6-Aminomethylphthalhydrazide as a highly sensitive chemiluminescence derivatization reagent for 5-hydroxyindoles in liquid chromatography

ELSEVIER Analytica Chimica Acta 346 ( 1997) 175- 18 1

ANALYTICA CHIMICA ACTA

6-Aminomethylphthalhydrazide as a highly sensitive chemiluminescence derivatization reagent for Shydroxyindoles in liquid chromatography

Junichi Ishida”, Tomohiro Yakabe”, Hitoshi Nohtab, Masatoshi Yamaguchi”.*

’ Faculty of Pharmaceutical Sciences, Fukuoka University, Nanakuma, .Johnun-ku, Fukuoka 814.80. Jqxm h Laboratory of Molecular Biophotonics, 5000 Hirakuchi, Hamukita. Shizuoka 434. Japm

Received 29 November 1996; accepted 5 February 1997

Abstract

6-Aminomethylphthalhydrazide was synthesized as a highly sensitive and selective chemiluminescence derivatization

reagent for 5hydroxyindoles in liquid chromatography. 5_Hydroxytryptophan, serotonin and 5-hydroxyindole-3-acetic acid were used as model compounds to optimize the derivatization conditions. The reagent reacts selectively with the indoles in the presence of potassium hexacyanoferrate(II1) to give highly chemiluminescent derivatives which produce chemiluminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(II1) in alkaline solution. The

chemiluminescent derivatives of the three Shydroxyindoles can be separated within 35 min by reversed-phase liquid chromatography with isocratic elution, followed by chemiluminescence detection. The detection limits (signal-to-noise ratio=3) for S-hydroxyindoles are in the range 0.74 fmol for a 20 pi injection.

Ke~w,o~/.\: Chemiluminescence: Liquid chromatography; 5-Hydroxyindoles: 6-Aminomethylphthalhydrazide

1. Introduction

Several methods have been reported for the determination of 5-hydroxyindoles including serotonin by liquid chromatography (LC) with native fluorimetric [ l-31 and electrochemical detections [4-61, gas chromatography-mass spectrometry [7] and radioimmu- nological [8,9] and radioenzymatic [ IO,1 l]

techniques. Chemiluminescence (CL) detection sys- tems have been also used in LC analysis owing to their high sensitivity. However, no CL-based methods have

Y’orresponding author.

0003-2670/97/$17.00 8; 1997 Elsevier Science B.V. All rights reserved. PI/ SOO03-2670(97)00122-O

been developed for the determination of 5-hydroxyindoles.

In a previous work [12,13], we have reported that aromatic methylamines react highly selectively and sensitively with 5-hydroxyindoles in weakly alkaline media in the presence of potassium hexacyanoferrate(II1) to produce highly fluorescent benzoxazole derivatives. Benzylamine was found to be a suitable derivatization reagent for the post-column fluorimetric

LC determination of 5-hydroxyindoles. However, for the determination of bioactive indoles such as serotonin occurring at low concentrations in small amounts of biological samples. a more sensitive method is required.

176 J. Ishida et al./Analytica Chimica Acta 346 (1997) 175-181

Only a few derivatization reagents, N-(4-aminobu- tyl)-N-ethylisoluminol (ABEI) for carboxylic acids [ 14,151 and 4-isothiocyanatophthalhydrazide for

amino acids [16], have been reported for the luminol CL system. These luminol type reagents have a 4-N- substituted aminophthalic acid as a light emitter. The

intensity of the luminescence generated from the CL derivatization reagents is known to be partially depen-

dent on the fluorescence quantum yield of the light

emitter. Therefore, in order to increase the sensitivity of the reagent, highly fluorescent compounds should be used as an emitter.

We have investigated new luminol-type CL reagents which have higher efficiency than previous reagents. As described above, one of our strategies to obtain higher sensitivity is to introduce a more highly

fluorescent skeleton than luminol. Quinoxalinone and quinoxaline derivatives were found to be highly fluorescent compounds. Thus, some new luminol type CL reagents with those skeletons have been developed

[17,18].

