Determination of Total Taurine in Pet Foods by Liquid Chromatography of the Dansyl Derivative: Collaborative Study

784 MCCARTHY ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 4, 2000 AGRICULTURAL MATERIALS Determination of Total Taurine in Pet Foods by Liquid Chromatography of the Dansyl Derivative: Collaborative Study MCCARTHY ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 4, 2000 KIERAN MCCARTHY 1 Pedigree Masterfoods, Mill St, Melton Mowbray, Leicestershire, LE13 1BB, United Kingdom CLAUDIA HISCHENHUBER Nestlé Research Centre, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland NEIL JOYCE Eclipse Scientific Group, Aspland & James House, Medcalfe Way, Bridge St, Chatteris, Cambs, PE16 6QZ, United Kingdom Collaborators: G. Cherix; C. Hischenhuber; N. Joyce; S. Kuhr; J. McDonald; S. McKnight; P. McNamee; L. Mevissen; B. Müller; S. Revitt A liquid chromatographic (LC) method for the determination of total taurine in pet foods was evaluated in a collaborative study. Ten laboratories assayed 6 blind duplicate pairs of wet and dry pet foods. The taurine in the 6 sample pairs ranged from low (170 mg/kg) to high (2250 mg/kg) concentrations as is. Collaborators also assayed a sample of known taurine concentration for familiarization purposes. Samples were hydrolyzed to release bound taurine, which was subsequently converted to the dansyl derivative and quantitated by gradient-elution LC with fluorescence detection. Repeatability relative standard deviations, RSD r, ranged from 3.2 to 10.0%; reproducibility relative standard deviations, RSD R, ranged from 6.1 to 16.1%. The method has been adopted Official First Action status by AOAC INTERNATIONAL. Submitted for publication December 1999. The recommendation was approved by the Methods Committee on Feeds, Fertilizers, and Related Agricultural Topics, and was adopted by the Official Methods Board of AOAC INTERNATIONAL. See Official Methods Board Actions, (1999) Inside Laboratory Management, November/December issue. 1 Deceased. Taurine is an amino sulfonic acid synthesized in mammals as an end product of sulfur amino acid metabolism. The cat is in a special position because, unlike other species, it cannot synthesize enough taurine to meet its total requirement and consequently is dependent on a dietary supply of this nutrient. Taurine is important in many aspects of metabolism and has been shown to be essential for retinal function (1 3), reproduction (4 6), growth, and bile salt formation (7) in the cat. Taurine (total taurine determined after acid hydrolysis) may be separated by cation-exchange chromatography using ninhydrin detection (via amino acid analyzer) and quantitated by reference to external/internal standards (Pharmacia-LKB Biochrom, Bromma, Sweden, unpublished data, 1992). The specificity and sensitivity for taurine in this system are not always satisfactory, and the chromatography is often affected by coeluting compounds. In addition, the process is time consuming (particularly if determination of all the other amino acids is also required) and expensive. More recent publications (8, 9) have proposed the use of 2,4-dinitrofluorobenzene (FDNB) pre-column derivatization followed by liquid chromatography (LC). Interference from cysteic acid as well as the safety concerns associated with FDNB can limit the use of this method. With the proposed LC method, the taurine dansyl derivative elutes after about 6 18 min without any interfering compounds. Collaborative Study Ten laboratories participated in this study. Six samples comprising 1 dog and 5 cat products (3 wet and 3 dry) representing the range of taurine levels found in commercially available pet foods were selected. Wet samples were submitted in cans (400 g), and dry samples, in plastic pots (ca 25 g). A single familiarization sample of known taurine concentration was included. The requirement was that the analytical result had to be within ±10% of the stated concentration before the trial samples could be analyzed. Taurine for the preparation of standard solutions was provided by the organizer of the collaborative study. On receipt of the materials, the collaborators were instructed to store the samples and standard at room temperature until needed. Before analysis, all samples were removed from the original containers and fully homogenized by using a food processor or equivalent. After homogenization, all wet samples were stored at 4 C until they were analyzed. Collaborators were instructed to perform single determinations for each sample.

