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Determination of Femtomole Concentrations of Catecholamines by High-performance Liquid Chromatography with Peroxyoxalate Chemiluminescence Detection

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Sakae Higashidate and Kazuhiro Imai* Branch Hospital Pharmacy, University of Tokyo, 3-28-6 Mejirodai, Bunkyou-ku, Tokyo 1 12, Japan

A highly sensitive method for determination of the plasma catecholamines, norepinephrine(NE), epinephrine (E) and dopamine (DA) is described. The method consists of the extraction of the catecholamines, using 3,4-dihydroxybenzylamine as internal standard, from plasma with alumina (5 mg), followed by a reversed-phase column separation, on-column fluorogenic derivatization with ethylenediamine (ED) and post-column peroxyoxalate chemiluminescent reaction detection utilizing bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate (TDPO) and hydrogen peroxide. In order to optimize the reaction conditions for high-performanceliquid chromatographyto obtain highly sensitive detection, the effects of changing reagent compositions on the chemiluminescenceyield were investigated. The following are the optimized conditions. Eluent, a mixture of 50 mmol 1-1 potassium acetate (pH 3.20)-50 mmol 1-1 potassium phosphate (pH 3.20)-acetonitrile (90.15 4.85 3 v/v/v) containing 1 mmol l-1 sodium hexanesulfonate(40"C) and flow rate, 0.5 ml min-1. Fluorogenic reagent solution, 105 mmol 1-1 ED and 175 mmol 1-1 imidazole in acetonitrile-ethanol (90 10 v/v) and flow rate, 0.25 ml min-I. Reaction coil (15 m x 0.5 mm i.d.) heated a t 80 "C. Chemiluminogenic reagent solution, 0.25 mmol 1-1 TDPO, 150 mmol 1-1 hydrogen peroxide and 110 mmol 1-1 trifluoroacetic acid in dioxane-ethyl acetate (50 : 50 v/v) and flow rate, 1.4 ml min-1. The detection limits for all the catecholamineswere 1 fmol (signal-to-noiseratio at 2). The standard deviations of the method for the determination of NE, E and DA added to rat plasma (2.5 nM) were 3, 3 and 4%, respectively. The concentrations of rat plasma catecholaminesmeasured by the proposed method were NE 1.21, E 0.12 and DA 0.63 pmol ml-1 and were lower than those obtained previously by radioenzymic-paper chromatography.

+ +

+

Keywords: Plasma catecholamine; high-performance liquid chromatography; eth ylenediamine condensation; peroxyoxalate chemiluminescence; bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate

Catecholamines (CAs) play an important role in higher animals as neurotransmitters between cells. Their overand underproduction in disease states result in the breakdown of the homeostasis of the body. Thus, the determination of CAs in body fluids is required for the diagnosis of their related diseases such as pheochromocytoma, neuroblastoma, idiopathic oedema and hypertension. Quantification is also needed in the fields of pharmacological and biological sciences for the investigation of the fate or metabolism of CAs in cells, tissues and organs. The level of CAs in biological specimens is very low and a sensitive assay is required. There have been many papers published on the determination of CAs, such as radioenzymic,' high-performance liquid chromatography (HPLC) with electrochemical* or fluorimetric detection.% Recently, a modified method for pre-column derivatization of CAs with 1,2-diphenylethylenediamine (DPE) was reported where emphasis was focused on an automated analysis and the lower detectabilities of norepinephrine (NE), epinephrine (E) and dopamine (DA) as 0.6, 0.9 and 2 pg m1-1, respectively.7 However, several interfering peaks appeared on the chromatogram, one of which obstructed the detection of the internal standard (isoproterenol) and the method was, therefore, less selective. This paper describes a selective and sensitive method for the determination of CAs utilizing peroxyoxalate chemiluminescence (PO-CL) detection after HPLC. The HPLC-PO-CL detection has enabled determination at the femtomole level of biological substances or drugs such as amino acids, steroids and drugs with or without fluorescent derivatization.8-16Both NE and DA were detected at the femtomole level by the HPLC-PO-CL method after derivatization with fluorescaminel7 or naphthalene-2,3-dicarboxaldehyde (NDA) .I8 However, E was not determined as it did not fluoresce with

* To whom correspondence should be addressed.

fluorescamine or NDA.17,18 On the other hand, condensation of ethylenediamine (ED) with CAs gave fluorescence for all the CAs with possibly higher selectivity than NDA.19 Therefore, in this experiment, ED was used as a post-column derivatization reagent for CAs, which were then sensitively determined using PO-CL detection.

