Simultaneous High-performance Liquid Chromatography Research Articles

Simultaneous high-performance liquid chromatography analysis of anthraquinones in sicklepod sprouts with α-glucosidase inhibitory activity.

Sicklepod [Cassia obtusifolia L. syn Senna obtusifolia (L.) H.S. Irwin & Barneby, Fabaceae] sprouts are promising ingredients with health-promoting benefits. Notwithstanding, the pharmacologically active compounds in sicklepod sprouts have not been studied or analysed in detail. This study aimed to isolate and structurally identify phytochemicals showing α-glucosidase inhibitory activity in sicklepod sprouts and simultaneously quantify the compounds in the sprouts to determine the optimal cultivation method and germination time to maximise active compounds. A simultaneous high-performance liquid chromatography-ultraviolet (HPLC-UV) method with high sensitivity and accuracy was developed and used to analyse time-dependent changes in anthraquinone content during sicklepod germination. Thirteen anthraquinones were isolated and identified, of which six-chrysoobtusin, emodin, 1-O-methyl-2-methoxychrysophanol, 7-O-methylobtusin, chrysophanol, and physcion-showed moderate α-glucosidase inhibitory activity. The maximum content of anthraquinones in a sprout was observed on Day 5 under both light and dark conditions. The findings of this study revealed that sicklepod sprouts which are promising functional food materials contain a variety of anthraquinones.

Comprehensive Review on Chiral Stationary Phases in Single-Column Simultaneous Chiral-Achiral HPLC Separation Methods.

This comprehensive review explores the utilization of chiral stationary phases (CSPs) in the context of single-column simultaneous chiral-achiral high-performance liquid chromatography (HPLC) separation methods. While CSPs have traditionally been pivotal for enantioselective drug analysis, contemporary CSPs often exhibit notable chemoselective properties. Consequently, there is a discernible trend towards the development of methodologies that enable simultaneous enantio- and chemoselective separations utilizing a single CSP-based chromatographic column. This review provides an exhaustive overview of reported HPLC methods in this domain, with a focus on four major CSP types: cyclodextrin-, glycopeptide antibiotic-, protein-, and polysaccharide-based CSPs. This article delves into the diverse applications of CSPs, encompassing various chromatographic modes such as normal phase (NP), reverse phase (RP), and polar organic (PO). This review critically discusses method development, emphasizing the additional chemoselective separation mechanisms of CSPs. It also explores possibilities for method optimization and development, concluding with future perspectives on this evolving field. Despite the inherent challenges in understanding the retention mechanisms involved in chemoselective separations, this review highlights promising trends and anticipates a growing number of simultaneous enantio- and chemoselective methods in pharmaceutical analyses, pharmacokinetic studies, and environmental sample determinations.

A green perspective on simultaneous HPLC and UV spectrophotometric estimation of Udenafil and Dapoxetine hydrochloride in pharmaceutical formulations

Clinical research has affirmed that the combination of Udenafil and Dapoxetine hydrochloride yields greater improvement in erectile dysfunction compared to the use of individual medications. There is a requirement of precise quantitative method for accurate determination of Udenafil and Dapoxetine when taken together. Consequently, our interest lies in developing and validating analytical methods for the simultaneous estimation of Udenafil and Dapoxetine - Various strategies, such as UV spectrophotometric approaches and an innovative, straightforward, accurate, sensitive, and robust High-Performance Liquid Chromatography (HPLC) procedure, have been explored. An Isocratic HPLC approach was employed using reversed-phase CHEMSIL ODS C18 column (4.6 mm x 250 mm, 5 µm) as a stationary phase and detection wavelength (λ max) 230 nm for the two analytes in the chromatographic column. The mobile phase comprised of (70:30, v/v) mixture of Acetonitrile and 10 mM Ammonium formate buffer having pH 5, adjusted with formic acid. The elution was carried out at a constant flow rate of 1 mL/min. The UV simultaneous equation technique estimated the medicine's quantities and derived a simultaneous equation for DAP and UDN using 2 wavelengths, 230 nm and 298 nm. Furthermore, the two advanced methods underwent rigorous statistical validation, guided by the valuable ICH Q2R1 guidelines, affirming their effectiveness as a successful analytical tool for the concurrent analysis of both tablets in bulk and the synthetic mixture fabricated in the laboratory. To assess the environmental impact of contemporary UV spectrophotometric and HPLC techniques, the Green Analytical Greenness Metric (AGREE) software and the Green Analytical System Index (GAPI) were employed to evaluate the greenness profile. This examination confirmed the method's environmental friendliness concerning solvent usage, chemical ingredients, energy consumption, and waste production. The developed UV method is more sustainable and greener compare to HPLC method for simultaneous estimation of compound, which can be utilised by industry for quantification of each individual drug in their combination products.

