Determination of norfloxacin in pharmaceuticals

c 2007 Institute of Chemistry, Slovak Academy of Sciences DOI: 10.2478/s11696-007-0047-y

Determination of Norfloxacin in Pharmaceuticals, Human Serum, and Urine Using a Luminol—Dissolved Oxygen Chemiluminescence System X. D. SHAO, H. Y. LIU, X. F. GAO, W. Q. CHEN, and Z. H. SONG*

Department of Chemistry, Northwest University, Xi’an, 710069, China e-mail: [email protected] Received 2 April 2007; Revised 21 May 2007; Accepted 29 May 2007

A sensitive chemiluminescence method, based on the enhancive effect of norfloxacin on the reaction between luminol and dissolved oxygen in a flow injection system, was proposed for the determination of norfloxacin. The increment of the chemiluminiscence intensity was proportional to the concentration of norfloxacin, giving a calibration graph linear over the concentration from 0.4 ng mL−1 to 400.0 ng mL−1 (r2 = 0.9988) with the detection limit of 0.1 ng mL−1 (3 × σ noise ). At the flow rate of 2.0 mL min−1 , a complete determination of norfloxacin, including sampling and washing, could be accomplished in 30 s with the relative standard deviation lower than 3.0 %. The proposed method was applied successfully to determine norfloxacin in pharmaceuticals, human urine, and serum. Possible mechanism of the reaction was also discussed. Keywords: norfloxacin, chemiluminescence, luminol, serum, urine, flow-injection

INTRODUCTION

O

O

F

Norfloxacin (1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7(1-piperazinyl)quinoline-3-carboxylic acid, Chart 1) with molecular mass 319.24 (C16 H18 FN3 O3 ) is a fluoroquinolone antibacterial agent which exhibits high antimicrobial activity in vitro against a wide variety of gram-negative and gram-positive bacteria, including gentamicin-resistant Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus species [1]. Norfloxacin has a remarkably broad spectrum of activity and excellent pharmacokinetics allowing for oncedaily dosing [2]. It is widely used to treat human and veterinary diseases and also to prevent diseases in animals [3, 4]. Its main excretion pathway is urinary, and low amounts are found also in plasma after a single oral dose of 400 mg norfloxacin [5]. On the other hand, there is concern about the possibility of exposure to low levels of these compounds resulting in the development of resistance of human pathogens to antibiotics [6]. Different methods were employed for the determination of norfloxacin in capsules or biological samples including high performance liquid chromatogra-

OH

N

N

HN

CH3 Chart 1

phy (HPLC) [7—9], spectrophotometry [10], fluorimetry [11], mass spectrometry [12], square-wave adsorptive voltammetry [13], and atomic absorption spectroscopy [14]. Progress in flow-injection (FI) chemiluminescence (CL) analysis has received much attention in pharmaceutical analysis thanks to the high sensitivity, rapidity, and simplicity of this method [15—17].For the determination of norfloxacin, different CL systems have been reported, including Ce(IV)—Na2 S2 O3 , Tb(III)—nitrate, and H2 O2 —luminol ones [18—20]. In our previous work, determinations of risperidone, roxithromycin, and clindamycin using different CL systems were reported [21—23]. However, no report

*The author to whom the correspondence should be addressed.

Chem. Pap. 61 (5) 353—358 (2007)

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353

X. D. SHAO, H. Y. LIU, X. F. GAO, W. Q. CHEN, Z. H. SONG

Pump

Luminol Carrier Flow Cell

Valve

Sample

Detector

NaOH

Mixing Tubing

Recorder Waste

Fig. 1. Schematic diagram of FI-CL for determination of norfloxacin.

