Ioannis N. Papadoyannis

Development and validation of an HPLC-method for determination of free and bound phenolic acids in cereals after solid-phase extraction

Whole cereal grains are a good source of phenolic acids associated with reduced risk of chronic diseases. This paper reports the development and validation of a high-performance liquid chromatography-diode array detection (HPLC-DAD) method for the determination of phenolic acids in cereals in either free or bound form. Extraction of free phenolic acids and clean-up was performed by an optimised solid-phase extraction (SPE) protocol on Oasis HLB cartridges using aqueous methanol as eluant. The mean recovery of analytes ranged between 84% and 106%. Bound phenolic acids were extracted using alkaline hydrolysis with mean recoveries of 80-95%, except for gallic acid, caffeic acid and protocatechuic acid. Both free and bound phenolic extracts were separated on a Nucleosil 100 C18 column, 5 μm (250 mm × 4.6 mm) thermostated at 30 °C, using a linear gradient elution system consisting of 1% (v/v) acetic acid in methanol. Method validation was performed by means of linearity, accuracy, intra-day and inter-day precision and sensitivity. Detection limits ranged between 0.13 and 0.18 μg/g. The method was applied to the analysis of free and bound phenolic acids contents in durum wheat, bread wheat, barley, oat, rice, rye, corn and triticale.

Simultaneous determination of phenolic acids and flavonoids in rice using solid-phase extraction and RP-HPLC with photodiode array detection

An analytical method based on an optimized solid-phase extraction procedure and followed by high-performance liquid chromatography (HPLC) separation with diode array detection was developed and validated for the simultaneous determination of phenolic acids (gallic, protocatechuic, 4-hydroxy-benzoic, vanillic, caffeic, syringic, p-coumaric, ferulic, sinapic, and cinnamic acids), flavanols (catechin and epicatechin), flavonols (myricetin, quercetin, kaempferol, quercetin-3-O-glucoside, hyperoside, and rutin), flavones (luteolin and apigenin) and flavanones (naringenin and hesperidin) in rice flour (Oryza sativa L.). Chromatographic separation was carried out on a PerfectSil Target ODS-3 (250 mm × 4.6 mm, 3 μm) column at temperature 25°C using a mobile phase, consisting of 0.5% (v/v) acetic acid in water, methanol, and acetonitrile at a flow rate 1 mL min(-1) , under gradient elution conditions. Application of optimum extraction conditions, elaborated on both Lichrolut C(18) and Oasis HLB cartridges, have led to extraction of phenolic acids and flavonoids from rice flour with mean recoveries 84.3-113.0%. The developed method was validated in terms of linearity, accuracy, precision, stability, and sensitivity. Repeatability (n = 5) and inter-day precision (n = 4) revealed relative standard deviation (RSD) <13%. The optimized method was successfully applied to the analysis of phenolic acids and flavonoids in pigmented (red and black rice) and non-pigmented rice (brown rice) samples.

Simultaneous Determination of Nine Water and Fat Soluble Vitamins After SPE Separation and RP-HPLC Analysis in Pharmaceutical Preparations and Biological Fluids

Abstract An automated reversed phase high performance liquid-chromatography (RP-HPLC) method is described, for the simultaneous analysis of water soluble [ascorbic acid (C), nicotinic acid, nicotinamide, folic acid, cyanocobalamine (B12), and riboflavin (B2)] and fat soluble (retinol, α-tocopherol, α-tocopherol acetate) vitamins. The compounds are separated after solid-phase extraction (SPE) on C18 cartridges, where water soluble vitamins pass unretained, while fat soluble vitamins are retained on the sorbent. After isolation of the two fractions, water soluble vitamins are separated on a Lichrosorb RP-18 250x4.0 mm, 5 μm analytical column, using a gradient elution system consisting of CH3OH-0.05 M CH3COONH4 (5:95 v/v) changing to (30:70 v/v) over a period of 20 min at a flow rate 1 mL/min. Fat soluble vitamins are separated on a Spherisorb RP-18 220x4.6 mm, 5 μm analytical column with an isocratic mobile phase consisting of CH3OH-CH3CN (30:70 v/v) at a flow rate of 1.5 mL/min. A UV-vis detector operated ...

