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Chemical Reagents and Derivatization Procedures in Drug
Analysis
Neil D. Danielson, Patricia A. Gallagher, and James J. Bao
in
Encyclopedia of Analytical Chemistry
R.A. Meyers (Ed.)
pp. 7042–7076
 John Wiley & Sons Ltd, Chichester, 2000
CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS
Chemical Reagents and
Derivatization Procedures
in Drug Analysis
Neil D. Danielson and Patricia A. Gallagher
Miami University, Oxford, USA
James J. Bao
Procter and Gamble Pharmaceuticals, Mason,
USA
1 Introduction
2 Gas Chromatography
2.1 Alkylation
2.2 Acylation
2.3 Silylation
2.4 Sample Handling
3 High-performance Liquid
Chromatography
3.1 Alkaloids
3.2 Amines
3.3 Antibiotics
3.4 Barbiturates
3.5 Carbonyl Compounds and Carboxylic Acids
3.6 Catecholamines
3.7 Hydroxy Compounds
3.8 Steroids
3.9 Sulfur Compounds
1
2
2
3
5
7
14
16
16
17
18
4
19
8
8
10
13
14
Capillary Electrophoresis
4.1 Derivatization for Ultraviolet
Detection
4.2 Derivatization for Fluorescence
Detection
4.3 Special Applications
4.4 Derivatization Modes
5 Conclusion
20
21
22
23
Abbreviations and Acronyms
Related Articles
References
23
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19
This article describes both pre- and postcolumn derivatization chemistry used in conjunction with either chromatography or capillary electrophoresis (CE) to facilitate
the determination of drugs. Generally, only prederivatization is used in gas chromatography (GC), principally
Encyclopedia of Analytical Chemistry
R.A. Meyers (Ed.) Copyright  John Wiley & Sons Ltd
1
to enhance the volatility, temperature stability, and/or
detectability. The GC section considers derivatization of
drugs by reagent class: alkylation, acylation, and silylation.
The GC sample handling section describes an approach
which combines the extraction and derivatization steps
together. Both pre- and postcolumn derivatization are
common approaches for high-performance liquid chromatography (HPLC) and many of these methods have
been adapted for CE. Derivatization for HPLC is often
directed toward aliphatic amines, carboxylic acids, or alcohols that are difficult to detect at low levels by absorbance,
luminescence, or electrochemical means. In addition, small
hydrophilic molecules upon prederivatization are often
converted into larger more hydrophobic compounds,
making reversed-phase HPLC easier or even feasible.
The HPLC section considers derivatization of drugs based
on type: alkaloids, amines, antibiotics, barbiturates, carbonyl compounds and carboxylic acids, catecholamines,
hydroxy compounds, steroids, and sulfur compounds. For
the GC and HPLC sections, tables are included giving
the structures of the more important derivatizing agents,
the analytes, and the corresponding reaction products. A
brief rationale for derivatization chemistry with CE concludes this article. For CE, derivatization is often done to
improve detectability since the path length for absorbance
detection is very short and fluorescence can be effective
using laser-based systems.
1 INTRODUCTION
Derivatization in conjunction with chromatography or
CE can be done either in an off-line mode, often prior
to the separation step (prederivatization), or in an online mode (postcolumn derivatization). Generally, only
prederivatization is used in GC, principally to enhance
the volatility, temperature stability, and/or detectability.
Both pre- and postcolumn derivatization are common
approaches for HPLC and many of these methods
have been adapted for CE. Derivatization for HPLC
is often directed toward aliphatic amines, carboxylic
acids, or alcohols that are difficult to detect at low
levels by absorbance, luminescence, or electrochemical
means. In addition, small hydrophilic molecules upon
prederivatization are often converted into larger more
hydrophobic compounds, making reversed-phase HPLC
easier or feasible. For CE, derivatization is often done to
improve detectability since the path length for absorbance
detection is very short and fluorescence can be effective
using-laser based systems.
The desirable conditions to be met for precolumn
derivatization are (1) the reaction stoichiometry and
product structure should be known, (2) the reaction
2
should be reasonably fast and proceed quantitatively (or
at least reproducibly), and (3) the derivative should be
stable and readily separable and distinguishable from the
starting material. One advantage of the prederivatization
approach is that simple equipment is commercially
available to allow for the reaction chemistry to be done
in the batch mode. Subsequent sample analysis using an
autoinjector and a standard unmodified chromatograph
is straightforward. Alternatively, the use of a robot to
carry out the entire prederivatization chemistry and an
HPLC instrument equipped with an autosampler means
that the sample throughput will usually be limited by the
chromatographic step.
Because postcolumn derivatization involves mixing the
column effluent with a reagent or passing it through
a reactor to form the derivative before it is detected,
the automation step is built in. Sample handling before
analysis is minimal, reducing the chance of error from
sample loss. The conditions desirable to be met for
postcolumn derivatization are (1) although the reaction
must be rapid, within about 2 min, and be reproducible,
the reaction products do not have to be very stable,
(2) the reagent itself must have no or a very low detection
response, and (3) the reactor volume must be small to
minimize dilution and band broadening.
The remainder of the article is divided into three major
sections, GC, HPLC, and CE. The GC section considers derivatization of drugs by reagent class: alkylation,
acylation, and silylation. In addition, a short discussion of sample handling is included. The HPLC section
considers the derivatization of drugs based on type:
alkaloids, amines, antibiotics, barbiturates, carbonyl compounds and carboxylic acids, catecholamines, hydroxy
compounds, steroids, and sulfur compounds. A brief
rationale for derivatization chemistry with CE concludes
this article.
2 GAS CHROMATOGRAPHY
Prior to analysis by GC, compounds containing functional
groups with active hydrogens such as COOH, OH, NH,
and SH need to be protected. Compounds with these
functional groups tend to form intermolecular hydrogen
bonds, reducing volatility. They are also thermally
unstable and can interact with either fused silica or
the stationary phase, causing peak broadening. Several
good review sources describing derivatization reagents,
reactions, and applications for GC are available. At least
one commercial company provides a good overview of the
commercial reagents and the apparatus available..1/ Two
other references are fairly comprehensive in describing
many of the published methods for GC derivatization
either by reaction class.2/ or type of organic compound..3/
PHARMACEUTICALS AND DRUGS
Alkylation, acylation, and silylation reaction chemistries
are the most common approaches to derivatization for
GC and will be the primary focus in this article. A specific
example and an overview for each of these reaction classes
will be described below.
2.1 Alkylation
Alkylation represents the replacement of an active hydrogen, found in an organic acid or amine, by an aliphatic or
aliphatic – aromatic (e.g. benzyl) group. Alkylation reactions can also be used to prepare ethers from alcohols
such as phenol, thioethers from sulfur compounds, and
N-alkylamines, amides, and sulfonamides from amines.
One principal chromatographic use is the conversion of
carboxylic acids into esters that are stable and volatile
with good chromatographic characteristics. Esterification
of carboxylic acids has traditionally been done through
reaction with an alcohol in the presence of an acid catalyst. Generally, a 1 – 2-mg sample of the acid is heated with
100 µL of the alcohol (methanol or ethanol) containing
3 M HCl for 30 min at 70 ° C. The alcohol is evaporated,
leaving the ester as a residue. A faster reaction in only
about 2 min using a boiling water-bath is possible with
a BF3 – methanol solution (Table 1, reagent 1). Generally the esters are extracted into n-heptane followed
by evaporation of the solvent..4/ DMFDA is a rapid
methylating agent which provides quantitative yields
upon mixing in a nonaqueous solvent with the sample
of interest (Table 1, reagent 2). Carboxylic acids are converted to methyl esters, thiols to thioethers, and phenols
to methyl ethers. Primary amines are converted into
their N,N-dimethylaminoethylene derivatives and amino
acids are simultaneously modified as this derivative at
the amino group and as the methyl ester at the carboxyl
group. Aliphatic hydroxyl groups are not methylated.
Ethyl, propyl, n-butyl, and t-butyl acetals may be used
analogously.
Pyrolysis of a quaternary ammonium salt (QUAT) in
the presence of the organic analyte can be a convenient
methylation procedure..5/ Upon injection of the QUAT
mixed with the drug having a reactive amino, hydroxyl, or
carboxyl group, into the hot injection port (250 – 300 ° C),
formation of the appropriate methyl derivative will
occur (Table 1, reagent 4). The derivative is immediately
swept onto the analytical column for separation and
detection. TMAH is the most common reagent used but
trimethyl (a,a,a-trifluoro-m-tolyl)ammonium hydroxide
can be used at a lower injection temperature, which is
important for labile unsaturated fatty acids. This method
is particularly good for barbiturates, sedatives, xanthine
bases, phenolic alkaloids, and dilantin.
PFB-Br can convert carboxylic acids, phenols, sulfonamides, and thiols into halogenated derivatives that can
3
CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS
Table 1 Common GC derivatives – alkylation
Reagenta
No.
Compounds
Derivatives
1.
F H
F B: O CH3
F
(BF3−methanol)
R COOH
R COOCH3
2.
O CH3
(CH3)2N C H
O CH3
(DMFDA)
R COOH
R COOCH3
R NH2
R NH (CH2)2 N(CH3)2
R OH
R O CH3
R SH
R S CH3
R COOH
O
R C O (PFB)
R OH
R O (PFB)
Sulfonamides
(PFB) HN
R SH
R S (PFB)
R OH
R O CH3
R NH2
R NH CH3
R COOH
R COOCH3
3.
F
F
CH2Br
F
4.
F
F
(PFB-Br)
CH3
N+ −OH
H3C CH3
SO2NHR
(TMAH)
a
DMFDA, N,N-dimethylformamide dimethylacetal; PFB-Br, pentafluorobenzyl bromide; TMAH, trimethylanilinium hydroxide.
be easily detected by electron capture (EC) (Table 1,
reagent 3). For carboxylic acids, the reaction is run in an
organic solvent in the presence of a base such as a tertiary
amine for 5 min at 40 ° C. Interestingly, tertiary amines
have been derivatized with pentafluorobenzyl chloroformate prior to GC with EC detection..6/ The analysis of
cannabidiol (CBD) and the internal standard tetrahydrocannabidiol in blood plasma involved derivatization with
PFB-Br followed by purification on a Florisil column..7/
The derivatization procedure involved refluxing a hexane extract with PFB-Br overnight with stirring. Acetone
washings of this solution were evaporated to dryness and
the residue was dissolved in hexane. A portion of this
hexane solution was injected into a gas chromatograph
equipped with an EC detector. A typical chromatogram
taken on a packed OV-225 glass column is shown in
Figure 1. Although a packed column has limited separation capability, it can handle larger amounts of sample. A
detection limit of 50 ng mL 1 of plasma was possible.
2
1
inj.
0
2.2 Acylation
Acylation is the use of a carboxylic acid or carboxylic acid
derivative to convert compounds that have active hydrogens (OH, SH, NH) into esters, thioesters, and amides,
respectively. Acyl derivatives tend to direct the mass
4
8
12
16
24
28
Time (min)
Figure 1 Typical chromatogram from a plasma sample containing 2.0 µg of the internal standard tetrahydrocannabidiol (1)
and calculated to contain 1.5 µg of the CBD (2). [Reproduced
with permission from Jones et al..7/ ]
4
PHARMACEUTICALS AND DRUGS
(MBTFA)
a
R NH2
O
R NH C CF3
R2
R1 NH
R2 O
R1 N C CF3
R OH
O
R O C CF3
R SH
O
R S C CF3
TFAA, trifluoroacetic anhydride; PFAA, pentafluoropropionic anhydride; HFAA, heptafluorobutyric anhydride; MBTFA,
N-methylbistrifluoroacetamide.
(b)
9.50
10.00
9.00
8.50
8.00
7.50
7.00
6.50
10.50
10.50
R2 O
R1 N C R′
9.50
R2
R1 NH
1
9.00
OH
O
R O C R′
O
O C R′
2
10.00
O
O
F3C C N C CF3
CH3
R OH
550 000
500 000
450 000
400 000
350 000
300 000
250 000
200 000
150 000
100 000
50 000
0
8.50
3.
