Ultra-fast Liquid Chromatography Method Research Articles

Method development and validation of an analytical quality by design ultrafast liquid chromatographic method for the determination of bedaquiline from pharmaceutical bulk and nanoemulsions.

Bedaquiline (BDQ) is a drug used to treat multidrug-resistant tuberculosis (MDR-TB). It exhibits exposure-dependent efficacy in eliminating Mycobacterium tuberculosis (Mtb). An easy, efficient and precise reverse-phase ultrafast liquid chromatography (RP-UFLC) method was developed to validate the free base of the antitubercular medication BDQ. BDQ was separated using a 10:90 v/v mobile phase of ammonium acetate buffer solution (pH = 5.4) and high-performance liquid chromatography-grade methanol, with a flow rate of 1.5mL/min and a UV detection wavelength of 226 nm. By using the Box-Behnken design (BBD) and response surface methodology (RSM), the method was optimised by varying critical analytical attributes (CAA) and critical performance attributes (CPAs) namely ammonium acetate fraction (%), flow rate (ml/min), buffer system molarity (M) and pH. BDQ was eluted at 7.5min utilising isocratic elution. The method was linear in the concentration range of 0.5-300 μg/mL with limit of detection values of 0.039 μg/mL and limit of quantification of 0.12 μg/mL. The results indicate that this validated method can be used as an alternative method for assay of BDQ.

Analysis of omarigliptin forced degradation products by ultra-fast liquid chromatography, mass spectrometry, and in vitro toxicity assay.

Omarigliptin (OMG) is an antidiabetic drug indicated for the treatment of type 2 diabetes mellitus. Forced degradation studies are practical experiments to evaluate the stability of drugs and to establish degradation profiles. Herein, we present the investigation of the degradation products (DPs) of OMG formed under various stress conditions. OMG was subjected to hydrolytic (alkaline and acidic), oxidative, thermal, and photolytic forced degradation. A stability-indicating ultra-fast liquid chromatography method was applied to separate and quantify OMG and its DPs. Five DPs were adequately separated and detected in less than 6 min, while other published methods detected four DPs. MS was applied to identify and obtain information on the structural elucidation of the DPs. Three m/z DPs confirmed previously published research, and two novel DPs were described in this paper. The toxicity of OMG and its DPs were investigated for the first time using in vitro cytotoxicity assays, and the sample under oxidative conditions presented significant cytotoxicity. Based on the results from forced degradation studies, OMG was found to be labile to hydrolysis, oxidation, photolytic, and thermal stress conditions. The results of this study contribute to the quality control and stability profile of OMG.

Pharmacokinetic analysis of febuxostat in human serum by UFLC based on fluorimetric derivatization

An ultra fast liquid chromatographic (UFLC) method using fluorimetric detection for the quantitation of Febuxostat (FEB) in human serum was developed by precolumn derivatization with 4-bromomethyl-7-methoxy coumarin (BrMmC). The derivatization was catalyzed with dibenzo-18-crown-6-ether to produce a fluorescent derivative which was determined through fluorescence measurement with excitation at 320 nm and emission at 395 nm wavelengths. A C18 column with 100 cm x 4.6 mm, 3 µm particule size was used for the UFLC separation procedure. A mixture of acetonitrile: methanol: 0.05 M aqueous ammonium acetate (pH 5.0) (40:40:20, v/v/v) was used as mobile phase. During the procedure, the flow rate was maintained at 0.5 mL/min. A calibration curve was then plotted as 0.005–3.0 µg/mL with a detection limit of 0.0022 µg/mL and quantification limit of 0.0073 µg/mL. The mean recovery to reveal the accuracy and relative standard deviation (RSD) that indicates the precision of the method were found out to be 85.20 % and less than 3.52 %, respectively. The utility of the new method has been demonstrated by measuring the pharmacokinetics of FEB by administration of 80 mg tablets to a healthy female volunteer, aged 42.

