Simple High-performance Liquid Chromatographic Method Research Articles

Development of an HPLC Method with an ODS Column to Determine Low Levels of Aspartame Diastereomers in Aspartame.

α-L-Aspartyl-D-phenylalanine methyl ester (L, D-APM) and α-D-aspartyl-L-phenylalanine methyl ester (D, L-APM) are diastereomers of aspartame (N-L-α-Aspartyl-L-phenylalanine-1-methyl ester, L, L-APM). The Joint FAO/WHO Expert Committee on Food Additives has set 0.04 wt% as the maximum permitted level of the sum of L, D-APM and D, L-APM in commercially available L, L-APM. In this study, we developed and validated a simple high-performance liquid chromatography (HPLC) method using an ODS column to determine L, D-APM and D, L-APM in L, L-APM. The limits of detection and quantification, respectively, of L, D-APM and D, L-APM were found to be 0.0012 wt% and 0.004 wt%. This method gave excellent accuracy, repeatability, and reproducibility in a recovery test performed on five different days. Moreover, the method was successfully applied to the determination of these diastereomers in commercial L, L-APM samples. Thus, the developed method is a simple, useful, and practical tool for determining L, D-APM and D, L-APM levels in L, L-APM.

Development of a simple HPLC–UV method for the determination of the hepatitis C virus inhibitor simeprevir in human plasma

A simple high-performance liquid chromatography method for the determination of the hepatitis C virus protease inhibitor simeprevir in human plasma was developed and validated. The method involved a rapid and simple solid-phase extraction of simeprevir using Oasis HLB 1cc cartridges, isocratic reversed-phase liquid chromatography on an XTerra RP18 (150mm×4.6mm, 3.5μm) column, and ultraviolet detection at 225nm. The mobile phase consisted of phosphate buffer (pH 6, 52.5mM) and acetonitrile (30:70, v/v). This assay proved to be sensitive (lower limit of quantification of 0.05μg/mL), linear (correlation coefficients ≥0.99), specific (no interference with various potentially co-administrated drugs), reproducible (both intra-day and inter-day coefficients of variation ≤8.3%), and accurate (deviations ranged from −8.0 to 1.2% and from −3.3 to 6.0% for intra-day and inter-day analysis, respectively). The method was applied to therapeutic monitoring of patients undergoing simeprevir treatment for hepatitis C and proved to be robust and reliable. Thus, this method provides a simple, sensitive, precise and reproducible assay for dosing simeprevir that can be readily adaptable to routine use by clinical laboratories with standard equipment.

A selective and sensitive pre-column derivatization HPLC method for the trace analysis of genotoxic impurity hydroxylamine in active pharmaceutical ingredients

A simple and robust high-performance liquid chromatographic (HPLC) method for the trace analysis of genotoxic impurity hydroxylamine in Vorinostat and Zileuton, two active pharmaceutical ingredients, is described.

Development of simple and sensitive HPLC method for determination of glyphosate residues in soybean

The purpose of this study was to develop a simple and sensitive high performance liquid chromatography (HPLC) method for determination of glyphosate (GP) residues in soybean grains. From soybean matrix, glyphosate was extracted with a mixture of water and methanol (4:1, v/v) from soybean samples followed by protein precipitation with equal volume of methanol. No preconcentration and further clean up of the sample were required. Pre-column derivatization was carried out with excess amount of 9- fluorenylmethyl chloroformate (FMOC-Cl) in the presence of borate buffer. The gradient program developed in this method was successfully applied to a reverse phase HPLC system with a C18 column (ACE 5 μm 4.6 x 250 mm), and eluted with a mobile phase consisting of 50 mM phosphate buffer, pH 2.5, and acetonitrile at the flow rate of 0.8 ml/min and fluorescence detection. Parameters and conditions affecting extraction, derivatization reaction and chromatographic separation were systematically examined. Linearity of the method ranged from 0.005 - 1.0 μg/ml. The correlation coefficient (r2) of calibration curve for glyphosate in soybean sample was found to be 0.99929. The limit of detection (LOD) and limit of quantitation (LOQ) values were determined to be 0.125 mg/kg and 0.25 mg/kg, respectively. Average recovery was 95.2%. Repeatability and intermediate precision calculated on the basis of peak area were excellent and showed relative standard deviation ranged from 0.15 - 1.29% and 1.15 - 3.87%, respectively. The developed method has been successfully applied for determination of glyphosate residues in soybean grains obtained from Thailand and Nepal. Soybean samples (53) from two different lots were analyzed and glyphosate residues ranged from 0.23 mg/kg to 5.06 mg/kg. Almost 50% soybean samples contained nearly consistent residue levels in both lots but in remaining samples there was a significant variation of glyphosate levels between two lots. Relatively higher residues were detected in samples from Thailand (0.27-5.06 mg/kg) compared to Nepal (0.23-0.99 mg/kg). The results suggest that the proposed method can be used to determine glyphosate residues in foods derived from soybean and other crops such as corn, cotton, wheat, etc. where glyphosate is widely applied to these crops.

