Cadmium telluride quantum dots as pH-sensitive probes for tiopronin determination

a n a l y t i c a c h i m i c a a c t a610(2008)50–56

Cadmium telluride quantum dots as pH-sensitive

probes for tiopronin determination

Yun-Qing Wang a,b,Chao Ye a,b,Zheng-Hui Zhu a,b,Yu-Zhu Hu a,b,?

a Key Laboratory of Drug Quality Control and Pharmacovigilance,Ministry of Education,Nanjing210009,China

b Department of Analytical Chemistry,China Pharmaceutical University,Nanjing210009,China

a r t i c l e i n f o

Article history:

Received30September2007

Received in revised form

18December2007

Accepted7January2008

Published on line16January2008

Keywords:

Cadmium telluride

pH sensitive

Fluorescence quenching

Tiopronin

Determination

a b s t r a c t

The pH-sensitive cadmium telluride(CdTe)quantum dots(QDs)were used as proton probes

for tiopronin determination.Based on the?uorescence quenching of CdTe QDs caused by

tiopronin,a simple,rapid and speci?c quantitative method was proposed.Under the optimal

conditions,the calibration plot of ln(F0/F)with concentration of tiopronin was linear in

the range of0.15–20?g mL?1(0.92–122.5?mol L?1)with correlation coef?cient of0.998.The

limit of detection(LOD)(3 /k)was0.15?g mL?1(0.92?mol mL?1).The content of tiopronin in

pharmaceutical tablet was determined by the proposed method and the result agreed with

that obtained from the oxidation–reduction titration method and the claimed value.

1.Introduction

Quantum dots(QDs)have been attracting much attention

owing to their ideal optical properties in the past decade.In

comparison to traditional organic dyes and?uorescent pro-

teins,QDs offer advantages in many aspects such as a narrow,

tunable,symmetric emission from visible to infrared wave-

lengths,high level of brightness and photochemical stability

[1].Since1998,QDs with variable surface capping ligands have

extensively been used as?uorescent species for cell label-

ing,tumor imaging and clinical diagnosis[2–4].They have

also been applied in quantitative determination of biological

macromolecules[5,6]and drugs[7,8]based on their?uores-

cence quenching which may be due to the changes of the

surface states of QDs.

?Corresponding author at:Department of Analytical Chemistry,China Pharmaceutical University,Nanjing210009,China.

Tel.:+862583271280;fax:+862583271280.

E-mail address:njhuyuzu@https://www.360docs.net/doc/4c16590411.html,(Y.-Z.Hu).

Another area QDs can be utilized is the development of

?uorescence sensors for analytical detection of chemical and

biological ionic species.It was found that Cu2+[9]Ag+[10,11]

and CN?[12]affected the?uorescence intensity of QDs dra-

matically and methods for determination of these ions by

water-soluble QDs had been proposed.In previous researches,

it was noted that pH changes of the medium could result in

pronounced?uorescence changes of QDs,indicating H+is also

correlated with their?uorescence properties[13,14].Susha et

al.found that the?uorescence intensity of CdTe QDs was pH

sensitive and went through an almost linear change within

a certain pH range.Accordingly,they?rstly proposed that

the water-soluble CdTe QDs could be developed as a promis-

ing hydrogen ion indicator[15].So far,the pH-sensitive QDs

had been introduced to practical biological application.For

doi:10.1016/j.aca.2008.01.015

a n a l y t i c a c h i m i c a a c t a610(2008)50–5651

example,Zhang et https://www.360docs.net/doc/4c16590411.html,ed CdTe QDs as proton sensors for the detection of proton?ux driven by ATP synthesis in chro-matophores[16].With the aid of pH-sensitive CdTe–ZnS QDs, Yu et al.successfully monitored the hydrolysis of glycidyl butyrate catalyzed by porcine pancreatic lipase[17].Sun et al. also reported that?uorescence of mercaptoacetic acid(MPA) capped CdSe QDs was pH dependent in SKOV-3human ovarian cancer cells[18,19].However,information about the applica-tion of pH sensitivity of QDs was still limited,especially in pharmaceutical analysis area.

