Improved HPLC Method Research Articles

Simultaneous determination of biogenic amines and estrogens in foodstuff by an improved HPLC method combining with fluorescence labeling

Biogenic amines (BA) and estrogens (ES) have attracted increasing attentions due to their significance on food safety and food quality. In this study, a novel HPLC-FLD method using ethyl-acridine-sulfonyl chloride (EAC) as fluorescence labeling reagent has been developed for simultaneous determination of BA and ES in food samples. The labeling conditions including labeling time, pH of borate buffers and amount of EAC were optimized. The simultaneous labeling of BA and ES can be finished in as little as 6 min. This method without complex pre-treatment offered the low LOD of 0.27–0.69 ng/mL. It was applied to analyze several food samples (beer, cheese, fish, sausage and shrimp), and showed excellent applicability.

A Simple and Improved HPLC Method for the Analysis of Dithianon in Red Pepper with Tandem Mass Spectrometry Confirmation

A simple and rapid method was developed for the analysis of dithianon in red pepper using HPLC-UV. Sample extraction was carried out using acidic acetonitrile as a solvent and sodium chloride as a salting out agent. The extract was then purified through solid phase extraction procedure. The method was validated using standard calibration. A linear range of standards were determined with excellent correlation coefficients (r2) >0.999. Limits of detection and quantification were 0.01 and 0.03 mg/kg, respectively. Recovery was assessed by spiking blank red pepper samples at two different concentrations (0.3 and 1.5 mg/kg) with three replicates. A consistent recovery was determined (72.2 and 79.1 %) according to SANCO guidelines for repeatability, and based on the relative standard deviation which was <4 within 3 days (intra- and inter-days) of experiment. The method was applied to field samples to determine the dissipation and pre-harvest residue interval (PHRL). From the PHRL curve, it can be predicted that if the residual amount of dithianon is below 8.5 mg/kg at 10 days or below 5.51 mg/kg at 7 days before harvest, then the residue of dithianon will be below the MRL during the harvest time. The levels of residues in field samples were confirmed using LC-MS/MS in negative electron spray ionization mode.

An Improved HPLC Method with the Aid of a Chemometric Protocol: Simultaneous Determination of Atorvastatin and Its Metabolites in Plasma

The aim of the present study was to optimize a chromatographic method for the analysis of atorvastatin (acid and lactone forms), ortho- and para-hydroxyatorvastatin by using an experimental design approach. Optimization experiments were conducted through a process of screening and optimization. The purpose of a screening design is to identify the factors that have significant effects on the selected chromatographic responses, and for this purpose a full 23 factorial design was used. The location of the true optimum was established by applying Derringer’s desirability function, which provides simultaneously optimization of all seven responses. The ranges of the independent variables used for the optimization were content of acetonitrile in mobile phase (60–70%), temperature of column (30–40 °C) and flow rate (0.8–1.2 mL min−1). The influences of these independent variables were evaluated for the output responses: retention time of first peak (p-hydroxyatorvastatin) and of last peak (atorvastatin, lactone form), symmetries of all four peaks and relative retention time of p-hydroxyatorvastatin. The primary goal of this investigation was establishing a new simple and sensitive method that could be used in analysis of biological samples. The method was validated and successfully applied for determination of atorvastatin (acid and lactone forms) and its metabolites in plasma.

HPLC法によるHDL一コレステロールの定量: 超遠心法, 沈澱法, 直接法との比較

HPLC法によるHDL一コレステロールの定量: 超遠心法, 沈澱法, 直接法との比較

レムナント様リボ蛋白 (RLP) 分画試薬のLDL, HDLとの結合能に関するHPLC法を用いた検討

レムナント様リボ蛋白 (RLP) 分画試薬のLDL, HDLとの結合能に関するHPLC法を用いた検討

An improved high performance liquid chromatography-fluorescence detection method for the analysis of major phenolic compounds in cigarette smoke and smokeless tobacco products