A new validated HPLC method for the determination of sulforaphane: application to study pharmacokinetics of sulforaphane in rats

A simple, accurate and reproducible high-performance liquid chromatography (HPLC) method has been developed and validated for the quantification of sulforaphane (SF) in rat plasma. The method involves a simple liquid-liquid extraction procedure to extract both SF and 7-hyrdoxycoumarin, the internal standard. The chromatographic analysis was achieved on a Shimadzu LC 20A HPLC system equipped with a Zorbax Eclipse XDB C18 column and an isocratic mobile phase consisting of 10 mm KH2 PO4 (pH 4.5) and acetonitrile HPLC grade (40:60, v/v) run at a flow rate of 1 mL/min for 10 min. The UV detection wavelength was set at 202 nm. The method exhibited good linearity (R(2) > 0.999) over the assayed concentration range (0.05-2 μg/mL) and demonstrated good intra- and inter-day precision and accuracy (relative standard deviations and the deviation from predicted values were <15%). This method was also successfully applied for studying the pharmacokinetics of SF in spontaneously hypertensive rats following single oral dietary doses of SF. The pharmacokinetics of SF show linear behavior at the dose range investigated in this study. Copyright © 2015 John Wiley & Sons, Ltd.

Rapid determination of parabens in seafood sauces by high-performance liquid chromatography: A practical comparison of core-shell particles and sub-2 μm fully porous particles.

In this work, the chromatographic performance of superficially porous particles (Halo core-shell C18 column, 50 mm × 2.1 mm, 2.7 μm) was compared with that of sub-2 μm fully porous particles (Acquity BEH C18 , 50 mm × 2.1 mm, 1.7 μm). Four parabens, methylparaben, ethylparaben, propylparaben, and butylparaben, were used as representative compounds for calculating the plate heights in a wide flow rate range and analyzed on the basis of the Van Deemter and Knox equations. Theoretical Poppe plots were constructed for each column to compare their kinetic performance. Both phases gave similar minimum plate heights when using nonreduced coordinates. Meanwhile, the flat C-term of the core-shell column provided the possibilities for applying high flow rates without significant loss in efficiency. The low backpressure of core-shell particles allowed this kind of column, especially compatible with conventional high-performance liquid chromatography systems. Based on these factors, a simple high-performance liquid chromatography method was established and validated for the determination of parabens in various seafood sauces using the Halo core-shell C18 column for separation.

Comparative fingerprint analyses of extracts from the root bark of wild Hippocratea excelsa and "Cancerina" by high-performance liquid chromatography.

A simple high-performance liquid chromatography method was developed for the identification and comparison of quinone-methide triterpenes in wild Hippocratea excelsa and "cancerina" to establish the chromatographic profile of these compounds in root bark. The essential chromatographic conditions for this method are based on a gradient system with a reversed-phase column (C18 ) using proportions of water, methanol, and tetrahydrofuran as mobile phases to correctly separate the signals at 254 and 420 nm and compare the signals to those reported in the literature. The chromatograms exhibit good resolution and precision. Statistical analysis showed that the chromatographic profiles of wild H. excelsa and cancerina do not exhibit significant differences (p≥0.05) in their area proportions or relative retention times. The method developed in this study is suitable for the identification of the major chemotaxonomic markers of the Celastraceae family and can be used for quality control of this herbal root bark, which has uses today in Mexican folk medicine.

