DETERMINATION OF CANTHAXANTHIN IN PREMIXTURE AND COMPOUND FEED BY THE LIQUID CHROMATOGRAPHY METHOD
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1 Bull Vet Inst Pulawy 53, , 2009 DETERMINATION OF CANTHAXANTHIN IN PREMIXTURE AND COMPOUND FEED BY THE LIQUID CHROMATOGRAPHY METHOD JOLANTA RUBAJ, GRAŻYNA BIELECKA, WALDEMAR KOROL, AND KRZYSZTOF KWIATEK 1 National Research Institute of Animal Production, National Feed Laboratory, Lublin, Poland 1 National Veterinary Research Institute, Pulawy, Poland jrubaj@clpp.lublin.pl Received for publication August 25, 2009 Abstract The analytical procedure of canthaxanthine quantification in premixes and compound feed by the HPLC method was presented. The principle of this method is based on the hydrolysis of powdered formulation of canthaxanthine with trypsin and pepsin in a water solution of ammonia and its purification in an aluminium oxide filled column. The canthaxantine content is determined by high performance liquid chromatography in normal phase with the usage of a DAD detector. The limit of quantification of this method was 1.0 mg/kg. The coefficient of variation (CV) of canthaxantine quantification results was 3.3% in premixtures and 4.68% in compound feed. The Horrat values (Hor) were 0.78, in premixtures and 0.52, in compound feeds, which proved the satisfactory precision of the applied method. The recovery rate of canthaxantine added as standard solution into premixture was 98.2%, and in case of compound feed was 97.3%. Key words: premixture, compound feed, canthaxanthin, quantification, HPLC. Canthaxantine (β, β-carotene - 4,4 -dione; C 40 H 52 O 2 ) is one of the colourants used as feed additives for poultry and the salmon fish family. Its introduction into feed is imposed by consumers tastes. First of all, it concerns the colour of the egg yolk. In the present circumstances, consumers willingly buy eggs with an intense yellow or orange colour of the egg yolk. Moreover, yellow-fleshed broilers are easier to sell. Compound feed for poultry, with maize and dried grass, contains in general a sufficient amount of pigments to colour egg yolk or poultry tissue. Currently, colourants can contain two kinds of pigments: natural - extracts from herbs - and synthetic. By chemical synthesis are made canthaxantine, astaxanthine, and ethyl ester of ß- apo-8'-carotenic acid. The usage of the colourants is justified primarily for compound feed, which are poor as regards natural colourant content (Polish grains) and in regions where consumers look for coloured egg yolk or poultry meat. According to Regulation 1831/2003 of the European Parliament and of the Council on feed additives for use in animal nutrition, canthaxanthine belongs to group 2a sensory additives colourants. As regards canthaxanthine the maximum content was established for compound feed as follows: for salmon and trout 25 mg/kg; and for poultry 25 mg/kg, except for laying hens - 8 mg/kg. In the case of feed for salmon and trout, it is admissible to use canthaxanthine and astaxanthine together, on condition that the whole amount of them in the feed will not be higher than 100 mg/kg. In case of compound feed for poultry, except laying hens, it is permissible to use canthaxanthine with other carotenoids and xanthophylls, on condition that the whole amount of them in feed will not be higher than 80 mg/kg (1). For cathaxathine, the tolerance limits for compound feed and premixtures were established in the Polish Regulation of the Ministry of Agriculture and Rural Development (8). The tolerance limits are from ±20% for cathaxantine content in compound feed in the range of 1 50 mg/kg, and to ±5% for canthaxanthine content in premixtures above 1,000 mg/kg. Recently, the maximum residue levels of canthaxanthine in food of animal origin were established (2) for poultry other than laying hens - 15 mg of canthaxanthine/kg of liver (wet tissue) and 2.5 mg of canthaxanthine/kg of skin/fat (wet tissue); laying hens - 30 mg of canthaxanthine/kg of egg yolk (wet tissue); salmon - 10 mg of canthaxanthine/kg of muscle tissue (wet tissue); trout - 5 mg of canthaxanthine/kg of muscle tissue (wet tissue). Traditional methods of carotenoids quantification rely on spectrophotometric measurements after their extraction and chromatography separation from feed material by using classic chromatography methods, i.e. column and planar mode chromatography. Such a procedure is characterised by low resolution,