Development and Validation of Single Laboratory Ultra Fast Liquid Chromatography Method for Quantification of Buprofezin in Suspension Concentrate (SC) Formulation

A novel ultra fast liquid chromatography (UFLC) method has been developed and validated for quantification of Buprofezin in Suspension Concentrate (SC) formulation, using Shimadzu, packed with C18 (250 mm × 4.6 mm, 5 μm) column. Mixture of acetonitrile:water (80:20 v/v) was used as mobile phase. The flow rate was kept 1.5 ml/min and detection was carried out at 250 nm. The linearity of proposed method was investigated in the range of 200-1002 PPM (r2=0.999). The percentage recovery found to be 99.87%. The % RSD value for precision study was <0.68 as per modified Horwitz equation as requirements by CIPAC. The developed method was found to be specific, linear, precise and accurate. This method is also useful for quantification of Buprofezin in their single or combination formulated products, environmental samples (soil, water), and agricultural products for pesticide residue analysis.

Role of Oxalic Acid in Expression of Resistance against the Pod Borer Helicoverpa armigera (Lepidoptera: Noctuidae) in Chickpea Varieties

Background: Pod borer is most serious problem to cultivation of chickpea across the world. In India, Madhya Pradesh rank first in both area and production of chickpea. This region is considered most affected yield of chickpea due to H. armigera. Host plant resistance is an important component for managing this pest. To develop cultivars with resistance to insects, it is important to understand the role of oxalic acid associated with resistance to this pest. The current study aimed to study the role of oxalic acid in expression of resistance against the pod borer Helicoverpa armigera (Lepidoptera: Noctuidae) in chickpea varieties under climate change. Methods: This investigation was carried out at Research Farm of Soybean Seed Production-Unit, College of Agriculture, JNKVV (MP) during Rabi season 2019-20 and 2020-21. The field experiment was laid out in split split plot design with 24 treatments and three replications. The treatments included three date of sowing i.e. 15th November, 30th November and 15th December as a main plots, two irrigation levels i.e. I0-no irrigation and I1- irrigation at 35 DAS as a sub plots and four chickpea varieties i.e. JG 12, JG 36, JG 14 and JG 24 as sub sub plots. The observation on number of eggs and larval population of H. armigera were recorded from one-meter row length (mrl). At five randomly selected places were averaged separately for each plot and made in to number of eggs and larval population per meter row length (mrl). The amount of oxalic acid in chickpea leaves was determined by UFLC method. The correlation was worked out with population of pod borer and amount of oxalic acid in chickpea varieties. Result: In this investigation, the ultra-fast liquid chromatography (UFLC) profiles of the leaf exudates of chickpea varieties exhibited amounts of oxalic acid significantly negative correlation with egg count and larvae incidence of H. armigera.

Enantioselective RP-UFLC Method for the Simultaneous Estimation of Sitagliptin (STG) Enantiomers (R and S) in the Racemic Mixture and their Pharmacokinetic Assessment in Male Wistar Rats

The present work aims to develop and validate a simple, rapid, and reproducible reverse phase ultra-fast liquid chromatography method with a UV detector (RP-UFLC-UV) was developed for the separation and determination of sitagliptin (STG) enantiomers (S-(STG) and R-(STG)) simultaneously. Baseline separation was achieved on Lux cellulose-1 column, (cellulose tri-(3,5-dimethyl phenyl carbamate (Chiralcel OD-RH, 250 mm × 4.6 mm, 5 µm) column and mobile phase consisted of 20mM borate buffer solution (pH = 9±0.05) and ACN in the ratio of (60:40, v/v) at a flow rate of 0.8 mL/min was used. Detection of peaks was monitored at 262 nm. For analyzing the STG enantiomers in the rat serum and pure API, a method was developed using the chiral RP-UFLC-UV method was validated. The single oral dose of 2.5 mg/kg of STG racemate was administered to a group of 6 healthy rats for a comparative pharmacokinetic study of both the enantiomers. The linear range of the calibration curve for each enantiomer was 0.5–64 µg/mL. The precision of this method at concentrations between 0.5–48 µg/mL was within 8.65% and the % recovery (accuracy) of both sitagliptin (STG) enantiomers (S-(STG) and R-(STG) were 98.47–101.02% and 98.93- 100.52%. The precision at LLOQ (0.5 µg/mL) was between 8.65%-7.09%, which was poor than that at QC levels, and the average extraction recovery was higher than 85% for both sitagliptin (SGT) enantiomers at QC levels, and its pharmacokinetics of enantiomers was found to be stereoselective.

