O-Phthalaldehyde derivatives

Serum carnosinase activity is readily measured as a marker for carnosinosis, homocarnosinosis and in instances of jS-alanine elevation in physiological fluids [7]. For quantification, carnosine is incubated with sera samples, and the histidine liberated in the reaction is quantified as the fluorescent o-phthalaldehyde derivative [19]. To estimate the activity of methylmalonate semialdehyde dehydrogenase (direct enzyme determination methods have not been reported), fibroblast extracts are incubated with l-14C-/ -alanine and trapping of 14C02. 

LA Allison, GS Mayer, RE Shoup. o-Phthalaldehyde derivatives of amines for high-speed liquid chromatography/electrochemistry. Anal Chem 56 1089-1096, 1984. 

The reaction is followed by separation of the substrate, lactose-lysine, from the product, fructose-lysine, on a cation-exchange resin (Durrum DC6A) using an isocratic mobile phase of pyridine-acetic acid-water (6 60 176, v/v). o-Phthalaldehyde derivatives were formed and detected by fluorescence. 

Bisulfite addition products are readily formed at wine pHs (1, 23, 24). The bisulfite addition product is thought to be a more sensory-neutral compound and may be exploited by winemakers as a means of decreasing the aldehydic character of wines (1). Bisulfite addition has also been used to mask the stale flavor of beer which is thought to be largely due to the formation of trans-l-noneml (25). Kaneda et al. (25) used HPLC with fluorescent detection of an o-phthalaldehyde derivative to quantitate and identify individual aldehyde-bisulfite products, however, only acetaldehyde-bisulfite adducts were observed in commercial beers with this method. Hydrolysis of the adducts occurs at pHs greater than 8, therefore by adjusting the pH prior to analysis, total aldehydes (free plus bisulfite bound) can be estimated. At low pHs accurate estimation of free aldehydes is complicated however, by analysis conditions which alter the equilibrium between bound and free forms (temperature, dilution, solvent extraction, analysis time, etc.). 

The Separation of Some Amino Acids by Monitoring their o-Phthalaldehyde Derivatives with a Fluorescence Detector Courtesy of Supelco Inc. 

Allison, L.A. Myer, G.S. Shoup, R.E. The o-phthalaldehyde derivatives of amines for high-speed liquid chromatography/ electrochemistry. Anal. Chem. 1984, 56, 1089-1096. 

Figure 2. Upper Gradient separation of o-phthalaldehyde derivatives of 11 thiols and sodium sulfite according to Mopper and Delmas Peaks (1) sodium sulfite (100 pmol) (2) glutathione (7 pmol) (3) thloglycollate (200 pmol) (4) N-acetylcysteine (7 pmol) (5) 2-mercaptoethanesulfonate (Co-M) (10 pmol) (6) 3-mercaptoproplonate (10 pmol) (8) monothloglycerol (10 pmol) (9) 2 mercaptoethanol (10 pmol) (10) methanethiol (15 pmol) (11) ethanethlol (10 pmol) (12) 2-propanethlol (15 pmol) (13) 1 propanethlol (15 pmol). Middle Thiols In porewater in reducing sediment slurry from Biscayne Bay. Porewater water was filter-sterilized prior to derlvatlzatlon. Peak 7 sulfide (Note response factor is about 200 times lower than for thiols). Lower reagent blank in porewater matrix.

T.P. Davis, High-performance liquid chromatographic analysis of hiogenic amines in biological materials as o-phthalaldehyde derivatives, J. Chromatogr., 162, 293-310 (1979). 

The o-phthalaldehyde derivatives of himonisins A and B were well resolved on a Cjg column (A = 335 nm, ex 440 nm, em) using an 80/20 methanol/water (0.1 M sodium phosphate at pH 3.35) mobile phase. Good peak shape was reported [323]. For com extracts, detection limits of 50 ng/g were rqwrted. 

Naphthalenedialdehyde was used as a precolumn derivatization reagent in the determination of desmosine, isodesmosine, and 17 other amino acid residues [468]. More stable complexes (as compared with o-phthalaldehyde derivatives) were cited as the analytical advantage. Detection limits of lOOfmol (S/N = 2) were reported and the analysis was completed in <35 min. A C 8 column (A = 420nm, ex 490nm, em) and a 10/5/85-> 63/1/36 methanoj/THF/water (5mM sodium citrate) gradient were used. Additionally, the amino acids were monitored electro-chemically (+750 mV vs. Ag/AgCl). Peak shape was good, but complete separation of all residues was not achieved. 

Twenty-six methyl carbamate residues and metabolites (e.g., aldicarb-sulfoxide and -sulfone, aldicarb, caibaryl, methiocarb and its sulfoxide and sulfone, etrofolan, baycarb, butocarboxim, thiofanox) were extracted from plant and soil matrices and analyzed as their o-phthalaldehyde derivatives using a C,g column (A = 340 nm, ex 455 nm, em). A 15-min 20/80 80/20 acetonitrile/water gradient was used [972]. 

