DETERMINATION OF RIBOFLAVIN IN PREMIXTURE AND COMPOUND FEED BY LIQUID CHROMATOGRAPHY METHOD

Transcription

1 Bull Vet Inst Pulawy 5, , 008 DETERMINATION OF RIBOFLAVIN IN PREMIXTURE AND COMPOUND FEED BY LIQUID CHROMATOGRAPHY METHOD JOLANTA RUBAJ, GRAŻYNA BIELECKA, WALDEMAR KOROL, AND KRZYSZTOF KWIATEK 1 National Feed Laboratory, National Research Institute of Animal Production, Lublin, Poland 1 National Veterinary Research Institute, Pulawy, Poland jrubaj@clpp.lublin.pl Received for publication September 10, 008 Abstract The analytical procedure of riboflavin quantification in premixtures and compound feedingstuffs by high performance liquid chromatography (HPLC) analysis is presented. Riboflavin was extracted from feed sample in autoclave with 0.1M sulphuric acid. The ester bonds with phosphoric acid were hydrolysed by the Taka-diastase enzyme. Riboflavin content was determined by HPLC with reversed-phase and usage of fluorescence detector. The limit of quantification of this method is 1.0 mg/kg. The coefficient of variation of riboflavin quantification results in premixtures was.9% and in compound feedingstuffs %. The Horrat value for premixtures reached 0.61, whereas in case of compound feedingstuffs 0.40, thus confirming an acceptable precision of the applied procedure. The recovery rate for riboflavin added in the form of a reference solution into premixtures was 98.3%, as regards compound feedingstuffs it was 98.0%. Key words: premixtures, compound feedingstuffs, riboflavin, quantification, HPLC analysis. Riboflavin [7,8-dimethyl-10 (1'-D-ribityl) isoalloxazine] is usually present in cells of live organisms in cofactor form, such as flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), in phosphoric ester forms, or in protein combinations. Riboflavin plays a key role in redox reactions in cells. It takes a part in proper functioning of the nervous system, participates in amino acids and lipid transformations, enhances the functions of eyes. Riboflavin and vitamin A participate in appropriate functioning of mucous membranes, epithelium of blood vessels, skin and respiratory tract. In feed materials, riboflavin occurs in various quantities. The content of this vitamin in grain is 1- mg/kg, in the bran of grains mg/kg, in DDGS (Dried Distillers Grains with Solubles) 3-5 mg/kg. The riboflavin level in seeds of leguminous plants is -3 mg/kg, in soybean and rapeseed meals 3-4 mg/kg. Feed of animal origin contains more riboflavin, from 5 mg/kg in meat-bone-meals to 7 mg/kg in fishmeal and 0 mg/kg in milk powder. The significant content of riboflavin is in hay (10 mg/kg), dried clover (15 mg/kg), or dried lucerne (1-17 mg/kg). The good source of riboflavin is feed yeast, from 35 to 70 mg/kg (13). The recommended riboflavin doses to compound feedingstuffs for poultry are 4-8 mg/kg in case of feed for laying hens, 4-6 mg/kg in feed for broiler chickens, 6-10 mg/kg in feed for turkey, and 0 mg/kg for breeding turkeys (1). The recommended riboflavin doses to compound feedingstuffs for swine are about 6 mg/kg in case of piglets, 5 mg/kg in case of sow, and 4 mg/kg as regards fatteners (16). From many years in commercial feedingstuff production, the riboflavin supplements are adjusted to requirements of particular animal species. Riboflavin is sensitive to light, but resistant to heat and oxidation. Due to that, thermal and pressure processing employed in commercial feedingstuff production have insignificant effect on its stability (17). Albers (1) examined the stability of vitamins in compound feedingstuffs after expanding and -months storage, and stated high level of riboflavin retention equal 95%, similar to other vitamins from group B with the exception of thiamine (90%). The stability increases with acidity, and resistance of riboflavin for heat degradation is the highest at ph from to 5. Isoalloxasine ring destruction occurs at ph above 7; moreover, FMN and FAD are hydrolysed to free form of riboflavin when ph is below 5 (4). The necessity of riboflavin monitoring in feed materials, compound feedingstuffs, complementary feedingstuffs and premixtures requires the validation of analytical methods. The interfering substances problem may be solved by applying of HPLC methods for the

