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Binding constants of oxytetracycline to animal feed divalent cations
Journal of Food Engineering 78 (2007) 69–73 www.elsevier.com/locate/jfoodeng
Binding constants of oxytetracycline to animal feed divalent cations M. Arias a, M.S. Garcı´a-Falco´n b, L. Garcı´a-Rı´o c, J.C. Mejuto d, R. Rial-Otero b, J. Simal-Ga´ndara b,* a
c
Area de Edafologı´a y Quı´mica Agrı´cola, Facultad de Ciencias de Ourense, Campus de Ourense, Universidad de Vigo, E-32004 Ourense, Spain b Area de Nutricio´n y Bromatologı´a, Facultad de Ciencias de Ourense, Campus de Ourense, Universidad de Vigo, E-32004 Ourense, Spain Departamento de Quı´mica Fı´sica, Facultad de Quı´mica, Campus de Santiago de Compostela, Universidad de Santiago de Compostela, E-15706 Santiago de Compostela, Spain d Departamento de Quı´mica Fı´sica, Facultad de Ciencias de Ourense, Campus de Ourense, Universidad de Vigo, E-32004 Ourense, Spain Received 6 June 2005; accepted 12 September 2005 Available online 3 November 2005
Abstract The binding constants of different divalent cations (Ca+2, Mg+2, Fe+2, Hg+2, Ni+2 and Co+2) to oxytetracycline (OTC) has been estimated. OTC can be used in veterinary formulations for the prevention and control of disease and added to feed for such a purpose at a dosage rate of 25–700 mg/kg. Feeds contain Ca+2 and Mg+2 carbonates as raw materials (at a percentage level), and Fe+2 and Co+2 as additives (at a ppm level), but also can contain Hg+2 as a contaminant from fish-based raw materials, or Ni+2 as a residual catalyser used in the partial hydrogenation of seed oils. It is key to develop reliable methods for OTC determination, paying special attention to OTC extraction from feed matrices containing those divalent cations, since the particular structure of OTC contains electron-donor groups able to form strong complexes with such metal ions. The values obtained for KOTC show that OTC exhibits a large affinity for Ca+2 and Mg+2 and decreases on the increase of the charge/ionic radius ratio for the other divalent cations, what means that Ca+2 and Mg+2 can make more difficult OTC extraction from feeds. 2005 Elsevier Ltd. All rights reserved. Keywords: Oxytetracycline; Divalent cations; Binding constants; Synchronous spectrofluorimetry
1. Introduction Oxytetracycline (OTC; Scheme 1) is one of the important members of the tetracycline group of antibiotics which is routinely used in animal husbandry. It is used for the prophylaxis and treatment of a great number of diseases since this antibiotic possesses a broad spectrum activity against many pathogenic organisms. OTC can be used in veterinary formulations for the prevention and control of disease and added to feed for such a purpose. It is licensed for use in a wide variety of food-producing animals such as cattle, pigs, sheep, poultry and it is a principal antibiotic used in fish farming too (Coopper et al., 1998; Farrington,
*
Corresponding author. Tel.: +34 988387060; fax: +34 988387001. E-mail address: [email protected] (J. Simal-Ga´ndara).
0260-8774/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2005.09.016
Tarbin, Bygrave, & Shearer, 1991; McCracken, Blanchflower, Haggan, & Kennedy, 1995; Pouliquen, Keita, & Pinault, 1992; Stubbings, Tarbin, & Shearer, 1996). Its usage may result in residues in food products of animal origin, often due to the improper observance of withdrawal periods. For this reason, OTC is often determined in those foods; for example, some authors found this antibiotic in honey (Salinas, Berzas, & Espinosa, 1989; Xie, Dong, Fen, & Liu, 1997), in milk (Boatto, Pau, Palomba, Arenare, & Cerri, 1999; Carson & Breslyn, 1996; Carson, Ngoh, & Hadley, 1998; Furusawa, 1999a; Pena, Lino, & Silveira, 1999; Tjørnelund & Hansen, 1997), in eggs (Coopper et al., 1998; Croubels, Vanoosthuyze, & Van Peteghem, 1997; Furusawa, 1999b, 1999c; Omija, Mitema, & Maitho, 1994), and in different animal tissues (Carson et al., 1998; Coopper et al., 1998; Croubels et al., 1997; De Wasch et al., 1998; Farrington et al., 1991; McCracken et al.,
70
M. Arias et al. / Journal of Food Engineering 78 (2007) 69–73
HO
NMe2
OH
Me H
H OH
S
S
S
R
R S
NH2 OH
OH
O
OH
O
O
present in the samples to be extracted, which makes difficult to extract OTC from animal tissues (Pouliquen et al., 1992; Sokol & Matisova, 1994) or from feeds that contain a large amount of calcium, magnesium and other cations (Ferna´ndez-Gonza´lez et al., 2002). In the present paper, the binding constant of different divalent cations (Ca+2, Mg+2, Fe+2, Hg+2, Ni+2 and Co+2) to the OTC has been estimated with the intention of providing information about the potential difficulties to extract OTC from divalent cation-containing feeds.
