Development of Method to Simultaneous Determination of Some Water and Fat Soluble Vitamins in Feeding Additives

Transcription

1 Journal of Selçuk University Natural and Applied Science Online ISSN: Development of Method to Simultaneous Determination of Some Water and Fat Soluble Vitamins in Feeding Additives Semahat KUCUKKOLBASI a*, Necla IRES b, Hüseyin KARA a a Selcuk University, Faculty of Science, Department of Chemistry, 42075, Campus-Konya/Turkey b Province Control Laboratory, 42040, Konya, Turkey Abstract This study was designed in orderr to develop a new method to simultaneously determine the level of water and fat soluble vitamins in feed additives. In this study, we tried to measure the levelsls of vitamins C, B 2, B 6, B 12 nicotinic acid folic acid (i.e., water soluble vitamins) and vitamins A, D 3, K 3 3, E (i.e., fat soluble vitamins). Several extraction procedures were performed in order to extract vitamins from tablets and the most appropriate solution for this purpose was determined as 0.01% aqueous trifluoroacetic acid, TFA (ph 3.9) and methanol, MeOH solution (20:80, v/v). Afterwards, the separation was achieved at 30ºC with retention times less than 40 min on a Phenomenex Gemini C18 110A column (150 x 4.6 mm, 3.0µm particle size) in a single run with a gradient elution of mobile phase consisting of 0.01% TFA ph 4.0 (solvent A) and 100% MEOH (solvent B) at a flow rate of 0.7 ml min -1 and the detection was performed with diode array (DAD) at different the wavelengths which are obtained maximumm signal of analyzed vitamins. Identification and quantification of compounds was achieved by comparing their retention times and peak areas with those of the vitamin standards. The lowest limit of detection (LOD) and limit of quantification (LOQ) were determined and the accuracy of the method was tested by measuring average recovery. Keywords: Water- and fat-soluble vitamins, RP-HPLC determination, feed. Introduction Vitamins are a broad group of organic compounds that are essential constituents from plant and animal sources and are required in minute amounts for the normal growth and maintenance of human and animal bodies (Poongothai et al., 2010). These compounds can be classified into the following two main groups: water soluble and fat soluble (Moreno & Salvado, 2000). Vitamins are essential for normal metabolism. They are responsible for various specific and vital functions in many metabolic processes, and their deficiency or excess can cause specific diseasess (Li & Chen, 2001). Knowing and controlling the amount of vitamins in feeds is very important because of their necessity to sustaining health and life in living creatures. As with humans, vitamins are necessary for cells and organs to * Corresponding author. Tel.: ; fax: ; ksemahat@gmail.com

2 31 KUCUKKOLBASI et al. work properly in animals. Thus, animals must get vitamins from their feed or feed additives. Vitamins stability can be affected by storage, moisture, temperature, light, ph, oxidation, etc. Unfortunately, many of the firms that produce animal feeds do not care properly for these factors. Therefore, the actual amounts of vitamins in feeds often do not match the amounts declared by feed companies. Often, the vitamins found in feeds are not sufficient to meet the animal s needs. Therefore, the effects associated with a lack of vitamins can sometimes later be seen in the animals, thereby reducing their strength against illness. The standard (Strohecker & Henning, 1965) and official analytical methods for vitamin analyses (United States Pharmacopeia, 1990), are tedious, sometimes nonspecific, time-consuming, and involve a pre-treatment of the sample through complex chemical, physical or biological reactions to eliminate interferences that are commonly found. Additionally, this pre-treatment is typically followed by individual methods to determine the amounts each specific vitamin present. These methods include spectrophotometric, polarographic, fluorimetric, enzymatic, and microbiological procedures (Moreno and Salvado, 2000). Indeed, UV-Vis spectrophotometry (Morelly, 1995; Ortega-Barrales et al., 1998), fluorimetry (Li & Chen, 2000; Garcia et al., 2001), chemiluminiscence (Zhou et al., 1991; Song and Hou, 2003) capillary electrophoresis (Schiewe et al., 1995; Okamoto et al., 2003) microbiology (Kothari &Taylor, 1982; Han & Tyler, 2003) and high-performance liquid chromatography (Li & Chen, 2000; Barnett et al., 1980; Fallon et al., 1987) have been proposed for the determination of vitamins. Nevertheless, no single analytical approach has been developed to date to determine the amounts of water and fat soluble vitamins within a complex matrix in a single run (Klejdus et al., 2004). Vitamin analysis can be done using various laboratory methods; indeed, fat soluble and water soluble vitamin determination can be done with different approaches as well. The extraction phase and chromatographic phase for fat soluble versus water soluble vitamins require different methods, so the implementation of these two methods to measure fat and water soluble vitamins requires more time and solvent. This study proposes a method to simultaneously determine fat soluble and water soluble vitamins and details its application in feed and its components.

