Derivatization with fluorescamine

The HPLC methods mainly use UV detectors, but one uses ampero-metric (18) and one uses fluorescent detection (25), Fluorescent detection after derivatization with fluorescamine is the method most commonly used for detection on TLC plates, Vilim (24) used TLC to... 

Simeonidou et al. (238) reported an ion-pair liquid chromatographic method for the determination of sulfadiazine, sulfamethazine, sulfadimethoxine, and sulfaquinoxaline residues in chicken muscle. According to this method, a 3 g ground tissue sample is extracted with 30 ml chloroform. Following centrifugation the supernatant is filtered and a 10 ml aliquot is extracted with 1 ml 3N hydrochloric acid and submitted to precolumn derivatization with fluorescamine. Liquid chro-... 

Aerosol Camphor HPLC Derivatized with fluorescamine G2000SWXL Supelco O.IMPO, buffer, pH 7.5-CHjCN-MeOH (6 3 1) EX 370 nm EM 420 nm Autosampler [320]... 

Newcomb, R. (1992). High-sensitivity detection of peptides by liquid chromatography using postcolumn derivatization with fluorescamine. LC-GC 10(1), 34-39. 

Aromatic nitro compounds can be determined after catalytic hydrogenation to the corresponding PAA, derivatizing with fluorescamine (93) according to equation 7 (Section III.B.3) and measuring the luminescence in the 490 to 520 nm region, obtained on excitation at 390 to 410 nm237,238. 

Fluorimetry Amino groups in an enzyme derivatize with fluorescamine or o-phthalaldehyde, which is not fluorescent, and the resulting derivative is measured fluorimetrically 0.005-10(ig ml" ... 

Some fluorimetric methods for the individual determination of amino acids in foods have also been reported. Thus, the native fluorescence of tryptophan has been used for its determination in food and feed hydrolysates using ion-exchange chromatography. Also, 3-methylhistidine has been determined in meat and meat products using LC and precolumn derivatization with fluorescamine or postcolumn derivatization with OPA and 2-ME. 

Residual monomers of epoxy resins (i.e., m-xylylenediamine and bisphenol A diglycidyl ether) were extracted from cured epoxy resins and analyzed on a C g column (A = 275 nm, ex 300 nm, em for m-xylylenediamine) using a complex 20-min 30/70 -> 75/25 acetonitrile/water gradient [1019]. Excellent resolution of bisphenol F, bisphenol A, three bisphenol F diglycidyl ether isomers, and bisphenol A diglycidyl ether resulted. Peak sh s were also excellent. Linear concentration curves from 20 to 1000 pg/L were obtained. The m-xylylenediamine was derivatized with fluorescamine and analyzed with the same column and gradient as for bisphenol (but with different excitation/emission wavelengths) and linear curves from 10-800 pg/L were obtained. 

Amino-3 -deoxythymidine, a catabolite of AZT, was determined in plasma by derivatization with fluorescamine and reversed-phase h.p.lx. with fluorescence detection. ... 

For many years, automated amino acid analyzer using lEC and postcolumn derivatization with nin-hydrin (or less frequently with fluorescamine) has been the most popular technique for amino acid determination. Amino acids are separated in their free form by employing stepwise elution with sodium- or lithium-based buffers. 

Whenever only primary amines need to be derivatized, fluorescamine often constitutes the reagent of choice. Fluorescamine, although nonfluorescent itself, can react with primary amines forming highly fluorescent pyrrolinones (139-144). Aliphatic primary amines favor derivatization reaction at pH 8-9, whereas primary aromatic amines exhibit optimal reactivity at pH 3-4. Secondary amines are also fully reactive with fluorescamine but their products do not fluoresce. However, secondary amines can be detected with fluorescamine if they are converted to primary amines by oxidation with N-chlorosuccinimide prior to their fluorescamine derivatization (145, 146). Alcohols can also interact with fluorescamine but this reaction is reversible as a result, alcohols just slow down the reaction rate of fluorescamine with primary amines. On the other hand, tertiary amines and guanidines are not reactive at all with fluorescamine. 

