OPA derivatives of amino acids

Figure 10.6 HPLC of OPA derivatives of amino acids. Chromatographic details column, 4.6 X 150 mm (5jim) ODS-Hypersil (Shandon) detector. Fluorescence (Kratos FS 970 E 230 nm 360 nm) eluent, linear gradient from 50 mM phosphate (pH 5.5) to 100% aceto-...

Fig. 6. HPLC separations of OPA derivatives of amino acids. Upper chromatogram shows a standard run employing gradient elution with phosphate-methanol (details given by Lindroth and Mopper, 1979). Each peak represents 30 pmol of an individual amino acid. Lower chromatogram shows the separation of amino acids from a sample of 100 fxl of Baltic surface water, unfiltered and injected after derivatisation. Peak 15 (internal standard) represents 2 pmol.

Pre-column derivatization—RPC analysis of phenylisothiocyanate (PITC), o-phthaldehyde (OPA), 9-fluorenylmethyl chloroformate (FMOC), or other derivatives of amino acids with UV or fluorescence detection is most common. This is the preferred methodology for life science research because of its higher sensitivity. More examples of precolumn derivatization of amino acids are shown in the life science section of this chapter. 

Re appears as a distinct peak, indicates that derivatization took place in precolumn mode. The presence of altered or metabolized derivatives of the 20 essential amino acids is characteristic of actual biological samples. The top trace is of a standard mixture of 200 pM concentration amino acids, while the lower one is from fingerprint oils extracted by water and ethanol from a glass surface. This illustrates impressive sensitivity for HPLC with UV/VIS detection. Twenty-fold greater sensitivity is possible when using fluorescent detectors with OPA (cf. Table 13.2) derivatives of amino acids. 

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 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-... 

The mixture of free amino acids is reacted with OPA (Fig. 7-8) and a thiol compound. When an achiral thiol compound is used, a racemic isoindole derivative results. These derivatives from different amino acids can be used to enhance the sensitivity of fluorescence detection. Figure 7-9 shows the separation of 15 amino acids after derivatization with OPA and mercaptothiol the racemic amino acids may be separated on a reversed-phase column. If the thiol compound is unichiral, the amino acid enantiomers may be separated as the resultant diastereomeric isoindole compound in the same system. Figure 7-10 shows the separation of the same set of amino acids after derivatization with the unichiral thiol compound Wisobutyryl-L-cysteine (IBLC). 

The reaction of amines and amino acids with orthophthaldehyde has been widely used in postcolumn and precolumn derivatization in analyses of foods (99-104) and in analyses of peptides from biological samples. Figure 2 (87) presents a chromatogram for OPA derivatives of tryptic peptides from two proteins. The sensitivity of the method was on the order of picomoles. The authors have themselves performed postcolumn OPA derivatization of low-molecular-weight peptides from blue cheeses separated by reversed-phase chromatography (86). 

The instability and chemical conversion of some OPA derivatives imply that a denvatized compound may, in fact, result in one fluorescent and two radioactive peaks (Simson and Johnson, 1976, Fig. 1). The chemical rearrangement of the derivatives may, however, be a minor factor with respect to retention and the fluorescent and nonfluorescent derivatives may coelute. The use of more chemically stable amino acid derivatives, i.e. those formed by reaction with FMOC chloride, eliminates this problem. When the radioactivity of an amino acid is measured, it is often desirable and necessary to inject larger concentrations of amino acids than in a routine expenment. With the OPA method it is then critical to (a) make sure that OPA is present in the required molar excess (Lindroth and Mopper, 1979), (b) lower the pH of the reagent mixture to spare the top of the column, and (c) use the same or lower proportion of organic solvent in the sample as in the beginning of the gradient in order to obtain a concentration of the derivatives on the column top. 

