Download Chromatographic Methods for Analysis of Aminoglycoside Antibiotics

ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 1017
DRUGS, COSMETICS, FORENSIC SCIENCES
Chromatographic Methods for Analysis of Aminoglycoside
Antibiotics
ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999
NINA ISOHERRANEN
University of Helsinki, Department of Chemistry, Laboratory of Analytical Chemistry, PO Box 55, Helsinki 00014, Finland
STEFAN SOBACK 1
Kimron Veterinary Institute, National Residue Control Laboratory, PO Box 12, Beit Dagan, Israel
Aminoglycosides are antimicrobial agents used
frequently in treatment of human and animal diseases caused by aerobic, gram-negative bacteria.
Because of the toxicity of these compounds, considerable effort has been attributed to analysis of
aminoglycoside content in drug preparations, in
serum and urine specimen in therapeutic drug
monitoring, and in edible animal tissues in residue
control. The present review emphasizes the analytical problems associated with aminoglycoside
analysis. Screening methods based on microbiological and immunological procedures were briefly
discussed. Gas chromatography and especially
high-performance liquid chromatography appeared
the most widely used chemical methods for the
analysis of these compounds. Due to lack of volatility, chromophore, and hydrophility of
aminoglycosides, most methods applied
derivatization for enhancement of their chromatographic characteristics. The applicability and advantages of the various derivatization procedures were
discussed in detail. A wide variety of detection
methods, including mass spectrometry have been
used. Packed column separation was generally
used for gas chromatographic separation. In liquid
chromatography, reversed phase, ion pair, ion exchange, and normal phase separation has been employed. Mass spectrometry, as a detection method,
was discussed in detail. Extraction procedures from
body fluids and tissues were emphasized. The performance and the operational conditions of the
methods were described and detailed information of
the data was provided also in table format.
minoglycosides have been used to treat infections
caused by aerobic gram-negative and some
gram-positive bacteria (1). They resemble each other
in chemical structure, antimicrobial activity, pharmacokinetic
A
Received November 2, 1998. Accepted by JM March 24, 1999.
1
Author to whom correspondence should be addressed.
characteristics, and toxicity (2). The first aminoglycoside,
streptomycin, was identified in 1944 during the search for water-soluble and stable compounds active against
gram-negative bacteria (1). Kanamycin was isolated in 1957,
and at the time, it was active against streptomycin-resistant bacteria (1). Subsequently, bacteria resistant to kanamycin were isolated and the mechanism of resistance was resolved. Understanding the mechanism of resistance led to the development of
semisynthetic aminoglycosides, of which more than 150 have
been described (1). Amikacin, dibekacin, gentamicin,
kanamycin, neomycin, netilmicin, paromomycin, sisomicin,
streptomycin, dihydrostreptomycin, and tobramycin are currently in therapeutic use. In veterinary therapy, neomycin,
gentamicin, kanamycin, streptomycin, and dihydrostreptomycin
are the most common aminoglycosides (3).
The antimicrobial activity of aminoglycosides is based on
their ability to inhibit bacterial protein synthesis (4). Current
primary use of these drugs in human medicine is for treatment
of infections caused by aerobic gram-negative bacteria (4).
The most important pathogens treated with aminoglycosides
are pseudomonads, enterococci, coliforms, and salmonellae.
Aminoglycosides are also used substantially in the treatment
of 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 compound 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 unbound, except in the renal cortex where they tend to concentrate. 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 toxicity. 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.
1018 ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999
Figure 1. Structures of the therapeutically used aminoglycosides (1).
ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 1019
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
S. rimosus
Parke Davis, 1959
M. inoyoensis
Schering Plough, 1970
Streptomycin
S. griseus
Waksman and Schatz, 1944
Tobramycin
S. tenebrarius
Eli Lilly, 1967
S. griseus and S. humidus
Takeda Chemicals, 1957
Paromomycin
Sisomicin
Dihydrostreptomycin
a
b
From reference 1, unless indicated otherwise.
From reference 4.
Structures and Chemical Characteristics
The word aminoglycoside is used to describe an
amino-function-containing carbohydrate linked via a
glycoside bond to an aminocyclitol (3). Therapeutically used
aminoglycosides usually contain a 1,3- or 1,4-diaminocyclitol
(1). The aminoglycosides are divided into 2 groups according
to the aminocyclitol: streptamine and 2-deoxystreptamine.
The deoxystreptamine group, the larger group (1, 3), is further
divided into 2 subgroups according to the number and position
of the substituents in the deoxystreptamine moiety. The best
known subgroups are (1) neomycins and paromomycins, in
which substituents are in adjacent positions (positions 4 and
5), and (2) gentamicins and kanamycins, in which the sub-
stituents are in nonadjacent positions (positions 4 and 6) of the
aminocyclitol moiety (3). The streptamine group contains
streptomycin and dihydrostreptomycin (3). Structures of therapeutically used aminoglycosides are presented in Figure 1.
Aminoglycosides are produced by Streptomyces or
Micromonospora fungi. Semisynthetic aminoglycosides,
such as dibekacin, amikacin, and netilmicin were developed to
reduce toxicity and increase antimicrobial activity (1).
Dibekacin (1) and amikacin (6) are synthesized from
kanamycin B, and netilmicin, from sisomicin (7). Tobramycin
can be synthesized from kanamycin or extracted from
Streptomyces (1). The fungi producing the various
aminoglycosides are presented in Table 1. Aminoglycosides
Table 2. Physical and chemical characteristics of aminoglycosidesa
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
Aminoglycoside
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
b
c
Data from references 12 and 101.
Depends on pH.
Intravenous.
1020 ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999
produced by Streptomyces have the suffix “mycin” and the ones
produced by Micromonospora have the suffix “micins” (3).
Aminoglycosides are water soluble and heat-, acid-, and
base-stable polar compounds. Their solubility in methanol is
limited, and they are practically insoluble in hydrophobic organic solvents. In water solutions, aminoglycosides are usually positively charged because of their amino groups. The
number of amino groups varies from 4 in kanamycin to 6 in
neomycin, and the pKa values of the amino groups vary between 7 and 8.8. The physical and chemical characteristics of
the therapeutic aminoglycosides are presented in Table 2.
Aminoglycosides are typically drug complexes formed by
several closely related components. The neomycin complex is
formed by 2 stereoisomers: neomycin B and C (8). Only
neomycin B is antimicrobial. Paromoamine and
paromomycins I and II occur as impurities of the neomycin
complex (8). Commercially available neomycin products include 85–90% neomycin B (8). The gentamicin complex comprises at least 4 main components and several minor components (9). Separation of up to 7 different components of
gentamicin has been reported (10), and proportions of
gentamicin components in various products differ (11).
Paromomycin is a complex of 2 stereoisomers: paromomycin I
and II. Kanamycin is a mixture of 3 isomers, A, B, and C (1). The
kanamycin components differ markedly in their toxicity and,
therefore, commercial mixtures are required to contain at least
75% kanamycin A and not more than 5% kanamycin B (1).
Screening of Aminoglycosides
Aminoglycosides have been analyzed in tissues and urine
by microbiological, radioenzymatic assay (REA), and
radioimmunoassay (RIA) methods and by paper chromatography (12). These methods are still widely used but often lack
quantitative or qualitative performance.
Microbiological assays use methods based on agar diffusion of the drug and concentration-dependent growth inhibition (inhibition zone) of the test organism inoculated in the
agar (12). The assays require 12–48 h incubation, after which
inhibition of bacterial growth is measured. Several factors influence the accuracy of these methods (2). Incubation temperature, agar pH and ion concentration, depth of the agar on the
plate, test strain, incubation time, and presence of other antibiotics in the sample can affect the assay (2). Additionally, different agar pHs need to be used for different aminoglycosides.
Although microbiological methods are versatile, simple, and
relatively cheap, they are inaccurate and subject to interferences caused by nonspecific inhibitors or other antimicrobial
drugs (13, 14). Additionally, repeatability and reproducibility
of results are generally poor (12), and the assays usually lack
sensitivity below 2 µg/mL (2).
RIA represents a major improvement over microbiological
assays (2). RIA methods are sensitive (2, 12) and specific, but
other aminoglycosides might cause interferences (12).
Gentamicin, tobramycin, amikacin, netilmicin, and sisomicin
can be determined by RIAs (12). Analysis using an RIA
method requires complicated parameter optimization and specialization of the analyst (12). Selection and preparation of
suitable specific antibodies is difficult and time consuming
(12). In addition, handling of radioactive materials and radioactive waste and high cost are inhibitory factors (2, 12). Correlations between RIA and other methods are variable (2).
REA offers similar advantages as do RIAs. The instability
of enzymes and the presence of other antibiotics in the sample
Table 3. GLC methods used for aminoglycoside analysis
Derivatization
Column phase,
length (m) × id (mm)
Temperature
programa
Carrier gas,
flow rate
(mL/min)
Matrix
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 tg
10EC /min
150–310EC
He, 40
Standard
FID
20
Kanamycin,
paromomycin
TMSDEA+ TrisilZ
0.75% OV-1 or 3% OV-1
GasChromQ, 1.83 × 3
it 290EC or it
300EC
He, 70
Standard
FID
21
Neomycin
TMSDEA+ TrisilZ
3% OV-1 GasChromQ, 0.61–0.91 × 3
it 300EC
He
Standard
FID
18
Kanamycin ,
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
Analyzed compounds
b
a
b
it = isothermal; tg = thermal gradient.
Internal standard.
Detector Ref.
ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 1021
can cause false results (2, 12). An REA method has been developed for kanamycin, tobramycin, amikacin, and sisomicin
(12). However, occasionally poor correlation with results
obtained with other methods has been observed (12).
The 2 most common assays for aminoglycosides are the
homogenous enzyme immunoassay and the fluorescence polarization immunoassay (FPIA; 2). These methods have similar sensitivity and precision as RIAs but are less expensive and
do not require handling of radioactive material (2).
Chromatographic Analysis
Chromatographic methods for aminoglycoside analysis are
needed for simultaneous qualitative and quantitative determinations. However, separations are difficult to achieve because
of the structural similarity of aminoglycosides (15, 16). Paper
chromatography of gentamicin components was the first reported chromatographic method for aminoglycosides (16).
Gas Chromatography
Gas chromatography (GC), with its high separation capacity and efficiency, is a popular technique for volatile,
heat-stable compounds. However, direct analysis of intact
aminoglycosides by GC is impossible because of the hydrophilic, basic, and nonvolatile nature of these molecules (17).
By derivatizing the amino and hydroxyl groups, volatile derivatives can be produced (18–24). The gas–liquid chromatographic (GLC) methods for aminoglycosides are presented in Table 3.
Trimethyl silyldiethyl amine (TMSDEA), a reagent that
silylates both amino and hydroxyl groups, has been used as a
derivatizing agent (18–21). The products are moisture sensitive and, therefore, unstable (18–21). Consequently, results
are nonlinear and poorly repeatable, and the derivatization
yield is low. Freeze drying of samples prior to derivatization
has been used to eliminate variations in sample moisture
content and solubility (18). Sealed sample vials, removal of
metal parts from the chromatographic system, and
on-column injection have been tried to improve repeatability
and quantitation (18). Nonetheless, linearity and
derivatization yield remain poor.
It is important to know the ratio of drug components or
stereoisomers that differ in their pharmacological characteristics in a pharmaceutical product. Stereoisomers of
neomycin B and C (18, 20) and paromomycin I and II (19, 21)
have been separated by GLC as TMSDEA–silylated derivatives. Stereoselective separation using a nonchiral column
has been suggested to result from stronger retention of the
equatorial than the axial epimer (21). On the basis of this assumption, it has been suggested that paromomycin I must
elute before paromomycin II and neomycin B before C. The
kanamycin components A, B, and C have been separated as
their trimethylsilyl (TMS) derivatives (21). The TMS derivatives of neomycin (19, 20), kanamycin (21), and
paromomycin (21) also have been identified by mass spectrometry (MS). Derivatization results in silylation of all
amino and hydroxyl groups.