An improved HPLC method has been developed for the determination of major phenolic compounds in cigarette smoke. A novel reversed phase column with a pentafluorophenylpropyl (PFP) ligand in the stationary phase was chosen to separate the positional isomers (p-, m-, and o-cresols). Methanol instead of acetonitrile was used as the organic mobile phase component to improve the separation of the isomers and cope with the crisis of global acetonitrile shortage in 2009. A shorter analytical column with smaller particle size was used to further increase separation efficiency and reduces solvent consumption. These improvements have led to a new HPLC method that is simpler and faster than the GC–MS method and more sensitive, selective and efficient than the widely used traditional HPLC method. The limit of detection (LOD) and limit of quantification (LOQ) of this method are at the ng/mL level for most of the phenols with good linearity (R2≥0.999) and precision (RSD<15%). The method has been validated using reference tobacco products. The mainstream and sidestream cigarette smoke yields of phenolic compounds obtained by this method are comparable to those obtained by traditional HPLC method with the advantage that p-, m-, and o-cresols can be determined and reported separately by the new method. The method can also be applied for analysis of phenols in smokeless tobacco product.

Quantification of dabsylated di- and tri-peptides in fermented milk

An improved HPLC method using pre-column dabsyl chloride derivatisation for the separation and quantification of antihypertensive di- and tri-peptides in fermented milk products was established. The dabsylated peptides Val-Pro-Pro (VPP), Ile-Pro-Pro (IPP), Leu-Pro-Pro (LPP) and Phe-Pro (FP) were separated on a C18-column coupled to UV/VIS and mass spectrometric detector, respectively. Due to the derivatisation of the peptides, an HPLC base line separation was achieved and the peak width was improved. The VIS-spectrometry did not allow a good quantification of these peptides since more than one peptide co-eluted under one single peak. In contrast applying LC–ESI–MS with a single quadrupole much better quantification of the dabsylated peptides was done. In Evolus® (Valio Ltd., Finland), a fermented milk drink, 6.9mgL−1 for VPP, 6.1mgL−1 for IPP, 0.8mgL−1 for LPP and 3.2mgL−1 for FP were determined. In fermented reconstituted milk (Lactobacillus helveticus, 37°C, 48h) lower concentrations of these peptides were determined (0.7, 0.6, 0.0 and 2.2mgL−1, respectively).

Four phenolic acids determined by an improved HPLC method with a programmed ultraviolet wavelength detection and their relationships with lignin content in 13 agricultural residue feeds

Lignification-associated phenolic acids are widely distributed in graminaceous plant cell walls. This study developed a rapid and sensitive reversed-phase method for the simultaneous quantification of protocatechuic (PRA), vanillic (VA), ferulic (FA) and p-coumaric (PCA) acids and investigated the relationship between these compounds and lignin contents in 13 fibrous feeds. The phenolic acids were identified at a column temperature of 15 °C in a single run, in which the wavelength was programmed at 260 nm for PRA and VA, then switched to 310 nm for FA and PCA determinations. Satisfactory precision, recovery, and linearity were obtained with this method. Among 13 feeds, PCA was most abundant, followed by FA, VA and PRA. Great variations in phenolic acid and lignin contents were found. FA content was much richer than PCA content in maize and wheat brans, and the highest PCA content occurred in maize stalks. Lignin content was correlated with proportions of FA (r = - 0.95) and PCA (r = 0.90) in the summed phenolic acids and the PCA:FA ratio (r = 0.91). The improved method appears to be useful for simultaneous quantification of target phenolic compounds. Both FA and PCA may be good indicators for plant cell wall lignification associated with feed digestibility.

Simultaneous analysis of irbesartan and hydrochlorothiazide: an improved HPLC method with the aid of a chemometric protocol.

Experimental design method was used for HPLC determination of irbesartan and hydrochlorothiazide in combined dosage forms. The traditional approach for optimization of experiments is time-consuming, involves a large number of runs and does not allow establishing the multiple interacting parameters. The main advantages of the experimental design method include the simultaneous screening of a larger number of factors affecting response and the estimation of possible interactions. On the basis of preliminary experiments, three factors-independent variables were selected as inputs (methanol content, pH of the mobile phase and temperature) and as dependent variables, five responses (resolution, symmetry of irbesartan peak, symmetry of hydrochlorothiazide peak, retention factor of irbesartan and retention factor of hydrochlorothiazide) were chosen. A full 23 factorial design, where factors were examined at two different levels (“low” and “high”) was used to determine which factors had an effect on the studied response. Afterwards, experimental design was used to optimize these influent parameters in the previously selected experimental domain. The novelty of our method lies in the optimization step accomplished by Derringer′s desirability function. After optimizing the experimental conditions a separation was conducted on a Supelcosil C18 (150 mm × 4.6 mm, 5 μm particle size) column with a mobile phase consisting of methanol-tetrahydrofuran-acetate buffer 47:10:43 v/v/v, pH 6.5 and a column temperature of 25 °C. The developed method was successfully applied to the simultaneous separation of these drug-active compounds in their commercial pharmaceutical dosage forms.