3 32 KUCUKKOLBASI et al. In this work, we developed and optimized a HPLC method using diode array detection for the determination of the amounts of water and fat soluble vitamins within a complex matrix via a single run. The method was successfully applied to the determination of six vitamins in feed and feed additives and provides a high degree of chromatographic resolution, sensitivity and reproducibility. Materials and Methods Chemicals and reagents All vitamin standards were purchased from Supelco and Sigma Aldrich. HPLC grade methanol, acetic acid, trifluoroacetic acid (TFA), and potassium hydroxide were purchased from Merck. Stock standard solutions of each vitamin (i.e., 1000 mg/l) were prepared in water or methanol and stored in darkness at 4 C. The deionized water used in the analyses was produced by Millipore Elix-3. The mobile phase was filtered using a vacuum pump with a 0.45 µm Durapore membrane filter (Millipore). All solutions were filtered through 0.45 µm filters prior to HPLC analyses. The ph value of the aqueous solution was controlled using a WTW 330İ ph meter that was calibrated using a set of buffer solutions at ph 4 and ph 10. The samples were mixed with a vortex (Velp Scientifica) and centrifuged (Hettich). Chromatographic apparatus An Agilent liquid chromatographic system was equipped with a degasser (1200-G1379 B), an iso pump (1100-G1310A), an auto sampler (1200- G1329A), a column thermostat (1200-G1316A), and a DAD detector (1100-G1315B) working at nm. The parameters for the chromatographic analysis are as follows: Mobile phase: Mobile phase A % 0.01 TFA, ph 3.9; Mobile phase B Methanol The ph value of the mobile phase was optimized with KOH or CH 3 COOH solutions. Column: Phenomenex Gemini 3u C18 110A, 150 x 4.60 mm Flow rate: 0.7 ml/mincolumn Temperature: 20 C Gradient elution: Isocratic and gradient elution Detector: Diode Array Detector

4 33 KUCUKKOLBASI et al. Optimization of chromatographic conditions for sample analysis To optimize the chromatographic conditions in the simultaneous detection of the vitamins, the subsequently detailed processes were undertaken. Wavelength selection In order to determine the optimum wavelength for the determination of the studied vitamins, chromatograms of standard vitamin solutions were taken at different wavelengths ranging from 265 nm to 325 nm, and the wavelength that revealed the peak area and provided the most proportional area for each vitamin was determined. The peak area that belongs to the determined wavelength was recorded. Column selection In order to choose the column that most successfully separates the peaks for simultaneous determination of vitamins in feed and feed additives, we studied two different columns. Reversed phase chromatographic (RPC) columns, i.e., Phenomenex Gemini 3µ C18 110A 150x4.60 mm and Hichrom 5µ C18-250A 25cm x 4.6mm, were tested for their ability to separate the vitamins. Gradient elution For the simultaneous separation of fat soluble and water soluble vitamins, studies on the changes in flow rate, retention times, and mobile phase ratios (A: TFA, %0,01 and B: methanol) were evaluated. After all of the studies were reviewed, the most suitable gradient elution was determined and is listed in Table 6. Sample preparation Extraction solvent ratio In order to determine the proper method to dissolve the vitamins from the feed additive samples into the solution for the HPLC study, studies were done with different ratios of TFA and methanol. Specifically, we studied samples using the following ratios of % TFA to % methanol: 100-0, 80-20, 50-50, 20-80, Then to achieve better extraction, a second extraction was done on the residue. These processes are detailed as follows:

