Research article Received: 19 October 2013,
Revised: 17 March 2014,
Accepted: 19 March 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2678
Determination of vitamin B6 using an optimized novel TCPO–indolizine–H2O2 chemiluminescence system M. J. Chaichi,* M. Ehsani, S. Asghari and V. Behboodi ABSTRACT: Indolizine derivatives are of great interest as fluorescent emitters for peroxyoxalate chemiluminescence. The reaction of peroxyoxalates such as bis-(2,4,6-trichlorophenyl) oxalate (TCPO) with H2O2 can transfer energy to fluorescer via the formation of dioxetanedione intermediate. Four indolizine derivatives were used as a novel fluorescer in the chemiluminescence (CL) systems in this study. The relationship between CL intensity and the concentration of fluorescer, peroxyoxalate, sodium salicylate and hydrogen peroxide was investigated. Optimum conditions were obtained for four fluorescers and it was found that the indolizine can be used as an efficient green fluorescence emitter. Vitamin B6 induces a sharp decrease in the CL intensity of the TCPO–hydrogen peroxide–sodium salicylate system. A simple, rapid and sensitive CL method for the determination of vitamin B6 has been developed. The results showed a linear relationship between vitamin B6 concentration and peroxyoxalate CL intensity in the range 7.0 × 108–1.0 × 104. A detection limit of 2.3 × 108 M and relative standard deviation (RSD) of < 4.5% were obtained. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: peroxyoxalate; hydrogen peroxide; chemiluminescence; indolizine derivatives; vitamin B6
Introduction Organic compounds containing two condensed rings (5- and 6-membered) and a bridging nitrogen atom are known as indolizines. This system is isoelectronic with indole and represents a group of heterocyclic compounds structurally related to purines. Therefore, indolizines can be considered as a 10-π electron system (1,2). Indolizine derivatives have been used as a key scaffold in the pharmaceutical industry due to the broad spectrum of biological activities associated with this structure. In addition, indolizine derivatives have been actively investigated as anti-inflammatory (3), antiviral (4), analgesic (5) and antitumor (6) agents. All these features make indolizines an important synthetic target in developing new pharmaceuticals for the treatment of cancer (6), cardiovascular disease (7) and HIV infection (8). In addition, indolizine-bearing polycyclic compounds have been found to have long wavelength absorption and strong fluorescence in the visible region (9). Considering these important properties and the steadily increasing importance of fluorescers in biolabeling and environmental trace analysis, the synthesis of these types of compounds is of great interest to chemists for the development of new drugs, novel classes of fluorescent dyes or chemosensors. Chemiluminescence (CL) detection has been successfully applied in analytical chemistry because it has significant advantages such as: relatively simple instrumentation, very low detection limits and a wide dynamic range (10,11). The peroxyoxalate (PO)–CL system is one of the most regularly used analytical CL schemes (12,13). PO–CL is based on the reaction of H2O2 with peroxyoxalate derivatives such as bis-(2,4,6trichlorophenyl) oxalate (TCPO), which results an unstable intermediate (14). The excited intermediate transfers its energy to an efficient fluorescer (15–19) through a chemically initiated electron-exchange luminescence (CIEEL) mechanism (20).
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Indolizine derivatives are of great interest as fluorescent emitters for peroxyoxalate CL. This reaction has been successfully used as a highly sensitive detection technique in several procedures developed for the low-level determination of various analytes (21,22), proteins (23), hydrogen peroxide (24,25), fluorescer-labeled amino acids (26) and prostate-specific antigen (27), as well as different quencher species (28,29). Four indolizine derivatives have been found to be intense and useful fluoropher compounds containing aromatic functional group with low-energy п → п* transition level (green light emission). Vitamin B6 is a water-soluble vitamin and includes a group of 3-hydroxy-2-methyl-pyridine derivatives that can exist in the C4′ position as an alcohol (pyridoxine) (PN), an aldehyde (pyridoxal) (PL) and an amine (pyridoxamine) (PM). Vitamin B6 is involved in different metabolic processes such as amino acid, glucose and lipid metabolism, neurotransmitter synthesis, histamine synthesis, hemoglobin synthesis and function, and gene expression (30–34). The available data suggest that PN predominates in most plant foods (35). Pyridoxine deficiency may lead to sideroblasticanemia, dermatitis, cheilosis and neurological symptoms such as peripheral neuritis and convulsions. In recent years, various analytical methods have been proposed for the determination of vitamin B6, involving UV/vis spectrophotometry (35,36), spectrofluorimetry (37), high-performance liquid chromatography (HPLC) with spectrophotometric detection
* Correspondence to: M. J. Chaichi, Department of Chemistry, University of Mazandaran, Postcaode 4741695447, Babolsar, Iran. Tel./Fax: +98-1125342350. E-mail: [email protected] Department of Chemistry, University of Mazandaran, Babolsar, Iran
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M. J. Chaichi et al. (38–41), voltammetry (42), turbidimetry (43) and flow injection with electrochemical (44) or optical detection (45,46). Most of these methods have several disadvantages such as poor selectivity, the requirement for tedious sample pretreatment (purification or pre-concentration steps), use of toxic reagents, or even complex chemometric treatment of the analytical results. In this work, we studied and compared influence of four indolizine derivatives of: (a) dimethyl 1-acetyl-7(dimethylamino)indolizine-2,3-dicarboxylate; (b) diethyl 1-acetyl-7-(dimethylamino)indolizine-2,3-dicarboxylate; (c) dimethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate and (d) diethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate (Fig. 1) as efficient fluorescers on a TCPO–H2O2 CL system in the presence of sodium salicylate as a base catalyst. Under optimal conditions, the best fluorescer was selected and we report a simple and fast CL method for the determination of pyridoxine in pharmaceutical products and plant foods using its quenching effect on TCPO–indolizine–H2O2 CL system intensity.
Experimental Reagents and solutions TCPO was prepared from the reaction of 2,4,6-trichlorophenol with oxalylchloride in the presence of triethylamine, as described elsewhere (47). Hydrogen peroxide (Merck; Perhydrol Suprapur, 30% in water) was assayed using permanganate potassium titration (48) and diluted in methanol. Vitamin B6 (pyridoxine hydrochloride) was purchased from Merck. Stock solutions of sodium salicylate (0.005 M in methanol), TCPO (0.005 M in ethyl acetate) and pyridoxine hydrochloride (0.001 M in water) were prepared shortly before use. The fluorescers were synthesized and purified in organic laboratories. A stock solution of fluorescer (0.005 M) was prepared in a calibrated 50 mL flask by dissolving an appropriate amount of fluorescer in ethyl acetate and protecting it from light. Phosphate buffer solutions (0.1 M) of varying pH were prepared by dissolving an appropriate amount of K2HPO4 in water and adjusting the pH with 0.1 M HCl or NaOH solutions. All other chemicals were of analytical grade. The preparation of the indolizine ring was by reaction of 4dimethylaminopyridine with dialkylacetylenedicarboxylate in the
Figure 1. Molecular structure of (a) dimethyl 1-acetyl-7-(dimethylamino) indolizine-2,3-dicarboxylate, (b) diethyl 1-acetyl-7-(dimethylamino)indolizine-2,3dicarboxylate, (c) dimethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate and (d) diethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate.
