a lanthanide sensitized chemiluminescence method of flow-injection for the determination of...

ISSN 1061-9348, Journal of Analytical Chemistry, 2009, Vol. 64, No. 1, pp. 75–81. © Pleiades Publishing, Ltd., 2009.

75

1

Prulifloxacin (PUFX), 6-fluoro-1-methyl-7-(4-[5-methyl-2-oxo-1, 3-Dioxolen-4-yl] methyl-1-piprazi-nyl)-4-oxo-4H-(1,3) thiaceto (

3,2-

α

) quinolone-3-Car-boxylic acid (Fig. 1b), is the prodrug of ulifloxacin (UFX,Fig. 1a). Ulifloxacin is a new broad-spectrum antibacterialagent that belongs to the family of fluoroquinolones.Fluoroquinolones

FQs

have gained global popularity inclinical practices owing to their potent activity against awide range of gram-positive and gram-negative bacteriaand good pharmacokinetic profile [1]. With the increasinguse of these antibiotics, the change in their chemical struc-ture to improve antibacterial activity and to avoid side-effects is necessary. The chemical structure of ulifloxacincontains the skeletal quinolone with a four-member ring inthe 1,2-position including a sulfur atom to increase anti-bacterial activity [2]. Ulifloxacin has potent and broad-spectrum antibactrrial activity, but unluckily, it has apoor oral absorption. So, in order to overcome this disad-vantage, an oxodioxolenylmethyl group is added to the7-piperazine ring, and then prulifloxacin is formed. Afteroral administration, prulifloxacin is immediately metab-olized and quantitatively transferred to ulifloxacin, theactive moiety, to play antibacterial activity [3]. There-fore, prulifloxacin exists in orally pharmaceutical prepa-rations, but the metabolite product, ulifloxacin, exists inbiological fluids.

Because fluoroquinolones have been widely used inclinics, many methods have been reported for the deter-mination of these synthetic agents either in pharmaceu-

1

The text was submitted by the authors in English.

tical preparations or in biological fluids; the most fre-quently employed is HPLC [4–7]. Other methodsinclude spectrophotometry [8, 9], fluorimetry [10–12],voltammetry [13, 14], capillary elcetrophoresis [15,16], and chemiluminescence (CL) [17–19]. However,reported analytical literature about the determination ofulifloxacin and prulifloxacin is seldom seen. There area few papers that describe HPLC methods [20, 21] forthe analysis of ulifloxacin in biological fluids. In ourstudy, we found that the oxidation-reduction reactionbetween KMnO

4

and Na

2

S

2

O

4

can produce very weakCL signal. The separate introduction of UFX (PUFX)and Tb(III) ion could increase the CL signal, but notvery obviously. The simultaneous addition of UFX(PUFX) and Tb(III) ion could enhance the CL signalgreatly. Based on this phenomenon, a flow-injection(FI) lanthanide sensitized chemiluminescence methodto determine UFX and PUFX was developed. The asso-ciation of chemiluminescence with the use of lan-thanide ion has offered some attractive characteristics,such as high sensitivity, wide dynamic ranges, largeStokes shift, narrow emission bands and long lifetimeof the luminescence. The proposed method was appliedsuccessfully to the determination of UFX in spikedhuman serum and urine and PUFX in tablets. On thebasis of experimental phenomena, the possible mecha-nism of the sensitized CL reaction was also discussed.

ARTICLES

A Lanthanide Sensitized Chemiluminescence Method of Flow-Injection for the Determination of Ulifloxacin

and Prulifloxacin

1

X. L. Wang

a

, A. Y. Li

a,b

, H. C. Zhao

a

, and L. P. Jin

a

a

College of Chemistry, Beijing Normal University, Beijing 100875, China

b

Department of Basic Theory, Shandong Institute of Physical Education, Ji’nan 250063, China

Received October 18, 2006; in final form, May 19, 2008

Abstract

—A lanthanide sensitized chemiluminescence method of flow-injection was developed for the deter-mination of a new fluoroquinolone, ulifloxacin (

