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Published byDorthy Elisabeth Ford Modified over 6 years ago
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LC-MS/MS Analysis of Tetracycline antibiotics in Honey using Advance UHPLC-EVOQ™
SIMA LAB PVT LTD
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Objective To develop “simple, robust and effective LC-MS/MS method is developed for simultaneous detection & quantification of Tetracycline (TC), Chlortetracycline (CTC), Oxytetracycline (OTC) and Doxycycline (DC) in food grade honey
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Tetracycline Drugs Oxytetracycline (M.W: 460.4)
Doxycycline (M.W: 444.4) Chlortetracycline (M.W: 478.8)
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Experimental Conditions
EVOQ – LC-MS/MS Source parameters Ionization Probe : HESI (Heated Electrospray Probe) Polarity : Positive HESI : V Probe Temperature : 450 °C Probe gas : 45 units Nebulizer gas : 50 units Cone gas temp : 350 °C Cone gas : 20 units Active exhaust : On Collision gas: Argon : 1.5 mTorr
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Experimental Conditions
Chromatography (Advance UHPLC) Column : Bruker Intensity C18 (50 mm x 2.1 mm x 3 µm) Column temperature : ambient Flow rate : 0.3 mL/min Mobile phase A : Water with 0.1 % Formic acid Mobile phase B : Methanol with 0.1 % Formic Acid Gradient conditions: 0.00 min 20 % B 0.50 min 60 % B 2.50 min 80 % B 3.50 min 80 % B 4.00 min 20 % B 5.00 min 20 % B Injection volume : 10 μL
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MRM transitions Compound Parent Ion Product Ion Collision Energy (V)
Tetracycline 445 a427 10 154 25 Chrolotetracycline 461.7 a426 20 443 Oxytetracycline 479.8 a444 462 17 Doxytetracycline 445.7 a428 32 427 22
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Sample Preparation 1 part of honey mixed with 4 part of
0.1 mol/L of Na2EDTA-Mcllvaine buffer Centrifugation at 3000 rpm for 3 min Extract was passed through the conditioned SPE cartridge TCs were eluted using Methanol containing 5mM oxalic acid Dried the residue, reconstituted with diluent and inject to LC-MS/MS
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Chromatograms of TCs at 1 ppb
Figure 1
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Calibration Curve Figure 2
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Summary of the validation results
Compound R2 % RSD for 1 ppb (n=10) % RSD for 10 ppb (n=10) 20 ppb(n=10) % Recovery Tetracycline 0.999 8.58 6.33 5.56 82-85 Chrolotetracycline 0.998 8.84 5.07 4.32 79-95 Oxytetracycline 5.79 4.16 6.31 87-94 Doxytetracycline 6.78 3.09 3.46 88-104 Repeatability results for tetracycline’s in honey
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Summary Fast reliable and robust method to analyze Tetracycline drug (Tetracycline, Chrolotetracycline, Oxytetracycline and Doxytetracycline) The use of advance UHPLC reduced total LC-MS/MS run time and enable analysis of 10 samples per hour. The average recoveries at the investigated levels were all in the range of 79 to 104 %, indicating good trueness. Both the intra-day and inter-day RSD’s were below 10 %, revealing that the precision and accuracy of the method were acceptable as per European Commission Decision 2002/657/EC. The validation results revealed that the method can be used for the screening and quantification of above TCs in honey as per the EU and other requirement.
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