MCCARTHY ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 4, 2000 785 METHOD 999.12 Taurine in Pet Food First Action 1999 (Applicable to the determination of 150 2000 mg total taurine/kg wet or dry cat or dog foods.) See Table 999.12A for the results of the interlaboratory study supporting acceptance of method. A. Principle The test portion is hydrolzyed with HCl and the extracted taurine reacted with dansyl chloride to form a fluorescent derivative determined by reversed-phase HPLC. B. Apparatus (a) Liquid chromatograph. Gradient elution system, automatic sampler with 20 µl loop. (b) Fluorescence detector. Excitation wavelength, 298 nm; emission wavelength, 550 nm. (c) HPLC column. 4 or 5 µm silica C 8 or C 18 reversed-phase column. (Note: A variety of specific column types may be accommodated with appropriate manipulation of the gradient elution profile.) (d) Filtration system. PVDF disposable syringe filters, 0.2 µm (Gelman Acrodisc, or equivalent). (e) Vortex mixer. C. Reagents (a) Dilute hydrochloric acid. 6M. Add slowly 500 ml concentrated HCl to 500 ml water. (b) Sodium carbonate solution. 0.2M, ph 9.7. Dissolve 21.2 g Na 2 CO 3 in ca 800 ml water. Adjust ph to 9.7 with 6M HCl, (a), using ph meter and dilute to 1 L. Solution is stable at least 6 months at room temperature. (c) Acetonitrile. HPLC grade. (d) Acetonitrile water solution. 70 + 30 (v/v). (e) Acetone. Minimum 99.5%. (f) Dansyl chloride solution. (1) Stock solution (100 mg/ml). Dissolve 1.000 g dansyl chloride (Sigma Chemical Co., 95%) in acetone, (e), in 10 ml volumetric flask and dilute to volume. Solution is stable 1 month when stored in the dark. (2) Working solution. Prepare immediately before use. Dilute 1 ml stock solution, (1), to 10 ml with acetonitrile water solution, (d). (g) Orthophosphoric acid solution. 2% (v/v). Dilute 2.35 ml 85% H 3 PO 4 to 100 ml with water. (h) Phosphate buffer solution. 0.5M, ph 6.2. Dissolve 30.6 g KH 2 PO 4 and 4.36 g K 2 HPO 4 in ca 400 ml water and adjust ph to 6.2 with H 3 PO 4. Dilute to 500 ml with water. (i) Taurine standard solutions. (1) Stock solution (1 mg/ml). Dissolve 100.0 mg taurine (Sigma Chemical Co., 99%) in water and dilute to 100 ml. Solution is stable 1 week at 4 C. (2) Working standard solutions. Dilute aliquots of stock solution with water to prepare solutions containing 0, 10, 20, 50, 80, and 100 µg/ml. Solutions are stable 1 week at 4 C. (j) Mobile phase. Solvent A. 0.02M phosphate buffer, ph 3.0. Dissolve 2.72 g KH 2 PO 4 in ca 800 ml water and adjust ph to 3.0 with H 3 PO 4. Dilute to 1 L with water. Filter through membrane, B(d). Solvent B. Mix 600 ml acetonitrile with 400 ml mobile phase A. D. Isolation and Derivatization Grind representative quantity (dry pet food) to a fine powder. Homogenize the content (wet pet food) of one unit with a homogenizer. (a) Hydrolysis. Weigh 400 mg ground dry food or 800 mg homogenized wet food into a 25 ml screw cap reagent bottle. Add 10 ml 6M HCl, C(a). Cap bottle and hydrolyze mixture in oven at 110 C for 16 h. Cool to room temperature, transfer hydrolysate quantitatively to a 25 ml volumetric flask, and dilute to mark with water. Filter ca 2 ml diluted hydrolysate through 0.2 µm disposable syringe filter. Pipet 250 µl filtered hydrolysate into 2 ml reaction vial and evaporate to dryness under gentle stream of N. Keep temperature <70 