UFX

), and its prodrug prulifloxacin (

PUFX

). The proposedmethod was based on the remarkable chemiluminescence enhancement effect of UFX (PUFX) on KMnO

4

–Na

2

S

2

O

4

–Ln(III). Tb(III) ion was chosen from lanthanides because it showed the best sensitizing effect. Underoptimized experimental conditions, the relative chemiluminescence intensity was in linear relationship withUFX and PUFX concentrations in the ranges of 1.0

×

10

–8

−

5.0

×

10

–6

M and 9.0

×

10

–9

−

5.0

×

10

–6

M, respec-tively. The minimum detectable value and relative standard deviation were 5.5

×

10

–9

M, 1.5% for UFX and7.0

×

10

–9

M, 2.9% for PUFX, respectively. The proposed method was applied to the determination of UFX inspiked human serum and urine, and of PUFX in tablets with satisfactory results. The possible mechanism ofchemiluminescence was also proposed.

DOI:

10.1134/S1061934809010158

76

JOURNAL OF ANALYTICAL CHEMISTRY

Vol. 64

No. 1

2009

WANG et al.

EXPERIMENTAL

Apparatus.

The flow-injection CL analyzer systemused for the determination of UFX and PUFX is illus-trated schematically in Fig. 2. It consists of two basicparts, flow-injection analyzer and luminescence detec-tor. FI-2100 flow-injection system (Beijing HaiguangInstrument Company, China) was used as the flow-injection analyzer. It contains two parts, i.e., two peri-staltic pumps, which are used to drive the reactants atthe same flow rate (70

r

/min), and a sixteen-way injec-tion valve with a sample loop (25-cm for both UFX andPUFX). A PTFE tube (0.8-mm i.d.) was used to connectall reagents in the flow system. The CL signal was mea-sured by a BPCL ultra-weak luminescence detector(Institute of Biophysics, Academia Sinica, China). Thesensitivity of the photomultiplier tube depends on thevalue of the negative voltage (0–1000 V) used. CLspectra were recorded with a RF-5301 PC spectrofluo-

rimeter (SHIMADZU, Japan), which was obtainedwith the Xe lamp off. Kinetic characteristics of the CLsystem were performed on the BPCL ultra-weak lumi-nescence analyzer.

Reagents.

All reagents used were of analyticalgrade. Double-distilled water was used throughout theexperiment. UFX (Beijing Synthesis Sky ChemicalTechnology Co. Ltd., China) standard stock solution(

5.0

×

10

–4

M) was prepared by dissolving 17.4 mg UFXin 1 M NaOH solution and diluting with water to100 mL. PUFX (Shenyang Jvying PharmaceuticalTechnology Co. Ltd., China) standard stock solution(

1.0

×

10

–3

M) was prepared by dissolving 23.1 mgPUFX in 0.2 M HCl and diluting to 50 mL. Standardstock solution of the Tb(III) ion (

1.0

×

10

–2

M) was pre-pared by dissolving 934.5 mg Tb

4

O

7

in 15.0 mL of HCl(11.6 M) at

100°

C, evaporating the solution to bealmost dry, then diluting it to 500 mL with water. Fur-ther diluted solutions were freshly prepared by dilutingthe stock solution. KMnO

4

solution and Na

2

S

2

O

4

solu-tion were prepared daily according to the required con-centration.

Procedures.

As shown in Fig. 2, flow lines (A, B, Cand D) were connected with UFX (PUFX), Tb(III) ion,Na

2

S

2

O

4

and KMnO

4

solutions, respectively. All solu-tions were continuously pumped into the manifold atthe same flow rate (about 2.2 mL/min) by two peristal-tic pumps. A certain amount of UFX (PUFX) solution(

120

µ

L) was injected into the Tb(III) carrier stream bythe sixteen-way injection valve every 10 s, then mergedwith Na

2

S

2

O

4

before being mixed with KMnO

4

solu-tion. All of these solutions were mixed at M. The fullymixed solution flowed through a 15-cm PTFE tube, andthen was transferred into the CL flow cell, and gave riseto an intensive CL signal immediately. The CL signalwas then recorded by BPCL ultra-weak luminescencedetector. Peak height was measured for each signal.Calibration graphs were constructed by plotting the rel-ative CL intensity (sample output minus blank) versusthe concentration of UFX (PUFX).