Scheme 1.
2. Experimental 1995; Pouliquen et al., 1992; Sokol & Matisova, 1994; Stubbings et al., 1996; Walsh, Walker, & Webber, 1992). OTC has been measured too in other matrices different from food, like plasma (Iwaki, Okumura, & Yamazaki, 1993), urine (Sharma, Koritz, Perkins, & Bevill, 1977; Weimann & Bojesen, 1999; Xie et al., 1997), feeds (Ferna´ndez-Gonza´lez, Garcı´a-Falco´n, & Simal-Ga´ndara, 2002; Hasselberger, 1993; Houglum, Larson, Mutchler, & Wetzler, 1998; Markakis, 1996; Martı´nez & Shimoda, 1988; Weng, Sun, Roets, & Hoogmartens, 2003), premixes and pharmaceutical formulations (Chappell, Houglum, & Kelley, 1986; Croubels et al., 1997; Ferna´ndez-Gonza´lez et al., 2002; Houglum et al., 1998; Iwaki, Okumura, & Yamazaki, 1992; Katz & Fassbender, 1973; Kazemifard & Moore, 1997; Weng et al., 2003), etc. In the matrix of the samples analysed exist lots of components that can interfere with OTC determination. In samples like feeds, interfering substances such as pigments, additives, fatty acids and mineral cations occur naturally (Salvatore & Katz, 1993). It is of paramount importance to achieve a selective OTC extraction. Recently, our research group (Ferna´ndez-Gonza´lez et al., 2002) has demonstrated that synchronous spectrofluorimetry is a versatile analytical technique ideal for OTC determination in analytical quality control and for research and development laboratories in the food and beverage industry. This method (Ferna´ndez-Gonza´lez et al., 2002) is reliable (with good response linearity, high recovery and precision, and low detection and quantification levels), simple and fast. The particular structure of this antibiotic, as in the case of other tetracyclines, contains electron-donor groups able to form strong complexes with metal ions (Carson & Breslyn, 1996; Lambs, Decock-Le Re´ve´rend, Kozlowski, & Berthon, 1988). Therefore, some authors use different OTC extraction procedures based on liquid–liquid extraction with calcium (Sharma et al., 1977) or clean-up with metal chelate affinity chromatography, which consists in one column preloaded with divalent cations where OTC is specifically adsorbed by chelation with the metal ions (Carson & Breslyn, 1996; Carson et al., 1998; Coopper et al., 1998; Croubels et al., 1997; De Wasch et al., 1998; Farrington et al., 1991; Stubbings et al., 1996). We must also take into consideration that these ions can be naturally
2.1. Reagents Oxytetracycline hydrochloride (OTC) was purchased from Riedel-de Hae¨n (Seelze, Germany). Because OTC is not very stable and is susceptible of photochemical and oxidative degradation, work solutions were freshly prepared every day. Divalent metal salts included calcium chloride anhydrous (CaCl2), supplied by Panreac (Barcelona, Spain); nickel chloride hexahydrate (NiCl2 Æ 6H2O) from Riedel-de Hae¨n (Seelze, Germany); cobaltous chloride hexahydrate (CoCl2 Æ 6H2O), magnesium chloride hexahydrate (MgCl2 Æ 6H2O), mercuric chloride anhydrous (HgCl2) and ferrous chloride tetrahydrate (FeCl2 Æ 4H2O), from Fluka (Steinheim, Germany). All solutions were prepared in highly pure water (R P 18 MX) obtained from a Millipore water purification system. 2.2. Apparatus and operating conditions All spectrofluorometric measurements were taking by means of a Jasco P-750 luminescence spectrophotometer equipped with a xenon lamp, Monk-Gillieson monochromators and 1 cm quartz cuvettes. The spectral data were collected and processed using the Spectra Manager software. The optimised instrumental parameters for synchronous scanning of OTC complexes were as in Table 1. 2.3. Procedure Association constants were measured by spectrofluorometry, using reported procedures in the literature (Garcı´a-Rı´o, Herve´s, Mejuto, Parajo´, & Pe´rez-Juste, 1998), by making use of different synchronous spectra of
Table 1 Optimised instrumental parameters for synchronous scanning of OTC complexes Parameters
Spectrofluorometric conditions
Spectral range Excitation and emission slits Scan speed Excitation-emission wavelength difference (Dk)
400–600 nm 10 and 5 nm respectively 125 nm/min 115 nm
M. Arias et al. / Journal of Food Engineering 78 (2007) 69–73
free and complexed OTC solutions. The complexes were formed by adding to fix OTC concentration (4 · 106 M) different aliquots of concentrated divalent cation solutions with the intention of increasing cation concentrations in the 3 mL final volume. In all cases the concentration of cation was at least 10 times bigger than the concentration of OTC to be sure that the assumption of the total concentration of cation non-bounded to OTC is approximately equal to the initial cation concentration. The pH of these OTCdivalent cation solutions varies with the addition of the cations. Since the pH of OTC solutions has a significant effect on the fluorescence intensity measured in the case of Fe+2, Ni+2, Co+2 and Hg+2, we corrected for the fluorescence intensity of OTC solutions caused only by effect of pH between 4.0 and 5.0 in the fluorescence intensity of these OTC-divalent cation solutions. 3. Results and discussion The determination of the binding constant of cations to OTC is based in the fact that changes in the chemical environment of OTC will modify its fluorescence spectrum. The association of cations to the OTC will imply changes in the position and in the intensity of their fluorescence peaks. We can write then that the total intensity measured (I) corresponds with the sum of OTC intensity in the absence of cations (If) and in the presence of cations (Ib). I ¼ If þ Ib
