Research article Received: 27 August 2013,
Revised: 19 December 2013,
Accepted: 20 January 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2651
Flow injection chemiluminescence determination of lercanidipine based on N-chlorosuccinimide–eosin Y post-chemiluminescence reaction Guowei Wang, Fang Zhao* and Ying Gao ABSTRACT: A novel post-chemiluminescence (PCL) reaction was discovered when lercanidipine was injected into the CL reaction mixture of N-chlorosuccinimide with alkaline eosin Y in the presence of cetyltrimethylammonium bromide (CTAB), where eosin Y was used as the CL reagent and CTAB as the surfactant. Based on this observation, a simple and highly sensitive PCL method combined with a ﬂow injection (FI) technique was developed for the assay of lercanidipine. Under optimum conditions, the CL signal was linearly related to the concentration of lercanidipine in the range 7.0 × 10-10 to 3.0 × 10-6 g/mL with a detection limit of 2.3 × 10-10 g/mL (3σ). The relative standard deviation (RSD) was 2.1% for 1.0 × 10-8 g/mL lercanidipine (n = 13). The proposed method had been applied to the estimation of lercanidipine in tablets and human serum samples with satisfactory results. The possible CL mechanism is also discussed brieﬂy. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: ﬂow injection; post chemiluminescence; N-chlorosuccinimide; eosin Y; lercanidipine
Introduction Lercanidipine, 2-[(3,3-diphenylpropyl) (methyl) amino]-1, 1-dimethylethyl methyl 2,6-dimethyl-4-(3-nitrophenyl)-1, 4-dihydropyridine-3,5-dicarboxylate (Fig. 1), is a new thirdgeneration amphiphilic drug. It belongs to the well-known pharmacological active compound series classiﬁed as 1,4dihydropyridine calcium channel antagonists, which block calcium entry into L-type calcium channels present in smooth muscle cells, thereby causing peripheral vasodialation and a reduction in blood pressure (1,2). Lercanidipine exhibits a short plasma half-life compared with a long pharmacological effect due to its high liposolubility (3,4). It is currently used alone or in addition to an angiotensin-converting enzyme inhibitor, to treat hypertension and chronic stable angina pectoris. Therefore, the estimation of lercanidipine is important for clinical medicine and pharmacology. Several analytical techniques have been devised for the determination of lercanidipine in pharmaceutical formulations and biological ﬂuids, such as spectrophotometry (5–7), voltammetry (8–10), capillary electrophoresis (11), thin-layer chromatography (TLC) (12), high-performance thin-layer chromatography (HPTLC) (13), high-performance liquid chromatography (HPLC) (14–17) and HPLC-mass spectrometry (MS) (18–20). However, these methods have several drawbacks such as low sensitivity, the requirement for sample pretreatment, environmentally unfriendly solvents, expensive instrumentation and, in some cases, long analysis time which makes them unsuitable for routine analysis. Chemiluminescence (CL) coupled with ﬂow injection (FI) analysis has the advantages of excellent sensitivity with relatively simple and inexpensive instrumentation, a wide linear response range and rapid signal detection, so this technique has been extensively used in many ﬁelds
(21–25). In order to reduce the background signals that increase the CL emission, Lv and co-workers (26) found that a new CL reaction is generated when some substances are added to the solution after the former CL reaction has ﬁnished; such a reaction was called post-chemiluminescence (PCL) reaction. To the best of our knowledge, there is no written information concerning the analytical assay of lercanidipine from pharmaceuticals or biological media using a FI-PCL method. Eosin Y, a xanthene ﬂuorescent dye, has been used as a chemiluminescence reagent in CL analysis (27–30). In this study, it was found that a strong PCL signal was given out when a trace amount of lercanidipine was injected into the reaction mixture of N-chlorosuccinimide and eosin Y solution, and the PCL signal was strongly dependent on the lercanidipine concentration when cetyltrimethylammonium bromide (CTAB) is present. Based on this phenomenon, a novel, simple and sensitive FI-PCL methodology was proposed for the estimation of lercanidipine. Compared with the above-mentioned techniques, the present assay offers much a lower detection limit, a wider linear range and a simple and rapid procedure. Under optimum conditions, the successful determination of lercanidipine in pharmaceutical formulations and biological samples was found with satisfactory results. The possible CL mechanism of this study is presented and discussed brieﬂy.
