region revealed differences ranging from 11 to 117 bp, while psbA-trnH showed variances of 3 to 95 bp, respectively. Phylogenetic analysis grouped all samples except Angelica sinensis into the same cluster, with some counterfeits forming separate clusters. Verification using the NCBI database confirmed the feasibility of species identification. For chemical identification, a robust quantitative HPLC analysis method was developed for 46 marker compounds. Subsequently, two A. reflexa-specific and seven A. biserrata-specific marker compounds were identified, alongside non-specific markers. Moreover, chemometric clustering analysis reflecting differences in chemical content between species revealed that most samples formed distinct clusters according to the plant species. However, some samples formed mixed clusters containing different species. These findings offer crucial insights for the standardization and quality control of medicinal Angelica species.

Impurity assessment, development and validation of an RP-HPLC method for the determination of eleven potential impurities of eltrombopag precursor

Eltrombopag is an oral non-peptide thrombopoietin receptor (TPO-R) agonist indicated for the treatment of thrombocytopenia in patients with persistent or chronic immune thrombocytopenia (idiopathic thrombocytopenic purpura, ITP) or chronic hepatitis C infection and the treatment of severe aplastic anemia. The purpose of this research was to assess the possible impurities that may carry over to eltrombopag from its precursor Eltro-1 (3′-amino-2′-hydroxy-[1,1′-biphenyl]-3-carboxylic acid) and to develop a specific analytical method for the determination of these impurities. Eltro-1 samples synthesized by two different synthesis routes were investigated during the evaluation and method development studies. Besides the expected process-related impurities (Eltro-1A – Eltro‐1J), e.g., starting materials, intermediates, and/or compounds formed from their further reactions, an unknown impurity detected above 0.10% was identified by LC-MS, synthesized and fully characterized by NMR, MS and FTIR (Eltro‐1K). Accordingly, an HPLC-RP method for the determination of eleven impurities (Eltro-1A – Eltro‐1K) in Eltro-1 was developed and validated according to ICH Q2. The control limits for impurities in Eltro-1 were set at ≤ 0.15% for Eltro-1A – Eltro‐1J and ≤ 1.0% for Eltro‐1K based on fate, spike-purge and carryover studies and in accordance with the ICH M7 classification for impurities in drug substance. Eltro-1 and eleven impurities at the specification limit were separated from each other and the diluent peaks with sufficient resolution without interference. Separation was performed on a Waters XBridge C18 column (150 × 4.6 mm, 3.5 μm) at 40 °C with a 10 µL injection volume at a detection wavelength of 220 nm and 15 °C sample temperature. The gradient elution is performed at a flow rate of 1.0 mL/min for 40 min with mobile phase A (0.1% orthophosphoric acid in water) and B (acetonitrile) according to the following program: Time (min) / Acetonitrile (%): 0/0, 35/70, 36/0, 40/0. Test and standard solutions were prepared at a concentration of 1.0 mg/mL and 1.0 µg/mL, respectively, using a mixture of mobile phase A and acetonitrile (75/25) as diluent. This is the first specific, selective, sensitive, linear, precise, accurate, and robust HPLC method for the determination of Eltro-1A – Eltro‐1K in Eltro-1, which showed no significant degradation under thermal stress, photostability (UV and VIS), and standard accelerated and long-term stability conditions.

A Robust HPLC Approach for Quantitation of Camptothecin in Mesoporous Silica Nanoparticles Matrix and in the Presence of Its Degradation Products.

Camptothecin is a potent anticancer drug used for the treatment of various cancers. The goal of this research investigation was to develop and validate a new stability-indicating HPLC technique for the quantitative assessment of camptothecin in in-house developed mesoporous silica nanoparticles, a novel nanoformulation matrix for the treatment of cancer. The Waters Inertsil® HPLC column (C18) was used for the chromatographic separation, with a flow rate of 1 mL/min, a column oven temperature of 40°C, an injection volume of 10 µL, a detection wavelength of 216 nm, and a 10 min runtime overall. An isocratic blend of phosphate buffer (10 mM, pH7.0) and acetonitrile (60:40, v/v) served as the mobile phase. Various stress conditions including acid, alkali, oxidative, photolytic, thermal, and humidity environments were tested for the quantitative estimation of the camptothecin through the proposed method. The results demonstrated that the proposed method is specific (peak purity ≥0.999), accurate (99.69-100.64% w/w), precise (RSD, % <2.0), and sensitive (LOD-0.17 µg and LOQ-0.56 µg) in accordance with ICH guideline Q2 (R1). Any unidentified degradation products did not interfere with the drug's estimation. Furthermore, the current method of analysis has eliminated any excipient interference from the matrix effect caused by the numerous excipients of the formulation matrix. To quantify camptothecin for routine assay purposes, this research work offers a novel and straightforward HPLC methodology with optimized chromatographic parameters, contributing to the research and development community while ensuring an appropriate and efficient use of the drug through a variety of nanoformulation for cancer treatment. The stability-indicating HPLC method was found to be specific and suitable for routine analysis of camptothecin. The absence of any interference from excipients was confirmed by forced degradation studies.

