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

The calcium antagonist nicardipine (17) and its pyridine metabolite M-5 were determined in plasma after LLE and concentration. End analysis was by capillary GC-NPD with temperature gradient; LOD 0.5 mg/L for both compounds93. See also reaction 27 in Section IV.H for electrochemical processes undergone by similar compounds.

CH2 Ph

The sensitivity of FID and ECD toward perfluoroacyl derivatives of amino, hydroxy and mercapto compounds was investigated. Thus, the FID signal of the perfluorovaleryl derivative of a compound increased with the size of the alkyl substituent, but was reduced as compared with the signal of an analogous hydrocarbon. The sensitivity of ECD toward the derivatives varied within two orders of magnitude, depending on the size of the alkyl substituents94.

Determination of biogenic amines in aqueous medium was based on a scheme consisting of derivatization with 3,5-bis(trifluoromethyl)benzoyl chloride (18), LLE, hydrolysis of phenolic esters present in the extract, silylation of the free hydroxy groups and GC combined with negative ion chemical ionization (NICI) MS. It happened that the molecular ion carried more than 60% of the ionic current, making the method highly specific, with potential LOD below the picogram level. This method revealed that the principal amines in bovine retina are dopamine (19b), tyramine (5) and serotonine (20)95, and in the

thoracic nervous system of a locust species are the same ones in addition to norepinephrine (21a) and octopamine (22)96.

The presence of hexamethylenediamine (4c) in hydrolyzed human urine is indicative of exposure to hexamethylene diisocyanate. The diamine was determined after derivatization with heptafluorobutyric anhydride followed by GC-CI-MS, using ammonia as the ionizing reagent and deuterated hexamethylenediamine as internal standard; LOD 0.5 mg/L urine97.

A screening program was proposed for the analysis of cocaine (23a) and its metabolite benzoyl ecgonine (23b) in the meconium of newborn infants, of mothers suspect of cocaine use during pregnancy. The method consists of SPE from a methanolic extract of the meconium, silylation of 23b with N,O-bis(trimethylsilyl)trifluoroacetamide (24) and analysis by GC-MS. The method was sensitive to less than 0.25 ppm of 23a and 0.5 ppm of 23b in the meconium, and is preferable for screening to the more involved fluorescence polarization immunoassay determination of these compounds98. See also the beginning of Section IV.D.2 for an alternative analysis of cocaine.

Attention should be paid to the appearance of spurious peaks in the fragmentation patterns of amines determined by GC-MS, when the analytes came into contact with methanol or ethanol as solvents. Thus, for example, Schiff bases may be formed on condensation of a primary amine with traces of formaldehyde or acetaldehyde present in the solvent. Although the peaks of such product may be unresolved in the chromatogram, they may appear as ions with mass increments of C12 or C26 in the mass spectrogram, complicating the identification of the analyte, as was the case with some amphetamine drugs99.

Simultaneous screening and determination of benzodiazepines (e.g. diazepam, 25a) and other anxiolytic drugs in plasma were carried out on 1 mL samples by SPE onto a C8 RPsorbent, reextraction with AcOH/MeOH and GC-NPD-ECD analysis with twin columns, using prazepam (25b) as internal standard. Application of SPE instead of the usual LLE proved to be of advantage in the case of imidazopyridine drugs (e.g. alpidem 26a and zolpidem 26b). The LOQ of the method allowed toxicological and pharmacological determinations, except for buspirone (27) that allowed determinations only at toxic blood levels100.

Volatile amines from C1 to C6 and ammonia were separated on a PoraPLOT column, with or without a temperature gradient, depending on volatility. The method is applicable to determination of the purity of manufactured amines. Trace analysis of these amines can be performed by capillary GC-FID and of ammonia by GC-ELCD101.

Ph

(25)

N

X

N

X

CH2 CONR2

(a)R = Pr, X = Cl

(b)R = X = Me

(26)

O N

N N N

N

O

(27)

Determination of the lower tertiary aliphatic amines in environmental samples, such as river water and bottom sediments, may be performed easily and with good selectivity by distillation of the amines followed by headspace GC-MS; LOD in mg/L for 40 mL samples were 1.25 for Me3N, 0.25 for Et3N, 0.125 for All3N, 0.25 for Pr3N and 0.125 for Bu3N, with recovery over 70% and standard deviation of the recoveries below 12% n D 5 102. A method for determination of volatile methylamines in urine, proposed as an aid for detection of the fish odor syndrome, is based on headspace GC analysis103. Volatile amines dissolved in water or sediments were determined by preconcentration in a Cavett diffusion flask, by adding strong alkali and cyclopropylamine as internal standard to the water or the solution in the pores of the sediment. The evolved amines were collected in a small volume of HCl. After neutralizing the acid, the amines were determined by GC-NPD, using cyclobutylamine as internal standard; LOD 7.3, 54.0 and 703 ng/L of

MeNH2, Me2NH and Me3N, respectively, for 5 mL injection; linear range 1 ð 10 6 to 7 ð 10 4 M104.

