Current Methodologies for the Analysis of Aminoglycosides

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Journal of Chromatography B, 747 (2000) 69±93 www.elsevier.com/locate/chromb

Review Current methodologies for the analysis of aminoglycosides

David A. Stead 14 Waite Close, Pocklington, York, YO42 2YU, UK

Abstract

The aminoglycosides are a large and diverse class of antibiotics that characteristically contain two or more aminosugars linked by glycosidic bonds to an aminocyclitol component. Structures are presented for over 30 of the most important members of this family of compounds. The use of aminoglycosides in clinical and veterinary medicine and in agriculture is described. Qualitative methods for aminoglycoside analysis include X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). The major part of this article comprises a comprehensive review of quantitative methods for the determination of aminoglycosides. These are microbiological assay, radiochemical assay, radioimmunoassay, enzyme immunoassay, ¯uoroimmunoassay and other immunoassays, spectrophotometric and other non-separative methods, gas chromatography (GC), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and capillary electrophoresis (CE). Simple spectrophotometric methods may be adequate for the assay of bulk pharmaceuticals and their formulations. Microbiological assays make useful semi-quantitative screening tests for the analysis of veterinary drug residues in food, but rapid enzyme immunoassays are more suitable for accurate measurements of aminoglycosides in complex matrices. Automated immunoassays are the most appropriate methods for serum aminoglycoside determinations during therapeutic drug monitoring. HPLC techniques provide the speci®city and sensitivity required for pharmacokinetic and other research studies, while HPLC±MS is employed for the con®rmation of veterinary drug residues. The potential for further development of chromatographic and CE methods for the analysis of biological samples is outlined.  2000 Elsevier Science B.V. All rights reserved.

Keywords: Reviews; Aminoglycosides

Contents

1. Introduction ...... 70 1.1. Classi®cation and structure ...... 70 1.2. Source...... 70 1.3. Use as therapeutic agents in clinical and veterinary medicine ...... 73 1.4. Use in agriculture...... 75 1.5. Need for analytical methodologies ...... 75 2. Qualitative methods for identi®cation/characterisation of aminoglycosides ...... 76 2.1. X-ray crystallography...... 76 2.2. Nuclear magnetic resonance spectroscopy...... 76 2.3. Mass spectrometry ...... 76 3. Quantitative methods for the determination of aminoglycosides ...... 76 3.1. Microbiological assay...... 76 3.2. Radiochemical assay ...... 78 3.3. Radioimmunoassay ...... 78

3.4. Enzyme immunoassay ...... 79 3.4.1. Heterogeneous EIA ...... 79 3.4.2. Homogeneous EIA ...... 81 3.5. Fluoroimmunoassay ...... 81 3.6. Direct chemiluminescence immunoassay ...... 82 3.7. Nephelometric and turbidimetric immunoassays...... 83 3.8. Immunohistochemical techniques...... 83 3.9. Spectrophotometric and other non-separative physicochemical methods...... 83 3.10. Gas chromatography...... 84 3.11. Thin-layer chromatography ...... 84 3.12. High-performance liquid chromatography ...... 85 3.12.1. Ion-exchange and ion-pair HPLC...... 86 3.12.2. Reversed-phase HPLC ...... 86 3.12.3. Sample preparation for HPLC analysis...... 86 3.13. Capillary electrophoresis ...... 88 4. Conclusions ...... 90 Acknowledgements...... 90 References ...... 90

1. Introduction 1.2. Source

1.1. Classi®cation and structure Many aminoglycosides occur naturally as products of various Actinobacteria (Actinomycetes), particu- The aminoglycosides are a large and diverse class larly members of the genera Streptomyces (in which of antibiotics [1] that characteristically contain two case they are named -mycins) and Micromonospora or more aminosugars linked by glycosidic bonds to (-micins) [2]. These organisms often produce a an aminocyclitol component. The cyclitol is 2-deox- number of structurally related antibiotics simultan- ystreptamine in most cases, one exception being eously [3] and the therapeutic product may contain a streptomycin, which has a streptidine moiety (Fig. mixture of active compounds, e.g. the gentamicin C 1). complex. Some aminoglycoside antibiotics are semi- The aminoglycosides may be categorised according to the pattern of substitution of the cyclitol (Table 1). The most important subclasses are the 4,5-disubstituted deoxystreptamines (Fig. 2), which include the neomycins, and the 4,6-disubstituted deoxystreptamines (Fig. 3), which include the gen- tamicins, kanamycins, and tobramycin. The structures of some other important aminoglycosides are shown in Fig. 4. These include apramycin (a 4- monosubstituted 4-deoxystreptamine), streptomycin (a 4-monosubstituted streptidines), and hygromycin B (a 5-monosubstituted (1)-N-methyl-2-deoxystreptamine). Spectinomycin is included although it is not strictly an aminoglycoside as it has neither an aminosugar nor a glycosidic bond. It contains the aminocyclitol N,N9-dimethyl-2-epi-streptamine Fig. 1. Aminocyclitol components of aminoglycosides. Num- linked at both the 4- and 5-positions to a single bering of the ring atoms, shown for 2-deoxystreptamine, begins carbohydrate moiety. with the nitrogen-bearing carbon atom having the R con®guration. D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93 71

Table 1 Nomenclature and sources of aminoglycosides

Aminoglycoside Systematic name Source

4,5-Disubstituted deoxystreptamines Butirosin A (S)-O-2,6-Diamino-2,6-dideoxy-a-D-glucopyranosyl-(1→4)-O-[b-D-xylofuranosyl-(1→5)]-N1- Mucoid strains of Bacillus circulans; (4-amino-2-hydroxy-1-oxobutyl)-2-deoxy-D-streptamine major component of butirosin Butirosin B (S)-O-2,6-Diamino-2,6-dideoxy-a-D-glucopyranosyl-(1→4)-O-[b-D-ribofuranosyl-(1→5)]-N1- Mucoid strains of B. circulans; (4-amino-2-hydroxy-1-oxobutyl)-2-deoxy-D-streptamine minor component of butirosin Neomycin B O-2,6-Diamino-2,6-dideoxy-a-D-glucopyranosyl-(1→4)-O-[O-2,6-diamino-2,6-dideoxy-b-L- Streptomyces fradiae; idopyranosyl-(1→3)-b-D-ribofuranosyl-(1→5)]-2-deoxy-D-streptamine major component of neomycin complex Neomycin C O-2,6-Diamino-2,6-dideoxy-a-D-glucopyranosyl-(1→4)-O-[O-2,6-diamino-2,6-dideoxy-a-D- S. fradiae; glucopyranosyl-(1→3)-b-D-ribofuranosyl-(1→5)]-2-deoxy-D-streptamine minor component of neomycin complex Paromomycin I O-2-Amino-2-deoxy-a-D-glucopyranosyl-(1→4)-O-[O-2,6-diamino-2,6-dideoxy-b-L- Various Streptomyces; (neomycin E) idopyranosyl-(1→3)-b-D-ribofuranosyl-(1→5)]-2-deoxy-D-streptamine minor component of neomycin complex Paromomycin II O-2-Amino-2deoxy-a-D-glucopyranosyl-(1→4)-O-[O-2,6-diamino-2,6-dideoxy-a-D- Various Streptomyces; (neomycin F) glucopyranosyl-(1→3)-b-D-ribofuranosyl-(1→5)]-2-deoxy-D-streptamine minor component of neomycin complex Ribostamycin O-2,6-Dimino-2,6-dideoxy-a-D-glucopyranosyl-(1→4)-O-[b-D-ribofuranosyl-(1→5)]- S. ribosidi®cus 2-deoxy-D-streptamine

4,6-Disubstituted deoxystreptamines Amikacin (S)-O-3-Amino-3-deoxy-a-D-glucopyranosyl-(1→6)-O-[6-amino-6-deoxy-a-D-glucopyranosyl- Semi-synthetic; derived from kanamycin A 1 (1→4)]-N -(4-amino-2-hydroxy-1-oxobutyl)-2-deoxy-D-streptamine Arbekacin (S)-O-3-Amino-3-deoxy-a-D-glucopyranosyl-(1→6)-O-[2,6-diamino-2,3,4,6-tetradeoxy-a-D- Semi-synthetic; derived from kanamycin B 1 (habekacin) erythro-hexopyranosyl-(1→4)]-N -(4-amino-2-hydroxy-1-oxobutyl)-2-deoxy-D-streptamine Dibekacin (S)-O-3-Amino-3-deoxy-a-D-glucopyranosyl-(1→6)-O-[2,6-diamino-2,3,4,6-tetradeoxy-a-D- Semi-synthetic; derived from kanamycin B erythro-hexopyranosyl-(1→4)]-2-deoxy-D-streptamine → Gentamicin C1 (R)-O-2,6-Diamino-2,3,4,6-tetradeoxy-6-(methylamino)-a-D-erythro-hexopyranosyl-(1 4)-O- Micromonospora purpurea or M. echinospora; [3-deoxy-4-C-methyl-3-(methylamino)-b-L-arabinopyranosyl-(1→6)]-2-deoxy-D-streptamine major component of gentamicin C complex → Gentamicin C1a O-3-Deoxy-4-C-methyl-3-(methylamino)-b-L-arabinopyranosyl-(1 6)-O-[2,6,-diamino- M. purpurea or M. echinospora; 2,3,4,6-tetradeoxy-a-D-erythro-hexopyranosyl-(1→4)]-2-deoxy-D-streptamine major component of gentamicin C complex → Gentamicin C2 (R)-O-2,6-Diamino-2,3,4,6-tetradeoxy-6-methyl-a-D-erythro-hexopyranosyl-(1 4)-O-[3-deoxy- M. purpurea or M. echinospora; 4-C-methyl-3-(methylamino)-b-L-arabinopyranosyl-(1→6)]-2-deoxy-D-streptamine major component of gentamicin C complex → Gentamicin C2a (S)-O-2,6-Diamino-2,3,4,6-tetradeoxy-6-methyl-a-D-erythro-hexopyranosyl-(1 4)-O-[3-deoxy- M. purpurea or M. echinospora; 4-C-methyl-3-(methylamino)-b-L-arabinopyranosyl-(1→6)]-2-deoxy-D-streptamine minor component of gentamicin C complex → Gentamicin C2b O-2-Amino-2,3,4,6-tetradeoxy-6-(methylamino)-a-D-erythro-hexopyranosyl-(1 4)-O-[3-deoxy- M. sagamiensis var. nonreductans; (micronomicin) 4-C-methyl-3-(methylamino)-b-L-arabinopyranosyl-(1→6)]-2-deoxy-D-streptamine minor component of gentamicin C complex Isepamicin (S)-O-6-Amino-6-deoxy-a-D-glucopyranosyl-(1→4)-O-[3-deoxy-4-C-methyl-3-(methylamino)-b- Semi-synthetic; derived from gentamicin B 1 L-arabinopyranosyl-(1→6)]-N -(3-amino-2-hydroxy-1-oxopropyl)-2-deoxy-D-streptamine Kanamycin A O-3-Amino-3-deoxy-a-D-glucopyranosyl-(1→6)-O-[6-amino-6-deoxy-a-D-glucopyranosyl- Streptomyces kanamyceticus; (1→4)]-2-deoxy-D-streptamine major component of kanamycin complex Kanamycin B O-3-Amino-3-deoxy-a-D-glucopyranosyl-(1→6)-O-[2,6-diamino-2,6-dideoxy-a-D-glucopyranosyl- S. kanamyceticus; (bekanamycin) (1→4)]-2-deoxy-D-streptamine minor component of kanamycin complex Kanamycin C O-3-Amino-3-deoxy-a-D-glucopyranosyl-(1→6)-O-[2-amino-2-deoxy-a-D-glucopyranosyl- S. kanamyceticus; (1→4)]-2-deoxy-D-streptamine minor component of kanamycin complex Netilmicin O-3-Deoxy-4-C-methyl-3-(methylamino)-b-L-arabinopyranosyl-(1→6)-O-[2,6,-diamino-2,3,4,6- Semi-synthetic; derived from sisomicin 1 tetradeoxy-a-D-glycero-hex-enopyranosyl-(1→4)]-2-deoxy-N -ethyl-D-streptamine Sisomicin O-3-Deoxy-4-C-methyl-3-(methylamino)-b-L-arabinopyranosyl-(1→6)-O-[2,6,-diamino-2,3,4,6- M. inyoesis tetradeoxy-a-D-glycero-hex-enopyranosyl-(1→4)]-2-deoxy-D-streptamine Tobramycin O-3-Amino-3-deoxy-a-D-glucopyranosyl-(1→6)-O-[2,6-diamino-2,3,6-trideoxy-a-D-ribo- S. tenebrarius; hexopyranosyl-(1→4)]-2-deoxy-D-streptamine component of nebramycin complex

