Liquid Chromatographic Method For Determination Research Articles

Greenness profile assessment of selective liquid chromatographic methods for determination of a quaternary antimigraine combination along with three of their related official impurities

Two selective, sensitive and environmentally safe LC methods were developed and validated for determination of paracetamol, caffeine, ergotamine tartrate and metoclopramide in coformulated antimigraine tablets along with p-aminophenol, p-nitrophenol and theophylline as officially specified impurities. The first is based on high-performance thin-layer chromatography (HPTLC) coupled with densitometric quantitation. Separation was achieved on HPTLC silica gel 60 F254 plates as stationary phase using ethyl acetate:aqueous ammonium hydroxide solution:glacial acetic acid (10.0:0.4:0.1, by volume) as a developing system followed by scanning of the separated bands at 210.0 nm. The subsequent method depends on HPLC with diode array detection. The LC separation was accomplished on a Scharlau C18 (250 × 4.6 mm, 5 μm) column using a mixture of 20.0 mm sodium dihydrogen phosphate, pH 3.0, adjusted with o-phosphoric acid and methanol, at a flow rate of 1.3 mL/min in a gradient elution program. The separated peaks were detected at 210.0 nm. The proposed methods have been validated and proven to meet the requirements outlined in the International Council for Harmonisation (ICH) guidelines. The greenness profile evaluation was carried out using three tools, namely, the National Environmental Method Index, the Analytical EcoScale and the Green Analytical Procedure Index tool, and a comparative study was then conducted. Successful application of the developed methods for determination of the cited quaternary mixture in Metograine tablets confirms their suitability regarding the analytical performance and ecological impact in quality control assay and impurity profiling purposes.

Rapid and Sensitive Liquid Chromatographic Method for Determination of Anastrozole in Different Polymer–Lipid Hybrid Nanoparticles

Anastrozole, an aromatase inhibitor drug, is used for the treatment of breast cancer in pre- and postmenopausal women. Anastrozole's incorporation into nanoparticulate carriers would enhance its therapeutic performance. To perceive the exact loaded amount of drug in nanocarriers, a valid analytical method is required. The reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated by using the C18 column, 150 × 4.6 mm, 5 µm particle size, in isocratic mobile phase composed of 50:50 V/V (volume/volume) acetonitrile-phosphate buffer (pH 3) flowing at a rate of 1.0 mL/min, and a diode array detector (DAD) set at λmax = 215 nm. The validation parameters such as linearity, accuracy, specificity, precision, and robustness have proven the accuracy of the method, with the relative standard deviation percentage (% RSD) values < 2. The limit of detection of the method was found equal to 0.0150 µg/mL, and the limit of quantitation was 0.0607 µg/mL. The percent recovery of sample was in the range of 98.04-99.25%. The method has the advantage of being rapid with a drug retention time of 2.767 min, specific in terms of resolution of peaks void of interference with any of the excipients, and high reproducibility. This makes it highly applicable for quality control purposes.

An Improved Liquid Chromatographic Method for Determination of Aluminium in Sodium and Potassium Chloride Brine using Spectrophotometric Detection

The quality of feed brine solution required for a modern chlor-alkali plant equipped with membrane cell is much more stringent. The electrical efficiency of the membrane is easily compromised by the presence of various anionic and cationic impurities [1]. Aluminium is one the impurity which will form alumino silicate precipitate when reacts with silicate. This will affect the membrane performance and sometimes even damages the membrane. Hence monitoring and controlling aluminium concentration is very crucial. There are many techniques and methods available for aluminium determination [2-5]. However many techniques fails to quantify aluminium concentration in trace level, especially in brine sample matrix. Chelation chromatography is one of the liquid chromatography techniques which can be used to quantify Aluminium in trace level using spectrophotometric detection [6-8]. The proposed method can be used to quantify trace level of aluminium in brine sample matrix. Aluminium forms complex with 8-hydroxy quinoline and detected using spectrophotometric detection method. This method does not require any derivatization.

