Chapter-7. Levofloxacin is described chemically as (-)-(S)-9-fluoro-2,3-dihydro-3- methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7h-pyrido[1,2,3-de]-1,4-

Transcription

1 160 Chapter Introduction Levofloxacin is described chemically as (-)-(S)-9-fluoro-2,3-dihydro-3- methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7h-pyrido[1,2,3-de]-1,4- benzoxazine-6-carboxylic acid(figure 7.1). The drug is an antibiotic of the 3rd-generation fluoroquinolone family inhibits the bacterial enzymes DNA gyrase and topoisomerase IV [1]. The empirical formula is C18H20FN3O4 and the molecular weight is Levofloxacin is a off-white to yellow crystalline powder. (-)-(S)-9-Fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7Hpyrido [1, 2, 3-de]-1,4-benzoxazine-6-carboxylic acid (Mol. Wt.:361.37) Figure 7.1: Chemical structure of Levofloxacin 7.2. Literature Survey Levofloxacin is a synthetic chemotherapeutic antibiotic of the fluoroquinolone drug class [2-3] and is used to treat severe or lifethreatening bacterial infections or bacterial infections that have failed to respond to other antibiotic classes [4-5] It is sold under various brand names, such as Levaquin and Tavanic, the most common. In form of ophthalmic solutions it is known as Oftaquix, Quixin and Iquix.Levofloxacin

2 161 is a chiral fluorinated carboxyquinolone. Investigation of ofloxacin, an older drug that is the racemic mixture, found that the l form [the ( )-(S) enantiomer] is more active. This specific component is levofloxacin[6-7]. Stress testing is a part of developmental strategy under the ICH requirements and is carried out under more severe conditions than accelerated conditions. These studies serve to give information on drug s inherent stability and help in the validation of analytical methods to be used in stability studies. Validation of a Levofloxacin HPLC assay in plasma and dialysate for pharmacokinetic studies was published using fluorescence detection [8]. In one publication an hplc assay and a microbiological assay to determine levofloxacin in soft tissue, bone, bile and serum was described [9]. Analysis of Levofloxacin in pharmaceutical preparations by high performance thin layer chromatography was also described [10]. The high performance liquid chromatography tandem mass spectrometry method (HPLC/MS/MS) has been used to determine Levofloxacin in human plasma [11]. So far several articles were published for determination of Levofloxacin in metabolites and in biological fluids [12, 13]. As on date, no validated stability-indicating HPLC method for quality control testing of Levofloxacin in bulk drugs or drug products was published in any of the journals neither by the innovator nor by any other manufacturer. Attempts were made to develop a stability-indicating HPLC method for the related substance determination and quantitative estimation of Levofloxacin. This chapter mainly deals with the forced degradation of

3 162 Levofloxacin under stress conditions like water hydrolysis, acid hydrolysis, base hydrolysis, oxidation, heat and light. This chapter also deals with the validation of the developed method for the accurate quantification of impurities and assay of Levofloxacin in bulk samples Development and optimization of HPLC method Samples, Chemicals and Reagents Samples of Levofloxacin and its three process impurities (Figure 7.1 to Figure 7.4) were received from Bulk Actives, Unit-II of Dr. Reddy s Laboratories, Hyderabad, India. HPLC grade Methanol and Acetonitrile was purchased from Rankem, Mumbai, India. Ortho-phosphoric acid was purchased from Qualigens Fine Chemicals, Mumbai, India. Sodium dihydrogen ortho phosphate dihydrate was purchased from Qualigens Fine Chemicals, Mumbai, India. Triethylamine was purchased from Loba Chemie Mumbai, India. High pure water was prepared by using Millipore Milli Q plus purification system Equipment The LC method development, validation and forced degradation studies were done using Agilent 1200 series HPLC system with diode array detector. The data were collected and the peak purity of the Levofloxacin peak was checked using chemistation software. The photolytic degradation was carried out using Binder KBS240 photolytic chamber.