Rapid and sensitive high-performance liquid chromatographic determination of four cephalosporin antibiotics in pharmaceuticals and body fluids

A rapid, accurate and sensitive method has been developed and validated for the quantitative simultaneous determination of four cephalosporins, cephalexin and cefadroxil (first-generation), cefaclor (second-generation) and cefataxim (third-generation), in pharmaceuticals as well as in human blood serum and urine. A Spherisorb ODS-2 250 x 4-mm, 5-microm analytical column was used with an eluting system consisting of a mixture of acetate buffer (pH 4.0)-CH(3)OH 78-22% (v/v) at a flow-rate 1.2 ml/min. Detection was performed with a variable wavelength UV-Vis detector at 265 nm resulting in limit of detection of 0.2 ng for cefadroxil and cephalexin, but only 0.1 ng for cefotaxime and cefaclor per 20-microl injection. Hydrochlorothiazide (HCT) (6-chloro-3,4-dihydro-7 sulfanyl-2H-1,2,4-benzothiadiazine-1-1-dioxide) was used as internal standard at a concentration of 2 ng/microl. A rectilinear relationship was observed up to 8, 5, 12 and 35 ng/microl for cefadroxil, cefotaxime, cefaclor, cephalexin, respectively. Analysis time was less than 7 min. The statistical evaluation of the method was examined by means of within-day repeatability (n=8) and day-to-day precision (n=9) and was found to be satisfactory with high accuracy and precision. The method was applied to the determination of the cephalosporins in commercial pharmaceuticals and in biological fluids: human blood serum after solid-phase extraction and urine simply after filtration and dilution. Recovery of analytes in spiked samples was in the range from 76.3 to 112.0%, over the range of 1-8 ng/microl.

Use of novel solid-phase extraction sorbent materials for high-performance liquid chromatography quantitation of caffeine metabolism products methylxanthines and methyluric acids in samples of biological origin.

An automated reversed-phase high-performance liquid chromatographic (RP-HPLC) method, using a linear gradient elution, is described for the simultaneous analysis of caffeine and metabolites according to their elution order: 7-methyluric acid, 1-methyluric acid, 7-methylxanthine, 3-methylxanthine, 1-methylxanthine, 1,3-dimethyluric acid, theobromine, 1,7-dimethyluric acid, paraxanthine and theophylline. The analytical column, an MZ Kromasil C4, 250 x 4 mm, 5 microm, was operated at ambient temperature with back pressure values of 80-110 kg/cm2. The mobile phase consisted of an acetate buffer (pH 3.5)-methanol (97:3, v/v) changing to 80:20 v/v in 20 min time, delivered at a flow-rate of 1 ml/min. Paracetamol was used as internal standard at a concentration of 6.18 ng/microl. Detection was performed with a variable wavelength UV-visible detector at 275 nm, resulting in detection limits of 0.3 ng per 10-microl injection, while linearity held up to 8 ng/microl for most of analytes, except for paraxanthine and theophylline, for which it was 12 ng/microl and for caffeine for which it was 20 ng/microl. The statistical evaluation of the method was examined performing intra-day (n=6) and inter-day calibration (n=7) and was found to be satisfactory, with high accuracy and precision results. High extraction recoveries from biological matrices: blood serum and urine ranging from 84.6 to 103.0%, were achieved using Nexus SPE cartridges with hydrophilic and lipophilic properties and methanol-acetate buffer (pH 3.5) (50:50, v/v) as eluent, requiring small volumes, 40 microl of blood serum and 100 microl of urine.

Development and validation of an HPLC method for the simultaneous determination of tocopherols, tocotrienols and carotenoids in cereals after solid‐phase extraction

The increasing interest in antioxidant properties of cereal and cereal-based products has prompted the development of a simple and reliable HPLC method for the simultaneous determination of important phytochemicals like tocopherols (T), tocotrienols (T3) and carotenoids. Separation was carried out on a Nucleosil 100 C(18) column, 5 μm (250 mm × 4.6 mm) thermostated at 25 °C, using a linear gradient elution system starting with methanol and ending with a mixture of methanol-isopropanol-acetonitrile. All separated compounds including the internal standard (α-tocopherol acetate) were eluted within 16 min and detected by dual detection: fluorescence for tocopherols and tocotrienols at 290 nm excitation and 320 nm emission and UV-vis photodiode array detection for lutein and β-carotene at 450 nm. Detection limits ranged from 0.2 μg/g (β-carotene) to 1.60 μg/g (α-tocopherol). The intra- and inter-assay coefficients of variation were calculated by using cereals with different levels of lipophilic antioxidants. The extraction method involved sample saponification and clean-up by solid-phase extraction (SPE). The extraction recoveries obtained using OASIS HLB SPE cartridges and dichloromethane as eluent were in the range of 90.2-110.1%, with RSD lower than 10%. The method was successfully applied to cereals: durum wheat, bread wheat, rice, barley, oat, rye, corn and triticale.

