Eftichia G. Karageorgou

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.

Multiresidue LC–MS/MS analysis of cephalosporins and quinolones in milk following ultrasound‐assisted matrix solid‐phase dispersive extraction combined with the quick, easy, cheap, effective, rugged, and safe methodology

A sensitive and selective confirmatory method for milk-residue analysis of ten quinolones and eight cephalosporins by LC-MS/MS has been developed herein. For the chromatographic separation of target analytes, a Perfectsil ODS-2 (250 × 4 mm, 5 μm) analytical column was used and gradient elution was applied, using a mobile phase of 0.1% w/w TFA in water and 0.1% w/w TFA in ACN. Ultrasound-assisted matrix solid-phase dispersion procedure was applied for the extraction and clean-up procedure of antimicrobials agents from milk matrix using a mixture of Bond Elut Plexa sorbent and QuEChERS. The method was validated meeting the European Legislation determining selectivity, linearity response, trueness, precision (repeatability and between-day reproducibility), decision limit, detection capability, and ruggedness following the Youden approach. Recoveries of all antibiotics ranged from 81.7 to 117.9%, while RSD values were lower than 13.7%. Limits of quantification for all examined compounds ranged from 2.4 to 15.0 μg/kg, substantially lower than the maximum residue limits established by the European Union (30-100 μg/kg).

Ultrasound-assisted dispersive extraction for the high pressure liquid chromatographic determination of tetracyclines residues in milk with diode array detection

Ultrasound assisted matrix solid phase dispersive extraction was applied for the selective isolation and clean-up of tetracyclines (oxytetracycline, tetracycline, epi-chlorotetracycline, chlorotetracycline and doxycycline) from milk. Target analytes were determined by an accurate and sensitive chromatographic analytical method, which was validated to meet the European Legislation criteria. The separation was performed on a LiChroCART-LiChrospher® 100 RP-18 (5μm, 250×4mm) analytical column, operated at ambient temperature, followed by diode array detection. Validation included investigation of linearity, selectivity, stability, limits of detection and quantitation, decision limit, detection capability, trueness, precision and ruggedness according to the Youdens approach. Limits of quantitation of examined tetracyclines were from 14.5 to 56.6μg/kg significantly lower than respective Maximum Residue Limits, whereas recoveries ranged from 82.0% to 108%. The applicability of the method was evaluated using milk samples purchased from local market. Accuracy of the method was additionally proved by analysis of bovine milk certified reference material (BCR®-492).

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.

Youden test application in robustness assays during method validation.

Analytical method validation is a vital step following method development for ensuring reliable and accurate method performance. Among examined figures of merit, robustness/ruggedness study allows us to test performance characteristics of the analytical process when operating conditions are altered either deliberately or not. This study yields useful information, being a fundamental part of method validation. Since many experiments are required, this step is high demanding in time and consumables. In order to avoid the difficult task of performing too many experiments the Youden test which makes use of fractional factorial designs and has been proved to be a very effective approach. The main advantage of Youden test is the fact that it keeps the required time and effort to a minimum, since only a limited number of determinations have to be made, using combinations of the chosen investigated factors. Typical applications of this robustness test found in literature covering a wide variety of sample matrices are briefly discussed in this review.

An overview of the use of monoliths in sample preparation and analysis of milk.

Monolithic columns with a unique structure possess some exceptional characteristics, which make them an excellent tool in the hands of analytical chemists, not only for separation but also for sample preparation. Their much higher external porosity compared to conventional particle-packed columns results in higher permeability and low-pressure drop with higher separation efficiency. Till now, monolithic columns have been applied to the analysis of different analytical matrices: pharmaceuticals, biofluids, food matrices, environmental samples, biochemical species, proteomics, etc. Since they offer a great potential for the separation of complex mixtures, it can be expected that the interest in applying these columns will increase. As long as more experience is gained with monolithic column technology, the emergence of applications is expected to continue. The need of reduction in organic solvent consumption also justifies this trend, in the frame of environmentally friendly methods. More and more applications using monolithic columns arise. In the future, monolithic columns are expected to prevail especially in the field of miniaturization and in chip-based nano-LC systems. In this review, we try to summarize any attempt that has been reported in the analysis of a sample of great complexity like milk using the technology of the monoliths.

Simultaneous Determination of Testosterone and its Major Metabolite Epitestosterone in Biological Fluids by HPLC

Abstract A reversed‐phase high performance liquid chromatographic (HPLC) method is developed and validated for the simultaneous determination of anabolic steroids: testosterone and its major metabolite, epitestosterone. The analytical column, Inertsil ODS‐2, 5 µm, 250×4 mm, was operated at ambient temperature. Isocratic elution was performed using 35% of A=buffer solution 0.11% CH3COOH‐CH3COONa 7.5 mmol/L, pH=4 and 65% of B=CH3CN, at a flow rate of 0.8 mL/min. Inlet pressure was 155 bar. UV detection was performed at 236 nm. The limit of detection was 0.02 ng per 20 µL injection volume, while linearity held up to 2 ng/µL. p‐Cresol was used as internal standard at a concentration of 2 ng/µL. Validation of the method was performed in terms of accuracy and precision: intra‐day assay (n=6) and inter‐day assay (n=3×5) and was found to be satisfactory, with high accuracy and precision results. The method was successfully applied to biological fluids after solid phase extraction (SPE) on Nexus cartridges. Recovery rates from blood plasma ranged between 92.0% and 107.5% for testosterone and between 82.5% and 98.8% for epitestosterone, while the analysis of urine provided recovery rates from 85.0% to 108.0% for testosterone and from 85.5% to 103.0% for epitestosterone. The developed method was applied to the analysis of urine samples of one female and four male volunteers.

Development of a Validated HPLC Method for the Simultaneous Determination of Anabolic Steroids in Biological Fluids

Abstract A reversed phase high performance liquid chromatographic (HPLC) method is developed and validated for the simultaneous determination of anabolic steroids: testosterone (TES), epitestosterone (EPI), and nandrolone (NAN). The analytical column, Inertsil C8, 5 µm, 250 × 4 mm, was operated at ambient temperature. Isocratic elution was performed using a mixture of 50% buffer solution CH3COOH 0.11% –CH3COONa 7.5 mmol/L, pH = 4, 45% CH3CN and 5% CH3OH, at a flow rate of 1.1 mL/min. UV detection was performed at 238 nm. The detection limits of the method were 2.4 ng for NAN, 3.6 ng for TES, and 2.6 ng for EPI in blood plasma, and 2.7 ng for NAN, 1.1 ng for TES, and 3.8 ng for EPI in urine, per 20 µL injection volume. Alprazolam was used as internal standard at a concentration of 2 ng/µL. Validation of the method was performed in terms of accuracy and precision: intra-day assay (n = 6) and inter-day assay (n = 3 × 6) and was found to be satisfactory, with high accuracy and precision results. Sample preparation involved solid phase extraction on Nexus cartridges with high recoveries. The developed method was successfully applied to the analysis of urine samples of one female and 9 male volunteers.