RP-HPLC Method Development and Validation for Determination of Tigecycline in Bulk and Pharmaceutical Dosage form

Aims: To develop and validate a new, simple, rapid, precise and accurate Reverse Phase High Performance Liquid Chromatographic (RP-HPLC) method for the quantitative determination of Tigecycline in bulk and pharmaceutical dosage form. Study Design: Place and Duration of the Study: RBVRR women's college of pharmacy, Barkatpura, Hyderabad, between june 2019 and july 2020. Methodology: The RP-HPLC method was developed on Sunsil C18 150 mm x 4.6mm x 5μ column using acetonitrile : water (pH maintained at 3.5 with acetic acid) [70:30] as mobile phase at flow rate 0.8 ml/min and UV detection at 250 nm. Results: Tigecycline exhibited linearity over the concentration range of 5-40 μg/mL (R2 > 0.999). The analytical method showed good precision with % RSD below 2. The method showed suitable accuracy and robustness. Conclusion: Validation of the developed method was done as per International Conference on Harmonization (ICH) Q2R1 guidelines. Original Research Article

Tigecycline inhibits protein translation in bacteria by binding to the 30S ribosomal subunit and interfering with the entry of amino molecules into the A site of the ribosome. This blocks incorporation of amino acid residues into elongating peptide chains, thereby preventing protein synthesis and eventually bacterial cell growth. Glycylcyclines appear to bind more effectively compared to tetracyclines. It has activity against a broad range of Gram and Gram-negative bacteria, including tetracycline-resistant organisms. This tetracycline analogue overcomes tetracycline resistance by two mechanisms namely resistance mediated by acquired efflux pumps and ribosomal protection. It is used for the intravenous treatment of complicated skin and skin structure infections caused by susceptible organisms [2 The present research work describes the development and validation of a simple accurate and precise RP-HPLC [4 estimation of Tigecycline in bulk and pharmaceutical formulation.

Instruments
Shimadzu HPLC (LC-20AD Multi system, SPD-20A UV-Visible detector, LC solution software). Labman sonicator was used  Tigecycline is the first drug clinically available under the class of Glycylcyclines which are a new class of antibiotics derived from tetracycline. Tigecycline is a new glycylcycline with broad spectrum antibiotic activity. It is chemically butylamino)acetami 3,10,12,12a-tetrahyd 1,4,4a,5,5a,6,11,12a-octahydrot Tigecycline inhibits protein translation in bacteria by binding to the 30S ribosomal subunit and interfering with the entry of amino-acyl tRNA molecules into the A site of the ribosome. This blocks incorporation of amino acid residues into elongating peptide chains, thereby preventing protein synthesis and eventually bacterial cell growth. Glycylcyclines appear to bind more to tetracyclines. It has activity against a broad range of Gram-positive negative bacteria, including resistant organisms. This tetracycline analogue overcomes tetracycline resistance by two mechanisms namely resistance mediated by quired efflux pumps and ribosomal protection. It is used for the intravenous treatment of complicated skin and skin structure infections caused by susceptible organisms [2][3].

20AD Multi-solvent delivery
Visible detector, LC tion software). Labman sonicator was used for sonication of the sample solution. Thermo scientific pH meter was used to measure pH. Vacuum pump filter was used for filtration of mobile phase solvents and they were provided by RBVRR women's college of pharm barkatpura, Hyderabad, India.

Chemicals
Tigecycline pure drug was obtained as gift sample from Gland Pharma Tigecycline formulation (TGKEM) was purchased from local drug store. HPLC grade water, methanol, acetonitrile and glacial acetic acid were purchased form SD Fine Chemicals, Mumbai, India.

CHROMATOGRAPHIC CONDITIONS
The isocratic mobile phase consisted of Acetonitrile: Water (pH adjusted to 3.5 with Acetic acid) [70:30], flowing through the column at constant flow rate 0.8 ml/min. Sunsil C18 column (150 mm x 4.6mm x 5µm) was used as the stationary phase. 250 nm was selected as the detection wavelength for UV detector.

Preparation of Standard Stock Solution
Accurately 10 mg of Tigecycline standard drug was weighed and transferred into a 10 mL volumetric flask. The volume was made up to the mark using the mobile phase resulting in 1 mg/mL concentration primary stock sol From this, 1mL was pipetted out and transferred into a 10 mL volumetric flask and diluted to obtain 100 μg/mL secondary stock solution. for sonication of the sample solution. Thermo scientific pH meter was used to measure pH. Vacuum pump filter was used for filtration of mobile phase solvents and they were provided by RBVRR women's college of pharmacy, barkatpura, Hyderabad, India.
Tigecycline pure drug was obtained as gift sample from Gland Pharma Hyderabad, India. Tigecycline formulation (TGKEM) was purchased from local drug store. HPLC grade water, and glacial acetic acid were purchased form SD Fine Chemicals,

CHROMATOGRAPHIC CONDITIONS
The isocratic mobile phase consisted of Acetonitrile: Water (pH adjusted to 3.5 with Acetic acid) [70:30], flowing through the column t flow rate 0.8 ml/min. Sunsil C18 column (150 mm x 4.6mm x 5µm) was used as the stationary phase. 250 nm was selected as the detection wavelength for UV-Visible

Preparation of Standard Stock
Accurately 10 mg of Tigecycline standard drug was weighed and transferred into a 10 mL volumetric flask. The volume was made up to the mark using the mobile phase resulting in 1 mg/mL concentration primary stock solution. From this, 1mL was pipetted out and transferred into a 10 mL volumetric flask and diluted to obtain 100 μg/mL secondary stock solution.