ð1Þ
71
Eqs. (4) and (5) imply eapp l½OTCt ¼
ef l½OTCt þ eb l½OTCt K OTC ½Cþ2 1 þ K OTC ½Cþ2
ð6Þ
Because eappl[OTC]t is equal to the total intensity (I), efl[OTC]t is the intensity in the absence of divalent cation (If), and ebl[OTC]t corresponds with the intensity when all the substrate is bound to divalent cation (Ib), Eq. (6) can be written as I¼
I f þ I b K OTC ½Cþ2 1 þ K OTC ½Cþ2
ð7Þ
Eq. (7) can be linearised I If ¼ K OTC ½Cþ2 Ib If
ð8Þ
If we measure the fluorescence intensity at various divalent cation concentrations at a fixed concentration of OTC, the binding constant can be calculated from Eq. (7) or Eq. (8). Fig. 1 shows, as example, the observed behaviour of fluorescence intensity of OCT in the presence of two divalent cations (Ca+2 and Mg+2). Similar behaviours (increase or decrease of intensity on the increase of the concentration of divalent cations) are observed for the other divalent cations—data not shown. The solid lines represent the fit of the experimental data of fluorescence intensity to Eq. (7). In Table 2, the binding constant of the different divalent cations to OTC is listed.
Taking into account Beer–Lambert law, Eq. (1) can be rewritten as a function of total OTC concentration, [OTC]t, and the respective concentrations of OTC free, [OTC]f, and OTC–Cation complex, [OTC]b eapp l½OTCt ¼ ef l½OTDf þ eb l½OTCb
ð2Þ
where l is the light pathway (in a standard cell is equal to 1 cm). e corresponds with the attenuation coefficient or extinction coefficient. The three concentrations of Eq. (2) are related to the constant of complex formation, KOTC, defined as K OTC ¼
½OTCb ½OTCf ½Cþ2
ð3Þ
where [C+2] is the non-bounded divalent cation concentration. Because the initial concentration of cation is at least 10 times bigger that OTC concentration, we can assume that the non-bounded divalent cation concentration is equal to the initial divalent cation concentration. Using Eq. (3), and the corresponding matter balance ([OTC]t = [OTC]f + [OTC]b), Eqs. (4) and (5) can be easily deduced ½OTCf ¼ ½OTCb ¼
½OTCt 1 þ K OTC ½Cþ2
ð4Þ
½OTCt K OTC ½Cþ2 1 þ K OTC ½Cþ2
ð5Þ
Fig. 1. Influence of divalent cation concentration upon the fluorescence intensity of OTC. Solid line represents the fit of experimental data to Eq. (7). (s) Mg+2, (d) Ca+2. Table 2 Binding constant of divalent cations to OTC obtained from Eq. (7) Cation +2
Ca Mg+2 Hg+2
KOTC 55 ± 9 66 ± 9 36 ± 3
Cation +2
Ni Fe+2 Co+2
KOTC 23 ± 5 8±1 7±3
72
M. Arias et al. / Journal of Food Engineering 78 (2007) 69–73
The value obtained for KOTC shows that OTC exhibit a large affinity for Ca+2 and Mg+2 and decreases on the increase of the charge/ionic radius ratio for the other divalent cations. The capacity of complex formation between OTC and divalent cations is clearly related to the electron acceptor capacity of cations. According with Pearson classification (1968a, 1968b), Ca+2 and Mg+2 are considered hard electron acceptors and present KOTC values of 55 and 66 while Ni+2, Co+2, Fe+2 and Hg+2 are intermediate or soft electron acceptors and present values of KOTC in the range of 7–36. This means that Ca+2 and Mg+2 can make more difficult OTC extraction from feeds. This problem can be solved by adding a chelating agent, like ethylenediamine tetraacetate (EDTA), which forms complexes with these ions and avoids the formation of OTC-metal complexes. Acknowledgements We gratefully acknowledge support of this research by the Spanish Ministry of Science and Technology through Ramo´n y Cajal research contracts awarded to Drs. Arias and Garcı´a-Falco´n. References Boatto, G., Pau, A., Palomba, M., Arenare, L., & Cerri, R. (1999). Monitoring of oxytetracycline in ovine milk by high-performance liquid chromatography. Journal of Pharmaceutical Biomedical Analysis, 20, 321–326. Carson, M. C., & Breslyn, W. (1996). Simultaneous determination of multiple tetracycline residues in milk by metal chelate affinity chromatography: collaborative study. Journal of AOAC International, 79(1), 29–42. Carson, M. C., Ngoh, M. A., & Hadley, S. W. (1998). Confirmation of multiple tetracycline residues in milk and oxytetracycline in shrimp by liquid chromatography-particle beam mass spectrometry. Journal of Chromatography B, 712, 113–128. Chappell, G. S., Houglum, J. E., & Kelley, W. N. (1986). Determination of oxytetracycline in premixes and veterinary products by liquid chromatography. Journal Association of Official Analytical Chemists, 69(1), 28–30. Coopper, A. D., Stubbings, G. W. F., Kelly, M., Tarbin, J. A., Farrington, W. H. H., & Shearer, G. (1998). Improved method for the on-line chelate affinity chromatography-high-performance liquid chromatographic determination of tetracycline