Porphyrin–metalloporphyrin composite based optical fiber sensor for the determination of berberine

Porphyrin–metalloporphyrin composite based optical fiber sensor for the determination of berberine

Analytica Chimica Acta 439 (2001) 65–71 Porphyrin–metalloporphyrin composite based optical fiber sensor for the determination of berberine Xiao-Bing ...

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Analytica Chimica Acta 439 (2001) 65–71

Porphyrin–metalloporphyrin composite based optical fiber sensor for the determination of berberine Xiao-Bing Zhang a , Zhi-Zhang Li b , Can-Cheng Guo a , Si-Hai Chen a , Guo-Li Shen a , Ru-Qin Yu a,∗ a

Institute for Chemometrics and Chemical Sensing Technology, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China b Teacher’s College, Yonzhou, Hunan 425006, PR China Received 12 December 2000; received in revised form 29 March 2001; accepted 3 April 2001

Abstract A berberine (BE)-sensitive optical fiber sensor has been prepared using a composite active material consisting of tetraphenylporphine (TPPH2 ) and chloro(tetraphenyl-porphinato)manganese (TPPMnCl) in a PVC matrix. The response of the sensor is based on the fluorescence quenching of TPPH2 –TPPMnCl by BE. The proposed sensor shows response to BE in the concentration range 7.5 × 10−7 to 5.6 × 10−4 mol l−1 , with a wide working pH range from 5.4 to 12.7, and a fast response time of less than 20 s. The sensor shows fair selectivity towards BE with respect to common co-existing species. The sensor based on TPPH2 –TPPMnCl composite shows better response characteristics comparing to that of the single TPPH2 . The effect of the composition of the sensor membrane has been studied and the experimental conditions optimized. The contents of BE in pharmaceutical tablets were determined by the proposed sensor and the results agreed with values obtained by the pharmacopoeia method. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Optical fiber sensor; Fluorescence; Berberine; Porphyrin–metalloporphyrin

1. Introduction Searching for sensing agents with reversibility, selectivity and sensitivity is of current interest as optical chemical sensors. Porphyrins or metalloporphyrins have been studied for ionophores for ion-sensitive electrodes [1–6]. Less attention has been paid to their use as sensing agents for optical chemical sensors [7–9]. Further exploration of the possibilities of application of these compounds as optical sensing materials is of considerable interest. In a preliminary ∗ Corresponding author. Tel.: +86-731-8822-710; fax: +86-731-8824-525. E-mail address: [email protected] (R.-Q. Yu).

experiment we observed the quenching of the fluorescence of tetraphenylporphine (TPPH2 ) by a metal complex of porphyrin. In a search of agents which could restore the quenched fluorescence, we discovered that a quaternary ammonium drug would further quench the fluorescence of the TPPMnCl–TPPH2 composite. The observed quenching reaction has a distinguish property of excellent reversibility. This experimental observation encouraged us to study the possibility as using it for the determination of quaternary ammonium drugs. Berberine (BE, structure was shown in Fig. 1) as a quaternary ammonium compound is the main active component contained in the rhizome of Chinese goldthread which has been used for centuries as

0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 1 ) 0 1 0 2 3 - 6


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Chloro(tetraphenyl-porphinato)manganese (TPPMnCl) was prepared according to documented procedures [23,24]. The porphyrins synthesized were identified by elementary analysis, UV–VIS spectroscopy and IR spectra. Fig. 1. Structure of berberine.

2.2. Apparatus traditional Chinese medicine. It is an anti-inflammatory drug for heart and intestinal disorders [10,11]. More recently it has been reported to possess anti-tumor promoting activities [12,13] and anti-lipase effects [14]. Analytical chemistry of BE has been the subject of numerous investigations. For the determination of BE, a number of methods have been proposed including extraction spectrophotometry [15], flow-injection [16], HPLC [17], ion-pair fluid chromatography [18], fluorometry [19], ion-sensitive electrode [20] and capillary electrophoresis [21]. This paper describes the use of the TPPH2 –TPPMnCl composite as sensing materials for the preparation of BE-sensitive optical fiber sensors. A bifurcated optical fiber and a micrometer-sized flow-cell were chosen for the determination of BE based on the fact that BE can quench the fluorescence of the TPPH2 –TPPMnCl composite. A sensor based on TPPH2 –TPPMnCl with a molar ratio of 1:1 shows response towards BE with a wide linear concentration range, fair selectivity and fast response time. The sensor can be used for the direct assay of BE in pharmaceutical preparations with satisfactory results.

