Phthaldialdehyde

The number of reagents for derivatization of amino compounds is larger than for any other class of compounds. Two of the most useful reagents for precolumn fluorescence labeling m liquid chromatography are dansyl chloride and o-phthaldialdehyde (OPA). 

Dansyl chloride was, for a long time, the most widely used fluorogenic derivatization reagent for amino acid determination. However, there are certain problems connected with the dansyl reaction at low ammo acid concentrahons (Neadle and Pollitt, 1965). Since dansyl chloride and its byproducts are fluorescent, serious interference can occur at high sensitivity levels 

The use of OPA for the determination of primary ammo acids was originally reported by Roth in 1971 In the presence of a reducing agent, such as 2-mercaptoethanol, OPA reacts in alkaline medium with primary amines, to form highly fluorescent thioalkyl-substituted isoindoles (Simons and Johnson, 1976, Fig. 1) OPA as a derivatization reagent has several advantages  

The OPA reaction, however, may be used to detect the secondary amines hydroxyprolme and proline if they are oxidized 

The poor fluorescence of the cysteine, lysine, and ornithine derivatives may be a drawback of the technique. Cysteine yields weakly fluorescent properties due to its sulfydryl group (Cooper and Turnell, 1982). However, these sulfydryl groups can be blocked with iodoacetic acid, lodoacetamine, or acrylonitrile, with the result that fluorescent isoindoles can then be formed with OPA (Cooper and Turnell, 1982). Cysteine may also be oxidized to cysteic acid, which forms a highly fluorescent product with OPA. Following the oxidation of cysteine, however, it may be difficult to obtain high reproducibility with the OPA derivatization of amino acids, because the conditions for these two reac- 

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FLUORIMETRIC DETERMINATION OE HISTAMINE WITH o-PHTHALDIALDEHYDE IN THE PRESENCE OE REDUCING AGENTS... 

Several methods were reported for the analysis of histamine, but the fluorimetric determination with o-phthaldialdehyde (OPA) the most widely used. It was shown that adducts, formed in the reaction of histamine with OPA in the presence of reducing agent, is more stable and gives high relative fluorescence intensity. The influences of different tiols on the fluorimeric determination histamine with OPA have been investigated. 

Dipping solution Make 0.1 g o-phthalaldehyde (phthaldialdehyde, OPA) and 0.1 ml 2-mercaptoethanol (2-hydroxy-l-ethanethiol) up to 100 ml with acetone. 

Adapted from Jones, B. V., Pddbo,. S ., and. Stein,. S ., 1981. Amino aeid analysis and enzymie seqnenee determination of peptides by an improved, o-phthaldialdehyde preeolnmn labeling pro-Journal of Liquid. Chromatography 4 56—586.)... 

Separation of Amino Acid Enantiomers after Derivatization with Or/ho-Phthaldialdehyde (OPA) and a Unichiral Tliiol Compound... 

Due to the high a-C,H acidity in the alkoxyethylidene complexes 6 (e.g.,piCa=8 (R=Me)) [ 16], transformations via an enolate analog are possible and have been used to introduce additional functionality into the carbene side chain to access various Fischer carbene complexes [3]. The a,/J-unsaturated complex 8 could be obtained from 6 (R=Et) by an aldol-type condensation with benzaldehyde 7 in the presence of triethylamine and trimethylsilyl chloride (Scheme 2) [17]. This reaction proceeds completely diastereoselectively to yield only the trans-isomer. Analogously, binuclear complexes have been prepared from 6 and 1,3-and 1,4-phthaldialdehyde in good yields [17]. This type of condensation has... 

The intramolecular approach of Staab and Graf, shown in Scheme 4, precluded formation of 5, but was considerably more involved [12]. The cyclic dienyne 6 was afforded by Wittig reaction of o-phthaldialdehyde with the corresponding bis(ylide) derived from tolane. Bromination of 6 and subsequent treat-... 

Bartok, T., Szalai, G., Lorincz, Zs., Borcsok, G., and Sagi, F., High-speed RP-HPLC/FL analysis of amino acids after automated two-step derivatization with o-phthaldialdehyde/3-mercaptoproprionic acid and 9-fluorenylmethyl chloroformate, /. Liq. Chromatogr., 17, 4391, 1994. 

A drawback to conventional amino analysis by chromatography is the need for pre- or post-column derivatization to improve sensitivity. Ninhy-drin, the reagent originally applied for detection, has been increasingly displaced by other reagents such as phenylisothiocyanate,71 9-fluorenylethyl chloroformate,72 and o-phthaldialdehyde (OPA). OPA allows fluorimetric detection, which offers the potential for greater sensitivity.73 A limitation of OPA is that it doesn t derivatize secondary amines, so an additional reaction must be added for proline detection. And, as noted for amine analysis in section A5.4.2, such derivatization adds to the analysis time and may yield unstable products. 

Jones, B.N., and Gilligan, J.P. (1983) o-Phthaldialdehyde precolumn derivatization and reversed phase high-performance liquid chromatography of polypeptide hydrolysates and physiological fluids. J. Chromatogr. 266, 471-482. 

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... 

Phthalazinone, 19 332 o-Phthaldialdehyde-2-mercaptoethanol, chiral derivatizing reagent, 6 76t Phthalein dyes, 19 304-305 Phthalic acid, 19 332... 

Certainly, a vast amount of experience has been gained by the widespread use of conventional amino acid analysers. They offer high reliability, accuracy, reproducibility and can separate complex samples. Because conventional analysers can be fully automated, they are widely used in routine analysis. However, the method is limited by the sensitivity which can be achieved using ninhydrin as the derivatizing agent. Sensitivity can be increased by using ortho-phthaldialdehyde (OPA) instead, but where extremely high sensitivity is required, HPLC is the method of choice. 

DW Aswad. Determination of d- and L-aspartate in amino acid mixtures by high-performance liquid chromatography after derivatization with a chiral adduct and o-phthaldialdehyde. Anal Biochem 137, 405, 1984. 

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. 

Elevated temperatures up to 100°C have also been used with integrated HPLC chips that include both a column and an electrochemical detector [83]. This system used a resistive heater and a mobile phase preheater and was used to separate o-phthaldialdehyde amino acid derivatives. 

Dichloroacetamide present in chlorinated and chloraminated drinking waters was determined (at detection level of 23 pg L ) through an LC/fluorimetric method and a post-column derivatization reaction with o-phthaldialdehyde in the presence of sulfite at pH 11.5, that leads to the formation of a highly fluorescent isoindole fluorophore [225]. 

Recently, Yekkala et al. [144,145] have proposed an HPLC method with fluorescence detection. The method involves a rather laborious sample preparation due to the peculiar nature of the substrate involved (teeth), including pulverization, demineralization, hydrolysis of dentin and derivati-zation with o-phthaldialdehyde and A-acetyl-L-cysteine in order to obtain the enantioseparation of aspartic acid. Using a similar procedure, Benesova et al. [146] found that, in comparison with GC, HPLC provides shorter analysis time and higher sensitivity. 

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