CHEM. RES. CHINESE UNIVERSITIES 2012, 28(4), 604—608

Determination of Biotin in Pharmaceutical Formulations by Potassium Permanganate-luminol-CdTe Nanoparticles Chemiluminescence System TRAORE Zoumana Sékou and SU Xing-guang* College of Chemistry, Jilin University, Changchun 130012, P. R. China Abstract A sensitive flow-injection chemiluminescence method was developed for the determination of biotin in the pharmaceutical formulations. The affinity between avidin and biotin was used to adsorb biotin on the polystyrene, with subsequent quantification of biotin based on its ability to enhance the chemiluminescence(CL) signal generated by the redox reaction of potassium permanganate-luminol-CdTe nanoparticles CL system. The investigations prove that apart from 3-aminophthalate, the CdTe quantum dots(QDs) play both catalytic and emitter roles. Under optimum conditions, the linear range for the determination of biotin was 0.01―25 ng/mL with a detection limit of 7.3×10–3 ng/mL(S/N=3). The relative standard deviation of 5 ng/L biotin was 2.06%(n=7). The proposed method was used to determine the biotin concentration in the pharmaceutical formulations and the recovery was between 96.4% and 104%. The proposed method is simple, convenient, rapid and sensitive. Keywords Flow-injection; Chemiluminescence; Biotin; CdTe nanoparticle Article ID 1005-9040(2012)-04-604-05

1

Introduction

Biotin is a water soluble vitamin belonging to the B-complex group, which plays an important role in the metabolism in a number of living organisms. It acts as a co-enzyme in carboxylation-decarboxylation, and participates in biosynthesis of fatty acids and proteins[1,2]. The previous research[3] has proved its role in gene expression and DNA replication. Its deficiency is rare but triggers dermatitis, conjunctivitis, loss of hair and appetite, hallucination, depression and developmental delay[4]. A number of methods have been reported for biotin detection in different food products, pharmaceutical preparations and biological fluids[5―7]. However, most of them use radiolabelled compounds[5]. The traditional methods for biotin determination are based on microbiological analysis. Since biotin is one of the essential nutrients for micro-organisms, the inoculation of some micro-organism culture with standard solution or sample and the subsequent determination of the culture development lead to quantifying the biotin[6,7]. These methods are sensitive but their specificities are disputed, they are time consuming[8], and generally radiolabelled compounds are used[9]. The uses of physicochemical methods which include colorimetric, polarographic, chromatographic, capillary zone electrophoresis methods are limited due to their low sensitivity[10]. So the development of new rapid and sensitive methods without radioactive reagents could be very beneficial. The flow-injection(FI) chemiluminescence(CL) is characterized by its simplicity, wide dynamic linear range, low

determination limit and accessibility[11,12]. In recent years, the CL studies have been extended from traditional molecular systems to nanoparticles systems[13,14]. The suspension array based on the particles like microspheres which provide a large assembling surface and the minimal diffusion path way for binding molecules to binding site has intrigued more and more interests. Luminol is traditionally known as a powerful luminescence agent, and has got a widespread application in modern analytical chemistry[15,16]. Its association with nanoparticles gives an impeccable tool for CL analysis. Herein we present a sensitive flow-injection CL method for the determination of biotin in the pharmaceutical formulations. The method is based on the ability of biotin to enhance the CL signal generated by potassium permanganate-luminolCdTe nanoparticles CL system. The method is simple, cheap and without using radioactive compounds. The method has been applied to the determination of biotin in pharmaceuticals and the results agree well with those by standard method based on the spectrometric determination.

2 2.1

Experimental Reagents and Apparatus

Mercaptosuccinic acid(MSA) and sulfo-NHS(N-hydroxysulfo-succinimide) were purchased from J & K Chemical Co.; tellurium powder, CdCl2 and NaBH4 were purchased from Aldrich Chemical Co.; poly(dimethyldiallylammonium chloride)(PDDA, Mw=82000), poly(4-sodium sulfonatestyrene)

——————————— *Corresponding author. E-mail: [email protected] Received October 14, 2011; accepted March 10, 2012. Supported by the National Natural Science Foundation of China(No.21075050) and the Science and Technology Development Project of Jilin Province, China(No.20110334).

