Accepted Manuscript Silver nanoparticles enhanced flow injection chemiluminescence determination of gatifloxacin in pharmaceutical formulation and spiked urine sample Saikh mohammad Wabaidur, Seikh Mafiz Alam, Zeid A. Alothman PII: DOI: Reference:

S1386-1425(15)00215-2 http://dx.doi.org/10.1016/j.saa.2015.02.051 SAA 13353

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

15 December 2014 5 February 2015 12 February 2015

Please cite this article as: S.m. Wabaidur, S.M. Alam, Z.A. Alothman, Silver nanoparticles enhanced flow injection chemiluminescence determination of gatifloxacin in pharmaceutical formulation and spiked urine sample, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa. 2015.02.051

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1 Silver nanoparticles enhanced flow injection chemiluminescence determination of gatifloxacin in pharmaceutical formulation and spiked urine sample Saikh mohammad Wabaidur1,*, Seikh Mafiz Alam2,*, Zeid A Alothman1

1

Advanced Materials Research Chair, Department of Chemistry, College of Science,

King Saud University, Riyadh 11451, Saudi Arabia 2

Department of Chemistry, Aliah University, West Bengal, India

*Author to whom correspondence should be addressed: E-mail: [email protected] (S. M. Wabaidur); [email protected] (S. M. Alam); Phone: +966545141396; Fax: +96614675992

2 Abstract Silver nanoparticles have been utilized for the enhanced chemiluminogenic estimation of fluoroquinolone

antibiotic

gatifloxacin.

It

has

been

found

that

the

weak

chemiluminescence intensity produced from the reaction between calcein and KMnO4 can further be strengthened by the addition of silver nanoparticles in the presence of gatifloxacin. This phenomenon has been exploited to the quantitative determination of gatifloxacin. Under the optimum experimental conditions, the calibration curves are linear over the range of 8.9×10-9˗4.0×10-6 M, while the limits of detections were found to be 2.6×10-9 M with correlation coefficient value (r2) 0.9999. The relative standard deviation calculated from six replicate measurements (1.0×10-4 M gatifloxacin) was 1.70 %. The method was applied to pharmaceutical preparations and the results obtained were in reasonable agreement with the amount labeled on the formulations. The proposed method was also used for the determination of gatifloxacin in spiked urine samples with satisfactory results. No interference effects from some common excipients used in pharmaceutical preparations have been found. Keywords Silver nanoparticles; calcein; gatifloxacin; chemiluminescence; flow injection analysis.

3 1. Introduction Gatifloxacin (GFLX) chemically named as 1-cyclopropyl-6-fluoro- 8-methoxy-7-(3methylpiperazin-1-yl)-

4-oxo-quinoline-3-carboxylic

acid,

is

an

8-methoxy

fluoroquinolones (FQs) with a 3-methylpiperazinyl substituent at C7 used as antibacterial agent with a broad spectrum of activity against Gram-positive and Gram-negative organism. It’s application is effective in a range of clinical infections, including community-acquired pneumonia, acute exacerbations of chronic bronchitis, acute sinusitis and genitor urinary tract infections [1, 2]. It exhibits enhanced activity against clinically relevant pathogens, including such common respiratory pathogens as Streptococcus

pneumoniae,

Haemophilus

influenzae,

Mycoplasma

pneumoniae,

Chlamydia pneumoniae, Moraxella catarrhalis, and Legionella [3]. Clinical cure rates in all trials of patients treated with GFLX were 90% or higher. Like other new FQs, GFLX has a dual mechanism of action, inhibiting both bacterial DNA gyrase and topoisomerase IV [4]. GFLX is a metabolically stable compound; >80% of the drug is excreted in the urine unchanged. Up to 72h after administration of single-dose GFLX (400mg), the amounts of ethylenediamine and methylethylenediamine metabolites recovered were each 0.03% of the total administered dose [5]. The extensive need for clinical and pharmacological study require fast and sensitive analytical techniques for the determination of their presence in biological and pharmaceutical preparations [6]. Several methods have been reported in the literature for determination of GFLX. Li et al. [7] developed a spectrophotometric method for the determination of GFLX. A first and second derivative spectrophotometric method and validation for the determination of GFLX in bulk and pharmaceutical dosage forms have been reported [8]. The quantitative

