Cerium(IV)-based chemiluminescence of phentolamine sensitized by rhodamine 6G

Analytica Chimica Acta 506 (2004) 183–187

Cerium(IV)-based chemiluminescence of phentolamine sensitized by rhodamine 6G Weibing Liu, Yuming Huang∗ Department of Chemistry, Institute of Analytical Sciences, Southwest Normal University, Beibei, Chongqing 400715, PR China Received 28 July 2003; received in revised form 5 November 2003; accepted 10 November 2003

Abstract A new chemiluminescence (CL) method is proposed for the determination of phentolamine, which is based on the reaction between studied drug and Cerium(IV) (Ce(IV)) in a nitric acid medium and measurement of the CL intensity produced by rhodamine 6G used as a sensitizer. In the optimum conditions, CL intensities are proportional to concentrations of the studied drug over the range 1 × 10−9 –1×10−6 g/ml with a detection limit of 4 × 10−10 g/ml. The relative standard deviation (R.S.D.) is 3.4% for 1 × 10−7 g/ml phentolamine (n = 11). The method has been applied to the determination of studied drug in injections and biological fluids with satisfactory results. © 2003 Elsevier B.V. All rights reserved. Keywords: Chemiluminescence; Flow-injection analysis; Cerium(IV); Phentolamine; Biological fluids

1. Introduction Phentolamine is (Fig. 1) an ␣-adrenergic blocking agent with a slight ␤ stimulating effect. Traditionally, it has been used to treat the hypertension and as a valuable research tool in the study of adrenergic receptor-mediated processes. It has been reported as a useful agent for therapy in congestive heart failure, myocardial infarction, arrhythmia, angina pectoris, shock, and bronchial asthma [1]. Because of the recent interest for use of this drug in the treatment of erectile dysfunction [2–7], the therapeutic importance of this drug requires the development of rapid, sensitive, simple, and industrial quality control and clinical monitoring. For its measurement, several methods have been reported, such as spectrophotometry [8], thin-layer chromatography [9,10], spectrofluorimetry [11], gas chromatography (GC) with electron capture detection [12], high performance liquid chromatography (HPLC) with UV [10,13,14], electrochemical [15,16] or mass spectrometric detection [10,17]. However, most of them have one or more drawbacks. Because of low sensitivity, spectrophotometry could not meet the need of trace analysis. Although the low detection limits are generally provided by electrochemical detection,

∗

Corresponding author. Fax: +86-23-68866796. E-mail address: [email protected] (Y. Huang).

0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2003.11.012

one serious pitfall of this type of determination arises from the electrode fouling, which causes the instability of signal detection. Also, many methods require time-consuming sample preparation techniques, particularly in the case of using GC and HPLC. Transient emission produced by chemiluminescence (CL) reactions which forms the basis of analytical methods with the advantages of simplicity, rapidity and sensitivity of detection, has been used for the analysis of pharmaceutical compounds [18–20]. Regarding the determination of phentolamine by chemiluminescence, however, there are no literatures dealing with the issue to date. Phentolamine is a derivative of imidazoline, which is a strong base. Besides the nitrogen atom in the imidazoline, phentolmaine contains another basic site and a phenolic group. Based on above characteristics, in this work, a different approach is constructed. It is discovered that chemiluminescence increases when the phentolamine solution is injected into mixture of acidic cerium(IV) (Ce(IV)) sulfate and rhodamine 6G solution. A simple, sensitive and rapid flow-injection analysis (FIA)–CL assay for phentolamine based on the above mentioned discovery was developed and it was applied to the determination of phentolamine in the commercial preparations and biological fluids with satisfactory results. To the best of our knowledge, this is the first report on the chemiluminescent determination of phentolamine.

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Fig. 1. Molecular structure of phentolamine.

