Fluorescence derivatization

Although various fluorescence-labeling derivatization reagents have been used in drug residues analysis, those replacing active hydrogens of hydroxyl, sulfydryl, or amino groups account for most applications. 

One of the most widely used reagents for introduction of a fluorophor into primary and secondary amines is 5-dimethylaminonaphthalene-l-sulfonyl chloride, com- 

Animal tissues Bovine hair and urine Bovine liver 

Drug Matrix Derivatizing agent Detection mode Ref. 

Ivermectin Animal tissues Methylimidazole, acetic LC-Fluorometric 47-49 

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Fluorescent derivatizing reagents with benzofuran skeleton for HPLC with spec-trometric detection 98YZ483. 

Reports on the use of fluorescent derivatives abound (5). Some reagents have become widely used. The dansyl group is probably the most thoroughly studied. Dansyl chloride has been widely used as a fluorescent derivatizing reagent for HPLC (6,7). It reacts readily with primary and secondary amino groups (7) and with phenols (8), but forms derivatives of alcohols very slowly (9). The lower detection limit for dansyl derivatives of aliphatic amines is in the range of 300 femtomoles per injection. 

Pyridone Acid Chlorides as Fluorescent Derivatizing Reagents. A second derivatizing reagent, a fluorescent acid chloride, was synthesized from the sodium salt of 3-phenyl-2(lH)pyridone (IV). 

The examples that are treated below are those sequences where all steps - except the last — are preparations for a color or fluorescence derivatization reaction which is carried out in the last step, i. e. they can be regarded as a sort of selective in situ pretreatment for a final detection reaction. Such reaction sequences are frequently necessary because all the reagents cannot be mixed together in a single solvent, or because it is necessary to dry, heat or irradiate with UV light between the individual reaction steps. The detection of aromatics by the reaction sequence nitration — reduction — diazotization - couple to form an azo dye is an example of this type (Fig 21). 

The course of the reaction has not been fully claritied. Hydrolytic and aromatizatioiij processes are probably responsible for the formation of colored or fluorescent derivat tives (cf. Potassium Hydroxide Reagent). For instance, sevin is converted to a-naphth(d [11] and paraoxon to the yellow 4-nitrophenolate anion [13]. In the case of a-pyrone j derivatives it is assumed that the alkali metal salt of the o-hydroxycinnamic acid pro- duced by hydrolytic cleavage of the pyrone ring is converted from the non-fluorescent ] CIS- to the fluorescent trans-form by the action of long-wavelength UV light (X = 365 nm) [2]. ... 

The application of the fluorescence derivatization technique in an HPLC method involves utilization of a post column reaction system (PCRS) as shown in Figure 3 to carry out the wet chemistry involved. The reaction is a 2-step process with oxidation of the toxins by periodate at pH 7.8 followed by acidification with nitric acid. Among the factors that influence toxin detection in the PCRS are periodate concentration, oxidation pH, oxidation temperature, reaction time, and final pH. By far, the most important of these factors is oxidation pH and, unfortunately, there is not one set of reaction conditions that is optimum for all of the PSP toxins. The reaction conditions outlined in Table I, while not optimized for any particular toxin, were developed to allow for adequate detection of all of the toxins involved. Care must be exercised in setting up an HPLC for the PSP toxins to duplicate the conditions as closely as possible to those specified in order to achieve consistent adequate detection limits. 

Coquet, A., Veuthey, J.-L., and Haerdi, W., Comparison of post-column fluorescence derivatization and evaporative light-scattering detection to analyse saccharides selectively by LC, Chromatographia, 34, 651, 1992. 

Mitsui, A., Nohta, H., and Ohkura, Y., High-performance liquid chromatography of plasma catecholamines using 1,2-diphenylethylenediamine as precolumn fluorescence derivatization reagent, /. Chromatogr., 344, 61, 1985. 