4 determination of the drug was carried out using the first derivative values measured at 276 nm and the second derivative values measured at 287 nm (n = 6). A terbiumsensitized spectrofluorometric method using an anionic surfactant, sodium dodecyl benzenesulfonate (SDBS), was developed by Guo et al. [9] for the determination of GFLX. A coordination complex system of GFLX-Tb(III)-SDBS was studied. SDBS significantly enhanced the FL intensity of the complex. A HPLC method was developed for the assay of GFLX in tablets [10]. Motwani et al. [11] has reported a stability indicating high-performance thin-layer chromatographic method for the determination of GFLX. The method employed thin-layer chromatographic aluminum plates precoated with silica gel 60F-254 as the stationary phase and the mobile phase consisted of npropanol-methanol-concentrated ammonia solution. Salgado et al. [12] developed a specific agar diffusion bioassay for the antibacterial GFLX using a strain of Bacillus subtilis ATCC 9372 as the test organism. Except aformentioned method, different others analytical method have also been put forward such as HPLC with diode-array and FL detection [13], thin layer chromatography [14] and chemiluminescence (CL) [15]. In this study, Silver nanoparticles (AgNP) have been utilized in order to quantitative estimation of the essential fluoroquinolone antibiotics, namely, GFLX. AgNPs were prepared based on aqueous-gaseous phase reaction of silver nitrate solution and ammonia gas [16, 17]. Few other methods for synthesis of AgNP were reported in the literature by reduction of silver nitrate with other drug, including ascorbic acid, [18, 19], b-D-glucose [20], heparin with glucose in the presence of n-hexadecyltrimethylammonium bromide [21, 22] and adrenaline [23]. AgNPs were found to enhance intensity of the CL reaction between calcein and KMnO4. AgNP displayed a good catalysis effect, by which the CL

5 intensity of calcein-KMnO4 was strongly increased in the presence of GFLX. Based on this phenomenon, a flow-injection CL assay was developed to detect GFLX in pharmaceutical formulations and biological samples.

2. Chemicals and sample preparations 2.1. Chemicals and reagent All chemicals were of analytical reagent grade and were used without further purification. Distilled deionized (DI) water (Millpore, MilliQ Water System, USA) was used throughout. GFLX were purchased from Sigma-Aldrich (St. Louis, USA). Stock solutions (1.0×10−3 M) of GFLX was prepared in deionized water. Working solutions of desired concentrations were freshly prepared by appropriate dilution of each stock solution with DI water. Calecin was purchased from Sigma Corporation (Steinheim, Germany). The 1.0×10−4 M working solution of calcein was prepared by dissolving 0.0310 g of calcein and then diluting to 500 ml with DI water Silver nitrate and ammonia solution were purchased from Sigma (Louis, USA). 2.2. Sample preparations 2.2.1. Pharmaceutical drug Sample solutions for analysis were prepared as follows. The average tablet weights were calculated from the weight of each of 10 tablets which were selected from the same group randomly. An accurately weighed portion of each homogenized sample containing 400 mg of GFLX (Gatizen) (Ulticare, Alkem Laboratories Ltd, Mumbai, India) was transferred into 1000 ml calibrated dark flask containing 500 ml of water and dissolved in ultrasonic bath for 20 min and diluted with DI water to mark. The dissolved sample was

6 filtered through Millipore membrane filter paper (MF-Millipore™, 0.8µm pore size and 150 µ m thickness, USA) and diluted with water to volume to obtain the appropriate concentration for analysis. 2.2.2. Urine sample collection Blank urine samples were kindly provided by several volunteers of ages between 25 to 45 years. Immediately after collection, 25 ml aliquots of urine samples from 5 volunteers were spiked with GFLX at variable concentration levels, in order to calculate the recoveries of the proposed method. From these pools, each 0.5 ml aliquots were distributed to 0.5 ml Eppendorf and stored at -18ºC until analysis. 2.3 Preparation of NP AgNP were prepared as follows with modifications [24]. The synthesis was based on the aqueous-gaseous phase reaction of silver nitrate solution and ammonia gas [16]. To a 100 ml two neck round bottom flask 50 ml of silver nitrate solution (1.0×10-3M) was added and the flask was placed into a constant temperature oil bath on a magnetic stirrer. 50 ml ammonia solution (1 M) was added to another 500 ml flask kept in a water bath at room temperature. The flasks were connected with glass tubes through which ammonia gas volatilized and diffused slowly into the flask of silver nitrate. Ammonia solution then reacted with silver nitrate. The whole system was exposed to the light of daylight lamp. AgNP were prepared in the five steps: (1) Silver nitrate containing flask was kept under stirring (~700C oil bath) for 6 hours, (2) settling the flask for 6 hours without stirring and heating, (3) following step 1 for 5 hours, (4) Repeating the step 2, (5) following step 1 for 3 hours. 2.4 Analytical procedure

7 The flow injection analysis (FIA) configuration consisted of a three-channel manifold using two pumps. For calcein based CL system, prior to the CL measurement acquisition, calcein stream was mixed with KMnO4 solution stream in a three-way “T1” connector. Pump P1 delivers AgNPs solution which is then mixed with sample solution through injection valve. The two streams were merged in the second “T2” connector and then reached the flow cell in the fluorimeter, accompanying the increase of CL intensity. The increase in the CL intensity produced when a solution containing the AgNP was incorporated into the carrier stream, in relation to the original CL signal corresponding to a blank, was proportional to the sample concentration and was used as analytical signal. FlA system for both the two systems was schematically shown in Figure 1.