Tablets

sample

preparation

. Five PUFX tablets(provided by the Hefei Con-source Medicine Technol-ogy Corp., China) were weighed and calculated to

NN

F

O

COOH

S

CH3N

H

NN

F

O

COOH

S

CH3NO

O

O

CH3

CH2

(a) (b)

Fig. 1.

Chemical structure of ulifloxacin (a) and prulifloxacin (b).

P1

P2

V

M

HV

PMT Rec

W

F

W

D

C

B

A

Fig

. 2.

The

schematic

diagram

of

the

flow

-

injection

CL

an-alyzer

.

A

–

UFX

(

PUFX

);

B

–

Tb

(

III

)

ion

;

C

–

Na

2

S

2

O

4

;

D

−

KMnO

4

;

P

1,

P

2 –

peristaltic

pump

;

V

–

sample

injec-tion

valve

;

M

–

mixed

cell

;

W

–

waste

;

HV

–

high

voltage

;

PMT

–

photomultiplier

tube

;

Rec

–

recorder

; the rate of thetwo pumps is 70 r/min.

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 64 No. 1 2009

A LANTHANIDE SENSITIZED CHEMILUMINESCENCE METHOD 77

obtain the average mass per tablet. Then, the tabletswere finely ground to homogenized powder. An amountof the powder equivalent to 100 mg of PUFX wasweighted accurately and transferred into a small beaker,in which 0.2 M HCl was added to dissolve the powder.The solution was filtered and the residue was washedseveral times. The filtrate was then transferred into a250 mL volumetric flask and diluted to the scale withwater. The injection solution was directly diluted withwater so that the final concentration was within theworking range.

Serum and urine samples preparation. Blank serumand urine samples were spiked with proper amounts ofUFX stock solution according to the reported literature[2]. Serum samples needed deproteinization to elimi-nate the interference of proteins by adding 10% trichloro-acetic acid (CCl3COOH), then centrifugation for 15 minat 4000 r/min was performed. The centrifugate wasdiluted with water to a suitable concentration. The urinesamples needed no other pretreatments, and were dilutedwith water only.

RESULTS AND DISCUSSION

The kinetic characteristics of the CL reaction.The kinetic characteristics of the CL reaction wereexamined as displayed in Fig. 3 (taking UFX as exam-ple). The results showed that reaction was rapid and theCL intensity reached its peak 5 s after the reactionstarted, then decreased to a base line in less than 20 s.

Effect of flow rate and sample volume. The effect ofthe flow rate of reagent solutions was studied, whilekeeping all other conditions unchanged. It was foundthat the relative CL intensity increased with increasingflow rate. However, at too high flow rate, not only weremore reagents needed, but also the precision becamepoor. On account of these considerations, a flow rate of2.2 mL/min (the pump rotation speed was 70 r/min)was recommended. The sample volume also plays animportant role in the variables. The results showed thatthe relative CL maximum could not show up wheneverthe sample volume was too small, or too large. Themaximum relative intensity of CL appeared if theinjected sample volume was 120 µL, so this sample vol-ume was fixed.

Selection of sensitizer and the effect of its concen-tration. The KMnO4–Na2S2O4 system could only gener-ate very weak CL signal. When UFX or PUFX wasadded, the CL emission was enhanced a little, and stillvery weak. Some trivalent lanthanide ions and fluores-cence compounds (1.0 × 10–4 M) were tested as possible

sensitizers for the CL system of KMnO4–Na2S2O4–PUFX, the results are listed in Table 1. It can be seenfrom Table 1 that Tb(III) ion gets the best result. Similarresults were obtained for the KMnO4–Na2S2O4–UFXsystem. So, Tb(III) ion was chosen as the sensitizer insubsequent work. The effect of Tb(III) ion concentra-tion on the relative CL intensity was also studied in therange of 5.0 × 10–5–8.0 × 10–4 M, the relative CL signalenhances with increasing of Tb(III) ion concentrationwhen the concentration is below a certain value (4.0 ×10–4 M for UFX, 5.0 × 10–4 M for PUFX). At higher con-centration, the relative CL intensity decreases slowly.Hence, the Tb(III) ion working concentration was fixedat 4.0 × 10–4 M for UFX and 5.0 × 10–4 M for PUFX.