Simple and Sensitive Detection of Hydrazine in Industrial Wastewater Using High-Performance Liquid Chromatography with Fluorescence Detector after Anthracene-2,3-dicarbaldehyde Derivatization

A simple and sensitive high-performance liquid chromatographic (HPLC) method was developed to determine the amount of hydrazine in industrial wastewater after derivatization with anthracene-2,3-dicarbaldehyde. The best reaction conditions (a reagent dosage of 200 mg/L, a pH of 11, and reaction for 1 min at room temperature) were established by the variation of parameters. The formed derivative was injected into the HPLC without further extraction or purification procedures. Under the established conditions, the limits of detection and the limits of quantification were 0.02 µg/L and 0.05 µg/L, respectively, and the inter-day relative standard deviation was less than 9% at concentrations of 0.1 and 0.5 µg/L. The calibration curve showed good linearity with r2 = 0.9999. The method was successfully applied to the determination of hydrazine in various kinds of industrial wastewater.

A Simple High-Performance Liquid Chromatographic Method for the Simultaneous Determination of Monoamine Neurotransmitters and Relative Metabolites with Application in Mouse Brain Tissue

As a means to evaluate the role of organic solute carriers in CNS homeostasis, a simple and rapid high-performance liquid chromatographic method utilizing ultraviolet and electrochemical detectors was developed and validated for the simultaneous determination of the neurotransmitters dopamine, norepinephrine, and serotonin and their metabolites in mouse brain homogenate. For analyte separation, the method utilized a C18 column with a mobile phase of 75 mM sodium dihydrogen phosphate (monohydrate), 1.7 mM 1-octanesulfonic acid sodium salt, 25 µM EDTA, 10% acetonitrile, and 1% triethylamine with a final pH of 3.0 adjusted using phosphoric acid. The method was linear for dopamine, norepinephrine, serotonin, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid from 20–1000 ng/mL (r ≥ 0.993) with detection limits of 5 ng/mL for dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid, and of 20 ng/mL for serotonin and 5-hydroxyindoleacetic acid. The method was linear over the range of 10–200 µg/ml for quinolinic acid, xanthurenic acid, and nicotinic acid (r ≥ 0.993) with detection limits of 1 µg/mL for all components. At a flow rate of 1 mL/min, the analytical run time was less than 10 min. The method was successfully applied to the quantitation of these compounds in mouse whole brain homogenates.

Simple High-Performance Liquid Chromatography Method for the Simultaneous Analysis of Aflatoxins, Ochratoxin A, and Zearalenone in Dried and Ground Red Pepper

Aflatoxins B1, B2, G1, G2 (AFB1, AFB2, AFG1, AFG2), ochratoxin A (OTA), and zearalenone (ZEA) in dried and ground red pepper (Capsicum annuum) were simultaneously analyzed with high-performance liquid chromatography coupled to fluorescence detection after post–column derivatization. The analytical method was validated for specificity, selectivity, linearity, limits of detection and quantification, recovery, precision, and measurement of uncertainty. The limits of detection and quantification were 0.10 and 0.25 μg/kg for AFB1, 0.04 and 0.06 μg/kg for AFB2, 0.14 and 0.50 μg/kg for AFG1, 0.05 and 0.10 μg/kg for AFG2, 0.12 and 0.45 μg/kg for OTA, and 4.00 and 13.25 μg/kg for ZEA, respectively. The average recoveries ranged from 80.4 to 98.5% for different concentrations of AFB1, AFB2, AFG1, AFG2, OTA, and ZEA in spiked samples. The measurement uncertainties were 0.64 to 1.62 μg/kg for AFB1, 0.24 to 0.45 μg/kg for AFB2, 0.79 to 2.19 μg/kg for AFG1, 0.32 to 0.61 μg/kg for AFG2, 0.81 to 2.31 μg/kg for OTA, and 8.48 to 26.25 μg/kg for ZEA. This method was successfully applied for the simultaneous determination of mycotoxins for 78 red peppers collected from Korean and Indian markets. Aflatoxins (sum of AFB1, AFB2, AFG1, and AFG2) were detected in 2% of nonpacked samples (n = 23) and 43% of packed samples (n = 55), at levels of 0.04 to 38.03 μg/kg. OTA was detected in 4% of nonpacked samples and 48% of packed samples, at levels of 0.15 to 56.30 μg/kg. ZEA was not detected in any samples. These findings indicate that the analytical method described here is suitable for the routine determination of the amounts of AFs, OTA, and ZEA in dried and ground red pepper.

