Determination of Urea Content in Dairy Cattle Feed by High Performance Liquid Chromatography via Refractive Index Detector: Method Optimization, Validation and Comparison with Spectro-Colorimetric Method


Published: Jul 11, 2024
Keywords:
Feed additives dairy cattle feed urea HPLC spectrophotometry
H Umur
https://orcid.org/0000-0002-9824-1165
H Hanoğlu Oral
https://orcid.org/0000-0003-3626-9637
H Ekşi Karaağaç
S Koçer
https://orcid.org/0000-0003-1926-5167
HÖ Uçurum
E Altınçekiç
Abstract

The aim of this study was to analyze the urea content of Dairy Cattle Feed (DCF) using High Performance Liquid Chromatography via Refractive Index Detector (HPLC-RID), without derivatization, along with spectro-colorimetric method that is the official control method of European Union (EU). Towards that goal, the analysis procedure for the method was established, method optimization and validation were carried out by spiking urea at 2% level to DCF, which is the upper limit allowed for use in ruminant feeds in Türkiye. In method validation; R2: 0.9997 coefficient of determination at 25-100000 mg/kg linear range, 75 mg/kg limit of detection (LOD) and 250 mg/kg limit of quantification (LOQ), 1.08% repeatability RSD, 1.84% reproducibility RSD and 95.10% recovery were obtained. The applicability of the method was proven at 25-100000 mg/kg linear range with real samples by spiking 2% urea in Urea-Free Dairy Cattle Feed (UF-DCF), Low Urea Dairy Cattle Feed (LU-DCF), and High Urea Dairy Cattle Feed (HU-DCF). The presence of urea in UF-DCF was detected with the spectro-colorimetric (at 420 and 435 nm wavelength) method, while urea in UF-DCF was not detected by the in-house HPLC-RID method, (P<0.05). The amount of urea in LU-DCF without spiking urea was detected by the same level as HPLC-RID method and the SC-435 method (P>0.05). For HU-DCF blank samples, higher urea amounts were detected with the in-house HPLC-RID method as compared to the spectro-colorimetric methods used in the study (P<0.05). In DCF spiked with 2.0% urea, the urea amount found in the in-house HPLC-RID method were higher than that of the spectro-colorimetric method (P<0.05). As a result, the in-house HPLC-RID method that is developed in this study has shown great promise to be a potential, applicable and valid method for determining the urea amount in DCF.

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Author Biographies
H Umur, Central Research Institute of Food and Feed Control, Bursa, Türkiye

Central Research Institute of Food and Feed Control, Bursa

H Hanoğlu Oral, Muş Alparslan University, Faculty of Applied Sciences, Department of Animal Production & Technologies, Muş, Türkiye

 

Muş Alparslan University, Faculty of Applied Sciences, Department of Animal Production & Technologies

H Ekşi Karaağaç, Çilimli District Directorate of Agriculture and Forestry, Düzce, Türkiye

 

Çilimli District Directorate of Agriculture and Forestry, Düzce

S Koçer, Central Research Institute of Food and Feed Control, Bursa, Türkiye

Central Research Institute of Food and Feed Control, Bursa

HÖ Uçurum, Central Research Institute of Food and Feed Control, Bursa, Türkiye

Central Research Institute of Food and Feed Control, Bursa

E Altınçekiç, Central Research Institute of Food and Feed Control, Bursa, Türkiye

