Rade Dragan

Ales Mlinaric

Vukosava Djordjevic-Milic

Katarina Karljikovic-Rajic

Borut Strukelj

Rade ; Injac

Vukosava Djorjevic-Milic

Katarina Karljikovic- Rajic

Optimal Condition for Determination of Zinc Bacitracin, Polymyxin B, Oxytetracycline and Sulfacetamide in Animal Feed by Micellar Electrokinetic Capillary Chromatography

Keywords: Additives general, Veterinary drug residues -antibiotics, Veterinary drug residues -oxytetracyclin Food Types: Animal feed, Animal feedingstuffs Animal feed additives, Zn bacitracin, oxytetracycline, polymyxin B, sulfacetamide, micellar electrokinetic capillary chromatography (MECK)

published or not. The documents may come   L'archive ouverte pluridisciplinaire

INTRODUCTION

Feed additives are intended to improve feed quality, nutritional aspects, animal health and animal performance. According to Regulation (EC) No. 1831/2003(European Union, 2003) there are a wide range of substances considered as feed additives and classified as technological, organoleptic, nutritional and zootechnical (i.e. increasing animal production or performance).

Antibiotics have been used in the rearing of food-producing animals in livestock farm industries as active disease treatment agents for prophylactics and in cases of pulmonary, urinary and digestive infections as feed additives. The use of these drugs to control and treat animal disease and to promote fast, more efficient growth of livestock is a common practice. Improper use, as well as the systematic administration of such additives at sub-therapeutic doses can lead to the development of antibiotic-resistant bacteria that can be transferred to humans [START_REF] Witte | Medical consequences of antibiotic use in agriculture[END_REF]Smith and Coast, 2002). Since 1999, the European Union has forbidden the use of those antibiotics as additive in animal feed [START_REF] Molterer | [END_REF], but they are still in use in non EU countries.

Recently, Serratosa et al., [START_REF] Serratosa | Residues from veterinary medicinal products, growth promoters and performance enhancers in food-producing Page 23 of 30[END_REF] published an overview of the presence of residues from veterinary medicinal products, growth-promoting agents and performance enhancers in food-producing animals, as a result of legally or illegally administration of these substances, as well as the current In order to carry out an efficient official controls of the possible illegal use of these drugs in feeding stuffs, reliable methods of analysis need to be available with efficient separation and high sensitivity. [START_REF] Oka | Chromatographic analysis of tetracycline antibiotics in foods[END_REF] reviewed the literature for the assay of tetracycline antibiotics in food, among which oxytetracycline, tetracycline, chlortetracycline and doxycycline are commonly applied to food-producing animals. They have summarized the application of thin-layer chromatography, capillary electrophoresis, and HPLC for the separation and detection, as well as extraction and clean up procedures for real samples.

Capillary electrophoresis (CE) has become a very useful method for pharmaceutical analysis and in comparison with HPLC has many advantages such as: high resolution, speed, the exceptionally small sample volume required and low content of organic solvents in running buffer and short run time for the separation. [START_REF] Oka | Chromatographic analysis of tetracycline antibiotics in foods[END_REF] indicated that CE had less applications in the analysis of drugs in food due to low sample injection volume, and on the other hand authors pointed out that due to properties of tetracycline's these bind with silanol groups in the stationary phase interfere with the establishment of a simple analytical method. Recently, [START_REF] Kowalski | Capillary electrophoresis in analysis of veterinary drugs[END_REF] proposed a CE method with UV detection for eight of the most frequently used antibiotics (among those was doxycycline) with detection limit of drug residue in tissues at level below 20 µg/kg. One of the commonly used CE modes is micellar electrokinetic capillary chromatography (MEKC), which is efficient for the separation of both ionic and neutral analytes.

Success of the separation is based mainly on appropriate selection of the surfactant. The results of MECK application for Zn Bacitracin and nystatin separation and assay in animal feed, recently developed in our laboratories [START_REF] Injac | Micellar electrokinetic capillary chromatography determination of zinc bacitracin and nystatin in animal feed[END_REF] demonstrated that this technique could be the alternative to HPLC method, but also the simple extraction procedure was proposed. For zinc bacitracin in animal feed [START_REF] Capitán-Vallvey | Determination of the Antibiotic Zinc Bacitracin in Animal Food by High-Performance liquid Chromatography with Ultraviolet Detection[END_REF]2002) proposed HPLC methods with UV and fluorescence detection, but with difficult extraction procedures using LLE as well as SPE techniques.

In the continuation of our research studies we selected, beside Zn Bacitracin (Zn BAC), oxytetracycline (OXY), polymyxin B (POL B) and sulfacetamide (SUL) to investigate the separation applying MECK with the similar extraction procedure described in previously published paper [START_REF] Injac | Micellar electrokinetic capillary chromatography determination of zinc bacitracin and nystatin in animal feed[END_REF]. OXY, SUL, Zn BAC and POL B have been determined in pharmaceutical formulations by CZE and MEKC [START_REF] Gallego | Micellar Electrokinetic Capillary Chromatography as an Alternative Method for Determination of Hydrocortisone and its Most Important Associated Compounds in Local Pharmaceutical Preparations[END_REF]Gallego and Arroyo, 2003a;Gallego and Arroyo, 2003b;Gallego and Arroyo, 2003c;Gallego and Arroyo, 2003d;[START_REF] Mamani | Simultaneous determination of tetracyclines in pharmaceuticals by CZE using experimental design[END_REF][START_REF] Nozal | Rapid determination of trace levels of tetracyclines in surface water using a continuous flow manifold coupled to a capillary electrophoresis system[END_REF]. Kang et al. (Kang et al., 2001) Counter-current chromatography and high-performance liquid chromatography were used for the separation of Zn-bacitracin [START_REF] Oka | Separation of antibiotics by counter-current chromatography[END_REF][START_REF] Capitan-Vallvey | High-performance liquid chromatography determination of Zn-bacitracin in animal feed by post-column derivatization and fluorescence detection[END_REF]Sin et al., 2005). Tetracyclines and sulfonamides were analysed by liquid chromatography-electrospray ionization tandem mass spectrometry, as well as with HPLC-UV, LC/MS/MS in wastewaters, pharmaceuticals and food [START_REF] Yang | Simultaneous extraction and analysis of 11 tetracycline and sulphonamide antibiotics in influent and effluent domestic wastewater by solid-phase extraction and liquid chromatography-electrospray ionization tandem mass spectrometry[END_REF][START_REF] Zhao | Determination of tetracyclines in ovine milk by high-performance liquid chromatography with a coulometric electrode array system[END_REF][START_REF] Mandens | Quantitative analysis of twelve sulfonamides in honey after acidic hydrolysis by high-performance liquid chromatography with post-column derivatization and fluorescence detection[END_REF][START_REF] Rao | A reverse phase ion-pairing HPLC method for the stability monitoring of sulphacetamide ophthalmic preparations[END_REF].

Zn BAC interferes with bacterial cell wall synthesis by bloking the function of the lipid carrier molecule that transfers cell wall subunits across the cell membrane. It is active against many Gram-positive bacteria. Acquried bacterial resistance to bacitracin rarely occurs [START_REF] Martindale | The complete drug reference[END_REF]. Oxytetracycline is bacteriostatic antibiotic with a wide spectrum of activity and has been used in the treatment of a large number of infections caused by susceptible organisms. Polymyxin B and the other polymyxin antibacterials act primarily by binding to membrane phospholipids and disrupting the bacterial cytoplasmic membrane. The use of sulfacetamide and other sulfonamides has been limited by the increasing incidence of resistant organisms [START_REF] Wang | Analysis of sulphonamide residue in edible animal products: A review[END_REF]. Their main use has been in the treatment of acute, uncomplicated urinary-tract infections, particularly those caused by Escherichia coli [START_REF] Martindale | The complete drug reference[END_REF].

Selected analytes, OXY, SUL, Zn-BAC and POL used as additives in animal feed at levels 100-200 mg kg -1 , 100 mg kg -1 , 50-250 mg kg -1 and 50-150 mg kg The aim of this paper was to establish the optimal conditions for MECK separation and assay using the model mixture of OXY, Zn BAC and POL B in spiked feedstuff samples, antibiotics belonging to different cases of antibiotics, in combination with SUL as these can be found as feed additives. The model mixture is also of interest since, polymyxins have been reported to demonstrate antimicrobial synergy with a variety of other drugs, including chloramphenicol, tetracycline, and the sulfonamides and trimethoprim [START_REF] Martindale | The complete drug reference[END_REF], and as such the prophylactics in food could be covered with these representative drugs.

MATERIALS AND METHODS

Apparatus

The HP 3D Capillary Electrophoresis system (Hewlett Packard, Waldbronn, Germany) with a diode-array detector, controlled by HP ChemStation software, was used to perform MEKC. Compounds were separated on a 48 cm (40 cm to the detector) x 50 µm i.d. fused silica capillary (with bubble cell, 150 µm) (Agilent, Waldbronn, Germany).

A Crison MicropH 2002 pH meter (Barcelona, Spain) was used for pH measurement. Samples solutions were prepared with deionized water (Milli-Q-quality).

