Optimization of the Reaction Conditions for the Peroxyoxalate Chemiluminescence Detection System of Fluorescent Compounds in Ahigh-Performance Liquid Chromatography
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ANALYTICAL SCIENCES OCTOBER 1996, VOL. 12 713 Optimization of the Reaction Conditions for the Peroxyoxalate Chemiluminescence Detection System of Fluorescent Compounds in aHigh-Performance Liquid Chromatography Ryoya GoHDA, Kohsuke KIMOTO, Tomofumi SANTA, Takeshi FUKUSHIMA, Hiroshi HoMMA and Kazuhiro IMAIt Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Tokyo 113, Japan Using an instrument of automatic fluid injection (batch method), we optimized such reaction conditions as pH, concentration and kinds of buffer for the peroxyoxalate chemiluminscence (PO-CL) detection of fluorescent compounds for high-performance liquid chromatography (HPLC). The results were compared with those obtained by HPLC. Consequently, the usefulness of the batch method for investigating the reaction conditions for PO-CL detection in HPLC was demonstrated. With the optimized condition using a phosphate buffer for the eluent in HPLC, the detection limits for Dns-Val and dipyridamole were 70 amol and 20 amol on column (S/N=2), respectively. Keywords Peroxyoxalate chemiluminescence, batch method, high-performance liquid chromatography, bis[4-nitro-2- (3,6,9-t,rioxadecyloxycarbonyl)phenyl]oxalate, dipyridamole, dansylated valine Since introducing the peroxyoxalate chemilumines- extrapolation from zero time to the first part of the decay cence (PO-CL) reaction to the detection system for high- curve based on the obtained results. In order to solve performance liquid chromatography (HPLC)', this this problem, we tried to employ an instrument of method has been widely used for the sensitive determi- automatic fluid injection (batch method) in order to nation of a number of compounds, such as catechol- optimize the reaction conditions for the PO-CL detection amines2-4, steroids and drugs.5 system in HPLC. Although several investigators have studied the In the present work, we first investigated the effects of mechanism of the PO-CL reaction, the exact mechanism the pH and buffer concentration on the chemilumines- of this reaction has not yet been clarified because of its cence intensity of a fluorescent drug, dipyridamole (an complexity. Rauhut and his co-workers6 have reported anti-platelet aggregation drug), and a dansylated valine that the key intermediate formed from oxalate ester and (Dns-Val) by both the batch method and HPLC. hydrogen peroxide is 1,2-dioxetane-3,4-dione. On the Comparing these results, we found that the batch method other hand, kinetic studies by Catherall et al.' have is useful to optimize the reaction conditions for the PO- indicated the occurrence of other key intermediates CL detection system in HPLC. Finally, we developed a during the CL reaction. Givens et al.8 suggested that the highly sensitive HPLC-PO-CL detection system by hydroperoxy oxalate ester was the likely reactive optimizing the reaction conditions for the PO-CL intermediate based on an investigation with bis(2,6- detection system determined by the batch method. difluorophenyl)oxalate (DFPO) using 19F-NMR. It was reported that the PO-CL reaction was affected by several factors, such as the pH, water content, kinds of Experimental oxalates, salts, temperature and organic solvents9-14,and especially by a catalyst, such as imidazole. Although an Chemicals optimization of the reaction conditions for the PO-CL Bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl]- detection system in HPLC is most desirable for the oxalate (TDPO), hydrogen peroxide (30%), nitric acid sensitive detection of fluorescent compounds, it is (61%) and boric acid were purchased from Wako Pure complicated and troublesome for these reasons. Chemical Industries Ltd. (Osaka, Japan). Dns-L-Val Although Imai et a1.15predicted the detection ranges for and dipyridamole were from Sigma (St. Louis, MO, the HPLC-PO-CL detection system of fluorescent USA). Imidazole was from Merck (Darmstadt, compounds using a manual method, the CL reaction Germany). Tris(hydroxymethyl)aminomethane, 3-(N curves required many points to be measured as well as an morpholino)propanesulfonic acid (MOPS) and 2- [4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) were from Nacalai Tesque (Kyoto, Japan). 