antibiotics in animal products. Journal of Chromatography A, 812, 321–326. Croubels, S. M., Vanoosthuyze, K. E. I., & Van Peteghem, C. H. (1997). Use of metal chelate affinity chromatography and membrane-based ion-exchange as clean-up procedure for trace residue analysis of tetracyclines in animal tissues and egg. Journal of Chromatography B, 690, 173–179. De Wasch, K., Okerman, L., Croubels, S., De Brabander, H., Van Hoof, J., & De Backer, P. (1998). Detection of residues of tetracycline antibiotics in pork and chicken meat: correlation between results of screening and confirmatory tests. Analyst, 123, 2737–2741. Farrington, W. H. H., Tarbin, J., Bygrave, J., & Shearer, G. (1991). Analysis of trace residues of tetracyclines in animal tissues and fluids using metal chelate affinity chromatography/HPLC. Food Additives and Contaminants, 8(1), 55–64. Ferna´ndez-Gonza´lez, R., Garcı´a-Falco´n, M. S., & Simal-Ga´ndara, J. (2002). Quantitative analysis for oxytetracycline in medicated premixes
and feeds by second-derivative synchronous spectrofluorimetry. Analytica Chimica Acta, 455, 143–148. Furusawa, N. (1999a). Rapid liquid chromatographic determination of oxytetracycline in milk. Journal of Chromatography A, 839(1/2), 247–251. Furusawa, N. (1999b). High-performance liquid chromatographic determination/identification of oxytetracycline and sulphadimidine in meat and eggs. Chromatographia, 49(7/8), 369–373. Furusawa, N. (1999c). Rapid and simple determination of oxytetracycline in chicken products. Journal of AOAC International, 82(3), 770–772. Garcı´a-Rı´o, L., Herve´s, P., Mejuto, J. C., Parajo´, M., & Perez-Juste, J. (1998). Association constant of crystal violet in micellar aggregates: determination by spectroscopic techniques. Journal of Chemical Research Synopsis, 716–717. Hasselberger, M. L. (1993). Assay of oxytetracycline in animal feed by liquid chromatography and microbiological plate assay. Journal of AOAC International, 76(1), 39–43. Houglum, J. E., Larson, R. D., Mutchler, D. L., & Wetzler, L. (1998). Assay of oxytetracycline in animal feeds by liquid chromatography with fluorescence detection. Journal of AOAC International, 81(5), 919–922. Iwaki, K., Okumura, N., & Yamazaki, M. (1992). Determination of tetracycline antibiotics by reversed-phase high-performance liquid chromatography with fluorescence detection. Journal of Chromatography, 623(1), 153–158. Iwaki, K., Okumura, N., & Yamazaki, M. (1993). Rapid determination of tetracycline antibiotics in serum by reversed-phased high-performance liquid chromatography with fluorescence detection. Journal of Chromatography, 619(2), 319–323. Katz, S. E., & Fassbender, C. A. (1973). Antibiotics. Fluorometric determination of oxytetracycline in premixes. Journal of AOAC International, 56(1), 17–19. Kazemifard, A. G., & Moore, D. E. (1997). Evaluation of amperometric detection for the liquid-chromatographic determination of tetracycline antibiotics and their common contaminants in pharmaceutical formulations. Journal of Pharmaceutical Biomedical Analysis, 16(4), 689–696. Lambs, L., Decock-Le Re´ve´rend, B., Kozlowski, H., & Berthon, G. (1988). Metal ion-tetracycline interactions in biological fluids. 9. Circular dichroism spectra of calcium and magnesium complexes with tetracycline, oxytetracycline, doxycycline and chlortetracycline and discussion of their binding modes. Inorganic Chemistry, 27, 3001–3012. Markakis, P. K. (1996). Determination of tetracyclines in animal feeds in the presence of other drugs by thin-layer chromatography and microbiological method. Journal of AOAC International, 79(2), 375–379. Martı´nez, E. E., & Shimoda, W. (1988). Liquid chromatographic determination of tetracycline residues in animal feeds. Journal of AOAC International, 71(3), 477–480. McCracken, R. J., Blanchflower, W. J., Haggan, S. A., & Kennedy, D. G. (1995). Simultaneous determination of oxytetracycline, tetracycline and chlortetracycline in animal tissues using liquid chromatography, post-column derivatization with aluminium, and fluorescence detection. Analyst, 120, 1763–1766. Omija, B., Mitema, E. S., & Maitho, T. E. (1994). Oxytetracycline residue levels in chicken eggs after oral administration of medicated drinking water to laying chickens. Food Additives and Contaminants, 11(6), 641–647. Pearson, R. (1968a). Hard and soft acids and bases. Fundamental principles. Journal of Chemical Education, 45, 581–587. Pearson, R. (1968b). Hard and soft acids and bases. Underlying theories. Journal of Chemical Education, 45, 643–648. Pena, A. L., Lino, C. M., & Silveira, I. N. (1999). Determination of oxytetracycline, tetracycline and chlortetracycline in milk by liquid chromatography with postcolumn derivatization and fluorescence detection. Journal of AOAC International, 82(1), 55–60. Pouliquen, H., Keita, D., & Pinault, L. (1992). Determination of oxytetracycline in marine shellfish (Crassostrea gigas, Ruditapes philippinarum and Scrobicularia plana) by high-performance liquid
