Analytical methods - Gentamycin

US EPA residu analytical methods (RAM): Analytical method for the determination of gentamicin in pears (1993)

Legislation :

There is increasing awareness of food safety by the consumer with respect to antimicrobial resistance due to the discovery of new resistant strains of bacteria and others that are becoming increasingly resistant over time. As a result there is increasing pressure on laboratories responsible for food safety to monitor the use of these drugs and ensure the safety of food for human consumption. Legislation regarding the control of antibiotic residues in live animals and animal products is given in Coun-cil Directive 96/23/EC. In the context of this directive, details for methods and their performance criteria are described in Commission Decision 2002/657/EC. Residues are divided into two groups A and B for the purposes of monitoring and defining the legislation relating to them.

Group A refers to substances having an anabolic effect and unauthorised substances while group B refers to veterinary drugs and contaminants. The aminoglycosides and macrolides are both listed under group B1 (antibacterial substances). The various sub-divisions of these two groups are listed in Council directive 96/23/EC. The EU Council regulation 2377/90 lays down the Community procedure for the establishment of maximum residue limits (MRLs) of veterinary medicinal products in foodstuffs of animal origin.

Some aminoglycosides with established MRLs are shown in Table 1 and some aminoglycosides with provisional MRLs are shown in Table 2. EU Council regulation 2377/90 also dictates the analytical methods that can be used for confirmatory analysis and these are listed in Table 3.

Table 1

Pharmacologically  (actvie substances)	Marker residue	Animal species	MRLs (µg kg−1 )	Target tissues
Gentamicin	Gentamicin	Bovine Porcine	50	Muscle
			50	Fat
			200	Liver
			750	Kidney
		Bovine	100	Milk

2

Table 2 – Suitable confirmatory methods for  organic residues or contaminants [11]
Measuring technique	Compound group from 96/23/EC	Limitations
LC or GC with mass-spectrometric detection	Groups A and B	Only  if following either an on-line or an off-line chromatographic separation. Only if full  scan techniques are used or using at least 3 (group B) or 4 (group A) identification points for techniques that do not record the full  mass spectra
LC or GC with IR spectrometric detection	Groups A and B	Specific requirements for absorption in IR spectrometry have to be met
LC-full-scan DAD	Group B	Specific requirements for absorption in UV spectrometry have to be met
LC-fluorescence	Group B	Only  for molecules that exhibit native fluorescence and to molecules that exhibit fluorescence after either transformation or derivatisation
2-D TLC-full-scan UV/VIS	Group B	Two-dimensional HPTLC and co-chromatography are mandatory
GC-Electron capture detection	Group B	Only  if two columns of different polarity are used
LC-immunogram	Group B	Only  if at least two different chromatographic systems or a second, independent detection method are used
LC–UV/VIS (single wavelength)	Group B	Only  if at least two different chromatographic systems or second, independent detection method are used
Key: LC: liquid chromatography; GC: gas chromatography; IR: infrared spectrometry; DAD: diode array detection; TLC: thin layer chromatography; UV/VIS: ultraviolet/visible spectrophotometry; HPTLC: high-performance thin layer chromatography.

values (from

To ensure that aminoglycosides are used only in approved situations and to control their use in meat-producing animals, targeted samples are taken at the slaughterhouse and screened for the presence of residues. A positive screening result means that the sample must be subjected to confirmatory analysis. This assay must adhere to Commission Decision 2002/657/EC whereby suitable confirmatory methods are based on a required number of identification points. For the identity of group B compounds such as the aminoglycosides, a minimum of three identification points are required. As a consequence, methods that are based on chromatographic analysis followed by mass spectrometric detection are becoming the normal way of confirming identity and determining concentration.

Other methods available

Alternative methods from literature

esticides: Analyical