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

spermine (75), histamine (6), tyramine (5) and agmatine (39)226. Mixtures of industrial polyvalent amines (85) were determined by RP-HPLC-UVD after derivatization with benzoyl chloride or m-toluyl chloride227,228. Ferrocenecarboxylic acid chloride (86) was used for tagging primary and secondary amines, amino acids and peptides. End analysis was by LC with electrochemical detection for electro-oxidation of ferrocene; LOD 500 fmol229.

Compound 87 in acid-hydrolyzed urine serves as a tracer for occupational exposure to the corresponding diisocyanate. It was derivatized with pentafluoropropionic anhydride and determined by LC using TSP-MS and plasmaspray (PSP) MS (discharge-assisted TSP-MS). The [M 2] ion was measured; instrumental LOD 0.1 pg/mL; LOD about 0.2 mg/L urine, RSD 10% for 0.5 mg/L230. Another determination of 87 in urine is with isobutyl chloroformate231.

(87)

A determination of traces of low (C1 to C4) aliphatic amines in the atmosphere consists of passing air through an absorber containing phosphorous acid, derivatizing with m-toluyl chloride and end analysis by HPLC-UVD; LOD 1 5 pmol of amine, corresponding to concentrations lower than 0.1 mg/m3 of air, in a 300 L sample232.

Various benzoxadiazole reagents have been proposed for fluorescent labelling of alcohols, phenols, thiols and amines. Reagent 88 was used for derivatization of various substrates, including aliphatic and aromatic amines233,234. Derivatization of alcohols and amines with reagent 89 affords flourescent labeling of these compounds, when excitation and fluorescence were essentially the same for all alcohols and amines tested with both reagents. Using LIF (ArC emission at 488 nm) improves the sensitivity of the method (LOD 2 10 fmol) as compared with a conventional Xe lamp (LOD 10 500 fmol). See also Section IV.D.4 for other benzoxadiazole reagents.

(ca 100 L of air), desorbed with dilute acid and determined by injection of a 5 10 mL sample into a reactor containing 91, on-line with HPLC-FLD243. Reactive polymers were synthesized for tagging amines with 9-fluorenylmethoxycarbonyl, 4-nitrobenzoyl and acetylsalicyl groups. The reactive polymers were combined into a mixed bed reactor for on-line pre-column derivatization in HPLC analysis of amines. The objective of multiple pre-column tagging was to aid identification of unknown analytes in a complex matrix. The method was demonstrated for amphetamine (28) in human urine with acceptable accuracy and precision244.

Nineteen biogenic amines in wine were determined by derivatization with dansyl chloride (92), SPE and HPLC-UVD. Linearity was observed for amine concentrations in the 0.5 20 ppm range; LOD 50 150 mg/L (SNR 3), which is typical of dansyl derivatives. Addition of standard amines showed recoveries better than 85% for ethanolamine, phenethylamine (33), putrescine (4a), cadaverine (4b), tyramine (5) and histamine (6)245,246. The dansyl derivatives of the latter five biogenic amines, tryptamine (40), spermidine (38) and spermine (75) were separated by TLC on silica gel plates. None of the twelve solvent systems studied could resolve the mixture; however, application of two-dimensional TLC did, using extraction with acetonitrile and spectrofluorometry. The method was applied for analysis of fish and dry sausage samples247, and also for analysis of biogenic amines in fermented olives248. After post-column addition of tris(2,20- bipyridyl)ruthenium(II) complex (44) and electrochemical oxidation in a flow cell to Ru(III), this ion reacts with the dansyl derivatives in situ and the chemiluminescence of the reaction can be measured249. An HPLC-CLD method based on measurement of the chemiluminescence emitted by the pre-column dansyl derivatives of the analytes in the presence of bis(2,4,6-trichlorophenyl) oxalate (42) and hydrogen peroxide was proposed for determination of ultratrace amounts of methamphetamine (29), amphetamine (28), norephedrine (79), p-hydroxymethampetamine (93) and p-hydroxyamphetamine (80) in urine, using phenethylamine (33) as internal standard; LOD 1 3 ð 10 14 mol217. A simplified automated procedure for pre-column derivatization of amino acids with 92 was proposed250.