5 34 KUCUKKOLBASI et al. 1. Samples were extracted with a solvent that contained 70% TFA and 30% methanol, and then the residue was further extracted with a solvent that contained 10% TFA and 90% methanol. 2. Samples were extracted with a solvent that contained 80% TFA and 20% methanol, and then the residue was extracted using a solvent that contained 10% TFA and 90% Methanol. Finally, all of the extracts were combined. Vortex time After suitable solvent was added during extraction, vortex stirring times of 5, 15, and 25 min were studied. The most suitable time was chosen. Simultaneous vitamin determination in feed and feed additives Feed additive samples were weighed (2.000 g), placed in a centrifuge tube and dissolved in 8.0 ml of solution containing 20% of 0.01% TFA solution and 80% methanol. The mixtures were stirred for 15 min using a vortex. The solution was centrifuged (5000 g, 24 C). The supernatant was decanted and diluted with the TFA methanol solution and filtered through a 0.45 µm filter prior to HPLC analysis. For all chromatograms, calibration graphics were drawn based on the area of the peak in order to determine the concentration amounts of each vitamin. Method validation Method validation studies were performed by measuring basic parameters such as precision, accuracy, linear region, LOD, LOQ and recovery. The precision of the analytical method was estimated by the intra-day precision or repeatability, and the inter-day precision or intermediate precision. The repeatability was evaluated on results from the same day on ten independent solutions. The intermediate precision was determined by evaluating the repeatability of the analytical method on three different days. The precision was taken from calculating the relative standard deviation (RSD %). For the accuracy determination, a triplicate analysis was performed for all samples to allow measurement of the average deviation. To evaluate the linearity, six standard solutions were prepared for each calibration level and a linear equation was established

6 35 KUCUKKOLBASI et al. for each vitamin by plotting peak areas versus concentration. The limits of detection (LOD) and quantification (LOQ) under the present chromatographic conditions were calculated by Eqs. (1) and (2), respectively, where δ is the standard deviation of blank and s is slope of calibration curve (Revanasiddappa et al., 2001). δ LOD = 3.3 s δ LOQ = 10 s (1) (2) Recoveries for spiked test samples were calculated by comparison to the measured recovery of spiked diluent control. The average recoveries were counted by the formula: recovery (%)= (amount found original amount)/amount spiked 100%, and RSD (%)= (SD/mean) 100%. Results In our work we attempted to simultaneously determine water and fat soluble vitamins using a HPLC method coupled with a diode array detector. For the simultaneous determination of water and fat soluble vitamins, optimum circumstances were defined. Under these optimum circumstances, the minimum detectable vitamin concentrations were chosen. Evaluation of the vitamins chromatographic features To determine the chromatographic features of each vitamin, chromatograms were taken for each standard vitamin solution. Table 1 shows the retention times and wavelengths for the standard vitamins and Figure 1 shows their spectrums. Vit A Vit E Vit D 3 Vit K 3 Vit C Nicotinic Acid Vit B 2 Vit B 6 Vit B 12 Folic Acid Figure 1. Vitamins spectrum

7 36 KUCUKKOLBASI et al. Table 1. Vitamin standards retention times and wavelengths Vitamins Retention times (min) Wavelength (nm) Vitamin C Nicotinic acid Vitamin B Vitamin B Vitamin B Folic acid Vitamin K Vitamin A Vitamin D Vitamin E Figure 2. The standard vitamins mix chromatogram Evaluation of the optimum circumstances for detection of the vitamins Extraction solvent ratio Studies were done with solvents that contained TFA and methanol. Firstly, studies were done using solvents that only contained TFA or methanol. When using only TFA, we observed the disappearance of peaks belonging to fat soluble vitamins. Additionally, when using only methanol, the peaks for the water soluble vitamins disappeared.