presence of α,α-dichloroacetone in dichloromethane as solvent at room temperature. The reaction yield for the production of the indolizine derivatives was ~ 80-90% (58). The route for the preparation of indolizine derivatives is shown in Scheme 1. Apparatus A 3030 Jenway pH meter (Leeds, UK) was used for to measure pH. Absorption spectra were recorded using a UV/vis spectrophotometer (Cecil, CE5501). Steady-state fluorescence spectra were recorded on a Perkin–Elmer, LS-3B spectrofluorimeter instrument and using a 3 cm quartz cuvette. Excitation monochromators were set at 400 nm for all four indolizine derivatives. CL light intensity–time curves were obtained on Berthold detection systems (Sirius, Germany). Procedures Solution I was made by mixing 1 mL of TCPO (1.0 × 103 M) and 0.05 mL of fluorescer (various concentrations in ethyl acetate). Solution II contained 2 mL of hydrogen peroxide (7.5 × 102 M) and 1 mL of sodium salicylate (8.0 × 104 M) in methanol. Solution I was transferred into an instrument quartz cuvette via polypropene syringes. Then, 100 μL of solution II was injected into the quartz cuvette and the CL spectrum was recorded after mixing the reagents in the cell. Vitamin B6 was found to inhibit TCPO–H2O2–fluorescer (indolizine)–sodium salicylate CL emission on the basis of the Stern–Volmer equation (equation (1)). Therefore, the concentration of vitamin B6 was determined using the following equation due to linear quenching (49). I0 =I ¼ 1 þ K sv ½Q
(1)
Where, I0 and I are the CL intensity in the absence and presence of inhibitors, respectively, Ksv is Stern–Volmer constant and [Q] is the concentration of the inhibitors or quenchers. Procedure for the extraction of vitamin B6 from real samples Acid hydrolysis. Extraction of vitamin B6 (pyridoxine) was carried out by autoclaving 2 g of the samples in 50 mL HCl (0.44 and 0.055 M) at 121ºC for 30 min for tablet and banana, respectively (50). The extract solution was cooled in cold water at room temperature and the suspension was centrifuged at 3000 r.p.m (g-value 850) for 5 min. The supernatant was recovered and adjusted to pH 7 with 2 M NaOH solution and diluted to 250 mL with methanol/water (1 : 1 v/v). Aliquots were filtered through 0.45 μm nylon Millipore chromatographic filters and injected into the RP-HPLC system as a standard analytical method for qualification analysis. Chromatograms of standard pyridoxine and extracts of samples were presented in Fig. 2, and results show that extraction of pyridoxine from different real samples
Scheme 1. Mechanism for the preparation of indolizine derivatives.
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Determination of vitamin B6 using TCPO–indolizine–H2O2 CL
Figure 2. Chromatograms of (a) standard solution, (b) tablet sample and (c) banana sample after extraction using acid hydrolysis (submitted to acid hydrolysis). Conditions were as follows: mobile phase. methanol/water (75 : 24 v/v); flow rate, 1 mL/min; column, C18 (250 mm × 4.6 mm, 10 μm); injection volume, 10 μL; λ, 275 nm; room temperature.
was carried out by acid hydrolysis, successfully. The extracted samples were analyzed using CL system.
Results and discussion PO–CL is well known as one of the most efficient non-biological light-producing systems. The possible mechanisms of the PO–CL reaction, taking into consideration what is reported in the
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literature. The intensity, duration and color of emission of the PO–CL systems are of great importance (51,52). In the first step, an aryl oxalate ester such as TCPO reacts with H2O2 to produce a key chemical intermediate of 1,2dioxetandione (C2O4) as an excitation source. In the second step, the excited cyclic C2O4 intermediate transfers its energy to fluorescer (indolizine derivatives were used here). The final step is the emission of light energy on the return of the excited
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M. J. Chaichi et al. fluorescer molecule to the ground state (20). The florescence spectra of four indolizine derivatives are shown in Fig. 3. By comparing the fluorescence spectra of the four compounds (a, b, c and d) it can be deduced that spectrum a is more intense than the other compounds (hyperchromic effect). As expected, the intensity of the PO–CL emission was found to be affected by the initial concentration of the reactants (8–14). Thus, the influence of concentrations of fluorescer, TCPO, H2O2, catalyst and pH on the PO–CL system for four fluorescers were studied.
Effect of pH The pH of the buffer solution is an important parameter that affects the CL intensity and hence has to be optimized if a very sensitive assay is required. The effect of pH on CL intensity was investigated using 0.1 M phosphate buffer in the pH range 4.0–9.0 and constant concentrations of H2O2 (7.5 × 102 M), TCPO (1.0 × 103 M), sodium salicylate (8.0 × 104 M) and fluorescer (1.0 × 104 M). The optimum pH was ~ 7. It can also be concluded that the CL intensity decreases slightly at pH values > 8. Such an observation highlights the fact that the neutral form of fluorescer can change in acidic and basic conditions, resulting in the production of a undesirable reactive intermediate and electron transfer is not possible. Further study showed that the best precision was achieved at pH 7, and consequently all measurements were performed at this pH.
Figure 4. Effect of TCPO concentration on the CL intensity of TCPO–H2O2–sodium salicylate–dimethyl 1-acetyl-7-(dimethylamino)indolizine-2,3-dicarboxylate, (b) salicylate–diethyl 1-acetyl-7-(dimethylamino)indolizineTCPO–H2O2–sodium 2,3-dicarboxylate, (c) TCPO–H2O2–sodium salicylate–dimethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate and (d) TCPO–H2O2–sodium salicylate–diethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate systems.
However, at concentrations of TCPO > 1.0 × 10-3 M, the increase in the signal is no longer proportional to the increase in the TCPO concentration, and a small decrease in the signalto-noise ratio is achieved. Therefore, TCPO concentrations above the stoichiometric ratio have no significant effect on the CL signal. A concentration of 1.0 × 10-3 M was used through this study.
Effect of TCPO concentration
Effect of H2O2 concentration
The influence of varying TCPO concentrations in the presence of excess H2O2 on the CL signal for four fluorescers was investigated over the concentration range 2.0 × 104 to 3.0 × 103 M, keeping constant the concentration of H2O2 (1.0 × 10-1 M), fluorescer (8.0 × 10–5 M) and sodium salicylate (6.0 × 10–4 M) and at pH 7. As seen from Fig. 4, the peak intensity increases rapidly after mixing and reaches a maximum in a few seconds. However, decay of the light intensity from the maximum occurs over much longer periods. As shown in Fig. 3, there is a linear correlation between the CL intensity and the TCPO concentration for all fluorescers. The basis for this linear correlation has been discussed previously (53).
The influence of H2O2 concentration on the PO–CL of indolizine was studied at constant concentrations of TCPO (1.0 × 10-3 M), fluorescer (8.0 × 10–5 M) and sodium salicylate (6.0 × 10-4 M) and at pH 7. It was found that there is a direct linear relationship between the concentration of hydrogen peroxide and the CL intensity of the system over a concentration range of 1.0 × 102 to 7.5 × 102 M (Fig. 5). This observation highlights that CL reaction progression improves proportional to the increasing H2O2 concentration. At higher H2O2 concentrations, the photomultiplier
Figure 3. The fluorescence emission spectra of (a) dimethyl 1-acetyl-7(dimethylamino)indolizine-2,3-dicarboxylate, (b) diethyl 1-acetyl-7-(dimethylamino)indolizine-2,3-dicarboxylate, (c) dimethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate and (d) diethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate at the same –4 concentration of 5.0 × 10 M in ethyl acetate with λex = 400 nm.
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Figure 5. Effect of H2O2 concentration on the CL intensity of TCPO–H2O2–sodium salicylate–dimethyl 1-acetyl-7-(dimethylamino)indolizine-2,3-dicarboxylate, (b) TCPO– H2O2–sodium salicylate–diethyl 1-acetyl-7-(dimethylamino)indolizine-2,3-dicarboxylate, (c) TCPO–H2O2–sodium salicylate–dimethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate and (d) TCPO–H2O2–sodium salicylate–diethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate systems.
