1.0 INTRODUCTION:

Gemifloxacin mesylate is a synthetic broad-spectrum antibacterial agent for oral administration. Gemifloxacin, a compound related to the fluoroquinolone class of antibiotics, is available as the mesylate salt in the sesquihydrate form. Chemically, gemifloxacin is (R,S)-7- [(4Z)-3-(aminomethyl)-4-(methoxyimino)-1-pyrrolidinyl]-1-cyclopropyl-6-fluoro-1, 4-dihydro-4-oxo-1, 8-naphthyridine-3-carboxylic acid.

The mesylate salt is a white to light brown solid with a molecular weight of 485.49. Gemifloxacin is considered freely soluble at neutral pH (350 µg/mL at 37ºC, pH 7.0). Its empirical formula is C₁₈H₂₀FN₅O₄•CH₄O₃S and its chemical structure is^1-3

It is not official in any pharmacopoeia^4-8 and till now ,few liquid chromatography procedures have been reported for the determination of Gemifloxacin and their metabolites in biological fluids . Mr E. Doyle has reported rapid determination of Gemifloxacin in Human Plasma by HPLC –Tandem Mass spectrometry ^9-10.So there is still need of simple, sensitive method for analyzing the same in plasma . This paper describes a method of analyzing Gemifloxacin in plasma, which is very and simple as well as sensitive using UV detector. The method was validated according to procedures and acceptance criteria based on FDA guidelines and recommendations of ICH.

2.0 EXPERIMENTAL:^14-18

2.1 Instrumentation: The waters LC system equipped with 2489 pump and 2487 UV detector was used .The output signal was monitored and integrated using waters Empower 2 software

2.2 Solutions:

2.2.1 Preparation of pH 3.0 buffer solution:

10mL of Triethylamine was taken in 1000mL of milli-Q water and mixed well , pH of the solution was adjusted to 3.0±0.05 with ortho Phosphoric acid.

2.2.2 Preparation of Mobile phase:

A mixture of pH 3.0 buffer , Acetonitrile and Methanol in the ratio 75:17:8(v/v/v) was prepared and filtered through 0.45µm nylon membrane filter prior to use.

2.2.3 Preparation of 0.1% Ammonia in water: 0.1ml of Ammonia is dissolved in 100ml of water and mixed well

2.2.4 Preparation of Standard Stock solution (2mg/ml): About 20mg of Gemifloxacin(GEMI) working standard was accurately weighed and transferred in to a 10mL volumetric flask and dissolved in 5ml of methanol sonicated to dissolve the material and made upto volume with methanol and mixed well.

2.2.5 Preparation of Intermediate Standard solution: 1.5ml of standard stock solution was taken into 10ml volumetric flask and made upto volume with water and mixed well.

2.2.6 Preparation of Internal Standard solution (1mg/ml): 10mg of Venlafaxine was accurately weighed and transferred into 10ml volumetric flask and dissolved in 5ml of methanol and made upto volume with water and mixed well.

2.2.7 Preparation of Sample: sample was prepared by using Solid phase extraction technique.25µl of internal standard solution was admixed with 0.5ml of sample in ria vial to it 0.5ml of 0.1% Ammonia water is added .1ml of this premixed sample was loaded onto each strata extraction cartridge which was kept for equilibration .After loading sample on to cartridge, cartridge was washed with 5% methanol and drained out by applying vacuum . Finally the sample was collected in to a suitable vessel by selecting mobile phase as eluent.

2.2.8 Chromatographic Conditions:

A Purospher, RP18e( 150 x 4.6 mm, 5 µm packing) column was used for analysis at column Temperature 35ºC .The mobile phase was pumped through the column at a flow rate of 1.2mL/min. The Sample injection Volume was 50µL.The UV detector was set to a wavelength of 273 nm for the detection.

