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Physicochemical Methods for Identifying Antibiotic Residues in Foods
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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
14.
MOATS
Physicochemical Methods for Identifying Antibiotic Residues
155
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.
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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