All fluorescence measurements were carried out on a Hitachi M-850 fluorescence spectrometer with excitation slit set at 10 nm and emission at 20 nm. A home-made poly(tetrafluoroethylene) flow-cell (shown in Fig. 2) and a bifurcated optical fiber (30+30 quartz fibers, diameter 6 mm and length 1 m) were used for the BE sensing measurements. The excitation light was carried to the cell through one arm of the bifurcated optical fiber and the emission light collected through the other. A quartz glass plate (diameter 10 mm) covered with sensing membrane was fixed on the top of the flow chamber by the mounting screw nut with the membrane contacted with the sample solution. The sample solution was driven through the flow-cell by a peristaltic pump (Guokang Instruments, Zhejiang, China) at a flow rate of 1.4 ml min−1 . The standard solution of BE was obtained by serial dilution of 1.0 × 10−3 mol l−1 BE hydrochloride solution and buffered with NaAc–HAc (0.05 mol l−1 ), the pH of which was adjusted with a HCl or NaOH solution. The pH measurements were carried out on Model PHS-3E digital ion analyzer (Jiangshu Instruments, Jiangshu, China). IR spectra were recorded on a Perkin-Elmer model 783 IR spectrophotometer.

2. Experimental 2.1. Reagents All chemicals used were of analytical reagent grade. Doubly-distilled water was used throughout all experiments. Before use, dichloromethane and benzaldehyde were subjected to simple distillation from K2 CO3 . Pyrrole was distilled at atmospheric pressure from CaH2 . High molecular weight poly(vinyl chloride) (PVC), tetrahydrofuran (THF), di-iso-octyl phthalate (DIOP), di-nonyl phthalate (DNP), and dioctyl sebacate (DOS) were purchased from Shanghai Chemicals (Shanghai, China) and used as received. TPPH2 was synthesized by Adler’s method [22].

Fig. 2. Schematics for the flow-cell. (1) Bifurcated optical fiber; (2) mounting screw nut; (3) flow-cell body; (4) sensing membrane covered quartz glass plate; (5) detecting chamber; (6) inlet and outlet channel for sample solution.

X.-B. Zhang et al. / Analytica Chimica Acta 439 (2001) 65–71

Perkin-Elmer 2400 elementary analyzer was used for elementary analysis. 2.3. Preparation of optode membrane The optode membrane solution was prepared by dissolving a mixture of 3.1 mg of sensing material, 50 mg of PVC and 100 mg of plasticizer in 2 ml of freshly-distilled THF. A circular 10 mm diameter quartz plate was mounted on a spin-on device [25] and then rotated at a frequency of 600 rpm. Using a syringe, 0.1 ml of the membrane solution was sprayed to the centre of the plate. A membrane of about 4 ␮m thickness was then coated on the quartz plate and dried in ambient air at 30◦ C for 30 min before used. 2.4. Sample preparation After carefully peeling off the sugar coat, the pharmaceutical tablets of BE were ground into powder. An appropriate amount of powder was then dissolved in 80 ml of boiling water. After being cooled, it was transferred into a calibrated flask and then diluted to 100 ml for analytical determination. 2.5. Measurement procedure Two arm of the bifurcated optical fiber were fixed in the detecting chamber of the spectrofluorimeter to carry the excitation and emission light. The fluorescence measurements were carried out at the maximum excitation wavelength of 421 nm and the maximum emission wavelength of 652.0 nm. The sample solution of BE was fed through the detecting chamber of the flow-cell by the peristaltic pump at a rate of 1.4 ml min−1 . After each measurement, the flow-cell was washed with blank buffer solution until the fluorescence intensity of the optode reached the original blank value.

3. Results and discussion


fluorescence emission of Q(0–0) at 652 nm and Q(0–1) at 718 nm when excited at 421 nm with a fluorescence quantum yield Φ f = 0.13 [26]. TPPMnCl does not show any fluorescence emission, moreover it quenches the fluorescence of TPPH2 probably by forming a complex with the later agent. Experiments show that the TPPH2 –TPPMnCl composite immobilized in a plasticized PVC membrane can be utilized as a reversible sensing element for BE. Suppose a complexation equilibrium between BE (B) in the aqueous sample solution and TPPH2 – TPPMnCl (A) in the organic membrane phase is established with formation of a ternary complex with a complexing ratio of m:n, one has Kd

mB (aq)mB (org)

(1) β

mB (org) + nA (org)An Bm (org)


i.e. K

mB (aq) + nA (org)An Bm (org)


Here Kd , β and K are the distribution coefficient, the apparent complex formation constant of An Bm and the over-all equilibrium constant of the reaction, respectively. The relative fluorescence intensity α is defined as the ratio of free A, [A]f , to the total amount of A, [A]t in the membrane phase. It can be experimentally determined by measuring the fluorescence intensity of the optode α=