Tiopronin[N-(2-mercaptopropionyl)-glycine]is a weak acidy compound for the treatment of cystinuria,rheuma-toid arthritis,hepatic disorders as well as an antidote to heavy metal poisoning.A number of spectrometric[20],chro-matographic[21–25]and chemiluminescent[26,27]methods had been proposed for its determination.However,the above methods are expensive,time-consuming or requiring com-plex mathematic dealing.In the present work,using CdTe QDs as pH-sensitive probes,we developed a new method for the determination of tiopronin based on the?uorescence quench-ing of CdTe QDs caused by pH changes when adding tiopronin in aqueous medium.

2.Experimental

2.1.Apparatus

The absorption spectrum was acquired on a UV2100UV–vis spectrometer(Shimadzu,Japan).All?uorescence measure-ments were made with RF-5301spectro?uo-photometer (Shimadzu)equipped with a1cm quartz cell.The trans-mission electron microscopy(TEM)image of the QDs was acquired on a JEM-2100transmission electron microscope (JEOL,Japan).The colloidal solution of the QDs was dropped onto copper grids covered with a thin?lm of amorphous carbon and the excess solution immediately wicked away. The FT-IR spectra were obtained on a FTIR-8400S Spectropho-tometer(Shimadzu).TGL-16platform high-speed centrifuge (HengFeng Equipment Factory,Jintan,China)was applied for centrifugation operation.All pH measurements were made with a Model pHS-25meter(Leici Equipment Factory,Shang-hai,China).

2.2.Reagents

Cd(NO3)2·4H2O,tellurium powder and NaBH4were purchased from Shanghai Reagent Company(Shanghai,China);mercap-toacetic acid was acquired from Lingfeng Reagent Company (Shanghai,China);tiopronin tablets were obtained from Xinyi Medicine Company(Henan,China).All water used in the experiment was double distilled.

2.3.Synthesis of CdTe QDs

CdTe QDs were prepared according to method described previ-ously[28].Brie?y,100mL Cd precursor solution was prepared by mixing a solution of92.4mg Cd(NO3)2·4H2O in the presence of63?L MPA,and was then adjusted to pH11.2with1mol L?1 NaOH.The solution was deaerated with N2for30min.Under vigorous stirring,600?L0.5mol L?1oxygen-free NaHTe solu-tion prepared according to the reference was injected.The molar ratio of Cd2+:NaHTe:MPA was1:0.5:2.4.Then the pre-cursor solution was re?uxed under open-air condition.The size of the prepared QDs could be tuned by changing re?ux-ing time.In this work,we chose the re?uxing time for2h. QDs solution concentration was adjusted according to the Te2?concentration.

2.4.Sample treatment

1.00mg mL?1tiopronin standard solution was obtained by dis-solving10.00mg tiopronin by water in a10mL volumetric ?ask.T wenty tiopronin tablets were weighed and powdered in

a mortar and the average weight of one tablet was calculated.

33.04mg powder(equivalent to10mg tiopronin)was then dis-solved in10mL water,and insoluble excipients were removed with centrifugation at14,000rpm for5min.

2.5.Determination of tiopronin with CdTe QDs

One hundred microliter QDs solution was diluted with20mL pure water then adjusted to6.0with0.1mol L?1HAc.Transfer 2.00mL diluted solution into the quartz cell.Ten microliter tiopronin solution was added into the mixture with a micro-syringe.The?uorescence intensity was measured with the following settings of the spectro?uo-photometer(excitation wavelength( ex)350nm;excitation slit5.0nm;emission slit 5.0nm).