M. Arias et al. / Journal of Food Engineering 78 (2007) 69–73 chromatography using solid-phase extraction. Journal of Chromatography, 627, 287–293. Salinas, F., Berzas, J. J., & Espinosa, A. (1989). Determination of oxytetracycline and doxycycline in pharmaceutical compounds, urine and honey by derivative spectrophotometry. Analyst, 114(9), 1141–1145. Salvatore, M. J., & Katz, S. E. (1993). Solubility of antibiotics used in animal feeds in selected solvents. Journal of AOAC International, 76(5), 952–956. Sharma, J. P., Koritz, G. D., Perkins, E. G., & Bevill, R. F. (1977). Highpressure liquid chromatographic determination of tetracyclines in urine. Journal of Pharmaceutical Sciences, 66(9), 1319–1322. Sokol, J., & Matisova, E. (1994). Determination of tetracycline antibiotics in animal tissues of food-producing animals by high-performance liquid chromatography using solid-phase extraction. Journal of Chromatography A, 669, 75–80. Stubbings, G., Tarbin, J. A., & Shearer, G. (1996). On-line metal chelate affinity chromatography clean-up for the high-performance liquid chromatographic determination of tetracycline antibiotics in animal tissues. Journal of Chromatography B, 679, 137–145.
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Tjørnelund, J., & Hansen, S. H. (1997). Use of metal complexation in nonaqueous capillary electrophoresis systems for the separation and improved detection of tetracyclines. Journal of Chromatography A, 779, 235–243. Walsh, J. R., Walker, L. V., & Webber, J. J. (1992). Determination of tetracyclines in bovine and porcine muscle by high-performance liquid chromatography using solid-phase extraction. Journal of Chromatography, 596, 211–216. Weimann, A., & Bojesen, G. (1999). Analysis of tetracyclines in raw urine by column-switching high-performance liquid chromatography and tandem mass spectrometry. Journal of Chromatography B, 721(1), 47–54. Weng, N., Sun, H., Roets, E., & Hoogmartens, J. (2003). Assay and purity control of tetracycline, chlortetracycline and oxytetracycline in animal feeds and premixes by TLC densitometry with fluorescence detection. Journal of Pharmaceutical and Biomedical Analysis, 33(1), 85–93. Xie, H., Dong, C., Fen, Y., & Liu, C. (1997). Determination of doxycycline, tetracycline and oxytetracycline simultaneously by TLCfluorescence scanning densitometry. Analytical Letters, 30(1), 79–90.
Binding constants of oxytetracycline to animal feed divalent cations M. Arias a, M.S. Garcı´a-Falco´n b, L. Garcı´a-Rı´o c, J.C. Mejuto d, R. Rial-Otero b, J. Simal-Ga´ndara b,* a
c
Area de Edafologı´a y Quı´mica Agrı´cola, Facultad de Ciencias de Ourense, Campus de Ourense, Universidad de Vigo, E-32004 Ourense, Spain b Area de Nutricio´n y Bromatologı´a, Facultad de Ciencias de Ourense, Campus de Ourense, Universidad de Vigo, E-32004 Ourense, Spain Departamento de Quı´mica Fı´sica, Facultad de Quı´mica, Campus de Santiago de Compostela, Universidad de Santiago de Compostela, E-15706 Santiago de Compostela, Spain d Departamento de Quı´mica Fı´sica, Facultad de Ciencias de Ourense, Campus de Ourense, Universidad de Vigo, E-32004 Ourense, Spain Received 6 June 2005; accepted 12 September 2005 Available online 3 November 2005
Abstract The binding constants of different divalent cations (Ca+2, Mg+2, Fe+2, Hg+2, Ni+2 and Co+2) to oxytetracycline (OTC) has been estimated. OTC can be used in veterinary formulations for the prevention and control of disease and added to feed for such a purpose at a dosage rate of 25–700 mg/kg. Feeds contain Ca+2 and Mg+2 carbonates as raw materials (at a percentage level), and Fe+2 and Co+2 as additives (at a ppm level), but also can contain Hg+2 as a contaminant from fish-based raw materials, or Ni+2 as a residual catalyser used in the partial hydrogenation of seed oils. It is key to develop reliable methods for OTC determination, paying special attention to OTC extraction from feed matrices containing those divalent cations, since the particular structure of OTC contains electron-donor groups able to form strong complexes with such metal ions. The values obtained for KOTC show that OTC exhibits a large affinity for Ca+2 and Mg+2 and decreases on the increase of the charge/ionic radius ratio for the other divalent cations, what means that Ca+2 and Mg+2 can make more difficult OTC extraction from feeds. 2005 Elsevier Ltd. All rights reserved. Keywords: Oxytetracycline; Divalent cations; Binding constants; Synchronous spectrofluorimetry
1. Introduction Oxytetracycline (OTC; Scheme 1) is one of the important members of the tetracycline group of antibiotics which is routinely used in animal husbandry. It is used for the prophylaxis and treatment of a great number of diseases since this antibiotic possesses a broad spectrum activity against many pathogenic organisms. OTC can be used in veterinary formulations for the prevention and control of disease and added to feed for such a purpose. It is licensed for use in a wide variety of food-producing animals such as cattle, pigs, sheep, poultry and it is a principal antibiotic used in fish farming too (Coopper et al., 1998; Farrington,
*
Corresponding author. Tel.: +34 988387060; fax: +34 988387001. E-mail address: [email protected] (J. Simal-Ga´ndara).
