SIMULTANEOUS DETERMINATION OF TIGECYCLINE AND ITS POTENTIAL IMPURITIES BY A STABILITY-INDICATING RP-HPLC-UV DETECTION TECHNIQUE

Objective: Stability representing the RP-HPLC method was established for synchronized quantification of Tigecycline and its impurities. This method was confirmed for its applicability to both tablet dosage and bulk drug forms. Methods: Intended for an isocratic elution, a mobile phase containing methanol: 10 mmol Triethylamine Buffer mixture (75:25 v/v, pH 6.1) was used at 1 ml/min flow rate and Agilent ZORBAX Eclipse XDB C 18 (250 mm × 4.6 mm, 5 μm) column. Results: At 231 nm as wavelength, high-pitched peaks of Tigecycline (Tig) and its impurities (1and2) were detected at 6.55, 8.73 and 4.87 min correspondingly. The linearity of tigecycline and its impurities (impurity-1 and 2 and) were estimated with ranging from 75–450 µg/ml for Tigecycline and 1–6 µg/ml for both impurity 1 and 2. The corresponding recognition limits (LOD and LOQ) of the tigecycline and its impurities were originated to be (1.37,0.047 and 0.071 µg/ml) and (4.15, 0.143 and 0.126 µg/ml). Conclusion: The technique was effectively stretched for stability signifying studies under different stress conditions. Justification of the method was done as per the current ICH guidelines.


INTRODUCTION
Tigecycline ( fig. 1a) is a glycylcyclines member that belongs to tetracycline derivative antibiotic medication used for the treatment of a number of bacterial infections and is potent against gram+ve and gram-ve organisms, including multi-drug resistance organisms [1]. Tigecycline is prescribed for the treatment of several bacterial infections including difficult skin/intra-abdominal contaminations and community-acquired bacterial pneumoni [2].
Tigecycline is the tetracycline derivative having N,Ndimethyglycylamido group in the 9-position of tetracycline ring and due to this structural modification, it having high minimal inhibitory concentrations against microbes than other tetracyclines [3]. It works by binding bacterial 30S ribosomal subunit and blocks the interaction of aminoacyl-tRNA with the A site of the ribosome [4]. The side effects of Tigecycline are similar to that of the other tetracyclines. Vomiting and Nausea are the common side effects and swelling, pain, and irritation at the injection site are the less common side effects by the use of Tigecycline [5,6]. Every 5 ml Tygacil container holds 50 mg of tigecycline (web). This drug is used only in conditions where other different antibiotics are located not appropriate. Tigecycline and its impurities (fig. 1b-c) 1 and 2 are degraded. Which are separated and characterized by NMR, HRMS and IR spectral investigation. In antimicrobial action Impurities, 1and2 shows good activity in the direction Gram-negative and Gram-positive. That's why Impurities 1 and 2 show good activity than Tigecycline [7].
The literature survey for the estimation of Tigecycline confirms that few HPLC [8][9][10][11][12] and one UV spectrophotometer [13] assay methods reported for the estimation of Tigecycline in pharmaceutical formulations. One bio-analytical method was reported for the estimation of Tigecycline in rabbit plasma [14]. Liquid chromatography-mass spectrometry (LCMS) analysis methods were reported for the estimation of Tigecycline in plasma and applied for pharmacokinetic study [15][16][17][18].
The review of the literature confirms that no analytical method was reported for the determination and quantification of Tigecycline and its related impurities. Hence the technique aimed to develop a simple and precise method for the separation and quantification of impurities 1 and 2 in bulk drug and formulations. The molecular structure of impurities 1 and 2 were given in fig. 1b and 1c, respectively. The established HPLC system was used for the evaluation of the drug with their impurities by in vitro method. Various abstractions were tried to used Methanol, Triethylamine [11,19].

MATERIALS AND METHODS
Tigecycline standard drug with 98.73% purity and its Impurities studied were obtained from Lupin Ltd, Hyderabad. Methanol (HPLC Grade) and Acetonitrile (HPLC grade) were obtained as Merck chemicals, Mumbai. Ultra-Pure (Milli-Q ® ) Water was used during the study. All the other substances used during the study are of analytical substance grade and were purchased from Merck chemicals, Mumbai.

General procedures
Preparation of standard solutions: Standard stock solution of Tigecycline and its impurities were prepared by appropriately estimating about 10 mg (0.1 ml) of each drug 100 ml volumetric flask separately. Then the drug was liquified in solvent and filter through a 0.45µ filter. Standard stock solution concentrations of 1000µg/ml were obtained.

Forced degradation and method validation studies
According to the present rules [20], the last optimized conditions are validated. Diluted appropriately stress samples to prepare the closing concentration holding the planned method conditions (Tigecycline of 300 µg/ml) and related with blank and standard chromatograms.

Instrumentation
To develop a High-Pressure Liquid Chromatographic method for the estimation of Tigecycline with impurities, isocratic PEAK HPLC instrument with Agilent ZORBAX Eclipse XDB C18(250 mm × 4.6 mm, 5 μm id) column, gradient pump (LC 20AT), programmable UVdetector (LC-7000) and the dyne type inject port (20μl capacity) and PEAK software for data analysis.