A novel reverse phase high-performance liquid chromatography (RP-HPLC–UV) method for the estimation of alosetron HCl and its validation

A novel simple, sensitive, and rapid isocratic reverse phase high-performance liquid chromatographic (RP-HPLC) method was developed for the quantitative determination of alosetron HCl, a 5-HT3 antagonist used in the treatment of severe irritable bowel syndrome (IBS) in females. The optimized chromatographic separation was achieved using a stationary phase of Phenomenex® kromasil C-18 (250 mm × 4.6 mm; 5 μm particle size) column, and mobile phase of 0.025 M disodium hydrogen orthophosphate buffer, pH adjusted to 3.0 with orthophosphoric acid, and acetonitrile in the ratio of 65:35 (v/v) with a flow rate of 1 mL min−1. The UV detection was carried out at 217 nm. The developed method provided linear responses within the concentration range 100–2000 ng mL−1, and regression analysis showed a correlation coefficient value (r2) of 0.997. The HPLC method was validated as per International Conference on Harmonization (ICH) guidelines with respect to selectivity, precision, linearity, and robustness. Limit of detec...

Development and validation of simple, rapid and sensitive LC-PDA ultraviolet method for quantification of Nebivolol in rat plasma and its application to pharmacokinetic studies

A simple, sensitive and rapid high-performance liquid chromatography method with ultraviolet (UV) detector was developed and validated for the analysis of Nebivolol (NBL) in rat plasma. The plasma sample, spiked with raloxifene hydrochloride as an internal standard (IS), was subjected to single step protein precipitation method prior to analysis. Chromatographic separation was achieved on the Agilent C8 (150 mm × 4.6 mm, 5 μm) column and monitored at a wavelength of 280 nm. Elution was carried out, in an isocratic mode, using a mobile phase consisting of acetonitrile and potassium di-hydrogen orthophosphate buffer (pH 3.5 ± 0.1) in the ratio of 37:63 v/v. Retention times of IS and NBL were 5.1 ± 0.10 min and 8.01 ± 0.12 min, respectively. No interference was observed from plasma components in the analysis of NBL and IS. Calibration curve was linear over the range of 125–3000 ng/mL (r2 = 0.999). NBL was found to be stable under various processing and storage conditions. The developed method was applied in ...

Determination of Flurbiprofen in Human Plasma by High-Performance Liquid Chromatography.

A simple high-performance liquid chromatography method has been developed for determination of flurbiprofen in human plasma. The method was validated on an Ace C18 column using UV detection. The mobile phase was acetonitrile-0.05 M potassium dihydrogen phosphate solution (60:40, v/v) adjusted to pH 3.5 with phosphoric acid. The calibration curve was linear between the concentration range of 0.10-5.0 μg/mL. Intra- and inter-day precision values for flurbiprofen in plasma were <4.47, and accuracy (relative error) was better than 3.67%. The extraction recoveries of flurbiprofen from human plasma were between 93.0 and 98.9%. The limits of detection and quantification of flurbiprofen were 0.03 and 0.10 μg/mL, respectively. In addition, this assay was applied to determine the pharmacokinetic parameters of flurbiprofen in six healthy Turkish volunteers who had been given 100 mg flurbiprofen.

Determining Terbinafine in Plasma and Saline Using HPLC

A simple, sensitive, and accurate high-performance liquid chromatography (HPLC) method for the determination of terbinafine concentrations in small-volume plasma and saline has been developed. Following a liquid extraction using hexane, samples were separated by reversed-phase HPLC on a Symmetry Shield RP18 (5 µm) 4.6 mm × 100 mm column and quantified using ultraviolet detection at 224 nm. The mobile phase was a mixture of water, phosphoric acid, and triethylamine (pH 3.0), with acetonitrile (65:35, v/v), at a flow rate of 1.1 mL/min. The standard curve ranged from 5 to 1500 ng/mL for parrot plasma and 1 to 25 µg/mL for 0.9% saline. Intra- and inter-assay variability for terbinafine was less than 10% for both matrices, and the average recovery was greater than 90%. This method has been developed in parrot plasma and should be applicable to other species, making it useful to those investigators dealing with small sample volumes, particularly when conducting pharmacokinetic studies that require multiple sampling from the same animal. This method would also be valuable to aquariums that need to determine terbinafine concentrations in salt water.