Other important aminoglycosides Apramycin O-4-Amino-4-deoxy-a-D-glucopyranosyl-(1→8)-O-(8R)-2-amino-2,3,7-trideoxy-7-(methylamino)- Streptomyces tenebrarius; D-glycero-a-D-allo-octadialdo-1,5:8,4-dipyranosyl-(1→4)-2-deoxy-D-streptamine component of nebramycin complex Destomycin A O-6-Amino-6-deoxy-L-glycero-D-galacto-heptopyranosylidene-(1→2-3)-O-b-D-talopyranosyl- S. rimofaciens 1 (1→5)-2-deoxy-N -methyl-D-streptamine 72 D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93

Table 1. Continued

Aminoglycoside Systematic name Source

Other important aminoglycosides (continued) Fortimicin A 2,6-Diamino-2,3,4,6,7-pentadeoxy-b-L-lyxo-heptopyranosyl-(1→3)-4-amino-1- Micromonospora olivoasterospora; [(aminoacetyl)methylamino]-1,4-dideoxy-6-O-methyl-1L-chiro-inositol major component of fortimicin complex Fortimicin B 2,6-Diamino-2,3,4,6,7-pentadeoxy-b-L-lyxo-heptopyranosyl-(1→3)-4-amino-1,4-dideoxy-6-O- M. olivoasterospora; methyl-1-(methylamino)-1L-chiro-inositol major component of fortimicin complex Hygromycin B O-6-Amino-6-deoxy-L-glycero-D-galacto-heptopyranosylidene-(1→2-3)-O-b-D-talopyranosyl- 3 (1→5)-2-deoxy-N -methyl-D-streptamine S. hygroscopicus Kasugamycin 2-Amino-4-[(carboxyiminomethyl)amino]-2,3,4,6-tetradeoxy-D-arabino-hexose- S. kasugaensis (1→3)-1D-chiro-inositol Spectinomycin [2R-(2a,4ab,5ab,6b,7b,8b,9a,9aa,10ab)]-decahydro-4a,7,9-trihydroxy-2-methyl- S. spectabilis 6,8-bis-(methylamino)-4H-pyrano[2,3-b][1,4]benzodioxin-4-one Streptomycin O-2-Deoxy-2-(methylamino)-a-L-glucopyranosyl-(1→2)-O-5-deoxy-3-C-formyl-a-L- Certain strains of S. griseus lyxo-furanosyl-(1→4)-N,N9-bis(aminoiminomethyl)-D-streptamine Dihydro- O-2-Deoxy-2-(methylamino)-a-L-glucopyranosyl-(1→2)-O-5-deoxy-3-C-(hydroxymethyl)-a-L- Semi-synthetic derivative of streptomycin; streptomycin lyxofuranosyl-(1→4)-N,N9-bis(aminoiminomethyl)-D-streptamine also produced by S. humidus

synthetic derivatives of natural fermentation prod- The total chemical synthesis of many of the amino- ucts, e.g. amikacin (derived from kanamycin A), glycosides has been achieved [4] but fermentation arbekacin and dibekacin (from kanamycin B), remains the most economical route for their pro- isepamicin (from gentamicin B), netilmicin (from duction. Table 1 lists the sources of important sisomicin), and dihydrostreptomycin (from strep- aminoglycoside antibiotics together with their chemi- tomycin) although the last is also produced naturally. cal nomenclature.

Fig. 2. Structures of deoxystreptamine aminoglycosides, 4,5-disubstituted. D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93 73

Fig. 3. Structures of 4,6-disubstituted deoxystreptamine aminoglycosides.

1.3. Use as therapeutic agents in clinical and conditions [5]. Their clinical use has been limited by veterinary medicine side effects of nephrotoxicity and ototoxicity [6,7], and by the emergence of resistant strains of organ- The aminoglycosides are broad-spectrum antibiot- isms that produce aminoglycoside-modifying en- ics that have bactericidal activity against some zymes [8]. Gram-positive and many Gram-negative organisms. Streptomycin, discovered in 1944, was initially They are not active against anaerobic bacteria, successful in the treatment of tuberculosis and other possibly because their uptake is blocked under these serious infections, but after several years of wide- 74 D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93

