DERIVATIZATION AS A FORM OF SAMPLE PREPARATION
IN ANALYSIS OF FOOD AND DRUG SAMPLES
Renata GADZAŁA-KOPCIUCH 1), Stanisław GRYS 2), Bogusław BUSZEWSKI 1),
1)
Nicholas Copernicus University, Faculty of Chemistry,
Department of Environmental Chemistry and Ecoanalytics
7 Gagarin St, 87-100 Toruń, POLAND
2)
Research and Development Service
12/22 J. Bruna St; 02-594 Warsaw, POLAND
Liquid chromatography with spectrophotometric detection, similarly as other
chromatographic techniques, has some limitations resulting from poor detection abilities.
Used detectors are characterized often by low sensitivity and low selectivity to microtrace
amounts of analytes contained in analyzed sample. In contrary techniques such as gas
chromatography there is available the wide range of selective and very sensitive detectors,
however in spite of these advantages the economic barrier (high cost of single modulus)
makes very important role. The predominant majority of such compounds as aliphatic amines,
carboxylic acids or alcohols present in biological samples is difficult to identify even in these
cases, when high-sensitive fluorescence or electrochemical detectors are used. For this reason
in order to fulfill more and more greater requirements relating to sensitivity and selectivity of
detection techniques as well as to establishment and qualitative information of the nature of
some, often toxic xenobiotics there appeared the necessity of introduction of additional
derivatization step to liquid chromatography and related techniques (e.g. capillary
electrophoresis). This step permits to convert given analyte into one or more derivatives
showing significantly better chromatographic (performance, resolution, plate number, height
of theoretical plates etc.) and detection (linearity, quantification and detection limits etc.)
parameters (Fig.1).
Fig.1. Scheme of derivatization reaction.
This step provides also the structural information necessary for confirmation of
analyte identity. The examples of different derivatization process are listed in table 1
The transformation of primary structure to other molecule(s) may be realized by
means of simple thermal, photochemical, catalytic, enzymatic or chemical reactions (e.g. the
action of acids and bases not connected with ionic reactions). In more complex cases there
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occurs the rearrangement or change/exchange of the bonds or atoms or addition of another
molecules having desired physico-chemical properties [1].
Table 1. The possibilities of improvement of sensitivity and/or selectivity of detection in
HPLC by conversion of analyte into appropriate derivative.
Purpose
Example
Reduction
Increase of detection sensitivity in UVD
of prostaglandines to 15-oxo-derivatives with
pyridinium dichromate
Increase of detection sensitivity and
specifity UVD→FLD
of morphine to pseudomorphine dimmer and
thiamine to thiochrome with alkaline solution
potassium ferrocyanate
Increase of detection sensitivity and
sensitivity by conversion to fluorescence
phenol
of ivermectine MnO2 to 5-keto derivative,
dehydration of ketone with ammonium
acetate and tautomerisation
Increase of detection sensitivity by
potentiation (25x) of fluorescence intensity
of aflatoxinesB1 and G1 with iodine to
derivatives containing metoxy group and
iodine atom added to double bond
Obtainment of derivatives of better
chromatographic properties
Oxidation
of ketoestrogens (estrone, equiline) to
hydroxy derivatives
Increase of detection sensitivity by
conversion to fluorescence hydroxy
derivatives
Of quinones: 1,4-naphtoquinone (vit.K)
1,8-dihydroxyantraquinone (dantrone)
Confirmation of sensitivity of detection (the Of nitrophenyls: nitro-phenylacetamide
exchange of nitro group to amino group)
(chloroamphenicol) and nitrosalicylanilide
(niclosamide)
Hydrolysis
Simultaneous obtainment of derivatives of of quaternary bipyridinium bases e.g. diquate
better chromatographic and detection
and paraquate
properties
Liquidation of hypochromic effect
Esters of B1,B2 and B6 vitamins
Obtainment of more favorable conditions
for chromatographic analysis
Estrified estrogens
Conversion of compounds of nonprofitable
Detection properties to parent substances or
to derivatives of high detection sensitivity
similar for whole group of detection
sensitivity
Hydration (pH 1) of double bond in
dihydrofurane ring od B1 and G1 aflatoxines
in order to obtain of B2a and G2a of
fluorescence intensity comparable to this of
B2 and G2
Conversion degradation
Conversion of compounds not containing of Alkaline degradation of penicilines in the
chromophore group to derivatives of
presence of 1,2,3-triazole and mercurium
desired optical density
chloride do mercurium mercaptides of
penicillin acid
2
The necessity of conversion of analyte into corresponding derivatives results from
three widely known reasons, which result of turn from:
 the absence in the structure of analytically important compounds (i.e. aliphatic amines,
alcohols, sugars or carboxylic acids) the chromophore group showing sufficiently high
active optical density or undergoing easily the redox processes,
 requirements of significant reduction of detection level of toxic and mutagen substance to
the values defined by law regulations,
 necessity of improvement o chromatographic properties of analyte,
 necessity of obtainment of structural information necessary for confirmation of analyte
identity.
