Electrogenerated Chemiluminescence Derivatization Reagent, 3

Tris(2,2′-bipyridyl)ruthenium( ii ) chemiluminescence. Bree A. Gorman , Paul S. Francis , Neil W. Barnett. The Analyst 2006 131, 616-639 ...
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Anal. Chem. 2003, 75, 940-946

Electrogenerated Chemiluminescence Derivatization Reagent, 3-Isobutyl-9,10-dimethoxy-1,3,4,6,7,11bhexahydro-2H-pyrido[2,1-a]isoquinolin-2-ylamine, for Carboxylic Acid in High-Performance Liquid Chromatography Using Tris(2,2′-bipyridine)ruthenium(II) Hirotoshi Morita* and Masaharu Konishi

Shionogi Research Laboratories, Shionogi & Co., Ltd., 12-4 Sagisu 5-Chome, Fukushima-ku, Osaka 553-0002, Japan

3-Isobutyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2Hpyrido[2,1-a]isoquinolin-2-ylamine (IDHPIA) was found to be a selective and highly sensitive derivatization reagent for carboxylic acid by high-performance liquid chromatography (HPLC) with electrogenerated chemiluminescence detection using tris(2,2′-bipyridine)ruthenium(II). Free fatty acids and phenylbutylic acid were used as model compounds of carboxylic acids, and the derivatization conditions were optimized with myristic acid. Under the mild reaction conditions of room temperature for 45 min in acetonitrile containing 2-bromo-1-ethylpyridinium tetrafluoroborate and 9-methyl-3,4-dihydro2H-pyrido[1,2-a]pyrimidin-2-one, all the fatty acids tested were reacted with IDHPIA to produce highly sensitive derivatives. The chemiluminescence intensity was essentially the same for all fatty acids. The derivatives obtained from 10 free fatty acids were completely separated by reversed-phase chromatography under isocratic elution conditions. The on-column detection limit (signalto-noise ratio of 3) with proposed HPLC separation and chemiluminescence detection was 0.5 and 0.6 fmol for myristic acid and phenylbutylic acid, respectively. IDHPIA was 100-fold more sensitive than previously developed reagents (Morita, H.; Konishi, M. Anal. Chem. 2002, 74, 1584-1589). The free fatty acids in human serum were successfully determined using the present method. The electrogenerated chemiluminescent (ECL) system using tris(2,2′-bipyridine)ruthenium(II) [Ru(bpy)32+] has recently become a powerful tool for the determination of compounds that have tertiary amine or diketone groups because of its high sensitivity and selectivity. The reaction mechanism between Ru(bpy)32+ and tertiary amine has been investigated by many * Corresponding author. Phone: +81-6-6458-5861. Fax: +81-6-6458-0987. E-mail: [email protected].

940 Analytical Chemistry, Vol. 75, No. 4, February 15, 2003

workers,1-6 and the postulated reaction mechanism is reported by Zu and Bard to be as follows:4,5

Ru(bpy)32+ f Ru(bpy)33+ + e-

(1)

TA f TA•+ + e-

(2a)

Ru(bpy)33+ + TA f Ru(bpy)32+ + TA•+

(2b)

TA•+ f TA• + H+

(3)

TA• + Ru(bpy)33+ f TA fragment + [Ru(bpy)32+]* [Ru(bpy)32+]* f Ru(bpy)32+ + hν

(4) (5)

It was thought that Ru(bpy)33+, obtained by electrochemical oxidation, reacted with tertiary amine (TA) to give cation radical (TA•+). However, cation radical is supposed to be mainly produced by direct oxidation at the electrode (eq 2a) and catalytic amine oxidation by Ru(bpy)33+ (eq 2b) is less important, recently. It is thought to produce a neutral radical (TA•) by elimination of an R-proton. This unstable radical compound immediately reacts with Ru(bpy)33+ to give the excited state of the ruthenium complex, [Ru(bpy)32+]*. Its emission to the background state gives phosphorescence, with the maximal wavelength at 620 nm. On the other hand, a new and different reaction mechanism has also been reported by Bard and co-workers quite recently.6 The tertiary amine group can be selectively detected by this system, and a variety of drugs and endogenous compounds containing a tertiary amine have been sensitively determined with simple, minimal (1) Leland, J. K.; Powell, M. J. J. Electrochem. Soc. 1990, 137, 3127-3131. (2) Liang, P.; Sanchez, R. I.; Martin, M. T. Anal. Chem. 1996, 68, 2426-2431. (3) Brune, S. N.; Bobbitt, D. R. Anal. Chem. 1992, 64, 166-170. (4) Zu, Y.; Bard, A. J. Anal. Chem. 2000, 72, 3223-3232. (5) Zu, Y.; Bard, A. J. Anal. Chem. 2001, 73, 3960-3964. (6) Miao, W.; Choi, J.-P.; Bard, A. J. J. Am. Chem. Soc. 2002, 124, 1447814485. 10.1021/ac020377i CCC: $25.00