2 60 quantification of riboflavin. Reviews of HPLC methods for riboflavin, FMN and FAD quantification in food and biological samples were presented by Finglas and Faulks (5), Russell and Vanderslice (15), Nielsen (11), and Keller (10). Current EU feed law, international ISO, European EN, and Polish PN standards do not contain any standardised method for riboflavin determination, which is serious problem for feed analysis. The necessity of having validated method is also connected with carrying out the official control of feed production and producers self-control. The aim of this study was to check and validate the HPLC method with fluorimetric detection for the quantification of whole riboflavin content in premixtures and compound feedingstuffs. Material and Methods Materials. The study was carried out on premixtures and compound feedingstuffs with an addition of vitamin B. The applied method was also validated on Certified Reference Material: CRM 41 (milk powder). Methanol (HPLC grade), sulphuric acid and sodium acetate were provided by POCH Gliwice, Poland. Taka-diastase enzyme from Aspergillus oryzae was obtained from Fluka. Deionised water was prepared on Mili-Q system (Milipore, France). Standards and standard solutions. The standard stock solution of riboflavin at concentration of 100 µg/ml was prepared in 0.1 M sulphuric acid solution and stored at 4ºC in darkness up to two months. The working solutions from 0.17 to 0.67 µg/ml were prepared each time exactly before use. Liquid chromatography - fluorescence detection. High-pressure liquid chromatography (Dionex P-680) with fluorescence detector (Dionex RF 000), extinction length λ=453 nm, and emission wavelength λ=51 nm, was used for the analysis. The separation was done on reversed-phase column C 18 (50 mm x 4.6 mm x 5 µm). Mobile phase was the solution of methanol and citric acid 0. mg/l (30:70 v/v). That solution was mixed with methanol with ratio 1:1. An isocratic flow was 0.8 ml/min., at controlled temperature of 5ºC for the separation of interfering substances, which could be present in the sample extract. Extraction and hydrolyses. Vitamin B is sensitive to light; therefore, all procedures were under protection from daylight (by using amber glass flask or flask covered by aluminium foil). Depending on vitamin B content, the mass from g to 10 g of premixture or compound feedingstuff, with precision of 1 mg, was weighed into Erlenmayer flask. Afterwards, 50 ml of 0.1M sulphuric acid was added, and that solution was boiled for 15 min at temperature from 110ºC to 10ºC. After cooling to the room temperature, whole volume of the sample hydrolysed in deionised water was transferred to the 100 ml measuring flask, with 8 ml of M sodium acetate. Next, 5 ml of 10% Taka-diastase suspension was added to the flask, which was then placed into water bath at 45ºC for 0 min. The enzymatic reaction was stopped by the addition of 4 ml of 30% sulphuric acid. The sample solution was next cooled to a room temperature, and the volume was adjusted to 100 ml by addition of 0.1M sulphuric acid. Afterwards, the samples were mixed and filtered. If it was necessary, an additional dilution was done, in order to adjust the concentration of the solution to the calibration curve. The 5 ml of filtrate was measured out into a centrifuge tube with 5 ml of methanol, and the tube was shaken. Next, the tube was centrifuged to remove the sediment. The 4 ml of supernatant was then mixed with ml of deionised water, shaken, filtered through syringe filter and injected into column. In parallel with sample analysis, the standard solution analysis was done. Evaluation of the procedure. The precision of the method was checked by the evaluation of repeatability and within-laboratory reproducibility. As confirmation of the precision, the Horwitz coefficient called Horrat value (Hor) was calculated. The Horrat value is the ratio of the reproducibility of standard deviation SD r, calculated from the data, to the target standard deviation σ p,, calculated from the Horwitz formula σ p = 0,0 C 0,8495, where C means concentration expressed as denominated mass fraction (e.g. 1 mg/kg = 10-6 ) (8). Acceptable Horrat values, which describe precision of measures, are 0.5<Hor< (9). In order to adjust the results to the repeatability conditions, target standard deviation σ p was multiplied by 0.66 (SD r = 0.66 SD R ) (). The accuracy of the method was evaluated by recovery rate calculation and analysis of certified reference material CRM 41 (milk powder). The estimated expanded uncertainty of elaborated method was evaluated by within-laboratory approach, according to the technical report of Eurolab (6). Because the within-laboratory approach for standard uncertainty (u) estimation was used, it was necessary to evaluate within-laboratory reproducibility (s), as a measure of precision and systematic bias (bbias), calculated from CRM analyses. The standard uncertainty was estimated according to the formulae from technical report of Eurolab (6) and handbook of Nordtest (7). u + = s b [1] Within-laboratory reproducibility was obtained from the spread between repeated riboflavin analyses of feed samples. According to Nordtest handbook (7), for two individual riboflavin analyses of each sample, the mean value, the difference between analyses (range), the relative difference (%), and the mean relative difference (%) for every samples of the same kind of feed were calculated. The mean range divided by d coefficient (for two repetitions d=1.18) was equal to within-laboratory reproducibility standard deviation. The contribution of statistic bias (b) in the uncertainty measure was calculated from the mean standard deviation of the results CRM, the uncertainty CRM u ref, and the standard deviation of analysis CRM s ref, based on the following formula (6, 7):

