High Performance Using Bifunctional a Review Liquid Chromatography

ANALYTICAL SCIENCES AUGUST 1989, VOL. 5 371

Reviews

High Performance Liquid Chromatography of Bioactive Substances Using Bifunctional Fluorogenic Reagents for Derivatization A Review

Yosuke OHKURA

Faculty of Pharmaceutical Sciences, Kyushu University62, Maidashi, Fukuoka 812, Japan

Pre- and postco umn derivatization methods employing recently-developed Bifunctional fluorogenic reagents for high performance liquid chromatography have been demonstrated to be successfulregarding both sensitivityand selectivity in the determination of bioactive substances and their related enzymes. The reagents were designed mostly based on the principle that vicinal or geminal, homo- or hetero-Bifunctional (diamino, amino-imino, amino- sulfhydrylor amino-hydroxyl) compounds react with 1,2-dioxo compounds (1,2-ketols, glyoxals, 1,2-diketones or 1,2-quinones) or monooxo compounds (aldehydes) to yield fluorescent cyclic products. These reagents have been introduced into the quantification process of diverse bioactive substances in biological samples at the femtomole level.

Keywords Bifunctional f uorogenic reagent, bioactive substance, enzyme activity, serum, urine, animal tissue, high performance liquid chromatography, quantification, high sensitivity, femtomole level, high selectivity, simplicity, precolumn derivatization, postcolumn derivatization

analysis methods should be both selective and sensi- 1 Introduction tive. Selectivity and sensitivity have been achieved by means of traditional analytical separation methods, A great variety of bioactive substances that exhibit including: gas chromatography (often coupled with bioactivities, even at extremely low concentrations, mass spectrometry), liquid chromatography (coupled occur in biological fluids and tissues, and their with mass spectrometric, refractive index, UV, fluores- concentrations are generally controlled by their related cence and electrochemical detection and various other enzymes. Therefore, it is of importance in areas where detection systems) and elctrophoresis. Also, molecular life science is involved to measure these substances and recognition techniques based on bioaffinity, such as their biosynthetic and metabolizing enzyme activities in immunoassay and receptor assay, have been used. biological materials. Since such biosubstances and Derivatization techniques used to lead target analyte(s) enzymes are usually found in complex matrices, to isotopically labeled, UV-absorbing, fluorescent, 372 ANALYTICAL SCIENCES AUGUST 1989, VOL. 5 chemiluminescent or electroactive compound(s) are group (diamino, amino-imino, amino-sulfhydryl or very useful for sensitive and selective detection when amino-hydroxyl compounds) may combine with mono- combined with the various separation methods. oxo compounds (aldehydes) or 1,2-dioxo compounds High performance liquid chromatography (HPLC), (1,2-ketols, glyoxals, 1,2-diketones or 1,2-quinones) to one of the most operable separation methods, has been afford fluorescent cyclic derivatives (i.e. imidazoles, widely accepted as a potential analytical tool for thiazoles, oxazoles, quinoxalines orp-oxazines) (Fig. 1). routine use. Regarding its detection systems, fluores- These bifunctional compounds and 1,2-dioxo com- cence detection is not only highly sensitive but also pounds can be reagents and analytes, respectively, and selective; it has therefore been introduced in the deter- vice versa. In addition, on the basis of the chemical mination process of bioactive substances in biologi- structures of fluorophores derived from reactions cal samples. A non-fluorescent or weakly fluorescent between bifunctional reagents and 1,2-dioxo com- analyte must be converted into highly fluorescent pounds, some monofunctional reagents have been derivative(s), normally through its functional group(s) contrived. by reaction with a fluorogenic reagent. This article aims to review sensitive HPLC analyses Fluorescence derivatization HPLC can be categoriz- of bioactive substances of small molecules, including ed into precolumn (before separation) and post- therapeutic drugs, in biological materials by means of column (after separation) derivatization. A precolumn pre- or postcolumn fluorescence derivatization using derivatization reaction does not require a rapid termina- these reagents as well as some reagents reported so far. tion and its conditions can usually be optimized. A postcolumn derivatization reaction, on the other hand, should proceed rapidly, but not necessarily terminate, 2 Aromatic Aldehyde Precursors and Biogenic since a prolonged reaction time causes peak-broaden- Amine-Related Enzymes ing, thus yielding low resolution. Fluorogenic reagents for postcolumn derivatization need to be non-fluores- 2.1 Aromatic aldehydes and arylaliphatic aldehydes cent or markedly different from the derivatives in derivable from drugs fluorescence excitation and/or emission spectra in the 1,2-Diaminonaphthalene, an aromatic 1,2-diamino mobile phase; they are allowed to give multiple compound, was first found to react with aromatic fluorescent derivatives, provided that their reactions are aldehydes in an acidic medium9 to form 2-substituted reproducible. naphtho[l,2-d] imidazole derivatives.10 Since then, o- A number of fluorogenic reagents which meet the phenylenediamine11,1,2-diamino-4,5-dimethoxy benzene12 above-mentioned requirements have already been de- (DDB, Fig. 2),1,2-diamino-4,5-methylenedioxybenzene13 veloped, and are summarized in reviewsl-6 and books.7'8 (DMB, Fig. 2) and 1,2-diamino-4,5-ethylenedioxyben- Many of these reagents have been applied to pre- zene13 (DEB, Fig. 2) have been developed (in that and/ or postcolumn fluorescence derivatization in the order) as fluorogenic reagents for aromatic aldehydes, HPLC of biological compounds. We have also which all produce the corresponding fluorescent introduced several fluorogenic reagents based on the imidazole compounds in acidic media (Fig. 2). following principle: compounds having vicinal or DDB also reacts with such arylaliphatic aldehydes as geminal bifunctional groups, one of which is the amino phenylacetaldehyde and cinnamaldehyde and has, thus,

Fig. 1 Scheme of reactions between bifunctional-bifunctional, or bifunctional-monofunctional compounds. Ar., aromatic moiety; Aliph., aliphatic moiety. ANALYTICAL SCIENCES AUGUST 1989, VOL. 5 373

Fig.2 Aromatic 1,2-diamines and 1,2-aminothiols and their reactions with aromatic aldehydes.

useful for evaluating the placental and foetal functions. DEB is the most sensitive fluorogenic reagent for aromatic aldehydes, especially those having a phenolic group. Forphenicine [L-(4-formyl-3-hydroxyphenyl)- glycine], an inhibitor of alkaline phosphatase, has a formylphenolic group in the molecule and is readily derivatized with DEB into a fluorescent imidazole compound which is separable on a reversed-phase