2 708 usually not sufficient for the canthaxanthine separation form present in feed-natural carotenoids (xanthophylls), which originate from dried grass and/or maize, or other carotenoids additives commonly used in compound feed (ethyl ester of ß-apo-8'-carotenic acid, ß-carotene, astaxanthine, citraxanthine), which usually makes their quantification impossible. Nowadays, the HPLC technique makes possible the precise separation of single carotenoids, even isomers, and then quantitative determination. The stabilisation of carotenoids additives in e.g. a gelatine matrix, carries additional difficulties for their quantitative separation from compound feedingstuffs. The current EU feed law regulations, international (ISO), European (EN), and Polish standards (PN) do not contain a standardised method for canthaxanthine quantification, which makes difficult the monitoring of this colourant in feed. The need to possess of reliable method is also connected with conducting official Government control and the internal control of the feed industry. In the National Feed Laboratory, the HPLC method developed by Weber (11) was adapted for canthaxanthine quantification in premixtures and compound feed. However, the author of this method did not present the validation performance criteria of the method. Currently, it is required that methods used for official feed control and internal feed-industry control methods have to be validated within the scope of the analysis of the target. Moreover, in recent years, many new products used in animal feeding are very diverse as regards matrix. The purpose of this study was to determine the precision, accuracy, and uncertainty of canthaxanthine quantification results in premixtures and compound feed by HPLC method with DAD detector. Material and Methods The premixtures and compound feed, which contained canthaxanthine were used as samples for analysis. The reagents used in this method: n-hexane and acetone (HPLC grade), chloroform, diethyl ether, 99.8% ethyl alcohol, and ammonium, were provided by POCH Gliwice, Poland. Pepsin and trypsin enzymes were obtained from Merck. Deionised water was prepared on Mili-Q system (Milipore, France). Standards and standard solutions. The standard of canthaxanthine obtained from Dr Ehrenstorfer (Germany) was used. The standard stock solution of canthaxanthine, with 30 µg/ml concentration, was prepared in chloroform and n-hexane solution and stored at 4ºC in darkness up to 1 month. The working solutions (from 1.0 to 10.0 µg/ml) were prepared just before use. Liquid chromatography DAD detection. High-pressure liquid chromatograph (Perkin Elmer Series 200) with DAD detector, which enable canthaxanthine detection at wavelength λ=446 nm, was used for analysis. The separation was done in a normalmode column (LiChrospher Si x 250 mm x 5 µm). Mobile phase for LC was n-hexane:acetone 86:14 (v/v) solution. The isocratic flow of eluent was 1.3 ml/min., at controlled temperature 25ºC, done for the separation of interfering substances, which could be present in the sample extract. Extraction and hydrolyses. Premixtures. The mass of 5 g of homogenised premixture was weighed into a 100 ml flask (with known mass of empty flask), with a precision of g. Afterwards, about 50 mg of trypsin, about 50 mg of pepsin, 80 ml of deionised water, and 1 ml of 25% ammonium (diluted by water 1:1) were added. That solution was blended and put into an ultrasonic bath for 30 min. at 50ºC. After cooling to room temperature, the solution was completed to the volume of the flask and weighed. Then it was blended and 4 g of the solution was weighed into a 20 ml baker. The whole volume of that solution was transferred by 40 ml of 99.8% ethyl alcohol to a 100 ml flask, and completed to 100 ml volume by diethyl ether. Afterwards, the sample was mixed and 25 ml of the extract or its submultiples was carried out on column, which contained aluminium oxide. Compound feed. The mass of 5 g of homogenised compound feed was weighed into a 100 ml flask (with known mass of empty flask), with a precision of g. Afterwards, about 50 mg of trypsin, about 50 mg of pepsin, 15 ml of deionised water, and 1 ml of 25% ammonium (diluted by water 1:1) were added. That solution was blended and put into an ultrasonic bath for 30 min, at 50ºC. After cooling to room temperature, 40 ml of 99.8% ethyl alcohol was added and the solution was completed to 100 ml volume by diethyl ether. Afterwards, the sample was mixed and 50 ml of the extract or its submultiples was carried out on a column, which contained aluminium oxide. Preparation of aluminium oxide column. A hundred grammes of aluminium oxide were weighed (neutral, activity 1) into a 300 ml Erlenmayer flask with fitted cork and 12 ml of deionised water was added. Afterwards, the flask was closed and shaken till a homogeneous solution was made. Then it was left in room temperature for 24 h (minimum 2-3 h). Ready to use aluminium oxide was then suspended in n-hexane and slowly poured into a glass column, till a 3-4 cm layer was made. The column was permanently