R NH2
O
R NH C R′
Time (min)
(a)
8.00
R′=CF3 (TFAA),
C2F5 (PFAA), or
C3H7 (HFAA)
R1
R2 O
N C R′
7.50
O
O
R′ C O C R′
R2
R1 NH
7.00
2.
O
R O C R′
6.50
R′=CF3 (TFAI),
C2F5 (PFAI), or
C3H7 (HFBI)
R OH
6.00
O
N C R′
Derivatives
5.50
N
Compounds
2
1
5.50
1.
Reagenta
Abundance
No.
400 000
350 000
300 000
250 000
200 000
150 000
100 000
50 000
0
6.00
Table 2 Common GC derivatives – acylation
MBTFA, shown in Table 2 as reagent 3, trifluoroacetylates primary and secondary amines and carbohydrates
under mild nonacidic conditions..9/ For carbohydrates, a
single derivative for mono-, di-, tri-, and tetrasaccharides
was obtained. The N-methyltrifluoroacetamide byproduct is stable and volatile, being amenable to GC.
Perfluoro acid anhydrides [R(CDO) O (CDO)R],
such as TFAA, PFAA and HFAA, react readily with
alcohols, phenols, and urine for the preparation of
perfluoroacyl derivatives, which are particularly useful
for EC detection (Table 2, reagent 2). Triethylamine is
added as part of the reaction mixture to neutralize the
acidic by-products and drive the reaction to completion.
Drugs of abuse are often derivatized in this way
before gas chromatography/mass spectrometry (GC/MS)
confirmation. A GC/MS example of derivatization of
amphetamine and methamphetamine by HFAA.10/ is
shown in Figure 2(a) and (b). The splitting of the peaks
Abundance
spectrometry (MS) fragmentation patterns of compounds
providing useful structural information. Perfluoroacylimidazoles such as trifluoroacetylimidazole (TFAI),
pentafluoropropionylimidazole (PFAI), or heptafluorobutylimidazole (HFBI) can react with hydroxyl groups
and primary or secondary amines to generate the halogenated derivative and the relatively inert by-product imidazole (Table 2, reagent 1). Generally the reaction times
are 1 – 3 h at 75 ° C. Indoleamines and indole alcohols,
which are acid-sensitive compounds, have been modified
by this procedure. Metoclopromide, an antispasmodic
agent related to procainamide, has been derivatized with
HFBI for GC with EC detection..8/ These reagents are
often used in bifunctional derivatization schemes involving silylation chemistry. For example, phenolic amines
can be silylated with N-trimethylsilylimidazole (TMSI)
to protect the hydroxyl group and acylated to protect
the amine group. Other halogenated imidazoles such as
p-bromobenzyl- and p-chlorobenzylimidazole have been
used because the detectability of the EC detector is
enhanced. However, the volatility and stability of the
derivatives under GC conditions tend to be lower compared with the fluorinated types.
Time (min)
Figure 2 (a) Total ion current chromatogram of HFAA-derivatized amphetamine (1) and methamphetamine (2). The
overloaded peak at about 5.6 min is due to excess HFAA.
(b) Total ion current chromatogram of HFAA derivatized
amphetamine (1) and methamphetamine (2) after extraction
of the excess derivatizing agent. The baseline level in this
chromatogram is only about 10% of that in (a). The splitting
of the drug peaks in both chromatograms is due to the
presence of the deuterated internal standards (amphetamine-d5
and methamphetamine-d8 ). [Both figures reproduced with
permission from Jones and Mell..10/ ]
CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS
is due to the presence of deuterated internal standards.
Excess HFAA, which causes a high background level,
column contamination and eventual degradation of the
stationary phase, was removed by the procedure outlined
below. The drugs of interest were extracted from 2 mL
of urine by solid-phase extraction (SPE) columns with a
10% 2-propanol in chloroform elution solvent. Sodium
periodate oxidation of interfering drugs was carried
out if necessary. The extract was acidified with 10%
HCl in methanol and the solvent evaporated before
reconstitution of the residue in hexane with 0.1 M
trimethylamine. HFAA was added and the mixture was
heated in a sealed tube for 1 h at 70 ° C. After cooling,
deionized water was added to the tube, the tube was
shaken, and then the aqueous phase was discarded. A
4% ammonia solution was shaken with the remaining
hexane volume and then this organic layer was removed
for injection into the GC/MS system. The limit of
quantification and limit of detection were both of the
order of 50 ng mL 1 and the linearity was observed up to
at least 4000 ng mL 1 .
2.3 Silylation
Silylation is probably the most widely used derivatization scheme for GC. Active hydrogens from acid,
alcohol, thiol, amine, and amide groups can all be
protected, usually with trimethylsilyl (TMS) groups. The
ease of silylation of these functional groups follows
the order alcohol > phenol > carboxylic acid > amine >
amide. Steric hindrance is also a factor within a class
of organic compounds. For alcohols, the reactivity
order is primary > secondary > tertiary, and, for amines,
primary > secondary. In general, silylation reagents
are unstable and must be protected from moisture.
Trimethylchlorosilane (TMCS) is the simplest reagent
that can be used, often to derivatize carboxylic acids
(Table 3, reagent 1). TMCS is often used in conjunction
with HMDS (Table 3, reagent 2) to improve the silylation
of sugars and related compounds. GC of silyl derivatives
is generally straightforward, with only one major concern,
the presence of excess silylating agent. Often an excess
of the silylating agent is used in the derivatization reaction to minimize the problem of moisture or other acidic
components in the sample. If possible, it is recommended
that the excess silylation reagent be evaporated from
the sample using a stream of nitrogen before injection
into the GC column. This avoids several problems such
as large reagent blank peaks in the chromatogram and
fouling of the flame ionization detector by SiO2 deposits.
A polar stationary phase such as poly(ethylene glycol)
(Carbowax 20 M) and free fatty acid phase (FFAP) will
be derivatized by excess silylation reagent and cannot be
used.
5
Silylacetamides are represented in the most popular group of silylation agents, owing to their ability
to react quickly and quantitatively under mild conditions. BSA is a highly reactive TMS donor (Table 3,
reagent 3). BSTFA (Table 3, reagent 4) has the advantage of giving a more fluorinated by-product than BSA.
This permits derivatization of lower molecular weight
analytes without potential overlap of the trifluoroacetamide by-product. The addition of TMCS to BSTFA
will promote the derivatization of amides, secondary
amines, and hindered hydroxyl groups. A comprehensive article on drug detection methodology describes
the determination of cocaine, heroin, and metabolites
in hair, plasma, saliva, and urine samples after isolation by SPE and subsequent derivatization with BSTFA
with TMCS before analysis by GC/MS..11/ After spiking with internal standards, plasma, saliva, and hair
extracts were passed through SPE columns and the drugs
eluted with a methylene chloride – 2 propanol – ammonia
solvent. The eluate was evaporated to dryness, reconstituted with acetonitrile, and allowed to react with a
BSTFA – TMCS mixture at 60 ° C for 30 min before analysis by GC/MS. Representative chromatograms are shown
in Figure 3(a – c). Separations were effected on a relatively short 12 m ð 0.2 mm ID capillary column. For
urine, saliva, and plasma, the limit of detection was about
1 ng mL 1 and for hair 0.1 ng mg 1 .
MSTFA has similar donor strength to BSA and
BSTFA but generates an even more volatile by-product,
N-methyltrifluoroacetamide (Table 3, reagent 5). Both
excess MSTFA and the by-product often elute with the
solvent peak, eliminating the presence of extra peaks
in the chromatogram. Cocaine and its metabolites benzoylecgonine and ecgonine methyl ester were sequentially
derivatized with thyliodide to obtain the ester derivative
and then MSTFA to form the o-TMS derivatives..12/
The derivatized compounds were separated by a GC
instrument with a methylphenylsilicone column and
nitrogen – phosphorus detection. Again TMCS can be
added to MSTFA to promote the derivatization of amides
and hindered amines and hydroxyl groups.
The strongest reagent for silylation of hydroxyl groups
is TMSI (Table 3, reagent 6). It does not react with amines
or amides and is effective for most steroids, including
those with unhindered and highly hindered OH groups.
It also reacts quickly and smoothly with carboxyl groups.
Silyl compounds other than those that derivatize
with a TMS group have also been used in conjunction with GC. Methyltestosterone was derivatized using either the dimethylethylsilyl (DMES)imidazole or dimethylisopropylsilyl (DMIPS)-imidazole
to the corresponding silyl ether derivative..13/ Reaction conditions involve heating at 70 ° C for 1 h before
removal of the excess reagent by N2 and reconstitution
6
PHARMACEUTICALS AND DRUGS
Table 3 Common GC derivatives – silylation
Reagenta
Compounds
1.
CH3
H3C Si CH3
Cl
(TMCS)
R COOH
2.
CH3 CH3
H3C Si N Si CH3
H
CH3 CH3
No.
Derivatives
R O Si(CH3)3
R OH
O
R C O Si(CH3)3
(HMDS)
3.
4.
CH3
CH3
H3C Si O C N Si CH3
CH3 CH3 CH3
R OH
R O Si(CH3)3
R NH2
R N Si(CH3)3
Si(CH3)3
(BSA)
R1 NH
R2
R1 N Si(CH3)3
R2
O
R C NH2
O Si(CH3)3
R C N Si(CH3)3
R COOH
O
R C O Si(CH3)3
CH3
CH3
H3C Si O C N Si CH3
CH3
5.
6.
CF3
Same as BSA, leaving group
O H
F3C C N Si (CH3)3
CH3
(BSTFA)
more volatile than that of BSA
CH3
O
F3C C N Si CH3
H3C CH3
Same as BSA, leaving group
(MSTFA)
is very volatile
N
CH3
N Si CH3
CH3
(TMSI)
O H
F3C C N CH3
R O Si(CH3)3
R OH
(steroids)
R COOH
O
R C O Si(CH3)3
No reaction with amines
a
HMDS, hexamethyldisilazane; BSA, N,O-bis(trimethylsilyl)acetamide; BSTFA, N,O-bis
(trimethylsilyl)trifluoroacetamide; MSTFA, N-methyl-N-trimethylsilyltrifluoroacetamide.
of the residue in cyclohexane. Because the desired
application was the assay of methyltestosterone in
bulk powder and tablets, separation on a packed
column was adequate in the concentration range
0.1 – 1.5 mg mL 1 (Figure 4a – c). Alternatively, N-methylN-(t-butyldimethylsilyl)trifluoroacetamide (MTBSTFA)
will derivatize hydroxyl, carboxyl, thiol, primary and
secondary amines by adding t-butyldimethylsilyl
(TBDMS) groups. These TBDMS derivatives are more
stable to hydrolysis than TMS compounds and, when analyzed by GC/MS, a strong M 57 fragment is noted which
can be used to determine the original compound’s molecular weight. These advantages have been demonstrated
in the determination of short- and long-chain carboxylic
acids..14/ Pentafluorophenyldimethylsilyl (flophemesyl)
derivatives are often generated from steroids by reaction
with flophemesylamine at room temperature for 15 min.