on the luminol—dissolved oxygen CL system for the determination of norfloxacin was given so far. It is well known that the reaction between luminol and dissolved oxygen could emit CL [24, 25], this reaction has been applied in different research fields [26—29]. In this study, an accelerating effect of norfloxacin on the CL reaction between luminol and dissolved oxygen was observed. Based on this observation, a simple, sensitive, and rapid procedure was developed for the indirect determination of norfloxacin. The increment of the luminescence intensity was linear with norfloxacin concentration over the range from 0.4 ng mL−1 to 400.0 ng mL−1 with the relative standard deviations (RSD) less than 3.0 % and the detection limit was 0.1 ng mL−1 (3 × σ noise ). At the sample flow rate of 2.0 mL min−1 , a complete determination of norfloxacin, including sampling and washing, could be completed in 30 s, offering the sampling efficiency of 120 h−1 . The proposed procedure was applied successfully in the determination of norfloxacin in drug capsules, eyedrops, human urine, and serum samples without any pretreatment. EXPERIMENTAL A schematic diagram of the CL flow-injection analysis system is shown in Fig. 1. A peristaltic pump was utilized to deliver all flow streams. PTFE tubing (1.0 mm i.d.) was used as connection material in the flow system. A six-way valve with a loop of 100.0 µL was employed for sampling. The flow cell was made by coiling 30.0 cm of colorless glass tube (2.0 mm i.d.) into a spiral disk with the diameter of 2.0 cm and placed close to the photomultiplier tube (PMT) (Model IP28, Hamamatsu). The CL signal induced in the flow cell was detected without wavelength discrimination, and the PMT output was amplified and quantified by a luminosity meter (Model GD-1, Xi’an Remax Electronic Science-Tech. Co. Ltd.) connected to a recorder (Model XWT-206, Shanghai Dahua Instrument and Meter Plant). The maximum of the fast CL emission observed due to the above reaction at 425 nm could 354

be proved by a spectrofluorometer (Model F-4500, Hitachi Co. Ltd.). A UV-VIS spectrophotometer (Model Lambda-40P, PE Co. Ltd.) was used, while the optical path length was 1.0 cm and the wavelength span from 200 nm to 400 nm. All the reagents were of analytical grade and the water used was purified in a Milli-Q system (Millipore, Bedford, MA, USA). A stock solution of norfloxacin (Shaanxi Institute for Drug Control) was stored in a refrigerator (4 ◦C). Working standard solutions were prepared daily from the stock solution by appropriate dilution. Luminol (2.5 × 10−2 mol L−1 ) was prepared by dissolving 4.4000 g luminol (Fluka, Switzerland) in 1.0 L of a 0.1 mol L−1 NaOH solution. As shown in Fig. 1, PTFE tubing was used to conduct sample, luminol, water carrier stream, and sodium hydroxide solutions to the flow cell. Prior to each experiment, the pump was started at a constant flow rate of 2.0 mL min−1 to wash the whole system until a stable baseline was recorded. Then, 100.0 µL of luminol solution was injected into the water carrier stream using an injection valve, and mixed with the sample solution containing norfloxacin. This mixture entered the CL cell, producing CL emission, detected by the PMT and luminometer. The sample concentration was quantified on the basis of the increment of the CL intensity (∆I = Is − Io ), where Is and Io were the CL signals in the presence and in the absence of norfloxacin, respectively. Determination of Norfloxacin Not less than ten capsules of norfloxacin were weighed and ground to a fine powder using a pestle and mortar. The powder was dissolved in water and the resulting solution was filtered through an ordinary filter paper and diluted to the mark in a 100.0 mL brown calibrated flask. Suitable aliquots from this solution were taken for the determination of norfloxacin so that the concentration of norfloxacin was in the working range of its determination. Eyedrops’ sample was prepared by dissolving 0.5


Chem. Pap. 61 (5) 353—358 (2007)

DETERMINATION OF NORFLOXACIN IN URINE AND SERUM USING CHEMILUMINESCENCE

600

Relative CL Intensity

500 400 300 200 100 0 0

3

6

9 Time/s

12

15

18

Fig. 2. Kinetic CL–time profile in static system. CL intensity observed in the absence of norfloxacin (empty circles) and in the presence of 10.0 ng mL−1 of norfloxacin (full circles).

mL norfloxacin eyedrops in 100.0 mL pure water. Suitable aliquots from this solution were taken for the determination of norfloxacin so that the concentration of norfloxacin was in the working range of its determination. Urine samples collected from three volunteers and serum samples supplied by the Hospital of Northwest University were spiked before determination. To prepare the spiked samples, known quantities of standard solution of norfloxacin were spiked into 1.0 mL of urine or serum. After homogenization, dilution with the factor 5.0 × 104 for urine samples and 5.0 × 105 for serum samples, the samples were processed according to the proposed method.