[A]f F − F0 = [A]t Fb − F 0


Here Fb is the fluorescence intensity of the optode in the blank buffer solution and F0 represents the fluorescence intensity of the same membrane when TPPH2 –TPPMnCl is completely complexed with BE. F is the fluorescence intensity of the optode actually measured when contacting with a BE solutions of given concentration. The relationship between the a and BE concentration [B](aq) can be represented as

3.1. Operation principle

αn 1 = n−1 1−α nK[A](org) [B]m (aq)

Owing to the conjugated double bond system and the high mobility of its ␲-electrons, TPPH2 exhibits

The response of the optode for different concentrations of BE is shown in Fig. 3. The three curves



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3.2. Optimization of membrane composition The composition of the optode membrane has been optimized through a series of experiments. The results of these optodes are shown in Table 1. From Table 1 it can be seen that the response characteristics of the optodes improved with the increase of the content of TPPMnCl in TPPH2 –TPPMnCl composite. The response characteristics became worse when the TPPMnCl–TPPH2 molar ratio reached 2:1 as the fluorescence intensity became too low. The optode membrane with a 1:1 TPPH2 –TPPMnCl molar ratio shows best response characteristics. Fig. 3. Relative fluorescence intensity (α) as a function of logarithm of the berberine concentration. Theoretical response curves of berberine as predicated by Eq. (5). (– – –) 1:2 (m:n), 8.71×107 (K); (—) 1:1, 4.88×104 ; (·····)·2:1, 2.38 × 109 . Data points obtained in experiment (䊊).

are calculated using Eq. (5) with different ratios of BE, TPPH2 –TPPMnCl and K. It can be seen that the curve with 1:1 complex ratio and a reasonable K of 4.88 × 104 fits best to the experimental data. Eq. (5), therefore, can been expressed as follows: log[B] = log(1 − α) − log α − log K


From aforementioned results one could propose an interaction mode of TPPH2 –TPPMnCl with BE. The central metal ion of the TPPMnCl could co-ordinate with the lone pair electrons of one of the two pyrrol skeleton of TPPH2 , The lone pair electrons of another pyrrol could be co-ordinated with the BE cation, which weakened the mobility of ␲-electrons of TPPH2 causing fluorescence quenching. Eq. (6) applied to evaluate response behavior of the optode can serve as the basis of quantitative analysis.

3.3. The effect of plasticizer Optodes with different plasticizers, i.e. DIOP, DNP and DOS, were prepared using 1:1 TPPH2 –TPPMnCl composite as sensing membrane component. The optode with DOS as the plasticizer has obviously better response performance for BE compared to other plasticizers. This is attributed to the difference in the solubility of the sensing component in the plasticizers. In the optode preparation experiment one observes TPPH2 dissolves in all three tested solvents quite well, while DOS shows obvious greater solubility of TPPMnCl comparing to DIOP and DNP. 3.4. Effect of pH The fluorescence intensity versus pH plot for the TPPH2 –TPPMnCl optode shown in Fig. 4 was obtained by adjusting the solution pH with hydrochloric acid and sodium hydroxide and fixing the BE concentration at 1.0 × 10−5 mol l−1 . It can be seen that in the section of lower pH value, the fluorescence intensity of the optode decreased with decreasing pH value, which

Table 1 The effect of sensing materials to the response behaviors of optodesa Optode no.

Sensing material

1 2 3 4 5


Working concentration range (mol l−1 ) (3:1) (2:1) (1:1) (1:2)

5.3 1.2 3.0 7.5 3.3

× × × × ×

Note: membrane composition for each optode was 2% sensing material, 65% DOS and 33% PVC.

10−5 10−6 10−6 10−7 10−6

to to to to to

1.7 4.8 1.2 5.6 1.0

× × × × ×

10−4 10−4 10−4 10−4 10−5

X.-B. Zhang et al. / Analytica Chimica Acta 439 (2001) 65–71

Fig. 4. Effect of pH on the response of the optode (berberine concentration fixed at 1.0 × 10−5 mol l−1 ).

is similar to that obtained with single TPPH2 for determining surface-active species [8]. This phenomenon might be caused by extracting of H+ from aqueous solution into the optode membrane at high acidity, which might weaken the mobility of ␲-electrons of TPPH2 with the synergistic action of TPPMnCl. Moreover, the larger hydrophilicity of BE in solutions of lower pH owing to its protonation might be another reason for this phenomenon. H+


H2 TPP H3 TPP+ H4 TPP2+ The optimum pH values for the determination of BE were found to be 5.4–12.7. In subsequent experiments, a pH 6.86 NaAc–HAc buffer solution was selected. 3.5. Response characteristics of the optode The response curve of TPPH2 –TPPMnCl (1:1, mol:mol) based optode is shown in Fig. 3. The linear response range covers from 7.5 × 10−7 to 5.6 × 10−4 mol l−1 of BE in a pH 6.86 NaAc–HAc buffered solution. The response time for BE concentration lower than 5.0 × 10−5 mol l−1 is as fast as 10 s; with a concentration larger than 5.0 × 10−5 mol l−1 , it is less than 20 s. It seems that the response involves a rapid reversible complex process at the solution–membrane interface. The reproducibility and reversibility of the optode response were studied by alternatively recording the fluorescence intensity for 1.0×10−6 mol l−1 and 5.0×10−5 mol l−1 BE. The relative standard deviations of the fluorescence intensity


