OPA-derivatized amino acids

OPA-derivatized amino acids are usually separated on an ODS-II solid phase using a mobile phase of sodium phosphate buffer and an acetonitrile gradient. 

Fig. 2 Distributions of OPA-derivatized amino acids and peptides chromatographed by the automatic online OPA/2-mercaptoethanol system. A and B 50 pmol of tryptic peptide digest of proteins M and R, respectively. The peak marked with an asterisk is due to the derivatizing reagents. Column 5-/zm Resolve C, (15 cm X 3.9 mm). Emission at 425 nm and excitation at 338 nm. A comparison between the sequences of peptides M,5 and R,5 is also shown.

The Kennedy lab has been at the forefront of using rapid capillary electrophoresis to measure neurochemical changes. Optically gated injection of OPA-derivatized amino acids has been achieved in less than 2 s. However, the high salt concentrations of physiological samples, such as cerebral spinal fluid, can reduce EOF, and make separations slower. Still, 10 s temporal resolution for online monitoring of directly sampled and microdialysis samples were obtained. An instrument has been developed in the Kennedy lab for online analysis of microdialysis samples after precolumn... 

In the report on the separation of a mixture of OPA-derivatized amino acids, phosphate buffers in the pH range 6.3-7 7 were used (Lindroth and Mopper, 1979). Two amino acids of importance in neurotransmission, glutamine and GABA, coelute with histidine and alanine, respectively, in this system Complete separation of the ammo acids was obtained, however, at pH 5.25... 

A recent improvement builds on the fluorometric analysis of ammonium concentration (Holmes et al., 1999) with OPA (ortho-phthalaldehyde). Johnston et al. (2003) extracted the OPA-NH4+ derivative (1 -sulfonato-iso-indole) by soHd phase extraction and found that derivatized amino acids were not retained along with the indole on the column so that the method was specific. Since they developed the method for natural abundance work, the requisite high precision obhged analysis by CF-IRMS and its attendant high mass requirement (7.14 pmol N for a standard deviation of 0.5%o) that is difficult to achieve in oligotrophic waters. Since tracer experiments do not require such high precision, a lower mass could likely be used with the inherently lower precision GC—MS. 

The derivatization was performed by mixing an aliquot of the amino acid solution with equimolar OPA-NAC reagent (OPA-NAC amino acid... 

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.)... 

OPA precolumn derivatization is probably the most commonly used precolumn method. OPA precolumn derivatized amino acids are detected with fluorescent detectors using the same wavelengths used for postcolumn OPA. The derivatized sample can also be loaded directly on the reversed-phase column, allowing automation of the derivatization reaction with an antosampler. The sample, however, does need to be evaporated to dryness after hydrolysis for removal of the acid. The removal of the acid needs to be complete, usually requiring redissolving and redrying or strongly buffered to an alkaline pH. 

Separation of Amino Acid Enantiomers after Derivatization with Or/ho-Phthaldialdehyde (OPA) and a Unichiral Tliiol Compound... 

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. 7-8. Derivatization of amino acids with OPA and a thiol compound.

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).

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. 

Certainly, a vast amount of experience has been gained by the widespread use of conventional amino acid analysers. They offer high reliability, accuracy, reproducibility and can separate complex samples. Because conventional analysers can be fully automated, they are widely used in routine analysis. However, the method is limited by the sensitivity which can be achieved using ninhydrin as the derivatizing agent. Sensitivity can be increased by using ortho-phthaldialdehyde (OPA) instead, but where extremely high sensitivity is required, HPLC is the method of choice. 

PITC has been used extensively in the sequencing of peptides and proteins and reactions under alkaline conditions with both primary and secondary amino acids. The methods of sample preparation and derivatization follow a stringent procedure which involves many labour-intensive stages. However, the resulting phenylthio-carbamyl-amino acids (PTC-AA s) are very stable, and the timing of the derivatization step is not as critical as when using OPA. 

Derivatization of primary amino acids with o-phthalaldehyde (OPA) is simple and the poor reproducibility due to the instability of the reaction product can be improved by automation and the use of alternative thiols, e.g. ethanthiol in place of the 2-mercaptoethanol originally used. An alternative fluorimetric method using 9-fluoroenylmethylchloroformate (FMOC-CL) requires the removal of excess unreacted reagent prior to column chromatography. This procedure is more difficult to automate fully and results are less reproducible. However, sensitivity is comparable with the OPA method with detection at the low picomole or femtomole level, and it has the added advantage that both primary and secondary amino acids can be determined. 

There are two major approaches to achieve enantiomeric separation of d- and L-amino acids. The first involves precolumn derivatization with a chiral reagent, followed by RP-HPLC [226], while the second involves direct separation of underivatized enantiomers on a chiral bonded phase [227], Weiss et al. [209] determined d- and L-form of amino acids by applying derivatization with OPA and chiral /V-isobutyryl-L-cysteine. 

Orthophthalaldehyde (OPA) in combination with a thiol is the reagent of choice for derivatization, despite its inability to react with proline, hydroxyproline, and the sulfur-containing amino acids. Another drawback of the reagent is the instability of the reaction products, making an automated derivatization system coupled to an automated injector, and constant retention times an absolute necessity. Taking into account these considerations, the HPLC analysis will be of use to every biochemical genetics laboratory for biological fluids other than urine. The system has also a... 

Detection of amino acids is typically by UV absorption after postcolumn reaction with nin-hydrin. Precolumn derivatization with ninhydrin is not possible, because the amino acids do not actually form an adduct with the ninhydrin. Rather, the reaction of all primary amino acids results in the formation of a chromophoric compound named Ruhemann s purple. This chro-mophore has an absorption maximum at 570 nm. The secondary amino acid, proline, is not able to react in the same fashion and results in an intermediate reaction product with an absorption maximum at 440 nm. See Fig. 5. Detection limits afforded by postcolumn reaction with ninhydrin are typically in the range of over 100 picomoles injected. Lower detection limits can be realized with postcolumn reaction with fluorescamine (115) or o-phthalaldehyde (OPA) (116). Detection limits down to 5 picomoles are possible. However, the detection limits afforded by ninhydrin are sufficient for the overwhelming majority of applications in food analysis. 

Since then, there have been a number of reversed-phase separations employing precolumn derivatization. Interestingly, fluorescamine (not frequently employed for RP-HPLC of amino acids with precolumn reaction) has been reported for taurine analysis in milk (197) and human plasma (198). Precolumn derivatization with OPA/2-mercaptoethanol has been reported for the analysis of infant formula and human breast milk (199). Although not the principal focus of the study, Carratu et al. (200) report taurine values in parenteral solutions as determined by FMOC. In an excellent article, Woollard and Indyk (201) report the dansylation of taurine for its determination in a wide variety of dairy-related products. Subsequently, the same authors report the results of a large collaborative study (202) for the determination of taurine (again, by dansylation) in milk and infant formula. This study afforded an overall interlaboratory RSD of 7.0% and established a lower limit for determination at 5 mg taurine per 100 g of product. 

U Butikofer, D Fuchs, JO Bosset, W Gmur. Automated HPLC-amino acid determination of protein hydrolysates by precolumn derivatization with OPA and FMOC and comparison with classical ion exchange chromatography. Chromatographia 31 441-447, 1991. 

H Godel, P Seitz, M Verhoef. Automated amino acid analysis using combined OPA and FMOC-C1 precolumn derivatization. LC-GC Inti 5 44-49, 1992. 

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). 

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