Fig. 3 Fluorescence derivatization of phenolic compounds (ODS) column. Thus, an HPLC method has been involving the Reimer-Tiemann reaction followed by the DDB established for monitoring forphenicine in mouse serum reaction. and muscle.18

2'2 Amine-metabolizing enzyme activities 2,2'-Dithiobis(1-aminonaphthalene)19 (DTAN) reacts been applied to a precolumn derivatization reversed- with high sensitivity with aromatic aldehydes in an phase (ODS column) HPLC determination of bestatin acidic medium in the presence of tributylphosphine, [(2S,3 R)-3-amino-2-hydroxy-4-phenylbutanoyl-S-leu- which serves to reduce the reagent to 1-amino-2- cine], an aminopeptidase inhibitor, in human14 and thionaphthol, to produce 2-substituted naphtho[1,2- mouse15 sera and mouse muscle15, which can be derived d]thiazole compounds20 (Fig. 2). The reaction is to phenylacetaldehyde by periodate oxidation. similar to that of 2-aminothiophenol2l (Fig. 2), but p-Hydroxybestatin, an active metabolite of bestatin, occurs under much milder conditions (above 0° C) and has a phenolic group in the molecule; the compound in can afford more intense fluorescence. human16 and mouse15 sera and mouse muscle15 can be DTAN have been applied to the highly sensitive monitored by reversed-phase (ODS column) HPLC assay of dopamine-/3-hydroxylase (DBH) activity in rat with precolumn fluorescence derivatization with DDB serum and adrenal medulla22 and human serum:23 after converting the phenolic group into an o-formyl- octopamine enzymatically formed from the substrate phenolic group by the Reimer-Tiemann reaction (see tyramine is oxidized with periodate to p-hydroxybenzal- Fig. 3 for the reaction scheme). Oestrogens [oestriol dehyde, which is determined by normal-phase HPLC (oesta-1,3,5(10)-trien-3,16a,16f 3-triol), oestrone (3-hy- on an alumina column after conversion into the DTAN droxyoesta-1,3,5(10)-trien-l7-one) and oestradiol (oesta- derivative. 1,3,5(10)-trien-3,17 f3-diol)] have a phenolic group in Monoamine oxidases A and B in rat brain mito- their molecules; the free and total (the sum of free and chondria can be assayed:24 p-sulfamoylbenzaldehyde conjugated) oestrogens in pregnancy urine can be formed enzymatically from p-sulfamoylbenzylamine (a simultaneously measured based on the same principle newly developed substrate for monoamine oxidase A25) as shown in Fig. 3.17 The concentration values are and benzaldehyde produced enzymatically from benzyl- 374 ANALYTICAL SCIENCES AUGUST 1989, VOL. 5

diamine

Fig. 5 Reactions of o- phenylenediamine, DDB and DMB with a-keto acids.

Fig. 4 DNT and its reaction catalyzed by COMT.

amine (a known substrate for monoamine oxidase B) are simultaneously converted into fluorescent compounds with DTAN and separated by reversed-phase HPLC on a cyanopropyl-bonded column. Catechol-0-methyltransferase (COMT) activity in rat liver can be determined by using 3,4-dihydroxybenz- aldehyde as a substrate: the m-methylation product (vanillin) and the p-methylation product (isovanillin) are measured by normal-phase HPLC on a silica gel column after conversion into the DTAN derivatives.26 On the basis of studies of the COMT-mediated reaction, a highly sensitive fluorogenic substrate for COMT, 2-(3,4-dihydroxyphenyl)naphtho[ 1,2-d]thiazole (DNT, Fig. 4) has been introduced.27 DNT has a high affinity for COMT (the Michaelis constant values for human erythrocytes COMT, 2.1 µM in m-methylation and 3.2 µM in p-methylation). A highly sensitive assay Fig. 6 Chromatograms of (A) the DMB derivatives of a-keto method of COMT in human erythrocytes27, human and acid in a normal urine (24 h) and (B) the reagent blank.39 rat erythrocyte membranes and soluble fractions, as well Peaks and concentrations (nmol/ml urine) in parentheses: as various rat tissues28 (all extremely low in COMT 1=a-ketoglutaric acid (98); 2=glyoxylic acid (36); 3=pyruvic activities) has been established based on normal-phase acid (115); 4=a-keto-y-methylthiobutyric acid (1.5); 5p- hydroxyphenylpyruvic acid (17.2); 6=a-ketobutyric acid (silica gel column) HPLC of the m- and p-methylated (1.5); 7=a-ketovaleric acid (0.2); 8=a-ketoisovaleric acid products (Fig. 4) (detection limits for the m- and p- (2.7); 9=a-ketoisocaproic acid (1.8); 10=f3-phenylpyruvic methylated products, 60 fmol each on column). acid (0.4);11=a-ketocaproic acid (0.1);12=a-keto-f3-methyl- valeric acid (4.7). The detector sensitivity was increased 40 times after elution for 6 min. 3 Glyoxal Precursors and a-Keto Acids

3.1 21-Hydroxycorticosteroids DMB (see Section 2.1) also reacts with glyoxal 3.2 a-Keto acids compounds to form fluorescent quinoxaline deriva- Biogenic a-keto acids that are important as biosyn- tives.29 Thus, a sensitive and simple precolumn HPLC thetic intermediates can be derivatized with o-phenyl- method has been devised for the simultaneous deter- enediamine31, DDB32,33and DMB34 to give fluorescent mination of 21-hydroxycorticosteroids (i.e. hydrocor- 2-quinoxalinone compounds (Fig. 5). The derivatiza- tisone, cortisone and corticosterone) in human and rat tion reactions occur in a rather strong acidic medium sera.30 This method is based on the conversion of the under conditions that are more intense than those for corticosteroids by cupric sulfate oxidation into the aromatic aldehydes (reaction temperatures and times: corresponding glyoxal compounds followed by reactions 80°C, 2 h, or 100°C, 30 min for o-phenylenediamine; with DDB and separation of the resulting products on 100°C, 2 h for DDB; 100°C, 45 - 50 min for DMB). an ODS column. The concentration values are useful These reactions have been employed in precolumn for evaluating the adrenal and pituitary functions. derivatization reversed-phase (ODS column) HPLCs of ANALYTICAL SCIENCES AUGUST 1989, VOL. 5 375

V, Ylll~ Lh1L UV V J.

Fig. 7 Reactions of sialic acids with DDB and DMB.

various biogenic a-keto acids and then applied to the simultaneous quantification of these acids in human serum and urine.35-39 The method with DMB is the most sensitive; eight and twelve a-keto acids in human serum and urine, respectively, can be determined with the detection limits of 6 - 44 fmol on column.39 A typical chromatogram obtained with normal urine is shown in Fig. 6.