Fig. 5. Reproducibility and reversibility for the optode after step changes of berberine concentration between 1.0 × 10−6 and 5.0 × 10−5 mol l−1 .

for the two solutions were found to be 2.3 and 3.1%, respectively. The recorded results are shown in Fig. 5. All these results indicate satisfactory reversibility and reproducibility of the optode. The short-time stability of the optode was tested by recording the fluorescence intensity of 5.0 × 10−5 mol l−1 BE over a period of 12 h for 20 measurements. A relative standard deviation of fluorescence reading of 3.6% was appeared. The fluorescence intensity of the optode decreased 8% after 100 measurements. The optode could be stored in wet conditions without large change of the fluorescence intensity for at least 1 month, which implies that the porphyrin and metalloporphyrin used are stable in a membrane contacting with water. The proposed optode seems to possess satisfactory response characteristics compared with sensors reported earlier. The BE-sensitive electrode [20], for instance, showed a higher detection limit (an order of 10−6 mol l−1 ) and a longer response time (3 min). As we mentioned in the introduction section, BE can be determined by using some other analytical methods. The proposed optode has an advantage of simplicity, high analysis speed and repeated reusability of the prepared sensor. It does not require any sophisticated instrumentation. 3.6. Interference The interference for a number of common species on the fluorescence determinations of BE was


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Table 2 Interference of different species to the fluorescence determination of berberine with the proposed optode Interferant

Concentrationa (mol l−1 )

NaCl KCl CaCl2 FeCl2 HgCl2 NH4 Cl Vitamin B1 Ascorbic acid Salicylate Ethacrynic acid Atropine Colchicine Lidocaine Caffein

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

× × × × × × × × × × × × × ×

10−2 10−2 10−2 10−2 10−2 10−2 10−3 10−3 10−3 10−3 10−3 10−3 10−3 10−3

Fluorescence change ( F = F − F0 )b

Relative error % ( F/F0 ) × 100

−0.5 −0.3 0.7 −1.1 −5.3 0.9 −0.8 0.5 −0.2 −2.9 1.5 −1.9 −0.9 0.3

−1.2 −0.7 1.7 −2.7 −13.2 2.2 −2.0 1.2 −0.5 −7.2 3.7 −4.7 −2.2 0.7

The concentration of berberine is fixed at 1.0 × 10−5 mol l−1 buffered with a NaAc–HAc solution (pH = 6.86). F and F0 are the fluorescence intensities of the optode contacting with 1.0 × 10−5 mol l−1 berberine solution with and without adding the interferant (F0 = 40.2). a


investigated. The experiments were carried out by fixing the concentration of BE at 1.0 × 10−5 mol l−1 and then recording the change of the fluorescence intensity before and after adding the interferant into the BE solution buffered with a NaAc–HAc solution (pH = 6.86). The results presented in Table 2 reveal that the ethacrynic acid and HgCl2 of high concentrations have effect to the determination of BE, which, fortunately, are rarely co-existing with BE in practical samples. The relative error of other interferants such as alkali and alkaline metal ions as well as many common pharmaceutical species, was less than ±5% which is recognized as tolerated. The TPPH2 –TPPMnCl (1:1, mol:mol) based sensor exhibited fairly selectivity towards BE with respect to other co-existing interferants, which making it feasible for practical applications in pharmaceutical analysis.

4. Preliminary analytical application The biomedical effect of the BE containing pharmaceutical tablets especially some composite preparations is related with BE content in these drug formulations. The proposed sensor was applied to the direct determination of BE in pharmaceutical tablets. The sample solutions prepared as described in the experimental section were diluted with a NaAc–HAc buffered solution of pH 6.86 and then analyzed using the TPPH2 –TPPMnCl (1:1, mol:mol) based optode. Results in Table 3 showed that the concentration of BE as determined by the optode method was in good agreement with that obtained by the pharmacopoeia method [27]. The present sensor seems useful for the determination of BE in real samples.

Table 3 Comparison of results of optode and pharmacopoeial method for determination of berberine (mg g−1 of tablet) in commercial tablets (n = 5) Sample




Optode method Pharmacopoeial method

271.1 ± 3.5 270.3 ± 1.8

543.2 ± 2.8 545.2 ± 2.3

716.9 ± 3.7 714.9 ± 2.0

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