UPLC Method Research Articles

An UPLC Method for Determination of Structural Analogues of DM1: the Payload of Trastuzumab Emtansine (T-DM1)

An UPLC Method for Determination of Structural Analogues of DM1: the Payload of Trastuzumab Emtansine (T-DM1)

A Green Approach: Optimization of the UPLC Method Using DoE Software for Concurrent Quantification of Pioglitazone and Dapagliflozin in a SNEDDS Formulation for the Treatment of Diabetes

A Green Approach: Optimization of the UPLC Method Using DoE Software for Concurrent Quantification of Pioglitazone and Dapagliflozin in a SNEDDS Formulation for the Treatment of Diabetes

An integrated paradigm between green analytical chemistry and quality-by-design for robust analysis of alcaftadine and ketorolac tromethamine in aqueous humor via sustainable chromatographic methods

The field of pharmaceutical industry has witnessed the launch of a new co-formulated eye drop, composed of alcaftadine and ketorolac tromethamine, for alleviating allergic conjunctivitis. Such new formulation evokes the need for a sensitive, robust and fast analytical method capable of their quantification in their dosage form, in aqueous humor for pharmaco-kinetics studies as well as in presence of plausible degradation products. In this work, two validated stability-indicating chromatographic methods were developed for alcaftadine and ketorolac assaying with good adherence to values of green analytical chemistry. Drugs stabilities were tested under various stress conditions where drugs showed liability for oxidative degradation only. For the first chromatographic method, an integrated merger between analytical quality-by-design and green analytical chemistry was implemented via a UPLC method coupled with photo-diode array detector. A short C18 analytical column of 100 mm was utilized with mobile phase of ethanol: aqueous solution pH 4.0 containing 0.1% triethylamine (40: 60, by volume). Second method was thin layer chromatographic one using HPTLC silica plates as a stationary phase and a developing system of dichloromethane: ethanol (5:4, by volume). Both methods achieved to selectively quantify the studied drugs with high accuracy and precision without any interference from degradation products or related excipients in eye drops. The methods were also successfully exploited for ALF and KTC assaying in rabbit aqueous humor, with no need for prior extraction procedures, for further pharmacokinetic studies. Finally, usage of green solvents gives us the advantage of applying various evaluation tools to conclude method greenness.

Validation of an UPLC-PDA Method for the Simultaneous Quantification of Phenol and Iomeprol in a Sterile Parenteral Preparation Used for Coeliac Plexus Block, and Its Application to a Pharmaceutical Stability Study.

Objectives: A commonly applied analgesic therapy for patients with severe abdominal pain due to cancer-related pain in the upper abdomen, is coeliac plexus neurolysis (CPN). Herein, a combination product of phenol and an iodine contrast agent are injected simultaneously. The chemical stability of such a combination product is unknown, and no chromatographic method is yet available that describes the simultaneous quantification of phenol and iomeprol. The aim of this study was to develop and validate a stability-indicating UPLC method for the simultaneous quantification of both phenol and iomeprol and to determine the chemical stability of a sterile 100 mg/mL phenol in 350 mg I/mL iomeprol solution for injection during shelf life. Methods: The product was compounded and sterilized in a GMP certified facility. The pharmaceutical analysis was validated by determination of the accuracy, precision, specificity, selectivity, carry-over and linearity. Pharmaceutical product stability was determined before and after sterilization, and during shelf life of 36 months at 25°C ± 2°C. Results: The accuracy for phenol and iomeprol was 97.1% to 99.3% and 100.0% to 100.2%, respectively. The RSD for repeatability and reproducibility for phenol were 0.65% and 1.17%, and for iomeprol 0.61% and 1.49%, respectively. All other tested parameters met the predefined validation criteria. All concentrations at all tested time points remained within ±2% of the initial concentrations for phenol and ±4% for iomeprol. No additional peaks were visible on the chromatograms. Conclusion: A stability-indicating method for the simultaneous quantification of phenol and iomeprol in a parental pharmaceutical preparation was developed and validated. This method was used to demonstrate the chemical stability of a newly developed sterile solution of 100 mg/mL phenol and 350 mg I/mL iomeprol. Chemical product stability was demonstrated during shelf life of up to 36 months.

