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Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar

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20232
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Subject: Dairy Chemistry × End date: 2023 × Start date: 2023 ×
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    Development and validation of HPLC method for detection of oxytetracycline residues in Khoa
    (LUVAS,Hisar, 2023-11) Priyanka; Siwach, Ruby
    The present study aimed to develop and validate a reliable RP-HPLC method for simultaneous detection of oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC) in Khoa. The method development included optimization of HPLC operating conditions, development of extraction method and evaluation of several validation parameters viz., system precision, system linearity, method linearity, sensitivity, specificity, intra-day precision, inter-day precision, accuracy, limit of detection (LOD) and limit of quantitation (LOQ). The HPLC analysis was carried out using a C18 reversed phase column (4.0 mm x 250.0 mm, 5 μm particle size) with a gradient flow using acetonitrile, methanol and 0.01 M oxalic acid as mobile phases. The flow rate was set to 0.5 mL/min. and the UV detection wavelength of 355 nm was used. After application of various extraction strategies, a solid phase extraction (SPE) method was developed with following steps – protein digestion, buffer addition, centrifugation, SPE cartridges elution, evaporation and reconstitution in methanol for simultaneous extraction of OTC, TC and CTC from Khoa. The method exhibited excellent chromatographic separation of OTC, TC and CTC with retention times (RT) of 16.7, 17.7 and 22.3 min. respectively. The linearity of the method was assessed over a concentration range of 50-1000 μg/kg and correlation coefficients (R2) exceeding 0.99 for each antibiotic were obtained. The accuracy of the method was determined by performing recovery studies at six different concentrations (50, 100, 250, 500, 750 and 1000 μg/kg), yielding recovery values ranging from 80.4 to 106.2% for OTC, 81.7 to 109.3 for TC and 80.2 to 102.2% for CTC. The relative standard deviations (%RSD) for intra-day were found to be 1.01, 2.56 and 3.28 for OTC, TC and CTC respectively, while the % RSD values for inter-day precision of OTC, TC and CTC varies from 2.30- 6.14, 2.54-4.47 and 1.25-4.01, indicating excellent repeatability and reproducibility of the method. The limit of detection (LOD) and limit of quantitation (LOQ) values (μg/kg) were found to be 0.13 and 0.44 for OTC, 0.29 and 0.99 for TC and 0.69 and 2.32 for CTC, respectively. The developed and validated RP-HPLC method was further used to study the effect of processing conditions during Khoa manufacturing on OTC, TC and CTC present in milk. Values of % degradation for OTC, TC and CTC ranged from 4.85-52.58, 5.66-54.86 and 18.21-56.39 respectively. The degradation % of each antibiotic increased with increase in spiking concentration in milk. In conclusion, the developed HPLC-UV method demonstrated excellent performance characteristics for the simultaneous detection of OTC, TC, and CTC in Khoa. The method exhibited good linearity, accuracy, precision, and low limits of detection and quantification, making it suitable for routine analysis and quality control of Khoa for tetracycline residues determination.
  • Thumbnail Image
    ThesisItemEmbargo
    Development and validation of HPLC method for detection of oxytetracycline residues in khoa
    (LUVAS Hisar, 2023-11) Priyanka; Siwach, Ruby
    The present study aimed to develop and validate a reliable RP-HPLC method for simultaneous detection of oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC) in Khoa. The method development included optimization of HPLC operating conditions, development of extraction method and evaluation of several validation parameters viz., system precision, system linearity, method linearity, sensitivity, specificity, intra-day precision, inter-day precision, accuracy, limit of detection (LOD) and limit of quantitation (LOQ). The HPLC analysis was carried out using a C18 reversed phase column (4.0 mm x 250.0 mm, 5 μm particle size) with a gradient flow using acetonitrile, methanol and 0.01 M oxalic acid as mobile phases. The flow rate was set to 0.5 mL/min. and the UV detection wavelength of 355 nm was used. After application of various extraction strategies, a solid phase extraction (SPE) method was developed with following steps – protein digestion, buffer addition, centrifugation, SPE cartridges elution, evaporation and reconstitution in methanol for simultaneous extraction of OTC, TC and CTC from Khoa. The method exhibited excellent chromatographic separation of OTC, TC and CTC with retention times (RT) of 16.7, 17.7 and 22.3 min. respectively. The linearity of the method was assessed over a concentration range of 50-1000 μg/kg and correlation coefficients (R2) exceeding 0.99 for each antibiotic were obtained. The accuracy of the method was determined by performing recovery studies at six different concentrations (50, 100, 250, 500, 750 and 1000 μg/kg), yielding recovery values ranging from 80.4 to 106.2% for OTC, 81.7 to 109.3 for TC and 80.2 to 102.2% for CTC. The relative standard deviations (%RSD) for intra-day were found to be 1.01, 2.56 and 3.28 for OTC, TC and CTC respectively, while the % RSD values for inter-day precision of OTC, TC and CTC varies from 2.30- 6.14, 2.54-4.47 and 1.25-4.01, indicating excellent repeatability and reproducibility of the method. The limit of detection (LOD) and limit of quantitation (LOQ) values (μg/kg) were found to be 0.13 and 0.44 for OTC, 0.29 and 0.99 for TC and 0.69 and 2.32 for CTC, respectively. The developed and validated RP-HPLC method was further used to study the effect of processing conditions during Khoa manufacturing on OTC, TC and CTC present in milk. Values of % degradation for OTC, TC and CTC ranged from 4.85-52.58, 5.66-54.86 and 18.21-56.39 respectively. The degradation % of each antibiotic increased with increase in spiking concentration in milk. In conclusion, the developed HPLC-UV method demonstrated excellent performance characteristics for the simultaneous detection of OTC, TC, and CTC in Khoa. The method exhibited good linearity, accuracy, precision, and low limits of detection and quantification, making it suitable for routine analysis and quality control of Khoa for tetracycline residues determination.