Analyrrca Chrmrca Acra, 231 (1990) l-6

Elsevler Science Pubhsbers B.V., Amsterdam - Prmted m The Netherlands

4,SDiaminophthalhydrazide as a highly sensitive chemiluminescence reagent for cw-keto acids

in liquid chromatography

JUNICHI ISHIDA, MASATOSHI YAMAGUCHI *, TOSHIHIRO NAKAHARA and MASARU NAKAMURA

Faculty ofPharmaceutrca1 Scrences, Fukuoka Umuersrfy, Nanukuma, Johnan-ku, Fukuoka 814-01 (Japan)

(Recetved 11 th September 1989)

ABSTRACT

4,5-Dlammophthalhydravde dthydrochlonde IS studled as a highly sensltlve and selective chemdummescence denvatlzatlon reagent for a-keto actds m hquld chromatography (‘LO The reagent reacts selectively with a-keto acids

m ddute hydrochlonc acid to @ve denvatlves which produce chemllummescence by reactlon with hydrogen peroxlde

and potassmm hexacyanokrrate(H1). ‘The derlvatlves m the reactlon mixture of eight bIologIcally Important a-keto

acids are separated wtthm M mm by reversed-phase LC with IsocratIc elutlon, followed by chemtlummescence

detectlon. i-he detection hmtts for the actds are m the range 4-50’ fmol for a 2&(7_yI InJectIon

Several denvatization reagents have been re- ported for the determination of cY-keto acids by

liquid chromatography (LC) with spectrophoto- metric and fluorimetric detection. The spectropho- tometric method using 2,6dmitrophenylhydrazine [l] is neither sensitive nor selective. Although fluo-

rimetric methods based on 4’-hydrazino-2-stilba- zole [2] and o-phenylenediamine [3,4] are fairly

sensitive, they do not allow the assay of a-keto acids at fmol levels.

In previous work we synthesized 1,2-diammo- 4,5-dimethoxybenzene [5,6] and 1,2-diammo-4,5- methylenedioxybenzene [7] as highly sensitive and selective fluorogenic reagents for cY-keto acids, and applied the reagents to LC, thus permitting the determination of the acids at fmol levels. These reagents have been used for the determination of the acids m human urine and serum [8].

Recently, the use of chemiluminescence (CL) detection has been introduced into LC methodol-

ogy and some precolumn chemiluminescence de- rivatization reagents for ammo acids [9], ammes

[lo] and carboxyhc acids [lo-121 have been re- ported. No reagents, however, have been devel- oped for a-keto acids.

In the work reported here, it was found that

3,4- and 4,5-diaminophthalhydrazide dihydrochlo- rides (3,4- and 4,5-DPH.2HC1, respectively) react selectively with cu-keto acids m dilute hydrochloric

acid to give compounds which produce chemi- luminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(II1) in alkaline media (Fig. 1) The reaction was ap- plied to the LC determination of eight cY-keto acids of biological importance.

EXPERIMENTAL

Chemrcals and solvents All chemicals and solvents were of the highest

purity available and were used as received. Dis- tilled water, purified with a Milh-Q II system

(M&pore), was used for all aqueous solutions.

J ISHIDA ET AL

4,5-DPH.2HCI ot-Keto acids Qutnoxaltnone

dertvatives 1 II

fig f Denvattzatton reactton of a-keto acids wtth 4,5-dtammophthalhydravde

Hydrogen peroxide (30%, v/v) was purchased from Mttsubishr Gas Kagaku (Tokyo).

Synthem of reagents Both 3,4- and 4,5-DPH were synthesized from

4-mtrophthalic acid by the method of Williams

and Shalaby [13]. Briefly, 4nitrophthalic acid was refluxed m methanol and concentrated sulphunc

acid to give dimethyl 4-mtrophthalate. The phtha- late was reduced to dimethyl 4-aminophthalate, and subsequently converted to the acetamide de- rivative. Nitration of the amide resulted m a mix- ture of dimethyl 4-acetamido-5-mtrophthalate and dimethyl 4-acetamido-3-mtrophthalate, which

were separated by fractional crystallization and column chromatography. Hydrolysis of the tso- merit acetamide derivatives gave the correspond- mg nitroamines, from which the isomerrc di- aminophthalates (dimethyl 4,5- and 3,4-diammo-

phthalates) were obtained by catalytic reduction. Condensation of the individual diaminophthalates with hydrazine hydrate and triethylamine in methanol gave 4,5- and 3,4-DPH. The individual DPHs were mixed with a small portion of con- centrated hydrochloric acid and the resulting pre- cipitates were recrystallized from ethanol to give the correspondmg dthydrochlorides. The struc- tures of the DPHs and their dihydrochlorides were