Phytochemical Profiles and Biological Activities of Five Wild Camellia Species from Ta Dung, Vietnam.

The C. luuana Orel & Curry (TD3), C. furfuracea (Merr.) Cohen-Stuard (TD4), C. bidoupensis Truong, Luong & Tran (TD6), C. sinensis (L.) Kuntze (TD7), and C. kissii var. spp (TD8), have been traditionally used as a health-promoting beverage by local people in Ta Dung, Dak Nong. Despite their potential health benefits, further scientific data on biological and phytochemical properties of these plants is needed. To address this issue, this study was conducted to investigate phytochemical and biological properties of five Camellia species extracts, using DPPH, ABTS radical scavenging, copper chelating (Cu-chelator), and tyrosinase inhibition (TI), α-amylase (Al-AI), and α-glucosidase (Al-GI) analyses. As results, ten compounds were identified using UPLC method, in which catechins (mainly EGCG and catechin (Cat)), were the most prevalent, and followed by chlorogenic acid (ChlA), quercitrin (Querci), rutin, and quercetin (Querce). Additionally, multiple factor analysis (MFA) also revealed that TD7, TD3, and TD4 containing high TPC, TFC, high concentrations of EGCG, ChlA, and caffeine were responsible for their high DPPH, ABTS radical scavenging activities, as well asTI, Al-AI and Al-GI. Furthemore, TD6 and TD8, possessing elevated levels of Apig, Querci, Rutin, Querce, Cat, and EA, exhibited a high Cu-chelator property, but a weak enzyme inhibition. From all above-mentioned results, the antioxidative and enzyme inhibitory potentials of Camellia species extracts collected in Dak Nong province in Vietnam were scientifically demonstrated paving a pathway to develop health supplement in further studies.

UPLC-PDA factorial design assisted method for simultaneous determination of oseltamivir, dexamethasone, and remdesivir in human plasma

A green and simple UPLC method was developed and optimized, adopting a factorial design for simultaneous determination of oseltamivir phosphate and remdesivir with dexamethasone as a co-administered drug in human plasma and using daclatasvir dihydrochloride as an internal standard within 5 min. The separation was established on UPLC column BEH C18 1.7 μm (2.1 ×\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\ imes $$\\end{document} 100.0 mm) connected to UPLC pre-column BEH 1.7 μm (2.1 ×\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\ imes $$\\end{document} 5.0 mm) at 50 °C with an injection volume of 10 μL. The photodiode array detector (PDA) was set at three wavelengths of 220, 315, and 245 nm for oseltamivir phosphate, the internal standard, and both dexamethasone and remdesivir, respectively. The mobile phase consisted of methanol and ammonium acetate solution (40 mM) adjusted to pH 4 in a ratio of 61.5:38.5 (v/v) with a flow rate of 0.25 mL min−1. The calibration curves were linear over 500.0–5000.0 ng mL−1 for oseltamivir phosphate, over 10.0–500.0 ng mL−1 and 500.0–5000.0 ng mL−1 for dexamethasone, and over 20.0–500 ng mL−1 and 500.0–5000.0 ng mL−1 for remdesivir. The Gibbs free energy and Van't Hoff plots were used to investigate the effect of column oven temperatures on retention times. Fluoride-EDTA anticoagulant showed inhibition activity on the esterase enzyme in plasma. The proposed method was validated according to the M10 ICH, FDA, and EMA’s bioanalytical guidelines. According to Eco-score, GAPI, and AGREE criteria, the proposed method was considered acceptable green.