2. Experimental 2.1. Reagents All the reagents were of analytical-reagent grade unless specified otherwise; doubly distilled water was used for the preparation of solutions. Phentolamine was purchased from the Shanghai Sixth Pharmaceutical Factory (Shanghai, China). Ce(SO4 )2 ·4H2 O was from the Shanghai Colored Metal Company Ltd. (Shanghai, China). Concentrated sulfuric acid, concentrated hydrochloric acid, concentrated nitric acid, concentrated phosphoric acid, quinine hydrochloride, fluorescein, Triton X-100, OP, Tween 80, cetyltrimethylammonium bromide (CTMAB), and sodium dodecylbenzenesulfonate (SDBS) were obtained from the Chongqing Chemical Reagents Company (Chongqing, China). Rhodamine 6G and Rhodamine B were from Merck (Schuchardt, Germany). Phentolamine stock solution (5 × 10−4 g/ml) was prepared and stored in the refrigerator. More diluted solutions were used immediately after preparation. 2 × 10−3 mol/l cerium(IV) solution from Ce(SO4 )2 ·4H2 O in 1.8 mol/l nitric acid was prepared daily. 2.2. Apparatus The flow system employed in this work is shown in Fig. 2. A peristaltic pump delivered all follow streams at a flow rate of 3.5 ml/min (per tube). PTFE tubing (0.8 mm i.d.) was used to connect all components in the flow system. 75 ␮l of sample solution was injected into water stream by an eight-way injection valve and then mixed with the mixture of acidic Ce(IV) solution and rhodamine 6G solutions. The emitted

Fig. 2. Schematic diagram of the flow system for the determination of phentolamine: (a): H2 O; (b): 2×10−3 mol/l cerium(IV) sulfate in 1.8 mol/l nitric acid; c: 2 × 10−5 mol/l rhodamine 6G; S: sample; P: peristaltic pump; V: injection valve; F: flow cell; W: waste liquid; D: PMT; PC: personal computer.

CL was collected with a photomultiplier tube (operated at −900 V) of the Type IFFL-D Flow-Injection Chemiluminescence Analyzer (Reike, Xi’an, China). The signal was recorded using an IBM-compatible computer, equipped with a data acquisition interface. Data-acquisition and treatment were performed with REMAX software running under Windows 98. Chemiluminescence spectrum was monitored using a RF-540 fluorescence spectrometer (Shimazhu, Japan). A U-2000 UV-spectrophotometer (Hitachi, Japan) was used. 2.3. Procedure 2.3.1. Procedure for calibration Working standard solutions containing phentolamine in the range of 1 × 10−9 –1×10−6 g/ml were prepared by dilution of a concentrated fresh standard solution of phentolamine (5 × 10−4 g/ml). The CL signal was measured by injection 75 ␮l of working standard solution into the water carrier stream, which then joined the mixture of acidic Ce(IV) solution and rhodamine 6G solution. The CL emission intensities versus phentolamine concentration were used for the calibration. 2.3.2. Procedure for pharmaceutical preparations Injection samples (10 mg/ml, the Shanghai Xudong Haipu Pharmaceutical Company, Shanghai, China) were diluted with doubly distilled water to the working range of the determination of phentolamine, and then used for an analysis. 2.3.3. Procedure for biological fluids 2.3.3.1. Procedure for spiked plasma. Add an aliquot of standard aqueous solution of phentolamine (5 × 10−4 g/ml) to 5.0 ml of plasma in a plastic tube and shake the tube for mixing. Add 1 ml of 6% (w/v) perchloric acid for each milliliter of the plasma for deproteination. Blend on a vortex mixer and centrifuge at 3000 r/min for 10 min. Transfer 2.5 ml of the protein-free supernatant into a 50 ml of volumetric flask and dilute to volume with water. Proceed as described above. A blank value was determined by treating phentolamine-free plasma in the same way. 2.3.3.2. Procedure for spiked urine. Add an aliquot of standard solution of phentolamine (5 × 10−4 g/ml) to the urine to obtain a concentration of 1 × 10−5 g/ml. Transfer 1 ml of this solution into a 100 ml of volumetric flask and dilute to volume with water. Proceed as described above. A blank value was determined by treating phentolamine-free urine in the same way.

