Precolumn derivatives

In recent years, the wide diffusion of precolumn derivation agents able to increase analyte hydro-phobicity and hence its retention on an apolar phase allowed a gradual replacement of dedicated amino acid analyzer with more versatile and less expensive RP-HPLC systems. 

KL Woo, QC Hwang, HS Kim. Determination of amino acids in the foods by reversed-phase high-performance liquid chromatography with a new precolumn derivative, butylthiocarbamyl amino acid, compared to the conventional phenylthiocarbamyl derivatives and ion-exchange chromatography. J Chromatogr A 740 31-40, 1996. 

M. Aycard, S. Letellier, B. Maupas, and F. Guyon, Determination of (R) and (S) warfarin in plasma by HPLC using precolumn derivation, J. Liquid Chromatogr., 75 2175 (1992). 

Precolumn derivatives are more tolerant to lipid-rich samples. Changing the guard-column routinely is recommended to avoid column buildup, especially for reversed-phase systems. 

In addition to more or less complete amino acid separations, suitable chromatographic systems for the rapid determination of selected amino acids [4-amino-butyric acid (GABA) [336], histidine and 3-methylhisti-dine [367] or a group of selected amino acids, such as those involved in the urea cycle (arginine, citrulline, ornithine, agmatine) [368], have been reported. Precolumn derivative formation with OPA/R-SH reagents is also suited to the automated establishment of peptide maps [369]. A number of authors have published methods for the sensitive determination of aminoglycoside antibiotics [365,370—373]. The method has also found application for the determination of histamine and its methylation products [374—376], catecholamines and serotonin [377—379] and polyamines [380]. 

Unlike ninhydrin, the reaction of amino acids with OPA does not destroy the amino acids, but adds the fluorescent tag to the primary amine. This allows OPA to also be used as a precolumn derivative, since the derivatization product is unique for each amino acid. 

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). 

Cooper, J. D. H., Ogden, G., McIntosh, J., and Tumell, D. C., The stability of the o-phthaldehyde/2-mercaptoethanol derivatives of amino acids an investigation using high-pressure liquid chromatography with a precolumn derivatization technique, Anal. Biochem., 142, 98, 1984. 

Amines are another important group of analytes. Mellbin and Smith [72] compared three different fluorescent reagents, dansyl chloride, 4-chloro-7-nitrobenzo-1,2,5-oxadiazole, and o-phthaldialdehyde, for derivatization of alkylamines. The dansyl tag was found to be the most effective. Hamachi et al. [73] described the application of an HPLC-POCL method for determination of a fluorescent derivative of the synthetic peptide ebiratide. Another comparative study was done by Kwakman et al. [74], where naphthalene-2,3-dialdehyde and anthracene-2,3-dial-dehyde were evaluated as precolumn labeling agents for primary amines. The anthracene-2,3-dialdehyde derivatives were not stable, especially in the presence of hydrogen peroxide, and the POCL detection of these derivatives was therefore... 

There is no necessity to remove excess OPA prior to sample injection since OPA itself will not interfere with separation or detection. However, since OPA-amino acid derivatives are unstable, complete automation of the precolumn reaction with accurate control of reaction time is essential for reproducible results. 

Petrovic and coworkers [6] used PLE applying MeOH acetone (1 1) for the extraction of APEOs, APECs, APs and their halogenated derivatives from sediment samples. In combination with column-switching LC, using a restricted access material precolumn, hours of sample treatment were saved. 

Several other methods have been published using RP-HPLC for the determination amphetamines and related derivatives. Studies have shown the determination of amphetamine and related derivatives in plasma, urine, and hair by RP-HPLC with precolumn derivatization and either UV/VlS or fluorescence detection. Various methods are employed by SPE technologies using Cl8 cartridges for sample cleanup prior to derivatization. The derivatized compounds were separated on analytical columns of various Cl 8 bonded phase materials. The methods generally used water/acetonitrile mobile phases operated in gradient mode. All studies reported extraction recoveries of 85-102% for all the analytes, with LLOQs ranging from 5 to 60 ng/ml (Tedeschi et al., 1993 Ealco et al., 1996 Hernandez et al., 1997 Al-Dirbashi et al., 1997 Al-Dirbashi et al., 2000 Soares et al., 2001). 

V- Much effort has been expended in the development of more sensitive methods for the analysis and detection of catecholamines. They have been analyzed as the dansyl derivatives (376) or after precolumn derivati-zation with o-phthalaldehyde (377, 378). Postcolumn derivatization followed by fluorometric analysis have been described in which the fluoro-phore was formed with o-phthalaldehyde (379) or with 9,10-dimethoxyanthracene-2-sulfonate as the ion-pair (380). Several laboratories have shown the sensitivity and specificity in electrochemical detection methods (381 -383). 

Biotin does not exhibit UV absorbance. It neither shows fluorescence nor electrochemical activity. Therefore, it needs to be derivatized. 4-Bromomethylmethoxiycoumarin (BMMC) [590], 9-anthryldiazomethane (ADAM) [591], and 1-pyrenyldiazomethane (PDAM) [592] have been used as precolumn reagents to convert biotin to fluorescent absorbing derivatives. Instead, to obtain derivatives that are UV detectable, hydrazines are used, such as 2-nitrophenylhydrazine hydrochloride... 

When the amount of thiamine is relatively high, U V detection at 254nm can be applied. Fluorometric detection, however, enables to reach higher sensitivity, when coupled with precolumn or postcolumn derivatization. The derivatization is carried out by using potassium ferricyanide under alkaline conditions to convert thiamine to highly fluorescent thiochrome derivatives. 

The separation mechanism in the LC analysis of aminoglycosides is usually highly dependent on the applied derivatization technique, either precolumn or postcolumn. This is due to the fact that a prerequisite of aminoglycosides analysis is most often suitable derivatization to produce fluorescent derivatives the presence of primary amine groups in most of the aminoglycoside antibiotics enables a number of derivatives to be readily formed. 

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