Effect of KMnO4 concentration. The concentrationof KMnO4, as the oxidant of the CL reaction, is a vitalfactor that affects the relative CL intensity. So, a studywas carried out in 2.0 × 10–5–6.0 × 10–4 M of KMnO4.For both UFX and PUFX, the maximum signalappeared when a 1.0 × 10–4 M KMnO4 solution wasused. Therefore, this concentration was used for subse-quent experiments.

Effect of Na2S2O4 concentration. Na2S2O4 is thereductant in the CL reaction, and the relative CL inten-sity also depends on its concentration. The effect ofNa2S2O4 concentration was studied in the range of 1.0 ×10–4–8.0 × 10–4 M for UFX and 2.0 × 10–4–1.0 × 10–3 Mfor PUFX. The relative CL intensity for UFX increasedevidently with the increase of Na2S2O4 concentration inthe beginning, the peak signal appeared when theNa2S2O4 concentration was 4.0 × 10–4 M, after which anotable decrease emerged. Regarding PUFX, the simi-

4000

3000

2000

1000

0 5 10 15 20Time, s

Inte

nsity

Fig. 3. Kinetic curve of the KMnO4–Na2S2O4–Tb(III)-UFX CL reaction. Conditions: KMnO4 – 1.0 × 10–4 M,Na2S2O4 – 4.0 × 10–4 M, Tb(III) ion – 4.0 × 10–4 M,UFX − 1.0 × 10–7 M.

Table 1. The effect of different sensitizers on relative CL intensity

Senditizer Tb(III) Eu(III) La(III) Sm(III) Dy(III) Rhodamine B Acridine orange

Relative CL Intensity 5297 1652 58 1030 770 –33 290

78


WANG et al.

lar result was obtained, but the maximum relative signalwas achieved when using 6.0 × 10–4 M Na2S2O4. There-fore, in the further measurements, 4.0 × 10–4 M Na2S2O4was selected for UFX and 6.0 × 10–4 M Na2S2O4 forPUFX.

Effect of acidity. The acidity of the solutions playsa considerable role in the CL reaction. The effect of dif-ferent acidity on the relative CL intensity was testedthrough regulating pH of KMnO4 solution by addingHCl or NaOH. The obtained results reveal that, withregard to UFX, the optimal pH is 5.4 and no HCl orNaOH was needed to add, but as for PUFX, the best pHis 5.8, thus 1.0 × 10–5 M NaOH in KMnO4 was used.

Interference studies. The influence of some metalions in human body, some common excipients used inpharmaceutical preparations and some organic com-pounds, on the relative CL intensity was investigatedfor the determination of 1.0 × 10–7 M UFX (PUFX).The experiments were performed by comparing the CLsignals obtained using UFX (PUFX) solution alone andUFX (PUFX) with foreign substances added. A foreignspecies is considered to interfere if it produces an errorgreater than ±5% in the determination of UFX (PUFX).Since UFX exits in biological fluids and PUFX exits inpharmaceutical preparations, so different kinds of for-eign species were tested for UFX and PUFX. As sum-marized in Table 2, most foreign species have no inter-ference, but some do. Since the content of proteins inhuman serum is very high, proteins will interfere in thedetermination of UFX in serum. Thus, a deproteiniza-tion procedure was performed by adding trichloroaceticacid in serum samples before determination. However,the interference of these substances in urine can be min-imized by diluting the urine samples properly, as thesespecies become much less common and do not interferewhen diluted. Thus, urine samples were used withoutpretreatments. The excipients used in tablets, such asstarch and dextrin, could be removed by filtration.

Analytical characteristics. Under optimum condi-tions described above, the calibration graphs for thedetermination of UFX and PUFX were conducted. Theresults are given in Table 3.