This is a selective reaction with only primary and
secondary hydroxyl groups in steroids reacting in the presence of unprotected ketone groups. GC with EC detection
provides detection limits in the nanograms – picograms
range. Flophemesyl derivatives also have favorable
7
CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS
advantages for GC/MS, producing diagnostic ions which
carry more of the current than TMS derivatives..15/
R2
R1
2.4 Sample Handling
1
3
9 10
6
R1
2
20
11
2
12
19
21
18
14
7
5
23
22
17
13
(a)
24
16
2880
(a)
1
7
2
2960
2
9
5
R2
(1) CH2 CH3
(2) H
C 2H 5
15
8
4
1
N
OCH 3
2770
2840
Generally, drugs are found in aqueous matrices such as
urine and plasma; extraction with an organic solvent is
14
(b)
16 19 20
23
2980
(b)
1
×75
×5
×1 ×5 ×75
10
14
6
9
7
11
8
2
1
12
3
23
20
19
15
2
17
4
5
3060
×15
21
16
2
24
(c)
4.0
(c)
5.0
6.0
7.0
8.0
9.0
10.0
11.0
Time (min)
Figure 3 Single ion monitoring recordings of extracts from
(a) standard cocaine/opiate hair, (b) drug-free control hair,
and (c) a hair sample collected from a heroin user. Analytes are identified as follows: anhydroecgonine methyl ester
(1), [2 H-3]ecgonine methyl ester (2), ecgonine methyl ester
(3), ecgonine ethyl ester (4), [2 H-3]cocaine (5), cocaine (6),
[2 H-3]cocaethylene (7), cocaethylene (8), [1 H-3]benzoylecgonine (9), benzoylecgonine (10), norcocaine (11), norcoceathylene (12), benzoylnorecgonine (13), [2 H-3]codeine (14), codeine
(15), [2 H-3]morphine (16), morphine (17), norcodeine (18),
[2 H-6]-6-acetylmorphine (19), [2 H-3]-6-acetylmorphine (20),
6-acetylmorphine (21), normorphine (22), [2 H-9]heroin (23),
heroin (24). [Reproduced with permission from Wang et al..11/ ]
4
6
Time (min)
Figure 4 Typical gas chromatograms of (1) methyltestosterone
and (2) norethandrolone as (a) TMS, DMCS (b) and (c)
DMIPS derivatives. Note the bulkier derivatives are shifted
to a cleaner area in the GC trace. [Reproduced with permission
from Zakhari et al..13/ ]
required not only to isolate the drug in a nonaqueous
solvent which is compatible with most derivatization
reagents but also to remove inorganic salts which
are not compatible with GC. An alternative approach
called direct derivatization combines the extraction and
derivatization steps together. Direct derivatization of
drugs in untreated biological samples for GC analysis
has the primary advantages of improved extractability of
8
a derivatized polar compound and avoidance of stability
problems. Direct derivatization of drugs in untreated
biological samples can mean not only derivatization in
the sample matrix followed by extraction, but also a twophase reaction where the derivatization takes place in the
organic phase while extraction of the analyte is continuing
from the aqueous phase..16/ Extractive alkylation has
been applied to valproic acid and ketoprofen, in which
the tetrabutylammonium ion acted as the anion-pairing
agent to pull the compound into the organic phase, where
alkylation to generate the methyl or phenacyl derivative
was possible. Phenolic compounds such as elioquinol
have been determined in an analogous fashion. Acylation
involving perfluorinated anhydrides and chloroformates
has also been used for the determination of drugs such as
metanephrine and normetanephrine. Chloroformates can
de-alkylate tertiary amines to form stable carbamates.
3 HIGH-PERFORMANCE LIQUID
CHROMATOGRAPHY
The variety of chemistries that can be adopted for
either pre- or postcolumn derivatization in conjunction
with HPLC is large in part because either aqueous
or organic solvent-compatible reactions are possible.
Most of the books reviewing fundamental reactions
for pre- and postcolumn derivatization chemistry in
conjunction with HPLC were published in the 1980s,
which appears to have been the most active time for
research in this field..17 – 23/ Many review articles have
focused on postcolumn derivatization with HPLC..24 – 31/
Some specific review articles on derivatization chemistry
of pharmaceutical compounds with HPLC have also been
published..32 – 36/ Although the other articles do not try to
be comprehensive, Ahuja.35/ gave a virtually complete
review of the topic up to 1979. Danielson et al..36/
published another fairly complete review covering the
period 1979 – 87, which is the basis for this part of this
article. Again, a specific example and an overview for each
major class of pharmaceutical compounds will be given.
3.1 Alkaloids
Separation of atropine and ergotamine by normal-phase
liquid chromatography (LC) followed by postcolumn
ion-pair extraction with an aqueous solution of 9,10dimethoxyanthracene-2-sulfonate has been reported..37/
The organic mobile phase was monitored fluorimetrically with detection limits of 40 – 100 ng. Conversely, methadone, phencyclidine, and their metabolites were separated by reversed-phase HPLC using
9,10-dimethoxyanthracene-2-sulfonic acid in the mobile
phase..38/ The resultant ion pairs (Table 4, reagent 3)
PHARMACEUTICALS AND DRUGS
were extracted with chloroform postcolumn on-line
and detected fluorimetrically with detection limits of
1 – 6 ng mL 1 in plasma. A precolumn ion-pair derivatization method for atropine, hyoscyamine, scopolamine, and
ergotamine, involving picric acid and normal-phase chromatography, provided ultraviolet (UV) detection limits
of about 200 ng..39,40/
Morphine has been detected fluorimetrically after
postcolumn oxidation with alkaline potassium hexacyanoferrate(II) to form the dimer pseudomorphine..41/
Morphine can be oxidized to the fluorescent dimer pseudomorphine using alkaline hexacyanoferrate(III). Other
opiates, such as normorphine, naborphine, codeine, norcodeine, and others, were also reactive. The excitation and
emission wavelengths were 323 and 432 nm, respectively.
A comparison of the UV and fluorescence response was
made using the instrumentation shown in Figure 5. The
mobile phase, a 12.5 : 87.5 methanol – 0.1 M KBr solution,
was propelled through a C18 reversed-phase column. The
derivatizing agent was 50 mg of K3 Fe(CN)6 in 250 mL
of 4 M ammonia solution delivered at 0.4 mL min 1 . The
presence of 4% of the nonionic surfactant Triton X100 at the micellar level in the derivatization reagent
solution increased the signal about twofold. The reason
for the improvement was credited to an increase in the
actual dimer formation yield. Morphine could be determined at the 2 – 30 µg mL 1 level in biological samples.
The selectivity advantage of the fluorescence detector for
the determination of morphine in a urine extract was
shown and urine and serum samples were analyzed with
detection limits of about 10 ng. Fluorescence detection of
morphine and related opiates was improved after postcolumn derivatization with alkaline hexacyanoferrate(III)
and micelle formation with Triton X..42/ A detection limit
of 0.2 pmol was possible for morphine after precolumn
dansylation..43/ The heroin metabolite 6-acetylmorphine
has been determined in urine by reversed-phase HPLC
with fluorescence detection after automated precolumn
oxidation with hexacyanoferrate(III)..44/ A detection
limit of 1 ppb was reported. Reserpine, an antihypertensive agent, was detected fluorimetrically after postcolumn
reaction with nitrous acid and UV irradiation..45,46/ Using
this UV photochemical reaction, a 20-fold increase in
signal over the native fluorescence of reserpine was found.
Physostigmine, an acetylcholinesterase inhibitor, was
separated from its degradation products and reacted with
coulometrically generated bromine..47/ Electrochemical
detection of unreacted bromine was inversely proportional to the amount of drug, and a detection limit
of 0.5 ng could be attained. An on-line photochemical reaction detector caused decreased fluorescence of
ergot alkaloids, permitting the identification of these compounds in complex chromatograms..48/ A similar system
has been shown to convert cannabinol into a fluorescent
9
CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS
Table 4 Common fluorescent (F) and electrochemical (E) derivatives for HPLC
No.
1.
Compounds
Reagent(s)
Derivative
CHO
CHO
, R′′SH
R′ NH2 (F)
S R′′
N R′
CN
2.
CHO
, CN−
RNH2 (F)
N R
CHO
OCH3
3.
OCH3
SO3−
+
R3NH (F)
OCH3
RSH (F)
H3C
BrH2C
5.
RCOOH (E)
6.
Steroid (E)
H2N
H2NHN
OCH3
O
O
4.
SO3− +NHR3
N
N
O
O
CH3
CH3
H3C
N
N
CH3
RSH2C
OH
NO2
O
RCHN
OH
NHN
NO2
O
OH
HO
Mobile
phase
reservoir
Pump
I
Injector Column
UV
detector
3-Way
valve
Waste
Tee
Dual-pen
recorder
Reaction
coil
Derivatization
reagent
reservoir
Pump
II
Gauge
Waste
Fluorescence
detector
Figure 5 HPLC system for morphine using postcolumn derivatization and fluorescence detection. [Reproduced with permission
from Nelson et al..41/ ]
10
derivative providing detection limits of less than 1 ng
in urine..49/ Post-column photochemical derivatization
has the advantage of no sample handling and without
the problems of pumping a reagent or detection background from a reagent. On-line reaction of cannabinoids
with Fast Blue Salt B produced colored derivatives for
detection at 490 nm..50/ Pilocarpine was quaternized with
p-nitrobenzyl bromide before reversed-phase HPLC separation and detection at 254 nm..51/
3.2 Amines
Primarily fluorescent methods have been developed for
amino acids and peptides. The o-phthalaldehyde (OPA),
mercaptoethanol (or other thiol) reaction (Table 4,
reagent 1) has been well studied in both pre- and
postcolumn modes. Specific articles describing common amino acids are cited in general references..17 – 20/
Other amino acids such as s-carboxymethyl-L-cysteine,.52/
baclofen,.53,54/ and melphalan.55/ have also been derivatized with OPA. A more recent modification of this
method is to use naphthalene-2,3-dicarboxyaldehyde with
cyanide ion (Table 4, reagent 2)..56/ Detection limits down
to 200 fmol with improved stability of the derivatives were
stated advantages. g-Aminobutyric acid was modified
with dansyl chloride before reversed-phase HPLC and fluorescent detection..57/ Detection at 360 nm was possible
for N-acetylcysteine after reaction with 2,4-dinitro-1fluorobenzene..58/ 1,2-Diamino-4,5-dimethoxybenzene
was used to form a fluorescent derivative of phydroxybestatin..59/ The peptide leupeptin was reacted
through the guanidino moiety with benzoin to form a fluorescent derivative..60/ Felypressin, a nonapeptide, was
derivatized with fluorescamine, providing detection limits of 0.3 ng..61/ A dinitrophthalic anhydride reaction of
peptides permitted either electrochemical or absorbance
detection..62/ An ion-pair detection technique has been
applied to hydrophobic amino acids and peptides..63/
A variety of other fluorescent methods have also
been reported for specific drugs. Tranexamic acid has
been detected using OPA..64/ Biogenic amines such
as tyramine, tryptamine, and serotonin were reacted
with OPA in a post-.65/ or precolumn.66/ mode. The
OPA precolumn fluorescent reaction has also been
applied to L-buthionine-(S,R)-sulfoximine in plasma.67/
and mexiletine..68/ Fluorescent derivatization with fluorescamine has been applied to tocainide.69/ and in
the postcolumn mode for sulfapyridine..70/ Enantiomers
of mexiletine have also been resolved..71/ After conversion of the drug panthenol to aminopropanol, the
fluorescamine reaction provided detection limits of
0.4 µg..72/ The determination of debrisoquine and its
hydroxy metabolites was possible by reaction through
the guanidine moiety to form fluorescent compounds..73/
PHARMACEUTICALS AND DRUGS
A postcolumn photochemical reactor caused the cleavage of methotrexate to form the highly fluorescent
2,4-diaminopteridine-6-carboxyaldehyde..74/ Indolethylamines were condensed with an aldehyde or a-keto acid
to form fluorescent carboline derivatives..75/ Thiamine
(vitamin B1 ) was oxidized with hexacyanoferrate(III)
in base to give the fluorescent thiochrome in either
the precolumn.76/ or postcolumn.77/ mode. Riboflavin,
already fluorescent, and thiamine, after precolumn oxidation to thiochrome using hexacyanoferrate(III), were
determined by HPLC in a variety of food powders such
as flour, milk, and beans..78/ Hydrazine compounds, such
as isoniazid, were reacted with m-fluorobenzyl chloride before reversed-phase separation and detection at
220 nm..79/ A precolumn coumarin reaction permitted fluorescent detection of 5-fluoro-20 -deoxyuridine..80/ Fluorescein isothiocyanate (FITC) modified a bronchodilator
before HPLC – fluorescence analysis..81/
Some UV methods have been developed for aliphatic
amines. Phenyl isothiocyanate has been used as a precolumn derivatizing agent for amino acids in conjunction
with reversed-phase HPLC. Both primary and secondary amines can react (Table 5, reagent 1B) and the
aromatic tag provides good retention for the smaller
amino acids..82/ Both primary and secondary amines
were derivatized with 9-fluorenylmethyl chloroformate
(FMOC) before separation by reversed-phase HPLC
with UV detection at 254 nm. After SPE of a urine sample, b-phenethylamine was determined with a detection
limit of 0.1 ng mL 1 ..83/ Amphetamine and methamphetamine have been reacted with 4-methoxybenzoyl
chloride (Table 5, reagent 1A) and other acid chlorides for subsequent UV detection..84/ A comparison
of methods for amphetamines has been made..85/ The
cardiotonic drug heptaminol has been derivatized with
aminoazobenzene-4-isothiocyanate for UV detection at
420 nm.86/ or OPA for electrochemical detection..87/ Secondary amines, such as piperazines, were converted into
UV derivatives with m-toluoylacyl chloride..88/ Enantiomers of metoprolol have been separated after reaction
with the chiral reagent (S)-( )-phenylethyl isocyanate..89/
The determination of isophenindamine in the presence
of phenindamine tartrate has been achieved by forming
charge transfer complexes using AgNO3 and reversedphase HPLC..90/
Antihistamines and other pharmaceuticals with a
tertiary amine moiety have been derivatized to provide for luminescence detection. An ion-pair extraction detector using dimethoxyanthracene sulfonate
(Table 4, reagent 3) has permitted the fluorescent determination of tertiary amines such as bromopheniramine and chlorpheniramine,.91,92/ ephedrine,.93/ and
hyoscyamine..94,95/ Detection limits of 200 – 500 pg were
possible. Precolumn reaction of chlorpheniramine with
11
CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS
Table 5 Common UV derivatives for HPLC
No.
