Food Analytical Methods

, Volume 12, Issue 2, pp 338–346 | Cite as

Validation of HPLC Multi-residue Method for Determination of Fluoroquinolones, Tetracycline, Sulphonamides and Chloramphenicol Residues in Bovine Milk

  • Pallavi MoudgilEmail author
  • Jasbir Singh Bedi
  • Rabinder Singh Aulakh
  • Jatinder Paul Singh Gill
  • Amit Kumar


The occurrence of antibiotic residues in milk constitutes a potential risk to the health of consumers. The present study describes the optimisation and validation of a high-performance liquid chromatographic (HPLC) method for the simultaneous determination of sulphadiazine (SDZ), sulphamethoxazole (SMX), oxytetracycline (OTC), doxycycline (DOX), tetracycline (TC), enrofloxacin (ENRO) and chloramphenicol (CLP) residues in bovine milk using colchicine (COL) as internal standard. The determination of these antimicrobials was carried out on C18 analytical column using high-performance liquid chromatographic-diode array detection (HPLC-DAD). The extraction method involving deproteinisation of the milk sample followed by a solid-phase extraction (SPE) clean-up of antibiotic residues has been developed. The method was validated according to the European Commission Decision 2002/657/EC and applied for the analysis of antibiotic residues in 21 raw milk samples collected from Ludhiana, Punjab, India. The recoveries for the studied antibiotics ranged from 83.3–111.8% with relative standard deviations between 3.5 and 16.2%. The limits of quantification for these antibiotics, with the exception of chloramphenicol, were below the maximum residue limits (MRLs), making the method suitable for performing routine analysis.


Antibiotic residues HPLC-DAD Maximum residue limits Milk Solid-phase extraction Validation 



Authors are thankful to “Rashtriya Krishi Vikas Yojana” (RKVY), Government of India, for providing funds for undertaking the study through project entitled “Studies on current scenario of antibiotic residues in food of animal origin in Punjab and prevention of antibiotic residue risks”.

Compliance with Ethical Standards

Conflict of Interest

Pallavi Moudgil declares that she has no conflict of interest. Jasbir Singh Bedi declares that he has no conflict of interest. Rabinder Singh Aulakh declares that he has no conflict of interest. Jatinder Paul Singh Gill declares that he has no conflict of interest. Amit Kumar declares that he has no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals.

Informed Consent

Not applicable.


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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.School of Public Health and ZoonosesGuru Angad Dev Veterinary and Animal Sciences UniversityLudhianaIndia
  2. 2.Department of Veterinary Public Health and EpidemiologyLala Lajpat Rai University of Veterinary and Animal SciencesHisarIndia

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