Development of a HPLC Method for Analysis of a Combination of Clofazimine, Isoniazid, Pyrazinamide, and Rifampicin Incorporated into a Dermal Self-Double-Emulsifying Drug Delivery System.

We describe the development and validation of a new high performance liquid chromatography (HPLC) method for analysis of a combination of the first-line anti-tubercular drugs isoniazid, pyrazinamide, and rifampicin together with clofazimine. This is a unique challenge since clofazimine and rifampicin are relatively highly lipophilic drugs, whereas isoniazid and pyrazinamide are considerably more hydrophilic. Thus, clear separation of peaks and quantification of four individual drugs can present difficulties during the development of an analytical method. Detection was established at two wavelengths-254 nm for isoniazid and pyrazinamide and 320 nm for clofazimine and rifampicin. Gradient elution was employed using 0.1% aqueous formic acid (A) and acetonitrile (B); clear separation of the four drugs was achieved within 10 min. A linear relationship was indicated by a correlation coefficient (r2) of 0.9999 for each anti-tubercular drug, respectively. The limit of detection (LOD) for the individual drugs was 0.70 µg/mL (isoniazid), 0.30 µg/mL (pyrazinamide), 0.20 µg/mL (rifampicin) and 0.20 µg/mL (clofazimine). Precision experiments rendered a mean recovery percentage of 101.25% (isoniazid), 98.70% (pyrazinamide), 99.68% (rifampicin) and 97.14% (clofazimine). This HPLC method was validated and is reliable, repeatable, and accurate for the purpose of conducting simultaneous HPLC analyses of the four anti-tubercular drugs.

Simultaneous HPLC Determination of Arbutin, Niacinamide and 3-O-Ethyl Ascorbic Acid in Whitening Cream Products in the Presence of Parabens.

Arbutin, niacinamide and 3-O-ethyl ascorbic acid (a new generation of vitamin C derivatives) are compounds that have a whitening effect on skin and are widely used in whitening cream products wherein parabens such as methyl paraben, ethyl paraben, propyl paraben and butyl paraben are also often added as preservatives. This study aims to develop a validated high-performance liquid chromatography (HPLC) method that can be used to determine arbutin, niacinamide and 3-O-ethyl ascorbic acid simultaneously in whitening cream products without interference from the parabens. The optimum conditions for the HPLC system were obtained using ODS-3 RP-C18 Inertsil column, mobile phase consisting of a mixture of aquabides, methanol and acetonitrile with gradient elution mode. Detection was carried out using a UV detector at 220nm. Validation studies demonstrated a good linearity for all analytes over each range concentration with a correlation coefficient >0.999 and Vx0 < 2%. The accuracy test also met the requirements with the recoveries being 96.93-99.55%, 98.60-99.73% and 97.88-100.63% for arbutin, niacinamide and 3-O-ethyl ascorbic acid, respectively. Intra-day and inter-day precision test gave a relative standard deviation (% RSD) of <2% along with a HorRat value of <2 for all analytes. The results of this study indicate that the developed HPLC method has a good selectivity, linearity, accuracy and precision. Due to its simplicity, the method can be used to analyze arbutin, niacinamide and 3-O-ethyl ascorbic acid in the presence of parabens in whitening cream products simultaneously.

