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

N

H

(47)

spectra to yield the second-order derivative. Comparison of the derivative spectra with those of standards consisting of the aromatic amino acids and their heterodipeptides, using spectral features such as amplitude and wavelength of primary and secondary minima and intersection with the abscissa, helps corroborating and quantifying their presence151.

The series of regioisomeric amines 48 50, methamphetamine (29) and phentermine (31), can be identified in forensic screening analyses by RP-HPLC-UVD (254/280 nm dual accessory) using a C18 stationary phase and a mobile phase buffered at pH 3.0. The capacity factors and retention times increase in the order 48 < 49 < 29 < 31 < 50. Other methods for identifying these compounds failed; for example, the base peak in MS is m/z D 58 for all five compounds, corresponding to a loss of a benzyl group from the molecular peak; also their IR and UVV spectra are too similar to be useful for this purpose152.

Analysis of a wide range of amines, in dialysate aliquotes taken from experimental animals, was carried out by isocratic HPLC with detection on a series of eight coulometric electrodes, measuring from 0 to 0.490 V with increments of 0.070 V. A parallel analysis was carried out after pre-column derivatization according to reaction 7 (Section IV.D.3.a), isocratic elution on a different column and measurement with a series of four electrodes set at 0.250, 0.450, 0.550 and 0.650 V. Compounds were identified by retention time and electrochemical profile along the arrays. Analyses were complete within 25 min. Among the compounds examined were isoproterenol (21c), phenylephrine (51), methoxamine (52), hydralazine (53), apomorphine (54), morphine (55) and its 3-glucoronide metabolite; for these compounds LOD 0.215 10.65 mg/L, at SNR 3, with linearity in the 0.5 500 mg/L range. For amino acids LOD ca 0.75 mg/L, at SNR 3, with linearity in the 0.25 20 mg/L range153.

RP-HPLC determination of trace impurities of the toxic 4-aminopyridine in the central system-stimulating drug 3,4-diaminopyridine can be performed on condition that the impurity has a lower retention time. This was accomplished on applying ion pairing with dodecanesulfonate to maximize selectivity; LOD 50 ppm of the impurity in the drug154.

(54)

CH3

N

OH

H

O

HO

(55)

Methods were described for HPLC determination of the mutagenic and carcinogenic ˛- carbolines (56,57), -carbolines (58,59)155 160, and other products of amino acid pyrolysis found in cigarette smoke, diesel exhaust and cooked foods and phenazines (60, 61) present as impurities of certain pesticides161. These compounds were also determined in human plasma, urine and bile161,162.

The use in consumer products of azo dyes that yield carcinogenic amines under reductive conditions is illegal in Germany. Detection of such carcinogenic amines in textiles is problematic, and a method was proposed combining TLC, automatic multiple development

and assessment by means of a scanner163. Azo dyes present in sludges can be determined after extraction with dichloromethane, reduction with sodium hydrosulfide or tin(II) chloride and HPLC-MS164. See also discussion of reaction 3 in Section IV.C.

A sensitive HPLC method used coulometric detection for the simultaneous determination of catecholamines, indoleamines and related metabolites. Oxidative and redox modes were applied to the various analytes, using arrays containing one to four coulometric working electrodes165. Pulsed amperometric detection following HPLC of the underivatized amino acid is sensitive but has a limited range of linear response. Application of ELCD before amperometric detection extends the dynamic range of amino acid determination166. Pulsed amperometric detection after IEC was found to be more sensitive (0.01 1.2 mM) than ninhydrin derivatization with UVD (4.5 55.0 mM)167.

Determination of the amino saccharides glucosamine, mannosamine and galactosamine in microbial polymers, chitin, animal waste, sewage, plant residues and soil was performed by HPLC using a strong ion-exchange column, an alkaline eluent and a pulsed amperometric detector. The latter was superior to RID. More than 3% of the total nitrogen in alfalfa and 20% in straw stems from amino saccharides168.

A glassy carbon electrode (GCE) modified by electrodeposition of Ru(III,IV) oxides was used for the amperometric determination of cystine, cysteine, methionine, glutathione and glutathione disulfide after HPLC using a strong cation exchange column. Unmodified electrodes are unfit for analysis of these compounds. For methionine, sensitivity was 20 š 0.3 nA/mM/cm2; linearity over the range of 0.6 180 mM at pH 2, 7.5 mL sample, 1 mL/min flow169,170. A constant potential amperometric detector was used in a FIA system for the determination of carbohydrates and amino acids. The working electrode is an Eastman-AQ electrode chemically modified by Ni(II) ions. The mechanism shown in reactions 4 6 was proposed, where reaction 5 is rate limiting171.