A Pl

(Tj w Column _rD Ml M2

E P2 P3

Ftg. 2 Schemattc flow dtagram of the LC-CL system P,, LC pump (Httacht 635), Pz and Ps, LC pumps (Jasco 880 PU), I, uqectlon

valve (Rheodyne 7125, 20 nl), D, detector (ATT0 AC-2220), G, guard column (TSK gel ODS-120T). column, TSK gel ODS-12OT

(250 X 4 6 mm id), Ml and M,, rmxmg devices; Rec. recorder, A, stamless-steel tube (5 cm X 0 5 mm 1 d ), E, mobtle phase

[acetomtnle-50 mM phosphate buffer (pH 7 0)(13 87, v/v)], R,, hydrogen peroxtde solutton, R,, potassmm hexacyanoferrate(II1)

solutton. Flow-rates: E, 1 0, R,, 1 0; R,, 2 0 ml mn-’

REAGENT FOR a-KETO ACIDS IN LC 3

verified by routine MS, IR and NMR data. Both DPH.2HCl compounds obtained were stable in the crystalline state at room temperature for at least 6 months m a desiccator containing silica gel.

Reagent solutions Solutions of 3,4- and 4,5-DPH.2HCl (1.2 mM)

were prepared in 0.6 M hydrochloric acid contain-

mg 0.6 M P-mercaptoethanol. These solutions were used within 5 h. Hydrogen peroxide (50 mM) and

potassium hexacyanoferrate(II1) (30 mM) solu- tions were prepared m water and 2.0 M sodium hydroxide, respectively.

LC condltlons and chemllummescence detectron system

Figure 2 shows a schematic diagram of the LC-CL system. Chromatography was performed with a Hitachi 635 liquid chromatograph equipped with a Rheodyne 7125 syringe-loading sample in- Jector valve (20+1 loop). The DPH derivatives of

the cy-keto acids were separated on a TSK gel ODS-120T (5 pm) reversed-phase column (250 X

4.6 mm i.d.) (Tosoh, Tokyo) by isocratic elution with acetonitrile-50 mM phosphate buffer (pH 7.0) (13 : 87, v/v) at a flow-rate of 1 ml mini. The column temperature was ambient (23 + 1” C).

The eluate was nuxed with the hydrogen per- oxide and potassium hexacyanoferrate solutions, delivered by two 880 PU pumps (Jasco, Tokyo) at flow-rates of 1.0 and 2.0 ml min-‘, respectively. The CL was monitored by an AC-2220 lummomonitor (ATTO, Tokyo) equtpped with a 60-~1 flow cell. Stainless-steel tubing (0.5 mm i.d.) was used for the LC system.

Derwatlzatlon procedure To a loo-p.1 portion of a test solution of a-keto

acids placed in a screw-capped tube (100 X 15 mm 1.d.) was added 100 ~1 of 1.2 mM DPH solution. The tube was tightly closed and heated at 100 o C for 45 min. A 2O+l portion of the final reaction

mixture was inJected into the chromatograph. For the reagent blank, 100 ~1 of water m place of test solutton were subJected to the same procedure.

RESULTS AND DISCUSSION

Evaluation of dlamrnophthalhydrazldes and their

dlhydrochlondes as CL derwatlzatron reagents 3,4- and 4,5-DPH and their dihydrochlorides

were evaluated as CL derivatization reagents for

a-keto acids. The CL intensities obtained with 4,5-DPH.2HCl were about 30 times higher than

those obtained with 3,4-DPH.2HCl for all cY-keto acids tested. One reason for the weakness of the

CL from the 3,4-DPH derivatives may be the effect of the steric hmdrance at the C-3 position [14]. The DPHs were only slightly soluble in water and dilute hydrochloric acid and gave some small interfering peaks in the chromatogram, which were due to impurities m the reagents. Therefore, 4,5- DPH.2HCl was selected for further mvestigation to estabhsh a general procedure for the determma- tion of a-keto acids.

4,5-DPH.2HCl itself gave only extremely weak CL (ca. 2% of that obtained with cY-ketoisovaleric acid at equimolar concentration) under the test

conditions.

