Victoria F. Samanidou

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.

Multi‐residue methods for confirmatory determination of antibiotics in milk

The prevalent use of antibiotics, e. g. sulphonamides, tetracyclines, beta-lactams, macrolides, aminoglycosides, etc., in dairy farming to prevent and treat microbial infections e. g. mastitis, can be a potential source of veterinary drug residues in milk. Antibiotic residues constitute a risk to human health, since they can cause allergic reactions in hypersensitive individuals or they may lead to the appearance of drug-resistant bacteria. Analysis of these residues plays a key role in ensuring food safety. Regulatory agencies in the European Union and in other countries have established maximum residue limits and requirements concerning the performance of analytical methods and the interpretation of the results. This review describes the state of the art in the analytical strategies concerning the multi-class as well as the multi-residue analysis of antibiotics in milk. Since milk is a complex matrix due to its high protein and fat content, which often interfere in analytical procedures, special focus has been placed on sample preparation: extraction and clean-up. Confirmation of antibiotic residues according to European Decision 657/2002/EC has been also discussed.

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 ...

Multi-residue determination of seven quinolones antibiotics in gilthead seabream using liquid chromatography–tandem mass spectrometry

A sensitive and selective confirmatory analytical method for the multi-residue determination of seven quinolones (ciprofloxacin, enrofloxacin, sarafloxacin, danofloxacin, oxolinic acid, nalidixic acid and flumequine) in gilthead seabream (Sparus aurata) was developed. The sample pre-treatment involves extraction with 0.1 M NaOH and purification by solid-phase extraction (SPE) on Waters Oasis HLB cartridges followed by the determination of all compounds in a single LC-electrospray ionization MS/MS run. Separation was achieved on a Perfectsil ODS-2, 5 microm, 250 mm x 4 mm, analytical column (MZ Analysentechnik) by gradient elution using a mixture of 0.2% (v/v) formic acid, methanol and acetonitrile within 30 min. Multiple reaction monitoring (MRM) was used for selective detection of each quinolone. Accuracy was evaluated through recovery studies at three different fortification levels. The mean recoveries are between 90 and 132% for the selected levels with RSD values lower than 20%. The method presents satisfactory results for linearity, precision and limits of quantification. The latter are much lower than the maximum residue limits (MRLs) established by the European Union for quinolones in fish tissues (6-8 microg/kg).

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.

Development and validation according to European Union Decision 2002/657/EC of an HPLC‐DAD method for milk multi‐residue analysis of penicillins and amphenicols based on dispersive extraction by QuEChERS in MSPD format

A precise and reliable method for milk residue analysis regarding five penicillins and three amphenicols by HPLC-diode array detection has been developed herein. The chromatographic separation was performed using a mobile phase of CH3 COONH4 (0.05 M) and ACN delivered by gradient program on a Kinetex(™) -C18 core-shell, 2.6 μm column, starting at a volume ratio of 95:5 and ending at 60:40 after 17 min, remaining stable for 3 more min. A modified matrix solid phase dispersion procedure was applied for the extraction and clean-up procedure of antibiotics using a mixture of Strata by Phenomenex and QuEChERS as a sorbent. The method was validated at the respective 0.5× MRL, MRL and 1.5 ×MRL level for each compound. Results were quantitated against the internal standard paracetamol (2 ng/μL) according to the matrix-matched approach. The method was validated in line with the EC guidelines as cited in the Decision 2002/657/EC. The within-laboratory reproducibility, expressed as a RSD, never exceeded 16%. All decision limit (CCα) values lay in the range between 35.2 and 56.3 μg/kg and the corresponding results for detection capability (CCβ) were 39.9 and 61.9 μg/kg. Ruggedness was estimated according to the Youden approach.

Application of ultrasound-assisted matrix solid-phase dispersion extraction to the HPLC confirmatory determination of cephalosporin residues in milk

Ultrasound-assisted matrix solid-phase dispersion (MSPD) was applied to isolate eight cephalosporins (cefadroxil, cefaclor, cephalexin, cefotaxime, cefazolin, cefuroxime, cefoperazone and ceftiofur) from milk. Multi-residue analysis was subsequently performed by HPLC-diode array detection. Extraction yield by matrix solid-phase dispersion using Nexus sorbent was higher than various investigated SPE protocols. Three analytical columns, two conventional silica based and one monolithic, were compared based on resolution, peak shape and retention time. The optimum method using Chromolith RP-18e (100×4.6 mm) achieved separation in less than 16 min. Method validation was performed according to the European Union Decision 2002/657/EC, determining linearity, selectivity, stability, decision limit, detection capability, accuracy and precision. RSD values observed were lower than 15.3%. Recovery rates of examined antimicrobials from milk ranged from 93.8 to 101.9% for cefadroxil, from 94.7 to 103.6% for cefaclor, from 93.4 to 106.6% for cephalexine, from 104.1 to 115.3% for cefotaxime, from 97.1 to 105.6% for cefazolin, from 97.4 to 108.6% for cefuroxime, from 98.8 to 103.4% for cefoperazone and from 95.5 to 103.6% for ceftiofur. Correlation coefficients ranged from 0.9926 to 0.9999. CC(b) values were in the range from 103.5 to 112.3 μg/kg for analytes with a maximum residue limit of 100 μg/kg and from 54.4 to 56.3 μg/kg for those with a maximum residue limit of 50 μg/kg.

Partitioning of heavy metals into selective chemical fractions in sediments from rivers in northern Greece

Abstract A five-step sequential extraction technique was used to determine the chemical association of heavy metals (Pb, Cd, Cu, Fe, Mn, Zn, Cr) with major sedimentary phases (exchangeable cations, easily reducible compounds, organic sulfidic phases, carbonates and residual components) in samples from rivers in northern Greece (Axios and Aliakmon). From the obtained data it can be seen that the surplus of metal contaminants introduced into the aquatic system from anthropogenic sources usually exists in relatively unstable chemical forms. A high proportion of the elements studied remains in the residual fraction for the Axios river and in the organic fraction for the Aliakmon. Most of the non-residual portion is bound to ferromanganese oxides and to organic matter.

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.