Determination of λmax
The absorption spectrum for Tigecycline was recorded by scanning 10μg/ml working standard solution using UV-Visible spectrophotometer in the range of 200-400nm. λmax was found to be 250nm. Fig. 2 shows the spectrum of Tigecycline.

RP-HPLC Method Development
Based on the drug solubility and pKa value the following chromatographic conditions have been selected to initiate the method development trials for determination of Tigecycline.

mins 781779
Inference: Improper baseline and bad peak shape were observed. Tailing Factor

1.299
Improper baseline and bad peak shape were observed.  Table 3.

mins 941127
Inference: Baseline was straight and also good peak shape was observed. Considering this, further method optimization was done.

METHOD OPTIMIZATION:
Optimized    Tailing Factor

1.346
Better peak shape compared to the initial trial but improper baseline and delayed retention Tailing Factor

1.243
Baseline was straight and also good peak shape was observed. Considering this, further Acetonitrile: Water (pH adjusted to 3.5 with Acetic acid) [70:30]  Table 4.

mins 1121869
Inference: Good peak shape and Rt were observed. Also system suitability parameters plate count and tailing factor were within the limits.

METHOD VALIDATION:
The developed method was validated according to ICH Guideline Q2 (R1). The following parameters were evaluated: Specificity: It is the ability to assess the analyte unequivocally in the presence of other components which may be expected to be present. A blank (only diluent without drug) was injected into HPLC. No peaks were observed.
Linearity: Linearity was performed by injecting Tigecycline working standard solutions in the range of 5 to 40μg/ml in HPLC and response was recorded. Calibration curve was obtained by plotting concentration against respective peak area values. R 2 value was determined which was found to be 0.9995.
Precision: Precision was assessed by injecting six replicates of 10μg/ml Tigecycline solution, on the same day, and under the same experimental conditions. Peak area of six replicates of standard solution was obtained from chromatograms. % RSD was determined.
Accuracy: The accuracy of the proposed method was assessed by recovery  Good peak shape and Rt were observed. Also system suitability parameters plate count and tailing factor were within the The developed method was validated according (R1). The following It is the ability to assess the analyte unequivocally in the presence of other components which may be expected to be without drug) was injected into HPLC. No peaks were observed. Linearity was performed by injecting Tigecycline working standard solutions in the range of 5 to 40μg/ml in HPLC and response was recorded. Calibration curve was obtained by g concentration against respective peak value was determined which was Precision was assessed by injecting six replicates of 10μg/ml Tigecycline standard solution, on the same day, and under the same experimental conditions. Peak area of six replicates of standard solution was obtained from chromatograms. % RSD was determined.
The accuracy of the proposed method was assessed by recovery studies.
Tigecycline standard solution was spiked to sample solution at three concentration levels (50 %, 100 % & 150 %). Three replicates of each concentration level were prepared. These solutions were injected in HPLC and response was recorded. % Recovery was determined.

Limit of Detection (LOD) and Limit of Quantitation (LOQ):
The LOD and LOQ were calculated according to ICH guidelines, where the factors 3.3 (for LOD) & 10 (for LOQ) were multiplied by the ratio of standard deviation (σ) and the slope obtained from calibration curve. LOD (Detection Limit) = 3.3 σ/ Slope LOQ (Quantitation Limit) = 10 σ/Slope Tailing Factor

1.351
Tigecycline standard solution was spiked to sample solution at three concentration levels (50 %, 100 % & 150 %). Three replicates of each concentration level were prepared. These solutions were injected in HPLC and response ry was determined.

Limit of Detection (LOD) and Limit of
The LOD and LOQ were calculated according to ICH guidelines, where the factors 3.3 (for LOD) & 10 (for LOQ) were multiplied by the ratio of standard deviation (σ) btained from calibration curve.
LOD (Detection Limit) = 3.3 σ/ Slope LOQ (Quantitation Limit) = 10 σ/Slope Robustness of the method was determined by injecting three replicates of 10 μg/ml Tigecycline standard solution by varying the flow rate in chromatographic ± 0.1ml/min and pH 3.5 ± 0.1. Chromatograms were obtained and % RSD was calculated.
System Suitability Test: Tigecycline working standard solution was prepared as per the  Tigecycline working standard solution was prepared as per the procedure and five replicates were injected into the HPLC system. The system suitability parameters were evaluated from the obtained chromatograms by calculating the % RSD of Rt, peak areas, tailing factor and theoretical plates, which were found to be within range.  procedure and five replicates were injected into The system suitability parameters were evaluated from the obtained chromatograms by calculating the % RSD of Rt, peak areas, tailing factor and theoretical plates, which were found to be within range.

HPLC Method
Inter Day Precision 1098493.8

ASSAY
Preparation of Tigecycline Sample Solution for RP-HPLC Method: Tigecycline lyophilized powder formulation equivalent to 10 mg was transferred into a 10 mL volumetric flask. It was dissolved in sufficient amount of mobile phase and sonicated. Then using mobile phase the volume was made up to the mark. The solution was filtered and from this 0.1 ml was pipetted out into a 10 mL volumetric flask and diluted using mobile phase. This solution was injected into HPLC and chromatogram was obtained. % Assay was calculated using the peak area.

CONCLUSION
A new, simple, rapid, precise and accurate RP-HPLC method was developed and validated as per the ICH guidelines. All the validation parameters were found to be within the limits. Therefore this method can be used for routine quality control tests of Tigecycline in bulk drug and pharmaceutical formulation.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.

ACKNOWLEDGEMENT
Authors are thankful to Gland Pharma, Hyderabad for providing Tigecycline pure drug as a gift sample and management of RBVRR Women's College of Pharmacy for providing facilities to carry out this research work.