3.3 Sialic acids and dehydroascorbic acid Sialic acids [i.e. N-acetyl- and N glycolyl-neuramic acids (NANA and NGNA, respectively)] occur as their glycosides, mostly at the termini of the saccharide chains in the glycoproteins and glycolipids of mammals and are related to immunity. The concentration of NANA has been found to increase in the sera of cancer patients. These acids behave as a-keto acids and can be derivatized with DDB or DMB under conditions similar to those for biogenic a-keto acids (Fig. 7). Fig. 8 Chromatograms of the DMB derivatives of NANA NANA and NGNA in human serum and urine and and NGNA in (A) human, (B) rat and (C) mouse sera.a' Peaks animal sera can be measured by a reversed-phase (ODS and concentrations (µmol f ml serum) in parentheses: column) HPLC method involving sulfuric acid- or 1=NANA (A, 1.75; B, 1.82; C, 0.06); 2=NGNA (A, not neuraminidase-mediated hydrolysis of samples followed detected; B, 0.06; C, 2.65); 3=DMB; 4=unknown, probably by fluorescence derivatization.4o,41 DMB is more 0-acetylated sialic acids. sensitive than DDB (detection limits, 25 fmol each on column). Chromatograms of the DMB derivatives of NANA and NGNA in normal human, rat and mouse sera are given in Fig. 8. by means of precolumn HPLC on a reversed-phase Dehydroascorbic acid fluoresces when treated with (phenyl) column, where ascorbic acid is derived to DDB under mild conditions (37°C, 30 min) in an dehydroascorbic acid by iodine oxidation (detection acetate buffer (pH 4.5).42 A highly sensitive quantifica- limit, 46 fmol on column).43 tion of the total ascorbic acid (the sum of dehydroascorbic acid and ascorbic acid) has thus been achieved 376 ANALYTICAL SCIENCES AUGUST 1989, VOL. 5

1 \ L

Fig. 9 Main biosynthetic pathway of catecholamines.

Fig. 10 Fluorescence derivatization of catechol and 4-hydroxy-3-methoxyphenyl corn- pounds with DPE.

of potassium hexacyanoferrate(III) to produce fluores- 4 Catecholamines and Related Compounds and cence.49 Of the compounds, 1,2-bis(3,4-dimethoxy- Enzymes phenyl)ethylenediamine, 1,2-bis(4-methoxyphenyl)ethylenediamine and 1,2-bis(4-ethoxyphenyl)ethylenediamine, 4.1 Catecholamines and their biosynthetic enzyme all in meso form,, are most sensitive to all the catechol- activities amines. Several HPLC methods with fluorescence detection For economical reasons, however, meso-diphenyl- have been reported for the determination of catechol- ethylenediamine (DPE, Fig. 10), the simplest 1,2- amines [epinephrine (E), norepinephrine (NE) and diarylethylenediamine, was most acceptable for routine dopamine (DA), Fig. 9].44 Of the methods, the well- use. DPE reacts with high selectivity with catechol known hydroxyindole method, based on postcolumn compounds under mild conditions to yield a single derivatization, is highly sensitive for NE and E, but not fluorescent compound from each catechol compound50 for DA;45 the methods with fluorescamine46 and o- (Fig. 10); it is therefore applicable to both pre- and phthalaldehyde47, based on pre- and postcolumn deriva- postcolumn derivatization in HPLC. tization, respectively, can detect NE and DA but not E, Precolumn HPLC on an ODS column permits a because the reagents do not react with E. The con- simultaneous determination of all the catecholamines in densation reaction between catecholamines and ethyl- plasma51, urine52,, erythrocytes and plateletsS3 as low as enediamine has been utilized only for postcolumn 1- 2 fmol on column using isoproterenol (IP; 1V HPLC of urinary catecholamines.48 isopropylnorepinephrine) as an internal standard. The The DL- or meso-diarylethylenediamines (28 species), DPE reaction is so specific for the catecholamines that vicinal arylaliphatic diamino compounds that have been no complicated procedure is necessary for the clean-up investigated for detecting fluorogenic reagents for of the plasma: a solid-phase extraction with a cation- catecholamines, all react with catecholamines under exchanger (sulfopropyl) resin cartridge is most satisfac- mild conditions (pH 6.5 - 6.8, 37 - 50° C) in the presence tory and provides good and reproducible recoveries of ANALYTICAL SCIENCES AUGUST 1989, VOL. 5 377 the catecholamines (87% for NE, 85% for E and 92% treatment can be used51 (Fig. 11B, C and D). Liquid- for DA; Fig. 11A), although ultrafiltration, deprotein- liquid extraction has also been proposed for the clean- ization with perchloric acid and traditional alumina up.54 The selectivity allows urinary catecholamines to be measured without sample clean-up, provided no protein is present in large amount in the sample urine.55 The HPLC method can also quantify L-DOPA in plasma and urine using a-methyldopa as an internal standard (detection limit, 10 fmol on column).56 Catecholamines in biological matrices also occur as conjugates, mostly 3-0-sulfates; their concentrations are higher than those that are free. The total amount (sum of free and conjugated) of catecholamines in plasma, erythrocytes, platelets53 and urine57 can be assayed by the DPE method after sulfatase-catalyzed hydrolysis of samples. IP administered to a human as a bronchodilator is found in serum as free and conjugated forms, and the free and total concentrations can be monitored in the same manner.S8 Four enzymes are involved in the biosynthesis of catecholamines from tyrosine (Fig. 9). They are tyrosine hydroxylase (TH, conversion of tyrosine into L-DOPA), aromatic amino acid decarboxylase (AADC, Fig. 11 Chromatograms of the DPE derivatives of cate- decarboxylation of L-DOPA to give DA), DBH cholamines in a normal human plasma added with IP.S1 Clean-up procedures for plasma: A=solid-phase extraction (introduction of /3-hydroxyl group to DA to form NE; cf. Section 22) and phenylethanolamine N methyl- with a sulfopropyl resin cartridge (Toyopak SP); B= ultrafiltration through an ultrafilter (30000D, UFO Mini- transferase (PNMT, introduction of a methyl group 30); C=deproteinization with perchloric acid; D=adsorption onto the amino group of NE to give E). These enzyme on alumina. Plasma sample sizes (ml): A and B=0.5; C and activities in human and rat plasmas (or sera) and rat D=1.0. Peaks and concentrations (pmol/ml plasma) in tissues have been assayed by measuring L-DOPA (for parentheses: 1=NE (1.72); 2=E (0.56); 3=DA (0.21); 4=1P TH)59, DA (for AADC)6o,61,NE (for DBH)62 and E (for (0.5, internal standard); 5=unknown (endogenous com- PNMT)63 enzymatically formed from the respective pounds). physiological substrates by means of precolumn de-

