Patai S., Rappoport Z. 1996 The chemistry of functional groups. The chemistry of amino, nitroso, nitro and related groups. Part 2 / 1041 1168

O

PhCH2 O2 CNHC*H(i-Pr)CONHCMe2 CO2 N

O

(122)

as chiral modifier298. The Cu(II) complex of (R,R)( )-N,N0 -dicyclohexyl-trans-1,2- cyclohexanediamine (123) was also proposed as chiral modifier. This was applied to the RP-HPLC resolution of free or dansylated amino acids and to further assist the separation of diasteroisomers299.

NHHex-c

NHHex-c

(123)

The concept of two-dimensional chromatography was applied to LC in columns for determination of enantiomeric composition of complex mixtures of amino acids, as occurring in biological fluids and foods. The first run performed was IEC with LiCl Li citrate buffer. Each eluted peak corresponding to an amino acid was reinjected into an RP- C18 column and eluted with an aqueous solution containing chiral Cu(II) complexes with various derivatives of L-phenylalanine (124 126), which undergo partial ligand exchange with the amino acid enantiomers and perform chiral discrimination. Advantages of the method are that only the chiral mixtures of interest are separated and each one of these can be treated with a different chiral reagent. Detection was by fluorometry, after post-column derivatization with 4-chloro-7-nitrobenz-2,1,3-oxadiazole (127a) for proline and hydroxyproline and, according to reaction 7, for the other amino acids. It was possible to detect chiral impurities as low as 0.1% in the nM concentration range300. Precolumn derivatization of amino acids with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (127b) followed by HPLC-FLD ( ex 470 nm, fl 530 nm) showed separation factors of 1.27 and 1.17 for the derivatives of the enantiomers of phenylalanine and leucine, respectively; LOD ca 30 fmol301; phenylcarbamylated ˇ-cyclodextrin stationary phases were also used301,303. The fluorogenic benzoxadiazolyl isothiocyanate Edman reagents (128) were proposed for pre-column derivatization of amino acids, followed by HPLC-FLD. No racemization took place on derivatization304. Pre-column derivatization of alcohols and amines with the chiral proline derivative reagent 129 affords fluorescent labeling of these compounds for LC-FLD. Excitation at about 450 nm and fluorescence at about 560 nm were essentially the same for all alcohols and amines tested; instrumental LOD (SNR 2) and detection using a conventional Xe lamp was 10 500 fmol, and 2 10 fmol with LIF, using the ArC emission at 488 nm234. See other benzoxadiazole reagents above (88, 89).

epimerization takes place, and the enantiomeric ratio can be correlated with age. This has some advantages over 14C radioisotope dating, as several easily resolved tracers (amino acids) are available for mutual reconfirmation or discovery of unusual features. The rate of racemization is affected by temperature, pH, catalysts etc., but these factors can be eliminated by correlating D:L ratios in fossils of a locality with 14C dating. This was done for about one-hundred known fossil specimens collected in Hungary, and the calibration curves were applied to estimate the age of specimens, based on two to three amino acids309. See also end of Section IV.B.

Besides providing a dating tool for samples older than the limit of radiocarbon dating (4 5 ð 104 year), enantiomer ratio can be applied to dating of recent samples, where the 14C method is also insensitive. Thus, the rate of epimerization of aspartic acid allows dating of deposits less than 350 years old310. Assessment of indigeneity of fossil samples can be carried out by analyzing the soluble organic matter. Each peptide is separated and submitted to amino acid analysis and differentiation using multivariate statistics. Besides dating, also the molecular phylogeny of the fossils can be asserted311. Determination of the ‘I/A’ ratio (L-isoleucine to D-alloisoleicine) requires very small samples, e.g. snail shells were individually analyzed and shown to belong to a mixed-aged deposit, aided by 14C dating312.

The effect of temperature on the rate of racemization of amino acids in fossils was investigated and the implications of the findings on fossil dating were analyzed313. The high rate of conversion of L-aspartic acid into its D-isomer, observed in uncontaminated bone samples taken from catacombs in Rome (IV century BC) was attributed to collagen decomposition due to the humidity of the catacombs314.

E. Electrophoresis

Refractive index may afford a sensitive universal detection method for capillary electrophoresis (CE). An important part of the setup is a refractive index matching fluid in which the capillary is submerged. The method was tested for a saccharide mixture including N-acetylglucosamine and N-acetylgalactosamine315. An intensity-modulated 257 nm pump laser-induced refractive index changes inside a 25 mm bore capillary used in CE. These changes were monitored with the aid of a probe laser beam oscillating at 663 nm. This RID was demonstrated for dansyl (92) derivatives of amino acids: total sample 350 pg, with detection volume <10 pL316.

The effectiveness of nine background electrolytes, providing both buffering action and background absorbance, was assessed for CE separation of twenty common amino acids. p-Aminosalicylic acid and p-(N,N-dimethylamino)benzoic acid were the best317. Low molecular weight amines can be separated and determined by CE using an electrolytic system based on Cu(II)318. Potential-amperometric detection of amino acids and peptides separated by CE was carried out by electro-oxidation at a Cu electrode in alkaline medium. The method was applied to determination of amino acids in urine, L-aspartyl- L-phenylalanine methyl ester (aspartame) in soft beverages and pentapeptides from a solid-phase synthesis process319.