A specific chiral HPLC method for lifitegrast and determination of enantiomeric impurity in drug substance, ophthalmic product and stressed samples

Lifitegrast is a lymphocyte function-associated antigen-1 (LFA-1) antagonist used to treat the indications and symptoms associated with dry eye disease (DED), one of the most common ocular surface diseases. Lifitegrast has a chiral center, and the S-enantiomer (S-Lif) is responsible for the therapeutic effects, while the R-enantiomer (R-Lif) lacks efficacy in the treatment of DED. Lifitegrast ophthalmic solution containing 5% lifitegrast was approved by the United States Food and Drug Administration (FDA) in July 2016 for the treatment of DED in patients 17 years of age and older. The objective of this study was to develop a chiral HPLC method for the determination of the enantiomeric impurity of lifitegrast in the drug substance and in the ophthalmic product. In addition, we aimed to investigate the effect of stress and stability conditions on the enantiomeric purity of lifitegrast in both drug substance and ophthalmic solution. During the method development studies, four known lifitegrast impurities (Lif. Imp. A-D) and stressed lifitegrast samples were injected to ensure the specificity of the developed method. The enantiomers of lifitegrast are well separated with a resolution of higher than 4.0. They are also well separated from the peaks of the diluent, impurities, and the placebo used to prepare the ophthalmic solution without interference in 20 min. Chiral separation was achieved using a Chiralpak AD-H column (250 × 4.6 mm, 5.0 μm) at 40 °C with a mobile phase consisting of a mixture of n-hexane, 2-propanol, and formic acid (500:500:2, v/v/v) at a flow rate of 1.0 mL/min and a detection wavelength of 260 nm. Methanol was used as the diluent, and the drug substance solution was found to be stable for 48 h at 15 °C. The optimized chiral HPLC method for lifitegrast was validated according to ICH Q2, and the calibration curves showed excellent linearity for R-Lif (0.0369 – 1.816 µg/mL). This is the first stability-indicating, specific / selective, sensitive, linear, precise, accurate, and robust chiral HPLC method for the determination of R-Lif in S-Lif. The amount of enantiomeric impurity R-Lif in S-Lif increased under all stress and photostability test conditions without exceeding the acceptable impurity limit, with the most significant increase observed at elevated temperatures (105 °C) for both the drug substance in powder form and the ophthalmic drug solution.

Development of the RP-HPLC Method for Simultaneous Determination and Quantification of Artemether and Lumefantrine in Fixed-Dose Combination Pharmaceutical Dosage Forms.

Developing countries face enormous challenges with substandard and falsified antimalarial drugs. One specific issue is the lack of a simple, cost-effective, and robust HPLC method to simultaneously determine and quantify the active pharmaceutical ingredients (APIs) in fixed-dose artemether-lumefantrine pharmaceutical dosage forms. The current study developed a novel, simple, sensitive, precise, accurate, and cost-effective RP-HPLC method for the simultaneous determination and quantification of artemether and lumefantrine in pharmaceutical dosage forms. The HPLC analysis was carried out on an Agilent 1260 Infinity Series HPLC system equipped with an ODS Intersil-C8 (150 × 4.6 mm) 5.0 µm column, by isocratic elution. The mobile phase composition consisted of acetonitrile and 0.05% orthophosphoric acid buffer of pH 3.5 in the ratio of 70 : 30 v/v. The analysis was performed at a 1 mL/min flow rate and a column temperature of 25°C. The total run time was 6 minutes. The detection was done with a variable wavelength detector (VWD) at an isosbestic point wavelength (λ) of 210 nm. The developed method was validated according to the ICH guidelines concerning system suitability, specificity, linearity, accuracy, precision, and robustness. The system suitability of the developed method revealed satisfactory theoretical plates and symmetry factors. The method proved to be specific, with no interference of mobile phase or excipients. The calibration plot exhibited linearity over the concentration range of 275-1925 μg/mL with R2 = 0.9992 for artemether and a range of 150-1050 μg/mL with R2 = 0.9985 for lumefantrine. The accuracy of the method, determined by the recovery study, was 99.79-100.16% for artemether and 99.04-99.50% for lumefantrine. The % RSD values for intraday precision were 0.175 and 0.203, while interday precision values were 0.340 and 0.554 for artemether and lumefantrine, respectively. The method demonstrated robustness when subjected to slight modifications in the flow rate, column temperature, and mobile phase composition. The developed analytical method proved satisfactory as per ICH guidelines and hence can be used for the determination and quantification of artemether and lumefantrine in bulk drug and pharmaceutical dosage forms.