The minimum detectable value according IUPAC[22] is 5.5 × 10–9 M for UFX and 7.0 × 10–9 M forPUFX. The relative standard deviations (RSD) were 1.5and 2.9% for 11 determinations of 1.0 × 10–7 M UFXand PUFX, respectively. An example of output peaks isgiven in Fig. 4.

Table 2. Tolerance of coexisting substances

Analyte Substance Concentration ratio, substance/analyte

Change of relative CL intensity, %

UFX K+ 1000 +1.64

Na+ 1000 –2.56

1000 +3.43

Mg2+ 1000 –1.83

Ca2+ 150 +4.98

Zn2+ 20 +4.90

Cu2+ 10 +3.95

Co2+ 10 –2.75

Ni2+ 10 –3.72

Pb2+ 10 +4.88

Mn2+ 10 –3.50

Fe3+ 100 –4.55

Hemoglobin 50 +3.24

Myoglobin 20 +2.28

Vitamin B1 1.5 –3.56

β-Alanine 20 –4.02

Starch 20 –2.11

PUFX β-CD 20 –3.86

Glucose 50 –2.37

Note: Concentration of Tb(III) ion, 1.0 × 10–4 M; KMnO4, 1.0 ×10–4 M; Na2S2O4, 4.0 × 10–4 M for UFX, 6.0 × 10–4 M for

PUFX; UFX, 1.0 × 10–7 M; PUFX, 1.0 × 10–7 M; pH, 5.4 forUFX, 5.8 for PUFX.

NH4+

Table 3. Analytical parameters for the determination of UFX and PUFX

Analyte Linear range, ×10–6 M Linear regression equation*, ×10–8 M Correlation coefficient, r ×10–9 M

UFX 0.01–1.0 ∆I = 282.73c – 186.62 0.9992 5.5

1.0–5.0 ∆I = 361.47c – 10791 0.9987

PUFX 0.009–0.1 ∆I = 390.81c – 230.61 0.9986 7.0

0.1–5.0 ∆I = 303.91c + 1127.1 0.9998

Note: * ∆I is the relative CL intensity. ** Minimum detectable value.

cmin** ,



Determination of PUFX in tablets. The proposedmethod was applied to the determination of PUFX intablets. The labeled contents and the experimentalresults are summarized in Table 4. Recovery was alsoperformed and ranged from 96.9 to 98.3%.

Determination of UFX in spiked serum and urinesamples. It was reported [2] that after oral administra-tion PUFX was rapidly absorbed. The maximum con-centration of UFX in the serum was between 1 µg/mL(2.87 × 10–6 M) and 1.6 µg/mL (4.59 × 10–6 M) after asingle oral administration of 300, 450 and 600 mgPUFX, while the concentrations of UFX in urine werevery high up to 48 h after dosing, reaching more than60 µg/mL (1.72 × 10–4 M).

Following the previously described procedure, themethod was applied to the determination of UFX inspiked human serum and urine samples. Deproteiniza-tion was carried out for serum samples. Diluting pro-cesses were required for all the samples to make theultimate concentrations of UFX in the linear range. Thestandard addition method [23] was used in determina-tions. The results are summarized in Tables 5 and 6.

Possible mechanism. It has been reported that thechemiluminescence of the permanganate−sulfite sys-tem arises from excited sulfur dioxide (S ) [24]. Adismutation reaction for Na2S2O4 can take place and

generate S in water. Thus, we deduce that thechemiluminescence of KMnO4–Na2S2O4 also arisesfrom S Since the luminescence efficiency of Sis low, the CL intensity is very weak. In our experi-ments, UFX (PUFX) or Tb(III) ion was added to thechemiluminescence system of KMnO4–Na2S2O4,respectively, and both resulted in an enhancement onthe chemiluminescence intensity, but neither was nota-ble, suggesting that energy transfer in both cases

