Amino precolumn derivatization, reagent

Wen-Chen Z, Ling-Jun L, Xian-En Z et al (2008) Application of 2-(l l//-benzo[ i]carbazol-11 -yl) ethyl carbonochloridate as a precolumn derivatization reagent of amino acid by high performance liquid chromatography with fluorescence detection. Chin J Anal Chem 36 1071-1076... 

This reagent has been used as a precolumn derivatizing reagent in the analysis of phenyl-isothiocarbamoyl derivatives of amino acids (89). The Waters Chromatography Division of Milli-pore (90) developed an automatic method (PICO-TAG) permitting analysis of the phenylthiocar-bamoyl derivatives of amino acids in under 12 min, with a detection limit of picomoles. 

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. 

Inagaki S, Tano Y, Yamakata Y, Higashi T, Min JZ, Toyo oka T. Highly sensitive and positively charged precolumn derivatization reagents for amines and amino acids in liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 2010 24 1358-64. 

In 2009, Shimbo et al. designed a new precolumn derivatization reagent, 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS Eigure 6.2), for the LC—MS/MS determination of amino compounds [36]. Amino compounds easily react with APDS imder mild alkaline conditions at 55°C within 10 min and can be directly injected into the reversed-phase LC system. The derivatized molecules are monitored by the common fragment ion mfz 121) derived from the aminopyridyl moiety... 

Shimbo K, Oonuki T, Yahashi A, Hirayama K, Miyano H. Precolumn derivatization reagents for higji-speed analysis of amines and amino acids in biological fluid using liquid chromatography/ electrospray ionization tandem mass spectrometry. Rapid Comm Mass Spec 2009 23 1483-92. 

Prados P, Fukushima T, Santa T et al (1997) 4-/V,/V-Dimethylam inosu 1 lbnyl-7 -N-(2-aminoethyl)amino-benzofurazan as a new precolumn fluorescence derivatization reagent for carboxylic acids (fatty acids and drugs containing a carboxyl moiety) in liquid chromatography. Anal Chim Acta 344 227-232... 

Direct and indirect chromatographic methods were developed and compared in systematic examinations for the enantioseparation of P-amino acids direct separation of underivatized analytes involved the use of commercially available Crownpak CR(-I-), teicoplanin, and ristocetin A CSPs [148], while indirect separation was based on precolumn derivatization with 2,3,4,6-tetra-G-acetyl-f)-D-glucopyranosyl isothiocyanate (GITC) or A - a-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (EDAA, Marfey s reagent), with subsequent separation on a nonenantioselective column. 

Nimura N, Fujiwara T, Watanabe A, Sekine M, Furuchi T, et al. 2003. A novel chiral thiol reagent for automated precolumn derivatization and high-performance liquid chromatographic enantioseparation of amino acids and its application to the aspartate racemase assay. Anal Bio-chem 315 262-269. 

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. 

Fig. 8 Separation of standard mixture employing precolumn derivatization with AQC. Gradient elution with acetonitrile and acetate buffer (pH 5.0) was employed with a C18 column. Standard three-letter abbreviations for amino acids were used also, CA = cysteic acid, AMQ = hydrolyzed excess reagent, and nle = norleucine. Data was supplied by Stephen D. Smith, Ross Products Division of Abbott Laboratories, Columbus, OH.

Since BAs occurring in food do not exhibit satisfactory absorbance or fluorescence in the visible or ultraviolet range, chemical derivatization, either pre- (35-37) or postcolumn (38), is usually used for their detection in HPLC. The most frequently employed reagents for precolumn derivatization are fluorescamine, aminoquinolyl-lV-hydroxysuccinimidyl carbamate (AQC) (39, 40), 9-fluorenylmethyl chloroformate (FMOC) (41-43), 4-dimethylaminoazobenzene-4 -sul-fonyl chloride (dabsylchloride, DBS) (44), N-acetylcysteine (NAC) (45,46), and 5-dimethyl-amino-1-naphthalene-1-sulfonyl chloride (dansylchloride, DNS) (47,48), phthalaldehyde (PA), and orf/to-phthaldialdehyde (OPA) (49-51), together with thiols such as 3-mercaptopropionic acid (MPA) (37) and 2-mercaptoethanol (ME) (35,49). 