An improved HPLC method for the quantitation of 3′-phosphoadenosine 5′-phosphate (PAP) to assay sulfotransferase enzyme activity in HepG2 cells

Sulfotransferase-catalyzed (SULT-catalyzed) sulfation is responsible for hormone regulation and xenobiotic detoxification. In the current study, a sensitive and widely applicable method of reversed-phase HPLC–UV was developed for the determination of 3′-phosphoadenosine 5′-phosphate (PAP), a common product of different subtypes of sulfation reactions, in HepG2 cells. The analyte was separated on a ZORBAX Extend-C18 column with the mobile phase of methanol and water containing 75mM KH2PO4, 100mM NH4Cl, and 1mM 1-octylamine (pH 4.55), at a flow rate of 1.0ml/min. The assay exhibited linearity over the range of 0.1–20μM for PAP with a correlation coefficient of 0.9995. The total time per run was under 10min. The intra- and inter-day precision was less than 7.2%, with accuracy in the range 82.6–102.0%. The method was applied to the determination of kinetic parameters Km, Vm, and Kcat, of three different SULTs (SULT1A1, SULT2A1, and SULT1E1) in HepG2. This universal HPLC–UV method was easy to perform, economically feasible, and suitably efficient for the investigation of the enzyme kinetics of the SULT family using multiplex substrates.

Simultaneous determination of four nucleosides in bee pollens of various floral origin by HPLC-UV detector

An improved HPLC method was developed for the simultaneous determination of nucleosides in bee pollen samples of various floral origins. Bee pollen samples were dissolved in 35 mL pure water by agitation on a vortex mixer followed by ultrasonic-assisted extraction prior to quantitative analysis. HPLC conditions were optimized and good linearity (r2 > 0.999) was obtained over the investigated concentration ranges. Precision was evaluated by intra- and inter-day assays and RSD values were below 3.15%. Accuracies ranged from 82.7% to 124.7%, respectively. Finally, the method was successfully applied to the analysis of nucleosides in bee pollen samples and could be used for routine analysis as the standard method.

Comparative Bioavailability Study of Amisulpride Tablets in Healthy Indian Volunteers

An improved HPLC method was developed and validated for the determination of concentration of amisulpride (CAS 71675-85-9) in human plasma, an attempt to compare the bioavailability of two amisulpride tablet formulations (reference and test) containing 200 mg of amisulpride. Both the formulations were administered orally as a single dose, separated by washout period of 1 week. This HPLC method validated by examining the precision and accuracy for the inter-day and intra-day runs in a linear concentration range of 50-1200 ng/ml. Bioequivalence of two formulation were determined on 12 healthy Indian male volunteer in a single-dose, two-period, two-sequence, two-treatment crossover study. The content of amisulpride in plasma was determined by a validated HPLC method with UV detection. The formulations were compared using the parameters like area under the plasma concentration-time curve (AUC0-t), area under the plasma concentration-time curve from zero to infinity (AUC0-infinity), peak plasma concentration (Cmax), and time to reach peak plasma concentration (tmax). The results indicated that there were no statistically significant differences (P > 0.05) between the logarithmic transformed AUC0-infinity and Cmax, values, between test and reference formulation. The 90% confidence interval for the ratio of the logarithmic transformed AUC0-t, AUC0-infinity, and Cmax were within the bioequivalence limit of 0.8-1.25 and the relative bioavailability of test formulation was 96.82% to that of reference formulation.

Bioequivalence study of levofloxacin tablets in healthy Indian volunteers using HPLC