© 2003 American Chemical Society Published on Web 01/21/2003

pretreatment of biological samples.7-13 Some applications for highperformance liquid chromatography (HPLC) or flow injection analysis (FIA) by Ru(bpy)33+ chemiluminescence (CL) have been developed in recent years.3,7-23 Nevertheless, the compounds detectable by this method are still limited. Therefore, we attempted to extend the system to a wider range of compounds containing nonsuitable functional groups by converting a variety of compounds into derivatives suitable for ECL detection by tagging appropriate molecules with an analyte. Many derivatization reagents for fluorescence, ultraviolet absorbance, electrochemical, and chemiluminescence detection have been developed. However, only a few derivatization reagents for ECL detection such as dansyl chloride21 or divinylsulfone23 have been developed. We have developed some reagents such as N-(3-aminopropyl)pyrrolidine for carboxylic acid24 and 3-(diethylamino)propionic acid for primary amine24 and alcohol.25 These reagents reacted with the analytes to produce sensitive derivatives in the presence of suitable catalysts at room temperature for 30-120 min and showed the detection limit of 40-70 fmol at a signal-to-noise ratio of 3. In this study, we examined a more sensitive derivatization reagent and developed a 100-fold more sensitive reagent for carboxylic acid than previously developed reagents.24,25 To our knowledge, this reagent is one of the most sensitive derivatization reagents developed for HPLC analysis. Moreover, this reagent showed the optimum detection pH at 1.5-2.0 in the ECL system, making it possible to omit the pH-conditioning pump and simplifying the equipment of the detection system. EXPERIMENTAL SECTION Reagents. Tris(2,2′-bipyridine)ruthenium(II) chloride pentahydrate (Ru(bpy)3Cl2‚5H2O) and ibuprofen were purchased from Sigma Chemical Co. (St. Louis, MO). Phenylbutylic acid (PB) was obtained from Kanto Chemical Co. Inc. (Tokyo, Japan). Fatty acids were obtained from Wako Pure Chemical Indutries, Ltd. (Osaka, Japan), Sigma Chemical Co., Nacalai Tesque Inc., or GL Sciences Inc. (Tokyo, Japan). 1,2,3,4,6,7-Hexahydrobenzo[a]quinolizine-2,4dione was purchased from Bionet Research Ltd. (Cornwall, U.K.). 2-Bromo-1-ethylpyridinium tetrafluoroborate (BEPT) and 9-methyl(7) Song, Q.; Greenway, G. M. Analyst 2001, 126, 37-40. (8) Ridlen, J. S.; Skotty, D. R.; Kissinger, P. T.; Nieman, T. A. J. Chromatogr., B 1997, 694, 393-400. (9) Skotty, D. R.; Nieman, T. A. J. Chromatogr., B 1995, 665, 27-36. (10) Koike, K.; Li, Y.; Seo, M.; Sakurada, I.; Tezuka, K.; Uchikura, K. Biol. Pharm. Bull. 2000, 23, 101-103. (11) Monji, H.; Yamaguchi, M.; Aoki, I.; Ueno, H. J. Chromatogr., B 1997, 690, 305-313. (12) Holeman, J. A.; Danielson, N. D. J. Chromatogr., A 1994, 679, 277-284. (13) Uchikura, K.; Kirisawa, M. Anal. Sci. 1991, 7, 971-973. (14) Jackson, W. A.; Bobbitt, D. R. Anal. Chim. Acta 1994, 285, 309-320. (15) He, L.; Cox, K. A.; Danielson, N. D. Anal. Lett. 1990, 23 (2), 195-210. (16) Targove, M. A.; Danielson, N. D. J. Chromatogr. Sci. 1990, 28, 505-509. (17) Ridlen, J. S.; Klopf, G. J.; Nieman, T. A. Anal. Chim. Acta 1997, 341, 195204. (18) Downey, T. M.; Nieman, T. A. Anal. Chem. 1992, 64, 261-268. (19) Martin, A. F.; Nieman, T. A. Anal. Chim. Acta 1993, 281, 475-481. (20) Skotty, D. R.; Lee, W.-Y.; Nieman, T. A. Anal. Chem. 1996, 68, 1530-1535. (21) Lee, W.-Y.; Nieman, T. A. J. Chromatogr., A 1994, 659, 111-118. (22) Danielson, N. D.; He, L.; Noffisinger, J. B.; Trelli, L. J. Pharm. Biomed. Anal. 1989, 7, 1281-1285. (23) Uchikura, K.; Kirisawa, M.; Sugii, A. Anal. Sci. 1993, 9, 121-123. (24) Morita, H.; Konishi, M. Anal. Chem., 2002, 74, 1584-1589. (25) Morita, H.; Konishi, M. J. Liq. Chromatogr. Relat. Technol. 2002, 25 (16), 2413-2423.