Fig. 4. Structures of some other important aminoglycosides. D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93 75 spread use resistant strains began to appear in 1.4. Use in agriculture hospital patients in the late 1950s. The same pattern was repeated with kanamycin during the following Kasugamycin has been used for many years in decade. This stimulated a search for new amino- Japan for crop protection. It has a narrow spectrum glycosides and semi-synthetic derivatives that re- of activity against phytopathogenic microbes and tained activity against the resistant organisms. Dur- certain strains of pseudomonads, and has been ing the 1970s, these efforts were rewarded with the applied in the control of Magnaporthe grisea discovery of many novel agents, of which several (Pyricularia oryzae), the rice blast fungus [2,17]. were successfully developed for therapeutic use. However, no aminoglycosides have moved into 1.5. Need for analytical methodologies development since 1980 and it seems reasonable to conclude that there is little potential for improving In a broad sense, methods of analysis have appli- upon the properties of existing antibiotics of this cations in the characterisation of new amino- class [9]. glycosides from bacterial fermentations. Such tech- Despite the introduction of newer, less toxic niques include thin-layer chromatography (TLC) of antimicrobials, aminoglycosides continue to serve a fragments released by chemical degradation, X-ray useful role as therapeutic agents [10,11]. Their crystallography, nuclear magnetic resonance (NMR) clinical importance is being reassessed in the light of spectroscopy and mass spectrometry (MS). the increasing resistance of pathogenic organisms to Quantitative analyses of aminoglycosides in blood other antimicrobials [12]. Gentamicin is currently the are routinely used in therapeutic drug monitoring as ®rst choice aminoglycoside for the treatment of a guide to dosing, to prevent toxicity and to ensure serious infections, alternatives being amikacin, netil- ef®cacy [7]. Automated immunoassays (Sections micin, and tobramycin [13]. They are all poorly 3.3±3.7) are the most appropriate methods for these absorbed from the gut and are administered by serum aminoglycoside determinations. Levels of intramuscular or intravenous injection. Monotherapy aminoglycosides are also measured in biological is indicated for less serious infections, e.g. those of ¯uids and tissues during pharmacokinetic and other the urinary and biliary tracts. Combination therapy research studies. For these applications, the method- with a penicillin may be used to treat more serious ologies must provide high speci®city and sensitivity, infections, e.g. bacterial endocarditis, hospital-ac- and it is advantageous if any metabolites can also be quired pneumonia, and septicaemia. Streptomycin detected. High-performance liquid chromatography still has a role in the treatment of multidrug-resistant (HPLC, Section 3.12) currently best meets these tuberculosis. Neomycin may be given orally for requirements. sterilisation of the gut prior to surgery. Spec- Sensitive methods are necessary for the analysis of tinomycin is indicated for the treatment of gonor- veterinary drug residues, including aminoglycosides, rhoea when penicillin is inappropriate. Arbekacin has in food products of animal origin. They are used by been used to combat the increasing problems caused statutory agencies responsible for ensuring food by methicillin-resistant Staphylococcus aureus safety and for enforcing the regulations governing (MRSA) [14]. the use of drugs in food-producing animals. Mi- In veterinary medicine and animal husbandry, crobiological assays (Section 3.1) are preferred as aminoglycosides are widely used in the treatment of screening tests for residue analysis because they can bacterial infections, e.g. bacterial enteritis (scours) detect all of the different classes of antibiotics used and mastitis, and have been added to feeds for and are simple and inexpensive to perform. Gas prophylaxis and to promote growth [15,16]. No chromatography±mass spectrometry (GC±MS, see aminoglycosides are currently permitted for use as Section 3.10) and more commonly HPLC±MS (see growth promoters in the EU. Those most commonly Section 3.12) are the techniques employed for con- used as therapeutic agents are gentamicin, neomycin, ®rmation of veterinary drug residues. dihydrostreptomycin and streptomycin. Others may Analytical methodologies are applied to bulk include apramycin, hygromycin B, and spec- pharmaceuticals and their formulations for stability tinomycin. studies, quality control procedures, and for authenti- 76 D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93 cation purposes. In many such cases the analyte is the identi®cation of a range of aminoglycosides [40]. present in a simple matrix without interfering com- The authors suggested that the technique could be pounds. Simple non-speci®c spectrophotometric developed into a multiresidue method for the identi®- methods (see Section 3.9) are often the most appro- cation of aminoglycosides in food products of animal priate in these situations. origin. A combination of FAB-MS and NMR spectroscopy has been used to analyse residues of neomycin and its metabolites in calf kidney after oral 2. Qualitative methods for identi®cation/ dosing [41]. characterisation of aminoglycosides MS is well established as a powerful detection method when coupled with gas chromatography 2.1. X-ray crystallography (GC±MS) or liquid chromatography (LC±MS) (see Sections 3.10 and 3.12). Preparations of puri®ed aminoglycosides tend to form amorphous solids that are not amenable to X-ray diffraction studies. For this reason, the stereo- 3. Quantitative methods for the determination chemistry of most of the aminoglycosides has been of aminoglycosides derived by chemical and NMR spectroscopic methods. However, the structures of kanamycin A [18], 3.1. Microbiological assay streptomycin [19], spectinomycin [20], apramycin [21], kasugamycin [22], and fortimicin B [23] have Microbiological assays (bioassays) historically been con®rmed by X-ray studies. have been used for the assay of aminoglycosides and other antibiotics. They are the methods of choice 2.2. Nuclear magnetic resonance spectroscopy when an estimate of biological potency is required. Potency is estimated by comparing the inhibition of Proton- and13 C-NMR spectoscopic methods have growth of a sensitive microorganism produced by been particularly useful for determining the structural known concentrations of the antibiotic being ex- con®guration of aminoglycosides [8,24,25]. amined and a reference substance. Microbiological NMR spectroscopy has been used recently to assays are relatively inexpensive to perform, require study the interaction between aminoglycosides and little in the way of sophisticated equipment other RNA [26±30], yielding important information about than that used for handling the microorganisms, and the mechanism of action of these drugs on the are suitable for testing large numbers of samples. ribosome. Similar techniques have also been used to Microbiological assay is the method prescribed by look at the interaction between aminoglycoside- British, European and US pharmacopoeias for the modifying enzymes and their substrates [31±34]. determination of aminoglycosides in bulk pharma- Such studies provide a structural basis for the design ceuticals and their formulations. The of®cial assay of novel antimicrobial agents that are resistant to may be performed either by agar diffusion or tur- inactivation by these bacterial enzymes. bidimetrically [42,43]. These methods were recently tested for neomycin assay in a multicentre study 2.3. Mass spectrometry [44]. Of the six laboratories that returned results using the agar diffusion method, two failed to MS has also been applied to the structural analysis achieve the required precision and the accuracy of aminoglycosides [35±38] and their enzyme-modi- ranged from 96.9 to 115.3% of the target value for a ®ed products [8,32]. A recent study showed the neomycin B standard. Three laboratories returned utility of fast-atom bombardment (FAB) or electro- results from the turbidimetric method: all achieved spray ionisation (ESI) in combination with tandem acceptable precision and the accuracy varied from MS for determining the positional isomers of amino- 99.8 to 112.2% for the same standard. It was noted acyl derivatives of kanamycin A [39]. Californium- that the reproducibility of the microbiological assays 252 plasma desorption MS has also been applied to deteriorated as the content of neomycin C in the D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93 77 sample increased. Because of these problems, the aminoglycosides with detection limits of 0.08±0.3 authors recommended that the microbiological assay mg/ml and the methodology applied to the determi- of neomycin be replaced in of®cial monographs by a nation of residual drugs in bovine kidney [55]. liquid chromatographic method [45] (see Section Microbiological assays in a variety of formats are 3.12.1). also used as screening tests for the detection of Inaccuracy was also reported in the determination veterinary drug residues in foods of animal origin of tobramycin by agar diffusion assay when com- [16,56,57]. The procedure commonly used for pared with HPLC and enzyme-linked immunosorbent screening meat samples in the EU is the four-plate assay (ELISA) [46]. The microbiological technique test (FPT). The minimal inhibitory concentration overestimated the actual quantity of tobramycin in (MIC) of neomycin detectable in the FPT was 0.2 pure solutions, particularly at or near the detection mg/g tissue [16]. This method was used to screen limit of 5 mg/ml when the error was of the order of nearly 5000 meat samples from retail outlets in the 500%. Another disadvantage with the standard agar EU for the presence of antibiotic residues. Positive diffusion method is the length of time before the results were obtained in 2% of samples, most of results can be read (18±24 h). Incorporation of the which were con®rmed as containing tetracycline redox indicator triphenyltetrazolium chloride in the antibiotics, and no aminoglycoside residues were agar medium allowed measurement of the inhibition reported [58]. No signi®cant differences were found zones after 7-h incubation [47]. between monolayer and bilayer plates used in the Microbiological assays, when combined with suit- AOAC method for the analysis of antibiotic residues able extraction techniques, may be used for the in meat extracts and monolayer plates were rec- determination of aminoglycosides and other drugs in ommended as being more ef®cient [59]. Where animal feeds [48±50]. Aminoglycosides are very combinations of penicillin-G and an aminoglycoside hydrophilic compounds that do not bind strongly to such as neomycin or dihydrostreptomycin have been proteins in the matrix. They are easily extracted with used in animal husbandry, it may be useful to be able aqueous solutions from ®nely ground feeds. Spec- to determine the residue levels of the individual tinomycin was extracted from dry meal and pelleted drugs in food products. Simple modi®cation of an feeds using a tri¯uoroacetic acid solution (containing agar diffusion method, in which the sample was 40% methanol to increase wetting ef®ciency) with an incubated with penicillinase prior to assay, allowed average recovery of 98% [51]. The Association of various aminoglycosides to be quanti®ed in the Of®cial Analytical Chemists (AOAC) cylinder-plate presence of penicillin-G [60]. assay for neomycin has been the subject of two In therapeutic drug monitoring and phar- validation studies. The analyses showed considerable macokinetic investigations, microbiological assays variation in recovery, ranging from 69 to 129% of have been widely used to measure levels of amino- the target value [52]. The analytical performance was glycosides in human serum or plasma [6,61]. Al- not substantially improved by the use of more than though bioassays used to be popular for monitoring two replicate plates [53]. aminoglycosides during therapy, there has been a A single procedure has been developed to separate massive decline in their use over the past 20 years mixtures of antibiotics in animal feeds and to enable due to the introduction of the more convenient their identi®cation [54]. The system used a solvent enzyme-multiplied immunoassay technique (EMIT) pre-wash step to differentiate groups of antibiotics, (see Section 3.4.2) and more recently the ¯uores- agarose gel electrophoresis for their separation and cence polarisation immunoassay (FPIA) (see Section identi®cation, and bioautography using a self-con- 3.5) [62]. The current situation is illustrated by the tained modi®cation of the agar diffusion assay for fact that, for gentamicin, bioassay was used by only detection of the antibiotics. Limits of detection were two out of the 280 laboratories that participated in 0.25 mg/ml for hygromycin B, 8.0 mg/ml for the UK National External Quality Assessment neomycin, 1.0 mg/ml for streptomycin, and 32.0 Scheme (UK NEQAS) for Antibiotic Assays during mg/ml for spectinomycin. Electrophoresis±bioautog- 1997±98. The remainder employed immunoassays, raphy has been used similarly to determine seven with FPIA accounting for 84% of all returns [63]. 78 D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93

Agar diffusion assay was used for the determi- the surface of microbial cells that are added to the nation of isepamicin in plasma in a recent phar- sample. The amount of radiolabelled tracer binding macokinetic study [64]. Comparison with HPLC and to the receptor sites is measured using a dedicated radioimmunoassay (RIA) methods showed that re- analyser or liquid scintillation counter. The Charm II sults obtained with the microbiological assay were tests can detect gentamicin, streptomycins, and 20±30% lower than those by HPLC and RIA where- neomycin with good sensitivity, e.g. gentamicin at as the last two were in good agreement. Mi- 0.02 mg/ml in milk and 0.4 mg/g in tissue and crobiological assay suffered from non-speci®city streptomycin at 0.15 mg/g in tissue, ®sh and eggs which would be a problem in the case of combina- [56]. In a comparison with thin-layer chromatog- tion therapy using an aminoglycoside plus another raphy±bioautography for screening meat samples, antibiotic, or in the situation where active metabo- Charm II Tests were found to be faster, more lites of the drug are present. Microbiological assay convenient, and more sensitive [68]. also had the disadvantages of a narrow linear range (0.5±24 mg/ml isepamicin) and poor sensitivity 3.3. Radioimmunoassay (limit of quanti®cation 0.5 mg/ml). In a study to determine the effectiveness of experimental routes of RIA has been used successfully for the determi- drug delivery in the treatment of eye infections, nation of aminoglycosides in biological ¯uids [6,61]. bioassays were used to quantify tobramycin in The technique involves competition between free aqueous humour [65]. The optimal sensitivity of 0.24 drug in the sample and a ®xed amount of125 I- mg/ml tobramycin was obtained by applying the labelled drug (tracer), for a limited number of sample in wells rather than on paper discs, and by antibody binding sites. Separation of bound and free using thinly poured agar plates. Sensitivity was drug traditionally has been achieved by adding a found to be dependent on the ionic strength of the second antibody directed against the ®rst antibody to sample, and improved with increasing salt concen- precipitate the antigen±antibody complex. Following trations. It was suggested that the higher solute centrifugation and removal of the supernatant (free content reduced ionic interactions between the drug drug), the amount of radioactivity in the precipitate and the agar medium, thereby enhancing antibiotic is measured using a gamma counter. Most RIAs are diffusion. The concentration of cations in the agar now automated with separation assisted by the use of medium is thought to affect both the rate of diffusion antibody bound to a solid phase, commonly the of aminoglycosides and the growth of test organisms. polystyrene assay tubes or magnetic particles. In- Since agar is highly variable in its composition, this creasing the amount of free drug in the sample is likely to contribute to the reliability and accuracy causes less tracer to bind to the antibody and the problems with of this type of microbiological assay measured radioactivity to decrease. The methodology [66]. is highly sensitive and precise, but the equipment is costly and there are problems associated with hand- 3.2. Radiochemical assay ling radioactivity. Like all immunoassays, there may be cross-reactivity between different amino- The Charm II Tests (Charm, Malden, MA, USA) glycosides, and the technique depends on the availa- are a series of microbial receptor assays that are bility of a suitable antibody and tracer, or the widely used for the rapid detection and identi®cation expertise, facilities and time required to produce of b-lactam antibiotics, sulphonamides, tetracyclines, them. The RIA used to measure plasma isepamicin macrolides, aminoglycosides, novobiocin, and in a recent pharmacokinetic study was very sensitive chloramphenicol in milk [67]. A modi®cation of the (limit of quanti®cation 1 ng/ml), had a reasonable procedure may also be used for screening these drugs linearity range (100-fold), and was highly speci®c in other dairy products, meats, feeds, eggs and urine. (,0.4% cross-reactivity with gentamicin) [64]. By The methodology is based on competition between suitably varying the assay conditions, plasma the drugs in the sample and radiolabelled tracers for isepamicin concentrations could be determined up to speci®c receptor sites. The receptors are present on 72 h after the drug was administered. D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93 79