4 163 de]-1,4-benzoxazine-6-carboxylic acid (Mol. Wt.: ) Figure 7.2: Chemical structure of impurity-1 oxo-7h-pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylate(mol. Wt.: ) Figure 7.3: Chemical structure of impurity-2 (-)-(S)-9-Fluoro-2,3-dihydro-3-methyl-10-piperazinyl-7-oxo-7H-pyrido[1,2,3- Ethyl(-)-(S)-9-Fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7- (-)-(S)-9,10-Difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de]-1,4- benzoxazine-6-carboxylic acid (Mol. Wt.: ) Figure 7.4: Chemical structure of impurity Sample preparation

5 164 A working solution of 300 µg ml -1 of Levofloxacin was prepared for the determination of assay and related substances analysis. Separate stock solutions of impurities (impurity-1, impurity-2 and impurity-3) at 300 µg ml - 1 were also prepared in diluent Specificity of the test method and generation of stress samples Specificity is the ability of the method to assess unequivocally the analyte in presence of components, which may be expected to present. Typically, these might include impurities, degradants, matrix, etc. [14]. The specificity of the developed LC method for Levofloxacin was carried out in the presence of its impurities. One lot of Levofloxacin drug substance was chosen for stress study experiment. From the ICH Stability guideline: stress testing is likely to be carried out on a single batch of material [15]. Various kinds of stress conditions (i.e., heat, humidity, acid, and base, water, oxidative and light) were employed on one lot of Levofloxacin drug substance based on the guidance available from ICH stability guideline (Q1AR2). The details of the stress conditions applied are as follows: a) Acid hydrolysis: drug substance in 0.5 N HCl solution was exposed at 70 C for 7 days. b) Base hydrolysis: drug substance in 0.5 N NaOH solution was exposed at 70 C for 7 days. c) Oxidative stress: drug substance in 0.01% v/v H2O2 solution was exposed at room temperature for 12 hours.

6 165 d) Water hydrolysis: drug solution in water at 70 C for 7 days. e) Thermal stress: bulk drug was subjected to dry heat at 100 C for 5 days. f) Photolytic degradation: bulk drug was subjected to ICH Q1B conditions. Stress testing of the drug substance can help to identify the likely degradation products, which can in turn help to establish the degradation pathways and the intrinsic stability of the molecule. Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities. All stress degradation studies were performed at an initial drug concentration of 300 µg ml -1. Acid hydrolysis was performed in 0.5 N HCl at 70 C for 7 days. The study in basic solution was carried out in 0.5 N NaOH at 70 C for 7 days.for study in neutral solution, the drug dissolved in water and was kept at 70 C for 7 days. Oxidation studies were carried out at ambient temperature in 0.01% hydrogen peroxide for 12 hours. Photo degradation studies were carried out according to Option 2 of Q1B in ICH guidelines [16].The drug sample was exposed to light for a overall illumination of 1.2 million lux hours and an integrated near ultraviolet energy of 200 W h m2. The drug sample was exposed to dry heat at 100 C for 5 days. Samples were withdrawn at appropriate times and subjected to LC analysis after suitable dilution (300 µg ml -1 ) to evaluate the ability of the proposed method to separate Levofloxacin from its degradation products. Photodiode array detector was employed to check and to ensure the

7 166 homogeneity and purity of Levofloxacin peak in all the stressed sample solutions. Assessment of mass balance in the degraded samples was carried out to confirm the amount of impurities detected in stressed samples matches with the amount present before the stress was applied. Quantitative determination of Levofloxacin was carried out in all the stressed samples against qualified working standard and the mass balance (% assay + % sum of all impurities + % sum of all degradation products) was tabulated in table Method development The main target of the chromatographic method is to get the separation of impurity-1, impurity-2, impurity-3 and the degradation products generated during stress studies from the analyte peak. Impurities were co-eluted by using different stationary phases like C8, Cyno, XTerra and Phenyl and different mobile phases containing buffers like phosphate, sulphate and acetate with different ph (4-10) and using organic modifiers like acetonitrile, methanol and ethanol in the mobile phase. Apart from the co-elution of impurities, poor peak shapes for some impurities and degradation products were also noticed. Sodium dihydrogen orthophosphate buffer with ph 6.0 and methanol at 1.0 ml min -1 flow was chosen for initial trail with a 250 mm length X 4.6 mm ID column and 5 µm particle size C18 stationary phase. When impurity spiked sample was injected the resolution between impurities and analyte was poor.