Development and Validation of Stability Indicating UPLC method of Memantine and Donepezil Fixed Dosage Form Combination in Marketed Dosage form

Development and Validation of Stability Indicating UPLC method of Memantine and Donepezil Fixed Dosage Form Combination in Marketed Dosage form

"Optimization of UPLC Method for Quantification of Molnupiravir: Stationary Phase, Mobile Phase, Organic Modifier, and Flow Rate Considerations"

"Optimization of UPLC Method for Quantification of Molnupiravir: Stationary Phase, Mobile Phase, Organic Modifier, and Flow Rate Considerations"

Development and Validation of an UPLC Method for Quality Control of Plantago major

Development and Validation of an UPLC Method for Quality Control of Plantago major

Development of a Reversed-Phase UPLC Method for Assay of Fipronil Including Determination of Its Related Substances in Bulk Batches of Fipronil Drug Substance.

Fipronil is a commonly used pesticide in the agricultural and animal health industries for the protection of crops and control of fleas, ticks, and chewing lice. It is difficult to obtain reproducible retention time and relative retention time (RRT) for a common hydrolytic degradation product of fipronil with the current European Pharmacopeia (EP) monograph for assay and estimation of related substances of fipronil. This situation causes misidentification, mislabeling, and/or false out-of-specification results for this hydrolytic degradation product of fipronil in bulk commercial batches during batch release and/or in the stability samples during the shelf life of a released batch. This study aimed to develop a reversed-phase ultra performance liquid chromatography (UPLC) method for assay and identification of fipronil including identification and estimation of its related substances in bulk drug substance batches of fipronil and provide consistent retention time of the hydrolytic degradation product. Fipronil and its related substances were separated by gradient elution on a Halo C18 column (50 mm × 2.1 mm id, 2.0 µm particle size) maintained at 40°C with 0.1% H3PO4 in H2O as mobile phase-A and acetonitrile-methanol (50 + 50, v/v) as mobile phase-B. Fipronil and its related substances were detected and quantified at 280 nm with a quantitation limit of 0.05% of the target (analytical) concentration. The UPLC method was able to separate all analytes of interest by gradient elution with a total run time of 7 min (approximately 40% faster than EP). In this paper, we report the development and validation of a fast, stability-indicating reversed-phase UPLC method for assay and estimation of related substances of fipronil in stability samples and bulk batches of fipronil. The new UPLC method is approximately 40% faster than the current Ph. Eur. monograph for fipronil assay and the new method provides reproducible retention of a common hydrolytic degradation product of fipronil.

A UPLC method development and validation study of Upadacitinib and its impurities in extended – release oral tablet dosage forms

The primary objective was to develop a concomitant isocratic ultra-performance liquid chromatographic photo-diode array detection method to estimate Upadacitinib and its process-related impurities: impurity-1 and impurity-2. Further validation was conducted and studied for possible degradants under stress environments. All the chemicals and reagents used were of HPLC (acetonitrile, methanol) and analytical grade (trifluoro acetic acid). The ultra-performance liquid chromatography (Agilent 1290 Infinity II LC system) consists of a quaternary pump, a BEH C18 (50×2.1mm, 1.7μ) column, and photo-diode array detector. The method was developed with acetonitrile: methanol: 0.1% v/v trifluoro acetic acid (50:20:30 v/v/v) mobile phase at 0.2mL/min flow rate within a run time of 5.5min The detection was carried at 231.2nm. The respective retention times achieved were 2.289min (Upadacitinib), 0.972min (Upadacitinib impurity-1), and 3.508min (Upadacitinib impurity-2). The optimized method was validated further, and the linearity range was best fit at 15.0-180.0μg/mL for Upadacitinib and 1.0-12.0μg/mL for both Upadacitinib impurity-1 and 2 respectively. The detection and quantification limits were 4.50μg/mL, 15.00μg/mL (Upadacitinib) and 0.30μg/mL, 1.0μg/mL (Upadacitinib impurity-1 and 2). A fast, isocratic, specific, and reproducible ultra-performance liquid chromatographic method was developed and validated for various parameters according to the ICH Q2 (R1) guidelines studies. Stress studies were conducted exposing the sample dilution to various treatments (acid, alkali, peroxide, HPLC water, heat, and UV light). The degradants were well-separated apart from the peaks of the active substance. The stability indicating nature was observed during the degradation. The optimized method can be applied for the separation and estimation of Upadacitinib and its process-related impurities in pharma sector in tablet dosage forms.

