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DRUGS, COSMETICS, FORENSIC SCIENCES

Chromatographic Methods for Analysis of AminoglycosideAntibiotics

ISOHERRANEN& SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999NINA ISOHERRANEN

University of Helsinki, Department of Chemistry, Laboratory of Analytical Chemistry, PO Box 55, Helsinki 00014, FinlandSTEFAN SOBACK

1

Kimron Veterinary Institute, National Residue Control Laboratory, PO Box 12, Beit Dagan, Israel

Aminoglycosides are antimicrobial agents usedfrequently in treatment of human and animal dis-eases caused by aerobic, gram-negative bacteria.Because of the toxicity of these compounds, con-siderable effort has been attributed to analysis ofaminoglycoside content in drug preparations, inserum and urine specimen in therapeutic drugmonitoring, and in edible animal tissues in residuecontrol. The present review emphasizes the analyt-ical problems associated with aminoglycosideanalysis. Screening methods based on microbio-logical and immunological procedures were brieflydiscussed. Gas chromatography and especiallyhigh-performance liquid chromatography appearedthe most widely used chemical methods for theanalysis of these compounds. Due to lack of vola-tility, chromophore, and hydrophility ofaminoglycosides, most methods appliedderivatization for enhancement of their chromato-graphic characteristics. The applicability and advan-tages of the various derivatization procedures werediscussed in detail. A wide variety of detectionmethods, including mass spectrometry have beenused. Packed column separation was generallyused for gas chromatographic separation. In liquidchromatography, reversed phase, ion pair, ion ex-change, and normal phase separation has been em-ployed. Mass spectrometry, as a detection method,was discussed in detail. Extraction procedures frombody fluids and tissues were emphasized. The per-formance and the operational conditions of themethods were described and detailed information ofthe data was provided also in table format.

Aminoglycosides have been used to treat infectionscaused by aerobic gram-negative and somegram-positive bacteria (1). They resemble each other

in chemical structure, antimicrobial activity, pharmacokinetic

characteristics, and toxicity (2). The first aminoglycoside,streptomycin, was identified in 1944 during the search for wa-ter-soluble and stable compounds active againstgram-negative bacteria(1). Kanamycin was isolated in 1957,and at the time, it was active against streptomycin-resistant bacte-ria (1). Subsequently, bacteria resistant to kanamycin were iso-lated and the mechanism of resistance was resolved. Understand-ing the mechanism of resistance led to the development ofsemisynthetic aminoglycosides, of which more than 150 havebeen described (1). Amikacin, dibekacin, gentamicin,kanamycin, neomycin, netilmicin, paromomycin, sisomicin,streptomycin, dihydrostreptomycin, and tobramycin are cur-rently in therapeutic use. In veterinary therapy, neomycin,gentamicin, kanamycin, streptomycin, and dihydrostreptomycinare the most common aminoglycosides (3).

The antimicrobial activity of aminoglycosides is based ontheir ability to inhibit bacterial protein synthesis (4). Currentprimary use of these drugs in human medicine is for treatmentof infections caused by aerobic gram-negative bacteria (4).The most important pathogens treated with aminoglycosidesare pseudomonads, enterococci, coliforms, and salmonellae.Aminoglycosides are also used substantially in the treatmentof tuberculosis.

Aminoglycosides are rapidly absorbed from an injection

site (2) but poorly absorbed after oral or rectal administration

(4). They are not inactivated in the intestine and are eliminated

quantitatively in feces (4). Systemically available

aminoglycosides are excreted almost entirely as parent com-

pound by glomerular filtration (2, 4). Aminoglycosides bind

to tissue proteins and macromolecules via ionic bonds, but

binding to plasma proteins is low (<25%). Aminoglycosides

in tissues are usually found in low concentrations and un-

bound, except in the renal cortex where they tend to concen-

trate. Half-lives of aminoglycosides in plasma are 2–3 h but in

tissues, bound aminoglycosides range from 30 to 700 h.

The major concern in aminoglycoside therapy is their toxic-

ity. All members of the group are both nephrotoxic and ototoxic

(1, 2). The problems are pronounced in patients with impaired

kidney function. Because of the narrow therapeutic range of

aminoglycosides, therapeutic drug monitoring is essential.

ISOHERRANEN& SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 1017

Received November 2, 1998. Accepted by JM March 24, 1999.1 Author to whom correspondence should be addressed.

1018 ISOHERRANEN& SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999

Figure 1. Structures of the therapeutically used aminoglycosides (1).

Structures and Chemical Characteristics

The word aminoglycoside is used to describe anamino-function-containing carbohydrate linked via aglycoside bond to an aminocyclitol (3). Therapeutically usedaminoglycosides usually contain a 1,3- or 1,4-diaminocyclitol(1). The aminoglycosides are divided into 2 groups accordingto the aminocyclitol: streptamine and 2-deoxystreptamine.The deoxystreptamine group, the larger group (1, 3), is furtherdivided into 2 subgroups according to the number and positionof the substituents in the deoxystreptamine moiety. The bestknown subgroups are (1) neomycins and paromomycins, inwhich substituents are in adjacent positions (positions 4 and5), and (2) gentamicins and kanamycins, in which the sub-

stituents are in nonadjacent positions (positions 4 and 6) of theaminocyclitol moiety (3). The streptamine group containsstreptomycin and dihydrostreptomycin (3). Structures of ther-apeutically used aminoglycosides are presented in Figure 1.

Aminoglycosides are produced byStreptomycesorMicromonospora fungi. Semisynthetic aminoglycosides,such as dibekacin, amikacin, and netilmicin were developed toreduce toxicity and increase antimicrobial activity (1).Dibekacin (1) and amikacin (6) are synthesized fromkanamycin B, and netilmicin, from sisomicin (7). Tobramycincan be synthesized from kanamycin or extracted fromStreptomyces (1). The fungi producing the variousaminoglycosides are presented in Table 1. Aminoglycosides


Table 1. Organisms that produce specific aminoglycosides

Aminoglycoside Organism(s) producing the compound Finder and year

Gentamicin Micromonospora purpurea and M. echinospora Schering Plough, 1963

Kanamycin Streptomyces kanamyceticus Umezawa, 1957b

Neomycin S. fradiae and S. albogriseolus Umezawa and Waksman, 1949

Paromomycin S. rimosus Parke Davis, 1959

Sisomicin M. inoyoensis Schering Plough, 1970

Streptomycin S. griseus Waksman and Schatz, 1944

Tobramycin S. tenebrarius Eli Lilly, 1967

Dihydrostreptomycin S. griseus and S. humidus Takeda Chemicals, 1957

a From reference 1, unless indicated otherwise.b From reference 4.

Table 2. Physical and chemical characteristics of aminoglycosides a

Aminoglycoside Molecular formula Molecular weight Melting point, EC Optical rotation [α]D LD50 (mg/kg) in mice

Amikacin C22H43N5O13 585.6 203–204 +99E 340–560b

Dihydrostreptomycin C21H41N7O12 583.6 — Levorotatory 200

Dibekacin C18H37N5O8 451.54 — +132E 63–72

Gentamicin C1 C21H43N5O7 — 94–100 +158E 81 (iv)c

Gentamicin C2 C20H41N5O7 — 107–124 +160E 183 (iv)c

Gentamicin C1a C19H39N5O7 — — +165.8E —

Kanamycin A C18H36N4O11 — — +146E 583

Kanamycin B C18H37N5O10 — 178–182 +114E 136

Kanamycin C C18H36N4O11 — — +126E —

Neomycin C23H46N6O13 — — +80E (B) +120E (C) 36

Netilmicin C21H41N5O7 475.6 — +164E 40

Sisomicin C19H37N5O7 447.55 198–201 +189E —

Streptomycin C21H39N7O12 581.58 — Levorotatory 200

Tobramycin C18H37N5O9 467.54 — +128E 118

a Data from references 12 and 101.b Depends on pH.c Intravenous.

produced byStreptomyceshave the suffix “mycin” and the onesproduced byMicromonosporahave the suffix “micins” (3).

Aminoglycosides are water soluble and heat-, acid-, andbase-stable polar compounds. Their solubility in methanol islimited, and they are practically insoluble in hydrophobic or-ganic solvents. In water solutions, aminoglycosides are usu-ally positively charged because of their amino groups. Thenumber of amino groups varies from 4 in kanamycin to 6 inneomycin, and the pKa values of the amino groups vary be-tween 7 and 8.8. The physical and chemical characteristics ofthe therapeutic aminoglycosides are presented in Table 2.

Aminoglycosides are typically drug complexes formed byseveral closely related components. The neomycin complex isformed by 2 stereoisomers: neomycin B and C (8). Onlyneomycin B is antimicrobial. Paromoamine andparomomycins I and II occur as impurities of the neomycincomplex (8). Commercially available neomycin products in-clude 85–90% neomycin B (8). The gentamicin complex com-prises at least 4 main components and several minor compo-nents (9). Separation of up to 7 different components ofgentamicin has been reported (10), and proportions ofgentamicin components in various products differ (11).Paromomycin is a complex of 2 stereoisomers: paromomycin Iand II. Kanamycin is a mixture of 3 isomers, A, B, and C (1). Thekanamycin components differ markedly in their toxicity and,therefore, commercial mixtures are required to contain at least75% kanamycin A and not more than 5% kanamycin B (1).

Screening of Aminoglycosides

Aminoglycosides have been analyzed in tissues and urineby microbiological, radioenzymatic assay (REA), and

radioimmunoassay (RIA) methods and by paper chromatog-raphy (12). These methods are still widely used but often lackquantitative or qualitative performance.

Microbiological assays use methods based on agar diffu-sion of the drug and concentration-dependent growth inhibi-tion (inhibition zone) of the test organism inoculated in theagar (12). The assays require 12–48 h incubation, after whichinhibition of bacterial growth is measured. Several factors in-fluence the accuracy of these methods (2). Incubation temper-ature, agar pH and ion concentration, depth of the agar on theplate, test strain, incubation time, and presence of other antibi-otics in the sample can affect the assay (2). Additionally, dif-ferent agar pHs need to be used for different aminoglycosides.Although microbiological methods are versatile, simple, andrelatively cheap, they are inaccurate and subject to interfer-ences caused by nonspecific inhibitors or other antimicrobialdrugs (13, 14). Additionally, repeatability and reproducibilityof results are generally poor (12), and the assays usually lacksensitivity below 2µg/mL (2).

RIA represents a major improvement over microbiologicalassays (2). RIA methods are sensitive (2, 12) and specific, butother aminoglycosides might cause interferences (12).Gentamicin, tobramycin, amikacin, netilmicin, and sisomicincan be determined by RIAs (12). Analysis using an RIAmethod requires complicated parameter optimization and spe-cialization of the analyst (12). Selection and preparation ofsuitable specific antibodies is difficult and time consuming(12). In addition, handling of radioactive materials and radio-active waste and high cost are inhibitory factors (2, 12). Cor-relations between RIA and other methods are variable (2).

REA offers similar advantages as do RIAs. The instabilityof enzymes and the presence of other antibiotics in the sample


Table 3. GLC methods used for aminoglycoside analysis

Analyzed compounds DerivatizationColumn phase,

length (m) × id (mm)Temperature

programa

Carrier gas,flow rate(mL/min) Matrix Detector Ref.

Neomycin TMSDEA+ TrisilZ 3% OV-1 GasChromQ, 0.61 × 3 it 290EC He, 70 Standard FID 19

Neomycin TMSDEA+ TrisilZ 0.75% OV-1 GasChromQ, 1.83 × 3 it 290EC or tg10EC /min

150–310EC

He, 40 Standard FID 20

Kanamycin,paromomycin

TMSDEA+ TrisilZ 0.75% OV-1 or 3% OV-1GasChromQ, 1.83 × 3

it 290EC or it300EC

He, 70 Standard FID 21

Neomycin TMSDEA+ TrisilZ 3% OV-1 GasChromQ, 0.61–0.91 × 3 it 300EC He Standard FID 18

Kanamycinb,gentamicin,tobramycin

TMSI/HFBI 3% OV-101, 2 × 3 it 272EC N2, 60 Plasma ECD 22

DNT, tobramycin,netilmicin

TMSI/HFBI 3% OV-101 ChromosorbW, 2 × 3 it 272EC N2, 60 Plasma ECD 23

Amikacin,paromomycin

TMSI/HFBI 1% OV-17, 2 × 3 it 277EC N2, 80 Plasma ECD 23

Kanamycin HFBI/TMSI 4% OV-101, 1.5 × 3 it 270EC — Muscle ECD 24

a it = isothermal; tg = thermal gradient.b Internal standard.

can cause false results (2, 12). An REA method has been de-veloped for kanamycin, tobramycin, amikacin, and sisomicin(12). However, occasionally poor correlation with resultsobtained with other methods has been observed (12).

The 2 most common assays for aminoglycosides are thehomogenous enzyme immunoassay and the fluorescence po-larization immunoassay (FPIA; 2). These methods have simi-lar sensitivity and precision as RIAs but are less expensive anddo not require handling of radioactive material (2).

Chromatographic Analysis

Chromatographic methods for aminoglycoside analysis areneeded for simultaneous qualitative and quantitative determi-nations. However, separations are difficult to achieve becauseof the structural similarity of aminoglycosides (15, 16). Paperchromatography of gentamicin components was the first re-ported chromatographic method for aminoglycosides (16).

Gas Chromatography

Gas chromatography (GC), with its high separation ca-pacity and efficiency, is a popular technique for volatile,heat-stable compounds. However, direct analysis of intactaminoglycosides by GC is impossible because of the hydro-philic, basic, and nonvolatile nature of these molecules (17).By derivatizing the amino and hydroxyl groups, volatile de-rivatives can be produced (18–24). The gas–liquid chro-matographic (GLC) methods for aminoglycosides are pre-sented in Table 3.

Trimethyl silyldiethyl amine (TMSDEA), a reagent thatsilylates both amino and hydroxyl groups, has been used as aderivatizing agent (18–21). The products are moisture sensi-tive and, therefore, unstable (18–21). Consequently, resultsare nonlinear and poorly repeatable, and the derivatizationyield is low. Freeze drying of samples prior to derivatizationhas been used to eliminate variations in sample moisturecontent and solubility (18). Sealed sample vials, removal ofmetal parts from the chromatographic system, andon-column injection have been tried to improve repeatabilityand quantitation (18). Nonetheless, linearity andderivatization yield remain poor.

It is important to know the ratio of drug components orstereoisomers that differ in their pharmacological characteris-tics in a pharmaceutical product. Stereoisomers ofneomycin B and C (18, 20) and paromomycin I and II (19, 21)have been separated by GLC as TMSDEA–silylated deriva-tives. Stereoselective separation using a nonchiral columnhas been suggested to result from stronger retention of theequatorial than the axial epimer (21). On the basis of this as-sumption, it has been suggested that paromomycin I mustelute before paromomycin II and neomycin B before C. Thekanamycin components A, B, and C have been separated astheir trimethylsilyl (TMS) derivatives (21). The TMS deriva-tives of neomycin (19, 20), kanamycin (21), andparomomycin (21) also have been identified by mass spec-trometry (MS). Derivatization results in silylation of allamino and hydroxyl groups.