2.3 Method development: ^11-13

To develop a suitable and robust LC method for the determination of Gemifloxacin in plasma different mobile phases and columns were employed to achieve the best separation and resolution. The method development was started with a C18 column using a mobile phase of pH 3.0 buffer: Acetonitrile: Methanol in the ratio 50:35:15.Early elution was observed . Further studies were conducted using a mobile phase of pH 3.0 buffer: Acetonitrile : Methanol in the ratio 75:10:15.In the above condition elution was very broad for Gemifloxacin. Finally the mobile phase consisting of pH 3.0 buffer: Acetonitrile: Methanol in the ratio 75:17:8 was found to be appropriate ,allowing good separation and symmetrical peak at a flow rate of 1.2mL/min using Purospher, RP18e( 150 x 4.6 mm, 5 µm packing) column. The Chromatogram of Gemifloxacin sample spiked in plasma using the proposed method is shown in Fig. 2.In the proposed method the resolution is more than 2 between the Gemifloxacin and internal standard. Gemifloxacin and its related compounds show significant UV absorbance at Wavelength 273 nm .Hence this wavelength has been chosen for detection in the analysis of Gemifloxacin.

2.3.1 Column Selection:

Based on the retention time and separation Purospher, RP18e (150 x 4.6 mm, 5 µm packing) column was selected as suitable column for the analysis of Gemifloxacin in Human plasma.

3.0 Method Validation:¹⁹

The developed bioanalytical method for determination of Gemifloxacin in Human plasma was Extensively validated as per FDA guidelines and recommendations of ICH.

Stock solutions of Gemifloxacin and internal standard were prepared in acetonitrile and kept at -20°. Working solutions were prepared by diluting the stock solutions of standard with mobile phase. For calibration curve eleven different concentrations (50, 100, 150, 300, 600, 900, 1200, 1800, 2100, 2400 and 3000ng/ml) in plasma were prepared by adding required volume of working solution of analyte to blank plasma. For internal standard the final concentration in plasma was 25µg/ml. The plasma samples were subjected to the sample preparation procedure and injected onto HPLC. Plasma calibration curve was prepared by taking area ratio of analyte to internal standard as Y-axis and concentration of analyte (ng/ml) as X-axis. Six replicates of calibration curve were prepared taking each concentration for six times.

3.1 Extraction recovery:

The extraction recovery of analyte was determined by measuring the peak areas of the drug from the prepared plasma quality control samples. 150 ng/ml, 900 ng/ml and 2100 ng/ml plasma samples were taken as LQC (low quality control), MQC (medium quality control) and HQC (high quality control) samples respectively. The peak areas of extracted LQC, MQC and HQC were compared to the absolute peak area of the unextracted samples containing the same concentration of the drug as 100%. To obtain good extraction efficiency the extraction recovery of Gemifloxacin was determined using five replicates of each QC samples.

3.2 Accuracy and precision:

Precision and accuracy were also determined from LQC (150 ng/ml), MQC (900 ng/ml) and HQC (2100 ng/ml). Five replicates of each concentration were analyzed on the same day to determine the within-run accuracy and precision of the method. To confirm the between-run accuracy and precision five replicates of each concentration on the first day and four replicates of each concentration on second day and third day were analyzed.

3.3 Stability study:

The stability of Gemifloxacin in plasma was evaluated with four studies; a short-term stability study, a long-term stability study, a freeze thaw study and stability in processed sample. Plasma blank samples were spiked with Gemifloxacin at concentration of 150 ng/ml (LQC), 900 ng/ml (MQC) and 2100 ng/ml (HQC) and each concentration was carried out for five times. Plasma samples were extracted and subsequent HPLC analysis was carried out as described previously.

Short-term stability test was performed at room temperature. Plasma samples spiked with Gemifloxacin were kept at room temperature for 6 h, extracted and then analyzed. The long term stability study was carried out with plasma blank samples spiked with Gemifloxacin, which were stored -20° and they were analyzed periodically 2months against a standard curve prepared on the analysis day. For freeze thaw stability spiked samples were analyzed immediately after preparation and on a daily basis after repeated freeze thaw cycles at -20° on three consecutive days. Finally the stability in the processed sample ready for injection was determined at two level of concentration, 150 ng/ml (LQC) and 2100 ng/ml (HQC). The processed QC samples ready for injection were kept for 6h before HPLC analysis.

3.4 The Lower limit of quantitation (LLOQ) and limit of detection (LOD):

The Lower limit of quantitation (LLOQ) and Limit of detection (LOD) were determined from the peak signal and noise level (S/N) as ten and three times the baseline noise, respectively.