A 2-step derivatization using trimethyl silylimidazole
(TMSI) for silylation of hydroxyl groups and
heptafluorobutyric imidazole (HFBI) for heptafluorobutyrylation of amino groups has been reported (22–24).
Using this technique, Mayhew and Gorbach (23) analyzed
concentrations of gentamicin, tobramycin, netilmicin,
amikacin, and paromomycin in serum. For each aminoglycoside, another one was used as an internal standard. Results are acceptable in accuracy and precision, and the method
is linear for the concentration range investigated. TMS–
heptafluorobutyryl (HFB) derivatives are more stable than
TMSDEA derivatives, but protection of the samples from
moisture still is crucial. Gentamicin elutes from the column as
2 peaks: the first is gentamicin C1, and the second, as a combination of gentamicins C1a and C2. All the other aminoglycosides analyzed produce 1 peak. Mayhew and Gorbach
(22) also reported an improved GLC method for gentamicin
with kanamycin as internal standard. The accuracy and repeatability of the method are improved, and the correlation coefficient (r) for the calibration curve is 0.979 (22). GLC analysis
of kanamycin in muscle tissue has been reported (24). In this
study, the 2-step TMSI–HFBI derivatization described by
Mayhew and Gorbach (23) is used in reversed order. This
procedure produces better results than the original
TMSI–HFBI derivatization.
In comparison, the 2-step derivatization with TMSI and
HFBI appears superior to silylation with TMSDEA, the main
advantage being the higher derivatization yield and the more
stable derivatives. The 2-step derivatization method is also applicable to larger number of aminoglycosides and more internal standards are available compared with the TMSDEAbased method. Unlike in TMSDEA methods, individual
aminoglycosides do not disturb the assays of other
aminoglycosides in the TMSI–HFBI methods. The HFBI
derivatization allows the use of an electron capture detector
(ECD), providing increased selectivity and sensitivity of the
method compared with methods using a flame ionization detector (FID). The main weakness of GC methods for
aminoglycosides is the lack of capillary column applications.
Liquid Chromatography
As water-soluble, polar, and relatively large compounds,
aminoglycosides typically would be analyzed by liquid chromatography (LC; 1). Most high-performance liquid chromatographic (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 relevant to HPLC analysis are the lack of a chromophore and the
high hydrophilicity. To improve detectability and separation,
the aminoglycosides are usually derivatized prior to or after
chromatographic separation. Methods used to separate
aminoglycosides by HPLC are presented in Table 4.
Reversed-Phase Chromatography
Because of their polar and ionic nature, aminoglycosides
usually are not partitioned on reversed-phase columns (15, 26).
1022 ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999
Table 4. HPLC methods used for separation of aminoglycosides
Column phase,
id (mm) × length (cm) ×
particle size (µm)
Flow rate of
mobile
phase,
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 fos
pH 3
—
PED
neo
8
Cellulose phosphate
PII, 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% aca
0.02M 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, 0.056M
Na2SO4, 0.007M aca
OPA pc
fl, ex 340, em 455
neo
51
Supelcosil LC-8-DB,
4.6 × 15 × 5
—
0.011M pns 0.007M aca,
1.5% MeOH
OPA pc
fl, ex 340, em 455
neo
25
PhaseSep Sperisorb
ODS2, 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
LiChrosorb RP8, 3.0 ×
25 × 5
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
ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 1023
Table 4. (continued)
Flow rate of
mobile
phase,
L/min
Mobile phasea
Derivatizationb
Detector,
wavelengths (nm)c
Aminoglycosidesd
Ref.
Merck RP18, 3.9 × 30 ×
10
1.3
ACN–H2O–aca, 70 + 29 + 1
FDNB
UV, 365
tob
37
LiChrosorb RP18, 3.9 ×
30 × 10
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
LiChrosorb RP8, 4.6 ×
25 × 5
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
Column phase,
id (mm) × length (cm) ×
particle size (µm)
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 + 65
2g/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
Spherisorb ODS-2,
2.0 × 10 × 5
0.2
ACN–H2O, 8–16 + 92–84 40 mM
HFBA
—
PAD or MS
str, dhs
49
Spherisorb ODS-2,
4.6 × 15 × 5
—
0.0056M Na2SO4, 0.007M aca
0.011M pns, 18.5% MeOH
OPA pc
fl, ex 340, em 455
neo
53
Supelcosil LC-8-DB,
4.6 × 15 × 5
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, 17.4M
aca, 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
LiChrosorb RP8, 3.2 ×
25 × 10
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/L
hxs
—
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
1024 ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999
Table 4. (continued)
Flow rate of
mobile
phase,
L/min
Mobile phasea
Derivatizationb
Detector,
wavelengths (nm)c
Aminoglycosidesd
Ref.
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, 0.1M
eds-ocs
OPA pc
fl, ex 365, em 440
gnt (3)
62
PRP-1 polymer, 4.6 ×
25 × 10
—
ACN–H2O, 7 + 1, different ion
pairs
—
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
Column phase,
id (mm) × length (cm) ×
particle size (µm)
a
b
c
d
OCS = octane sulfonate; fos = phosphate buffer; pns = pentane sulfonate; aca = acetic acid; DEA = diethylamine; HFBA = heptafluorobutyric
acid; 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.
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.
fl = fluorescence detector; ex = excitation wavelength; em = emission wavelength; PED = pulsed electrochemical detector; RI = refractive
index detector; MS = mass spectrometry; TSP = thermospray; UV = ultraviolet; MS/MS = tandem mass spectrometry; APCI = atmospheric
pressure chemical ionization; PAD = pulsed amperometric detector.
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.
ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 1025
Partition to C18 columns can be improved by using acetate
buffer in the mobile phase. The acetate ion acts as a counterion,
forming ion pairs with aminoglycosides (26). Usually
aminoglycosides are derivatized with a nonpolar group prior to
reversed-phase analysis to improve the partition to column and
the separation characteristics. The most commonly used
derivatization reagents are o-phthalaldehyde (OPA) and
1-fluoro-2,4-dinitrobenzene (FDNB). The separation columns
usually contain C8 or C18 materials, and mobile phases consist
of acidic buffers with methanol and/or acetonitrile.
The reversed-phase method reported by Essers in 1984
(26) apparently remains the method able to simultaneously
separate the largest number of aminoglycosides. On-line
precolumn derivatization of the aminoglycosides is used. The
mobile phase consists of methanol–acetic acid (80 + 20).
Dibekacin, sisomicin, tobramycin, and 4 components of
gentamicin can be separated in one chromatographic run. The
gentamicin 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 be
separated. Netilmicin requires an incresed proportion of methanol. For each aminoglycoside, one of the others was used as
an internal standard.
The separation of gentamicin components is of interest in
the quality control of pharmaceutical preparations,
pharmacokinetics, and toxicological studies. Gentamicin
components can separate to 1–5 peaks in reversed-phase columns 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 elution orders of dansyl and dinitrophenyl derivatives are similar
but separation of components differ. Retention of gentamicin
components is assumed to increase with the number of methyl
groups in the gentamicin structure (30). As dansyl derivatives,
gentamicins C1a and C2 elute together, followed by
gentamicin C1 as a separate peak (28). As 2,4-dinitrophenyl
derivatives, gentamicin C1a elutes before the coeluting C1 and
C2 components (29, 30).
Both dansyl chloride and FDNB derivatize all amino groups
in gentamicin and, therefore, the relative functionalities do not
change as a result of the derivatization reagent used. In the separation of 2,4-dinitrophenyl derivatives, the lack of a methyl
group in gentamicin C1a appears to be the major factor affecting
separation. The mechanism of separation of dansyl derivatives
has not been discussed in the literature. It can be assumed that
the separation of the dansylated gentamicins is based on the
functionality of the amine on C-6′. After derivatization, the
components (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 derivatives on C8 or C18 columns by use of acetonitrile–tris (31) or
methanol–ethylenediamine tetraacetic acid (EDTA)–water
(32, 33) mobile phases, correspondingly. In these studies,
identification of the different gentamicin components is based
on standards of individual components but the structures of
the derivatives are not discussed. Strangely, the elution order
of the gentamicin components is C1a, C2, and C1 with a C8 column but C1, C1a, and C2 with a C18 column. The retention
mechanism is not discussed.
The retention of gentamicin C1 relative to other gentamicin
components is of special interest because as an OPA derivative, it contains one derivatizable amine less than the other
components. The derivatized C1 component is much smaller
and has one basic secondary amine more than the other components. The retention of the gentamicin C1 component is
more sensitive to changes in mobile phase pH than the other
components, and its retention increases rapidly when the pH
of the mobile phase increases (31). This or derivatization differences may explain why gentamicin C1 elutes as the last
component from a C8 column and as the first component from
a C18 column. Addition of EDTA to the mobile phase yields
sharper peaks (32).
The gentamicin 9-fluorenyl methoxycarbonyl chloride
(FMOC–Cl) derivatives are separated as 4 separate peaks on a
C18 column with an acetonitrile–water mobile phase (34). The
elution order is C1a, C2, x, and C1. The component x is believed to
be the stereoisomer of gentamicin C2 and gentamicin C2a. The reported elution order corresponds to the increasing methylation of
gentamicin components. On the basis of the elution order of the
components, it is assumed that all the primary and secondary
amino 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 column
with methanol–water (45 + 55) elution (35). The
3,5-dinitrophenyl derivatives elute as 5 peaks, of which only 4
could be identified. The fifth peak is assumed to be one of the
minor gentamicin components. The elution order of the
gentamicins is C2a, C1, C1a, and C2. No explanation for the
separation of the stereoisomers C2a and C2, in such a manner
that 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 been
analyzed as 3,5-dinitrophenyl derivatives, while tobramycin
(30, 36, 37), sisomicin (30), and amikacin (38, 39) have been
analyzed as 2,4-dinitrophenyl derivatives. C18 columns have
been used with a mobile phase containing acetonitrile and water for 3,5-dinitrophenyl derivatives or acetonitrile and acetic
acid for 2,4-dinitrophenyl derivatives. The separation of different aminoglycosides as their 3,5-dinitrophenyl derivatives
has not been investigated. As 2,4-dinitrophenyl derivatives,
tobramycin and gentamicin components can be separated in
one chromatographic run (36), but when sisomicin, a dehydro
analogue of gentamicin C1, is added to the same run, it does
not separate from gentamicin (30). Gentamicin has been used
as the internal standard for tobramycin (36, 37). Apparently
sisomicin can be quantitated more easily as an internal standard than gentamicin because it produces only one chromatographic peak.
1026 ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999
The separation of different 2,4-dinitrophenyl derivatives of
aminoglycosides is assumed to be related to the different
amounts of hydroxyl groups in the different aminoglycosides
(30). Tobramycin has 2 hydroxyl groups more than sisomicin
and gentamicin and is more polar. Thus, tobramycin should
elute first. Sisomicin elutes before gentamicin, probably because of the polar double bond in its structure, and the
gentamicins elute according to increasing degree of
methylation (30). The dinitrophenyl derivative of amikacin is
more polar than the corresponding derivatives of other
aminoglycosides (38). As a consequence, in reversed-phase
chromatography (RP), it requires a weaker eluent than the others. By reducing the ratio of acetonitrile in the mobile phase,
separation of amikacin has been achieved (38). Kanamycin is a
suitable internal standard for amikacin, with its dinitrophenyl
derivative eluting after the amikacin derivative (38).
2,4,6-Trinitrobenzene sulfonic acid (TNBS) has also been
used for precolumn derivatization of aminoglycosides
(40–42). TNBS forms trinitrophenyl derivatives with
aminoglycosides that are very similar to dinitrophenyl derivatives in their polarity and in derivatized groups. Amikacin (41)
and tobramycin (42) have been analyzed as their
trinitrophenyl derivatives with a C8 column, an acetonitrile–
phosphate buffer mobile phase, sisomicin as the internal standard for tobramycin, and kanamycin as the internal standard
for amikacin. Because of their different polarities, amikacin
and tobramycin are analyzed with different mobile phases.