714 ANALYTICAL SCIENCES OCTOBER 1996, VOL. 12 Fig. 1 Experimental apparatus of the batch method. L, Fig. 2 Flow diagram for the HPLC-PO-CL detection system. luminescencer (Luminescencer AB2000 (ATTO, Tokyo)); E, eluent (buffer/CH3CN (60/40, v/v)); CL reagent, TS, test solution (500 nM Dns-Val or dipyridamole in chemiluminogenic reagent (TDPO and H2O2 in CH3CN); buffer/CH3CN); CL reagent, chemiluminogenic reagent P1, pump for the eluent; P2, pump for the CL reagent; I, (0.25 mM TDPO and 25 mM H2O2 in CH3CN (60/40, v/v)); injector; C, analytical column (TSK-gel ODS-80TM (4.6 PMT, photomultiplier; P, pump; DP, data processor. mm i.d.X 150 mm)); CO, column oven (40°C); M, mixing device; D, chemiluminescence detector; DP, data processor. Acetonitrile (HPLC grade), sodium dihydrogenphos- phate 2-hydrate and disodium hydrogenphosphate 12- hydrate were from Kanto Chemical (Tokyo, Japan). Water was purified by a Milli-Q reagent system (Millipore, Bedford, MA, USA). Preparation of buffers The pHs of imidazole and Tris buffer were adjusted with HNO3, which did not quench the PO-CL reaction.16 The pHs of borate, MOPS and HEPES buffer were adjusted with NaOH. The phosphate buffers were obtained by mixing NaH2PO4 and Na2HPO4 buffer. Fig. 3 Chemiluminescence profile obtained by mixing the Batch method test solution and the CL reagent solution in batch method. A scheme of the experimental apparatus of the Test solution: 500 nM Dns-Val in 25 mM imidazole buffer batch method is shown in Fig. 1. The chemilumines- (pH 6.0)/CH3CN (60/40, v/v); CL reagent: 0.25 mM TDPO cence was measured by a Luminescencer AB2000 and 25 mM H2O2 in CH3CN. (ATTO, Tokyo, Japan). The obtained data were processed by a Macintosh LC 630 (Apple Computer, Inc., CA, USA). The peroxyoxalate chemilumines- cence was produced by mixing the test solution (500 nM output was recorded by a Chromatocorder 12 (SIC, dipyridamole or Dns-Val in buffer/CH3CN (6/4, v/v)) Tokyo, Japan) (DP). and the chemiluminogenic reagent (CL reagent) (TDPO and H2O2 in acetonitrile) on a poly(tetrafluoroethylene) Preparation of standard solutions well. The test solution had been previously put into the Stock standard solutions of 10 nM dipyridamole and well; the CL reagent was pumped later. 100 nM Dns-Val were respectively prepared by dis- solving a few mg of dipyridamole and Dns-Val in water/ High-performance liquid chromatography acetonitrile (6/4, v/v) and successively diluting in A schematic flow diagram of HPLC is shown in Fig. 2. the same solution. A 10 µl (100 fmol and 1 pmol, The HPLC system consisted of a PU-880 pump (JASCO, respectively) aliquot of the mixture was subjected to Tokyo, Japan) for the eluent (P1), a PU-980 pump HPLC. (JASCO) for the CL reagent (P2), a 20 µl Rheodyne 7125 injector (Cotani, CA, USA) (I), a TSKgel ODS-80TM (4.6 mm i.d.X150 mm, 5 µm) (Tosoh Co., Tokyo, Japan) Results and Discussion (C) and a 825-CL Intelligent CL Detector (JASCO) (D), the cell of which comprised a wound poly(tetrafluoroeth- Effectof thepHand buffer concentration on the chemilumines- ylene)tube. The analytical column and mixing device cence intensity in the batch method (M) were set in a 860-CO column oven (JASCO) (CO) The effects of two parameters, pH and the buffer maintained at 40° C. The eluent was buffer/ acetonitrile concentration, on the chemiluminescence intensity of (60/40, v/v) at a flow rate of 1.0 ml/min. The CL dipyridamole and Dns-Val were first examined by the reagent was TDPO and H2O2in acetonitrile. The signal batch method. In this study, the pH was varied from 4.0 ANALYTICAL SCIENCES OCTOBER 1996, VOL. 12 715 Fig. 4 Effect of pH and imidazole concentration on the signal (S) values (A) and the signal/noise (S/N) values (B) by the batch method and the peak area (C) and the peak area/noise (D) by HPLC. [Batch Method] Test solution: 500 nM Dns-Val in imidazole buffer/CH3CN (60/40, v/v); CL reagent: 0.25 mM TDPO and 25 mM H2O2 in CH3CN. [HPLC] Sample: Dns-Val (1 pmol); eluent: imidazole buffer/CH3CN (60/40, v/v); CL reagent: 0.25 mM TDPO and 25 mM H2O2 in CH3CN; flow rate: eluent (1.0 ml/min), CL reagent (1.3 ml/min). Table 1 Conditions which gave the maximal S and S/ N values in various buffers by the batch method to 8.0, and the concentration of buffer was varied from (0.25 mM TDPO and 25 mM H202 in acetonitrile, 5 mM to 100 mM. The buffers used were imidazole, 250 µl), a typical chemiluminescence profile was obtained borate, Tris, MOPS, HEPES and phosphate. Using the (Fig. 3). The N value means an integral value of blank test solution (500 nM dipyridamole or Dns-Val in buffer/ emission for 3 s, from 1 to 4 s after mixing the test acetonitrile (6/4, v/v), 192 µl) and the CL reagent solution and the CL reagent. An integral value of the 716 ANALYTICAL.SCIENCES OCTOBER 1996, VOL. 12 chemiluminescence of Dns-Val for the same 3 s minus the We then investigated the concentrations of oxalate and N value is the S value. H2O2, the ratio between the test solution and the CL Figures 4A) and 4B) showed the effect of the pH and reagent and the cell volume by the batch method when a imidazole concentration on the S value and the S/N phosphate buffer was used for the test solution. So far, value of Dns-Val by the batch method. A slight the CL reagent conditions (0.25 mM TDPO and 25 mM difference was observed in the two figures.