* Correspondence to: F. Zhao, School of Chemistry and Chemical Engineering, Shihezi University, Shihezi, Xinjiang 832003, People’s Republic of China. Tel: +86-9932057159; Fax: +86-9932057270. E-mail: [email protected]
Key Laboratory for Chemical Materials of Xinjiang Uygur Autonomous Region, Engineering Center for Chemical Materials of Xinjiang Bingtuan, School of Chemistry and Chemical Engineering, Shihezi University, Xinjiang 832003, People’s Republic of China
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G Wang et al.
Figure 1. Molecular structure of lercanidipine.
Injection occurred via a six-way valve equipped with a 90 μL sample loop. A coil of glass tubing was used as the ﬂow cell placed in front of the detection window of the photomultiplier tube (PMT). CL signal acquisition and treatment were handled by a personal computer employing CL analysis software. The ﬂuorescence spectra were collected by a Hitachi F-4500 ﬂuorescence spectroﬂuorometer (Tokyo, Japan). The absorption spectra were made on a Shimadzu UV-2401 PC spectrophotometer (Kyoto, Japan). Analytical procedures
Reagents and chemicals All reagents were of analytical grade and were used without further puriﬁcation. Doubly distilled water was used throughout. Lercanidipine was obtained from the National Institutes for Food and Drug Control (Beijing, China). N-Chlorosuccinimide (> 98.5%) and eosin Y were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). CTAB was purchased from Sigma Aldrich Corp. (Steinheim, Germany). A stock standard solution containing 3.0 × 10-4 g/mL of lercanidipine was prepared by dissolving 30.0 mg of lercanidipine in 10 mL of methanol and diluting to 100 mL with water. This solution was found to be stable for at least one week when protected from light and stored at 4 °C in the refrigerator. Lercanidipine working solutions were freshly prepared by making suitable dilutions of the stock solution with water. The 5.0 × 10-2 mol/L N-chlorosuccinimide solution was prepared daily by dissolving 0.67 g of N-chlorosuccinimide in 100 mL of water. The 1.0 × 10-2 mol/L eosin Y stock solution was prepared by dissolving the required amount of eosin Y in 0.07 mol/L sodium hydroxide solutions. The eosin Y working solution was prepared from the stock solution by appropriate dilution with the same concentration of sodium hydroxide solution. The 0.01 mol/L CTAB stock solution was prepared in doubly distilled water.
Apparatus All CL measurements were conducted with an IFFM-E-type FI-CL analyzer (Xi’an Remex Analysis Instrument Co. Ltd, China) and a schematic diagram of the instrumental set-up is shown in Fig. 2. A peristaltic pump was employed to carry all reagents at a ﬂow rate of 2.0 mL/min. Polytetraﬂuoroethylene (PTFE) tubing (0.8 mm i.d.) was used for the connections in the ﬂow system. The reaction coil used was made from PTFE tubing (0.8 mm i.d. and 130 cm long) for the recommended conﬁguration (Fig. 2).
The FI-PCL manifold used in this work is shown in Fig. 2. Prior to the CL measurement acquisition, the N-chlorosuccinimide stream, eosin Y basic stream, CTAB stream and water were continuously transferred to the manifold until good reproducibility of the signal and the stable baseline were recorded. The lercanidipine standard or sample solution was injected into the carrier stream (water), mixed with the reaction mixture of N-chlorosuccinimide, eosin Y and CTAB solution and then reached the ﬂow cell accompanied by CL. Quantiﬁcation of the lercanidipine concentration was based on changes in the CL peak intensity between solutions with and without lercanidipine. Preparation of samples Lercanidipine tablets from different manufacturers were bought from the local market. Ten tablets of lercanidipine were weighed and crushed to a ﬁne powder. An accurate weight equivalent to 10 mg of lercanidipine was dissolved in 10 mL methanol in a small beaker. This solution was sonicated for ~ 15 min to aid dissolution and was ﬁltered. The residue was washed with 10 mL methanol three times. It was then transferred to a 100 mL volumetric ﬂask and completed to the mark with water. Working solutions were prepared by the appropriate dilution of this stock solution so that the ﬁnal concentration of lercanidipine was within the working range. Pharmaceutical sample.