Development and Validation of a Quantification Method for L-DOPA in Plants and Pharmaceutical Materials

L-DOPA is an antiparkinsonian drug and is used per oral application. A reliable liquid chromatographic method for determination of L-DOPA in leaves, flowers and seeds of Vicia faba L. grown in Turkish habitats and pharmaceutical dosage forms has been developed. The analysis has been carried out using Ace C18 (5 µm, 4.6 x 250 mm,) column, and the separation was carried out using a mobile phase consisting of 50 mM potassium dihydrogen phosphate (pH 2.3) at a flow rate of 1.2 mL min-1 with UV detection at 280 nm. The method has been validated according to the acceptance criteria of the ICH guidelines. The method demonstrated good linearity (R2 &amp;gt; 0.999) over the assayed concentration range and showed good intra-day and inter-day precision. The detection limit (LOD) and the quantification limit (LOQ) values were determined as 1.70 and 5.13 μg mL-1 for L-DOPA. This method has been successfully used for the determination of L-DOPA in pharmaceutical formulations and plant materials.

Determination of mifepristone in workplace air by ultra-performance liquid chromatography

Objective: To establish an ultra-high performance liquid chromatography method for the determination of mifepristone in the air of workplaces. Methods: From January to July 2018, glass fiber filter membrane was used to collect mifepristone in the workplace air, eluted with acetonitrile/water solution, and determined by ultra-high performance liquid chromatography-UV method. Results: The detection limit of mifepristone determination method was 0.005 μg/ml, the lower limit of quantification was 0.015 μg/ml, and the lowest detection concentration was 0.14 μg/m(3) (calculated by collecting 75 L air samples) . The concentration of mifepristone has a good linear relationship between 0.015-40.24 μg/ml, r=0.9999. The accuracy and elution efficiency of the method were calculated based on the recovery rate, ranging from 92.7% to 100.0%. The average sampling efficiency was 99.6%-100.0%. The intra-assay precision of the method was 0.92% to 5.41%, and the inter-assay precision was 0.89% to 6.25%. The sample could be stored stably for 3 days at 4°C. Conclusion: The ultra-high performance liquid chromatography method for determination of mifepristone is easy to operate, and its indicators meet the requirements of GBZ/T 201.4-2008 "Guidelines for the Establishment of Occupational Hygiene Standards Part 4: Methods for Determination of Chemical Substances in Workplace Air", which is suitable for the detection of mifepristone in the air of the workplace.

Development and Validation of Stability Indicating High-Performance Liquid Chromatographic Method for Determination of Cyproheptadine Hydrochloride, its Impurity And Degradation Product.

This work presents a sensitive, accurate and selective RP-HPLC method for simultaneous determination of cyproheptadine HCl (CPH), its impurity B (dibenzosuberone) and CPH oxidative degradation product (10,11-dihydroxy-dibenzosuberone) in bulk powder and in pharmaceutical formulation. The RP-HPLC method depends on isocratic elution using C8 column and mobile phase consisting of 0.05M KH2PO4 buffer:methanol (35:65, v/v, pH=4.5) at a flow rate of 2mL/min, and the eluant was monitored at 245nm. Good resolution was obtained with tR values of 3.05, 7.54 and 6.17min for CPH, impurity and oxidative degradate, respectively. The proposed method has been validated as per ICH guidelines using pure forms of CPH, its impurity and degradation product in pharmaceutical formulation with an accuracy of 100.48, 100.16 and 100.11, respectively. Additional spiking experiments yielded an accuracy of 100±1.6%. Repeatability and intermediate precision results indicated acceptable low <2% RSD values. Moreover, the developed method's statistical results were favorably compared to the previously reported method results regarding both accuracy and precision. The developed method can be applied for analysis of the three components in quality control laboratories.