8 167 To get the good resolution of impurities from analyte, in the phosphate buffer Triethyl amine added from 0.1% to 0.5% (v/v) then ph adjusted to 6.0 at each level of Triethyl amine and injected impurity spiked sample solution, from Figure 7.5 at 0.5% Triethyl amine level the resolution was good among impurities and analyte. At low concentrations of Triethyl amine the resolution between oxidative drgradant, impurity-1 and also the resolution between impurity-2, impurity-3 was poor although they were well separated from analyte. At 0.5 %( v/v) level of Triethyl amine at ph 6.0 all the impurities and degradation products were well separated amongst as well as from anlayte. Figure 7.5: Effect of Triethyl amine in mobile phase on the resolution between Levofloxacin; Impurity-1; oxidative degradant; Impurity-2; Impurity-3. The effect of buffer ph (Figure 7.6) was also studied under the above

9 168 conditions and it was found that at higher and lower ph the tailing of the Levofloxacin peak was more and also resolution was poor between impurities and degradants and also from the analyte. Figure 7.6: Effect of mobile phase ph on the resolution between Levofloxacin; Impurity-1; oxidative degradant; Impurity-2; Impurity-3. The effect of Buffer concentration on the retention of Levofloxacin and its impurities was also studied (Represented in Figure 7.7). At low concentration of buffer the retention time of analyte as well as impurities was very high to decrease the retention time of analyte and impurities buffer concentration was increased to 25 mm without changing other conditions.

10 169 Figure 7.7: Effect of Buffer concentration on the retention of Levofloxacin; Impurity-1; Impurity-2; Impurity-3. At these chromatographic conditions all the impurities and degradants were well separated amongst and also from Levofloxacin. The effect of solvent B was also studied, when Acetonitrile used instead of Methanol the resolution between imp-1, oxidative degradant and resolution between imp-2, imp-3 was very poor when 100% Methanol used as solvent B all the impurities were well separated. The results clearly indicated that on ACE C18 column 250 mm length X 4.6 mm ID with 5 µm particle size and solvent A as 0.5% Triethyl amine in Sodium dihydrogen orthophosphate dihydrate (25 mm; ph 6.0), solvent B as Methanol with a gradient programme: Time(t)/ % solvent B: 0/30, 20/50, 25/80, 30/80 with a post run time of 5 minutes at detection wavelength 294 nm was successful in separation of drug from its impurities and degradation products. Under the above conditions, results were as follows, retention time of Levofloxacin was around 9.7 min, with a tailing factor of 1.1, number of theoretical plates (N) for the Levofloxacin peak was and % RSD for 5 replicate injections was 0.1% the typical retention times imp-1,imp-2,imp-3 were about 3.4,12.3,13.5 min respectively (Figure 7.22). Peak purity of stressed samples of Levofloxacin was checked by using a photodiode array detector of Agilent 1200 series, the purity factor is within the threshold limit in all the stress samples, demonstrating the homogeneity of analyte peak. Accelerated and long term stability study results as per ICH Q1A (R2) for

11 170 Levofloxacin were generated for 12 months by using the developed LC method and the results were well within the limits, this further confirms the stability indicating of the developed LC method. Optimized chromatographic conditions Column : ACE C18, 250mm x 4.6mm, 5 m particle size Mobile phase A : ph6.0 Buffer* Mobile phase B : Methanol HPLC program : Gradient Gradient Programme : T/%B: 0/30, 20/50, 25/80, 30/80 Post run time Flow rate : 5 minutes : 1.0 ml/min Column temperature : 40 ± 2 C Wavelength of detection Injection volume : 294 nm : 20 L for related substances 10 L for assay determination Diluent : Water :Acetonitrile (60 :40) Run time Retention time : 35 min : Levofloxacin about 9.7 min. Relative Retention Time : Impurity-1 about 0.35 Impurity-2 about 1.27 Impurity-3 about 1.39

12 171 ph6.0 Buffer*: 0.5% Triethyl amine in Sodium dihydrogen orthophosphate dihydrate (25 mm; ph 6.0) Figure 7.8 to Figure 7.19 is the typical HPLC chromatograms showing the degradation of Levofloxacin in various stress conditions and also the corresponding peak purity plots. X-axis: Retention time in min and Y-axis: Peak response in mau Figure 7.8: Typical HPLC chromatograms of acid hydrolysis Figure 7.9: Peak purity plot of acid hydrolysis