Here, we report a novel luminol-type CL derivatization reagent, 6-aminomethylphthalhydrazide (6- AMP)(Fig. 1). 6-AMP has a benzylamine skeleton as a reaction site and a cyclic phthalhydrazide as an

::

“7 ,c

HN ‘C

6

6-AMP

emission site. 6-AMP reacts selectively with 5-hydro-

xyindoles in the presence of potassium hexacyanoferrate(II1). The resulting derivatives, which fluoresced intensely, produce CL by reaction with hydrogen peroxide in the presence of potassium hexacyanofer-

rate(II1) in alkaline medium. In this work, we examined the optimum derivatization and CL reaction

conditions, and developed a sensitive and selective

method for the determination of 5-hydroxyindoles using 6-AMP, based on LC with CL detection.

2. Experimental

2.1. Apparatus

Infrared spectra were recorded with a Jasco (Tokyo) IRA-2 spectrophotometer using potassium bromide pellets. Fast atom bombardment mass spectra (FAB-MS) were taken with a JEOL (Tokyo) DX-300 spectrometer. High resolution FAB-MS measure-

ment was made on a JEOL JMS-HXllO instrument.

Uncorrected melting points were measured with a Gallenkamp (Loughborough, UK) melting point apparatus.

K3Fe(CNhj

ii Chemiluminescent derivatives

I.3202

KPe(CNhj *

+ hv

Fig. 1. CL derivatization of 5-hydroxyindoles with 6-AMP, and CL reaction of the derivatives.

J. Ishida et al./Anul~ticn Chimica ,~lcro 346 (1997) 17.S181 177

2.2. Chrmicals and .solutions

Distilled water, purified with a MU-Q II system (Millipore), was used for all aqueous solutions. Hydrogen peroxide (3 I%, v/v) was purchased from Mitsubishi Gas Kagaku (Tokyo). 5-Hydroxytrypto-

phan (SHTP). serotonin (SHT), 5-hydroxyindole-3- acetic acid (SHIAA), S-hydroxytryptophol (5HOL),

S-hydroxyindole-3-acetamide (5HA) and N-acetyl-S- hydroxytryptamine (N-AcSHT) were purchased from

Sigma (St. Louis, MO). Standard solutions of these

compounds were prepared in water and kept frozen ( ~-20 C) in amber colored test-tubes. Other chemicals

were of the highest purity available and were used as received.

.?._<. Synthrsi.s of b-AMP

&AMP was synthesized via compounds I-III from 4-methyl-N-phenylphthalimide in satisfactory yields

by the following methods (Fig. 2). 4-Methyl-N-phenylphthalimide was obtained by a known method [ 191.

3-Methyl-N-phenylphthalimide (10.0 g) was dis- sol\;ed in 190 ml of tetrachloromethane. To the solu-

tion. N-bromosuccinimide (8.3 g) and benzoyl peroxide (0.4 g) were added successively. The suspen- Gon was refluxed for 4 h and evaporated to dryness

under reduced pressure. The residue was dissolved in 30 ml of benzene, purified by column chromatography

(25 x 3.5 cm i.d. column) on silica gel 60 (ca. 120 g,

70-230 mesh; Japan Merck, Tokyo) with benzene-n-

Fig. 2. Synthesis of 6-AMP.

hexane (3 : 2, v/v) as eluent. The fractions correspond-

ing to compound I were collected and evaporated to dryness under reduced pressure to give compound I (ca. 5.7 g) as a pale yellow crystalline solid. m.p. 176.2-177.9 C (decomp.). IRv,,,,,: 1700 cm ’ (lactam C=O). FAB-MS showed in/:= 3 16. 3 18 [Mvt I 1’

2.3.2. Cornpound II Compound I (5.0 g) and hexamethylenetetramine

(2.5 g) were dissolved in 250 ml of chloroform and left at room temperature overnight. The resulting

precipitate, collected by tiltration. was rinsed with much chloroform and dried to yield compound II

(ca. 7.0 g) as a pale yellow crystalline solid, m.p. 224.3-226.9 C (decomp.). ~Ru,,,;,,: 1700 cm ’ (Iac- tam C=O). FAB-MS, nz/:-376 [ Mt 11 ‘.