3.Results and discussion

3.1.Characterization of CdTe QDs

Fig.1(a)shows the absorption spectrum of CdTe QDs with a shoulder centered at495nm.The emission spectrum of CdTe QDs is shown in Fig.1(b).The maximum emission wavelength is535nm.The narrow?uorescence spectrum indicates that as-prepared CdTe QDs are nearly monodisperse and homo-geneous.The TEM image of CdTe QDs also shows much less agglomerated particles with average size of3.0nm(Fig.2

Fig.1–Absorption(a)and emission(b)spectra of CdTe QDs at room temperature.

a n a l y t i c a c h i m i c a a c t a 610(2008)

50–56

Fig.2–TEM image of the CdTe QDs re?uxed for 2h.The scale bar is 10

nm.

Fig.3–Relationship between ?uorescence intensity of QDs and pH values.Here 20?L of CdTe QDs was added into

20mL 0.05mol L ?1phosphate buffer solutions with various pH values (3.3,3.62,3.93,4.18,4.35,4.63,4.72,4.96,5.21,5.6,6.03,6.93,8.07,8.3,9.07,10.07,11.72and 12.62).The excitation wavelength of the measurements was set at 350nm.

3.2.Relationship between ?uorescence intensity of CdTe QDs and pH

It is known that QDs are pH sensitive.However,the effect of pH on ?uorescence intensity of QDs varies with different reports [13–15].In the present study,the relationship between ?uorescence intensity and pH was investigated by diluting as-prepared QDs with 0.05mol L ?1phosphate buffer solu-tions adjusted to different pH values.The results indicated that the maximum emission of CdTe QDs did not show any pH dependence.However,the ?uorescence intensity of QDs changed dramatically.As shown in Fig.3,the ?uorescence

intensity reached a maximum value at pH 6.03.With further pH decrease,the ?uorescence intensity decreased sharply and became nearly undetected at pH 3.6.So the pH range of 3.6–6.0could be selected as the most pH-sensitive range that was suitable for application.

3.3.Fluorescence quenching caused by tiopronin

Firstly,100?L CdTe QDs was diluted with 20mL pure water,and then adjusted pH to 6.0with 0.1mol L ?1HAc,which was the initial value of pH-sensitive range.Fluorescence intensi-ties before (F 0)and after (F )adding tiopronin were recorded.As shown in Fig.4(a),in pure water medium QDs solution,a sig-ni?cant decrease of QDs ?uorescence emission was observed,causing F 0/F increase remarkably.

In our study,it has been noticed that tiopronin is a weak acid with a sulphydryl group.The sulphydryl group may also have an effect on the ?uorescence of QDs.In order to identify whether sulphydryl group or pH was the dominant in?u-ence factor,0.05mol L ?1NaH 2PO 4–Na 2HPO 4,NaAc–HAc and Tris–HCl buffers with pH at 6.0were further selected to run the assay.As shown in Fig.4(b–d),the ?uorescence intensity was nearly unchanged with the addition of the tiopronin in all these three buffers.Therefore,we deduced that the ?uo-rescence quenching was mainly due to the pH change of the QDs solution.

3.4.Effect of the QDs concentration

It was found that concentration of QDs affected not only the ?uorescence intensity but also sensitivity of the assay.As shown in Fig.5,when the concentration of QDs increased,the sensitivity decreased signi?cantly.High concentration of QDs may also result in self-quenching of the QDs ?uorescence.However,if the concentration was too low,when a given emis-sion slit was settled,the ?uorescence intensity was also very low,which may sacri?ce the linear range.Considering these factors,7.5×10?6mol L ?1of CdTe QDs was

applied.

Fig.4–Fluorescence response of CdTe QDs to addition of tiopronin in water and buffer systems.(a)Pure water,(b)NaAc–HAc,(c)Tris–HCl,and (d)NaH 2PO 4–Na 2HPO 4buffer solutions with pH 6.0.The concentration of CdTe QDs was 7.5×10?6mol L ?1.

a n a l y t i c a c h i m i c a a c t a 610(2008)50–56

Fig.5–Effect of QDs concentration.The concentration of CdTe was (a)7.50×10?6,(b)1.88×10?5,(c)3.75×10?5and,(d)7.50×10?5mol L ?1with the initiative pH

6.0.