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(PSS, Mw=70000) and polystyrene(PS) particles(average diameter 3 µm) were purchased from Sigma-Aldrich Co.; bovine serum albumin(BSA) was purchased from Genview; potassium permanganate and sodium hydroxide were purchased from Tianjin Bohai Chemical Reagent Factory(China); luminol was purchased form Tianjin Fuchen Chemical Reagent Factory, China. All the chemicals used were of analytical grade and were used without further purification. PDDA and PSS were prepared at a concentration of 2 mg/L in 0.2 mol/L sodium chloride solution and stored at 4 °C before use. The CL analysis was performed on an FIA-3110 flow injection analysis processor(Beijing Titan Instruments Co., Ltd., China) system. The schematic diagram of the system is shown in Fig.1. The injection system consists of two peristaltic pumps PA and PB, a sixteen-hole eight-ways valve for reagents and samples delivering, a digital system for flow time control, an ultra-weak luminescence analyzer(type BPCL manufactured by Beijing Institute of Bio-physique, Chinese Academy of Sciences), a photomultiplier tube operated at 1080 V and 30 °C for CL amplification. The temperature in the CL reaction chamber was automatically adjusted by the temperature control system. An FIA monitor/data processing mechanism served to record the CL signal. The flow cell was homemade coil, made by coiling 30 cm of colorless glass tube(1 mm i.d. and 2 mm o.d.) into a spiral disk shape. Polytetrafluoroethylene(PTFE) tubing(0.5 mm i.d., Shenyang Zhaofa Institute of Automatic Analysis, China) and 150 μL loop were used as connecting material in the FI-CL system.

2.3

Coating Procedure of Microspheres

First, 0.1 g of polystyrene microsphere was dissolved in 2 mL of distilled water and stirred for 5 min. The negative charged microspheres were then incubated with 400 µL of a PDDA solution for 30 min with gently shaking. The mixture was centrifuged at 3500 r/min for 10 min and washed two times with 1 mL of phosphate buffer solution(PBS, pH=7.4). Then it was incubated with 400 µL of PSS solution and washed as described above. These operations were repeated to get two alternate layers of PDDA and PSS on the microsphere surface via electrostatic self assembling method(Fig.2). The increase of the number of the layers led to the increase of global active surfaces of the coated microspheres, and consequently its assembling capacity increased. Finally, the coated microspheres were incubated in 100 µL(4 nmol) of an avidin aqueous solution and 400 µL(0.01 mol/L) of NaCl for 4 h. After washing twice with PBS, the unoccupied sites were blocked with BSA, and then it was diluted to 2 mL with PBS and stored at 4 °C before use.

Fig.2

2.4

Fig.1

Schematic diagram of flow-injection chemiluminescence detection system

PA: pump A; PB: pump B; V: eight-way valve; RC: reaction chamber; PMT: photomultiplier tube; S: power supplier; R: recorder; QDs: quantum dots.

2.2 Synthesis of Water-Soluble CdTe Nanoparticles Water-soluble CdTe nanoparticles(quantum dots, QDs) used were synthesized by refluxing method with MSA as stabilizer according to our previous paper[17]. Briefly, the precursor solution of CdTe nanoparticles was formed in water by adding fresh NaHTe solution to 20 mmol/L N2-saturated CdCl2 solution(pH=11.2) in the presence of MSA as stabilizing agent. The molar ratio of Cd2+: MSA: HTe− was fixed at 1:1.5:0.2. Then, the resulting solution mixture was subjected to a reflux at 100 o C under open-air conditions with a condenser attached. Stable water compatible MSA capped CdTe nanoparticles with emission maximum at about 560, 568, 570, 614 and 648 nm were obtained with different refluxing time.