Development and validation of a new stability indicating RP-UFLC method for the estimation of Perampanel in presence of internal standard

Perampanel is chemically α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor antagonist. Perampanel is used for the treatment of seizures which involve one part of brain both in adults as well as in children who are above four years. In the present study a new stability indicating reverse phase ultrafast liquid chromatographic method has been proposed for the quantification of Perampanel in presence of an internal standard, Rufinamide using water: acetonitrile as mobile phase. Linearity was observed over the concentration range 0.05-50 μg/mL with linear regression equation y = 1.3839x - 0.0302 (r2 = 0.9999). The LOD and LOQ were found to be 0.01451 μg/mL and 0.0437 μg/mL. Perampanel was exposed to different stress conditions such as acidic hydrolysis, alkaline hydrolysis, oxidation and thermal treatment and the proposed method was validated.

Validated Chiral Ultra Fast Liquid Chromatographic Method for Quantitative Analysis of Enantiomeric Vildagliptin

A rapid, accurate, and precise chiral Ultra fast liquid chromatography (UFLC) method was developed and validated for enantiomeric separation of racemic vildagliptin and S-vildagliptin according to the guidelines of the International Conference on Harmonization (ICH). The chiral chromatographic separation was achieved with a mobile phase consisting of 20 mM borax buffer (pH 9.0 ± 0.05), ACN, and 0.1% Triethylamine (50:50:0.1, v/v/v) at a flow rate of 1 ml/min using a chiralcel OD-RH column, tris(3,5-dimethyl phenyl carbamate) (250 mm × 4.6 mm, 5 μm) column. The UFLC analysis was monitored at 210 nm. The method showed good linearity with a regression coefficient (r2) of 0.999 in the range of 1 - 12 μg/ml for S-vilda. The detection limit (LOD), quantitation limit (LOQ), and the average percentage recovery for S-vilda were found to be 0.024, 0.075 μg/mL, and 99.19% to 100.4%, respectively. The percentages of relative standard deviation (% RSD) for intra- and inter-day precision were found to be 0.346% and 0.364%, respectively. The developed method proved to be reproducible as % RSD was S-vilda was found to be 99.19 w/w. The proposed chiral method can be put in application for the enantiomeric purity determination of S-vilda formulations.

Development and Validation of a New Stability Indicating Ultra-Fast Liquid Chromatographic (RP-UFLC) Method for the Quantification of Luliconazole

Development and Validation of a New Stability Indicating Ultra-Fast Liquid Chromatographic (RP-UFLC) Method for the Quantification of Luliconazole

A new stability indicating ultra-fast liquid chromatographic (RP-UFLC) method for the quantification of Nilotinib – A drug for blood cancer

Nilotinib hydrochloride monohydrate is a tyrosine kinase inhibitor approved for the treatment of chronic myelogenous leukemia. A new stability indicating ultra-fast liquid chromatographic (RP-UFLC) method was developed for the quantification of Nilotinib and validated. Mobile phase consisting of a mixture of sodium acetate (pH 5.0): acetonitrile (40: 60, v/v) was used with flow rate 0.8 mL/min (UV detection at 254 nm) for the chromatographic study. Nilotinib obeys Beer-Lambert’s law over the concentration range of 0.2-80 μg/mL (R2 = 0.9999) with linear regression equation y = 175336x +20675. The LOQ was found to be 0.1897 μg/mL and the LOD was found to be 0.0619 μg/mL. Forced degradation studies were performed such as acidic, alkaline, oxidation and thermal degradations and the method was validated as per ICH guidelines.