3.4. Enzyme immunoassay immunospeci®cally bound is determined by addition of a chromogenic enzyme substrate. In all competi- Enzyme immunoassays (EIAs) may be classi®ed tive ELISAs the intensity of the colour formed in the as heterogeneous (the enzyme-labelled antigen or ®nal step is inversely proportional to the concen- antibody is separated from the antigen±antibody tration of antigen in the sample. The antibody-cap- complex before the enzyme activity is measured in ture method has the advantage that a conjugated either fraction), or homogeneous (the enzyme activi- reagent speci®c to each assay is not required. ty of the labelled antigen is measured in the presence Non-competitive ELISAs usually involve binding of the antigen±antibody complex). of the antigen in the sample to an excess of immobilised antibody. The extent of binding is measured 3.4.1. Heterogeneous EIA with a second (enzyme-labelled) antibody directed Heterogeneous EIAs are best exempli®ed by against a different epitope. In this procedure, which ELISA. These assays are commonly performed as is also known as a sandwich ELISA or two-site quantitative tests using 96-well microtitre plates, but immunometric assay, the measured signal is directly rapid qualitative tests have also been developed that proportional to the amount of antigen in the sample. use test tube, dipstick and membrane (immuno®ltra- ELISAs that have been developed for drugs and tion) formats [56,69]. ELISAs may be competitive or other small-molecular-mass compounds are mostly non-competitive. Competitive assays may be de- of the competitive type because such molecules scribed as antigen-capture or antibody-capture de- generally possess only one antibody binding site. pending on whether the solid phase is coated with Examples of competitive ELISAs that have been antibody or antigen, respectively. In the former, developed for aminoglycosides are given in Table 2. competition occurs between antigen in the sample The gentamicin ELISA used to screen bacterial and tracer (enzyme-labelled antigen) for a limited fermentations for the production of aminoglycosides number of immobilised antibody binding sites. In the is typical of the standard antigen-capture technique latter, soluble antigen in the sample competes with [70]. The antibody employed as immunosorbent immobilised antigen for binding the primary anti- showed cross-reactivity with other aminoglycosides, body. In this case the tracer is an enzyme-labelled but this was a useful attribute for this application. An secondary antibody that is directed against the ELISA was reported for the determination of strep- primary antibody. The amount of tracer that is tomycins in milk in which a double antibody solid-

Table 2 Competitive microtitre plate ELISAs for aminoglycosidesa

Type Analyte Sample (pretreatment) Enzyme Substrate Sensitivity Ref. label

Antigen-capture Gentamicin Fermentation broth (none) ALP PNPP 10 pg/ml [70] Antigen-capture, Streptomycin, Milk (defatted, diluted 1/10) HRP TMB 6, 0.8 ng/ml respectively (milk) [71] DASP Dihydrostreptomycin Antigen-capture, Gentamicin Milk (defatted, diluted 1/10) or kidney HRP TMB 0.7 ng/ml (milk), 4 ng/g (kidney) [72] DASP Neomycin (deproteinised extract, diluted to 2% w/v) 3.6 ng/ml (milk), 25 ng/g (kidney) Streptomycin 5.1 ng/ml (milk), 28 ng/g (kidney)

Antigen-capture Streptomycin Honey (puri®ed by C18 solid-phase extraction HRP TMB 10 ng/g (honey) [74] and immunoaf®nity chromatography) Antibody-capture Streptomycin, DHS Milk (defatted) ALP PNPP 1.6 ng/ml (milk) [76] Antibody-capture Spectinomycin Liver, kidney (2.5% w/v aqueous extract) ALP PNPP 0.5 ng/ml (tissue extract) [79] Antibody-capture Gentamicin Human serum (diluted 1/1000) HRP OPDb 0.5 ng/ml (diluted serum) [77] Antibody-capture Spectinomycin Chicken plasma (diluted 1/20) b-Gal MUG 2 ng/ml (diluted plasma) [78] a Abbreviations: DASP, double antibody solid-phase; DHS, dihydrostreptomycin; ALP, alkaline phosphatase; HRP, horseradish peroxidase; b-Gal, b-galactosidase; PNPP, p-nitrophenyl phosphate; TMB, 3,39,5,59-tetramethylbenzidine; OPD, o-phenylenediamine; MUG, 4-methylumbelliferyl-b-galactoside. b This was not stated, but detection of the coloured product at 490 nm suggested that OPD was used as substrate. 80 D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93 phase (DASP) technique was used [71]. The DASP ter, NY, USA) were used to develop a competitive technique signi®cantly reduces the consumption of antibody-capture ELISA for streptomycins in milk speci®c antiserum since most of the reagent used to [76]. Small molecules in general adsorb very poorly coat a solid phase by non-speci®c adsorption remains to solid phases. This proprietary modi®cation, which unbound and is discarded. The assay was speci®c for results in a polystyrene surface with high af®nity for streptomycin and dihydrostreptomycin, and their polar groups, enabled dihydrostreptomycin to be recoveries from milk were generally .80%. Similar used directly as immunosorbent. Streptomycin and ELISAs have recently been developed for the de- dihydrostreptomycin gave similar responses in the tection of gentamicin, neomycin and streptomycin in test, but little (,2%) cross-reactivity with other milk and kidney [72]. Limits of detection for gen- aminoglycosides was observed. Reproducibility of tamicin, neomycin and streptomycin were far below this ELISA appeared to be good although the results the corresponding maximum residue levels (MRLs) were only reported semi-quantitatively. of 100, 500, and 200 mg/l in milk and 1000, 5000 Conjugating the antigen to a carrier protein has and 1000 mg/kg in kidney that are permitted in the also been used to facilitate its adsorption to the solid European Union [73]. The gentamicin ELISA phase. Care must be taken not to use the same carrier showed 25% cross-reactivity with sisomicin, which and cross-linking chemistry that was used during the has a closely related structure. Similarly, dihydro- production of the antiserum otherwise high back- streptomycin had high cross-reactivity (150%) in the ground signals are likely to be obtained. Examples of streptomycin ELISA. No other cross-reactions be- this strategy include ELISAs for gentamicin in tween aminoglycosides were detected in the three human serum [77] and spectinomycin in chicken assays. However, the assays were only suitable for plasma [78]. The gentamicin ELISA showed good screening purposes because of their inaccuracy (re- reproducibility (RSD,12%), accuracy (recovery coveries ranged from 47 to 78% in milk and 70 to varied from 91 to 106%) and correlated well with a 96% in kidney) and poor reproducibility (relative ¯uorescence polarisation immunassay technique. standard deviation, RSD, ranged from 23 to 60% for Cross-reactivity of the primary antiserum with other milk and 10 to 38% for kidney analyses). The assay aminoglycosides was less than 1%. Dilution of developed by Schnappinger et al. was recently serum by 1/1000 was necessary to reduce interfer- applied to the screening of streptomycin residues in ence by the sample matrix on the assay measure- honey [74]. The two-stage sample preparation pro- ments [77]. Performance of the spectinomycin cedure eliminated interference from the sample ELISA was similar, with RSD,12% and recovery of matrix with excellent recovery of streptomycin, and spectinomycin in spiked plasma varying from 97 to therefore allowed the required sensitivity to be 110%. However, interference from the sample matrix achieved. did not appear to be as high with this method, which A rapid test for streptomycins in milk has been may be due to the use of a ¯uorogenic enzyme developed using a related enzyme-linked immuno- substrate. It was observed that spectinomycin bound ®ltration assay (ELIFA) format [75]. This employed strongly to protein which prevented accurate mea- a nylon membrane as the support for a DASP similar surement of the total drug in the sample. This to that used in the ELISA described earlier [71]. problem was overcome by the incorporation of Subjective detection limits were 5 ng/ml for strep- suf®cient streptomycin in the assay buffer to saturate tomycin and 2 mg/l for dihydrostreptomycin. No the plasma protein drug binding sites [78]. No such cross-reactivity was observed with nine different dif®culties were reported during the development of aminoglycosides at high concentrations. This sensi- a spectinomycin ELISA for the analysis of residues tive test was simple to perform, could be completed in food animal tissues, in which a simple aqueous within 10 min, and because no equipment was extraction provided recoveries of 83±99% [79]. required it would be suitable for screening milk Heterogeneous, competitive ELISAs for gen- samples in the ®eld. tamicin and tobramycin have been developed by Microtitre plates with a specially modi®ed surface Bayer Diagnostics for the automated Technicon (Nunc-ImmunoE MaxiSorpE, Nalge Nunc, Roches- Immuno 1E system (Bayer, Tarrytown, NY, USA). D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93 81