Derivatization consists not only in simple substitution of labile hydrogen atom but
includes also all reaction utilized in organic chemistry. Basic principles of derivatization in
organic chemistry and chromatography are the same and include the principle of availability
of derivatization („Corpora non agunt nisi activata”), the principle of optimum of this
process („Reactio posterior generati non derogat priori reactio speciali”) and principle of
exclusion of conjunction reaction („Cessante ratione coniugationis, eo ipso coniugato
cessat”).
In analytical practice there distinguishes in the respect of type of sample and analyte as
well as in the respect of aim of dramatization process three ways of realization of this process
namely pre- and postcolumn derivatization in on-line or off-line system and derivatization on
solid phase. The success of this technique depends in predominant degree on selection of
appropriate derivatizing agent.
Table 2. Specifity of conjunction of different functional group [1-4].
Conjuncting agent
Fluorescamine
OPA
NBD-Cl
DNS-Cl
Main
functional
group
NH2
NH2
NH2
NH
NH2, NH
Phenols
SH (aliph)
SH (aliph)
Alcohols
Phenols
SBD-F
Phenylisothiocyanate
Benzoyl chloride
p-metoxybenzoyl chloride
p-nitrobenzoyl chloride
3,5-dinitrobenzoyl chloride
p-bromophenacetyl bromide Organic
2-naphtacyl bromide
acids
ADAM
4-bromomethyl-7metoxycumarine
DNPH
Aldehydes
and
ketones
Detection
FL 390/475 nm
FL 340/455 nm
FL 480/520 nm
FL 365/500
FL 395/520 nm
FL 230/315 nm
UV 230 nm
UV 260 nm
UV 260 nm
UV 350 nm
UV 258 nm
FL 246/445 nm
FL 254(365)
FL 375/417 nm
UV 360 nm
FL 220/470 nm
Another
reactive
groups
NH
Thiols
Phenols
Thiols
Imidazole
Detection
UV
FL
UV 258
FL 2%
Phenols
NH2, NH
NH2, NH
NH2, NH
NH2, NH
As near
Diols
As near
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The main purpose of this lecture is the possibilities of utilization and presentation of
different derivatizing agents depending on the nature of analyte and on the aim of introduction
of additional step of sample preparation to analytical procedure. There were made also the
attempts to explanation of some problems connected with derivatization of diethylenediamine
widely used in pharmacy either as a native compound called piperazine, or as starting material
to synthesis of methyl and hydroxyl derivatives. This agent of turn a raw materials to
synthesis of many drugs of different actions and different application (estropine, clozapine,
cinerazine).
.
NH
NH
Fig. 2. Ultraviolet spectrum and chemical structure of diethylenediamine.
This compound should be determined during industrial production as intermediate
product and as technological contamination of final product, well as in investigations of
metabolism of mentioned above drugs and in environmental analyses.. Piperazine molecule
not contains the chromophore groups which is confirmed by obtained spectrum (Fig.2).
A)
B)
6000
y = 212.26x
R2 = 0.9999
Peak area [mAU]
5000
4000
3000
2000
1000
0
0
5
10
15
20
25
30
Concentration of DNS-diethylenediamine
Fig. 3. Separation of DNS-diethylenediamine for the application of different gradient elution (A)
and the calibration graph used for a series of standard solution of
DNS-diethylenediamine conditions: mobile phase: A = hexane/2-propanol (95/5 %); B = hexane/2propanol (75/25 %); gradient: start with 100 % A; at 15 min 100 % B; at 20 min 100 % A; at 25 min 100
% A; at 30 min 100 % A.
In order to precise determine of its content (on the level below 1 mg/kg) it is necessary
to converse to derivative showing significantly high optical density [2]. For this reason there
were taken up the studies on possibilities of selection of chemically modified adsorbents of
different properties [5,6] used as column packings in HPLC, and permitting to separation of
DNS-diethylenediamine and potential coeluents. The starting point for these studies made the
fact that on the one side the separation of diethylenediamine from aliphatic amines containing
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few carbon atoms during the step of sample preparation is labor-consuming and o the other
side determination of this compound in examined sample may be sometimes necessary.
References:
1.
2.
3.
4.
5.
I.S. Krull, Z. Deyl, H. Lingeman, J. Chromatogr. B, 659 (1994) 1-17.
B. D. McGarvey, J. Chromatogr. B, 659 (1994) 243-257.
Y. Yasaka, M. Tanaka, J. Chromatogr. B, 659 (1994) 139-155.
C.A. Lau-Cam, R.W. Ross, J. Liq. Chromatogr., 18 (1995) 3347-3357.
B. Buszewski, R. Gadzała-Kopciuch, R. Kaliszan, M. Markuszewski, M.T. Matyska, J.J. Pesek,
Chromatography, 48 (1998) 615-622.
6. R. Gadzała-Kopciuch, B. Buszewski, J. Chin. Chem. Soc., 45 (1998) 249-256.
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