An improved HPLC method was developed and validated for the determination of concentration of levofloxacin (CAS 100986-85-4) in human plasma. This paper is an attempt to compare the bioavailability of two levofloxacin tablet formulations (reference and test) containing 500 mg of levofloxacin. Both the formulations were administered orally as a single dose, separated by a washout period of 1 week. The HPLC method was validated by examining the precision and accuracy for the inter-day and intra-day runs in a linear concentration range of 0.10-10.00 microg/ml. Bioequivalence of two formulations were determined in 12 healthy, Indian, male volunteers in a single-dose, two-period, two-sequence, two-treatment crossover study. The content of levofloxacin in plasma was determined using HPLC with UV detection. The formulations were compared using the following pharmacokinetic parameters: area under the plasma concentration-time curve (AUC(o-t)), area under the plasma concentration-time curve from zero to infinity (AUC(o-infinity)), peak plasma concentration (C(max)), and time to reach peak plasma concentration (t(max)). The results indicated that there were no statistically significant differences (P > 0.05) between the logarithmically transformed AUC(o-infinity), and C(max) values of the test and reference formulations. The 90% confidence interval for the ratio of the logarithmically transformed AUC(o-t), AUC(o-infinity) and C(max) were within the bioequivalence limit of 0.8-1.25 and the relative bioavailability of the test formulation was 99.98% of that of the reference formulation.

Production of phytase under solid-state fermentation using Rhizopus oryzae: Novel strain improvement approach and studies on purification and characterization

Present study introduces linseed oil cake as a novel substrate for phytase production by Rhizopus oryzae. Statistical approach was employed to optimize various medium components under solid state fermentation (SSF). An overall 8.41-fold increase in phytase production was achieved at the optimum concentrations (w/w, mannitol, 2.05%; ammonium sulfate, 2.84% and phosphate, 0.38%). Further enhancement by 59% was observed due to a novel strain improvement approach. Purified phytase (∼34kDa) showed optimal temperature of 45°C, dual pH optima at 1.5 and 5.5 and possesses high catalytic efficiency (2.38×106M−1s−1). Characterization study demonstrates the phytase as highly thermostable and resistant to proteolysis, heavy metal ions, etc. Furthermore, an improved HPLC method was introduced to confirm the ability of phytase to degrade phytic acid completely and was found to be an efficient method.

Improved HPLC Method Using 2,3-naphthalenedicarboxaldehyde as Fluorescent Labeling Agent for Quantification of Histamine in Human Immunoglobulin Preparations

ObjectivesTo develop and optimize quantitative HPLC method using 2,3-naphthalenedicarboxaldehyde (NDA) after simple and efficient solid phase extraction to determine the histamine in a biopharmaceutical (Histobulin™). MethodsThe HPLC method was established using NDA-induced Histobulin and compared with the recently reported HPLC method using o-phthaldehyde (OPA). The validated NDA-applied HPLC method was adjusted to 15 lots of Histobulin and compared by the current lot-release-test method using fluorimetry in recovery of histamine and reproducibility. ResultsAnalyses of six HPLC chromatograms using NDA and OPA each were compared. NDA produced a more stable chromatogram baseline than OPA, and showed better stability. The HPLC analysis was validated in accuracy (91–103%), precision (interday/intraday assay CV ≤2.30%), and linearity of dose–response curve (R2 ≥ 0.9919). The detection limit was 0.0076 μg/mL and the quantitative limit was 0.0229 μg/mL. The amount of histamine per 12 mg of immunoglobulin was determined to be 0.17 ± 0.016 μg by the HPLC and 0.025 ± 0.013 μg by the current lot-release-test method using fluorimetry. ConclusionNDA derivatization showed better stability compared with the OPA method. Therefore the newly established NDA-derivatizated HPLC method may be more suitable than the fluorimetric method in lot-release-tests of biopharmaceuticals.

New and Improved HPLC Methods for Drug Formulations and Clinical Analysis

New and Improved HPLC Methods for Drug Formulations and Clinical Analysis

Improved HPLC method for the evaluation of the major steviol glycosides in leaves of Stevia rebaudiana

A simple reversed-phase high-performance liquid chromatographic method has been developed for the determination of the major steviol glycosides, the diterpene sweeteners derived from Stevia rebaudiana. The method is based on a water extraction step and a solid-phase extraction (SPE) clean-up. Different SPE cartridges and two HPLC columns were tested in the separation of the main steviol glycosides stevioside and rebaudioside A. A good separation was performed on a Luna HILIC analytical column with a mobile phase consisting of acetonitrile/water (85:15 v/v). The calibration curves were linear from 10 to 800 µg/mL and the recoveries for stevioside and rebaudioside A were 99 ± 4.4 and 100 ± 5.0%, respectively. The applicability of this method was demonstrated in the analysis of stevioside and rebaudioside A from Stevia plants grown in two different areas in Germany. Stevioside and rebaudioside A contents showed statistically significant differences (F- and t-test) between the two harvests. Nevertheless, the total concentrations (>12%) and the ratio of stevioside to rebaudioside A (6:4) were similar to those found in the countries in which Stevia rebaudiana originates. Based on a comparison of yields from different harvests, we discussed whether Stevia rebaudiana can be economically grown in the temperate zones of the northern European hemisphere.