C to avoid loss of taurine. (b) Derivatization. Dissolve the residue obtained from D(a)in100µL water and Vortex mix. Add 0.5 ml Na 2 CO 3 solution, C(b), and Vortex mix for 10 s. Add 0.5 ml dansyl chloride working solution, C(f)(2), and Vortex mix for 10 s. Cap the vial and place in oven or heating block at 65 C for 30 min. Cool to room temperature and add 100 µl 2%H 3 PO 4 solution. Vortex mix for 10 s. Add 0.5 ml 0.5M phosphate buffer and 0.3 ml water and Vortex mix for 10 s. Transfer reaction mixture toa2ml syringe and pass through a 0.2 µm disposable syringe filter into Table 999.12A Interlaboratory study results for total taurine in cat and dog foods Product No. of labs No. outliers excluded Avg., mg/kg RSD r, % r, mg/kg RSD R, % R, mg/kg Wet cat food 1 8 1 526 4.1 61 6.1 90 Wet cat food 2 7 2 514 3.9 56 6.6 94 Wet dog food 3 9 0 167 10.0 47 10.2 48 Dry dog food 4 9 0 333 7.4 69 16.1 150 Dry cat food 5 7 2 936 3.2 84 8.9 233 Dry cat food 6 8 0 2256 5.7 361 13.3 839

786 MCCARTHY ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 4, 2000 Table 999.12B Gradient program F. Calculation Time, min HPLC autosampler vial (solution may be stored for up to 2 days at 20 C until ready for analysis). Treat 100 µl of each working standard solution, C(i)(2), the same as the reconstituted residue above. E. HPLC Determination A Percent 0 80 20 21 71 29 25 20 80 40 20 80 42 80 20 47 80 20 Use flow rate, 1.5 ml/min and gradient program, percent solvents A and B, C(j), and Table 999.12B. Optimize separation conditions to achieve satisfactory separation of the taurine peak. Establish stable HPLC performance by repeatedly injecting a 20 µl aliquot of a calibrant derivative using the gradient elution profile, Table 999.12B, which may be varied to suit individual analytical column characteristics. Inject 20 µl aliquots of calibrant and test dervatives and establish peak response (area or height) using an electronic integrator or computer package. Construct a multiple level calibration with forced origin at 0. Reject the analysis if the 0.00 µg/ml calibrant fails to give a zero response and determine the cause of the interference. Calibration line should be linear. B Calculate total taurine (mg/kg) as follows: Taurine, mg/kg = R S test 100 250 25 W where R test = peak response (area or height) of taurine in test extract; S = slope of calibration curve; 100 = volume of reconstituted extract (µl); 250 = volume of extract taken for derivatization (µl); 25 = final volume of test extract (ml); W = weight of test portion (g). To convert a result from mg/kg into g/100 g, divide the figure obtained by 10 000. Ref.: J. AOAC Int. 83, 785 786(2000) Collaborators Comments The collaborators reported the use of a variety of commercial C 8 or C 18 columns (Table 1). Consequently, the gradient was modified in several cases to obtain retention times that allowed a good separation of taurine from matrix components. The protocol indicated that the mobile phase should be adjusted to obtain sufficient separation. Those laboratories using C 8 columns reported retention times that were significantly lower than those reported by laboratories using C 18 columns. All participants used fluorescence detection. One laboratory reported additional results obtained with UV detection that agreed well with those obtained with fluorescence detection. Two