Acetic anhydride is a useful GC pre-column derivatizing reagent for amines, phenols and alcohols. In the presence of aqueous base only amines and phenols are derivatized, but

under anhydrous conditions also alcohols undergo acetylation. The acetylated derivatives are useful for GC and GC-MS analysis of biogenic amines, antidepressants, antipsychotics and some of their metabolites105.

A comparison was made between variations of the full scan GC ion-trap MS method for detection of amphetamine (28) and similar drugs in urine. Thus, the fragmentation patterns obtained by methane-CI-MS of underivatized methamphetamine (29), ephedrine (30), pseudoephedrine (30) and phentermine (31) have more characteristic peaks that help making positive identifications, than those obtained by EI-MS of the N-(heptafluorobutyryl) or N-( -(ethoxycarbonyl)hexafluorobutyryl) derivatives; LOD 2.4 and 2.6 mg/L of 28 and 29 for CI-MS vs 0.7 and 1.4 mg/L for EI-MS, respectively; also LOQ are slightly higher for CI-MS than EI-MS106. A polymeric reagent was proposed for derivatizing primary and secondary amines, consisting of a polystyrene matrix with attached pentafluorobenzoyl groups via anhydride moieties. Analysis of amines at picogram levels was by capillary GC with thermionic specific detection (TSD) on N mode or ECD107 or with NICI-MS-SIM; LOD 1 mg BuNH2/L for 2 mL injection, with linearity in the 5 250 mg/L range108. Detection of benzidine (32) and its conjugates in urine can be performed after hydrolysis of the conjugates, LLE, adding benzidine-d8 as internal standard, derivatizing with pentafluoropropionic anhydride and end analysis by GC-NICI-MS-SIM; LOD 0.5 mg/L urine; linearity between 2 and 200 mg/L. This test was applied for toxicological monitoring of workers in polyuretane manufacture109.

CH2 CHCH3 PhCH2 CH(Me)NHMe PhCH(OH)CH(Me)NHMe

A sensitive method for primary amines is shown in reaction 2, leading to the corresponding N-benzenesulfonyl-N-trifluoroacetyl derivatives. These can be determined by GC-ECD using SE-30 columns; LOD 1 5 pg, which is about 200 times more sensitive than GC-FID. The method was applied for determination of phenethylamine (33) in urine110. This analysis was performed also by LLE into n-pentane, derivatization to the benzenesulfonamide and GC-FPD using a capillary column; recoveries of aliphatic primary amines in urine were 91 107%, RSD 0.2 4.5%111,112. Amines in environmental waters and sediments were determined after LLE with dichloromethane, derivatization with benzenesulfonyl chloride and GC-SIM-MS; LOD 0.02 2 mg/L of water and 0.5 50 ng/g of sediment113.

PhCH2CH2NH2

(33)

More extensive derivatization schemes than reaction 2 are necessary for GC analysis of amino acids. A method was proposed for detection of amino acids and dipeptides at the femtomol level. After LLE the analytes were N-alkylated with pentafluorobenzyl bromide, N-acylated with heptafluorobutyric anhydride and esterified with N,O- bis(trimethylsilyl)trifluoroacetamide (24). Of the twenty amino acids studied, only glutamic acid (34b) and arginine (8) could not be detected by this scheme. Dipeptides with neutral side groups were more easy to derivatize. End analysis was by GC-NICI- MS. Recoveries of phenylalanine, lysine and threonine were 76, 55 and 34% respectively; LOD was less than 150 fg for MS-SIM at signal-to-noise ratio (SNR) 80. The method was applied to urine samples114. A sensitive and specific method for detecting the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5,b]pyridine (35) adducted to DNA in living tissues consists of alkaline hydrolysis of the tissue, LLE, production of the bis(pentafluorobenzyl) derivative and GC-MS combined with ECD; LOD 0.03 fmol of 35/mg DNA (1 adduct/108 nucleotides). Evidence for the adduct was found in samples of human colon but not of human pancreas or urinary bladder115.