3.5 Pharmacokinetic parameters:

The above mentioned bioanalytical method was used in bioequivalence study of Gemifloxacin. It was an open, randomized crossover study to assess relative bioavailability of Gemifloxacin in twelve healthy male volunteers following single dose administration of Gemifloxacin 320 mg tablet. Test preparation was Gemifloxacin 320 mg tablet manufactured by Orchid Healthcare. Tablet Factive containing 320 mg of Gemifloxacin, manufactured by Oscient Pharmaceuticals was used as Reference preparation. The pharmacokinetic parameters like area under the plasma-concentration–time curve from time zero to the last measurable Gemifloxacin sample time and to infinity (AUC_0-tand AUC_0-inf), maximum concentration (C_max), time to maximum concentration (T_max), elimination rate constant (K_el) and elimination half-life (t_1/2) were determined for the period of 0 to 24h.

RESULTS AND DISCUSSION:

Representative chromatogram of blank plasma and plasma spiked with Gemifloxacin and internal standard are shown in figs. 1 and 2, respectively. Retention time for the Gemifloxacin and internal standard were 6.710 min and 9.444 min, respectively. No interfering peaks at these times were found in the chromatogram obtained from blank plasma. Good separation and baselines with low background noise were observed. The chromatographic run time was 15 minutes for plasma sample analysis. The linearity of the calibration curve was evaluated by calculating the r²(regression coefficient) value. The standard curves of Gemifloxacin in human plasma were linear over the ranges of 50 to 3000 ng/ml and the regression coefficients (r²) were over 0.998 from each standard curve of six separate runs.

The limit of detection defined as three times the base noise was 10 ng/ml for this analytical method and the lower limit of quantitation defined as ten times the base noise was 50 ng/ml.

Within- and between-run precision and accuracy of the method was assessed by analyzing the QC samples spiked with known amount of Gemifloxacin according to the procedure described in the previous section. Results are shown in Table 1 and 2. The accuracy of this bioanalytical method for within- and between-run was from 92.53 to 107.94% and from 96.54 to 104.36%, respectively. The %RSD within- and between-run precision ranged from 1.44 to 7.44% and from 5.84 to 7.84%, respectively.

TABLE 1: WITHIN-RUN ACCURACY AND PRECISION OF THE ANALYTICAL METHOD FOR GEMIFLOXACIN

Q. C. Samples	LQC	MQC	HQC
Concentration spiked (ng/ml)	150	900	2100
Concentration found	144.97	939.724	2131.828
S.D.	8.174	66.331	162.231
C.V.%	5.639	7.059	7.610
% Nominal	96.644	104.414	101.516

LQC, MQC, HQC are low quality control, medium quality control, high quality control samples of 150, 900 and 2100 ng/ml. Data obtained are average of five observations (n= 6) where SD means Standard Deviation, CV% (precision) means coefficient of variation calculated as (SD/Mean concentration found)×100 and% nominal means accuracy which is calculated as (Concentration found/ Concentration spiked)×00.

TABLE 2: BETWEEN-RUN ACCURACY AND PRECISION OF THE ANALYTICAL METHOD FOR GEMIFLOXACIN

Q. C. Samples	LQC	MQC	HQC
Concentration spiked (ng/ml)	150	900	2100
Concentration found	150.582	932.918	2210.448
S.D.	4.694	20.480	50.435
C.V.%	3.168	2.215	2.305
% Nominal	100.388	103.055	104.588

LQC, MQC, HQC are low quality control, medium quality control, high quality control samples of 150, 900 and 2100 ng/ml. Data obtained are average of 6 observations (n= 6) where SD means Standard Deviation, CV% (precision) means Coefficient of Variation calculated as (SD/Mean concentration found) ×100 and % nominal means accuracy which is calculated as (Concentration found/ Concentration spiked) ×100.

The recovery of Gemifloxacin from plasma was determined in accordance with the method described in the previous section. The recoveries (mean) of Gemifloxacin from plasma were found to be 84.150% at 150 ng/ml, 93.105% at 900ng/ ml and 100.626% at 2100 ng/ml.

The stability of Gemifloxacin in plasma was determined under various conditions according to the procedure described in the earlier section. Short-term stability test performed at room temperature showed that three QC samples were stable for 48h (mean recoveries were 102.473%, and 94.711% at LQC and HQC, respectively).

The long- term stability results indicated that Gemifloxacin samples were stable for a period of 2 months, with an average recovery of 94.08%. No significant decrease of Gemifloxacin concentration in plasma was detected after exposing samples to three freeze/thaw cycles and mean recovery was found to be 95.079% and 94.251% at LQC and HQC, respectively. Finally, the stability in the processed sample ready for injection was also determined. Result showed that three QC samples were stable at least for 6h with loss not higher than 10%.