Determination of 13 chemical components of Epimedii Folium in pharmacopoeia by UPLC method combined with quantitative analysis of multicomponents by single-marker

The quantitative analysis of multicomponents by single-marker(QAMS) was established for 13 chemical components of Epimedii Folium, including neoglycolic acid, chlorogenic acid, cryo-chlorogenic acid, magnolidine, hypericin, epimedin A, epimedin B, epimedin C, icariin, baohuoside Ⅱ, sagittatoside A, icariin subside Ⅰ, and baohuoside Ⅰ, so as to investigate the feasibility and accuracy of this method in evaluating the quality of Epimedii Folium materials from different origins and different varieties. Through the scientific and accurate investigation of the experimental method, the external standard method was used to determine the content of 13 chemical components in epimedium brevieornu. At the same time, icariin was used as the internal standard, and the relative correction factors of icariin with neoglycolic acid, chlorogenic acid, cryo-chlorogenic acid, magnolidine, hypericin, epimedin A, epimedin B, epimedin C, icariin, baohuoside Ⅱ, sagittatoside A, icariin subside Ⅰ, and baohuoside Ⅰ were established, respectively. The contens of neoglycolic acid, chlorogenic acid, cryo-chlorogenic acid, magnolidine, hypericin, epimedin A, epimedin B, epimedin C, icariin, baohuoside Ⅱ, sagittatoside A, icariin subside Ⅰ, and baohuosideⅠ in Epimedii Folium were calculated by QAMS. Finally, the difference between the measured value and the calculated value was compared to verify the accuracy and scientific nature of QAMS in the determination. The relative correction factor of each component had better repeatability, and there was no significant difference between the results of the external standard method and those of QAMS. With icariin as the internal standard, QAMS simultaneously determining neoglycolic acid, chlorogenic acid, cryo-chlorogenic acid, magnolidine, hypericin, epimedin A, epimedin B, epimedin C, icariin, baohuoside Ⅱ, sagittatoside A, icariin subside Ⅰ, and baohuoside Ⅰ can be used for quantitative analysis of Epimedii Folium.

Stability-indicating UPLC assay coupled with mass spectrometry for the analysis of vilanterol degradation products in human urine

Vilanterol is a once-daily dose inhaler prescribed for asthma and chronic obstructive pulmonary disease. This study involved an investigation of vilanterol stability under acidic, basic, oxidative, thermal, and photolytic stress conditions. UPLC method was developed and validated for the analysis of vilanterol with its degradants. The drug was stable under photolytic and thermal stress conditions and degraded under acidic, basic, and oxidative stress conditions. Degradation kinetics was performed for acidic, basic and oxidative stress conditions. Kinetics parameters, K, half-life time (t1/2) and shelf-life time (t90) were assessed, and the degradation followed first order reaction. The method was linear from 0.10 to 100.00 µg mL−1 with accuracy, inter-day and intra-day precision from 99.45 to 100.02%, 0.391–0.694 and 0.041–0.345, respectively. Mass spectrometry was employed to elucidate the structure of the degradants, and the results revealed that certain degradation products were comparable to vilanterol metabolites. The World Anti-Doping Agency has prohibited the presence of vilanterol and its metabolites in athletes’ urine except for exercise bronchoconstriction with limited dose. So, quantification of vilanterol in the presence of its degradants was performed in human urine. The results revealed that the method was linear in range of 1.00 to 100.00 µg mL−1. Samples collection and experimental protocol was performed according to the guidelines of the Research Ethics Committee of the Faculty of Pharmacy, the British University in Egypt with approval No. CH-2305.