8 37 KUCUKKOLBASI et al. Then, in order to obtain the best separation of the peaks, five different extraction solvent ratios were studied (Table 2). The number 1 extraction ratio was 80% TFA- 20% MeOH and the number 2 was 50% TFA -50% MeOH and the number 3 was 20% TFA-80% MeOH. For extractions number 4 and 5 (Table 2), a second extraction was done on the residues. In number 4, the samples were extracted using a solvent that contained 70% TFA and 30% methanol and the supernatant was taken in to tube. Then the residue was extracted again with a solvent that contained 10% TFA and 90% methanol and the supernatant was combined in to the same tube. In number 5, the samples were extracted using a solvent that contained 80% TFA and 20% methanol and the supernatant was taken in to tube. Then the residue was extracted again with a solvent that contained 10% TFA and 90% methanol and the supernatant was combined in to the same tube. The results indicated that the recovery of the fat soluble vitamins was reduced. The results in Table 2 show that the best extraction solvent ratio for the simultaneous separation of all vitamins is 80% methanol and 20% TFA (Extraction number 3). When comparing the two chromatograms in Figures 3 (for extraction number3) and Figure 4 (for extraction number4), the peaks for vitamins K 3, D 3, and E were detectable in Figure 3, but these peaks were absent in Figure 4. Also, the peak area of vitamin A was reduced in Figure 4. Table 2. Comparing the extraction solvents ratio % Solvent Area Vitamin A E D B Extraction Number: 1. 80% TFA- 20% MeOH, 2. 50% TFA -50% MeOH, 3. 20% TFA - %80 MeOH, 4. 70% TFA-30% MeOH then residue 10% TFA-90% MeOH, 5. 80% TFA-20% MeOH then residue 10% TFA-90% MeOH

9 38 KUCUKKOLBASI et al. Figure 3. Feed additives chromatogram (extraction number 3) Figure 4. Feed additives chromatogram (extraction number 4)

10 39 KUCUKKOLBASI et al. Based on the results, we determined that the best separation was achieved when a solvent with a ratio of 80% methanol and 20% TFA was used. Vortex time After a suitable solvent was added during extraction, different vortex stirring times (i.e., 5, 15, and 25 min) were evaluated, and 15 min was chosen. Table 3 shows that for the shorter stirring time of 5 min, recovery was reduced. Also, we can see that no difference in recovery was observed between the stirring times of 15 and 25 min. Therefore, 15 min was chosen for vortex stirring time. The chromatograms for vitamin B 12 using various vortex times are shown in Figure 5. Table 3. The peak area of some vitamins at various vortex time Time(min) Vitamin A E D B Figure 5. B 12 vitamin s chromatograms for various vortex times

11 40 KUCUKKOLBASI et al. Evaluation of the chromatographic conditions for sample analysis Wavelength A primary focus of this study was the selection of the most suitable wavelength for the simultaneous determination of water and fat soluble vitamins. Several wavelengths for the detection of water soluble vitamins (i.e., Vitamin C, B 12, folic acid, B 2, nicotinic acid, and B 6 ) and fat soluble vitamins (K 3, A, E, and D 3 ) were tested. According to our measurements, we selected three suitable wavelengths (i.e., 265, 280, and 325 nm) for the subsequent experiments. Vitamin peak areas at various wavelengths are shown in Table 4. The most suitable detection wavelength for each vitamin is shown in Table 5. Additionally, Figures 6 and 7 show the chromatograms for vitamins K 3 and D 3, respectively, at various wavelengths. Table 4. Vitamins area at various wavelengths (vitamin concentration: 10 mg/l) Wavelength Peak Area (nm) A E D 3 B ,5 12,9 476,2 89, ,8 32,4 343,7 99, ,8 18,0 168,4 76, ,1-2,9 52,3 Table 5. The wavelengths in vitamin analyses Wavelength (nm) Vitamins 265 K 3, D E, B 2, B 12, C, Nicotinic acid, B 6, Folic acid 325 A

