INTRODUCTION:

Linezolid (LNZ) is chemically (S)-N-({3-[3-fluoro-4-(morpholin-4-yl) phenyl] - 2-oxo-1,3-oxazolidin-5-yl}methyl) acetamide.^1-5 It is member of oxazolidi-none class. It is used for the treatment of serious infection caused by Gram positive bacteria that resistance to other antibiotics. The main uses are infections of the skin and pneumonia although it may be use for a variety of other infections.^6-10 Oxazo-lidinone bind to the 50S subunit of the prokaryoticribosome, preventing it from complexing with the 30S subunit, mRNA, initiation factors and formyl-methionyl-tRNA. The net result is to block assem-bly of a functional initiation complex for protein synthesis, thereby preventing translation of the mRNA. ^11-15

There are number of methods are reported for the estimation of Linezolid but forced degradation studies of Linezolide from formulation has not been studied extensively hence here and RP-HPLC method will be developed and validated for the estimation of Linezolid from formulation. ^16-20

Fig.No. 1: Chemical structure of Linezolid

MATERIAL AND METHODS:

Materials:

The chemicals were purchased from local market of Amaravati; all the chemicals are of HPLC grade.

Methods:

Selection of wavelength:

Standard solution of Linezolid (10 μg/mL) was scanned between 200-400 nm using UV-visible spectrophotometer. Wavelength was selected from the spectra of Linezolid show reasonably good response at 249 nm.

Preparation of mobile Phase:

The mobile phase selected for the RP-HPLC method was Methanol: 1% Acetic acid (50:50 v/v). Sonicate the solution for 15 min.

Preparation of standard stock solution:

Standard solution of linezolid (100μg/ml) was prepared by transferring accurately weighed linezolid (10 mg) in 100 ml volumetric flask separately and dissolving in methanol. The solution was diluted to 100 ml with mobile phase in separate volumetric flask to inject in chromatographic system.

Preparation of standard solution:

Take 1 mL from Linezolid stock solution and transferred to 10 mL volumetric flask and volume made up to the mark by mobile phase.

Preparation of sample solution:

Take Tablet Powder equivalent to 10 mg of Linezolid was transferred to a 100 ml volumetric flask, and made up volume up to the mark with mobile phase. The solution was filtered through whatman filter paper no. 42 and first few drops of filtrate were discarded. 1 ml of this solution was diluted to 10 ml with mobile phase.

Validation Studies ^21-23

The following parameters were considered for the analytical method validation for the quantification of Linezolid in tablet dosage form as per ICH guideline.

System suitability test ^24-27

System suitability was performed by preparing solutions per the test method and analysed before performing any validation parameters to verify that the system is adequate for the analysis. The parameter used to verify in this test were retention time, theoretical plate, tailing factor and resolution. Acceptance criteria: %RSD of area of five replicate standard injection should not be more than 2.0; Theoretical plates for the analysis peak should not be less than 2000; Tailing factor for the analyte peak should not be more than 2.0.

Specificity ²⁸

Specificity of an analytical method is its ability to measure the analyte accurately and specificity in the presence of component that may be expected to be present in the sample matrix. Chromatogram of standard and sample solution of Linezolid was compared, and peak purity spectra obtained from using UV detector was recorded in order to provide an indicated of specificity of the method. Chromatograms are Shows in Figure no. 02. Acceptance criteria: There must be no interference

Fig. No. 2: Chromatogram of Linezolid at a concentration of 10 ppm

Linearity and Range ²⁹

Linearity response was determined by analysing different concentration for calibration curve in the range 10-50 ppm for Linezolid. Peak area was measured at each level. Peak area was plotted against concentration and equation of straight line and correlation co-efficient was determined. Acceptance criteria: value of R2should be ≥0.99. Linearity data are given in table no. 01. calibration curve in shown in Figure no. 03.

Fig. No.3: Linearity curve of Linezolid

Precision:

The precision of an analytical expresses the closeness of agreement (degree of scatter) between a series of measurement obtained from multiple sampling of the same homogeneous sample under the prescribed condition. Precision considered at three levels: Repeatability, intermediate (intraday) Precision and Reproducibility (Interday) Precision. Repeatability Method precision of experiment was performed by preparing the standard solution of Linezolid (10 μg/ml) for six times and analysed as per proposed method and % RSD was calculated

Accuracy:

The accuracy of the method was determined at 80%, 100%, and 120% by calculating recoveries of Linezolid by the standard addition method. Known amount of standard solution of Linezolid (5, 10, 15 ppm) were added to prequalified sample solution of Linezolid (10 g/ml). Each solution was injected in triplicate and the percentage recovery was calculated by measuring the peak areas and fitting these values into the regression equation of the respective calibration curves. Acceptances Criteria: % Recovery at each should be between 98.00% to 102.00%.