5.0 × 10−5 mol L−1 was selected on the basis of norfloxacin content determination in a series of standard solutions (analyte content from 4.0 × 10−10 g mL−1 to 4.0×10−7 g mL−1 ). Selected optimum luminol concentration was used in all subsequent experiments. Owing to the nature of luminol CL reactions, which are favored in an alkaline medium, sodium hydroxide was introduced into the CL cell through a flow line to improve the detection sensitivity. A series of sodium hydroxide solutions from 5 × 10−3 mol L−1 to 1.0 × 10−1 mol L−1 were tested in the presence of 1.0 × 10−8 g mL−1 norfloxacin and 5.0 × 10−5 mol L−1 luminol. The plot of CL intensity (I ) versus the sodium hydroxide concentration showed the maximum at the sodium hydroxide concentration of about 5.0 × 10−2 mol L−1 .Thus, this concentration was employed in the subsequent experiments. Mixing tubes with the length ranging from 0.5 cm to 10.0 cm were employed in order to asses the length at which maximum CL intensity in the cell is achieved. By comparing the CL intensities measured in the presence of 10.0 ng mL−1 norfloxacin, the most intense signal was detected using tubes with the length of 5.0 cm. Accordingly, this length was selected for the following experiments. The influence of the reactants flow rate on the determination of norfloxacin was examined by investigating the signal-to-noise ratio under different flow rates. The effect of flow rate on the CL intensity was examined in the range from 0.5 mL min−1 to 5.0 mL min−1 . It was found that the flow rate of about 2.0 mL min−1 offered the highest signal-to-noise ratio and, therefore, it was chosen as a suitable condition for the subsequent measurements. Performance of the Detection System

RESULTS AND DISCUSSION Optimization of Conditions for Norfloxacin Determination Kinetics of the reaction of luminol with dissolved oxygen was examined by a static method using 5.0 × 10−5 mol L−1 luminol in a 0.05 mol L−1 sodium hydroxide solution. As shown in Fig. 2, the CL signal reached the maximum intensity 5 s after the reaction was initiated, and then vanished within the following 20 s. Scintillescent CL signal was detected also in the presence of norfloxacin (10.0 ng mL−1 ) and the CL intensity reached maximum 3 s after mixing the reactants, then, the CL signal disappeared within the following 16 s. It was also demonstrated that norfloxacin enhanced the CL reaction and increased the CL intensity importantly. The influence of luminol on the intensity of the CL signal was examined by changing the luminol concentration in solution from 1.0 × 10−7 mol L−1 to 1.0 × 10−4 mol L−1 . An optimum luminol concentration of

Chem. Pap. 61 (5) 353—358 (2007)

100.0 µL of luminol was flow-injected into the system in the presence of 1.0 ng mL−1 , 10.0 ng mL−1 , and 100.0 ng mL−1 norfloxacin solution and the CL intensity ∆I = Is − Io was recorded to test the operational stability of the system. The experiment lasted for 6 days and the flow system was regularly used over 5 h per day. The results of the determination precision at three different concentrations of norfloxacin are listed in Table 1, with the average value of the RSD for the norfloxacin concentrations of 1.0 ng mL−1 , 10.0 ng mL−1 and 100.0 ng mL−1 of 2.88 %, 2.01 %, and 2.02 %, respectively. A series of standard solutions of norfloxacin were injected into the manifold depicted in Fig. 1. The increment of the CL intensity was found to be proportional to the concentration of norfloxacin. It was found that the calibration graph corresponds to a straight line within the norfloxacin concentration range from 0.4 ng mL−1 to 400 ng mL−1 with the detection limit of 0.1 ng mL−1 (3 × σ noise ). The respective regression equation was ∆I = 1.171 × ρnorfloxacin + 38.01 (r2 =


355


Table 1. Precision of Norfloxacin Determination at Three Different Contentsa Norfloxacin nominal content/(ng mL−1 ) Time/day

