Photochemical Derivatization

An elegant approach to increase the selectivity and specificity of the analyte after its elution from a chromatographic column is to apply an online photochemical reaction for which suitable reactors are now commercially available. Photochemical reactions can be applied for various purposes, as, for example, to increase the detectability by converting weakly or nonfluorescing analytes into highly fluorescent products or to create electroactive products that can be detected with 

When using photochemical reactions, a gain in fluorescence output or electroactivity not only lowers detection limits but also contributes to confirm an analytical result. Examples are the conversion of diethylstilbestrol to a fluorescent hexahydrophenanthrene (277), and the conversion of fenbendazole to fluorescent species (278). Tlie fluorescence of photoconverted diethylstilbestrol can further be enhanced by a subsequent online postchromatographic derivatization with bisulfite to the highly fluorescent phenanthrenediol (279). Another example of photolytic derivatization is the postchromatographic conversion of penicillins and cephalosporins into electroactive species that can be detected by an amperometric detector (280). 

This type of derivatization seems promising for the detection of drugs and metabolites with phenolic groups. A recent application on the determination of chlorophenols in surface water showed that the dansyl derivatives of phenols are readily convertible to the highly fluorescent dansyl-OH and dansyl-OCHs products after postchromatographic irradiation (281). Fluorescence gain factors of up to 8000-fold were obtained for chlorophenol derivatives with a low native fluorescence. 

in Residues of Veterinary Drugs in Food, Proc. Euroresidue Conf., Noordwijkerhout, May 21-23, 1990 (N. Haagsma, A. Ruiter, and P.B. Czedik-Eysenberg, Eds.), Fac. Vet. Med., Univ. Utrecht, The Netherlands, p. 394 (1990). 

Malisch, U. Sandmeyer, and K. Kypke-Hutter, Lebensmittelchem. Gerichl. Chem., 38 11 (1984). 

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Post-column in-line photochemical derivatization permits fluorescence detection of the common aflatoxins Bl, B2, Gl, and G2 (60). Chromatographic evidence indicates that photolysis causes the hydration of the nonfluorescent Bl and Gl components to B2a and G2a components, respectively. Analysis of naturally contaminated com samples show no interfering peaks and permits the deterrnination of 1 and 0.25 ppb for Bl and B2, respectively. 

Fernandez Garcia-Borregon, R, Lores, M., and Cela, R., Analysis of barbiturates by micro-high-performance liquid chromatography with post-column photochemical derivatization, /. Chromatogr. A, 870 (1 2), 39, 2000. 

Detection can be performed using UV, UV-Vis, DAD, fluorometric detector (ELD) or ED. Gatti et al. [444] report an on-line photochemical derivatization to enhance the selectivity. A photo reactor system was inserted on-line between the column and the ELD. The eluate was irradiated at 254 and 366 nm inducing alterations that modified spectral properties of the analytes. Recently an increasing number of studies have reported the use of LC-MS for the identification of both retinoids and carotenoids [426,433,445-447],... 

Starting with a description of the analytical challenge in Chapter 19, the third part, which is devoted to analytical attitudes, proceeds with a detailed description in Chapter 20 of modern sample preparation procedures including solid-phase extraction, matrix solid-phase dispersion, use of restricted-access media, supercritical fluid extraction, and immunoaffinity cleanup. Flexible derivatization techniques including fluorescence, ultraviolet-visible, enzymatic, and photochemical derivatization procedures are presented in Chapter 21. 

Di Pietra, A.M. Gatti, R. Andrisano, V. Cavrini, V. Application of high-performance liquid chromatography with diode-array detection eind on-hne post-column photochemical derivatization to the determination of analgesics. J.ChromatognA, 1996, 729, 355-361... 

Post-chromatographic photochemical derivatization Solid-phase reactors Electrochemical methods... 

Such reactions are of practical use only if the products are more readily detectable than the starting materials, although in some cases detectability has been achieved by demonstrating the disappearance of peaks on irradiation. In general, it is essential first to establish that a photochemical reaction does occur, and to work out the optimal conditions, such as wavelength of irradiation and mode of detection. One can then go on to employ a photochemical derivatization system for a post-chromatographic application when the process works, photochemical detection is capable of dramatic successes. 