Quantitative analysis of paeoniflorin, geniposide and glycyrrhizin in Jing-Jieh-Lian-Chyau-Tong by high performance liquid chromatography

High Performance Liquid Chromatographic methods for determination of paeoniflorin in Pasoniae Radix, geniposide in Gardenia Radix and glycyrrhizin in Glycyrrhizae Radix were developed for the quality control of traditional Chinese medicinal prescriptions Jing-Jieh-Lian-Chyau-Tong. Samples were extracted with 70% methanol by ultrasonic shaking for 30 minutes. The extract was separated by Inertsil 5 ODS-2 column with water-acetonitrile (85:15) as the mobile phase at a flow-rate of 1.0 ml/min. for paeoniflorin and geniposide, while for glycyrrhizin, it was separated by Lichrospher 100 RP-18 column with 1% acetic acid-acetonitrile (65:35) as the mobile phase at a flow-rate of 1.0 ml/min. The detecting wavelength was 254 nm with methylparaben and butylparaben used as the internal standards respectively. The calibration curves were linear in the range of 0.025-0.200 mg/ml for paeoniflorin; 0.02-0.10 mg/ml for geniposide and 0.016-0.150 mg/ml for glycyrrhizin. The recovery for each constituent from this prescription was 100.0% for paeoniflorin; 99.6% for geniposide and 99.9% for glycyrrhizin and the coefficient of variation was lower than 1.0%. The contents of these preparation constituents were not affected by treatment with concentration under reduced pressure, spray-drying or lyophilization respectively. The difference between the highest and lowest content of paeoniflorin, geniposide and glycyrrhizin were 4.2, 4.8 and 4.6-fold respectively.

Liquid chromatographic method for determination of calcium pantothenate preparations and related stability studies

This report describes a fast, simple and reliable HPLC method for the assay and quantitative determination of calcium pantothenate in commercial products. The samples were analyzed on a C18 column with the mobile phase of acetonitrile and potassium dihydrogen phosphate solution (adjusted pH = 2.5 with phosphoric acid) at a flow rate of 1.0 mL/min and UV absorbance defection at 204 nm. Ampicillin was used as an internal standard. The retention times of calcium pantothenate and ampicillin were 5.3 and 6.5 min, respectively. An equation was presented for linear relationship between peak height ratios of calcium pantothenate to ampicillin and the calcium pantothenate concentration over a range of 10-50 μg/mL (r = 0.9999). Standard addition recoveries were greater than 98.96% with twelve commercial products. The relative standard deviations were between 0.1 and 0.9% in inter-day assays, 0.1 and 0.7% in intra-day assays. The results obtained from the HPLC assay method which we developed and the microbiological assay of USP were paired at 95% confidence level. There were no significant differences between these two methods. The proposed HPLC method was a suitable substitute for microbiological method for quantitative assays of calcium pantothenate in commercial products.

Simple high-performance liquid chromatographic method for determination of Donepezil HCl in pharmaceutical formulations

Simple high-performance liquid chromatographic method for determination of Donepezil HCl in pharmaceutical formulations

Validation of High-Performance Liquid Chromatography Method for Determination of Vitamin B1 in Powder Milk

Vitamin B1 plays an important role in the co-enzymatic reactions for energy-rich compounds called ATP (Adenosine Tri Phosphate). Therefore, it should be added to various food products, for example, milk powder. One method that can be used to determine vitamin B1 is SNI number 3751: 2009, but the method is intended for wheat flour. If the method is to be used for the analysis from other samples, such as milk powder, optimization, and validation, are needed. This experiment was carried out using HPLC, C18 column, and UV detector with a wavelength of 254 nm. The mobile phase used is methanol: acetic acid: bi-distilled water = 32:1:67 (v/v/v), flow rate = 1 mL/minute, isocratic, and reverse phased technique. Method validation parameters include tests of system suitability, linearity, the limit of detection, the limit of quantitation, precision (repeatability), and accuracy. The results showed that the system suitability test was obtained relative standard deviations (% RSD) for retention time and peak area, tailing factor, resolution, separation factor was 0.297%, 1.476%, 1.113, 6.693, and 4.406 respectively. The validation test gets a correlation coefficient (R) of 0.9996, the limit of detection and limit of quantitation were 0.0122 mg/100 mL and 0.0244 mg/100 mL, respectively. The precision test obtained Horwitz's ratio of 0.27%. Accuracy test using CRM obtained % recovery of 93.79-97.77%. All these results meet the requirements of method validation, so it can be concluded that the method of SNI number 3751: 2009 is valid for the determination of vitamin B1 in milk powder and can be used for routine analysis procedure.