Fig.6–Effect of reaction time on the ?uorescence intensity of CdTe QDs-tiopronin solution system (CdTe:7.5×10?6mol L ?1;c tiopronin :2.5?g mL ?1).

3.5.Effect of reaction time

Initial experiments demonstrated that the tiopronin quench-ing of the QDs ?nished very soon and the ?uorescence signals were stable for more than 80min (Fig.6).In this work,we recorded the ?uorescence intensity immediately after the tio-pronin solution was added.

3.6.Calibration curve and detection limit of the method

Under the optimal condition mentioned above,the emis-sion spectra of CdTe QDs with different amount of tiopronin were recorded.The results were shown in Fig.7.As is well known,in a homogeneous medium having only a single component exponential decay,the quencher concen-tration could be obtained from the well-known Stern-Volmer relationship:F 0

=1+K SV [Q

]Fig.7–Fluorescence quenching of CdTe QDs to addition of different amount of tiopronin in water.The ?nal tiopronin concentration was (a)0,(b)2.5,(c)5.0,(d)7.5,(e)10.0,(f)

12.5,(g)15.0,(h)17.5,and (i)20.0?g mL ?1. ex =350nm.The concentration of CdTe QDs was 7.5×10?6mol L ?1.

where F 0is the intensity of the ?uorescence phenomena from the ?uorophore in absence of quencher,and F is the intensity of the ?uorophore when the quencher is present at concen-tration [Q ].Interestingly,the plots of F 0/F versus tiopronin concentration did not ?t a conventional linear Stern-Volmer equation.A steep upward curvature was observed (Fig.8).The obtained experimental data for tiopronin determination could be ?tted,however,to the following empirical equation:ln

F 0

=0.0421x ?0.0514,

obtaining a linear relationship in the range of 0.15–20?g mL ?1(0.92–122.5?mol L ?1)with correlation coef?-cient of 0.998,which could be used to develop a determination method for tiopronin.The limit of detection (LOD)is

de?ned

Fig.8–Stern-Volmer plot of tiopronin concentration dependence of the ?uorescence intensity of QDs.The standard deviations (S.D.)of F 0/F for each dot from left to right were 0.008,0.011,0.058,0.063,0.066,0.066,0.031and 0.081,respectively (n =3).The concentration of CdTe QDs was 7.5×10?6mol L ?1.

54a n a l y t i c a c h i m i c a a c t a610(2008)50–56 Table1–Effects of several metal ions and excipients on the?uorescence of CdTe QDs

Metal ions Coexisting concentration

(?mol L?1)Change of F(%)Excipients Coexisting concentration

(?g mL?1)

Change of F(%)

K+(Br?)2500 2.90Magnesium stearate Saturated solution0.42

Zn2+(NO3?)25 2.29L-HPC Saturated solution 1.58

Mg2+(NO3?)25?1.37Starch Saturated solution?1.79

Ca2+(Cl?)25 2.43MCC Saturated solution0.22

Cd2+(Cl?) 1.25?4.04Lactose200 1.63

Al3+(NO3?)0.25 4.81Mannitol2000 2.73

Ag+(NO3?)0.125 3.04d-Glucose200 4.16

Pb2+(NO3?)0.125 2.47SDS50 2.16

Cu2+(NO3?)0.05 4.26?-CD250.23

Table2–Results for the determination of tiopronin in synthetic samples(n=3)

Number Amount(?g mL?1)Main interferents Amount found(?g mL?1)R.S.D.(%)

1 5.00K+,Zn2+,L-HPC,?-CD 4.99±0.10 2.1

2 5.00Mg2+,Pb2+,starch,d-glucose 5.23±0.040.7

3 5.00Mg2+,Ag+,MCC,SDS 5.04±0.06 1.1

4 5.00Al3+,Cu2+,lactose,mannitol 5.19±0.16 3.0

The concentration of all metal ions was0.1?M.The concentration of?-CD,d-glucose,SDS and lactose was5.0?g mL?1.L-HPC starch and MCC were saturated in mixture solution.

by the equation LOD=(3 /k),where is the standard deviation of blank measurements(n=10)and k is the slope of calibration graph.Here LOD was0.15?g mL?1(0.92?mol L?1).