A comparison was made between Ag, Au, Co, Cu, Ni and Pt electrodes for constantpotential amperometric detection of carbohydrates, amino acids and related compounds in FIA systems. Cu electrodes showed the best performance as for their range of linear response, LOD, stability and long life172. Two electro-oxidation processes of amino acids at a Cu electrode are possible, depending on the applied potential and the conditions of the solution: In neutral or slightly basic solution, at very low potentials measured against an Ag/AgCl electrode, the process is related to complex formation between the amino acid and Cu(II) ions. In a strongly alkaline solutions, at 0.4 0.8 V the process involves electrocatalytic oxidation. The latter process is better for constant-potential amperometric detection of underivatized amino acids and peptides in FIA chromatographic systems; LOD 1 10 pmol for most amino acids and simple peptides173.

Nitrogen-containing analytes in a FIA system, e.g. amino acids, as they emerge from the LC column, are introduced into a pyrolysis oven under argon atmosphere. The products

are converted to nitrate by potassium peroxidisulfate, and determined with malachite green using a UVD at 650 nm174.

Determination of iodo amino acids by HPLC with inductively coupled plasma (ICP)- MS detection had LOD 35 130 pg of I, which is about one order of magnitude lower than with UVD usually applied for these compounds175. Amino acids and peptides containing sulfur, such as cysteine, cystine, methionine and glutathione, can be determined after HPLC separation by pulsed electrochemical detection, using gold electrodes176.

It is usually a very difficult task to introduce chemical modifications in the solid phase filling the chromatographic column. On the other hand, the possibilities of modifying the carrying fluid used for elution are practically unlimited. This includes solvent composition in isocratic and gradient regimes, buffers and other additives. For RP-HPLC on C18 columns, ion interaction reagents such as octylammonium salycilate or orthophosphate are used to modify the properties of the absorbing surface, improving the resolution of many mixtures. Thus, trace levels of aromatic amines can be determined without derivatization. Applying this method to the analysis of a commercial brown hair dye revealed the presence of more than 7000 ppm of p-phenylenediamine177,178. The same two ion interaction reagents were applied to the RP-HPLC-UVD determination of the food-related biogenic amines tyramine (5), histamine (6), 2-phenethylamine (33) and tryptamine (40). The elution sequence was different for both additives; LOD 400 ppb for 5 ( 230 or 280 nm), 900 ppb for 6 ( 230 nm), 500 ppb for 33 ( 254 nm) and 20 ppb for 40 ( 280 nm)179. RP-HPLC using alkylammonium salicylates as ion interaction reagents is effective in the separation of amines from inorganic analytes such as nitrite and nitrate ions. However, care should be taken that alkylamine analytes should be of shorter chain-length than the ion interaction reagent. By this method 0.50 ppm of p- phenylenediamine and 0.20 ppm of nitrate could be determined in seawater180. RP-ion pair chromatography of amino acids was performed using 1-naphthylamine as ion-interaction reagent with sodium heptanesulfonate as hydrophobic counterion, to enhance the capacity ratio of the column for all amino acids tested181. Amino acids and peptides yield under physiological conditions carbamates of structure 62, which are presumed to be neurotoxic agents. A correlation was found between the propensity of these compounds to undergo such transformation and their RP-LC behavior in the presence of cetrimide (63), a cationic surfactant that makes the separation technique sensitive to the negative charge on 62182.

Determination of halogenated 2-aminobenzophenones (64a c), which are metabolites of psychotropic drugs, was performed by HPLC with amperometric detection (GCE vs Ag/AgCl); LOQ 750 ng of metabolite/L of biological fluid (urine or serum), with recovery better than 97%183.

The impurities of H-acid (65), an intermediate for various synthetic dyes, include Koch’s acid (66), omega acid (67), chromotropic acid (68) etc. They were determined by RP-HPLC-UVD at 235 nm, using as mobile phase a 0.3 M aqueous solution of sodium sulfate184.