A 2-step derivatization using trimethyl silylimidazole(TMSI) for silylation of hydroxyl groups andheptafluorobutyric imidazole (HFBI) for heptafluoro-butyrylation of amino groups has been reported (22–24).Using this technique, Mayhew and Gorbach (23) analyzedconcentrations of gentamicin, tobramycin, netilmicin,amikacin, and paromomycin in serum. For each amino-glycoside, another one was used as an internal standard. Re-sults are acceptable in accuracy and precision, and the methodis linear for the concentration range investigated. TMS–heptafluorobutyryl (HFB) derivatives are more stable thanTMSDEA derivatives, but protection of the samples frommoisture still is crucial. Gentamicin elutes from the column as2 peaks: the first is gentamicin C1, and the second, as a combi-nation of gentamicins C1a and C2. All the other amino-glycosides analyzed produce 1 peak. Mayhew and Gorbach(22) also reported an improved GLC method for gentamicinwith kanamycin as internal standard. The accuracy and repeat-ability of the method are improved, and the correlation coeffi-cient (r) for the calibration curve is 0.979 (22). GLC analysisof kanamycin in muscle tissue has been reported (24). In thisstudy, the 2-step TMSI–HFBI derivatization described byMayhew and Gorbach (23) is used in reversed order. Thisprocedure produces better results than the originalTMSI–HFBI derivatization.

In comparison, the 2-step derivatization with TMSI andHFBI appears superior to silylation with TMSDEA, the mainadvantage being the higher derivatization yield and the morestable derivatives. The 2-step derivatization method is also ap-plicable to larger number of aminoglycosides and more inter-nal standards are available compared with the TMSDEA-based method. Unlike in TMSDEA methods, individualaminoglycosides do not disturb the assays of otheraminoglycosides in the TMSI–HFBI methods. The HFBIderivatization allows the use of an electron capture detector(ECD), providing increased selectivity and sensitivity of themethod compared with methods using a flame ionization de-tector (FID). The main weakness of GC methods foraminoglycosides is the lack of capillary column applications.

Liquid Chromatography

As water-soluble, polar, and relatively large compounds,aminoglycosides typically would be analyzed by liquid chro-matography (LC; 1). Most high-performance liquid chro-matographic (HPLC) methods concern gentamicin analysis(25), but because of the chemical similarity within the group,these methods are often applicable to other aminoglycosides.The most significant characteristics of aminoglycosides rele-vant to HPLC analysis are the lack of a chromophore and thehigh hydrophilicity. To improve detectability and separation,the aminoglycosides are usually derivatized prior to or afterchromatographic separation. Methods used to separateaminoglycosides by HPLC are presented in Table 4.

Reversed-Phase Chromatography

Because of their polar and ionic nature, aminoglycosidesusually are not partitioned on reversed-phase columns (15, 26).



Table 4. HPLC methods used for separation of aminoglycosides

Column phase,id (mm) × length (cm) ×particle size (µm)

Flow rate ofmobilephase,L/min Mobile phasea Derivatizationb

Detector,wavelengths (nm)c Aminoglycosidesd Ref.

LiChrospher 100 RP18,4.0 × 12.5 × 5

0.2 MeOH–H2O, 55 + 45,0.05 cs-EDTA

OPA pc fl, ex 340, em 440 gnt (1), neo, kan,ami, dhs

5

PLRP-S 1000Å, 4.6 ×25 × 8

1.0 70 g/L Na2SO4 1.4 g/L ocs fospH 3

— PED neo 8

Cellulose phosphatePII, 0.9 × 15 × —

0.2 2.1M NaCl — Polarography gnt (4) 10

Lichrosphere C8, 3.0 ×25 × 7

1.5 0.015M pns aca OPA pc fl, ex 350, em 450 gnt, ntl, neo, sis, kan 13

µPartisil, 3.9 × 30 × — 1.0 H2O–MeOH–DEA, 60 + 40 + 0.5 OPA pc RI gnt, ntl, neo, kan, sis 13

Ultrasphere C18, 4.6 ×25 × 5

1.0 MeOH–H2O HFBA — RI str, kan, sis, gnt 15

ODS II, 4.6 × 10 × 3 1.0 MeOH–H2O TFA — MS TSP gnt (4) 16

Hypersil C18, 5 × 12.5 ×5 or Spherisorb ODS,5 × 25 × 5

0.25–3.0 MeOH–H2O, 70:30 aca,.02M hps

OPA UV, 330 gnt (4) 63

Zorbax 18, 4.6 × 15 × 5 0.8 25 mM tls, perchloric acid pH 2.0 OPA pc fl, ex 360, em — sis, ntl, neo, par 14

Spherisorb ODS-2,2.0 × 10 × 5

0.2 ACN–H2O, 5 + 95, 20 mM PFPA — MS/MS str, dhs, neo, gnt 17

µBondapak C18, 3.9 ×30 × 5

2.0 MeOH–H2O, 3 + 97, 0.1% aca0.02M pns 0.2M Na2SO4

OPA pc fl, ex 340, em 418 gnt, sis, ntl 50

Supelcosil LC-8-DB,4.6 × 15 × 5

— 1.5% MeOH, 0.01M pns, 0.056MNa2SO4, 0.007M aca

OPA pc fl, ex 340, em 455 neo 51


— 0.011M pns 0.007M aca,1.5% MeOH


PhaseSep SperisorbODS2, 4.6 × 15 × 5

1.5 MeOH–H2O, 82 + 18 0.01M pns,0.0056M Na2SO4, 0.1% aca

OPA pc fl, ex 340, em 420 gnt (4) 52

Nucleosil C18, 4.6 ×12.5 × 5

1.0 MeOH–H2O, 80 + 20 aca OPA on line fl nea, ami, dib,gnt (4), sis, tob, ntl

26

LiChrosorb RP18, 4.6 ×25 × 10

4.0 ACN–DCM–H2O–MeOH,80 +10 + 8 + 2

BSC UV, 230 nm gnt (1), ntl 27

µBondapak C18, 4.0 ×30 × —

1.0 ACN–H2O, 95 + 5 DansCl fl, ex 220, em 470 gnt (2) 28

µBondapak C18, 3.9 ×30 × 10

3 ACN–tris, 70 + 30 FDNB UV, 365 gnt (3) 29

µBondapak C18, 3.9 ×30 × 10

1.5 ACN–tris (pH 7), 70 + 30 FDNB UV, 365 gnt (3), sis, tob 30


0.5 ACN–tris (pH 3), 70 + 30 OPA fl, ex 340, em 418 gnt (3) 31

µBondapak C18, 4.0 ×30 × —

— MeOH–H2O, 79 + 21 2g/L EDTA OPA fl, ex 360, em 430 gnt (3) 32

µBondapak C18, — — MeOH–H2O, 79 + 21 2g/L EDTA OPA fl gnt (3) 33

µBondapak C18, — — MeOH–H2O, 75 + 25 tris, TEA,H2SO4 pH 7

OPA fl tob, ntl 33

Hypersil ODS, 4.6 ×20 × 3

1.0 ACN–H2O, 90 + 10 FMOC–Cl fl, ex 260, em 315 gnt (3) 34

Partisil5 ODS-3, 4.6 ×10 × 5

1.5 ACN–H2O, 40 + 60 DNBC UV, 254 ami, tob, kan 35

Partisil5 ODS-3, 4.6 ×25 × 5

1.5 MeOH–H2O, 70 + 30 DNBC UV, 254 gnt (4) 35

µBondapak C18, 3.9 ×30 × 10

3 ACN–H2O–aca, 60 + 39 + 1 FDNB UV, 365 tob, gnt 36


Table 4. (continued )




Merck RP18, 3.9 × 30 ×10

1.3 ACN–H2O–aca, 70 + 29 + 1 FDNB UV, 365 tob 37


2.5 ACN–H2O–aca, 47 + 52 + 1 FDNB UV, 365 ami, kan 38

Ultrasphere ODS C18,4.6 × 25 × 5

— ACN–H2O, 68 + 32 FDNB UV, 360 ami 39

µBondapak, 3.9 × 30 ×10

2.0 MeOH–fos (pH 6), 78 + 22 TEA OPA fl, ex 260, em 418 gnt (3) 71


1.5 0.02M fos (pH 7.5), KOH, ACN,MeOH

TNBS UV, 350 gnt, tob, sis, ami,kan

40

Ultrasphere octyl C8,4.6 × 25 × 5

2.0 ACN–fos, 52 + 48 TNBS UV, 340 ami, kan 41

Ultrasphere octyl, 4.6 ×25 × —

3.0 ACN–fos (pH 3.5), 70 + 30 TNBS UV, 340 tob, sis 42

µBondapak C18, — 1.0 ACN–H2O, 95 + 5 DansCl fl, ex 220, em — ntl 43

µBondapak C18, 4.0 ×30 × —

1.0 ACN–H2O–MeOH, 5 + 30 + 652g/L EDTA

OPA fl, ex 350, em 450 ami 44

Hisep, 4.0 × 15 × 5 1.7 MeOH–H2O TCA, EDTA OPA fl, ex 340, em 418 neo 45

Amine bonded, 4.7 ×25 × 5

0.7 ACN–H2O–aca, 70 + 30 + 0.1 — MS moving belt kan, tob, nea 46

Hitachi gel ODS, 4.0 ×15 × 5

1.0 100 mM NH4 acetate — MS APCI kan 47

Hitachi gel ODS, 4.0 ×15 × 5

— MeOH–H2O NH4 acetate — MS APCI kan, gnt (3) 48

Ultraspher C18, 4.6 ×25 × 5

1.0 MeOH–H2O different ion pairs — MS tai RI gnt, sis, kan, str, neo 69


0.2 ACN–H2O, 8–16 + 92–84 40 mMHFBA

— PAD or MS str, dhs 49


— 0.0056M Na2SO4, 0.007M aca0.011M pns, 18.5% MeOH



0.9 0.01M pns, 0.056M Na2SO4,0.007M aca, 1.5% MeOH

OPA pc fl, ex 340, em 455 neo, par, str, dhs 54

µBondapak C18, 3.9 ×30 × 10

1.0 0.05M pns, 0.2M Na2SO4,0.1% aca

— Electrochemical gnt (3) 55

Nucleosil C18, 4.6 ×15 × 10

1.2 0.02M pns aca OPA pc fl, ex 340, em 418 ami, tob 56

µBondapak C18, 3.9 ×30 × —

2.0 0.2M Na2SO4, 0.02M pns, 17.4Maca, 3% MeOH

OPA pc fl, ex 340, em 418 gnt, ami, tob 57

Ultrasphere ODS, 4.6 ×25 × 5

1.5 MeOH–H2O, 80 + 20 5% aca,5 g/L hxs

OPA UV, 330 gnt (4) 59


1.7 MeOH–H2O–aca, 9 + 90 + 1,5 g/L hxs

OPA fl, ex 340, em 418 gnt (3) 59

Lichrosorb RP18, 4.0 ×25 × 5

1.0 ACN–fos (pH 3), 8 + 92 3.76 g/Lhxs

— UV, 195 str, dhs 61

µBondapak C18, 3.9 ×30 × 5

1.0 ACN–H2O, 8 + 92, 0.02M hxs,0.025M fos

— UV, 195 str, dhs 58

Supelcosil LC8-DB,4.6 × 25 × 5

0.5 ACN–H2O, 17 + 83 hxs NQSA fl, ex 347, em 418 str, dhs 89

Supelcosil LC8-DB,4.6 × 25 × 5

0.5 ACN–H2O, 17 + 83 hxs NQSA fl, ex 365, em 418 str, dhs 60






Ultrasphere ODS, 4.6 ×15 × —

1.5 MeOH–H2O, 70 + 30, aca, hps OPA UV, 330 gnt (4) 64

Zorbax SB C18, 2.1 ×15 × —

0.5 MeOH–H2O, 70 + 30, aca, hps OPA UV, 330 gnt (4) 65

ODS Hypersil, 5.0 ×10 × 5

1.0 MeOH–H2O–aca, 70 + 25 + 5,5 g/L hps

OPA UV, 330 gnt (4) 66

Nucleosil C18, 4.0 ×20 × 5

0.9 MeOH–H2O, 80 + 16, aca, hps OPA fl, ex 340, em 450 sis, ntl 86

Ultremex C18, 4.6 ×25 × 5

1.6 MeOH–H2O, 70 + 30 aca, hps OPA fl, ex 340, em 418 gnt (3) 67

Hypersil ODS, 4.6 ×10 × 5

1.0 MeOH–H2O, 72 + 28 hps OPA UV, 350 gnt 11

Supelcosil LC-8, 4.6 ×25 × 5

1.8 MeOH–H2O 10–60% MeOH, aca,hps

OPA fl, ex 340 em 448 gnt (3) 68

Radicalpak C18, 8 ×10 × 10

1.5 ACN–H2O, 80 + 20, aca, ocs-eds NQS fl, ex 351, em 420 str 88

µBondapak C18, 3.9 ×30 × 10

1.5 ACN–H2O, 15 + 85 aca, 0.1Meds-ocs

OPA pc fl, ex 365, em 440 gnt (3) 62

PRP-1 polymer, 4.6 ×25 × 10

— ACN–H2O, 7 + 1, different ionpairs

— RI tob, kan, str, neo 70

Dowex AG 1×2, 0.9 ×15 × —

4.5 H2O — — neo 74

Carbopak PA1, — 1.0 0.5–50M NaOH — PAD tob 75

MPIC–NSI polystyrene,4.0 × 25 × —

0.6 0.25M NaOH — PAD tob, kan 76

Partisil SCX, 3.6 ×25 × 10

2.0 ACN–fos, 70 + 30 Fluorescamine fl, ex 275, em 418 gnt 77

Zipak SCX, 2.1 ×100/50 × 37-44

0.8 0.01M EDTA (pH 9.5) OPA pc/fluorescamine

fl and RI kan (3) 78

Amberlite IRC 50,13.9 × 29 × —

— For elution, 0.5M Na2SO4 Dihydrolutidine fl, ex 421, em 488 tob, neo, sis, kan,ami

79

Zorbax Sil, 4.6 × 25 × — — Dichloroethane–heptane–MeOH–H2O–DEA, 79 + 15 + 5.5 + 3.6 +

1.5

FDNB UV, 365 neo (3) 80

P-EHS5 Silica, 4.6 ×12.5 × 5

1.5 Chl–MeOH–aca, 95 + 2 + 3 NSCl UV, 254 neo, gnt, kan 81

LiChrosorb SI-100, 4.6 ×25 × 5

— Chl–THF–H2O, 60 + 39 + 1 FDNB UV, 254 or 350 neo, gnt, kan, par 82

Nucleosil C18, 4.0 ×20 × 5

0.6 MeOH–H2O, 60 + 40 EDTA OPA fl, ex 340, em 450 sis 87

a OCS = octane sulfonate; fos = phosphate buffer; pns = pentane sulfonate; aca = acetic acid; DEA = diethylamine; HFBA = heptafluorobutyricacid; TFA = trifluoroacetic acid; hps = heptane sulfonate; t/s = toluene sulfonate; ACN = acetonitrile; PFPA = pentafluoropropionic acid; DCM= methylene chloride; TEA = triethylamine; TCA = trichloroacetic acid; hxs = hexane sulfonate; eds = ethane disulfonate; chl = chloroform.

b OPA = o-phthalaldehyde; pc = postcolumn derivatization; NQSA = $-naphthoquinone-4-sulfonic acid; NQS = $-naphthoquinone-4-sulfonate;FDNB = 1-fluoro-2,4-dinitrobenzene; NSCl = naphthalene sulfonyl chloride; BSC = benzenesulfonyl chloride; DansCl = dansyl chloride;FMOC–Cl = 9-fluorenylmethoxycarbonyl chloride; DNBC = dinitrobenzoylchloride; TNBS = 2,4,6-trinitrobenzene sulfonic acid.

c fl = fluorescence detector; ex = excitation wavelength; em = emission wavelength; PED = pulsed electrochemical detector; RI = refractiveindex detector; MS = mass spectrometry; TSP = thermospray; UV = ultraviolet; MS/MS = tandem mass spectrometry; APCI = atmosphericpressure chemical ionization; PAD = pulsed amperometric detector.

d Numbers in parentheses indicate the number of components; gnt = gentamicin; neo = neomycin; kan = kanamycin; ami = amikacin; dhs =dihydrostreptomycin; ntl = netilmicin; sis = sisomicin; str = streptomycin; par = paromomycin; tob = tobramycin; dib = dibekacin; nea =neamine.