3. Results and discussion As an effective drug in the treatment of male and female sexual dysfunction, increasing interest has been paid

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to phentolamine analysis recently. For example, a liquid chromatography–tandem mass spectrometry system was developed for high-throughout screening of drugs in rat plasma including phentolamine [17]. More recently, another liquid chromatography–mass spectrometry system together with thin-layer chromatography and HPLC/photo-diode-array was reported to identify and determine phentolamine and sildenafil as adulterants in soft drinks advertising roborant nutrition [10]. The aim of the present study is to use a flow-injection analysis system with Ce(IV)-based chemiluminescent detection for determination of phentolamine. Ce(IV)-based CL has been reported for the determination of pharmaceutical compounds such as cysteine [20], paracetamol [21], catopril [22], phenothiazines [23]. In order to study the possible FIA chemiluminescence determination of phentolamine, different oxidants, for example, potassium permanganate, hydrogen peroxide, cerium(IV) sulfate and N-bromosuccinimide in acidic or basic media was tested. The results show that the CL of phentolamine was obtained when Ce(IV) or potassium permanganate was used as an oxidant in an acidic medium. However, the CL emission produced by potassium permanganate oxidation is relatively weak. Ce(IV) was selected as an oxidant in the present work. 3.1. Optimization of chemiluminescence reaction 3.1.1. Effect of different acid concentrations Ce(IV) reacts with phentolamine to produce light emission in acidic solution. The nature and concentration of the acid used in the reaction had a very strong influence on the CL emission. Therefore, four different acids (i.e. HCl, HNO3 , H2 SO4 , and H3 PO4 ) of different concentration in the range 5 × 10−2 –2.4 mol/l were tested in order to ascertain which was the most suitable. As can be seen from Table 1, in the phosphoric acid medium, no CL response was observed. The reason for this may be that Ce(IV) and phosphoric acid can form a precipitation product. Nitric acid gave the best signal. The effect of nitric acid concentration was studied over the range 0.5–2.4 mol/l. As shown in Fig. 3, 1.8 mol/l nitric acid was found to give the highest intensity. Therefore, 1.8 mol/l of nitric acid was selected for the present work.

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Fig. 3. Effect of nitric acid concentration. Error bars represent one standard deviation for three measurements.

sults are shown in Fig. 4. Maximum emission intensity was obtained at 2 × 10−3 mol/l Ce(IV). So, 2 × 10−3 mol/l Ce(IV) was adopted as the working solution in future experiment. 3.1.3. Effect of senitizers Potentially chemiluminescent molecules can transfer their excitation energy to a fluorophore with subsequent emission of energy by the fluorophore, often leading to an enhancement of the intensity. This type of process, named sensitization, has been used to determining pharmaceuticals [20,24] and sensitizer [25] using strong oxidants such as Ce(IV), permanganate. In order to study their potential effect as sensitizers on present CL reaction, a few different fluorophores were selected and examined in this study. As shown in Table 2, in the fluorophores examined, only rhodamine dyes as sensitizers show enhancement of the CL intensity. As compared to rhodamine B, rhodamine 6G gave the highest CL efficiencies and it was selected as sensitizer for further investigation. The effect of rhodamine 6G concentration was studied in the range 1×10−5 –6×10−5 mol/l, showing an increase in the CL intensity with increasing rhodamine 6G concentrations up to 2 × 10−5 mol/l, thereafter remaining almost constant up to 5 × 10−5 mol/l, above 5 × 10−5 mol/l, CL intensity decreases sharply probably because of self-absorption

3.1.2. Effect of Ce(IV) concentration The concentration effect of Ce(IV) upon the CL behavior of phentolamine was examined over the range 5 × 10−4 –4×10−3 mol/l in 1.8 mol/l nitric acid. The reTable 1 Effect of different acid concentrations on the CL intensity of phentolamine Acid

Optimized concentration (mol/l)

Relative CL intensity

Hydrochloric acid Sulfuric acid Nitric acid Phosphoric acid

0.5 0.8 1.8 –

0.4 0.2 1.0 0

Fig. 4. Effect of cerium(IV) sulfate concentration. Error bars represent one standard deviation for three measurements.