Kanamycin and amikacin elute in the solvent front in
tobramycin analysis (42). The elution order of the
aminoglycosides in these methods is amikacin, kanamycin,
tobramycin, and sisomicin, similar to the elution order of
dinitrophenyl derivatives. Therefore, the number of hydroxyl
groups can be assumed to determine also the elution order of
the trinitrophenyl derivatives. The mobile phase is optimized
by increasing the proportion of acetonitrile, adding
tetrahydrofuran (THF), or changing the pH. The separation of
tobramycin and sisomicin is insufficient when acetonitrile exceeds 75% (42). Addition of THF reduces tailing (41, 42), but the
same results can be achieved also by reducing the pH (41). When
the pH of the mobile phase is 3 or less, no tailing is observed and
optimal conditions can be achieved without THF (41).
Tobramycin, sisomicin, amikacin, the 4 components of
gentamicin, and kanamycins A, B, and C have been analyzed
as their trinitrophenyl derivatives with a C8 column and an
acetonitrile–phosphate buffer–methanol mobile phase (40).
The solvent ratio of the mobile phase has been varied according to the polarity of the analyzed aminoglycosides. The ratio
of the organic phase is highest for gentamicin and lowest for
amikacin. Kanamycin elutes before tobramycin when they are
in the same run. When kanamycin is added to the amikacin
analysis, it elutes clearly after amikacin. The possibility of
separating all aminoglycosides in one chromatographic run or
the possible interference caused by other aminoglycosides has
not 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 almost
simultaneously with netilmicin and interferes in the analysis.
The separation of tobramycin and netilmicin as their OPA
derivatives has been investigated with a C18 column and a
methanol–EDTA–water mobile phase. Retention times are
too long, and the separation is unsuccessful (33). Reversed-phase separation of amikacin as its OPA derivative
with an EDTA–methanol–water–acetonitrile mobile phase
has been reported (44). Neomycin has been analyzed as an
OPA derivative with a methanol–EDTA–water mobile phase
and a Supelco HISEP column that has a silica-based stationary
phase containing hydrophobic regions shielded by a hydrophilic network (45).
Nonderivatized kanamycin, tobramycin, and neamine have
been separated by RP using amino-bonded column, an
acetonitrile–acetate buffer (70 + 30) mobile phase, and a mass
spectrometer as a detector (46). Underivatized kanamycin
also has been separated with a C18 column and a methanol–acetate buffer mobile phase (47, 48). The underivatized
kanamycin isomers A and C (47) and 3 gentamicin components (48) can be separated and identified by RP and MS.
Ion-Pair Chromatography
Ion-pair chromatography (IP) is well-suited to
aminoglycosides because of their polar, charged, and basic
characteristics under conditions usually used for this technique. IP seems the most popular chromatographic method
for aminoglycoside analysis. The most widely used
counterions are the negatively charged pentane, heptane, and
hexane sulfonates. However, alkylsulfonates are not suitable
for HPLC/MS applications because of their nonvolatile characteristics (16, 49). Instead, the volatile fluorinated
carboxylic acids are used as counterions in HPLC/MS applications for aminoglycosides (49), as well as in preparative
work (15). The counterions used for IP of aminoglycosides
are presented in Table 5.
The mechanisms of retention of aminoglycosides by use of
alkylsulfonates as ion-pairing reagents have not been discussed in the literature. However, separation and retention are
assumed to result from ion-pair formation between positively
charged
protonated
aminoglycoside
and
anionic
alkylsulfonate ions and the different interactions between the
ion pair and the hydrophobic column phase (50).
Derivatization markedly affects the polarity and acid–base
characteristics of the analyzed compound and, it is assumed,
also the IP separation. OPA has been the main derivatization
reagent in IP of aminoglycosides. In postcolumn
derivatization, ion-pair formation is not affected. But in
precolumn derivatization, effects are likely to be observed.
The protonated primary and secondary amino groups of
aminoglycosides responsible for ion-pair formation are the
same ones that undergo OPA derivatization. Thus, retention
mechanisms
and
ion-pair
formation
for
precolumn-derivatized and nonderivatized aminoglycosides
must differ. Unfortunately, these differences have not been
noted in the literature, and no hypothesis of the ion-pair formation between derivatized aminoglycoside and
ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 1027
Table 5. Counterions used in ion-pair chromatographic analysis of aminoglycosides
Ion-pair reagenta
pns
hxs
hps
ocs
ocs and eds
Column phase
Aminoglycosidesa
Derivatizationa
References
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
C18
gnt (3)
OPA pc
59
C8
gnt (4)
OPA
59
C18
str, dhs
—
58, 61
C8
str, dhs
NQS pc
60, 89
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
PLRPc
neo
—
8
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
TFA
HFBA
PFPA
a
b
c
C18
gnt (3)
OPA
71
C18
tob, ntl
OPA
33
C18
str, kan, sis, gnt (4)
—
15
gnt (4)
—
16
str, dhs
—
49
str, dhs, neo, gnt
—
17
C18
For definitions of abbreviations, see footnotes to Table 4.
Numbers in parentheses indicate the number of gentamicin components separated.
Poly(styrenevinylbenzene) copolymer column.
alkylsulfonate has been presented. Different mercaptans are
used in OPA derivatizations, but their effect on the charge and
polarity of the derivatives and on ion-pair formation has not
been discussed.
In most methods, Na2SO4 is added with the alkylsulfonate
to the mobile phase (8, 13, 29, 50–57). The Na2SO4 concentration is the most important factor affecting the retention of
aminoglycosides (8). In IP, the sulfate ion reduces retention
times of aminoglycosides (13) and decreases k′ (8), apparently
because the sulfate ion is more hydrophilic than the sulfonates
used as counterions (8). The ionic strength of the mobile phase
is also affected by the sulfate ion, and without sulfate in the
mobile phase, an increase in the alkylsulfonate concentration
reduces the retention of aminoglycosides, resulting in poor
separation (50).
Pentane, hexane, and heptane sulfonates have been compared in their ability to separate streptomycin and
dihydrostreptomycin (58). The retention of streptomycin increases with the length of the carbon chain in the
alkylsulfonate. Hexane sulfonate provides optimal separation
in a relatively short time. For other aminoglycosides, the use
of different alkylsulfonates has not been compared.
In IP, the mobile-phase buffer should not form ion pairs but
should have sufficient buffering capacity to maintain constant
pH. Acetate buffer is the most widely used with
aminoglycosides (25, 51–57, 59, 60) but phosphate buffer
1028 ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999
also has been investigated (8). With octane sulfonate as
counterion, phosphate buffer is more advantageous than acetate buffer (8). Phosphate buffer is the choice when
underivatized aminoglycosides are detected by UV absorption
(195 nm) because it does not absorb UV light at this wavelength (58, 61). The proportions of phosphate buffer and
alkylsulfonate affect separation characteristics (58). When the
alkylsulfonate concentration exceeds the phosphate concentration, the retention of aminoglycosides can be increased by
adding alkylsulfonate. When phosphate concentration is
greater than alkylsulfonate concentration, the retention cannot
be 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 positively charged at pH < 5, and pH changes do not affect its retention. 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 report concludes that pH 6 is optimal for separation (58).
The pH and the ion strength of the injected sample have an
effect on IP results (17, 25, 54, 57, 62). To achieve the best results, the sample should be dissolved in the mobile phase prior
to injection (54, 57). When the sample is injected in basic solution after sample preparation, split peaks of paromomycin
(54), neomycin (54), and gentamicin (57) are observed, resulting in nonuniform gentamicin chromatography (62). The
inhomogenities, caused by sample addition into the mobile
phase, move more slowly than the solvent front and result in
nonuniform retention of the sample components (57). To
avoid this effect, ion-pair concentrate and mobile-phase buffer
are added to the sample before injection. Improved peak
shape, separation, and repeatability are recorded (54, 57). Apparently, the effect varies among compounds. For example,
using pentafluoropropionic acid (PFPA) as a counterion, sulfuric acid, HCl, and trifluoroacetic acid (TFA) causes peak
splitting but formic acid does not (17).
All IP methods for neomycin concern separation of the
underivatized drug (8, 25, 51, 53, 54). Neomycin stereoisomers
B and C have been separated with a polymer column with octane
sulfonate as counterion (8). Neomycin B (25, 51, 53, 54) and
paromomycin (54) have been analyzed with pentane sulfonate
and Na2SO4 in the mobile phase. Separation is done with a C8
column and a methanol–acetate buffer (1.5 + 98.5) mobile
phase. The life span of the C8 column is limited with this mobile phase (53). Column stability can be improved by increasing the proportion of methanol to 18%, reducing the concentration of Na2SO4, and changing the column to C18. These
changes improve retention time repeatability (53). However,
the C18 column cannot be used for simultaneous analysis of
multiple aminoglycosides.
Derivatization affects separation of gentamicin components with hexane sulfonate as counterion (59). Precolumn
derivatization of gentamicin results in separation of 4 components, whereas only 3 components are separated with
postcolumn derivatization. Similar results are achieved with
other ion-pair reagents. Postcolumn derivatization usually results in 3 gentamicin peaks when pentane or octane sulfonates
are used (50, 57, 62), but 4 peaks also are observed (52). Four
gentamicin components can be separated after precolumn
derivatization when hexane or heptane sulfonate is used as
ion-pairing reagent (59, 63–66). The elution order of the
OPA-derivatized gentamicins is C1, C1a, C2a, and C2. The separation of 3 gentamicin components is achieved with heptane
sulfonate as counterion (11, 67, 68). The elution order of the
components is C1, C1a, and C2 when the stereoisomers C2a and
C2 elute together.
Simultaneous elution of all gentamicin components is advantageous when a quantitative and sensitive method is required. Simultaneous elution has been achieved with the use
of camphor sulfonate as the ion pair and methanol as the organic solvent in the mobile phase (5). Use of acetonitrile instead of methanol results in gentamicin eluting as 3 peaks.
When camphor sulfonate is the counterion and EDTA is
added to the mobile phase, the elution order of the
aminoglycosides studied is amikacin, kanamycin, gentamicin,
and neomycin (5). Dihydrostreptomycin elutes in the solvent
front. The elution order coincides with the order of increasing
amount of amino groups.
Octane sulfonate, when used as a counterion with
2,2-ethane disulfonate in gentamicin analysis, causes
gentamicin to elute after compounds with fewer amino
groups. The use of 1,2-ethane disulfonate increases the separation of gentamicin components (62). Toluene sulfonate has
been used as a counter ion for sisomicin, netilmicin, and
tobramycin analysis, providing selective separation and lacking interference from other aminoglycosides (14).
Perfluorocarboxylic acids have been used as volatile
ion-pairing reagents in the analysis of aminoglycosides
(15, 49, 69, 70). The applicabilities of the different fluorinated counterions for aminoglycoside analysis have been
compared, and the separation mechanism has been investigated in detail (15, 69, 70). Inchauspé and Samain (15) concluded that PFPA and heptafluorobutyric acid (HFBA), despite their short carbon chains, are suitable as aminoglycoside
counterions, except for gentamicin for which only poor separation could be achieved. TFA is especially selective to
gentamicin because other aminoglycosides are not retained on
reversed-phase columns with TFA (15, 69, 70). The retention
of gentamicin depends on the concentration of TFA, and baseline separation of the different gentamicin components can be
achieved (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 different components are partially separated (69). For other
aminoglycosides, TFA, when used with HFBA or PFPA,
serves as an inorganic salt reducing the formation of ion pairs
and decreasing kN (70). HFBA and TFA have opposite effects,
ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 1029
and therefore, the chromatographic mechanisms of these 2 ion
pairs are believed to be different (70). Inchauspé et al. assumed that the cause for the ability of TFA to selectively retain gentamicin is the unique methyl group in α-position relative to an amino group in the gentamicin structure, allowing
simultaneous interaction of the amino group with the ion pair
and the methyl group with the hydrophobic column phase
(69). However, the methyl group can have an effect only when
short-chain counterions are used (69). When HFBA and
PFPA are compared, PFPA can be considered better because
it does not adsorb to the stationary phase as strongly as HFBA
and retention is clearly through an ion-pair mechanism. The
best reagent combination is TFA and PFPA (70).