3. Results and discussion 3.1. TEM image and UV spectral characteristics of AgNPs The TEM image of AgNP shows that the average size is about 13 nm and the NP dispersion is good, which is shown in Figure 2 [16, 17]. The UV-vis absorption spectrum of the obtained AgNP is provided in Figure 3. There is an absorption peak near 400 nm, corresponding to the characteristic wavelength of silver absorbance. The narrow absorption peak indicates a high level of monodispersity of the AgNP [25, 26]. 3.2. Optimizastion of experimental parameters 3.2.1. Effect of oxidizing agents on Calcein-KMnO4 system In order to investigate the effect of oxidizing agents on CL intensity, the effects of four different oxidizing agents on the CL intensity were investigated. The oxidizing agents tested in this study were KMnO4, K3Fe(CN)6, KIO3, Ce(SO4)2, KBrO3. 1.0×10−4 M

8 solutions of the following oxidants were prepared in 1.0×10−2 M H2SO4: KMnO4, Ce(SO4)2, KBrO3 and KIO3, and K3 Fe(CN)6 was made in 0.1 M NaOH. The experimental results obtained are shown that the CL intensity was strongly dependent on oxidizing agents. The highest CL intensity was observed when KMnO4 was used. Hence KMnO4 was chosen for further experimentation. 3.2.2. Calcein concentration To find the optimum concentration of calcein used for the chemiluminogenic determination of GFLX, the effect of calcein concentration on CL intensity was investigated. The CL intensity was examined over the calcein concentration in the range from 1.0×10-6 to 1.0×10-3 M. The concentration of GFLX was kept at 1.0×10-4 M. The results indicate that the CL intensity of the calcein-KMnO4 system is strongly dependent on calcein concentration (Fig. 4). The CL intensity increased upto 1.0×10-4 M and then become almost constant. Hence 1.0×10-4 M was chosen as optimum calcein concentration for the determination of GFLX. The increased CL intensity with increasing calcein concentration can be attributed by the higher CL reaction rate of calcein. 3.2.3. KMnO4 concentration In order to find the optimum concentration of KMnO4 used for the chemiluminogenic reaction for the determination of GFLX, the effect of KMnO4 concentration on the CL intensity was investigated. KMnO4 was utilized as the oxidant in this CL system. As the oxidant, the concentration of KMnO4 could affect the CL intensity of the systems and the corresponding experiments were carried out under the fixed amount of 1.0×10-4 M calcein and the variable concentration of KMnO4 in the range of 1.0×10-6–1.0×10-3 M. The concentration of GFLX was kept at 1.0×10-4 M. The experimental results show that

9 the CL intensity increased upto 7.0×10-4 M and then started to decrease gradually (Fig. 5). Hence 7.0×10-4 M was chosen as optimum concentration of KMnO4 for the determination of GFLX. 3.2.4. NaOH concentration The CL reaction of calcein-KMnO4 performs in alkaline medium. The alkalinity of reaction medium was adjusted by varying the concentration of NaOH in potassium permanganate solution. The concentration of GFLX was kept 1.0×10-4 M. The effect of NaOH concentration on the CL reaction was examined in the range 1.0×10-3–1.0 M. The concentration of the other reagents was kept constant. The highest CL intensity was observed when the concentration of NaOH was 0.05 M. Hence 0.05 M chosen was optimum NaOH concentration for future experiment. 3.2.5. Flow-rate The flow rate of reagent solutions was studied, keeping all other conditions constant, over the range 1.0–4.5 ml/min, with equal flows in each channel. The results obtained show that 2.2 ml/min, is the best total flow rate for GFLX determination. 3.2.6. AgNP concentration The effect of concentration of AgNP on CL reaction of calcein was investigated. The concentration used for GFLX was 1.0×10-4 M. The concentration of calcein was selected as 1.0×10-4 M. The FL intensity was examined over the AgNP concentration range from 1.0×10-4 to 1×10-3 M. The results showed that the suitable concentration of AgNP was 5.0×10-4 M for both the analytes. Further increase in the NPs concentration results in decrease of the intensity. This might be because when the concentration of AgNP was too