Anions of weak acids can be problematic for detection in suppressed IEC because weak ionization results in low conductivity and poor sensitivity. Converting such acids back to the sodium salt form may overcome this limitation. Caliamanis et al. have described the use of a second micromembrane suppressor to do this, and have applied the approach to the boric acid/sodium borate system, using sodium salt solutions of EDTA.88 Varying the pH and EDTA concentration allowed optimal detection. Another approach for analysis of weak acids is indirect suppressed conductivity IEC, which chemically separates high- and low-conductance analytes. This technique has potential for detection of weak mono- and dianions as well as amino acids.89 As an alternative to conductivity detection, ultraviolet and fluorescence derivatization reagents have been explored 90 this approach offers a means of enhancing sensitivity (typically into the low femtomoles range) as well as selectivity. 

Mukherjee, P. S. and Karnes, H. T., Ultraviolet and fluorescence derivatization reagents for carboxylic acids suitable for high performance liquid chromatography A review, Biomed. Chromatogr., 10, 193, 1996. 

Fluorescence. - A further report on the use of naphthalene phosphorus compounds for fluorescence derivatization concerns the dependence of fluorescence on the type and position of substituents on the naphthalene rings.141 It has been found that the... 

Guo, X., Fukushima, T., Li, F., Imai, K. (2002). Determination of fluoxetine enantiomers in rat plasma by precolumn fluorescence derivatization and column-switching high-performance liquid chromatography. Analyst 127, 480-484. 

Prados P, Fukushima T, Santa T et al (1997) 4-/V,/V-Dimethylam inosu 1 lbnyl-7 -N-(2-aminoethyl)amino-benzofurazan as a new precolumn fluorescence derivatization reagent for carboxylic acids (fatty acids and drugs containing a carboxyl moiety) in liquid chromatography. Anal Chim Acta 344 227-232... 

Table 2 Application of Fluorescence Derivatization Reagents to F1PLC with PO-CL Detection ... 

Figure 14 Representative fluorescence derivatization reagents used for PO-CL detection. DNS-CI, dansyl chloride DBD-F, 4-(A,A-dimethylaminosulphonyl-7-fluoro-2,l, 3-benzoxadiazole NDA, naphthalene-2,3-dicarboxaldehyde DNS-H, dansyl hydrazine DBD-H, 4-(iV,iV-dimethylaminosulphonyl-7-hydrazino-2,l,3-benzoxadiazole).

Lindroth, P., and K. Mopper. 1979. High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolumn fluorescence derivatization with o-phthaldialdehyde. Analytical Chemistry 51 1667-1674. 

Determination of serum cortisone by reversed-phase liquid chromatography using pre-column sulfuric acid-ethanol fluorescence derivatization and column switching. (159)... 

Similar dicholoro precursor 205 was utilized by Katoh and co-workers in the discovery of a new fluorescence derivatizing agent for fatty acids <99H299>. The conversion of 205 to fatty acid-coupled 206 proceeded through the displacement of both chloro groups with morpholine in several high-yielding steps. 

Other reagents used for fluorescence derivatization are the so-called Edman-type isothiocyanate compounds, namely fluoresceinisothiocyanate (163), 9-isothi-ocyanatoacridine (164), 4-dimethylamino-l-naphthylisothiocyanate (165), or... 

SA Cohen, DP Michaud. Synthesis of a fluorescent derivatizing reagent, 6-aminoquinolyl-A -hydroxysuccinimidyl carbamate, and its application for the analysis of hydrolysate amino acids via high-performance liquid chromatography. Anal Biochem 211 279-287, 1993. 

P Kucera, H Umagat. Design of a post-column fluorescence derivatization system for use with microbore columns. J Chromatogr 255 563-579, 1983. 

P Vinas, CL Erroz, N Campillo, M Hernandez Cordoba. Detennination of sulfonamides in foods by liquid chromatography with post-column fluorescence derivatization. J Chromatogr A 726 125 -131, 1996. 

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