Forced degradation studies and stability-indicating liquid chromatography method for determination of tirofiban hydrochloride and synthetic impurities

This study aimed to develop and validate a stability-indicating liquid chromatography method for the determination of tirofiban hydrochloride and two synthetic impurities (impurity A and impurity C). The method utilizes a RP-18 column (250 mm × 4.6 mm; 5 μm) with the PDA detector for quantitation. A mixture of triethylamine 0.1% (acidified to pH 5.5 with phosphoric acid) and acetonitrile was used as the mobile phase at a flow rate of 1 mL min−1 with gradient elution. The method presented satisfactory linearity, precision, accuracy and robustness, as well as low limits of detection and quantification, which demonstrate sensitivity in the determination of tirofiban and impurities A and C. It was selective for the determination of the drug and impurities analysed, without interference of the degradation products generated under forced conditions, demonstrating the stability-indicating capacity of the proposed method. Tirofiban showed to be practically stable to oxidative (30% H2O2 for 24 h) and thermal (75 ºC for 24 h) conditions, but presented degradation to UVA light and acid hydrolysis, obeying the first order kinetics for both. In this way, it can be used as a stability-indicating method in the quality control of the raw material of tirofiban hydrochloride, as well as of the finished product. The obtained results demonstrate the importance of deepening the studies in this area, in order to guarantee the quality of commercialized pharmaceutical products.

Analytical QbD Approach for Development and Validation of RP-High Performance Liquid Chromatography Method for Determination of Tofisopam in Pure Form and Tablets

A novel, simple, optimized reversed-phase chromatography method for assay of Tofisopam in pure and tablet form is developed. The experimental trial was by Box Behnken design using the Design Expert® software 10 version. The attributes selected were peak symmetry, number of theoretical, and peak purity. The predicted data satisfied with actual experimental data. The optimized chromatographic conditions required a quaternary pump with a mobile phase of Water: Acetonitrile 25:75 v/v at 1 mL/min, oven temperature at 25oC at 310 nm using C18(250 × 4.6 mm Id, 5μm) column and PDA detector with a run time of 5 min. The method was validated for linearity, precision, accuracy, and specificity. The method produced a linear response over a concentration range of 4–24 ppm with an overall average accuracy of 99.98%. The method was robust, reproducible, and specific with respect to the retention time of tofisopam.

Isolation and Characterization of Novel Degradation Products of Eliglustat Tartrate Using 2D-NMR and HRMS: Development and Validation of Stability Indicating RP-UPLC Method for Quantification of Assay and Characterized Impurities

Eliglustat tartrate is an oral inhibitor of glucosylceramide synthase used in the therapy of Gaucher’s disease. The drug was subjected to forced degradation study using various stress parameters like acidic, basic, oxidation, photolysis and thermal conditions. The drug showed significant degradation under acidic and peroxide stress conditions, however the drug remained stable under basic, neutral, thermal and photolytic stress conditions. The present research reports the characterization of degradation products of Eliglustat by liquid chromatography-tandem mass spectrometry(HRMS) and 2D-NMR techniques along with the development and validation of stability indicating ultra performance liquid chromatographic method for determination and quantification of Eliglustat drug in presence of characterized impurities. Two novel degradation products (DP1 & DP-2) were found under stress conditions. These degradation products were isolated using preparative high performance liquid chromatography. The structure of the DP-1 was identified as the cis- diastereoisomer of Eliglustat and DP-2 as the N-Oxide impurity of Eliglustat which were not reported in any of the literature. The chromatographic method was developed on UPLC to achieve separation of degradation products and Eliglustat using Acquity; BEH; C-18; 100 x 2.1 mm; 1.7 μm column within a short run time of 8.0 minutes. The method was validated for specificity, precision, linearity and accuracy. The quantitation limits obtained were in the range of 0.3-3.0 μg ml-1 for the characterized impurities. This method is compatible to LCMS analysis which enables to identify the unknown impurities formed in the process.