filled with n- hexane. Purification of the extract by chromatography on aluminium oxide. The submultiples of the extract (25 ml) were moved into an aluminium oxide column and washed in a ml of n-hexane:diethyl ether (1:1) solution. The eluate was collected in a 300 ml Erlenmayer flask. The solvent was then evaporated into the vacuum evaporator into a water bath at a temperature no higher than 50 C. Drought remains were then dissolved into known volume of n-hexane:acetone (96:4) solution. Afterward, this solution was filtered in a syringe filter and dosed on a column. In parallel, an analysis of the standard was done. Evaluation of the procedure. The precision of this method was checked by the evaluation of its
3 709 repeatability and reproducibility. In terms of precision, the Horwitz coefficient, called the Horrat value (Hor), was calculated. The Horrat value is the ratio of reproducibility relative to standard deviation SD r, calculated from data, to target standard deviation σ p, calculated from Horwitz formula σ p =0.02 C , where C means concentration expressed as a denominated mass fraction (e.g. 1 mg/kg = 10-6 ) (5). In order to adjust the results to the repeatability conditions, the target standard deviation σ p was multiplied by 0.66 (SD r = 0.66 SD R ). Acceptable Horrat values, which describe the precision of measures, are 0.5 < Hor < 2 (6). Within-laboratory reproducibility was obtained from the spread between repeated canthaxanthine analyses of feed samples. According to the Nordtest guidance (4), for two individual canthaxanthine analyses of each sample mean value, difference between analyses (range), relative difference (%), and mean relative difference (%), were calculated. The mean range divided by the d coefficient (for two repetitions d=1.128) was equal to withinlaboratory reproducibility standard deviation. The expanded uncertainty as double within-laboratory reproducibility U = 2 S Rw was evaluated (3). For comparison, the expanded uncertainty was also evaluated as regards the Horwitz equation RSD R (%) = 2C -0.15, where C means concentration as non-nominated mass cofactor. Results The characteristic chromatograms of the reference canthaxanthine solution, premixture extract, and compound feed, are presented in Fig. 1. The retention time of canthaxanthine was about 6 min. For the applied analytical procedure and chromatography conditions of separation there were not observed any interference substances. Table 1 contains the validation performance parameters and precision of the method, which was based on repeatability and expressed as coefficient of variation ( %), and Horrat values (Hor = ). Intermediate precision, expressed as withinlaboratory reproducibility (6.04% 7.97%), and Horrat value (Hor = ) are presented in Table 2. These data are the objective measures of the precision of the applied analytical procedure. The calibration curve possessed good linearity in the range µg/ml, with correlation coefficient r = The recovery rate of canthaxanthine addition to analysed samples ranged from 97.3% to 98.2% (Table 1). The limit of the quantification of the applied method was 0.8 mg/kg. The expanded uncertainty, evaluated from within-laboratory reproducibility, was 15.9% as regards compound feed, and 12.1% as regards premixtures. The achieved results of the expanded uncertainties were lower than those evaluated from the Horwitz equation (26.2% and 13.7%) and similar to the VDLUFA data (15%) (9), which proved the sufficient precision of this method (Table 2). Discussion In the presented analytical procedure highperformance liquid chromatography (HPLC) was applied, which allowed the separation and quantification of canthaxanthin in premixtures and compound feed. The chromatograms of standard solution and analysed samples indicated the presence of one, single peak of canthaxanthine, which represented all isomers (all E+Z canthaxantine). A similar chromatogram was achieved as regards cathaxanthine determination in fish flesh by HPLC method with normal phase (10). Table 1 Validation parameters of the HPLC method for canthaxanthin determination Validation parameters Limit of quantification 1.0 mg/kg Standard calibration curve r= y=192,459.2x +2,028.3 Linearity range, µg/ml Precision CV, % (n=16) (repeatability) Compound feed 3.11 mg/kg 4.68 Premixture 470 mg/kg 3.30 Hor (compound feed) 3.11 mg/kg 0.52 Hor (premixture) 470 mg/kg 0.78 Recovery rate, % (n=6) Compound feed 3.11 mg/kg 97.3 Premixture 470 mg/kg 98.2
4 710 a) b) c) Fig. 1. Characteristic chromatograms of canthaxanthin: a) chromatogram of standard extract (concentration µg/ml); b) chromatogram of compound feedingstuff extract (concentration µg/ml); c) chromatogram of premixture extract (concentration µg/ml), KS canthaxanthin.
