1. LC-MS/MS Analysis of Tetracycline antibiotics in Honey using Advance UHPLC-EVOQ™
SIMA LAB PVT LTD

2. 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

3. Tetracycline Drugs Oxytetracycline (M.W: 460.4)
Doxycycline (M.W: 444.4)
Chlortetracycline (M.W: 478.8)

4. 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 

5. 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

6. 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

7. 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

8. Chromatograms of TCs at 1 ppb
Figure 1

9. Calibration Curve
Figure 2

10. 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

11. 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.