0260-8774/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2005.09.016
Tarbin, Bygrave, & Shearer, 1991; McCracken, Blanchflower, Haggan, & Kennedy, 1995; Pouliquen, Keita, & Pinault, 1992; Stubbings, Tarbin, & Shearer, 1996). Its usage may result in residues in food products of animal origin, often due to the improper observance of withdrawal periods. For this reason, OTC is often determined in those foods; for example, some authors found this antibiotic in honey (Salinas, Berzas, & Espinosa, 1989; Xie, Dong, Fen, & Liu, 1997), in milk (Boatto, Pau, Palomba, Arenare, & Cerri, 1999; Carson & Breslyn, 1996; Carson, Ngoh, & Hadley, 1998; Furusawa, 1999a; Pena, Lino, & Silveira, 1999; Tjørnelund & Hansen, 1997), in eggs (Coopper et al., 1998; Croubels, Vanoosthuyze, & Van Peteghem, 1997; Furusawa, 1999b, 1999c; Omija, Mitema, & Maitho, 1994), and in different animal tissues (Carson et al., 1998; Coopper et al., 1998; Croubels et al., 1997; De Wasch et al., 1998; Farrington et al., 1991; McCracken et al.,
70
M. Arias et al. / Journal of Food Engineering 78 (2007) 69–73
HO
NMe2
OH
Me H
H OH
S
S
S
R
R S
NH2 OH
OH
O
OH
O
O
present in the samples to be extracted, which makes difficult to extract OTC from animal tissues (Pouliquen et al., 1992; Sokol & Matisova, 1994) or from feeds that contain a large amount of calcium, magnesium and other cations (Ferna´ndez-Gonza´lez et al., 2002). In the present paper, the binding constant of different divalent cations (Ca+2, Mg+2, Fe+2, Hg+2, Ni+2 and Co+2) to the OTC has been estimated with the intention of providing information about the potential difficulties to extract OTC from divalent cation-containing feeds.
Scheme 1.
2. Experimental 1995; Pouliquen et al., 1992; Sokol & Matisova, 1994; Stubbings et al., 1996; Walsh, Walker, & Webber, 1992). OTC has been measured too in other matrices different from food, like plasma (Iwaki, Okumura, & Yamazaki, 1993), urine (Sharma, Koritz, Perkins, & Bevill, 1977; Weimann & Bojesen, 1999; Xie et al., 1997), feeds (Ferna´ndez-Gonza´lez, Garcı´a-Falco´n, & Simal-Ga´ndara, 2002; Hasselberger, 1993; Houglum, Larson, Mutchler, & Wetzler, 1998; Markakis, 1996; Martı´nez & Shimoda, 1988; Weng, Sun, Roets, & Hoogmartens, 2003), premixes and pharmaceutical formulations (Chappell, Houglum, & Kelley, 1986; Croubels et al., 1997; Ferna´ndez-Gonza´lez et al., 2002; Houglum et al., 1998; Iwaki, Okumura, & Yamazaki, 1992; Katz & Fassbender, 1973; Kazemifard & Moore, 1997; Weng et al., 2003), etc. In the matrix of the samples analysed exist lots of components that can interfere with OTC determination. In samples like feeds, interfering substances such as pigments, additives, fatty acids and mineral cations occur naturally (Salvatore & Katz, 1993). It is of paramount importance to achieve a selective OTC extraction. Recently, our research group (Ferna´ndez-Gonza´lez et al., 2002) has demonstrated that synchronous spectrofluorimetry is a versatile analytical technique ideal for OTC determination in analytical quality control and for research and development laboratories in the food and beverage industry. This method (Ferna´ndez-Gonza´lez et al., 2002) is reliable (with good response linearity, high recovery and precision, and low detection and quantification levels), simple and fast. The particular structure of this antibiotic, as in the case of other tetracyclines, contains electron-donor groups able to form strong complexes with metal ions (Carson & Breslyn, 1996; Lambs, Decock-Le Re´ve´rend, Kozlowski, & Berthon, 1988). Therefore, some authors use different OTC extraction procedures based on liquid–liquid extraction with calcium (Sharma et al., 1977) or clean-up with metal chelate affinity chromatography, which consists in one column preloaded with divalent cations where OTC is specifically adsorbed by chelation with the metal ions (Carson & Breslyn, 1996; Carson et al., 1998; Coopper et al., 1998; Croubels et al., 1997; De Wasch et al., 1998; Farrington et al., 1991; Stubbings et al., 1996). We must also take into consideration that these ions can be naturally