LC condttlons

The separation of DPH derivatives of eight cx-keto acids was studied on a TSK gel ODS-120T

reversed-phase column with a acetomtrile-50 mM phosphate buffer (pH 7.0) as eluent. The con- centration of acetonitrile in the mobile phase af- fected the separation of the peaks of the reagent blank and cY-keto acids. At acetomtrile concentra- tions > 15%, the peak for the a-ketobutyric acid partially overlapped with that for the reagent blank, whereas acetorutrile concentrattons of < 10% caused a delay in elution, with peak broad- ening, especially for phenylpyruvic acid. The con- centration (lo-100 mM) and pH (2.5-7.5) of the phosphate buffer m mobile phase did not affect the separation and CL intensity of a-keto acids. When aqueous methanol was used as the mobile phase, the peaks for eight derivatives and reagent

blank were not separated at any methanol con- centration. Therefore, LC was carried out with acetonitrile-50 mM phosphate buffer (pH 7.0)

(13 : 87, v/v). Figure 3 shows a typical chromato- gram obtained with a standard mixture of eight cu-keto acids of biological importance. The DPH

9

1

2 Ii “i

7

6

!

8

- r I I I I I I 0 a 16 24 32 40 48

Time (mln)

Fig. 3 Chromatogram of the DPH denvattves of a-keto acids

Peaks (pmol per mjectlon volume). 1 = a-ketobutync actd (2 5),

2 = p-hydroxyphenylpyruwc actd (7 5). 3 = n-ketovalenc actd

(2 5). 4 = a-ketotsovalenc acid (7.5). 5 = a-ketolsocaprolc acid

(7 5), 6 = a-keto-/?-methylvalenc acid (15 0), 7 = a-ketocaprolc

acid (2 5). 8 = phenylpyruwc acid (2.5). 9 = reagent blank.

derivatives of the eight cu-keto acids were success- fully separated within 50 min. The individual (Y- keto acids gave single peaks m the chromatogram. This indicates that 4,5-DPH.2HCl can be used as a precolumn derivatization reagent for a-keto acids.

Derlvatlzatlon of cy-keto acids with 4,5-

DPH.ZHCl a-Keto acids reacted with 4,5-DPH.2HCl in

dilute hydrochloric acid, but not m neutral or alkaline solution. Hydrochloric acid at 0.4-0.8 M in the DPH solution gave maximum peak heights; 0.6 M was adopted for the preparation of the DPH solution. The greatest peak heights were achieved at > ca. 1.0 mM 4,5-DPH.2HCl m the reagent solution; 1.2 mM was adopted. /3- Mercaptoethanol was required for the stabilization of 4,5-DPH.2HCl during the derivatization. In its

absence, the reagent blank (peak 9 in Fig. 3) became larger in area, which interfered with the assay of cY-ketobutyric and p-hydroxyphenyl-

J ISHIDA ET AL

pyruvic acids. P-Mercaptoethanol (0.6 M m the DPH solution) was therefore included m the re- commended procedure.

The derivatization reaction of 4,5-DPH.2HCl with Lu-keto acids occurred more rapidly as the reaction temperature was increased. An example for p-hydroxyphenylpyruvrc and a-ketovaleric acids is shown m Fig. 4. The peak heights became maximum and constant after heating at 100 o C for 40 mm. Therefore, heating at 100” C for 45 mm was adopted m the recommended procedure.

The DPH derivatives in the final solution were stable for at least 3 h in daylight at room tempera- ture.

The reaction products between a-keto acids and 4,5-DPH.2HCl seem to be quinoxalinone derivatives (Fig. 1) [5-S]. Studies of the structural and CL characteristics of the products are in

progress.

Chemllumrnescence reactlon The optimum conditions for the CL reaction

were examined by setting the flow-rates of the hydrogen peroxide and potassium hexacyanofer- rate(II1) solutions to 1.0 and 2.0 ml mm-‘, respec- tively.

I I I I I 0 20 40 60 60

Time (man)

Fig 4 Effect of reactton time and temperature on the denvatl-

zatlon reaction of a-keto actds with 4,5-DPH.2HCl. Tempera-

tures (1 and 2) 100°C. (3 and 4) 80 o C, (5 and 6) 50°C

a-Keto actds (1, 3 and 5) p-hydroxyphenylpyruvc acid, (2, 4

and 6) a-ketovalenc aad.