3,4-dihydro-2H-pyrido[1,2-a]pyrimidin-2-one (MDPP) were from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Acetonitrile and methanol of HPLC grade were obtained from Kanto Chemical Co. Inc. Tetrabenazine was synthesized in this laboratory but is commercially available. All the other chemicals were of guaranteed grade and used without further purification. Twice-distilled deionized water was used throughout the study. Instrumentation. Melting points were recorded on a Yanagimoto micro melting point apparatus (Tokyo, Japan) and are uncorrected. 1H NMR spectra were recorded on an ASM-100 (300 MHz) spectrometer (Varian) using tetramethylsilane as an internal standard. Mass spectra (electron impact ionization) were determined with a GCMS-QP1000(A) spectrometer (Shimadzu). Highresolution (HR) mass spectra (FAB-MS) were determined with a SX/SX102A (JEOL) using m-nitrobenzyl alcohol as a matrix. Elemental analyses were performed by CHN corder MT-6 (Yanako). The ECL intensity was observed by modifying a commercially available system. The HPLC system is shown in Figure 1. All the solutions were purged with two types of degassers (DGU10B for helium gas purge type and DGU-3A for membrane type, Shimadzu) and were delivered with pumps (LC-10AD, Shimadzu). Electrochemical oxidation was performed with a porous graphite working electrode (guard cell, model 5020, ESA), and the current was controlled with a potentio-galvanostat (NPGS-2501, Nikkoh Keisoku). The chemiluminescence detector used was a Shimadzu CLD-10A equipped with an 80-µL spiral flow cell and R374HA photomultiplier tube (Hamamatsu Photonics). Shimadzu SIL-10A was used as the autosampler. CLASS LC-10 (Shimadzu) was used as the data processor. To avoid the permeation of atmospheric oxygen, a metal tube was used for the connection. A low-volume tee tube was used for mixing of the mobile phase and the reagent solution. Synthesis of 1,3,4,6,7,11b-Hexahydro-2H-pyrido[2,1-a]isoquinolin-2-ol (HPI). To a solution of 1,2,3,4,6,7-hexahydrobenzo[a]quinolizine-2,4-dione (1) (1.86 g, 8.64 mmol) in 25 mL of ethyl acetate/methanol (2:3, v/v) in an ice bath was added NaBH4 (17.3 mmol), and the mixture was stirred for 2 h at room temperature. The reaction mixture was evaporated to dryness in vacuo. The residue was dissolved with chloroform and washed with water. The organic layer was dried with anhydrous MgSO4, and the filtrate was evaporated in vacuo. The residue was purified by column chromatography on silica gel with ethyl acetate/ methanol (10:1, v/v) as the elution solvent to afford 1.58 g of 2-hydroxy-1,2,3,6,7,11b-hexahydropyrido[2,1-a]isoquinolin-4-one (2) as a yellow powder, followed by recrystallization from ether to give colorless crystal: 1H NMR (CDCl3) δ 1.2-3.5 (6H, m), 4.24.3 (1H, m, 2-H), 4.6-4.8 (2H, m, 3-H), 7.1-7.3 (4H, m, phenylH); MS m/z 217 ([M]+). To a solution of 2 (1.40 g, 6.44 mmol) in 25 mL of tetrahydrofuran in an ice bath was added LiAlH4 (25.8 mmol), and this was refluxed for 2 h at 100 °C. After cooling, a small portion of methanol and water was added to the reaction mixture to decompose the excess reagent, followed by filtration, and the filtrate was evaporated to dryness in vacuo. After addition of 10% NaOH, the residue was extracted with chloroform. The organic layer was dried with anhydrous MgSO4, and the filtrate was Analytical Chemistry, Vol. 75, No. 4, February 15, 2003

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Figure 1. Schematic flow diagram of ECL detection.