2.1. Reagents Oxytetracycline hydrochloride (OTC) was purchased from Riedel-de Hae¨n (Seelze, Germany). Because OTC is not very stable and is susceptible of photochemical and oxidative degradation, work solutions were freshly prepared every day. Divalent metal salts included calcium chloride anhydrous (CaCl2), supplied by Panreac (Barcelona, Spain); nickel chloride hexahydrate (NiCl2 Æ 6H2O) from Riedel-de Hae¨n (Seelze, Germany); cobaltous chloride hexahydrate (CoCl2 Æ 6H2O), magnesium chloride hexahydrate (MgCl2 Æ 6H2O), mercuric chloride anhydrous (HgCl2) and ferrous chloride tetrahydrate (FeCl2 Æ 4H2O), from Fluka (Steinheim, Germany). All solutions were prepared in highly pure water (R P 18 MX) obtained from a Millipore water purification system. 2.2. Apparatus and operating conditions All spectrofluorometric measurements were taking by means of a Jasco P-750 luminescence spectrophotometer equipped with a xenon lamp, Monk-Gillieson monochromators and 1 cm quartz cuvettes. The spectral data were collected and processed using the Spectra Manager software. The optimised instrumental parameters for synchronous scanning of OTC complexes were as in Table 1. 2.3. Procedure Association constants were measured by spectrofluorometry, using reported procedures in the literature (Garcı´a-Rı´o, Herve´s, Mejuto, Parajo´, & Pe´rez-Juste, 1998), by making use of different synchronous spectra of
Table 1 Optimised instrumental parameters for synchronous scanning of OTC complexes Parameters
Spectrofluorometric conditions
Spectral range Excitation and emission slits Scan speed Excitation-emission wavelength difference (Dk)
400–600 nm 10 and 5 nm respectively 125 nm/min 115 nm
M. Arias et al. / Journal of Food Engineering 78 (2007) 69–73
free and complexed OTC solutions. The complexes were formed by adding to fix OTC concentration (4 · 106 M) different aliquots of concentrated divalent cation solutions with the intention of increasing cation concentrations in the 3 mL final volume. In all cases the concentration of cation was at least 10 times bigger than the concentration of OTC to be sure that the assumption of the total concentration of cation non-bounded to OTC is approximately equal to the initial cation concentration. The pH of these OTCdivalent cation solutions varies with the addition of the cations. Since the pH of OTC solutions has a significant effect on the fluorescence intensity measured in the case of Fe+2, Ni+2, Co+2 and Hg+2, we corrected for the fluorescence intensity of OTC solutions caused only by effect of pH between 4.0 and 5.0 in the fluorescence intensity of these OTC-divalent cation solutions. 3. Results and discussion The determination of the binding constant of cations to OTC is based in the fact that changes in the chemical environment of OTC will modify its fluorescence spectrum. The association of cations to the OTC will imply changes in the position and in the intensity of their fluorescence peaks. We can write then that the total intensity measured (I) corresponds with the sum of OTC intensity in the absence of cations (If) and in the presence of cations (Ib). I ¼ If þ Ib
ð1Þ
71
Eqs. (4) and (5) imply eapp l½OTCt ¼
ef l½OTCt þ eb l½OTCt K OTC ½Cþ2 1 þ K OTC ½Cþ2
ð6Þ
Because eappl[OTC]t is equal to the total intensity (I), efl[OTC]t is the intensity in the absence of divalent cation (If), and ebl[OTC]t corresponds with the intensity when all the substrate is bound to divalent cation (Ib), Eq. (6) can be written as I¼
I f þ I b K OTC ½Cþ2 1 þ K OTC ½Cþ2
ð7Þ
Eq. (7) can be linearised I If ¼ K OTC ½Cþ2 Ib If
ð8Þ
If we measure the fluorescence intensity at various divalent cation concentrations at a fixed concentration of OTC, the binding constant can be calculated from Eq. (7) or Eq. (8). Fig. 1 shows, as example, the observed behaviour of fluorescence intensity of OCT in the presence of two divalent cations (Ca+2 and Mg+2). Similar behaviours (increase or decrease of intensity on the increase of the concentration of divalent cations) are observed for the other divalent cations—data not shown. The solid lines represent the fit of the experimental data of fluorescence intensity to Eq. (7). In Table 2, the binding constant of the different divalent cations to OTC is listed.