Human serum sample. Serum samples were obtained from three healthy volunteers. A known amount of lercanidipine standard solution and 5.0 mL of serum sample were added to a centrifuge tube, which was thoroughly vortex-mixed for 3 min. The protein in the serum sample was eliminated with 10.0 mL of acetonitrile. The mixture was centrifuged at 3000 rpm for 15 min and the protein-free supernatant was collected. The acetonitrile was evaporated to dryness under a stream of nitrogen at room temperature. The dry residue was dissolved in methanol and diluted to 5.0 mL with water as a sample solution for CL analysis (30). A control was set up by treating lercanidipine-free serum in the same way.
Results and discussion Kinetic characteristics of the CL reaction
Figure 2. Schematic diagram of CL ﬂow system. P, peristaltic pump; V, injection valve; RC, reaction coil; F, ﬂow cell; HV, high voltage; PMT, photomultiplier tube; W, waste; COM, computer; S, lercanidipine solution. a, water; b, Nchlorosuccinimide solution; c, eosin Y (in 0.07 mol/L sodium hydroxide) solution; d, CTAB solution.
The CL kinetic curves of the N-chlorosuccinimide–eosin Y–CTAB reaction and N-chlorosuccinimide–eosin Y–lercanidipine–CTAB reaction were investigated by using the static system of the CL analyzer. It can be observed from Fig. 3 that when N-chlorosuccinimide was injected into eosin Y in a micellar medium, a CL signal occurred (Fig. 3a). After ~ 35 s, the CL reaction
Copyright © 2014 John Wiley & Sons, Ltd.
Post-chemiluminescence determination of lercanidipine Effect of sodium hydroxide concentration Alkaline conditions are needed for the N-chlorosuccinimide– eosin Y–lercanidipine–CTAB system. The alkalinity of the reaction medium was adjusted by varying the concentration of sodium hydroxide in the eosin Y solution. The effect of sodium hydroxide concentration on the PCL signal was examined between 1.0 × 10-3 and 0.1 mol/L. The results indicated that the strongest CL signal was obtained at 0.07 mol/L.
Effect of surfactant on the CL intensity
Figure 3. CL kinetic curves of N-chlorosuccinimide–eosin Y–CTAB (a) and Nchlorosuccinimide–eosin Y–lercanidipine–CTAB (b). Conditions: N-chlorosuccinimide, -4 0.05 mol/L; eosin Y (in 0.07 mol/L sodium hydroxide), 7.0 × 10 mol/L; lercanidipine, -8 -3 1.0 × 10 g/mL; CTAB, 5.0 × 10 mol/L.
ﬁnished and the CL signal decreased to the baseline. Afterwards, a new stronger CL emission signal appeared (Fig. 3b) when the lercanidipine solution was injected into a reaction mixture of N-chlorosuccinimide–eosin Y–CTAB. The CL signal reached a maximum value within 2 s. The CL signal decreased to the baseline after ~ 30 s. Under the same experimental conditions, no CL emission signal appeared when using the water instead of the lercanidipine solution.
Surfactants are commonly used to increase the efﬁciency of CL and the sensitivity of the measurement. Therefore, the inﬂuence of various surfactants including cationic surfactants such as CTAB and cetyltrimethylammonium chloride (CTAC), anionic surfactants such as sodium dodecylbenzenesulfonate (SDBS) and sodium dodecylsulfonate (SDS) and non-ionic surfactants such as Tween-80 and Triton X-100, on the PCL signal from the reaction of N-chlorosuccinimide with lercanidipine in the presence of eosin Y in alkaline medium was studied. The results indicated that the maximum PCL signal was obtained by using CTAB as the micellar medium. So, CTAB was employed for this research. The effect of CTAB concentration on the CL signal was investigated from 1.0 × 10-4 to 1.0 × 10-2 mol/L. The greatest PCL signal appeared at a concentration of 5.0 × 10-3 mol/L CTAB, namely under or above 5.0 × 10-3 mol/L, a decrease of the CL signal was caused. Hence, 5.0 × 10-3 mol/L CTAB was used.