laboratories measured peak heights, and 8 laboratories measured the peak areas. Results and Discussion Eleven laboratories were initially invited to participate in the study. Ten laboratories agreed to participate, submitting a total of 119 individual values (60 blind duplicates). The whole data set is listed in Table 2. One laboratory reported the loss of a sample during analysis. Consequently, 1 value is missing. Table 1. LC operating conditions used by collaborators Lab a Column Gradient Retention time of taurine, min Peak measurement 1 Superspher 60, RP8, 250 4mm Permethod 6.9 8.6 Area 2 Nucleosil-100, C 8,125+10 4mm,5µm Modified 5.6 7.0 Area 3 LiChrospher 100, RP18, 5 µm Modified 18.0 18.2 Area 4 Ultrasphere C 18, 250 4.6 mm, 5 µm Modified 13.1 13.3 Height 5 Partisphere C 18, 250 4.6 mm Modified 9.2 9.3 Area 6 Ultrasphere Octyl, 150 + 45 4.6 mm Modified 11.5 11.6 Height 7 Spherisorb ODS-1, 150 4.6 mm, 5 µm Per method 12.4 Area 8 Phenomenex Phenosphere C 8 150 4.6mm, 5 µm Per method 6.7 6.9 Area 9 Ultrasphere Octyl, 150 + 45 4.6 mm, 5 µm Per method 16.6 16.8 Area a Operating conditions for Laboratory 10 are not shown because all of its data were excluded from the statistical evaluation.

MCCARTHY ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 4, 2000 787 Table 2. Collaborative results (mg/kg) for determination of total taurine in pet foods (blind duplicates) by liquid chromatography Sample a Lab 1 2 3 4 5 6 1 591.4 b 349.3 b 489.6 b 970.9 b 184.3 129.5 352.7 328.9 884.6 880.6 1898.6 1980.3 2 540.4 515.4 537.9 496.6 173.1 172.2 369.2 392.0 1149.1 c 1571.5 c 2381.4 2749.9 3 537.4 557.3 536.0 546.9 192.4 190.6 342.4 345.7 909.7 900.4 2400.0 2380.3 4 536.9 561.2 515.0 554.0 159.0 159.7 254.5 289.7 977.8 1051.8 2076.1 2287.1 5 483.8 522.4 478.3 484.7 149.3 153.7 255.4 290.8 834.1 880.1 2229.6 2462.8 6 481.3 475.9 460.4 464.5 157.1 158.9 431.2 411.4 956.1 950.0 1716.0 1813.8 7 544.0 588.4 325.6 b 563.6 b 154.6 198.5 263.8 334.5 822.4 b 1048.9 b 2406.5 d 8 511.6 518.2 561.3 519.3 170.0 162.2 327.3 281.6 850.0 886.1 2349.7 2227.6 9 545.0 496.6 511.8 530.1 170.9 171.3 365.2 363.7 1099.0 1040.4 2577.8 2566.0 10 e 698 717 652 697 317 284 524 472 1222 1483 3518 3512 a b c d e Samples 1, 2, and 3 = wet cat and dog food; samples 4, 5, and 6 = dry cat and dog food. Cochran outlier. Grubbs outlier. Loss of sample. All data of this laboratory are invalid (data for training sample not within required range). All participants analyzed the training sample to check whether they encountered methodological problems or obtained a result within the expected range. Nine of 10 collaborators reported results for the training sample that lay within the indicated range of 1042 ± 104 mg taurine/kg. Although Laboratory 10 found as much as 1579 mg taurine/kg in the training sample, the test samples were analyzed without any corrective action. Because a significant bias for all results was observed, the data from Laboratory 10 were excluded from the statistical evaluation. In addition, Laboratory 10 did not report a standard calibration. All the other laboratories returned acceptable standard calibration parameters based on linear regression, with r ranging between 0.9985 and 1; typically r was >0.9990. The remaining valid data were statistically analyzed by using AOAC guidelines. Overall mean, within-laboratory standard deviation (s