A small-scale method of sample preparation suitable for simultaneous HPLC-UV assay of capecitabine and its 5’-DFCR metabolite in mouse blood plasma

The objective of the study was to develop an easy, cheap, effective, and safe, small-scale method for sample preparation suitable for the simultaneous high-performance liquid chromatography (HPLC)-ultraviolet (UV) assay of capecitabine and its 5′-deoxy-5-fluorocytidine (5′-DFCR) metabolite in mouse blood plasma. The suitability of the proposed method of sample preparation was verified by the optimal effectiveness and efficiency achieved in the overall analytical workflow. The chromatographic separation of capecitabine and its first metabolite was performed on a Hypersil GOLD aQ column with a mobile phase consisting of 1% formic acid, methanol, and water, and run in a gradient elution mode. The absence of interfering endogenous components at the retention times of each analyte was confirmed by the chromatographic analysis of blank matrices and matrices spiked with the corresponding standards. The absence of any tactile matrix effect was also recorded. For the first time, the effect of the vacutainer’s anticoagulant on the extraction efficiency of both analytes was evaluated. The method was found to be accurate, precise, and specific. The estimated mean “extraction” efficiencies were ≥90% for each analyte. The lower limit of quantitation for both capecitabine and 5′-DFCR was 0.05 μg/mL.

Simultaneous HPLC estimation of Amphetamine and Caffeine abuse drugs in Iraqi human addicts

The main aim of this research is to establish and validate a high performance liquid chromatography (HPLC) process for the separation and estimation of Amphetamine (AM) and Caffeine (CAF) in its illegal formula and in sera of addicts. This method is established on the HPLC separation of the two drugs on the ZORBAX ODS column (250×4.6×5µm particle size). The mobile phase contained 1% ortho-phosphoric acid 85% and 1%of diethyl amine 99%, acetonitrile and methanol ratio was 85:10:5 v/v/v. The flow rate is 1.2 mL.min-1, buffer value pH of 2.5 via isocratic elution also UV detection at 210 nm. The retention times for the two drugs AM and CAF were obtained at 4.425 and 6.456 min, respectively. The calibration curves founded that the linear regression analysis data gave a good linear relationship for the concentration range 1 to 100 µg.mL-1 for AM and CAF. The values achieved for correlation coefficient, slope and intercept were 9999, 8104.2 and 5012 for AM and 0.9999, 9698.5 and 6342.9 for CAF, whereas the LOD and LOQ was 0.51, 1.64 µg.mL-1 for AM and 0.60, 1.32 µg.mL-1 for CAF  

High Performance Liquid Chromatography–Tandem Mass Spectrometry Method for Correlating the Metabolic Changes of Lactate, Pyruvate and L-Glutamine with Induced Tamoxifen Resistant MCF-7 Cell Line Potential Molecular Changes

Breast cancer is one of the most prevalent cancers worldwide usually treated with Tamoxifen. Tamoxifen resistance development is the most challenging issue in an initially responsive breast tumor, and mechanisms of resistance are still under investigation. The objective of this study is to develop and validate a selective, sensitive, and simultaneous high performance liquid chromatography–tandem mass spectrometry method to explore the changes in substrates and metabolites in supernatant media of developed Tamoxifen resistance MCF-7 cells. We focus on the determination of lactate, pyruvate, and L-glutamine which enables the tracking of changes in metabolic pathways as a result of the resistance process. Chromatographic separation was achieved within 3.5 min. using a HILIC column (4.6 × 100 mm, 3.5 µm particle size) and mobile phase of 0.05 M acetic acid–ammonium acetate buffer solution pH 3.0: Acetonitrile (40:60 v/v). The linear range was 0.11–2.25, 0.012–0.227, and 0.02–0.20 mM for lactate, pyruvate, and L-glutamine, respectively. Within- and between-run accuracy was in the range 98.94-105.50% with precision (CV, %) of ≤0.86%. The results revealed a significant increase in both lactate and pyruvate production after acquiring the resistant. An increase in L-glutamine levels was also observed and could be attributed to its over production or decline in its consumption. Therefore, further tracking of genes responsible of lactate, pyruvate, and glutamine metabolic pathways should be performed in parallel to provide in-depth explanation of resistance mechanism.