Partition to C18 columns can be improved by using acetatebuffer in the mobile phase. The acetate ion acts as a counterion,forming ion pairs with aminoglycosides (26). Usuallyaminoglycosides are derivatized with a nonpolar group prior toreversed-phase analysis to improve the partition to column andthe separation characteristics. The most commonly usedderivatization reagents areo-phthalaldehyde (OPA) and1-fluoro-2,4-dinitrobenzene (FDNB). The separation columnsusually contain C8 or C18 materials, and mobile phases consistof acidic buffers with methanol and/or acetonitrile.

The reversed-phase method reported by Essers in 1984(26) apparently remains the method able to simultaneouslyseparate the largest number of aminoglycosides. On-lineprecolumn derivatization of the aminoglycosides is used. Themobile phase consists of methanol–acetic acid (80 + 20).Dibekacin, sisomicin, tobramycin, and 4 components ofgentamicin can be separated in one chromatographic run. Thegentamicin components elute in the order C1, C1a, C2a, and C2,with the stereoisomers C2 and C2a separated from each other.By reducing the proportion of methanol in the mobile phase,the more polar aminoglycosides amikacin and neamine can beseparated. Netilmicin requires an incresed proportion of meth-anol. For each aminoglycoside, one of the others was used asan internal standard.

The separation of gentamicin components is of interest inthe quality control of pharmaceutical preparations,pharmacokinetics, and toxicological studies. Gentamicincomponents can separate to 1–5 peaks in reversed-phase col-umns depending on derivatization and separation conditions(27–35). When gentamicin is derivatized with benzene–sulfonyl chloride (BSC), the derivatives elute as a single peak(27). After dansyl chloride (28) or FDNB (29, 30)derivatization, gentamicin elutes as 2 separate peaks. The elu-tion orders of dansyl and dinitrophenyl derivatives are similarbut separation of components differ. Retention of gentamicincomponents is assumed to increase with the number of methylgroups in the gentamicin structure (30). As dansyl derivatives,gentamicins C1a and C2 elute together, followed bygentamicin C1 as a separate peak (28). As 2,4-dinitrophenylderivatives, gentamicin C1aelutes before the coeluting C1 andC2 components (29, 30).

Both dansyl chloride and FDNB derivatize all amino groupsin gentamicin and, therefore, the relative functionalities do notchange as a result of the derivatization reagent used. In the sepa-ration of 2,4-dinitrophenyl derivatives, the lack of a methylgroup in gentamicin C1aappears to be the major factor affectingseparation. The mechanism of separation of dansyl derivativeshas not been discussed in the literature. It can be assumed thatthe separation of the dansylated gentamicins is based on thefunctionality of the amine on C-6′. After derivatization, thecomponents (C1a and C2) that have similar amino groups,HNR2, elute together before the gentamicin component C1

which contains an NR3 amino group.Three gentamicin components are separated as OPA deriv-

atives on C8 or C18 columns by use of acetonitrile–tris (31) ormethanol–ethylenediamine tetraacetic acid (EDTA)–water(32, 33) mobile phases, correspondingly. In these studies,

identification of the different gentamicin components is basedon standards of individual components but the structures ofthe derivatives are not discussed. Strangely, the elution orderof the gentamicin components is C1a, C2, and C1 with a C8 col-umn but C1, C1a, and C2 with a C18 column. The retentionmechanism is not discussed.

The retention of gentamicin C1relative to other gentamicincomponents is of special interest because as an OPA deriva-tive, it contains one derivatizable amine less than the othercomponents. The derivatized C1 component is much smallerand has one basic secondary amine more than the other com-ponents. The retention of the gentamicin C1 component ismore sensitive to changes in mobile phase pH than the othercomponents, and its retention increases rapidly when the pHof the mobile phase increases (31). This or derivatization dif-ferences may explain why gentamicin C1 elutes as the lastcomponent from a C8 column and as the first component froma C18 column. Addition of EDTA to the mobile phase yieldssharper peaks (32).

The gentamicin 9-fluorenyl methoxycarbonyl chloride(FMOC–Cl) derivatives are separated as 4 separate peaks on aC18 column with an acetonitrile–water mobile phase (34). Theelution order is C1a, C2, x, and C1. The component x is believed tobe the stereoisomer of gentamicin C2and gentamicin C2a. The re-ported elution order corresponds to the increasing methylation ofgentamicin components. On the basis of the elution order of thecomponents, it is assumed that all the primary and secondaryamino functions of the gentamicins are derivatized. Otherwise,the component C1 is expected to elute first (34).

After 3,5-dinitrobenzoyl chloride (DNBCl) derivatization,the gentamicin components are separated on a C18 columnwith methanol–water (45 + 55) elution (35). The3,5-dinitrophenyl derivatives elute as 5 peaks, of which only 4could be identified. The fifth peak is assumed to be one of theminor gentamicin components. The elution order of thegentamicins is C2a, C1, C1a, and C2. No explanation for theseparation of the stereoisomers C2a and C2, in such a mannerthat the 2 other components eluted in between, is provided.The reported elution order is different from that reported(29, 30) for the nitrophenyl derivatives of gentamicin.

Amikacin, tobramycin, and kanamycin (35) also have beenanalyzed as 3,5-dinitrophenyl derivatives, while tobramycin(30, 36, 37), sisomicin (30), and amikacin (38, 39) have beenanalyzed as 2,4-dinitrophenyl derivatives. C18 columns havebeen used with a mobile phase containing acetonitrile and wa-ter for 3,5-dinitrophenyl derivatives or acetonitrile and aceticacid for 2,4-dinitrophenyl derivatives. The separation of dif-ferent aminoglycosides as their 3,5-dinitrophenyl derivativeshas not been investigated. As 2,4-dinitrophenyl derivatives,tobramycin and gentamicin components can be separated inone chromatographic run (36), but when sisomicin, a dehydroanalogue of gentamicin C1, is added to the same run, it doesnot separate from gentamicin (30). Gentamicin has been usedas the internal standard for tobramycin (36, 37). Apparentlysisomicin can be quantitated more easily as an internal stan-dard than gentamicin because it produces only one chromato-graphic peak.


The separation of different 2,4-dinitrophenyl derivatives ofaminoglycosides is assumed to be related to the differentamounts of hydroxyl groups in the different aminoglycosides(30). Tobramycin has 2 hydroxyl groups more than sisomicinand gentamicin and is more polar. Thus, tobramycin shouldelute first. Sisomicin elutes before gentamicin, probably be-cause of the polar double bond in its structure, and thegentamicins elute according to increasing degree ofmethylation (30). The dinitrophenyl derivative of amikacin ismore polar than the corresponding derivatives of otheraminoglycosides (38). As a consequence, in reversed-phasechromatography (RP), it requires a weaker eluent than the oth-ers. By reducing the ratio of acetonitrile in the mobile phase,separation of amikacin has been achieved (38). Kanamycin is asuitable internal standard for amikacin, with its dinitrophenylderivative eluting after the amikacin derivative (38).

2,4,6-Trinitrobenzene sulfonic acid (TNBS) has also beenused for precolumn derivatization of aminoglycosides(40–42). TNBS forms trinitrophenyl derivatives withaminoglycosides that are very similar to dinitrophenyl deriva-tives in their polarity and in derivatized groups. Amikacin (41)and tobramycin (42) have been analyzed as theirtrinitrophenyl derivatives with a C8 column, an acetonitrile–phosphate buffer mobile phase, sisomicin as the internal stan-dard for tobramycin, and kanamycin as the internal standardfor amikacin. Because of their different polarities, amikacinand tobramycin are analyzed with different mobile phases.Kanamycin and amikacin elute in the solvent front intobramycin analysis (42). The elution order of theaminoglycosides in these methods is amikacin, kanamycin,tobramycin, and sisomicin, similar to the elution order ofdinitrophenyl derivatives. Therefore, the number of hydroxylgroups can be assumed to determine also the elution order ofthe trinitrophenyl derivatives. The mobile phase is optimizedby increasing the proportion of acetonitrile, addingtetrahydrofuran (THF), or changing the pH. The separation oftobramycin and sisomicin is insufficient when acetonitrile ex-ceeds 75% (42).Addition of THF reduces tailing (41, 42), but thesame results can be achieved also by reducing the pH (41). Whenthe pH of the mobile phase is 3 or less, no tailing is observed andoptimal conditions can be achieved without THF (41).

Tobramycin, sisomicin, amikacin, the 4 components ofgentamicin, and kanamycins A, B, and C have been analyzedas their trinitrophenyl derivatives with a C8 column and anacetonitrile–phosphate buffer–methanol mobile phase (40).The solvent ratio of the mobile phase has been varied accord-ing to the polarity of the analyzed aminoglycosides. The ratioof the organic phase is highest for gentamicin and lowest foramikacin. Kanamycin elutes before tobramycin when they arein the same run. When kanamycin is added to the amikacinanalysis, it elutes clearly after amikacin. The possibility ofseparating all aminoglycosides in one chromatographic run orthe possible interference caused by other aminoglycosides hasnot been investigated.

Netilmicin has been detected as its dansyl derivative with a C18

column and an acetonitrile–water (95 + 5) mobile phase (43).Netilmicin elutes after gentamicin, suggesting that derivatized

netilmicin is relatively nonpolar. Gentamicin C1 elutes almostsimultaneously with netilmicin and interferes in the analysis.

The separation of tobramycin and netilmicin as their OPAderivatives has been investigated with a C18 column and amethanol–EDTA–water mobile phase. Retention times aretoo long, and the separation is unsuccessful (33). Re-versed-phase separation of amikacin as its OPA derivativewith an EDTA–methanol–water–acetonitrile mobile phasehas been reported (44). Neomycin has been analyzed as anOPA derivative with a methanol–EDTA–water mobile phaseand a Supelco HISEP column that has a silica-based stationaryphase containing hydrophobic regions shielded by a hydro-philic network (45).

Nonderivatized kanamycin, tobramycin, and neamine havebeen separated by RP using amino-bonded column, anacetonitrile–acetate buffer (70 + 30) mobile phase, and a massspectrometer as a detector (46). Underivatized kanamycinalso has been separated with a C18column and a methanol–ac-etate buffer mobile phase (47, 48). The underivatizedkanamycin isomers A and C (47) and 3 gentamicin compo-nents (48) can be separated and identified by RP and MS.

Ion-Pair Chromatography

Ion-pair chromatography (IP) is well-suited toaminoglycosides because of their polar, charged, and basiccharacteristics under conditions usually used for this tech-nique. IP seems the most popular chromatographic methodfor aminoglycoside analysis. The most widely usedcounterions are the negatively charged pentane, heptane, andhexane sulfonates. However, alkylsulfonates are not suitablefor HPLC/MS applications because of their nonvolatile char-acteristics (16, 49). Instead, the volatile fluorinatedcarboxylic acids are used as counterions in HPLC/MS appli-cations for aminoglycosides (49), as well as in preparativework (15). The counterions used for IP of aminoglycosidesare presented in Table 5.

The mechanisms of retention of aminoglycosides by use ofalkylsulfonates as ion-pairing reagents have not been dis-cussed in the literature. However, separation and retention areassumed to result from ion-pair formation between positivelycharged protonated aminoglycoside and anionicalkylsulfonate ions and the different interactions between theion pair and the hydrophobic column phase (50).Derivatization markedly affects the polarity and acid–basecharacteristics of the analyzed compound and, it is assumed,also the IP separation. OPA has been the main derivatizationreagent in IP of aminoglycosides. In postcolumnderivatization, ion-pair formation is not affected. But inprecolumn derivatization, effects are likely to be observed.The protonated primary and secondary amino groups ofaminoglycosides responsible for ion-pair formation are thesame ones that undergo OPA derivatization. Thus, retentionmechanisms and ion-pair formation forprecolumn-derivatized and nonderivatized aminoglycosidesmust differ. Unfortunately, these differences have not beennoted in the literature, and no hypothesis of the ion-pair for-mation between derivatized aminoglycoside and


alkylsulfonate has been presented. Different mercaptans areused in OPA derivatizations, but their effect on the charge andpolarity of the derivatives and on ion-pair formation has notbeen discussed.

In most methods, Na2SO4 is added with the alkylsulfonateto the mobile phase (8, 13, 29, 50–57). The Na2SO4 concen-tration is the most important factor affecting the retention ofaminoglycosides (8). In IP, the sulfate ion reduces retentiontimes of aminoglycosides (13) and decreasesk′ (8), apparentlybecause the sulfate ion is more hydrophilic than the sulfonatesused as counterions (8). The ionic strength of the mobile phaseis also affected by the sulfate ion, and without sulfate in themobile phase, an increase in the alkylsulfonate concentration

reduces the retention of aminoglycosides, resulting in poorseparation (50).

Pentane, hexane, and heptane sulfonates have been com-pared in their ability to separate streptomycin anddihydrostreptomycin (58). The retention of streptomycin in-creases with the length of the carbon chain in thealkylsulfonate. Hexane sulfonate provides optimal separationin a relatively short time. For other aminoglycosides, the useof different alkylsulfonates has not been compared.

In IP, the mobile-phase buffer should not form ion pairs butshould have sufficient buffering capacity to maintain constantpH. Acetate buffer is the most widely used withaminoglycosides (25, 51–57, 59, 60) but phosphate buffer


Table 5. Counterions used in ion-pair chromatographic analysis of aminoglycosides

Ion-pair reagenta Column phase Aminoglycosidesa Derivatizationa References

pns C8 gnt (3)b OPA pc 13

C8 neo OPA pc 25, 51

C18 neo OPA pc 53

C8 neo, par, str, dhs OPA pc 54

C18 gnt (3) — 55

C18 gnt (4) OPA pc 52

C18 ami, tob OPA pc 56

C18 gnt (3), sis, ntl OPA pc 50

C18 gnt (3), ami, tob OPA pc 57

hxs C18 gnt (3) OPA pc 59

C8 gnt (4) OPA 59

C18 str, dhs — 58, 61

C8 str, dhs NQS pc 60, 89

hps C18 gnt (4) OPA 11, 63–66

C18 sis, ntl OPA 86

C18 gnt (3) OPA 67

C8 gnt (3) OPA 68

C8 ami, kan OPA pc 102

ocs PLRPc neo — 8

ocs and eds C18 str NQS pc 88

C18 gnt (3) OPA pc 62

tls C8 sis, ntl, tob OPA pc 14

CS C18 gnt (1), neo, kan, ami, dhs, str OPA pc 5

TEA C18 gnt (3) OPA 71

C18 tob, ntl OPA 33

TFA C18 str, kan, sis, gnt (4) — 15

gnt (4) — 16

HFBA C18 str, dhs — 49

PFPA str, dhs, neo, gnt — 17

a For definitions of abbreviations, see footnotes to Table 4.b Numbers in parentheses indicate the number of gentamicin components separated.c Poly(styrenevinylbenzene) copolymer column.

also has been investigated (8). With octane sulfonate ascounterion, phosphate buffer is more advantageous than ace-tate buffer (8). Phosphate buffer is the choice whenunderivatized aminoglycosides are detected by UV absorption(195 nm) because it does not absorb UV light at this wave-length (58, 61). The proportions of phosphate buffer andalkylsulfonate affect separation characteristics (58). When thealkylsulfonate concentration exceeds the phosphate concen-tration, the retention of aminoglycosides can be increased byadding alkylsulfonate. When phosphate concentration isgreater than alkylsulfonate concentration, the retention cannotbe increased and tailing is observed.