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Table 2 Effect of different fluorophores on the CL emission from Ce(IV)- phentolamine Fluorophores

Concentration range (mol/l)

Optimized concentration (mol/l)

Relative CL intensity

None Rhodamine B Fluorescein Quinine hydrochloride Rhodamine 6G

– 2 5 5 1

– 8 × 10−6 – 5 × 10−5 2 × 10−5

1.0 50.0 Suppression 1.0 573

× × × ×

10−6 –1 10−6 –5 10−6 –8 10−5 –6

× × × ×

10−5 10−4 10−5 10−5

of the emission by rhodamine 6G. Hence, 2 × 10−5 mol/l of rhodamine 6G was used through out in further experiments. The effect of some organized systems, including three neutral surfactants (Triton X-100, OP, Tween 80), one cationic surfactant CTMAB, and one anionic surfactant SDBS on the present CL reaction was also investigated. The results show that all the tested systems could not enhance CL emission. Therefore, the micellar solutions were not used in the present work. 3.1.4. Performance of the proposed method for phentolamine measurements Under the selected conditions given above, the calibration graph of emission intensity versus phentolamine concentration was linear in the 1 × 10−9 –1×10−6 g/ml range (I = 258.6 × 107 [phentolamine] (g/ml) + 180; r = 0.9958, n = 9) with a detection limit (3σ) of 4 × 10−10 g/ml. Relative standard deviation (R.S.D.) (n = 11) was 3.4% for 1 × 10−7 g/ml phentolamine. 3.2. Interference study The effect of foreign substances was tested by analyzing a standard solution of phentolamine (1 × 10−6 g/ml) to which increasing amounts of interfering substances were added. The tolerable concentration ratios with respect to 1 × 10−6 g/ml phentolamine for interference at 5% level were over 800 for KBr; 700 for CaCl2 ; 500 for KNO3 and glucose; 200 for (NH4 )2 SO4 and NaCl; 150 for urea; 100 for ␤-cyclodextrin; 50 for amylum and Al(NO3 )3 ; 20 for CuSO4 and NaHCO3 ; 10 for ZnSO4 ; 5 for Mg(NO3 )2 , FeCl3 , Na2 CO3 , maltose and tartaric acid; 2 for MnSO4 and EDTA; 0.004 for ascorbic acid, respectively.

3.3. Application of the method 3.3.1. Analysis of pharmaceutical preparations Following the procedure detailed under Section 2, the proposed method was applied to the determination of phentolamine preparations. The results for phentolamine concentrations measured using the proposed method were compared with those given by using UV spectrophotometry [26]. The recovery tests were also carried out on the samples. As shown in Table 3, the results given by the proposed method are in good agreement with those given by UV spectrophotometry and the obtained recoveries were satisfactory. 3.3.2. Analysis of spiked urine and plasma samples The method presented here has a low detection limit and, therefore, the proposed method allows the determination of phentolamine in biological fluids. The mean peak serum concentrations of phentolamine after administration of an oral 40 mg dose was about 3 × 10−8 g/ml [15]. Thus the proposed method proved to be satisfactory for the routine estimation of phentolamine in human urine and plasma. For plasma only a deproteination process was carried out using perchloric acid as a sample pretreatment, an extraction procedure was not necessary. Phentolamine-free urine samples produced a relatively high CL intensity, so a dilution of 1:100 (v/v) was required to minimize this interference. The diluted urine gave a very low CL intensity. Table 4 shows the results of the recovery studies of phentolamine from spiked plasma and urine. 3.4. Characteristics of the chemiluminescence reaction The determination of phentolamine proposed in this work is based on the amplification of rhodamine 6G of the weak

