Estimation of Related Substances in Tigecycline by rp-hplc Method

Estimation of related substancesby usinghigh-performance liquid chromatographic method was developed and validated for the determination of Tigecycline in the present work. Reversedphase chromatography was performed on Waters 2489 UV 2695 pump, Waters 2998 PDA 2695 pump Software Empower2 photodiode array detector using Zorbax Eclipse plus C18 (100 mm × 4.6 mm, 1.8 μm particle size) column with eluent-A: pH 6.50 buffer: acetonitrile: DMSO (90:5:5 %v/ v/v) and eluent-B: pH 6.50 buffer: acetonitrile: DMSO (71:24:5 %v/v/v) as mobile phase at a flow rate of 1.0 mL/min. with UV detection at 270 nm.Linearity was observed in the concentration range of Tigecycline LOQ–1.13% (R2 = 1.000), the concentration range of di-MA-TIG impurity 0.04– 0.23% (R2 = 1.000), the concentration range of CMI 0.05-0.23% (R2 = 0.999). The limit of quantitation (LOQ) and limit of detection (LOD) were found to be di-MA-TIG impurity 0.0001 and 0.0004mg/ mL, CMI impurity 0.0001and 0.0004μg/mL, Tigecycline 0.0001and 0.0005mg/mL respectively. The method was validated as per ICH guidelines. The %RSD precision was found to be less than 1.0 %. The percentage recovery was in good agreement with the labeled amount in the pharmaceutical formulations and the method is simple, specific, precise and accurate for the determination of Tigecycline in pharmaceutical formulations.


INTRODUCTION
Tigecycline [TIG] is (4S,4aS,5aR,12aS)-9 -[ 2 -( t e r t -b u t y l a m i n o ) a c e t a m i d o ] -4 , 7 -b i s (dimethylamino)-1,4,4a, 5, 5a, 6, 11, 12 a-octahydro 3 , 1 0 , 1 2 , 1 2 a t e t r a h y d r o x y -1 , 1 1 -d i o x o -2naphthacenecarboxamide.Tigecycline is a new glycylcycline with an expanded broad spectrum antibiotic, including inhibition of Gram positive, anaerobic and antibiotic resistant organisms.Studies have demonstrated that Tigecycline is superior to the treatment of complicated skin infections as well as complicated intra-abdominal infections.Tigecycline is only available as an intravenous injection 1 .
There are only limited reports regarding determination of Tigecycline in pharmaceutical R e t r a c t e d R e t r a c t e d dosage forms and biological fluids such as spectrophotometric [2][3][4] and HPLC methods 5-7 to determine Tigecycline in pharmaceutical dosage forms.The assay of Tigecycline in the human bone is also reported by LC-MS method 8 .Tigecycline is not official in any pharmacopoeia and there is no monograph containing methods to characterize or quantify Tigecycline.Such methods could offer official parameters to guarantee the validity of the assay.Hence, there is a need for simple, rapid and reproducible method for the routine analysis of Tigecycline in pharmaceutical dosage forms.There is not even a single method estimation of impurities in TIG by using RP-liquid chromatographic method in pharmaceutical dosage forms.In the present work a simple estimation of impurities in TIG reverse phase liquid chromatographic method has been developed for the determination of TIG and validated as per ICH guidelines [9][10][11] .

R e t r a c t e d
Preparation of buffer 1.54g of Ammonium acetate, 3.72g of Ethylenediaminetetraacetic acid, disodium dihydrate and 0.66gms of Sodium sulfitein 1000ml HPLC grade water sonicated to dissolve then adjusted top H 6.50 with 25% aqueous ammonia solution.Filtered through 0.45µmembrane filter paper and degassed.Transferred above buffer, acetonitrile and of dimethyl sulfoxide in the ratio of (90:5:5 %v/v/v).Filtered through 0.45µmembrane filter paper and degassed.

Chromatographic conditions
Chromatographic separation was achieved by using a Waters 2489 UV 2695 pump, Waters 2998 PDA 2695 pump Software Empower 2 photodiode array detector using Zorbax Eclipse plus C18 (100 mm×4.6 mm, 1.8µm particle size) column with eluent-A: pH 6.50 buffer: acetonitrile:DMSO (90:5:5 %v/v/v) and eluent-B: pH 6.50 buffer: acetonitrile: DMSO (71:24:5 %v/v/v) as mobile phase at a flow rate of 1.0 mL/min.with UV detection at 270 nm.Column maintained at temperature 30 ºC, sample temperature 2-5ºC.The overall run time was 26 min.and the flow rate was 1.0 mL/min.10µl of sample was injected into the HPLC system.Retention times of impurities were 13.50 min for di-MA-TIG impurity, 16.75min for CMI and 14.35min for Tigecycline.

Method validation System suitability
The system suitability was performed by analyzing the reference solution three times.Calculate % RSD for replicate injections of each component from reference solution.Preparations of Tigecycline, di-MA-TIG and CMI standard at c o n c e n t r a t i o n s : 2 8 .5 0 0 × 1 0 -3 m g / m l ( 0 .7 % ) , 5.0516×0 -3 mg /ml 0.15% and 5.0516×10 -3 mg /ml 0.15% of the nominal concentration of sample required by the method were analyzed in triplicate for each solution according to the method.The details of summary of system suitability from reference solution were incorporated in the Table 1.1.