3 61 b = + = u ref Results + n s ref ( bias i ) n [] [3] The characteristic chromatograms of standard riboflavin solution, premixture extract, and compound feedingstuffs are presented in Fig. 1.The retention time was about 3.8 min. In the applied analytical procedure and chromatography conditions of the separation, there were not observed any of the interference substances. Table 1 contains the validation parameters such as precision of the method, which was based on repeatability and expressed as coefficient of variation ( %), and Horrat values (Hor = ). The intermediate precision, expressed as within-laboratory reproducibility ( %), and Horrat values (Hor = ) are presented in Table 3. This data is the objective measure of precision of the applied analytical procedure. The calibration curve had good linearity in the range of µg/ml, with correlation coefficient r= The recovery rate of riboflavin addition to the analysed samples ranged from 98.0 to 98.3% (Table 1). The limit of quantification was 1.0 mg/kg, combined standard uncertainty was 6.4%, and expanded uncertainty was 1.5% (Table 3). In order to verify the applied analytical procedure, the levels of riboflavin in certified reference material was determined. The results are listed in Table. The bias of the method was evaluated on the basis of the results from CRM analyses and was expressed as the relative difference between achieved results and certified reference value. The bias of the method was 0.7%. Therefore, the accuracy of the method was 99.3% (Table ). Table 1 Validation parameters for riboflavin determination by HPLC method Validation parameters Limit of quantification Standard calibration curve 1.0 mg/kg r= y=.8337x Linearity range, µg/ml Precision CV, % (n=10) (Repeatability) Compound feedingstuff 4.56 mg/kg 3.39 Premixture 944 mg/kg.9 Hor (compound feedingstuffs) 4.56 mg/kg 0.40 Hor (premixture) 944 mg/kg 0.61 Recovery rate, % (n=6) Compound feedingstuff 4.56 mg/kg 98.0 Premixture 944 mg/kg 98.3 Combined uncertainty u (%) 6.4 Expanded uncertainty U= x u (%) (k=) 1.5 Table Concentration of riboflavin in certified reference material Certified reference material CRM 41 Milk powder Declared concentration of riboflavin (uncertainty) mg/kg dry matter Achieved results mg/kg dry matter 14.5 (±0.6) 14.4

4 6 Standard 7.00 mv a) riboflavin Emission EM:51 nm min b) Compound feedingstuff extract Emission 10.0 mv EM:51 nm 8.0 riboflavin min c) 9.0 Premixture extract mv riboflavin Emission EM:51 nm min Fig. 1. Characteristic chromatograms of riboflavin: a) chromatogram of standard extract (concentration µg/ml), b) chromatogram of compound feedingstuff extract (concentration 0.44 µg/ml), c) chromatogram of premixture extract (concentration µg/ml).

