High-pressure liquid chromatography Derivatization

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. 

Off line derivatization has also been utilized for the derivatization of pyrolysate to be analyzed by high-pressure liquid chromatography (HPLC) techniques [6,10, 28]. 

As mentioned above in the context of the analysis of hgnin degradation products, gas chro-matography/mass spectrometry and related methods have been developed as extremely powerful tools for the identification of phenolic compounds. Use of high-pressure liquid chromatography in combination with mass spectrometry adds to the analytical arsenal with respect to the detection of polar, non-volatile compounds but, in particular, the advent of modem ionization techniques, such as ESI and MALDI mass spectrometry, have continued to broaden the analytically governable field of organic chemistry. The latter methods diminish the need of derivatization of polar phenolics to increase the volatility of the analyte. In this section, a more or less arbitrary selection of examples for the application of mass spectrometric techniques in analytical chemistry is added to the cases already discussed above in the context of gas-phase ion chemistry. 

Bawdon, R.E. Leveno, K.L. Cunningham, F.G. Nobles, B. Nelson, S. Intrapartum pharmacokinetics of ticarcillin and clavulanic acid in serum as determined by high-pressure liquid chromatography. Adv.Therapy, 1984, 1, 419-426 [derivatization SPE serum pharmacokinetics]... 

Because the presence of anatoxin-a and homoanatoxin-a in the environment represents a risk for animals and humans, several analytical methods have been designed to detect these toxins as well as their natural derivatives (Fig. 3.2). GC-MS was first used to detect and quantify anatoxin-a or its A -acetyl derivative [51,52,66]. High Pressure Liquid chromatography (HPLQ coupled to UV detection was also used, although this detectirm method is not veiy sensitive [67]. Thus, to improve the sensitivity, several authors have used pre-derivatization with a fluorophore, which reacts on the amine of anatoxin-a or of homoanatoxin-a, followed by separation by HPLC coupled to a fluorescence detector [56, 68, 69]. However, the derivatization might lead to false positives even if the technique was improved to remove primary amines present in the sample [70] or by crmcentratirm by extraction of anatoxin-a prior to analysis [71,72]. It is now accepted that the best analytical technique relies on the use of HPLC coupled to tandem mass spectrometry (LC-MS, or even LC-MS") without derivatization to avoid... 

Some compounds can be easily detected by both CGC and high-pressure liquid chromatography (HPLC), for instance, cocaine, which has characteristic electron-impact mass and UV fluorescence spectra (Fernandez et al., 2009). Inert nonpolar or low-polar silicone phases have chromatographic features suitable for separation and analysis by CGC. In addition, dedicated base-treated stationary phases are now used to improve the identification and quantification of compoimds. HPLC analyses are done by reversed-phase elution with buffered water/organic solvent mixtures. In contrast, numerous native drags and almost all metabolites require chemical derivatization before analysis [e.g., with trifluoroacetic anhydride, A -methyl-7V-trimethylsilyltrifluoroacetamide, or iV-(r-butyldimethylsilyl)-Af-methyltrifluoroacetamide, which is usually used for cannabinoids] this improves the analytical performance of the method and drastically reduces the risk of misinterpretation and misidentification of the compoimds. 

Other methods employed. The activity of OAD in patient leukocytes was determined by a method described by Ohnuma and Holland (17). Quantification of orotic acid and orotidine was made by column chromatography (8). Urinary uric acid was measured by the technique of Pileggi al. (18). Orotic acid, orotidine, and urinary uric acid were also separated by high pressure liquid chromatography (19) using a u-C 3 column and O.IM formic acid at a flow rate of 0.5 ml/min. Constituents were collected, evaporated to dryness with dry nitrogen, derivatized into the trimethylsilyl derivative as described above and positively identified by mass spectrometry. 

Homish, R. E. and Wiest, J. R., Quantitation of spectinomycin residues in bovine tissues by ion-exchange high-performance liquid chromatography with post-column derivatization and confirmation by reversed-phase high performance chromatography-atmospheric pressure chemical ionization tandem mass spectrometry, /. Chromatogr. A, 812, 123, 1998. 

Desai, M.J. and Armstrong, D.W., Analysis of derivatized and underivatized thean-ine enantiomers by high-performance liquid chromatography/atmospheric pressure ionization-mass spectrometry. Rapid Commun. Mass Spectrom., 18, 251,2004. 

Peters R, Hellenbrand J, Mengerink Y, Wal Van der S. 2004. On-line determination of carboxylic acids, aldehydes and ketones by high-performance liquid chromatography-diode array detection-atmospheric pressure chemical ionization mass spectrometry after derivatization with 2-nitrophenylhydrazine. J Chromatogr A 1031 35. 

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