Reagents and solutions

Sulfacetamide sodium was obtained from Vetprom (Belgrade, Serbia) and Znbacitracin from Sigma (Deisenhofen, Germany). The quality of the both complied with BP requirements. Oxytetracycline hydrochloride and polymyxin B sulphate were obtained from Sigma (Deisenhofen, Germany) and Fluka (Buchs, Switzerland), respectively (USP quality).

Animal diet and feedstuff mixtures for cow K-19 (protein min 19 %, crude fat max 15 %, ash max 15 %, crude fiber max 10 %, calcium 0.8 %, phosphorus 0.45 %, sodium 0.25 %, moisture max 13.5 %, vitamins A, D 3 , E, zinc, manganese, selenium, cobalt, copper, iron and iodine), young cattle TL-TIP (protein min 14 %, ash max 10 %, crude fiber max 12 %, moisture max 13.5 %, vitamins A, D 3 , E, zinc, manganese, selenium, cobalt, copper and magnesium), pigs from 25 to 60 kg BEK-1 (protein 17 %, ash max 8 %, crude fiber max 7 %, moisture max 13.5 %, vitamins A, D 3 , E, B-complex, zinc, selenium, manganese, iodine, cobalt, copper, iron and antioxidants Sanox), chicken feed NSK-1 (protein min 15 %, moisture max 13.5 %, crude fiber max 8 %, ash max 12 %, vitamins A, D 3 , E, K 3 , B-complex, zinc, selenium, manganese, iodine, cobalt, copper, iron, carophyll red and antioxidants Sanox), and minerals and vitamins for cattle BOVISAL (calcium 16 %, phosphorus 11 %, sodium 10 %, magnesium 0.6 %, moisture max 7 %, vitamins A, D 3 , E, zinc, manganese, selenium, iodine, cobalt and copper), were manufactured by JATA EMONA (Ljubljana, Slovenia). Sodium dodecylsulphate (SDS) was from Riedel Line AG (Seelze,Germany).

The background electrolyte (BGE) was 20 mmol L -1 borate + 20 mmol L -1 phosphate buffer, pH 8.4, containing 20 mmol L -1 SDS and 10 % (v/v) methanol.

Preparation of standard stock solutions

Stock solutions of antibiotics were prepared by weighing 50 mg of the drugs and dissolving in 50 mL methanol -deionized water (1:1; v/v). The stock solutions were diluted with running buffer to obtain the concentration ranges required (linearity was check in range of 0.1 -100 mg kg -1 for each drug).

Sample preparation and extraction

Spiked animal feed were prepared by grinding 500 g of feedstuff and adding 50 mg of each of the antibacterial. Blank feed or spiked feed with added antibiotics, were weighed (100 g) and extracted with methanol (5 x 20 mL), first by shaking, then in an ultrasonic bath for 15 min. The extracts were combined, filtered (0.22 µm), transferred to 100 mL volumetric flask, and made up with methanol.

Different known volumes were placed in 1.5 mL calibrated vials and diluted to volume with running buffer.

Operating conditions

The capillary was conditioned prior to its first use by flushing with 0.1 mol L -1 NaOH for 20 min and then with water for 10 min. The capillary was conditioned, in the optimized method, at the beginning of each day with methanol under high pressure for 3 min, than rinsed for 2 min with 0.1 mol L -1 NaOH and 3 min with background electrolyte. This was followed by hydrodynamic sample injection at 600 mbars. Separations were performed at 25 kV and 25°C (under applied pressure of 10 mbar) in 12 min; under these conditions the current was 97-98 µA.

UV detection was at 215 nm.

RESULTS AND DISCUSSION

Preliminary studies

Preliminary investigations of Zn-BAC, SUL, OXY and POL B were carried out, to optimize the separation. MEKC methods were published for the determination of hydrocortisone and its most important associated compounds (including Zn-BAC, POL B and OXY) in topical pharmaceutical preparations [START_REF] Gallego | Micellar Electrokinetic Capillary Chromatography as an Alternative Method for Determination of Hydrocortisone and its Most Important Associated Compounds in Local Pharmaceutical Preparations[END_REF]Gallego and Arroyo, 2003c) SUL were determined in pharmaceuticals by MEKC with others associated compounds (Gallego and Arroyo, 2003a). Some modifications of the SDS concentration, organic modifier and applied pressure were assessed to obtain a shorter run time and better resolutions in separation of of all analytes from animal feed. The published literature [START_REF] Gallego | Micellar Electrokinetic Capillary Chromatography as an Alternative Method for Determination of Hydrocortisone and its Most Important Associated Compounds in Local Pharmaceutical Preparations[END_REF]Gallego and Arroyo, 2003a) pointed out the effect of pH values (6 -11) of phosphate-borate (1:1) buffer, buffer component concentrations (10 to 60 mmol L -1 ) and effect of organic modifier (methanol, acetonitrile; 3 -12 %). In our research the presence of 10 % methanol in the electrolyte resulted, in well resolved peaks and the shoulders disappeared. The results demonstrated the separation is better when the pH is between 8 and 9. A 40 mmol L -1 (20 mmol L -1 borate/20 mmol L -1 phosphate buffer) concentration was considered suitable for its good resolution and peak shape. Higher concentration of buffer resulted in peak broadening.

Running voltages effects in the range 5-30 kV were tested using a background electrolyte of 20 mmol L -1 borate/20 mmol L -1 phosphate buffer, pH 8.4, containing 10-80 mmol L -1 sodium dodecylsulphate (SDS) and 10% methanol, without running pressure, at 25°C. The best results were at 25 kV, and an acceptable level of baseline noise was achieved by performing experiments at 25°C.

The influence of SDS in the electrolyte on the migration time (without running pressure) is very significant. The results show that the SDS concentration dramatically affects the migration time of the Zn-BAC and POL B, but not that of SUL and OXY. A concentration of 70 mmol L -1 was selected for the experiment as to give the best resolution, but without a short analysis time (20 min) and without good symmetry and widths of Zn-BAC and POL B peaks. Running pressure was tested in the range 5-30 mbar, using the above experimental conditions. Migration times were decreased with increasing running pressure, and the value of 10 mbar was selected as optimum. It gives the best resolution and symmetric peaks in all cases, and also the analysis time is shorter. Under such experimental conditions the concentration of 20 mmol L -1 SDS was selected as the most advantageous. It gave the best separation without broadened and deformed peaks, and acceptable resolution for Zn-BAC and POL B (Figure 1).

The best results were with applying pressure of 10 mbar at 25 kV. UV detection was at 215 and 254 nm for Zn-BAC, SUL and OXY, 205 and at 215 nm for POL B, for simultaneous detection 215 nm was selected. The electropherograms obtained in the separation under selected conditions are presented in Figure 2. It is remarkable that all peaks have good resolutions in a run time of 12 min (OXY 3.1; SUL 3.7; POL B 9.3; Zn-BAC 10.6). The conditions of MECK with SDS was exceptionally suitable for OXY, since it is eluted as the first peak without any interferences, confirming that this technique could be more efficient in comparison to HPLC, since [START_REF] Oka | Chromatographic analysis of tetracycline antibiotics in foods[END_REF] pointed out that EDTA and oxalic acid should be used during each analytical step (extraction, clean up and HPLC separation). Our recently published paper [START_REF] Injac | Precision of micellar electrokinetic capillary chromatography in the determination of seven antibiotics in pharmaceuticals and feedstuffs[END_REF]) denoted sufficient difference in migration time for two tetracycline antibiotic OXY and doxycycline. The characteristics and the procedures used for validation were those described in USP 24 (USP 24, 2000), the International Conference of Harmonization (ICH) Guidelines (ICH Q2A, 1995;ICH Q2B, 1997) and other literature [START_REF] Heyden | Determination of system suitability limits with a robustness test[END_REF]Shabin, 2003;[START_REF] Brown | Analytical Procedures and Method Validation: Highlights of the FDA's Draft Guidance[END_REF]Toro et al., 2004;[START_REF] Altria | Validation of a capillary electrophoresis method for the determination of a quinolone antibiotic and its related impurities[END_REF][START_REF] Injac | Precision of micellar electrokinetic capillary chromatography in the determination of seven antibiotics in pharmaceuticals and feedstuffs[END_REF].

Validation of the method

Selectivity

The selectivity of the method was investigated by observing interfering peaks from matrix present in the feedstuffs. Five different feedstuff mixtures were tested for the interferences. There was no interference in MEKC results by the feed's ingredients in any of the tested mixtures, which indicates that the method is selective.

Linearity

The linearity of the assay was determined by analysis of a series of standards at five different concentrations in range of 0.1 to 100 mg kg -1 for each compound.

The linearity of calibration curves (peak area vs. concentration) for OXY, SUL, POL B and Zn-BAC over the concentration ranges of 6.5 -77.2 mg kg -1 for OXY, 0.3 -84.5 mg kg -1 for SUL, 18.5 -85.0 mg kg -1 for POL B and 12.1 -62.4 mg kg - 1 for Zn-BAC gave correlation coefficients of 0.9994, 0.9997, 0.9987 and 0.9991, respectively. LODs and LOQs were estimated by the baseline noise method. Baseline noise was evaluated by recording the detector response over a period of ten times the peak width. LOD and LOQ, respectively, were defined as the analyte concentrations resulting in peaks of height three and ten times the baseline noise level ((ICH Q2A, 1995;ICH Q2B, 1997;[START_REF] Heyden | Guidance for robustness/ruggedness tests in method validation[END_REF]. Thus, LODs/LOQs are 1.6/6.2, 0.1/0.3, 6.0/17.5 and 3.7/12.3 mg kg -1 for OXY, SUL, POL B and Zn-BAC, respectively.

