Agricultural Uses of Antibiotics - American Chemical Society

Meat Science Research Laboratory, Agricultural Research Service, U.S. Department ... types of screening methods, although they can be used for direct ...
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Physicochemical Methods for Identifying Antibiotic Residues in Foods

Downloaded by UNIV OF MASSACHUSETTS AMHERST on June 6, 2017 | http://pubs.acs.org Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch014

William A. Moats Meat Science Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705 The physicochemical methods are needed f o r i d e n t i f i c a t i o n and quantitation of a n t i b i o t i c residues i n milk and tissues of animals. Methods successfully employed include high voltage electrophoresis with detection by bioautography and chromatographic procedures. Gas-liquid (GLC), thin-layer (TLC) and high performance l i q u i d chromatography (HPLC) have all been used f o r residue a n a l y s i s . A number of chromatographic methods have been described f o r chloramphenicol and the sulfonamides using all three chromatographic modes. Less work has been reported with residues of other a n t i b i o t i c s . S a t i s f a c t o r y physicochemical confirmatory tests are not available for some compounds. The work on residue monitoring has been divided into microbiological methods covered i n the preceding chapter and physicochemical methods which i s the topic of t h i s chapter. The d i v i s i o n between the two approaches i s somewhat a r b i t r a r y since many methods include elements of both approaches. Physicochemical methods are commonly used f o r i d e n t i f i c a t i o n and/or quantitation of residues detected by various types of screening methods, although they can be used for d i r e c t testing f o r residues. Successful methods mainly employ either high voltage electrophoresis or chromatography f o r separation of compounds and I w i l l discuss a p p l i c a t i o n of these two approaches to residues i n food substrates. For the present discussion, sulfonamides are also included, since they are used i n a s i m i l a r manner to a n t i b i o t i c s . Electrophoresis High voltage electrophoresis (HVE) i n agar gel with detection by bioautography has been used with considerable success i n some laboratories f o r i d e n t i f i c a t i o n of residues (1-6). This procedure has the advantage that a l l a n t i b i o t i c substances detectable by bioautography can be c l a s s i f i e d on the basis of electrophoretic mobility. Further t e s t i n g may be required f o r quantification and to This chapter not subject to U.S. copyright. Published 1986, American Chemical Society

Moats; Agricultural Uses of Antibiotics ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Downloaded by UNIV OF MASSACHUSETTS AMHERST on June 6, 2017 | http://pubs.acs.org Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch014

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MOATS

Physicochemical Methods for Identifying Antibiotic Residues

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distinguish compounds with s i m i l a r electrophoretic m o b i l i t i e s , especially i f only one buffer i s used (7,8). Natural microbial i n h i b i t o r s found i n some animal tissues form a streak unlike any a n t i b i o t i c compound. Smither et a l (9) examined 5442 UK-produced meat samples using the four plate test (FPT) of the European community. Of these, 34 were i n i t i a l l y p o s i t i v e . However, electrophor e s i s demonstrated that only two of the p o s i t i v e s were recognizable a n t i b i o t i c s . On r e t e s t i n g , 20 of the samples o r i g i n a l l y p o s i t i v e were negative and 12 samples were found to contain natural microbial i n h i b i t o r s . Van Schothorst and Van Leusden (5) reported good agreement between electrophoresis and bioassays f o r confirmation of residues found i n kidney. Engel et a l (10) found that of residues detected i n kidney and muscle by the European four-plate test (FPT), only 50% and 37%, respectively, could be confirmed by HVE. They concluded that HVE i s l e s s s e n s i t i v e than the FPT. Chromatographic Methods Enough chromatographic methods f o r a n t i b i o t i c s have been described to warrant a book on the subject (11). These are, however, mainly f o r formulations and c l i n i c a l applications and a p p l i c a t i o n to residue analysis has been rather l i m i t e d . Residue analysis requires greater s e n s i t i v i t y and i s o l a t i o n from more complex substrates than i s the case with other a p p l i c a t i o n s . However, considerable progress has been reported i n recent years, e s p e c i a l l y with chloramphenicol and the sulfonamides. Thin layer chromatography (TLC), high performance l i q u i d chromatography (HPLC), and gas l i q u i d chromatography (GLC) have a l l been used. The applications of GLC f o r analysis of drug residues i n tissues were recently reviewed by Petz (12). Chromatographic methods are frequently suitable f o r determination of residues of a number of compounds i n a single procedure. They also have the potential to detect metabolites. Further confirmation by spectrophotometry and/or mass spectrometry i s possible. A discussion of the a p p l i c a t i o n to s p e c i f i c a n t i b i o t i c residues follows. Sulfonamides Rapid progress has been reported i n the development of methods f o r sulfonamide residues i n t i s s u e s , milk, and eggs since the subject was reviewed by Horwitz (13) i n 1981. The colorimetric method of T i s h l e r et a l (14) has i n the past been used to detect v i o l a t i v e l e v e l s of sulfonamide residues i n animal t i s s u e s . The lack of s p e c i f i c i t y and the variable background l e v e l s produced by t h i s method have been discussed by Horwitz (13), Matusik et a l (15), and Lloyd et a l (16). Recently, a number of s p e c i f i c chromatograpKic methods have been described f o r determination of residues of a v a r i e t y of sulfonamides. These are summarized i n Table I and suggest that HPLC i s emerging as the method of choice followed by GLC and TLC methods. The methods l i s t e d do not include a number described f o r blood and/or urine only. The HPLC methods mainly use UV detectors, but one uses amperometric (18) and one uses fluorescent detection (25). Fluorescent detection a f t e r d e r i v a t i z a t i o n with fluorescamine i s the method most commonly used f o r detection on TLC plates. V i l i m (24) used TLC to

Moats; Agricultural Uses of Antibiotics ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

156

AGRICULTURAL USES OF ANTIBIOTICS

Downloaded by UNIV OF MASSACHUSETTS AMHERST on June 6, 2017 | http://pubs.acs.org Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch014

Table I,

Chromatographic Methods for Determination of Sulfonamide Residues i n Tissue, Milk, and Eggs

Method

Substrate

Compounds

Detection

Sensi- Refert i v i t y ence (ppb)

HPLC

Chicken, tissue eggs

SFX, SMMf/ SDM, SQX

UV

5

(17)

Liver, kidney muscle

Several

Amperometric

10

(18)

Chicken tissue

SQX

UV

10

(19)

Chicken tissue

SMM, SDN, SQX

UV

10-30

(20)

Swine l i v e r

Glycopyranosyl SMZ

UV

10

(21)

Chicken tissue, eggs

SQX

UV

10-30

(22)

Beef tissue

SMZ

UV

100

(23)

Pork tissue

SMZ

UV

50

(24)

Chicken tissue, eggs

SMM, BDM, SQX

Fluorescamine Derivative

TLC



(25)

Eggs, meat, milk SMR, SDZ SDD, SMX, SQX

UV

100

(26)

Swine tissue

SMZ

UV

50

(27)

Swine tissue

5

UV

50

(28)

Pork l i v e r

SMZ, STH

Colorimetric

100

(29)

Liver, muscle

SMZ, SDM STH, SQX SBM

Fluorescamine Quantity