Effect of the reaction coil
Effect of ﬂow rate
The length of the reaction coil is an important factor in completion of the CL reaction of N-chlorosuccinimide–eosin Y–CTAB. For this reason, a reaction coil (0.8 mm i.d.), in which the oxidation of eosin Y by N-chlorosuccinimide took place, was placed in this manifold. The effect of the reaction coil length on the relative PCL signal was tested over the range of 20–250 cm when the ﬂow rate of each channel was ﬁxed at 2.0 mL/min. The experimental results showed that the maximum change in PCL signal was obtained at 130 cm. Therefore, a reaction coil length of 130 cm was chosen to ensure high sensitivity and a high measurement rate.
The ﬂow rate is a crucial factor that inﬂuences not only analytical efﬁciency, but also the sensitivity of the system in FI-PCL analysis. With the reaction coil length ﬁxed at 130 cm, the effect of ﬂow rate on the PCL signal was investigated over the range 1.0–4.0 mL/min. The PCL signal increased with increasing ﬂow rate up to 2.0 mL/min, above which the PCL signal decreased. A ﬂow rate of 2.0 mL/min was therefore adopted for the determination of lercanidipine because it allows enough time for the N-chlorosuccinimide–eosin Y–lercanidipine–CTAB reaction to reach completion with the highest PCL emission signal. Consequently, a ﬂow rate of 2.0 mL/min was chosen.
Effect of N-chlorosuccinimide concentration
N-Chlorosuccinimide was the oxidant in the PCL reaction. The effect of the N-chlorosuccinimide concentration on the PCL signal was examined from 5.0 × 10-3 to 0.1 mol/L. The results showed that the CL intensity rapidly increased with increasing N-chlorosuccinimide concentration up to 0.05 mol/L, above which it decreased. Therefore, 0.05 mol/L N-chlorosuccinimide was adopted in the present study.
With the ﬂow system depicted in Fig. 2 and under the optimum experimental conditions mentioned above, the calibration curve of relative CL intensity (ΔI = Isample – Iblank) versus lercanidipine concentration (C) over range 7.0 × 10-10 to 3.0 × 10-6 g/mL was linear; typical CL signals are shown in Fig. 4. The regression equation was ΔI = 19.27 + 93.99C (C = 10-9 g/mL) with a correlation coefﬁcient of 0.9996. The limit of detection (LOD) was determined as 3S/m, where S is the standard deviation of replicate blank readings and m is the slope of the calibration graph. The LOD is calculated as 2.3 × 10-10 g/mL. The intraday (n = 11) and interday (n = 7) precisions of the assay for lercanidipine were 2.1 and 3.0%, respectively. The relative standard deviation (RSD) was found to be 2.1% after 13 repeated measurements of 1.0 × 10-8 g/mL lercanidipine. The merits of comparable methods for the detection of lercanidipine are shown in Table 1. Compared with previous results, the presented
Effect of eosin Y concentration Eosin Y was used as an important CL reagent in this work. The effect of eosin Y concentration on the PCL signal was tested in the range of 1.0 × 10-5–1.0 × 10-3 mol L-1. At ﬁrst, the CL signal increased rapidly with increasing eosin Y concentration, but then decreased at eosin Y concentrations over 7.0 × 10-4 mol/L. Therefore, 7.0 × 10-4 mol/L was chosen as the eosin Y concentration.
Copyright © 2014 John Wiley & Sons, Ltd.
G Wang et al. polyethylene glycol, urea, uric acid, stearic acid and Zn2+; 2+ 100-fold for PO34 , Ba , EDTA, sucrose, sorbitol, magnesium stearate, galactose, saccharose, tryptophan, casein and dextrin; 30-fold for Mg2+ and ascorbic acid; 10-fold for NH+4 , Mn2+, Al3+ and Fe3+; and 5-fold for Co2+ and Cu2+. The results indicated that the foreign substances did not signiﬁcantly affect the CL signal of the system for the determination of lercanidipine.