SO2 Cl

4-HOC6 H4 CH2 CHMe

NHMe

NMe2

A good correlation between experimental retention times and calculated selectivities and molecular connectivities was found using the PRISMA model for seventeen dansylated biogenic amines present in foodstuffs and animal fodder251.

Derivatization with dabsyl chloride (94) was applied for the separation of primary amino acids in physiological samples, prior to determination of their specific radioactivity. The derivatization is easy to perform and the derivatives are stable252.

N-Acylation can be preformed with esters of N-hydroxysuccinimide; N-succinimidyl 4-nitrophenylacetate (95) was used to derivatize the primary and secondary amines conferring bad odor to water253. Derivatization of amines with the ester of N- hydroxysuccinimide with N-(quinolin-6-yl)carbamic acid (96) gives excellent yields of

(94)

unsymmetric ureas. The selective fluorescence of the derivatives allows direct injection of the reaction mixture with no interference of the excess reagent; LOD from 40 fmol for phenylalanine to 800 fmol for cystine, linear response in the 2.5 200 mM range. Good amino acid analyses could be obtained from protein hydrolysates containing as little as 30 ng of sample254. By derivatization with reagent 96 nineteen amino acids were separated in 35 min with resolution of at least 1.6, seventeen of which showed linearity at concentrations of 25-500 mM255. A study of the long-term repeatability and consistency of amino acid analyses showed that derivatization with 96 was superior to that with phenyl isothiocyanate (reaction 11)256. See also Section IV.E for other acylating reagents derived from N-hydroxysuccinimide (134, 135).

d. Reaction with isothiocyanates. Primary and secondary amines in general react with isothiocyanates (97) to yield the corresponding thioureas, as in reaction 10; however, ˛- amino acids can undergo further cyclization to the corresponding thiohydantoins (98), the classical Edman reaction 11.

Derivatization with phenyl isothiocyanate (97, R D Ph) followed by HPLC was compared with IEC followed by the ninhydrin reaction for over ninety compounds. The former method was favored for speed, sensitivity and equipment versatility257. Phenylthiocarbamyl derivatives of amino sugars and amino sugar alcohols (reaction 10) were

resolved by RP-HPLC258. Twenty-two protein amino acids underwent derivatization with butyl isothiocyanate (97, R D Bu) and were determined by RP-HPLC-UVD; RSD of the molar response relative to the methionine peak was less than 5% for all except cysteine; asparagine and serine were not resolved from one another259. RP-HPLC on a C18 column with a multistep linear gradient of two solutions was investigated for the phenyl isothiocyanate derivatives of twenty-six amino acids260, and also for determination of amino acids in deprotenized blood261. Modifications were proposed to make it faster, for the analysis of nutritionally important amino acids in serum and internal organs262. It was claimed that RP-HPLC of the phenyl isothiocyanate derivatives of plasma and urine amino acids, followed by electrochemical detection, virtually eliminates interferences of other components in the sample and enables the determination of secondary amino acids263. Methods based on derivatization with phenyl isothiocyanate were proposed for determination of free amino acids in wine and must264 and infant food265,266. The derivatives obtained on treatment of amino acids with 4-nitrophenyl isothiocyanate are stable and suitable for subsequent HPLC-UVD analysis267.

A study was made of RP-HPLC with constant-potential (1.2 V vs SCE) and pulsedpotential amperometric detection using platinum or gold electrodes, of the derivatives of the common amino acids, obtained from phenyl and methyl isothiocyanates. All the thiohydantoins (98) were oxidized at both electrodes; LOD was less than 0.2 mM for lysine and glycine, for 50 mL injection268.

e. N-Arylation. A comparative study was carried out for the analysis of amino acids in serum by pre-column derivatization with o-phthalaldehyde (73) and N,N-diethyl- 2,4,dinitro-5-fluoroaniline (99) followed by HPLC, and IEC followed detection by the ninhydrin method (37) in an amino acid analyzer. Good agreement was found for the three methods, but pre-column derivatization was more sensitive and faster. Good resolution was found for thirty amino acids with 73 and thirty-eight with 99269. See also reagents 127 in Section IV.D.4.