1.
Compounds
RNH2
Reagent(s)
(A)
Cl
Derivative
(A)
R
O
C
N
H
O
C
OCH3
OCH3
(B)
2.
R2NH
3.
R3N
NCS
(B)
NHCSNHR
SO2Cl
SO2NR2
(A) O
H
C
C
Cl
O
CH2
N
H
(B)
4.
O
C
RCOOH
RSH
O
C
CH2Br
Cl
C
H2C C
C2H5
R
O
C
C
H2 O
Br
Cl
O
6.
NHR2
C C N C O
H 2 H2
Br
5.
O
C
OCH2COOH
Cl
O
C
RS
H2C CH
C2H5
Cl
OCH2COOH
Penicillin
R
C
O
H
N
N
O
S CH3
CH3
NaOH, HgCl2, EDTA
COOH
benzyl chloroformate gives a fluorescent derivative with
a detection limit of 0.1 ng mL 1 ..96/ Antihistamines, such
as diphenhydramine, were converted through the tertiary
amine group into fluorescent derivatives using 2-naphthyl
chloroformate..97/ Aliphatic tertiary amines can be determined at the picomole level by postcolumn chemiluminescent detection, using tris(bipyridyl)ruthenium(III)..98/
Antihistamines in urine have also been separated by
HPLC and detected with good selectivity using this ruthenium metal complex (Figure 6a – c)..99/ Tamoxifen and its
metabolites in human serum can be UV photochemically
activated to form fluorescent phenanthrenes..100/ Postcolumn UV irradiation of tamoxifen and its derivatives
R
C
O
H
N
−OOC
N
H
S CH3
CH3
COO−
caused rearrangement to a substituted phenanthrene,
permitting fluorescent detection with 0.1 ng mL 1 detection limits..101/
Derivatives of nicotine, cotinine, and other metabolites can be detected at 530 nm in urine after HPLC,.102/
by using diethylthiobarbituric acid as a color-forming
agent. Racemic phenothiazines were N-demethylated
with vinyl chloroformate to form their secondary amines,
which could then be reacted with (R)-(C)-1-phenylethyl
isocyanate (Table 5, reagent 3), and these compounds
were separated by reversed-phase HPLC..103/ Tertiary
tetrahydroisoquinolines, such as diclofensine, were oxidized before photochemical conversion to fluorescent
12
PHARMACEUTICALS AND DRUGS
Analytical
column
Pump 1
Pump 2
K 2Cr2O 7
(0.1 mL min –1)
Mobile phase
(0.7 mL min –1)
A
∆t1
B
A
C
Detector
B
λ = 290 nm
∆t2
Pump 3
0
6
12
18
24
(a)
0
6
12
18
NaHSO 3
(0.1 mL min –1)
24
(b)
Figure 7 Schematic representation of the Pt HSO3 reaction
detector for cisplatin-type compounds. [Reproduced with
permission from Marsh et al..106/ ]
C
0
6
12
18
24
Time (min)
(c)
Figure 6 Chromatograms of an undiluted urine sample spiked
pheniramine, (B) 0.26 µg mL
browith (A) 0.15 µg mL
mopheniramine, and (C) 0.29 µg mL 1 diphenhydramine taken
with (a) UV (214 nm), (b) UV (254 nm), and (c) Ru(bpy)3 3C
chemiluminescence detection. [Reproduced with permission
from Holeman et al..99/ ]
1
1
2
1
3
0
5
2
0.05 au
Absorbance (290 nm)
B
0.0025 au
A
Absorbance (300 nm)
1
0
3
5
10
.104/
isoquinolinium derivatives.
After postcolumn ion-pair
extraction of secoverine, peroxylate chemiluminescence
detection was carried out..105/
Postcolumn detection of platinum(II) antineoplastic
agents such as cisplatin is possible, as shown in Figure 7.
The cisplatin-derived species are first reacted with a
oxidant such as dichromate to form an activated species,
which then combines rapidly with bisulfate to give a UVabsorbing complex. Using knitted open-tubular reactors,
delay times of 26 s for the dichromate reaction and 4.7 min
for the bisulfate reaction were found to be optimal.
Figure 8(a) and (b) shows the improvement in response
over conventional UV detection using the derivatization
(a)
Time (min)
(b)
Time (min)
Figure 8 Comparison of UV detection (300 nm) after HPLC
separation (a) and reactor response (290 nm) to derivatized
platinum (b) for a mixture of cis-dichloroplatinum complexes: (1) cis-dichloro(1,2-diaminocyclohexane)platinum(II),
(2) cis-dichloro(ethylenediamine)platinum(II), and (3) cis-dichlorodiammineplatinum(II). [Reproduced with permission
from Marsh et al..106/ ]
scheme. Detection limits of 5 – 10 µg mL 1 were possible.
Cisplatin was also determined in a plasma ultrafiltrate
sample at the 5-ng level..106/
CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS
3.3 Antibiotics
This class of pharmaceuticals has received major attention
for both pre- and postcolumn derivatization. Fluorescent derivatization of the primary amine group of
many antibiotics with OPA and a sulfhydryl compound,
often mercaptoethanol, has been commonly performed
(Table 4, reagent 1). Gentamicin,.107,108/ penicillin V after
enzymatic conversion to 6-aminopenicillanic acid,.108/
penicillin N,.109/ cephalosporin C,.110/ cycloserine,.111/
and fludalanine.112/ were assayed by using an OPA
postcolumn reactor. Spectinomycin after postcolumn
oxidation with hypochlorite could also be derivatized
with OPA in a second reaction coil..113/ Reversedphase separation was employed for all these separations
because the OPA reaction is carried out in an alkaline buffer solution. Precolumn reaction with OPA for
gentamicin,.114/ sisomicin,.115/ phosphinothricin and its
alanine analog.116,117/ before reversed-phase separation,
have all been reported. Amikacin isomers were first
adsorbed on a silica gel column before reaction with
OPA; the derivatives were eluted with ethanol before
separation by reversed-phase HPLC..118/ A comparison
of pre- and postcolumn OPA reaction conditions for
gentamycin has been made..119/ For all these OPA fluorescence methods, detection limits are about 1 µg mL 1 .
For example, b-lactams found in microbial fermentation broths were separated on a C18 HPLC column and
detected fluorimetrically with excitation at 350 nm and
emission at 450 nm after reaction with OPA and mercaptoethanol. Cephamycin, penicillin N, cephalosporin
C, and 6-aminopenicillanic acid were separated in about
12 min at a flow rate of 1.5 mL min 1 with postcolumn
conditions involving the OPA reagent at pH 12, a reaction coil of 12 m, a temperature of 90 ° C, and a reagent
flow rate of 0.8 mL min 1 . Detection limits of less than
0.5 and 1.0 µg mL 1 were achieved for penicillin N and
cephalosporin C, respectively..120/
Numerous other postcolumn UV, fluorimetric, and
electrochemical methods have been reported for
tetracycline-type antibiotics and related compounds. Formation of a mercuric mercaptide of penicillins permits
UV detection at 310 nm with detection limits of 10 ng..121/
Sulfonamides in egg, milk, and meat samples have
been detected at 450 nm after derivatization with pdimethylaminobenzaldehyde..122/ Monensin, narasin, and
salinomycin in animal feeds have been reacted with
vanillin to give products detectable at 520 nm..123/ Penicillins, such as amoxicillin, ampicillin, and others, can
be separated by reversed-phase HPLC with alkaline
degradation in the presence of mercuric chloride.124/
(Table 5, reagent 6). Methanol promotes this reaction,
forming the ring-cleaved product, which absorbs at
274 nm, giving a detection limit of 50 ng mL 1 . It was
13
discovered later that sodium hypochlorite could replace
the mercuric chloride reagent while maintaining a 1-min
hydrolysis time..125/ Application of this latter method to
penicillins in biological samples has been made..126,127/
Fluorescent detection of streptomycin reacted through
the guanidino groups with naphthoquinone-4-sulfonate in
alkaline solution has been reported in serum samples..128/
Photothermal derivatization of ciprofloxacin and its
metabolites permits fluorescent detection throughout a
linear range of about 2 – 1000 ng mL 1 ..129/ Photolysis
with electrochemical detection of penicillins and cefoperazone has provided detection limits of about 6 ng..130/
Electrochemically generated bromine was used as an oxidizing agent for cephalosporins and their decomposition
products..131/ The excess bromine was detected at 0.4 V
using a glassy carbon electrode. A comparison of this
method with UV detection and postcolumn fluorescamine
derivatization has recently been summarized..132/ Acidification of a serum sample sometimes improved recovery
of cephalosporins when micellar chromatography was
used with sodium dodecyl sulfate (SDS) in the mobile
phase..133/ Fluorescamine has been used in an automated
system for amoxycillin in biological fluids..134/ Chemiluminescence detection of clindamycin phosphate using
tris(bipyridyl)ruthenium(III).135/ gave detection limits of
8 ppb compared with 970 ppb using UV detection at
214 nm.
A variety of reagents are also available for precolumn
derivatization of antibiotics and UV or fluorimetric detection. Phenacyl esters of some natural penicillins were
prepared using dibromoacetophenone before reversedphase HPLC with UV detection..136/ Nitrobenzene
derivatives of neomycin B and C can be separated
by normal-phase HPLC and detected at 350 nm..137/
Similar methods using 1-fluoro-2,4-dinitrobenzene to
form 2,4-dinitrophenyl derivatives of neomycin B and
C.138,139/ and amikacin.140,141/ have also been published.
