Analytica Chimica Acta, 223 (1989) 319-326 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

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3,4-DIHYDRO-6,7-DIMETHOXY-4-METHYL-3-OXOQUINOXA- LINE-2-CARBONYL CHLORIDE AS A SENSITIVE FLUORES- CENCE DERIVATIZATION REAGENT FOR AMINES IN LIQUID CHROMATOGRAPHY

JUNICHI ISHIDA, MASATOSHI YAMAGUCHI*, TETSUHARU IWATA and MASARU NAKAMURA

Faculty of Pharmaceutical Sciences, Fukuoka University, Nanakuma, Johnan-ky Fukuoka 814-01 (Japan)

(Received 27th January 1989)

SUMMARY

A rapid and sensitive method for the fluorescence derivatization of primary and secondary amines is described, based on the reaction of the amines with 3,4-dihydro-6,7-dimethoxy-4-methyl- 3-oxoquinoxaline-2-carbonyl chloride. Cyclohexylamine, n-hexylamine and di-n-butylamine were used as model compounds to optimize the derivatization conditions. The reagent reacts with the amines in acetonitrile in the presence of potassium carbonate very rapidly to give the correspond- ing fluorescent amides, which can be separated on a reversed-phase column, TSKgel ODS-80TM, with aqueous acetonitrile as eluent. Alcohols and amino acids did not give any fluorescent products under the derivatization conditions. The detection limits are in the range 5-50 fmol per 20-~1 injection. Reactions with other amines are also discussed.

Several derivatization reagents having a carbonyl chloride group have been introduced for the determination of amines by liquid chromatography (LC) with spectrophotometric and fluorimetric detection. The spectrophotometric LC methods using benzoyl chloride [ 1,2], 4-biphenylcarbonyl chloride [ 31 and m-toluoyl chloride [ 41, which have been widely used, are neither selective nor sensitive. A fluorimetric LC method based on reaction with phthalimidylben- zoyl chloride [ 51 offers high sensitivity at the lo-fmol level.

In previous work, 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxoquinoxaline-2- carbonyl chloride [ 61 (DMEQ-COCl) was developed as a fluorescence deri- vatization reagent for primary and secondary alcohols. Recently, it was found that primary and secondary amino compounds also react with DMEQ-COCl under conditions different from those for alcohols to give highly fluorescent amides. Thus we have developed a sensitive, rapid and selective method for the determination of amines using DMEQ-COCl, based on LC with fluorime- tric detection. Cyclohexylamine, n-hexylamine and di-n-butylamine were used as model compounds to establish reaction conditions suitable for a more gen-

0003-2670/89/$03.50 0 1989 Elsevier Science Publishers B.V.

era1 method. The reactivities of DMEQ-COCl with other amines were also investigated.

EXPERIMIiNTAL

Reagents and materials All chemicals were of the highest purity available and were used as received.

Distilled water, purified with a Milli-QII system, was used for all aqueous so- lutions. DMEQ-COCl was prepared as described previously [ 61. DMEQ-COCl solution (2 mM) was prepared by dissolving 2.8 mg of DMEQ-COCl in 5 ml of acetonitrile; this solution was used within 3 h. Test solutions of amines were prepared in acetonitrile containing 2.0% (w/v) Triton X-405.

Preparation of fluorescent compounds from cyclohexylamine, n-hexylamine and di-n-butylamine

DMEQ-COCl (45 mg, 0.16 mmol) and the individual amine (0.2 mmol) were dissolved in 20 ml of acetonitrile and the solution was allowed to stand at room temperature ( 10-25’ C ) for 30 min. The reaction mixture was evaporated to dryness under reduced pressure. The residue, dissolved in 5 ml of chloro- form, was chromatographed on a silica gel 60 column (25 x 2.7 cm i.d.; ca. 120 g, 70-230 mesh; Japan Merck) with chloroform-acetone (17+3, v/v). The main fraction was evaporated to dryness under reduced pressure and the resi- due was recrystallized from ethyl acetate to give the corresponding product (I, II or III, Tables 1 and 2 ) .

Apparatus and LC conditions Uncorrected fluorescence spectra and intensities were measured with a Hi-

tachi 650-60 spectrofluorimeter in 10 x lo-mm quartz cells; spectral band- widths of 5 nm were used for both the excitation and emission monochroma- tors. Ultraviolet spectra and absorbances were measured with a Hitachi 200- 20 spectrophotometer in lo-mm quartz cells. Infrared spectra were recorded with a Shimadzu 430 spectrophotometer using potassium bromide pellets. ‘H nuclear magnetic resonance spectra were obtained with a Hitachi R-90H spec- trometer at 90 MHz using a ca. 5% (w/v) solution of [ 2H] chloroform contain- ing tetramethylsilane as an internal standard. Electron-impact mass spectra were taken with a JEOL DX-300 spectrometer. Uncorrected melting points were measured with a Yazawa melting point apparatus.