A Modified and Cost-Effective HPLC Method for Determination of Plasma Concentrations of Rifampicin in Pulmonary TB Patients

Objective: To evaluate the pharmacokinetics of standard doses of rifampicin (RMP) in fixed dose combination in pulmonarytuberculosis patients by a modified high performance liquid chromatography (HPLC) methodMaterials and Methods: This descriptive study was conducted after approval from Ethical Committee, Army Medical CollegeRawalpindi and was funded in part by National University of Sciences and Technology (NUST), Islamabad. Twenty adultpatients with newly diagnosed pulmonary TB consented to participate in the study. RMP plasma concentrations were assayedby a simple and sensitive HPLC method in the initial phase of pulmonary tuberculosis treatment. The method was modifiedto use naproxen as an internal standard and validated according to International Conference on Hormonization (ICH) guidelines.Results: The calibration curve of rifampicin was linear within the range of 0.781–50 μg/ml. Both intra-day and inter-dayvariability and accuracy demonstrated good reproducibility at all quality control levels. The developed method was found tobe simple, precise and accurate for estimation of rifampicin in plasma. The pharmacokinetic parameters of RMP showed markedinter-individual differences and sub-therapeutic levels.Conclusion: Evaluation of pharmacokinetics of standard doses of rifampicin in fixed dose combination in pulmonary tuberculosispatients by a modified high performance liquid chromatography (HPLC) method is precise, accurate and cost-effective. It maybe used for monitoring plasma RMP levels in TB patients who are slow to respond or are non-responders and have less availabilityof resources.Keywords: Tuberculosis,

Determination of Metalaxyl in Potatoes and Soil by Dispersive Solid-Phase Extraction and High-Performance Liquid Chromatography

A simple and sensitive high-performance liquid chromatography method with photodiode array detection combined with dispersive solid-phase extraction was developed to quantify metalaxyl residues in potatoes and soil. The limits of detection and quantification of metalaxyl were 0.0015 and 0.005 mg kg−1. The recoveries of metalaxyl extracted from potato, potato foliage, and soil ranged from 81.53–95.50%, 90.12–94.72%, 93.16–103.0%, respectively, with relative standard deviations less than 9.74%. The method was applied to determine metalaxyl in potatoes and soil; the results showed that the dissipation rates were described by first-order kinetics. The half-lives in potato foliage and soil were 0.75 days and 5.87 days to 8.45 days, respectively. At harvest, the residue of metalaxyl in potatoes was less than 0.05 mg kg−1 following the recommended dosage at an interval of seven days from the final application. These results may be used to help establish appropriate application frequencies and harvest intervals for metalaxyl on potatoes.

Effects of unbound mycophenolic acid on inosine monophosphate dehydrogenase inhibition in pediatric kidney transplant patients.

Mycophenolic acid (MPA) is a key immunosuppressive drug that acts through inhibition of inosine monophosphate dehydrogenase (IMPDH). MPA is commonly measured, as part of therapeutic drug monitoring, as the total concentration in plasma. However, it has been postulated that the free (unbound) fraction of MPA (fMPA) is responsible for the immunosuppressive effects. In this study, a sensitive low volume high-performance liquid chromatography (HPLC) assay was developed to measure fMPA concentrations to explore the relationship between fMPA and IMPDH activity. To obtain fMPA concentrations, plasma samples were filtrated using Centrifree ultrafiltration devices. The ultrafiltrate was analyzed by HPLC using a Kinetex C18 column (2.6 μm, 3.0 × 75 mm). fMPA concentrations were compared with the total MPA concentrations available in 28 pediatric kidney transplant patients at 3 consecutive occasions after transplantation. The relationship between fMPA and IMPDH activity was analyzed using an Emax model. The HPLC assay, using 25 μL of the ultrafiltrates, was validated over a range from 2.5 to 1000 μL with good accuracy, precision, and reproducibility. Total and free MPA concentrations were well correlated (R = 0.85, P < 0.0001), although large intraindividual and interindividual variability in the bound MPA fractions was observed. The overall relationship between fMPA concentrations and IMPDH inhibition using the Emax model was comparable with that of total MPA, as previously reported. The model estimated EC50 value (164.5 μL) is in good agreement with reported in vitro EC50 values. This study provides a simple HPLC method for the measurement of fMPA and a pharmacologically reasonable EC50 estimate. The good correlation between the total and free MPA concentrations suggests that routine measurement of fMPA to characterize mycophenolate pharmacokinetic and pharmacodynamic does not seem warranted, although the large variability in the bound fractions of MPA warrants further study.