Development and validation of a new stability indicating ultra-fast liquid chromatographic (RP-UFLC) method for the quantification of Rosuvastatin

Development and validation of a new stability indicating ultra-fast liquid chromatographic (RP-UFLC) method for the quantification of Rosuvastatin

Determination of Coumarins from Pterocaulon balansae by an Ultra-Fast Liquid Chromatography Method in Topical Applications

Determination of Coumarins from Pterocaulon balansae by an Ultra-Fast Liquid Chromatography Method in Topical Applications

Development and Validation of Spectrophotometric and Liquid Chromatographic Methods for Estimation of Tigecycline in Injections

Three new analytical methods were optimized and validated for the estimation of tigecycline (TGN) in its injection formulation. A difference UV spectroscopic, an area under the curve (AUC), and an ultrafast liquid chromatographic (UFLC) method were optimized for this purpose. The difference spectrophotometric method relied on the measurement of amplitude when equal concentration solutions of TGN in HCl are scanned against TGN in NaOH as reference. The measurements were done at 340 nm (maxima) and 410nm (minima). Further, the AUC under both the maxima and minima were measured at 335-345nm and 405-415nm, respectively. The liquid chromatographic method utilized a reversed-phase column (150mm×4.6mm, 5µm) with a mobile phase of methanol: 0.01M KH2PO4 buffer pH 3.5 (using orthophosphoric acid) in the ratio 80:20 %, v/v. The flow rate was 1.0ml/min, and diode array detection was done at 349nm. TGN eluted at 1.656min. All the methods were validated for linearity, precision, accuracy, stability, and robustness. The developed methods produced validation results within the satisfactory limits of ICH guidance. Further, these methods were applied to estimate the amount of TGN present in commercial lyophilized injection formulations, and the results were compared using the One-Way ANOVA test. Overall, the methods are rapid, simple, and reliable for routine quality control of TGN in the bulk and pharmaceutical dosage form.

Anti-Inflammatory and Chemopreventive Effects of Bryophyllum pinnatum (Lamarck) Leaf Extract in Experimental Colitis Models in Rodents.

Inflammatory bowel diseases, mainly ulcerative colitis and Crohn’s disease are characterized by chronic inflammation in the intestine. Currently several therapeutic strategies available to treat inflammatory bowel diseases. Though, most treatments can be associated with serious adverse effects what justifies the search for new treatments. In this sense, we highlight the interest in herbal products rich in bioactive compounds which immunomodulatory and antioxidant properties as is the case of Bryophyllum pinnatum (Crassulaceae). This plant is used in traditional medicine in Brazil for treating inflammatory diseases. We hypothesized that hydroethanolic B. pinnatum leaf extract has intestinal anti-inflammatory effects on two experimental colitis models: 2.4-dinitrobenzene sulfonic acid (DNBS) in rats, and dextran sulfate sodium (DSS) in mice. Ultra-fast liquid chromatography method used for the quantification of the main compounds indicated good linearity, specificity, selectivity, precision, robustness and accuracy. The major flavonoids (mg/g of the extract) quantified were: quercetin 3-O-α-L-arabinopyranosyl-(1→2)-α-L-rhamnopyranoside (35.56 ± 0.086 mg/g), kaempferol 3-O-α-L-arabinopyranosyl-(1→2)-α-L-rhamnopyranoside (4.66 ± 0.076 mg/g) and quercetin-3-O-rhamnopyranoside (4.56 ± 0.026 mg/g). The results obtained in the DNBS and DSS models indicate that extract has both chemopreventive and anti-inflammatory effects, observing a significant reduction in the disease activity index score, and less macroscopic and microscopic damage. The extract promoted downregulation of Toll-like receptor and kappa B p65 nuclear factor gene expression, leading to a reduction in pro-inflammatory and oxidative mediators, chemokines, and cell adhesion molecules. This immunomodulatory property was proposed that one of the possible action mechanisms of extract. An improvement in intestinal damage was also associated with a reduction in oxidative stress and infiltration of leukocytes, as evidenced by the reduction in malonaldialdehyde and myeloperoxidase activity and increase in total glutathione in the colonic tissue. Moreover, the extract improved the cytoarchitecture of the colonic tissue and the integrity of the intestinal epithelial barrier by restoring the expression of the proteins associated with mucosa protection. In view of the beneficial effects showed by the B. pinnatum leaf extract in preclinical rodent models of colitis there is the potential to conduct some future clinical studies to ensure safe and effective development of a phytotherapeutic treatment for human inflammatory bowel diseases.