Improved Sensitive High Performance Liquid Chromatography Assay for Glucosamine in Human and Rat Biological Samples with Fluorescence Detection

An improved HPLC method with fluorescence detection was developed and validated for determination of glucosamine in human and rat biological samples. Aliquot of 0.1 mL plasma was spiked with mannosamine HCl as the internal standard (IS); proteins were precipitated with acetonitrile; the clear layer was derivatized with 9-fluorenylmethyl chloroformate (8 mM/acetonitrile) in presence of borate 0.2 M buffer at 30 degrees C for 30 min. The excess derivatizing agent was removed with 1-aminoadamantane HCl (300 mM in acetonitrile-water 1:1). Chromatographic separation was achieved on a C18 (100 mm X 4.6 mm, id 3 microm) reversed phase column using 0.1% acetic acid/acetoniltrile gradient mobile phase at 1 mL/min flow rate. Glucosamine was determined in the plasma of a human and rats and also in rat urine. The analytes were detected at excitation and emission wavelengths of 263 and 315 nm, respectively. The assay was linear over the range of 0.05-20 microg/mL with a typical correlation coefficient of 0.999 and intra-day and inter-day coefficient of variation of <15%. The lowest limit of quantification was set at 50 ng/mL. The recovery for glucosamine and mannosamine was 98 and 96%, respectively. We were able to improve glucosamine assay suitable to quantify glucosamine in both human and rat plasma and rat urine.

Improved HPLC method for vitamin C analysis in serum

Vitamin C (VC) is necessary for development of normal connective tissue and human health. VC HPLC separations have remained nearly the same for 25 yrs. Recent advances in column technology have permitted some improvements in the methodology.ObjectiveTo improve serum VC separation, sensitivity and reproducibility w/o ion‐pairing agents or gradients.MethodsSeveral HPLC columns, possible internal standards and two buffers at different concentrations were examined. Both UV and electrochemical (EC) detection was used.ResultsMost columns offered poor retention and peak shape of VC. Phenomenex Synergi Hydro‐RP column provided good retention (k=1.3) symmetry (A=1.1) and tailing (T=1.15). Tris(2‐carboxyethyl)phosphine (TCEP) was used as an alternate reducing agent to dithiothreitol. Hypoxanthine, selected as an internal standard, eluted isocratically at k=3.2 with UV absorbance at 245nm but no EC.DiscussionThe HPLC method modifications yield good retention and peak shape. TCEP provides a cost effective and superior reducing agent. Efficiency and reproducibility were improved by incorporation of an internal standard. The method is amenable to short narrow bore columns.

Improved HPLC Method for Determination of Four PPIs, Omeprazole, Pantoprazole, Lansoprazole and Rabeprazole in Human Plasma

To develop a simple and rapid HPLC method for measuring of four proton-pump inhibitors (PPIs), omeprazole (OPZ), pantoprazole (PPZ), lansoprazole (LPZ) and rabeprazole (RPZ) concentrations in human plasma. Following a single step liquid-liquid extraction analytes along with an internal standard (IS) were separated using an isocratic mobile phase of phosphate buffer (10 mM)/acetonitrile (53/47, v/v adjusted pH to 7.3 with triethylamine) at flow rate of 1 mL/min on reverse phase TRACER EXCEL 120 ODS-A column at room temperature. Total analytical run time for selected PPIs was 10 min. The assays exhibited good linearity (r(2)>0.99) over the studied range of 20 to 2500 ng/mL for OPZ, 20 to 4000 ng/mL for PPZ, 20 to 3000 ng/mL for LPZ and 20 to 1500 ng/mL for RPZ. The recovery of method was equal or greater than 80% and lower limit of quantification (LLOQ) was 20 ng/mL for four PPIs. Coefficient of variation and error at all of the intra-day and inter-day assessment were less than 9.2% for all compounds. The results indicated that this method is a simple, rapid, precise and accurate assay for determination of four PPIs concentrations in human plasma. This validated method is sensitive and reproducible enough to be used in pharmacokinetic studies and also is time- and cost-benefit when selected PPIs are desired to be analyzed.