Derivatization, chemical precolumn

The identification and quantification of potentially cytotoxic carbonyl compounds (e.g. aldehydes such as pentanal, hexanal, traw-2-octenal and 4-hydroxy-/mAW-2-nonenal, and ketones such as propan- and hexan-2-ones) also serves as a useful marker of the oxidative deterioration of PUFAs in isolated biological samples and chemical model systems. One method developed utilizes HPLC coupled with spectrophotometric detection and involves precolumn derivatization of peroxidized PUFA-derived aldehydes and alternative carbonyl compounds with 2,4-DNPH followed by separation of the resulting chromophoric 2,4-dinitrophenylhydrazones on a reversed-phase column and spectrophotometric detection at a wavelength of378 nm. This method has a relatively high level of sensitivity, and has been successfully applied to the analysis of such products in rat hepatocytes and rat liver microsomal suspensions stimulated with carbon tetrachloride or ADP-iron complexes (Poli etui., 1985). 

Increased use of liquid chromatography/mass spectrometry (lc/ms) for structural identification and trace analysis has become apparent. Thermo-spray lc/ms has been used to identify by-products in phenyl isocyanate precolumn derivatization reactions Liquid chromatography/thermospray mass spectrometric characterization of chemical adducts of DNA formed during in vitro reaction lias been proposed as an analytical technique to detect and identify those contaminants in aqueous environmental samples which have a propensity to be genotoxic, t.e.. to covalently bond to DNA. 

Since BAs occurring in food do not exhibit satisfactory absorbance or fluorescence in the visible or ultraviolet range, chemical derivatization, either pre- (35-37) or postcolumn (38), is usually used for their detection in HPLC. The most frequently employed reagents for precolumn derivatization are fluorescamine, aminoquinolyl-lV-hydroxysuccinimidyl carbamate (AQC) (39, 40), 9-fluorenylmethyl chloroformate (FMOC) (41-43), 4-dimethylaminoazobenzene-4 -sul-fonyl chloride (dabsylchloride, DBS) (44), N-acetylcysteine (NAC) (45,46), and 5-dimethyl-amino-1-naphthalene-1-sulfonyl chloride (dansylchloride, DNS) (47,48), phthalaldehyde (PA), and orf/to-phthaldialdehyde (OPA) (49-51), together with thiols such as 3-mercaptopropionic acid (MPA) (37) and 2-mercaptoethanol (ME) (35,49). 

Chemical derivatization of an analyte is performed to improve the selectivity and sensitivity of that analyte. There are two approaches to the use of derivatizing agents in HPLC. Precolumn derivatization, which is performed before the analytes are separated, is most commonly used for small molecules such as amino acids, whereas postcolumn derivatization, which is performed after the separation but before the analytes reach the detector, is often used for larger molecules such as peptides. 

In precolumn derivatization, the derivatization process alters the chemical nature of the analytes. Therefore, it may be necessary to develop new chromatographic methods. However, the separation can be optimized for the particular analytes, and any excess reagent can be removed so that it does not interfere with detection. The selection of precolumn derivatization reagents is therefore less restricted than is the choice of postcolumn derivatization reagents, and rapid kinetics are not particularly important. The stability of the derivative is important, however, as is the percent derivatization, which should be as near to 100% as possible. It is also important that the reaction yield only one derivative per analyte, so that coelutions of extra peaks does not occur, and so solute identification and quantitation are accurate. 

One of the weak points of fluorescence is that relatively few compounds fluoresce in a practical range of wavelengths. However, chemical derivatization allows many nonfluorescent molecules containing derivatiz-able functional groups to be detected, thus expanding the number of applications. Fluorescence derivatization can be accomplished either via precolumn or postcolumn methods. 

Derivatization means changing the sample in a specific way be chemical reaction and can be carried out prior to chromatographic injection (precolumn derivatization) or between the column and detector (postcolumn derivatization). 

Ephedrine and pseudoephedrine have been measured in guinea pig plasma using HPLC with fluorescence detection, following precolumn derivatization with 5-dimethylamino-napthalene-1-sulfonyl chloride in acetonitrile. The mobile phase was 0.6% phosphate buffer (pH 6.5)-methanol (3 8 v/v). Jacob et al. developed an LC-atmospheric pressure chemical ionization MS-MS method for the quantitaion of various alkaloids found in ephedra-containing dietary supplements and also in plasma and urine from subjects using these supplements. Using this method, the concentrations of ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine, methylephedrine, methylpseudoephedrine and caffeine were determined in low nanogram quantities in plasma and urine. The analytical cycle time for this method was 12 min. 