Fig. 12 Schematic diagram of a postcolumn derivatization HPLC system for the determination of catecholamines and their metabolites.65,66 378 ANALYTICAL SCIENCES AUGUST 1989, VOL. 5 rivatization HPLC with DPE. compounds(Fig. 10). There exist many catechol-related An automated catecholamine analyzer system has compounds in urine and plasma, which results in the been constructed based on the DPE reaction applied to formation of a~complex chromatogram. Therefore, the postcolumn HPLC.64 A deproteinized biological compounds of interest in deproteinized sample solu- sample is injected into the system and the compounds tions from urine and plasma spiked with 3,4-dihy- of interest are adsorbed on a short column packed with droxybenzylamine (DHBA) and 4-hydroxy-3-methoxy- an ether-bonded resin, transferred by column switching cinnamic acid (ferulic acid) as internal standards are to a short column of a cation-exchanger (sulfopropyl) fractionated by solid-phase extraction using a cation- resin for further purification, switched again onto a exchanger (sulfopropyl) resin cartridge into two frac- conventional-sized column of the cation-exchanger and tions: amine [L-DOPA, NE, E, DA, normetanephrine chromatographed. The catecholamines in the effluent (NM), metanephrine (M), 3-methoxytyramine (3MT) is automatically derivatized with DPE and detected and DHBA] fraction, and acid [3,4-dihydroxymandelic fluorometrically. The system enables NE, E and DA in acid (DOMA), 3,4-dihydroxyphenylacetic acid (DOPAC), plasma and urine to be quantified as low as 20 -40 fmol vanillylmandelic acid (VMA), homovanillic acid (HVA) on column. and ferulic acid]-alcohol [3,4-dihydroxyphenylethyl- eneglycol (DOPEG) and 4-hydroxy-3-methoxyphenyl- 4.2 Catecholamines and their metabolites ethyleneglycol (MOPEG)] fraction. Each fraction is For the quantification of catecholamines and their subjected to HPLC. The detection limits vary from metabolites in urine and plasma, an ion-pair reversed- compound to compound (20 fmol - 4 pmol on column). phase HPLC system with postcolumn derivatization Typical chromatograms of an amino fraction and an involving coulometric oxidation followed by fluores- acid-alcohol fraction obtained from a human urine are cence reaction with DPE has been established65,66 depicted in Fig. 13. (Fig. 12). Catechol compounds and 4-hydroxy-3- methoxyphenyl compounds are oxidized by the use of a coulometric device to the corresponding o-quinone

Fig. 13 Chromatograms of (A) amine and (B) acid-alcohol fractions of catecholamines and their metabolites in normal human urine (0.5 ml).66 Peaks and concentrations (pmol/ml urine) in parentheses: 1=NE (211); 2=L-DOPA (208); 3=E (46); 4=DHBA (500, internal standard); 5=NM (401); 6=DA(1820); 7=M (117); 8=3MT(111); 9=DOMA (21200);10=DOPEG (418); 11=VMA (18900);12=MOPEG (282);13=DOPAC (4590);14=HVA (26600);15=ferulic acid (20000, internal standard); others=unidentified, but attributable to catechol-related compounds. All the peaks disappeared upon omitting DPE from the HPLC system illustrated in Fig. 12. ANALYTICAL SCIENCES AUGUST 1989, VOL. 5 379

kaline and subsequent acidic conditions69 (Fig. 14A); it 5 Guanidino Compounds, and Arginine-Con- has therefore been employed only for the postcolumn taining Peptides and Related Enzymes HPLC of biogenic guanidino compounds.70 On the other hand, DL-benzoin, a 1,2-ketoalcoholic 5.1 Biogenic guanidino compounds compound, yields a single fluorescent derivative, 2-substituted amino-4,5-diphenylimidazole, for each Ninhydrin reacts with guanidino compounds in an monosubstituted guanidino compound, geminal amino- alkaline medium to give fluorescent compounds;67 this imino compound, when reacted in an alkaline me- reaction has been applied to the postcolumn derivatiza- dium71 74 (Fig. 14B); the reaction can, thus, be adapted tion HPLC of some guanidino compounds of biological for both pre- and postcolumn derivatization in HPLC. interest.68 9,1o-Phenanthraquinone, a 1,2-quinone A cation-exchange (sulfonated resin column) HPLC compound, produces a sole fluorescent compound from system with postcolumn derivatization using DL- all monosubstituted guanidino compounds under al- benzoin has been established for the determination of

Fig. 14 Reactions of monosubstituted guanidino compounds with (A) 9,10-phenanthraquinone and (B) DL-benzoin.

Table 1 Amino acid sequences of some arginine- and tyrosine-containing peptides 380 ANALYTICAL SCIENCES AUGUST 1989, VOL. 5 biogenic guanidino compounds including toxic com- trypsin results in the production of arginine-containing pounds such as methylguanidine, guanidinosuccinic peptide fragments.80 Substance P (Table 1), a neuro- acid and taurocyamine in serum and urine.75,76 This peptide, in hypothalamus tissue of rat brain can be system also permits the determination of disubstituted measured by this postcolumn HPLC system using [D- gaunidino compounds, such as creatine and creatinine, Phe11]-neurotensin (cf Table 1) as an internal stan- since these compounds decompose in part to guanidine dard.81 under the conditions of postcolumn clerivatization. Precolumn clerivatization, however, leads to a more Precolumn clerivatization with DL-benzoin leads to a sensitive HPLC method for the arginine-containing 50 -100 times more sensitive reversed-phase (phenyl- peptides, which makes possible detection at as low as bonded column) HPLC method for the quantification 100 fmol on the column.79 The benzoin derivatives of of the biogenic guanidino compounds (detection limits, angiotensins, vasocontracting peptides, and their analog- 50 -100 fmol on column).77,78 ues can be successfully resolved on a reversed-phase (ODS) column (Fig. 16).82 5.2 Arginine-containing peptides and related enzyme The precolumn HPLC method can be applied to activities enzyme assays in a renin-angiotensin system: e.g., renin Arginine-containing peptides (Table 1) can be rapidly (liberation of angiotensin I from angiotensinogen) converted to their respective fluorescent derivatives by activity in human plasma is assayed by measuring the condensation of the guanidino moiety of an arginyl angiotensin I produced enzymatically from a synthetic residue with DL-benzoin in an alkaline solution (100°C, substrate, tridecapeptide of human angiotensinogen 90 s)79(Fig. 14B). Then, a selective detection based on (Table 1), using [Val5]-angiotensin I as an internal postcolumn clerivatization has been achieved in HPLC standard;83 a highly sensitive assay of the angiotensin for the analysis of arginine-containing peptides.80 converting enzyme (cleavage of the carboxy-terminal Intact peptides are separated on a reversed-phase His-Leu of angiotensin I, generating angiotensin II) in (ODS) column. Figure 15 shows chromatograms human serum is made based on the quantification of obtained with seven arginine-containing peptides, an angiotensin II or the analogous peptides formed arginine-free peptide and eight biological substances enzymatically from angiotensin I or the analogous other than peptides, using simultaneous fluorescence peptides as substrates, respectively. 82 and UV detection. Fluorescence detection is sufficient- Leupeptin (acetyl-Leu-Leu-arginal) is an inhibitor of ly specific for arginine-containing peptides, although proteinases. Although leupeptin has a guanidino the sensitivity is not so high (detection limits for the moiety, it does not react with DL-benzoin, probably peptides, approximately 10 pmol on column). This because of a Schiff-base formation by reaction of the HPLC can also be applied to tryptic mapping of a large guanidino moiety with the formyl group in the peptide or protein, since enzymatic digestion with molecule. Leupeptin, however, forms a fluorescent derivative when it is converted to leupeptinol (acetyl- Leu-Leu-arginol) by reduction with sodium borohy-