The chromatographic system consisted of a Waters 6000A liquid chroma- tograph equipped with a Rheodyne 7125 syringe-loading sample injector valve (20-~1 loop) and a Shimadzu RF-530 fluorescence spectrometer fitted with a 12-~1 flow cell operating at an emission wavelength of 483 nm and an excita- tion wavelength of 400 nm. The column was a TSK gel ODS-80TM (150 x 4.6 mm i.d.; particle size 5 pm) (Tosoh, Tokyo). Aqueous 44% (v/v) acetonitrile

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was used as a mobile phase. The flow-rate was 1.0 ml min-’ throughout. The column temperature was ambient (H-25’ C ).

Derivatization procedure To a lOO-~1 portion of a test solution of amines placed in a test-tube were

added ca. 3 mg of potassium carbonate and 50 ~1 of DMEQ-COCl solution and the mixture was allowed to stand at room temperature for ca. 1 min. A 20-~1 portion of the final reaction mixture was injected into the chromatograph. For the reagent blank, 100 ,ul of acetonitrile containing 2.0% (w/v) Triton X-405 in place of the test solution was subjected to the same procedure.

RESULTS AND DISCUSSION

Fluorescent products of reaction between amines and DMEQ-COCI The reaction products from cyclohexylamine, n-hexylamine and di-n-butyl-

amine were confirmed as the corresponding DMEQ-carboxyamide (com- pounds I, II and III, respectively) from elemental analysis data (Table 1) and spectral data (Table 2).

The fluorescence properties of compounds I-III in acetonitrile, methanol and water, which have been widely used as components of the mobile phase in reversed-phase LC, were examined (Table 3). The wavelengths of maximum fluorescence excitation and emission of III (the DMEQ derivative of a second- ary amine) were shorter than those of I and II (those of primary amines ). The

TABLE 1

Analytical data for products I-III

R

Compound R Yield M.p.

(%) (“C) Formula Elemental data, talc.

(found) (%)

C H N

I CONH u 21 225 C18HzzN304 62.59 6.71 12.17 (62.44 6.69 12.05)

II CONHCGH,B 23 199 C18HxNsO4 62.23 7.25 12.10 (62.19 7.36 11.98)

III CON(C4H& 18 129 Cz0Hz9NsO4 63.98 7.79 11.19 (64.17 7.96 11.36)

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TABLE 2

Spectral data for compounds I-III

Compound CV, A,, (nm) IR, u,,. (cm-‘) MS, ‘H NMR, S (ppm)

(loge) ml2 C-O Arom. C-C NH (M+)

and/or C=N

I 405

(4.2) 320

(4.0)

II 404

(4.2) 320

(3.9)

III 380

(4.1) 305

(3.7)

1670 1520 3300 345 9.83-9.73 (lH, d, NH), 7.60 and 6.71 1630 (1H each, s each aromatic H), 4.06 and

3.95 (3H each, s each, OCH,), 3.78 (3H s, NCH3), 2.13-1.26 (llH, m, &Hi,)

1680 1540 3300 347 9.74-9.73 (lH, t, NH), 7.56 and 6.67

1630 (1H each, s each, aromatic H), 4.02 and

3.92 (3H each, s each, OCH,), 3.73 (3H,

s, NCHa), 3.60-3.39 (2H, q, NH+CH,),

1.75-0.81 (llH, C,H,,)

1620 1540 375 7.29 and 6.72 (1H each, s each, aromatic

1580 H), 4.03 and 3.94 (3H each, s each,

OCH,), 3.71 (3H, s, NCH,), 3.55 and

3.19 (2H each, t each, NCH,CH,), 1.72-

0.70 [14H, m, (CH,CH,CH,),]

TABLE 3

Fluorescence properties of the products in acetonitrile (A), methanol (M ) and water ( W)

Compound Excitation maximum Emission maximum RFI”

(nm) (nm)

A M W A M W A M W

I 397 401 400 462 480 486 100 125 100 II 397 400 400 465 481 485 153 200 155 III 382 385 383 443 466 467 42 78 98

“Relative fluorescence intensity. The fluorescence intensity was measured at the excitation and emission maxima. The intensity of compound I in acetonitrile was taken as 100.

maxima in acetonitrile were slightly blue-shifted compared with those in meth- anol and water for all the compounds. The fluorescence intensities in aceto- nitrile were lower than those in methanol and water.