Compound II (I.0 g) was dissolved in 750 ml of

ethanol containing S7r hydrochloric acid with warm-

ing at SO-60 C and then left at room temperature overnight. The solution was evaporated under reduced

pressure. The residue was recrystallized from ethanol to afford compound III (ca. 0.36 g) as a yelloN crystalline solid, m.p. 286.2-288.X C (decomp. ). IRv,,,: 1700 cm ~~’ (lactam C=O). FAR-MS. nr/-

253 [Mtlj

2.3.4. &AMP

Compound III (0.45 g) was dissolved in 60 ml 01‘

ethanol with warming. To the solution. I ml (I .03 g) of hydrazine monohydrate was added, and the mixture

was refluxed for 40 min. The precipitate which was formed was washed with ethanol and dried to give 6 AMP (ca. 0.16 g) as a pale yellow crystalline solid.

m.p. 290.8--293.7 C (decomp.). FAB-MS, nl/:= 192 IM+ I ] ‘. High-resolution FAB-MS. ~v/z= 192.0774

(as calcd. for CgHIoOZN-,). IRo,,,;,,: 1700 cm ’ (lx- tam C=O): 3 I SO cm-’ (lactam NH).

The total yield of the 6-AMP synthesis was ca. IO%. 6-AMP was stable in the crystalline state at room temperature for at least 6 months in a deslccatot

containing \ilica gel.

Test solutions of S-hydroxyindoles were prepared in water. 6-AMP (0.5 mM) solution was prepared in


dimethylsulphoxide and used within a week. Hydro-

gen peroxide (5 mM) and potassium hexacyanoferrate(III) (5 n&I) solutions were prepared in water and 0.5 M sodium hydroxide, respectively.

2.5. Derivatization procedure

To a 100 pl portion of a test solution of 5-hydroxyindoles placed in a test tube (100x 15 mm id.) were

added successively 100 ul each of the 6-AMP solution, 20 mM borate buffer (pH 9.0) and 100 mM potassium hexacyanoferrate(II1) solution. The mixture was allowed to stand at room temperature for 2 min. A 20 ~1 portion of the final reaction mixture was injected

into the chromatograph. For the reagent blank, 100 ul of water in place of a test solution were subjected to

the same procedure.

2.6. LC conditions and chemiluminescence detection system

Chromatography was performed with a Hitachi

(Tokyo) L-6200 liquid chromatograph equipped with a Rheodyne 7125 syringe-loading sample injector vaive (20 ul loop). The 6-AMP derivatives of 5-hydroxyindoles were separated on a reversed-phase column

(Shiseido Capcell Pat C18, 5 pm, 250x4.6 mm id., Shiseido, Tokyo) by isocratic elution with aceto-

nitrile- mM acetate buffer (pH 4.3) (1 : 4, v/v) at a flow-rate of 0.8 ml min-‘. The column temperature was ambient (23f2”C).

The eluate from the LC column was mixed with 5.0 mM hydrogen peroxide solution by the first T-type mixing device and then with 5.0 mM potassium hexacyanoferrate(II1) in 0.5 M sodium hydroxide by the second T-type mixing device, delivered by two Hitachi L-6000 pumps at flow-rates of 0.7 and 1.5 ml mini’, respectively. The generated CL was

monitored by a 825-CL intelligent CL detector (Jasco)

equipped with a 90 yl flow cell. Stainless-steel tubing (0.5 mm id.) was used for the LC-CL system.

3. Results and discussion

SHTP, 5HT and SHIAA were used as model compounds to establish LC and derivatization conditions suitable for a more general method.

L

I I I 0 15 30

Time ( min )

Fig. 3. Chromatogram of the 6-AMP derivatives of 5-hydroxy-

indoles. Peaks (2.5 pmol each on column): 1=5HTP; 2=5HT;

3=5HIAA; 4=reagent.

