Detection-Oriented Derivatization Techniques in Liquid Chromatography

Henk Lingeman, Willy J. M. Underberg

• Éditeur: CRC Press
• Année: 2021
• ISBN:
  9781000447354
• Édition: 1
• Sujet: Naturwissenschaften
• Sous-sujet: Chemie

This book covers the principles, kinetics, and applications of derivatization reactions in liquid chromatography. It emphasizes detection-oriented derivatization procedures that can be used to enhance to detectability of a wide variety of solutes using absorbance and fluorescence detection.

1
Derivatization in Liquid Chromatography: Introduction

HENK LINGEMAN* Leiden University, Leiden, The Netherlands

WILLY J. M. UNDERBERG State University of Utrecht, Utrecht, The Netherlands

* Present affiliation: Free University, Amsterdam, The Netherlands

INTRODUCTION

Accurate, sensitive, and selective bioanalytical determination methods for drugs and drug products (e.g., degradation products and metabolites) are of major importance in a number of pharmaceutical research fields: therapeutic drug monitoring, metabolic profiling, toxicology, drug pharmacology and pharmacokinetics, and stability [1]. The concentration of drugs and metabolites in whole blood, plasma, urine, saliva, or tissue homogenates is in many cases in the nanogram (ng/ml) or even the picogram (pg/ml) range, necessitating the use of selective and sensitive separation and detection methods after isolation and cleaning of the analyte from excess of endogenous and exogenous components present in the biological matrix.

Chromatographic separation techniques such as high-performance liquid chromatography (HPLC or LC) and gas chromatography (GC) in combination with selective and sensitive detection methods have gained an enormous popularity in the analysis of compounds of pharmaceutical and environmental interest [2].

The chemical structure of the analyte and the physicochemical properties of the matrix together with the desired selectivity and sensitivity determines the best suitable isolation and quantitation method. The required clean-up procedure strongly depends on the chosen chromatographic separation-detection combination. In the case of a very selective detection technique for the analyte under investigation, isolation from the matrix may be superfluous. However, even with the variety of currently available separation-detection systems it is not always possible to achieve the desired detection limit with the necessary precision without a chemical manipulation, derivatization, of the drug [3–7].

However, derivatization is a necessary evil. In developing accurate and precise methods the number of manipulations should be kept as low as possible. Every derivatization procedure consists of several manipulations which are potential error sources. Therefore, the analytical chemist must be sure, for instance, that no impurities are present in the solvents and the reagents. Blank derivatization procedures are an essential part in the development of every derivatization procedure. Furthermore, a great number of derivatives are not very stable, e.g., hydrolysis under the influence of moisture as well as other degradative processes may occur.

The main reasons for derivatization before or after the chromatographic separation are:

• Improvement of the detectability of the analyte
• Introduction or improvement of the resolution of closely related compounds during chromatography
• Establishment of analyte identity
• Improvement of the selectivity for the solute in a complex matrix
• Improvement of the chromatographic behavior of the drug under investigation
• Prevention of decomposition during chromatography and/or detection of the drug
• Achievement of better chemical and/or physical properties (e.g., volatility of polar compounds, solubility) of the solute to be chromatographed
• Introduction of an additional sample clean-up step

Derivatization reactions necessary to improve the chromatographic properties of analytes must be performed before the chromatographic separation (prechromatographic), but structure manipulation reactions to improve the sensitivity and/or the selectivity can be performed either before or after (postchromatographic) the separation. Prechromatographic derivatization techniques are far more often applied than postchromatographic methods because of their simplicity in comparison with the latter.

In general, derivatization reactions can be performed off-line with the chromatographic separation and on-line with the chromatography. The result is that four different derivatization principles can be distinguished (Scheme 1).

Scheme 1

The two most widely applied derivatization procedures are the prechromatographic off-line and the postchromatographic on-line principles. Both procedures have their own advantages and disadvantages as discussed below. The postchromatographic off-line mode (D) is becoming more important after the introduction of the solid phase isolation cartridges (see Chap. 3). The principle is that a chromatographic system is used to clean up the samples (e.g., biological samples), followed by a derivatization step and a second separation step in order to quantitate the resulting derivatives. The prechromatographic on-line procedure (B) is also more widely used during the last few years because of the use of sample processors, which allow the automation of mixing solvents and analysis (see Chap. 3).

In choosing the proper derivatization technique for a certain problem, one of the questions is whether to perform the derivatization before or after the chromatographic separation.

Prechromatographic Derivatization

Prechromatographic derivatization is the most widely used derivatization technique with many advantages and only a few disadvantages [4,6,8,9]. The major advantages are:

• There are no restrictions on the reaction kinetics, provided that the reaction goes to completion within a reasonable time, yielding one derivatization product for the compound in question without the formation of side products.
• There is a free choice in varying the conditions in order to optimize the reaction time and reaction yield.
• The solvent in which the prechromatographic reaction takes place need not be compatible with the chromatographic system (e.g., the mobile phase of the LC system).
• Chromatographic properties (e.g., capacity factors, resolution) can be optimized.
• Side products formed during the derivatization, degradation products of the reagent, and the excess of the reagent will be separable from the derivative, either on the chromatographic system or by a prechromatographic clean-up step.

The main disadvantages of the prechromatographic derivatizations are sometimes:

• There is formation of side products and artifacts, which may interfere in the chromatographic analysis or in the reproducibility of the derivatization reaction.
• Each sample needs individual handling (e.g., laborious, time consuming).
• There is a need for an internal standard in case the derivatization reaction is incomplete.
• Target compounds may be more alike after derivatization.

One of the most frequently encountered problems in derivatization procedures is the occurrence of interfering peaks in the chromatograms due to degradation products or impurities of reagents and/or solvents. If an LC separation is used in combination with a prechromatographic derivatization step, the possibility exists that the derivative will be unstable in the mobile phase and will be partly degraded during the chromatographic process or during the pre-treatment [10]. In these cases a postchromatographic derivatization method may be advantageous.

Ideally the components of the derivatization mixture do not change the chromatographic system through chemical reactions, precipitation, or demixing of the eluent upon injection or otherwise, and do not interfere with the detection of the derivatized compounds. If these conditions are fulfilled, aliquots of the derivatization mixture can be directly injected into the chromatographic system (Fig. 1.1). However, it is frequently necessary to remove the excess reagent or the solvent or other components of the reaction mixture prior to the chromatographic analysis.

Figure 1.1 Chromatograms obtained after the derivatization of α-naphthyl-β-D-glucuronide, β-estradiol-17-β-D-glucuronide, and mentholglucuronide with N-(1-naphthyl)ethylenediamine (NED) according to the 2-bromo-l-methylpyridinium iodide method. (A) blank chromatogram. (B) derivatization of 10 μg of the three glucuronides. x, amide of mentholglucurohide and NED. y, amide of estradiolglucuronide and NED. z, amide of mentholglucuronide and NED. (Reprinted with permission from Lingeman, H., U. R. Tjaden, C. M. B...