Comprehensive ethoxymer characterization of complex alcohol ethoxy sulphate products by mixed-mode high-performance liquid chromatography coupled to charged aerosol detection

The present work describes a simultaneous mixed-mode high performance liquid chromatography (HPLC) method combined with a universal and non-selective-response detector for the complete ethoxymer profiling of alcohol ethoxy sulphate mixtures. The optimized HPLC methodology combines the dual hydrophilic (HILIC) and reversed-phase selectivity of a surfactant-type column in order to render a comprehensive and simultaneous separation of more than 50 endogenous ethoxymers in a single analysis. Furthermore, an accurate quantitation of every single analyte was achieved using a final universal charged aerosol detector (CAD) including specific mathematical processing tools. Results obtained helped describing a complete alkyl chain and ethoxymer distribution of the investigated AES samples. Method validation evidences provided reliability of the individual ethoxymer contributions determined with the proposed HPLC−CAD methodology. Regarding accuracy including independent nuclear magnetic resonance (NMR) experiments, an excellent correlation was found between the structural information provided by a COSY NMR spectrum and the CAD results regarding the mono/polyethoxylated and the non-ethoxylated/ethoxylated distribution. Additional calculations including the average molecular weight and the degree of ethoxylation for the reference AES sample showed minimum differences (relative error < 1 %) between the two considered techniques. An outstanding precision and linearity along the working concentration range (r2>0.999) was also observed. The individual limit of detection for the target sulphate ethoxymers was determined to be in the low ppm range. Further validated distribution profiles for a large number of AES samples demonstrated the applicability of the optimized HPLC−CAD methodology to routine surfactant screenings. Therefore, the hereby developed methodology provided extensive information regarding the detailed individual ethoxymer profile of AES formulations, which can be extremely useful for the surfactant industry in order to gain information on specific synthesis routes and/or detergency properties.

Simultaneous HPLC Quantification of Four Lignan Glycosides in Sesame Seeds with the Single Reference Standard Method

Seven lignans including four lignan glycosides and three lignans were isolated from 70% methanolic extract of defatted sesame seeds. The chemical structures of the isolated compounds were identified by UV, 1H and 13C NMR, and HR-MS spectra. To overcome the difficulty and high cost for the preparation of lignans, the single reference standard method for the simultaneous determination of multi-components (SSDMC) with high-performance liquid chromatography (HPLC) was developed for simultaneous quantification of four lignin glycosides in sesame seeds. All four lignin glycosides had good linear concentration-measurement relationships (r2 ≥ 0.9992). The relative deviation of the method was less than 4.9% for intra- and inter-day assays and the average recovery varied from 93.3 to 101.2%. The method was also successfully applied to determine four lignan glycosides in eighteen white sesame seeds and ten black sesame seeds. The results demonstrated that the single reference standard method could be used for quality control of sesame seeds and related products since the reference standards are not commercially available.

STABILITY-INDICATING METHOD DEVELOPMENT AND VALIDATION OF ITRACONAZOLE AND TERBINAFINE HCL IN BULK AND PHARMACEUTICAL TABLET DOSAGE FORM

Objective: The objective of the present work was to develop and validate the stability-indicating method for the simultaneous estimation of itraconazole and terbinafine HCl in bulk and pharmaceutical tablet dosage form by reversed-phase high-performance liquid chromatography (HPLC). This combination of drugs is not reported for simultaneous HPLC analysis as of now.&#x0D; Methods: The analysis of the developed method was carried on Shimadzu LC Prominence-i 2030 model with Lab Solution software and the separation was done on Shim-pack C18 GIST (250 mm×50 mm, 5 μm) column with a flow rate of 1.2 ml/min and run time of 12 min. The injection volume was 10 μl and mobile phase consisted of acetonitrile and 0.1% triethylamine in the ratio of 90:10 and 225 nm was used as a detection wavelength.&#x0D; Results: The retention time was found to be 3.464 min and 8.705 min for itraconazole and terbinafine HCl, respectively. The calibration curve was found to be linear and r2 values were 0.9989 and 0.9995 for itraconazole and terbinafine HCl, respectively.&#x0D; Conclusion: The stability-indicating method was developed by subjecting itraconazole and terbinafine HCl marketed formulation to various stress conditions such as acidic, basic, oxidative, thermal, and water hydrolysis degradation conditions and the degraded product peaks were well resolved from sample peaks.

Hwangryunhaedok-Tang Exerts Neuropreventive Effect on Memory Impairment by Reducing Cholinergic System Dysfunction and Inflammatory Response in a Vascular Dementia Rat Model.