evaporated in vacuo. The residue was purified by column chromatography on silica gel with chloroform/methanol (35:1, v/v) as the elution solvent to afford 0.96 g of HPI (3) as a colorless crystal: mp 142-143 °C; 1H NMR (CDCl3) δ 1.4-3.2 (11H, m), 3.7-3.9 (1H, m, 2-H), 7.0-7.2 (4H, m, phenyl-H); MS m/z 203 ([M]+). Anal. Calcd for C13H17NO: C, 76.81; H, 8.43; N, 6.89. Found: C, 76.52; H, 8.40; N, 7.00. Synthesis of 1,3,4,6,7,11b-Hexahydro-2H-pyrido[2,1-a]isoquinolin-2-ylamine (HPIA). Thionyl chloride (6.9 mmol) was added dropwise to a solution of 3 (500 mg, 2.46 mmol) in 36 mL of toluene in an ice bath, and the resultant mixture was stirred for 1.5 h at 80 °C. After cooling, a small portion of water was added to the reaction mixture to decompose the excess reagent. After addition of 10% NaOH, the reaction mixture was extracted with chloroform. The organic layer was dried with anhydrous MgSO4, and the filtrate was evaporated in vacuo to afford 495 mg of 2-chloro-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinoline (4) as a brown oil. Compound 4 was used without further purification. To a solution of 4 (495 mg, 2.19 mmol) in 10 mL of N,N-dimethylformamide (DMF) was added sodium azide (6.57 mmol) for 7 h at 100 °C. After evaporation, 10% NaOH was added to the residue and the reaction mixture was extracted with chloroform. The organic layer was dried with anhydrous MgSO4, and the filtrate was evaporated in vacuo. The residue was purified by column chromatography on silica gel with chloroform/ methanol (20:1, v/v) and n-hexane/ethyl acetate (1:1, v/v) as the elution solvent to afford 349 mg of 2-azido-1,3,4,6,7,11b-hexahydro2H-pyrido[2,1-a]isoquinoline (5) as a brown oil. Compound 5 (99 mg, 0.43 mmol) was hydrogenated in ethanol (3 mL) at room temperature and 1 atm in the presence of 5% Pd/C (30 mg) for 3.5 days. After filtration, the filtrate was evaporated in vacuo. The residue was purified by column chromatography on silica gel with chloroform/methanol (10:1, v/v) and n-hexane/ ethyl acetate (1:1, v/v) as the elution solvent to afford 51 mg of HPIA (6) as a white powder: mp 199-200 °C (dec); 1H NMR (CDCl3) δ 1.4-3.4 (12H, m), 7.1-7.3 (4H, m, phenyl-H); MS m/z 942

Analytical Chemistry, Vol. 75, No. 4, February 15, 2003

202 ([M]+); HR-MS m/z calcd for C13H19N2 [M + H]+: 203.1548, found 203.1557. Synthesis of an HPI Derivative of Ibuprofen. After stirring of a mixture of ibuprofen (10 mg, 48 µmol), DMF (1 drop), and oxalyl chloride (48 µmol) in 1 mL of dichloromethane for 1 h, HPI (48 µmol) and (dimethylamino)pyridine (96 µmol) were added and the mixture was kept for 4 days at room temperature. After addition of 5% NaHCO3, the reaction mixture was extracted with chloroform. The organic layer was dried with anhydrous MgSO4, and the filtrate was evaporated in vacuo. The residue was purified by column chromatography on silica gel with n-hexane/ethyl acetate (9:2, v/v) as the elution solvent to afford the ester: 1H NMR (CDCl3) δ 0.88 (6H, d, J ) 4.4 Hz, (CH3)2CHCH2), 1.4-3.3 (11H, m), 1.50 (3H, d, J ) 4.8 Hz, CH(CH3)COO), 2.44 (2H, d, J ) 4.6 Hz, (CH3)2CHCH2), 3.70 (1H, q, J ) 5.0 Hz, CHCOO), 4.85.0 (1H, m, 2-H), 7.0-7.3 (8H, m, phenyl-H); MS m/z 391 ([M]+). Synthesis of 3-Isobutyl-9,10-dimethoxy-1,3,4,6,7,11bhexahydro-2H-pyrido[2,1-a]isoquinolin-2-ylamine (IDHPIA). To a solution of tetrabenazine (500 mg, 1.58 mmol) in 30 mL of methanol were added ammonium acetate (15.8 mmol) and sodium cyanoborohydride (1.1 mmol) followed by stirring for 22 h at room temperature. The reaction mixture was evaporated to dryness in vacuo. The residue was dissolved with chloroform and washed with 10% NaOH. The organic layer was dried with anhydrous MgSO4 and the filtrate was evaporated in vacuo. The residual oil was purified by column chromatography on silica gel with chloroform/methanol (40:1, v/v) and alumina with ethyl acetate as the elution solvent to afford 96 mg of IDHPIA as a white powder: mp 97-98 °C; 1H NMR (CDCl3) δ 0.91 (3H, d, J ) 6.0 Hz, (CH3)2CH), 0.94 (3H, d, J ) 6.0 Hz, (CH3)2CH), 1.0-3.4 (14H, m), 3.84 (6H, s, 2 × CH3O), 6.57 (1H, s, phenyl-H), 6.68 (1H, s, phenyl-H); MS m/z 318 ([M]+); HR-MS m/z calcd for C19H31N2O2 [M + H]+ 319.2386. found 319.2395. Synthesis of IDHPIA Derivatives of PB and Myristic Acid (C14:0). To a solution of IDHPIA (2.0 mg, 6.28 µmol) in 0.3 mL of acetonitrile were added PB (7.5 µmol), BEPT (12 µmol), and