A Green UPLC Method for the Simultaneous determination of Tenofovir and Amoxicillin in Biological Fluids and Dosage Forms

This paper elucidates the process of developing and validating the UPLC technique, aiming to enable the concurrent measurement of tenofovir and amoxicillin. This UPLC method is accurate and sensitive. The experimental setup involved utilizing a Kinetex 1.7μ C18 100A (2.1-mm × 50-mm) column maintained at a temperature of 30°C. The mobile phase employed a combination of methanol and phosphate buffer (0.1% orthophosphoric acid in water, pH 3.5) in a ratio of 30:70 (v/v). The flow rate was set at 0.2mL/min, and the detection wavelength for analysis was 230nm. All these conditions resulted in optimum chromatographic separation. This developed method of UPLC took less than 4 minutes to separate this mixture. The detection limits for two medications were determined to be 0.45µg/mL for tenofovir and 0.26µg/mL for amoxicillin. The calibration plots of both substances exhibited satisfactory linearity within the concentration range of 1.0 - 2.0 µg/mL. Remarkably, the method demonstrated excellent performance with high percentage recoveries ranging from 96.8% to 102.92% and low percentage relative standard deviation (%RSD) values below 2%. Consequently, the proposed methodology proved to be highly effective for detecting the targeted drugs in their respective dosage forms.

Study on the Active Components and Inhibiting Effect of Melastoma dodecandrum Lour. on Human Cervical Cancer Cells Based on Spectrum–Effect Relationship Analysis

Background The ethnic drug Melastoma dodecandrum Lour. (MDL) is an important medicinal plant with potential antitumor properties, which have been proposed as promising anticancer agents, but the material basis for its therapeutic effect is still unclear and lacks in-depth research. Methods In this study, the in vitro antitumor effects of different MDL extracts in different gynecological tumor cells were investigated by CCK-8 assay. The active components of 21 batches of MDL samples collected from different product areas and markets were determined and compared by the UPLC method. On this basis, the data was processed with the fingerprint similarity software, and the correlation between MDL fingerprint and antihuman cervical cancer activity was further analyzed by a multivariate statistical method. Results Five kinds of MDL extracts, including the aqueous extract, the crude polysaccharide, the total polyphenol, the ethyl acetate fraction of the aqueous extract and 50% ethanol extract, had inhibitory effects on human cervical cancer HeLa and SiHa cells, among which total polyphenols of MDL showed stronger inhibition effect on human cervical cancer cells. In addition, the fingerprint of the total polyphenols of MDL had sixteen common peaks, and six common peaks were checked by using diode array and UV spectra, among which peak 1 (gallic acid), peak 4 (protocatechuic acid), peak 12 (unknown), and peak 15 (ellagic acid) were negatively correlated with the IC50 value of the SiHa cells, and all variable importance values were greater than 1. Conclusion Our data demonstrated that the main active components of MDL inhibiting human cervical cancer SiHa cells may be the synergistic effect of gallic acid, protocatechuic acid and ellagic acid, and the further spectrum-effect relationship analysis provides an important reference and basis for the quality control and the development of new anticervical cancer drug of MDL.