10 high, the interactions among particles were strong and the CL energy was transferred among particles which were in small distance. 3.3. CL spectra of the systems The CL intensity of calcein-KMnO4 system in the absence of and in the presence of GFLX was recorded with the emission monochromator, respectively, and the obtained CL curves were shown in Figure 6. The experimental results indicated that calceinKMnO4 reaction system has an ultra-weak CL. Adding the analytes into the system can largely enhance its CL intensity and the value of enhancement is proportional to the concentration of the substance added. By this property, GFLX can be determined sensitively with CL method. On addition, once the AgNPs were added into the calceinKMnO4 system, the CL intensity was further intensified by several folds. 3.4. Possible CL reaction mechanism The mechanism of the proposed calcein–potassium permanganate (KMnO4) CL reaction can be described as follows. Firstly, KMnO4 reacts with calcein in presence of NaOH and releases energy. Then the unreacted calcein present in the solution absorbs the energy and enters into the excited state (calcein*). Then the excited calcein returns to the ground state, accompanied by CL emission. The mechanism can be expressed simply as: Potassium permanganate + calcein + OH− → Products + energy (E)

(1)

Calcein + E → Calcein*

(2)

Calcein* → Calcein + hν (λmax = 545 nm)

(2)

3.5. Analytical features

11 In order to obtain calibration curves for GFLX, sets of standard solutions were used and CL spectra were recorded. Calibration curve for GFLX run under the optimum conditions such as [calcein] = 1.0×10-4 M, [KMnO4] = 6.2×10-4 M, [NaOH] = 0.05 M, [AgNP] = 5.0×10-4 M, was obtained by using a series of eight standard solutions. The calibration curves was found to be linear over the concentration of GFLX 8.9×10-9˗ 4.0×10-6M (r2=0.998). The detection limit was 2.6×10-9 M, while the relative standard deviation (RSD) (n=6) for 1.0×10-4 M of GFLX was 1.7%. 3.6. Interfernce studies In a real sample, the analyte under investigation will be in the presence of interferents. They may suppress or enhance the synchronous signal, although they have no significant effect on the intensity. If interference occurred, the concentration was progressively reduced until interference disappeared. The tolerance level was defined as the amount of foreign species that produce an error not exceeding ±5 in the determination of the analyte. The effect of some foreign compounds and ions on the assay of GFLX was studied by analyzing synthetic sample solutions containing 1.0×10-5 M of GFLX together with various excess amounts of these compounds. The results are shown in the Table 1. From the table it could be seen that most interferents were found to show no influence on GTFX determination by proposed CL method. 3.7. Analytical applications In order to investigate the validity of the proposed method, the commercial pharmaceutical formulation Gatizen was analyzed (containing 400 mg of GFLX). The contents of 10 tablets or finely ground tablets were weighed and mixed. An equivalent amount of one tablet was weighed and dissolved it in de-ionized water with shaking for

12 20 min in an ultrasonic bath. The solution was filtered through Millipore membrane filter, and the filtrates were diluted to the 1000 ml flask. Appropriate dilution was made from this solution to meet the linear range. The results obtained are given in Table 2. As can be seen from the table, overall recovery of GFLX was about 95.0–98.2%. Thus, it could be concluded that, there were no significant differences between the labeled contents and those obtained by the proposed CL method. The proposed method was also applied to the determination of GFLX in spiked human urine samples. The results are shown in Table 3. The percentage recovery of GFLX was found to be between 97.0% and 98.0%. From the presented results, it can be concluded that the proposed procedure is sufficiently sensitive, selective and simple for the determination of GFLX in urine sample.

4. Conclusions In the proposed method AgNPs were prepared by aqueous-gaseous phase reaction of silver nitrate solution and ammonia gas. AgNP was found to enhance the CL intensity of the reaction between calcein and KMnO4. The weak CL from the reaction of calcein– KMnO4 was further strengthened by the addition of silver nanoparticle in the presence of GFLX. This phenomenon was exploited to the quantitative determination of GFLX. Under the optimum conditions, the calibration curves are linear over the concentration range 8.9×10-9˗4.0×10-6 M for GFLX. The limit of detection was found to be 2.6×10-9 M. The correlation coefficient for the calibration curves for GFLX was 0.9999. The relative standard deviation calculated from six replicate measurements was 1.70 % for 1.0×10-4 M GFLX. The method was successfully applied to the determination of GFLX in

13 pharmaceutical formulations and urine samples. No interference effects were observed from some common excipients used in pharmaceutical preparations. The proposed work expands the field of analytical application of AgNP.