Hwangryunhaedok-tang (HRT) is a traditional oriental herbal formula used in Asian countries for treating inflammatory diseases and controlling fever. Our present study aimed to determine whether HRT has therapeutic effects for patients with vascular dementia (VaD) using a bilateral common carotid artery occlusion (BCCAO) rat model and assessing spatial memory impairment and activation of neuroinflammation. BCCAO was performed in male Sprague Dawley rats to induce VaD, and oral HRT was administered daily for 30 d. Our data showed that HRT ameliorated BCCAO-induced memory and cognitive impairment in behavioral tests. In addition, HRT reversed cholinergic dysfunction and neuronal damage in the hippocampus of BCCAO rats. Furthermore, HRT attenuated microglial activation and reduced the phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase (JNK) induced by BCCAO. Simultaneous high-performance liquid chromatography analysis of HRT using index compounds from the herbal composition revealed that both HRT ethanol extract and commercial HRT granules primarily comprise geniposide, baicalin, and berberine. Our study showed that HRT administration resulted in the prevention of neuronal injury induced by BCCAO through improvement of cholinergic dysfunction and inhibition of neuroinflammatory responses, suggesting that HRT may have potential as a treatment for VaD.

Hepatoprotective effects of different combinations of sweet orange, Unshiu mikan, and mini tomato juice powders againsttert-butyl hydroperoxide-induced oxidative stress in HepG2 cells

The aim of the present study was to evaluate the ability of raw mini tomato (Solanum lycopersicum L.) juice powder to attenuate cytotoxicity in combination with juice powders from two orange species [Citrus sinensis (L.) Osbeck and Citrus unshiu Marcow]. To this end, the hepatoprotective activities of these combinations against tert-butyl hydroperoxide (t-BHP)-induced oxidative stress in HepG2 cells were evaluated, and the most suitable ratios for optimal flavonoid availability were identified. The levels of intracellular reactive oxygen species (ROS) and glutathione (GSH) and the extent of upregulation of phase-ІІ proteins such as heme oxygenase-1 (HO-1) were quantified to assess the hepatoprotective effects of four different ratios of these powders. Three main compounds–hesperidin, narirutin, and rutin were analyzed by simultaneous high-performance liquid chromatography. The total phenolic and flavonoid contents of the 2:1:3 powder mixture were 8.69 mg/g GAE and 2.56 mg/g QE, respectively. The levels of these contents were correlated with the decrease in ROS, increase in GSH level, and restoration of HO-1. Furthermore, the hepatoprotective efficacy of each of the four ratios was attributed to its flavonoid content. These results indicate that combinations of juice powders, particularly at a ratio of 2:1:3, are a potentially useful therapeutic source of phenolic compounds for the treatment of oxidative stress-related hepatotoxicity. Practical applications Sweet orange (Citrus sinensis (L.), Unshiu mikan (Citrus unshiu Marcow) and mini tomato (Solanum lycopersicum L.) are rich sources of phenolic compounds which are consumed frequently in Korea. This research provides proper understanding of the consumption of these fruits in combination to deliver effective amount of phenolic compounds to promote human health and fight against oxidative stress related diseases. The present study demonstrates the hepatoprotective effect through prevention of oxidative stress, suggesting that combinations of juice powders may have beneficial effects in preventing hepatic diseases.

Morphological and chemical analyses of Eriocauli Flos sold in Taiwan markets

Eriocauli Flos (Gujingcao; EF), the dried capitulum with the peduncle of Eriocaulon buergerianum Koern. (Eriocaulaceae), is a Chinese herbal medicine for treating eye diseases and inflammation. However, several species of the Eriocaulon genus are used as substitutes in different areas. To examine the species of EF used in Taiwan and to establish the quality control platform, morphological and chemical analyses have been performed. Ten major compounds, including apigenin (7) and its 7-O-β-D-glucopyranoside (1) and 7-O-(6-O-E-coumaroyl)-β-D-glucopyranoside (6), hispidulin (8) and its 7-O-β-D-glucopyranoside (2) and 7-O-(6-O-E-coumaroyl)-β-D-glucopyranoside (5), jaceosidin (9) and its 7-O-β-D-glucopyranoside (3), and toralactone (10) and its 9-O-β-D-glucopyranosyl(1→6)-β-D-glucopyranoside (4), were isolated and identified from commercially available EF. Morphological investigation showed that two kinds of EFs and most of the EFs sold in Taiwan herbal markets are capitulum without the peduncle. A simultaneous high performance liquid chromatography and ultra performance liquid chromatography analyses of multiple components (1–10) in commercially available EFs, collected from different areas of Taiwan, was conducted. Results showed wide variations in morphology and chemical profiles between capitulum with and without the peduncle. In comparison with an authentic E. buergerianum, we found not only the morphology but also the chemical profile was different from both collected samples. In terms of the morphological examination, the samples without peduncle are closer to the authentic one. To ensure the correct EF materia medica is used in Taiwan so as to guarantee their therapeutic efficacy in clinical practice, further monitoring is necessary.