Experimental Reagents

The catecholamines (NE, E and DA) , 3,4-dihydroxybenzylamine (DHBA, the internal standard for CAs) and alumina (WA-4) purchased from Sigma (St. Louis, MO, USA). Trifluoroacetic acid (TFA) was obtained from Pierce (Rockford, IL, USA). Acetonitrile, ethanol, dioxane, ethyl acetate and distilled water, were all of HPLC grade and were purchased from Wako (Osaka, Japan). Hydrogen peroxide, bis[6ni tro-2-(3,6,9-trioxadecyloxycarbony1)phen yl J oxalate (TDPO) and ethylenediaminetetraacetic acid disodium salt (EDTA) were also from Wako. Sodium hexanesulfonate and imidazole (zone-refined) were obtained from Tokyo Kasei (Tokyo, Japan). The purified ED for washing semi-conductors was a gift from Wako. A11 the other reagents were of analytical-reagent grade. Male Sprague-Dawley rats weighing from 320 to 560 g were obtained from Nihon Seibutu Zairyo Centre (Tokyo, Japan). Fluorescence spectroscopic measurements were made with a Hitachi Model 650-10sinstrument (Hitachi, Tokyo, Japan). Alumina Purification

Alumina was purified as previously described.20 Briefly, 20 g of alumina was immersed in 200 ml of 2 moll-' hydrochloric acid and heated at 100 "C for 1h with gentle mixing to prevent bumping. After the supernatant was decanted, the alumina

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was washed with 20 x 200 ml of distilled water, filtered with a filter paper (Toyo Roshi No. 2; Toyo Roshi, Tokyo, Japan) and dried at 120 "C in an oven overnight.

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Human and Rat Plasma Treatment Human blood was obtained from the antecubital vein, EDTA was added (at a ratio of 1 mg to 1 ml blood) and the sample centrifuged at 15OOg for the collection of the plasma fraction. The rats were anaesthetized with sodium pentobarbital (50 mg kg-I), and 200 pl blood was collected from the cervical vein and treated as for human blood. To 100 pl of human or rat plasma in a 2 ml Eppendorf conical tube (Eppendorf, Hamburg, Germany), 5 mg acid-washed alumina, 10 p1 internal standard solution (10 pmol l-1 DHBA in 0.01 moll-1 perchloric acid) and 100 pl 1.5 moll-1 tris hydrochloric acid buffer (pH 8.7) were added successively. The tube was stirred with a multi-tube mixer (Jasco Model MT-30) for 10 min, centrifuged at 3000g for 1rnin and the supernatant discarded. The alumina ,was washed with 500 pl of distilled water twice and then 100 pl of 0.1 moll-1 perchloric acid was added to the tube. The tube was mixed for 1 min, with the mixer, then centrifuged at 3000g for 1 min. A 50 p1 aliquot of the supernatant was subjected to analysis by HPLC. Calibration Curve

The same pre-treatment as above was performed for 100 p1 of rat plasma to which 10 pl of a standard mixture containing 0.25, 0.50, 0.75 or 1 pmol each of NE, E and DA, and 100 pmol DHBA has been added. Recovery Experiment To 100 p11 of rat plasma was added 10 p1 of standard mixture, containing 0.25 pmol NE, E and DA. The sample was treated as described under Human and Rat Plasma Treatment. HPLC-PO-CL Detection System

The HPLC detection system, shown in Fig. 1, consisted of a pump (JASCO Model 880-PU) (JASCO, Tokyo, Japan) for delivering the eluent, a pump (JASCO Model 885-PU) for the fluorogenic reagent solution, a pump (JASCO Model 885-PU) for the chemiluminogenic reagent solution, a thermostat (JASCO Model TC-100), a chemiluminescence detector