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Determination of vitamin B6 using TCPO–indolizine–H2O2 CL tube detector was saturated. It is noteworthy that further increases in H2O2 concentration were found to have no measurable effect on the PO–CL intensity. Effect of sodium salicylate concentration The PO–CL intensity of a 6.0 × 10–5 M solution of fluorescer, under optimal constant concentrations of TCPO and H2O2 was found to increase significantly in the presence of sodium salicylate, which is clearly indicative of the catalytic effect of the salt on the PO–CL system studied (53). In order to investigate the optimal concentration of sodium salicylate, the CL response of the H2O2–TCPO-fluorescer system was measured against the varying concentrations of the base and the resulting plot is shown in Fig. 6. The PO–CL intensity increased sharply with the increasing concentration of sodium salicylate until values of 8.0 × 10–4 M (a, b and c) and 1.0 × 10–3 (c) were reached; the observed intensity enhancement was indicative of the catalytic effect of the base. However, further addition of sodium salicylate revealed a gradual decrease in the CL intensity. This is most probably due to the quenching effect of the base at higher concentration, which begins to decompose the reactive intermediate, dioxetanedione, and hence reduces the CL signal. It should be noted that maximum rise and fall rate constant is also observed at the optimum sodium salicylate concentration. Effect of fluorescer concentration The influence of fluorescer concentration on the PO–CL system was studied at constant concentrations of H2O2 (7.5 × 10-2 M), TCPO (1.0 × 103 M) and sodium salicylate (8.0 × 104 M) was studied and the results are shown in Fig. 7. As it is seen from Fig. 7, the peak intensity increases with increasing fluorescer concentrations until 1.0 × 104 M, however, the peak intensity of H2O2–TCPO–sodium salicylate–fluorescer system decreases at higher concentrations. This is most probably due to the selfabsorption effect and the formation of a dimer structure at high concentrations, which causes decreases or inhibits electron transfer of the intermediate, dioxetanedione, and hence reduces the CL signal.
Figure 7. Effect of fluorescer concentration on the CL intensity of (a) TCPO–H2O2– sodium salicylate–dimethyl 1-acetyl-7-(dimethylamino)indolizine-2,3-dicarboxylate, (b) TCPO–H2O2–sodium salicylate–diethyl 1-acetyl-7-(dimethylamino) indolizine-2,3-dicarboxylate, (c) TCPO–H2O2–sodium salicylate–dimethyl 1-acetyl7-aminoindolizine-2,3-dicarboxylate and (d) TCPO–H2O2–sodium salicylate–diethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate systems.
Possible CL mechanism The mechanism of the TCPO–H2O2 CL reaction has been studied previously. It is suggested that TCPO can be oxidized by H2O2 to generate an energy-rich intermediate, 1,2-dioxetanedione. For a fluorescent molecule, 1,2- dioxetanedione could interact with the fluorescent molecule to yield a charge-transfer complex, which decomposed on electron transfer from the intermediate back to the fluorescent molecule, generating its excited state (54). The results of all optimization experiments for four indolizine derivatives showed that the CL intensities for fluorescer a are higher than for the other compounds in the TCPO–H2O2–CL system, under the same conditions. This observation may be attributed to the presence of dimethyl amine (–NMe2) as a stronger electron-donor substituent in comparison with (–NH2) on the benzene ring in compounds c and d. Also, displacement of the ethyl group by a methyl group on the five-member ring in compound b led to increasing steric hindrance in the indolizine structure and the ethyl group is a better electron donor than methyl group (in –COOR group) in that it can decrease the π conjugation in compound b. Decreasing the π conjugation in compound b can lead to a decrease in the resonance forms and lower fluorescence intensity. With increasing π conjugation, the energy difference between highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) decreases, suggesting the feasibility of absorption and fluorescence of compound a. The applicability of the optimized TCPO–H2O2–fluorescer a (dimethyl 1-acetyl-7(dimethylamino)indolizine-2,3-dicarboxylate) CL system was tested for the determination of vitamin B6 in pharmaceutical and plant (herbal) products. The most likely mechanism for the quenching of TCPO–H2O2–indolizine CL by vitamin B6, as an easily oxidizable compound (54), may be an electron transfer quenching pathway (55,56). The crucial step in luminescence quenching is assumed to be the reaction of the quencher Q, with the highly energetic intermediate C2O4 to give non-chemiluminescent products, in competition with the reaction of fluorescer as follows (57):
Figure 6. Effect of sodium salicylate concentration on the CL intensity of TCPO– salicylate–dimethyl 1-acetyl-7-(dimethylamino)indolizine-2,3H2O2–sodium dicarboxylate, (b) TCPO–H2O2–sodium salicylate–diethyl 1-acetyl-7-(dimethylamino)indolizine-2,3-dicarboxylate, (c) TCPO–H2O2–sodium salicylate–dimethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate and (d) TCPO–H2O2–sodium salicylate–diethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate systems.
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M. J. Chaichi et al. According to this mechanism, after the reduction of indolizine, the resulting indolizine radical anion may react with C2O4 to produce C2O4– (anion) and free indolizine. The net result would be a diminished amount of the reactive intermediate in solution and, consequently, reduced CL intensity of the TCPO– H2O2–indolizine system. The quencher, vitamin B6, may also undergo a reduction reaction with the excited state indolizine to further decrease the CL of the system. Also, in the TCPO–H2O2 CL reaction, the formation of hydroperoxide was catalyzed under alkaline conditions. Addition of phenolic compounds such as vitamin B6 (containing a -OH group) to the TCPO– H2O2–fluorescer (indolizine) CL system increases its acidic properties, leading to decreased production of the hydroperoxide anion, and followed by its nucleophilic attack of TCPO. Therefore, the results show that vitamin B6 acts as a quencher, and decreases light production of the TCPO–H2O2–fluorescer (dimethyl 1-acetyl-7-(dimethylamino)indolizine-2,3-dicarboxylate) in the CL reaction. A linear relationship between vitamin B6 concentration and peroxyoxalate CL intensity according the Stern–Volmer plot is observed (Fig. 8).
Analytical performance of the CL system in vitamin B6 measurements Under optimum conditions, the ratios of I0/I were linear with the concentrations of a series of standard solutions of vitamin B6 7.0 × 108 to 1.0 × 104 M. Three replicate injections were performed for each standard solution. The Stern–Volmer plots for vitamin B6 are shown in Fig. 8. The regression equations were I0/I = 37049.0 C + 1.7865 with correlation coefficients (r) of 0.998 for vitamin B6 and the limit of detection (3σ) was 2.3 × 108 M. The reproducibility of the peak height was determined by measuring seven replicates of a 5 × 106 M pyridoxine solution; the relative standard deviation (RSD) was < 3.8%. The results indicate that the PO–H2O2–indolizine CL system is a rapid, simple and sensitive method and is suitable for highthroughput and real-time vitamin B6 analysis. Real sample analysis The proposed PO–CL system was successfully applied to the determination of vitamin B6 in two different samples of pharmaceutical product (tablet) and plant product (banana). To evaluate the accuracy and reliability of the method, known amounts of standard vitamin B6 solution at two concentrations (5 and 10 mg/L) were added to the real sample and analyzed in triplicate (n = 3) under optimal conditions according to the proposed method. The recovery of each measurement was calculated by comparing results obtained before and after addition of the standard vitamin B6 solution. The results are been listed in Table 1. In order to examine its applicability, the optimized TCPO–H2O2–sodium salicylate–dimethyl 1-acetyl-7-(dimethylamino)indolizine-2,3-dicarboxylate CL system, was applied for determination of vitamin B6 in pharmaceutical and plant (herbal) products.