MDPP (15 µmol) for 3.5 h at room temperature. The reaction mixture was applied and purified by column chromatography on silica gel with ethyl acetate as the developing solvent to afford 2.8 mg of the IDHPIA derivative of PB as a white powder: mp 186-187.5 °C; 1H NMR (CDCl3) δ 0.89 (3H, d, J ) 6.6 Hz, (CH3)2CH), 0.90 (3H, d, J ) 6.3 Hz, (CH3)2CH), 1.0-3.4 (20H, m), 3.79 (3H, s, CH3O), 3.84 (3H, s, CH3O), 6.59 (2H, s, phenyl-H), 7.27.3 (5H, m, phenyl-H); MS m/z 465 ([M + H]+). The IDHPIA derivative of C14:0 was also synthesized in the same manner and afforded 2.3 mg of the product as a white powder: mp 47-48 °C; 1H NMR (CDCl3) δ 0.85-0.90 (9H, m, 3 × CH3), 1.1-3.2 (38H, m), 3.82 (3H, s, CH3O), 3.84 (3H, s, CH3O), 6.57 (2H, each s, phenyl-H); MS m/z 528 ([M]+). Optimization of Detection pH. The detection pH was optimized by an FIA method. The carrier solution of 0.5 M Britton-Robinson (BR) buffer (pH 1.5-9.0) and the reagent solution of 0.8 mM Ru(bpy)3Cl2 in 10 mM H2SO4 were pumped at the flow rates of 1.0 and 0.3 mL min-1, respectively. The BR buffer was prepared as an acid solution with 61.3 g of boric acid, 56.5 mL of acetic acid, and 68 mL of phosphoric acid in 2 L of water. The pH was adjusted with 2.5 M NaOH to prepare pH 1.5-9.0 buffers. Aliquots of 5 µL of 10 or 100% methanol solutions containing 100 pmol of synthetic samples were injected triplicate, and mean values were used throughout the study. Relative standard deviations of most of the points were within 10% except for the points that showed very weak ECL intensity. The electrochemical oxidation was done by controlled-current mode with the current maintained at 200 µA. The oxidation potential was approximately +1.3 V versus R-hydrogen/palladium reference electrode. The photomultiplier applied was biased at 0.5 kV. Derivatization Procedure for PB. To a solution of 10 µM PB in 50 µL of acetonitrile were added 50 µL each of 5 mM BEPT, 0.3 mM MDPP, and 1 mM IDHPIA solution in acetonitrile. After mixing with a vortex mixer for a few seconds, the reaction mixture was allowed to stand for 60 min at room temperature, and then 800 µL of 50% methanol containing 0.5% TFA was added to stop the reaction. Of the resulting mixture, 10 µL was injected into the chromatograph. Derivatization Procedure for Fatty Acids in Human Serum. Blood samples were obtained from healthy human volunteers (male, aged from 21 to 51 years) in our laboratories after overnight fasting. A 5-µL aliquot of human serum was mixed with 200 µL of 0.5 M phosphate buffer (pH 6.5), 50 µL of the 0.4 µM margaric acid (C17:0, internal standard) solution in acetonitrile, and 2.0 mL of a mixture of chloroform/n-hexane (1:1, v/v). The mixture was vortexed for ∼5 min and centrifuged at 2000 rpm for 5 min, and the organic layer was evaporated under nitrogen stream. The residue was dissolved in 50 µL of acetonitrile, and then 50 µL each of the 5 mM BEPT, 1 mM MDPP, and 1 mM IDHPIA solution in acetonitrile was added. After mixing with a vortex mixer for a few seconds, the reaction mixture was allowed to stand for 45 min at room temperature, and then 800 µL of 80% acetonitrile was added to stop the reaction. Of the resulting mixture, 10 µL was injected into the chromatograph. HPLC Conditions for Fatty Acids. The mobile phase of 52% acetonitrile containing 0.05% trifluoroacetic acid (TFA) and reagent solution of 0.8 mM Ru(bpy)3Cl2 in 10 mM H2SO4 were delivered at the flow rates of 1.0 and 0.3 mL min-1, respectively.