Method development
On the basis of absorption maxima, experimental condition for tigecycline and its impurities the detector λmax was set at 231 nm ( fig. 2) for simultaneous determination of Tigecycline and its related impurities (impirity 1 and imp 2). λmax is lower in the present study (231 nm) compared to the studies carried out by other researchers like De Silva et al. [8] Suneetha et al. [9] Hua XI et al. [11] Mohan et al. 2017 [12] and Zorpas et al. [14] where λmax was in the range of 247 to 270 nm.
The iso-absorption wavelength observed for Tigecycline and its impurities 1 and 2 was considered as the optimum wavelength for the simultaneous detection of standard and both the impurities studied. The initial method development trials and the results observed in the studied conditions were summarized in table 1 and fig. 3 (a-d). The systematic trails of method development for the separation of Tigecycline and its impurities with acceptable system suitability were achieved using stationary phase is Agilent ZORBAX Eclipse XDB C18 (250 mm × 4.6 mm, 5 μm) column, Methanol and 10 mmol Triethylamine Buffer at pH 6.1 in the ratio of 75:25 (v/v) as mobile phase at a flow rate of 1.0 ml/min, UV detection was supported at a wavelength of 231 nm and the analysis was completed with a run time of 15 min. In these conditions, acceptable system suitability was observed for both impurities and Tigecycline ( fig. 4). In these conditions, retention times of Tigecycline and its impurities are 6.55, 8.73 and 4.87 min through a tailing factor of 1.18, 0.95 and 1.07 and the number of theoretical plates were found 4148, 3370 and 5935, which indicates the column's fruit full output the % RSD for six duplicate injections was around 0.138, 0.367 and 0.515 and the proposed method recommends that it is very precise. Hence these methods were validated through ICH Q2 (R1) [20] guidelines. These optimized chromatographic conditions are observed in table 2. In the present study, eco-friendly methanol is used a solvent in the mobile phase, whereas acetonitrile was a solvent in other studies [8, 9, 11, 12 and 14].  Three peaks corresponds to three compounds studied were identified but the separation was found to be very less and broad peaks were observed ( fig. 2b).
Method  Sample volume 20 μl

Stability indicating studies
To determine the stability representing specificity and power of the planned method forced degradation conditions were studied. ( fig. 6 (a-e)) Completely degradation mixtures under pressure situations were fine disconnected from each other as well as typical indicating of non-interference after somewhat degradation product. It gives information about the applicability of the establish method even for both unknown and known products. Understanding of the TIG in the direction of the acidic (10.15) and photolytic (7.96) situations is recognized from the high % of degradation (table 4).

System suitability and specificity
Detected system suitability parameters and tabulated parameters were satisfactory and reported theoretical plate count values, tail factor and resolution values are tabulated in (table 3). Retention time in the present study is comparable to the studies of De Silva et al. [8] and Suneetha et al. [9] but less than Mohan et al. 2017 [12].

Specificity
In this way and standard, sample and placebo solutions were investigated separately to study the interference. Fig. 5 expressions the dynamic elements were well separated from blank and their excipients. There is no interference of placebo with the major peak. So these technique is specific.

Method Precision (M. P.) and Intermediate Precision (I. P.)
Six average trials (comprising a combination of 300 µg ml -1 of Tigecycline and 4 µg ml -1 of impurities) were inserted and the mean values of system suitability parameters were noted (  (table 7). The precision of the method is established from the numerical results (% RSD is less than1 for both Tigecycline and impurities 1 and 2). Six standard replications of the combined to normal solution confirm that the analytical system is working correctly [26]. Values are given in mean±SD; n=6 Values are given in mean±SD; n=6

Accuracy
In the accuracy study, the % recovery (50, 100 and 150%) values were found to be in the range of 100.76-97.30%, 100.57-97.01%, 99.58-98.10% was observed for Tigecycline and both impurities studied and the % RSD in different level was found to be less than 1 (table 8) which is the acceptable limit. Hence the method was found to be accurate. % Recovery range is comparable to the work reported by other researchers [8, 9, 11, 12 and 14].

LOD and LOQ
The least quantification and detection values (Tigecycline and its impurities 1 and 2) were originated to be (4.15, 0.143 and 0.126) and (1.37,0.047 and 0.071). The technique is penetrating as the LOQ and LOD values were originated to be lower the specified limit. The following formula is used for the calculation of LOD and LOQ (26). LOD and LOQ values are lower in the present study compared to the other studies [8,9].

Robustness
The parameters studied are mobile phase ratio, mobile phase pH, and wavelength. The mobile phase ratio was observed at±5 from the optimized mobile phase ratio, mobile pH was studied at±0.1 units and wavelength was studied at±5. The reply factors experiential through the robustness study are USP resolution, relative retention time, tail factor, plate count, and %change of peak area (table 9). The % change in the peak area values of Tigecycline and impurities 1 and 2 in the robustness study confirms that there is no considerable change was observed. This confirms that the method is found to be robust as there is no considerable change in the separation and detection of Tigecycline and impurities when a small change in the developed method conditions.

CONCLUSION
The present work establishes the chromatographic situations to concurrent resolve of Tigecycline and both impurities 1 and 2 is precise, robust, linear, specific, and accurate. The degradationrelated compounds do not combine Tigecycline and its impurities; thus the technique is stability-indicating. In the present technique, appraised the factors which unusually managed the resolution of the peaks. Good theoretical plates indicates the column's fruit full output. These method conditions are effective authenticated and create an effectively submission of the method for Tigecycline investigation and stability representative studies.