Jarret, H.W., Cooksy, K.D., Ellis, B., and Anderson, J.M., The separation of o-phthalaldehyde derivatives of amino acids by reversed-phase chromatography on octylsilica columns. Anal. Biochem., 153. 189, 1986. 

Note If netilmicin is to be chromatographed alone it is recommended that the methanol content of the mobile phase be increased (e.g. to 23 -I- 7), in order to increase the value of the hRf. The detection limit for the substances in the application tested was more sensitive using DOOB reagent on RP layers than when NBD chloride, fluorescamine or o-phthalaldehyde were employed. The derivatives so formed were stable and still fluoresced after several weeks if they were stored in the dark. 

In the presence of 2-mercaptoethanol o-phthalaldehyde reacts with primary amines to yield fluorescent isoindole derivatives [20] ... 

Another reagent that readily forms fluorescent derivatives with primary amines is o-phthalaldehyde (trade name "Fluoropa"). The reaction proceeds in aqueous solution in the presence of a mercaptan at a pH of 9-11 producing an isoindole. 

The derivatives have an optimum fluorescence at an excitation wavelength of 340 nm and an emission wavelength of 455 nm. The adduct is relatively stable at a pH of 9-11 but it rapidly degrades to a non-fluorescent residue at low pH values. Consequently, when used as a pre-column derivatizing reagent the pH of the mobile phase should be kept fairly high, o-phthalaldehyde has been employed for derivatization in the analysis of dopamine (29), catecholamines (30) and histamines (31). 

The reaction of or o-phthalaldehyde and a thiol compound with an amino acid to form an isoindole derivative can be used to enhance the detection sensitivity for the normally only weakly UV-detectable amino acid compounds, and to introduce an... 

Amino acid analysers based on ion exchange resins are available commercially. These achieve good separations of amino acid mixtures. Fluorescent derivatives of separated amino acids constitute a very sensitive means of detecting these compounds in seawater [256,258]. Fluorescent derivatives that have been studied include o-phthalaldehyde [259], dansyl [260], fluo-rescamine [261], and ninhydrin [261]. 

A comparative study was made of the RP-HPLC analysis of free amino acids in physiological concentrations in biological fluids, with pre-column derivatization by one of the four major reagents o-phthalaldehyde (73) in the presence of 2-mercaptoethanol, 9-fluorenylmethyl chloroformate (90), dansyl chloride (92) and phenyl isothiocyanate (97, R = Ph) (these reagents are discussed separately below). Duration of the analysis was 13-40 min. Sensitivity with the latter reagent was inferior to the other three however, its use is convenient in clinical analysis, where sample availability is rarely a problem. The derivatives of 73 were unstable and required automatized derivatization lines. Only 92 allowed reliable quantation of cystine. All four HPLC methods compared favorably with the conventional ion-exchange amino acid analysis188. 

A method for analysis of A-nitroso-A-alkylureas (288b) has been developed by forming fluorescent derivatives with sodium sulfide, taurine (77) and o-phthalaldehyde (73)... 

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

V- Much effort has been expended in the development of more sensitive methods for the analysis and detection of catecholamines. They have been analyzed as the dansyl derivatives (376) or after precolumn derivati-zation with o-phthalaldehyde (377, 378). Postcolumn derivatization followed by fluorometric analysis have been described in which the fluoro-phore was formed with o-phthalaldehyde (379) or with 9,10-dimethoxyanthracene-2-sulfonate as the ion-pair (380). Several laboratories have shown the sensitivity and specificity in electrochemical detection methods (381 -383). 

Fluorometric detection has also been employed for the determination of sulfonamides in edible animal products, because it confers the advantages of selectivity and sensitivity. Although sulfonamides possess weak native fluorescence, their sensitive lluorometric detection necessitates use of precolumn or postcolumn derivatization producing the corresponding fluorescent derivatives. The most commonly used derivatizing reagent for precolumn derivatization is fluorescamine (217, 228, 230, 238, 239), while for postcolumn derivatization fluorescamine (231), and o-phthalaldehyde (OPA), and -mercaptoethanol (219) are most often used. 

The benzo[c]furan (126) is probably formed on treatment of the tetra-Ione derivative 125 with o-phthalaldehyde in the presence of acetic anhydride/ sulfuric acid. ... 

It has been suggested that the benzo[c]furan (245) is formed from the tetralone derivative (244) on treatment with o-phthalaldehyde in acetic acid-sulfuric acid (71JCS(C)2175). 

If only very small samples of amino acids are available for analysis, fluorescence is used for detection. One of the most sensitive methods of microanalysis is based on the reaction of amino acids with o-phthalaldehyde and /3-mercaptoethanol (Equation E2.1). The isoindole derivative is fluorescent and amounts as small as 10-12 mole may be measured. 

C Cloete. Automated optimized high performance liquid chromatographic analysis of pre-column o-phthalaldehyde-amino acid derivatives. J Liq Chromatogr 7 1979-1990, 1984. 

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