The detection pH was ∼2.1. The column was an Inertsil C8 (4.6 × 150 mm; GL Sciences Inc., Tokyo, Japan). The column temperature was ambient (23 ( 2 °C). The photomultiplier applied was biased at 0.7 kV. RESULTS Candidates for the Derivatization Reagent. In this system, hydroxide ion reacts with Ru(bpy)33+; therefore, the background level rises with an increase of pH. A lower detection pH leads to a more stable analytical condition; therefore, the compounds that can be detected at acid pH with strong intensity should serve as good derivatization reagents. As most of the tertiary amines showed maximum ECL intensity at alkaline pH, we searched for suitable compounds. We focused on sparteine and dihydrocodeine, which showed maximum ECL intensity at pH 3.0 and detection limits of 50 and 2.5 fmol, respectively, at the signal-to-noise ratio of 3. To develop more sensitive derivatization reagents, we examined compounds closely related to sparteine and dihydrocodeine. The results suggested that compounds fulfilling the following criteria would show good signal-to-noise ratios: (i) rigid amino compounds such as quinolizidine-derived compounds; (ii) compounds containing a phenyl or mono- or dimethoxyphenyl group; (iii) compounds showing high ECL intensity at acid pH with high signal-to-noise ratio. Selection of Derivatization Reagent and Optimization of Detection pH. We evaluated the ECL intensity including the background noise level defined as normalized ECL intensity (I), which was calculated with the following equation: I ) A/B, where A is peak area per nanomole (µV‚s/nmol) and B is background current (nA). We examined some amino compounds with quinolizidine or a rigid structure. In the study of ECL intensity, we have found that the compounds containing an alkoxyphenyl group tended to have maximum ECL intensity at lower pH than the compounds without an alkoxyphenyl group. No effect was observed with phenolderived compounds and these compounds showed optimum pH at 7-9 (e.g., 9a-phenylquinolizidine). Quinolizidine-derived compounds bonded with the phenyl group at a long distance also showed no effect on optimum pH (e.g., 9a-(4-methoxybenzyl)quinolizidine) (data not shown). Consequently, we selected benzoquinolizidine- and dimethoxybenzoquinolizidine-derived compounds as candidates for derivatization reagents. We synthesized HPI and HPIA as benzoquinolizidine-derived compounds and IDHPIA as a dimethoxybenzoquinolizidin-derived compound. The structure of benzoquinolizidines is shown in Figure 2. Initially, HPI was synthesized by a one-step reduction reaction with 1 in the presence of LiAlH4 but HPI was obtained in low yield. HPI was synthesized by reducing 1 with NaBH4 to give 2, followed by reduction with LiAlH4. HPIA was synthesized from HPI. IDHPIA was synthesized by a reductive amination reaction of tetrabenazine with NaBH3CN. This reaction gave a ∼1:1 mixture of diastereoisomers. Although we examined the reaction with dimethylamine borane, which is a reductive amination reagent reacting by different mechanisms with NaBH3CN, the proportion of the two products did not change. We used the less polar compound of the diastereoisomers as the derivatization reagent. The pH-dependent ECL intensity was examined for benzoquinolizidine-derived compounds. As shown in Figure 3, benzoAnalytical Chemistry, Vol. 75, No. 4, February 15, 2003

943

Figure 2. Structure of benzoquinolizidine-derived compounds.

Figure 3. pH profile of benzoquinolizidine-derived compounds: O, tetrabenazine; 0, IDHPIA; 4, HPI; 2, HPIA; b, N,N-diisopropylethylamine. Samples: 100 pmol in 10% methanol solution. Each point was measured in triplicate, and standard deviation is indicated as error bars.

