Detection with OPA

Because of high selectivity of the postcolumn fluorescence detection with OPA, no sample pretreatment other than deproteinization was required for the assay of his-... 

Because of the high selectivity and sensitivity of the postcolumn fluorescence detection of histidine with OPA, the present HPLC method is applicable to a specific and rapid assay of histidine in human semm, blood, and urine after simple pretreatment. A recent paper demonstrated that the postcolumn detection with OPA was applicable to the simultaneous assays of histidine and its major metabolites cis- and frawi-urocanic acids) in human stratum corneum. " The postcolumn detection system was also applicable to the flow injection analysis (FIA) method for the assay of histidine in semm and urine. The FIA method enabled us to determine histidine in blood after pretreatment of the sample with A-ethylmaleimide (masking reagent of glutathione).These methods are useful in the diagnosis of histidinanemia, one of hereditary metabolic disorders characterized by a virtual deficiency of histidine ammonia-lyase. 

Por IR-Raman experiments, a mid-IR pump pulse from an OPA and a visible Raman probe pulse are used. The Raman probe is generated either by frequency doubling a solid-state laser which pumps the OPA [16], or by a two-colour OPA [39]. Transient anti-Stokes emission is detected with a monocliromator and photomultiplier [39], or a spectrograph and optical multichannel analyser [40]. 

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). 

Fig. 7-9. Separation of amino acids after derivatization 5 with OPA and mercaptoethanol. Column Superspher 100 RP-18 (4 pm) LiChroCART 250-4, mobile phase 50 mM sodium acetate buffer pH 7.0/methanol, flowrate 1.0 ml min temperature 40 °C detection fluorescence, excitation 340 nm/emission 445 nm. Sample amino acid standard sample (Merck KGaA Application note W219180).

The first step in analysing a data table is to determine how many pure factors have to be estimated. Basically, there are two approaches which we recommend. One starts with a PCA or else either with OPA or SIMPLISMA. PCA yields the number of factors and the significant principal components, which are abstract factors. OPA yields the number of factors and the purest rows (or columns) (factors) in the data table. If we suspect a certain order in the spectra, we preferentially apply evolutionary techniques such as FSWEFA or HELP to detect pure zones, or zones with two or more components. 

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. 

UV absorption occurs only below 220nm, thereby it is affected by the interference from mobile phase and from artifacts in complex foods. A multiwavelength UV detection has been experimented successfully for unambiguous evaluation of pantothenic acid [609]. However, UV detection presents a low sensitivity, compared to other techniques, like FLD or MS. FLD is applied by using a postcolumn derivatization. Pantothenic acid is converted to 3-alanine by hot alkaline hydrolysis and a reaction with OPA [610]. Also MS is successfully applied to increase the sensitivity of pantothenic acid analysis. 

OPA (ortho-phthalaldehyde) Aex = 340 nm, Aem = 455 nm. Derivatives are not stable (minutes). OPA is best used as a postcolumn derivatizing reagent. Precolumn use necessitates well-controlled and automated (mechanized) handling of the sample/reagents to ensure uniformity of time elapsed from reaction to injection. It reacts with primary amines only. Proline can be detected only if first converted to primary amine by oxidative reagents like chloramine T or sodium hypochlorite. Conversely, the concomitant reaction with OPA followed by FMOC obviates this problem and is the basis of the AminoQuant system marketed by Hewlett-Packard... 

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... 

A method for the determination of 15 BAs in different foodstuffs, namely, Swiss cheese, salami, milk, beer, and wine, was developed by Petridis and Steinhart it is based on an automated precolumn derivatization of BAs [after acidification with TCA and solid-phase extraction (SPE) on Amberlite resin] with OPA/ME and separation on a Spherisorb ODS-2 column with gradient elution (ACN-MeOH-phosphate buffer) and fluorescence detection (71). 

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. 

Figure 4.10. On-line coupling of USAL and detection with derivatization (A) in the pre-column mode for the determination of coiistin A and B in feeds (B) in the post-column mode for the determination of N-methyicarbamates in soil and foods, AC — analytical column, C — carrier, DC — derivatization coil, EL — elution loop, F — filter, FD — fiuorimetric detector, HC — hydrolysis coil, HPiV — high-pressure injection valve, HPP — high-pressure pump, IV — injection valve, L — leachant, LC — leaching chamber, MP — mobile phase, OPA — o-phthaldiaidehyde, PC — preconcentration column, PL — propagating liquid, PP — peristaltic pump, S / — switching valve, UP — ultrasonic probe, W — waste and WB — water bath. (Reproduced with permission of Elsevier, Refs. [48,49].)...

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