CLM

rtr

RC

rn

Y

Y

MDI

MD2

CL

(JASCO Model 825-CL) with or without cut-off filters (Kenko Models Y-46 and Y-48), a data processor (JASCO Model 807-IT), an injector (Rheodyne Model 7125), a separation column (JASCO Model Catecholpak), two mixing devices21 (Kyowa Model KZU-1,25 pl) and a knitted type reaction coil made of poly(tetrafluoroethy1ene) (PTFE) tubing (1.6 mm 0.d. X 0.5 mm i.d. x 15 m). Mobile Phase Selection

At first, to prevent the formation of precipitates in the flow line, the buffers (pH 3.20) for the eluent were examined in a static system, the buffers tried included potassium malonate, potassium tartrate, potassium acetate, potassium 3,3dimethylglutarate, ammonium formate, sodium formate, ammonium acetate and potassium glycine. The volumes of the buffers, acetonitrile, ethanol, dioxane and ethyl acetate in the static system were 0.5, 0.225, 0.025, 0.7 and 0.7 ml, respectively, calculating from the volume ratios and the flow rates of the eluent (50 mmol 1-1 buffer, 0.5 ml min-I), fluorogenic reagent solution (acetonitrile-ethanol, 90 + 10 v/v, 0.25 ml min-1) and chemiluminogenic reagent solution (dioxane-ethyl acetate, 50 50 v/v, 1.4 ml min-1). Then the formation of precipitates was observed. In order to choose the buffers that were suitable for the fluorogenic reaction, 50 mmol 1-1 potassium acetate, potassium 3,3-dimethylglutarate, ammonium formate, sodium formate, ammonium acetate and potassium glycine (pH 3.20) were examined in a static system. A mixture of 2 ml of each buffer, 1 nmol D A (as a typical CA) in 10 pl of 0.01 mol 1-1 perchloric acid solution and 1ml of different concentrations of E D (50-200 mmol 1-1) in acetonitrile-ethanol (90 + 10 v/v) was heated at 80 "C for 4 rnin and cooled in an iced-water bath for 1 min. The fluorescence intensities were measured at 480 nm with excitation at 380 nm in a static system.

+

Optimization of Fluorogenic Reaction Conditions

The effect of organic solvents on the fluorogenic reaction of CAs was examined in the following HPLC conditions: eluent, 50 mmol 1-1 potassium acetate (pH 3.20)-50 mmol 1-1 potassium phosphate (pH 3.20) (95 k 5 v/v) containing 1 mmol 1-1 sodium hexanesulfonate, and flow rate 0.5 ml min-1; fluorogenic reagent solutions, 120 mmol 1-1 E D dissolved in mixtures of acetonitrile-water (0 + 100, 50 + 50, 80 + 20, 90 + 10 and 95 k 5 v/v) and in acetonitrile-ethanol (0 + 100,SO + 50,80 + 20,90 10 and 95 f 5 v/v), and flow rate 0.25 ml min-1. Then, CAs (10 pmol of each dissolved in 10 p10.01 moll-' perchloric acid solution) was injected onto the column. The peak heights of the CAs on the chromatogram, detected at 500 nm with excitation at 410 nm, were measured. The examination of E D and imidazole concentrations were performed in a static system under the following conditions: 2 ml of 50 mmol 1-1 potassium acetate (pH 3.20)-50 mmol 1-1 potassium phosphate (pH 3.20) (95 k 5 v/v), 1 nmol D A in 10 pl of 0.01 mol 1-1 perchloric acid solution and 1 ml of acetonitrile-ethanol solution (9 + 1 v/v) containing E D (95-140 mmol 1-1) and imidazole (0-200 mmol 1-l) were mixed, heated at 80 "C for 4 rnin and cooled in an iced-water bath for 1 min. The fluorescence at 480 nm with excitation at 380 nm was measured.