A much more general application of OPA was introduced when Roth [275] showed that nearly all amino acids can form fluorescent condensation products in the presence of an alkylthiol, such as 2-mercaptoethanol. Continuous reaction of the column effluent with OPA/ 2-mercaptoethanol or other thiol at pH 10, followed by recording of fluorescence intensity (excitation at 340-345 nm, emission at 455 nm), became a very widely used method for the determination of amino acids [276-298], peptides [280,291, 294—299], biogenic amines and polyamines [300-317], antibiotics [318-323] carbamates [324] and other primary amino group-containing compounds. OPA/R-SH reagents have to a considerable extent replaced ninhydrin for post-column derivative formation. 

Isoindoles of o-phthalaldehyde (OPA)/N acetyl-L-cysteine (NAC) are commonly used for detection of amino acids (RiCH(COOH)NH2) RPLC. A particular hydrophobicity scale was established with the retention data of these derivatives in mobile phases of SDS at pH 3, using the glycine derivative as a reference [20], Linear relationships were obtained between the ratios of log k of each amino acid derivative to log k of the glycine derivative (which was called quantitation of hydrophobicity index, QH), and log Pq for the Ri substituents (tTri). 

The chromatographic analysis of amino acids with spectrophotometric detection usually requires the formation of derivatives, because of httle absorption of UV light above 210 run. Precolumn deiivatization is usually preferable. o-Phthalaldehyde (OPA) is the deiivatization reagent that probably has the best characteristics. It reacts with primary amino groups in the presence of a thiol at pH 9.5 and room temperature to form l-alkylthio-2-alkyl substituted isoindoles (Fig. 10.5). The derivatives show maximum absorption at 335 nm and are highly fluorescent, with excitation wavelength at 340 nm and emission at 445 nm. Mercaptoethanol has been more extensively used than other thiols for the derivatization, but the OPA-mercaptoethanol isoindoles are unstable. The stability of isoindoles is improved when A-acetyl-L-cysteine (NAC) is used instead of mercaptoethanol [12]. 

OPA can also be employed for the detection of mixtures of amino acids. It reacts with primary amines in the presence of mercaptoethanol or other thiols to produce highly fluorescent isoindole derivatives (excitation wavelength 340 nm, emission 455 nm). In contrast to that with ninhydrin, this... 

Acetonitrile has found wide use in the separation of amino acids, peptides and proteins. A mainstay separation is that of derivatized amino acids. Classic precolumn derivatization methods include phenyl isothiocyanate (PITC) to give the PTH (phenylthiohydantoin) derivative, dimethylaminonapthalenesulfonyl (dansyl) chloride, o-phthalaldehyde (OPA) and 9-fluoromethylchloroformate (FMOC). From there, many variations on a theme have been developed. (The reader is referred to Chapter 4 for some of them.)... 

The reaction of amino acids with o-phthaldialdehyde (OPA) and mercaptoethanol leads to fluorescent isoindole derivatives (Aex = 330 nm, Aem = 455 nm) (Reaction 1.44a). 

Unlike ninhydrin, the reaction of amino acids with OPA does not destroy the amino acids, but adds the fluorescent tag to the primary amine. This allows OPA to also be used as a precolumn derivative, since the derivatization product is unique for each amino acid. 

In light of the improved host properties we found for modified CD, we investigated the potential effects of HP- -CD on a wide array of amino acid OPA-derived isoindoles, to see if significant fluorescence enhancements could be observed, and in addition, whether a stabilization of the isoindoles could be obtained (this was not observed in the above-mentioned previous studies) [99]. Our measurements showed modest fluorescence enhancements for some of the derivatives, including lysine and glycine, with values between 2 and 3. However,... 

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. 

A number of drawbacks in the application of the 0PA/2-ME reagent system include the instability of the fluorescent isoindole derivative (5-7) the use of the noisome reagent 2-mercaptoethanol the low and solvent-dependent fluorescence efficiencies (8,9) of the isoindole and—perhaps the most limiting—the effective restriction of the OPA assay to primary aliphatic amines and to amino acids. 