REAGENT FOR a-KETO ACIDS IN LC

I 1 I 0 50 100

H202 (mM)

Fig 5 Effect of hydrogen peroxlde concentration on the CL

peak heights Curves I = p-hydroxyphenylpyruwc acid, 2 = a-

ketovalenc acid

The concentrations of hydrogen peroxide,

potassium hexacyanoferrate(II1) and sodium hy- droxide are very important in determmmg the intensity of the CL (Figs. 5-7). The concentra-

tions of these three reagents were varied one at a time to establish the maximum intensity obtaina- ble. Based on these experiments, concentrations of 50 mM hydrogen peroxide, 30 mM potassium hexacyanoferrate(II1) and 2.0 M sodium hydrox- ide were selected.

The CL is generated immediately after mixing

the eluate and the potassium hexacyanoferrate(II1) solution. Therefore, the length of tubing between

0 30 50 80

K3Fe(CN)6 (mM)

Fig 6 Effect of potassmm hexacyanoferrate(II1) concentration

on the CL peak heights Curves as m Fig. 5

r L I I 1 0 1 2 3 4

NaOH (MI

Fig 7 Effect of sodmm hydroxide concentration on the CL

peak heights. Curves as m Fig 5

the second mixmg device (M, in Fig. 2) and the detector affected the CL response. Figure 8 shows the effect of tube length on the peak height. The peak heights for all cY-keto acids increased on decreasing this length; 5-cm was selected tenta- tively. This shows that the CL reaction occurs very

rapidly and is complete m a short period.

Cahbratton, precwon and detectron hmlts

The relationships between the peak heights and

the amounts of the mdividual cu-keto acids were linear from 20 fmol to at least 100 pm01 per 2Oq1.1 qection volume (corresponding to 200 fmol to 1 nmol m 100 ~1 of test solution).

0 510 50 100

Tube length (cm)

Fig 8 Effect of the tube length between the second mlxmg

dewce and CL detector on the peak heights Curves as m Fig

5

6

The precision was established by repeated de- termmation (n = 6) using a mixture of the eight cY-keto acids (500 pmol ml-’ each). The relative standard deviation did not exceed 4.0% for any acids.

The detection linuts (fmol per lo-p.1 mlection volume, signal-to-noise ratio = 3) for the eight (Y- keto acids are as follows: a-ketobutync acid 5.0, p-hydroxyphenylpyruvtc acid 22.0, ol-ketovalenc acid 4.0, a-ketoisovaleric acid 15.0, cy-ketoiso-

caprotc acid 12.0, cY-keto-/?-methylvalenc acid 50.0, a-ketocaproic acid 6.0 and phenylpyruvic acid 5.0. The sensitivity 1s ca. 2-20 times higher than that of the fluonmetnc LC method using 1,2-diamino-

4,5_methylenedioxybenzene [7]. In particular, the present method is useful for the assay of p-hy- droxyphenylpyruvic acid, which can be de- termined only at 0.5-1.0 pmol even by the most sensitive fluorimetric LC procedure.

Reactlon of other substances with 4,5-DPH.2HCl

Ascorbic acid reacted with 4,5-DPH.2HCl un- der the recommended procedure to give several peaks on the chromatogram (retention times 7-13 mm). However, the intensities were less than one tl-urd of those from the cr-keto acids. None of the other biologically important substances examined generated CL under the recommended conditions at a concentration of 10 nmol ml-‘. The com- pounds tested were 2-, 3- and 4-methoxyben- zaldehyde, 4_methylbenzaldehyde, seventeen L-

amino acids, histamme, tyramine, tryptamine, 2-

phenylethylamine, glutathione, thiamine, uracil,

adenme, uric acid, urea, bilirubin, acetone,

acetophenone, lactic acid, malic acid, butyric acid, mositol, D-glucose, D-xylose, D-fructose, D-man-

nose, D-ribose, D-lactose, aldosterone, epi- androsterone, cortisone and cholesterol. The re- sults suggest that the present derivatization method is usefully selective for a-keto acids.

J ISHIDA ET AL

Conclustons 4,5-DPH.2HCl is the first CL derivatization

reagent for cY-keto acids. The LC method using this reagent may be applicable to the determina- tion of biogenic cY-keto acids in small amounts of serum and urine. The method should be particu- larly useful for the assay of p-hydroxyphenyl- pyruvic acid, which is present in only trace amounts in normal urine; further studies are in progress.

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