Amines, aminoalcohols and amino acids in aqueous medium can be derivatized before GC analysis by treatment with alkyl chloroformates116. A method proposed for protein amino acids uses ethyl chloroformate which, under optimal conditions, reacts within a few seconds with all the reactive moieties of the molecule. Amino acid GC analysis is finished within five minutes in a capillary column, including pre-column derivatization time. Arginine (8) fails to react to completion and is not eluted from the column117,118. Of the alkyl chloroformates investigated for derivatization of amino acids, isobutyl chloroformate gave the most sensitive derivatives for determination by GC-FID and GC-MS119. A procedure proposed for protein and nonprotein amino acids consists of pre-column isobutyloxycarbonylation of amino, alcohol and mercapto groups with isobutyl chloroformate, SPE, producing the t-butyldimethylsilyl ester with t-butylchlorodimethylsilane

(7) and end analysis by GC-MS. Temperature-programmed retention indexes for DB-5 and DB-17 capillary columns were determined. The method was applied to determination of amino acids in almond, walnut and sunflower seeds120. The analytical and synthetic applications of reagent 7 were reviewed121,122.

Azo dyes extracted from waste sludges can by identified by GC-MS after H2/Pd cleavage to aromatic amines according to reaction 3, in a microreactor mounted on the injector123.

The offensive odor emitted by fish meal processing plants is due mainly to aliphatic amines. The degree of environmental damage can be measured from a correlation between

an odor organoleptic test and the results of GC fitted with a semiconductor sensor, with trimethylamine serving as reference compound124.

A combination of column adsorption chromatography on basic alumina and GC of the eluate served for characterization of the trace fraction of nitrogen-containing compounds in hydroprocessed naphtha. These were subdivided into groups of four types, namely pyridines, pyrroles (the most abundant), anilines and indoles125.

D. Liquid Chromatography

1. General

Intense research activity is taking place at all times on LC separation of biologically active amines in general and amino acids in particular. All aspects of the analytical problem are focused in these studies, such as sampling, nature of sample, preand postcolumn treatment with well established and new reagents, separating phases, carrying liquids and their composition gradients, and detection methods.

Various important LC methods for amino acid, peptide and protein analysis were reviewed and evaluated126,127. A review of HPLC methods for the analysis of selected biogenic amines in foods appeared, including methods for extraction and for elimination of interfering compounds128.

A study comprising five laboratories was carried out on the accuracy and precision of protein amino acid analysis. An important conclusion reached was that it is necessary to examine both accuracy and precision to achieve maximum improvement in either129. A commercially available single-cell protein, Pruteen, was proposed as reference material for the determination of amino acids and other substances in food. This recommendation was the result of a five-year-long study on the stability of this particular protein130.

Some recovery problems encountered during hydrolytic extraction of amino acids from environmental samples were discussed. A way was proposed for compensating for differential losses of neutral, acidic and basic amino acids, consisting of adding various nonprotein amino acids before the hydrolysis, that act as charge-matched recovery standards131.

The stability under long-term storage of biogenic amines dissolved in Krebs Ringer Henleit saline solution, usually employed for studying the release of such compounds, was examined by LC with electrochemical detection, with 3,4- dihydroxybenzylamine (19a) serving as reference compound. Although every amine shows a peculiar behavior, all are affected by temperature, pH of the solution and length of storage. Catecholamines such as dopamine (19b), epinephrine (21b) and norepinephrine (21a) were stable for weeks in acidic solution under refrigeration, while indolamines such as serotonine (20) underwent fast degradation under the same conditions. At room temperature and pH 7.81, marked reductions in the concentration of the catecholamines were observed, but not of serotonin. Under freezing, at pH 1.96, the catecholamines remained intact and serotonin disappeared after two weeks132. At the low concentrations required for detection by thermal lens spectrometry, catecholamines can undergo immediate oxidative cyclization with hexacyanoferrate ions to aminochromes (36), at pH 7. These are intensely colored quinonoid dyes. At higher concentrations a lower pH is required to avoid polymerization of the dyes, and the process becomes slower and inadequate for HPLC detection; LOD ca 1 mg/L in urine133. See Section IV.D.3.g for an alternative application of aminochromes to the analysis of catecholamines.

A universal eluent system was proposed for analysis of amino acids in biological fluids by IEC on the cation exchange resin Ostion LG ANB134. A study was carried out on the effect of the carrier pH on RP-HPLC of amines, using a C8 column and octyl sulfate as ion pairing reagent. Optimal results were obtained for the analysis of catecholamines

(36)

in urine by a pre-column cleanup with alumina and chromatography with the mobile phase at pH 5.4135.

A comparative study of the analysis of aliphatic amines by GC-FID, GC-TSD and HPLC with refractive index detector (RID), using isopropylamine as internal standard, gave good results in all cases. Determination of trimethylamine oxide by HPLC with a pulsed amperometric detector was problematic136.

A study involving twenty-six laboratories was carried out to assess the quality of amino acid analysis, using samples of urine and lyophilized plasma. Coefficients of variation ranged from 13% for glycine to 65% for methionine. Automated IEC followed by ninhydrin detection (37) seemed to perform better than other methods; however, there was no clearly superior method and no analyzer clearly outperformed the others. This seems to point to the importance of personal proficiency and expertise in the performance of such analyses137.