+

Optimized HPLC Conditions

The eluent was a mixture of 50 mmol 1-1 potassium acetate (pH 3.20)-50 mmol 1-1 potassium phosphate (pH 3.20)acetonitrile (92.15 + 4.85 + 3 v/v/v) containing 1 mmol 1-* sodium hexanesulfonate as an ion-pair reagent. The flow rate was adjusted to 0.5 ml min-1. The catecholpak separation column (150 x 4.6 mm i.d., JASCO) was kept at 40 "C. The

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100

125 150150

175 200125

150

175 200100

125 150100

125

150 17550

75

100

Concentration of ED/mmol I-' Fig. 2 Effect of buffers on fluorogenic reaction of DA with varying ED concentrations. Buffers (50 mmol I-l, pH 3.20): ( a ) potassium acetate; (b) potassium 3,3-dimethylglutarate; (c) ammonium formate; (d) sodium formate; (e) ammonium acetate; and U, potassium glycine. The mixture of 2 ml each buffer, 10 1.11 D A solution (1 nmol) and 1ml ED solution (50-200 mmol l-1) was heated at 80 "C for 4 min, cooled in an iced-water bath for 1 min and measured at 480 nm with excitation at 380 nm

75

fluorogenic reagent solution was prepared by dissolving 105 mmol 1-1 E D and 175 mmol 1-1 imidazole in acetonitrileethanol (90 + 10 v/v), at a flow rate of 0.25 ml min-1. The reaction coil (15 m x 0.5 mm i.d.) was heated at 80 "C. The chemiluminogenic reagent solution was prepared by dissolving 0.25 mmol 1-1 TDPO, 150 mmol 1-1 hydrogen peroxide and 110 mmol l-1 TFA in dioxane-ethyl acetate (50 + 50 v/v), at a flow rate of 1.4 ml min-1.

Results and Discussion

(a)

50

r r

I O

Mobile Phase Selection Catecholamines are usually separated on a reversed-phase column with an eluent containing phosphate ion such as potassium dihydrogen phosphate-phosphoric acid. However, the usual concentration of phosphate used for the separation of CAs (100 mmol l-1) causes precipitates to form in the flow line of the final PO-CL detection system, which contains organic solvents such as acetonitrile-ethyl acetate (50 + 50 v/v). Hence, the eight other buffers, including potassium malonate, potassium tartrate, potassium acetate, potassium 3,3-dimethylglutarate7 ammonium formate, sodium formate, ammonium acetate and potassium glycine were examined in a static system to test for the formation of precipitates. None of the buffers, except potassium malonate and potassium tartrate, gave precipitates. In order to select a suitable buffer for the fluorogenic reaction, the six buffers that gave no precipitates were examined in a static system. Among the buffers examined, acetate gave the highest yield as shown in Fig. 2. Therefore, subsequent separations were carried out with a mobile phase of 50 mmol 1-1 acetate buffer (pH 3.20). According to the preliminary experiment, phosphate ion was found to catalyse the fluorogenic reaction of CAs with ED. Thus, a compromise concentration of phosphate ion was investigated for inclusion in the final chemiluminogenic reaction solution [SO mmol 1-1 potassium acetate buffer (pH 3.20)-acetonitrile-ethanol-dioxane-ethyl acetate, (20 + 9 + 1 + 28 + 28, v/v/v/v/v)]. Fifty mmol l-* phosphate at up to 7% in the acetate buffer caused no precipitates. Therefore, the final buffer composition of the mobile phase selected was 50 mmol 1-1 potassium acetate buffer (pH 3.20)-50 mmol I-' potassium phosphate buffer (pH 3.20) (95 k 5 v/v). The other constituents of the eluent were sodium hexanesulfonate (an ion-pair reagent) and acetonitrile; 1 mmol 1-1 sodium hexanesulfonate was added to the final buffer to increase the selectivity of the separation and 3% acetonitrile to shorten the separation time.