Simultaneous separation of 5 aminoglycosides has been
achieved by IP with PFPA as counterion (17). The
aminoglycosides are streptomycin, dihydrostreptomycin,
tobramycin, gentamicin as its 4 components, and neomycin.
Detection and identification are by MS. Gradient elution is
needed for satisfactory separation within reasonable time.
With an ordinary C18 column, both gentamicin and neomycin
peaks tail. YMC basic-column improves the peak shapes.
Triethylamine (TEA) is the only positively charged
counterion used for IP separation of aminoglycosides in
gentamicin (71), tobramycin, and netilmicin (33) analyses.
Gentamicin has been analyzed with a mobile phase containing
phosphate buffer at pH 6.2 (71). EDTA and TEA in the mobile
phase are compared. Alteration of the reagent does not change
retention times, but separation and column stability are better
with TEA. Basic tris-buffer–methanol (pH 7.9) mobile phase in
tobramycin and netilmicin analysis causes netilmicin to elute first
and the compounds separate well (33). Removal of tris-buffer
from the mobile phase results in a very broad netilmicin peak.
The chromatographic pattern is improved by TEA, eliminating
peaks of the blank samples (33). Attempts to analyze gentamicin
with this method have been unsuccesful because of the lack of retention (33). The effects of analyte charges and of pH on ion-pair
formation and retention are not discussed.
Ion-Exchange Chromatography
Ion-exchange chromatography (IE) should be a popular
method for analysis of polar aminoglycosides that are ionized
in solutions. However, IE requires careful regulation of pH,
temperature, and ionic strength, and it has not become widely
used in aminoglycoside analysis.
One of the earliest chromatographic methods for
aminoglycoside analysis is anion-exchange chromatography
(72–74). Kanamycin (72), paromomycin (72), and neomycin
(72–74) have been analyzed with a column containing a
strongly basic Dowex 1×2 anion exchanger and water elution.
After separation, aminoglycosides have been detected as their
ninhydrin derivatives (72, 73) or polarometrically (74). The
authors assume that separation is based on adsorption of
analytes to the ion-exchange resin according to their molecular weight/amino group content ratio (72). Neomycins B and
C; kanamycins A, B, and C; and paromomycins I and II can be
separated (72). Tobramycin also has been analyzed with a
polymeric CarboPac PA1 anion exchanger and NaOH gradi-
ent for elution (75). Pulsed amperometry is used for detection.
The aminoglycosides are like other aminosugars, anionic at
high 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 method
before 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 been
analyzed with a strong-cation exchanger (Partisil SCX) and
elution with acetonitrile–phosphate buffer after precolumn
derivatization with fluorescamine, but the different components are not separated (77). The fluorescamine derivatives
have a free carboxyl group. Therefore, an attempt has been
made to analyze gentamicin by anion exchange at pH 6–8. A
wide asymmetric peak indicates the presence of unreacted
amine groups. These amino groups are positively charged and
can be adsorbed to cation exchangers. Fluorescamine and
OPA have been used as alternative postcolumn derivatization
reagents for analysis of kanamycin with an SCX (78).
A cellulose phosphate PII column has been used to separate gentamicin components and analyze the purity of the drug
(10). Tobramycin, neomycin, sisomicin, kanamycin, and
amikacin have been separated with an Amberlite IRC 50 column (79). Aminoglycosides have been eluted with sulfuric
acid and postcolumn derivatized with dihydrolutidine (79).
Normal-Phase Chromatography
Normal-phase chromatography (adsorption chromatography) is usually used for simple compounds that are not ionized
and that dissolve readily in organic solvents. The
hydrophilicity and poor solubility in organic solvents of
aminoglycosides makes normal-phase chromatographic analysis impossible without derivatization. However, normal-phase chromatography has been used for analysis of
aminoglycoside drug preparations when separation of
stereoisomers was of interest. The aminoglycosides are
derivatized prior to separation.
Isocratic methods for simultaneous analysis of
neomycins B and C and neamine have been developed
(80–82). Simultaneous analysis was achieved with neomycin
derivatized with nephthalene sulfonyl chloride (NSCl; 81) or
FDNB (80, 82). Helboe and Kryger (80) stated that in analysis
of
dinitrophenyl
derivatives
of
neomycin
1,2-dichloroethane–methanol–water–diethylamine elution
gives better and faster separation than chloroform–THF–water elution. Three gentamicin components and kanamycin can
be analyzed by normal-phase chromatography with NSCl
precolumn derivatization (81), and 3 gentamicin components,
paromomycin, and kanamycin can be analyzed as their
dinitrophenyl derivatives (82). When normal- and reversed-phase separations of dimethyl–phenyl derivatized
amikacin are compared, the normal-phase method is more
sensitive, although retention time variations are higher and repeatability is poor (39).
1030 ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999
Detection Methods in Aminoglycoside Analysis
In GC, aminoglycosides have been determinated with either
FID or ECD. ECD is used mainly for fluorinated aminoglycoside
derivatives. Because GC methods are few, HPLC detection
methods are the primary focus of this discussion.
UV–Vis and refractive index detectors are popular HPLC
detectors. Refractive index detectors are generally applicable,
but they are sensitive to pressure and temperature changes.
They also lack selectivity and sensitivity. However, refractive
index detection is good for preparatory methods when sample
derivatization is impossible and concentrations are high
(15, 70). Refractive index detectors have been used mainly for
preparatory separations of tobramycin, kanamycin, amikacin,
sisomicin, streptomycin, neomycin, and gentamicin (15, 70).
However, refractive index detection is too sensitive to
changes in the environment for kanamycin analysis (78).
Aminoglycosides lack UV-absorbing chromophores and
have to be derivatized to gain UV detectability (12). In process
control, when fast and specific chemical assay is needed, UV
detection at 195 nm has been used for streptomycin and
dihydrostreptomycin (58, 61). It has been assumed that the
method would be applicable also to neomycin and
paromomycin (58).
Optical rotation has been used to detect nonderivatized
aminoglycosides (10, 74, 83). Neomycin components A, B,
and C are all optically active (74), and gentamicin includes
7 optically active components, of which C1, C1a, C2a, and C2
are biologically active (10). The optical activity allows their
detection with polarimetry. The advantages of polarimetry
over other direct detection methods are selectivity and sensitivity. Polarimetry has been considered a promising method
for aminoglycoside applications (83).
Derivatization in Detection Enhancement
Derivatization facilitates extraction, analysis, and identification of poorly soluble and ionized compounds and improves
sensitivity. In LC, derivatization can be performed pre- or
postcolumn and on- or off-line. Most derivatizations improve
UV, fluorescence, or electrochemical detectability. Fluorescing derivatives usually are more desirable because fluorescence detection generally provides better sensitivity and
selectivity 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 is
possible (44). The reactive hydroxyl groups further increase
the possibility to form different derivatives (37). The reagents
for aminoglycoside derivatization are presented in Table 6.
In addition, a reagent called dihydrolutidine has been used
to derivatize neomycin, sisomicin, kanamycin, tobramycin,
and amikacin (79). Dihydrolutidine derivatives are formed
when the amino group of the aminoglycoside reacts with the
keto and enol forms of acetylacetone. The derivatives are fluorescent and can be detected with an excitation wavelength of
421 nm and an emission wavelength of 488 nm.
The most widely used reagent is OPA, which reacts in the
presence of mercaptan or other strong reducing agents in basic
conditions with primary amines to form fluorescent derivatives (85). Its popularity is due to the fast rate of the reaction at
room temperature and the possibility to perform the reaction
in water solution (31, 78). In addition, OPA is stable in different buffers (78). Borate buffer (pH 9.5–10.5) has been used
predominantly with OPA derivatization of aminoglycosides.
In fluorescence detection, the excitation wavelength generally
is 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 wavelengths are 450 (13, 44), 430 (32), and 440 nm (62). Background noise is lowest with an excitation wavelength of
260 nm and an emission wavelength of 340 nm (71). UV absorption at 330 (59, 63–66), 350 (11, 82), or 254 nm (82) is
also used. The first reported OPA derivatization for
aminoglycosides is postcolumn derivatization of kanamycin
(78). The reducing agent is mercaptoethanol and the basic
buffer is borate.
In postcolumn derivatization of aminoglycosides, OPA
usually
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 in
the detector (13). Triton-X-100 in lieu of Brij-35 enhances fluorescence (5, 56). However, Triton-X-100 is more temperature sensitive than Brij-35 (5).
Derivatization temperature, reactor geometry, and flow
rate of the derivatization reagent have been optimized
(5, 54, 56, 57). The results are similar for the different
aminoglycosides tested. Reactor geometry affects both sensitivity and separation (56). The reactor coil length is usually
1–2 m, but to separate gentamicin components properly, the
coil must be less than 2 m (57). Resolution of amikacin suffers
when the coil is more than 1 m (56). Reducing the internal diameter of the coil increases sensitivity (13, 57).
The most common reaction temperatures for OPA
postcolumn derivatization are 45E and 50EC. The effect of
reaction 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), and
for neomycin at 33EC (51). Partial derivatization occurs with
amikacin at temperatures below 50EC, and fluorescence
yield decreases at reaction temperatures over 60EC (56).
This result has led to the assumption that fluorescence is temperature dependent.
Reagent flow affects the observed fluorescence (5, 56, 57).
However, the optimum reagent flow rate depends on other
chromatographic parameters as well, and no absolute values
can be determined. Some authors have stated that the optimum
flow rate is half the flow rate of the mobile phase (54). Optimal flow rates ranging from 0.30 to 0.55 mL/min have been
reported (5, 56, 57). If the flow is too high, the eluate is diluted
to such an extent that the signal becomes too weak (56).
Gentamicin components react differently to changes of reagent flow (57), which does not affect the fluorescence of C1a
ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 1031
Table 6. Reagents used for aminoglycoside derivatizationa
Aminoglycoside
ami
Derivatization reagent
Detection method
References
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
kan
neo
ntl
par
sis
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
OPA pc
fl
5, 13, 78
FDNB
UV
38, 82
NSCl
UV
81
TNBS
UV
41
Fluorescamine
fl
78
Ninhydrine
UV
72
OPA pc
fl
5, 13, 25, 51, 53, 54
OPA
fl
45
FDNB
UV
80, 82
NSCl
UV
81
Ninhydrine
UV
72, 73
OPA pc
fl
13, 14, 50
OPA
fl
26, 33, 86
OPA
UV
65
BSC
UV
27
Dansyl chloride
fl
43
OPA pc
fl
54
FDNB
UV
82
Ninhydrine
UV
72
OPA pc
fl
13, 14, 50
26, 86, 87
OPA
fl
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
OPA pc
fl
56
tob
a
OPA
fl
26, 33
FDNB
UV
30, 36, 37
TNBS
UV
40, 42
For definitions, see footnotes to Table 4.
1032 ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999
but increases the fluorescence of C1 and C2 when flow rate is
increased. This result suggests that the reaction of C1a is faster
than the reactions of the other 2 components.
A weakness of postcolumn derivatization is the increase of
baseline noise as a consequence of reagent pumping (26, 71).
Reagent consumption is also greater than with precolumn
derivatization, and an extra pump is needed for the
derivatization reagent. Improving the sensitivity in
postcolumn derivatization is difficult because derivatization
time can be increased only by increasing the dead volume of
the system, which, in turn, results in broadening of bands.
Precolumn OPA derivatization of aminoglycosides also
has been reported (31–33, 44, 45, 64, 66–68, 86, 87). Advantages are simplicity of equipment and effective separation of
interfering 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 precolumn
OPA derivatizations. However, information concerning their
applicability is contradictory. Mercaptoethanol has been
widely used and other reagents have been compared to it.
Nonetheless, mercaptoethanol–OPA derivatives are not stable
and, therefore, unsuitable for precolumn derivatization (87).