Limit of detection (LOD) and limit of quantification (LOQ)

Accuracy

The accuracy of the method was determined by analyzing a solution of known concentration (working standard solution -spiked feed samples with standards) and comparing the measured and known values. Fifteen determinations were carried out for five concentration levels. The mean recovery was 99.7 ± 0.3, 99.9 ± 0.9, 99.81 ± 1.0 and 99.5 ± 0.4 for OXY, SUL, POL B and Zn-BAC, respectively, proving a good accuracy of the method.

Precision

Precision can be measured as repeatability, reproducibility, and intermediate precision. In this work only repeatability and intermediate precision were studied.

Repeatability

A repeatability test was performed to determine intra-day variation in corrected areas and migration times. Working solutions of concentrations 20, 40 and 60 mg L -1 (relative to 20, 40 and 60 mg kg -1 ) were analyzed (n = 6). The RSD values for migration times (0.29 % for OXY, 0.13 % for SUL, 0.94 % for POL B and 0.82 % for Zn-BAC) and for peak areas (0.92-1.76 % OXY, 0.57-0.77 % SUL, 1.12-1.53 % POL B and 0.85-1.72 % Zn-BAC) indicate that the repeatability of the method is acceptable.

Intermediate Precision

Intermediate precision was evaluated over three days by performing working solutions of concentrations 20-50 mg L -1 (relative to 20-50 mg kg -1 ). These were injected on each of three days under the same conditions and the results were used for the repeatability study. When stored in the dark refrigerated, the recovery ranged from 100.2 to 99.1 % over three days. The RSD values (0.12-0.34 % for OXY, 0.09-0.25 % for SUL, 0.39-0.83 for POL B and 0.42-0.81 % for Zn-BC) indicate that the intermediate precision is acceptable. When they were stored at room temperature in sunlight, decreasing recovery values from 100.15 to 94.81 % for Zn-BAC, from 100.40 to 95.03 % for POL B and from 100.01 to 92.98 % for OXY were observed for the standards in BGE.

Robustness

The optimum MEKC conditions set for this method have been slightly modified in order to evaluate the robustness. The effects of different concentrations of SDS (20 ± 1 mmol L -1 ) and organic modifier (10 ± 0.5 % methanol) in the mobile phase, as well as the effects of buffer pH (8.4 ± 0.06), capillary temperature (25 ± 5°C), applied pressure (10 ± 1 mbar), running voltage (25 ± 1 kV) and detection wavelength (± 3 nm), and were determined. The design applied was the [START_REF] Heyden | Guidance for robustness/ruggedness tests in method validation[END_REF]. No significant variations in accuracy, specificity and precision were found over the tested ranges, which indicated that the method conditions are robust.

Stability of antibiotics

The stability of OXY, SUL, POL B and Zn-BAC in acidic (pH < 5) and basic (pH > 10) solutions was checked in test samples at room temperature for 24 and 48 h and the recoveries were 98.2 ± 2.3 % at 24 h and 95.8 ± 3.8 % at 48 h. The stability in the BGE was also checked at 24 h. Recoveries of each compounds were ≥ 99.3 %. It was indicating good stability.

Application

The present method was tested by determining OXY, SUL, POL B and Zn-BAC in animal feedstuffs. When analyzing spiked commercial products, the amounts and recoveries obtained were determined by comparing the results with a standard solution containing the same concentration as expected in the spiked commercial products. The results presented in Table I show good agreement between the claimed and found values. Recovery values also confirmed that, due to the sufficient solubility of investigated compounds, extraction procedure was efficient.

According to LOQ values for each analyte, the minimum quantifiable amounts with the proposed method are 6.2, 0.3, 17.5 and 12.3 mg kg -1 (ppm) of OXY, The obtained results are in correlation that CE is already an official method in USP will also be valuable method as the alternative to HPLC (S. [START_REF] Görög | The changing face of pharmaceutical analysis[END_REF] and the precision of MECK [START_REF] Injac | Precision of micellar electrokinetic capillary chromatography in the determination of seven antibiotics in pharmaceuticals and feedstuffs[END_REF] confirmed the applicability in routine quality control.

The advantage of proposed method over the HPLC and TLC methods described in literature for analysis OXY, SUL, POL B and Zn-BAC in different samples is its lower running costs and higher environmental friendliness. In the proposed method, 20-30 analyses with MEKC require 2 mL of phosphate/borate buffer containing SDS and 10 % (v/v) methanol, while 20 analyses by HPLC require 300 to 1000 mL of mobile phase with different proportion of most commonly used organic solvent (methanol, acetonitrile, acetone, THF). The advantage of LC (MS; MS/MS) methods over MECK is evident for the analysis of residues in samples of different tissues [START_REF] Bogialli | A Liquid Chromatography-Mass Spectrometry Assay for Analyzing Sulfonamide Antibacterials in Cattle and Fish Muscle Tissues[END_REF].

CONCLUSION

The MEKC method presented here is a useful technique for rapid separation (within 12 min) of zinc bacitracin, polymixin B, oxytetracycline and sulfacetamide at low concentration of surfactant (SDS 20 mmol L -1 ) and pH 8.4 (phosphateborate buffer). A pressure of 10 mbar was applied and it gives the best resolution Effect of SDS concentration on resolution with a applied pressure of 10 mbar.

The electrolyte solution was 20 mmol L -1 phosphate + 20 mmol L -1 borate buffer, pH 8.4, containing 10% methanol, and the temperature and voltage were 25°C and 25 kV, respectively. SDS and 10% methanol, the temperature and voltage were 25°C and 25 kV, respectively with applied pressure of 10 mbar.

Table I.

Application results for spiked feedstuff samples.

  and practical challenges for uncovering illegal uses and prevention public health risks.

  All solvents and reagents were of analytical grade unless indicated otherwise.

  Buffer solutions were prepared by dissolving the appropriate amount of NaH 2 PO 4 (20 mmol L -1 ) and Na 2 B 4 O 7 (20 mmol L -1 ) in deionized water and the pH was adjusted to 8.4 with HCl. Na 2 B 4 O 7 x 10H 2 O was p.a. from Kemika (Zagreb, Croatia) and NaH 2 PO 4 x H 2 O was p.a. from J.T. Baker Inc. (Phillipsburg, USA).

  B and Zn-BAC, respectively in feed. The sensitivity of the proposed method is acceptable in relation to the efficiency levels of OXY, SUL, POL B and Zn-BAC as additives and can be utilized for routine control of animal feedstuffs.

  in all cases, special for POL B and Zn-BAC. This system was also applied successfully to their identification and assay in animal feedstuff, with different matrixes, spiked with zinc bacitracin, polymixin B, oxytetracycline and sulfacetamide. Union perspective, Revue scientifique et technique -Office international des epizooties 25(2): 637-653. Shabir GA. 2003.Validation of high-performance liquid chromatography methods for pharmaceutical analysis Understending the differences and similarities between validation requirements of the US Food and Drug Administration, the US Pharmacopeia and the International Conference on Harmonization. Journal of Chromatography A 987: 57-66. Sin DW, Ho C, Wong Y, Ho S, Ip AC. 2005. Analysis of major components of residual bacitracin and colistin in food samples by liquid chromatography tandem mass spectrometry. Analytical Chimica Acta 535: 23-31. Smith RD, Coast J. 2002. Antimicrobial resistance: a global response. Bulletin of the World Health Organization 80: 126-133. muextension.missouri.edu. Accessed 2006 October 7. Toro I, Dulsat JF, Fabregas JL, Claramunt, J. 2004. Development and validation of a capillary electrophoresis method with ultraviolet detection for the determination of the related substances in a pharmaceutical compound. Journal of Chromatography A 1043: 303-315.

  Figure captions (R. Injac et al.)

Figure 2 .

 2 Figure 2. Electropherograms obtained A) 100 mg kg -1 of Zn-BAC, POL B, SUL and OXY standards, and B) 50 mg kg -1 of Zn-BAC, POL B, SUL and OXY from spiked

Figure

  Figure 1 R. Injac et al.

  have used MEKC to separate bacitracin components, with Brij 35 and PAPS as surfactants.

Table I R. Injac et al.

 I

	Page 27 of 30			Food Additives and Contaminants
	Tested	Amount	OXY	OXY	SUL	SUL	POL B	POL B	Zn-BAC	Zn-BAC
	Sample	expected	Amount	Recovery	Amount	Recovery	Amount	Recovery	Amount	Recovery
	(n = 10)	(mg)	found	(%)	found	(%)	found	(%)	found	(%)
			(mg)		(mg)		(mg)		(mg)
	K-19 TL-TIP BEK-1 NSK-1 BOVISAL	50 50 50 50 F o 50 r	49.9±0.1 49.9±0.2 50.0±0.1 49.8±0.1 50.1±0.3	99.8 99.8 100.0 99.6 100.2	48.9±0.5 49.8±0.3 49.9±0.1 49.7±0.4 49.8±0.2	97.8 99.6 99.8 99.4 99.6	48.9±0.4 48.8±0.3 49.3±0.8 49.5±0.5 49.1±0.6	97.8 97.6 98.6 99.0 98.2	49.3±0.3 49.2±0.4 50.2±0.3 50.3±0.1 49.7±0.7	98.6 98.4 100.4 100.6 99.4
		P
		e e r
				R e v i e w
					O n
						l y

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Acknowledgment

This work was financed in part by the Ministry of Science and Environment protection, Belgrade, Serbia, as part of Project no. 142072.