3.1. LC conditions

Fig. 3 shows a typical chromatogram obtained with

a mixture of the three 5-hydroxyindoles. A good separation of the 6-AMP derivatives was achieved on the reversed-phase column by isocratic elution with the acetonitrile-20 mM acetate buffer. The individual compounds each gave a single peak. In the case of a peroxyoxalate chemiluminescence system, alco- holic solvents such as methanol, which is widely used in reversed-phase LC, can not be used because of their quenching effect on the CL [20,21]. Methanol did not affect the peak heights of the derivatives in this

J. Ishida et al. /Analytica Chimica Acts 336 (199’) 175%INI

25

0

fl 5HTP

q 5HT

SHIAA

DMSO H20 Ethanol Methanol Acetonitrile Acetone DMF 1,4-Dioxane THF

Fig. 4. Effects of solvents for the derivatization reaction on the CL intensit).

proposed CL system. Therefore, a greater variety of separation conditions are applicable for the determi-

nation of the CAMP derivatives

.3.2. Derivatization qf 5-hydroxyindoles with 6-AMP

Solvents in the reaction medium affected the derivatization yield (Fig. 4). Dimethylsulphoxide gave maxi-

mum peak heights. However, the derivatization reaction was limited in methanol, ethanol, acetone, acetonitrile dimethylformamide, I ,4-dioxan and tetrahy- drofuran. Dimethylsulphoxide as solvent allowed the

most intense peaks at concentrations between 12.5 and 40% (v/v) in the reaction mixture; 25% dimethyl-

sulphoxide was selected tentatively. Dimethylsulph- oxide was therefore used for the preparation of 6-AMP.

The derivatization reaction proceeded in the presence of potassium hexacyanoferrate(II1). The potas-

sium hexacyanoferrate(II1) solution gave the most intense peaks at a concentration greater than 80 mM: 100 mM was used as a sufficient concentration.

The derivatization reaction of 6-AMP with Shydro- xyindoles occurred very rapidly even at 0°C. The peak

heights for all Shydroxyindoles reached maximum and constant values after standing at room temperature

for >I min. Therefore, standing at room temperature for 2 min was selected in the recommended procedure.

The 6-AMP solution gave the most intense and constant peaks at concentrations ~0.4 mM: 0.5 mM was adopted for the recommended procedure. The h-

AMP derivatives in the final solution were stable and still gave constant peak heights after standing for al least 3 d in daylight at room temperature.

3.3. Cherniluminescence reaction

The optimum conditions for the CL reaction were examined by setting the flow rates of the hydrogen peroxide and potassium hexacyanoferrate(II1) solu-

tions to 0.7 and I .5 ml min- ‘. respectively. The CL intensities were affected by the concentra-

tions of hydrogen peroxide, potassium hexacyanoferrate(II1) and sodium hydroxide. The effects of the concentrations of the reagents on the CL reaction were examined. The concentrations of the reagents were varied one at a time to establish the maximum


(a) (b)

50

Hz02 (mM)

100 0 25 5” 0 1 .o 2.0 3.0

K,Fe(CN)c (mM) NaOH (M)

Fig. 5. Effects of (a) hydrogen peroxide, (b) potassium hexacyanoferrate(II1). and (c) sodium hydroxide concentration on the CL peak heights.

Curves: 1 =SHTP; 2=5HT; 3=5HIAA.

intensity obtainable. Based on these experiments (Fig. 5), 5.0 mM hydrogen peroxide, 5.0 mM potas-

sium hexacyanoferrate(II1) and 0.5 M sodium hydroxide were selected.

The CL reaction occurred immediately after the

eluate from the column was mixed with potassium hexacyanoferrate(II1) in the second mixing device. Therefore, the length of tubing between the second mixing device (M2) and the detector affected the CL response. The peak heights for all amines increased

with decreasing length of the tubing; 5 cm was used as

the shortest practicable length.

3.4. Calibration graph, precision and detection limits

The relationships between the peak heights and the

amounts of the individual 5-hydroxyindoles were linear up to at least 25 pmol per 20 pl injection

volume; the linear correlation coefficients were

20.996 (n=6) for all the indoles. The precision was established by repeated determi-

nation (12=9) using mixtures of the three 5-hydroxyindoles at different concentrations. The relative standard deviations did not exceed 2.3% at 2.5 pmol each per 20 pl injection and were 6.0-7.5% at 50 fmol each per 20 pl injection.