Neomycin and other aminoglycosides have been published. Neomycin and other aminoglycosides have been
converted to benzoyl derivatives before UV detection at
230 nm..142/ Aminoglyosides have also been reacted with
2,4,6-trinitrobenzenesulfonic acid, permitting UV detection at 350 nm..143/ Ion-pair formation of methscopolamine bromide with an aromatic anion during reversedphase HPLC permitted UV detection..144/ The secondary
amine group of spectinomycin was derivatized with 2naphthalenesulfonyl chloride (Table 5, reagent 2) before
normal-phase HPLC and UV detection at 254 nm..145/
The reagent FMOC formed fluorescent derivatives of
natural penicillins, cephalosporins, and their precursors
in biological samples..146/ b-Lactams were prederivatized with FMOC for 5 min at 20 ° C in borate buffer
at pH 7.7 before injection of the derivatives on to
a C18 reversed-phase column. Excitation and emission
14
PHARMACEUTICALS AND DRUGS
D
B
3.4 Barbiturates
100
Acetonitrile (%)
Relative fluorescence
C
A
0
0
10
20
30
40
50
Retention time (min)
Figure 9 Reversed phase HPLC of FMOC derivatives of
cephalosporins (5 µg mL 1 ) in a fermentation broth using
a borate buffer – acetonitrile gradient mobile phase. (A)
Deacetylcephalosporin C; (B) Deacetoxycephalosporin C;
(C) cephalosporin C; (D) ampicillin. [Reproduced with permission from Shah and Adlard..146/ ]
wavelengths for the fluorescent detection were 260 and
313 nm, respectively. A typical chromatogram is given in
Figure 9 showing the modest acetonitrile gradient until
after elution of ampicillin in which the acetonitrile is taken
to 100%. Detection limits were 0.01 and 0.05 µg mL 1
for 6-aminopenicillanic acid and isopenicillin N, respectively. Application of the method to fermentation
broths was made over a range of 0.05 – 100 µg mL 1 ..147/
A maleimide reagent gave a fluorescent derivative
for penicillamine..148/ An imidazole – mercuric chloride
reagent can convert penicillins into mercury-stabilized
penicillinic acids after reaction at 50 ° C for 50 min prior to
UV detection at 325 nm..149/ A comparison of this method
with the postcolumn OPA fluorescence method has been
made..110/ A similar method using 1,2,4-triazole and
Hg(II) for ampicillin, amoxicillin, and other antibiotics
has been studied..150 – 152/ Two precolumn derivatization
studies with UV detection were checked with real samples. Tobramycin plus impurities neamine and kanamycin
and also the degradation product nebramine were derivatized with 2,4-dinitrofluorobenzene for 20 min at 70 ° C in
0.8 mM sulfuric acid. Separation of the derivatives on a
C18 column with detection at 365 nm was carried out and
the stability of tobramycin in ophthalmic solutions was
determined..153/
Precolumn methods primarily include reaction with
various alkylating reagents. The highly reactive chlorine
of N-chloromethylphthalimides permits derivatization of
OH and NH functional groups to form UV-absorbing
compounds..154/ Detection limits of 5 ng for phenobarbital
have been reported..154,155/
2-Naphthacyl bromide forms strongly absorbing derivatives of barbiturates at 254 nm with detection in plasma
or serum below the therapeutic range..156/ An online solid-phase anion-exchange extraction provided
enhanced chromatographic selectivity of barbiturates
in urine..157/ Postcolumn UV detection of barbiturates
can be conveniently enhanced by mixing with a pH 10
borate buffer..158,159/ A wavelength shift for maximum
absorbance from 220 to 240 nm provides for detection
limits of about 6 µg for butabarbital.
The UV detection of barbiturates can be significantly
enhanced through postcolumn photochemical derivatization. Barbiturates have no significant absorbance above
230 nm and detection in the 200 – 220-nm range can be
complicated by the presence of interfering peaks, particularly in biological samples such as serum. Using a
25 m ð 0.25 mm ID Teflon knitted open tubular reactor
mounted around a mercury lamp, which provided an irradiation time of about 190 s, excellent signal enhancement
at 270 nm was possible for barbiturates (Figure 10a and
b). It was determined by HPLC that de-alkylation at the
5-position to give ethylbarbituric acid was the mechanism
of the photochemical reaction..160/
3.5 Carbonyl Compounds and Carboxylic Acids
Most of these methods refer to carboxylic acids;
short-chain carboxylic acids were modified using pbromophenacyl bromide (Table 5, reagent 4) to form
esters that could be easily detected by UV absorbance..161/
However, first a short summary of the methods involving
the reduction of quinone compounds to form fluorescent hydroxy derivatives will be given. Vitamin K1 in
human plasma was electrochemically reduced at 0.4 V
and the fluorescent derivative detected at levels as low as
25 – 50 pg..162/ Comparison of this method with UV and
electrochemical oxidation detection systems has been
made..163/ Danthron (1,8-dihydroxyanthraquinone) has
been reduced with dithionite and determined by either
flow injection analysis in Modane tablets or HPLC in
urine..164/ A comparison of UV and fluorescence chromatograms for danthron spiked in a urine sample showed
the selectivity advantage of fluorescence (Figure 11a
and b). Dansylhydrazine formed fluorescent derivatives
of tetraphenylacetone isomers before separation..165/
5-Bromomethylfluorescein was studied for the prederivatization of carboxylic acids for detection by either
15
CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS
3
1
2
5
0.04 AUFS
4
UV
Lamp on
Lamp off
0
(a)
0.15%
Relative
fluorescence
10
Time (min)
Danthron
0
(b)
10
Time (min)
Figure 10 Chromatogram of a standard sample of barbiturates
detected at 270 nm, (a) without and (b) with on-line photochemical reaction. (1) Aprobarbital; (2) butethal; (3) pentobarbital;
(4) mephobarbital; (5) secobarbital. [Reproduced with permission from Wolf and Schmid..106/ ]
UV absorbance or fluorescence (standard and laser
induced). Model analytes included prostaglandins (unsaturated carboxylic acids) and the drug cefuroxime, and also
standard aliphatic and aromatic carboxylic acids. Dicarboxylic acids did not react with bromomethylfluorescein,
possibly owing to solubility problems in the organic reaction medium..166/ Fatty acids and prostaglandins were
converted into p-hydroxanilides using p-aminophenol
(Table 4, reagent 5). These hydroxy derivatives were
then oxidized through electrochemical detection after
reversed-phase HPLC..167/ A UV-absorbing naphthacyl
ester of the prostaglandin carboprost has been formed
before normal-phase HPLC..168/ Fluorescent derivatives
of prostaglandins using 9-anthryldiazomethane provided detection limits of 100 pg after reversed-phase
HPLC..169/ The prostaglandin arbaprotstil was derivatized with panacyl bromide before column switching using
fluorescent derivatization..170,171/ Prostaglandins have
also been derivatized with p-(9-anthroyloxyl)phenacyl
bromide..172/ Oxidation of prostaglandins to the corresponding 15-oxo derivatives using pyridinium dichromate
0
0 2 4 6 8
(a)
Time (min)
(b)
4
8
Time (min)
Figure 11 Separation of danthron spiked in urine (2 ng mL 1 ).
(a) UV detection (254 nm). Column, 250 ð 4.6 mm C18 ; mobile
phase, 60 : 40 methanol – water at 1.2 mL min 1 . (b) Fluorescence
detection (388 nm excitation, 510 nm emission). A solution of
90 mM dithionite in 2% borate buffer was pumped into the
effluent at 0.4 mL min 1 . [Reproduced with permission from
Miller and Danielson..164/ ]
permitted UV detection at 228 nm and picomole detection
limits..173,174/ Prostaglandins have been labeled with the
fluorescent reagent 9-anthoyldiazomethane through the
carboxyl group. This esterification reaction can be carried
out under mild conditions such as 40 ° C for 30 min. Five
prostaglandin derivatives were separated by reversedphase HPLC with fluorescent detection with excitation at
365 nm and emission at 418 nm at the 8-ng level..175/
Ascorbic acid from 5 to 800 mg L 1 was detected
by chemiluminescence after postcolumn reaction with
lucigenin..176/ After derivatization with 1,2-phenylenediamine both ascorbic acid and dehydroascorbic acid were
separated by reversed-phase ion-pair HPLC..177/ The
same two compounds were separated by reversed-phase
HPLC, and the dehydroascorbic acid was reduced to
ascorbic acid with dithiothreitol for UV detection at
267 nm..178/ UV detection of bis(dinitrophenyl)hydrazine
derivatives of ascorbic and dehydroascorbic acid was
16
possible at 497 nm..179/ Four forms of ascorbic acid,
the previous two plus isoascorbic acid and its dehydro form, were detected postcolumn using benzamidine and fluorescence..180/ Total ascorbic acid was
determined fluorimetrically after reaction with diaminodimethoxybenzene..181/
Finally, a variety of methods directed toward pain
relievers and other compounds have been published.
Indomethacin formed a fluorescent derivative after postcolumn alkaline hydrolysis, giving a detection limit of
5 pg..182/ Postcolumn alkaline hydrolysis has also been
applied to aspirin in plasma in order to form the fluorescent salicylic acid..183/ The enantiomeric composition of
ibuprofen in human plasma has been resolved after prederivatization with (S)-( )-1-(naphthenyl)ethylamine.184/
or ethyl chloroformate – leucinamide..185/ Flunoxaprofen
enantiomers have been separated after reaction with (S)( )-1-phenylethylamine..186/ Artesunic acid was determined after derivatization with o,p-nitrobenzyl-N,N 0 -diisopropylisourea by HPLC with UV detection..187/ Choline, betaine, and related compounds have been reacted to
form either 40 -bromophenacyl esters.188/ or p-nitrobenzyl
oxines.189/ to permit UV detection at 254 nm. A new
acridinium sulfonylamide label permits the chemiluminescent detection of carboxylic acids such as the test
compound ibuprofan. This alkylation reaction takes place
in dry acetonitrile for 20 min at 50 ° C and separation of
the derivative is possible on a C18 column with an acetonitrile – water – tetrahydrofuran mobile phase with the
ion-pairing agent tetrabutylammonium ion. Chemiluminescence detection of the acridinium label is possible
by postcolumn addition of potassium hydroxide solution.
A detection limit of 3 pg of derivatized ibuprofen was
found..190/
3.6 Catecholamines
Precolumn fluorescent derivatization of catecholamines
with OPA and a thiol has been well established
(Table 4, reagent 1). Norepinephrine, dopamine, and
normetanephrine have been measured at the low
picogram level after reversed-phase HPLC..191,192/ An
electrochemical cell placed between the injector and
column was used for oxidation of adrenaline and levodopa to the corresponding quinones for detection
in the visible region..193/ Resolution of norephedrine
enantiomers after derivatization with either acetylglycosyl isothiocyanates.194/ or 4-methyl-5-phenyl-2-oxazolidone.195/ has been reported. Fluorescent catecholamine
derivatives have been generated using 1,2-diphenylethylenediamine, providing detection limits in the femtomole
range..196/
Postcolumn fluorescent reaction of norepinephrine and
epinephrine with trihydroxyindole provided 1-pg detection limits..197/ This method was compared with the
PHARMACEUTICALS AND DRUGS
OPA – thiol reaction in the postcolumn mode..198,199/ The
same two catecholamines can be converted into fluorescent products by heating in an alkaline borate buffer
after ion-exchange chromatography..200/ Dopamine isomers in serum and urine have been separated before
hydrolysis and reaction with p-aminobenzoic acid to
form fluorescent products..201/ Glycylglycine as an alternative postcolumn reagent to glycinamide increased
the rate of formation of fluorescent derivatives after
ion-exchange HPLC..202,203/ Catecholamines catalyze the
reaction between formaldehyde and o-dintrobenzene and
permit their detection at 560 nm..204/
3.7 Hydroxy Compounds
Most precolumn methods focus on enhancing UV detection. Isotachysterol derivatives of vitamin D improved
detection at 290 nm after normal-phase HPLC..205/ Oncolumn periodate oxidation of ephedrine sulfate, a
nasal decongestant, to form benzyl alcohol has been
carried out in a seven-laboratory study..206/ Indirect
photometric detection using n-heptyl p-aminobenzoate
in the mobile phase and a C18 column has been
applied to menthol..207/ Enantiomers of propranolol
and 4-hydroxypropranolol were formed upon derivatization with (C)-1-phenylethyl isocyanate.208/ or (C)tetraacetyl b-D-glucopyranosyl isothiocyanate.209,210/ and
separated by reversed-phase HPLC. Enantiomers of
derivatized or underivatized propranolol have been
separated..211/ Diastereomeric derivatization of 1-methyl3-pyrolidinol.212/ and proxyphylline.213/ for UV detection
has been accomplished. Qinghaosu, an antimalaria component in a Chinese herb, can be converted into a
UV-absorbing compound using a sodium hydroxide solution before HPLC separation..214/ A derivative of qinghaosu has been esterified with diacetyldihydrofluorescein
before HPLC and UV detection..215/ Cholic acid and
its derivatives have been labeled with 1-anthroylnitrile
before HPLC and fluorescent detection at the femtomole level..216/ Trospium has been converted into the
corresponding spiro alcohol before fluorescent derivatization with benoxaprofen chloride and reversed-phase
HPLC..217/
Derivatization can be an effective method to facilitate the chiral separation of pharmaceuticals. Nadolol
diastereomers were derivatized with (R)-( )-1-(1naphthyl)ethyl isothiocyanate to chiral urea derivatives
through the secondary amine group by reaction for 5 min
at 45 ° C. Separation of the RS, SR, RR, and SS diastereomers was straightforward on a C18 column using a
60 : 40 water – acetonitrile mobile phase (Figure 12a – c).