12 41 KUCUKKOLBASI et al. Minute Figure 6. K 3 vitamins chromatogram at various wavelengths Minute Figure 7. D 3 vitamins chromatogram at various wavelengths Column To choose the column that separates the peaks most effectively for the simultaneous determination of vitamins in feed and feed additives, we studied two different columns. Specifically, two RPC columns, i.e., Phenomenex Gemini 3u C18 110A (150 x 4.60 mm) and Hichrom 5 C18-250A (25 cm x 4.6 mm, 5 micron) were tested for their ability to separate the vitamins.

13 42 KUCUKKOLBASI et al. While vitamin E s retention time was 39 min with a peak area of 16.3 on the Hichrom column, the retention time and the peak area for the same vitamin was 28 min and 214, respectively, on the Phenomenex column. Similar results were obtained for vitamin D 3 and other vitamins. The Phenomenex column was the most suitable for the separation of both fat and water soluble vitamins in a single run; therefore, this column was used for all other experiments. In Figure 8, the chromatograms from both of these columns for vitamin A are shown. A B Figure 8. Peaks of vitamin A with two different column. a) Hichrom b) Phenomenex Gradient elution To develop a method for the simultaneous separation of fat soluble and water soluble vitamins, we evaluated studies on the changes in flow rate, retention times, and mobile phase ratios (Mobile phase A: TFA, 0.01% and mobile phase B: methanol). Based on all of the previous studies, the most suitable gradient elution, which was used in this study, is shown in Table 6 and Figure 9. The gradient profile for simultaneous vitamin separation started at 95:5 (0.01% TFA: methanol) and was constant in the first 4 min, then decreased to 2:98 during the next 10 min, and finally linearly increased to 95:5 from 30 to 35 min as shown in Figure 9. Figure 9. Mobile phase B s flow graphic against to the minute

14 43 KUCUKKOLBASI et al. Table 6. Gradient elution Time (Min) Mobile phase A(TFA)% Mobile phase B(MeOH)% Flow (ml/min) LOD, LOQ and Recovery Based on the limit of detection (LOD) studies, the lowest amount of each vitamin that can be seen as a peak in HPLC is detailed in Table 7. LOD and limit of quantitation (LOQ) were calculated using the following respective formulas: LOD = 3 x S/N (signal/noise), LOQ = 10 x S/N. The recovery studies for fat soluble and water soluble vitamins in feed and feed additives are shown in table 8. Clearly, the percent recovery of the fat soluble vitamins A, E, D 3, and K 3 and the water soluble vitamins B 2 and B 12 is good. The statistical data for all vitamins is shown in Table 9. Because the recovery is very low for folic acid, vitamin C, vitamin B 6, and nicotinic acid, these vitamins are not represented in Table 3.8. In this study, the better recovery results were observed for vitamins B 12, B 2, A, D 3, K 3, and E. Therefore, the method was considered successful for the water soluble vitamins B 12 and B 2 and the fat soluble vitamins A, D 3, K 3, and E. Table 7. LOD studies of vitamins Vitamin K 3 Concentration (mg/l) Area Vitamin A Concentration (mg/l) Area

15 44 KUCUKKOLBASI et al. Vitamin E Concentration(mg/L) Area Vitamin D 3 Concentration(mg/L) Area Vitamin C Concentration(mg/L) Area Nicotinic Acid Concentration(mg/L) Vitamin B 6 Concentration(mg/L) Area Vitamin B Area 0.5 8, , Concentration (mg/l) Vitamin B 2 Area Concentration (mg/l) Area 0.1 4,