UPLC METHOD DETERMINATION AND QUANTIFICATION OF BELUMOSUDIL AND ITS IMPURITIES IN THE HUMAN PLASMA SAMPLES

This current analysis demonstrates the development of the related substances (RS) method for separating and quantifying BSL and its related substances from plasma using the UPLC technique. The solid phase extraction (SPE) method was employed to extract the belumosudil (BSL) and related impurities from plasma. This SPE method has been shown to have high recovery values for BSL and its related impurities. The method employed a column from Waters, specifically the X-Bridge BEH C18 150x4.6mm, 3.5µm, with a mobile phase flow rate (0.8 mL/min) and a UV-wavelength (250 nm). In this method, BSL and its impurities show good peak shapes and high resolutions. This developed method shows high sensitivity, accuracy, and precision values. While performing the stability studies, the drug was stable and it showed less than 4.53% degradation. Six different concentration solutions (BSL (25-250µg/mL) and impurities (3-18µg/mL)) were prepared for constructing linearity plots. The obtained linearity plot shows the accepted correlation coefficient (R2) value (≥0.999). The Limit of detection (LOD) and Limit of quantification (LOQ) were determined using the signal-to-noise (S/N) ratio method. This UPLC method was validated according to ICH guidelines.

Validation of an UPLC method for the determination of Pioglitazone Hydrochloride active substance in Pharmaceutical Dosage Forms

Validation of an UPLC method for the determination of Pioglitazone Hydrochloride active substance in Pharmaceutical Dosage Forms

Development and Validation of a UPLC Method for the Determination of Docetaxel and Its Related Substances in Pharmaceutical Dosage Forms, an Antineoplastic Agent

Development and Validation of a UPLC Method for the Determination of Docetaxel and Its Related Substances in Pharmaceutical Dosage Forms, an Antineoplastic Agent

Stability Indicating UPLC Method Development and Validation for the Quantitative Estimation of Pazopanib in Pure form and Marketed Pharmaceutical Dosage form.

An analytical, accurate, precise, specific, efficient and simple Ultra-Performance Liquid Chromatography method has been developed and validated for the determination of Pazopanib in bulk and was applied on marketed Pharmaceutical Dosage form. The mobile phase used for the chromatographic runs consisted of 0.1% OPA Buffer and Acetonitrile in the ratio of 30:70% v/v. The separation was achieved on a BHEL UPLC column using isocratic mode. Pazopanib Drug peak were well separated and were detected by a PDA detector at 256 nm. The developed method was linear at the concentration range 6-14 μg/ml for Pazopanib. The method has been validated according to ICH guidelines with respect to system suitability, specificity, precision, accuracy and robustness. The LOD and LOQ for the Pazopanib were found to be 0.5853 µg/ml and 1.7738µg/ml respectively. The developed method is simple, precise, specific, accurate and rapid, making it suitable for estimation of Pazopanib in bulk and marketed pharmaceutical dosage form dosage form.

Quality by design tool evaluated green stability-indicating UPLC content determination method for the Olanzapine and Samidorphan dosage form

A green, environmentally friendly, quick, sensitive, cost-effective, stability-indicating, and analyst-friendly UPLC method for determining the content of Olanzapine and Samidorphan tablets formulation was developed since no method was reported in published articles. Waters CHS (50 × 2.1) mm, 1.7 um column, flow rate of 0.3 mL/min, injection volume of 1.0 µL, column temperature of 30 °C, detection wavelength of 232 nm, and a total runtime of 2 min were used to quantify the analytes. The mobile phase was composed of a solution containing 0.01 % di sodium phosphate and acetonitrile in the ratio of (30:70, v/v). The optimised technique has been validated, and the results are met the regulatory criteria. Olanzapine's accuracy results found from 99.5 % to 99.8 %, whereas Samidorphan accuracy results found from 99.4 % to 100.4 %. Olanzapine's precision and intermediate precision %RSD results found from 0.3 % to 0.7 %, whereas Samidorphan found from 0.5 % to 0.7 %. The correlation coefficient value for both analytes was found to be greater than 0.999 for the linearity range from 2.5 µg/mL to 15.0 µg/mL. Solution stability established for 24 hrs and filter compatibility studied. By studying forced degradation, the method stability indicating nature is proven. The method's robustness was assessed by utilising a design expert software tool. The method greenness was evaluated by using National method environmental index (NEMI), Green analytical procedure index (GAPI), Analytical greenness (AGREE) tools and Analytical Eco-Scale. AGREE value found 0.66 and eco-scale value found 81.