Luis ; Asensio

Isabel ; González

Miguel A Pavón

Teresa García

Rosario Martín

Luis Asensio Gil

Application of an indirect ELISA and a PCR technique to detect grouper (Epinephelus marginatus) adulteration in the fish market

Keywords: Immunoassays, Molecular biology -PCR grouper, adulteration, multiplex PCR, ELISA .manuscriptcentral

Application of an indirect ELISA and a PCR technique to detect grouper (Epinephelus marginatus) adulteration in the fish market

Introduction

Fresh and frozen fish fillets, where only a portion of flesh is available, are difficult to identify since many of the distinguishing morphological features are no longer detectable. This opens the possibility of fraudulent adulteration and substitution of the expected fish species with others of less value.

In this context, grouper (Epinephelus marginatus) is a highly appreciated and expensive fish in the Spanish fish market. Due to its high cost, popularity and demand, grouper fillets are subjected to substitution using cheaper fish species such as Nile Perch (Lates niloticus). Also, grouper can be substituted by closely related species such as wreck fish (Polyprion americanus); this fish species is renamed as grouper to make it sound more appealing to consumers. Therefore, analytical approaches for the specific identification of commercial fish species are required for detection of inappropriately labelled products (Woolfe and Primrose 2004).

Different protein-based methods have been reviewed for fish species identification, such as electrophoretic, chromatographic and immunological techniques [START_REF] Dreyfuss | Agar-gel immunodiffusion test for rapid identification of Pollock surimi in raw meat products[END_REF][START_REF] Knuutinen | Identification of fish species by reversed-phase high-performance liquid chromatography with photodiode-array detection[END_REF][START_REF] Berrini | Identification of freshwater fish commercially labelled "perch" by isoelectric focusing and two-dimensional electrophoresis[END_REF][START_REF] Martinez | High resolution two-dimensional electrophoresis as a tool to differentiate wild from farmed cod (Gadus morhua) and to assess the protein composition of klipfish[END_REF]). Nevertheless, in food analysis, the Enzyme-Linked ImmunoSorbent Assay (ELISA) has been the most widely used technique for regulatory purposes in detecting food authenticity because of its specificity, simplicity and sensitivity, among other advantages [START_REF] Asensio | Determination of food authenticity by enzyme-linked immunosorbent assay (ELISA)[END_REF]. However, work relating to fish species identification is scarce, partly due to the variety of fish species that are commercialized. To date, enzyme immunoassays have been used for rock shrimp [START_REF] An | Development of monoclonal antibody for rock shrimp identification using enzyme-linked immunosorbent assay[END_REF], sardines [START_REF] Taylor | An immunoassay for verifying the identity of canned sardines[END_REF], red snapper [START_REF] Huang | Development of monoclonal antibodies for red snapper (Lutjanus campechanus) identification using enzyme-linked immunosorbent assay[END_REF], flat fish [START_REF] Céspedes | Indirect enzyme-linked immunosorbent assay for the identification of Sole (Solea solea), European plaice (Pleuronectes platessa), flounder (Platichthys flesus), and Greenland Halibut (Reinhardtius hippoglossoides)[END_REF], or grouper identification (Asensio et al. 2003a), among others.

In recent years, significant attention has been turning towards DNA-based approaches, which have proven to be reliable, sensitive and fast for many aspects of food authentication. Among them, polymerase chain reaction (PCR) based techniques are undoubtedly the most common genetic methods used for tracing the species origin in food [START_REF] Lockley | DNA-based methods for food authentication[END_REF]. PCR-based methods commonly used for fish species identification include random amplified polymorphic DNA (RAPD) [START_REF] Bartfai | Genetic analysis of two common carp broodstocks by RAPD and microsatellite markers[END_REF], PCR-restriction fragment length polymorphism (PCR-RFLP) [START_REF] Santaclara | Development of a method for genetic identification of four species of anchovies: E. encrasicolus, E. anchoita, E. ringens and E. japonicus[END_REF], PCR-single strand conformational polymorphism (PCR-SSCP) [START_REF] Colombo | Identification of tuna species by computer-assisted and cluster analysis of PCR-SSCP electrophoretic patterns[END_REF], multiplex-PCR [START_REF] Infante | PCR-based methodology for the authentication of the Atlantic mackerel Scomber scombrus in commercial canned products[END_REF]) and PCR lab-on-a-chip [START_REF] Dooley | Improved fish species identification by use of lab-on-a-chip technology[END_REF], among others.

On the basis of this information, this paper reports the application of two alternatives for the detection of inappropriately labelled grouper fillets in the marketplace: a multiplex PCR technique relying on the 5S rDNA gene [START_REF] Asensio | Identification of Nile perch (Lates niloticus), grouper (Epinephelus guaza), and wreck fish (Polyprion americanus) fillets by PCR amplification of the 5S rDNA gene[END_REF], and an indirect ELISA assay that uses two monoclonal antibodies against grouper muscle proteins (Asensio et al. 2003a;Asensio et al. 2003b).

Both methodologies provide a specific, sensitive and reliable means to detect adulteration of grouper, and may assist the regulatory agencies to enforce

Materials and methods

Fish samples collection

Grouper (Epinephelus marginatus), wreck fish (Polyprion americanus), Nile perch (Lates niloticus) and other fish species (Table 1) were obtained from local markets from Madrid. Every species was morphologically identified according to the keys of [START_REF] Muus | Peces de mar del Atlántico y del Mediterráneo. Guía de identificación[END_REF] and [START_REF] Froese | Editors of FishBase[END_REF].

In addition, 70 commercial fish fillets samples (Table 2) labelled as grouper were collected at random in different local markets and supermarkets from Madrid.

Preparation of fish samples for ELISA analysis

Fish muscle samples (1 g) were homogenized in 5 mL of saline solution (8.5 g of NaCl/liter) and maintained for 10 min in a stomacher. Extracts containing soluble muscle proteins were filtered through a Whatman no. 1 filter paper.

Protein extracts were diluted 1/50 in phosphate buffered saline (PBS) before they were analyzed by the ELISA technique described bellow.

Indirect ELISA

Flat-bottomed micro-ELISA plates (Costar, Corning, NY) were filled with 0.1 mL of the diluted muscle protein extracts and incubated for 1 h at 37 ºC. After washing with PBST (PBS containing 1% Tween 20), 40 µL of the 3D12 and 1A4 monoclonal antibodies (MAbs) developed previously (Asensio et al. 2003a; Asensio et al. 2003b) were diluted in 60 µL of PBS and the total volume (100 µL) was added to the wells. The plates were shaken for 1 h at room temperature and, after washing with PBST, 0.1 mL aliquots of peroxidaseconjugated rabbit antimouse immunoglobulins (DAKO, Glostrup, Denmark) diluted 1/2000 in PBST were added to the wells. The plates were incubated with shaking for 1 h at room temperature and the wells were washed with distilled water before addition of 0.05 mL of a ready-to-use substrate solution of 3,3´,5,5´,-tetramethylbenzidine (Boehringer Mannheim GmbH, Mannheim, Germany). After 10 min of incubation, the reaction was stopped by addition to each well of 0.025 mL of 1 M H 2 SO 4 . The yellow color developed by conversion of the substrate was measured at 450 nm with an iEMS Reader MF Spectrophotometer (Labsystems, Oy, Helsinki, Finland).

DNA extraction procedure

Genomic DNA was extracted from fish samples using a Wizard DNA cleanup kit (Promega, Madison, WI, USA). The extraction procedure was performed as follows: Samples (0.2 g) were digested in 860 µL of extraction buffer, pH 8.0 (10 mM Tris, 150 mM NaCl, 2 mM EDTA and 1% SDS), 100 µL of 5 M guanidine hydrochloride and 40 µL of 20 mg mL -1 proteinase K (Boehringer Manheim GmbH, Mannheim, Germany). Digests were incubated overnight at 55 ºC with shaking at 70 x g, and were left to cool at room temperature. 500 µL of chloroform were added to the lysate before centrifugation at 10.000 x g for 10 min. The clear aqueous supernatant obtained after the centrifugation (500 µL) was used to purify the DNA using the Wizard DNA cleanup system kit (Promega) with a vacuum manifold, according to the manufacturer´s

Results and discussion

Food quality and safety are priority research areas that include traceability along the production chain. Then, it is necessary to develop reliable traceability techniques to establish the origin and mode of manufacturing fishery products needed to prevent fraud and consequently to assure consumer confidence and to enable rapid product withdrawal [START_REF] Santaclara | Development of a method for genetic identification of four species of anchovies: E. encrasicolus, E. anchoita, E. ringens and E. japonicus[END_REF]. In this context, analytical tools such as immunoenzymatic assays (ELISA) are being increasingly demanded by law enforcement agencies for fish and fishery products identification [START_REF] Asensio | Determination of food authenticity by enzyme-linked immunosorbent assay (ELISA)[END_REF]. Moreover other approaches include PCR-based methods, which have also proved successful for fish species authentication [START_REF] Mackie | Challenges in the identification of species of canned fish[END_REF][START_REF] Gil | PCR-based methods for fish and fishery products authentication[END_REF]. In the present study, we

ELISA analysis

Among immunological methods, the enzyme-linked immunosorbent assay (ELISA) has been the most widely used technique for regulatory purposes in detecting fish species adulteration, because of its simplicity, specificity and sensitivity [START_REF] Céspedes | Indirect enzyme-linked immunosorbent assay for the identification of Sole (Solea solea), European plaice (Pleuronectes platessa), flounder (Platichthys flesus), and Greenland Halibut (Reinhardtius hippoglossoides)[END_REF]).