The methodology involves the mixing of sample monitoring aminoglycosides [62]. The EMIT princi- (drug), tracer (drug±enzyme conjugate), and mono- ple involves competition between drug in the sample clonal anti-drug antibody that is labelled with and drug labelled with the enzyme glucose-6-phos- ¯uorescein. Antigen±antibody complexes are cap- phate dehydrogenase (G6PDH) for antibody binding tured on magnetic solid-phase particles coated with sites. Enzyme activity decreases when the labelled anti-¯uorescein antibodies, allowing the rapid sepa- drug is bound to the antibody because of steric ration of bound and free tracer. Bayer gentamicin hindrance and is inversely related to the concen- and tobramycin immunoassays are currently used by tration of drug in the sample. Enzyme activity is some clinical chemistry laboratories in the UK and, measured spectrophotometrically by the conversion although not as accurate as FPIA, appear to be of nicotinamide adenine dinucleotide (NAD) reliable [63]. coenzyme from the oxidised form (NAD1 ) to the A non-competitive sandwich ELISA in the form of reduced form (NADH) [81]. Reagent kits are avail- a dipstick test for gentamicin in milk has been able for amikacin, gentamicin, netilmicin and tobra- reported [80] despite theory suggesting that such mycin EMIT assays which may be used with the two-site immunoassays are not possible for small Viva automated dedicated EMIT analyser (Dade molecules. The test detected gentamicin at concen- Behring) or with automated clinical chemistry trations of 0.1 mg/ml or above and, because of its analysers from different manufacturers. Now largely simplicity, could be used for ®eld studies. superseded by FPIAs (see below), EMIT amino- A non-competitive EIA for tobramycin has recent- glycoside assays are still used by several clinical ly been reported which, although described as an chemistry laboratories in the UK for routine thera- ELISA, used a non-immunological coating reagent, peutic drug monitoring [62]. The EMIT gentamicin Alcian blue, to capture the antigen non-speci®cally assay was found to be rapid, very precise, reliable onto the microtitre plate surface [46]. Tobramycin and correlated well with FPIA and two other com- could be detected at 0.05 mg/ml with a working mercial automated immunoassays over the concen- range up to 6 mg/ml. The anti-tobramycin antibody tration range 0.3±16.3 mg/ml [82]. showed strong cross-reactivity with the closely re- Substrate-labelled immuno¯uorescent assay lated aminoglycoside, kanamycin. Results from the (SLIFA) is analogous to EMIT but in this case the analysis of pure kanamycin solutions were not as enzyme substrate generates a ¯uorescent product accurate as by HPLC but more accurate than by a [56]. Miles Labs. (Elkhart, IN, USA) developed microbiological assay. It was also reported that solid aminoglycoside SLIFAs for use with the Ames phase coated with Alcian blue could bind a wide Fluorostat [83] but it is not clear to what extent, if range of molecules including peptides and phos- any, these assays are still in use. pholipids. The tobramycin ELISA was not tested on samples with complex matrices, for example tissue 3.5. Fluoroimmunoassay extracts or plasma. It is doubtful whether this assay would be suitable for such applications since many Heterogeneous ¯uoroimmunoassay (FIA) is analo- molecules in the sample would compete with the gous to RIA except that the label is a ¯uorophore analyte for the non-speci®c binding sites. such as rhodamine or ¯uorescein rather than a radioisotope. Competitive FIAs for gentamicin and 3.4.2. Homogeneous EIA tobramycin are currently available in a multilayer Homogeneous EIAs are best exempli®ed by the dry-®lm format for use with the OpusE Plus auto- enzyme multiplied immunoassay technique (EMIT). mated immunoassay analyser (Dade Behring). The  EMIT assays were developed and ®rst introduced Opus multilayer ®lm system was originally de- in 1972 by Syva (now part of Dade Behring, veloped by PB Diagnostics and ®rst introduced in Deer®eld, IL, USA). This major breakthrough in 1990. It consists of a transparent polyester base on immunoassay technology was successfully applied to top of which are an agarose signal layer containing therapeutic drug monitoring in the late 1970s and immobilised monoclonal anti-drug antibody and caused a rapid decline in the use of bioassays for rhodamine-labelled drug (tracer), an iron oxide opti- 82 D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93 cal screen, a top coat containing buffers, detergents Basel, Switzerland) also developed FPIA methodolo- etc. and ®nally a grooved sample applicator [84]. gy for their Cobas FaraE II centrifugal clinical Sample spread over the top layer mixes with buffer chemistry analyser. Cobas-FpE assays had similar and surfactant, and low-molecular-mass components performance to Abbott TDx for the analysis of serum diffuse downwards through the iron oxide layer. amikacin and gentamicin, but were even more rapid When liquid meets the signal layer, dried antibody (1 min per test) and cost effective [86]. Sensitivity of and tracer are reconstituted and competition occurs the TDx FPIA for serum tobramycin (working range between drug from the sample and tracer for anti- 0.1±10.0 mg/ml) could theoretically be increased body binding sites. Tracer not bound by the im- 10-fold by using a reverse dilution technique. Nor- mobilised antibody diffuses upwards into the iron mal operation of the TDx system causes sample to be oxide layer which screens it from detection by the diluted during the automated procedure, but if buffer ¯uorimeter positioned below. The Opus assay for is replaced by sample in the predilution well of the serum gentamicin, although reliable and reproduc- instrument, dilution of the sample is avoided. Using ible, performed less well than two alternative auto- such a modi®cation it was possible to quantify serum mated immunoassays [82]. According to the UK tobramycin down to 0.025 mg/ml with good preci- NEQAS, the Opus assay is used by few laboratories sion and little interference, with the exception of and was the least accurate method for monitoring lipaemic samples which gave falsely elevated results gentamicin [63]. [87]. FPIA has been shown to be suitable for the Homogeneous FIA is exempli®ed by the ¯uores- analysis of other biological ¯uids. Performance of cence polarisation immunoassay (FPIA). Abbott the netilmicin TDx assay was found to be similar for  (Abbott Park, IL, USA) introduced the TDx system serum, peritoneal dialysate and urine samples and the in 1981 and rapidly became the market leader in results correlated well with an HPLC method [88]. In therapeutic drug monitoring. This batch analyser another study, FPIA using the TDx system was  system, and its updated version the TDxFLx (with found to be suitable for the measurement of amino- random access capability), uses FPIA methodology glycosides in cerebrospinal ¯uid [89]. Using a modi- for a wide range of analytes, including the amino- ®ed calibration procedure with the TDxFLx system, glycosides amikacin, gentamicin, netilmicin and another group were able to quantify very low tobramycin. The principle of FPIA is as follows. concentrations (down to 0.02 mg/ml) of tobramycin Sample (drug), tracer (¯uorescein-labelled drug), and in bronchoalveolar lavage ¯uid with good accuracy anti-drug antibody are incubated together in the and precision [90]. FPIA has also been used to reaction cell until competition for the limited number quantify gentamicin residues extracted from tissues of antibody binding sites reaches equilibrium (3 of food-producing animals using a one-step alkali min). Illumination of the reaction cell with vertically- digestion procedure [91]. The method provided good polarised light causes the ¯uorescein label to emit recovery and reproducibility (RSDs,10%) and a light at longer wavelength that is detected through sensitivity of 17 ng/g (ppb) for kidney tissue. another vertical polarising ®lter. Free tracer molecules rotate rapidly in solution; their emitted light is 3.6. Direct chemiluminescence immunoassay orientated in different planes from the incident light and is not detected. Tracer that is bound to antibody This is analogous to RIA, except the label is a has a much slower rotation; its emitted light is in chemiluminescent acridinium ester. Signal is gener- almost the same plane as the incident light and is ated by cleavage of the ester bond, releasing N- therefore detected. Increasing the concentration of methylacridone which decomposes with the emission drug in the sample limits the binding of tracer to the of an intense ¯ash of light [92]. The ACS:180, antibody and therefore causes a decrease in the launched in 1991 by Chiron Diagnostics (East measured ¯uorescence [85]. The Abbott TDx system Walpole, MA, USA and recently acquired by Bayer) has been widely used and evaluated for serum was the ®rst automated immunoassay system to use aminoglycoside analyses and found to be accurate, this methodology. Heterogeneous competitive assays precise (RSDs,5%), rapid and simple to use. Roche are available for amikacin, gentamicin and tobramy- Diagnostics (a division of Hoffmann-La Roche, cin and are currently used by some clinical chemistry D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93 83 laboratories for therapeutic drug monitoring. Sample FPIA [82]. It has been suggested that the latex (drug), drug±acridinium ester (tracer) and solid- agglutination reaction does not require bridging of phase antibody reagent are incubated together for the particles by divalent antibody to form a classical about 7 min. Following the separation step, in which immune complex. Colloidal stability of the analyte- the antibody-coated ferric oxide microparticles are modi®ed latex particles may instead be decreased by sedimented in a magnetic ®eld, the amount of speci®c binding of antibody causing agglutination antibody-bound tracer is measured upon addition of without cross-linking [94]. an oxidising reagent by photomultiplier detection of the ¯ash emission within 5 s. Sensitivity is at least as 3.8. Immunohistochemical techniques high as RIA and better than most other non-isotopic immunoassays [93]. In order to study the distribution of drugs in tissues, methods are required that can detect the 3.7. Nephelometric and turbidimetric analyte in situ. Such studies are particularly relevant immunoassays for drugs that are known to concentrate in speci®c organs causing damage to particular cells, for exam- Homogeneous immunoassays for proteins and ple the ototoxic and nephrotoxic effects of amino- other macromolecules have been developed using the glycosides. principle that large, multivalent antigens form insolu- Gentamicin was localised in inner ear ¯uid com- ble complexes with antibodies that can be detected partments using an immunohistochemical method by their optical properties. In nephelometry, a beam related to the heterogeneous non-competitive EIAs of light is shone into the sample and the amount of described above [95]. Cochlea tissue sections were light scattered by the immune complexes is measured placed onto nitrocellulose membranes and gen- at an angle from the beam. Turbidimetry is similar tamicin was adsorbed to the surface of the membrane except that in this case the amount of light trans- corresponding to its location in the tissue. Incubation mitted through the solution and not lost due to of the membrane with anti-gentamicin antibody adsorption, scattering or re¯ection is measured. followed by a secondary peroxidase-labelled anti- These techniques have been developed into competi- body and a chromogenic substrate allowed gen- tive assays for small analytes by using tracer that tamicin to be detected at concentrations down to 10 consists of latex particles coated with analyte mole- mg/ml. Alternatively, use of a chemiluminescent cules. Formation of insoluble complexes between substrate increased the sensitivity of detection 100- anti-analyte antibody and tracer is inhibited by the fold. presence of univalent analyte in the sample [92]. Tobramycin has been quanti®ed with extremely Examples of automated immunoassays currently high sensitivity in renal tubular epithelial cells of available for monitoring gentamicin and tobramycin kidney following an immunostaining technique [96]. using these methodologies are as follows. The Tissue sections were incubated with anti-tobramycin  Array rate nephelometric assay (Beckman Coulter, antiserum followed by a secondary antibody labelled Fullerton, CA, USA), the particle-enhanced tur- with colloidal gold. Detection of the gold label by bidimetric inhibition immunoassay (PETINIA) for photo-thermal microscopy allowed the quanti®cation  the Dimension system (Dade Behring, Newark, DE, of tobramycin in individual cells down to 10 zmol USA), and the latex agglutination (LA) assay for the (10221 mol). Technicon Immuno 1E system (Bayer). The Dimen- sion assay for serum gentamicin was very precise, 3.9. Spectrophotometric and other non-separative linear to 12 mg/ml, had a sensitivity of 0.3 mg/ml, physicochemical methods and correlated well with FPIA [94]. The antibody reagent was derived from a monoclonal anti-gen- Simple, non-speci®c methods may be appropriate tamicin antibody that had minimal cross-reactivity when there is a requirement for the assay of amino- with structurally related aminoglycosides. The Tech- glycosides in the absence of interfering compounds, nicon latex agglutination assay was reported to have for example the quality control of bulk and formu- similar performance and gave comparable results to lated pharmaceuticals, and dissolution studies. 84 D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93