605

Preparing process of avidin coated microspheres

Preparation of Standard Solution and Sample

Standard stock solution of biotin was prepared by dissolving 1 mg of biotin in a volumetric flask of 100 mL with distilled water and 1 mL PBS(pH=7.4). Then the solution was completed up to the mark and stocked at 4 °C. The standard work solutions were prepared by diluting the required amounts of standard stock solution in distilled water. The tablets or capsules of the drug were accurately weighted and powdered if necessary. Then 10 mg of drug was accurately transferred into the conical flask with 1 mL of 0.1 mol/L NaOH and 50 mL of distilled water and sonicated for 15 min in the ultrasonic bath. Then the solution was filtered with 0.22 µm filter into the calibrated flask of 100 mL. The conical flask was washed a few times and the combined washing was filtered into the calibrated flask, and the solution was completed up to the mark. The stock sample solution was used to prepare the working sample solutions.

2.5

Flow Injection CL Measurement

The scheme of flow injection CL measurement is represented in Fig.1. The luminol solution(5 μmol/L in 1 mmol/L NaOH) was carried to the loop collectively with the standard or sample solution by pump B at a flow rate of 1.6 mL/min and a running time of 10 s. KMnO4 solution of 40

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μmol/L and 10 μmol/L CdTe nanoparticles solution were carried by pump A at a flow rate of 4.5 mL/min and the running time of 30 s. All the solutions are mixed in the mixing tube prior to the reaction chamber. The CL intensities were recorded and the relative CL intensity ΔI(defined as the difference between the CL intensities in the presence and absence of biotin, respectively) was recorded. The sample concentration was determined via the calibration graph. To plot the calibration graph, a series of standard working solutions was prepared in the graduated essay tubes of 4 mL at concentrations from 1×10–3 ng/mL to 35 ng/mL. To each solution was added 200 µL of avidin coated microspheres that was incubated for 4 h. Further the coated microspheres were separated by centrifugation at 3500 r/min and washed two times with 1 mL of PBS and then diluted to 2 mL. A certain amount of diluted sample was incubated with 200 µL of avidin coated microspheres for 4 h and then centrifuged and washed as described above, and then diluted to 2 mL.

3

Fig.4

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Effect of luminol concentration on net CL intensity

Conditions: 40 μmol/L KMnO4, 1 mmol/L NaOH, 1 μmol/L CdTe nanoparticles, and 5 ng/mL biotin.

Results and Discussion

3.1

Optimization of Analytical Parameters

3.1.1

Effect of KMnO4 Concentration

The effect of KMnO4 concentration on CL intensity generated by KMnO4-luminol-biotin-CdTe nanoparticles system was studied at concentrations from 30 μmol/L to 70 μmol/L, with the results shown in Fig.3. It can be seen from Fig.3 that the CL net intensity increased with increasing the concentration of KMnO4 up to 40 μmol/L, and then decreased with the further increasing of KMnO4 concentration. Thus, 40 μmol/L was chosen as the optimal concentration of KMnO4.

Fig.3

Effect of KMnO4 concentration on net CL intensity

Fig.5

Effect of NaOH concentration on net CL intensity

Conditions: 40 μmol/L KMnO4, 2 μmol/L luminol, 1 μmol/L CdTe nanoparticles, and 5 ng/mL biotin.

3.1.3 Effect of CdTe Nanoparticles Size and Concentration To investigate the influence of nanoparticles size on net CL intensity of KMnO4-luminol-biotin-CdTe nanoparticles system, five kinds of CdTe nanoparticles with diameter sizes of 9.69, 4.30, 4.04, 3.68 and 3.10 nm were studied. The highest net CL intensity was generated by the smallest size of 3.10 nm (Fig.6). The decrease of net CL intensity with increase of CdTe nanoparticles size could be attributed to the fact that the increased size lead to the decrease of active surface area[18,19]. Further investigation prove that the net CL intensity of the system increases with the increase of CdTe nanoparticles concentration up to 1 μmol/L, and then the net CL intensity

Conditions: 2 μmol/L luminol, 1 mmol/L NaOH, 1 μmol/L CdTe nanoparticles, and 5 ng/mL biotin.