OPA derivatives of aminoglycosides are stable for 8 h at
–20EC (33), and OPA derivatives of neomycin are formed
even during the first 5 min of freezing (45). When
mercaptoethanol–OPA derivatives are kept at –4EC, the fluorescence yield decreases 5% in 1 h (33). At room temperature,
the derivatives decompose exponentially, causing a 30% decrease in fluorescence in 10 min (78).
Mercaptopropionic acid is used for OPA derivatization of
sisomicin and netilmicin when better stability of derivatives is
needed (86, 87). The steric group in mercaptopropionic acid is
bigger than that in mercaptoethanol, and this is believed to improve the stability of the derivatives (87). The solution where
derivatives are kept also affect stability (86). Unlike what happens with mercaptoethanol, the extra mercaptan does not
destabilize the derivatives when mercaptopropionic acid is
used (87). A reaction time of 10 min is satisfactory at 60EC,
but at 20EC, 1 h is needed. Partial derivatization of sisomicin
results in formation of 3 different derivatives. The reaction is
accompanied 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 stable than those formed with mercaptoethanol (65), but they decompose under acidic conditions (63). A 16–43% decomposition occurs during the first hour after derivatization if
derivatives 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 of
components are different. The relationship between response
and number of derivatized groups is not discussed.
Only mercaptoethanol has been used as reducing agent in
OPA 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 used
for amikacin (44), gentamicin (32, 67, 68), and neomycin
(45). The stability of derivatives is not discussed, except that
neomycin needs to be frozen for complete derivatization (45).
Detector responses of OPA derivatives of the different
gentamicin components have been compared and results differ. On the basis of the number of derivatized groups, the responses of C1a and C2 should be equal and that of C1 smaller.
Same responses indeed are reported for C1a and C2, but the response is much smaller than that recorded for C1 (71). Another
study finds equal C2 and C1 responses but a smaller C1a response (32). Moreover, different detector responses are reported for all the components (26, 66). Reaction kinetics and
reasons for the observed differences are not discussed. Furthermore, most authors use a gentamicin standard that contains an unknown mixture of the components. Consequently,
identification of components is not based on individual standards. The variation in detector responses indicates that different derivatives are formed with the various methods, and they
probably also affect the elution order. However, the same elution order is suggested in different reports.
In partial OPA derivatization of amikacin (44) and
neomycin (45), 2 peaks are observed instead of one for
neomycin, and several peaks are produced for amikacin if the
derivatization solution is not heated. Discussion of partial
derivatization has been limited, and most authors do not take
this possibility into account.
FDNB reacts with primary and secondary amines in basic
conditions (36), producing highly UV absorbing derivatives
(37). The UV absorption maximum is at 365 nm. FDNB has
been used for derivatization of almost all aminoglycosides
(29, 30, 36–39, 80, 82), and a comprehensive study of its reaction kinetics with aminoglycosides has been published with
tobramycin as model compound (37). Derivatization conditions have been developed with borate (39, 80, 82) or tris
(29, 30, 36, 37) buffers. Phosphate, phthalate, acetate, and bicarbonate buffers also have been investigated (37, 39). FDNB
reacts with the amine or hydroxyl groups of most buffers, producing 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. The
main problem of FDNB derivatization of aminoglycosides is
the different solubilities of the reagent and the analytes (82).
Organic solvents must be used in the derivatization solution to
dissolve FDNB. Derivatization buffers, organic solvents, temperatures, times, and pH conditions have been varied to optimize derivatization conditions. Also a big problem with
FDNB is its toxicity (37). FDNB irritates the skin and may
cause blistering dermatitis and severe allergic reactions. Absorption of FDNB by inhalation, ingestion, or through the skin
can be lethal. The different methods used for aminoglycoside
derivatization with FDNB are presented in Table 7.
Tobramycin analysis has revealed that yield and speed of
the FDNB reaction, as well as occurrence of several side reactions, are strongly pH dependent (37). Derivatization of
tobramycin amine groups and of hydroxyl groups thereafter
follow second-order kinetics. Hydrolysis of FDNB in basic
ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 1033
Table 7. Reaction conditions for FDNB derivatization of aminoglycosidesa
Aminoglycoside
gnt, tob
gnt, sis, tob
Solvent
Buffer
Temperature, EC
Time, min
References
ACN
Tris pH 9.0
80
45
29, 36
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.
solutions follows pseudo first-order kinetics and is faster the
more basic the derivatization solution is. Hydrolysis of the
tobramycin
derivative
decreases
with
increasing
derivatization temperature, but increasing the solution pH results in faster dissociation. The optimum pH range of the reaction is very narrow because of the high reactivity of the
aminoglycoside hydroxyl groups with FDNB and the
pH-dependent dissociation of the derivatives (37).
Best results for gentamicin, sisomicin, and tobramycin
derivatization with FDNB have been achieved with tris buffer
at pH > 9.5 and 80EC (29). Higher reaction temperature decreases the repeatability, and reaction time of 90 min results in
incomplete derivatization (29). Molar absorptivity and elemental analysis have confirmed derivatization of all amino
groups of tobramycin (36). Gentamicin derivatization is more
sensitive to derivatization conditions than tobramycin.
Proton nuclear magnetic resonance spectrometry of the
amikacin–FDNB derivative indicates that amino groups are
derivatized (38, 39). Derivatization yields of 92% for standards
and 77% for serum have been achieved with NaOH (39).
NaOH gives slightly higher yields than borate buffer (38). Inorganic salts such as Na2SO4 interfere with derivatization.
Side products of aminoglycoside derivatization with
FDNB 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-prop
anediol (the reaction product of tris and FDNB), and
2,4-dinitrophenyl, the hydrolysis product FDNB (36). Generally, these side products can be seen in chromatograms, and
they may interfere with analysis.
FMOC–Cl is a very reactive derivatization reagent, reacting with both primary and secondary amines (34). The reaction is very fast in basic solutions at room temperature. The
derivatives are very stable and fluorescent. However, during
derivatization FMOC–OH is formed, which can interfere with
chromatography. For gentamicin derivatization (34), the best
derivatizating solution is borate–acetonitrile (1 + 1) at
pH 7.5–9.0. Acetonitrile is needed to dissolve FMOC–Cl.
Derivatization is complete in 10 min. The detector responses
of the FMOC derivatives of gentamicin components are equal.
Naphthoquinone sulfonate (NQS) reacts with guanidine
compounds in basic conditions to produce fluorescent derivatives (88). It is suitable for derivatization of streptomycin and
dihydrostreptomycin. NQS decomposes rapidly in basic solutions. Therefore, it is not suitable for postcolumn
derivatization in a basic environment. Postcolumn
derivatization with NQS would require 2 extra pumps, one for
NQS and one for the base. NQS has been added as a solution
to the acidic mobile phase before separation (88). Optimal
conditions for streptomycin analysis include a 5 m
postcolumn reactor coil at 65EC, NQS concentration of
0.4 mM, and NaOH concentration of 0.5M (88, 89). A
high-quality postcolumn system is essential for maintaining
baseline stability and constant reactor temperature (89). For
fluorescence detection, excitation wavelengths of 347, 351,
and 365 nm have been used with emission wavelengths of
418, 420, and 418 nm, respectively (60, 88, 89).
TNBS reacts with primary amines in basic aqueous solution at room temperature without interfering side reactions
(41, 42). The molar absorptivity of the TNBS derivatives is
highest at 340 nm. TNBS has been used to derivatize
gentamicin (40), tobramycin (42), and amikacin (41) with
kanamicin (41) and sisomicin (42) as internal standards. The
aminoglycosides are dissolved in acetonitrile to speed up the
derivatization and to dissolve the nonpolar derivatives (41).
At room temperature, the reaction requires 19–20 h (42). Increasing the temperature improves the results, and 70EC is
best temperature for derivatization (41, 42). At lower temperatures, several derivatives are formed when derivatization
time is less than 30 min. At temperatures higher than 80EC,
tobramycin derivatives decompose (42). TNBS derivatization
of gentamicin is complete in 15 min at 70EC (40). The
trinitrophenyl derivatives have absorption maxima at 350 and
420 nm and give different detector responses.
TNBS derivatization of amikacin and tobramycin is affected 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 performed within pH 9.5–10.0. Carbonate, borate, and phosphate
buffers are not satisfactory because of their low buffering capacities in this pH range and solubility problems resulting in
1034 ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999
complex formation with aminoglycosides. Therefore, tris
buffer produces the best results (41, 42).
Fluorescamine reacts rapidly in a basic aqueous solution at
room temperature with primary amines to produce strongly
fluorescent derivatives (90). The reagent itself and its degradation products are not fluorescent. Kanamycin (78) and
gentamicin (77) have been derivatized with fluorescamine.
For kanamycin, postcolumn derivatization in acetone has
been used (78). The derivatives are stable, and the sensitivity
of the method is good. For precolumn derivatization of
gentamicin, best results are achieved with 0.03M or stronger
phosphate buffer at pH 7.2–8.0 (77). The fluorescamine concentration must exceed the gentamicin concentration by
10 times. The fluorescence responses of the different
gentamicin components are equal, and the method is accurate
and repeatable.
Dansyl chloride has been used for precolumn
derivatization of netilmicin (43) and gentamicin (28, 91).
Dansyl chloride reacts with amines to form fluorescent derivatives (92). The derivatization solution for aminoglycosides is
acetonitrile–phosphate buffer at pH 11 (28, 43, 91). Reaction
temperature and time are 75EC and 5 min, respectively. Buffer
volume, pH, reaction temperature, and time affect results (28).
At 100EC, the derivatives decompose rapidly, and at low temperatures, reaction is not complete. The possibility of partial
derivatization of gentamicin has been investigated but could
not be detected (28). Furthermore, dansyl chloride reagents
from different producers differ in solubility, and a too low
dansyl chloride amount gives incorrect results (91).
DNBCl has been used to derivatize gentamicin,
tobramycin, kanamycin, and amikacin (35). Derivatives are
detected with UV at 254 nm. The reaction is repeatable, and
the derivatives are stable. However, an unidentified derivative
is formed with gentamicin.
BSC has been used to derivatize netilmicin and
gentamicin (27). Derivatization must be at pH 7–9 and 75EC
for 10 min. Too high a temperature or too long a reaction
time destroys the derivatives.
NSCl reacts by nucleophilic substitution with hydroxyl and
amine groups, forming UV-absorbing (254 nm) derivatives
(93). Neomycin, gentamicin, and kanamycin have been
derivatized with NSCl at pH 8.0–9.0 and 100EC for 10 min
(81, 93). Phosphate buffer is best for NSCl derivatization of
aminoglycosides.
It is difficult to compare the different derivatization reagents used in aminoglycoside analysis because most reports do not give derivatization yields and method sensitivities. Fluorescent derivatives do not seem to be detectable at
lower concentrations than UV-absorbing derivatives, as
would be expected.
Postcolumn derivatization with OPA appears preferable
when analysis speed is critical because the derivatization time
is negligible. However, because of the limitations of
postcolumn derivatizations, use of an OPA precolumn
derivatization could be a better option when reaction time is
0–15 min. Other fast-reacting reagents are FMOC–Cl, dansyl
chloride, and BSC, which all react in <10 min. The disadvan-
tage of dansyl chloride is that the derivative has to be extracted
from the derivatization solution before chromatography.
Dansyl chloride has been applied to gentamicin (28) and
netilmicin (43); FMOC–Cl, to gentamicin (34); and BSC, to
gentamicin and netilmicin (27). No studies about the applicability of these reagents to other aminoglycosides have been reported. In terms of derivatization time, FDNB is the most
problematic 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 a
basic environment. FDNB reagent is the most limited in terms
of buffer selection because of its side reactions with buffer ions
and its narrow pH optimum for the reaction. The limited solubility of aminoglycosides demands use of reagents that are soluble in water or aqueous solutions of methanol or acetonitrile.
The most problematic reagents in terms of solubility are dansyl
chloride and FDNB. The dinitrophenyl derivatives of
aminoglycosides also are poorly water soluble. OPA has the
best solubility characteristics among derivatization reagents.