Aminoglycosides absorb light poorly in the UV± extract was puri®ed sequentially on Amberlite XAD- Visible range and various indirect spectrophotomet- 2 and activated carbon columns, then derivatised ric methods have been developed using different with a 1:1:1 mixture of N,O-bis(trimethylsilyl)-acet- derivatisation reagents giving limits of detection of amide (BSA), 1-(trimethylsilyl)-imidazole (TMSI), around 1 mg/ml [97±103]. Streptomycin, however, and trimethylchlorosilane (TMCS). Recoveries for have been determined directly with comparable aminoglycosides from meat ranged from 82 to 94% sensitivity using derivative spectroscopy [104,105]. and the limits of detection were 25±50 mg/kg using Aminoglycosides have been determined by stop- a ¯ame ionisation detector. ped-¯ow ¯uorimetry using o-phthaldialdehyde as A chemometric experimental design has been used derivatisation reagent, with a sensitivity of 0.02 mg/ to optimise the derivatisation of gentamicin and ml [106]. The interaction of lanthanide ions (Eu31 ) kanamycin prior to analysis by GC±MS [112]. In with aminoglycosides has also been used as a basis this case the aminoglycosides were derivatised using for their ¯uorimetric determination at levels of 5± a two-step procedure involving trimethylsilylation of 100 mg/ml [107]. the hydroxyl groups with TMSI and acylation of the Kanamycin was determined by its inhibitory effect amino groups with N-(hepta¯uorobutyryl)-imidazole on the chemiluminescent reaction between lucigenin (HFBI). Following liquid±liquid extraction the de- and hydrogen peroxide in basic solution [108]. rivatives were analysed by capillary GC±MS using Precise determinations were possible over a very on-column injection and the detector in electron wide linear dynamic range (0.06 pg/ml±6 mg/ml) ionisation mode. Gentamicin was separated into using a ¯ow-injection system. Flow-injection chemi- three major components (C11a , C and C 2 ). Speci®c luminescence has also been used to measure strep- fragment ions were selected for monitoring each tomycin at levels of 2±30 mg/ml [109]. The method analyte because the limited range of the MS system was based on the chemiluminogenic oxidation of prevented detection of the molecular ions. This streptomycin by N-bromosuccinimide in alkaline resulted in a substantial loss in sensitivity compared solution. to electron-capture detection, although the actual Titration with tetrabutylammonium hydroxide or sensitivities were not reported. The method showed barium acetate has also been reported for the assay good linearity between 30 and 200 mg gentamicin of netilmicin in acetic acid solution [110,111]. and 10 and 200 mg kanamycin.

3.10. Gas chromatography 3.11. Thin-layer chromatography

Few GC methods have been developed for the TLC, or planar chromatography, has been used as analysis of aminoglycosides [15,61,112,113]. These a qualitative method for the identi®cation of amino- compounds are non-volatile and require lengthy glycosides and for routine quality testing. Examples derivatisations at elevated temperatures with silylat- are the methods prescribed by the British and ing agents to make them amenable to analysis. GC is European Pharmacopoeias for the identi®cation and the prescribed method of the US Pharmacopoeia for quality control of kanamycin and neomycin the assay of spectinomycin and a modi®cation of this [116,117]. has been reported [114]. Spectinomycin was deriva- Advances in TLC technology, including automa- tised with hexamethyldisilazane then analysed by tion and the application of scanning densitometry, packed-column GC with ¯ame ionisation detection. have occurred during the past decade which have The method was linear over the concentration range increased the speed and reliability of this technique 75±125% and the limit of quanti®cation for the and extended its use to quantitative determinations. determination of impurities was 0.1%. Amino- Many of these advances in TLC instrumentation glycosides and a range of other drug residues in meat have been introduced by Camag (Muttenz, Switzer- were simultaneously determined by a packed-column land). High-performance thin-layer chromatography GC method [115]. Meat was homogenised and (HPTLC) is a term that has been introduced to deproteinised with trichloroacetic acid. The crude describe the improved sensitivity, resolution and D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93 85 accuracy that may be obtained using thinner layers tion of netilmicin at concentrations down to 0.2 (100±200 mm) of stationary phases with smaller (e.g. mg/ml [124]. Post-chromatographic reaction with a 5 mm), more uniform particle sizes comparable to mixture of diphenylborinic anhydride and salicylal- those used in HPLC. dehyde produced a highly ¯uorescent netilmicin A mixture of nine aminoglycosides was almost derivative. The assay had good precision (RSD,5%) fully separated by HPTLC [118]. Gentamicin was but a limited linear range of 1±5 mg/ml. Comparison partially resolved into three peaks (C1a , C 2 and C 1 ) with FPIA revealed a reasonably good correlation, and impurities in a netilmicin preparation were also although the HPTLC method was less sensitive and detected by varying the composition of the solvent subject to greater interference by other drugs and system. Detection limits ranged from 4 to 100 ng per also heparin. In another study, hygromycin B was spot using a ninhydrin reagent and scanning den- adsorbed and concentrated from deproteinised bovine sitometry. Neomycin A, B and C were quanti®ed in plasma by solid-phase extraction on silica gel co- pharmaceuticals by HPTLC [119]. Fluoroden- polymerised with C8 and benzene sulphonic acid sitometry of the ¯uorescamine derivatives gave a functional groups prior to TLC [125]. Hygromycin B detection limit of 20 ng per zone. The relative was quanti®ed by ¯uorodensitometry following de- amounts of neomycins B and C in commercial rivatisation with ¯uorescamine. The lowest amount samples were determined in a similar study [120]. of hygromycin B detectable was 5 ng per spot, Detection was performed after derivatisation with giving a limit of detection in plasma of 25 ng/ml, 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). which is similar to some immunoassays. This meth- Sensitivity for the assay of neomycin C content was od was further developed for the analysis of hy- about 1% (0.04 mg for an application of 4 mg sample gromycin B in plasma, serum or milk [126]. The to the plate). HPTLC was used to determine same copolymeric solid-phase extraction columns amikacin in parenteral dosage forms [121]. Quanti®- were used to extract the drug from the biological cation of the ninhydrin derivative at 498 nm was ¯uid prior to further puri®cation by af®nity chroma- very precise (RSD,2%) and was linear over the tography on agarose±lysozyme columns. Lysozyme, range 0.5±3 ng/ml. In a comparative study of which normally catalyses hydrolysis of (b1→4) methods for the determination of gentamicin in glycosidic bonds, presumably binds aminoglycosides pharmaceuticals, an automated HPTLC procedure at the active site without hydrolysis taking place. using ninhydrin derivatisation was found to be faster Af®nity chromatography proved useful in reducing (total analysis time of 45 min compared with 100 the amount of ¯uorescent derivatives from plasma min) but 10 times less sensitive than HPLC [122]. that interfered in the TLC analysis, but gave incom- TLC has been tried in the past as an alternative to plete recovery of hygromycin B from milk. microbiological assay for the determination of aminoglycosides in biological samples, but its sen- 3.12. High-performance liquid chromatography sitivity was comparatively poor [113]. Gentamicin has been recently quanti®ed in plasma and urine by HPLC, because of the speci®city and sensitivity HPTLC [123]. Fluorodensitometry of the gentamicin that may be achieved, is the analytical technique NBD-Cl derivatives gave a linear quanti®cation most used in pharmacokinetic [127] and drug stabili- range of 40±200 ng per spot, which equated to ty studies. When coupled with mass spectrometry 20±100 mg/ml in plasma and 10±50 mg/ml in urine. (MS), it becomes the most powerful method for the This is above the therapeutic range for gentamicin of con®rmation of antibiotic residues in food [113,128± 5±12 mg/ml [7] and the method would not be 131]. The literature contains a vast number of HPLC suitable for monitoring plasma levels without modi- methods for the analysis of aminoglycosides and ®cation. other antibiotics. Several conclusions may be drawn Sensitivity can be improved by pre-treatment of from this fact. HPLC methods are relatively easy to the sample prior to TLC analysis. Thus, solid-phase develop and are very suitable for the analysis of extraction of serum on C18 cartridges followed by small-molecular-mass compounds. Some of the pub- HPTLC and ¯uorodensitometry allowed the detec- lished methods, however, are not robust and are 86 D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93 therefore not transferable to different laboratories available spectrophotometric detectors. This ap- without modi®cation. Continual development of proach has been widely adopted using o-phthaldial- HPLC as an analytical technique has resulted in a dehyde (OPA) [64,135,136,144±147,151], 1,2-naph- wide range of columns from different manufacturers thoquinone-4-sulphonic acid (NQS) [138,140,143, exhibiting varying selectivities. Separations using 148] and ninhydrin [142] as derivatisation reagents. different column types have been combined with various detection systems, resulting in the publi- 3.12.2. Reversed-phase HPLC cation of a large number of methods. Advances in An alternative strategy is to use pre-column sample preparation procedures, particularly the intro- derivatisation, which has several advantages duction of disposable solid-phase extraction (SPE) [152,153]. The derivatives formed are invariably less columns, have also been reported and have greatly polar than the parent aminoglycosides and are amen- improved the extraction of antibiotics from biomat- able to separation by the preferred technique of rices [132]. reversed-phase HPLC [154±161] (see Table 3 for Analysis of aminoglycosides by HPLC has been details). No extra pump, mixing apparatus and dealt with in a number of reviews, which adequately reaction coil are required for the derivatisation cover the literature up to 1995 [15,61,113,133,134]. reagent. Sensitivity is higher because derivatisation The present work will review those methods that is complete and the chromatographic peak is not have been developed and applications reported since diluted or chromatographically degraded prior to then. detection. It has also been suggested that the pre- HPLC methods may be subdivided by the type of column derivatisation technique is more likely to separation (normal-phase, reversed-phase, ion-pair, remove interfering substances [153]. Pre-column or ion-exchange) and by the mode of detection derivatisation with OPA plus 2-mercaptoethanol is (ultraviolet (UV) absorption, ¯uorescence, electro- not recommended because the derivatives formed are chemical or MS detection). not stable. Stability may be improved by using 2- mercaptopropionic acid as the thiol [134]. More 3.12.1. Ion-exchange and ion-pair HPLC suitable reagents that have been used for pre-column Aminoglycosides are polybasic cations at low pH derivatisation of aminoglycosides include 1-¯uoro- and have either been separated on strong cation- 2,4-dinitrobenzene (DNFB) [154,157] and 2,4,6-tri- exchange (SCX) columns [135±137] or more com- nitrobenzene-1-sulphonic acid (TNBS) [155] for UV monly by ion-pair HPLC, typically on C reversed- 18 detection, and 9-¯uorenylmethyl chloroformate phase columns using alkylsulphonate ion-pair re- (FMOC-Cl) [156,158,159] which is ¯uorescent. agents [45,64,138±148]. Details of these methods are given in Table 3. Ion-pair is generally preferred to ion-exchange HPLC because of the higher ef®cien- 3.12.3. Sample preparation for HPLC analysis cies obtainable, easier control over selectivity and Biological samples contain many matrix compo- resolution, and greater stability and reproducibility of nents that may interfere with the HPLC analysis of reversed-phase columns [149]. Aminoglycosides lack aminoglycosides. The aim of sample preparation is chromophores and are therefore not amenable to to extract, and where necessary to concentrate, the direct UV or ¯uorescence detection. They may be analyte from the matrix, leaving behind proteins and analysed using electrochemical [150] or MS other components that may interfere with subsequent [128,129] detectors. A volatile mobile phase is derivatisation, separation, and detection steps. In required for MS detection, which may be achieved order to minimise the potential for interference it is using ion-exchange with per¯uorinated carboxylic sensible to select different principles for the ex- acids and ammonium salts, or ion-pairing with a traction and analytical separations, for example ion- volatile reagent such as hepta¯uorobutyric acid exchange extraction followed by reversed-phase [113,137,139]. Aminoglycosides may be derivatised HPLC. after separation with UV absorbent or ¯uorescent The use of SPE columns and cartridges has greatly agents, which allows analysis with more generally increased the convenience and performance of sam- D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93 87