Primary amines are derivatized readily and quantitatively as illustrated in reaction 15.

CE and detection by LIF had LOD in the low attomol 1 ð 10 18 range for amino acids and amino sugars320,321.

CO2 H

O

+ Η2 NCHRCO2 H

N CHO

(15)

CO2 H

NCHRCO2 H

N

Amino acids derivatized with 9-fluorenylmethyl chloroformate (90) were separated by CE and determined by LIF with a pulsed laser; LOD 0.5 nM (SNR 2)322. A sensitive technique for amino acids is capillary zone electrophoresis (CZE) combined with LIF of their fluorescein isothiocyanate (133) derivatives. Not all amino acids give good resolution. LOD for proline and arginine were 0.3 and 0.5 nM, respectively323.

N=C=S

CO2 H

(133)

The chiral purity of amino acids at large enantiomeric excess can be determined automatically by derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (127b) followed by CE with cyclodextrin chiral selectors and detection of the LIF excitation at 488 nm. Lod 140 ppm of L-phenylalanine in D-phenylalanine324.

isoproterenol (21c), epinephrine (21b), norepinephrine (21a), pseudoephedrine (30) and octopamine (22)326.

Micellar techniques can increase the concentration of a component in the disperse phase and the ionic mobility due to lower specific surface for a given specific charge. Pre-column derivatization with 90 was followed by micellar electrokinetic chromatography (MEKC) and LIF detection. Combined derivatization with 73 and 90, to attach fluorescent markers to primary and secondary amino acids in biological samples, was investigated327. Derivatization with 78 in the presence of cyanide afforded fluorescent derivatives of amino acids that were separated by MEKC; LOD of 0.9 amol (attomol, 10 18 mol) at SNR 2, using LIF, at 200 mM concentrations, with injections of ca 2.5 nL328. The analysis of thirty dansyl (92) derivatized amino acids by MEKC was investigated. Sodium dodecylsulfate micelles were used for neutral and acidic amino acids, attaining separation efficiency between 210,000 and 343,000 theoretical plates; LOD was 3 6 fmol, RSD 0.09 0.70% for migration times and 0.85 3.41% for peak area. Sodium cholate micelles were used for basic amino acids. The method was demonstrated for determination of amino acid composition in foodstuffs and skin329.

Applicability of CZE to the Edman phenylthiohydantion derivatives of amino acids (140) is limited because the neutral amino acids cannot be resolved by this method and by the reduced thickness of the sample requiring relatively high concentrations of the fluorescent material for detection. These limitations may be overcome by a micellar technique that confers mobility to neutral 140 species and by application of thermotropic

detection that allows one to detect a few tens of fmol of the derivative, obtained after injecting ca 0.5 nL, at a concentration of ca 1 mM330.

The thiohydantoin derivatives of amino acids obtained from 4-(4-dimethyaminophenyl- azo)phenyl isothiocyanate (141) and fluorescein isothiocyanate (133) can be separated by CZE. Lowering the absolute detection limits of thiohydantoin derivatives of the amino acids is a basic requirement for the development of highly sensitive protein sequencer based on Edman-like processes. Thus, the absolute LOD of thiohydantoin derivatives are at present of the order of 10 16 mol for 141 and 10 21 mol for 133331.

The basic material in seeds that is extractable with trichloroacetic acid solutions is ascribed to nonprotein nitrogen when the acid is in the 0.4 1.0 M concentration range. Gel electrophoresis on a sodium dodecylsulfate polyacrylamide medium pointed to the presence of 12 kDa polypeptides in soybean meal and 7, 10, 12 and 28 kDa in almond meal332.

F. Spectrophotometric Methods

Aromatic amines can be determined by measuring the difference of their UVV absorption spectra, taken at identical concentrations but different pH of the solution. Also, standard mixtures and samples of the amines isolated from coke processing products were tested; LOD 0.1 1 ppm. The procedure is potentially useful for waste waters and industrial effluents, where techniques such as GC and nonaqueous titrations may prove difficult to apply333. A determination of certain metabolites symptomatic of pancreatitis

consists of basic hydrolysis of urine, followed by spectrofluorimetric determination of p-aminobenzoic acid and p-aminosalicylic acid334.

o-Aminophenol undergoes oxidative dimerization followed by hydrolysis, yielding the intensively colored 2-hydroxy-3H-phenoxazin-3-one, as shown in reaction 16. Halogensubstituted o-aminophenols in urine were determined by the same reaction335.

Modifications of the Bratton Marshall method mentioned in Section IV.D.3.g can be used for sensitive spectrophotometric detection and determination of primary aromatic and heterocyclic amines. Thus, a simple spectrophotometric determination of the cardioprotective agent acadesine (142) was developed, to measure concentrations of the drug in plasma during intravenous infusion to patients undergoing coronary artery bypass graft surgery336.