Development and Validation of an New High Performance Liquid Chromatography Method for Determination of Apixaban Isomers

The purpose of this research study is to develop a novel, simple, precise, accurate and economical method for determination of Apixaban API isomers. Apixaban API has three isomer as ortho, meta and para. In reverse phase and normal phase chromatography it was very difficult to separate these isomer; hence new chiral technique was adopted. This chromatographic method was developed on chiralpak IA column (250×4.6×5µm) with isocritic technique. The detection of isomeric impurities were observed at wavelength 290nm. This analytical method was validated as per ICH guideline and regression analysis showed R value (correlation coefficient) > 0.999 for Apixaban API and its isomeric impurities. A solution of Apixaban in dichloromethane was found stable up to 48 hrs. The degradation study was done within the given guidelines prescribed by ICH. The method is validated for Linearity, Accuracy and Precision.

Development and Validation of Reverse-Phase High-Performance Liquid Chromatographic Method for Determination of Resveratrol in Human and Rat Plasma for Preclinical and Clinical Studies

Development and Validation of Reverse-Phase High-Performance Liquid Chromatographic Method for Determination of Resveratrol in Human and Rat Plasma for Preclinical and Clinical Studies

Rapid resolution liquid chromatography method for determination of malathion in pesticide formulation

This paper presents a new, simple, precise, and accurate rapid resolution liquid chromatography (RRLC) method for determination of an active ingredient malathion in the pesticide formulation. The analysis was performed on a Poroshell EC 120-C18 (50 mm × 3 mm, 2.7 μm) analytical column using isocratic elution with a mobile phase consisted of acetonitrile–water (50:50, v/v), a flow rate of 1 mL/min, a constant column temperature at 25 °C, and ultraviolet-diode array detection (UV-DAD) at 220 nm. The specificity, selectivity, linearity, precision, and accuracy were tested for the method validation according to the Collaborative International Pesticides Analytical Council (CIPAC) and Directorate General Health and Consumer Protection (SANCO) guidelines and all tested parameters were found within acceptance criteria. The obtained results indicated that the proposed method can be used for routine analysis of the active ingredient malathion in the pesticide formulation “Etiol tecni” following the CIPAC and SANCO rules. The run time of analysis under the stipulated chromatographic conditions was about 2.5 min, meaning that the proposed method requires a small volume (&lt; 1.5 mL) of the organic solvent (acetonitrile) making it cost-effective.