The above mentioned bioanalytical method was used in the plasma analysis of a bioequivalence study of Gemifloxacin as described in the earlier section. Fig.3 shows the mean plasma level of Gemifloxacin for test and reference preparation after the oral administration of a single dose 320 mg of Gemifloxacin in 12 healthy human volunteers. Maximum plasma concentration (C_max) ranged from 810 to 1970 ng/ml at 0.75 to 1.50 h (t _max). The half life (t_1/2) ranged from 4.30 to 7.65h. Also the mean value of area under the concentration time curve (AUC_0-t) obtained was 6469 ± 1790 ng x h/ml and AUC_0-infwas found to be 6833 ± 1958ng x h/ml. Relative bioavailability of test preparation was 102.78% to that of reference preparation and both the products were bioequivalent.

Fig. 3: Mean Plasma Concentration Time Curve for Test and Reference Product.

From the above discussion it is found that the analytical method for analysis of Gemifloxacin in plasma is simple, rapid and sensitive. The main advantage of this method is the use of soild extraction procedure for sample preparation, which shows less interference from plasma. It also uses UV detection, which is less costly than LC-MS (Liquid chromatography coupled with mass spectrometry). It can be used as a reliable assay method in the study of Pharmacokinetics of Gemifloxacin as well as bioavailability / bioequivalence study.

ACKNOWLEDGEMENTS:

The authors would like to acknowledge the Orchid Healthcare in providing the samples and to conduct the study

REFERENCES:

1. The Merck index,13 th edition., Merck Research Laboratories., Division of Merck and CO,INC, White House station,NJ,2003,

2. The Merck Index, 12^th Edition, Merck & Co Inc, New York, 1996.

3. PDR: Physician’s Desk Reference, 54^th Edition, 2000.

4. Indian Pharmacopeia, Vol I, II, 1996 & Addendum 2000 and Vet, Government of India, Ministry of Health and Family Welfare, Controller of Publications, Delhi, 2000.

5. British Pharmacopeia, Vol. I & II and Vet 1993, HMSO, London, 1999.

6. United States Pharmacopeia USP XXIV & NF IXX, 2000 and 4 Addendum, USP convention Inc., Rockville, 2002.

7. European Pharmacopeia, 3^rd Edition, 1997 and Supplement, Council of Europe, Strasbourg, 1999.

8. Japan Pharmacopoeia, XIII^th Edition, 1996

9. A.R. Rote, S.P. Pingle , Journal of Chromatography B, Volume 877, Issue 29, 1 November 2009, Pages 3719-3723

10. E. Doyle, S. E. Fowles, D. F. McDonnell, R. McCarthy, S. A. White , Journal of Chromatography B: Biomedical Sciences and Applications, Volume 746, Issue 2, 15September2000,Pages191-198

11. Chattan, L.G., Edt., Pharmaceutical Chemistry, Vol I & II, Moral Dekker Inc, New York, 1966.

12. Beckett, A.H., Stenlake, J.B., Practical Pharmaceutical Chemistry, Vol. I & II, CBS Publishers and Distributors, New Delhi, 1986.

13. Higuchi, T., Brochman – Hansen, E., Edt., Pharmaceutical Analysis, Interscience, London, 1961.

14. Donnely M.T., Hawkey C.J., Aliment Pharmacol. Ther., 1997, 11, 227.

15. Jouzeau J.Y., Terlain . B., Abid. A., Drugs, 1997, 53,563.

16. UR Manglani, IJ Khan, K Soni, P Loya, MN Saraf , Indian Journal of Pharmaceutical Sciences, Year 2006 , Volume 68, Issue 4

17. Mitchell J.A., Akarasereenont. P., Thiemermann. C., Proc. Natl. Acad. Sci. USA, 1994, 90, 11693.

18. G Sowmiya, M Gandhimathi, TK Ravi, KR Sireesaa , Indian Journal of Pharmaceutical Sciences, Year 2007, Volume 69, Issue 2

19. ICH Harmonized Tripartite Guidance for validation of Analytical Procedures ., Methodology 1996

Received on 08.11.2009 Modified on 27.12.2009

Asian J. Research Chem. 3(1):Jan.-Mar. 2010 page 192-196