Figure 4. pH profile of HPI derivative of ibuprofen and IDHPIA derivatives of PB and C14:0: O, IDHPIA derivative of PB; 4, C14:0; 0, HPI derivative of ibuprofen. Samples: 100 pmol in 10 or 100% methanol solution. Each point was measured in triplicate, and standard deviation is indicated as error bars.

quinolizidine-derived compounds such as HPI and HPIA showed the maximum normalized ECL intensity at pH 5.0. The values of the normalized ECL intensity increased 10-41-fold for dimethoxybenzoquinolizidine-derived compounds such as tetrabenazine and IDHPIA compared to the benzoquinolizidine-derived compounds and showed maximum intensities at pH 1.5-2.0 in the tested range. These compounds gave 3-120-fold higher normalized ECL intensities than N,N-diisopropylethylamine, which is one of the most responsive compounds with Ru(bpy)33+ and showed a detection limit of 50 fmol at the signal-to-noise ratio of 3 (unpublished data). The pH-dependent normalized ECL intensity was examined for HPI, HPIA, and IDHPIA derivatives of some carboxylic acid compounds with the synthetic samples. These derivatives showed the same optimum pH as the unbound derivatization reagents as shown in Figure 4. The normalized ECL intensities of IDHPIA derivatives of PB and C14:0 were 153 and 50% relative to that of unbound IDHPIA, respectively. The normalized ECL intensity of the HPI derivative of ibuprofen was 83% relative to that of unbound HPI. Only a small effect was observed for the intensity with condensation of the analyte; therefore, these results show that a wide range of compounds could be used without significant decrease of ECL intensity. The detection limits of HPI and HPIA

derivatives of ibuprofen were both 15 fmol with the proposed HPLC separation (data not shown). IDHPIA was more sensitive than HPI and HPIA. IDHPIA was used for the following experiment. Optimization of the Derivatization Reaction. For the derivatization of carboxylic acids, BEPT and MDPP were selected as the condensation reagent and the acid-capturing reagent for HBr and HBF4 formed in the reaction, respectively. These reagents allowed the reaction to proceed with carboxylic acids to produce the corresponding amides at room temperature within 1 h (Figure 5), and electrogenerated chemiluninescence is based on the reaction between Ru(bpy)32+ and tertiary amine of the benzoquinolizidine-derived compounds. Moreover, chromatographic separation of the reagent and the conjugate was easy and no interference was observed for the analysis of the analyte. C14:0 was used as a representative fatty acid to optimize the reaction condition. The effect of the concentration of IDHPIA, BEPT, and MDPP on the reactivity to C14:0 was examined at 0-5 mM. The maximum peak area was obtained at 1 mM for IDHPIA and MDPP (50 equiv) and 5 mM for BEPT (250 equiv). Figure 6 shows the time course of the reaction under these conditions. A quantitative reaction was observed at more than 45 min. The derivatization condition for PB was also optimized at 1 mM IDHPIA (100 equiv),

944 Analytical Chemistry, Vol. 75, No. 4, February 15, 2003

Figure 5. Reaction scheme of fatty acids with IDHPIA. Table 2. Concentration of Fatty Acids in Human Serum (nmol mL-1) subject C12:0 C14:1 C14:0 C18:3 C16:1

C18:2

C16:0

C18:1

C18:0

1 2 3 4 5 6 7

1.2 8.1 3.8 0.3 0.2 0.3 0.1

0.6 0.6 0.7 1.8 0.8 0.8 1.1

4.1 8.4 12.7 1.4 2.5 1.2 3.1

15.3 7.8 6.3 9.3 9.8 5.3 7.9

3.8 2.8 12.0 12.5 11.6 10.4 17.6

89.0 78.6 48.5 62.1 61.0 71.9 76.2

88.4 85.3 89.5 63.9 98.0 90.2 107.3

83.0 88.8 93.1 90.4 141.8 133.1 159.0

22.5 24.2 21.4 13.5 33.6 23.1 27.7

mean SD

2.0 3.0

0.9 0.4

4.8 4.3

8.8 3.3

10.1 5.2

69.6 13.4

88.9 13.3

112.7 30.9

23.7 6.1

meana 1.5 1.5 7.9 4.4 4.7 59.4 91.9 90.3 21.2 (SD) (1.8) (1.1) (4.9) (2.4) (1.5) (23.4) (36.3) (42.1) (9.1) Figure 6. Time course of the derivatization reaction of IDHPIA with C14:0 in the presence of BEPT and MDPP. Conditions: 20 µM C14:0, 5 mM BEPT, 1 mM MDPP, and 1 mM IDHPIA in acetonitrile, room temperature. Each point was measured in duplicate.

meanb 5.8 (SD) (0.6) a

ndc

nd

3.9 18.5 52.1 80.7 93.7 31.5 (1.1) (1.9) (15.5) (26.4) (38.9) (7.8)

Quoted from ref 26. b Quoted from ref 27. c nd, not determined.