Precolumn derivatization methods include 1,2-diphenylethylenediamine treatment, dansylation of E, NE and DA, derivatization of NE and DA by o-phthalaldehyde and mercaptoethanol and derivatization of catecholamines with 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl). Derivatization with o-phthalaldehyde increases the sensitivity of NE and DA, but E is not measured because only primary amines are derivatized. Co-analysis of catecholamines, metanephrines and other related compounds by combined electrochemical oxidation and fluorescence derivatization had also been reported. ° This approach involves sequential chromatographic separation, coulometric oxidation and final chemical derivatization with 1,2-diphenylethylenediamine to fluorescent products. 

Derivatization reactions can be performed either pre- or postcolumn. As outlined by Brinkman, there are important advantages to using the postcolumn techniques whenever possible (68). First, the analytes can be separated in their original form, which often permits the adoption of published separation procedures. Second, artifact formation is generally not a serious problem, in contrast to precolumn derivatization, where it increases the separation difficulty and causes problems with quantitation. Third, the reaction does not need to be complete and the reaction products need not be stable the only requirement is reproducibility. Several reaction principles have been extensively applied. These include true chemical derivatization such as with dansyl chloride or o-phthalaldehyde UV irradiation, which can convert the analyte of interest into a more easily detectable species solid-phase reactions, including catalytic reactions such as with the use of immobilized enzymes and chemiluminescence techniques. 

It is felt that the precolumn sampling techniques deserve much attention in future studies, as they can serve a double function in biochemical investigations (a) removal of solvents or derivatization agents and (b) protection of the analytical column from non-volatile impurities. Chemical nature of the precolumn packing can also be varied to suit a particular sample type. Further investigations aiming at the optimization and automation of the precolumn sampling techniques appear desirable. 

The choice of a fluorescent tag depends on the type of derivatization (e.g., precolumn or postcolunui) because the stability of analyte-tag chemical bonds varies greatly. Therefore, for precolunm derivatization it is important to determine the time delay between derivatization and analysis as well as the stability of the reaction product in the mobile phase prior to HPLC method development. For postcolunm derivatization, compatibility of the reaction solvent with the chromatographic system as well as the rate of reaction are variables that must be considered. 

Naphthalenedialdehyde was used as a precolumn derivatization reagent in the determination of desmosine, isodesmosine, and 17 other amino acid residues [468]. More stable complexes (as compared with o-phthalaldehyde derivatives) were cited as the analytical advantage. Detection limits of lOOfmol (S/N = 2) were reported and the analysis was completed in <35 min. A C 8 column (A = 420nm, ex 490nm, em) and a 10/5/85-> 63/1/36 methanoj/THF/water (5mM sodium citrate) gradient were used. Additionally, the amino acids were monitored electro-chemically (+750 mV vs. Ag/AgCl). Peak shape was good, but complete separation of all residues was not achieved. 

Chemical derivatization can be carried out in two ways. The sample can be derivatized and then injected onto the column which has been termed precolumn derivatization. Conversely, the column eluent can be mixed with the derivatizing reagent prior to the detector, the reaction allowed to complete by the use of a suitable reactor and the products then passed through the... 

Capillary GC-MS is an extremely powerful approach, combining the high separation efficiency of the capillary GC column with the identification power of the MS in electron-ionization mode. However the applicability range of GC is limited to relatively volatile compounds. In order to widen the applicability range, precolumn analyte derivatization strategies are often applied to enhance the volatility of the analytes. Methylation, silylation and acetylation reactions are most often applied for the analysis of compounds with amine, (poly-) hydroxy and/or carboxylic acid functional groups. Furthermore, derivatization to pentafluorobenzyl derivatives is applied to enhance the sensitivity of analytes in electron-capture negative-ion chemical ionization. 

A typical chromatogram of OPA amino acid derivatives at the nanogram level is shown in Fig. 29. Dansylation reactions to form fluorescent derivatives of amines or carbonyl compounds have also been applied to HPLC. Com-plexation of transition metals with pyridylazoresorcinol (PAR) either in the precolumn or postcolumn mode has been a popular method. Precolurrm derivatization has been shown by Ian Blair and co-workers to be useful even for LC/MS. Atmospheric pressure chemical ionization commonly used in LC/MS can provide a source of electrons... 

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