r ), repeatability relative standard deviation (RSD r ), repeatability value (r = 2.8 s r ), reproducibility standard deviation (s R ), reproducibility relative standard deviation (RSD R ), and reproducibility value (R = 2.8 s R ) were calculated. Outliers were detected by performing Cochran and Grubbs (single and double) tests. Four duplicates were detected as Cochran outliers and 1 duplicate, as a Grubbs outlier. The statistical data are presented in Table 999.12A. RSD R values estimated for wet pet food samples ranged from 6.1 to 10.2% (mean, 7.6%); for dry pet food samples, they ranged from 8.9 to 16.1% (mean, 12.8%). RSD r values ranged from 3.2 to 10.0% (mean, 5.7%); the sample with the lowest taurine content, 167 mg/kg, gave the highest RSD r value. Recommendation The statistical data obtained in this collaborative study confirm the suitability of the method for the determination of taurine in both wet and dry cat or dog food in a concentration range of ca 150 2400 mg/kg. On the basis of the results of this study, it has been recommended that this LC method for the determination of total taurine in pet foods be adopted First Action by AOAC INTERNATIONAL. Acknowledgments The co-authors and members of the FEDIAF (European Pet Food Producers Association) Analytical Working Party would like to acknowledge the contribution made by Kieran McCarthy, coordinator of the study, who died before the paper was completed. We also thank Jean-Marc Aeschlimann for helping in the statistical analysis, and the other members of the FEDIAF Analytical Working party, Willy de Smedt, Heike Schnüll, Jacques Debraekeleer, Rainer Hesse, and Peter McNamee. We also thank the following collaborators for their active participation in this study: Lutz Mevissen, Gesellschaft für Lebensmittel-Forschung GmbH, Berlin, Germany Susanne Kuhr, Institut Prof. Nehring, Braunschweig, Germany Bernd Müller, Dr. Wiertz - Dipl. Chem. Eggert - Dr. Jörrison GmbH, Hamburg, Germany Genevi ve Cherix, Nestlé Quality Assurance Laboratory, Dublin, OH John McDonald, Ralston Purina Co., St. Louis, MO

788 MCCARTHY ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 4, 2000 Rick McKnight, Friskies R&D Center, St. Joseph, MO Neil Joyce, Eclipse Scientific Group, Chatteris, Cambs, UK Peter McNamee, Pedigree Masterfoods, Division of Mars Ltd, Melton Mowbray, Leicestershire, UK Claudia Hischenhuber, Nestlé Research Centre, Lausanne, Switzerland Steve Revitt, Eclipse Scientific Group, Chatteris, Cambs, UK References (1) Schmidt, S.Y., Berson, E.L., & Hayes, K.C. (1976) Invest. Ophthalmol. 15, 47 52 (2) Berson, E.L., Hayes, K.C., Rabin, A.R., Schmidt, S.Y., & Watson, G. (1976) Invest. Ophthalmol. 15, 52 58 (3) Schmidt, S.Y., Berson, E.L., Watson, G., & Huang, C. (1977) Invest. Ophthalmol. 16, 673 678 (4) Sturman, J.A., Moretz, R.C., French, J.H., & Wisniewski, H.M. (1985) J. Neurosci. Res. 13, 405 416 (5) Sturman, J.A., Gargano, A.D., Messing, J.M., & Imaki, H. (1986) J. Nutr. 116, 655 667 (6) Sturman, J.A., Palackal, T., Imaki, H., Moretz, R.C., French, J., & Wisniewski, H.M. (1987) Adv. Exp. Med. Biol. 217, 113 124 (7) Rabin, B., Nicolosi, R.J., & Hayes, K.C. (1976) J. Nutr. 106, 1241 1246 (8) Chen, Z.L., Xu, G., Specht, K., Yang, R.J., & She, S.W. (1994) Anal. Chim. Acta 296, 249 253 (9) Polanuer, B., Ivanov, S., & Sholin, A. (1994) J. Chromatogr. 656, 81 85