An endangered medicinal plant, Plumbago zeylanica L. and its substitute, Dyerophytum indicum (Gibbs ex Wight) Kuntze: A comparative HPTLC, HPLC study, and screening of antioxidant activity

Plumbago zeylanica L. (PZ) is a significant medicinal plant in Ayurveda, so is used for the treatment of various disorders. The main active part of this plant is the root, and due to its inherent uses, it has been exploited hence becoming an endangered species. Dyerophytum indicum (Gibbs ex Wight) Kuntze (DI), mainly considered as a substitute of PZ, is also an important folklore medicine, used in many health problems. Both plants are much similar in their physical as well as chemical properties. However, an effective validation is required before declaration of substitution. In the present study, quantitative and qualitative estimations were performed on both plants with the help of modern analytical techniques. Simultaneous high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC) and quantitative determinations of β-sitosterol have been performed for comparing both plants, i.e., PZ and DI. HPTLC fingerprinting analysis was also performed comparatively in diffe...

Quality evaluation of cortex berberidis from different geographical origins by simultaneous high performance liquid chromatography combined with statistical methods

Purpose: To develop an effective method for evaluating the quality of Cortex berberidis from different geographical origins. Methods: A simple, precise and accurate high performance liquid chromatography (HPLC) method was first developed for simultaneous quantification of four active alkaloids (magnoflorine, jatrorrhizine, palmatine, and berberine) in C ortex berberidis obtained from Qinghai, Tibet and Sichuan Provinces of China. Method validation was performed in terms of precision, repeatability, stability, accuracy, and linearity. Besides, partial least squares discriminant analysis (PLS-DA) and one-way analysis of variance (ANOVA) were applied to study the quality variations of Cortex berberidis from various geographical origins. Results: The proposed HPLC method showed good linearity, precision, repeatability, and accuracy. The four alkaloids were detected in all samples of Cortex berberidis . Among them, magnoflorine (36.46 - 87.30 mg/g) consistently showed the highest amounts in all the samples, followed by berberine (16.00 - 37.50 mg/g). The content varied in the range of 0.66 - 4.57 mg/g for palmatine and 1.53 - 16.26 mg/g for jatrorrhizine, respectively. The total content of the four alkaloids ranged from 67.62 to 114.79 mg/g. Moreover, the results obtained by the PLS-DA and ANOVA showed that magnoflorine level and the total content of these four alkaloids in Qinghai and Tibet samples were significantly higher ( p < 0.01) than those in Sichuan samples. Conclusion: Quantification of multi-ingredients by HPLC combined with statistical methods provide an effective approach for achieving origin discrimination and quality evaluation of Cortex berberidis . The quality of Cortex berberidis closely correlates to the geographical origin of the samples, with Cortex berberidis samples from Qinghai and Tibet exhibiting superior qualities to those from Sichuan. Keywords: Tibetan medicine, Cortex berberidis , Origin discrimination, Quality evaluation, Magnoflorine, Palmatine, Berberine, Jatrorrhizine

Simultaneous High Performance Liquid Chromatography Assay of Pentoxifylline, Bupivacaine HCl, Levocetirizine HCl, Tranilast, and Fluticasone Propionate in Humco™ Sanare Advanced Scar Base

This article details the elements used in the verification method for the simultaneous high performance liquid chromatography (HPLC) assay of Pentoxifylline, Bupivacaine HCl, Levocetirizine HCl, Tranilast, and Fluticasone Propionate in Humco™ Sanare Advanced scar base. The method was proven to be linear over 50%–150% of the nominal concentration of the standard. The method was proven to be accurate over 50%–150%, with 98%–102% recovery of the actives from spiked placeboes over that range. The method exhibited specificity to the analytes listed, and it was shown to be precise, yielding acceptable results for system reproducibility and method repeatability. The method, as written, is considered to have been verified.

