EN   EN 
 
 
 EUROPEAN  
COMMISSION   
Brussels, XXX   
SANTE/PLAN/2023/1707 ANNEX CIS 
(POOL/G5/2023/1707/1707 -EN 
ANNEX CIS.docx)  
[…](2023) XXX  draft  
ANNEX  
 
ANNEX  
to the  
Commission Delegated Regulation  
supplementing Regulation (EU) 2019/4 of the European Parliament and of the Council 
by establishing specific maximum levels of cross -contamination of anti -microbial active 
substances in non -target feed and methods of analysis for these substances    
EN 1  EN ANNEX  
Reference methods of analysis for the quantification of the levels of cross -contamination of antimicrobial active substances in feed  in 
accordance with Article 3 
Chemical class  Name of substance  CAS 
number1 EU number2 Multi -analyte 
methoda,b,c Reference  analytical method3,4,5 Maximum acceptable 
method quantification  limit 
(LOQ)   
(µg/kg)  
Polymixins 
(polypeptide 
antibiotics)  Colistin  1264 -72-8 - (b) LSE - A - C - SPE - E - LC-MS/MS  150 (Colistin A )  
300 (Colistin B ) 
Pyrimidine inhibitor 
of dihydrofolate 
reductase  Trimethoprim  738-70-5 212-006-2 (c) LSE - US - A - C - F - LC-MS/MS or 
LC-HRMS  25 
Macrolides  Tylvalosin  63409 -12-1 - (c) LSE - US - A - C - F - LC-MS/MS or 
LC-HRMS  100 
Tilmicosin  108050 -54-0 639-676-2 (c) LSE - US - A - C - F - LC-MS/MS or 
LC-HRMS  100 
Tylosin  1401 -69-0 215-754-8 (c) LSE - US - A - C - F - LC-MS/MS or 
LC-HRMS  100 
Lincosamides  Lincomycin  154-21-2 205-824-6 (c) LSE - US - A - C - F - LC-
MS/MS or LC -HRMS  25  
EN 2  EN Pleuromutilins  Tiamulin  55297 -96-6 259-580-0 (c) LSE - US - A - C - F - LC-
MS/MS or LC -HRMS  10 
Valnemulin  101312 -92-
9 - (c) LSE - US - A - C - F - LC-
MS/MS or LC -HRMS  50 
Penicillins  Amoxicillin  26787 -78-0 612-127-4 (c) LSE - US - A - C - F - LC-MS/MS or 
LC-HRMS  150 
Penicillin V  1098 -87-9 - (c) LSE - US - A - C - F - LC-MS/MS or 
LC-HRMS  50 
Aminoglycosides  Apramycin  65710 -07-8 265-890-7 
253-460-1 (a) LSE - A - C - SPE - LC-MS/MS 50 
Neomycin  1404 -04-2 1404 -04-2 (a) LSE - A - C - SPE - LC-MS/MS 50 
Paromomycin  1263 -89-4 - (a) LSE - A - C - SPE - LC-MS/MS 50 
Spectinomycin  1695 -77-8 - (a) LSE - A - C - SPE - LC-MS/MS 500 
Amphenicols  Florfenicol  73231 -34-2 642-986-0 (c) LSE - US - A - C - F - LC-MS/MS or 
LC-HRMS  150 
Tiamfenicol  15318 -45-3 239-355-3 (c) LSE - US - A - C - F - LC-MS/MS or 
LC-HRMS  200 
Coccidiostats  Amprolium  137-88-2 204-458-4 (c) 
 LSE - US - A - C - F - LC-MS/MS or 
LC-HRMS  100 
   (a) LSE - A - C - SPE - LC-MS/MS 5  
EN 3  EN Fluoroquinolones  Flumequine  42835 -25-6 255-962-6 (c) 
 LSE - US - A - C - F - LC-MS/MS or 
LC-HRMS  25 
Oxolinic Acid  14698 -29-4 238-750-8 (c) 
 LSE - US - A - C - F - LC-MS/MS or 
LC-HRMS  25 
Sulfonamides  Sulfamonomethoxine  1220 -83-3 624-483-8 (c) 
 LSE - US - A - C - F - LC-MS/MS or 
LC-HRMS  25 
Sulfadimethoxine  122-11-2 204-523-7 (c) 
 LSE - US - A - C - F - LC-MS/MS or 
LC-HRMS  25 
Tetracyclines  Chlortetracycline  57-62-5 200-341-7 (c) 
 LSE - US - A - C - F - LC-MS/MS or 
LC-HRMS  100 
Doxycycline  564-25-0 209-271-1 (c) 
 LSE - US - A - C - F - LC-MS/MS or 
LC-HRMS  100 
Tetracycline  60-54-8 200-481-9 (c) LSE - US - A - C - F - LC-MS/MS or 
LC-HRMS  100 
Oxytetracycline  79-57-2 - (c) LSE - US - A - C - F - LC-MS/MS or 
LC-HRMS  100 
 
(1) Chemical Abstracts Service  
(2) European Union number – not available for all substances  
(3) Extraction methods:  
• LSE – liquid solid  extraction   
EN 4  EN • US – Ultrasonication  
• A – Agitation  
(4) Clean -up methods  
• C – Centrifugation  
• SPE – Solid Phase Extraction  
• E – Evaporation, re -dissolution  
• F – Filtration  
(5) Analytical methods:  
• LC-MS/MS – Liquid chromatography tandem mass spect rometry  
• LC-HRMS – Liquid chromatography coupled to high resolution mass spectrometry  
(a): multi -analyte method for the aminoglycosides and amprolium  
(b): multi -analyte method for the polymixins colistin  A and B  
(c): multi -analyte method for trimethoprim, a mprolium, the macrolides, the penicillins, the amphenicols, the fluoroquinolones, the sulphonamides and 
the tetracyclines