3.7.Precision and accuracy

To assess the precision and accuracy of the method,deter-minations were carried out for a set of10measurements of 5.08?g mL?1tiopronin under optimal condition.The average result for10determinations was5.10?g mL?1with the relative standard deviation of2.2%.These values indicated that this method had good accuracy and precision.Owing to the exper-imental variables in the procedure of the QDs synthesis,the ?uorescence emission/absorption pro?les and?uorescence intensity of each batch of QDs would be a little different.Thus, a calibration curve must be constructed for each batch of QDs.

3.8.Effect of interferences

In order to investigate the possibility of practical application in determination of pharmaceutical preparation,the interfer-ence from some metal ions and excipients often contained in tablets,such as lactose,microcrystalline cellulose(MCC), low-substituted hydroxypropyl cellulose(L-HPC),starch,mag-nesium stearate,sodium dodecyl sulphate(SDS),d-glucose,?-cyclodextrin(?-CD)and mannitol,was tested in the cho-sen condition.Because MCC,L-HPC,starch and magnesium stearate are barely soluble in water,their saturated solutions were selected to study the effects.The results in Table1indi-cated that the tolerant concentrations of these excipients were much larger than those in the as-prepared tablet sample solu-tion.Accordingly,we can conclude that these excipients did not quench QDs?uorescence emissions which in turn would give rise to erroneous tiopronin levels being determined.How-ever,some heavy metal ions,such as Cu2+,Ag+and Pb2+,can quench the?uorescence of CdTe QDs dramatically.But their contents in drugs and common pharmaceutical excipients are strictly limited,usually in ppm level.So the amount of heavy metal ions introduced into the CdTe QDs solution was so small that they could hardly cause any interference to the?uores-cence intensity.

3.9.Application

Four synthetic samples of tiopronin with ions and excipients were determined under the optimal condition.As presented in Table2,the determination results were satisfactory.The proposed method was then applied to determine tiopronin in commercial tablets.The recovery of the method was acquired using the standard addition method by adding a known amount of standard to the pre-analyzed tablet sample in three different levels.Results were given in Table3.The recoveries

Table3–Results of recovery studies by standard addition method(n=3)

Background(?g mL?1)Added(?g mL?1)Found(?g mL?1)Recovery(%)R.S.D.(%)

5.084.00 3.849

6.07 1.2

5.00 4.9398.66 3.7

6.00 6.04100.70.4

a n a l y t i c a c h i m i c a a c t a 610(2008)50–56

Table 4–Assay results for the determination of tioproninin in commercial tablets (labeled value:100mg tablet ?1)Tiopronin tablets

Repeated determination (mg tablet ?1)

Average (mg tablet ?1)

R.S.D.(%)

Titration method (n =5)102.5100.7101.7101.7102.6101.80.8This method (n =5)

101.8

103.2

105.4

101.5

105.1

103.4

1.8

Statistical t -test revealed no signi?cant difference between the content found by both methods (P =0.08).

were in the range of 96.07–100.7%.Oxidation–reduction titra-tion was also performed for the determination,with iodine as the titrant [29].As shown in Table 4,the content of tio-pronin in tablets assayed by the present method agreed with the titration result and the labeled value.