13 172 X-axis: Retention time in min and Y-axis: Peak response in mau Figure 7.10: Typical HPLC chromatograms of alkali hydrolysis Figure 7.11: Peak purity plot of alkali hydrolysis X-axis: Retention time in min and Y-axis: Peak response in mau Figure 7.12: Typical HPLC chromatograms of oxidative degradation

14 173 Figure 7.13: Peak purity plot of oxidative degradation X-axis: Retention time in min and Y-axis: Peak response in mau Figure 7.14: Typical HPLC chromatograms of thermal degradation

15 174 Figure 7.15: Peak purity plot of thermal degradation X-axis: Retention time in min and Y-axis: Peak response in mau Figure 7.16: Typical HPLC chromatograms of water hydrolysis

16 175 Figure 7.17: Peak purity plot of water hydrolysis X-axis: Retention time in min and Y-axis: Peak response in mau Figure 7.18: Typical HPLC chromatograms of photolytic degradation

17 176 Figure 7.19: Peak purity plot of photolytic degradation 7.4. Comments on the stress degradation of Levofloxacin No considerable degradation observed when the Levofloxacin sample was subjected to acid, base, water, thermal and photolytic stress. Considerable degradation was observed during oxidative degradation (Figure 7.12) at 0.50 RRT which was identified by LCMS/MS. However, the developed method is able to well resolve all the degradants from the analyte peak (i.e. Levofloxacin) generated from oxidative degradation and the Levofloxacin peak was observed to be pure and homogeneous when checked under DAD and also assay of levofloxacin was unaffected by impurities and degradation products, thus establishes the stability-indicating power of the developed method Identification of major degradation product (at 0.50 RRT) formed in oxidative stress condition LCMS/MS analysis was carried out for the oxidative stress sample of Levofloxacin using Agilent 6410 QQQ mass spectrometer with suitable volatile buffer ammonium acetate(10 mm, ph=6.0) as mobile phase. The degradation product formed at 0.50 RRT shows the mass of 377 which is 16 higher mass than Levofloxacin mass 361.The fragmentation for the degradant was also carried out for degradation product and Levofloxacin using product ion scan by LCMS/MS with optimum collision energy of 25. The fragmentation pattern (Figure 7.20) clearly indicates that formed degradant was N-Oxide of Levofloxacin which was supported by chemical properties of Levofloxacin. The fragment formed from the cleavage of

18 177 CO-OH bond in acid group and the fragment results from the cleavage of N-Oxide ( mass of 16) followed by loss of carbonyl group( mass of 28).Due to steric hindrances and localization of lone pair on nitrogen the N-Oxide will form in piperazine ring at N-Methyl position. So the probable structure as shown in Figure The N-Oxide was formed due to oxidation so this impurity was reduced by adding antioxidant during purification of Levofloxacin. Figure 7.20 Fragmentation mass spectrum of 0.50 RRT degradation product formed in oxidative degradation of Levofloxacin.

19 178 Figure 7.21 Structure of 0.50 RRT degradation product (M.Wt:377.37) formed in oxidative degradation of Levofloxacin 7.5. Validation of Analytical method and its results The developed and optimized HPLC method was taken up for validation. The analytical method validation was carried out in accordance with ICH guidelines [17] System Suitability Test (SST) A mixture of Levofloxacin standard, impurity-1, impurity-2 and impurity-3, were injected into HPLC system and good resolution was obtained between impurities and Moxifloxacin. A typical blank, pure Moxifloxacin and spiked HPLC chromatograms were presented below (Figure 7.22). These results are tabulated in table 7.1.