50

100

Volume ratio of acetonitrile and water (%)

Q,

K

50

25

I

I 50

0

I 100

Volume ratio of acetonitrile and ethanol (%) Fig. 3 Effect of organic solvents on fluorogenic reaction of CAs with ED. A, NE; B, E; and C, DA. A 10 p1 volume of a mixture of standard CAs (10 pmol each) was injected onto the column. HPLC conditions: column, catecholpak (150 X 4.6 mm i.d.); column tem erature, 40"C; eluent, 50 mmol 1-1 potassium acetate ( H 3.2Or-50 mmol 1-1 potassium phosphate (pH 3.20) (95 + 5 vhycontaining 1 mmol 1-l sodium hexanesulfonate; flow rate, 0.5 ml min-1; fluorogenic reagent solution, 120 mmol l-l ED in the organic solvents; reagent flow rate, 0.25 ml min-l; reaction coil for fluorogenic reaction, 15 m x 0.5 mm i.d.; and reaction temperature, 80 "C. The fluorescence intensity was monitored at 500 nm with excitation at 410 nm

Fluorogenic Reaction In a previous paper,19 ED in aqueous solution was utilized. However, the organic solvent rich solutions in the postcolumn PO-CL reaction gave higher chemiluminescence yields. Therefore, the effect of organic solvents (ethanol and acetonitrile) on the fluorogenic reaction of CAs with ED was examined using the conditions described in the Experimental section. Among the co-solvents, a mixture of acetonitrile and ethanol gave the best results, as shown in Fig. 3 and, hence acetonitrile-ethanol (90 + 10 v/v) was used hereafter.

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(9)

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al

.-

+a

-al ~

a

100

110

120

130 140120 130

120

130 140100 110

120

100

110

120

130100 110

120

I 100

I

110

Concentration of ED/mmol I-' Fig. 4 Effect of imidazole on fluorogenic reaction of ED with DA. Concentrations of imidazole: ( a ) 0; (b) 25; (c) 50; ( d ) 100; (e) 150; U, 175; and (g) 200 mmol 1-1. The mixture of 2 ml of 50 mmol 1-1 potassium acetate ( H 3.20)-50 mmol 1-I potassium phos hate (pH 3.20) (95 + 5 v/v), 10 p1 DA solution (1 nmol), and ED (95-140 mmol l-1) and imidazole 6 2 0 0 mmol l-1) in 1 ml acetonitrile-etkanol (90 + 10) was heated at 80 "C for 4 min, cooled in an iced-water bath for 1 min and measured at 480 nm with excitation at 380 nm

The HPLC-PO-CL reaction requires imidazole buffer (pH 6-7) as a catalyst.22 Imidazole was therefore added to the E D solution. The fluorescence reaction yield of CAs with E D depended on the pH of the reaction medium.19 The optimum E D and imidazole concentrations were examined using the static system under the conditions described in the Experimental section. As shown in Fig. 4, the highest yield was obtained with 175 mmol l-1 imidazole and 105 mmol 1-1 ED. 80

ChemiluminogenicReaction

In a previous paper,l6 acetohitrile-ethyl acetate (50 + 50 v/v) was used as the solvent for the chemiluminogenic solutions. However, this solvent composition separated the final solution (a mixture of eluent, fluorogenic reagent solution and chemiluminogenic reagent solution) into two phases; then two phases resulted in considerable baseline noise. Thus, in the present experiment, instead of this solvent composition, dioxane-ethyl acetate (1 + 1v/v) was used for the CL solution in order to bring the final mixture into one phase. The PO-CL reaction using TDPO gives the highest CL intensity when the pH of the reaction medium is around 6-7 and imidazole buffer (pH 6-7) is included in the eluent to catalyse the PO-CL reaction.22 In this experiment, imidazole (105 mmol l-1) was added to the E D solution beforehand, the reaction medium of which was pH 9. Thus, pH adjustment of the final solution of the fluorogenic reaction medium was required before the PO-CL reaction in order to obtain maximum CL intensity. To perform this pH adjustment, two different ways of delivering the acids were chosen: firstly, the acid solution was delivered using an additional pump; or secondly, the acid was dissolved in the chemiluminogenic reagent solution beforehand. Trifluoroacetic, acetic and citric acids were tested. The delivery of the acid solutions using an additional pump made the system more complicated. Also, the acid containing chemiluminogenic reagent solution [0.25 mmol 1-1 TDPO and 150 mmol 1-1 hydrogen peroxide in dioxane-ethyl acetate (1 + 1v/v)], especially TFA, resulted in facile pH adjustment and a stable baseline. The results are shown in Fig. 5. A concentration of 110 mmol l-1 TFA gave maximum sensitivity. Under the selected conditions containing 0.25 mmol 1-1 TDPO, 150 mmol 1-1 hydrogen peroxide and 110 mmol l-1 TFA in dioxane-ethyl acetate (50 + 50 vh), the CL intensity increased five times as compared with that without imidazole addition. Examination of the concentration of hydrogen peroxide showed that a '12 times higher concentration of hydrogen peroxide (150 mmol 1-1) than reported,l6 with a TBPO concentration of 0.25 mmol 1-1 and a TFA concentration of 110 mmol l-1, gave the highest CL intensity with DA (Fig. 6).