Specificity
Solutions of TIG-6-ene impurity, di-MA-TIG impurity, epi-TIG impurity, CMI impurity, AMC impurity and Tigecycline each were prepared and analysed individually.A spiked solution of each potential impurity to the Tigecycline drug substance was preapred and analyzed.Performed the analysis using PDA detector and the peak purity was determined.This study showed that all the known impurities of Tigecycline are adequately

Limit of Detection
A solution containing 0.8152×10 -3 mg/ml of Tigecycline standard (0.02% of the nominal concentration of a sample), 0.8152×10 -3 mg/ml of di-MA-TIG standard (0.02% of the nominal

R e t r a c t e d R e t r a c t e d
concentration of a sample) and 0.8368×10 -3 mg / ml of CMI standard (0.02% of the nominal concentration of a sample), was injected three times.The worst found signal to noise ratio for each

Linearty and Range
The linearity is determined by injecting the solutions in duplicate containing known impurities and Tigecycline ranging from 0.05 to 1.13% and impurities ranging from 0.05% to 0.22% of the specified limit.Regression analysis was performed and the correlation coefficient and residual sum of squares were determined.The response factor for each impurity with respect to Tigecycline was determined.Linearity range as the range for determining the impurities were reported.Results obtained are incorporated in Table1.5-Table 1 The linearity results for Tigecycline and all the impurities in the specified concentration range are found satisfactory, with a correlation coefficient greater than 0.99.

Accuracy
Tigecycline solution spiked with a known amount of each impurity at five levels each in triplicate (in total 15 determinations) was prepared and analyzed as per the method.Summary of % recoveries for Tigecycline and impurities such as di-MA-TIG and CMI were presented in the tables from Table 1.8-1.10.The percentage recovery values obtained for each impurity are in the range of about 99.6-104.3,which are within the specified criteria.The relative standard deviation values of recoveries obtained for all impurities are in the range of 0.04%-0.23.

System precision
The analysis of reference solution six times was performed and determined the percentage relative standard deviation of peak area of replicate injections of each impurity and Tigecycline.Datails of results were mentioned in the Table 1.11.

Method precision
The precision of the method is determined by analyzing a sample of Tigecycline solution spiked with impurities at 100% of the specification limit.The precision of the method for Tigecycline and its impurities were shown in the Table 1.12.The relative standard deviation observed for Tigecycline and R e t r a c t e d impurities are less than 10%.The results comply with the acceptance criteria and indicate acceptable precision of the system.

RESULTS AND DISCUSSION
A simple, economic, accurate and precise HPLC method was successfully developed by using Zorbax Eclipse plus C18 (100 mm × 4.6 mm, 1.8 µm particlesize).Injection volume of 10μl is injected and eluted with the mobile phase eluent-A: pH 6.50 buffer: acetonitrile: DMSO (90:5:5 %v/v/v) and eluent-B: pH 6.50 buffer: acetonitrile: DMSO (71:24:5 %v/v/v), which is pumped at a flow rate of 1.0 ml/ min.Detection, was carried out at 270 nm.The results obtained were accurate and reproducible.The method developed was statistically validated in terms of Selectivity, accuracy, linearity, precision, robustness, and stability of solution.
For Selectivity, the chromatograms were recorded for standard and sample solutions of Tigecycline and its related substances.Selectivity studies reveal that the peak is well separated from each other.Therefore the method is selective for the determination of related substances in Tigecycline.The limit of detection (LOD) and limit of quantitation (LOQ) for di-MA-TIG impurity 0.0001 and 0.0004mg/ml, CMI impurity 0.0001 and 0.0004µg/ml, Tigecycline 0.0001 and 0.0005 mg/ ml respectively.Using the optimized chromatographic conditions, the retention times of impurities were 13.50 for di-MA-TIG impurity, 16.75 for CMI, and 14.35 for Tigecycline.The linearity results for Tigecycline and all the impurities in the specified concentration range are found satisfactory, with a correlation coefficient greater than 0.99.Calibration curve was plotted and correlation co-efficient for Tigecycline and its impurities found to be 1.000, 1.000, and 0.9999 respectively.
The accuracy studies were shown as % recovery for Tigecycline and its impurities at specification level .The limit of % recovered shown is in the range of 90 and 110% and the results obtained were found to be within the limits.Hence the method was found to be accurate.The accuracy studies showed % recovery of the Tigecycline and its related substances in the range 99.6 to104.30respectively.For Precision studies six replicate injections were performed.% RSD was determined from the peak areas of Tigecycline and its impurities.The acceptance limit should be not more than 10, and the results were found to be within the acceptance limits.

CONCLUSIONS
A simple and precise RP-HPLC method has been developed by the author for the estimation of related impurities present in the Tigecycline and it was observed that the chromatographic method developed for Tigecycline and its related substances are rapid, sensitive, precise, and accurate.Therefore, the proposed method can be successfully applied for the routine analysis of the active pharmaceutical ingredients for assurance of its quality during its formulation.

Table : 1.3 Limit of detection (LOD) for Tigecycline and impurities.
than 3 in each injection.All the peaks were detected in all the three injections.Results of LOD for Tigecycline and impurities were shown in Table1.3.The limit of detection values obtained for each impurity and Tigecycline are within the acceptance criteria.