20 179 X-axis: Retention time in min and Y-axis: Peak response in mau Figure 7.22: System Suitability Test chromatogram Table 7.1: System Suitability Test results Compound (n=3) USP Resolution (R S) USP Tailing factor (T) No. of theoretical plates (N) / USP Levofloxacin Impurity Impurity Impurity n = Number of determinations Precision The precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogenous sample under the prescribed conditions [18]. Assay method precision study was evaluated by carrying out six

21 180 independent assays of Levofloxacin test sample against qualified reference standard and RSD of six consecutive assays was 0.2% (Table 7.2). Table 7.2: Precision results of the assay method Preparation Assay (% w/w) %RSD 0.4 The precision of the related substance method was checked by injecting six individual preparations of Levofloxacin (0.3 mg/ml) spiked with 0.10% of Impurity-1, Impurity-2 and Impurity-3 with respect to Levofloxacin analyte concentration. The % RSD of area of Impurity-1, Impurity-2 and Impurity-3 for six consecutive determinations was 4.3%, 3.7% and 6.0%. (Table 7.3). Table 7.3: Precision results of the RS method Preparation Peak area of Impurity-1 Peak area of Impurity-2 Peak area of Impurity

22 %RSD Limit of quantification (LOQ) and limit of detection (LOD) LOQ and LOD established for Impurity-1, Impurity-2 and Impurity-3 based on signal to noise ratio method [19,20]. Limit of quantification (LOQ) The quantitation limit (LOQ) of an analytical procedure is the lowest amount of analyte in a sample, which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities. A series of diluted solutions of impurities at low concentrations were prepared and injected the LOQ concentrations and their signal to noise ratios were tabulated in table 7.4. Table 7.4: LOQ values of the impurities S.No. Impurity Name Concentration Signal to Noise ratio 1 Impurity-1 90 ng/ml Impurity-2 60 ng/ml Impurity ng/ml 10.1 Limit of detection (LOD)

23 182 The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample, which can be detected but not necessarily quantitated as an exact value. A series of diluted solutions of impurities at low concentrations were prepared and injected the LOD concentrations and their signal to noise ratios was tabulated in table 7.5. Table 7.5: LOD values of the impurities S.No Impurity Name Concentration Signal to Noise ratio 1 Impurity-1 27 ng/ml Impurity-2 18 ng/ml Impurity-3 64 ng/ml Linearity Linearity of the assay method The linearity of an analytical procedure is its ability to obtain test results, which are directly proportional to the concentration of analyte in the test sample [21]. The linearity of the assay method was established by injecting test sample at 50%, 75%, 100%, 125% and 150% of Levofloxacin assay concentration (i.e.300 µg/ml). Each solution was injected twice (n=2) into HPLC and calculated the average area at each concentration (Table 7.6). Calibration curve was drawn by plotting average area on the Y-axis and concentration on the X-axis (Figure 7.23). Table 7.6: Linearity results of the assay method Concentration (%) Mean peak area

24 Correlation coefficient P e a k A r e a Linearity curve for Assay y = 801.3x R² = Concentration in % Figure 7.23: Linearity graph of assay method Linearity of the related substances method Linearity experiments were carried out by preparing the Levofloxacin sample solutions containing impurity-1, impurity-2 and impurity-3, from LOQ to 200% (i.e. LOQ, 50%, 75%, 100%, 150% and 200%) with respect to their specification limit (0.10% w/w) Please refer Table 7.7 below. Calibration curve was drawn by plotting mean area of the individual impurity (Impurity-1, Impurity-2 and Impurity-3) on the Y-axis and concentration on the X-axis (Figure 7.24 and Figure 7.26).

25 184 Table 7.7: Linearity results of the RS method S.No Concentration (in %) Impurity-1 (Mean peak area) Impurity-2 (Mean peak area) Impurity-3 (Mean peak area) 1 LOQ Slope Intercept Correlation coefficient P e a k A r e a Linearity curve for Impurity-1 y = 0.657x R² = Concentration % Figure 7.24: Linearity graph of Impurity-1

26 185 P e a k A r e a Linearity curve for Impurity-2 y = 0.755x R² = Concentration % Figure 7.25: Linearity graph of Impurity-2 P e a k A r e a Linearity curve for Impurity-3 y = 1.093x R² = Concentration % Figure 7.26: Linearity graph of Impurity Accuracy / Recovery The accuracy of an analytical procedure expresses the closeness of agreement between the value, which is accepted either as a conventional true value or an accepted reference value and the value found [22] Accuracy of the assay method Accuracy of the assay method was established by injecting test sample at 50%, 100% and 150% of analyte concentration (i.e., 0.3mg/mL). Each