A stability-indicating ultra-fast liquid chromatography method for the assay of the main flavonoids of Achyrocline satureioides (Marcela) in porcine skin layers and nanoemulsions.

Achyrocline satureioides (marcela or macela) is a plant widely used in folk medicine in South America. Recently, there has been increasing interest for the development of skin care products containing A. satureoides extracts, due to its well-documented antioxidant, antiherpetic, and wound healing properties. The present study aimed to develop and validate a yet unexplored stability-indicating and robust ultra-fast liquid chromatography (UFLC) method for the simultaneous quantification of the main flavonoids of A. satureioides in extracts, nanoemulsions, and porcine skin layers. The chromatographic separation of flavonoids quercetin, luteolin, and 3-O-methylquercetin was performed on a Luna C18 analytical column (100 mm × 2.0 mm i.d.; particle size 2.5 μm) using isocratic elution with methanol/phosphoric acid 1% (48:52 v/v) with a flow rate of 0.3 mL/min at 40°C. The method was found to be specific, linear (R > 0.998), precise, accurate, and robust for all flavonoids assayed in A. satureioides extract, nanoemulsions, and porcine ear skin. A low matrix effect was noted for all complex matrices. The stability-indicating UFLC method was evaluated by submitting isolated flavonoids, a mixture of standards, and A. satureioides extract to acidic, alkaline, oxidative, UV-A/UV-C light, and thermal stress conditions. No peaks were found co-eluting with the flavonoids of interest in all matrices. The robustness of the method was confirmed using Plackett-Burman experimental design. The short run time (8 min) and reliability of the method could be useful for the determination of A. satureioides flavonoids in topical product development since extracts of this medicinal plant have been used to treat various skin disorders.

Analytical method development and validation of teneligliptin by rp-uflc

To estimate Teneligliptin by Ultra-Fast Liquid Chromatographic (RP-UFLC), a fast, reliable, and cost-effective UFLC method was developed. The approximate flowing rate was 1.0 mL/minfor the Phenomenex Kinetex column C18 (250mmx 4.6mm. 5 Micron) with MeOH and ACN (60:40 v/v) as a portable stream. A PDA detector at 246 nm was used to track the eluent. Teneligliptin elutes at 4.938 minutes of the processing period. The proposed method has a linearity of 10 to 50μg/ml concentration range; the calibration diagram is continuous and 0.9942 percent for the correlations variable (r2). For the suggested procedure, both LOD and LOQ are measured as 0.097 and 1.023μg/mL, respectively. In the pharmaceutical product, the drug has been under the states of stress conditions acidic, alkaline, degradation, Ultraviolet, and heat stress. The degradation product from the Teneligliptin was well solved, and thus the stability of this method was demonstrated. The process was tested in compliance with ICH criteria of device suitability, linearity, reliability, consistency, and robustness.