The effect of the mobile-phase buffer pH on the separation

of neomycin (8), streptomycin, and dihydrostreptomycin

(58, 61) has been investigated at pH 3–6 with octane (8) or

hexane sulfonate (58, 61) as counterion. Neomycin is posi-

tively charged at pH < 5, and pH changes do not affect its re-

tention. Thus, separation depends mainly on the sulfonate

concentration (8). When hexane sulfonate is the ion-pairing

reagent, streptomycin and dihydrostreptomycin cannot be

separated at all at pH 6. Only when pH is reduced to 3 or less is

satisfactory separation observed (61). However, another re-

port concludes that pH 6 is optimal for separation (58).

The pH and the ion strength of the injected sample have aneffect on IP results (17, 25, 54, 57, 62). To achieve the best re-sults, the sample should be dissolved in the mobile phase priorto injection (54, 57). When the sample is injected in basic so-lution after sample preparation, split peaks of paromomycin(54), neomycin (54), and gentamicin (57) are observed, result-ing in nonuniform gentamicin chromatography (62). Theinhomogenities, caused by sample addition into the mobilephase, move more slowly than the solvent front and result innonuniform retention of the sample components (57). Toavoid this effect, ion-pair concentrate and mobile-phase bufferare added to the sample before injection. Improved peakshape, separation, and repeatability are recorded (54, 57). Ap-parently, the effect varies among compounds. For example,using pentafluoropropionic acid (PFPA) as a counterion, sul-furic acid, HCl, and trifluoroacetic acid (TFA) causes peaksplitting but formic acid does not (17).

All IP methods for neomycin concern separation of theunderivatized drug (8, 25, 51, 53, 54). Neomycin stereoisomersB and C have been separated with a polymer column with octanesulfonate as counterion (8). Neomycin B (25, 51, 53, 54) andparomomycin (54) have been analyzed with pentane sulfonateand Na2SO4 in the mobile phase.Separation is done with a C8

column and a methanol–acetate buffer (1.5 + 98.5) mobilephase. The life span of the C8 column is limited with this mo-bile phase (53). Column stability can be improved by increas-ing the proportion of methanol to 18%, reducing the concen-tration of Na2SO4, and changing the column to C18. Thesechanges improve retention time repeatability (53). However,the C18 column cannot be used for simultaneous analysis ofmultiple aminoglycosides.

Derivatization affects separation of gentamicin compo-nents with hexane sulfonate as counterion (59). Precolumnderivatization of gentamicin results in separation of 4 compo-nents, whereas only 3 components are separated withpostcolumn derivatization. Similar results are achieved withother ion-pair reagents. Postcolumn derivatization usually re-sults in 3 gentamicin peaks when pentane or octane sulfonatesare used (50, 57, 62), but 4 peaks also are observed (52). Fourgentamicin components can be separated after precolumnderivatization when hexane or heptane sulfonate is used asion-pairing reagent (59, 63–66). The elution order of theOPA-derivatized gentamicins is C1, C1a, C2a, and C2. The sep-aration of 3 gentamicin components is achieved with heptanesulfonate as counterion (11, 67, 68). The elution order of thecomponents is C1, C1a, and C2 when the stereoisomers C2aandC2 elute together.

Simultaneous elution of all gentamicin components is ad-vantageous when a quantitative and sensitive method is re-quired. Simultaneous elution has been achieved with the useof camphor sulfonate as the ion pair and methanol as the or-ganic solvent in the mobile phase (5). Use of acetonitrile in-stead of methanol results in gentamicin eluting as 3 peaks.When camphor sulfonate is the counterion and EDTA isadded to the mobile phase, the elution order of theaminoglycosides studied is amikacin, kanamycin, gentamicin,and neomycin (5). Dihydrostreptomycin elutes in the solventfront. The elution order coincides with the order of increasingamount of amino groups.

Octane sulfonate, when used as a counterion with2,2-ethane disulfonate in gentamicin analysis, causesgentamicin to elute after compounds with fewer aminogroups. The use of 1,2-ethane disulfonate increases the sepa-ration of gentamicin components (62). Toluene sulfonate hasbeen used as a counter ion for sisomicin, netilmicin, andtobramycin analysis, providing selective separation and lack-ing interference from other aminoglycosides (14).

Perfluorocarboxylic acids have been used as volatileion-pairing reagents in the analysis of aminoglycosides(15, 49, 69, 70). The applicabilities of the different fluori-nated counterions for aminoglycoside analysis have beencompared, and the separation mechanism has been investi-gated in detail (15, 69, 70). Inchauspé and Samain (15) con-cluded that PFPA and heptafluorobutyric acid (HFBA), de-spite their short carbon chains, are suitable as aminoglycosidecounterions, except for gentamicin for which only poor sepa-ration could be achieved. TFA is especially selective togentamicin because other aminoglycosides are not retained onreversed-phase columns with TFA (15, 69, 70). The retentionof gentamicin depends on the concentration of TFA, and base-line separation of the different gentamicin components can beachieved (15). With camphor sulfonate as counterion,gentamicin does not separate at all. But with HFBA,gentamicin separates as a wide peak, and with PFPA, the dif-ferent components are partially separated (69). For otheraminoglycosides, TFA, when used with HFBA or PFPA,serves as an inorganic salt reducing the formation of ion pairsand decreasing kN (70). HFBA and TFA have opposite effects,


and therefore, the chromatographic mechanisms of these 2 ionpairs are believed to be different (70). Inchauspé et al. as-sumed that the cause for the ability of TFA to selectively re-tain gentamicin is the unique methyl group inα-position rela-tive to an amino group in the gentamicin structure, allowingsimultaneous interaction of the amino group with the ion pairand the methyl group with the hydrophobic column phase(69). However, the methyl group can have an effect only whenshort-chain counterions are used (69). When HFBA andPFPA are compared, PFPA can be considered better becauseit does not adsorb to the stationary phase as strongly as HFBAand retention is clearly through an ion-pair mechanism. Thebest reagent combination is TFA and PFPA (70).

Simultaneous separation of 5 aminoglycosides has beenachieved by IP with PFPA as counterion (17). Theaminoglycosides are streptomycin, dihydrostreptomycin,tobramycin, gentamicin as its 4 components, and neomycin.Detection and identification are by MS. Gradient elution isneeded for satisfactory separation within reasonable time.With an ordinary C18 column, both gentamicin and neomycinpeaks tail. YMC basic-column improves the peak shapes.

Triethylamine (TEA) is the only positively chargedcounterion used for IP separation of aminoglycosides ingentamicin (71), tobramycin, and netilmicin (33) analyses.Gentamicin has been analyzed with a mobile phase containingphosphatebuffer at pH 6.2 (71). EDTA and TEA in the mobilephase are compared. Alteration of the reagent does not changeretention times, but separation and column stability are betterwith TEA. Basic tris-buffer–methanol (pH 7.9) mobile phase intobramycin and netilmicin analysis causes netilmicin to elute firstand the compounds separate well (33). Removal of tris-bufferfrom the mobile phase results in a very broad netilmicin peak.The chromatographic pattern is improved by TEA, eliminatingpeaks of the blank samples (33). Attempts to analyze gentamicinwith this method have been unsuccesful because of the lack of re-tention (33). The effects of analyte charges and of pH on ion-pairformation and retention are not discussed.

Ion-Exchange Chromatography

Ion-exchange chromatography (IE) should be a popularmethod for analysis of polar aminoglycosides that are ionizedin solutions. However, IE requires careful regulation of pH,temperature, and ionic strength, and it has not become widelyused in aminoglycoside analysis.

One of the earliest chromatographic methods foraminoglycoside analysis is anion-exchange chromatography(72–74). Kanamycin (72), paromomycin (72), and neomycin(72–74) have been analyzed with a column containing astrongly basic Dowex 1×2 anion exchanger and water elution.After separation, aminoglycosides have been detected as theirninhydrin derivatives (72, 73) or polarometrically (74). Theauthors assume that separation is based on adsorption ofanalytes to the ion-exchange resin according to their molecu-lar weight/amino group content ratio (72). Neomycins B andC; kanamycins A, B, and C; and paromomycins I and II can beseparated (72). Tobramycin also has been analyzed with apolymeric CarboPac PA1 anion exchanger and NaOH gradi-

ent for elution (75). Pulsed amperometry is used for detection.The aminoglycosides are like other aminosugars, anionic athigh pH and retained to anion exchangers (75).

Cation exchange has been used to separate tobramycin(76), gentamicin (77), and kanamycin (78). With tobramycin,cation exchange serves primarily as a concentrating methodbefore the main separation in a polystyrene column (76).Tobramycin is adsorbed to the column from phosphate buffer(pH 5.2) and elutes with 0.25M NaOH. Gentamicin has beenanalyzed with a strong-cation exchanger (Partisil SCX) andelution with acetonitrile–phosphate buffer after precolumnderivatization with fluorescamine, but the different compo-nents are not separated (77). The fluorescamine derivativeshave a free carboxyl group. Therefore, an attempt has beenmade to analyze gentamicin by anion exchange at pH 6–8. Awide asymmetric peak indicates the presence of unreactedamine groups. These amino groups are positively charged andcan be adsorbed to cation exchangers. Fluorescamine andOPA have been used as alternative postcolumn derivatizationreagents for analysis of kanamycin with an SCX (78).

A cellulose phosphate PII column has been used to sepa-rate gentamicin components and analyze the purity of the drug(10). Tobramycin, neomycin, sisomicin, kanamycin, andamikacin have been separated with an Amberlite IRC 50 col-umn (79). Aminoglycosides have been eluted with sulfuricacid and postcolumn derivatized with dihydrolutidine (79).

Normal-Phase Chromatography

Normal-phase chromatography (adsorption chromatogra-phy) is usually used for simple compounds that are not ionizedand that dissolve readily in organic solvents. Thehydrophilicity and poor solubility in organic solvents ofaminoglycosides makes normal-phase chromatographic anal-ysis impossible without derivatization. However, nor-mal-phase chromatography has been used for analysis ofaminoglycoside drug preparations when separation ofstereoisomers was of interest. The aminoglycosides arederivatized prior to separation.

Isocratic methods for simultaneous analysis ofneomycins B and C and neamine have been developed(80–82). Simultaneous analysis was achieved with neomycinderivatized with nephthalene sulfonyl chloride (NSCl; 81) orFDNB (80, 82). Helboe and Kryger (80) stated that in analysisof dinitrophenyl derivatives of neomycin1,2-dichloroethane–methanol–water–diethylamine elutiongives better and faster separation than chloroform–THF–wa-ter elution. Three gentamicin components and kanamycin canbe analyzed by normal-phase chromatography with NSClprecolumn derivatization (81), and 3 gentamicin components,paromomycin, and kanamycin can be analyzed as theirdinitrophenyl derivatives (82). When normal- and re-versed-phase separations of dimethyl–phenyl derivatizedamikacin are compared, the normal-phase method is moresensitive, although retention time variations are higher and re-peatability is poor (39).


Detection Methods in Aminoglycoside Analysis

In GC, aminoglycosides have been determinated with eitherFID or ECD. ECD is used mainly for fluorinated aminoglycosidederivatives. Because GC methods are few, HPLC detectionmethods are the primary focus of this discussion.

UV–Vis and refractive index detectors are popular HPLCdetectors. Refractive index detectors are generally applicable,but they are sensitive to pressure and temperature changes.They also lack selectivity and sensitivity. However, refractiveindex detection is good for preparatory methods when samplederivatization is impossible and concentrations are high(15, 70). Refractive index detectors have been used mainly forpreparatory separations of tobramycin, kanamycin, amikacin,sisomicin, streptomycin, neomycin, and gentamicin (15, 70).However, refractive index detection is too sensitive tochanges in the environment for kanamycin analysis (78).

Aminoglycosides lack UV-absorbing chromophores andhave to be derivatized to gain UV detectability (12). In processcontrol, when fast and specific chemical assay is needed, UVdetection at 195 nm has been used for streptomycin anddihydrostreptomycin (58, 61). It has been assumed that themethod would be applicable also to neomycin andparomomycin (58).

Optical rotation has been used to detect nonderivatizedaminoglycosides (10, 74, 83). Neomycin components A, B,and C are all optically active (74), and gentamicin includes7 optically active components, of which C1, C1a, C2a, and C2

are biologically active (10). The optical activity allows theirdetection with polarimetry. The advantages of polarimetryover other direct detection methods are selectivity and sensi-tivity. Polarimetry has been considered a promising methodfor aminoglycoside applications (83).

Derivatization in Detection Enhancement

Derivatization facilitates extraction, analysis, and identifi-cation of poorly soluble and ionized compounds and improvessensitivity. In LC, derivatization can be performed pre- orpostcolumn and on- or off-line. Most derivatizations improveUV, fluorescence, or electrochemical detectability. Fluo-rescing derivatives usually are more desirable because fluo-rescence detection generally provides better sensitivity andselectivity than UV absorption.

Aminoglycosides lack chromophores and fluorescence,making derivatization essential for their UV detection (84).Aminoglycosides have several primary amino groups and,therefore, are easily derivatized (14). Partial derivatization ispossible (44). The reactive hydroxyl groups further increasethe possibility to form different derivatives (37). The reagentsfor aminoglycoside derivatization are presented in Table 6.

In addition, a reagent called dihydrolutidine has been usedto derivatize neomycin, sisomicin, kanamycin, tobramycin,and amikacin (79). Dihydrolutidine derivatives are formedwhen the amino group of the aminoglycoside reacts with theketo and enol forms of acetylacetone. The derivatives are fluo-rescent and can be detected with an excitation wavelength of421 nm and an emission wavelength of 488 nm.

The most widely used reagent is OPA, which reacts in thepresence of mercaptan or other strong reducing agents in basicconditions with primary amines to form fluorescent deriva-tives (85). Its popularity is due to the fast rate of the reaction atroom temperature and the possibility to perform the reactionin water solution (31, 78). In addition, OPA is stable in differ-ent buffers (78). Borate buffer (pH 9.5–10.5) has been usedpredominantly with OPA derivatization of aminoglycosides.In fluorescence detection, the excitation wavelength generallyis 340 nm, and the emission wavelengths, 450 (86, 87),430 (52), 440 (5), 418 (50, 56, 57, 67), 448 (68), or 455 nm(25, 51, 53, 54). Other excitation wavelengths used are 350,360, and 365 nm, and the corresponding emission wave-lengths are 450 (13, 44), 430 (32), and 440 nm (62). Back-ground noise is lowest with an excitation wavelength of260 nm and an emission wavelength of 340 nm (71). UV ab-sorption at 330 (59, 63–66), 350 (11, 82), or 254 nm (82) isalso used. The first reported OPA derivatization foraminoglycosides is postcolumn derivatization of kanamycin(78). The reducing agent is mercaptoethanol and the basicbuffer is borate.