The stability of the aminoglycoside derivatives has been
subject to limited investigation. Because most reagents are
used for precolumn derivatization, the stability of derivatives
is very important in consideration of the best derivatization
option especially when automatic equipment is not available
and quantitative results are needed. Furthermore, the structures of aminoglycoside derivatives formed with different reagents have been inadequately investigated. The number of
derivatized groups is rarely specified. When several different
derivatives are formed, attention should be paid to the
derivatization yield and to the number of different derivatives
formed. Of the reagents used for aminoglycoside
derivatization, only FDNB has been investigated with respect
to derivatization kinetics and the structures of the
aminoglycoside derivatives formed.
Electrochemical Detection
Amines, thiols, and hydroxyl groups oxidize readily at
electrodes such as the carbon electrodes used for electrochemical detection (55). The multiple amino and hydroxyl groups
in the aminoglycoside structures are apparently the source of
their electrochemical activity (55). The advantage of electrochemical detection in aminoglycoside analysis is that it does
not require derivatization. However, the structure of the detected compound cannot be identified, and confirmation of a
specific chromatographic peak is not possible (16). The inability to do these is a particular disadvantage when retention
times and elution orders change.
All gentamicin components have been detected electrochemically with a carbon electrode (16, 55). A
voltammogram measured manually for gentamicin and a detection voltage of +1.3 V was selected. When the detection potential was changed to a more positive potential, the signal increased but the baseline deteriorated. The detector response
for gentamicin was linear from 16 to 30 µg with r = 0.999.
Substantial changes in the detector response were observed
ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 1035
Table 8. Pulsed electrochemical methods for aminoglycoside detectiona
Aminoglycoside
Working
electrode
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
–0.2
125
125
400
0.25
76
Method
a
b
Counter
electrode
E1, V
E2, V
–1.3
E3, V
t1, ms
t2, ms
t3, ms
NaOH, Mb
Ref.
For definitions, see footnotes to Table 4.
Concentration after column.
with changes of Na2SO4 concentration in the mobile phase. A
change from 0.2 to 0.3M reduced peak areas by 40%.
Pulsed methods for aminoglycoside detection are presented in Table 8. All aminoglycosides react via a common
mechanism in pulsed amperometry (76). The sensitivity of detection in neomycin analysis with pulsed electrochemical detection (PED) improves by adding NaOH to the eluent to increase the pH to 13 (8). In tobramycin analysis, a high pH is
required to oxidize tobramycin, and a gold electrode is used
(75). The sensitivity of the tobramycin method (75) is similar to
or better than that reported for different derivatization methods.
With an injection volume of 10 µL, the limit of detection of the
method is 2 ng/injection, and the detector response is linear
from 10 ng to 1 µg/injection with r = 0.999 (75). Stability problems have been encountered when neomycin was analyzed
with PED, and specialization and experience are required from
the analyst to achieve accurate and repeatable results (8).
Mass Spectrometry
In theory, all drug compounds can be detected by MS but
the use is limited by the complexity of the equipment and high
costs (83). Regulatory methods require confirmatory identification at the action level (94). Because of the sensitivity and
possibility for confirmatory identification, MS is often the
only method that offers the required sensitivity and selectivity
for confirmation of different analytes.
No GC/MS application for aminoglycosides has been published. Available methods have been limited to LC/MS instruments and to the MS analysis of pure standards. LC/MS interfaces used in aminoglycoside analysis have been thermospray
(16), moving belt (46), ion spray (17, 49), and heated pneumatic nebulization with atomspheric pressure chemical ionization (APCI; 47, 48). For aminoglycoside ionization in MS
analysis, plasma (95) and field (69) desorption, chemical ionization (CI; 46, 96), and APCI (47, 48) have been used.
The ion spray interface (IS) is well adapted to
aminoglycosides that are ionized in solutions (49). Desorption
of ions from the liquid phase to the gas phase is caused by an
electric field without heating or other severe conditions. The
advantage of IS–MS in quantitative work is that the ion flow
concentrates one specific species, improving sensitivity. A
disadvantage of IS is that the ionic strength of the mobile
phase has to be minimized while the organic solvent content is
maximized. This is a limitation especially for aminoglycosides,
which are usually analyzed by IP. For this reason, the concentration of the ion-pair reagent must be carefully optimized.
However, the counterion concentration does not have significant effects on mass spectra (49). Gentamicin, neomycin,
tobramycin, streptomycin, and dihydrostreptomycin have been
analyzed with LC/IS–MS (17, 49).
Better sensitivity has been obtained with positive-ion than
with negative-ion monitoring when IS–MS is used in
aminoglycoside analysis. Thus, positive-ion spectra have
been investigated in more detail (17). Selected-ion monitoring
does 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 for
confirmative analysis. With limited heating in IS, only a few
fragments can be observed. Collision-induced activation
(CAD) allows formation of daughter ions from various ions of
the spectra, enabling confirmation of the structure (17). In
MS/MS, use of singly charged parent ions of aminoglycosides
provides a lower signal-to-noise (S/N) ratio in the
chromatograms than use of doubly charged parent ions, partly
because of the smaller absolute intensity of singly charged
species and partly because of the mutual repulsion of
protonated groups in doubly charged species. In the mass
spectra of aminoglycosides, doubly charged species dominate, and single or triple charging rarely occurs (17). However, when the ionization potential is increased, the quantity of
singly charged ions increases (17, 49).
With heated pneumatic nebulization systems, APCI is usually preferred (47, 48). LC–APCI is generally applicable for
nonvolatile compounds that are unsuitable for GC analysis.
The main advantage of APCI–MS is that it can be performed
under usual HPLC separation conditions with flow rates of
0.3–2.0 mL/min (47). The most important parameters to be
optimized in analysis of nonvolatile compounds are the temperatures of the nebulizer and vaporizer (48). Kanamycin
(47, 48) and gentamicin (48) have been detected by
LC–APCI. The mass spectra of gentamicin components are
different from each other in APCI–MS (48). The kanamycin
isomers A, B, and C can be separated by LC–APCI, and the
mass spectra of the different components are also different.
1036 ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999
Thermal decomposition of several aminoglycosides has been
observed in LC–APCI interfaces (17). APCI spectra resemble
CI spectra but with less fragmentation. The limited fragmentation frequently offers insufficient structural information (48)
and, therefore, CAD is used with APCI–MS to obtain structure-confirming fragment ions. Most ions obtained with CAD
also can be seen in the original APCI spectra.
The thermospray interface (TS; 75) has been used for
gentamicin analysis (16). Three gentamicin components (C1,
C1a, and C2) and another component assumed to be
gentamicin C2a have been detected. Instead of strong molecular ions [M + H], gentamicin components produce high intensity fragments with TS–MS. Identification of the individual components is possible on the basis of the mass spectral
information, and changes in elution order can be detected immediately. The greatest advantage of the MS detection apart
from identification is the lack of need for derivatization. In addition, the sensitivity of LC–TS for gentamicin is much better
than the sensitivity obtained with an electrochemical or
UV–Vis detector (16).
The suitability of moving-belt interfaces for
aminoglycoside analysis has been investigated (46).
Kanamycin, tobramycin, and neamine have been studied with
chemical ionization with ammonia. Different belt types have
an effect on the spectra. The molecular weight and the order of
amino sugars in the molecule can be determined for all
aminoglycosides studied with this method (46).
Plasma desorption MS (PD–MS) yields useful information
about the structure of nonvolatile or thermolabile
aminoglycosides (95). The mass spectra of neomycin,
kanamycin, paromomycin, tobramycin, streptomycin,
dihydrostreptomycin, amikacin, netilmicin, sisomicin, and
gentamicin have been determined with time-of-flight (TOF)
PD–MS and positive-ion monitoring (95). This method facilitates confirmative analysis of aminoglycosides presumably
because the resulting spectra are very repeatable. Very strong
molecular or quasimolecular ions are obtained for
aminoglycosides studied with positive-ion monitoring, but
much more fragmentation occurs with negative-ion monitoring. Extraction of strongly polar aminoglycosides from biological matrixes can be difficult. Therefore, more easily
extractable dinitrophenyl derivatives have been prepared, and
their ionization and detectability have been studied. Except for
derivatives of streptomycin and dihydrostreptomycin, the molecular weights and mass spectra of all the dinitrophenyl derivatives can be recorded with positive- and negative-ion
monitoring. The best spectra are obtained with positive-ion
monitoring (95). Negative ionization produces only little fragmentation for the derivatized aminoglycosides and [M–H]– or
M− C is the main peak formed.
The molecular ion is either very weak or does not exist at
all in aminoglycoside mass spectra when electron impact ionization (EI) is used (95). Field desorption MS (FD–MS) has
been used to determine gentamicin from LC fractions (69).
However FD–MS is usually applicable only to molecular
weight determination because practically no fragmentation
occurs (96). Thus, emitter chemical ionization (CI), a tech-
nique characterized by use of the activated FD emitter as a
solid probe, has been used for aminoglycosides to obtain both
molecular ions and characteristic fragments (96). Gentamicin,
kanamycin, and dibekacin have been analyzed with ammonia
or isobutane as reagent gas. The molecular peak and 9 characteristic ions resulting from the dissociation of the glycoside
bonds can be detected with both reagent gases. The molecular
weights of kanamycin and dibekacin cannot be determined
from MS using conventional CI. Thus, emitter CI can be considered a better method. Generally, aminoglycosides fragment
between the glycosidic oxygen and the anomeric carbon when
the hydrogen is transferred to oxygen (96).
Extraction and Analysis of Aminoglycosides from
Different Matrixes
Pure Drug Preparations
The quality of drug preparations is continuously monitored
(1) during preparation by the producer and later in quality control according to requirements of pharmacopoeias.
Confirmative methods of analysis are preferred. When the
drug is formed from several components, limits for component ratios are given (63). Fast, specific chromatographic
methods are needed for quality control of aminoglycoside
preparations and injectable solutions (64).
Accurate methods for measuring gentamicin components
are required because the different components apparently
have different toxicity (40). Quality control chromatographic
methods for gentamicin have to be in agreement with the official microbiological method (59). Compatible results between
bioassay and chromatographic methods can be achieved only
when gentamicin components C2a and C2 elute together (59).
Remarkable differences (up to 100%) in the ratios of different
components have been found among gentamicins from different producers (11).
Gentamicin component ratio (10, 11, 13, 16, 55, 59, 63,
64) and stability (66) have been extensively studied. Occasionally, when gentamicin preparations are analyzed, unsubstantiated assumptions have been made. These include the
presumptions that equimolar amounts of the different components give the same detector response as OPA derivatives
(59, 63, 64) or that the microbiological activities of the different components are equal (64). Some authors have used individual
gentamicin
components
for
quantitation
(10, 11, 13, 55). The area response factors of the
4 OPA-derivatized gentamicin components differ (11). Consequently, a quantitative method based on the assumption that
the area response factors of OPA-derivatized gentamicins are
similar gives results that are not representative of the actual
component ratio (11). In quantitation of the 4 gentamicin components, it is necessary to know their specific responses in the
chromatographic procedure (11). Different references give divergent analytical results, and the component C2a has been determined differently in different papers. Some authors consider it as an individual component (16, 63–66), while others
include it in the C2 component (29–33, 48, 55, 67, 68, 71),
making comparison of methods difficult.
ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 1037
Neomycin in ointments has been analyzed by dissolving
samples in chloroform and separating neomycin by
centrifugation (19). The neomycin is then dissolved in water
and freeze dried for GC analysis. Recovery of the method is
98–100%. Dissolution of ointments in chloroform also has
been used for gentamicin analysis (66). Gentamicin is extracted from the chloroform solution with phosphate buffer.
The gentamicin in the phosphate buffer is derivatized with
OPA and analyzed by HPLC. The recovery is 94–97%.
Plasma and Serum
The response of a patient to a specific drug depends on various factors, such as age, sex, renal and liver function, and simultaneous use of other drugs (97). Concentration in serum
after drug administration is often difficult to predict (33). Correlation between the achieved concentration in serum and
pharmacological and toxicological effects is substantial, and
knowledge of the concentration in serum is of great help in
treatment, especially when the therapeutic range is narrow.