3.1.2

Effect of Luminol Concentration

The effect of luminol concentration on net CL intensity of KMnO4-luminol-biotin-CdTe nanoparticles system was studied in a range of 0.9―9 μmol/L , and the results are shown in Fig.4. It can be seen that the maximum net CL intensity was obtained at 2 μmol/L of luminol concentration. Therefore 2 μmol/L luminol was used in further experiments. Luminol CL reaction is optimum in an alkaline medium, so NaOH was used to provide an optimum condition for it and the effect of NaOH concentration on net CL intensity generated by the system was also investigated in a range of 0.01―3 mmol/L. As shown in Fig.5, the maximum net CL intensity was obtained at an NaOH concentration of 1 mmol/L.

Fig.6

Effect of CdTe nanoparticles size and concentration on net CL intensity

Nanoparticle size/nm: a. 3.10; b. 3.68; c. 4.04; d. 4.30; e. 9.69. Conditions: 40 μmol/L KMnO4, 2 μmol/L luminol, 1 mmol/L NaOH, and 5 ng/mL biotin.

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becomes almost stable. Therefore, 1 μmol/L CdTe nanoparticles was chosen in further experiments.

3.1.4

Effect of Flow Rate

The flow rate and sample volume are two important parameters in term of sensitivity, time consuming and reagent consumption. Actually the high flow rate causes the reduction of signal intensity due to the decrease of contact time between reactants. The slow flow rate provides a good contact time of reagents, however, the sample throughput slows down. According to this fact, the influence of flow rate was studied simultaneously for KMnO4 and CdTe nanoparticles in a range of 0.5―5 mL/min. The sufficient signal-to-noise ratio with minimum reagent consumption and time waste was observed at a rate of 4.5 mL/min for these solutions; meanwhile the maximal signal-to-noise ratio was achieved at 1.2 mL/min for luminol and sample. Therefore these values were employed in further experiments.

3.2

Reaction Mechanism

The CL mechanism of KMnO4-luminol system has been extensively studied in the literature[20], and it is accepted that the emitter species in luminol oxidation by KMnO4 is 3-aminophthalate. Besides, the catalytic, reducing and emitter roles of QDs in the CL reactions have been confirmed in previous reports[20,21]. Previous studies[9,22―24] have also elucidated that the two regions of biotin are capable of undergoing oxidation. In fact, the valeric chain is able to be oxidized to bisnorbiotin or tetranorbiotin. It is accepted that the oxidation of valeric chain pass through the formation of unstable ketone compounds[22], which could transfer their energy to emitter species. Besides, the sulfur atom of the heterocyclic ring can undergo the oxidation to form biotin-sulfoxide or biotin-sulfone. Considering these facts, we proposed the hypothesis that the major emitter is 3-aminophthalate produced in KMnO4-luminol oxidation, while the CdTe nanoparticles play both or one of catalytic and emissive roles(by resonance energy transfer)[21]. To investigate the major emitter species and the possible role of each participating reagent in the CL system, the CL emission spectra of different combinations of reagents were examined over the visible wavelengths(Fig.7). It can be seen that for KMnO4-luminol-CdTe nanoparticles-biotin system (Fig.7 curve a), there are two emission peaks. The emission peak at around 425 nm corresponds to the 3-aminophthalate emission wavelength[15]. The second one is at about 575 nm, which is the emission wavelength of used CdTe nanoparticles. Compared with KMnO4-luminol system(Fig.7 curve d) and KMnO4-CdTe nanoparticles-biotin system(Fig.7 curve e), curve b(KMnO4-luminol-CdTe nanoparticles system) and curve c(KMnO4-luminol-biotin system) confirm the enhancing effects of biotin and the catalytic role of CdTe nanoparticles on the peaks of KMnO4-luminol CL system(first peak at 450 nm on both curves and the second peak at 575 nm on curve b). The presence of the first peak on curve d and its absence on curve e(where luminol is absent) confirm the major emitter role is played by 3-aminophthalate. The presence of the second peak on curves a, b and e and its absence on others(where the CdTe

607

nanoparticles is absent) confirm this peak is due to the CdTe nanoparticles. In conclusion, 3-aminophthalate is the major emitter of the CL system. The CdTe nanoparticles play the role of catalytic reagent as well as emitter species.