Detection of amino acids is typically by UV absorption after postcolumn reaction with nin-hydrin. Precolumn derivatization with ninhydrin is not possible, because the amino acids do not actually form an adduct with the ninhydrin. Rather, the reaction of all primary amino acids results in the formation of a chromophoric compound named Ruhemann s purple. This chro-mophore has an absorption maximum at 570 nm. The secondary amino acid, proline, is not able to react in the same fashion and results in an intermediate reaction product with an absorption maximum at 440 nm. See Fig. 5. Detection limits afforded by postcolumn reaction with ninhydrin are typically in the range of over 100 picomoles injected. Lower detection limits can be realized with postcolumn reaction with fluorescamine (115) or o-phthalaldehyde (OPA) (116). Detection limits down to 5 picomoles are possible. However, the detection limits afforded by ninhydrin are sufficient for the overwhelming majority of applications in food analysis. 

Since then, there have been a number of reversed-phase separations employing precolumn derivatization. Interestingly, fluorescamine (not frequently employed for RP-HPLC of amino acids with precolumn reaction) has been reported for taurine analysis in milk (197) and human plasma (198). Precolumn derivatization with OPA/2-mercaptoethanol has been reported for the analysis of infant formula and human breast milk (199). Although not the principal focus of the study, Carratu et al. (200) report taurine values in parenteral solutions as determined by FMOC. In an excellent article, Woollard and Indyk (201) report the dansylation of taurine for its determination in a wide variety of dairy-related products. Subsequently, the same authors report the results of a large collaborative study (202) for the determination of taurine (again, by dansylation) in milk and infant formula. This study afforded an overall interlaboratory RSD of 7.0% and established a lower limit for determination at 5 mg taurine per 100 g of product. 

The derivatization reactions were chosen to improve the UV detection of SAs (reactions with DMBA, p-aminobenzoic acid) or to yield derivatives with high fluorescence intensity (reactions with fluorescamine, o-phthaldehyde). 

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). 

Current methods of detection for CAs in biological fluids (urine, plasma, and serum) involve chromatographic separation coupled to either electrochemical 119,120) or optical 121) techniques. However, most optical methods rely purely on the native fluorescence of CAs (lex 280 nm, lem 310 nm) 122), which have small Stokes shifts and suffer from signal losses due to reabsorption others involve pre- or postcoliunn derivatization with various fluorophores, such as naphthalene-2,3-dicarboxaldehyde 123), 1,2-diphenylethylenediamine 124,125), or fluorescamine 126). These methods all require significant time for separation using expensive instrumentation and thus are not feasible for rapid CA detection. 

Aromatic amines undergo condensation with fluorescamine (93), presumably according to equation 7, to yield fluorescent products (94). The process was applied to derivatize PAA mixtures prior to determination by MEKC with FLD (Section IV.C)189,190. This derivatizing procedure was used for detection of unreacted isocyanate residues in the... 

ISO-DALT uses protein-specific detection methods such as autoradiography and dye-binding, e.g., with Coomassie Brilliant Blue. Iso-tachophoresis and HPLC both use UV-absorption for the detection of proteins, the equipment for the former being usually at a fixed wavelength and for the latter a variable wavelength. These detection methods are sensitive but not specific. With HPLC, there is the possibility of pre-or post-column derivatization, e.g., with fluorescamine, which may improve both sensitivity and specificity. 

R Zhu, WT Kok. Postcolumn derivatization of peptides with fluorescamine in capillary electrophoresis. J Chromatogr A 814 213-221, 1998. 

Takeda, N. Akiyama, Y. Pre-column derivatization of sulfa drugs with fluorescamine and high-performance liquid chromatographic determination at their residual levels in meat and meat products. J.Chromatogr., 1991, 558, 175-180... 

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