Taking into account Beer–Lambert law, Eq. (1) can be rewritten as a function of total OTC concentration, [OTC]t, and the respective concentrations of OTC free, [OTC]f, and OTC–Cation complex, [OTC]b eapp l½OTCt ¼ ef l½OTDf þ eb l½OTCb
ð2Þ
where l is the light pathway (in a standard cell is equal to 1 cm). e corresponds with the attenuation coefficient or extinction coefficient. The three concentrations of Eq. (2) are related to the constant of complex formation, KOTC, defined as K OTC ¼
½OTCb ½OTCf ½Cþ2
ð3Þ
where [C+2] is the non-bounded divalent cation concentration. Because the initial concentration of cation is at least 10 times bigger that OTC concentration, we can assume that the non-bounded divalent cation concentration is equal to the initial divalent cation concentration. Using Eq. (3), and the corresponding matter balance ([OTC]t = [OTC]f + [OTC]b), Eqs. (4) and (5) can be easily deduced ½OTCf ¼ ½OTCb ¼
½OTCt 1 þ K OTC ½Cþ2
ð4Þ
½OTCt K OTC ½Cþ2 1 þ K OTC ½Cþ2
ð5Þ
Fig. 1. Influence of divalent cation concentration upon the fluorescence intensity of OTC. Solid line represents the fit of experimental data to Eq. (7). (s) Mg+2, (d) Ca+2. Table 2 Binding constant of divalent cations to OTC obtained from Eq. (7) Cation +2
Ca Mg+2 Hg+2
KOTC 55 ± 9 66 ± 9 36 ± 3
Cation +2
Ni Fe+2 Co+2
KOTC 23 ± 5 8±1 7±3
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M. Arias et al. / Journal of Food Engineering 78 (2007) 69–73
The value obtained for KOTC shows that OTC exhibit a large affinity for Ca+2 and Mg+2 and decreases on the increase of the charge/ionic radius ratio for the other divalent cations. The capacity of complex formation between OTC and divalent cations is clearly related to the electron acceptor capacity of cations. According with Pearson classification (1968a, 1968b), Ca+2 and Mg+2 are considered hard electron acceptors and present KOTC values of 55 and 66 while Ni+2, Co+2, Fe+2 and Hg+2 are intermediate or soft electron acceptors and present values of KOTC in the range of 7–36. This means that Ca+2 and Mg+2 can make more difficult OTC extraction from feeds. This problem can be solved by adding a chelating agent, like ethylenediamine tetraacetate (EDTA), which forms complexes with these ions and avoids the formation of OTC-metal complexes. Acknowledgements We gratefully acknowledge support of this research by the Spanish Ministry of Science and Technology through Ramo´n y Cajal research contracts awarded to Drs. Arias and Garcı´a-Falco´n. References Boatto, G., Pau, A., Palomba, M., Arenare, L., & Cerri, R. (1999). Monitoring of oxytetracycline in ovine milk by high-performance liquid chromatography. Journal of Pharmaceutical Biomedical Analysis, 20, 321–326. Carson, M. C., & Breslyn, W. (1996). Simultaneous determination of multiple tetracycline residues in milk by metal chelate affinity chromatography: collaborative study. Journal of AOAC International, 79(1), 29–42. Carson, M. C., Ngoh, M. A., & Hadley, S. W. (1998). Confirmation of multiple tetracycline residues in milk and oxytetracycline in shrimp by liquid chromatography-particle beam mass spectrometry. Journal of Chromatography B, 712, 113–128. Chappell, G. S., Houglum, J. E., & Kelley, W. N. (1986). Determination of oxytetracycline in premixes and veterinary products by liquid chromatography. Journal Association of Official Analytical Chemists, 69(1), 28–30. Coopper, A. D., Stubbings, G. W. F., Kelly, M., Tarbin, J. A., Farrington, W. H. H., & Shearer, G. (1998). Improved method for the on-line chelate affinity chromatography-high-performance liquid chromatographic determination of tetracycline antibiotics in animal products. Journal of Chromatography A, 812, 321–326. Croubels, S. M., Vanoosthuyze, K. E. I., & Van Peteghem, C. H. (1997). Use of metal chelate affinity chromatography and membrane-based ion-exchange as clean-up procedure for trace residue analysis of tetracyclines in animal tissues and egg. Journal of Chromatography B, 690, 173–179. De Wasch, K., Okerman, L., Croubels, S., De Brabander, H., Van Hoof, J., & De Backer, P. (1998). Detection of residues of tetracycline antibiotics in pork and chicken meat: correlation between results of screening and confirmatory tests. Analyst, 123, 2737–2741. Farrington, W. H. H., Tarbin, J., Bygrave, J., & Shearer, G. (1991). Analysis of trace residues of tetracyclines in animal tissues and fluids using metal chelate affinity chromatography/HPLC. Food Additives and Contaminants, 8(1), 55–64. Ferna´ndez-Gonza´lez, R., Garcı´a-Falco´n, M. S., & Simal-Ga´ndara, J. (2002). Quantitative analysis for oxytetracycline in medicated premixes
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