Development and Validation of a Robust HPLC Method for Simultaneous Quantitative Analysis of Quercetin and β-sitosterol in Plant Extract

Development and Validation of a Robust HPLC Method for Simultaneous Quantitative Analysis of Quercetin and β-sitosterol in Plant Extract

Development and validation of the RP-HPLC method for quantification of tavaborole.

The stability-indicating approach for tavaborole quantification was developed and validated to establish a precise, linear, accurate, and robust HPLC method. The development section includes optimizing the detection wavelength, the mobile phase ratio, and the type of column used to achieve the best possible separation and sensitivity for analysis. The chromatographic conditions were established, considering peak symmetry, resolution, and retention time. The mobile phase composition, comprising a buffer: acetonitrile (75 : 25, %v/v), with an injection volume of 15 μL, showed suitable elution and recovery at 265 nm. A constant column oven temperature of 35 °C and a 1 mL min-1 flow rate were maintained. The pH of the buffer was changed to 3.0 by using orthophosphoric acid. Linearity was observed from 5 to 1000 ppm (r2 = 1.00000). The capacity (retention) factor (k) of 3.43 was observed, indicating significant interaction and good separation. Forced degradation (FD) or stress tests were performed for chemical and physical photolytic stress conditions, and the results observed were within the specified limits. The stability in the analytical solution was observed for up to 35 hours at 5 °C, confirming the stability of the solution. Validation of the developed HPLC method confirmed the system's suitability, precision, linearity, accuracy, FD, robustness, and results. All validation criteria for the technique were within acceptable limits.

Nova HPLC metoda za istovremeno određivanje empagliflozina i dapagliflozina - razvoj, validacija, testiranje robusnosti i procena ekološke prihvatljivosti

Empagliflozin (EMPA) and Dapagliflozin (DAPA) are mainly recommended for the treatment of type 2 diabetes mellitus and heart failure. Based on the principles of green analytical chemistry, a simple, rapid and robust HPLC method was developed for the determination of both analytes in bulk. An isocratic protocol was developed using a C18 column, with a mobile phase consisting of just 30% organic modifier (ACN), while the remaining 70% was HPLC water. The run was completed within 4 min, with a flow rate of 1.20 mL/min, while UV detection took place at 230 nm. The method was then validated according to the ICH Q2 (R1) guidelines, and all the parameters examined were within the specified limits. Furthermore, the robustness of the method was evaluated by employing a factorial experimental design. Finally, the environmental friendliness of the proposed method was assessed by using the Analytical GREEness (AGREE) metric tool. The proposed method can be used for analysis of EMPA and DAPA in bulk, with potential application to the relevant pharmaceuticals.

Quality Control Analysis of Neomycin, Gramicidin and Triamcinolone in Ointment Formulation using HPLC Methodology: Methodology Development, Validation and Application

The combination of gramicidin (GMN), a bactericidal agent; neomycin (NMN), an aminoglycoside; and triamcinolone (TAA), a glucocorticoid, is used for treating eye infections that are accompanied by inflammation. The contents of GMN, NMN, and TAA in eye ointments must be monitored for quality. In this research, a quick, selective, and robust stability-indicating HPLC method has been developed for the concurrent assay of GMN, NMN, and TAA in bulk and ointment formulations. Analysis was performed using ODS, a250mm × 4.6mm column and mobile phase having 0.1% aqueous formic acid-acetonitrile with a ratio of 20:80 v/v; PDA analysis was performed at 225nm. The elution times for GMN, NMN, and TAA were 2.597min, 4.440min, and 3.251 min, respectively. The GMN, NMN, and TAA linear ranges were 0.625 µg/ml to 3.75 µg/ml, 6.25 µg/ml to 37.50µg/ml and 2.5µg/ml to 15.0µg/ml, respectively. The method is precise with 0.68-0.96% (GMN), 0.52-1.02% (NMN), and 0.26-0.62% (TAA) RSD values. The method is accurate with 98.75-101.25% (GMN), 98.61-100.33% (NMN), and 98.67-100.23% (TAA) recovery values. In degradation investigation, the degradant’s peak elution times are different from the elution times of GMN, NMN, and TAA. Thus, the proved specificity and stability indicating the power of the method. Finally, this developed analytical approach was efficaciously applied to a commercial ointment formulation containing afixed dose of GMN, NMN, and TAA, demonstrating its usefulness for quality control and degradation investigations on GMN, NMN, and TAA.