16 45 KUCUKKOLBASI et al. Table 8. Vitamin analyses in feed additives and recovery results (n=6) Vitamin Amount (mg/kg±sd*) Added (mg/kg) Found (mg/kg±sd*) Recovery (%±SD) Vitamin A ± ± ± 1.5 Vitamin E ± ± ± 0.6 Vitamin D ± ± ± 1.6 Vitamin K ± ± ± 2.2 Vitamin B ± ± ±1.3 Vitamin B ± ± 1.4 * Average of five determinations Vitamin Table 9. Validation data for fat and water soluble vitamins (n=6) Retention Time (min) LineerRate (mg/l) LOD (mg/l) LOQ (mg/l) %RSD (mg/l) Regression equation Regression coefficients (r 2 ) L-ascorbic Acid y=44.7x Cyanocobalamin y=9.9x Folic acid y=3.1x Riboflavin y= x Nicotinic acid y=3.9x Pyridoxine y=19.5x Vitamin K y=83.6x Vitamin A y= x Vitamin E y= x Vitamin D y=47.5x Discussion Vitamin analysis can be done using various routine laboratory methods, but fat soluble and water soluble vitamins are typically analyzed separately. To determine the concentrations of vitamins using two different methods requires more time and more organic solvent (especially ether). This work proposed a new method for the simultaneous separation and quantification of six water soluble vitamins (i.e., nicotinic acid, folic acid, and vitamins C, B 2, B 6, and B 12 ) and four fat soluble vitamins (i.e., vitamins A, E, D 3, and K 3 ) using HPLC coupled with a diode array detector. This method was optimized and applied to the analyses of samples of feed and feed additives. The combined isocratic and linear gradient profile of the mobile phase (i.e., 0.01% TFA at ph 3.9 and methanol) allowed for the successful determination of some vitamins. A baseline separation with good resolution of all vitamins in each group was

17 46 KUCUKKOLBASI et al. achieved in a single run. HPLC provided a fast, accurate, and reliable method for their determination with recoveries ranging from 88.5% to 100.3%. Low detection limits, good sensitivity, and resolution with a minimum analysis time of 10 min for some of the studied water soluble vitamins and 18 min for the fat-soluble vitamins were achieved. Indeed, our results were in very good agreement with the declared values. Our chromatographic method allowed for the very easy and fast separation of vitamins from both types. This study provides important information about the simultaneous determination of fat soluble and water soluble vitamins and the application of such a method to feed and feed components. Also, the proposed method requires less extraction time. Therefore, this method saves both time and money as compared to more traditional methods. This proposed methodology can be applied to the determination of the concentration of vitamins in feed and feed additives. Acknowledgments We gratefully acknowledge the financial support from the Selcuk University Research Fund (Project No: ). References Barnett, S.A., Frick, L.W., Baine, H.M., (1980): Simultaneous determination of vitamins A, D2 or D3, E and K1 in infant formulas and dairy products by reversed-phase liquid chromatography. Anal. Chem., 52, Fallon, A., Booth, R.F.G., Bell, L.D., (1987): Applications of HPLC in Biochemistry, Elsevier, Amsterdam, New York. Garcia, L., Blazquez, S., San Andres, M.P.,. Vera, S., (2001): Determination of thiamine, riboflavin and pyridoxine in pharmaceuticals by synchronous fluorescence spectrometry in organized media. Anal. Chim. Acta., 434, Han, J.Y., Tyler, R.T., (2003): Determination of Folate Concentrations in Pulses by a Microbiological Method Employing Trienzyme Extraction. J. Agric. Food. Chem., 51, Klejdus, B., Petrlova, J., Potesil, D., Adam, V., Mikelova, R., Vacek, J., Kizek, R., Kuban, V., (2004): Simultaneous determination of water- and fat-soluble vitamins in pharmaceutical preparations by high-performance liquid chromatography coupled with diode array detection. Analytica Chimica Acta., 520, Kothari, R.M. & Taylor, M.W., (1982): Analysis of oxidized and reduced pyridine dinucleotides in rat liver by high-performance liquid chromatography. J. Chromatogr., 247,

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