In postcolumn derivatization of aminoglycosides, OPAusually has been used with mercaptoethanol(5, 13, 50, 54, 56, 57, 62, 78) in borate buffer at pH 9.5–10.5.Brij-35 has been used to prevent polysulfide precipitation inthe detector (13). Triton-X-100 in lieu of Brij-35 enhances flu-orescence (5, 56). However, Triton-X-100 is more tempera-ture sensitive than Brij-35 (5).

Derivatization temperature, reactor geometry, and flowrate of the derivatization reagent have been optimized(5, 54, 56, 57). The results are similar for the differentaminoglycosides tested. Reactor geometry affects both sensi-tivity and separation (56). The reactor coil length is usually1–2 m, but to separate gentamicin components properly, thecoil must be less than 2 m (57). Resolution of amikacin sufferswhen the coil is more than 1 m (56). Reducing the internal di-ameter of the coil increases sensitivity (13, 57).

The most common reaction temperatures for OPApostcolumn derivatization are 45E and 50EC. The effect ofreaction temperature has been investigated in the range 20E

to 75EC (5, 56). Best results are obtained for gentamicin (5)and tobramycin (56) at 45EC, for amikacin at 50EC (56), andfor neomycin at 33EC (51). Partial derivatization occurs withamikacin at temperatures below 50EC, and fluorescenceyield decreases at reaction temperatures over 60EC (56).This result has led to the assumption that fluorescence is tem-perature dependent.

Reagent flow affects the observed fluorescence (5, 56, 57).However, the optimum reagent flow rate depends on otherchromatographic parameters as well, and no absolute valuescan be determined. Some authors have stated that the optimumflow rate is half the flow rate of the mobile phase (54). Opti-mal flow rates ranging from 0.30 to 0.55 mL/min have beenreported (5, 56, 57). If the flow is too high, the eluate is dilutedto such an extent that the signal becomes too weak (56).Gentamicin components react differently to changes of re-agent flow (57), which does not affect the fluorescence of C1a



Table 6. Reagents used for aminoglycoside derivatization a

Aminoglycoside Derivatization reagent Detection method References

ami OPA fl 26, 44

OPA pc fl 56, 57, 102

FDNB UV 38, 39

TNBS UV 40, 41

dib OPA fl 26

gnt OPA fl 26, 31–33, 59, 67, 68, 71

OPA UV 11, 59, 63–66

OPA pc fl 5, 13, 50, 52, 57, 62

FDNB UV 29, 30, 82

FMOC–Cl fl 34

BSC UV 27

Fluorescamine fl 77

NSCl UV 81

Dansyl chloride fl 28

DNBCl UV 35

TNBS UV 40

kan OPA pc fl 5, 13, 78

FDNB UV 38, 82

NSCl UV 81

TNBS UV 41

Fluorescamine fl 78

Ninhydrine UV 72

neo OPA pc fl 5, 13, 25, 51, 53, 54

OPA fl 45

FDNB UV 80, 82

NSCl UV 81

Ninhydrine UV 72, 73

ntl OPA pc fl 13, 14, 50

OPA fl 26, 33, 86

OPA UV 65

BSC UV 27

Dansyl chloride fl 43

par OPA pc fl 54

FDNB UV 82

Ninhydrine UV 72

sis OPA pc fl 13, 14, 50

OPA fl 26, 86, 87

FDNB UV 30

TNBS UV 40, 42

str OPA pc fl 54

NQS fl 60, 88, 89

dhs OPA pc fl 54

NQS fl 60, 89

tob OPA pc fl 56

OPA fl 26, 33

FDNB UV 30, 36, 37

TNBS UV 40, 42

a For definitions, see footnotes to Table 4.

but increases the fluorescence of C1 and C2 when flow rate isincreased. This result suggests that the reaction of C1ais fasterthan the reactions of the other 2 components.

A weakness of postcolumn derivatization is the increase ofbaseline noise as a consequence of reagent pumping (26, 71).Reagent consumption is also greater than with precolumnderivatization, and an extra pump is needed for thederivatization reagent. Improving the sensitivity inpostcolumn derivatization is difficult because derivatizationtime can be increased only by increasing the dead volume ofthe system, which, in turn, results in broadening of bands.

Precolumn OPA derivatization of aminoglycosides alsohas been reported (31–33, 44, 45, 64, 66–68, 86, 87). Advan-tages are simplicity of equipment and effective separation ofinterfering compounds (32).

Different mercaptans, such as mercaptoethanol(31–33, 44, 45, 67, 68), mercaptoacetic acid (11, 59, 63–66),and mercaptopropionic acid (86, 87) are used in precolumnOPA derivatizations. However, information concerning theirapplicability is contradictory. Mercaptoethanol has beenwidely used and other reagents have been compared to it.Nonetheless, mercaptoethanol–OPA derivatives are not stableand, therefore, unsuitable for precolumn derivatization (87).OPA derivatives of aminoglycosides are stable for 8 h at–20EC (33), and OPA derivatives of neomycin are formedeven during the first 5 min of freezing (45). Whenmercaptoethanol–OPA derivatives are kept at –4EC, the fluo-rescence yield decreases 5% in 1 h (33). At room temperature,the derivatives decompose exponentially, causing a 30% de-crease in fluorescence in 10 min (78).

Mercaptopropionic acid is used for OPA derivatization ofsisomicin and netilmicin when better stability of derivatives isneeded (86, 87). The steric group in mercaptopropionic acid isbigger than that in mercaptoethanol, and this is believed to im-prove the stability of the derivatives (87). The solution wherederivatives are kept also affect stability (86). Unlike what hap-pens with mercaptoethanol, the extra mercaptan does notdestabilize the derivatives when mercaptopropionic acid isused (87). A reaction time of 10 min is satisfactory at 60EC,but at 20EC, 1 h isneeded. Partial derivatization of sisomicinresults in formation of 3 different derivatives. The reaction isaccompanied by interfering side reactions.

Mercaptoacetic acid–OPA derivatization of gentamicin(11, 63–66) and netilmicin (63) is performed either at 90EC(64) or at 60EC (66) for 15 min. The derivatives are more sta-ble than those formed with mercaptoethanol (65), but they de-compose under acidic conditions (63). A 16–43% decomposi-tion occurs during the first hour after derivatization ifderivatives are kept in acidic buffer or in the mobile phase(63). This decomposition does not occur in basic solutions.Gentamicin yields 4 derivatives with mercaptoacetic acid,compared with 3 with mercaptoethanol. Detector responses ofcomponents are different. The relationship between responseand number of derivatized groups is not discussed.

Only mercaptoethanol has been used as reducing agent inOPA derivatization of aminoglycosides within the precolumn.Silica (32, 44, 67, 68) and Amberlite CG-50 ion exchange

(45) precolumns have been used. The technique has been usedfor amikacin (44), gentamicin (32, 67, 68), and neomycin(45). The stability of derivatives is not discussed, except thatneomycin needs to be frozen for complete derivatization (45).

Detector responses of OPA derivatives of the differentgentamicin components have been compared and results dif-fer. On the basis of the number of derivatized groups, the re-sponses of C1aand C2 should be equal and that of C1 smaller.Same responses indeed are reported for C1aand C2, but the re-sponse is much smaller than that recorded for C1 (71). Anotherstudy finds equal C2 and C1 responses but a smaller C1a re-sponse (32). Moreover, different detector responses are re-ported for all the components (26, 66). Reaction kinetics andreasons for the observed differences are not discussed. Fur-thermore, most authors use a gentamicin standard that con-tains an unknown mixture of the components. Consequently,identification of components is not based on individual stan-dards. The variation in detector responses indicates that differ-ent derivatives are formed with the various methods, and theyprobably also affect the elution order. However, the same elu-tion order is suggested in different reports.

In partial OPA derivatization of amikacin (44) andneomycin (45), 2 peaks are observed instead of one forneomycin, and several peaks are produced for amikacin if thederivatization solution is not heated. Discussion of partialderivatization has been limited, and most authors do not takethis possibility into account.

FDNB reacts with primary and secondary amines in basicconditions (36), producing highly UV absorbing derivatives(37). The UV absorption maximum is at 365 nm. FDNB hasbeen used for derivatization of almost all aminoglycosides(29, 30, 36–39, 80, 82), and a comprehensive study of its re-action kinetics with aminoglycosides has been published withtobramycin as model compound (37). Derivatization condi-tions have been developed with borate (39, 80, 82) or tris(29, 30, 36, 37) buffers. Phosphate, phthalate, acetate, and bi-carbonate buffers also have been investigated (37, 39). FDNBreacts with the amine or hydroxyl groups of most buffers, pro-ducing compounds that interfere with separation and detection(39). However, FDNB does not react with tertiary amines.Thus use of such buffers seems the preferred approach. Themain problem of FDNB derivatization of aminoglycosides isthe different solubilities of the reagent and the analytes (82).Organic solvents must be used in the derivatization solution todissolve FDNB. Derivatization buffers, organic solvents, tem-peratures, times, and pH conditions have been varied to opti-mize derivatization conditions. Also a big problem withFDNB is its toxicity (37). FDNB irritates the skin and maycause blistering dermatitis and severe allergic reactions. Ab-sorption of FDNB by inhalation, ingestion, or through the skincan be lethal. The different methods used for aminoglycosidederivatization with FDNB are presented in Table 7.

Tobramycin analysis has revealed that yield and speed ofthe FDNB reaction, as well as occurrence of several side reac-tions, are strongly pH dependent (37). Derivatization oftobramycin amine groups and of hydroxyl groups thereafterfollow second-order kinetics. Hydrolysis of FDNB in basic


solutions follows pseudo first-order kinetics and is faster themore basic the derivatization solution is. Hydrolysis of thetobramycin derivative decreases with increasingderivatization temperature, but increasing the solution pH re-sults in faster dissociation. The optimum pH range of the reac-tion is very narrow because of the high reactivity of theaminoglycoside hydroxyl groups with FDNB and thepH-dependent dissociation of the derivatives (37).

Best results for gentamicin, sisomicin, and tobramycinderivatization with FDNB have been achieved with tris bufferat pH > 9.5 and 80EC (29). Higher reaction temperature de-creases the repeatability, and reaction time of 90 min results inincomplete derivatization (29). Molar absorptivity and ele-mental analysis have confirmed derivatization of all aminogroups of tobramycin (36). Gentamicin derivatization is moresensitive to derivatization conditions than tobramycin.

Proton nuclear magnetic resonance spectrometry of theamikacin–FDNB derivative indicates that amino groups arederivatized (38, 39). Derivatization yields of 92% for standardsand 77% for serum have been achieved with NaOH (39).NaOH gives slightly higher yields than borate buffer (38). Inor-ganic salts such as Na2SO4 interfere with derivatization.

Side products of aminoglycoside derivatization withFDNB in tris buffer include 1-hydroxy-2,4-dinitrobenzene(the reaction product of FDNB and water),2-[N-(2,4-dinitrobenzene)amino]-2-hydroxymethyl-1,3-propanediol (the reaction product of tris and FDNB), and2,4-dinitrophenyl, the hydrolysis product FDNB (36). Gen-erally, these side products can be seen in chromatograms, andthey may interfere with analysis.

FMOC–Cl is a very reactive derivatization reagent, react-ing with both primary and secondary amines (34). The reac-tion is very fast in basic solutions at room temperature. Thederivatives are very stable and fluorescent. However, duringderivatization FMOC–OH is formed, which can interfere withchromatography. For gentamicin derivatization (34), the bestderivatizating solution is borate–acetonitrile (1 + 1) atpH 7.5–9.0. Acetonitrile is needed to dissolve FMOC–Cl.Derivatization is complete in 10 min. The detector responsesof the FMOC derivatives of gentamicin components are equal.

Naphthoquinone sulfonate (NQS) reacts with guanidinecompounds in basic conditions to produce fluorescent deriva-tives (88). It is suitable for derivatization of streptomycin anddihydrostreptomycin. NQS decomposes rapidly in basic solu-tions. Therefore, it is not suitable for postcolumnderivatization in a basic environment. Postcolumnderivatization with NQS would require 2 extra pumps, one forNQS and one for the base. NQS has been added as a solutionto the acidic mobile phase before separation (88). Optimalconditions for streptomycin analysis include a 5 mpostcolumn reactor coil at 65EC, NQS concentration of0.4 mM, and NaOH concentration of 0.5M (88, 89). Ahigh-quality postcolumn system is essential for maintainingbaseline stability and constant reactor temperature (89). Forfluorescence detection, excitation wavelengths of 347, 351,and 365 nm have been used with emission wavelengths of418, 420, and 418 nm, respectively (60, 88, 89).

TNBS reacts with primary amines in basic aqueous solu-tion at room temperature without interfering side reactions(41, 42). The molar absorptivity of the TNBS derivatives ishighest at 340 nm. TNBS has been used to derivatizegentamicin (40), tobramycin (42), and amikacin (41) withkanamicin (41) and sisomicin (42) as internal standards. Theaminoglycosides are dissolved in acetonitrile to speed up thederivatization and to dissolve the nonpolar derivatives (41).At room temperature, the reaction requires 19–20 h (42). In-creasing the temperature improves the results, and 70EC isbest temperature for derivatization (41, 42). At lower temper-atures, several derivatives are formed when derivatizationtime is less than 30 min. At temperatures higher than 80EC,tobramycin derivatives decompose (42). TNBS derivatizationof gentamicin is complete in 15 min at 70EC (40). Thetrinitrophenyl derivatives have absorption maxima at 350 and420 nm and give different detector responses.

TNBS derivatization of amikacin and tobramycin is af-fected by the pH and the buffer capacity of the solution(41, 42). At pH < 9, the reaction is not complete, and at pH >10, the reagent decomposes. Derivatization, therefore, is per-formed within pH 9.5–10.0. Carbonate, borate, and phosphatebuffers are not satisfactory because of their low buffering ca-pacities in this pH range and solubility problems resulting in


Table 7. Reaction conditions for FDNB derivatization of aminoglycosides a

Aminoglycoside Solvent Buffer Temperature, EC Time, min References

gnt, tob ACN Tris pH 9.0 80 45 29, 36

gnt, sis, tob ACN Tris pH 9.0 80 45 30

tob Ethylisopropylamine Tris pH 8 70 ? 37

neo MeOH Borate 0.02M pH 9 60 60 80

ami MeOH NaOH 100 45 38

neo MeOH Borate 0.02M pH 9 100 45 82

ami MeOH Borate 0.1M pH 9.3 80 30 39

a For definitions, see footnotes to Table 4.

complex formation with aminoglycosides. Therefore, trisbuffer produces the best results (41, 42).

Fluorescamine reacts rapidly in a basic aqueous solution atroom temperature with primary amines to produce stronglyfluorescent derivatives (90). The reagent itself and its degra-dation products are not fluorescent. Kanamycin (78) andgentamicin (77) have been derivatized with fluorescamine.For kanamycin, postcolumn derivatization in acetone hasbeen used (78). The derivatives are stable, and the sensitivityof the method is good. For precolumn derivatization ofgentamicin, best results are achieved with 0.03M or strongerphosphate buffer at pH 7.2–8.0 (77). The fluorescamine con-centration must exceed the gentamicin concentration by10 times. The fluorescence responses of the differentgentamicin components are equal, and the method is accurateand repeatable.