Developed and Validated for the Estimation of Tapinarof in Topical Formulation and Active Pharmaceutical Ingredients.

Its broad applicability and capacity to separate numerous components in a single chromatographic run led to the initial recognition of reversed-phase (RP)-HPLC as an analytical technique. The objective of this study was to create a straightforward and reliable method for accurately and precisely measuring the amount of tapinarof in both the topical formulation and the active pharmaceutical ingredient. Additionally, a robust HPLC assay was developed specifically for analyzing the topical formulation. In this study, chromatographic analysis was conducted using a Kromosil C18 column with dimensions of 250 × 4.6 mm and a particle size of 5 microns. The mobile phase consisted of a phosphate buffer and methanol in a ratio of 100:900 (v/v). The flow rate was set at 1.0 mL/min, with an injection volume of 10 µL and a run time of 6 min using isocratic elution. UV detection was performed at a wavelength of 313 nm, and the temperature was maintained at 30°C. The analysis showed well-separated peaks with a high number of theoretical plates, a low tailing factor, and consistent retention time. Validation of the method was conducted, and all validation parameters were found to be within the acceptable limits. A method that is simple, accurate, and precise has been developed to estimate the amount of tapinarof in a topical formulation and active pharmaceutical ingredient. The optimized method involved the use of a column temperature set at 30°C, 90% methanol as the mobile phase, and a flow rate of 1.0 mL/min. The retention time for tapinarof was determined to be 2.88 min. The method exhibited linearity in the concentration range of 5 to 30 µg/mL (with an R2 value greater than 0.999) for tapinarof. The topical formulated cream and active pharmaceutical ingredient showed more than 90% dissolution within 5 min. The method developed in this study utilized photo diode array (PDA) for peak integrity and purity confirmation, making it suitable for the quantification of tapinarof in both topical formulations and active pharmaceutical ingredients. The method was validated and can be recommended for routine analysis in QC laboratories.

HPLC analysis of biomarkers of Toluene and Xylene in human urine samples

Introduction: Urinary Hippuric acid (HA) and Methylhippuric acid (MHA) are considered reliable biomarkers for monitoring exposure to toluene and xylenes. A simple and robust HPLC method for simultaneous determination of HA and MHA in urine samples was developed and validated as per Internation Conference on Harmonization (ICH) Q2 guidelines. Methods: Mobile phase consisting of two solvents A &amp; B, solvent A was 0.1% TFA in water, and solvent B was 0.1% TFA in acetonitrile. Separation was performed in gradient elution mode and the time program was as follows - Time (min)/%B: 0/20, 4/20, 11/80, 11.1/90, 16/90, 16.1/20, 25/20. The detection wavelength was 205 nm. Results: The limits of detection (LOD) and quantification (LOQ) of HA in urine were 0.3 and 1.0 mg/L, and for MHA were 0.25 mg/L and 1.0 mg/L respectively and the recovery for HA and MHA were 95% and 98.9% respectively. Conclusion: The developed method was successfully applied for the analysis of urine samples.

Development and antibacterial investigation of Linezolid loaded SPIONs and HPLC method development for quantitative analysis of Linezolid.

Linezolid (LNZ) is extremely prone to resistance. The development of resistance to LNZ should be taken into consideration when selecting this drug as a therapeutic option. By considering the hypothesis that reactive oxygen species (ROS) generated by iron oxide nanoparticles (MNPs) could kill the infecting bacteria. So, we hypothesized the synergistic antibacterial effect of iron oxide nanoparticles and LNZ. To study the release and antibacterial effects of LNZ loaded superparamagnetic iron oxide nanoparticles (SPIONs) on Staphylococcus aureus and Streptococcus pneumoniae. Ferrofluid containing SPIONs was synthesized via chemical co-precipitation method and stabilized by sodium lauryl sulphate (SLS). SPIONs were then loaded with LNZ and characterized for particle size, FT-IR, XRD, and entrapment efficiency. Further antibacterial activity of SPIONs and LNZ-loaded SPIONs was investigated. For the in-vitro release findings, a HPLC analytical method development and validation was performed. Isolation of LNZ was accomplished on a C-18 column with methanol: TBHS (Tetra butyl ammonium hydrogen sulphate, 50:50 v/v). The eluate was monitored at 247 nm with retention time of 4.175 min. The MNP's DLS measurement revealed monodispersed particles with an average size of 16.81 ± 1.07 nm and PDI 0.176 ± 0.012. In optimized formulation, 25 ± 1.75% (w/w) of the drug was found to be entrapped. XRD revealed uniform coating of oleic acid covering the entire magnetic particles surface with no change in its crystallinity. An effective antimicrobial activity was observed at the lowered dose of drug. A robust HPLC method was developed to quantify the LNZ in MNPs and outcomes showed that the reduced dose of LNZ incorporated in SPIONs was able to show similar activity as the marketed product. Successfully reduction of dose of LNZ was established with the aid of biocompatible MNPs to attain the equivalent antibacterial activity.