Analytical lifecycle management oriented development and validation of a new stability-indicating ultra-fast liquid chromatographic method: case study of Glycopyrrolate

A new stability-indicating liquid chromatographic method was developed and validated for the estimation of glycopyrrolate in pharmaceutical formulations. A contemporary approach to analytical life-cycle management was followed to develop a robust and reliable chromatographic method. Scouted method variables such as % methanol, the strength of tetra butyl ammonium hydrogen sulfate and mobile phase flow rate were optimized using the design of experiment approach and their effect on critical quality attributes was studied. The critical quality attributes viz. retention time, theoretical plate count and symmetry factor were highly influenced by the three critical method variables. Optimum chromatography was attained on a C-18 column with a mobile phase methanol: 10 mM tetra butyl ammonium hydrogen sulfate (80:20, v/v) flowing at 1.0 mL.min-1. Chromatographic method specificity was ensured by degrading the drug forcefully. Validation studies postulated method acceptability and suitability for estimating glycopyrrolate in both bulk as well as injection formulation. Results for parameters viz. linearity (5-250 µg.mL-1), accuracy (>99%) and precision (<2%) advocated method reliability. Overall the method was reliable and of optimum quality and, possess the potential of application in routine and bio-analytical purposes.

Ultra-fast liquid chromatographic method for the determination of ticagrelor in pharmaceutical formulations and spiked plasma samples

Ultra-fast liquid chromatographic method for the determination of ticagrelor in pharmaceutical formulations and spiked plasma samples

Sensitive ultra-fast liquid chromatography method for rosmarinic acid determination in Wistar rat’s plasma and brain

Rosmarinic acid (RA) is a polyphenolic compound recently associated to a neuroprotective potential. Nevertheless, besides the RA poor bioavailability and availability in the central nervous system (CNS) some alternatives to RA delivery, protection, and release been investigated. Recently, our research group optimized chitosan-coated nanoemulsions for RA nasal administration and demonstrated the glioprotective effect against LPS-induced damage in astrocytes. In this context, this study aimed to validate a fast, simplified and stability-indicating UFLC method previously reported by our research group for RA determination in Wistar rat’s plasma and brain, to be employed in further in vivo studies, since the nasal route for RA are not completely understood. The method was validated in terms of specificity, linearity, matrix effect, stability, precision, accuracy and extraction recovery for rat plasma and brain, according to the official guidelines. The method was sensitive, linear (0.1 - 10 µg.mL−1), precise and accurate, and showed RA recovery higher than 85% in plasma and brain. Overall results demonstrated that method was successfully validated for determination of RA in rat plasma and brain matrices with high sensibility and with high recovery using simple extraction processes.

Development and Validation of a Rapid Solid-Phase Extraction: Ultrafast Liquid Chromatographic Method for the Estimation of Azithromycin and Its Major Related Substances in Human Plasma and Dosage Forms Using a Novel Polyfunctional Silyl Reagent-Bonded Core–Shell Column

In this study, a rapid reversed-phase liquid chromatography method for the determination of azithromycin and its related substances was developed and validated on a novel polyfunctional silyl reagent [hexanhexamethyloctadecyltetrasiloxane (HMODTS–C18)] bonded core–shell RP C-18 column (100 × 4.6 mm, 2.6 µm) as per ICH guidelines. A binary gradient elution programme with a mixture of solvent A (phosphate buffer, pH 8.9) and solvent B (ACN: MeOH, 3:1) as mobile phase, at a flow rate of 1.5 mL min−1 and detection at 210 nm was finally optimized for the study. The validation results showed that the method to be specific, selective, highly sensible (0.004 mg mL−1), precise (% RSD ≤ 10), linear and accurate in a concentration range of 0.004–0.032 mg mL−1. Simple SPE method was performed for extraction and checked for repeatability. The result showed that the method is precise (%RSD < 2). The validated chromatographic method was applied to the solid-phase extracted samples of azithromycin and its four major related substances. Also, docking study was carried out using AutoDock Tools to find out the binding affinities, number of hydrogen bonds and residues involved in hydrogen bonds for azithromycin and its four major related impurities with the human plasma proteins. The results confirmed the applicability of the proposed method on the extracted human plasma samples. Finally, the study demonstrates that the impurities of azithromycin have strong binding affinities with the plasma proteins compared to that of azithromycin and half life of these impurities may be higher than azithromycin which can cause major health risk on repeated doses of azithromycin.