DAFTAR ISI
DAFTAR ISI..................................................................................................................................................... 1
1. Pre-treatment for Real Sample Analysis and Choice of Suitable Reagent Akihiko Nakagawa, and
Yukinori Kawahara ........................................................................................................................................ 2
1.1. Derivatization for Drugs ..................................................................................................................... 2
1.1.1. Introduction ................................................................................................................................ 2
1.1.2. Application of Derivatization of Drugs ........................................................................................ 2
1.2. Derivatization for Agrochemicals ....................................................................................................... 2
1.2.1. Introduction ................................................................................................................................ 2
1.2.2. Analysis of Nutrients ................................................................................................................... 2
1.2.2.1. Carbohydrates ...................................................................................................................... 2
1.2.2.2. Amino Acids and Peptides ................................................................................................... 2
2. Reagent for UV-VIS Detection ................................................................................................................... 3
3. Reagent for FL Detection .......................................................................................................................... 3
3.1. Introduction ....................................................................................................................................... 3
3.2. Reagents for Amines and Amino Acids .............................................................................................. 3
4. Reagent for CL Detection .......................................................................................................................... 3
4.1. Introduction ....................................................................................................................................... 3
4.2. Label of Amines (-NH2, -NH) .............................................................................................................. 3
5. Reagents for Electrochemical Detection................................................................................................... 4
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1. Pre-treatment for Real Sample Analysis and Choice of Suitable Reagent Akihiko
Nakagawa, and Yukinori Kawahara
1.1. Derivatization for Drugs
1.1.1. Introduction
Drug analysis is mainly divided into three fields; materials, formulations and bioanalysis of
specimens obtained in vitro and in vivo. The former two are controlled by regulations such as
UPS in the USA, and the physico-chemical properties of drugs are summarized in a series of
books entitled 'Analytical
Profiles of Drug Substances', published periodically by Academic Press Inc. from 1972. In this
section, methods for derivatization for drugs, in order to monitor their levels in biological
samples, are described.
1.1.2. Application of Derivatization of Drugs
For HPLC separation of DNS-ED-derivatized pravastatin, a column-switching technique was
used to remove excess reagents and by-products. The HPLC system was described elsewhere [5].
In this case, a combination of two reversed-phase columns of different lipophilicity were
employed: A C4 column was used as a preseparation column (first column) to delete the major
peaks derived from the reagents and the plasma components, and a fraction containing
derivatized pravastatin was introduced into the analytical C18 column (second column). A
comparison between the chromatograms obtained with the ODS column 30/09/2003
1.2. Derivatization for Agrochemicals
1.2.1. Introduction
Chemical compounds found in foods can be divided into two categories: essential nutrients that
maintain human lives and substances which have unfavorable effects on man. The former
includes saccharides, fats, amino acids, proteins, vitamins, and minerals. The latter contains
environmental hazardous compounds such as organic mercury and PCB.
1.2.2. Analysis of Nutrients
1.2.2.1. Carbohydrates
Carbohydrates are structurally classified into monosaccharides, oligosaccharides and
polysaccharides. Monosaccharides and some oligosaccharides taste sweet. Compounds whose
aldehyde group in aldose is substituted by primary alcohol are called sugar alcohol. This chapter
describes the analysis of carbohydrates including sugar alcohol.
1.2.2.2. Amino Acids and Peptides
Proteins are the main constituents of organisms, and about 20 amino acids build polypeptide
chains with peptide bonding. Molecular weights of proteins range from 4000 (protamine etc.) to
some ten millions (virus proteins etc.).
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2. Reagent for UV-VIS Detection
Whenever a new or a more sensitive and selective method has been developed for the detection
and determination of biologically important compounds, drugs or their metabolites, biopolymers,
and also environmental pollutants, important scientific knowledge has evolved to a greater or
lesser extent. In the 1970s and 1980s, various derivatization methods for ultraviolet and visible
light (UV-VIS) absorption detection have been developed [1-5] for high-performance liquid
chromatography (HPLC), and many of the fundamental UV-VIS derivatizing reagents
30/09/2003
3. Reagent for FL Detection
3.1. Introduction
High-performance liquid chromatography (HPLC) has been one of the most widely used of the
effective separation methods, Capillary electrophoresis (CE) is rapidly becoming an accepted
routine analytical technique, characterized by short run times and high efficiencies. Of the
various detection methods used in HPLC and CE, fluorescence detection has been frequently
utilized for the determination of trace levels of bioactive compounds in complicated matrices
such as biological and environmental samples, owing to its high selectivity and sensitivity.
However, most biologically and environmentally important substances are weakly fluorescent or
nonfluorescent. Thus, various reagents have been developed for the fluorescence detection of
their substances in HPLC and CE.
3.2. Reagents for Amines and Amino Acids
The reagents utilized for fluorescence detection are divided into two groups: 'fluorogenic reagent'
and 'fluorescence labeling reagent'. The fluorogenic reagents are generally non-fluorescent and
react with target compounds to form conjugatedring molecules, resulting in the generation of
fluorescence. The fluorescence labeling reagents are composed of a highly fluorescent aromatic
moiety (fluorophore) and a reactive group, and the reactive group attaches to an analyte to give a
fluorescent derivative.
4. Reagent for CL Detection
4.1. Introduction
In the analysis of environmental contaminants using chromatography, the analysis of GC
methods which are superior to LC methods in separation characteristics and sensitivity have,
together with the rapid popularization of GC/MS, been applied far more widely than in the
analysis of bio-chemicals due to the applicability of many of the analytes to GC.
4.2. Label of Amines (-NH2, -NH)
The detection and determination of amines are concerned not only with biochemical but also
industrial analysis. Thus various derivatization reagents for the chromatographic determination
of primary and secondary amines as well as tertiary amines, are available. In many biologically
important compounds, an amino groups exists as amino acids and polyamines. When HPLC or
CE analysis of these amino compounds with direct UV-VIS detection is difficult because of the
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weak UV-VIS absorption of the compounds, derivatization of amines with a reagent having a
strong UV-VIS absorbing structure is one of the considerable methods to choose from.
5. Reagents for Electrochemical Detection
Electroanalytical methods are categorized by their mode of measurement into amperometric
(measurement of current), potentiometric (measurement of voltage), and conductimetric
(measurement of conductivity) methods. This chapter deals with amperometric electroanalysis in
flowing streams [1-3]. The major advantages of amperometric detection over classical
spectroscopic measurements are its high selectivity, low detection limits, and the simplicity of
the instrument.
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