Fluorescence detection with excitation at 285 nm and
emission at 340 nm provided selectivity for the determination of nadolol in plasma samples. The limit of detection
CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS
Cyclodextrins in biological fluids were assayed by
negative colorimetric detection after postcolumn complexation with phenolphthalein..223/ Vitamin B6 (pyridoxine) has been postcolumn derivatized with 2,6dibromoquinone-4-chlorimide to form a colored product
with a maximum absorbance at 650 nm..224/
200
180
160
140
120
100
80
60
40
20
(a) 0
3.8 Steroids
200
180
160
140
120
100
80
60
40
20
(b) 0
1
200
180
160
140
120
100
80
60
40
20
0
2 3 4
1
2
0
(c)
17
5
10
15
20
25
30
3
35
4
40
45
Time (min)
Figure 12 Typical chromatograms of (a) blank control dog
plasma, (b) plasma spiked with 50 ng mL 1 of each diasteromer and (c) plasma obtained 2 h after oral administration
of 1 mg kg 1 of racemic nadolol. Peaks: 1 D .SR/-nadolol;
2 D .RS/-nadolol; 3 D .RR/-nadolol; 4 D .S, S/-nadolol. [Reproduced with permission from Hoshino et al..218/ ]
was 2.5 ng mL 1 , representing 50 pg injected. This chiral
derivatization method has been used for the determination of enantiomers of other b-blocker drugs..218/
A few of the post-column approaches are outlined
below. Using a photochemical reactor, diethylstilbestrol
(DES) has been converted into a fluorescent derivative and determined at the low parts per billion level
in biological matrixes..219,220/ Anabolic stilbenes, such as
DES, have been measured in urine by HPLC and an offline chemiluminescence immunochemical assay..221/ The
antihypertensive agent fenoldopam was formed using
a postcolumn enzyme reactor from the corresponding glucuronide, and detected electrochemically..222/
Esterified estrogens were converted into their free phenolic forms by acid hydrolysis, and separation could be
achieved by reversed-phase HPLC..225/ The chromatographic behavior of estrogen carbonyls, such as equilin,
equilin, and estrone, was improved upon reduction to the
17-a-hydroxy compounds using sodium borohydride..226/
Prederivatization of conjugated estrogens with dansyl
chloride permitted fluorescent detection after separation by normal-phase HPLC (Table 4)..227/ Ketosteroids, such as androsterone, dehydroepiandrosterone,
epiandosterone, and etiocholanolone, were derivatized
with p-nitrophenylhydrazine (Table 4, reaction 6) and
detected electrochemically at levels as low as 200 pg..228/
Isonicotinoyl hydrazine was used to tag steroids,
such as corticosterone, to permit fluorescent detection after normal-phase HPLC..229/ Further work using
this method with reversed-phase HPLC and optimized
reaction conditions permitted detection limits as low
as 7 – 10 ng..230,231/ 17-Oxosteroids were labelled with
dansylhydrazine and then chromatographed on a silica
column with subsequent fluorescent detection from 60 to
100 pg..232/ A similar method for this class of compounds
using 3-chloroformyl-7-methoxycoumarin and reversedphase HPLC has been published..233/ Hydroxysteroids
were derivatized with anthroylnitrile, showing the feasibility of the reaction for fluorescent detection after
HPLC..234/
Six corticosteroids were separated within 25 min
and reacted with a lucigenin – KOH solution for
chemiluminescence detection..235/ The reactive site is
an a-hydroxycarbonyl group, and detection limits of
2 pg were comparable to those with precolumn fluorescent methods. Corticosteroids were also postcolumn
derivatized with glycinamide in the presence of hexacyanoferrateIII) before fluorimetric detection at the 5-ng
level..236/ Digoxin, a widely used cardiac glycoside, has
been detected fluorimetrically by postcolumn reaction
with a solution of ascorbic acid, peroxide, and HCl..237,238/
A detection limit less than 1 ng was attained.
Digoxin and its metabolites, such as digoxigenin, were
derivatized with 1-naphthoyl chloride before separation
and fluorescent detection.239/ and 5-ng amounts could be
determined in urine or feces. A similar method using
4-nitrobenzoyl chloride has been reported..240/ Dihydrodigoxin, a major metabolite of digoxin, has been
18
separated from digoxin with dual UV and postcolumn
fluorescent derivatization detection..241/ The photoreduction of anthroquinone-2,6-disulfonate to the fluorescent
9,10-dihydroxyanthracene-2,6-disulfonate occurs only in
the presence of hydrogen atom-donating substrates, such
as alcohols, aldehydes, amines, ethers, and saccharides.
Using a knitted Teflon reactor, cardiac glycosides, such as
digoxin, digoxigenin, and diginatin, have been determined
in the range 50 – 500 ng..242/
PHARMACEUTICALS AND DRUGS
Waste
AS
P(A)
MP
(A)
VAL
1
MP
(B)
P(B)
C2
C1
3.9 Sulfur Compounds
Most methods employ a precolumn reaction to form
fluorescent or UV derivatives. Dansylaziridine.243/ was
used to determine cysteine and other thiols. Biological thiols, such as glutathione and ergothioneine, were
reacted with monobromobimane (Table 4, reagent 4)
before ion-exchange chromatography..244/ This method
was also adopted for the determination of dithiols, such as 2,3-dimercaptopropane-1-sulfonic acid,
in urine at levels down to 10 pmol..245/ Fluorescent
derivatization of 2-mercaptopropionylglycine using N-(7dimethylamino-4-methyl-3-coumarinyl)maleimide could
probably be extended to other thiol compounds..246/
Resolution of the optical isomers of diltiazem was accomplished using UV detection after derivatization with
optically pure 2-naphthylsulfonyl-2-pyrollidinecarbonyl
chloride..247/ Three N-substituted maleimides were compared for precolumn derivatization of thiols such as
penicillamine before electrochemical detection at the
picogram level..248/ Ethacrynic acid (Table 5, reagent 5)
forms UV-detectable derivatives of thiols, such as captopril, N-acetyl-L-cysteine, and mercaptopropionylglycine
with detection limits down to 0.5 µg mL 1 ..249/
Postcolumn derivatization of thioethers, such as ampicillin and ranitidine, was accomplished using on-line
generated bromine and electrochemical detection of the
excess bromine..250/ An analogous method has been
reported for phenothiazines, such as thioridazine; however, the resultant products from the oxidation reaction
with bromine are detected fluorimetrically..251/ Photochemical activation of phenothiazines and demoxepam
in 2 min and subsequent fluorescence detection provided detection limits a factor of 10 better than UV
detection..252/ Applications of photochemical reactors to
a variety of pharmaceuticals, such as phenothiazines, have
been summarized..253,254/ A precolumn derivatization
method for phenothiazine involves desulfurization with
Raney nickel to produce diphenylamine, which is electrochemically active..255/ A detection limit of 10 pg was
found. The reagent pyrenemaleimide provided derivatization of N-acetylcysteine with a detection limit of
10 pmol..256/ Postcolumn complexation of disulfiram and
two of its metabolites using Cu2C allowed colorimetric
CO
DET
(A)
Waste
DET
(B)
Waste
VAL
2
MP
(C)
P(C)
C3
Figure 13 Schematic diagram of the HPLC system. P(A),
P(B), and P(C) D pumps; AS D autosampler; VAL-1 and
VAL-2 D six-port valves 1 and 2; C0 D cleanup column; Cl,
C2, and C3 D columns 1, 2, and 3; DET(A) and DET.B/ D UV
detectors A and B; MP(A), MP(B), and MP .C/ D mobile
phases A, B, and C. The solid and dotted lines in the
six-port valves indicate valve positions 0 and 1, respectively.
[Reproduced with permission from Funakoshi et al..259/ ]
detection at 435 nm with detection limits in the low parts
per billion range..257/ Quenched peroxalate chemiluminescence has been employed using immobilized reagents
in a postcolumn reactor for thioridazin, sulforidazine, and
methimazole..258/
On-line precolumn derivatization to improve reproducibility has been demonstrated for the determination
of busulfan in human serum. A schematic diagram of the
HPLC system is shown in Figure 13. All three columns
C1, C2, and C3 were C18 types with different lengths
and/or diameters. After extraction of busulfan from
serum, the residue reconstituted in water was injected
on to C1, where it was derivatized with diethyldithiocarbamate (DCC) in mobile phase A for 5 min. Then
the busulfan – DCC derivative from C1 was backflushed
on to C2, where it was separated using mobile phase
B. Because the background interference from mostly
excess DCC overlapped completely the busulfan– DCC
derivative peak, valve 2 was used to take a heart cut
of this peak of interest and inject it on to C3. The
resulting separation using mobile phase C is shown in
Figure 14(a) and (b). Detection was at 278 nm and the
lower limit of quantitation in serum was 10 ng mL 1 .
The total time for the derivatization and separation was
33 min..259/
0
(a)
8
16
Time (min)
24
0
(b)
8
16
19
0.0006 a.u.
the detectability. Since UV/VIS and LIF detection are
the most common detection methods used in CE for
pharmaceutical analysis, we shall discuss the most recent
work related to this area.
Inj.
Inj.
Busul fan/16.150
CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS
24
Time (min)
Figure 14 Typical chromatograms of (a) drug-free serum and
(b) serum spiked with busulfan (100 ng mL 1 ) obtained with
columns Cl, C2, and C3. The arrow indicates the retention time
of the busulfan derivative. [Reproduced with permission from
Funakoshi et al..259/ ]
4 CAPILLARY ELECTROPHORESIS
CE has developed into a versatile separation technique
well suited for the determination of pharmaceutical
and biomedical samples. The major advantages of CE
over chromatographic separation techniques are its
simplicity and efficiency. However, CE suffers from
poor sensitivity in detection owing to the extremely
small sample volume involved. One way to improve the
sensitivity of CE detection is to derivatize the analytes
with more favorable detection characteristics by adding
either an ultraviolet/visible (UV/VIS) chromophore or a
fluorophore.
Derivatization in conjuction with GC and HPLC
is a well-developed field. With the advent of CE,
derivatization chemistry previously developed for HPLC
is often applicable to CE..260/ At least one comprehensive
review specifically focused on the derivatization in
CE has appeared..261/ Several other review articles
dealt with more specific topics, such as postcolumn
derivatization,.262/ diastereomer derivatization,.263/ and
dyes used to derivatize molecules for CE with laserinduced fluorescence (LIF) detection in drug analysis..264/
The pros and cons of the various modes of derivatization
and a detailed comparison of this topic between LC and
CE can be found in the literature..261/ Instead of listing
the derivatization reagents suited for labeling amino,
aldehyde, keto, carboxyl, hydroxyl, and sulfhydryl groups,
we intend to focus on the purpose of derivatization
in CE from a practical point of view, i.e. enhancing
4.1 Derivatization for Ultraviolet Detection
Although most organic compounds have UV/VIS chromophores, derivatization is still important, and sometimes
necessary, for UV/VIS detection in CE. Very often,
UV/VIS detection of the chromophores does not give
a satisfactory response owing to the very short light
pathlength (50 – 100 µm) in CE. For example, the anticancer drug prospidin is a piperazinium derivative with
chloroxypropyl groups but no UV/VIS chromophore.