1541. A Novel and Fast Liquid Chromatography Method for Determination of Fluoroquinolones in Human Plasma

BackgroundFluoroquinolones (FQs) are frequently used antimicrobial agents. Considering that the FQs exhibit concentration-dependent bactericidal activity, concentrations of FQs in the biological fluids must be monitored to ensure treatment success. The literature search revealed that there is no method for the determination of levofloxacin (LEV), ciprofloxacin (CIP), moxifloxacin (MOX), and gemifloxacin (GEM) in plasma up to date. Consequently, the aim of this study was to develop and validate a new high-performance liquid chromatography (HPLC) method for determination of these FQs in plasma and evaluate effects of concomitant drugs on plasma FQ concentrations of patients.MethodsBlank plasma samples spiked with FQs were employed for method validation studies. Validation studies were conducted in accordance with the recommendations of the US FDA. In order to demonstrate feasibility of method, 5 patients with polypharmacy, receiving orally CIP, LEV, or MOX as part of their treatment were included in the study. Blood samples were collected at two different times, just before and 2 hours after the second drug administration.ResultsThe separation of FQs was accomplished within 7.5 minutes. The method was linear in the range of 0.1–10 µg/mL with the correlation coefficient >0.99. The RSD at four concentration levels (0.1, 0.3, 4, and 8 µg/mL) was less than 7% with accuracy in the range of 91.8–111.9%.The method was applied to the determination of CIP, LEV, and MOX levels in plasma samples of 5 patients with polypharmacy. Determined CIP and LEV levels were in accordance with literature. On the other hand, MOX concentration 2 hours after administration in plasma of one patient was found to be 6.1 ± 0.1 µg/mL which was higher than previously reported maximum plasma concentration of MOX (4.5 µg/mL). The patient had hypoalbuminemia and MOX is approximately 50% bound to serum proteins. Due to low level of albumin, the level of free MOX in plasma may be increased.ConclusionA simple, fast, and reliable HPLC method was developed and validated for the determination of LEV, CIP, MOX, and GEM in plasma. It is suitable therapeutic drug monitoring of these FQs and can be applied to other pharmacokinetic and toxicological studies.Disclosures All authors: No reported disclosures.

Determination of retinoic acid in human serum and plasma by high-performance liquid chromatography

All-trans retinoic acid (ATRA) is the main derivative of retinol. It is formed in the intestinal mucosa from retinaldehyde. The aim was to establish a high-performance liquid chromatography method for determination of ATRA in human serum and plasma. Stability of ATRA in serum stored under various conditions was tested as well. ATRA was extracted from serum (plasma) after deproteination with acetonitrile by hexan:ethylacetate mixture (50:50). The extract was then evaporated under nitrogen at room temperature and dissolved in methanol. ATRA was detected after separation on C18 column at 355 nm. This method uses acitretin as internal standard. Limit of detection was 0.21 µg/dm3 and limit of quantification 0.70 µg/dm3. The method is linear for ATRA concentration 0.78–25 µg/dm3. Based on our results, ATRA has the same concentration in serum and in plasma. Repeatability of the method measured within run is given by variation coefficient 4.31% for serum and 1.86% for plasma. Reproducibility of the method measured between runs is given by variation coefficient 9.55% for serum and 8.00% for plasma. The worst storage conditions were light and room temperature, ATRA decreased by 0.69 µg/dm3, i.e., by 39%. Simultaneously, with ATRA, retinol is measured as well. We have introduced a fast, simple, and reliable method for determination of ATRA in human serum and plasma.

Liquid Chromatographic Separation and Thermodynamic Investigation of Ethyl Nipecotate Enantiomers on Immobilized Amylose-Based Chiral Stationary Phase.

Ethyl nipecotate enantiomers are widely used as chiral building blocks in the synthesis of drug substances. An efficient and economic chiral high-performance liquid chromatographic method for determination of enantiomeric purity of ethyl nipecotate is developed and validated. Chiral separation was achieved on immobilized amylose-based stationary phase using a mixture of n-hexane: ethanol: diethylamine (80:20:0.1, v/v/v) as mobile phase. Resolution between R-(-)-ethyl nipecotate (REN) and S-(+)-ethyl nipecotate (SEN) peaks was found to be 3.59. The detector response of SEN exhibited an excellent linearity over the concentration range of 4.5-120μgmL-1. The limit of detection and limit of quantification for SEN were 0.016 and 0.045μg and REN were 0.015 and 0.043μg, respectively. The influence of column oven temperatures on chiral retention and separation was studied in the range of 25°C to 50°C; the thermodynamic parameters ΔH°, ΔS° and ΔG° were evaluated from van't Hoff plots and used to explain the strength of interaction between analyte and stationary phase.

High Performance Liquid Chromatographic Method for Determination of Tadalafil in Tablets and Wastewater

High Performance Liquid Chromatographic Method for Determination of Tadalafil in Tablets and Wastewater