OPA reagent

The advantages of this method are a short reaction time and the nonfluorescence of the OPA reagent. Therefore, excess reagent must not be removed before the chromatography stage. Using this method, it is possible to measure tryptophan, but not secondary amino acids such as proline or hydroxyproline. Cysteine and cystine can be measured, but because of the low fluorescence of their derivatives, they must be detected using an UV system, or alternatively oxidized to cysteic acid before reaction. 

Merino-Merino et al. [32] used the OPA reagent (o-phthaldehyde condensed with 2-mercaptoethanol) to separate penicillamine enantiomers after their derivatization. Racemic and (/q-penicillamine were dissolved in aqueous 0.5 M NaOH, and treated with the derivatizing solution (methanolic o-phthaldehyde and 2-mercaptoethanol in 0.4 M potassium borate buffer solution of pH 10). The reaction mixture was set aside for 2 min at room temperture, whereupon a portion of solution was analyzed by HPLC. The method used a Cyclobond column (25 cm x 4.6 mm) maintained at 5 °C, a mobile phase of ethanol/1% triethylammonium acetate (1 1 pH 4.5) eluted at... 

To a set of labeled tubes, add 2 ml of OPA reagent (Thermo Fisher) and 200 pi of the appropriate standard or sample. Mix well. If using a microplate format, scale back these quantities 10-fold to fit in the microwells. 

Reagents OPA-reagent 5 mM o-phtalic dialdehyde, 0.020 M N-acetylcysteine, 7 mM sodium lauroylsarcosinate, 0.5 M potassium borate, pH 10 acidifying reagent 0.067 M sodium citrate, pH 2.0. 

The OPA reagent for HPLC is prepared according to the method of Benson and Hare (21). The fluorescence reaction is performed in a 55 C water bath. OPA reacts with the guanidino group of TTX presumably to form a fluorescent product, l-alkylthio-2-alkylisoindole (22, 23). TTX is monitored at 453 nm with 332-nm excitation. Peak areas are calculated by a data processing system of the analyzer. 

In brief, a 200-pl sample will be deproteinized with 20 pi 5% SSA containing internal standard. Following precipitation and centrifugation, 150 pi supernatant is diluted with 150 pi of the lithium citrate buffer and placed in the autosampler. The derivatization step takes 40 pi of sample and 20 pi of the OPA reagent, which are mixed and left to react for 30 s. A 20-pl aliquot is injected. 

Because the reaction temperature markedly influences the rates of formation and degradation of the fluorescent adduct, its precise control is an essential factor for the reproducibility of the postcolumn fluorescence detection. Therefore preheating of both the eluent and OPA reagent to a constant temperature of 40°C is required before their mixing, and these was achieved by insertion of preheater tubes for both the eluent and OPA reagent into the hne. As described in the section HPLC System and Conditions, the preheater tubes, as well as colunms, resistor tube, and the reactor tube were placed in a column oven maintained at 40°C. 

Fig. 6.3 Interaction between phthalic dialdehyde, mercaptan group (OPA reagents) and primary amine (I) thioacetal formation (II) formation of fluorescent complex between hemithioacetal and primary amine (III).120 Reproduced with permission...

The development of fluorescent derivatives of amino acids and their chromatography on reversed-phase columns yield a significant gain in sensitivity. Many fluorescent derivatives of amino acids are available that greatly enhance the sensitivity of detection. 0-phthaldialdehyde/mercaptoethanol (OPA) reagent reacts with most of the common amino acids (but not proline) to form fluorescent derivatives (14). Because OPA derivatives are not very stable, it is essential to chromatograph the OPA derivatives within a few minutes. In order to achieve consistent analytical results, it is necessary to automate or to time accurately the derivatization step. Amino acid analysis with pre-column OPA takes less than 15 minutes including the derivatization step. It has become a popular technique wherever prollne values are not necessary. 

The HPLC system comprised an L-6000 pump (Hitachi, Tokyo, Japan) and an LC-9A pump (Shimadzu, Kyoto, Japan) for deliveries of an eluent and the OPA reagent, a DGU-12A degasser (Shimadzu), a Rheodyne Model 77251 sample injector (Rheodyne, Cotati, CA, USA), aCTO-lOA column oven (Shimadzu), an F-1050 fluorescence detector equipped with a 12 p,l square flow cell, and a D-2500 data processor (Hitachi). Separation was performed at 40°C... 

Amino acid standards (10, 25, and 100 pmole, Cat. Nos. 5061-3334, 5061-3333, and 5061-3332), OPA reagent (Cat. No. 5061-3335), FMOC reagent (Cat. No. 5061-3337), and 0.4 N borate buffer (Cat. No. 5061-3339) were obtained from Hewlett-Packard. Sodium acetate (Cat. No. 6267) and 37% hydrochloric acid (Cat. No. 13386) were from Merck, and methanol, acetonitrile, and tetrahydrofuran from Baker. Triethylamine (Cat. No. 90340) was obtained from Fluka. 

The UV detector has been the most widely used in the LC determination of pesticides [59,63,69,71,77,106,107,109,111,122,147,148]. However, at present, the diode array detector is usually preferred to obtain the UV spectrum for each individual compound and confirm e presence of target analytes [35 8,45,48,50,71,74,76,108,149]. Carbamate pesticides are usually determined with fluorescence detector, following LC-postcolumn derivatization of methylamine (formed in the previous hydrolysis of pesticide) with OPA reagent [68,74]. Recently, a postcolumn detection system was used for the... 

The OPA reagent was first reported in 1971 by Roth as a postcolimm fluorogenic reagent for amines [5] and has been widely used for the sensitive determination of primary amino compounds. However, the fluorescent derivatives are not sufficiently stable, and it is sometimes difficult to obtain reproducible results using the postcolumn derivatization system. A precolumn derivatization technique has also been developed using OPA in the presence of alkylthiol compounds such as 2-mercaptoethanol. OPA rapidly reacts with primary amino compounds within 2 min at room temperature, and the derivatives can be separated by reversed-phase liquid chromatography [20]. Fluorescence detection of the derivatives is performed at 440 nm (emission wavelength) with excitation at 330 nm. Because OPA does not react with secondary amino compounds, proline and hydroxyproline can not be determined by this method. Replacement of 2-mercaptoethanol with other thiols, such as 2-ethanethiol [21] and 3-mercaptopropionic acid [22], produced more stable fluorescent derivatives. 

After this first step, the stream passes into a first mixing tee, where the acidified and diluted sample encounters alkaline OPA reagent (P2), and the alkaline part of the histamine-OPA reaction proceeds for a time determined by the OPA reactor volume (R2). 

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