Table 3 Results of the determination of phentolamine in injections Sample

Label (mg/ml)

Proposed methoda (mg/ml)

UV spectrometrya (mg/ml)

Added (mg/ml)

Founda (mg/ml)

Recoverya (%)

Injection 1

10

9.2 ± 3.1

9.9 ± 0.1

5 10

14.2 ± 1.0 19.6 ± 0.3

100.0 ± 1.0 103.5 ± 0.3

Injection 2

10

10.2 ± 2.5

10.5 ± 0.4

5 10

15.7 ± 1.3 20.9 ± 1.5

108.0 ± 1.3 106.0 ± 1.5

Injection 3

10

10.0 ± 2.5

10.4 ± 0.3

5 10

14.6 ± 0.7 20.2 ± 0.5

92.0 ± 0.7 102.0 ± 0.5

a

Average of three measurements (±R.S.D.%).

W. Liu, Y. Huang / Analytica Chimica Acta 506 (2004) 183–187 Table 4 Results of the determination of phentolamine in spiked urine and plasma Concentration added (10−6 g/ml)

Found (%) (R.S.D.%, n = 3)

0.05 0.1 0.2 0.5 0.8 1.0

91.5 102.0 104.0 106.0 96.1 100.0

Urine

Plasma (1.4) (1.0) (3.7) (1.3) (2.1) (2.2)

93.8(3.6) 102.5(1.7) 104.0(2.4) 105.0(3.0) 99.0(1.9) 91.2(4.9)

emission produced by the oxidation of above compound by Ce(IV) in nitric acid medium. The fluorescent and CL emission spectra were recorded in order to clarify the reaction product generating the CL. A fluorescent emission was observed at 360 nm as a result of the reaction of Ce(IV) with phentolamine in the absence of rhodamine 6G, due to the appearance of Ce(III). In the presence of rhodamine 6G, the fluorescent emission occurred at 560 nm (the maximum fluorescence peak of rhodamine 6G) and did not appear at the wavelength of Ce(III). We examined the CL spectra by a RF-540 Fluoreimetry, which showed only one peak at 560 nm (same as the maximum emission spectra of rhodamine 6G). Based on above discussion, the possible mechanism of the CL reaction may be that the reaction of Ce(IV) with phentolamine in acidic medium will cause the excited Ce(III)∗ . When the reaction occurred in the presence of rhodamine 6G, energy is transferred from Ce(III)∗ to rhodamine 6G to form rhodamine 6G in an electronically excited state, which emits its characteristic radiation corresponding to the peak at 560 nm. Because rhodamine 6G has a higher quantum efficiency than Ce(III), more light is emitted. In its simple form, the possible mechanism stated above can be expressed as following: Ce(IV) + PHEred → Ce(III)∗ + PHEOx Ce(III)∗ → Ce(III) + hν Ce(III)∗ + Rh6G → Ce(III) + Rh6G∗ Rh6G∗ → Rh6G + hν(strong CL) where PHE, Rh6G, hν, Red, and Ox are the phentolamine, rhodamine 6G, the light emission, the reduced form and the oxidized form of phentolamine, respectively.

4. Conclusion The proposed method, with Ce(IV) as oxidant, rhodamine 6G as energy transfer reagent and chemiluminescence detection, was found to be useful for the determination of phentolamine, a drug recently utilized to treat the erectile dysfunction in clinical medicine. With limit of detection for phentolamine of 4 × 10−10 g/ml in the presence of

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rhodamine 6G, this is the first chemiluminescence method for phentolamine molecular measurement reported up to now. The proposed method is simple and sensitive. The method has been successfully applied to analysis of phentolamine in the commercial preparations and spiked biological fluids with satisfactory results.

Acknowledgements Financial support was provided by the Natural Science Foundation of Chongqing (Project no.2003-7982).

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