5 63 Table 3 Selected validation parameters of HPLC method for the determination of riboflavin content in premixtures and compound feedingstuffs evaluated on the basis of within-laboratory approach Validation parameters Results/Horrat values Relative within-laboratory reproducibility s for compound feedingstuffs (6.48 mg/kg) evaluated from the range (n=35) 6.5 % H=0.81 Relative within-laboratory reproducibility s for complementary feedingstuffs (37.84 mg/kg) evaluated from the range (n=1) 5.8 % H=0.94 Relative within-laboratory reproducibility s for 5.09 % premixtures (1444 mg/kg) evaluated from the range H=1.4 (n=6) Relative within-laboratory reproducibility s for all analysed feed evaluated from the range (n=73) 5.38 % Uncertainty of statistic bias of CRM B analysis evaluated from formula [] 3.16 % Recovery rate (%) as trueness measure on the basis of CRM - 41 analysis (n=6) 99.3 Combined standard uncertainty evaluated from formula [1] 6.4 % Expanded uncertainty, for k=; U = x u 1.5 % H = Horrat value Discussion In the presented analytical procedure, HPLC was applied, which allows the separation and quantification of vitamin B in premixtures and compound feedingstuffs. Previously, the HPLC method with fluorescence detection was successfully used for the quantification of thiamine in feedingstuffs (14). The achieved coefficients of variation,.3% in case of premixtures, 3.3% in case of compound feedingstuffs and Horrat values, which were 0.61 and 0.40, respectively, confirmed that the applied analytical procedure offers acceptable precision. According to Horwitz (9), acceptable Horrat values, which are the measure of precision of analysis results, should range between 0.5 and. The Horrat values below 1 gave evidence of high precision of the results. The accuracy of measures was also excellent, and was expressed by riboflavin recovery rate in the range from 98.0% to 98.3%. The intermediate precision of riboflavin determination by HPLC method, which is expressed by within-laboratory reproducibility, reached the mean value of 5.38%, and the mean Horrat value was Hor=1.06, which proved the stability of the achieved validation parameters at acceptable level for a long period (inter-laboratory reproducibility was evaluated from result of 3-year study). This illustrated a high robustness of the method. Britton et al., (3) reached the relative withinlaboratory reproducibility of riboflavin in premixture (3,000 mg/lb) at the level of 3.03% within 4-year study. These authors also stated that variation of results can be higher in case of lower content of riboflavin. Similar precision of riboflavin determination results at the level of 4% (Hor 0.80) and recovery rate in the range 88%-103% achieved Wollard and Indyk (18) during hydrophilic vitamin analysis of baby-food ( 13.6 mg/kg) by liquid chromatography with fluorescence detector. In inter-laboratory collaborative trials for the determination of whole riboflavin content in pet foods and compound feedingstuffs for turkey by HPLC method with fluorescence detection, repeatability results were achieved in the range of 1.4%-3.9%, with acceptable Horrat value Hor=0.93, as regards pet foods, and 0.89 in the case of compound feedingstuffs for turkeys (). The obtained chromatographic separation, precision parameters and accuracy defined by recovery rate illustrated that the applied analytical procedure was correct. The accuracy of the developed procedure was additionally confirmed in analyses performed on certificated reference material (CRM) and reached the value 99.3% (bias 0.7%). This method allows for the determination of the whole amount of riboflavin in premixtures and compound feedingstuffs. Because of this, this method can be applied for an official control, which allows verifying the producer declarations in case of riboflavin addition to premixtures and compound feedingstuffs.

6 64 References 1. Albers N.: The influence of the production process and the composition of the mixture on vitamin stability. Feed Compounder 1996, 4, Anonimus: Analytical Methods Committee. Determination of thiamine and riboflavin in pet foods and animal feedingstuffs. Analyst 000, 15, Britton N.L., Riter K.L., Smallidge R.L., Hillebrandt J.: Reversed-phase liquid chromatographic determination of riboflavin in feeds. J AOAC Int 003, 86, Eitenmiller R.R., Lin Ye, Landen W.O., Jr.: Riboflavin. In: Vitamin Analysis for the Health and Food Sciences. CRC Press, Boca Raton, FL, 008, pp Finglas P.M., Faulks R.M.: Critical review of HPLC method for the determination of thiamine, riboflavin and niacin in food. J Micronutr Anal 1987, 3, Eurolab Technical Report 1/007. 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 198, 54, 67A- 76A. 9. Horwitz W. (Editor). Official Methods of Analysis of AOAC International, 17 th Edition. AOAC International, Gaithersburg, USA, Keller H.E.: Analytical Methods for Vitamins and Carotenoids in Feed, Department of Vitamin Reaserch and Development Roche Basle, Nielsen P.: Flavins. In: Modern Chromatographic Analysis of Vitamins. Edited by A.P. DeLeenheer, W.L. Lambert, H.J. Nelis, Dekker, New York, 199, p Nutrient Recommendation and Nutritive Value of Feedingstuffs for Poultry. Poultry Feeding Standards (in Polish). Edited by S. Smulikowska and A. Rutkowski, Institute of Physiology and Animal Feeding, Jabłonna, Raw Material Compendium. A compilation of worldwide data sources. Novus Europe S.A. Brussels, 1996, p Rubaj J., Korol W., Bielecka G., Kwiatek K.: Determination of thiamine in premixture and compound feed by liquid chromatography method. Bull Vet Inst Pulawy 008, 5, Russell L.F., Vanderslice J.T.: A comprehensive review of vitamin B analytical methodology. J Micronutr Anal 1990, 8, Swine Feeding Standards. Nutritive Value of Feedingstuffs (in Polish). Institute of Physiology and Animal Feeding, Jabłonna, Van der Poel A.F.B., Gadient M.: Effects of expander processing on the recovery of feed additives. In: Expander Processing of Animal Feeds. Edited by A.F.B Van der Poel. Feed Processing Centre, Wageningen, 1997, pp Woollard D., Indyk H.E.: Rapid determination of thiamine, riboflavin, pyridoxine and niacinamide in infant formulas by liquid chromatography. J AOAC Int 00, 85,