The pre-requisite for an ELISA is the availability of amounts of antibodies sufficient to detect analytes. Both polyclonal and monoclonal antibodies (MAbs) can be used in ELISA methods for fish identification. Monoclonal antibodies, compared to polyclonal antibodies, are a homogeneous population of antibodies produced by hybridoma technology that have defined biological activity, consistent specificity and their production is not limited [START_REF] Goding | Monoclonal Antibodies: Principles and Practice[END_REF].

In two previous publications, we reported the development of two monoclonal antibodies specific to grouper (3D12 MAb) (Asensio et al. 2003a) and grouper and wreck fish (1A4 MAb) (Asensio et al. 2003b). Firstly, to study the specificity of these MAbs, other fish species were analysed by ELISA using these MAbs.

As it can be seen in the MAbs were compared, we could distinguish wreck fish from grouper samples, so that, we could identify specifically 13 samples of wreck fish out of 70 (Table 2).

This ELISA procedure, which can be performed in less than 4 h, has proved to be specific, sensitive, cheap and very simple to develop. It could be very useful as routine analysis tools in food control laboratories for enforcing labeling regulations in the authentication of these fish species.

Multiplex PCR

Detailed sequence information has become available for many species and consequently phylogenetically informative single base polymorphisms may be identified that enable species-specific primers to be designed. Under suitably stringent reaction conditions, such primers generate a product only in the presence of DNA from a given species. Complete sequence information permits the size of the product to be predicted, so that identification is confirmed if an appropriately sized amplicon is seen on a gel [START_REF] Lockley | DNA-based methods for food authentication[END_REF]. Recent works have reported the application of specific primers for fish and fishery products authentication [START_REF] Taylor | Species-specific TaqMan probes for simultaneous identification of cod (Gadus morhua L.), haddock (Melanogrammus aeglefinus L.) and whiting (Merlangius merlangus L.)[END_REF][START_REF] Hsieh | Application of DNA technique for identifying the species of different processed products of swordfish meat[END_REF].

In this context and in agreement with the results obtained in a previously reported work [START_REF] Asensio | Identification of Nile perch (Lates niloticus), grouper (Epinephelus guaza), and wreck fish (Polyprion americanus) fillets by PCR amplification of the 5S rDNA gene[END_REF], the combination of different primers specific for grouper (5SG), Nile perch (5SP) and wreck fish (5SW), along with the 5S1 oligonucleotide (forward primer), allowed the amplification of specific regions of the 5S rDNA gene: a 323 bp fragment from grouper DNA, a 471 bp fragment from wreck fish DNA and a 185 bp fragment from Nile perch DNA (Figure 1).

To determine the specificity of this procedure, other fish species were analysed by using these markers, and no amplification was obtained (Table 1). Figure 1 shows an example of this specificity study. Moreover, to avoid false negative PCR results and verify positive amplification we used an internal control based on the 18S rRNA gene. All fish species studied in this work were amplified by this marker (Table 1), which is eukaryotes specific.

Also, the commercial samples collected from the fish shops were analysed by using the multiplex PCR. PCR amplifications results matched unequivocally with ELISA results showing a complete agreement of both methods allowing the authentication of grouper (Table 2). In addition, the multiplex PCR allowed to detect 34 samples of Nile perch out of the 70 fish samples. Nile perch is the fish species most used as a substitute for grouper in Spain. Likewise, 11 samples analysed were unknown: no amplification was obtained. Probably other cheap fish species could be used to substitute grouper. Also, to avoid false negatives The application of this multiplex PCR is a powerful approach for the specific identification of grouper fillets. Since it is very specific, rapid, reliable and easy to perform, this methodology is an useful alternative for detection of grouper adulteration in the market of fish fillets.

Conclusions

  -national laws and regulations (Council Regulation (EC) 104/2000; Resolution of 27 th of February 2007 of the Spanish Secretary General of Maritime Fishing).

8

  , the DNA was eluted in 100 µL of sterile deionized water and its concentration was determined by spectrophotometry at 260 nm(Eppendorf Biophotometer, Hamburg, Germany).Polymerase Chain Reaction (PCR)The set of primers used for PCR amplification of DNA were the forward primer 5S1 (5´-TACGCCCGATCTCGTCCGATC-3´), designed byPendás et al. (1994), ) designed by[START_REF] Asensio | Identification of Nile perch (Lates niloticus), grouper (Epinephelus guaza), and wreck fish (Polyprion americanus) fillets by PCR amplification of the 5S rDNA gene[END_REF]. These primers were mixed in the same ratio and used together for the multiplex PCR of this study. Double-stranded amplifications were carried out in a final volume of 25 µl containing 2 mM MgCl2, 10 pmol of each primer (5S1, 5SG, 5SW and 5SP), 20 ng of template DNA, and 2 U of DNA polymerase (Roche, Mannheim, Germany) in a reaction buffer containing 75 mM Tris-HCl, pH 9.0, 50 mM KCl, 20 mM (NH 4 ) 2 SO 4 and 0.001% bovine serum albumin (BSA).PCR amplification was performed in a Progene Thermal Cycler (Techne Ltd, Cambridge, UK). Thirty-five cycles of amplification with the following step-cycle profile were carried out: strand denaturation at 94 ºC for 45 s, primer annealing at 66 ºC for 45s and primer extension at 72 ºC for 45 s. An initial denaturation at 94 ºC for 3 min, and a final extension at 72 ºC for 5 min, improved the product yielded.To avoid false negative PCR results and verify positive amplification of fish species analysed in this study we designed an eukaryote-specific primer pair: 18Seu-fw (5´-GGTAGTGACGAAAAATAACAATACAGGAC-3´)/18Seu-inv (5´-ATACGCTATTGGAGCTGGAATTACC-3´) flanking a 141 bp fragment from 18S rRNA gene was used as an internal control of the assay. Amplification was performed with the following steps: a denaturation step of 93 ºC for 2 min, followed by 35 cycles with a strand denaturation at 93 ºC 30 s, primer annealing at 60 ºC for 30 s, and primer extension at 72 ºC for 45 s. The final extension step was performed at 72 ºC for 5 min.PCR results were determined by visualization of amplicons on 2% agarose gels stained with ethidium bromide (1 µg/mL). The sizes of the DNA fragments were estimated by comparison with a commercial 1Kb plus DNA ladder (Gibco BRL, Life Technologies Inc., Rockville, MD).

  of an indirect ELISA by using two MAbs and a multiplex PCR assay relying on the 5S rDNA gene, to detect grouper adulteration in the fish shops.

  amplified the internal control used in this study (data not shown). Figure2shows an example of electrophoresis of various commercial samples analysed by this technique.

Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .

 2 Figure 2. Electrophoretic analysis of the 5S rDNA amplicons from the multiplex

Table 1 ,

 1 3D12 MAb recognized specifically the grouper and did not show cross-reactivity with the other species analysed in this work.

	Furthermore, 1A4 MAb only recognized grouper and wreck fish specimens. The
	absorbance values of negative samples were similar to those of the background

  Results obtained in this work suggest that both the ELISA and PCR techniques may provide a reliable means for the specific detection of inappropriately labelled grouper fillets. PCR-based methods are the most specific and sensitive methods for fish species identification, although they require some expensive laboratory equipment and a certain degree of knowledge. On the contrary, ELISA, being sensitive and specific, are quicker than genetic methods for routine analysis of large sample numbers. In spite of ELISA technique was unable to recognise Nile perch from other unknown species used to adulterate the grouper, both procedures reported here may represent two effective and straightforward tools for grouper authentication and this work is a good example of applied research and its direct impact on the lives and health of consumers.Woolfe M, Primrose S. 2004. Food forensics: using DNA technology to combat misdescription and fraud. Trends in Biotechnology 22:222-226.

	F o r F o r F o r F o r F o r F o r
	P P P P P P
	e e e e e e
	e r e r e r e r e r e r
	R R R R R R
	e e e e e e
	v i e v i e v i e v i e v i e v i e
	w w w w w w
	O n l O n l O n l O n l O n l O n l
	y y y y y y
	18

Table 1 .