O r

1 1111G/ 111111 Fig. 15 Chromatograms of arginine-containing peptides and several biological substances in postcolumn HPLC with (A) fluorescence detection and (B) UV detection.80 Peaks and concentrations (nmol/injection volume of 50 µl) in parentheses: 1=kyotorphin (1.0); 2=kallidin (0.5); 3= angiotensin II (0.25); 4=angiotensin III (0.25); 5=angio- Fig. 16 Chromatogram of the benzoin derivatives of angiotensin I (0.5); 6=f3-melanocyte stimulating hormone (1.0); tensins and their analogues (200 pmol each).82 Peaks: 1= fluorescent by-products yielded from benzoin itself during 7=substance P (0.5); 8=tyrosine (1.0); 9=propionic acid the clerivatization; 2=[Val5]-angiotensin II; 3=angiotensin (10.0); 10=phenylalanine (2.0); 11=tryptophan (0.5); 12= II; 4=[Va15]-angiotensin I; 5=[Asn',Va15]-angiotensin II; phenylpyruvic acid (10.0); 13=methionine-enkephalin (1.0); 6=angiotensin 1;7=[Asn',Va15]-angiotensin I; 8=angiotensin 14=sorbic acid (2.5); 15=estriol (1.5); 16=estron-3- III. sulfate (3.0). ANALYTICAL SCIENCES AUGUST 1989, VOL. 5 381 dride prior to the benzoin reaction; precolumn derivat- applied to both pre- and postcolumn derivatization ization reversed-phase (ODS) HPLC has, thus, been systems in HPLC. introduced for monitoring leupeptin in mouse serum Basic conditions of precolumn HPLC on a reversed- and muscle.84 Antipain (N-[(s)-1-carboxy-2-phenylethyl] phase (ODS) column have been established for the carbamoyl-arginal-Val-arginal), an inhibitor of protein- separation of N-terminal tyrosine-containing peptides.90 ases, in mouse serum can be monitored in the same A chromatogram, thus obtained, with a standard manner. 85 mixture of N-terminal tyrosine-containing peptides involving the known opioid peptides is shown in Fig. 18. Although Tyr-Gly and Tyr-Gly-Gly-Phe are 6 Tyrosine-Containing Peptides and Related En- zymes

6.1 Tyrosine-containing peptides The selective detection of tyrosine-containing peptides is also useful for HPLC analysis of complex peptide mixtures. A fluorescence derivatization of tyrosine-containing peptides, based on the formylation of the phenolic moiety in the tyrosyl residue by means of the Reimer-Tiemann reaction and the subsequent conversion of the aldehyde thus formed into a fluorescent derivative with DDB (Fig. 3), has been developed and applied to precolumn HPLC on a reversed-phase (ODS) column for oligopeptides such as angiotensins I, II and III, methionine- and leucine-enkephalins (Table 1) and their tyrosine-containing fragments (Tyr-Gly-Gly- Phe, Tyr-Gly-Gly and Tyr-Gly).86,87 This provided the first HPLC method with fluorescence detection that is Fig. 18 Chromatogram of a standard mixture of N-terminal sufficiently sensitive to permit an assay of the endoge- tyrosine-containing peptides.91 Peaks and concentrations nous leucine-enkephalin in rat brain tissue (such as the (pmol/injection volume of 100µl) in parentheses: 1= striatum, cortex and hypothalamus) at concentrations kyotorphin (40); 2=dynorphin 1- 7 (40); 3=Tyr-Gly (20) and Tyr-Gly-Gly (20); 4=leucine-enkephalin-Arg (40); as low as 5.6 pmol/ g in the tissues.88 5=Tyr-Gly-Gly-Phe (20) and methionine-enkephalin-Arg- Gly-Leu (20); 6=dynorphin 1- 8 (40); 7=methionine- 6.2 N-Terminal tyrosine-containing peptides (opioid enkephalin-Arg-Phe (40); 8=methionine-enkephalin (40); peptides) and related enzyme activities 9=leucine-enkephalin (40); 10=a-endorphin (40); 11=[D- Mammalian brain tissues contain several opioid A1a2'3]-methionine-enkephalin (40); 12=y-endorphin (40). peptides, such as methionine-enkephalin and leucine- enkephalin, that play a role in the control of pain sensation. Most of the opioid peptides known have a tyrosyl residue at the N-terminus in their amino acid sequence. Such NVterminal tyrosine-containing peptides can be derivatized into the corresponding fluorescent compounds by a unique reaction: heating at 100°C for 1- 5 min in a weakly alkaline (pH 8 - 9) solution in the presence of hydroxylamine, cobalt(II) ion and borate (Fig. 17).89 The chemical structures of the fluorescent compounds remain unknown. The reaction can be

Fig. 19 Chromatogram of opioid peptides in cortex tissue of rat brains.91 Peaks and concentrations (pmol/g) in parentheses: 1=methionine-enkephalin (103); 2=leucine- Fig. 17 Fluorescence derivatization of N-terminal tyrosine- enkephalin (36); 3=[D-A1a2'3]-methionine-enkephalin (200, containing peptides. internal standard). 382 ANALYTICAL SCIENCES AUGUST 1989, VOL. 5