Figure 4. Representative recorder outputs for blank and lercanidipine standards under the proposed conditions by FI-CL system.
methodology has relatively lower LOD and a wider linear range (6,9,13,15,17).
Interference To evaluate possible analytical applications of the CL method described above, the effect of some common excipients in drugs and several organic compounds on the PCL signal was investigated. The experiments were carried out by comparing with the intensities obtained without the potentially interfering substances and with them added. Tolerable concentration ratios with respect to 1.0 × 10-8 g/mL lercanidipine for interference at the 5% level were > 1000-fold for K+, Na+, Cl-, HCO-3, 2+ 2CO23 and citric acid; 500-fold for Ca , SO4 , glucose, lactose, starch, mannitol and β-cyclodextrin; 200-fold for arabic gum,
Determination of lercanidipine in tablets. Following the procedure detailed in the experimental section, the method was applied to the determination of lercanidipine in commercial pharmaceutical preparations after the appropriate sample pretreatments. At the same time, lercanidipine in these sample solutions was determined by spectrophotometry (6). The experimental results are summarized in Table 2. Statistical analysis using the t-test showed no signiﬁcant differences between the novel FI-PCL method presented herein and the previously proposed method at the 95% conﬁdence level. Using the standard addition method, the recoveries for the determination lercanidipine were in the range 98.53–101.4%, indicating the accuracy of the developed method. Determination of lercanidipine in human serum. A single oral administration of 20 mg of lercanidipine leads to peak plasma concentration levels of 7.6 ng/mL (19). The high sensitivity and low LOD achieved with the proposed FI-PCL method allows the estimation of lercanidipine in serum samples. Serum samples were used to compare recovery by the proposed method with that using standard addition method. The experimental results are given in Table 3. The recoveries for human serum were 98.0–104% with an RSD of 1.3-2.1%, showing that the developed method was adequate for the analysis of lercanidipine.
Table 1. Figures of merit for the determination of lercanidipine Method
Linear range (g/mL)a
Spectrophotometry Voltammetry HPTLC HPLC RP-HPLC UPLC/ESI-MS/MS Chemiluminescence
1 × 10-6–2.0 × 10-5 2.45 × 10–8-4.65 × 10-6 3 × 10-8–2.10 × 10-7 5 × 10-6–1 × 10-4 5 × 10-7–2.5 × 10-5 5 × 10-11–3.0 × 10-8 7.0 × 10-10-3.0 × 10-6
Detection limit (g/mL) 1 × 10-8 1.2 × 10-8 9.3 × 10-7 3.8 × 10-8 5 × 10-11 2.3 × 10-10
Tablets Tablets, serum, urine Tablets Tablets Tablets Human plasma Tablets, serum
(6) (9) (13) (15) (17) (19) This study
HPTLC linear range measured in g/band.
Table 2. Determination results of lercanidipine in tablets (mg/tablet) Proposed method a
UV-vis method (6)
Found ± RSD (%) (n = 7)
Found ± RSD (%) (n = 5)
Tablet 1 Tablet 2 Tablet 3
10 10 10
9.87 ± 1.9 10.21 ± 2.0 9.92 ± 1.6
1.00 5.00 10.0
11.07 14.78 20.28
100.6 98.53 101.4
9.83 ± 0.29 10.3 ± 0.39 9.95 ± 0.47
Relative standard deviation
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Post-chemiluminescence determination of lercanidipine Table 3. Determination results of lercanidipine in serum samples (10-8 g/mL, n = 5) Sample
Serum Serum Serum Serum Serum
0.5 1.0 5.0 10.0 30.0
0.49 1.03 5.21 9.83 29.9
1 2 3 4 5
Recovery ± RSD (%) 98.0 ± 1.6 100.3 ± 1.7 104 ± 1.9 98.3 ± 2.1 99.7 ± 1.3