Figure 6. Simple arrangement for photochemical derivatization. Reprinted with permission from X.-D. Ding and I. S. Krull, ]. Agric. Food Chem., 32, 622 (1984). Copyright (1984) American Chemical Society.

Table 5 Factors contributing to successful photochemical derivatization...

The insertion of a photochemical derivatization system between the chromatographic column and the detector carries the risk of band broadening. To minimize the danger of this various different designs have been tried out [54,55], and the latest, a commercially available photochemical derivatization reaction module, consists of Teflon tubing formed into a KOT reactor wrapped round the UV lamp (Figure 7). 

The improved commercial availability of solid-phase reaction systems, immobilized enzyme reactors and photochemical derivatization instruments is one such direction for a brief outline of some of these see [34]. There will undoubtedly be new developments in the application of analytical methods, particularly in liquid chromatography, that will exploit the practical advantages of these devices when they are linked to the most sensitive detection modes, particularly the fluorescence and electrochemical types of detector. 

Fluorescence spectroscopy plays an important function in modern food analysis as can be seen from its wide use in the determination of numerous food components, contaminants, additives, and adulterants. This technique has made available very sensitive and selective methods that satisfy the requirements of food analysis, which are usually very complex, taking into account the large number of species to be determined, frequently at very low concentrations, and the wide variety of foodstuffs available. Initially, the use of fluorescence spectroscopy in food analysis was limited to the determination of species with intrinsic fluorescence (e.g., vitamins, aflatoxins, and some polycyclic aromatic hydrocarbons (PAHs)), but now it is widely applied to nonfluorescent species, using several physicochemical means such as chemical or photochemical derivatization reactions. Numerous techniques involve fluorescence detection in liquid chromatography (LC), frequently using pre- or postcolumn derivatization. In addition to conventional fluorime-try, which is commonly chosen for this purpose, other fluorimetric techniques such as laser-induced... 

Woolf, E. Fu, I. Matuszewski, B. Determination of rofe-coxib, a cyclooxygenase-2 specific inhibitor, in human plasma using high-performance liquid chromatography with post-column photochemical derivatization and fluorescence detection. J. Chromatogr. B, Biomed. Appl. 1999, 730 (2), 221-227. 

Waltking, A. and Wilson, D. 2006. Liquid chromatographic analysis of aflatoxin using postcolumn photochemical derivatization Collaborative study. I. AOAC Int. 89 678-692. 

I. S. Krull and W. R. LaCourse, PostCoJ-umn Photochemical Derivatizations, in PCR Detectorsfor HPLC, vol. 37, Marcel Dekker New York, 1986. 

Garcia Sanchez, F., Navas Diaz, A., and Fernandez Correa, M. R. (1993). Image analysis of photochemically derivatized and charge-couple-device-detected phenothiazines separated by thin layer chromatography. J. Chromatogr. 655 31-38. 

Niederberger, E. Tegeder, I. Schafer, C. Seegel, M. Grbsch, S. Geisslinger, G. Opposite effects of rofe-coxib on nuclear factor-kappaB and activating protein-1 activation, J.Pharmacol.Exp.Ther., 2003,304, 1153-1160. [etoricoxib is internal standard post-column photochemical derivatization]... 

Andrisano, V. Bertucci, C. Battaglia, A. Cavrini, V. Photostability of drugs photodegradation of melatonin and its determination in commercial formulations, J.Pharm.Biomed.Anal., 2000, 23, 15-23. [post-column photochemical derivatization]... 

Halpin, R.A. Geer, L.A. Zhang, K.E. Marks, T.M. Dean, D.C. Jones, A.N. Melillo, D. Doss, G. Vyas, K.P. The absorption, distribution, metabolism and excretion of rofecoxib, a potent and selective cyclooxygenase-2 inhibitor, in rats and dogs, Drii Metafe.Dispos., 2000,1244-1254. [LOQ 1 ng/mL plasma post-column photochemical derivatization fluorescence detection]... 

Hsieh, J.Y.-K. Lin, L. Matuszewski, B.K. High-throughput liquid chromatographic determination of rofecoxib in human plasma using a fully automated on-line solid-phase extraction system, J.Liq.Chromatogr.Rel.TechnoL, 2001, 24, 799-812. [LOQ 0.5 ng/mL plasma post-column photochemical derivatization fluorescence detection SPE]... 

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