90

100

110

Concentration of TFkrnrnol

120 1-1

Fig. 5 Effect of TFA concentration on the detection limit of DA with chemiluminescent detection. A 10 pl volume of a mixture of standard CAs (1 pmol each) was injected onto the column. Conditions: column, catecholpak (150 x 4.6 mm i.d.); column temperature, 40 'C; eluent, 50 mmol 1-1 potassium acetate ( H 3.20)-50 mmol 1-1 potassium 4.85 + 3 v/v/v) containing phosphate ( H 3.20)-acetonitrile ($2.15 1 mmol 1- sodium hexanesulfonate; flow rate, 0.5 ml min-'; fluorogenic reagent solution, 105 mmol 1-1 ED and 175 mmol 1-1 imidazole in acetonitrile-ethanol(90 + 10 v/v); reagent flow rate, 0.25 ml min-1; reaction coil for fluorogenic reaction, 15 m x 0.5 mm i.d.; reaction temperature, 80 "C; chemiluminogenic reagent solution, 0.25 mmol l-1 TDPO, 150 mmol l-l hydrogen peroxide and TFA (80-120 mmol 1-1) in dioxane-ethyl acetate (50 50 v/v); and reagent flow rate, 1.4 ml min-1. The DA was detected under the conditions above. The signal-to-noise ratio = 2

+

P

+

75

100

125

150

175

200

Concentration of hydrogen peroxide/mmol I-' Fig. 6 Effect of hydrogen peroxide concentration on the detection limit of DA with chemiluminescent detection. The conditions were the same as in Fig. 5 except the chemiluminogenic reagent solution: 0.25 mmol l-1 TDPO, hydrogen peroxide (75-200 mmol l-l) and 110 mmol l-1 TFA in dioxane-ethyl acetate (50 + 50 v/v)

According to the preliminary experiment, the CL reagent solution containing TDPO, hydrogen peroxide and TFA after storage at room temperature for 3 d gave the highest sensitivity. Thus, the stock reagent solution was used hereafter.

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Other Factors Influencing the PO-CL Detection System

HPLC-PO-CL Detection of CAs

The knitted reaction coil, made of PTFE tubing (15 m X 0.5 mm i.d.), reduced the peak broadening of NE and E due to their diffusion in the coil by a factor of about 2 as compared with that obtained using a rolled coil of the same length. The two pumps with a short stroke plunger (2 mm) used to deliver the fluorogenic and chemiluminogenic reagent solutions diminished the baseline noises due to their small delivery pulses. A mixing device for the CL reagent solution and the mixture of the eluent and the E D solution was installed in the column oven and kept at 40 "C to maintain a stable baseline. The purity of the reagents and solvents is very important to attain a high signal-to-noise ratio in the PO-CL detection system.23 In this experiment, the purest materials were used; HPLC-grade acetonitrile, ethyl acetate, dioxane, ethanol and water were the purest solvents available. The E D for washing the semiconductors was also of the best quality obtainable. The TDPO was also purified. However, there was a baseline shift from zero to about 0.1 V, which corresponds to a peak height of about 0.2 pmol of D A fluorophore. The most probable impurities would originate from the hydrogen peroxide solution, a commercial product stored in a polyethylene bottle that contained many fluorescent materials. The impurities in the hydrogen peroxide solution proved difficult to remove. Therefore, a colour cut-off filter to remove the background CL was placed between the flow cell and the photomultiplier tube. The Y-46 filter reduced the background level by about half and the Y-48 filter reduced it to about one third. The Y-46 filter retained the same peak height for the D A fluorophore and thus the signal-to-noise ratio was improved by 1.5-2 times. The Y-48 filter also reduced the signal and thus no improvement in the signal-tonoise ratio was observed. These results were consistent with the previous findings that filters suitable for the chemiluminescence wavelengths of each of the fluorophores gave high signal-to-noise ratios. 10.24