Therapeutic drug monitoring is used frequently when
aminoglycosides or certain other drugs are used for treatment
(14, 25, 29, 40, 57, 97). The methods used for monitoring
aminoglycoside concentrations in plasma and serum are summarized in Table 9.
Because of their solubility characteristics, direct extraction
of aminoglycosides from plasma is difficult and impractical
(27, 28). Protein precipitation and separation of amino acids
from aminoglycosides prior to chromatography is necessary
because of interference both in chromatography and in
derivatization (14, 28). Interferences have been reported in
gentamicin, 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. Contradictory
information is available about the applicability of different
buffers and organic solvents for aminoglycoside extraction,
and different aminoglycosides are often extracted with different buffers or solvents. TCA and tungstic acid precipitate
gentamicin with the proteins (28). However, >90% recovery
of neomycin from serum has been achieved with TCA to precipitate proteins (25). However, neomycin recoveries from
plasma are lower than from water when TCA is used, perhaps
because of endogenic substances in the plasma. The pH of the
precipitation buffer affects streptomycin recovery (61). Recovery is maximum at pH 2 and decreases with increasing pH.
In gentamicin analysis, ultrafiltration is useless because the
compound is adsorbed to the membrane (28).
Tris buffer is well suited for extraction of tobramycin and
amikacin from serum and for protein precipitation (41, 42).
However, protein precipitation is completed with
TNBS−acetonitrile derivatization solution. Tris buffer together with acetonitrile results in considerably lower
tobramycin recovery than has been obtained for gentamicin or
sisomicin (36) and also less than observed for tobramycin
with tris alone. Phosphate buffer with acetonitrile provides
better recovery for netilmicin than for gentamicin, while
tobramycin recovery decreases with decreasing phosphate dilution volume (33). However, recoveries with phosphate
buffer and acetonitrile are higher than those with tris and
acetonitrile. Preference has been given to tris buffer in precipitation because inorganic buffers tend to precipitate from
acetonitrile–water solutions (29).
Acetonitrile is the best organic solvent for extracting
gentamicin from serum and for precipitating serum proteins
(28, 77). Other water-soluble organic solvents such as ethanol, methanol, and acetone do not provide satisfactory results
(28, 77). Use of methanol for protein precipitation and
gentamicin extraction from plasma has been reported (62). Diethyl ether, ethyl acetate, hexane, and methylene chloride
have been investigated for back extraction of the precipitation
mixture (28, 77). Best results are achieved with methylene
chloride (28, 77). Gentamicin recovery is poor (77), but it
could be improved by adding phosphate buffer to acetonitrile.
Tobramycin recovery is also low after acetonitrile precipitation and cannot be improved (30).
Solid-phase extraction (SPE) has been used to extract TNBS
and FDNB derivatives of aminoglycosides (39, 41, 42). This
extraction prolongs column life and removes the large solvent
front and interfering peaks. Silica SPE has been used to extract
and clean up dinitrophenyl derivatives of amikacin (39). The
derivatives are eluted from silica with acetonitrile–water
(68 + 32). Trinitrobenzoyl derivatives of amikacin and
tobramycin (with kanamycin and sisomicin as internal standards) have been extracted with a Bond-Elut C18 solid-phase
column (41, 42).
SPE with C18 (61) and silica (32, 44, 71) columns and
CM-Sephadex C25 ion-exchange resins (14, 38, 50, 56, 57, 87)
has been used to extract aminoglycosides from plasma. SPE has
been used without precipitation of serum proteins, and
aminoglycosides have been separated from the matrix according to partition differences (32). Among organic solvents, SPE
and CM-Sephadex, CM-Sephadex gives the highest recoveries for sisomicin, tobramycin, and netilmicin (14).
Aminoglycosides are strongly basic compounds, and ion
exchange can be expected to be a good sample preparation
method. CM-Sephadex is applicable to all aminoglycosides
except streptomycin (14, 38, 50, 56, 57, 87). In extraction of
streptomycin from plasma, CM-Sephadex ion exchange has
been considered too complicated, and interfering compounds
in plasma cannot be separated from streptomycin (61). Silica
gel has been used for sample cleanup and for extraction of
gentamicin (32, 71) and amikacin (44). Gentamicin is charged
to the silica column in water-diluted serum, and amikacin, in
phosphate–buffer-diluted serum. These aminoglycosides
have been derivatized with OPA in the silica column before
elution with ethanol (32, 44) or methanol (71).
Ion exchangers with carboxylic acid functional groups
have worked well in extraction of gentamicin (34).
Gentamicin is retained on a carboxypropylsilica ion-exchange
column at pH 6–8 and eluted at pH > 9.5. The ion-exchange
method is simple and reliable.
Table 9. Methods used to monitor aminoglycoside levels in plasma and serum
Aminoglycosidea
sis, ntl, tob*
Extraction and protein
precipitation
Cleanup
Chromatography
derivatizationb
Intraday
CV, %
Interday
CV, %
r
Determination
Slope of
Linear range,
limit, µg/mL calibration curve
µg/mL
Recovery, %
Ref.
—
CM-Sephade
x
IP, OPA pc
—
1.9–3.1
0.999
—
0.034 (sis)
0.101 (ntl)
0.3–22 (sis)
0.2–11 (ntl)
—
14
0.5% H2SO4 incubation
80EC
Hexane
GC, TMSI/HFBI
—
—
0.979
<0.6
—
2.5–20
—
23
gnt, tob, ntl,
0.5% H2SO4 incubation
83EC
ami, par*, kan*
Hexane
GC, TMSI/HFBI
—
—
—
<0.6
—
2.5–20
—
23
gnt, tob, kan*
gnt
—
CM-Sephade
x
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
—
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 and
gnt C2)
0.5–16
84 (sis)
83–84 (gnt)
65 (tob)
30
ami, dib, gnt, ntl,
sis, tob
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
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
—
Carboxy
propylsilica
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-Sephade
x
RP FDNB
1.5–5.3
9
0.993
1
—
1–64
95 (ami)
92 (kan)
38
MeOH + borate
Silica
RP, FDNB
—
0.8–9.9
0.996
—
3.21
2–64
—
39
ntl, gnt, tob
gnt
ami
Table 9. (continued)
Aminoglycosidea
gnt
ami, kan*
Extraction and protein
precipitation
Cleanup
Chromatography
derivatizationb
Intraday
CV, %
Interday
CV, %
r
Determination
Slope of
Linear range,
limit, µg/mL calibration curve
µg/mL
Recovery, %
—
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
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
Ref.
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
tob, sis*
Incubation
Silica
RP, OPA
5
5–6
—
1.0
—
1–15
—
44
ami, tob*
—
CM-Sephade
x
IP, OPA pc
3.2–25 (RSD)
3.9–28
(RSD)
—
—
—
0.025–2
—
56
gnt, ami, tob
nag*
—
CM-Sephade
x
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
Drying and
extraction 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
sis, ntl
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
—
Amberlite
IRC 50
IE, dihydrolutidine
—
—
—
0.05
—
0–5
—
79
sis
—
CM-Sephade
x
RP, OPA
5.5–6.8
7.2–8.0
0.995
0.08
1.571
—
111
87
a
b
Compounds marked with * are internal standards.
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.
1040 ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999
No single method can be identified as superior on the basis
of method recoveries. Methods using acetonitrile and tris
buffer for extraction and protein precipitation generally give
10% lower recoveries than other methods. Comparisons are
difficult, however, because recoveries are not reported in
about half of the methods reviewed here. Most of the reported
methods cover the therapeutic concentration range of the
aminoglycosides within their linear range. Limits of determination are generally below therapeutic concentrations.
Urine
Analysis of aminoglycosides in urine generally does not
require complex sample preparation such as protein precipitation. Several methods used for aminoglycoside analysis in
plasma could also be applied to urine as such as has been
done with gentamicin extraction using acetonitrile and phosphate buffer (28).
Two methods concerning analysis of gentamicin in urine
have been reported. One uses only derivatization for sample
preparation (77), and the other is based on SPE (31). The SPE
column is Sep-Pak C18, and gentamicin is eluted with methanol containing ammonia. The eluent is evaporated to dryness
to 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 extractions
are cleaner chromatograms and prolonged analytical column
life (31). The sensitivity of the method can be improved easily
by 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). The
calibration curve is linear from 0 to 71 mg/L, and the method
is repeatable.
Analysis of neomycin in urine has been done by
centrifugation of samples and addition of counterion concentrate to the supernatant for chromatography. The supernatant
is then analyzed by IP with OPA postcolumn derivatization
(25). Recovery was 75% for a neomycin concentration of
1 µg/mL and 104% for a neomycin concentration of 5 µg/mL.
Muscle Tissue, Kidneys, and Liver
Neomycin,
gentamicin,
streptomycin,
and
dihydrostreptomycin are widely used as veterinary
therapeutics (95). The illegal or irresponsible use of these
drugs can cause residues in edible tissues (17). Characteristic
to aminoglycoside pharmacokinetics is their tissue binding,
especially to renal cortex (65). Therefore, aminoglycoside
residues in food products of animal origin are not uncommon, and continuous monitoring of these residues is necessary (54). Monitoring of aminoglycoside residues requires
confirmative methods (95).
Sample preparation for residue analysis usually includes
2 stages: (1) mixing tissue in a buffer or in a protein-precipitating agent using organic solvent, precipitator, or
alkaline or enzymatic digestion and (2) extraction of analytes
by liquid–liquid, liquid–solid, or immunoaffinity methods (5).
The methods used for sample preparation and extration of
aminoglycosides in tissues are presented in Table 10.
Matrix solid-phase dispersion (MSPD) has been used to
extract aminoglycosides from kidneys (17). The tissue homogenate is mixed with cyanopropyl packing material, and
the mixture is poured into a cartridge and compacted to a desired volume. During analysis of aminoglycosides, the stationary phase is washed with hexane, water, and methanol,
and then the analytes are eluted with water. The method has
been validated with fortified tissue samples because matrix
components affect the shape and retention times of chromatographic peaks. Coeluting matrix components affect the MS
performance. MS–PD also has been used for streptomycin and
dihydrostreptomycin (49).
Neomycin has been extracted from kidneys, liver, and
muscle tissue with phosphate buffer for homogenization and
protein precipitation (53, 54). Recovery and the effect of protein precipitation on recovery have been investigated with radioactively labeled neomycin, and recovery has been compared with that achieved from extractions with saline (54).
Neomycin recovery from kidneys is 60% with saline, and
drops to 30% after incubation and protein precipitation. With
phosphate buffer, incubation does not affect recovery. However, neomycin becomes adsorbed to glass, and incubation
and extraction must be performed in plastic.
Gentamicin recovery has been investigated with different
protein-precipitating buffers and CM-Sephadex extraction
(5). With the precipitation agents HClO4, TCA, and acidic
phosphate, recoveries are 78.5, 85.9, and 39.2%, respectively
(5). TCA has been chosen as precipitation buffer, with addition of EDTA, because gentamicin has been reported to chelate positively charged metal cations (98). No gentamicin precipitation is observed with this technique.
The effect of phosphate buffers on gentamicin recovery
from edible tissues also has been studied (67). Results are
contradictory to ones reported earlier because only basic
buffers are found to extract gentamicin. When the pH of the
phosphate 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 increases
gentamicin recovery. However, the effect decreases as pH
increases. Simultaneously, too high a sulfate concentration
decreases recovery. Best results are achieved with 0.1M
phosphate buffer when sulfate concentration is 0.1M and pH
is 8.8. Recovery is then 98%.
Separation of gentamicin from matrix components is
difficult because of the similarity of chemical properties
(65). Most of the methods used for sample preparation use
only one retention mechanism, and that usually is not satisfactory for cleanup of aminoglycosides from tissue samples. For example, when phosphate buffer precipitation and
Sephadex extraction are used, some interfering elements
elute with gentamicin, making it impossible to quantitate
all 3 components (67).