Simultaneous Determination of Famotidine and Flurbiprofen by High Performance Liquid Chromatography

Purpose: To develop a selective, sensitive and accurate simultaneous High Performance Liquid Chromatography (HPLC) method for the analysis of flurbiprofen and famotidine tablet dosage form and excipients.Methods: A simultaneous method for the determination of the two drugs was employed. The assay consisted of isocratic elution of flurbiprofen and famotidine in prepacked Column RT 250-4.6 Purospher® STAR RP-18 end-capped (5 μm), using a mobile phase composed of acetonitrile and phosphate buffer (pH 3.7) in a ratio of 1:1, respectively, as well as an ultraviolet (UV) detector. Flurbiprofen and famotidine were both detected at 265 nm at a flow rate of 1 ml/min following the guidelines of International Conference of Harmonization (ICH).Results: A sensitive and linear range of 10 to 100 ppm with 0.999 coefficient of correlation for famotidine and flurbiprofen were observed. Blank and placebo showed no interference with the peak retention time of flurbiprofen and famotidine. Recovery of 50 mg famotidine was 99.61 % after 12 h and 99.76 % after 7 days and for flurbiprofen 20 mg it were 99.60 % after 24 h and 99.85 % after 7 days.Conclusion: Good precision, recovery and accuracy data indicate the reliability of the proposed method. The method may also be suitable for use in bioequivalence studies.Keywords: Flurbiprofen, Famotidine, Retention time, Limit of detection, Limit of quantification

Quantification of vasaka alkaloids in in vitro cultures and in natural leaves from Indian subcontinents by reversed phase- high performance liquid chromatography

Background: The present study was designed to develop an efficient protocol for studying the enhancement of in vitro production of secondary metabolites in Adhatoda vasica leaves collected from the varied geographical locations of Indian sub-continents by a validated simultaneous high-performance liquid chromatography gradient method for the quantification of vasicine and vasicinone in the developed calli and crude samples. Materials and Methods: The analysis was carried out on a reverse phase C 18 column using 0.1% of orthophosphoric acid: Acetonitrile in gradient manner and carried out the detection at 280 nm wavelength keeping the flow rate of 1.0 mL/min. Results: The simultaneous method was found linear with regression coefficient r 2 = 0.991 in a wide range (100–500 μg/mL) precise, accurate, and robust for quinazoline alkaloids in samples. Results clearly showed a significant increase in the concentration of alkaloids in in vitro cultures as compared to natural ones. The proposed method was validated as per the International Conference on Harmonization guidelines for accuracy, precision, robustness, limit of detection, and limit of quantitation. Conclusion: The developed method was found suitable for quality control of A. vasica and for the analysis of vasicine and vasicinone in any type of sample.

Development and Application of an HPLC–UV Procedure to Determine Multiple Flavonoids and Phenolics inAcanthopanaxLeaf Extracts

Acanthopanax species are used in traditional medicine to treat numerous diseases. A simultaneous high-performance liquid chromatography (HPLC) method was developed and validated for the determination of the flavonoid and phenolic content of Acanthopanax leaves. HPLC analysis was performed on an AKZO NOBEL Kromasil 100-5C18 column (250 × 4.6 mm, 5 µm) using a gradient elution of acetonitrile-water containing 0.2% formic acid with a flow rate of 1.0 mL/min, monitored at 320 nm. The method was linear over the range 1-500 µg/mL (determination coefficients R(2) > 0.999). Satisfactory intraday and interday precision was achieved, with the relative standard deviation (RSD) <2.99%. The mean recoveries measured at the three concentrations were in the range of 90.11-104.83%, with RSD <2.91% for the targets. Twenty-four samples of Acanthopanax leaves from different species and locations were examined using this analytical method, and their chemical profiles provided information for the chemotaxonomic investigation. The established method is simple, rapid and reliable for the quality control of Acanthopanax leaves of various species from different collections. The complete phenolic and flavonoid profiles of Acanthopanax leaves of various species have been established.