Fig. 20 Enzymatic hydrolysis of enkephalins by brain tissue homogenates.

not separated from Tyr-Gly-Gly and methionine- enkephalin-Arg-Gly-Leu, respectively, leucine-enkephalin, methionine-enkephalin and [D-A1a2'3]-methionine-enkephalin are mutually separated from the other tested Fig. 21 Some f7uorogenic reagents for reducing carbohy- peptides (detection limits, 220 - 810 fmol on column). drates. Methionine-enkephalin and leucine-enkephalin in rat brain tissues such as cortex, striatum and hypothalamus have been simultaneously measured by the precolumn method using [D-A1a2'3]-methionine-enkephalin as an tional groups, one of which is amino group, such as internal standard (detection limits, 6 pmol/ g tissue).91 ethanolamine94, 2-aminoethylsulfonic acid (taurine)95, The clean-up of the sample homogenate is attained by 2-cycanoacetamide96,9' (Fig. 21), 2-aminopropionitrile98 deproteinization with perchloric acid followed by a (Fig. 21), arginine99, p-methoxybenzamidine'o0,l01 (Fig. solid-phase extraction with an ODS cartridge. A 21), ethylenediaminelo2,'03 and meso-l,2-bis(4-methoxy- chromatogram obtained with cortex tissue of rat brains phenyl)ethylenediamine104 (Fig. 21) can be reagents for is illustrated in Fig. 19. reducing carbohydrates (1,2-ketoalcoholic compounds), A postcolumn HPLC system has been constructed which react in an alkaline or neutral medium at 100 - for the assay of N-terminal tyrosine-containing peptides, 150° C to produce fluorescence. They are all used in including enkephalins.92 The peptides are automatically postcolumn derivatization HPLC, and an amino- converted into fluorescent derivatives by the reac- bonded column with aqueous acetonitrile as the mobile tion given in Fig. 17 after peptide separation on a phase94,97,101or an anion-exchanger column with borate reversed-phase (ODS) column, followed by passage buffer (pH 7.4 -- 8.6)94,95,98,i03_b05 has been employed for through a UV (215 nm) detector if it is so required to the separation of reducing carbohydrates. simultaneously detect peptides without an N-terminal Of the reagents, 2-cyanoacetamide, 4-methoxybenz- tyrosyl residue. The HPLC system permits the fluores- amidine and meso-1,2-bis(4-methoxyphenyl)ethylene- cence detection of peptides in as little as picomole diamine appear to be most favorable in terms of amounts. sensitivity and reactivity towards various reducing The endogenous enkephalins are physiologically carbohydrates including amino sugars, uronic acids and inactivated by enkephalin-degrading peptidases, such as sialic acids. 2-Cyanoacetamide probably requires the aminopeptidase and enkephalinases A and B, that are participation of the hydroxy group at the 2-position of present in brain tissue (Fig. 20). Amnnopeptidase does the carbohydrate in the reaction; therefore, it cannot not have a specific function for the enkephalin afford fluorescence for 2-deoxy sugars, one of which, degradation in vivo. Enkephalinase A, however, has a 2-deoxy-D-ribose, is a component of DNA. significant role in the inactivation of enkephalins. 4-Methoxybenzamidine offers the advantage of rapi- Enkephalinase B has not been sufficiently characteriz- dity in reaction over the other fluorogenic reagents: the ed. The activities of enkephalinases A and B can be reaction comes to completion within 3 min. This simultaneously assayed by measuring Tyr-Gly-Gly and reagent provides fluorescence for 2-deoxy sugars, Tyr-Gly formed enzymatically from the substrate though its intensity is 10 -15%0 of that given by D- methionine-enkephalin, respectively, in the presence of glucose. On the other hand, meso-l,2-bis(4-methoxy- bestatin as an inhibitor of aminopeptidase and captop- phenyl)ethylenediamine reacts with all kinds of reduc- ril as an inhibitor of angiotensin-converting enzyme.93 ing carbohydrates, especially 2-deoxy sugars such as The measurement of tri- and dipeptides is performed 2-deoxy-D-ribose and 2-deoxy-D-glucose. Typical chro- by the postcolumn HPLC system. matograms of normal human serum and urine obtained by an anion-exchange HPLC with postcolumn fluorescence derivatization using meso-l,2-bis(4-methoxyphen- 7 Reducing Carbohydrates yl)ethylenediamine are shown in Fig. 22 (detection limits, 10 -100 pmol on column), 105 Many compounds having vicinal or geminal bifunc- ANALYTICAL SCIENCES AUGUST 1989, VOL. 5 383

polyunsaturated, long-chain fatty acids [a-linoleic 8 Fatty Acids Including Prostaglandins (C18:2), a-linolenic (C18:3), dihomo-y-linolenic (C20:3), arachidonic (C20:4)and eicosapentaenoic (C20:5)acids] 8.1 Long-chain fatty acids are known to be the precursors of prostaglandins (PGs) Many free fatty acids are present in serum. Several and thromboxanes, and play important roles in the living body. Especially, C20:5acid is known to prevent arteriosclerosis. Although docosahexanoic (C22:6)acid occurs in human serum at a relatively high concentration, the physiological roles of the acid in the human body have still remained unknown. In addition to the polyunsaturated fatty acids, several saturated and unsaturated long-chain fatty acids, including lauric (C12:0), myristic (C14:0), myristoleic (C14:1), palmitic (C16:0), palmitoleic (C16:1),stearic (C18:0)and oleic (C18:1)acids, are found in serum (the total of the long-chain fatty acid mentioned, thirteen species). Various fluorogenic reagents for carboxylic acids have been developed so far6, which are all used in precolumn HPLC because they are fluorescent and require mostly a dried aprotic solvent in the derivatization reaction. Among the reagents, 106'107, 9-anthryldiazomethanelos-110 and 4-bromomethyl-7-acet- oxycoumarin111"12 (that work on long-chain fatty acids to yield the corresponding amides, esters and esters, respectively) have been applied to the HPLC determination of the fatty acids. However, these reagents are not Fig. 22 Chromatograms of reducing carbohydrates in (A) a sufficiently sensitive to simultaneously quantify the normal human urine (24 h) and (B) a normal human serum thirteen long-chain fatty acids in normal human serum (17 h after supper).'°5 Peaks; 1=N-acetylglucosamine; 2= at femtomole levels. cellobiose; 3=maltose; 4=lactose and/or L-rhamnose; 5= 3-Bromomethyl-6,7-dimethoxy- l -methyl-2(1 H)-quinox- D-fructose; 6=D-arabinose and/or L-fucose; 7=D-galactose; alinone (Br-DMEQ, Fig. 23) has been introduced as a 8=D-xylose; 9=D-glucose; 10=a-melibiose; 11=D-mannose; fluorogenic reagent for carboxylic acids on the basis of 12=unidentified, probably reducing carbohydrates; 13= the observations that the reaction of DDB with pyruvic endogeneous fluorescent compounds. Concentrations (nmol/ acid can afford a highly fluorescent product (6,7- ml): (A), 1=170; 2=130; 3=180; 5=200; 7=280; 8=230; 9=370; 10=400; (B), 5=40; 9=5010; 10=400; 11=40. dimethoxy-3-methyl-2(1 H)-quinoxalinone, cf. Section