Acknowledgements The authors extend their appreciation to the Deanship of Scientific Research, College of Science Research Center, King Saud University, Riyadh, Saudi Arabia for supporting this project.

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16 Figure caption: Fig. 1. Schematic diagram of the FIA CL manifold employed for the quantitative determination of GFLX. P1, P2: Peristaltic pumps; T1, T2: Y- pieces. Fig. 2. TEM image of AgNP. Fig. 3. UV-vis spectrum of AgNP. Fig. 4. Effect of calcein concentration on the CL intensity for the quantitative analysis of GFLX (1.0×10-4M). Fig. 5. Effect of KMnO4 concentration on the CL intensity for the quantitative analysis of of GFLX (1.0×10-4 M). Fig. 6. CL spectra for the quantitative analysis of GFLX; (1) calcein-KMnO4 system, (2) calcein-KMnO4-GFLX system and (3) calcein-KMnO4-GFLX-AgNP system; Conditions: [GFLX] = 1.0×10-5 M, [calcein] = 1.0×10-4 M, [KMnO4] = 7.0×10-4 M, [NaOH] = 0.05 M, [AgNP] = 5.0×10-4 M.

17

Fig. 1. Schematic diagram of the FIA CL manifold employed for the quantitative determination of GFLX. P1, P2: Peristaltic pumps; T1, T2: Y- pieces.

18

Fig. 2. TEM image of AgNP

19

Fig. 3. UV-vis spectrum of AgNP

20

Fig. 4. Effect of calcein concentration on the CL intensity for the quantitative analysis of GFLX (1.0×10-4M)

21

Fig. 5. Effect of KMnO4 concentration on the CL intensity for the quantitative analysis of of GFLX (1.0×10-4 M).

22

Fig. 6. CL spectra for the quantitative analysis of GFLX; (1) calcein-KMnO4 system, (2) calcein-KMnO4-GFLX system and (3) calcein-KMnO4-GFLX-AgNP system; Conditions: [GFLX] = 1.0×10-5 M, [calcein] = 1.0×10-4 M, [KMnO4] = 7.0×10-4 M, [NaOH] = 0.05 M, [AgNP] = 5.0×10-4 M.

23 Table 1. Maximum tolerable concentration ratios with respect to 1.0×10-5 M GFLX for quantitative determination Foreign species Foreign species : GFLX Na+, K+ ,NH4+

500

Al3+, Fe3+, Co2+

1000

Fructose, glucose

50

Sucrose, dextrin, ephedrine, galactose

45

24 Table 2. Assays of GFLX acid in pharmaceutical formulations by AgNP enhanced calcein-KMnO4 CL system. Sample

Gatizen

b

Active ingredient labeled 400 mg of GFLX

Mean of three measurements

Found±RSDb

Added (×10-7M)

Found (×10-7M)

Recovery (%) mean±RSDb

382.7±0.31

2.0

1.91

95.0±1.6

4.0

3.93

98.2±2.1

6.0

5.88

98.0±2.4

25 Table 3. Assays of GFLX acid in spiked urine samples by AgNP enhanced calceinKMnO4 CL system. Sample

GFLX

b

Added

Found

Recovery (%)

(×10-7M)

(×10-7M)

mean±RSDb

2.0

1.89

94.5±1.7

4.0

3.87

96.7±2.2

6.0

5.82

97.0±2.5

Mean of three measurements

26 Silver nanoparticles enhanced flow injection chemiluminescence determination of gatifloxacin antibiotics in pharmaceutical formulation and spiked urine sample Saikh mohammad Wabaidur1,*, Seikh Mafiz Alam2,*, Zeid A Alothman1 1

Advanced Materials Research Chair, Department of Chemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia 2 Department of Chemistry, Aliah University, West Bengal, India

CL spectra for the quantitative analysis of GFLX; (4) calcein-KMnO4 system, (5) calcein-KMnO4-GFLX system and (6) calcein-KMnO4-GFLX-AgNP system; Conditions: [GFLX] = 1.0×10-5 M, [calcein] = 1.0×10-4 M, [KMnO4] = 7.0×10-4 M, [NaOH] = 0.05 M, [AgNP] = 5.0×10-4 M.

27 ►Precise and reliable method was developed for the determination of gatifloxacin ►Silver nanoparticle was used as chemiluminescence enhancer ►This method is easily performed and affords good precision and accuracy ►The sensitivity of this technique does not affected by coexisting ions/compounds ►The method was successfully applied to analysis of drug and spiked urine