F

NO2

Et2 N

NO2

(99)

f. Schiff bases. Measurement of the chemiluminesce of Schiff base formation (reaction 12) can be applied for determination of primary amines, by post-column derivatization in a FIA system. Sodium bis(2-ethylhexyl) sulfosuccinate (100) is also added to form reversed micelles to accelerate the reaction270. The chemiluminesce of the oxidation of Schiff bases with the Fenton reagent in a FIA system was proposed; LOD 1.5 ð 10 8 M for hexylamine and 1.4 ð 10 7 for alanine, with linear behavior in the 10 5 10 3 M range. The mechanism depicted in reaction 13 was tentatively proposed, where PhCHOŁ is a benzaldehyde molecule in a triplet state271.

n-Bu NaO3 S CO2 CH2 CHEt

CO2 CH2 CHEt

n-Bu

(100)

PhCH2 CHO + H2 NR PhCH2 CH NR

(13)

Ph NHR

PhCHO + RNHCHO

O O

1-Pyrenecarboxaldehyde (101) was used for derivatization of primary aromatic amines to the corresponding Schiff bases, for their determination by HPLC-FLD; LOD 1 2 pmol272.

CH O

(101)

g. Miscellaneous reactions. Catecholamines were oxidized to aminochromes (36) with hexacyanoferrate(III) ion and the products were separated on a C18 column using a micellar mobile phase containing sodium dodecylsulfonate. Detection was by thermal lens spectrophotometry, using the 488 nm line of an ArC laser; LOD ca 4 mg/L. The method was applied for determination of unconjugated catecholamines in urine, using isoproterenol (21c) as internal standard273.

The PRISMA model274 and factorial experimental design were applied in the development of a one-dimensional overpressured layered chromatography separation method for the anti-neoplastic bis-indole alkaloids vincristine (102a), vinblastine (102b) and some derivatives275.

Diazotization and formation of diazo dyes affords a general approach to pre-column derivatization, to be followed by direct phase or RP-HPLC with UVD or FLD. Thus,

sulfanylamide (103), p-aminobenzoyl-ˇ-alanine (104) and p-aminobenzoic acid served as model compounds for diazotization and coupling with 2-aminoanthracene (105). The method was applied for determination of p-aminobenzoic acid in urine. The method was unsuitable for 4-hydroxy or alkyl derivatives of aniline276. Modifications of the Bratton Marshall method277 were proposed for precolumn derivatization, by which primary aromatic amines are diazotized and coupled with N-(1-naphthyl)ethylenediamine (106). The diuretics hydrochlorothiazide (107), bendroflumethazide (108) and furosemide (109) were determined in urine after hydrolysis, diazotization and coupling with 106, with p-aminobenzoic acid serving as model compound. Substituted indoles such as 5-hydroxyindole-3-acetic acid (110) and tryptophan (47) underwent N-nitrosation and interfered with the determination. End analysis was by HPLC with UVD or thermal lens spectrophotometry, using a micellar carrier; LOD for the diuretics was ca 5 nM for the

NH2

4-H2 NC6 H4 SO2 NH2 4-H2 NC6 H4 CONHCH2 CH2 CO2 H

(103)(104)

latter detector, which was 20 50-fold more sensitive than UVD278. The necessity of preparing fresh solutions of unstable nitrite is avoided in a FIA system where nitrate is reduced to nitrite in situ by Cd/Cu, followed by diazotization and coupling with reagent 106. This was applied to fast analysis of sulfadiazine (111), with a throughput of 72/h. UVD measurements at 542 nm were linear in the 0.5 50 ppm range279.

1-(2,4-Dinitrophenyl)pyridinium chloride (112) is a versatile display reagent after planar chromatography (e.g. TLC and paper chromatography), revealing as colored areas on a yellow background. The reagent can be applied for detection of nucleophilic analytes such as primary and secondary amines, thiols, thiolactones and carboxylic acids, as shown in reaction 14280.

NO2

NO2

(112)

(14)

N N

NO2

NO2

4. Chiral purity

See Section IV.I for alternative methods of chiral resolution. Partial chemical hydrolysis of proteins and peptides with hot 6 M HCl, followed by enzymatic hydrolysis with pronase, leucine aminopeptidase and peptidyl D-amino acid hydrolase, avoids racemization of the amino acids281. The problems arising from optical rotation measurements of chiral purity were reviewed. Important considerations are the nonideal dependence of optical rotation on concentration and the effect of chiral impurities282.