Central Research Institute of Food and Feed Control, Bursa

References
Akkoç B (2023) https://www.slideshare.net/
BerkayAKKO/hplc-96041390 [accessed 01 May 2023].
Anitha, T., Madhuri, S. B., Alexander, G., Chandra, A. S., & Venkateswarlu, M. (2023). Detection of Urea Adulteration in Oilseed Cakes using
-Dimethylaminobenzaldehyde Reagent and Foldscope. Asian Journal of Dairy and Food Research, 42(1), 65-69.
Anonymous (1970) AOAC 967.07. Urea in animal feed. Colorimetric
method.
Anonymous (2008) TS 10338. Hayvan yemleri- Üre Tayini.
Anonymous (2009) Commision Regulation (EC) No 152/2009 of 27 January 2009 laying down the methods of sampling and analysis for the
official control of feed. Off J Eur Union L. 54(52):27-29.
Anonymous (2011) Yemlerin piyasaya arzı ve kullanımı hakkında yönetmelik. R.G. 27.12.2011 tarih 28155 sayı.
Anonymous (2012) Commission Implementing Regulation (EU) No
/2012.
Anonymous (2014) Eurachem guide ‘The fitness for purpose of analytical methods’. A Laboratory Guide to Method Validation and Related
Topics. Second edition, pp70.
Anonymous (2017) Yemlerin resmî kontrolü için numune alma ve analiz
metotlarına dair yönetmelik. R.G. 21.1.2017 tarih 29955 sayı.
Ashri, N. Y., & Abdel-Rehim, M. (2011). Sample treatment based on extraction techniques in biological matrices. Bioanalysis, 3(17), 2003-
.
Chauhan Mahipalsinh, P., Agnihotri, A., Shaikh, A., Patel, S., & Aparnathi,
K. (2017). Evaluation of DMAB test for detection of urea mixed in
milk and improvement in its efficacy. IJCS, 5(6), 1572-1576.
Clark, S., Francis, P. S., Conlan, X. A., & Barnett, N. W. (2007). Determination of urea using high-performance liquid chromatography with
fluorescence detection after automated derivatisation with xanthydrol. Journal of Chromatography A, 1161(1-2), 207-213.
Czauderna, M., & Kowalczyk, J. (2012). Simple, selective, and sensitive
measurement of urea in body fluids of mammals by reversed-phase
ultra-fast liquid chromatography. Czech J Anim Sci, 57, 19-27.
Dai, X., Fang, X., Su, F., Yang, M., Li, H., Zhou, J., & Xu, R. (2010).
Accurate analysis of urea in milk and milk powder by isotope dilution
gas chromatography-mass spectrometry. Journal of Chromatography
B, 878(19), 1634-1638..
Dai, X., Zhao, Y., Li, M., Fang, X., Li, X., Li, H., & Xu, B. (2012). Determination of urea in milk by liquid chromatography-isotope dilution
mass spectrometry. Analytical letters, 45(12), 1557-1565.
Flannelly, L., Dillon, G. P., & Moran, C. A. (2019). A comparison of analytical methods for the determination of urea in yeast-based food and
feed ingredients. Food Additives & Contaminants: Part A, 36(10),
-1474..
Follmer, C. (2008). Insights into the role and structure of plant ureases.
Phytochemistry, 69(1), 18-28.
Galecio, J. S., Hernandis, V., Escudero, E., & Marín, P. (2022). Validation
of a LC-UV method for determination of tildipirosin in goat milk.
Journal of the Hellenic Veterinary Medical Society, 73(4), 4905-4912.
Giraldo, J. D., & Rivas, B. L. (2017). Determination of urea using p-N,
N-dimethylaminobenzaldehyde: Solvent effect and interference of
chitosan. Journal of the Chilean Chemical Society, 62(2), 3538-3542.
Hojjatie, M. M., & Abrams, D. (2015). Evaluation of the Determination
of Free Urea in Water-Soluble Liquid Fertilizers Containing Urea and
Ureaforms by Urease and HPLC Methods. Journal of AOAC International, 98(6), 1475-1482.
Hussain, I., Mahmood, Z., Yasmeen, R., Jahangir, M., Hammed, R., &
Nasir, R. (2002). Assay of urea with p-dimethylaminobenzaldehyde.
Journal-Chemical Society of Pakistan, 24(2), 122-128.
Kertz, A. F. (2010). Urea feeding to dairy cattle: A historical perspective
and review. The Professional Animal Scientist, 26(3), 257-272.
Krämer, M., Fry, H., & Kappenstein, O. (2021). Development and validation of two analytical methods for urea determination in compound
feed, including pet food, and yeast using high-performance liquid
chromatography coupled with fluorescence detection and tandem
mass spectrometry. Food Additives & Contaminants: Part A, 38(6),
-942.
Koebel, M., & Elsener, M. (1995). Determination of urea and its thermal
decomposition products by high-performance liquid chromatography.
Journal of Chromatography A, 689(1), 164-169.
Matsudo, T., & Sasaki, M. (1995). Determination of urea and citrulline in
fermented foods and beverages. Bioscience, biotechnology, and biochemistry, 59(5), 827-830.
Patra, A. K., & Aschenbach, J. R. (2018). Ureases in the gastrointestinal tracts of ruminant and monogastric animals and their implication
in urea-N/ammonia metabolism: A review. Journal of advanced research, 13, 39-50.
Pibarot, P. & Pillard S. (2012). Analysis of urea in petfood matrices: comparison of spectro-colorimetric, enzymatic and liquid chromatography electrospray ionization high resolution mass spectrometry methods. American Journal of Analytical Chemistry, 3(09), 613.
Phonchai, A., Rattana, S., & Thongprajukaew, K. (2020). A portable solgel urea colorimetric method for the determination of urea in feedstuffs. Food chemistry, 319, 126545.
SAS Institute (1998). Statistical analysis system institute: StatView reference Manuel. SAS Institute Cary NC.
Wang, R., Wu, H., Zhou, X., & Chen, L. (2014). Simultaneous detection
of ethyl carbamate and urea in Chinese yellow rice wine by HPLCFLD. Journal of Liquid Chromatography & Related Technologies,
(1), 39-47.
Wang, Y. Q., Wang, S. S., Zhu, J., Wang, L., Jiang, B. H., & Zhao, W. J.
(2016). Determination of urea content in urea cream by centrifugal
partition chromatography. journal of food and drug analysis, 24(2),
-405.
Wegh, R. S., Berentsen, R. J., Bolck, Y. J. C., Zuidema, T., & de Jong, J.
(2018, October). Determination of urea in animal feed with liquid
chromatography-mass spectrometry (LC-MS). In Feed 2018: The 6th
International Feed Conference..
Zeng, Q., Zhang, J., Xu, D., Zhang, Z., & Ke, Z. (2015). Determination of
urea in canned foods by high performance liquid chromatography-fluorescence detection coupled with precolumn derivatization. Se pu=
Chinese Journal of Chromatography, 33(1), 80-83
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