27 186 solution was injected twice into HPLC and the average peak area of Levofloxacin peak was calculated. % Recovery of assay method was carried out in triplicate at each concentration level (Table 7.8). Table 7.8: Recovery of the assay method S.No. Concentration (%) % Mean recovery (n=3) n= number of Determinations Accuracy of the RS method Accuracy of the related substances method established at 50%, 100% and 150% of the impurities specification limit (0.10% w/w). Accuracy at 50% impurity specification level Prepared test solution in triplicate (n=3) with impurities Impurity-1 and Impurity-2 at 0.05% level w.r.t. analyte concentration (i.e. 0.3mg/mL). Injected each solution once into HPLC. Calculated % mean recovery of impurities in the test solution using the peak area obtained in impurities solution injected without test solution (Table 7.9). Table 7.9: Recovery at 50% level S.No. Impurity Name % Mean recovery (n=3) 1 Impurity Impurity Impurity

28 187 Accuracy at 100% impurity specification level Prepared test solution in triplicate (n=3) with impurities Impurity-1, Impurity-2 and Impurity-3 at 0.10% level w.r.t. analyte concentration (i.e. 0.3mg/mL). Injected each solution once into HPLC. Calculated the %mean recovery of impurities in the test solution using the area obtained in impurities solution injected without test solution (Table 7.10). Table 7.10: Recovery at 100% level S.No. Impurity Name % Mean recovery (n=3) 1 Impurity Impurity Impurity Accuracy at 150% impurity specification level Prepared test solution in triplicate (n=3) with impurities Impurity-1, Impurity-2 and Impurity-3 at 0.15% level w.r.t. analyte concentration (i.e.0.3mg/ml). Injected each solution once into HPLC. Calculated the %mean recovery of impurities in the test solution using the area obtained in impurities solution injected without test solution (Table 7.11). Table 7.11: Recovery at 150% level S.No Impurity Name % Mean recovery (n=3) 1 Impurity Impurity Impurity

29 Solution and mobile phase stability Leaving both the test solutions of sample and reference standard in tightly capped volumetric flasks at room temperature for two days carried out the solution stability of Levofloxacin in the assay method. The same sample solutions were assayed at six hours interval up to the study period. The RSD of assay of Levofloxacin during solution stability experiments was within 1.0%. No significant change was observed in the content of Impurity-1, Impurity-2 and Impurity-3 during solution stability and mobile phase stability experiments up to the study period. The data obtained in both the above experiments proves that sample solutions and mobile phase used during assay and related substance determination were stable up to 48 h. Assay method Injected standard and test solution each at 0 h, 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h and 48 h. Table 7.12 summarizes assay content obtained at different interval. Table 7.12: Solution stability results of the assay method S.No. Interval Assay (%w/w) 1 0 h h 98.5

30 h h h h h h h 98.3 %RSD 0.7 Related substances method Solution and mobile phase stability was established for 48 h by injecting test solution at an interval of 6 h. The impurity profiles obtained at different interval were very consistent and matched with initial value Robustness In all the varied chromatographic conditions (flow rate, composition of organic modifier and column temperature) the resolution between critical pair i.e. Impurity-2 and Levofloxacin was greater than 3, illustrating the robustness of the developed method. The results obtained were captured in the below Table Table 7.13: Results of the robustness study S.No. Parameter Variation Resolution between Levofloxacin and Impurity-2 Impurity-2 and Impurity-3

31 190 1 Temperature (± 5 C of set temperature) a) At 35 C b) At 45 C a) At Flow rate (± 20% of set flow) ml/min b) At ml/min 3 ph of the buffer(± 0.5 of ph-6.0) a) At 6.5 b) At Mass balance The mass balance is a process of adding together the assay value and the levels of degradation products to see how closely these add up to 100% of the initial value, with due consideration of the margin of analytical error [23]. Its establishment hence is a regulatory requirement. The mass balance is very closely linked to the development of stability-indicating assay method as its acts as an approach to establish its validity. Mass balance is also important in understanding alternate degradation pathways. The stressed samples of Levofloxacin bulk drug were assayed against the qualified reference standard and the results of mass balance obtained were very close to 100%. The results of mass balance obtained in each condition are presented below (Table 7.14). Table 7.14: Summary of forced degradation results