3.10.Mechanism

As is well known,complexes of MPA and cadmium ions bond on the surface of QDs contribute to the ?uorescence enhance-ment and the stability of the CdTe QDs.When adding tiopronin into CdTe QDs solution,the increasing H +may cause the decomposition of the annulus of the Cd 2+-MPA complexes due to the protonation of the surface-binding thiolates [13].In addition,a portion of MPA dissociated from the nanoparticle surface at low pH,resulting in a lower surface charge,and the uncapped QDs trended to aggregate.This process will cause strong ?uorescence quenching of CdTe QDs [19].The presump-tion was veri?ed by FT-IR and resonance light scattering (RLS)information.T wo CdTe QDs samples were precipitated by the addition of isopropyl alcohol to the colloidal solution with pH 6.0and 3.0and then separated by centrifuging and dried before FT-IR measurement.The results were shown in Fig.9.Peaks at 1633and 1461cm ?1represented carbonyl stretching vibration (

C O )and methylene scissoring vibration (?CH 2)of MPA coated on the surface of https://www.360docs.net/doc/4c16590411.html,pared with CdTe QDs precipitated at

pH 6.0(Fig.9(a)),the intensity of both peaks decreased sig-ni?cantly for the sample obtained at pH 3.0(Fig.9(b)),which indicated the loss of MPA capping ligand of CdTe QDs in acidic atmosphere.The change of RLS signals of a serial of QDs colloid solutions at different pH adjusted by HAc,was also

Fig.9–FT -IR spectra of CdTe QDs precipitated by adding isopropyl alcohol to the colloidal solution adjusted to pH 6.0(a)and 3.0(b).

Fig.10–The changes of resonance light scattering (RLS)signals of CdTe colloid solution with pH (a)6.55,(b)5.78,(c)5.45,(d)5.11,(e)4.76,(f)4.52and (g)4.05.The insert was a plot of RLS intensity against pH values of the solution.

investigated.The RLS spectra of the QDs were recorded with synchronous scanning at ex = em (i.e., =0nm)with the slit (ex/em)was 5.0nm/5.0nm.As shown in Fig.10,in the pH range of 4.0–6.5,RLS intensity became stronger as pH decreased,and the shape became irregular,which implied the QDs went through an aggregation process and particles became larger in solution [30].Another reason is that CdTe QDs are synthesized with NaHTe and Cd 2+in the basic atmosphere,the addition of H +induces reversible reaction,and Te is precipitated from the unstable NaHTe which is easily oxidized by O 2[17].

4.Conclusion

CdTe QDs were found to be a satisfactory pH-sensitive probe that could have potential chemical and biochemical sensing ability.Here we attempted to establish a novel method for quantitative analysis of an acidy drug based on the quench-ing of the ?uorescence of CdTe QDs.The method was simple,rapid and speci?c.The content of tiopronin in commercial tablets determined by the present method agreed with the oxidation–reduction titration result and the labeled value.Clearly,the potential of QDs as pH probes has just begun to be realized and the application in analytical chemistry is still in its infancy.The works such as the fundamental quenching mechanism,the relationship between ?uorescence quench-ing and molecular structure of the acidy quencher as well as the synthesis and modi?cation strategy for pH sensitive QDs with better speci?c and sensitive properties,are need to be

56a n a l y t i c a c h i m i c a a c t a610(2008)50–56

investigated.Therefore,we believe there will be more work in this area in the coming years.

r e f e r e n c e s

[1]X.Michalet,F.F.Pinaud,L.A.Bentolila,J.M.Tsay,S.Doose,J.J.

Li,G.Sundaresan,A.M.Wu,S.S.Gambhir,S.Weiss,Science

307(2005)538.

[2]M.Bruchez Jr.,M.Moronne,P.Gin,S.Weiss,A.P.Alivisatos,

Science281(1998)2013.

[3]M.H.Igorl,E.G.Tetsuouyeda,M.Hedi,Nat.Mater.4(2005)

435.

[4]X.H.Gao,Lily Yang,J.A.Petros,F.F.Marshall,J.W.Simons,

S.M.Nie,Curr.Opin.Biotechnol.16(2005)63.

[5]L.Wang,H.Q.Chen,L.Y.Wang,L.Li,F.G.Xu,J.S.Liu,C.Q.

Zhu,Anal.Lett.37(2004)213.