O O

N

OH O

(37)

A general approach was described to the analysis of traces of nitrogenand phosphoruscontaining pesticides in environmental samples, using on-line and off-line SPE, followed by LC with TSP-MS-SIM detection. The assignments of various pesticides were reconfirmed by a variety of MS techniques138. Preconcentration of traces of thirty-four pesticides and various transformation products was performed on-line by SPE on extraction disks or a packed column, followed by LC-TSP-MS-SIM of the positive ions [M C H] C and either [M C NH4] C or [M C CH3CN] C or the negative ions [M H] and [M C HCO2] ; LOD 0.01 0.4 mg/L for 100 mL samples, depending on the analyte and mode of operation. The method was applied to determination of trace levels of pesticides in river waters139.

2. Underivatized analytes

The standard urine immunoassay for detection of cocaine (23a) abuse during the gestation period of newborn babies was frequently found to yield negative results in cases where positive results were shown by extraction of meconium with a solvent, followed by HPLC. The drug and metabolites such as norcocaine (23b) and cocaethyline (23c) were detected140. See Section IV.C for an alternative analysis of cocaine.

Results of high-performance silica gel, cellulose and RP-bonded silica gel for eighteen amino acids were compared with previous reports on IEC, RP-LC and paper chromatography. Determination was by scanning densitometry of the product (presumably 37) obtained after applying ninhydrin141,142. Determination of amino acids in pig plasma by IEC and detection by the ninhydrin method is influenced by the protein and lipophilic compounds present, causing lower resolution. This is avoided on addition of sulfosalicyclic acid followed by SPE. An increase was observed in the results for threonine, asparagine, glutamic acid, glutamine, glycine, alanine, valine and lysine, whereas those for phenylalanine and tryptophane showed a decrease with this modification143.

IEC was applied to determine biogenic polyamines such as putrescine (4a), cadaverine (4b), tyramine (5), histamine (6), spermidine (38), agmatine (39) and tryptamine (40), contained in aqueous trichloroacetic extracts of leafy vegetables, such as cabbage and lettuce. A cation exchange column loaded with potassium ions and a special buffer were used. Spermidine (38) was the major amine detected in this group (7 15 mg/g fresh weight)144.

N

H

(40)

Underivatized amino acids were analyzed in a FIA system including a high-performance IEC column and a CLD cell. The chemiluminescence generated on a GCE was measured when Ru(II) ions in the carrier solution were oxidized to Ru(III) and reacted in situ with the amino acid. LOD ranged from 100 fmol for proline to 22 pmol for serine (SNR 6)145.

A study of fifty-five aliphatic, aromatic and heteroclyclic amines showed that twentyeight of them could be detected in a FIA system at concentrations in the range of 1.0 ð 10 10 to 4.0 ð 10 6 M (SNR 3, 20 mL injection), without derivatization, by HPLC-CLD, taking profit of the chemiluminescence produced in the presence of aryl oxalate and sulforhodamine 101 (41). The method was applied to the determination of histamine (6) in fish146. See reaction 24 in Section IV.G.

HPLC on a Cosmosil 5 C18 column, using a perchloric acid acetonitrile eluent (pH 7.6), followed by CLD in the presence of hydrogen peroxide and bis(2,4,6-trichlorophenyl) oxalate (42), was applied to the determination of 1-aminopyrene (43a) and various diaminopyrenes (43b d). Ascorbic acid was added to avoid oxidative degradation of the aminopyrenes in the presence of metals; LOD in the sub-fmol range (SNR 3)147. A fast (less than 10 min) HPLC-ELCD method was proposed for determination of dopamine (19b) and its metabolites in microdialysates, using packed fused silica capillary columns; LOD 0.05 mg/L of dopamine in a 2 mL sample, RSD 3% (n D 10)148.

SO3 −

SO3 −

Tris(2,20-bipyridine)ruthenium(III) complex ions (44) produce a chemiluminescence in the presence of amino acids in a FIA system. Amino acids containing secondary amino groups have the strongest response; LOD 20 pmol for proline to 50 nmol for asparagine149.

Nitrogen-containing compounds are easily detected by CLND, without preor postcolumn derivatization. Thus, peptide mapping by RP-HPLC using CLND gave the correct results as for the chain size, against the results of UV visible (UVV) detection (UVD), which were biased by the presence of strong UV chromophores150.

A method for determination of the aromatic amino acid phenylalanine (45), tyrosine (46) and tryptophan (47) content of peptides at low microgram levels is based on sizeexclusion HPLC combined with UVD using a diode array, and data processing of the