The chemiluminescence of the reaction of hydrogen peroxide with luminol (69) is catalyzed by metalloporphyrins 70a and 70b. This chemiluminescence is quenched by

NH

NH

O

the presence of amines that form complexes with the metal ions and reduce the catalytic effect of the porphyrine complexes. This abatement is the working principle of a method proposed for determination of amino acids in a FIA system following LC185.

The mutagenic aminophenazines 71 are present as impurities of carbendazim (72) fungicides and its formulations. They were determined by HPLC-UVD (diode array), using 0.02% sulfuric acid in MeOH, and measuring at 270 and 453 nm186.

The neutral nitrogen-containing components in diesel oil were selectively retained by an alumina HPLC column and were eluted by a gradient mobile phase of hexane dioxan. Identification and determination was carried out by a combination of MS and UV diode array detectors, showing that these components were mainly alkylcarbazoles and alkylindoles187.

Aromatic and sulfur-containing amino acids were separated by HPLC, and subjected to post-column UV irradiation before electrochemical detection with GCE vs AgCl/Ag electrodes. The analytes showed different behavior during lamp off and on periods. Thus, for example, tyrosine (46) and tryptophan (47) showed inherent electrochemical response at C0.80 V, but none at C0.60 V; however, on turning on the UV lamp they showed sensitive response at both potentials126.

3. Pre-column and post-column derivatization

Pre-column derivatization procedures fulfil several important analytical functions. However, some problems are involved such as separation of the analytes from the matrix with the inherent recovery problems; modification of the analyte to improve chromatographic resolution with the inherent problems of functional specificity, derivatization yields of individual analytes, stability of the derivative and the necessity of removing excess reagents. Although automatization of pre-column treatment is now commonplace, this is not usually a requirement. Post-column derivatization is performed mainly for labelling the analyte to better fit the installed detector and is frequently applied in FIA systems. This requires fast and effective reactions with minimum excess reagent or a means for removal of excess to avoid interferences. Additional targets may also be sought in post-column derivatization, such as modification of retention times in a second chromatographic cycle.

A comparative study was made of the RP-HPLC analysis of free amino acids in physiological concentrations in biological fluids, with pre-column derivatization by one of the four major reagents: o-phthalaldehyde (73) in the presence of 2-mercaptoethanol, 9- fluorenylmethyl chloroformate (90), dansyl chloride (92) and phenyl isothiocyanate (97, R D Ph) (these reagents are discussed separately below). Duration of the analysis was 13 40 min. Sensitivity with the latter reagent was inferior to the other three; however, its use is convenient in clinical analysis, where sample availability is rarely a problem. The derivatives of 73 were unstable and required automatized derivatization lines. Only 92 allowed reliable quantation of cystine. All four HPLC methods compared favorably with the conventional ion-exchange amino acid analysis188.

Some examples of derivatization used in LC are shown here and in Sections IV.D.4 and IV.E.

a. Reaction with dicarboxaldehydes. Primary amines react with o-phthalaldehyde (73) in the presence of 2-mercaptoethanol, as shown in reaction 7, yielding fluorescent isoindole products (74)189,190. This reaction affords a very frequently used pre-column and postcolumn derivatizing scheme.

CH O

(7)

CH O

SCH2 CH2 OH

Some problems and solutions involved in the automatization of pre-column derivatization of amino acids applying reaction 7 to biological samples were discussed191. Solid media and conditions were examined for enrichment by SPE of the lower aliphatic amines in aqueous solution, prior to HPLC with fluorometric detection of the o-phthalaldehyde derivative. The best medium was the weak cation exchanger Spheron C 1000, and desorption with methanolic perchloric acid. Concentration limits for the determination were in the nM range, with enrichment factor of 240 in the preconcentration step192. The problem of the stability under laser-induced fluorescence (LIF) of the fluorescent species 74, derived from amino acids and peptides in biological samples, in the concentration range of 10 12 to 10 15 M was addressed193. N-Acetylcysteine was proposed as replacement for 2-mercaptoethanol, to avoid its unpleasant odor194; -glutamylcysteine provided its own mercapto group, as shown in reaction 8193. See also references to reaction 7 in Section IV.D.4.