[6]X.D.Chen,X.B.Wang,L.Liu,D.C.Yang,L.Fan,Anal.Chim.

Acta.542(2005)144.

[7]J.G.Liang,S.Huang,D.Y.Zeng,Z.K.He,X.H.Ji,X.P.Ai,H.X.

Yang,Talanta69(2006)126.

[8]Y.Ma,C.Yang,N.Li,X.R.Yang,Talanta67(2005)979.

[9]M.T.Fern′andez-Arg¨uelles,J.J.Wei,J.M.Costa-Fern′andez,

R.Pereiro,A.Sanz-Medel,Anal.Chim.Acta.549

(2005)20.

[10]J.G.Liang,X.P.Ai,Z.K.He,D.W.Pang,Analyst129(2004)619.

[11]K.M.Gatt′as-Asfura,R.M.Leblanc,https://www.360docs.net/doc/4c16590411.html,mun.(2003)

2684.

[12]J.J.Wei,J.M.Costa Fern′andez,R.Pereiro,A.Sanz-Medel,

Anal.Chim.Acta522(2004)1.

[13]M.Y.Gao,S.Kirstein,H.M¨ohwald,J.Phys.Chem.B102(1998)

8360.[14]H.Zhang,Z.Zhou,B.Yang,J.Phys.Chem.B107(2003)8.

[15] A.S.Susha,A.M.Javier,W.J.Parak,A.L.Rogach,Colloids Surf.

A Physicochem.Eng.Asp.281(2006)40.

[16]Y.Zhang,Z.T.Deng,J.C.Yue,F.Q.Tang,Q.Wei,Anal.

Biochem.364(2007)122.

[17] D.H.Yu,Z.Wang,Y.Liu,L.Jin,Y.M.Cheng,J.G.Zhou,S.G.

Cao,Enzyme Microb.Technol.41(2007)127.

[18]Y.H.Sun,Y.S.Liu,P.T.Vernier,C.H.Liang,S.Y.Chong,L.

Marcu,M.A.Gundersen,Nanotechnology17(2006)

4469.

[19]Y.S.Liu,Y.H.Sun,P.T.Vernier,C.H.Liang,S.Y.Chong,M.A.

Gundersen,J.Phys.Chem.C111(2007)2872.

[20]T.P′erez Ruiz,C.Mart′?nez-Lozano,V.Tom′as,C.Sidrach de

Cardona,J.Pharm.Biomed.Anal.15(1996)33.

[21]K.Matsuura,H.Takashina,J.Chromatogr.Biomed.Appl.616

(1993)229.

[22]T.M.Huang,B.Yang,Y.J.Yu,X.W.Zheng,G.L.Duan,Anal.

Chim.Acta565(2006)178.

[23]K.Matsuura,K.Murai,Y.Fukano,H.Takashina,J.Pharm.

Biomed.Anal.22(2000)101.

[24]J.F.Liu,H.H.Wu,Y.N.Hou,J.Chromatogr.B844(2006)

153.

[25]J.Y.Ma,Y.L.Gu,B.Chen,S.Z.Yao,Z.N.Chen,J.Chromatogr.A

1113(2006)55–59.

[26]T.P′erez-Ruiz,C.Mart′?nez-Lozano,Willy R.G.Baeyens,A.

Sanz,M.T.San-Miguel,J.Pharm.Biomed.Anal.17(1998)823.

[27]J.Z.Lu,https://www.360docs.net/doc/4c16590411.html,u,S.Yagisawa,K.Ohta,M.Kai,J.Pharm.

Biomed.Anal.33(2003)1033.

[28]H.Zhang,L.Wang,H.Xiong,L.Hu,B.Yang,W.Li,Adv.Mater.

15(2003)1712.

[29]Standards for New Drugs in Due Forms,Drug Speci?cations

Promulgated by the Ministry of Health,PR China,X30-106. [30] C.Z.Huang,Y.F.Li,Anal.Chim.Acta500(2003)105.