Determination of chiral purity using chromatographic methods has been reviewed283. The feasibility of using a circular dichroism spectrophotometer as LC detector for chromophore-bearing chiral molecules was critically examined. Using UVD in tandem with such spectrophotometer may be of advantage284. The sensibility of the chromatographic detectors and the good yields usually attained with derivatizing reagents make it possible to analyze small samples containing low concentrations of chiral amines, such as biological fluids and environmental samples. Usually, methods are based on precolumn derivatization with a chiral reagent, for example SC -Mosher’s acid chloride (113)285,286, followed by destruction of excess reagent, chromatographic resolution and detection of the diasteroisomers. Derivatization of amino acids with the C form of 1- (9-fluorenyl)ethyl chloroformate (114) affords dioasteroisomers that can be resolved and determined by RP-ion pair HPLC. Of the nineteen amino acids investigated tyrosine (46), tryptophan (47) and cysteine (115) could not be detected due to their weak reaction with the derivatizing reagent. The method was applied to the study of amino acids in the nervous system of crustaceans287.

The enantiomeric purity of protected amino acids used in peptide synthesis can be determined by pre-column partial deprotection followed by derivatization with Marfey’s reagent (116). The Marfey diastereoisomers can be easily resolved and determined by RP-HPLC using an ODS-Hypersil column288. Fifteen amino acids collected from mammalian tissues were derivatized with Marfey’s reagent and subjected to two-dimensional TLC. Each individual spot (enantiomeric mixture of a diasteroisomer) was then resolved by RP-HPLC. Except for tyrosine (46) and histidine (117), subnanomole quantities of enantiomers could be analyzed289,290.

A fully automated derivatization system for determination of enantiomeric purity of amino acids is based on derivatization with o-phthalaldehyde (73) in the presence of the chiral thiol N-isobutyryl-L-cysteine (118a) or its D enantiomer (see reaction 7). The diasteroisomeric isoindole derivatives of fourty-one amino acids were separated on a C18 RP-column. The fluorescence allowed detection of 1 2 pmol of an amino acid enantiomer; linearity was good in the 25 1000 pmol range291. Derivatization by reaction 7, using N-t-butyloxycarbonyl-L-cysteine (118b) as mercaptol, was applied to enantiomeric analysis of the free amino acids in brain tissue, revealing the presence of a large amount of free D-serine (0.22 mmol/g, 25% of the free serine found) while D-alanine and D-aspartate are present at trace levels292. Similarly, thiosugars were proposed as chiral thiol reagents for pre-column derivatization of enantiomeric mixtures of 2-amino-1-alcohols. The reaction takes place within 1 min and resolution by RP-HPLC with fluorescence detection is efficient; LOD is less than 1 mM for 10 mL injection293.

(118)

A comparative study was carried out of the effectiveness of three commercially available chiral columns and nonchiral derivatives of amino acids such as N-(3,5-dinitrobenzoyl) esters (119), phenylurea esters (120), hydantoins (121) and thiohydantoins (98). Although good separations were obtained, no column was universally effective294.

Derivatization of secondary amino acids with 9-fluorenylmethyl chloroformate (90), followed by HPLC using a modified cyclodextrin-bonded phase and a nonaqueous polar mobile phase, served to determine enantiomeric impurities which were in some cases as low as 1 ppm of the main enatiomer. The derivatizing group served both as a tracer and as a means for avoiding further racemization of imino acids295. The enantiomeric purity of esters of amino acids derivatized with reagent 90 could be separated on Chiralcel-OD with large separation factors (1.5 2.2). FLD had LOD below 0.05% down to the ppm range. Inversion of the elution order was observed for certain proline and tryptophan enantiomers296.

The chiral reagent 122 was proposed for derivatization of enantiomeric mixtures of amino acids. Good HPLC separations were obtained for the diasteroisomer derivatives of a series of amino acids, including some unusual ˛-amino acids with long or bulky side chains, aryl and hetaryl groups, and ˇ-substituted ˇ-amino acids297.

Nonchiral columns can be used with nonchiral derivatization for better detectability of the analytes, using chiral modifiers of the carrier solvent. For example, RP-HPLC resolution of dansylated (92) D,L-amino acids using L-phenylalanine Cu(II) complex