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.

Development and validation of an HPLC coupled with tandem mass spectrometry method for the determination of HSK7653, a novel super long-acting dipeptidyl peptidase-4 inhibitor, in human plasma and urine and its application to a pharmacokinetic study.

HSK7653 is a novel super long-acting dipeptidyl peptidase-4 inhibitor, which is promising for the treatment of type 2 diabetes mellitus with the twice-monthly dosing regimen. In this article, a robust and sensitive HPLC coupled with tandem mass spectrometry method for determining the concentration of HSK7653 in human plasma and urine was developed and validated for the first time. Plasma and urine samples were prepared by protein precipitation. After that, the extracts were analyzed using an LC-20A HPLC system coupled with API 4000 tandem MS equipped with an electrospray ionization source in positive mode. Separation was obtained using an XBridge Phenyl column (2.1 × 50 mm, 3.5 μm) with a gradient elution of acetonitrile and water containing 0.1% formic acid and 5% acetonitrile at room temperature. This bioanalysis method has been fully validated and the results showed good sensitivity and specificity. In brief, the standard curves were linear over the concentration range of 2.00-2000 ng/ml for plasma and 20.0-20,000 ng/ml for urine, respectively. In addition, the precisions of inter- and intra-run of HSK7653 were less than 12.7% and the accuracies were -3.3%to 6.3% for both plasma and urine. Finally, this method was successfully applied to explore the pharmacokinetic characteristics of HSK7653 in Chinese healthy volunteers in a first-in-human study.

DEVELOPMENT, OPTIMIZATION AND VALIDATION OF HPLC METHOD FOR ASSAY AND DISSOLUTION TESTING OF FUROSEMIDE TABLET FORMULATION UTILIZING DESIGN OF EXPERIMENT APPROACH

A robust HPLC method was developed and validated for the quantitative estimation of furosemide tablet and dissolution testing. The optimization of chromatographic conditions (% organic phase, flow rate and pH of aqueous phase) was done using the Box-Behnken experimental design. The creation of a reliable HPLC technique utilised the design of experiments methodology effectively. The furosemide peak was found to have a Rt value of 4.86 min under the optimal HPLC settings, which included a C18 (250 mm 4.6 mm, 5 m) column and a methanol: aqueous solution of formic acid buffer (0.2 % V/V, pH 2.5) as mobile phase.The optimized method was validated according to ICH Q2(R1) guideline. Using a fractional factorial design with six chromatographic components, the robustness of the approach was evaluated. The results showed that a multivariant approach was successfully used to improve a reliable HPLC technique for everyday quality monitoring of furosemide in tablet formulations

Structural Elucidation of Novel Degradation Impurity and Development, Validation of a Single HPLC Method for all Putative Impurities of Clobetasol Propionate in a Foam Drug Product.

Clobetasol Propionate is a highly strong corticosteroid that is used in a variety of topical medication formulations, including foam, ointment, lotion, spray and shampoo; with a dosage strength of 0.05% (w/w). The goal of this research was to identify and characterize a substantial unknown impurity (UK) detected during the stability testing of a Clobetasol Propionate foam pharmaceutical product in accelerated conditions (40°C and 75% relative humidity). Developing a single, robust and accurate HPLC method that is LC-MS compatible for quantifying all 14 potential Clobetasol Propionate impurities in therapeutic drugs is another goal. Preparative column chromatography was used to separate the impurity, and spectroscopic techniques like IR, NMR and MS were used to characterize its structure. The structure of isolated UK was effectively characterized and defined using spectroscopic data evaluation. The chromatographic method has also been validated according to the International Conference on Hominization's Q (2) quality guidelines. The isolated and characterized impurity had the same equivalence as the impurity found during stability testing. Precision, accuracy, linearity, robustness and ruggedness are all met in the method validation data.