Interference study Figure 8. Chemiluminescence emission intensity as a function of time for the TCPO–H2O2–dimethyl 1-acetyl-7-(dimethylamino)indolizine-2,3-dicarboxylate–sodium salicylate–vitamin B6 CL system with a constant concentration of TCPO -3 -4 -2 (1.0 × 10 M), fluorescer (1 × 10 M), H2O2 (7.5 × 10 M), sodium salicylate -4 -4 (8.0 × 10 M) and varying concentrations of vitamin B6 (a) 0.0, (b) 1.0 × 10 , (c) -5 -6 -7 -8 6.0 × 10 , (d) 1.0 × 10 , (e) 1.0 × 10 and (f) 7.0 × 10 M at pH 7. (Inset) Stern–Volmer plot Io/I vs. [vitamin B6].
To assess the possibility of applying the proposed method, the effect of foreign species was examined. Under optimal experimental conditions, interference by selected metal ions and organic compounds was evaluated. Most of the candidate compounds had no significant influence on the determination of vitamin B6 concentrations. Tolerable molar concentration ratios of foreign species to vitamin B6 were: 100 for glucose, folic acid,
Table 1. Determination of vitamin B6 in real samples Sample Tablet Banana
Claimed (mg)
Found (mg)
5.0 0.5
4.85 0.517
Spiked concentration (mg/L) Tablet 1 2 3 Banana 1 2 3
Found (mean ± SD) (mg/L)
% Recovery 97.0 103.4 % Recovery
% RSD (n = 3) 2.8 3.4 % RSD (n = 3)
0 5 10
1.06 ± 0.06 5.57 ± 0.15 12.03 ± 0.32
– 90.4 105.3
– 2.4 4.5
0 5 10
2.08 ± 0.04 6.64 ± 0.13 11.07 ± 0.08
– 91.2 88.0
– 3.5 4.2
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Determination of vitamin B6 using TCPO–indolizine–H2O2 CL fructose, sucrose, lactose, starch, bilirubin, vitamin C, Zn2+ and SO24 ; 50 for maltose, vitamin B2, vitamin B1, hydroquinone as a dihydroxyphenol, urea, Ca2+ and NO-3; 20 for Fe2+, Cu2+, Mg2+ and Cl-; and 10 for uric acid. As can be seen, the selectivity of the method is high.
Conclusion This study describes a novel CL system of H2O2–bis-(2,4,6trichlorophenyl) oxalate (TCPO) using indolizine derivatives as the fluorescer. In this system, the green fluorescence and CL signal of compound a is the highest. The influence of the concentrations of the components involved in the PO–CL system were also studied. The results showed that concentrations of H2O2, TCPO and indolizine derivatives have a direct relationship on the light intensity. The proposed CL system has good linearity, high sensitivity and precision, and the method has been successfully applied to the determination of vitamin B6 in pharmaceutical and plant products.
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17. Bowie AR, Sanders MG, Worsefold PJ. Analytical application of liquid phase chemiluminescence reactions – a review. Biolumin Chemilumin 1996;11:61–90. 18. Shamsipur M, Chaichi MJ. Quenching effect of dl(+/-)αmethylbenzylamine on peroxyoxalate chemiluminescence of 7-amino-4-trifluoromethylcumarin. J Photochem Photobiol A 2003;155:69–72. 19. Shamsipur M, Chaichi MJ. A study of chemiluminescence from reaction of bis(2,4,6-trichlorophenyl)oxalate, hydrogen peroxide and an optical brightener 5-(3-anilino-5-chloroanilino)-2-{(E)-2-[4-(3-anilino5-chloroanilino)-2-sulfophenyl]-1-ethenyl}-1-benzenesulfonic acid. Dyes Pigment 2007;72:113–8. 20. Schuster GB. Chemiluminescence of organic peroxides. Conversion of ground-state reactants to excited-state products by the chemically initiated electron-exchange luminescence mechanism. Acc Chem Res 1979;12:366–73. 21. Nishitan A, Tsukamoto Y, Kanda S, Imai K. Determination of the fluorescent drugs dipyridamole and benzydamine in rat plasma by liquid chromatography with peroxyoxalate chemiluminescence detection. Anal Chim Acta 1991;251:247–53. 22. Yan B, Lewis SW, Worsfold PJ, Lancaster JS, Gachanka A. Procedures for the enhancement of selectivity in liquid phase chemiluminescence detection. Anal Chim Acta 1991;250:145–55. 23. Alba FJ, Daban JR. Nonenzymatic chemiluminescent detection and quantitation of total protein on western and slot blots allowing subsequent immunodetection and sequencing. Electrophoresis 1997;18:1960–6. 24. Nakashima K, Kuroda N, Kawaguchi S, Wada M, Akiyama S. Peroxyoxalate chemiluminescent assay for oxidase activities based on detecting enzymatically formed hydrogen peroxide. J Biolumin Chemilumin 1995;10:185–91. 25. Kojo S, Tokumaru S, Kishida E, Tsukamoto I, Peroxyoxalate chemiluminescent assay in aqueous solution with gamma-cyclodextrin. Clin Chem 1992;38:788. 26. Quab U, Klochow D. Determination of Fe(II) and H2O2 in atmospheric liquid water by peroxyoxalate chemiluminescence. Int J Environ Anal Chem 1995;60:361–75. 27. Albrecht S, Brandl H, Steinke M, Freidt T. Chemiluminescent enzyme immunoassay of PSA based on indoxyl phosphate substrate. Clin Chem 1994;40:1970–1. 28. Gooijer C, Volthorst M. Low-level interferences in peroxyoxalate chemiluminescence. Biomed Chromatogr 1990;4:92–5. 29. Nakamura MM, Saraiva SA, Coichev N. A critical review of the transition metal ions influence on peroxyoxalate chemiluminescence. Anal Lett 2000;33:391–404. 30. Royal Pharmaceutical Society of Great Britain. The complete drug reference. 33rd ed. London: Royal Pharmaceutical Society of Great Britain, 2002:1384. 31. Van Niekerk PJ. Determination of vitamins. In: Macrae R, editor. HPLC in food analysis. 2nd ed. Reading, UK: Academic Press, 1988. 32. Coultate TP. Food: the chemistry of its components. 2nd ed. London: Royal Society of Chemistry, 1989. 33. Peter CH, Hollman JH, Slangen PT, Wahstaffe PJ, Southgate AT, Finglas PM. Intercomparison of methods for the determination of vitamins in foods. Part 2. Water-soluble vitamins. Analyst 1993;118:481–8. 34. Viñas P, Balsalobre N, López-Erroz C, Hernández-Córdoba M. Determination of vitamin B6 compounds in foods using liquid chromatography with post-column derivatization fluorescence detection. Chromatographia 2004;59:381–6. 35. Lopéz-de-Alba PL, Martinez LL, Cerdá V, Hernández I. Simultaneous determination and classification of riboflavin, thiamine, nicotinamide and pyridoxine in pharmaceutical formulations, by UV–visible spectrophotometry and multivariate analysis. J Braz Chem Soc 2006;17:715–22. 36. Ozdemir D, Dinc E. Determination of thiamine HCl and pyridoxine HCl in pharmaceutical preparations using UV–visible spectrophotometry and genetic algorithm based multivariate calibration methods. Chem Pharm Bull 2004;52:810–7. 37. Feng F, Wang K, Cheng Z, Cheng Q, Ling J, Huang S. Flow injection renewable drops spectrofluorimetry for sequential determinations of vitamins B1, B2 and B6. Anal Chim Acta 2004;527:187–93. 38. El-Gindy A, El-Yazib F, Mostafa A, Maher MM. HPLC and chemometric methods for the simultaneous determination of cyproheptadine hydrochloride, multivitamins, and sorbic acid. J Pharm Biomed Anal 2004;35:703–13.
