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The applications of various pre-column derivatization reactions are presented in this review. The
reactions are particularly applicable for various detection techniques [ultraviolet (UV), fluorescence,
chemiluminscence, enantiomeric determination and mass spectrometry (MS)] in liquid chromatographic
analysis of drugs and related compounds in various matrices. This review discovered that an
improvement in reaction conditions, reagent suitability and detection limits have evolved over the last
60 years. The challenge however, still remains in ensuring that a particular pre-column derivatization
step satisfies the requirement of high sensitivity, short reaction time, short work-up procedures and in
particular selectivity for the drug(s) of interest. The evolution of high performance liquid
chromatography-tandem mass spectrometry (HPLC-MS/MS) as an emerging technique in derivatization
is discussed.
Key words: Tandem mass spectrometry, ultraviolet, pre-column derivatization, liquid chromatography.
INTRODUCTION
Chemical derivatization is routinely employed to improve
the chromatographic and/or mass spectrometric
characteristics of an analyte. In gas chromatography
(GC), derivatization is often required to achieve the
necessary volatility. Specific derivatization of analytes
can simplify the separation from interferences in the
sample during liquid chromatography (LC), for example
by reversed-phase (RP) separation or ion-exchange
methods after the introduction of a nonpolar group or a
charged function, respectively. A permanent charge in a
molecule, such as the positive charge of a quaternary
ammonium moiety, increases the ionization efficiency
and thus the sensitivity in electrospray ionization (ESI)
mass spectrometry (MS). Furthermore, by modification
with a defined structural element, derivatization often
enables the prediction of specific fragmentation reactions
in tandem MS (MS/MS), which enhances the specificity of
the method (Kretschmer et al., 2011).
Derivatization has long been accepted as an effective
modification technique that can improve the overall
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internal standard were extracted from serum using liquidliquid partition. Upon exposure to UV light, the drug
undergoes a photocyclization reaction giving a species
with high absorbance which was determined by RPHPLC.
A highly sensitive and simple method for the
determination of prostaglandins (PGs) by HPLC with
fluorescence detection is described. PGs are converted
to the corresponding fluorescence derivatives by the
reaction
with
4-(N,N-dimethylaminosulphonyl)-7-(1piperazinyl)-2,1,3-benzoxadiazole (DBD-PZ) in the
presence
of
2,2-dipyridyl
disulphide
and
triphenylphosphine in acetonitrile. The reaction is
completed at room temperature after 30 min. The DBD
derivatives of nine PGs are separated within a single 45
min chromatographic run on a RP ODS column with a
linear gradient elution using water and acetonitrile. The
detection limits (signal-to-noise ratio of 3) calculated from
the standard mixture of PGs (6-keto-F1, F1, F2, E1, E2,
D2, limaprost, A1 and B1) are in the range 1.7 to 5.0 fmol.
The applicability of the proposed procedure is evaluated
to the detection of PGs added to rat plasma (Toyo'oka et
al., 1992).
A simple and selective method for the multiple residue
determination of eight sulphonamides in consumers' milk
was also reported. The drugs are sulphisomidine,
sulphadiazine,
sulphamerazine,
sulphadimidine,
sulphamonomethoxine,
sulphamethoxazole,
sulphadimethoxine and sulphaquinoxaline. The milk
sample was deproteinized with the same volume of
2 M hydrochloric acid and filtered. A 1-ml volume of the
titrate was mixed with 1 ml each of 1.25 M sodium
acetate solution and a buffer (pH 3.0) for derivatization
with 0.6 ml of 0.02% fluorescamine solution in acetone.
An HPLC analysis was carried out on a C18 column with a
mobile phase of acetonitrile-2% acetic acid (3:5) at 55C
using a fluorescence detector at an excitation wavelength
of 405 nm and an emission wavelength of 495 nm. The
method was applied to 25 milk samples and all appeared
to be free from the drugs (Takeda and Akiyama, 1992).
Aflatoxins have been widely derivatized for sensitive
fluorescence detection by HPLC. In a reciew by Kok
(1994), various derivatization methods for the fluorimetric
detection of aflatoxins after separation by HPLC were
reviewed. Optimum conditions for the reactions are
discussed. In terms of sensitivity, the three described
derivatization schemes give similar results. The methods
are compared with respect to experimental convenience,
selectivity, reproducibility and suitability for automation.
A new method for determining cycloserine in plasma
samples based on derivatization of cycloserine with pbenzoquinone was described. The reaction is reported to
take place at the same time as the process of plasma
deproteinization due to the presence of ethanol as
solvent in the solution of the derivatization reagent. Four
derivatives are obtained from this reaction. The main
derivative is well correlated with the cycloserine
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reagent,
(S)-(1)-1-methyl-2-(6,
7-dimethoxy-2,
3naphthalimido)ethyltrifluoromethanesulfonate was also
developed for the determination of the enantiomers of
carboxylic acids. By introducing the two methoxy groups
on the naphthalimido ring moiety, the red shift in the
fluorescence spectrum and a high resolution in reversedmode separation of the diastereomers of chiral carboxylic
acids were achieved (Yasaka et al., 1998).
PRE-COLUMN
DERIVATIZATION
SPECTROMETRIC DETECTION
FOR
MASS
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