Derivatization with DCC at 37 ° C for 90 min in a basic
solution, with loss of HCl, generates a product that
absorbs light at 254 nm. Separation of the desired product
from excess derivatizing agent is possible in 10 min by CE
with a detection limit of 1 ng L 1 ..265/
Rimantadine is a synthetic analog of amantadine. Both
are antiviral agents used for prophylaxis and treatment of
influenza A. Because rimantadine is almost transparent in
the UV/VIS range, either indirect detection or derivatization has to be used to detect this compound. The indirect
detection method used 5 mM 4-methylbenzylamine in
1 : 4 methanol – water as absorbing background electrolyte
for detection at 210 nm. The derivatization method used
rimantadine to react with 1,2-naphthoquinone-4-sulfonic
acid in alkaline medium. CE determination of the derivative at 280 nm was performed in an uncoated capillary
(44 cm ð 75 µm ID) using a 40 mM tetraborate buffer at
pH 9.2. The detection limits were 0.1 and 2 mg L 1 for
indirect detection and derivatization methods, respectively. The methods were used to determine rimantadine
in pharmaceutical products and for dissolution testing of
Flumadin tablets..266/
Most of the naturally occurring amino acids do not have
proper UV/VIS chromophores for CE analysis. Derivatization is a necessity for the determination of amino acids
at reasonable concentrations. By using dansyl chloride
to derivatize the amino acids, the enantiomeric forms
of novel depsipeptide antitumor antibiotic BMY-45012,
and its analogs, were determined by CE with UV/VIS
detection..267/ The compounds were subjected to total
hydrolysis in a vacuum hydrolysis tube with 6 M HCl at
110 ° C for 24 h. The hydrolyzed residue (about 7.8 mg)
was dissolved in water – acetonitrile solvent. For subsequent derivatization, the solution was further diluted to
about 1.7 mg mL 1 with water. Dansyl chloride dissolved
in acetonitrile (3.0 mg mL 1 ) was utilized for the derivatization of standard native amino acids and those present
in the hydrolyzate. A 50-µL sample solution was mixed
with 50 µL of dansyl chloride solution and an aliquot of
20
PHARMACEUTICALS AND DRUGS
borate buffer at pH 9.08. The mixture was held at room
temperature for 2 h and used directly for injection.
The aminocychitol antibiotic amikacin can be derivatized with 1-methoxycarbonylindolizine-3,5-dicarbaldehyde at room temperature for 15 min before determination in plasma by micellar CE with UV detection at
280 nm and standard fluorescence detection (excitation
at 414 nm, emission at 482 nm)..268/ However, there was
only a twofold gain in sensitivity on switching from UV to
fluorescence detection of the amikacin derivative. If laser
excitation can be used (as discussed in the next section),
the gain in sensitivity should be significantly higher.
H3C
H3C C
C CH2
+
N
CH3
N
(CH2 )4
X
I
O
SO3−
CY5.3a.IA: X = −NH −
H
HS
•
CH3
N
•
O
C
HO
O
Captopril
Relative peak height
1
2
4.2 Derivatization for Fluorescence Detection
In principle, almost any detection mode can be combined
with a derivatization procedure. In practice, fluorescence
monitoring is favored in most cases. The principles of fluorescence and the prerequisites for a good fluorophore,
including the potential of using diode lasers in combination with a labeling procedure, have been reviewed
previously..261/
LIF detection is now commercially available for CE,
and more and more industrial scientists are using LIF
detection to detect trace amounts of analytes in complex
matrices. LIF detection has several advantages. First, it
offers excellent sensitivity; under optimized conditions,
a level of 10 10 M is feasible..269/ This is critical to
pharmaceutical applications because sensitivity is often
the most important parameter to consider when analyzing
biological samples. Second, LIF offers a certain selectivity
because, unlike UV absorbance, LIF selectively detects
compounds that are fluorescent at certain wavelengths,
and most organic compounds do not fluoresce. On the
other hand, the fact that most organic compounds do not
fluoresce is also a major limitation of LIF detection.
Since the available excitation wavelengths of various
lasers are limited, derivatization is often required when
using CE with LIF detection. The following examples
demonstrate the applicability of derivatization in CE
with LIF detection for pharmaceutical analysis.
Captopril, an antihypertensive agent, was derivatized
through the thiol group with a dicarbocyamine label
to give a fluorescent derivative with a wavelength
maximum at 675 nm..270/ This wavelength was compatible
with a semiconductor laser at 670 nm, which was used
for LIF detection in conjunction with CE. Using a
methanol – borate running electrolyte, CE also separated
other labeled thiols such as cysteine and reduced
glutathione from derivatized captopril (Figure 15). The
detection limit of 2.5 ð 10 8 M for captopril is limited by
dilution due to the derivatization reaction.
Amines can easily be derivatized with FITC isomer
I and analyzed by CE using alkaline buffers with or
CH3
7
5
6 4
3
40
30
20
10
0
Migration time (min)
Figure 15 Electropherogram of a number of thiols labeled
with CY5.3a.IA: (1) unreacted label; (2) captopril; (3) DL-homocysteine; (4) L-cysteine; (5) 3-mercaptopropionic acid; (6) 2-mercaptoacetic acid; (7) reduced glutathione. [Reproduced with
permission from Couderc et al..264/ ]
without dodecyl sulfate micelles. Ramseier et al. reported
the determination of FITC-derivatized amphetamine,
methamphetamine,
3,4-methylenedioxymethamphetamine and b-phenylethylamine in human urine by CE
using chip-based and fused-silica capillary instrumentation with LIF detection..271/ The results obtained via
direct labeling of fortified urine were compared with
those generated after FITC labeling of urinary extracts
that were prepared by SPE. Using 5 mL of urine with a
‘spiked amine’ to FITC ratio of 1 : 250, the SPE extract
had a sensitivity of 200 ng mL 1 urine. That value is relevant for toxicology drug screening and confirmation.
In contrast, with direct labeling of 10 µL of urine that
had been alkalinized and diluted for derivatization, the
limit of identification was 10 µg mL 1 , a value that is too
high for practical purposes. Compared with fused-silica
capillaries, electrophoresis in microstructures is shown to
provide faster separation and higher efficiencies without
loss of accuracy and precision.
21
CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS
4.3 Special Applications
7-aminonaphthalene-1,3-disulfonic
acid
(ANDS)
(Scheme 1) in human urine allowed the on-column LIF
detection of the pseudo-oligosaccharides in human urine
in the nanomolar range..272/ Before derivatization, 0.5mL samples of urine or spiked urine were evaporated
to dryness. A 100-µL volume of 0.08 M ANDS solution
in acetic acid – water (3 : 17 v/v) and a 50-µL volume of
0.9 M NaCNBH3 solution in dimethyl sulfoxide (DMSO)
were added to each sample residue. The reagent solutions
were freshly prepared before derivatization. The reaction
tubes were vortex mixed and then incubated overnight
at 40 ° C. The efficient separation of these derivatives by
CE using 100 mM triethylammonium phosphate buffer at
pH 1.5 allowed the quantitation of acarbose and (3) in
human urine after application of 300 mg of acarbose.
4.3.1 Chiral Confirmation
With proper derivatization, CE has been used for the
chiral separation of enantiomeric forms of derivatized
amino acids. Liu et al. reported the determination of
enantiomeric forms of amino acids derived from the
novel depsipeptide antitumor antibiotic BMY-45012,
and its analogs, the proposed structure of which is
shown as (1) [reproduced with permission from Liu
et al..267/ ]. Amino acids were analyzed by complete
hydrolysis and the hydrolyzate was derivatized with either
dansyl chloride for UV absorbance detection or FITC
for LIF detection in CE. For fluorescence detection,
the fluorogenic reagent FITC was dissolved in acetone
(0.01 M) as a stock solution. Amino acids were derivatized
with FITC-derivatizing solution (5.0 ð 10 4 M) under
basic conditions (borate buffer, pH 9.08). The reaction
was allowed to proceed for 2 – 4 h in the dark at
room temperature and then stored at 20 ° C prior to
use. Both a metal chelate chiral CE method and a
cyclodextrin-mediated host – guest interaction approach
in micellar electrokinetic chromatography (MEKC) with
LIF detection confirmed the presence of several chiral
amino acids, such as D-serine and L-b-hydroxyl-Nmethylvaline, and the nonchiral amino acid sarcosine
in the proposed structure. These methodologies provide
a quick and sensitive approach for the determination
of amino acid racemization in pharmaceutical natural
products and have proven to be useful for structural
elucidation refinement.
4.3.3 Derivatization of Protein Samples
With the increased interest in the therapeutic use of the
recombinant monoclonal antibody (rMAb) technique, a
generic analytical approach for the analysis of size-based
rMAb variants is desired. CE/LIF with proper derivatization has been shown to be promising as a general method.
The rMAb can be derivatized with a neutral fluorophore,
e.g. 5-carboxytetramethylrhodamine succinimidyl ester
(5-TAMRSE) for CE/LIF analysis..273/ Samples containing 2.5 mg of rMAb were buffer exchanged into 800 µL of
0.1 M sodium hydrogencarbonate (pH 8.3) using a NAP-5
column. A 10-µL volume of 5-TAMRSE (1.4 mg mL 1 )
dissolved in DMSO was then added to 190 µL of rMAb
solution and the resultant mixture incubated for 2 h at
30 ° C. After incubation, 190 µL of the antibody – dye conjugate was loaded on to a second NAP-5 column and
collected in 700 µL of 0.1 M sodium hydrogencarbonate (pH 8.3). The labeled sample, after incubating with
4.3.2 Oligosaccharide Determination
The derivatization of the pseudo-oligosaccharide
acarbose (2) and its main metabolite (3) with
CH3O
N
H
N
N
O
O
N
N
O
O
O
H3C
HO
O
N
CH3 CH3
H
N
O
N
O
CH
OR1 3 O
R2O
CH3
O
N
O
CH3 CH3
OH
N
CH3
O
O
N
N
O
N
H
O
N
H
O
(1)
Compound
BMY45012
Analog-G439B
Analog-G451
Analog-G435
Analog-G439A
R1
R2
CH(CH3)2
CH2CH3
C(CH3)3
C(CH3)3
CH(CH3)2
CH(CH3)2
CH2CH3
C(CH3)3
CH(CH3)2
CH3
N
N
OCH3
22
PHARMACEUTICALS AND DRUGS
CH2OH
HO
HO
CH3
HO
O
HN
CH2OH
HO
HO O
O
CH2OH
HO
HO O
O
OH
HO
HO
(2)
CH2OH
HO
HO
CH3
HO
O
HN
CH2OH
HO
HO O
O
OH
HO
HO
(3)
O
HO
4.4 Derivatization Modes
CH2OH
O
CH2OH
OH
O
HO
C O
HO
H
OH
HO
Carbohydrate
SO3H
H2N
SO3H
O
HO
CH2OH
OH
C N
HO
H
of the bulk manufacture of a protein pharmaceutical and
in providing a size-based separation of product-related
variants and also nonproduct impurities. The CE/LIF with
derivatization demonstrated comparable resolution and
sensitivity to silver-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) but offered
the advantages of enhanced precision and robustness,
speed, ease of use, and on-line detection.
CE was used for the study of palmitoyl derivatization
of interferon-a2b (p-IFN-a)..274/ The derivative was
prepared by covalent attachment of the fatty acid to
lysine residues in the protein through a reaction with Nhydroxysuccinimide palmitate ester (Scheme 2). The first
step involved the preparation of an intermediate (NHSP),
which in turn was reacted with IFN-a. CE was able to
study the effect of reaction time and reagent/protein ratio.
In general, derivatization in CE can be accomplished by
either pre- or postcolumn derivatization. The precolumn
derivatization is similar to methods used for derivatization
in HPLC. Since CE has a much superior separation power,
the interference from the derivatization by-products may
be less of a problem than in LC. However, CE is very
sensitive to the ionic strength of the sample. Additional
sample preparation may be needed if the mixture in the
derivatization reaction has a high ionic strength.