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M. J. Chaichi et al. 39. Li K. Simultaneous determination of nicotinamide, pyridoxine hydrochloride, thiamine mononitrate and riboflavin in multivitamin with minerals tablets by reversed-phase ion-pair high performance liquid chromatography. Biomed Chromatogr 2002;16:504–7. 40. Chen Z, Chen B, Yao S. High-performance liquid chromatography/ electrospray ionization-massspectrometry for simultaneous determination of taurine and 10 water-soluble vitamins in multivitamin tablets. Anal Chim Acta 2006;569:169–75. 41. Marzall ML, Lebiedzìnska A, Czarnowski W, Szefer P. High-performance liquid chromatography method for the simultaneous determination of thiamine hydrochloride, pyridoxine hydrochloride and cyanocobalamin in pharmaceutical formulations using coulometric electrochemical and ultraviolet detection. J Chromatogr A 2005;1094: 91–8. 42. Teixeira MFS, Segnini A, Moraes FC, Marcolino-Júnior LH, FatibelloFilho O, Cavalheiro ETG. Determination of vitamin B6 (pyridoxine) in pharmaceutical prepartaions by cyclic voltammetry at a copper (II) hexacyanoferrate(III) modified carbon paste electrode. J Braz Chem Soc 2003;14:316–21. 43. Costa-Neto CO, Pereira AV, Aniceto C, Fatibello-Filho O. Flow injection turbidimetric determination of thiamine in pharmaceutical formulations using silicotungstic acid as precipitant reagent. Talanta 1999;48:659–67. 44. Shairadova LG, Davletshina LN, Budnikov GK. Flow-injection determination of water-soluble vitamins B1, B2, and B6 from the electrocatalytic response of a graphite electrode modified with a ruthenium(III) hexacyanoruthenate(II) film. Anal Chem 2006;61:544–51. 45. Zhu H, He Q, Fang Q, Chen H. Elimination of ascorbic acid interference with the determination of thiamine in pharmaceutical preparation by flow injection on-line photochemical spectrofluorimetry. Anal Lett 2002;35:707–20. 46. Barrales PO, Vidal AD, C´ordova MLF, Dı́az AM. Simultaneous determination of thiamine and pyridoxine in pharmaceuticals by using a single flow-through biparameter sensor. J Pharm Biomed Anal 2001;25:619–30.
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47. Mohan AG, Turro NJ. A facile and effective chemiluminescence demonstration experiment. J Chem Educ 1974;51:528–9. 48. Skoog DA, Leary JJ. Principles of instrumental analysis. 4th ed. New York: Saunders College, 1992. 49. Castanho MARB, Prieto MJE. Fluorescence quenching data interpretation in biological systems. Biochim Biophys Acta 1998;1373:1–16. 50. Ollilainen V. HPLC analysis of vitamin B6 in foods. Agr Food Sci Finland 1999;8:515–9. 51. Campbell AK. Chemiluminescence: principle and application in biology and medicine. New York: Ellis Horwood, 1988:510. 52. Givens RS, Schowen RL. The peroxyoxalate chemiluminescence reaction. In: Birks JW, editor. Chemiluminescence and photochemical reaction detection in chromatography. New York: VCH, 1989:125. 53. Catherall CLR, Palmer TF, Cundall RB. Chemiluminescence from reactions of bis(pentachloro-phenyl)oxalate, hydrogen peroxide and fluorescent compounds. Role of the fluor and nature of chemielectronic process(es). J Chem Soc Faraday Trans 1984;80: 837–49. 54. Van Zoonen P, Bock H, Gooijer C, Velthorst NH, Frei RW. Quenched peroxyoxalate chemiluminescence detection in aqueous liquid chromatographic separations. Anal Chim Acta 1987;200:131–40. 55. Van Zoonen P, Kamminga DA, Gooijer C, Velthorst NH, Frei RW. Quenched peroxyoxalate chemiluminescence as a new detection principle in flow-injection analysis and liquid chromatography. Anal Chem 1986;58:1245–8. 56. Lakowicz JR. Principles of fluorescence spectroscopy. New York: Plenum Press, 1999. 57. Hosseini M, Dehghan Abkenara S, Chaichi MJ, Ganjali M, Norouzi P. A study of quenching and enhancing effects of some amino acids on peroxyoxalate chemiluminescence of rhodamine 6G. Spectrochim Acta A 2009;72:484–9. 58. Asghari S, Qandalee M. Facile one-pot synthesis of amino furans using trans-cinnamaldehyde in the presence of nucleophilic isocyanides. Acta Chim Slov 2007;54:638–641.
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Luminescence 2014
Revised: 17 March 2014,
Accepted: 19 March 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2678
Determination of vitamin B6 using an optimized novel TCPO–indolizine–H2O2 chemiluminescence system M. J. Chaichi,* M. Ehsani, S. Asghari and V. Behboodi ABSTRACT: Indolizine derivatives are of great interest as fluorescent emitters for peroxyoxalate chemiluminescence. The reaction of peroxyoxalates such as bis-(2,4,6-trichlorophenyl) oxalate (TCPO) with H2O2 can transfer energy to fluorescer via the formation of dioxetanedione intermediate. Four indolizine derivatives were used as a novel fluorescer in the chemiluminescence (CL) systems in this study. The relationship between CL intensity and the concentration of fluorescer, peroxyoxalate, sodium salicylate and hydrogen peroxide was investigated. Optimum conditions were obtained for four fluorescers and it was found that the indolizine can be used as an efficient green fluorescence emitter. Vitamin B6 induces a sharp decrease in the CL intensity of the TCPO–hydrogen peroxide–sodium salicylate system. A simple, rapid and sensitive CL method for the determination of vitamin B6 has been developed. The results showed a linear relationship between vitamin B6 concentration and peroxyoxalate CL intensity in the range 7.0 × 108–1.0 × 104. A detection limit of 2.3 × 108 M and relative standard deviation (RSD) of < 4.5% were obtained. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: peroxyoxalate; hydrogen peroxide; chemiluminescence; indolizine derivatives; vitamin B6
Introduction Organic compounds containing two condensed rings (5- and 6-membered) and a bridging nitrogen atom are known as indolizines. This system is isoelectronic with indole and represents a group of heterocyclic compounds structurally related to purines. Therefore, indolizines can be considered as a 10-π electron system (1,2). Indolizine derivatives have been used as a key scaffold in the pharmaceutical industry due to the broad spectrum of biological activities associated with this structure. In addition, indolizine derivatives have been actively investigated as anti-inflammatory (3), antiviral (4), analgesic (5) and antitumor (6) agents. All these features make indolizines an important synthetic target in developing new pharmaceuticals for the treatment of cancer (6), cardiovascular disease (7) and HIV infection (8). In addition, indolizine-bearing polycyclic compounds have been found to have long wavelength absorption and strong fluorescence in the visible region (9). Considering these important properties and the steadily increasing importance of fluorescers in biolabeling and environmental trace analysis, the synthesis of these types of compounds is of great interest to chemists for the development of new drugs, novel classes of fluorescent dyes or chemosensors. Chemiluminescence (CL) detection has been successfully applied in analytical chemistry because it has significant advantages such as: relatively simple instrumentation, very low detection limits and a wide dynamic range (10,11). The peroxyoxalate (PO)–CL system is one of the most regularly used analytical CL schemes (12,13). PO–CL is based on the reaction of H2O2 with peroxyoxalate derivatives such as bis-(2,4,6trichlorophenyl) oxalate (TCPO), which results an unstable intermediate (14). The excited intermediate transfers its energy to an efficient fluorescer (15–19) through a chemically initiated electron-exchange luminescence (CIEEL) mechanism (20).