Standard CAs A chromatogram obtained from a mixture of standard CAs (500 fmol each) is shown in Fig. 7. The calibration curve for each of the CAs with DHBA as the internal standard exhibits linearity from 0.01 to 1 pmol. The detection limits for all CAs at a signal-to-noise ratio of 2 were 1fmol on column injection. The detection limits were about 3-6 times lower than those obtained by HPLC with electrochemical detection (NE, 5.7 fmol; E, 2.2 fmol; DA, 6.5 fm0l)zs and about 1-2 times lower than those obtained by HPLC with pre-column fluorescence derivatization using DPE (NE, 1 fmol; E, 1 fmol; DA, 2 fmol).6

DA

0

10

Recovery experiment The recoveries of CAs added to plasma were 69 f 3,67 k 3 and 69 _+ 4% for NE, E and DA, respectively (n = 5). The values are consistent with our previous data26 and those of other work27 except for D A (94 f5%). Concentration of CAs in human and rat plasma Chromatograms of CAs in a human plasma and SD rat plasma are shown in Fig. 8. The plasma concentrations of NE, E and D A in a healthy human (40 years old) were 4.0,0.28 and 0.24 pmol ml-1, respectively, which were within a normal range of CAs: NE 2.03 f 1.08, E 0.39 -t 0.22, and D A 0.07 f 0.06 pmoUml-1;6 and NE 4.2 f 1.7 and E 1.0 f 0.3 pmol ml-1.28 The values for CAs in rat plasma were 1.21, 0.12, and 0.63 pmol ml-1, respectively. The concentrations of NE, E and DA in the Sprague-Dawley rats (n = 8) were 0.99 2 0.56,0.26 f 0.37, and 0.48 k 0.33 pmol ml-1, respectively, which were lower than those obtained by radioenzymic-paper chromatography (NE, 2.72 f 0.47 and E, 0.98 k 0.13 pmol ml-').29 The sensitivity of the proposed method was sufficient to make the determination of CAs in 100-200 p1 of human plasma and 100 pl of rat plasma. The methods using the electrochemical detector required 1ml7 and 500 pl27 of human plasma and the radioenzymic-paper chromatographic method required 400 pl29 of rat plasma (also DA was not detected). The high sensitivity of the present method enables the use of only a single rat to monitor the changes in plasma CA concentration after administration of drugs affecting the

20

Retention time/min Fig. 7 Chromatogram of a mixture of standard CAs with peroxyoxalate chemiluminescence detection. A 50 p1 volume of the mixture prepared with 0.1 mmol 1-l perchloric acid was injected onto the column. Peaks (500fmol each): NE, norepinephrine; E, epinephrine; I, 3,4-dihydroxybenzylamine;DA, dopamine. Conditions: column, catecholpak (150 x 4.6 mm i.d.); column temperature, 40 "C; eluent, 50 mmol I-' potassium acetate ( H 3.20)-50 mmol 1-l potassium phosphate ( H 3.20)-acetonitrile 62.15 4.85 3 v/v/v) containing 1 mmol 1- sodium 1-hexanesulfonate; flow rate, 0.5 ml min-1; fluorogenic reagent solution, 105 mmol I-' E D and 175 mmol 1-1 imidazole in acetonitrile-ethanol(90 + 10 v/v); reagent flow rate, 0.25 ml min-l; reaction coil for fluorogenic reaction, 15 m x 0.5 mm i.d.; reaction temperature, 80 "C; chemiluminogenic reagent solution, 0.25 mmol 1-l TDPO, 150 mmol 1-1 hydrogen peroxide and 110 mmol 1-1 TFA in dioxane-ethyl acetate (50 + 50 v/v); and reagent flow rate, 1.4 ml min-1

P

+

+

0

10

20

Retention time/min Fig. 8 Chromatograms obtained from ( a ) human and (b) SpragueDawley rat plasma. A 50 pl volume of extract was injected onto the column. Reaction conditions were the same as in Fig. 7

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activity of the sympathetic nervous system. The study is in progress and the details will be published elsewhere. The authors thank Dr. C. K. Lim of the Medical Research Council Laboratories for his kind review of this manuscript.