Possible reaction mechanism In order to understand the possible mechanism of the CL reaction, the CL emission spectra of the N-chlorosuccinimide– eosin Y–CTAB system and the N-chlorosuccinimide–eosin Y–lercanidipine–CTAB system were scanned using a modiﬁed F-4500 ﬂuorescence spectrophotometer (Fig. 5). It was found that CL spectrum of N-chlorosuccinimide–eosin Y–CTAB and the PCL spectrum of N-chlorosuccinimide–eosin Y–lercanidipine–CTAB system had the same maximum CL wavelength at around 583 nm. Accordingly, the luminophore of both CL reactions was the same. The ﬂuorescence spectra of different systems were scanned using a F-4500 spectroﬂuorimeter (Fig. 6). It was observed that no ﬂuorescence emission of N-chlorosuccinimide–lercanidipine in alkaline medium was obtained over the range 500–650 nm. Eosin Y alone produced a ﬂuorescence emission with a broad peak at 537 nm, and when CTAB was added to the solution, the emission peak of eosin Y shifted from 537 to 552.5 nm. However, ﬂuorescence spectra taken from the reaction mixture of N-chlorosuccinimide–eosin Y–CTAB and Nchlorosuccinimide–eosin Y–lercanidipine–CTAB system showed a new ﬂuorescence emission with a maximum wavelength at 583 nm. Consequently, this suggested that the illuminant of the PCL reaction was attributed to the oxidation product of eosin Y and N-chlorosuccinimide in s micellar medium. Furthermore, the characteristic absorption peak of lercanidipine at 357 nm almost disappeared when N-chlorosuccinimide and lercanidipine were mixed in an alkaline medium.
Figure 6. The ﬂuorescence spectra. (a) Eosin Y; (b) eosin Y–CTAB; (c) Nchlorosuccinimide–eosin Y–lercanidipine–CTAB; (d) N-chlorosuccinimide–eosin Y– CTAB. Conditions: N-chlorosuccinimide, 0.05 mol/L; eosin Y (in 0.07 mol/L sodium -4 -8 -3 hydroxide), 7.0 × 10 mol/L; lercanidipine, 1.0 × 10 g/mL; CTAB, 5.0 × 10 mol/L; λex = 320 nm.
Based on the above experimental phenomenon and previously reported results (31), the following possible mechanism for this PCL reaction can be proposed as. NChlorosuccinimide hydrolyzed to produce hypochlorite with strong oxidation. Eosin Y was oxidized by hypochlorite producing the product of oxidation in the excited state. The oxidation product returned to the ground state accompanied by CL (λmax = 583 nm). When lercanidipine was injected into the reaction mixture of N-chlorosuccinimide and eosin Y, it was oxidized with an excess of N-chlorosuccinimide and released energy. The product of oxidation at the ground state received this energy and was again excited, accompanied by CL (31,32). The possible CL mechanism for this CL system may be proposed as follows: N-chlorosuccinimide + OH- → ClOClO- + eosin Y + OH- → product* product* → product + hv (λmax = 583 nm) ClO- + lercanidipine + OH- → energy product + energy → product* product* → product + hv (λmax = 583 nm) * refers to material in the excited state
Figure 5. CL spectra. (a) N-Chlorosuccinimide–eosin Y–CTAB; (b) Nchlorosuccinimide–eosin Y–lercanidipine–CTAB. Conditions: N-chlorosuccinimide, -4 0.05 mol/L; eosin Y (in 0.07 mol/L sodium hydroxide), 7.0 × 10 mol/L; lercanidipine, -8 -3 1.0 × 10 g/mL; CTAB, 5.0 × 10 mol/L.
In this study, a novel PCL phenomenon was observed when lercanidipine was added to the CL reaction mixture comprising N-chlorosuccinimide and eosin Y in a micellar medium. Based on this, a new FI-PCL method for the estimation of lercanidipine was established. The feasibility of the application of the PCL reaction to analyses of lercanidipine was evaluated and the possible mechanism of the PCL reaction was discussed brieﬂy. Compared with previously published articles, this new technique has the advantages of rapidity, simplicity, sensitivity and accuracy for the estimation of trace amounts of lercanidipine in pharmaceutical and human serum samples.
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G Wang et al. Acknowledgements This project was supported by the National Undergraduate Innovation Program (201310759026) and the Large Precious Instrument Share Test Foundation of Shihezi University (20130919) for ﬁnancial support.
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