 1 Different fish species analysed by ELISA and PCR techniques.

		ELISA by
	Fish species analysed	1A4 MAb

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Acknowledgments


Keywords: anabolic steroids, nutritional supplements, foodstuffs, two dimensional gas chromatography, time-of-flight mass spectrometry

Development of method employing comprehensive two dimensional gas chromatography with time-of-flight mass spectrometric detection for determination of anabolic steroids and related compounds in nutritional supplements

Radim Stepan, Petr Cuhra, Sona Barsova

To cite this version:

Introduction

Sport is a common world-wide leisure activity. During the last decades various nutritional supplements promoting muscle strength and supporting regeneration of human body after physical performance have been introduced on a market. These products are currently used not only by professional athletes, but also by many people for various leisure sport activities. [START_REF] Geyer | Positive Dopingfälle mit Norandrosteron durch verunreinigte Nahrungsergänzungsmittel[END_REF] reported a significant problem regarding the quality of some nutritional supplements was the cross-contamination with anabolic steroids. Additionally there are some producers that adulterate nutritional supplements i.e. illegally add anabolic steroids into their products to support a muscle growth effect and consequently increase sales. High amounts of anabolic steroids can be found in these samples [START_REF] Geyer | High doses of the anabolic steroid metandienone found in dietary supplements[END_REF][START_REF] Parr | High amounts of 17-methylated anabolic-androgenic steroids in effervescent tablets on the dietary supplement market[END_REF]. Anabolic steroids may enhance muscle growth, but their non-supervised use may lead to serious health problems (e.g. kidney or liver cancer) and therefore their application other than for therapeutic and medical usage is not allowed in the European Union. For this reason the Czech Agriculture and Food Inspection Authority (CAFIA) established a national monitoring programme to check for the presence of anabolic steroids in nutritional supplements and developed an analytical method to test for a spectrum of compounds preferably which are specified in the Prohibited list of substances published by World Anti-Doping Agency (WADA 2007).

Several analytical methods for the determination of anabolic steroids in nutritional supplements have been described [START_REF] Cock | Detection and determination of anabolic steroids in nutritional supplements[END_REF][START_REF] Thuyne | Validation of a GC-MS screening method for anabolizing agents in solid nutritional supplements[END_REF][START_REF] Thuyne | Validation of a GC-MS screening method for anabolizing agents in aqueous nutritional supplements[END_REF]Parr 2004). These methods usually include dissolution of the sample in an alkaline medium (e.g. solution of sodium hydroxide) followed by extraction with n-pentane and finally derivatization of target compounds before GC-MS analysis. In this paper, a simple and effective analytical approach employing ethyl acetate extraction, dispersive solid phase extraction (SPE) clean-up using primary secondary amine (PSA) followed by analysis of underivatised compounds using comprehensive two dimensional gas chromatography with time-of-flight mass spectrometric detection (GCxGC-TOF MS) is presented. This method was validated for the determination of 25 anabolic steroids and related compounds specified on the WADA list in two types of solid nutritional supplements: (i) protein concentrate and (ii) creatine monohydrate.

Materials and Methods

Reference standards and chemicals

A total 25 compounds were included in the method (Table I). Steroid standards were obtained from Steraloids Inc. (USA) except for metandienone (Cerilliant, USA), trenbolone acetate and testosteron 17-propionate (Dr. Ehrenstorfer, Germany), oxandrolone, oxymetholone and boldenone (Alltech -Applied Science Labs, USA) and nandrolone, progesterone, testosterone and 17α-methyltestosterone (Riedel-de Haën / Sigma-Aldrich, Germany). Organic solvents ethyl acetate and methanol for trace analysis (Lab-Scan, Ireland) were used. Primary secondary amine (Bondesil PSA, particle size 40 µm) for clean-up of ethyl acetate extracts was supplied by Varian Inc., USA.

[Insert Table I about here]

Materials

Two different types of solid nutritional supplements (protein concentrate and creatine monohydrate) that are commonly sold and/or used in fitness centres were selected for I) were prepared by dilution of solid standards in ethyl acetate except for fluoxymesterone, methylandrostenediol, oxandrolone and 5-androsten-3β,17β-diol where mixture ethyl acetate : methanol (1 : 1) was used. These stock solutions were diluted with ethyl acetate to obtain standard mixture STD1 (concentrations 1.090 -27.324 µg.ml -1 ).

Working solutions STD2 -STD6 were prepared by dilution STD1 with ethyl acetate as follows: STD2 = STD1:ethyl acetate, 1:1, v/v; STD3 = STD1:ethyl acetate, 1:4, v/v; STD4 = STD1:ethyl acetate, 1:9, v/v; STD5 = STD1:ethyl acetate, 1:24, v/v; and STD6 = STD1:ethyl acetate, 1:69, v/v. Sample preparation. A sample of protein concentrate (1 g) was weighed into a 10 ml conical -bottom polypropylene centrifuge tube with a screw cap. A 5 ml amount of ethyl acetate was added and a mixture was shaken vigorously for 0.5 min and then treated in an ultrasonic bath for 5 min. To separate insoluble solid particles of mixture tube was centrifuged at 8000 g for 5 min. Approximately of 5 ml liquid phase was transferred into a second centrifuge tube containing 0.25 g primary secondary amine (PSA). To the remaining part of the sample in the first tube second portion (5 ml) of ethyl acetate was added and mixture was subjected to the same procedure (shaker 0.5 min, ultrasonic bath 5 min, centrifuge 8000 g / 5 min). The combined crude extracts (approximately 10 ml in total) were shaken for 0.5 min with PSA and centrifuged (8000 g, 5 min). Clean extract was transferred into conical -bottom flask (50 ml) and ethyl acetate was evaporated (230 mbar, 38 o C) nearly to dryness. The residual solvent was removed with a gentle stream of nitrogen and the residue was re-dissolved in 1 ml ethyl acetate. Before injection (1 µl) into the GCxGC-TOF MS extract was filtrated through PTFE membrane filter. Because creatine monohydrate (commercial product) was a pure substance insoluble in ethyl acetate cleaning step with PSA was omitted in this case due to the absence of co-extracted matrix components. The other two types of nutritional supplements analysed within the monitoring programme were treated either with or without PSA clean-up. While nutritional supplements which contain synephrine (i.e. "fat burners") were extracted as protein concentrates because of their complex matrix composition, creatine pyruvate samples were prepared without PSA in the same way as creatine monohydrate because of their physical and chemical similarity.

Preparation of matrix-matched standards. To minimize bias resulting from different amounts of analyte transferred from injection liner to the 1st capillary column due to so-called matrix effects [START_REF] Erney | Matrix-induced peak enhancement of pesticides in gas chromatography: Is there a solution[END_REF][START_REF] Hajšlová | Review: Matrix effects in (ultra)trace analysis of pesticide residues in food and biotic matrices[END_REF]) matrix-matched standards were prepared for compensation of these effects and evaluation of analyte recovery associated with the whole analytical method and evaluation of detection limits. A 1 g amount of a corresponding blank matrix (e.g. protein concentrate without steroids) was subjected to the procedure described in the paragraph Sample preparation. Dry residue after clean-up of crude extract and removal of ethyl acetate was redissolved in 1 ml of corresponding working solution (STD3, STD5 or STD6) and filtrated through PTFE membrane filter prior to the injection (1 µl) into the GCxGC-TOF MS. Validation procedure. A series of ten samples spiked with 200 µl STD1 before addition of extraction solvent was realised for both types of nutritional supplements (i.e. protein concentrate and creatine monohydrate). Samples were processed by the analytical method described in above paragraph (concentration of target analytes before injection into the GCxGC-TOF MS corresponded to STD3 i.e. it was 0.218 -5.465 mg.kg -1 , see Table II ). In each series repeatability expressed as relative standard deviation (RSD, %) of sample concentration was calculated. For calculation of analyte recovery concentration in spiked samples was compared with that of in matrix-matched standard (concentration level STD3) injected into GCxGC-TOF MS before and after a series of ten spiked samples. The limits of detection (LODs) were evaluated from a chromatographic records of matrix-matched standards (STD5 -STD6) and represented the concentration of analyte that generated a response for which average signal-to-noise ratio S/N = 3 was achieved. Reporting limit represents the lowest level at which analytes are reported as absolute numbers [START_REF] Sanco | Quality control procedures for pesticide residue analysis. Guidelines for residues monitoring in the European Union[END_REF]. According to this document reporting limit was considered as practical limit of quantification (LOQ) i.e. represented the minimum concentration of analyte that can be quantified with acceptable accuracy and precision whereas it is not lower than the corresponding lowest calibrated level (LCL). LCL was assessed as the lowest concentration of analyte with which the determination system was successfully calibrated, i.e. analyte response remained measurable throughout the analysis batch, whereas LCL is at least equal or higher than 3 x LOD. As described in the mentioned document [START_REF] Sanco | Quality control procedures for pesticide residue analysis. Guidelines for residues monitoring in the European Union[END_REF] for EU monitoring purposes where samples for surveys are analysed over a 12-month period, the same reporting limit should be achievable throughout the whole year.