O2*

O32–

O2*. O2*

occurs, but not effectively. However, when we addedTb(III) ion and UFX (PUFX) simultaneously to the sys-tem of KMnO4–Na2S2O4, a conspicuous increase in thechemiluminescence intensity was observed. Thechemiluminescence spectra of KMnO4–Na2S2O4–Tb(III)–UFX and KMnO4–Na2S2O4–Tb(III)–PUFX sys-tems are shown in Fig. 5. The emission peaks of bothsystems are located at 490, 545, 585 and 620 nm, whichare the characteristic fluorescence peaks of terbium,indicating clearly that the excited Tb(III) ion is theemitter. Thus, we conclude that UFX (PUFX) gainedenergy through intermolecular energy transfer fromS followed by intramolecular energy transfer from[UFX*–Tb] ([PUFX*–Tb]) to [UFX–Tb*] ([PUFX–Tb*]) [25, 26].

O2*,

2470

Time, s

2170

1870

1570

1270

970200 40 60 80 100 120 140

Cou

nt

2

01

3

4

5

6

Fig. 4. The example of output peaks for different concentra-tions of UFX0 – Blank solution; 1 – UFX 1.0 × 10–8 M; 2 – UFX 2.0 ×10–8 M; 3 – UFX 4.0 × 10–8 M; 4 – UFX 6.0 × 10–8 M;5 – UFX 8.0 × 10–8 M; 6 –UFX 1.0 × 10–7 M.

Table 4. Results of the determination of PUFX in tablets

Labeled, mg

Found, mg

PUFX added,

nM

PUFX found,

nM

Recovery, %

RSD, %, (n = 5)

69.4 67.2 96.9 1.6

100 96.7 34.7 34.1 98.3 3.5

104.0 102.7 97.9 2.4

160

Wavelength, nm

120

80

40

0450 500 550 600 650

Inte

nsity

1

2

Fig. 5. The chemiluminescence spectra of following ob-jects:1 – KMnO4–Na2S2O4–Tb(III)–UFX; 2 – KMnO4–Na2S2O4–Tb(III)–PUFX.Conditions: UFX – 5.0 × 10–5 M; PUFX – 1.0 × 10–4 M;Tb(III) ion – 5.0 × 10–4 M; KMnO4 – 1.0 × 10–4 M;Na2S2O4 – 4.0 × 10–4 M.

80


WANG et al.

CONCLUSIONS

This paper first reports a flow-injection lanthanidesensitized chemiluminescence method for the determi-nation of ulifloxacin, a new fluoroquinolone and its pro-drug, prulifloxacin. From the experimental point ofview, the manipulation of the proposed method is verysimple; the apparatus is much cheaper than those ofother methods, such as HPLC. The ultra-weak chemilu-minescence analyzer combined with the flow-injectionsystem and the use of Tb(III) ion sensitizer provide sen-sitive and precise results for the measurement. Themethod also shows good results in applications to thedetermination of prulifloxacin in tablets and ulifloxacinin spiked human serum and urine samples.

ACKNOWLEDGMENTS

This project was supported by the National NatureScience Foundation of China (20331010).

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n n

ın

Table 5. Results of the determination of UFX in spiked hu-man serum samples

Serum sample

Added, ×10–7 M

Found, ×10–7 M

Recovery,%

RSD, %, ( n = 5)

1 20.00 21.04 105.2 3.2

2 10.00 9.33 93.3 1.9

3 8.00 7.54 94.2 2.0

4 6.00 5.62 93.7 1.7

5 4.00 4.06 101.4 3.9

Table 6. Results of the determination of UFX in spiked human urine samples

Urine sample

Amount added, ×10–5 M

Amount found, ×10–5 M

Added, ×10–8 M

Found, ×10–8 M Recovery, % RSD, %, ( n = 5)

1 10.00 10.70 4.00 4.30 107.5 1.7

8.00 8.49 106.1 1.8

10.00 10.60 106.0 2.3

2 8.00 8.39 4.00 4.22 105.5 2.5

6.00 6.28 104.1 3.8

8.00 8.41 105.1 1.9

3 5.00 5.12 4.00 4.12 103.0 1.9

6.00 6.19 103.2 2.2

8.00 8.08 101.0 4.1



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a lanthanide sensitized chemiluminescence method of flow-injection for the determination of...

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