Table 3 Recent HPLC methods for the analysis of aminoglycosidesa

Type Analyte Detection, Column Mobile phase Sensitivity Ref. derivatisation (5-mm unless stated)

IE Spectinomycin Fl. 340/460 nm, Spherisorb SCX, Potassium phosphate (0.15 M), pH 3.5±acetonitrile (80:20) 70 ng/ml [135] Post-col. OPA±ME 25034.6 mm (tissue extract)

IE Spectinomycin Fl. 340/460 nm, Spherisorb SCX, Sodium sulphate (0.1 M) pH 2.6±acetonitrile (80:20) 100 ng/ml [136] Post-col. OPA±ME 25034.6 mm (plasma extract)

IE Spectinomycin Fl. 340/455 nm, IonoSpher 5C, Sodium sulphate (0.05±0.19 M gradient)±acetonitrile (80:20) 100 ng/ml [137] Post-col. OPA±ME 15034.6 mm (tissue extract)

IP Neomycin Pulsed electrochemical 8-mm PLRP-S 1000A,Ê 1-Octanesulphonate (6.5 mM), sodium sulphate (0.49 M), 750 ng/ml [45] 25034.6 mm potassium phosphate (10 mM, pH 3) (aqueous soln.)

IP Gentamicin Electrochemical- or Symmetry C18 , Tri¯uoroacetic acid (0.11 M, pH 3.6)±acetonitrile ND [141] thermospray-MS 15033.9 mm (97:3±80:20 gradient)

IP Spectinomycin Electrospray-MS±MS PLRP-S, Hepta¯uorobutyric acid (8 mM) in 125±250 ng/ml [139] 15032.1 mm water±methanol (60:40) (milk extract)

IP DHS Fl. 270/420 nm, Supelcosil ABZ1 Plus, 1-Octanesulphonate (40 mM), NQS (0.4 mM), pH 3.2± 7 ng/ml [138] Post-col. NQS 15034.6 mm acetonitrile (68:32) (milk extract)

IP Streptomycin Fl. 260/435 nm, 3-mm Hypersil BDS, 1-Heptanesulphonate (10 mM), NQS (0.4 mM) in 20% 50 ng/ml [140] Post-col. NQS 10034 mm acetonitrile, pH 3.3±acetonitrile (97:3) (milk extract)

IP DHS Fl. 375/420 nm, Supelcosil ABZ1 Plus, 1-Octanesulphonate (40 mM), NQS (0.4 mM), pH 3.2 400 ng/ml [143] Post-col. NQS 15034.6 mm ±acetonitrile (68:32) (tissue extract)

IP Streptomycin, Fl. 263/435 nm, 4-mm Superspher RP 1-Hexanesulphonate (10 mM), NQS (3.5 mM),triethylamine 120 ng/ml [148] DHS Post-col. NQS select B, 12533 mm (0.15%), acetic acid (0.9%), pH 3.3±acetonitrile (88:12) (milk extract)

IP DHS Fl. 305/500 nm, Supelcosil LC-ABZ, 1-Octanesulphonate (40 mM), 1,2-ethanedisulphonate (20 mM), 375 ng/ml [142] Post-col. ninhydrin 15034.6 mm ninhydrin (5 mM), pH 3.2±triethylamine (0.3%) in (milk extract) acetonitrile±methanol (63:19:18)

IP Gentamicin Fl. 340/430 nm, Spherisorb ODS2, 1-Pentanesulphonate (11 mM), sodium sulphate (5.6 mM), 54 ng/ml [144] Post-col. OPA±ME 15034.6 mm acetic acid (0.1%) in water±methanol (82:18) (milk extract)

IP Isepamicin Fl. 338/450 nm, Hypersil ODS, 1-Hexanesulphonate (10 mM), sodium acetate (0.1 M), 100 ng/ml (plasma) [64] Post-col. OPA±ME 15034.6 mm acetic acid (17 mM) in water±methanol (86:14)

IP Paromomycin Fl. 340/455 nm, Kromasil C18 , 1-Pentanesulphonate (20 mM), sodium sulphate (0.2 M), ND [145] Post-col. OPA±ME 25034.6 mm pH 6.8±8.5 gradient

IP Various Fl. 355/415 nm, TSK ODS 120T, 1-Pentanesulphonate (10 mM), sodium sulphate (15 mM), 150±220 ng/ml [146] Post-col. OPA±ME 15034.6 mm acetic acid (10 mM)±tetrahydrofuran (97:3) (feed extract)

IP Paromomycin Fl. 340/440 nm, Inertsil C8 , 1-Heptanesulphonate (1.2 mM), sodium sulphate (0.2 M), 500 ng/ml [147] Post-col. OPA±ME 25034.5 mm acetic acid (0.1%) (aqueous soln.)

RP Spectinomycin APCI-MS±MS Zorbax SB-C18 , Acetic acid (1%)±methanol (92:8) 50 ng/ml [137] 15032.1 mm (tissue extract)

RP Paromomycin UV 350 nm, Zorbax SB-C18 , Water, pH 3±methanol (36:64) 150 ng/ml [154] Pre-col. DNFB 25034.6 mm (plasma extract)

RP Tobramycin UV 365 nm, 4-mm Nova-Pak C18 , Tris (17 mM), sulphuric acid (20 mM)±acetonitrile (45:55) 3 mg/ml [157] Pre-col. DNFB 15033.9 mm (aqueous soln.)

RP Amikacin UV 350 nm, Zorbax SB-C8 , Potassium phosphate (20 mM)±acetonitrile±methanol ND [155] Pre-col. TNBS 15034.6 mm (45:41:14), pH 7.7 88 D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93

Table 3. Continued

Type Analyte Detection, Column Mobile phase Sensitivity Ref. derivatisation (5-mm unless stated)

RP Neomycin Fl. 260/315 nm, Supelcosil LC-304 Sodium phosphate (11 mM), pH 5±acetonitrile (37:63) 170 ng/ml [156]

Pre-col. FMOC-Cl (C4 ), 25034.6 mm (muscle extract) RP Various Fl. 260/315 nm, 3-mm Hypersil ODS, Water±acetonitrile (10:90) 10 ng/ml [158,159] Pre-col. FMOC-Cl 20034.6 mm (aqueous soln.)

RP Netilmicin Fl. 337/437 nm, 10-mm LiChrosorb Acetic acid (10%), 1-heptanesulphonate (10 mM)±acetonitrile 7 ng/ml (dilute [151] Pre-col. OPA±ME RP18, 30034 mm (60:40±50:50 gradient) deprot. plasma)

RP Apramycin Fl. 230/389 nm, Nova-Pak C18 , Ammonium acetate or 1-octanesulphonate (5 mM) in 23 ng/ml (dilute [160] Pre-col. OPA±ME 30033.9 mm water±acetic acid±acetonitrile (60:2:40) tissue extract) a Abbreviations: IE, ion-exchange; IP, ion-pair; RP, reversed-phase; DHS, dihydrostreptomycin; Post-col., post-column; Pre-col., pre-column; APCI, atmospheric pressure chemical ionisation; OPA±ME, o-phthaldialdehyde±2-mercaptoethanol; NQS, 1,2-naphthoquinone- 4-sulphonate; DNFB, 1-¯uoro-2,4-dinitrobenzene; TNBS, 2,4,6-trinitrobenzene-1-sulphonic acid; FMOC-Cl, 9-¯uorenylmethyl chloroformate; Tris, Tris(hydroxymethyl)aminomethane; ND, not determined; deprot., deproteinised. ple preparation for HPLC analysis. Aminoglycosides 3.13. Capillary electrophoresis are retained most successfully on weak cation-ex- changers, or by ion-pairing on reversed-phase media Capillary electrophoresis (CE) is a general term [113,130±132]. Table 4 gives details of some recent covering the various electrophoretically driven capil- pre-treatment procedures that have been used for lary separation techniques of capillary zone electro- aminoglycosides in plasma and various food prod- phoresis (CZE), capillary isotachophoresis (CITP), ucts. capillary isoelectric focusing (CIEF), capillary gel A column-switching arrangement was developed electrophoresis (CGE), micellar electrokinetic capil- that allowed the extraction step to be performed lary chromatography (MECC) and capillary electro- automatically on-line [64]. Isepamicin was extracted chromatography (CEC). CZE and MECC in par- from deproteinised plasma on a guard column con- ticular are well established techniques that have been taining normal-phase (cyano-bonded) silica then applied to the analysis of a wide range of biological subjected to ion-pair chromatography on a reversed- substances with the advantages of very high res- phase (C18 ) analytical column. A minimum of 60 olution and minute sample volume requirement. plasma samples could be processed in this way Little has been published on the analysis of amino- before replacement of the guard column packing was glycosides using CE however, the main reason for required. this being the dif®culty in detecting these compounds Future developments in the HPLC analysis of by conventional spectrophotometric means [163]. aminoglycosides may include greater use of re- The problems of aminoglycoside detection discussed stricted access, or shielded, stationary phases for the earlier (Section 3.12.1) are even more severe for CE direct injection of serum and other biological sam- because the very short path length used in CE ples. This column technology has been developed for spectrophotometric detectors decreases the concen- the analysis of drugs in serum eliminating the need tration sensitivity that may be achieved. for laborious deproteinisation steps. A shielded hy- Sensitivity is not a problem for the analysis of drophobic phase column was used in the analysis of bulk pharmaceuticals and their formulations where neomycin in milk [162]. The sample, however, had the concentrations of aminoglycosides are in the ®rst been extracted on a weak cation-exchange 1±250 mg/ml range. Aminoglycosides were ®rst column, which would be expected to remove pro- determined by CZE using indirect UV detection with teins and fats. It is therefore not clear what advan- imidazole as background electrolyte [164]. Thirteen tage was gained by using a restricted access medium different aminoglycosides were analysed in the for the analytical separation in this case. anionic mode (anode at the detection side) with D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93 89