Io

RSD/% 1.0

10.0

100.0

Is

RSD/%

Is

RSD/%

Is

RSD/%

1 2 3 4 5 6

65 67 65 64 66 65

1.50 1.76 1.32 2.57 3.01 1.78

104 105 101 103 106 105

2.12 4.09 1.98 3.75 2.12 3.24

115 116 115 117 114 115

2.16 1.31 2.54 2.23 1.05 2.78

220 216 223 221 219 221

2.16 1.93 2.21 1.53 2.16 2.12

Average

65 ± 1.0

1.99 ± 0.66

104 ± 1.8

2.88 ± 0.93

115 ± 1.0

2.01 ± 0.69

220 ± 2.4

2.02 ± 0.26

a) Average of five determinations. Table 2. Results of Determination of Norfloxacin Content in Capsulesa Norfloxacin content/(ng mL−1 ) RSD/%

Sample

1 2 3 4 5 6

Content per capsuleb /mg

Added

Found

0 10 0 30 0 30 0 75 0 90 0 120

3.2 12.6 21.4 50.0 52.5 82.5 45.0 127.5 30.0 116.0 37.6 162.4

Recovery/%

1.56 1.32 0.95 0.89 1.83 1.18 1.80 1.68 2.23 2.52 2.23 2.38

Proposed method

UV

94.0

99.2

98.7

95.3

102.7

99.5

100.0

105.0

101.6

110.0

99.0

103.1

95.6

96.0

101.4

104.0

101.5

97.2

a) The average of five determinations. b) Label claim: 100 mg per capsule.

0.9988). At the flow rate of 2.0 mL min−1 , a complete determination of the analyte, including sampling and washing, could be accomplished in 0.5 min with an RSD lower than 3.0 %. Interference of foreign substances was tested by analyzing a standard solution of norfloxacin (1.0 ng mL−1 ) into which increasing amounts of interfering analytes were added. Assuming the interference at the level of 5.0 %, tolerable ratios of foreign species with respect to 1.0 ng mL−1 of norfloxacin were over 2− 3− − − − 10000 for Cl− , NO− 3 , Ac , I , SO4 , PO4 , BrO3 , amylum, glucose, borate, oxalate, malic acid, cephradine, azithromycin, roxithromycin, and levofloxacin; 2+ 5000 for NH+ , Ca2+ , methanol, ethanol, glutin, 4 , Mg Tween-80, and CTMAB; 1000 for Ba2+ , Pb2+ , urea, tartrate, and risperidone; 100 for uric acid; and 50 for Cu2+ , Zn2+ , Ni2+ , Cr3+ , and Fe2+ /Fe3+ . Common excipients, such as agar and cellulose in capsules, caused no obvious interference in the determination of norfloxacin. Determination of Norfloxacin Following the procedure detailed above, the pro356

posed method was applied in the determination of norfloxacin in capsules (Ginwa Enterprise Inc.) from the local market. The results are listed in Table 2 showing that the recovery rate ranged from 94.0 % to 110.0 %. The results are in a good agreement with the results obtained by UV spectrophotometry. Furthermore, six samples of eyedrops (Wuhan Wujing Medicine Co. Ltd.) were examined for norfloxacin by the standard addition method with the recovery rate from 92.1 % to 102.5 % (Table 3). The results obtained by the proposed method agree well with the results obtained by UV spectrophotometry. The proposed CL method was also applied to determine norfloxacin in spiked human urine and human serum at nanogram level. Six spiked urine samples were examined for norfloxacin by the standard addition method. The results of the determination are listed in Table 4. The observed recovery rate ranged from 92.0 % to 108.5 %. In order to evaluate the precision of the proposed method, recovery studies were carried out on samples of serum to which known amounts of norfloxacin were added. The results are presented in Table 5, while the recovery rate of 92.0 % to 108.0 % was found. The concentrations of


Chem. Pap. 61 (5) 353—358 (2007)

DETERMINATION OF NORFLOXACIN IN URINE AND SERUM USING CHEMILUMINESCENCE

Table 3. Results of Determination of Norfloxacin Content in Eyedropsa Norfloxacin content/(ng mL−1 )