The stability of the CBI derivative is sufficient for its isolation and complete characterization (11), an accomplishment that is not realized with most OPA adducts. Thus, the CBI derivatives of a number of representative amino acids and amines have been isolated and their fluorescent properties determined as a function of the media and other relevant parameters encountered in reverse-phase HPLC (RP-HPLC). 

Perhaps most encouraging in these discoveries was the observation that NDA/CN worked equally well for derivatization of dipeptides and higher homologues of the primary amino acid series. Again, a stable, fluorescent, isolatable derivative was obtained. One of the most important initial findings was the high fluorescence efficiency of the CBI adduct (12). Tables 1 and 2 list the efficiencies for a representative group of mono-, di-, and tripeptides and a limited comparison of the CBI efficiencies with the more traditional OPA (8) and dansyl (9) derivatives, respectively. 

There is no necessity to remove excess OPA prior to sample injection since OPA itself will not interfere with separation or detection. However, since OPA-amino acid derivatives are unstable, complete automation of the precolumn reaction with accurate control of reaction time is essential for reproducible results. 

Hardware requirements for the separation of PTC-amino acids are similar to those for OPA amino acid determination. Since the derivatives do not fluoresce the technique is limited to UV detection, which normally takes place at 245nm. 

OPA in combination with chiral thiols is one method used to determine amino acid enantiomers. A highly fluorescent diastereomeric isoindole is formed and can be separated on a reverse-phase column. Some of these chiral thiols include N-acetyl-L-cysteine (NAC), N-tert-butyloxy-carbonyl- L-cysteine (Boc-L-Cys), N-isobutyryl- L-cysteine (IBLC), and N-isobutyryl- D -cysteine (IBDC). Replacing OPA-IBLC with OPA-IBDC causes a reversal in the elution order of the derivatives of D- and L-amino acids on an ODS column (Hamase et al., 2002). Nimura and colleagues (2003) developed a novel, optically active thiol compound, N-(tert-butylthiocarbamoyl)- L-cysteine ethyl ester (BTCC). This reagent was applied to the measurement of D-Asp with a detection limit of approximately 1 pmol, even in the presence of large quantities of L-ASP. 

Figure 4B. Chromatogram of RP-HPLC gradient elution separation of OPA—Ac—Cys derivatives of standard protein amino acid enantiomers.

The reaction of OPA with amino acids (see Fig. 10) requires a mercaptan cofactor that is incorporated as part of the final derivative product. The choice of mercaptan can affect derivative stability and chromatographic selectivity (178). Mercaptoethanol is the most commonly used coreagent. Cysteine is not well detected, because this amino acid can react at the a-amine group or it can react via the side chain thiol. Thus, cysteine is determined only after conversion of the thiol group by oxidation or alkylation. Reaction time with OPA is very fast, 1 minute at room temperature. Detection limits are typically in the low picomole range. Representative references include... 

Amino acids are derivatized two ways to increase sensitivity. Free amino acids in solution are reacted with o-phthaldehyde (OPA) to form a fluorescent derivative that excites at UV,230nm, and emits at FL, 418 nm. These OPA derivatives are separated on Ci8 in a complex mixture of An/MeOU/ DMSO/water at pH 2.65. PTH amino acids are formed from the N-terminai end of peptides during Edman degradation for structure analysis of peptides and proteins. HPLC is used to identify which amino acids are released. PTH amino acids are separated at UV, 254 nm, on a Ci8 column with a gradient from 10% THF/water containing 5 mM acetic acid to 10% THF/AN.The separation with reequilibration takes 60min. Work with short 3-pm columns has reduced this separation to a 10-min gradient. 

Pre-column OPA derivatization was also employed to analyze biogenic amines prior to MEKC separation on a PDMS chip [654]. Pre-column OPA derivatization and MEKC were also performed on a glass chip to analyze amino acids. Usually, OPA was used for fluorescent detection. However, in this report, amperometric detection was used as the OPA derivatives were also electroactive. Voltage (needed for separation) programming was used to decrease the migration time of late migrating species [655]. 

---