Fig.7

Emission spectra of different systems

a. KMnO4-luminol-CdTe nanoparticles-biotin system; b. KMnO4luminol-CdTe nanoparticles system; c. KMnO4-luminol-biotin system; d. KMnO4-luminol system; e. KMnO4-CdTe nanoparticles-biotin system. Conditions: 40 μmol/L KMnO4, 2 μmol/L luminol, 1 mmol/L NaOH, 5 ng/mL biotin, and 1 μmol/L CdTe nanoparticles.

3.3

Calibration Graph and Analytical Features

Under the optimal conditions, the relation between biotin concentration and net CL intensity was studied. The linear range for biotin was found in the range of 0.01―25 ng/mL. The linear equation was ΔI=393.71+10.79lgρ(ng/L), the correlation coefficient was 0.999. Relative standard deviation of 7 replicate injections of 5 ng/mL biotin was 2.06%. The limit of detection (LOD) was 7.3×10–3 ng/mL.

3.4

Interferences Study

The influences of possibly coexisting species in pharmaceutical formulations were investigated at a biotin concentration of 5 ng/mL with various interfering species added. Tolerable concentrations defined as the concentrations of foreign species causing less than ±5% relative error were examined. The results show that no interference was observed from gelatin, glycerin, cellulose, stearate, Mg2+, K+, Na+, Cl–, glucose and sucrose even when they were 1000-fold excess over biotin. NH4+, SO42–, PO43–, dextrin and tartaric acid did not interfere with the determination up to 300-fold excess. Oxalic acid and urea did not affect the determination till 100-fold excess over biotin. Cu2+, Mn2+, Zn2+, Ba2+ and Fe2+ were tolerable in a limit of 10-fold.

3.5 Determination of Biotin in Pharmaceutical Formulations The proposed method was applied to determining biotin in five kinds of pharmaceutical formulations, and compared with the standard method based on the spectrometric determination[25]. The results are shown in Table 1. It can be seen from Table 1 that there is no significant difference between the results obtained by the two methods, which indicates that the proposed CL method can be used as a suitable and efficient alternative to other existing methods for the determination of

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Table 1 Comparison of the proposed method with the standard method for the determination of biotin in pharmaceutical formulations Sample

Declared amount/mg

Found by proposed method

Found by standard method

Formulation 1(5 mg/capsul) Formulation 2(5 mg/capsul)

5.0 5.0

Amount/mg 4.890 5.100

Recovery(%) 98.0 102.0

RSD(%, n=3) 2.2 2.0

Amount/mg 5.120 5.110

Recovery(%) 102.4 102.2

RSD (%, n=3) 2.4 2.2

Formulation 1(0.5 mg/tablet)

0.5

0.482

96.4

3.6

0.512

102.4

Formulation 2(0.5 mg/tablet)

0.5

0.493

98.6

1.4

0.517

103.4

2.4 3.4

Formulation 3(0.5 mg/tablet)

0.5

0.512

102.4

2.4

0.519

103.8

3.8

biotin in pharmaceutical formulations.

4

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Conclusions

A new flow-injection chemiluminescence method for the determination of biotin has been developed based on its enhancement effect on the CL signal generated by KMnO4luminol-CdTe nanoparticles system. The proposed method has been applied to determining the biotin in the pharmaceutical preparations. The method is simple, sensitive and does not require complicated equipments and radioactive compounds.

[1]

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