1 2 3 4 5 6

Content in eyedropsb /(mg mL−1 ) RSD/%

Sample Added

Found

0 30 0 60 0 60 0 75 0 90 0 120

19.4 47.6 40.7 98.6 14.6 76.1 39.7 115.8 36.7 127.3 29.7 140.2

2.76 1.09 1.81 1.68 1.91 1.60 1.18 0.91 1.77 1.54 1.79 1.32

Recovery/% Proposed method

UV

94.0

2.8

2.9

96.5

2.9

2.9

102.5

3.1

3.2

101.5

3.1

3.0

100.7

3.0

2.9

92.1

2.8

2.7

a) The average of five determinations. b) Label claim: 3 mg mL−1 . Table 4. Results of Determination of Norfloxacin Content in Spiked Human Urinea Norfloxacin content/(ng mL−1 )

1 2 3 4 5 6

Content in urine/(mg mL−1 ) RSD/%

Sample Added

Found

0 10 0 50 0 75 0 75 0 60 0 60

10.0 20.4 28.1 76.6 36.5 113.5 49.3 130.7 69.5 124.7 110.5 167.4

2.82 2.29 2.40 2.29 2.68 1.75 2.29 1.87 1.54 2.07 2.21 2.41


Spiked

104.0

0.50

0.50

97.0

1.41

1.50

102.7

1.83

1.80

108.5

2.47

2.50

92.0

3.48

3.50

94.8

5.53

5.50

a) The average of five determinations. Table 5. Results of Determination of Norfloxacin Content in Spiked Human Seruma Norfloxacin content/(ng mL−1 )

1 2 3 4 5 6

Content in serum/(mg mL−1 ) RSD/%

Sample Added

Found

0 10 0 60 0 75 0 90 0 90 0 120

19.2 30.0 37.8 95.2 52.8 133.3 57.6 140.4 86.8 178.4 86.7 211.1

1.86 2.60 2.79 1.70 2.91 1.58 2.91 2.68 2.56 1.77 2.78 1.77


UV

108.0

9.60

9.50

95.7

18.90

19.00

107.3

26.40

26.00

92.0

28.80

28.00

101.8

43.40

43.00

103.7

43.35

43.00

a) The average of five determinations.

norfloxacin determined by the proposed method were compared with the concentration of the spiked samples, and a good agreement was obtained.

Chem. Pap. 61 (5) 353—358 (2007)

Possible CL Mechanism Possible mechanism of the norfloxacin-enhanced


357


CL reaction was proposed on the basis of static CL and UV spectrophotometry measurements. The CL emission spectrum was examined using an F-4500 spectrofluorometer, showing the maximum CL emission wavelength of 425 nm. This observation affirms that luminol was oxidized to excited 3-aminophthalate by . O2− anion radicals in an alkali medium accompanied by the CL signal emission [24, 25, 30]. Moreover, it was found that the essential difference between the spectra of norfloxacin and luminol—norfloxacin solutions containing the same amount of analyte was the absorption intensity which increased from 0 to 0.4681 at 219 nm (absorption intensity of luminol at 219 nm was 0.1869). Thus, a reaction between luminol and norfloxacin was supposed. In Fig. 2 the CL intensity in the presence of 10.0 ng mL−1 of norfloxacin is compared to that obtained in the absence of this analyte. It was found that the reaction of luminol with dissolved oxygen in the presence of norfloxacin was accelerated from 20 s to 16 s, while the maximum CL intensity was observed in 3 s compared to that detected in the absence of norfloxacin (5 s). Therefore, the possible mechanism of norfloxacin-enhanced luminol—dissolved oxygen CL reaction could include the acceleration of the electron transfer yielding 3aminophthalate excited state capable to emit light. CONCLUSIONS Presented CL method combined with FI technique offers prominent advantages including instrumental simplicity, high sampling efficiency, reduced reagents consumption, analytical sensitivity, and selectivity comparable with the existing methods. The satisfactory performance in the assay of norfloxacin in pharmaceutical preparations and biological fluids demonstrated that the method is practical and suitable not only for quality control analysis but is also applicable in the analyte determination in complex biological samples, confirming the promise to pharmacological and clinical research. Acknowledgements. The authors gratefully acknowledge the financial support from the Shaanxi Province Nature Science Foundation and the Ministry of Education of China, Grants No. 2006B05 and No. 04JK145.

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Chem. Pap. 61 (5) 353—358 (2007)