The applicability for gentamicin extraction of SPE materials with TCA as precipitation buffer has been studied (5). Different nonpolar groups, such as C8, phenyl, and C18, bound to
silica have been studied. Good recoveries are achieved with
all of these (55–81%), but specificity to gentamicin is not sat-
ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 1041
Table 10. Sample preparation methods for aminoglycosides from edible tissues
Analyte
neo
Chromatography and
derivatizationa
Extraction and precipitationb
Cleanupc
Recovery,
%
Ref.
IP, OPA pc
fos 0.2M pH 8, incubation 100EC 10 min
—
80
53
neo
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
IP, —
MSPD, cyanopropyl
—
46–75
17
d
gnt, neo, str, dhs
e
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
GC, HFBI/TMSI
10% TCA
Amberlite CG 50
74–83
24
gnt
kan
a
b
c
d
e
For definitions, see footnotes to Table 4.
MSPD = matrix solid-phase dispersion.
IE = ion-exchange chromatography.
Paromomycin as internal standard.
Netilmicin as internal standard.
isfactory. When silica, weak cation exchanger (WCX), or polymeric resins are used, recovery is not sufficient. Thus
CM-Sephadex, which is specific to gentamicin and gives recoveries of 65% or more, has been chosen for analysis (5). Others have reported good results for cleanup of gentamicin samples
with WCXs (65). The method based on WCX–SPE can be used
for all tissues including liver, kidneys, and lungs (65).
Precipitation of proteins and extraction of streptomycin
and dihydrostreptomycin from kidney and muscle tissues with
3.6% perchloric acid has been done (89). Recoveries of the
drugs vary depending on tissues. For swine muscle, the lowest
dihydrostreptomycin recovery is obtained when recovery of
streptomycin is highest. Adding perchloric acid during extraction does not increase recovery, but it increases interferences
in chromatography.
Milk
Milk and milk product matrixes contain several compounds that might interfere in analysis (83). Analytical methods for antibiotics in milk include cleanups to remove proteins
and lipophilic compounds. Gentamicin (65, 68), neomycin
(45, 51), streptomycin, and dihydrostreptomycin (60) in milk
have been analyzed.
An Amberlite CG-50 ion-exchange column has been used
to extract neomycin from milk when no protein precipitation
is used (45). Neomycin is derivatized on column with OPA,
and the derivatives are eluted with basic methanol. Recovery
of the method is 94–102% at concentrations of
0.1–5.0 µg/mL. The limit of determination is 0.05 µg/mL. In
another study, neomycin is extracted from milk by protein
precipitation with 20% TCA (51). OPA postcolumn
derivatization and fluorescence detection are used. Recovery
of neomycin is 94–122%, and the method is linear in range
0.15–12 µg/mL.
CM-Sephadex C-25 ion exchanger has been used to extract
gentamicin from milk. Milk is added to the column in NaOH
solution (68). After elution from the column, gentamicin is
derivatized in a silica Sep-Pak SPE column with OPA. Attempts to precipitate the milk proteins before Sephadex extraction have been unsuccessful. However, the method is
quantitative and selective, and recoveries are 94–106% at
gentamicin concentrations of 0.2–10 µg/mL. A C18 COOH
ion-exchange column also has been used to extract gentamicin
from milk at concentrations of 0.625–80 µg/mL. The limit of
determination is 0.6 µg/mL (65). In another study, gentamicin
is extracted from milk with 30% TCA and a C18 SPE column
(52). Gentamicin is eluted from the SPE cartridge with ammonia–methanol. Recovery is 72–88%, and the limit of determination for individual components is 0.4 ng/mL.
Streptomycin and dihydrostreptomycin have been extracted from milk with 3.6% perchloric acid, followed by IP
and postcolumn derivatization with NQS (60). Recoveries
were 32.6–65.0%, depending on milk fat content.
Comparison and Evaluation of Methods
Chromatographic, Microbiological, and
Immunological Methods
Microbiological and immunological methods are still popular for aminoglycoside analysis and are used in both therapeutic monitoring (99) and screening for aminoglycoside residues (9). The reliability of results obtained with these methods
is, however, poorer than can be achieved with chromatographic methods. The slowness of microbiological methods is
a major disadvantage, particularly in therapeutic monitoring
where the speed of analysis is important. Immunoassays provide high throughput of samples but often give erroneous results (99). The advantages of chromatographic methods over
microbiological or immunological methods include greater
1042 ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999
accuracy, repeatability, and specificity of results. However,
when antimicrobial activity is of concern, only bioassays can
directly measure this property. Immunoassays based on enzyme-linked immunosorbent assay (ELISA) or RIA techniques generally measure cut-off points ranking result as positive or negative with reference to a concentration limit.
Maximum residue limits (MRLs) for aminoglycoside vary
widely among different aminoglycosides and different tissues.
Therefore, it appears extensively difficult to produce a general
assay that would monitor these drugs as a group relative to a
regulatory tolerance level. The structural similarity of the different aminoglycosides is likely to cause cross-reactivity between compounds, complicating quantitative determination.
Aminoglycoside bioassays and immunological methods
usually use the aqueous extract of most matrixes without need
for further concentration. This lack of need for further concentration is an advantage for compounds that are difficult to extract with organic solvents. However, bioassays do not detect
compounds bound to proteins, which makes results difficult to
quantitate. For example, in determination of neomycin concentrations in incurred tissue samples, the microbial assay shows
wider variation in results than HPLC as a function of standards
prepared in buffer or tissue homogenate (99). Purity or ratio of
drug components in drug preparations cannot be determined
microbiologically or immunologically. Thus, chromatographic
methods are necessary. Furthermore, when the importance of
judicial and economic consequences of results increases, the
reliability and accuracy of results become more significant.
When linearity, accuracy, precision, reliability, or diversity
of the method are considered, HPLC is at present the best
method for aminoglycoside analysis. Separation capacity and
low costs also contribute to HPLC as the method of choice. An
additional advantage of chromatographic methods is the possibility to use internal standards, which often increases reliability.
Usually, microbiological methods are compared with chromatography by the so-called blind-duplicate method. The purpose is to evaluate the correlation of results obtained by the
2 methods. Usually chromatographic results are presented as a
function of microbiological results, and the slope and r of the
plot are indicative of the correspondence. Both of the mentioned values should be as close as possible to 1. The results
from the use of this method are presented in Table 11.
Correlations of chromatographic methods with enzymological,
immunological, or microbiological methods differ depending on
the 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-phase
LC method using FDNB derivatization (31) and RIA. IP with
OPA postcolumn derivatization (62) correlates better with microbiological assays than a precolumn derivatization method (32)
with RP. Interestingly, both methods separate the 3 gentamicin
components and the quantitative differences could not be explained by different amounts of the separated components. It
appears that quantitation suffers when OPA postcolumn
derivatization is changed to precolumn derivatization.
Table 11. Comparison of the microbiological, immunological, and chromatographic methods used for
aminoglycoside analysis by blind duplicate method
Aminoglycosidea
ami
gnt
ntl
Derivatization and
chromatographya
Reference
methodb
Slope
r
Reference
TNBS, RP
RIA
1.047
0.999
41
FDNB, RP
MB
0.92
0.993
38
OPA, RP
MB
0.94
0.94
44
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
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
b
For definitions, see footnotes to Table 4.
RIA = radioimmunoassay; MB = microbiological assay; EMIT = enzyme immunoassay technique; FPIA = fluorescence polarization
immunoassay.
ISOHERRANEN & SOBACK: JOURNAL OF AOAC INTERNATIONAL VOL. 82, NO. 5, 1999 1043
Between the microbiological method and chromatographic
methods using OPA precolumn derivatization for analysis of
amikacin (44), gentamicin (32), and netilmicin (33), the best
agreement is for amikacin (Table 11). For netilmicin, the
agreement between RIA and the same chromatographic
method (33) is only marginally better than between the microbiological and chromatographic methods. The microbiological and RIA methods appear to correlate well.
Results of RP with FDNB derivatization are in good agreement with reference methods for gentamicin (30) and
tobramycin (36) and slightly less for amikacin (38) and
sisomicin (30; Table 11). The best correlations for tobramycin
and amikacin are obtained with reversed-phase methods with
TNBS derivatization (41, 42). For streptomycin, NQS postcolumn derivatization after IP (88) has better agreement with fluorescence polarization immunoassay (FPIA) than does the reversed-phase method with low-wavelength UV detection (61).
Chromatographic Methods
Most chromatographic determinations of aminoglycosides
are done by HPLC, and the aminoglycosides are derivatized to
improve detection. The choice of derivatization reagent has a
remarkable effect on quantitative and qualitative results. In
general, IP is used with OPA derivatization, MS, or electrochemical detection. When MS, electrochemical detection, or
postcolumn derivatization with OPA is used, IP as the method
of choice is justified because the analyzed aminoglycosides
are charged. However, when OPA is used for derivatization
prior to chromatography, it is not clear why IP is the method of
choice for separation. It is unclear if retention occurs through an
ion-pairing mechanism. Furthermore, when mercaptoacetic
acid (11, 63–66) or mercaptopropionic (86, 87) acid is used
with OPA, the derivatives include negatively charged groups
that could complicate ion-pair formation with sulfonates and facilitate ion-pair formation with positively charged counterions.
Unfortunately, this possibility is not discussed.
A good example of the lack of clarity associated with IP
and OPA precolumn derivatization is the analysis of sisomicin
by IP with heptane sulfonate as counterion and precolumn
derivatization with OPA (86). Sisomicin is separated with the
original method but when heptane sulfonate is replaced with
EDTA, the separation improves (87). This result suggests that
separation by the original method does not occur by an
ion-pair mechanism.
On the basis of promising results so far, FMOC–Cl will find
more applications in aminoglycoside analysis. The methods based
on TNBS and FDNB, which work for most aminoglycosides except for streptomycin and dihydrostreptomycin, will probably get
more attention, too. Comparison of FDNB and TNBS with
OPA as derivatization agents does not reveal marked differences in limits of determination even though OPA
derivatization allows fluorescence detection. Incomplete
derivatization of aminoglycosides with OPA may, at least
partly, explain this phenomenon.
In therapeutic monitoring and in the analysis of drug preparations, the chromatographic methods using derivatization and
IP or RP chromatographic separation will remain dominant be-
cause of their simplicity, short analysis time, and low cost. In
laboratories where confirmatory methods are needed, LC/MS
applications can be expected to replace other methods. In these
cases, IP with the use of fluorinated carboxylic acids such as
TFA, PFPA, and HFBA as counterions will probably become
the methods of choice. The advantage of these methods is that
the retention of aminoglycosides with these counterions has
been thoroughly studied (69, 70), and separation of several
aminoglycosides can be achieved in one run (15, 69, 70).
Conclusions
Aminoglycosides are a heterogenic group of antibiotics
characterized by a wide spectrum of activity and toxicity. Because of their toxicity, the monitoring of their concentrations
in plasma during therapy is necessary. The wide use of
aminoglycosides in veterinary medicine also requires suitable
routine and confirmatory methods for their detection in edible
tissues. In both cases, the methods must be accurate, sensitive,
and robust against interferences. However, the chemical characteristics of aminoglycosides such as polarity, water solubility, lack of volatility, and lack of chromophore make development of applicable methods difficult.
Chromatographic analysis almost always requires
derivatization to improve either detectability or separation.
However, derivatization is difficult when the aminoglycoside
structure incorporates several groups of different reactivity,
leading to possible partial derivatization. The most popular
method for aminoglycoside analysis is IP chromatography
with OPA derivatization. The interpretation of the results is,
however, unclear and contradictory, and the reliability and
reproducibility of results is questionable. The most promising
method seems to be LC/MS in combination with IP. The combination permits both quantitative and confirmatory analysis
for most aminoglycosides.
For the analysis and extraction of aminoglycosides from
different tissues, no generally applicable method has been reported. Extraction from edible tissues appears extremely difficult. However, promising results have been achieved with
methods using SPE. More work is needed to develop generally applicable and validated regulatory methods.
Acknowledgment
We thank James D. MacNeil for his constructive criticism
during preparation of this review.
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