Separation and detection. Sample analysis was performed using

Results and Discussion

Optimisation of sample preparation step

Due to the physico-chemical properties of the steroids, ethyl acetate was selected for sample extraction as a moderately polar solvent. Common laboratory procedures (shaking and ultrasonic treatment of sample) were used for improvement of dissolving of steroids in the solvent. For removing matrix components isolated together with target compounds from a sample a suitable clean-up procedure was searched. Analysis of crude extract of protein concentrate carried out using two dimensional gas chromatography with time-of-flight mass detection showed relatively large amount of fatty acids (palmitic acid, stearic acid), compounds related to saccharides (1,6-anhydro-D-glucopyranose, 1,6-anhydro-Dgalactofuranose) and aromatic compounds (vanillin, 4-hydroxy-2-methoxycinnamaldehyde) (Fig. 1A). All of these compounds were reliably identified, i.e. high match between measured and library spectra was achieved, using deconvolution algorithm of ChromaTOF software.

These coextractives (especially fatty acids) may create deposits in an injection liner and also in first part of capillary column and therefore negatively affect chromatographic separation as well as quantification of target compounds. There are several clean-up procedures that may help to remove co-extracted compounds from crude extracts. As described in previous studies liquid-liquid partition (Parr 2004[START_REF] Thuyne | Validation of a GC-MS screening method for anabolizing agents in solid nutritional supplements[END_REF] introduced as a part of so called QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) multiresidue method for determination of pesticide residues in plant matrices [START_REF] Anastassiades | Fast and easy multiresidue method employing acetonitrile extraction / partitioning and "dispersive solid-phase extraction" for the determination of pesticide residues in produce[END_REF]. As described by authors of the mentioned method dispersive SPE employing primary secondary amine (PSA) as sorbent can effectively remove (due to interactions with chemicals by hydrogen bonding) polar compounds like organic acids, fatty acids, sugars, pigments etc.

As shown in Fig. 1B, in case of our method PSA substantially reduced content of 1,6-anhydro-D-glucopyranose,1,6-anhydro-D-galactofuranose, 4-hydroxy-2-methoxycinnam aldehyde and partly also fatty acids and vanillin.

[Insert Figure 1 about here]

The potential of the GCxGC-TOF MS technique is fully demonstrated in the unique identification of steroids at trace concentration levels. Due to compression of elution zones during modulation, analyte peaks in a second dimension are narrower than in a conventional one dimensional GC system. Therefore signal-to-noise (S/N) ratios are substantially higher and limits of detection are lower (Fig. 2). Further important feature of two dimensional chromatography in comparison with a conventional GC employed in other studies [START_REF] Cock | Detection and determination of anabolic steroids in nutritional supplements[END_REF], Parr 2004[START_REF] Thuyne | Validation of a GC-MS screening method for anabolizing agents in solid nutritional supplements[END_REF]) is enhanced chromatographic resolution of compounds that is achieved as result of sample separation performed in two capillary columns with different polarities. For separation of steroids combination of non-polar DB-5 MS column (5% phenylmethylpolysiloxane) in the 1st dimension and moderately polar BPX-50 column (50% phenyl polysilphenylene -siloxane) in the second dimension was recognized as the most convenient. As shown in Fig. 3 steroids were successfully separated from co-extracted matrix components and also in spite of similar chemical structures and physico-chemical properties all of 25 target compounds were separated from each other. The essential and key advantage of time-of-flight mass spectrometry is a collection of full mass spectra during the whole analytical run. This fact together with data processing employing deconvolution algorithm that enable reveal identity of potentially co-eluted compounds substantially reduce a risk of false positive findings of target analytes.

Considerable feature of our method is also absence of derivatisation step employed usually in other studies [START_REF] Cock | Detection and determination of anabolic steroids in nutritional supplements[END_REF], Parr 2004, Thuyne 2003) and therefore possibility to realise socalled non-target analysis for potential detection of new unexpected substances by a comparison of mass spectra of underivatised compounds with a spectral library.

[Insert Figure 2 and Figure 3 about here]

Method validation

An in-house validation procedure was performed in accordance with IUPAC harmonized guidelines [START_REF] Thompson | Harmonised guidelines for single -laboratory validation of methods of analysis. IUPAC technical report[END_REF] and other relevant documents regulating this field [START_REF] Thompson | Harmonised guidelines for the use of recovery information in analytical measurement[END_REF][START_REF] Sanco | Quality control procedures for pesticide residue analysis. Guidelines for residues monitoring in the European Union[END_REF], ISO/IEC 17025). Repeatability expressed as relative standard deviation of analyte concentration (RSD, %), method recovery (%), calibration linearity and reporting limits were evaluated for each compound in two series of experiments (each series for one kind of the supplement).

As it is shown in Table II acceptable repeatability and recovery were obtained for all compounds except for oxymetholone in protein concentrate. In contrast to the other steroids oxymetholone has slightly different chemical structure, i.e. there are hydroxy-and oxogroups bonded to the same ring (Fig. 4). Due to this fact there are probably strong hydrogen interactions between oxymetholone and PSA resulting in a very low recovery as a result of [Insert Figure 4 about here]

[Insert Table II about here] Linearity of multi-point calibration was evaluated using regression coefficient. This parameter was calculated for individual compounds from peak areas (ions used for quantification are specified in Table II) of six calibration standards. Table III shows concentration ranges and regression coefficients obtained for individual compounds.

Acceptable reporting limits (see Table IV) were obtained for all target compounds. These reporting limits are low enough to determine concentrations of steroids that can be found in samples of nutritional supplements (Geyer 2004).

[Insert Table III from producers, importers and shops dealing with products for sport nutrition including fitness centres. Four samples of natural products containing extracts from Citrus aurantium were also analysed. An active compound extracted from Citrus aurantium called synephrine is chemically related to stimulants as ephedrine or caffeine and should enhance a cell metabolism. For this reason synephrine containing nutritional supplements are marketed as a natural "fat burners". These natural products were sent to our authority by different consumers suspecting them of contamination with prohibited substances. Besides monitoring programme CAFIA as a national food authority is responsible also for solution of consumer´s complaints so that we performed screening of these products for steroids.

Results from this monitoring programme showed a total 6.3 % (i.e. 3) positive samples. Nandrolone (0.022 mg.kg -1 ), testosterone (0.070 mg.kg -1 ) and DHEA (0.063 mg.kg -1 ) were found in a whey protein gainer, 5α-Androstan-3,17-dione (0.398 mg.kg -1 ) and 19-Norandrostendione (0.304 mg.kg -1 ) in creatine pyruvate and one sample of synephrine based "fat burner" contained progesterone (0.102 mg.kg -1 ). In contrast to anabolic androgenic steroids progesterone has an gestagenic activity. From this point-of-view the presence of progesterone in the sample was unreasonable and was probably due to cross-contamination during production.

Conclusions

This analytical method based on the comprehensive two dimensional gas chromatography with time-of-flight mass spectrometric detection provides an advantageous strategy for the determination of anabolic androgenic steroids and related compounds in nutritional supplements. Further development and extension of this method towards the other kinds of supplements that are commonly available on a market (e.g. mixtures of amino acids, a 3. Methylandrostenediol,5. Dromostanolone,7. Nandrolone,9. Mesterolon,10. Testosterone,11. Mibolerone,17dione,14. Boldenon,16. 7α,17. Metandienone,19. 1. DHEA,3. Methylandrostenediol,5. Dromostanolone,7. Nandrolone,9. Mesterolon,10. Testosterone,11. Mibolerone,17dione,14. Boldenon,16. 7α,17. Metandienone,19. Methenolone,20. Trenbolone acetate,21. Oxandrolone,22. Progesterone,23. Testosteron 17propionate,24. Oxymetholone,25. Fluoxymesterone

  Protein concentrate consisted of whey protein, vitamins, minerals and vanilla flavour, creatine monohydrate was declared as a pure substance.ApparatusA vibrating shaker Heidolph Reax Top (Heidolph, Germany) and a centrifuge Hermle Z 383 K (Hermle, Germany) were used for shaking and centrifugation of extracts. An ultrasonic compact cleaner UCC 4 (Powersonic, Slovakia) for ultrasonic treatment of sample during extraction was used. Ethyl acetate from purified extracts was evaporated using a vacuum rotary evaporator Büchi Rotavapor (Büchi, Switzerland). PTFE membrane filters (0.45 µm, Millipore, USA) were used for filtration of extracts prior injection into GCxGC-TOF MS.Two dimensional chromatographic analysis of steroids was performed using a gas chromatograph Agilent 6890 N (Agilent technologies, USA) equipped with an autosampler Agilent 7683 Series (Agilent technologies, USA), modulator and oven for second capillary column (both LECO Corporation, USA). A column for the second dimension was connected via a transfer line to a time-of-flight mass spectrometer LECO Pegasus III (LECO Corporation, USA). Dry air and nitrogen were used for (i) hot and (ii) cool stage of comprehensive sampling (i.e. modulation) of eluent from the first capillary column and its introduction to the second column. Compressed dry air was prepared using oil-free compressor Atlas Copco SF1 equipped with a moisture trap Atlas Copco CD 3 (Atlas Copco Airpower, Belgium). Liquid dry nitrogen (dew point < -196 o C ) was supplied by Messer Technogas (Czech Republic) and stored in a container Euro-Cyl 180/24 (Chart -Ferox, Preparation of standard solutions. Individual stock solutions of steroids (concentrations see Table