Table 1. Recovery of Fatty Acids (n ) 5) compound

recovery (%)

compound

recovery (%)

C12:0 C14:1 C14:0 C18:3 C16:1

98.8 ( 6.1 93.7 ( 5.0 102.0 ( 7.2 94.9 ( 5.8 96.1 ( 6.0

C18:2 C20:4 C16:0 C18:1 C18:0

94.7 ( 5.8 94.0 ( 4.9 105.0 ( 6.5 94.6 ( 5.6 97.7 ( 6.1

5 mM BEPT (500 equiv), and 0.3 mM MDPP (30 equiv) for 60 min. Determination of Fatty Acids in Human Serum. Lauric acid (C12:0), myristoleic acid (C14:1), myristic acid (C14:0), R-linolenic acid (C18:3), palmitoleic acid (C16:1), arachidonic acid (C20:4), R-linoleic acid(C18:2), palmitic acid (C16:0), oleic acid (C18:1), and stearic acid (C18:0) were used as free fatty acids and C17:0 was used as the internal standard. Free fatty acids were extracted with a mixture of chloroform and n-hexane (1:1, v/v). The recovery of all fatty acids tested was over 93% as shown in Table 1. The photomultiplier voltage was optimized for IDHPIA derivatives of fatty acids, and a bias of 0.7 kV was used for the determination of fatty acids. Figure 7 shows typical chromatograms of fatty acids in standard solution and extracted samples from human serum. Separation of the 11 fatty acid derivatives was satisfactorily achieved on the reversed-phase column by isocratic elution mode with the 0.05% TFA/acetonitrile. Each peak shape of IDHPIA derivatives of fatty acids was good and the theoretical numbers were between 29 000/ m (C12:0) and 59 500/m (C18:0). Each analyte was identified by comparison of its retention time to that of the fatty acid standard. All the fatty acid derivatives showed the same peak area; therefore, differences of ECL intensity between the compounds would be small. The excess reagent eluted earlier at the void and did not

interfere with the analyte peaks. Under these conditions, the detection pH was ∼2.1. The optimum detection pH was the same as that of the mobile phase; therefore, no pump was needed to deliver pH conditioning. Nieman et al. reported a versatile Ru(bpy)32+ ECL detection technique with the CL reagents added directly to the mobile phase.8,17,20 This technique requires just one pump to deliver the mobile phase containing the CL reagent. We examined the technique to try to develop more sensitive detection and simpler equipment. But more sensitive detection could not be achieved in our system. This result would be caused by the difference of the flow cell. They used their own apparatus with a laminar-type flow cell with a cell volume of 9.2 µL, and the potential was applied at the flow cell. Our system contains a spiral flow cell with a cell volume of 80 µL, and the potential was applied before the cell inlet. The detection limits of the HPLC analysis were found to be 0.6 and 0.5 fmol, respectively, for PB and C14:0 at the signal-tonoise ratio of 3. Standard solutions of different concentrations of PB, C12:0, and other nine fatty acids ranging from 0.25 to 500 (n ) 12), 0.5 to 1000 (n ) 8), and 2 to 2000 nM (n ) 8) were derivatized, respectively. All the calibration curves were linear for the range tested (correlation coefficients >0.999). Table 2 shows the concentrations of free fatty acids in sera from healthy volunteers determined by this method. The mean values for the individual free fatty acids in normal serum were in good agreement with the published data.26,27 (26) Yamaguchi, M.; Matsunaga, R.; Hara, S.; Nakamura M.; Ohkura, Y. J. Chromatogr., B 1986, 375, 27-35. (27) Kargas, G.; Rudy, T.; Spennetta, T.; Takayama, K.; Querishi, N.; Shrago, E. J. Chromatogr., B 1990, 526, 331-340.

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Figure 7. Chromatograms of IDHPIA derivatives of fatty acids of (a) standard solution (2 pmol each on column) and (b) extracts from human serum. HPLC conditions are given in the Experimental Section. Peaks: 1, C12:0; 2, C14:1; 3, C14:0; 4, C18:3; 5, C16:1; 6, C18:2; 7, C20:4; 8, C16:0; 9, C18:1; 10, C17:0; 11, C18:0.

CONCLUSIONS IDHPIA, a new electrogenerated chemiluminescent derivatization reagent for carboxylic acids, was specifically developed for the ECL system. The IDHPIA derivative showed optimum pH in the acid region and was 100-fold more sensitive than reagents reported previously. Also, because the optimum pH was the same for the mobile phase, the equipment could be simplified. This reagent could be synthesized with a one-step reaction from the commercially available compound and should be useful for a

946 Analytical Chemistry, Vol. 75, No. 4, February 15, 2003

wide range of drugs and endogenous compounds in biological fluids. ACKNOWLEDGMENT We thank Ms. R. Nishimura and Ms. Y. Nakano for measuring elemental analyses and high-resolution mass spectra, respectively. Received for review June 5, 2002. Accepted December 12, 2002. AC020377I