CONH2

N

NH2

H2 O3 PO CH2 O N

H H

H H

HO OH

(142)

Amino acids can be determined according to reaction 17. The resulting dithiocarbamates have two specific absorption bands at max 255 and 285 nm. Lysine and cysteine have almost double the molar aborbance because of reaction of the additional NH2 and SH group, respectively337. The kinetics of this reaction can be used for the determination of secondary amines, by measuring the absorbance of the Cu(II) complex with the N,N- dialkyldithiocarbamate, at max 440, in a stop-flow cell. The products of primary amines are unstable. The reaction rate of n-alkylamines is faster than that of isoalkylamines. Micelle formation by addition of Triton X-100 improved the method338.

A spectrophotometric method for determination of primary and secondary amines requires development for each particular compound, determining the kinetics of reaction of the amine with sodium 1,2-naphthoquinone-4-sulfonate (143) and the UVV absorption spectrum of the product, under a set of fixed conditions. The procedure was applied to determination of ephedrine (30) and amphetamine (28) in pharmaceutical samples339. Reagent 143 in a FIA system was used for the fast determination of lysine (144) in commercial feed samples by multivariate calibration techniques, without need of chromatographic separation340.

O

O

CH2 CH2 CH2 CH2 CHCO2 H

NH2 NH2

SO3 Na

Reaction 7 (Section IV.D.3.a) was applied for the automatic kinetic-fluorometric determination of primary amine pharmaceuticals, using N-acetylcysteine (118c) as thiol and a stop-flow technique for data acquisition194. A FIA system was designed for determining the total free amino acids in seawater, based on reaction 7 and measurement of LIF ( ex 337 nm, fl 455 nm) using a diode array. The signals of dissolved ammonia and urea are weak. Possible interference by ammonia can be eliminated by the time-resolved fluorescence technique, because the fluorescent lifetime of the ammonia derivative is 9 ns vs 21 ns for that of all the amino acid derivatives. Linearity was observed in the 1 500 nM range of alanine equivalent. The method is suitable for real-time analyses in on-board laboratories341. The results from this FIA method compared well with HPLC determination of the free amino acids. Only in the case when ammonia concentration strongly overbalanced that of the free amino acids (ratio × 10) did the FIA method fail342.

The electron acceptor 7,7,8,8-tetracyanoquinodimethane (TCNQ, 145) is capable of abstracting one electron from a donor molecule, yielding deeply colored solutions of a

NHCHCH2 CH2 CH2 NH2

Me

stable radical anion that can be measured spectrophotometrically. This was applied to the determination of the antimalarial drugs amodiaquin (146) hydrochloride, chlorodiaquin (147) phosphate and primaquin (148) phosphate in pharmaceutical formulations343.

A solution containing an amino acid is passed through a polymeric bed containing Cu(II) ions, forming a complex which is further reacted with zincon (149) and the blue color measured at 600 nm in a FIA system. This was applied for fast determination of amino acids in pharmaceutical formulations344.

SO3 −

(149)

A sensitive method for the spectrocolorimetric determination of primary or secondary amino functions attached to a solid support consists of derivatizing with either 2- iminothiolane (150, Traut’s reagent) or sulfosuccinimidyl 3-(4-hydroxyphenyl)propionate (151). The products contain one mercapto or phenolic OH group attached to each amino site, which are capable of reducing Cu(II) to Cu(I) in alkaline medium. Thus, the derivatized sample is incubated with the so-called 2,20-bicinchoninic acid copper protein reagent, containing 152 and Cu(II) ions, yielding an intensely colored chelate complex with Cu(I) ions345. Derivatization with 150 and 5,5-dithiobis(2-nitrobenzoic acid) (153, Ellman’s reagent) as the chelating agent was also recommended for determination of primary amino groups attached to the surface of solid supports346.

The lysine and hydroxylysine sites of gelatin were determined by combining their free amino groups with fluorescamine (154) and measuring the induced photoluminescence347. Primary amino groups covalently attached to the surface of glass were determined by derivatization with the same reagent and measurement of the fluorescence348.

O

O

O

O

(154)

Latent fingerprints on paper have been revealed by combining the amino acids present with reagents such as ninhydrin (see 37), dansyl chloride (92), fluorescamine (154), 4- chloro-7-nitrobenzofurazan (127a) and o-phthalaldehyde (see reaction 7). To avoid some problems encountered with these reagents it was proposed to use 1,8-diazafluorenone (155), leading to the formation of highly fluorescent ylides (156)349.

Amines form ion associates of the type [dye (amine HC )] or [dye (amine HC)2] with sulfonphthalein-type dyes, such as bromophenol blue (157a) or bromocresol green (157b). Spectrophotometry using these ion associates may give wrong results if the amines were once dissolved in halogenated solvents, due to quaternization between the amine and the solvent. This was demonstrated for a series of amines such as ephedrine (30), tropine (158), atropine (159), quinine (160) and ajmaline (161), and a series of solvents such as CH2Cl2, CHCl3, CCl4 and ClCH2CH2Cl350.

A series of calixarenes bearing azo groups, such as 162a c, are potential detectors of aliphatic primary amines, as they showed bathochromic shifts of 37 100 nm frommax 382 with these compounds. No shift was shown in the presence of aniline or p- nitroaniline351.