The effect of pH on the fluorescence intensities in a mixture of acetonitrile and 40 mM Britton-Robinson buffer (pH 2-11) (1 + 1, v/v) was investigated. The pH did not have any significant effect on the fluorescence intensities and spectra.

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Chromatographic conditions The separation of DMEQ derivatives of cyclohexylamine, n-hexylamine and

di-n-butylamine was studied on a reversed-phase column (TSKgel ODS-80TM) with aqueous acetonitrile. The concentration of acetonitrile in the mobile phase affects the separation of the peaks from those of the blank components. At acetonitrile concentrations > 50% (v/v) the peak for the cyclohexylamine de- rivative partially overlapped that for the reagent blank, whereas acetonitrile concentrations < 40% (v/v) caused a delay in elution with peak broadening. Optimum separation was obtained with 447 ( / ) o v v acetonitrile in water. When aqueous methanol was used as the mobile phase, the half-widths of the peaks about doubled.

Figure 1 shows a typical chromatogram obtained with a mixture of the three amines. The individual compounds each gave a single peak.

Fluorescence derivatization Acetonitrile as a solvent for the derivatization reaction provided the most

intense peaks for the amines examined, acetone and benzene gave less intense

I 5 1

2

0 4 8 12 16

Retention time (min)

Fig. 1. Chromatogram of DMEQ derivatives. Peaks: (1) cyclohexylamine; (2) n-hexylamine; (3) di-n-butylamine; (4-6) unknown products, probably decomposition products of DMEQ-COCl. An aliquot (0.1 ml) of a mixture of the amines (300 pmol ml-’ each) was treated as in the de- scribed procedure.

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peaks (ca. 20 and lo%, respectively, of those obtained in acetonitrile). On the other hand, the fluorescence reaction was very limited in chloroform, cyclo- hexane, dimethyl sulphoxide and dimethylformamide. Acetonitrile was there- fore chosen for the recommended procedure.

Reproducible peak heights were not obtainable without Triton X-405. This might be due to the adsorption of the amines on the glass wall [ 71. Triton X- 405 [LO-3.0% (w/v) in the test solution] gave maximum and constant peak heights for the amines tested; 2.0% was selected as the optimum concentration.

Potassium carbonate was used to facilitate the derivatization of amines with DMEQ-COCl. The peak heights for the amines were maximum and constant at amounts of potassium carbonate > 1 mg (Fig. 2); ca. 3 mg was employed in the procedure. Acceleration of the reaction was not observed in the presence of pyridine, triethylamine or 18-crown-6.

The derivatization reaction with the three amines proceeded rapidly inde- pendent of the temperature (O-100’ C ); the reaction was complete within 30 s even at 0°C. Therefore, standing for ca. 1 min at room temperature was used in the procedure. Even in the absence of potassium carbonate, the reaction occurred at O-100” C and was complete within 30 s. However, the peak heights were ca. 50% of those obtained with potassium carbonate. The reaction time

I I , I f

0 2 4 6 8

Potassium carbonate (mg)

1 100

. /’ . .

0 1 2 3 4 5

DMEQ-COC1 (“Ml

Fig. 2. Effect of the amounts of potassium carbonate on the fluorescence derivatixation. Curves: (1) cyclohexylamine; (2) n-hexylamine; (3) di-n-butylamine. Aliquots (0.1 ml) of a mixture of amines (300 pmol ml-’ each) were treated as in the recommended procedure at various amounts of potassium carbonate.

Fig. 3. Effect of DMEQ-COCl concentration on the fluorescence derivatization. Curves: (1) cy- clohexylamine; (2) n-hexylamine; (3) di-n-butylamine. Aliquots (0.1 ml) of a mixture of amines (300 pmol ml-‘) were treated as in the recommended procedure at various concentrations of DMEQ-COCl.

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is much shorter than those with other reagents having a carbonyl chloride group [ l-51.

The DMEQ-COCl solution gave the most intense and constant peaks at concentrations > 0.5 mM; 2.0 mM was used as a sufficient concentration (Fig. 3). The efficiencies of conversion of the three amines to the DMEQ derivatives were examined by comparing the peak heights obtained under the reaction conditions with,those given by the pure reaction products (compounds I, II and III); the extents of conversion (mean t s.d., n= 5) were 89.4 + 2.4% (cy- clohexylamine), 87.0 +- 2.9% (n-hexylamine) and 84.3 + 3.6% (di-n-butyl- amine). The DMEQ derivatives in the final solution were stable for at least 120 h in daylight at ambient temperature.