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Indolizine derivatives are of great interest as fluorescent emitters for peroxyoxalate CL. This reaction has been successfully used as a highly sensitive detection technique in several procedures developed for the low-level determination of various analytes (21,22), proteins (23), hydrogen peroxide (24,25), fluorescer-labeled amino acids (26) and prostate-specific antigen (27), as well as different quencher species (28,29). Four indolizine derivatives have been found to be intense and useful fluoropher compounds containing aromatic functional group with low-energy п → п* transition level (green light emission). Vitamin B6 is a water-soluble vitamin and includes a group of 3-hydroxy-2-methyl-pyridine derivatives that can exist in the C4′ position as an alcohol (pyridoxine) (PN), an aldehyde (pyridoxal) (PL) and an amine (pyridoxamine) (PM). Vitamin B6 is involved in different metabolic processes such as amino acid, glucose and lipid metabolism, neurotransmitter synthesis, histamine synthesis, hemoglobin synthesis and function, and gene expression (30–34). The available data suggest that PN predominates in most plant foods (35). Pyridoxine deficiency may lead to sideroblasticanemia, dermatitis, cheilosis and neurological symptoms such as peripheral neuritis and convulsions. In recent years, various analytical methods have been proposed for the determination of vitamin B6, involving UV/vis spectrophotometry (35,36), spectrofluorimetry (37), high-performance liquid chromatography (HPLC) with spectrophotometric detection
* Correspondence to: M. J. Chaichi, Department of Chemistry, University of Mazandaran, Postcaode 4741695447, Babolsar, Iran. Tel./Fax: +98-1125342350. E-mail: [email protected] Department of Chemistry, University of Mazandaran, Babolsar, Iran
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M. J. Chaichi et al. (38–41), voltammetry (42), turbidimetry (43) and flow injection with electrochemical (44) or optical detection (45,46). Most of these methods have several disadvantages such as poor selectivity, the requirement for tedious sample pretreatment (purification or pre-concentration steps), use of toxic reagents, or even complex chemometric treatment of the analytical results. In this work, we studied and compared influence of four indolizine derivatives of: (a) dimethyl 1-acetyl-7(dimethylamino)indolizine-2,3-dicarboxylate; (b) diethyl 1-acetyl-7-(dimethylamino)indolizine-2,3-dicarboxylate; (c) dimethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate and (d) diethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate (Fig. 1) as efficient fluorescers on a TCPO–H2O2 CL system in the presence of sodium salicylate as a base catalyst. Under optimal conditions, the best fluorescer was selected and we report a simple and fast CL method for the determination of pyridoxine in pharmaceutical products and plant foods using its quenching effect on TCPO–indolizine–H2O2 CL system intensity.
Experimental Reagents and solutions TCPO was prepared from the reaction of 2,4,6-trichlorophenol with oxalylchloride in the presence of triethylamine, as described elsewhere (47). Hydrogen peroxide (Merck; Perhydrol Suprapur, 30% in water) was assayed using permanganate potassium titration (48) and diluted in methanol. Vitamin B6 (pyridoxine hydrochloride) was purchased from Merck. Stock solutions of sodium salicylate (0.005 M in methanol), TCPO (0.005 M in ethyl acetate) and pyridoxine hydrochloride (0.001 M in water) were prepared shortly before use. The fluorescers were synthesized and purified in organic laboratories. A stock solution of fluorescer (0.005 M) was prepared in a calibrated 50 mL flask by dissolving an appropriate amount of fluorescer in ethyl acetate and protecting it from light. Phosphate buffer solutions (0.1 M) of varying pH were prepared by dissolving an appropriate amount of K2HPO4 in water and adjusting the pH with 0.1 M HCl or NaOH solutions. All other chemicals were of analytical grade. The preparation of the indolizine ring was by reaction of 4dimethylaminopyridine with dialkylacetylenedicarboxylate in the
Figure 1. Molecular structure of (a) dimethyl 1-acetyl-7-(dimethylamino) indolizine-2,3-dicarboxylate, (b) diethyl 1-acetyl-7-(dimethylamino)indolizine-2,3dicarboxylate, (c) dimethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate and (d) diethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate.
presence of α,α-dichloroacetone in dichloromethane as solvent at room temperature. The reaction yield for the production of the indolizine derivatives was ~ 80-90% (58). The route for the preparation of indolizine derivatives is shown in Scheme 1. Apparatus A 3030 Jenway pH meter (Leeds, UK) was used for to measure pH. Absorption spectra were recorded using a UV/vis spectrophotometer (Cecil, CE5501). Steady-state fluorescence spectra were recorded on a Perkin–Elmer, LS-3B spectrofluorimeter instrument and using a 3 cm quartz cuvette. Excitation monochromators were set at 400 nm for all four indolizine derivatives. CL light intensity–time curves were obtained on Berthold detection systems (Sirius, Germany). Procedures Solution I was made by mixing 1 mL of TCPO (1.0 × 103 M) and 0.05 mL of fluorescer (various concentrations in ethyl acetate). Solution II contained 2 mL of hydrogen peroxide (7.5 × 102 M) and 1 mL of sodium salicylate (8.0 × 104 M) in methanol. Solution I was transferred into an instrument quartz cuvette via polypropene syringes. Then, 100 μL of solution II was injected into the quartz cuvette and the CL spectrum was recorded after mixing the reagents in the cell. Vitamin B6 was found to inhibit TCPO–H2O2–fluorescer (indolizine)–sodium salicylate CL emission on the basis of the Stern–Volmer equation (equation (1)). Therefore, the concentration of vitamin B6 was determined using the following equation due to linear quenching (49). I0 =I ¼ 1 þ K sv ½Q
(1)
Where, I0 and I are the CL intensity in the absence and presence of inhibitors, respectively, Ksv is Stern–Volmer constant and [Q] is the concentration of the inhibitors or quenchers. Procedure for the extraction of vitamin B6 from real samples Acid hydrolysis. Extraction of vitamin B6 (pyridoxine) was carried out by autoclaving 2 g of the samples in 50 mL HCl (0.44 and 0.055 M) at 121ºC for 30 min for tablet and banana, respectively (50). The extract solution was cooled in cold water at room temperature and the suspension was centrifuged at 3000 r.p.m (g-value 850) for 5 min. The supernatant was recovered and adjusted to pH 7 with 2 M NaOH solution and diluted to 250 mL with methanol/water (1 : 1 v/v). Aliquots were filtered through 0.45 μm nylon Millipore chromatographic filters and injected into the RP-HPLC system as a standard analytical method for qualification analysis. Chromatograms of standard pyridoxine and extracts of samples were presented in Fig. 2, and results show that extraction of pyridoxine from different real samples
Scheme 1. Mechanism for the preparation of indolizine derivatives.
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Luminescence 2014
Determination of vitamin B6 using TCPO–indolizine–H2O2 CL
Figure 2. Chromatograms of (a) standard solution, (b) tablet sample and (c) banana sample after extraction using acid hydrolysis (submitted to acid hydrolysis). Conditions were as follows: mobile phase. methanol/water (75 : 24 v/v); flow rate, 1 mL/min; column, C18 (250 mm × 4.6 mm, 10 μm); injection volume, 10 μL; λ, 275 nm; room temperature.
was carried out by acid hydrolysis, successfully. The extracted samples were analyzed using CL system.