A study of residual analysis of thirty pesticides and their transformation products was based on SPE on-line with HPLC-UVD or post-column derivatization with o- phthalaldehyde (73) and fluorescence detection (FLD), according to EPA method 531.1 and others. The method allowed determination of many pesticides in river and well waters at 0.01 to 0.5 mg/L levels195. An automatized procedure was proposed for determination

of amino acids in plasma based on pre-column derivatization with this reagent that was claimed to be fast and stable for long series of analyses; the coefficient of variation was <3% for most of the thirty physiological amino acids tested196.

Biogenic amines such as -aminobutyric acid, spermidine (38), spermine (75) and hexamethylenediamine (4c) were determined by pre-column derivatization according to reaction 7, gradient elution LC and amperometric detection at C650 mV, using GCE vs Ag/AgCl electrodes. On column detection limits are at the low picomole range197. A method was evaluated for determination of amines in wine, such as polymethylene diamines (4a c), tryptamine (40) and 2-phenethylamine (33), based on pre-column derivatization with o-phthalaldehyde (73), RP-HPLC with gradient elution and coulometric detection with an array of sixteen electrodes at increasing potentials198. The same derivatization served for determination of biogenic amines in wine by RP-HPLC-FLD on a C18 column; ex 356 nm, fl 445 nm, recovery 84.0 108.3%, RSD 3.0 8.2%199. Amino acids in cultured cells were determined by RP-HPLC-FLD; ex 330 nm, fl 450 nm, LOD 0.5 pmol, linearity in the 1 800 pmol range200. Determination of -aminobutyric acid in cerebrospinal fluid requires special care, because of its relatively low concentration as compared to other amino acids and the possibility of distorting results on degradation of homocarnosine (76). A procedure was proposed consisting of deproteinization with sulfosalicyclic acid, separation by IEC, post-column derivatization according to reaction 7 and FLD measurement201. A method for determination of ε-aminocaproic acid in body fluids consists of deproteinization with zinc sulfate, addition of D-valine as internal standard, derivatization according to reaction 7 and HPLC-FLD using a mobile phase containing 2.5 mM of the Cu(II) complex of L-proline. As low as 50 mg/L of the analyte could be detected, using 100 mL samples of urine or plasma202.

Fast determinations of amino acids in plasma, cerebrospinal fluid and other media were based on reaction 7 and RP-HPLC. In one of them, taking 17 min per run, including times for derivatization and column re-equilibration, recovery of amino acids spiked into plasma was 96 106%, except for tryptophan (47, 89%). The method had within run precision of 1.8 6.4%, between run precision of 2.1 7.2% and was linear in the 5 800 mM range for all amino acids203. RP-HPLC with electrochemical detection, using a multistep polarity gradient, resolved the derivatives of twenty-three amino acids and physiological dipeptides in less than 25 min204,205. A fast variation claimed runs of less than 5 min for simultaneous determination of the neurotransmitter amino acids glycine, aspartic (34a), glutamic (34b), taurine (77) and -aminobutyric acid in brain tissue; LOD 2.5 pmol for all five amino acids206. A modification allowed improved resolution and sensitivity and shorter performance times. This was applied to determination of sixteen amino acids in plasma and cerebrospinal fluid207.

H2NCH2CH2SO3H

(77)

Amino acids and other nitrogen-containing compounds affect chlorine consumption in the treatment of drinking water. Such amino acids from three French rivers were determined in the raw samples and, after each treatment step in a plant, by pre-column derivatization by reaction 7 and HPLC-FLD. The main compounds detected were glycine, serine, alanine, aspartic acid, glutamic acid, threonine and valine. The global amino acid content was 20 90 mg N/L, causing a consumption of 0.4 1 mg Cl2/L208. Use of a basic citrate buffer for the pre-column derivatization step of amino acids in seawater avoids precipitation of Ca and Mg hydroxides209.