Calibration graph, precision and,detection limits The relationships between the peak heights and the amounts of the individ-

ual amines were linear from 20 fmol to at least 100 pm01 per 20-pl injection volume (corresponding to 150 fmol-750 pmol in 100 ~1 of a test solution). The precision was established by repeated analyses (n= 30) of a mixture of the three amines (300 pmol ml-l each). The relative standard deviations were 1.5, 1.9 and 2.0% for cyclohexylamine, n-hexylamine and di-n-butylamine, respec- tively. The detection limits (fmol per 20-~1 injection, signal-to-noise ratio = 3) were 12.4 (cyclohexylamine), 15.7 (n-hexylamine) and 18.1 (di-n-butyl- amine). The sensitivity is comparable to that of the fluorimetric method using phthalimidylbenzoyl chloride [ 51, and much higher than those of spectropho- tometric methods [l-4].

Reaction of other substances with DMEQ-COCZ Many amines reacted with DMEQ-COCl under the derivatization condi-

tions. Table 4 gives the detection limits and retention times for the DMEQ derivatives of the compounds. Some biogenic amines such as histamine, tyra- mine and 2-phenylethylamine can be converted into DMEQ derivatives, and single fluorescent peaks for these amines were observed in the chromatograms. Seventeen L-at-amino acids did not give fluorescent products under the deri- vatization conditions.

Primary and secondary alcohols react with DMEQ-COCl at higher temper- atures (100-130’ C ) over a prolonged period (60-100 min) to produce fluores- cent esters. However, under the present derivatization conditions, the alcohols gave no fluorescent products.

No other biologically important substance examined fluoresced under the recommended conditions at a concentration of 20 nmol ml-‘. The compounds tested were alloxan, ascorbic acid, glutathione, thiamine, citrulline, allantoin, uric acid, urea, bilirubin, acetone, cyclohexane, 4-methylcyclohexane, acetyl- acetone, acetophenone, benzil, lactic acid, 3-hydroxybutyric acid, acetoacetic acid, homogentisic acid, pyruvic acid, phenylpyruvic acid, N-acetylneuraminic

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TABLE 4

Detection limits and retention times for DMEQ derivatives of primary and secondary amines

Compound . Detection limit” Retention time (fmol) (min)

n-Propylamine 6.4 3.6 n-Butylamine 7.8 4.6 n- Amylamine 9.5 6.2 n-Hexylamine 15.7 10.4 n-Heptylamine 18.2 16.8 n-Octylamine 24.7 26.6 n-Nonylamine 57.2 47.8 Cyclohexylamine 12.4 6.5 Benzylamine 5.9 5.0 4-Methylbenzylamine 14.1 12.4 Di-n-propylamine 11.8 7.6 Di-n-butylamine 18.1 12.0 Diethylamine 15.1 3.3 N-Ethylbenzylamine 57.2 6.6 Dibenzylamine 76.2 35.8 Tyramine 8.6 3.4 Histamine 17.6 3.3 2-Phenylethylamine 7.6 6.2

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

acid, inositol, D-xylose, D-glucose, D-fructose, D-mannose, D-maltose, D-lac- tose, epiandrosterone, dehydroepiandrosterone, cortisone, cholesterol and methylglyoxal. These results suggest that the present derivatization method is usefully selective for amines.

Conclusions The method permits the highly sensitive and selective determination of pri-

mary and secondary amines. The fluorescence from DMEQ derivatives is not sensitive to the pH of the chromatographic mobile phase. The reaction is com- pleted very rapidly at room temperature and the resulting fluorescent deriva- tives are stable for at least 120 h. The proposed reagent can be applied to the determination of biogenic amines in body fluids; further studies are in progress.

REFERENCES

1 S. Asotra, P.V. Mladenov and R.D. Burke, J. Chromatogr., 408 (1987) 227. 2 E.S. Barreira, J.P. Parente and J.W. Alencar, J. Chromatogr., 398 (1987) 381. 3 F.B. Jungalwala, R.J. Turel, J.E. Evans and R.H. Mccluer, Biochem. J., 145 (1975) 517. 4 S.L. Wellons and M.A. Carey, J. Chromatogr., 154 (1978) 219. 5 Y. Tsuruta and K. Kohashi, Anal. Chim. Acta, 192 (1987) 309. 6 T. Iwata, M. Yamaguchi, S. Hara, M. Nakamura and Y. Ohkura, J. Chromatogr., 362 (1986)

209. 7 J.J. Biscan, V. Pravdic and W. Haller, J. Colloid Interface Sci., 121 (1988) 345.