Results and discussion PO–CL is well known as one of the most efficient non-biological light-producing systems. The possible mechanisms of the PO–CL reaction, taking into consideration what is reported in the
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literature. The intensity, duration and color of emission of the PO–CL systems are of great importance (51,52). In the first step, an aryl oxalate ester such as TCPO reacts with H2O2 to produce a key chemical intermediate of 1,2dioxetandione (C2O4) as an excitation source. In the second step, the excited cyclic C2O4 intermediate transfers its energy to fluorescer (indolizine derivatives were used here). The final step is the emission of light energy on the return of the excited
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M. J. Chaichi et al. fluorescer molecule to the ground state (20). The florescence spectra of four indolizine derivatives are shown in Fig. 3. By comparing the fluorescence spectra of the four compounds (a, b, c and d) it can be deduced that spectrum a is more intense than the other compounds (hyperchromic effect). As expected, the intensity of the PO–CL emission was found to be affected by the initial concentration of the reactants (8–14). Thus, the influence of concentrations of fluorescer, TCPO, H2O2, catalyst and pH on the PO–CL system for four fluorescers were studied.
Effect of pH The pH of the buffer solution is an important parameter that affects the CL intensity and hence has to be optimized if a very sensitive assay is required. The effect of pH on CL intensity was investigated using 0.1 M phosphate buffer in the pH range 4.0–9.0 and constant concentrations of H2O2 (7.5 × 102 M), TCPO (1.0 × 103 M), sodium salicylate (8.0 × 104 M) and fluorescer (1.0 × 104 M). The optimum pH was ~ 7. It can also be concluded that the CL intensity decreases slightly at pH values > 8. Such an observation highlights the fact that the neutral form of fluorescer can change in acidic and basic conditions, resulting in the production of a undesirable reactive intermediate and electron transfer is not possible. Further study showed that the best precision was achieved at pH 7, and consequently all measurements were performed at this pH.
Figure 4. Effect of TCPO concentration on the CL intensity of TCPO–H2O2–sodium salicylate–dimethyl 1-acetyl-7-(dimethylamino)indolizine-2,3-dicarboxylate, (b) salicylate–diethyl 1-acetyl-7-(dimethylamino)indolizineTCPO–H2O2–sodium 2,3-dicarboxylate, (c) TCPO–H2O2–sodium salicylate–dimethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate and (d) TCPO–H2O2–sodium salicylate–diethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate systems.
However, at concentrations of TCPO > 1.0 × 10-3 M, the increase in the signal is no longer proportional to the increase in the TCPO concentration, and a small decrease in the signalto-noise ratio is achieved. Therefore, TCPO concentrations above the stoichiometric ratio have no significant effect on the CL signal. A concentration of 1.0 × 10-3 M was used through this study.
Effect of TCPO concentration
Effect of H2O2 concentration
The influence of varying TCPO concentrations in the presence of excess H2O2 on the CL signal for four fluorescers was investigated over the concentration range 2.0 × 104 to 3.0 × 103 M, keeping constant the concentration of H2O2 (1.0 × 10-1 M), fluorescer (8.0 × 10–5 M) and sodium salicylate (6.0 × 10–4 M) and at pH 7. As seen from Fig. 4, the peak intensity increases rapidly after mixing and reaches a maximum in a few seconds. However, decay of the light intensity from the maximum occurs over much longer periods. As shown in Fig. 3, there is a linear correlation between the CL intensity and the TCPO concentration for all fluorescers. The basis for this linear correlation has been discussed previously (53).
The influence of H2O2 concentration on the PO–CL of indolizine was studied at constant concentrations of TCPO (1.0 × 10-3 M), fluorescer (8.0 × 10–5 M) and sodium salicylate (6.0 × 10-4 M) and at pH 7. It was found that there is a direct linear relationship between the concentration of hydrogen peroxide and the CL intensity of the system over a concentration range of 1.0 × 102 to 7.5 × 102 M (Fig. 5). This observation highlights that CL reaction progression improves proportional to the increasing H2O2 concentration. At higher H2O2 concentrations, the photomultiplier
Figure 3. The fluorescence emission spectra of (a) dimethyl 1-acetyl-7(dimethylamino)indolizine-2,3-dicarboxylate, (b) diethyl 1-acetyl-7-(dimethylamino)indolizine-2,3-dicarboxylate, (c) dimethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate and (d) diethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate at the same –4 concentration of 5.0 × 10 M in ethyl acetate with λex = 400 nm.
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Figure 5. Effect of H2O2 concentration on the CL intensity of TCPO–H2O2–sodium salicylate–dimethyl 1-acetyl-7-(dimethylamino)indolizine-2,3-dicarboxylate, (b) TCPO– H2O2–sodium salicylate–diethyl 1-acetyl-7-(dimethylamino)indolizine-2,3-dicarboxylate, (c) TCPO–H2O2–sodium salicylate–dimethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate and (d) TCPO–H2O2–sodium salicylate–diethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate systems.
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Luminescence 2014
Determination of vitamin B6 using TCPO–indolizine–H2O2 CL tube detector was saturated. It is noteworthy that further increases in H2O2 concentration were found to have no measurable effect on the PO–CL intensity. Effect of sodium salicylate concentration The PO–CL intensity of a 6.0 × 10–5 M solution of fluorescer, under optimal constant concentrations of TCPO and H2O2 was found to increase significantly in the presence of sodium salicylate, which is clearly indicative of the catalytic effect of the salt on the PO–CL system studied (53). In order to investigate the optimal concentration of sodium salicylate, the CL response of the H2O2–TCPO-fluorescer system was measured against the varying concentrations of the base and the resulting plot is shown in Fig. 6. The PO–CL intensity increased sharply with the increasing concentration of sodium salicylate until values of 8.0 × 10–4 M (a, b and c) and 1.0 × 10–3 (c) were reached; the observed intensity enhancement was indicative of the catalytic effect of the base. However, further addition of sodium salicylate revealed a gradual decrease in the CL intensity. This is most probably due to the quenching effect of the base at higher concentration, which begins to decompose the reactive intermediate, dioxetanedione, and hence reduces the CL signal. It should be noted that maximum rise and fall rate constant is also observed at the optimum sodium salicylate concentration. Effect of fluorescer concentration The influence of fluorescer concentration on the PO–CL system was studied at constant concentrations of H2O2 (7.5 × 10-2 M), TCPO (1.0 × 103 M) and sodium salicylate (8.0 × 104 M) was studied and the results are shown in Fig. 7. As it is seen from Fig. 7, the peak intensity increases with increasing fluorescer concentrations until 1.0 × 104 M, however, the peak intensity of H2O2–TCPO–sodium salicylate–fluorescer system decreases at higher concentrations. This is most probably due to the selfabsorption effect and the formation of a dimer structure at high concentrations, which causes decreases or inhibits electron transfer of the intermediate, dioxetanedione, and hence reduces the CL signal.
Figure 7. Effect of fluorescer concentration on the CL intensity of (a) TCPO–H2O2– sodium salicylate–dimethyl 1-acetyl-7-(dimethylamino)indolizine-2,3-dicarboxylate, (b) TCPO–H2O2–sodium salicylate–diethyl 1-acetyl-7-(dimethylamino) indolizine-2,3-dicarboxylate, (c) TCPO–H2O2–sodium salicylate–dimethyl 1-acetyl7-aminoindolizine-2,3-dicarboxylate and (d) TCPO–H2O2–sodium salicylate–diethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate systems.
