O-phthaldialdehydes

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. 

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.)... 

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... 

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. 

Tobramycin sulphate was determined spectrophotometrically using a derivatization procedure [6]. A 0.5-1 ml aliquot of the extracted solution was mixed with 1 ml of o-phthaldialdehyde reagent solution followed by addition of 1.5 ml of isopropanol to prevent precipitation. The volume was adjusted to 5 ml with distilled water and the absorbance was determined after 45 min on a Beckman DU-7 spectrophotometer at the maxima of 333 nm. The concentrations were obtained from a calibration curve of o-phthaldialdehyde-derivatized tobramycin. The assay had a coefficient of variation of less than 3% and a detection limit of 0.5 pg/ml. 

Treatment of o-phthaldialdehyde with triethylphosphite in the presence of Lewis acids yields 1-phosphoryl substituted isobenzofurans (96TL5963 see also 79JOC494). For the reaction of 3-chloro-3-phenylphthalide with trimethylphosphite see Groffits et al. [93ZOR(63)2245]. 

LC Liquid chromatography CB Contamination Bureau SPE solid-phase extraction TFA trifluoroacetic acid MS mass spectrometry OPA o-phthaldialdehyde IA immunoaffinity column FD fluorescence detector BF best food UV ultraviolet. 

O-Phthaldialdehyde (OPA) is an amine detection reagent that reacts in the presence of 2-mercaptoethanol to generate a fluorescent product (for preparation, see Section 4.1, 2-mercaptoethanol) (Fig. 91). The resultant fluorophore has an excitation wavelength of 360 nm and an emission point at 455 nm. OPA can be used as a sensitive detection reagent for the HPLC separation of amino acids, peptides, and proteins (Fried et al., 1985). It is also possible to measure the amine content in proteins and other molecules using a test tube or microplate format assay with OPA. Detection limits are typically in the microgram per milliliter range for proteins. 

Fried, V. A., Ando, M. E., and Bell, A. J. (1985) Protein quantitation at the picomole level An o-phthaldialdehyde pre-TSK column-derivatization assay. Anal. Biochem. 146, 271— 276. 

Fluorimetric methods for the determination of amino acids are generally more sensitive than colorimetric methods. Fluorescamine (4-phenyl-spiro[furan-2(3H),l -phthalan]-3,3 -dione) and o-phthaldialdehyde (OPA) substances are used for protein analysis. Fluorescamine reacts with amino groups to form fluorophores that excite at 390 nm and emit at 475 nm (Weigele et al., 1972). Applications of fluorescamine include monitoring the hydrolysis of K-casein (Beeby, 1980 Pearce, 1979) and quantification of proteins, peptides, amino acids in extracts (Creamer et al., 1985). OPA produces fluorescence on reaction with 2-mercaptoethanol and primary amines, with strong absorption at 340 nm. Lemieux et al. (1990) claimed that this method was more accurate, convenient, and simple for estimating free amino acids than the TNBS, ninhydrin, or fluorescamine methods. 

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