Table 4 Sample preparation for HPLC analysis of aminoglycosidesa

Matrix Analyte Pre-treatment Solid-phase extraction Recovery (%) Ref.

Plasma Spectinomycin Dilute, pH→5.5, centrifuge Ion-pair (dioctyl sulphosuccinate) on high 91±104 [136]

hydrophobic C18 (3 ml; J.T.Baker) Plasma Paromomycin Deprot. (PCA), centrifuge, pH→7.8, deriv. (DNFB), None 87 [154] LLE (toluene/acetonitrile)

Plasma Netilmicin Deprot. (TCA), centrifuge, pH→11, LLE None ND [151] (dichloromethane), deriv. (OPA), LLE (2-propanol)

Plasma Various None Ion-exchange on CBA (Isolute, 3 ml, 100 mg; 94±107 [158,159] International Sorbent Technology)

Tissue DHS Homogenise, deprot. (PCA), centrifuge, LLE Ion-exchange on SCX (3 ml, 500 mg; Varian) 67±83 [138] (dichloromethane)

Tissue Spectinomycin Homogenise, deprot. (TCA), centrifuge, ®lter, pH→6.7 Ion-exchange on CBA (3 ml, 500 mg; J.T.Baker) 74±97 [135]

Tissue Spectinomycin Homogenise, deprot. (TCA), LLE (dichloromethane), Ion-exchange on CBA (no details available) 81±94 [137] centrifuge, pH→6.7, centrifuge

Tissue Streptomycin Homogenise, deprot. (PCA), centrifuge, ®lter (1) Ion-exchange on SCX (200 mg; Appl. Sep.) 81 [140]

(2) Ion-pair (1-heptanesulphonate) on C18 (500 mg; J.T.Baker)

Tissue DHS Homogenise, deprot. (TCA), centrifuge, LLE Ion-pair (1-heptanesulphonate) on mixed-mode SAX/C8 73±83 [143] (dichloromethane), homogenise, centrifuge, pH→5.5 (Certify II, 6 ml, 500 mg; Varian)

Tissue Neomycin Homogenise, alkali digest, deprot. (PCA), pH→5 Ion-exchange on CBA (AccellE Plus, 6 ml, 1 g,; Waters) 78±120 [156]

Tissue Apramycin Homogenise, LE (alkali/methanol), centrifuge, ion-pair None 76±86 [160] LLE (ethyl acetate), LLE (dil. HCl), pH↑, LLE (toluene)

Tissue Gentamicin Homogenise, centrifuge, ®lter Ion-exchange on mixed-mode CBA/C18 (300 mg; .90 [161] Chemical Separations)

Milk DHS Deprot. (TCA), ®lter, pH→3.2, ultra®lter None 90±97 [138]

Milk Spectinomycin Deprot. (TCA), centrifuge, dilute Ion-pair (hepta¯uorobutyric acid) on trifunctional 69±93 [139]

C18 (Sep-Pak Vac, 3 ml; Waters) Milk Streptomycin None (1) Ion-exchange on SCX (200 mg; Appl. Sep.) 88 [140]

(2) Ion-pair (1-heptanesulphonate) on C18 (500 mg; J.T.Baker)

Milk DHS Deprot. (TCA), centrifuge, LLE (dichloromethane), Ion-pair (1-heptanesulphonate) on trifunctional 83 [142] → neutralise, centrifuge, pH 6 C18 (Sep-Pak Vac, 6 ml, 1 g; Waters)

Milk Gentamicin Defat (centrifuge), deprot. (TCA), centrifuge Reversed-phase on C18 (6 ml, 500 mg; Supelco) 72±88 [144]

Milk DHS, Defat (oxalic acid), centrifuge, deprot. (TCA), Ion-pair (1-heptanesulphonate) on C18 Streptomycin centrifuge, pH↑, centrifuge (Chromabond ec, 500 mg; Macherey-Nagel) 78±107 [148]

Milk Gentamicin None Ion-exchange on mixed-mode CBA/C18 .90 [161] (300 mg; Chemical Separations) a Abbreviations: DHS, dihydrostreptomycin; deprot., deproteinise; LLE, liquid±liquid extraction; LE, liquid extraction; OPA, o- phthaldialdehyde; PCA, perchloric acid; TCA, trichloroacetic acid; CBA, carboxylic acid-bonded weak cation-exchanger; SCX, strong cation-exchanger; ND, not determined.

electroosmotic ¯ow reversed by the addition of the rated under optimal conditions (pH 5). Addition of surfactant FC 135. Although the analyte peaks were cetyltrimethylammonium bromide (CTAB) to the very sharp, not all the components could be sepa- electrolyte allowed the simultaneous determination 90 D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93 of neomycin and hydrocortisone in eardrops by foresee major changes in this area. Further develop- combined CZE±MECC. Limits of detection were ment and commercialisation of simple visual im- less than 50 mg/ml for aminoglycosides. munoassay tests is desirable to facilitate the screen- Borate complexation was used for the direct UV ing of aminoglycoside residues in milk and animal detection of aminoglycosides in a CZE method tissues on the farm and at slaughter. Microbiological [165]. Twelve different aminoglycosides were almost assays however, because of their non-speci®city and completely separated by CZE in the cationic mode inaccuracy, cannot be recommended for analytical (cathode at the detection side) at pH 9. The method use when alternative methods of far superior per- showed excellent precision (RSD53%) using inter- formance are available. There is potential for further nal standardisation and the limit of quanti®cation development of chromatographic and CE methods was again in the region of 50 mg/ml. for the analysis of biological samples. In particular, Derivatisation of aminoglycosides to allow more greater use of restricted access columns for HPLC sensitive direct UV or ¯uorescence detection has also that allow direct injection of serum and other bio- been reported in a MECC method [166]. Amikacin logical samples may be anticipated. As MS detection in plasma ultra®ltrate was derivatised with 1-methox- becomes more widely available and its quantitative y-carbonylindolizine-3,5-dicarbaldehyde. The deriva- performance improves, this will probably replace tive was separated by MECC at pH 7 using so- spectrophotometric and electrochemical detection in diumdodecyl sulphate (SDS) as micellar reagent and many applications. Perhaps the greatest scope for detected by ¯uorescence. Sensitivity of this method development in this direction will be in CE±MS. was about 100-fold greater than for the non-derivatisation methods above, but the ability to separate different aminoglycosides appeared to be lost. Thus, amikacin, arbekacin, dibekacin and kanamycin de- Acknowledgements rivatives had almost identical migration times by MECC, whereas the parent aminoglycosides were Thanks are expressed to Dr. D.G. Durham and The well separated by the CZE methods described above. Robert Gordon University for supporting my position Electrochemical detection has been used with during the production of this article, and to Dr. A. some success in the analysis of aminoglycosides by Stead and Professor R.B. Taylor for their helpful HPLC (see Section 3.12.1) and its suitability for use comments on the manuscript. in CE has also been demonstrated. Seven aminoglycosides were separated by CZE in the cationic mode at high pH (100 mM NaOH as electrolyte) and detected down to 0.2±2 mg/ml using a nickel References electrode in wall-jet con®guration [167]. Similar CZE conditions were employed for the separation of [1] H. Umezawa, I.R. Hooper (Eds.), Aminoglycoside Anti- biotics, Springer±Verlag, Berlin, 1982. a different mixture of seven aminoglycosides, but in [2] I.R. Hooper, in: H. Umezawa, I.R. Hooper (Eds.), Amino- this case detection was achieved below 5 mg/ml with glycoside Antibiotics, Springer±Verlag, Berlin, 1982, p. 1. greater stability, reproducibility and simplicity using [3] T. Okuda, Y. Ito, in: H. Umezawa, I.R. Hooper (Eds.), a copper on-capillary electrode [168]. Aminoglycoside Antibiotics, Springer±Verlag, Berlin, 1982, p. 111. [4] H. Umezawa, T. Tsuchiya, in: H. Umezawa, I.R. Hooper (Eds.), Aminoglycoside Antibiotics, Springer±Verlag, Berlin, 4. Conclusions 1982, p. 37. [5] N. Tanaka, in: H. Umezawa, I.R. Hooper (Eds.), Amino- Despite the introduction of newer, less toxic glycoside Antibiotics, Springer±Verlag, Berlin, 1982, p. 221. antimicrobials, aminoglycosides continue to serve a [6] T. Koeda, K. Umemura, M. Yokota, in: H. Umezawa, I.R. Hooper (Eds.), Aminoglycoside Antibiotics, Springer±Ver- useful role as therapeutic agents. The ongoing need lag, Berlin, 1982, p. 293. for routine therapeutic drug monitoring is adequately [7] C.A. Hammett-Stabler, T. Johns, Clin. Chem. 44 (1998) met by automated immunoassays and it is dif®cult to 1129. D.A. Stead / J. Chromatogr. B 747 (2000) 69 ±93 91

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