The detection limits (fmol per 20 pl injection, signal-to-noise ratio=3) were 1.96 (SHTP), 2.50 (5HT) and 3.67 (SHIAA), respectively.

3.5. Reaction of other substances with 6AMP

Some other 5-hydroxyindoles reacted with 6-AMP under the proposed derivatization condition. Table 1 gives the detection limits and retention times for the 6-

AMP derivatives of the compounds. Each compound examined provided a single CL peak in the chromatogram. Highly sensitive detection in the range of 0.69-

3.67 fmol can be achieved. Catecholamines also reacted with 6-AMP with

different reaction conditions to give the CL derivatives. A study of this reaction is in progress. No other biologically important substances examined produced CL under the recommended procedure at a concentration of 10 nmol ml-‘. The compounds tested

were tryptophan, tryptamine, indole-3-acetic acid, melatonin, 5-methoxytryptophan, 5-methoxytrypta- mine, 5-methoxyindole-3-acetic acid, ascorbic acid, glutathione, thiamine, uric acid, urea, acetone, n-

decylaldehyde, benzaldehyde, 4-anisaldehyde, aceto- phenone, lactic acid, palmitic acid, caproic acid,

myristic acid, pyruvic acid, phenylpyruvic acid, Q- ketoisocaproic acid, ai-ketoisovaleric acid, cu-ketova- leric acid, N-acetylneuraminic acid, n-nonylalcohol, benzyl alcohol, cyclohexanol, cholesterol, o-glucose, o-xylose, o-fructose, o-mannose, o-maltose, o-lactose, inositol, epiandrosterone, dehydroepiandrosterone, cortisone and methylglyoxal. These results suggest that the present chemiluminescent derivatization method is usefully selective for 5-hydroxyindoles.

.I. Ishida et al. /Analytica Chimica Acta 346 (1997) 175-181 1x1

Table 1

Detection limits and retention times for 6-AMP derivatives of 5-hydroxyindoles

5-Hydroxyindole

5Hydroxy-L-tryptophan (SHTP)

S-Hydroxytryptamine (SHT) 5Hydroxyindole-3.acetic acid (SHIAA)

S-Hydroxyindole-3.acetamide

S-Hydroxytryptophol

N-Acetyl-S-hydroxytryptamine

Detection limit

(fmol) a

I .96

2.50

3.67

0.69

2.20

1.82

Retention time

(nun)

7.9

13.2

30. I

23. I 10.8 14.5

” The amount in the injection volume (20 ~1) giving a signal-to-noise ratio of 3

The chemical structure of the CL product in the reaction of 5hydroxyindoles with 6-AMP remains

unknown. However, Sundaramoorthi et al. have reported that 1 ,bdimethyl-6-hydroxycarbazole, which has a hydroxyindole moiety, reacted with N-

monosubstituted primary amines such as propylamine

and benzylamine to give the corresponding oxazole derivatives in the presence of manganese dioxide as an oxidizing agent [22]. Therefore, 2-phenyloxazolo[4,5-

elindole compounds having a cyclic phthalhydrazide are probably formed in the proposed derivatization

reaction (Fig. 1). The subsequent CL reaction might give the corresponding excited phthalate ions resulting

in CL emission.

4. Conclusions

&AMP was developed as a novel CL derivatization reagent for the highly sensitive and selective determination of 5-hydroxyindoles. The CL-LC method

using the reagent can be applied to the sensitive measurement of biological substances and drugs having S-hydroxyindoles in body fluids. Further studies are in progress.

Acknowledgements

The authors are grateful to Dr. M. Nakamura (Faculty of Pharmaceutical Sciences, Fukuoka University) for helpful suggestions, and Miss M. Tanaka and Mr. T. Yokoyama for their skilfull assistance.

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Documents

6-Aminomethylphthalhydrazide as a highly sensitive chemiluminescence derivatization reagent for 5-hydroxyindoles in liquid chromatography