  GCxGC-TOF MS under following operating conditions: the injection (1 µl) was carried out by splitless technique (inlet temperature 280 o C, splitless period 1 min); capillary columns DB5-MS (30 m x 0.25 mm x 0.25 µm; J&W Scientific, USA) and BPX-50 (2 m x 0.1 mm x 0.1 µm; SGE, USA) were used for separation in the 1st and 2nd dimension resp.; temperature programme (DB5-MS): 110 o C (1 min), 10 o C min -1 to 290 o C (10 min); temperature programme (BPX-50): 120 o C (1 min), 10 o C min -1 to 300 o C (10 min); modulator: modulation period 7.5 sec, cold jet min -1 (dry nitrogen), hot jet flow 20 l min -1 (dry air); TOF-MS: transfer line temperature 300 o C, ion source (temperature 220 o C, electron ionization), acquisition rate 50 spectra / sec, mass range 35 -500 amu, detector voltage 1550 V. Instrument control, data acquisition as well as data processing were performed using ChromaTOF 2.32 software (LECO Corporation, USA).

  or liquid chromatography[START_REF] Cock | Detection and determination of anabolic steroids in nutritional supplements[END_REF]) can be employed for clean-up of extracts, however in our opinion, these methods are relatively laborious and time-consuming. For this reason novel modification of SPE called dispersive solid phase extraction was considered as potential clean-up procedure. Dispersive SPE was

  protein concentrate, nevertheless repeatability is not affected.. Repeatability and recovery of oxymetholone in creatine monohydrate, where clean-up based on PSA in omitted, remains satisfactory. Based on these results acceptable determination of oxymetholone is restricted only to samples where the clean-up procedure can be skipped (e.g. creatine monohydrate, creatine pyruvate).

  Fig. 1. Surface plot i.e. 3D-view of two dimensional chromatogram (Total Ion Current, TIC)

Fig. 3 .

 3 Fig. 2. Peak of nandrolone (2.834 µg/ml, ion 274) obtained by a conventional (A) and two

Methenolone, 20 .

 20 Fig. 4. Comparison of oxymetholone (A) and 17α-Methyltestosterone (B) as a representative

Fig. 1 .Fig. 2 .Fig. 3 .

 123 Fig. 1. Surface plot i.e. 3D-view of two-dimensional chromatogram (Total Ion Current, TIC)

Fig. 4 .

 4 Fig. 4. Comparison of oxymetholone (A) and 17α-Methyltestosterone (B) as a representative

  about here] [Insert Table IV about here]

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	Monitoring programme
	During 2006 a preliminary monitoring programme focused on steroids in nutritional
	supplements was realised. Within this programme totally 29 samples of various protein
	concentrates and 15 samples of creatine monohydrate or creatine pyruvate were collected

Table I .

 I Target compounds and concentrations of individual stock solutions

	Page 21 of 31		Food Additives and Contaminants Food Additives and Contaminants			Page 20 of 31
	Table II. Repeatability (RSD, %) and recovery of target compounds in nutritional supplements
	Analyte 1-Dehydroandrostenedione 17α-Ethyl-19-nortestosterone 17α-Methyltestosterone 17α-Methyl-19-nortestosterone 19-Norandrostendione 4-Androsten-3,17-dione 5-Androsten-3β,17β-diol 5α-Androstan-3,17-dione 7α,17α-Dimethyltestosterone Boldenone DHEA (Dehydroepiandrosterone) F o r P e e r R e v i e w CAS RN * RSD (%) Concentration (µg.ml -1 ) Analyte 897-06-3 2280 Mesterolone 52-78-8 2260 Metandienone 58-18-4 1396 Methenolone 514-61-4 1160 Methylandrostenediol 734-32-7 1120 Mibolerone 63-05-8 2198 Nandrolone 521-17-5 1504 Oxandrolone 846-46-8 1445 Oxymetholone 1605-89-6 570 Progesterone 846-48-0 1643 Testosterone 53-43-0 1344 Testosteron 17-propionate O n l y CAS RN * 1424-00-6 72-63-9 153-004 521-10-8 3704-09-4 434-22-0 53-39-4 434-07-1 57-83-0 58-22-0 57-85-2 Quantification Concentration Analyte ion level (mg.kg -1 ) Protein concentrate Creatine monohydrate Protein Recovery (%) Concentration (µg.ml -1 ) 1350 1090 627 1360 1260 1417 762 1366 1853 1223 1396 concentrate Creatine monohydrate 1-Dehydroandrostenedione 122 2.280 6.4 7.7 90.9 78.7 17α-Methyltestosterone 302 0.838 5.5 9.3 89.5 82.3 17α-Ethyl-19-nortestosterone 302 0.452 8.8 9.9 84.4 78.3 17α-Methyl-19-nortestosterone 288 0.464 5.5 8.6 89.1 76.7 19-Norandrostendione 272 0.448 4.3 8.2 78.0 81.9 4-Androsten-3,17-dione 286 0.616 4.1 6.6 84.8 79.5 5-Androsten-3β,17β-diol 290 1.203 18.2 12.5 90.3 85.9 5α-Androstan-3,17-dione 288 0.405 16.2 7.3 90.3 84.0 7α,17α-Dimethyltestosterone 298 0.456 6.3 7.3 88.7 73.4 Boldenone 122 0.329 10.2 18.8 92.9 70.9 DHEA 288 0.806 8.6 7.7 93.7 82.4 Dromostanolone 245 0.440 4.9 8.6 90.1 77.2 Fluoxymesterone 336 0.572 11.4 8.6 72.9 80.9 F o r P e e r R e v i e w O n l y
	Dromostanolone Mesterolone	58-19-5 304	1100 0.540	Trenbolone Acetate 5.6	10.6	10161-34-9 81.5	1151 78.7
	Fluoxymesterone Metandienone	76-43-7 122	1430 0.218	10.1	20.5	122.6	79.2
	* Chemical Abstracts Service Registry Number Methenolone 302	1.003	9.2	12.7	86.5	78.3

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table IV .

 IV Reporting limits * of target compounds

	Table II. -continued Table III. Regression coefficients
			RSD (%)	Recovery (%)
	Analyte Methylandrostenediol Mibolerone Nandrolone Oxandrolone Oxymetholone Progesterone Testosterone Testosteron 17-propionate Trenbolone Acetate F o r P e e Quantification ion 253 284 274 291 332 314 288 147 252 r R Analyte Concentration range (µg.ml -1 ) 1-Dehydroandrostenedione 0.046 -11.400 17α-Methyltestosterone 0.017 -4.188 17α-Ethyl-19-nortestosterone 0.009 -2.260 17α-Methyl-19-nortestosterone 0.032 -2.320 19-Norandrostendione 0.009 -2.240 4-Androsten-3,17-dione 0.012 -3.078 5-Androsten-3β,17β-diol 0.024 -6.016 5α-Androstan-3,17-dione 0.028 -2.023 7α,17α-Dimethyltestosterone 0.009 -2.280 Boldenone 0.007 -1.643 DHEA 0.056 -4.031 Dromostanolone 0.009 -2.200 Fluoxymesterone 0.114 -2.860 Mesterolone 0.011 -2.700 Metandienone 0.044 -1.090 Analyte Reporting limit (mg.kg -1 ) 1-Dehydroandrostenedione 0.023 17α-Ethyl-19-nortestosterone 0.032 17α-Methyltestosterone 0.009 17α-Methyl-19-nortestosterone 0.032 19-Norandrostendione 0.009 4-Androsten-3,17-dione 0.012 5-Androsten-3β,17β-diol 0.024 5α-Androstan-3,17-dione 0.012 F o r P e e r R e v i e w Concentration level (mg.kg -1 ) Protein concentrate 0.544 11.0 0.504 5.6 0.567 5.3 1.525 10.0 5.465 15.2 1.260 8.3 1.223 8.0 0.391 15.5 0.322 6.9 Regression coefficient 0.999 0.999 0.999 0.999 0.999 0.999 0.997 1.000 0.999 1.000 0.997 1.000 0.998 0.998 1.000 Reporting limit Creatine monohydrate 10.9 9.2 10.3 7.2 9.0 11.7 10.0 9.6 12.5 Analyte (mg.kg -1 ) Mesterolone 0.011 Metandienone 0.008 Methenolone 0.020 Methylandrostenediol 0.011 Mibolerone 0.010 Nandrolone 0.011 Oxandrolone 0.006 Oxymetholone 0.109 7α,17α-Dimethyltestosterone 0.009 Progesterone 0.020 Boldenone 0.007 Testosterone 0.012 DHEA 0.028 Testosteron 17-propionate 0.027 O n l y Protein concentrate 99.0 81.5 83.6 88.6 8.7 78.9 87.6 76.3 100.2 Dromostanolone 0.009 Trenbolone Acetate 0.023 e Nandrolone 0.011 -2.834 0.999 w Mibolerone 0.010 -2.520 1.000 e Methylandrostenediol 0.011 -2.720 0.998 i v Methenolone 0.020 -5.016 0.998 Fluoxymesterone 0.114	Creatine monohydrate 79.9 80.7 70.0 78.5 92.5 75.7 78.1 73.6 117.1
	Oxandrolone Oxymetholone Progesterone Testosterone Testosteron 17-propionate	0.006 -7.263 0.109 -27.324 0.088 -6.299 0.024 -6.115 0.027 -1.954	0.998 0,999 O 0.999 n l 0.999 y 0.999
	Trenbolone Acetate	0.023 -1.611	0.999

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants