INTRODUCTION:

Ofloxacin (OFX), ±9-Fluoro-2, 3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido [1, 2, 3-de]-1, 4-benzoxazine-6-carboxy-lic acid is a member of quinolone class of antibacterial drugs. The bactericidal action results from the inhibition of DNA synthesis. This effect is due to the inhibition of bacterial DNA gyrase (topoisomerase II), an enzyme responsible for introducing negative supercoils into circular duplex DNA. OFX is absorbed rapidly after oral administration. It is widely distributed in body fluids, including CSF, and tissue penetration is good. A small amount of the dose is metabolised to desmethyl Ofloxacin (moderate activity) and N-oxide Ofloxacin. Excretion is mainly via the kidneys with around 80% of the dose being excreted as the unchanged drug in urine over 24 to 48 h and between 4 to 8% being excreted in faeces. It is broad-spectrum antimicrobial agent for oral administration which is active against both gram positive and gram negative bacteria.

OFX has been approved for the treatment of infections of the lower respiratory tract, including chronic bronchitis and pneumonia, caused by Gram- negative bacilli [1].

Literature survey revealed that there are number of methods for quantitation of OFX in biological fluid viz. HPLC with UV detection [2], HPLC with fluorescence detection [3-5], HPLC UV with using solid phase extraction [6], and HPLC with solid phase spectrofluorimetry [7]. Few methods have also been reported to determine the levels of OFX in biological fluids in combination with lomifloxacin [8]. HPTLC method has been reported for determination of OFX in bulk and pharmaceutical dosage form [9-12] and Spectrophotometric method for simultaneous estimation of OFX and other drugs [13-15]. To the best of our knowledge, no High Performance Thin Layer Chromatographic (HPTLC) method has been reported for determination of OFX in human plasma.

The present method describes a simple, selective and sensitive HPTLC method with a calibration range of 50-600 ng/band for OFX in human plasma. The method utilizes liquid-liquid extraction using chloroform as the sample preparation technique. Mirtazepine was used as an internal standard (IS). The mobile phase employed was mixture of n-butanol: ethanol: ammonia (5:5:4 v/v/v). The method was validated as per EMEA and CDER guidelines on validation of bioanalytical methods [16,17].

MATERIAL AND METHODS:

Chemicals and reagent :

OFX working standard was kindly supplied by Arbro Pharmaceuticals Ltd., Delhi and Mirtazapine (IS) by Matrix Laboratories, Hyderabad. The drugs were used as such without further purification. The structures for OFX and IS are described in Figure 1. AR grade methanol, ammonia, ethanol and chloroform were purchased from S. D.fine-chemical Laboratories, Mumbai, India.

(A)

(B)

Figure 1: Chemical structures of Ofloxacin (A) and Mirtazapine (B)

Instrumentation:

Chromatographic separation of drugs was performed on aluminium plates precoated with silica gel 60 F₂₅₄, purchased from E-Merck, Germany. Samples were applied on the plate as a band with 4 mm width using Camag 100 µl sample syringe (Hamilton, Switzerland) with a Linomat 5 applicator (Camag, Switzerland). Linear ascending development was carried out in a twin trough glass chamber (10 x 10 cm) at room temperature and a densitometric scanning was performed using Camag TLC scanner 3 in the range of 400-200 nm, operated by winCATS software (Version 1.4.3, Camag). Deuterium lamp was used as a radiation source. All weighing was done on Shimadzu balance (Model AY-120).

Chromatographic conditions:

Samples were applied on the 10 cm x 10 cm plate as band with a width of 4 mm and slit dimensions were kept as 3.00 x 0.45 mm. 25 μl volume of each sample was applied on TLC plate. Chamber saturation time was 15 minutes and the migration distance was 80 mm.

Sample preparation:

Stock solutions for OFX and IS were prepared by separately dissolving in methanol, to obtain a concentration of 1 mg/ml. Working solution for OFX was prepared by diluting suitably with methanol to get the concentration of 12.5 to 150 μg/ml. The stock solutions were stored at 4⁰ C. Working solution for IS was prepared by diluting suitably stock solution of IS with methanol to get the concentration of 125 μg/ml.

Q.C. (Quality Control) samples for OFX were prepared at concentration levels so as to get 100, 300 and 500 ng/band. Spiked plasma was prepared by taking 4.8ml plasma, 0.1ml solution of OFX and 0.1 ml stock solution of IS(125 μg/ml) were added. The contents of the tubes were vortexed for 3 min. 2ml of this spiked plasma was taken, 1ml phosphate buffer (pH7) and 5ml chloroform were added, vortexed for 5min. It was centrifuged for 10 minutes at 2500 rpm. After centrifugation, Chloroform layer was separated and transferred to separate test tubes, evaporated on a waterbath maintained at 60⁰C and reconstituted in 0.5ml methanol. 25μl of this was applied on TLC plate.

The calibration curve for OFX was obtained using seven calibration standard levels (50, 100,200, 300, 400, 500, 600ng/band). Linear regression analysis was done, considering the ratio of the peak area of analyte to internal standard versus concentration applied. A correlation coefficient of more than 0.99 was obtained for calibration curve.

Method Validation:

Selectivity:

Selectivity is the ability of an analytical method to differentiate and quantify the analytes in the presence of other components in the sample. The selectivity of the method was evaluated by analyzing pooled plasma samples obtained from different sources spiked at LLOQ (Lower Limit of Quantification - 50 ng/band corresponding to 2 μg/ml in plasma).

Calibration/standard curve:

Linearity was tested for the range of concentrations 50-600 ng/band. Each sample in five replicates was analyzed and peak areas were recorded. The response factor for each concentration was calculated by taking ratio of peak area of OFX and IS. The response factors were then plotted against the corresponding concentrations to obtain the calibration graphs.

Accuracy, precision and lower limit of quantification:

The accuracy and precision of the method were evaluated using the Q.C. samples. Intra-day accuracy and precision was measured by consecutively analyzing Q.C. samples in one single day. The procedure was repeated for three different days to test the inter-day accuracy and precision. Accuracy was calculated as percentage accuracy, whereas precision was measured in terms of relative standard precision (R.S.D.) of each calculated concentration. Lower limit of quantification (LLOQ) was found to be 50 ng/band, since the response obtained was five times the response compared to blank.

Recovery:

Recovery for OFX was evaluated at three concentration levels corresponding to three routine Q.C. samples (100, 300 and 500 ng/band) analyzed in triplicate. Recovery was determined by comparing the ratio of the peak area of OFX obtained after the application of the processed plasma calibration samples with those achieved by working standard solution in the methanol.

Stability:

As per EMEA and US CDER guidelines, stability was checked under different conditions viz.

1. Freeze- thaw stability

2. Short term stability

3. long term stability

4. Stock solution stability

5. Post preparative stability

Freeze-thaw stability of OFX was determined by assaying low and high Q.C. samples (100 and 500 ng/band) in triplicate over three freeze-thaw cycles. First freeze-thaw cycle consisted of 24 hrs freezing at -5⁰ C followed by a complete thaw at a room temperature. The next two freeze-thaw cycles were of 12 hrs each frozen state at -5⁰ C followed by a complete thaw at a room temperature. Short term stability consisted of two Q.C. samples stored for 4 hrs at room temperature and long term stability involved storage of two Q.C. samples for 14 days at 4⁰ C. For stock solution stability, the stock solutions of the drug and IS were stored for period of 5 days in refrigerator at 4⁰ C and then for 6 hrs at room temperature. Post preparative stability, where stability of the spiked samples for 300 ng/band of OFX and 500 ng/band of IS were determined after the storage for 5 hrs at room temperature. All these Q.C. samples were then evaluated in triplicate and the results were compared with the freshly prepared samples of same concentrations.

RESULTS:

Chromatographic characteristics:

Retention factor for OFX and IS were found to be 0.53 ± 0.03 and 0.82 ± 0.03 respectively. Representative densitograms of blank human plasma, blank plasma spiked with OFX (200 ng/band) and IS (500 ng/band) with OFX (200 ng/band) after Liquid- liquid extraction (LLE) are shown in figure 2, 3 and 4 respectively.

Figure 2: Densitogram of blank human plasma using LLE

Figure 3: Densitogram of human plasma spiked with ofloxacin 200 ng/band (Rf-0.53 ± 0.03) using LLE

Figure 4: Densitogram of human plasma spiked with ofloxacin, 200 ng/band (Rf-0.53 ± 0.03) and IS 500ng/ band (0.82± 0.03) using LLE

Selectivity:

The selectivity of the method was evaluated by analyzing pooled plasma samples obtained from different sources spiked at LLOQ (50 ng/band) in which no interference by endogenous components was noted. % RSD (Relative standard deviation) for 6 replicates spiked at LLOQ was found to be 4.31%.

Calibration/standard curve:

The data for linearity studies was found to be best fitted by linear equation y = mx + c in the range of concentration 50-600ng/band. The correlation coefficient was 0.996, with mean slope of 0.0012 and a mean y-intercept of 0.1354.

Accuracy, precision and lower limit of quantification:

The method showed good accuracy and precision in plasma samples. Table 1 shows the results for intra- and inter-day precision and accuracy for OFX in plasma samples. Intra- and inter-day (%R.S.D.) precisions were 2.90 ± 0.94 and 2.57 ± 0.34 respectively. Intra- and inter-day accuracies were 100.86 ± 0.58 and 100.18 ± 0.2501 respectively. LLOQ was found to be 50 ng/band (corresponding to 2 μg/ml in plasma).

Table 1: Intra-day, inter-day precision and accuracy of OFX in human plasma QC samples

Theoretical (ng/band)	Observed (mean ng/band ± SD)	Precision (%R.S.D.)	Accuracy (%)
Intra-day
100	101.1 ± 1.15	2.22	101.1
300	303.9 ± 3.04	2.51	101.3
500	501.0 ± 1.58	3.98	100.2
Average		2.90 ± 0.94	100.86 ± 0.58
Inter-day
100	99.93 ± 3.39	2.54	99.93
300	300.5 ± 2.09	2.24	100.19
500	502.15 ± 1.67	2.93	100.43
Average		2.57 ± 0.34	100.18±0.2501

% R.S.D. = SD/mean x 100, accuracy = observed/theoretical x 100

RECOVERY:

Table 2 shows the results of the recovery tests for the three Q.C. levels of OFX tested (100, 300 and 500 ng/band). The mean recovery for OFX was found to be 98.76%.

Table 2: Recovery of OFX and IS in human plasma Q.C. samples

Added (ng/band) (OFX)	% R.S.D. (OFX)	Recovery (%) (OFX)
100	1.30	98.16
300	1.37	98.83
500	1.03	99.29
Average	1.23%	98.76%

% R.S.D. = SD/mean x 100

Stability:

Plasma Q.C. at two concentrations (100 and 500 ng/band) was used for freeze-thaw, Short term and long term stability studies. Stock solution stability was performed at three concentrations (100, 300,500 ng/band).Post preparative stability was performed for the drug (300 ng/band) and IS (500 ng/band). It was performed to evaluate the influence of storage conditions from the sample collection to analysis. Table 3 represents the results of stability studies. Results indicated that OFX is stable in human plasma for the given stability conditions. The deviation of the mean test responses to the freshly prepared solutions was less than 5% at any of the stability condit

Table 3: Stability of OFX in human plasma Q.C. samples

Stability	Conc. (ng/band)	Mean Stability (%)	% R.S.D.
Freeze thaw stability (three cycles)	100	99.35	2.7
Freeze thaw stability (three cycles)	500	98.29	1.53
Short term stability (for 4h at RT)	100	98.43	1.61
	300	98.64	1.48
	500	98.66	1.14
Long term stability (for 14 days at 4⁰ C)	100	98.43	1.6
	300	98.61	1.15
	500	99.44	1.94
Stock solution stability (for 5 days at 4⁰ C, 6hrs at RT)	100	99.43	1.53
	300	99.64	1.94
	500	99.18	1.80
Post preparative stability (for 5hrs RT)	300	98.56	1.44
Post preparative stability (for 5hrs RT)	500 (IS)	99.84	2.11

% RSD = SD/mean x 100, RT (room temperature)

DISCUSSION :

Most published methods to quantify OFX in body fluids use tedious extraction, purification steps and sometimes evaporation under nitrogen stream. In this study, rapid and sensitive HPTLC method has been developed for the determination of OFX in human plasma by simple liquid-liquid extraction technique in which evaporation under nitrogen stream is not required. Validation results proved that the developed method performs well with selectivity, precision, accuracy, stability and linearity for the concentration range of OFX expected to be found in human plasma after oral administration of 200-400mg dose. The validated method covers the wide range of linearity over 50-600 ng/band and is therefore suitable for the determination of OFX in human plasma at different therapeutic dose levels. The mean recovery of OFX was found to be 98.76% .The separation between OFX and endogenous substances was satisfactory. HPTLC technique, offers advantage of high throughput. As compared to HPLC methods, the present method is economical, simple and fast. The proposed method can be used for therapeutic drug monitoring in order to optimize drug dosage on an individual basis. The developed method is able to measure concentration of OFX to monitor drug concentration in body fluid, determination of drug level in plasma for dose regulation and bioavailability.

ACKNOWLEDGEMENT:

Authors are thankful to Arbro Pharmaceuticals Ltd., Delhi and Matrix Laboratories, Hyderabad for providing gift samples of Ofloxacin and Mirtazapine respectively. Authors are also thankful to Akshay Blood Bank, Pune for providing pooled human plasma and the Management, AISSMS College of Pharmacy, Pune, India for providing required facilities to carry out research work.

ABBREVIATIONS:

IS: internal standard, OFX: Ofloxacin, LLOQ: lower limit of quantification, QC: quality control, SD: standard deviation, RSD: relative standard deviation, RT: room temperature, EMEA: European medicines agency, CDER: center for drug evaluation and research, LLE: Liquid liquid extraction.

REFERENCES :

1. Moffat A, Osselton MD and Widdop B. Clarke's Analysis of Drugs and Poisons. Pharmaceutical Press, London, UK. 2005; 3rd Ed.

2. Amini M, Abdi K, Darabi M. Determination of Ofloxacin in plasma by HPLC with UV detection. Journal of Applied Sciences. 2005; 5:1655-1657.

3. Fabre D, Bressolle F, Kinowski J M, Bouvet O and Paganin F. A Reproducible simple,and sensitive HPLC assay for determination of Ofloxacin in plasma and lung tissue. Application in pharmacokinectic studies. Journal of Pharmaceutical and Biomedical Analysis. 12(11); 1994: 1463-1469.

4. Garcia M A, Solans C, Calvo A, Royo M, Hernandez E. HPLC Seperation and quqntitation of Ofloxacin enantiomers in rabbit plasma. Application to pharmacokinetic studies. Chromatographia. 56(1-2); 2002: 39-42.

5. Gracia M et al. Analysis of ofloxacin in plasma samples by high performance liquid chromatography. Chromatograhia. 56(7-8); 2002: 43.

6. Mustarichie R, Indriyati W and Sopyan L. Ofloxacin analysis validation method in human plasma (in vitro) using solid phase extraction HPLC. Medical and Health Science Journal. 8; 2011:80-87.

7. Ballesteros, Vlchez J, Alberto N. Determination of the antibacterial ofloxacin in human urine and serum samples by solid-phase spectrofluorimetry. Journal of Pharmaceutical and Biomedical Analysis. 30; 2002: 1103- 1110.

8. D Zendelovska and Stafilov T. Development and validation of high-perfomance liquid chromatographic method for determination of ofloxacin and lomefloxacin in human plasma. J. Serb. Chem. Soc. 70 (12); 2005: 1451–1460.

9. Khandagle K, Gandhi S, Deshpande P and Gaikwad N. High Performance Thin Layer Chromatographic determination of Cefixime and Ofloxacin in combined tablet dosage form. International Journal of Pharmacy and Pharmaceutical Sciences. 3;2011:46-48.

10. Premanand D,Senthilkumar K, Senthilkumar B, Saravanakumar M and Thirumurthy R. A Validated RP-HPLC Method for Simultaneous Estimation of Nitazoxanide and Ofloxacin in Pharmaceutical Formulation. Der Chemica Sinica. 1 (2); 2010: 1-5.

11. Kasabe A, Shitole V, Waghmare V, Mohite V. Simultaneous Estimation of Metronidazole and Ofloxacin in Combined dosage form by Reverse Phase High Performance Liquid Chromatography Method. International Journal of ChemTech Research. 1(4); 2009: 244-1250.

12. Khandagle K, Gandhi S, Deshpande P, Kale A and Deshmukh P, A simple and sensitive RP HPLC method for simultaneous estimation of cefixime and ofloxacin in combined tablet dosage form. Journal of Chemical and Pharmaceutical Research .2(5); 2010: 92-96.

13. Patel S and Patel S. Development and Validation of First Order Derivative Spectrophotometric method for simultaneous estimation of Ofloxacin and Cefpodoxime Proxetil in tablet dosage form. Journal of Pharmaceutical Science and Biopharmaceutical Research. 1(2); 2011: 108-112.

14. Kumar A, Nanda S and Chomwal R. Spectrophotometric estimation of Cefixime and Ofloxacin in tablet formulation. Journal of Chemical and Pharmaceutical Research. 3(5); 2011: 705-709.

15. Patel N, Aravdiya A and Desai H. Development and validation of two spectrophotometric methods for simultaneous estimation of metronidazole and ofloxacin in suspension. International Journal of Science and Research. 2(12); 2011: 3202-3206.

16. European Medicines Agency.Guideline on validation of Bioanalytical method, London, Nov. 2011.Guidance for Industry; Bioanalytical Method Validation.

17. U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Veterinary Medicine (CVM) May 2001 BP.

Received on 12.04.2012 Modified on 22.04.2012

Research J. Pharm. and Tech. 5(5): May2012; Page 682-686