Dansyl chloride has been used for precolumnderivatization of netilmicin (43) and gentamicin (28, 91).Dansyl chloride reacts with amines to form fluorescent deriva-tives (92). The derivatization solution for aminoglycosides isacetonitrile–phosphate buffer at pH 11 (28, 43, 91). Reactiontemperature and time are 75EC and 5 min, respectively. Buffervolume, pH, reaction temperature, and time affect results (28).At 100EC, the derivatives decompose rapidly, and at low tem-peratures, reaction is not complete. The possibility of partialderivatization of gentamicin has been investigated but couldnot be detected (28). Furthermore, dansyl chloride reagentsfrom different producers differ in solubility, and a too lowdansyl chloride amount gives incorrect results (91).

DNBCl has been used to derivatize gentamicin,tobramycin, kanamycin, and amikacin (35). Derivatives aredetected with UV at 254 nm. The reaction is repeatable, andthe derivatives are stable. However, an unidentified derivativeis formed with gentamicin.

BSC has been used to derivatize netilmicin andgentamicin (27). Derivatization must be at pH 7–9 and 75ECfor 10 min. Too high a temperature or too long a reactiontime destroys the derivatives.

NSCl reacts by nucleophilic substitution with hydroxyl andamine groups, forming UV-absorbing (254 nm) derivatives(93). Neomycin, gentamicin, and kanamycin have beenderivatized with NSCl at pH 8.0–9.0 and 100EC for 10 min(81, 93). Phosphate buffer is best for NSCl derivatization ofaminoglycosides.

It is difficult to compare the different derivatization re-agents used in aminoglycoside analysis because most re-ports do not give derivatization yields and method sensitivi-ties. Fluorescent derivatives do not seem to be detectable atlower concentrations than UV-absorbing derivatives, aswould be expected.

Postcolumn derivatization with OPA appears preferablewhen analysis speed is critical because the derivatization timeis negligible. However, because of the limitations ofpostcolumn derivatizations, use of an OPA precolumnderivatization could be a better option when reaction time is0–15 min. Other fast-reacting reagents are FMOC–Cl, dansylchloride, and BSC, which all react in <10 min. The disadvan-

tage of dansyl chloride is that the derivative has to be extractedfrom the derivatization solution before chromatography.Dansyl chloride has been applied to gentamicin (28) andnetilmicin (43); FMOC–Cl, to gentamicin (34); and BSC, togentamicin and netilmicin (27). No studies about the applica-bility of these reagents to other aminoglycosides have been re-ported. In terms of derivatization time, FDNB is the mostproblematic reagent because the reaction requires 30–60 min.Long incubations may be a problem also for analyte stability,and some losses may occur.

Buffer selection is not critical because all reagents require abasic environment. FDNB reagent is the most limited in termsof buffer selection because of its side reactions with buffer ionsand its narrow pH optimum for the reaction. The limited solu-bility of aminoglycosides demands use of reagents that are sol-uble in water or aqueous solutions of methanol or acetonitrile.The most problematic reagents in terms of solubility are dansylchloride and FDNB. The dinitrophenyl derivatives ofaminoglycosides also are poorly water soluble. OPA has thebest solubility characteristics among derivatization reagents.

The stability of the aminoglycoside derivatives has beensubject to limited investigation. Because most reagents areused for precolumn derivatization, the stability of derivativesis very important in consideration of the best derivatizationoption especially when automatic equipment is not availableand quantitative results are needed. Furthermore, the struc-tures of aminoglycoside derivatives formed with different re-agents have been inadequately investigated. The number ofderivatized groups is rarely specified. When several differentderivatives are formed, attention should be paid to thederivatization yield and to the number of different derivativesformed. Of the reagents used for aminoglycosidederivatization, only FDNB has been investigated with respectto derivatization kinetics and the structures of theaminoglycoside derivatives formed.

Electrochemical Detection

Amines, thiols, and hydroxyl groups oxidize readily atelectrodes such as the carbon electrodes used for electrochem-ical detection (55). The multiple amino and hydroxyl groupsin the aminoglycoside structures are apparently the source oftheir electrochemical activity (55). The advantage of electro-chemical detection in aminoglycoside analysis is that it doesnot require derivatization. However, the structure of the de-tected compound cannot be identified, and confirmation of aspecific chromatographic peak is not possible (16). The in-ability to do these is a particular disadvantage when retentiontimes and elution orders change.

All gentamicin components have been detected electro-chemically with a carbon electrode (16, 55). Avoltammogram measured manually for gentamicin and a de-tection voltage of +1.3 V was selected. When the detection po-tential was changed to a more positive potential, the signal in-creased but the baseline deteriorated. The detector responsefor gentamicin was linear from 16 to 30 µg with r = 0.999.Substantial changes in the detector response were observed


with changes of Na2SO4 concentration in the mobile phase. Achange from 0.2 to 0.3M reduced peak areas by 40%.

Pulsed methods for aminoglycoside detection are pre-sented in Table 8. All aminoglycosides react via a commonmechanism in pulsed amperometry (76). The sensitivity of de-tection in neomycin analysis with pulsed electrochemical de-tection (PED) improves by adding NaOH to the eluent to in-crease the pH to 13 (8). In tobramycin analysis, a high pH isrequired to oxidize tobramycin, and a gold electrode is used(75). The sensitivity of the tobramycin method (75) is similar toor better than that reported for different derivatization methods.With an injection volume of 10 µL, the limit of detection of themethod is 2 ng/injection, and the detector response is linearfrom 10 ng to 1 µg/injection with r = 0.999 (75). Stability prob-lems have been encountered when neomycin was analyzedwith PED, and specialization and experience are required fromthe analyst to achieve accurate and repeatable results (8).

Mass Spectrometry

In theory, all drug compounds can be detected by MS butthe use is limited by the complexity of the equipment and highcosts (83). Regulatory methods require confirmatory identifi-cation at the action level (94). Because of the sensitivity andpossibility for confirmatory identification, MS is often theonly method that offers the required sensitivity and selectivityfor confirmation of different analytes.

No GC/MS application for aminoglycosides has been pub-lished. Available methods have been limited to LC/MS instru-ments and to the MS analysis of pure standards. LC/MS inter-faces used in aminoglycoside analysis have been thermospray(16), moving belt (46), ion spray (17, 49), and heated pneu-matic nebulization with atomspheric pressure chemical ion-ization (APCI; 47, 48). For aminoglycoside ionization in MSanalysis, plasma (95) and field (69) desorption, chemical ion-ization (CI; 46, 96), and APCI (47, 48) have been used.

The ion spray interface (IS) is well adapted toaminoglycosides that are ionized in solutions (49). Desorptionof ions from the liquid phase to the gas phase is caused by anelectric field without heating or other severe conditions. Theadvantage of IS–MS in quantitative work is that the ion flowconcentrates one specific species, improving sensitivity. Adisadvantage of IS is that the ionic strength of the mobile

phase has to be minimized while the organic solvent content ismaximized. This is a limitation especially for aminoglycosides,which are usually analyzed by IP. For this reason, the concen-tration of the ion-pair reagent must be carefully optimized.However, the counterion concentration does not have signifi-cant effects on mass spectra (49). Gentamicin, neomycin,tobramycin, streptomycin, and dihydrostreptomycin have beenanalyzed with LC/IS–MS (17, 49).

Better sensitivity has been obtained with positive-ion thanwith negative-ion monitoring when IS–MS is used inaminoglycoside analysis. Thus, positive-ion spectra havebeen investigated in more detail (17). Selected-ion monitoringdoes not provide sufficient selectivity for complex matrixes,but selectivity and sensitivity can be improved with MS/MS.For these reasons, MS/MS appears especially suitable forconfirmative analysis. With limited heating in IS, only a fewfragments can be observed. Collision-induced activation(CAD) allows formation of daughter ions from various ions ofthe spectra, enabling confirmation of the structure (17). InMS/MS, use of singly charged parent ions of aminoglycosidesprovides a lower signal-to-noise (S/N) ratio in thechromatograms than use of doubly charged parent ions, partlybecause of the smaller absolute intensity of singly chargedspecies and partly because of the mutual repulsion ofprotonated groups in doubly charged species. In the massspectra of aminoglycosides, doubly charged species domi-nate, and single or triple charging rarely occurs (17). How-ever, when the ionization potential is increased, the quantity ofsingly charged ions increases (17, 49).

With heated pneumatic nebulization systems, APCI is usu-ally preferred (47, 48). LC–APCI is generally applicable fornonvolatile compounds that are unsuitable for GC analysis.The main advantage of APCI–MS is that it can be performedunder usual HPLC separation conditions with flow rates of0.3–2.0 mL/min (47). The most important parameters to beoptimized in analysis of nonvolatile compounds are the tem-peratures of the nebulizer and vaporizer (48). Kanamycin(47, 48) and gentamicin (48) have been detected byLC–APCI. The mass spectra of gentamicin components aredifferent from each other in APCI–MS (48). The kanamycinisomers A, B, and C can be separated by LC–APCI, and themass spectra of the different components are also different.


Table 8. Pulsed electrochemical methods for aminoglycoside detection a

MethodAmino-

glycosideWorkingelectrode

Counterelectrode E1, V E2, V E3, V t1, ms t2, ms t3, ms NaOH, Mb Ref.

PAD tob Pt, rotating disc Ag/AgCl 0.55 0.7 –0.9 250 125 425 0.25 76

PAD str, dhs Au Not defined 0.1 0.6 –0.8 480 120 300 0.3 49

PAD tob Au Not defined 0.1 0.6 –0.8 300 120 300 0.5 75

PED neo Au Ag/AgCl 0.05 0.75 –0.15 400 190 390 0.5 8

PED tob Pt, rotating disc Ag/AgCl 0.7 –1.3 –0.2 125 125 400 0.25 76

a For definitions, see footnotes to Table 4.b Concentration after column.

Thermal decomposition of several aminoglycosides has beenobserved in LC–APCI interfaces (17). APCI spectra resembleCI spectra but with less fragmentation. The limited fragmenta-tion frequently offers insufficient structural information (48)and, therefore, CAD is used with APCI–MS to obtain struc-ture-confirming fragment ions. Most ions obtained with CADalso can be seen in the original APCI spectra.

The thermospray interface (TS; 75) has been used forgentamicin analysis (16). Three gentamicin components (C1,C1a, and C2) and another component assumed to begentamicin C2ahave been detected. Instead of strong molecu-lar ions [M + H], gentamicin components produce high in-tensity fragments with TS–MS. Identification of the individ-ual components is possible on the basis of the mass spectralinformation, and changes in elution order can be detected im-mediately. The greatest advantage of the MS detection apartfrom identification is the lack of need for derivatization. In ad-dition, the sensitivity of LC–TS for gentamicin is much betterthan the sensitivity obtained with an electrochemical orUV–Vis detector (16).

The suitability of moving-belt interfaces foraminoglycoside analysis has been investigated (46).Kanamycin, tobramycin, and neamine have been studied withchemical ionization with ammonia. Different belt types havean effect on the spectra. The molecular weight and the order ofamino sugars in the molecule can be determined for allaminoglycosides studied with this method (46).

Plasma desorption MS (PD–MS) yields useful informationabout the structure of nonvolatile or thermolabileaminoglycosides (95). The mass spectra of neomycin,kanamycin, paromomycin, tobramycin, streptomycin,dihydrostreptomycin, amikacin, netilmicin, sisomicin, andgentamicin have been determined with time-of-flight (TOF)PD–MS and positive-ion monitoring (95). This method facili-tates confirmative analysis of aminoglycosides presumablybecause the resulting spectra are very repeatable. Very strongmolecular or quasimolecular ions are obtained foraminoglycosides studied with positive-ion monitoring, butmuch more fragmentation occurs with negative-ion monitor-ing. Extraction of strongly polar aminoglycosides from bio-logical matrixes can be difficult. Therefore, more easilyextractable dinitrophenyl derivatives have been prepared, andtheir ionization and detectability have been studied. Except forderivatives of streptomycin and dihydrostreptomycin, the mo-lecular weights and mass spectra of all the dinitrophenyl de-rivatives can be recorded with positive- and negative-ionmonitoring. The best spectra are obtained with positive-ionmonitoring (95). Negative ionization produces only little frag-mentation for the derivatized aminoglycosides and [M–H]– orM− C is the main peak formed.

The molecular ion is either very weak or does not exist atall in aminoglycoside mass spectra when electron impact ion-ization (EI) is used (95). Field desorption MS (FD–MS) hasbeen used to determine gentamicin from LC fractions (69).However FD–MS is usually applicable only to molecularweight determination because practically no fragmentationoccurs (96). Thus, emitter chemical ionization (CI), a tech-

nique characterized by use of the activated FD emitter as asolid probe, has been used for aminoglycosides to obtain bothmolecular ions and characteristic fragments (96). Gentamicin,kanamycin, and dibekacin have been analyzed with ammoniaor isobutane as reagent gas. The molecular peak and 9 charac-teristic ions resulting from the dissociation of the glycosidebonds can be detected with both reagent gases. The molecularweights of kanamycin and dibekacin cannot be determinedfrom MS using conventional CI. Thus, emitter CI can be con-sidered a better method. Generally, aminoglycosides fragmentbetween the glycosidic oxygen and the anomeric carbon whenthe hydrogen is transferred to oxygen (96).

Extraction and Analysis of Aminoglycosides fromDifferent Matrixes

Pure Drug Preparations

The quality of drug preparations is continuously monitored(1) during preparation by the producer and later in quality con-trol according to requirements of pharmacopoeias.Confirmative methods of analysis are preferred. When thedrug is formed from several components, limits for compo-nent ratios are given (63). Fast, specific chromatographicmethods are needed for quality control of aminoglycosidepreparations and injectable solutions (64).

Accurate methods for measuring gentamicin componentsare required because the different components apparentlyhave different toxicity (40). Quality control chromatographicmethods for gentamicin have to be in agreement with the offi-cial microbiological method (59). Compatible results betweenbioassay and chromatographic methods can be achieved onlywhen gentamicin components C2a and C2 elute together (59).Remarkable differences (up to 100%) in the ratios of differentcomponents have been found among gentamicins from differ-ent producers (11).

Gentamicin component ratio (10, 11, 13, 16, 55, 59, 63,64) and stability (66) have been extensively studied. Occa-sionally, when gentamicin preparations are analyzed, unsub-stantiated assumptions have been made. These include thepresumptions that equimolar amounts of the different compo-nents give the same detector response as OPA derivatives(59, 63, 64) or that the microbiological activities of the differ-ent components are equal (64). Some authors have used indi-vidual gentamicin components for quantitation(10, 11, 13, 55). The area response factors of the4 OPA-derivatized gentamicin components differ (11). Con-sequently, a quantitative method based on the assumption thatthe area response factors of OPA-derivatized gentamicins aresimilar gives results that are not representative of the actualcomponent ratio (11). In quantitation of the 4 gentamicin com-ponents, it is necessary to know their specific responses in thechromatographic procedure (11). Different references give di-vergent analytical results, and the component C2ahas been de-termined differently in different papers. Some authors con-sider it as an individual component (16, 63–66), while othersinclude it in the C2 component (29–33, 48, 55, 67, 68, 71),making comparison of methods difficult.


Neomycin in ointments has been analyzed by dissolvingsamples in chloroform and separating neomycin bycentrifugation (19). The neomycin is then dissolved in waterand freeze dried for GC analysis. Recovery of the method is98–100%. Dissolution of ointments in chloroform also hasbeen used for gentamicin analysis (66). Gentamicin is ex-tracted from the chloroform solution with phosphate buffer.The gentamicin in the phosphate buffer is derivatized withOPA and analyzed by HPLC. The recovery is 94–97%.