RP-HPLC-DAD method for the identification of two potential antioxidant agents namely verminoside and 1-O-(E)-caffeoyl-β-gentiobiose from Spathodea campanulata leaves

A simple and reliable high-performance liquid chromatographic method was successfully developed for the study of fingerprint chromatograms of extract and fractions from the leaves of Spathodea campanulata (SC) using verminoside (1) and 1-O-(E)-caffeoyl-β-gentiobiose (2) as marker compounds. Antioxidant activity of SC was determined by using free radical of 2,2-diphenyl-1-picryl-hydrazyl-hydrate as an experimental model. The docking study of selected target, tyrosinase and ligands (ascorbic acid, compounds 1 and 2) was performed through Autodock Vina v0.8. Fingerprints of methanol, chloroform, ethylacetate, n-butanol and water extracts could resolve 13, 11, 22, 16 and 5 peaks, respectively. Extract, fractions and compounds 1 and 2 previously isolated from SC displayed remarkable antioxidant activity with radical-scavenging activity ranging from 2.5 to 6.7 μg/mL. In silico study identified compounds 1 and 2 as potential inhibitors of tyrosinase correlating with the observed antioxidant activity in vitro.

Stability Indicating RP-HPLC Studies for the Estimation of Irbesartan and Amlodipine Besylate in Pharmaceutical Formulations and Identification and Characterization of Degradants Using LC-MS

A simple, specific, accurate, and stability-indicating reversed-phase high-performance liquid chromatography method was developed for simultaneous estimation of irbesartan and amlodipine besylate in pharmaceutical formulations. The chromatographic separation was achieved on a Zorbax CN column using a mixture of 1 mM potassium dihydrogen phosphate (pH 3.0) and acetonitrile (70:30, v/v) as the mobile phase at a flow rate of 0.9 mL/min. Detection was carried out at 240 nm. The calibration curves were linear (R 2 ≥ 0.99) over a concentration range of 6–42 µg/mL for irbesartan and 2–14 µg/mL for amlodipine besylate. The retention times of irbesartan and amlodipine besylate were 9.6 and 7.6 min, respectively. For stability studies, irbesartan and amlodipine besylate stock solutions were subjected to acid, alkali hydrolysis, chemical oxidation, and dry heat degradation. The degraded product peaks were well resolved from the pure drug peak with significant difference in their retention times and resolution. To elucidate structures of degradation products, the LC-MS method was used. The proposed method was successfully validated in accordance to the ICH guidelines acceptance criteria.

Comparative assessment of extraction methods and quantitative estimation of luteolin in the leaves of Vitex negundo Linn. by HPLC

ObjectiveTo find out the ideal organic solvent and extraction technique for the isolation of luteolin from the leaves of Vitex negundo Linn. (V. negundo) by quantitative estimation of luteolin through high performance liquid chromatography (HPLC) method. MethodsThe leaves of V. negundo were identified by a botanist, cleaned, dried under shade and powdered. Maceration, reflux, Soxhlet and ultrasound assisted extraction techniques were used for the extraction of luteolin from the leaves by using four different solvents of varying polarity such as methanol, ethanol, chloroform, and dichloromethane. A simple HPLC method was used to determine the quantity of luteolin in each sample extract. ResultsThe calibration plot of standard luteolin showed a linear relationship in the concentration range of 100-500 μg/mL with a correlation coefficient, r2 of 0.998. The methanolic extract was found to contain highest amount of luteolin and among various techniques employed for extraction and isolation of luteolin, reflux technique was observed to be the most efficient. ConclusionBased on the HPLC results, it can be concluded that reflux technique using methanol is better than the other extraction techniques and should be preferred for the extraction and isolation of luteolin from V. negundo leaves extract in research labs or industries.