Fig. 23 Reactions of Br-DMEQ, Br-MMEQ and panacyl bromide with fatty acids. 384 ANALYTICAL SCIENCES AUGUST 1989, VOL. 5

8.2), a methylation product of which (6,7-dimethoxy- oxalinone (Br-MMEQ, Fig. 23) can determine saturat- 1,3-dimethyl-2(1 H)-quinoxalinone) exhibits a more ed C3 - C20 fatty acids with the detection limits of intense fluorescence, l 13,114 0.2 - 0.8 fmol on column in reversed-phase (octyl-bond- Br-DMEQ reacts with fatty acids in acetonitrile in ed column) HPLC.117 the presence of potassium carbonate and 18-crown-6 to produce the corresponding esters (Fig. 23). The 8.2 Prostagla.ndins reagent can detect long-chain fatty acids as low as 0.5 - Many kinds of PGs, structurally categorized in fatty 2 fmol on a column in reversed-phase HPLC on an acids, occur in biological materials, and play diverse octyl-bonded column, and permits the quantification of roles of physiological importance at trace levels in the all the thirteen fatty acids in human serum using living body. Although several precolumn HPLC margaric (C17:0)acid as an internal standard)15,116 methods with fluorescence detection have been reported A fluorogenic reagent analogous to Br-DMEQ, 3- for the assay of PGs, the methods utilizing p-(9- bromomethyl-6,7-methylenedioxy-l-methyl-2(1 H)-quin- anthroyloxy)phenacyl bromide (panacyl bromide)' 18, a fluorogenic reagent for carboxylic acids (for the reaction scheme, see Fig. 23), and Br-DMEQ are extremely sensitive and permit the quantification of femtomole levels of endogenous PGs in some biological materials. The panacyl derivatives of PGs are separable on normal (silica gel) and reversed-phase (ODS) columns19,'20 and the DMEQ derivatives on a reversed- phase (Octyl) column.121 Chromatograms obtained from a standard mixture of eleven PGs and normal human seminal fluid are shown in Fig. 24. The detection limits of the PGs are 10 -15 fmol on column, and PGE,, PGE2, PGFIa and PGF2a in human seminal fluid can be determined using 16-methyl-PGFIa as an internal standa.rd.121

9 Nucleic Acid Bases, Nucleosides and Nucleo- tides

Nucleosides having an active imino hydrogen in the molecule, such as uridine, deoxyuridine and thymidine, Fig. 24 Chromatograms of the DMEQ derivatives of PGs in combine with Br-DMEQ to yield the corresponding (A) a standard mixture and (B) normal seminal fluid.121 fluorescent products that are separable on a reversed- Peaks: U PGE,; 2=PGE2; 3=PGFIa; 4=PGFIa; 5=16- phase (ODS) column.114 methyl-PGFIa; 6=PGA, and PGBI; 7=PGA2 and PGB2; An antitumor agent, 5-fluorouracil (a pyrimidine 8=PGD2; 9=6-keto-PGF,a;10=Br-DMEQ;11=endogenous base) and its prodrug, 5-fluoro-2'-deoxyuridine (a carboxylic acids and Br-DMEQ. Concentrations: (A, nucleoside) can also be derived to fluorescent com- pmol/ 100 µl standard mixture), 1- 5, 8 and 9=10; 6=5 each; 7=5 each, (B, nmol/ml seminal fluid): 1=94.1; 2=84.1; pounds by reaction with Br-DMEQ (Fig. 25). These 3=8.2; 4=5.5; 5=150 (internal standard). findings have been applied to precolumn HPLC on a

Fig. 25 Reactions of Br-DMEQ with 5-fluorouracil and 5-fluoro-2'-deoxyuridine. Fig. 26 Reaction of phenylglyoxal with guanine and its nucleosides and nucleotides.

Fig. 27 Reactions of DMEQ-COCI and DMEQ-CONS with alcoholic compounds.

reversed-phase (ODS) column for the simultaneous secondary alcohols involving hydroxysteroids to the monitoring of these drugs in human serum (detection corresponding fluorescent esters (Fig. 27), which can be limits, 375 -575 fmol on column).122 separated on a reversed-phase (Octyl) column in HPLC Guanine and its nucleosides and nucleotides can be (detection limits, 2 - 3 fmol on column). derived to fluorescent compounds by reaction with A reagent analogous to DMEQ-COCI, 6,7-dimeth- phenylglyoxal in weakly acidic medium'23 (Fig. 26), oxy- l -methyl- 2(1H)-quinoxaline-3-carbonyl azide which may be used in postcolumn HPLC. The (DMEQ-CONS) is more active and reacts with not reaction has been applied to precolumn HPLC on a only primary and secondary alcohols but also tertiary reversed-phase (ODS) column for the assay of guano- alcohols to give the corresponding fluorescent car- sine-3',5'-cyclic monophosphate, a second messenger of bamates (Fig. 27), which can be resolved by reversed- 1 24 phase HPLC on an octyl-bonded column.126 The detection limits for aliphatic and arylaliphatic alcohols are 2 - 5 fmol on column and those for hydroxysteroids 10 Biogenic Alcoholic Compounds with primary, secondary and/ or tertiary alcoholic group(s) vary between 3 - 45 fmol on column, depend- A number of diverse fluorogenic reagents that are ing on the species of target hyroxysteroid. sensitive to alcoholic compounds of biological impor- The cholestanol present in human serum as free and tance have recently been reported.6 The reagents are esterified forms in trace amounts can be simultaneously all fluorescent and, therefore, have been used only for measured with the cholesterol that occurs at a relatively precolumn derivatization in HPLC. high concentration in human serum after conversion 6,7-Dimethoxy-l-methyl-2(1 H)-quinoxalinone-3-car- with DMEQ-CONS into the fluorescent carbamates, bonyl chloride (DMEQ-COCI) has been developed as a with the detection limits of 5 - 7 fmol on column.127 more sensitive reagent for alcoholic compounds, which This method should be a useful aid in the diagnosis of is analogous to Br-DMEQ in the fluorescent moiety'25 cerebrotendious xanthomatosis. 7-Dehydrocholesterol, (cf. Section 8.1). The reagent leads primary and a precursor of vitamin D3, is also present as free and

386 ANALYTICAL SCIENCES AUGUST 1989, VOL. 5 esterified forms in skin. DMEQ-CONS can derive 7- systems as described above will enable HPLC to be dehydrocholesterol to a highly fluorescent carbamate applied to ultrahigh sensitivity analysis of bioactive derivative that is separable on an ODS column, and the compounds. determination of the free and the total (the sum of free and esterified) 7-dehydrocholesterol in rat skin has been The author is grateful to Drs. M. Kai and H. Nohta of achieved (detection limits, 18 fmol on column).128 this laboratory for their help in the preparation of the manuscript.