Plasma and Serum

The response of a patient to a specific drug depends on var-ious factors, such as age, sex, renal and liver function, and si-multaneous use of other drugs (97). Concentration in serumafter drug administration is often difficult to predict (33). Cor-relation between the achieved concentration in serum andpharmacological and toxicological effects is substantial, andknowledge of the concentration in serum is of great help intreatment, especially when the therapeutic range is narrow.Therapeutic drug monitoring is used frequently whenaminoglycosides or certain other drugs are used for treatment(14, 25, 29, 40, 57, 97). The methods used for monitoringaminoglycoside concentrations in plasma and serum are sum-marized in Table 9.

Because of their solubility characteristics, direct extractionof aminoglycosides from plasma is difficult and impractical(27, 28). Protein precipitation and separation of amino acidsfrom aminoglycosides prior to chromatography is necessarybecause of interference both in chromatography and inderivatization (14, 28). Interferences have been reported ingentamicin, amikacin, tobramycin (57), and streptomycin(61) analyses. For protein precipitation, trichloroacetic acid(TCA; 25), acidic or basic phosphate buffer with acetonitrile(27, 28, 33, 43, 77), basic tris buffer with acetonitrile(29–31, 36), phosphate buffer (44), or tris buffer (41, 42)alone and acid incubation (23) have been used. Contradictoryinformation is available about the applicability of differentbuffers and organic solvents for aminoglycoside extraction,and different aminoglycosides are often extracted with differ-ent buffers or solvents. TCA and tungstic acid precipitategentamicin with the proteins (28). However, >90% recoveryof neomycin from serum has been achieved with TCA to pre-cipitate proteins (25). However, neomycin recoveries fromplasma are lower than from water when TCA is used, perhapsbecause of endogenic substances in the plasma. The pH of theprecipitation buffer affects streptomycin recovery (61). Re-covery is maximum at pH 2 and decreases with increasing pH.In gentamicin analysis, ultrafiltration is useless because thecompound is adsorbed to the membrane (28).

Tris buffer is well suited for extraction of tobramycin andamikacin from serum and for protein precipitation (41, 42).However, protein precipitation is completed withTNBS−acetonitrile derivatization solution. Tris buffer to-gether with acetonitrile results in considerably lowertobramycin recovery than has been obtained for gentamicin orsisomicin (36) and also less than observed for tobramycinwith tris alone. Phosphate buffer with acetonitrile provides

better recovery for netilmicin than for gentamicin, whiletobramycin recovery decreases with decreasing phosphate di-lution volume (33). However, recoveries with phosphatebuffer and acetonitrile are higher than those with tris andacetonitrile. Preference has been given to tris buffer in precipi-tation because inorganic buffers tend to precipitate fromacetonitrile–water solutions (29).

Acetonitrile is the best organic solvent for extractinggentamicin from serum and for precipitating serum proteins(28, 77). Other water-soluble organic solvents such as etha-nol, methanol, and acetone do not provide satisfactory results(28, 77). Use of methanol for protein precipitation andgentamicin extraction from plasma has been reported (62). Di-ethyl ether, ethyl acetate, hexane, and methylene chloridehave been investigated for back extraction of the precipitationmixture (28, 77). Best results are achieved with methylenechloride (28, 77). Gentamicin recovery is poor (77), but itcould be improved by adding phosphate buffer to acetonitrile.Tobramycin recovery is also low after acetonitrile precipita-tion and cannot be improved (30).

Solid-phase extraction (SPE) has been used to extract TNBSand FDNB derivatives of aminoglycosides (39, 41, 42). Thisextraction prolongs column life and removes the large solventfront and interfering peaks. Silica SPE has been used to extractand clean up dinitrophenyl derivatives of amikacin (39). Thederivatives are eluted from silica with acetonitrile–water(68 + 32). Trinitrobenzoyl derivatives of amikacin andtobramycin (with kanamycin and sisomicin as internal stan-dards) have been extracted with a Bond-Elut C18 solid-phasecolumn (41, 42).

SPE with C18 (61) and silica (32, 44, 71) columns andCM-Sephadex C25 ion-exchange resins (14, 38, 50, 56, 57, 87)has been used to extract aminoglycosides from plasma. SPE hasbeen used without precipitation of serum proteins, andaminoglycosides have been separated from the matrix accord-ing to partition differences (32). Among organic solvents, SPEand CM-Sephadex, CM-Sephadex gives the highest recover-ies for sisomicin, tobramycin, and netilmicin (14).

Aminoglycosides are strongly basic compounds, and ionexchange can be expected to be a good sample preparationmethod. CM-Sephadex is applicable to all aminoglycosidesexcept streptomycin (14, 38, 50, 56, 57, 87). In extraction ofstreptomycin from plasma, CM-Sephadex ion exchange hasbeen considered too complicated, and interfering compoundsin plasma cannot be separated from streptomycin (61). Silicagel has been used for sample cleanup and for extraction ofgentamicin (32, 71) and amikacin (44). Gentamicin is chargedto the silica column in water-diluted serum, and amikacin, inphosphate–buffer-diluted serum. These aminoglycosideshave been derivatized with OPA in the silica column beforeelution with ethanol (32, 44) or methanol (71).

Ion exchangers with carboxylic acid functional groupshave worked well in extraction of gentamicin (34).Gentamicin is retained on a carboxypropylsilica ion-exchangecolumn at pH 6–8 and eluted at pH > 9.5. The ion-exchangemethod is simple and reliable.


Table 9. Methods used to monitor aminoglycoside levels in plasma and serum

AminoglycosideaExtraction and protein

precipitation CleanupChromatography

derivatizationbIntradayCV, %

InterdayCV, % r

Determinationlimit, µg/mL

Slope ofcalibration curve

Linear range,µg/mL Recovery, % Ref.

sis, ntl, tob* — CM-Sephadex

IP, OPA pc — 1.9–3.1 0.999 — 0.034 (sis)0.101 (ntl)

0.3–22 (sis)0.2–11 (ntl)

— 14

gnt, tob, kan* 0.5% H2SO4 incubation80EC

Hexane GC, TMSI/HFBI — — 0.979 <0.6 — 2.5–20 — 23

gnt, tob, ntl,ami, par*, kan*

0.5% H2SO4 incubation83EC

Hexane GC, TMSI/HFBI — — — <0.6 — 2.5–20 — 23

gnt — CM-Sephadex

IP, OPA pc — — 0.997 — 4.72 1.0–10 95 50

neo 20% TCA — IP, OPA 4.4 9.8 0.981 <0.25 — 0.25–1.0 90–116 25

ami, dib, gnt, ntl,sis, tob

— Silica RP, OPA 5.9 — 0.999 (gnt) — 16.6 (gnt C1)9.5 (gnt C1a)7.2 (gnt C2)

0–16 — 26

gnt, ntl* ACN + fos, NaOH — RP, BSC 2.0–5.1 — — 0.2 — 2.5–10 — 27

gnt ACN + fos, NaOH DCM RP, DansCl 0.84–3.00 6.11–7.61 0.999 1.2 1.516 (gnt C1)0.832 (gnt C1a and

gnt C2)

0–40 — 28

gnt ACN + tris — RP FDNB — 1.7–5.9 — 0.33 — 1–16 83–84 29

gnt, sis, tob* ACN + tris — RP FDNB — 1–10 0.999 — 0.267 (sis)0.091 (C1a)

0.204 (gnt C1 andgnt C2)

0.5–16 84 (sis)83–84 (gnt)

65 (tob)

30

gnt ACN + tris chl RP, OPA 2.2–14.4 ≤8 0.99 0.5 — 0.5–40 ≥85 31

gnt Incubation 100EC Silica RP, OPA 6 3.9–7.5 — — — 0–20.0 80–105 32

ntl, gnt, tob ACN + fos DCM IP, OPA 1.7–7.9 (n) 3.6–7.4 (n) — 0.5 — 0–24 110–96.7 (ntl)94.1–98.3 (tob)91.5–91.8 (gnt)

33

gnt — Carboxypropylsilica

RP, FMOC 4.3–8.6 5.0–8.9 0.999 <0.05 72.7 (gnt C2a)120.7 (gnt C2)118.2 (gnt C1)92.9 (gnt C1a)

0–5 93.9 (gnt C2a)97.8 (gnt C2)96.8 (gnt C1)99.0 (gnt C1a)

34

tob, gnt* ACN + tris — RP FDNB — ≤3 0.998 0.25 0.398 0.5–16 75 (tob) 36

ami, kan* — CM-Sephadex

RP FDNB 1.5–5.3 9 0.993 1 — 1–64 95 (ami)92 (kan)

38

ami MeOH + borate Silica RP, FDNB — 0.8–9.9 0.996 — 3.21 2–64 — 39


AminoglycosideaExtraction and protein

precipitation CleanupChromatography

derivatizationbIntradayCV, %

InterdayCV, % r

Determinationlimit, µg/mL

Slope ofcalibration curve

Linear range,µg/mL Recovery, % Ref.

gnt — Silica IP, OPA 5.13–11.66 6.49–19.76

0.991–0.996

0.02 (gnt C1)0.08 (gnt C1a

and gnt C2)

0.311 (gnt C1)0.131 (gnt C1a)0.129 (gnt C2)

1–20 — 71

ami, kan* 2M tris Bond Elut C18 RP, TNBS 3.5–6.0 2.8–3.1 — <0.5 — 2.5–50 92.8–98.4(ami)

41

tob, sis* 2M tris Bond Elut C18 RP, TNBS 4.0–4.9 4.6–5.1 — — — 1–25 94–98.6 (tob) 42

ntl ACN + fos, NaOH DCM RP, DansCl 1.49–2.02 — 0.999 0.5 0.563 0–20 — 43

ami Incubation Silica RP, OPA 5 5–6 — 1.0 — 1–15 — 44

ami, tob* — CM-Sephadex

IP, OPA pc 3.2–25 (RSD) 3.9–28(RSD)

— — — 0.025–2 — 56

gnt, ami, tobnag*

— CM-Sephadex

IP, OPA pc 2.3 (tob)–4.0 (gnt) 2.8(ami)–3.6

(gnt)

0.999 — 0.136 (ami)0.278 (tob)0.395 (gnt)

1.0–12 (gnt)2.0–32 (ami)20–15 (tob)

89 57

str, dhs* fos Sep-Pak C18 IP, — 2.07–5.46 — 0.999 2 0.0476 5–50 80 61

sis, ntl Drying andextraction on

paper

IP, OPA 7.5–9.0 (sis)3.5–13.7 (ntl)

10.5 (sis)16.4 (ntl)

0.999 (sis)0.998 (ntl)

0.053 (sis)0.5 (ntl)

0.334 (sis)0.306 (ntl)

0.1–7.4 (sis)1.0–10 (ntl)

93–105 (sis)97–106 (ntl)

86

str 3.5% perchloric acid — IP, NQS 2.67–3.02 3.01–3.50 0.999 0.5 0.329 5–50 100 88

gnt MeOH — IP, OPA pc 2–2.5 2.3–3.2 0.999 0.5 0.792 2.7–16.5 97–103 62

gnt ACN + fos, NaOH DCM:fos IE, fluorescamine 2 3.5 0.999 1.0 2.4–3.8 0–40 93 77

tob, neo, sis,kan, ami

— AmberliteIRC 50

IE, dihydrolutidine — — — 0.05 — 0–5 — 79

sis — CM-Sephadex

RP, OPA 5.5–6.8 7.2–8.0 0.995 0.08 1.571 — 111 87

a Compounds marked with * are internal standards.b IP = ion-pair chromatography; TMSI = trimethylsilylimidazole; HFBl = heptafluorobutyric imidazole; RP = reversed-phase chromatography; BSC = benzenesulfonyl chloride; DansCl = dansyl

chloride; FDNB = 1-fluoro-2,4-dinitrobenzene; FMOC = 9-fluorenylmethoxycarbonyl chloride; TNBS = 2,4,6-trinitrobenzenesulfonic acid; NQS = $-naphthoquinone-4-sulfonate; OPA =o-phthalaldehyde; pc = postcolumn derivatization.

No single method can be identified as superior on the basisof method recoveries. Methods using acetonitrile and trisbuffer for extraction and protein precipitation generally give10% lower recoveries than other methods. Comparisons aredifficult, however, because recoveries are not reported inabout half of the methods reviewed here. Most of the reportedmethods cover the therapeutic concentration range of theaminoglycosides within their linear range. Limits of determi-nation are generally below therapeutic concentrations.

Urine

Analysis of aminoglycosides in urine generally does notrequire complex sample preparation such as protein precipi-tation. Several methods used for aminoglycoside analysis inplasma could also be applied to urine as such as has beendone with gentamicin extraction using acetonitrile and phos-phate buffer (28).

Two methods concerning analysis of gentamicin in urinehave been reported. One uses only derivatization for samplepreparation (77), and the other is based on SPE (31). The SPEcolumn is Sep-Pak C18, and gentamicin is eluted with metha-nol containing ammonia. The eluent is evaporated to drynessto remove ammonia, and gentamicin is derivatized with OPA.The method is linear from 0.5 to 5 mg/L, and recovery is>85%. The advantages of SPE over liquid–liquid extractionsare cleaner chromatograms and prolonged analytical columnlife (31). The sensitivity of the method can be improved easilyby increasing the sample amount (31). In the direct method,phosphate buffer (pH 7.35) and acetone are added to the urine,and the solution is derivatized with fluorescamine (77). Thecalibration curve is linear from 0 to 71 mg/L, and the methodis repeatable.

Analysis of neomycin in urine has been done bycentrifugation of samples and addition of counterion concen-trate to the supernatant for chromatography. The supernatantis then analyzed by IP with OPA postcolumn derivatization(25). Recovery was 75% for a neomycin concentration of1 µg/mL and 104% for a neomycin concentration of 5 µg/mL.

Muscle Tissue, Kidneys, and Liver

Neomycin, gentamicin, streptomycin, anddihydrostreptomycin are widely used as veterinarytherapeutics (95). Theillegal or irresponsible use of thesedrugs can cause residues in edible tissues (17). Characteristicto aminoglycoside pharmacokinetics is their tissue binding,especially to renal cortex (65). Therefore, aminoglycosideresidues in food products of animal origin are not uncom-mon, and continuous monitoring of these residues is neces-sary (54). Monitoring of aminoglycoside residues requiresconfirmative methods (95).

Sample preparation for residue analysis usually includes2 stages: (1) mixing tissue in a buffer or in a pro-tein-precipitating agent using organic solvent, precipitator, oralkaline or enzymatic digestion and (2) extraction of analytesby liquid–liquid, liquid–solid, or immunoaffinity methods (5).The methods used for sample preparation and extration ofaminoglycosides in tissues are presented in Table 10.

Matrix solid-phase dispersion (MSPD) has been used toextract aminoglycosides from kidneys (17). The tissue ho-mogenate is mixed with cyanopropyl packing material, andthe mixture is poured into a cartridge and compacted to a de-sired volume. During analysis of aminoglycosides, the sta-tionary phase is washed with hexane, water, and methanol,and then the analytes are eluted with water. The method hasbeen validated with fortified tissue samples because matrixcomponents affect the shape and retention times of chromato-graphic peaks. Coeluting matrix components affect the MSperformance. MS–PD also has been used for streptomycin anddihydrostreptomycin (49).