A chip-based integrated precolumn microreactor with 1 nl reaction volume has been explored by Jacobson et al. [67]. The reactor is operated in a continuous manner by electrokinetically mixing of sample (amino acids) and reagent (o-phthaldialdehyde) streams. The reaction time is adjusted via the respective flow velocities. By switching of potentials, small plugs of the reaction product were injected into a 15.4 mm separation channel in a gated injection scheme (< 1.8% RSD in peak area). The separation efficiency achieved was relatively poor, however, electrokinetic control of reaction time (and yield) permitted to monitor the kinetics of the derivatization under pseudo first-order conditions. A similar integrated precolumn reactor operated in a stopped flow mode has been described by Harrison et al. [68]. 

Most amino acids react with ninhydrin at ambient temperatures to form a blue color that becomes purple on heating. However, proline and hydroxyproline yield yellow compounds that are measured at a different wavelength. Other postcolumn derivatizations use fluorogenic reagents, such as o-phthaldialdehyde or fluorescamine. Precolumn derivatization techniques using o-phthaldialdehyde, dansyl, phenyl isothiocyanate, or 9-fluorenylmethyl chloroformate derivatives have been used with reversed-phase HPLC. Electrochemical detection has also been coupled with derivatization methods to enhance analytical sensitivity. 

Figure 22.4 for the successful separation of the propranolol enantiomers on a chiral stationary phase the molecule should have a rigid structure. This was obtained by a precolumn derivatization with phosgene. This reagent gives an oxazolidone ring from the alcohol and secondary amino groups. The reaction is fast at 0°C. 

The precolumn technique that is most frequently employed today was developed during the early 1980s [32,33]. For this method, The classical Edman reagent phenylisothiocyanate (PITC) is used for amino acid derivatization after hydrolysis. Separation of the PTC amino acids i then accomplished by HPLG, with detection at 254 nm. Although standard Cig columns available Irom a variety of vendors are suitable for separation of the PTC-derivatized amino acids, there are specific columns that have bqen optimized for this purpose (e.g.. Waters). Approximately 0.5 /ug of peptMe should be hydrolyzed for analyses using precolumn derivatization. ... 

Precolumn derivatization For separation by reversed-phase chromatography, precolmnn derivatization is a necessity. The derivatization step increases not only detectability of the analyte but also its hydrop-hobicity, which makes the separation of the amino acids by reversed-phase chromatography possible. Several precolmnn derivatization reagents are available. These include OPA, naphthalenedialdehyde (NDA), dimethylaminonaphthalene sulphonyl chloride (DANSYL), phenyl isothiocyanate (PITC), and N-(9-fluorenyl)methoxycarbonate (FMOC). 

Precolumn derivatization methods are not free from disadvantages. One is that side-products from the reaction can sometimes be difficult to separate from the peaks of interest. Also with precolumn derivatization, as its name implies, each sample must be derivatized prior to injection. This can add to the total analysis time. However, instrumentation is now available to derivatize several samples simultaneously in a closed system, which not only solves the sample preparation time problem but also results in highly reproducible chemistry. Since the reaction may not go to 100%, an internal standard is frequently necessary. Finally, the addition of a similar tag to all the amino acids may make them more difficult to separate. Table 1 summarizes the properties of several pre- and postcolumn derivatizing reagents employed for amino acid analysis. 

Luminol can be used to derivatize analytes (for immunoassay or HPLC) through substitution at the primary amine but this results in a 10- to 100-fold decrease in chemiluminescence efficiency. Isoluminol (6-amino-2,3-dihydro-l,4-phthalazinedi-one) is somewhat less efficient than luminol however, it does not suffer a decrease in efficiency upon binding. Aminobutylethylisoluminol (ABEI, (9) is a useful precolumn labeling reagent for amines and carboxylic acids because the hydrocarbon spacer isolates the chemiluminophore from the analyte. 

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