Possible CL mechanism The mechanism of the TCPO–H2O2 CL reaction has been studied previously. It is suggested that TCPO can be oxidized by H2O2 to generate an energy-rich intermediate, 1,2-dioxetanedione. For a fluorescent molecule, 1,2- dioxetanedione could interact with the fluorescent molecule to yield a charge-transfer complex, which decomposed on electron transfer from the intermediate back to the fluorescent molecule, generating its excited state (54). The results of all optimization experiments for four indolizine derivatives showed that the CL intensities for fluorescer a are higher than for the other compounds in the TCPO–H2O2–CL system, under the same conditions. This observation may be attributed to the presence of dimethyl amine (–NMe2) as a stronger electron-donor substituent in comparison with (–NH2) on the benzene ring in compounds c and d. Also, displacement of the ethyl group by a methyl group on the five-member ring in compound b led to increasing steric hindrance in the indolizine structure and the ethyl group is a better electron donor than methyl group (in –COOR group) in that it can decrease the π conjugation in compound b. Decreasing the π conjugation in compound b can lead to a decrease in the resonance forms and lower fluorescence intensity. With increasing π conjugation, the energy difference between highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) decreases, suggesting the feasibility of absorption and fluorescence of compound a. The applicability of the optimized TCPO–H2O2–fluorescer a (dimethyl 1-acetyl-7(dimethylamino)indolizine-2,3-dicarboxylate) CL system was tested for the determination of vitamin B6 in pharmaceutical and plant (herbal) products. The most likely mechanism for the quenching of TCPO–H2O2–indolizine CL by vitamin B6, as an easily oxidizable compound (54), may be an electron transfer quenching pathway (55,56). The crucial step in luminescence quenching is assumed to be the reaction of the quencher Q, with the highly energetic intermediate C2O4 to give non-chemiluminescent products, in competition with the reaction of fluorescer as follows (57):
Figure 6. Effect of sodium salicylate concentration on the CL intensity of TCPO– salicylate–dimethyl 1-acetyl-7-(dimethylamino)indolizine-2,3H2O2–sodium dicarboxylate, (b) TCPO–H2O2–sodium salicylate–diethyl 1-acetyl-7-(dimethylamino)indolizine-2,3-dicarboxylate, (c) TCPO–H2O2–sodium salicylate–dimethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate and (d) TCPO–H2O2–sodium salicylate–diethyl 1-acetyl-7-aminoindolizine-2,3-dicarboxylate systems.
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M. J. Chaichi et al. According to this mechanism, after the reduction of indolizine, the resulting indolizine radical anion may react with C2O4 to produce C2O4– (anion) and free indolizine. The net result would be a diminished amount of the reactive intermediate in solution and, consequently, reduced CL intensity of the TCPO– H2O2–indolizine system. The quencher, vitamin B6, may also undergo a reduction reaction with the excited state indolizine to further decrease the CL of the system. Also, in the TCPO–H2O2 CL reaction, the formation of hydroperoxide was catalyzed under alkaline conditions. Addition of phenolic compounds such as vitamin B6 (containing a -OH group) to the TCPO– H2O2–fluorescer (indolizine) CL system increases its acidic properties, leading to decreased production of the hydroperoxide anion, and followed by its nucleophilic attack of TCPO. Therefore, the results show that vitamin B6 acts as a quencher, and decreases light production of the TCPO–H2O2–fluorescer (dimethyl 1-acetyl-7-(dimethylamino)indolizine-2,3-dicarboxylate) in the CL reaction. A linear relationship between vitamin B6 concentration and peroxyoxalate CL intensity according the Stern–Volmer plot is observed (Fig. 8).
Analytical performance of the CL system in vitamin B6 measurements Under optimum conditions, the ratios of I0/I were linear with the concentrations of a series of standard solutions of vitamin B6 7.0 × 108 to 1.0 × 104 M. Three replicate injections were performed for each standard solution. The Stern–Volmer plots for vitamin B6 are shown in Fig. 8. The regression equations were I0/I = 37049.0 C + 1.7865 with correlation coefficients (r) of 0.998 for vitamin B6 and the limit of detection (3σ) was 2.3 × 108 M. The reproducibility of the peak height was determined by measuring seven replicates of a 5 × 106 M pyridoxine solution; the relative standard deviation (RSD) was < 3.8%. The results indicate that the PO–H2O2–indolizine CL system is a rapid, simple and sensitive method and is suitable for highthroughput and real-time vitamin B6 analysis. Real sample analysis The proposed PO–CL system was successfully applied to the determination of vitamin B6 in two different samples of pharmaceutical product (tablet) and plant product (banana). To evaluate the accuracy and reliability of the method, known amounts of standard vitamin B6 solution at two concentrations (5 and 10 mg/L) were added to the real sample and analyzed in triplicate (n = 3) under optimal conditions according to the proposed method. The recovery of each measurement was calculated by comparing results obtained before and after addition of the standard vitamin B6 solution. The results are been listed in Table 1. In order to examine its applicability, the optimized TCPO–H2O2–sodium salicylate–dimethyl 1-acetyl-7-(dimethylamino)indolizine-2,3-dicarboxylate CL system, was applied for determination of vitamin B6 in pharmaceutical and plant (herbal) products.
Interference study Figure 8. Chemiluminescence emission intensity as a function of time for the TCPO–H2O2–dimethyl 1-acetyl-7-(dimethylamino)indolizine-2,3-dicarboxylate–sodium salicylate–vitamin B6 CL system with a constant concentration of TCPO -3 -4 -2 (1.0 × 10 M), fluorescer (1 × 10 M), H2O2 (7.5 × 10 M), sodium salicylate -4 -4 (8.0 × 10 M) and varying concentrations of vitamin B6 (a) 0.0, (b) 1.0 × 10 , (c) -5 -6 -7 -8 6.0 × 10 , (d) 1.0 × 10 , (e) 1.0 × 10 and (f) 7.0 × 10 M at pH 7. (Inset) Stern–Volmer plot Io/I vs. [vitamin B6].
To assess the possibility of applying the proposed method, the effect of foreign species was examined. Under optimal experimental conditions, interference by selected metal ions and organic compounds was evaluated. Most of the candidate compounds had no significant influence on the determination of vitamin B6 concentrations. Tolerable molar concentration ratios of foreign species to vitamin B6 were: 100 for glucose, folic acid,
Table 1. Determination of vitamin B6 in real samples Sample Tablet Banana
Claimed (mg)
Found (mg)
5.0 0.5
4.85 0.517
Spiked concentration (mg/L) Tablet 1 2 3 Banana 1 2 3
Found (mean ± SD) (mg/L)
% Recovery 97.0 103.4 % Recovery
% RSD (n = 3) 2.8 3.4 % RSD (n = 3)
0 5 10
1.06 ± 0.06 5.57 ± 0.15 12.03 ± 0.32
– 90.4 105.3
– 2.4 4.5
0 5 10
2.08 ± 0.04 6.64 ± 0.13 11.07 ± 0.08
– 91.2 88.0
– 3.5 4.2
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Determination of vitamin B6 using TCPO–indolizine–H2O2 CL fructose, sucrose, lactose, starch, bilirubin, vitamin C, Zn2+ and SO24 ; 50 for maltose, vitamin B2, vitamin B1, hydroquinone as a dihydroxyphenol, urea, Ca2+ and NO-3; 20 for Fe2+, Cu2+, Mg2+ and Cl-; and 10 for uric acid. As can be seen, the selectivity of the method is high.
Conclusion This study describes a novel CL system of H2O2–bis-(2,4,6trichlorophenyl) oxalate (TCPO) using indolizine derivatives as the fluorescer. In this system, the green fluorescence and CL signal of compound a is the highest. The influence of the concentrations of the components involved in the PO–CL system were also studied. The results showed that concentrations of H2O2, TCPO and indolizine derivatives have a direct relationship on the light intensity. The proposed CL system has good linearity, high sensitivity and precision, and the method has been successfully applied to the determination of vitamin B6 in pharmaceutical and plant products.
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