Robustness ³⁰

The Robustness was studies by analysing the sample of Linezolid by deliberate variation in the method parameters. The change in the response of Linezolid was noted. Robustness of the method was studied by changing flow rate by + 0.2 ml/min,

Limit of Detection and Limit of Qualification

According to the ICH recommendation, the approach based on the standard deviation (SD) of the response and slop was use of the determining the LOD and LOQ value. LOD andLOQ values:

LOD = 3.3σ/S

LOQ = 10σ/

Where σ = standard deviation of response; S = slop of calibration curve; Data of LOD and LOQ are shown in Table.

Acid degradation studies ³¹

For study in acidic condition, aliquot quantity of drug was weigh and transferred in 100 ml volumetric flask. To this 2 ml of 1N HCL, was added and diluted to 50 ml with water. This solution was kept for room temp. for 1hr, cooled to room temperature, and volume made up to 100 ml with water. The solution was further diluted to give 10µg/ml and then subjected for RP-HPLC

Alkaline degradation studies:

Drug was weigh and transferred in 100 ml volumetric flask. To this 2 ml of 10% NaOH, was added and diluted to 50 ml with water. This solution was kept for room temp for1 hr, cooled to room temperature, and volume made up to 100 ml with water. The solution was further diluted to give 10µg/ml and then subjected for RP-HPLC

Oxidative studies:

For study in oxidative condition, of drug was weighed and transferred in 100ml volumetric flask. To this 2ml of 3% H₂O₂, was added and diluted in 100ml with water. This solution was kept in dark condition away from sunlight for 1 hrs. The solution was further diluted to give 10µg/ml and then subjected for RP-HPLC

Photolytic studies:

For photolytic study the bulk sample was exposed to sunlight for 24 hrs. by placing 100 mg of ITO in closed petri dish. Exposure was done by exposing to sunlight 6 hrs per day in petri dish. Further the solution dissolved in water and appropriate dilution of 10µg/ml and then subjected for RP-HPLC. Control samples were kept in dark for the same period.

Table No. 02: system suitability parameter

Sr. No.	Parameter	Linezolid
1	Detection Wavelength	249 nm
2	Linearity Range	10-50 ppm
3	Slope	98.07
4	Intercept	269.6
5	Correlation Coefficient	0.999
6	Retention Time ± SD	7.30
7	Theoretical Plate	7523.1
8	Tailing Factor	1.2917

Table No 03. Linearity statistical data of Linezolid

Sr. No.	Conc.	Area
1	10	1193.97
2	20	2331.475
3	30	3230.285
4	40	4080
5	50	5223.36

Table No. 04: Intra-day precision study of HPLC

Sr No.	Conc	Area	Area II	Mean	Amt Found	% Amt Found	SD	RSD
1	10	2311.14	2318.57	2314.86	10.08	100.80	5.25	0.23
2	15	3532.85	3538.04	3535.45	15.18	101.20	3.67	0.10
3	20	4822.3	4819.85	4821.08	20.55	102.75	1.73	0.04

Table No 05. Statistical data for recovery study of HPLC

Level (%)	Added Pure Drug(µg/ml)	Amount recover(µg/ml)	% Recovered	Mean*	SD*	%RSD
50	5	4.98	99.60	18.01	0.08	0.18
100	10	10.23	102.30	20.39	0.02	0.10
150	15	14.95	99.67	22.01	0.16	0.29

Table No. 06: Degradation studies of Linezolid

Sr.no.	Stress Condition	Optimum working condition	Time
1	Acid Hydrolysis	1mg/ml in 1 N HCL at room temp.	1 hours
2	Base Hydrolysis	1mg/ml in 10% NaOH at room temp.	1 hours
3	Oxidative solution	3%H2O2_at room temp. protected from Light (Dark condition)	1 hours
4	Photo degradation	Bulk sample exposed to sunlight for 6 Hrs. per day in Petri dish	24 hours
5	Neutral condition	This solution was Room temp.	1 hours

CONCLUSION:

The developed RP-HPLC method is a fast, simple and reliable analytical method for the determination of Linezolid in pharmaceutical preparation using RP-HPLC and also for its forced degradation studies, as there is no interference between blank and placebo at the retention time of Linezolid. It is very fast, with good reproducibility and good response. Validation of this method was accomplished, getting results meeting all requirements.

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Received on 26.09.2022 Modified on 24.02.2023

Asian J. Pharm. Ana. 2023; 13(4):273-277.

DOI: 10.52711/2231-5675.2023.00045

Sr no.	Conc.	Amount Fond	Area	Retention Time	Tailing Factor	Theoretical Plates	Resolution
1	20	20.33	4768.84	8.1833	7695.7	7695.7	0
2	20	20.15	4722.3071	8.6000	1.5350	7024.3	0
Mean		4766.50	4723.75
%RSD		0.07	0.04