Shear bond strength of AH-26 root canal sealer to dentine using three dentine bonding agents

OBJECTIVE The purpose of the study was to compare the bond strength of AH-26 root canal sealer to human root canal dentine exposed to different intracanal medications both with and without the use of three bonding agents. The materials used were AH-26 sealer, two one-bottle bonding agents (Single Bond, Bond-1) and one self-etching bonding agent (Clearfill SE Bond). METHODS The dentine substrate was obtained from single rooted human teeth. The dentine specimens were treated either with EDTA 15% or phosphoric acid 37% to achieve the removal of smear layer. The AH-26 sealer was placed on the dentine surfaces both with and without the use of the bonding agents. Bond strength was tested using a single plane shear test assembly. RESULTS The SBS values were (MPa): Group A (EDTA-AH-26) 3.678+/-0.853, Group B (Phosphoric acid-AH-26) 3.470+/-0.834, Group C (EDTA-Single Bond-AH-26) 4.8+/-0.865, Group D (Phosphoric acid-Single Bond-AH-26) 5.043+/-1.022, Group E (EDTA-Bond 1-AH-26) 4.939+/-0.877, Group F (Phosphoric acid-Bond 1-AH-26) 5.101+/-1.117, Group G (Clearfill SE Bond-AH-26) 6.975+/-1.694. The use of dentine bonding agents improved significantly (p<0.05) the adhesion of AH-26 sealer with the root canal dentine. However, the best results were obtained with the self-etching system. Similar results were observed from the pretreatment of dentine either with phosphoric acid 35% or EDTA 15%. CONCLUSIONS The use of dentine bonding agents gave higher shear bond strengths of AH-26 sealer to human root canal dentine.

A Simple and Quick Solid Phase Extraction and Reversed Phase HPLC Analysis of Some Tropane Alkaloids in Feedstuffs and Biological Samples

Abstract A simple and rapid reversed - phase high performance liquid chromatographic (HPLC) method using an ultraviolet detector for the analysis of tropane alkaloids in feedstuffs and biological samples is presented. Hyoscyamine, scopolamine and the internal standard bamifylline are adsorbed on a C18 cartridge employing the solid - phase liquid extraction technique for sample clean up and subsequent separation of those compounds and internal standard from endogenous interfering compounds and preconcentration. Chromatographic analysis is achieved on a Lichrosorb RP-18 10μm reversed phase column and the mobile phase is an isocratic mixture of acetonitrile, methanol and 0.05 M ammonium acetate (20.9 : 27.9 : 51.2) at a flow rate of 1.30 ml/min. The edited alkaloids are detected at 210 nm. The retention time is 3.990 min for scopolamine, 6.934 min for hyoscyamine and 8.750 min for the internal standard bamifylline. The correlation of the integrated peak area ratios of scopolamine and hyoscyamine to internal ...

Ultrasound‐assisted matrix solid phase dispersive extraction for the simultaneous analysis of β‐lactams (four penicillins and eight cephalosporins) in milk by high performance liquid chromatography with photodiode array detection

The application of ultrasound-assisted matrix solid phase dispersive extraction for the confirmatory analysis of 12 β-lactam antibiotics in milk by high performance liquid chromatography with photodiode array detection has been proposed herein. Four penicillins (cloxacillin, dicloxacillin, oxacillin, and amoxicillin) and eight cephalosporins (cefaclor, cefadroxil, ceftiofur, cefuroxime, cefoperazone, cefazolin, cephalexin, and cefotaxime) are effectively extracted using a mixed sorbent of Quick Easy Cheap Effective Rugged Safe technique and OASIS HLB providing a matrix free from any endogenous interference. Examined analytes were well resolved on an Inertsil ODS-3 analytical column with a mobile phase of CH(3)COONH(4) (0.05 M) and acetonitrile delivered under a gradient program. 1,7-Dimethyl-xanthine was used as internal standard. The method was validated meeting the European Legislation determining linearity, selectivity, stability, decision limit, detection capability, accuracy, precision, and ruggedness according to the Youden approach. Recoveries of all antibiotics rated from 85.0 to 115.7%, while RSD values were <12.7%. Finally, the method was successfully applied to milk samples purchased from local market.

Simultaneous determination of nitrite and nitrate in drinking water and human serum by high performance anion-exchange chromatography and UV detection

A rapid, accurate, and sensitive method has been developed for the simultaneous determination of nitrite and nitrate. A low capacity strong anion exchange PRP-X100 Hamilton, 150 × 4.1 mm, 10 μm, with exchange capacity 0.19 ± 0.02 meq/g, analytical column was used, with a mixture of 0.1 M NaCl-CH3OH, at a volume ratio 45:55. The flow rate of 2 mL/min, lead to a pressure of 150 kg/cm2. Detection was performed with a variable wavelength UV-visible detector, at 230 nm, resulting in detection limits of 0.1 ng and 0.2 ng for nitrite and nitrate, respectively, per 20 μL injection. For the quantitative determination, iodide was used as internal standard, at a concentration of 7.0 ng/μL. A rectilinear relationship was observed up to 12 ng/μL for nitrite and up to 25 ng/μL for nitrate. Analysis time was less than 6 min. The statistical evaluation of the method was examined performing intra–day (n = 8) and inter-day calibration (n = 8) and was found to be satisfactory, with high accuracy and precision results. The m...