11 Conclusion and Future Trend References Over the last two decades, pre- or postcolumn fluorescence derivatization HPLC has been demonstrat- 1. R. W. Frei and J. F. Lawrence, J. Chromatogr., 83, 321 ed to be a sensitive and selective analysis method and (1973). has been practically applied to the quantification of 2. N. Seiler, J. Chromatogr.,143, 221 (1977). bioactive substances. Some instances of the use of 3. J. F. Lawrence, J. Chromatogr. Sci., 17, 147 (1979). bifunctional reagents and related monofunctional re- 4. K. Imai, T. Toyo'oka and H. Miyano, Analyst [London], 109, 1365 (1984). agents for the fluorescence derivatization of bioactive 5. H. Lingeman, W. J. M. Underberg, A. Takadate and A. substances in HPLC are shown here. The reagents Hulshoff, J. Liq. Chromatogr., 8, 739 (1985). allow many substances to be detected at the femtomole 6 . Y. Ohkura and H. Nohta, in "Advances in Chromatog- or subfemtomole levels. Much more sensitive, selective raphy", Vol. 29, pp. 221- 258, ed. J. C. Giddings, E. and practical fluorogenic reagents have still been Grushka and P. R. Brown, Marcel Dekker, New York, required to be introduced in fluorescence derivatization 1989. HPLC for bioactive substances that are present in trace 7. D. R. Knopp, "Handbook of Analytical Derivatization amounts in biological materials. Detection at the Reaction", Wily-Interscience, New York, 1979. femtomole or subfemtomole levels, however, seems to 8. R. W. Frei and J. F. Lawrence, "Chemical Derivatization be the lower limit of sensitivity that can be obtained by in Analytical Chemistry", Plenum, New York, 1981. conventional HPLC with a fluorescence detector. 9. Y. Ohkura and K. Zaitsu, Talanta, 21, 547 (1974). 10. K. Zaitsu and Y. Ohkura, Chem. Pharm. Bull., 23, 1057 Higher sensitivity that enables bioactive substances to be quantified at the attomole level has become (1975). 11. T. Kaito, K. Sagara and K. Ikunaga, Chem. Pharm. Bull., necessary in various areas of life science. Therefore, 27, 3167 (1979). techniques based on instrumental principles, such as the 12. M. Nakamura, M. Toda, H. Saito and Y. Ohkura, Anal. use of a laser as the excitation source for fluorescence Chim. Acta,134, 39 (1982). detector and time-resolved fluorometry, have been 13. W.-F. Chao, M. Kai, J. Ishida, Y. Ohkura, S. Hara and introduced to the HPLC system. Laser-excitation M. Yamaguchi, Anal. Chim. Acta, 215, 259 (1988). offers not only enhanced fluorescence but also sup- 14. J. Ishida, M. Yamaguchi, M. Kai and Y. Ohkura, J. pressed background fluorescence, because of the high Chromatogr., 305, 381 (1984). intensities and well-defined monochromaticity of laser 15. J. Ishida, M. Kai and Y. Ohkura, Xenobio. Metabol. beams.129,130 Time-resolved fluorometry can be used to Dispos., 1, 397 (1986). selectively detect compounds having longer fluorescence 16. J. Ishida, M. Kai and Y. Ohkura, J. Chromatogr., 344, 267 (1985). lifetimes, and shorter-lifetime fluorescences from co- 17. J. Ishida, M. Kai and Y. Ohkura, J. Chromatogr., 431, 249 existing compounds; light-scattering does not cause (1988). interference. A combination of the two techniques, 18. W.-F. Chao, M. Kai and Y. Ohkura, J. Chromatogr., 430, laser-induced time-resolved fluorometry, offers a good 361 (1988). possibility of improving the detection limit in the 19. Y. Ohkura, K. Ohtsubo, K. Zaitsu and K. Kohashi, Anal. attomole range for the HPLC analysis of biosubstances. Chim. Acta, 99, 317 (1978). This requires the development of a more practical laser 20. K. Ohtsubo, Y. Okada, K. Zaitsu and Y. Ohkura, Anal. and a time-resolved fluorometer. On the other hand, Chim. Acta,110, 335 (1979). laser-induced time-resolved fluorometry requires re- 21. T. Uno and H. Taniguchi, Bunseki Kagaku, 21, 76 (1972). agents that can afford long-lifetime fluorescent deriva- 22. H. Nohta, K. Ohtsubo, K. Zaitsu and Y. Ohkura, J. tives with excitation maxima at wavelengths longer Chromatogr., 227, 415 (1982). 23. H. Nohta, M. Yamaguchi, K. Zaitsu and Y. Ohkura, J. than approximately 450 nm. Chromatogr., 233, 324 (1982). Microcapillary column HPLC techniques may be 24. H. Nohta, K. Zaitsu, Y. Tsuruta and Y. Ohkura, J. used to improve the detection sensitivity when coupled Chromatogr., 280, 343 (1983). with a special detection system, such as on-column 25. H. Nohta, K. Zaitsu, Y. Tsuruta and Y. Ohkura, Anal. laser-induced fluorescence detection.131 Peroxyoxalate Chim. Acta,156, 253 (1984). chemiluminescence detection, a method of chemical 26. K. Zaitsu, Y. Okada, H. Nohta, K. Kohashi and Y. excitation of fluorescent compounds, has improved the Ohkura, J Chromatogr., 211,129 (1981). detection sensitvity.132 27. H. Nohta, S. Noma and Y. Ohkura, J. Chromatogr., 308, In the near future, such fluorescence detection 93 (1984). ANALYTICAL SCIENCES AUGUST 1989, VOL. 5 387

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