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References 1 Engelman, K., and Portnoy, B., Circ. Res., 1970,26, 53. 2 Kissinger, P. T., Riggin, R. M., Alcorn, R. L., and Rau, L. D., Biomed. Med., 1975, 13,299. 3 Imai, K., Tsukamoto, M., and Tamura, Z., J. Chromatogr., 1977, 137, 357. 4 Seki, T., J. Chromatogr., 1978,155,415. 5 Yamatodani, A., and Wada, H., Clin. Chem. (Winston-Salem, N . C . ) , 1981, 27, 1983. 6 Mitsui, A., Nohta, H., and Ohkura, Y., J. Chromatogr., 1985, 344,61. 7 Kamahori, M., Taki, M., Watanabe, Y., and Miura, J., J. Chromatogr., 1991,567, 351. 8 Kobayashi, S., and Imai, K., Anal. Chem., 1980,52, 424. 9 Imai, K., Metho& Enzymol., 1986, 133B, 435. 10 Kobayashi, S . , Sekino, J., Honda, K., and Imai, K., Anal. Biochem., 1981, 112, 99. 11 Kawasaki, T., Imai, K., Higuchi, T., and Wong, 0. S., Biomed. Chromatogr., 1990,4, 113. 12 Nozaki, O., Ohba, Y., and Imai, K., Anal. Chim. Acta, 1988, 205, 255. 13 Higashidate, S., Hibi, K., Senda, M., Kanda, S., and Imai, K., J. Chromatogr., 1990,515,577. 14 Nishitani, A., Tsukamoto, Y., Kanda, S., and Imai, K., Anal. Chim. Acta, 1991, 251,247.

15 Takada, K., Oh-hashi, M., Yoshikawa, H., Muranishi, S., Nishiyama, M., Yoshida, H., and Hata, T., J. Chromatogr., 1990, 530,212. 16 Uzu, S., Imai, K., Nakashima, K., and Akiyama, S., Analyst, 1991, 116, 1353. 17 Imai, K., J. Chromatogr., 1975, 105, 135. 18 Kawasaki, T., Higuchi, T., Imai, K., and Wong, 0. S., Anal. Biochem., 1989, 180, 279. 19 Mori, K., and Imai, K., Anal., Biochem., 1985, 146,283. 20 Imai, K., Sugiura, M., and Tamura, Z., Chem. Pharm. Bull., 1971, 19, 409. 21 Kobayashi, S., and Imai, K., Anal. Chem., 1980,52, 1548. 22 Imai, K., Nishitani, A., Tsukamoto, Y., Wang, W. H., and Kanda, S., Biomed. Chromatogr., 1990, 4, 100. 23 Miyaguchi, K., Honda, K., Toyo’oka, T., and Imai, K., J. Chromatogr., 1986,352, 255. 24 Mellbin, G., J. Liq. Chromatogr., 1983, 6, 1603. 25 Meineke, I., Stuwe, E., Henne, E. M., Rusterberg, G., Brendel, E., and D e Mey C., J. Chromatogr., 1989,493, 287. 26 Wang, M. T., Imai, K., Yoshioka, M., and Tamura, Z., Clin. Chim. Acta, 1975,63, 13. 27 Ganhao, M. F., Hattingh, J., Hurwitz, M. L., and Pitts, N. I., J. Chromatogr., 1991, 564, 55. 28 Nohta, H., Yamaguchi, E., Ohkura, Y., and Watanabe, H., J. Chromatogr., 1989, 493, 15. 29 Poppwe, C. W., Chiueh, C. C., and Kopin, I. J., J. Pharmacol. Exp. Ther., 1977,202,144.

Paper 2102573F Received May 18, 1992 Accepted September 1, 1992

Determination of femtomole concentrations of catecholamines by high-performance liquid chromatography with peroxyoxalate chemiluminescence detection.

A highly sensitive method for determination of the plasma catecholamines, norepinephrine (NE), epinephrine (E) and dopamine (DA) is described. The met...
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