Neomycin has been extracted from kidneys, liver, andmuscle tissue with phosphate buffer for homogenization andprotein precipitation (53, 54). Recovery and the effect of pro-tein precipitation on recovery have been investigated with ra-dioactively labeled neomycin, and recovery has been com-pared with that achieved from extractions with saline (54).Neomycin recovery from kidneys is 60% with saline, anddrops to 30% after incubation and protein precipitation. Withphosphate buffer, incubation does not affect recovery. How-ever, neomycin becomes adsorbed to glass, and incubationand extraction must be performed in plastic.

Gentamicin recovery has been investigated with differentprotein-precipitating buffers and CM-Sephadex extraction(5). With the precipitation agents HClO4, TCA, and acidicphosphate, recoveries are 78.5, 85.9, and 39.2%, respectively(5). TCA has been chosen as precipitation buffer, with addi-tion of EDTA, because gentamicin has been reported to che-late positively charged metal cations (98). No gentamicin pre-cipitation is observed with this technique.

The effect of phosphate buffers on gentamicin recoveryfrom edible tissues also has been studied (67). Results arecontradictory to ones reported earlier because only basicbuffers are found to extract gentamicin. When the pH of thephosphate buffer is 4.5, gentamicin is not extracted at all.When the pH is 8–10, recoveries vary between 70 and 98%.Addition of Na2SO4 to the precipitation buffer increasesgentamicin recovery. However, the effect decreases as pHincreases. Simultaneously, too high a sulfate concentrationdecreases recovery. Best results are achieved with 0.1Mphosphate buffer when sulfate concentration is 0.1M and pHis 8.8. Recovery is then 98%.

Separation of gentamicin from matrix components isdifficult because of the similarity of chemical properties(65). Most of the methods used for sample preparation useonly one retention mechanism, and that usually is not satis-factory for cleanup of aminoglycosides from tissue sam-ples. For example, when phosphate buffer precipitation andSephadex extraction are used, some interfering elementselute with gentamicin, making it impossible to quantitateall 3 components (67).

The applicability for gentamicin extraction of SPE materi-als with TCA as precipitation buffer has been studied (5). Dif-ferent nonpolar groups, such as C8, phenyl, and C18, bound tosilica have been studied. Good recoveries are achieved withall of these (55–81%), but specificity to gentamicin is not sat-


isfactory. When silica, weak cation exchanger (WCX), or poly-meric resins are used, recovery is not sufficient. ThusCM-Sephadex, which is specific to gentamicin and gives re-coveries of 65% or more, has been chosen for analysis (5). Oth-ers have reported good results for cleanup of gentamicin sampleswith WCXs (65). The method based on WCX–SPE can be usedfor all tissues including liver, kidneys, and lungs (65).

Precipitation of proteins and extraction of streptomycinand dihydrostreptomycin from kidney and muscle tissues with3.6% perchloric acid has been done (89). Recoveries of thedrugs vary depending on tissues. For swine muscle, the lowestdihydrostreptomycin recovery is obtained when recovery ofstreptomycin is highest. Adding perchloric acid during extrac-tion does not increase recovery, but it increases interferencesin chromatography.

Milk

Milk and milk product matrixes contain several com-pounds that might interfere in analysis (83). Analytical meth-ods for antibiotics in milk include cleanups to remove proteinsand lipophilic compounds. Gentamicin (65, 68), neomycin(45, 51), streptomycin, and dihydrostreptomycin (60) in milkhave been analyzed.

An Amberlite CG-50 ion-exchange column has been usedto extract neomycin from milk when no protein precipitationis used (45). Neomycin is derivatized on column with OPA,and the derivatives are eluted with basic methanol. Recoveryof the method is 94–102% at concentrations of0.1–5.0µg/mL. The limit of determination is 0.05µg/mL. Inanother study, neomycin is extracted from milk by proteinprecipitation with 20% TCA (51). OPA postcolumnderivatization and fluorescence detection are used. Recoveryof neomycin is 94–122%, and the method is linear in range0.15–12µg/mL.

CM-Sephadex C-25 ion exchanger has been used to extractgentamicin from milk. Milk is added to the column in NaOHsolution (68). After elution from the column, gentamicin isderivatized in a silica Sep-Pak SPE column with OPA. At-tempts to precipitate the milk proteins before Sephadex ex-traction have been unsuccessful. However, the method isquantitative and selective, and recoveries are 94–106% atgentamicin concentrations of 0.2–10µg/mL. A C18 COOHion-exchange column also has been used to extract gentamicinfrom milk at concentrations of 0.625–80µg/mL. The limit ofdetermination is 0.6µg/mL (65). In another study, gentamicinis extracted from milk with 30% TCA and a C18 SPE column(52). Gentamicin is eluted from the SPE cartridge with ammo-nia–methanol. Recovery is 72–88%, and the limit of determi-nation for individual components is 0.4 ng/mL.

Streptomycin and dihydrostreptomycin have been ex-tracted from milk with 3.6% perchloric acid, followed by IPand postcolumn derivatization with NQS (60). Recoverieswere 32.6–65.0%, depending on milk fat content.

Comparison and Evaluation of Methods

Chromatographic, Microbiological, andImmunological Methods

Microbiological and immunological methods are still pop-ular for aminoglycoside analysis and are used in both thera-peutic monitoring (99) and screening for aminoglycoside resi-dues (9). The reliability of results obtained with these methodsis, however, poorer than can be achieved with chromato-graphic methods. The slowness of microbiological methods isa major disadvantage, particularly in therapeutic monitoringwhere the speed of analysis is important. Immunoassays pro-vide high throughput of samples but often give erroneous re-sults (99). The advantages of chromatographic methods overmicrobiological or immunological methods include greater


Table 10. Sample preparation methods for aminoglycosides from edible tissues

AnalyteChromatography and

derivatizationa Extraction and precipitationb CleanupcRecovery,

% Ref.

neo IP, OPA pc fos 0.2M pH 8, incubation 100EC 10 min — 80 53

neod IP, OPA pc fos incubation 100EC 5 min — 90 54

gnt IP, OPA pc 5% TCA, EDTA 1 mM IE, CM-Sephadex 68–98 5

gnt, neo, str, dhs IP, — MSPD, cyanopropyl — 46–75 17

gnte IP, OPA 0.1M fos, 0.1M Na2SO4, pH 8.8 IE, C18COOH 90 65

str, dhs IP, — MSPD, cyanopropyl 0.1M Na2SO4 — — 49

gnt IP, OPA fos incubation 100EC 5 min IE, CM-Sephadex 83–101 67

str, dhs IP, NQS 3.6% perchloric acid IE, (Bakerbond) aromatic sulfonic acid 53–66 89

kan GC, HFBI/TMSI 10% TCA Amberlite CG 50 74–83 24

a For definitions, see footnotes to Table 4.b MSPD = matrix solid-phase dispersion.c IE = ion-exchange chromatography.d Paromomycin as internal standard.e Netilmicin as internal standard.

accuracy, repeatability, and specificity of results. However,when antimicrobial activity is of concern, only bioassays candirectly measure this property. Immunoassays based on en-zyme-linked immunosorbent assay (ELISA) or RIA tech-niques generally measure cut-off points ranking result as posi-tive or negative with reference to a concentration limit.Maximum residue limits (MRLs) for aminoglycoside varywidely among different aminoglycosides and different tissues.Therefore, it appears extensively difficult to produce a generalassay that would monitor these drugs as a group relative to aregulatory tolerance level. The structural similarity of the dif-ferent aminoglycosides is likely to cause cross-reactivity be-tween compounds, complicating quantitative determination.

Aminoglycoside bioassays and immunological methodsusually use the aqueous extract of most matrixes without needfor further concentration. This lack of need for further concen-tration is an advantage for compounds that are difficult to ex-tract with organic solvents. However, bioassays do not detectcompounds bound to proteins, which makes results difficult toquantitate. For example, in determination of neomycin concen-trations in incurred tissue samples, the microbial assay showswider variation in results than HPLC as a function of standardsprepared in buffer or tissue homogenate (99). Purity or ratio ofdrug components in drug preparations cannot be determinedmicrobiologically or immunologically. Thus, chromatographicmethods are necessary. Furthermore, when the importance ofjudicial and economic consequences of results increases, thereliability and accuracy of results become more significant.

When linearity, accuracy, precision, reliability, or diversityof the method are considered, HPLC is at present the bestmethod for aminoglycoside analysis. Separation capacity andlow costs also contribute to HPLC as the method of choice. Anadditional advantage of chromatographic methods is the possi-bility to use internal standards, which often increases reliability.

Usually, microbiological methods are compared with chro-matography by the so-called blind-duplicate method. The pur-pose is to evaluate the correlation of results obtained by the2 methods. Usually chromatographic results are presented as afunction of microbiological results, and the slope and r of theplot are indicative of the correspondence. Both of the men-tioned values should be as close as possible to 1. The resultsfrom the use of this method are presented in Table 11.

Correlations of chromatographic methods with enzymological,immunological, or microbiological methods differ depending onthe aminoglycosides and the chromatographic methods(27, 30–33, 36, 38, 41–44, 61, 62, 88, 100). For gentamicin anal -ysis, for example, the best correlation is between reversed-phaseLC method using FDNB derivatization (31) and RIA. IP withOPA postcolumn derivatization (62) correlates better with micro-biological assays than a precolumn derivatization method (32)with RP.Interestingly, both methods separate the 3 gentamicincomponents and the quantitative differences could not be ex-plained by different amounts of the separated components. Itappears that quantitation suffers when OPA postcolumnderivatization is changed to precolumn derivatization.


Table 11. Comparison of the microbiological, immunological, and chromatographic methods used foraminoglycoside analysis by blind duplicate method

AminoglycosideaDerivatization andchromatographya

Referencemethodb Slope r Reference

ami TNBS, RP RIA 1.047 0.999 41

FDNB, RP MB 0.92 0.993 38

OPA, RP MB 0.94 0.94 44

gnt BSC, RP EMIT 0.86 0.995 27

OPA, RP MB 0.87 0.99 32

OPA pc, IP MB 1.02 0.934 62

FDNB, RP RIA 1.002 0.9996 31

ntl OPA, IP RIA 1.08 0.98 33

OPA, IP MB 1.13 0.99 33

DansCl, RP MB 1.048 0.949 43

sis FDNB, RP MB 0.91 0.99 30

str —, IP FPIA 0.931 0.969 61

NQS, IP FPIA 1.003 0.99 88

tob TNBS, RP RIA 0.971 0.968 42

TNBS, RP EMIT 0.981 0.971 42

FDNB, RP MB 1.04 0.997 36

a For definitions, see footnotes to Table 4.b RIA = radioimmunoassay; MB = microbiological assay; EMIT = enzyme immunoassay technique; FPIA = fluorescence polarization

immunoassay.

Between the microbiological method and chromatographicmethods using OPA precolumn derivatization for analysis ofamikacin (44), gentamicin (32), and netilmicin (33), the bestagreement is for amikacin (Table 11). For netilmicin, theagreement between RIA and the same chromatographicmethod (33) is only marginally better than between the micro-biological and chromatographic methods. The microbiologi-cal and RIA methods appear to correlate well.

Results of RP with FDNB derivatization are in good agree-ment with reference methods for gentamicin (30) andtobramycin (36) and slightly less for amikacin (38) andsisomicin (30; Table 11). The best correlations for tobramycinand amikacin are obtained with reversed-phase methods withTNBS derivatization (41, 42). Forstreptomycin, NQS post-column derivatization after IP (88) has better agreement with flu-orescence polarization immunoassay (FPIA) than does the re-versed-phase method with low-wavelength UV detection (61).

Chromatographic Methods

Most chromatographic determinations of aminoglycosidesare done by HPLC, and the aminoglycosides are derivatized toimprove detection. The choice of derivatization reagent has aremarkable effect on quantitative and qualitative results. Ingeneral, IP is used with OPA derivatization, MS, or electro-chemical detection. When MS, electrochemical detection, orpostcolumn derivatization with OPA is used, IP as the methodof choice is justified because the analyzed aminoglycosidesare charged. However, when OPA is used for derivatizationprior to chromatography, it is not clear why IP is the method ofchoice for separation. It is unclear if retention occurs through anion-pairing mechanism. Furthermore, when mercaptoaceticacid (11, 63–66) or mercaptopropionic (86, 87) acid is usedwith OPA, the derivatives include negatively charged groupsthat could complicate ion-pair formation with sulfonates and fa-cilitate ion-pair formation with positively charged counterions.Unfortunately, this possibility is not discussed.

A good example of the lack of clarity associated with IPand OPA precolumn derivatization is the analysis of sisomicinby IP with heptane sulfonate as counterion and precolumnderivatization with OPA (86). Sisomicin is separated with theoriginal method but when heptane sulfonate is replaced withEDTA, the separation improves (87). This result suggests thatseparation by the original method does not occur by anion-pair mechanism.

On the basis of promising results so far, FMOC–Cl will findmore applications in aminoglycoside analysis. The methods basedon TNBS and FDNB, which work for most aminoglycosides ex-cept for streptomycin and dihydrostreptomycin, will probably getmore attention, too.Comparison of FDNB and TNBS withOPA as derivatization agents does not reveal marked differ-ences in limits of determination even though OPAderivatization allows fluorescence detection. Incompletederivatization of aminoglycosides with OPA may, at leastpartly, explain this phenomenon.

In therapeutic monitoring and in the analysis of drug prepa-rations, the chromatographic methods using derivatization andIP or RP chromatographic separation will remain dominant be-

cause of their simplicity, short analysis time, and low cost. Inlaboratories where confirmatory methods are needed, LC/MSapplications can be expected to replace other methods. In thesecases, IP with the use of fluorinated carboxylic acids such asTFA, PFPA, and HFBA as counterions will probably becomethe methods of choice. The advantage of these methods is thatthe retention of aminoglycosides with these counterions hasbeen thoroughly studied (69, 70), and separation of severalaminoglycosides can be achieved in one run (15, 69, 70).

Conclusions

Aminoglycosides are a heterogenic group of antibioticscharacterized by a wide spectrum of activity and toxicity. Be-cause of their toxicity, the monitoring of their concentrationsin plasma during therapy is necessary. The wide use ofaminoglycosides in veterinary medicine also requires suitableroutine and confirmatory methods for their detection in edibletissues. In both cases, the methods must be accurate, sensitive,and robust against interferences. However, the chemical char-acteristics of aminoglycosides such as polarity, water solubil-ity, lack of volatility, and lack of chromophore make develop-ment of applicable methods difficult.

Chromatographic analysis almost always requiresderivatization to improve either detectability or separation.However, derivatization is difficult when the aminoglycosidestructure incorporates several groups of different reactivity,leading to possible partial derivatization. The most popularmethod for aminoglycoside analysis is IP chromatographywith OPA derivatization. The interpretation of the results is,however, unclear and contradictory, and the reliability andreproducibility of results is questionable. The most promisingmethod seems to be LC/MS in combination with IP. The com-bination permits both quantitative and confirmatory analysisfor most aminoglycosides.

For the analysis and extraction of aminoglycosides fromdifferent tissues, no generally applicable method has been re-ported. Extraction from edible tissues appears extremely diffi-cult. However, promising results have been achieved withmethods using SPE. More work is needed to develop gener-ally applicable and validated regulatory methods.

Acknowledgment

We thank James D. MacNeil for his constructive criticismduring preparation of this review.

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