Post-column application of reaction 7 was used to bind the ε-amino groups of lysine residues in a hemoregulatory peptide, for determination by HPLC-FLD; LOD 1 ng, LOQ 20 mg/L for 0.25 mL plasma samples; response was linear for 20 4000 mg/L of plasma210. Reaction 7 was also applied to amino acids in seawater in a FIA system. Determination of ammonia vs primary amines could be accomplished in the system by a buffer change from pH 7 to pH 10.5, respectively211. A fast method for determination of biogenic amines is important for assessing the freshness of sea food and its possible allergenic and toxic effects. The simultaneous detection of histamine (6), agmatine (39) and other polyamines poses a difficult problem. A method for determination of these compounds uses sodium hexanesulfonate as ion pairing agent in RP-HPLC and post-column derivatization with o-phthalaldehyde (73)212. Perchloric acid extraction, RP-HPLC-UVD (diode array) using post-column derivatization by reaction 7 was applied in the determination of the biogenic amines putrescine (4a), cadaverine (4b), tyramine (5), histamine (6) and phenethylamine (33) in meat products; LOD 0.5 mg/kg with 96 113% recoveries213. Biogenic amines 4a, 4b, 5, 6, 33, serotonine (20), tryptamine (40), spermidine (38), spermine (75) and agmatine (39) were determined simultaneously in beer after microfiltration, RP-HPLC, post-column derivatization with 73 and FLD; LOD 0.30 0.40 mg/L except for 20 and 75, which were slightly higher. Linearity, precision, sensitivity and recovery were satisfactory, and the interference from amino acids and other amines was assessed214. The effect of SPE was studied on the determination of thirty pesticides and their transformation products was based on derivatization by reaction 7, using UVD or FLD (EPA method 531.1). The method was used for analysis of river waters of Spain and France195.

A variant of reagent 73, naphthalene-2,3-dicarboxaldehyde (78), was used for determination of amino acids in tobacco215. Analytes containing more than one primary amino group show a much diminished quantum efficiency, which can be corrected by coordinated deposition of the LC effluent on a TLC plate and performing a second fluorometric measurement of the immobilized derivatives216. An HPLC-CLD method was proposed, based on measurement of the chemiluminescence emitted by the pre-column 78-derivatives of the analytes in the presence of a diaryl oxalate (42) and hydrogen peroxide, for determination of ultratrace amounts of amphetamine (28), norephedrine (79) and p-hydroxyamphetamine (80) in urine, using phenethylamine (33) as internal standard; LOD 0.1 1.5 ð 10 15 mol217. See discussion of reaction 24 in Section IV.G.

The painstaking procedures and special instrumentation required for manipulating nanoliter range volumes were described for the determination of amino acids in single cells218 and the amino acid analysis of subnanogram amounts of protein219. The amino acid extract

of a single cell was derivatized with 78 in the presence of cyanide ions, leading to products of probable structure 81, which were both fluorescent and electroreactive. Separation was by open tubular microbore LC and the derivatives were measured electrochemically218. Using norleucine as internal standard, and the various amino acids contained in the hydrolyzate of a 5 nL sample of protein solution were determined by the method just described219. A high-sensitivity LIF-based detector for HPLC was developed incorporating a HeCd laser, which responds specifically to amines derivatized with 78 in the presence of cyanide ions; LOD ca 10 12 M220.

CN

NR

(81)

Peptides containing a tryptophan residue at the N-end can be determined by pre-column derivatization with glyoxal and RP-HPLC-FLD, as shown in reaction 9, giving single fluorescent peaks; LOD 0.55 3.82 nM (SNR 3) for 100 mL injection volume221.

(9)

CONH peptide

N

N

H

CH2 OH

See also reaction 15 in Section IV.E.

b. Oxazole derivatives. Various oxazole-based reagents have been proposed for tagging amines and thiols. For example, 2-fluoro-4,5-diphenyloxazole (82a) and 2-chloro-4,5- bis(p-N,N-dimethylaminosulfonylphenyl)oxazole (82b) for pre-column derivatization of amino acids followed by HPLC-FLD; LOD for the 82a derivatives of thirteen amino acids is in the 19 64 fmol range (SNR 2). Amino acids with thiol substituents had LOD of one order of magnitude lower. Chemiluminescence with hydrogen peroxide/oxalate esters also afforded very sensitive determinations222,223. Sodium benzoxazole-2-sulfonate (83) itself is not fluorescent, but with amines and amino acids the derivatives exhibit an intense blue fluorescence224.

c. N-Acylation and N-sulfonation. By pre-column benzoylation in biological fluids, the hydroxy groups of sugars become esterified and neutral amino acids are converted to the corresponding 5-benzoyloxy-2-phenyloxazoles (84). No protein precipitation takes place and no pyridine or drying are required. Determination by HPLC-MS has LOD ca 1 pmol (SNR 2)225. Benzoylation followed by RP-HPLC on a C18 column detects biogenic amines in fish, including putrescine (4a), cadaverine (4b), phenethylamine (33), spermidine (38),