O
SO3H
R COOH + HO N
SO3H
Schiff base
NaCNCH3
CH2OH
OH
O
CH2 NH
HO
HO
O
O
DCC
R O N
+ DCU
O
Active ester (isolated)
+
NH2 IFN α
pH 7.2
SO3H
SO3H
Scheme 1 Reaction of (2) and (3) for derivatization with
ANDS by reductive amination. [Reproduced with permission
from Rethfeld and Blaschke..272/ ]
SDS, can be separated by CE using a hydrophilic polymer as a sieving matrix. Using these precolumn labeling
conditions, the detection of rMAb at a low-nanomolar
concentration (9 ng mL 1 ) is obtained with no apparent
decrease in resolution or changes to the distribution of
rMAb analyte species in comparison with an unlabeled
sample. This assay can be used in monitoring consistency
O
R C N IFN α
H
Scheme 2 Synthetic process for the fatty acrylation of IFN-a.
R COOH palmitic acid. [Reproduced with permission from
Foldvari et al..274/ ]
Postcolumn detection strategies have also been developed for CE. An overview of the advantages and limitations of postcolumn derivatization for CE can be found
in a recent paper..261/ The details about the instrumental
developments and applications of post-column derivatization in CE can also be found in the literature..262/ Various
systems to merge the reagent solution with the separation
medium have been developed, including coaxial capillary
reactors, gap reactors, and free-solution or end-column
systems. For all reactor types, the geometry of the system,
CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS
and the method used to propel the reaction mixture (by
pressure or by voltage) appear to be critical to preserve
the separation efficiency. To minimize peak broadening,
careful design in terms of connecting a reagent capillary to
form a tee is necessary. Because of the small (50 – 75 µm)
ID of the capillaries used, normal mixing through a diffusion process is sufficient. The most frequently applied
reactors are (1) co-axial, (2) gap, (3) free-solution, and
(4) sheath flow. With proper design, plate numbers of
over 100 000 could be realized. The strict requirements
on the reaction rate in postcolumn derivatization in CE
limit the number of different reagents that have been
used. For LIF detection, mainly OPA and its naphthalene analog have been used. With careful instrument
design, the detection limits of postcolumn derivatization
can be comparable to those of precolumn derivatization.
FFAP
FITC
FMOC
GC
GC/MS
HFAA
HFBI
HMDS
HPLC
LC
LIF
MBTFA
MEKC
MS
MSTFA
5 CONCLUSION
It can be argued that the advances made in MS
detection for GC, HPLC, and CE have diminished
the importance of chemical derivatization for these
techniques. However, chemical derivatization of drugs
is critical for GC because these samples, which often
contain multiple polar substituents, are simply not volatile
or thermally stable. MS detection for HPLC and CE
is still expensive and requires considerable operator
expertise. Chemical derivatization with HPLC to permit
fluorescence detection is certainly one of the more
desirable methods for routine use to solve selectivity and
detectability problems for drug samples not amenable to
GC. As the cost of laser technology comes down, the
same statement will eventually be true for CE also.
ABBREVIATIONS AND ACRONYMS
ANDS
BSA
BSTFA
CBD
CE
DCC
DES
DMES
DMFDA
DMIPS
DMSO
EC
7-Aminonaphthalene-1,3-disulfonic
Acid
N,O-Bis(trimethylsilyl)acetamide
N,O-Bis(trimethylsilyl)trifluoroacetamide
Cannabidiol
Capillary Electrophoresis
Diethyldithiocarbamate
Diethylstilbestrol
Dimethylethylsilyl
N,N-Dimethylformamide
Dimethylacetal
Dimethylisopropylsilyl
Dimethyl Sulfoxide
Electron Capture
MTBSTFA
OPA
PFAA
PFAI
PFB-Br
QUAT
rMAb
SDS
SDS/PAGE
SPE
TBDMS
TFAA
TFAI
TMAH
TMCS
TMS
TMSI
UV
UV/VIS
5-TAMRSE
23
Free Fatty Acid Phase
Fluorescein Isothiocyanate
9-Fluorenylmethyl Chloroformate
Gas Chromatography
Gas Chromatography/Mass
Spectrometry
Heptafluorobutyric Anhydride
Heptafluorobutylimidazole
Hexamethyldisilazane
High-performance
Liquid Chromatography
Liquid Chromatography
Laser-induced Fluorescence
N-Methylbistrifluoroacetamide
Micellar Electrokinetic
Chromatography
Mass Spectrometry
N-Methyl-N-trimethylsilyltrifluoroacetamide
N-Methyl-N-(t-butyldimethylsilyl)trifluoroacetamide
o-Phthalaldehyde
Pentafluoropropionic Anhydride
Pentafluoropropionylimidazole
Pentafluorobenzyl Bromide
Quaternary Ammonium Salt
Recombinant Monoclonal
Antibody
Sodium Dodecyl Sulfate
Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis
Solid-phase Extraction
t-Butyldimethylsilyl
Trifluoroacetic Anhydride
Trifluoroacetylimidazole
Trimethylanilinium Hydroxide
Trimethylchlorosilane
Trimethylsilyl
N-Trimethylsilylimidazole
Ultraviolet
Ultraviolet/Visible
5-Carboxytetramethylrhodamine
Succinimidyl Ester
RELATED ARTICLES
Pharmaceuticals and Drugs (Volume 8)
Eluent Additives and the Optimization of Highperformance Liquid Chromatography Procedures ž Gas
and Liquid Chromatography, Column Selection for, in
Drug Analysis ž Planar Chromatography in Pharmaceutical Analysis ž Solid-phase Extraction and Clean-up
Procedures in Pharmaceutical Analysis
24
PHARMACEUTICALS AND DRUGS
Gas Chromatography (Volume 12)
Gas Chromatography: Introduction ž Column Technology in Gas Chromatography ž Sample Preparation for
Gas Chromatography
13.
14.
Liquid Chromatography (Volume 13)
Liquid Chromatography: Introduction ž Capillary Electrophoresis ž Column Theory and Resolution in Liquid
Chromatography ž Normal-phase Liquid Chromatography ž Reversed Phase Liquid Chromatography
15.
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J.F. Lawrence, U.A.Th. Brinkman, R.W. Frei, ‘Extraction Detector for High-performance Liquid Chromatography Using Solvent Segmentation of the Column
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R.D. Barlow, P.A. Thompson, R.B. Stone, ‘Simultaneous Determination of Nicotine, Cotinine and Five
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G. Seidl, H.P. Nerad, ‘Gentamicin C: Separation of
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W. Buchberger, K. Winsauer, F. Nachtmann, ‘Trace
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D.G. Musson, S.M. Maglietto, W.F. Bayne, ‘Determination of the Antibiotic Fludalanine in Plasma and
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H.P. Fiedler, A. Plaga, ‘Separation of Amino Acids and
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H. Scholl, K. Schmidt, B. Weber, ‘Sensitive and Selective
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M.A. Targove, N.D. Danielson, ‘High Performance Liquid Chromatography of Clindamycin Antibiotics Using
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P.A. Hartman, ‘Liquid Chromatographic Determination
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L.T. Wong, A.R. Beaubien, A.P. Pakuts, ‘Determination of Amikacin in Microlitre Quantities of Biological
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by High-performance Liquid Chromatography and Its
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J. Haginaka, J. Wakai, ‘High-performance Liquid Chromatographic Assay of Carbenicillin, Ticarcillin and
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J. Haginaka, J. Wakai, H. Yasuda, T. Uno, T. Nakagawa,
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H. Russ, D. McCleary, R. Katimy, J.L. Montana, R.B.
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30
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W. Lindner, ‘N-Chloromethyl-4-nitro-phthalimid als
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A. Hulshoff, H. Roseboom, J. Renema, ‘Improved
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J. DeJong, M.W.F. Nielen, R.W. Frei, U.A.Th. Brinkman, ‘Selective On-line Sample Handling for the
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C.R. Clark, J.-L. Chan, ‘Improved Detectability of Barbiturates in High Performance Liquid Chromatography
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E.P. Scott, ‘Application of Postcolumn Ionization in the
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C.H. Wolf, R.W. Schmid, ‘Enhanced UV-detection
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R.L. Patience, J.D. Thomas, ‘Rapid Concentration and
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J.P. Langenberg, U.R. Tjaden, ‘Improved Method for
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J.P. Langenberg, U.R. Tjaden, ‘Determination of (Endogenous) Vitamin K1 in Human Plasma by Reversedphase High-performance Liquid Chromatography Using
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B.E. Miller, N.D. Danielson, ‘Fluorimetric Determination of Danthron in Pharmaceutical Tablets and in
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T. Seki, N. Hashida, T. Kanazawa, ‘Determination of
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P.S. Mukherjee, H.T. Karnes, ‘Reaction of 5-Bromomethylfluorescein (5-BMF) with Cefuroxime and Other
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L.W. Brown, B.E. Carpenter, ‘Comparison of Two Highpressure Liquid Chromatographic Assays for Carboprost, a Synthetic Prostaglandin’, J. Pharm. Sci., 69,
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M. Hatsumi, S.-I. Kimata, K. Hirosawa, ‘9-Anthryldiazomethane Derivatives of Prostaglandins for Highperformance Liquid Chromatographic Analysis’, J.
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J.W. Cox, R.H. Pullen, M.E. Royer, ‘Isolation of Plasma
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R.H. Pullen, J.W. Cox, ‘Determination of (15R)- and
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T.A. Stein, L. Angus, E. Borrero, L.J. Auguste, L. Wise,
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M. Hatsumi, S. Kimata, K. Hirosawa, ‘9-Anthryldiazomethane Derivatives of Prostaglandins for Highperformance Liquid Chromatographic Analysis’, J.
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R.L. Veazy, T.A. Nieman, ‘Chemiluminescence Highperformance Liquid Chromatographic Detector Applied
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R.W. Keating, P.R. Haddad, ‘Simultaneous Determination of Ascorbic Acid and Dehydroascorbic Acid
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S.J. Ziegler, B. Meier, O. Sticher, ‘Rapid and Sensitive
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T. Seki, Y. Yamaguchi, K. Noguchi, Y. Yanagihara,
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T. Iwata, M. Yamaguchi, S. Hara, M. Nakamura, ‘Determination of Total Ascorbic Acid in Human Serum by
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D. De Zeeuw, J.L. Leinfelder, D.C. Brater, ‘Highly Sensitive Measurement of Indomethacin Using a Highperformance Liquid Chromatographic Technique Combined with Post-column In-line Hydrolysis’, J. Chromatogr., 380, 157 – 162 (1986).
D.M. Seibert, F. Bochner, ‘Determination of Plasma
Aspirin and Salicylic Acid Concentrations After Low
Aspirin Doses by High-performance Liquid Chromatography with Post-column Hydrolysis and Fluorescence
Detection’, J. Chromatogr., 420, 425 – 431 (1987).
A. Avgerinos, A.J. Hutt, ‘Determination of the Enantiomeric Composition of Ibuprofen in Human Plasma by
High-performance Liquid Chromatography’, J. Chromatogr., 415, 75 – 83 (1987).
S. Bjorkman, ‘Determination of the Enantiomers of
Indoprofen in Blood Plasma by High-performance
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Means of Ethyl Chloroformate’, J. Chromatogr., 339,
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S. Pedrazzini, W. Zanoboni-Muciaccia, C. Sacchi,
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A.-Q. Zou, M. Xie, X.-D. Lou, ‘Determination of Artesunic Acid After Chemical Derivatization with o,pNitrobenzyl-N,N 0 -diisopropylisourea by High-performance Liquid Chromatography and Ultraviolet Absorption’, J. Chromatogr., 410, 217 – 221 (1987).
P.E. Minkler, S.T. Ingalls, L.S. Kormos, D.E. Weir, C.L.
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J. Gorham, E. McDonnell, ‘High-performance Liquid
Chromatographic Method for the Separation and Estimation of Choline, Glycinebetaine Aldehyde and
Related Compounds’, J. Chromatogr., 350, 245 – 254
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O.M. Steijger, D.A. Kamminga, H. Lingeman, U.A.Th.
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