32 191 S.No Stress condition Duration % Assay of active substance Mass balance (% Assay+ % impurities+ % degradants) Remarks 1 Acid hydrolysis (0.5 N HCl at70 C) 7 days Slight degradation observed 2 Alkali hydrolysis (0.5 N NaOH at 70 C) 7 days No degradation was observed 3 Oxidation (0.01% H 2O 2 at RT) 12 hours Major degradation was observed 4 Water hydrolysis at 70 C 7 days Mild degradation was observed 5 Thermal Stress (at 100 C) 5 days No degradation observed 6 Photolytic stress (ICH Q1B) 11 days No degradation observed 7.7. Quality control monitoring of Levofloxacin in three production batches Using the developed HPLC method Levofloxacin samples of three production batches was analyzed and results (Table 7.15) indicates that the method was able to determine the Levofloxacin without interference of other impurities as well as its related substances. Hence the developed was suitable for determination of Levofloxacin as well its impurities in bulk samples.

33 192 Table 7.15: Results of quality monitoring of Levofloxacin in three production batches Batch No: LP001E08 LP001G09 LP001H08 Description Yellowish white crystalline powder Yellowish white crystalline powder Yellowish white crystalline powder ND-Not detected Wate r Cont ent by KF Specific optical rotation ( ) Related substances by HPLC Imp-1 Imp-2 Imp- 3 Any Unknow n impurity Total impurit ies Assay on Anhydr ous basis ND ND ND ND ND ND Analysis of Levofloxacin stability samples USP states that stability testing for both drug substance and drug product should be performed by validated stability-indicating test method [24]. One manufacturing lot of Levofloxacin monohydrate was placed for stability study in chambers maintained at ICH set conditions. The analysis of stability samples was carried up to 12 months period using the above stability-indicating method. The stability data results obtained are presented in Table 7.16 and The developed HPLC method performed satisfactorily for the quantitative evaluation of stability samples.

34 193 Table 7.16: Accelerated stability data (storage conditions 40 C / 75% RH) Storage condition Temperature 40 C±2 C, Relative humidity 75±5% Product: Levofloxacin Accelerated Stability data Batch No:LP001F07 Packing conditions: Each sample placed in poly bag which is placed in triple laminated bag Period Initial 1 st month 2 nd month 3 rd month 6 th month Description Yellowish white crystalline powder Yellowish white crystalline powder Yellowish white crystalline powder Yellowish white crystalline powder Yellowish white crystalline powder Stability duration:6 months Water Conten t by KF Specific optical rotation( ) Related substances by HPLC Imp-1 Imp -2 Imp -3 Any Unknown impurity Total impuriti es Assay on Anhydrous basis ND ND ND ND 0.01 ND ND ND 0.01 ND ND ND 0.02 ND ND ND 0.02 ND Storage condition Temperature 30 C±2 C, Relative humidity 60±5% Product: Levofloxacin Long term Stability data Batch No:LP001F07 Packing conditions: Each sample placed in poly bag which is placed in triple laminated bag Period Initial 1 st month 2 nd month 3 rd month 6 th month 9 th month Description Yellowish white crystalline powder Yellowish white crystalline powder Yellowish white crystalline powder Yellowish white crystalline powder Yellowish white crystalline powder Yellowish white crystalline Stability duration:12 months Water Content by KF Specific optical rotation( ) Related substances by HPLC Imp -1 Imp -2 Imp -3 Any Unknown impurity Total impuritie s Assay on Anhydrou s basis ND ND ND ND ND ND 0.02 ND ND ND 0.02 ND ND ND 0.01 ND ND ND 0.02 ND

35 194 powder 12 th month Yellowish white crystalline powder ND ND 0.02 ND Table 7.17: Long-term stability data (storage conditions: 30 C / 60% RH) 7.9. Summary A simple specific validated stability indicating RP-HPLC method developed for the determination of related substances and assay of Levofloxacin drug substance for the first time. The developed method was precise, accurate and selective. The method was fully validated showing satisfactory data for all the method validation parameters tested. The developed method was stability-indicating and can be conveniently used by quality control department to determine the related substances and assay in regular Levofloxacin production samples and also stability samples.