5 711 Table 2 Selected validation parameters of the HPLC method for the determination of canthaxanthin in premixtures and compound feed evaluated on the basis of the within-laboratory approach Validation parameter Relative within-laboratory reproducibility s for compound feed (3.77 mg/kg) evaluated from the range (n=18) Relative within-laboratory reproducibility s for premixtures (272 mg/kg) evaluated from the range (n=26) Expanded uncertainty for compound feed U = 2 S Rw Expanded uncertainty for premixtures 12.1 U = 2 S Rw Expanded uncertainty for compound feed calculated from the Horwitz equation, 26.8 U = RSD R (%) = 2C Expanded uncertainty for premixtures calculated from the Horwitz equation, 12.6 U = RSD R (%) = 2C Result/Horrat value 7.97 % Hor = % Hor = The achieved coefficient variation, 3.30% in case of premixtures, 4.68% in the case of compound feed and Horrat values from 0.78 to 0.52, confirmed that the applied analytical procedure has sufficient precision. Additionally, the intermediate precision of the canthaxanthine determination by the HPLC method, which measure is within-laboratory reproducibility, was satisfactory and the obtained values were 7.97% in the case of compound feed, and 6.04% in the case of premixtures. The Horwitz values, as an intermediateprecision measure, were 0.91 and 1.31, which proved that the precision was stable for long enough at a satisfactory level (inter-laboratory reproducibility was determined on the basis of the previously-achieved results in 2-year studies). It gave indirect evidence about the high robustness of this method. According to Horwitz and Albert (7), achieved acceptable Horrat values, which are the measure of the precision of the achieved analysis results, should range between 0.5<Hor<2. The Horrat values below 1 gave evidence of the high precision of the achieved results. The expanded uncertainties of canthaxanthine determination in compound feed and premixtures, elaborated from the within-laboratory practical approach, were 16% and 12%. These values can be considered as satisfactory, because were lower than those evaluated from the Horwitz equation: 26% and 14%. Moreover, the accuracy of the canthaxanthine determination was also very satisfactory, and its measure - recovery rate - ranged from 97.3% to 98.2%. Therefore, one factor can influence accuracy of the method. Canthaxanthin standards and preparations may have different isomeric forms. To solve this problem CRL-FA applied the isobestic concept to determine all cis- and trans- isomers, using a wavelength where the absorption coefficient is identical for all isomers (12). The obtained chromatographic separation, precision parameters, and accuracy defined by the recovery rate, illustrated that the applied analytical procedure was correct. This method allows for the determination of the whole amount of canthaxanthin in premixtures and compound feedingstuffs. Because of this, the method can be applied to official control. It allows us to verify the producer declaration in the case of canthaxanthin addition in premixtures and compound feedingstuffs. References 1. Commission Information (2004/C 50/01). List of the authorized additives in feedingstuffs published in application of Article 9t (b) of Council Directive 70/524/EEC concerning additives in feedinstuffs. OJ C 50, , p Commission Regulation (EC) No. 775/2008 of 4 August 2008 establishing maximum residue limits for the feed additives canthaxanthin in addition to the conditions provided for in Directive 2003/7/EC. OJ L 207, , p Eurolab Technical Report 1/2007. Measurement uncertainty revisited: Alternative approaches to uncertainty evaluation, Eurolab, Paris, Handbook for Calculation of Measurement Uncertainty in Environmental Laboratories. Nordtest Project Horwitz W.: Evaluation of analytical methods used for regulation of food and drugs. Anal Chem 1982, 54, 67A- 76A. 6. Horwitz W.: Official Methods of Analysis of AOAC International, AOAC International, Gaithersburg, USA, Horwitz W., Albert R.: The Horwitz Ratio (HorRat): A useful index of method performance with respect to precision. J AOAC Int 2006, 89, Regulation of MARD: Regulation of Ministry of Agriculture and Rural Development on tolerance limits
6 712 of nutrients and feed additives (in Polish), Law Gazette, 2007, No. 20, pos Schönherr J., Peterhänsel M.: Zur Untersuchung von Futtermitteln Testing of feeds. Neue ausgabe der analysenspielräume des VDLUFA. Kraftfutter, 7-8/2008, pp Technical Report CEN/TC 275, Foodstaffs. Part 1. Determination of astaxanthin and canthaxanthin in fish flesh. 11. Weber S.: Determination of stabilized, added canthaxanthin in complete feeds and premixes with HPLC In.: Analytical Methods for Vitamins and Carotenoids in Feed. Edited by Keller H.E., Roche Basle, 1988, pp Vincent U., Mitrowska K., von Holst C.: Carotenoids in animal feedingstuffs. Joint CRL-FA, NRLs, and FEFANA Carotenoid Meeting. JRC, Brussels, 2009.
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