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VALIDATED STABILITY INDICATING RP-HPLC METHOD FOR THE ESTIMATIONOF LINEZOLID IN A PHARMACEUTICALDOSAGE FORMSharmistha Mohapatra a , M. Mathrusri Annapurna a , B. V. V. RaviKumar a , Mohammed Anwar b , Musarrat Husain Warsi c & SohailAkhter ca Department of Pharmaceutical Analysis & Quality Assurance,Roland Institute of Pharmaceutical Sciences, Berhampur, Ganjam,Orissa, Indiab Department of Quality Assurance, Faculty of Pharmacy, HamdardUniversity, New Delhi, Indiac Nanomedicine Research Lab, Hamdard University, New Delhi, India

Available online: 03 Nov 2011

To cite this article: Sharmistha Mohapatra, M. Mathrusri Annapurna, B. V. V. Ravi Kumar, MohammedAnwar, Musarrat Husain Warsi & Sohail Akhter (2011): VALIDATED STABILITY INDICATING RP-HPLCMETHOD FOR THE ESTIMATION OF LINEZOLID IN A PHARMACEUTICAL DOSAGE FORM, Journal of LiquidChromatography & Related Technologies, 34:18, 2185-2195

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VALIDATED STABILITY INDICATING RP-HPLC METHOD FORTHE ESTIMATION OF LINEZOLID IN A PHARMACEUTICALDOSAGE FORM

Sharmistha Mohapatra,1 M. Mathrusri Annapurna,1 B. V. V. Ravi Kumar,1

Mohammed Anwar,2 Musarrat Husain Warsi,3 and Sohail Akhter3

1Department of Pharmaceutical Analysis & Quality Assurance, Roland Institute ofPharmaceutical Sciences, Berhampur, Ganjam, Orissa, India2Department of Quality Assurance, Faculty of Pharmacy, Hamdard University,New Delhi, India3Nanomedicine Research Lab, Hamdard University, New Delhi, India

& A rapid high performance liquid chromatographic method was developed and validated for thedetermination and stability evaluation of linezolid in pharmaceutical dosage forms. Separation oflinezolid was successfully achieved on a C-18 column utilizing water: methanol (50:50 v=v) at aflow rate of 1mL=min and eluate was monitored at 254 nm, with a retention time of 5.117 minute.The method was validated and the response was found to be linear in the drug concentration rangeof 0.001mg=mL to 3.4mg=mL. The mean values�RSD of the slope and the correlation coefficientwere found to be 51169� 0.290 and 0.9999, respectively. The RSD values for intra- and inter-dayprecision were found to be 0.782 (<1.00%) and 0.610 (<0.90%), respectively. The forced degra-dation kinetic study of linezolid by using HCl (0.5N), NaOH (0.02M), H2O2 (6%v=v), thermal(80� 2�C), and UV radiation (365 nm) were observed to be very specific.

Keywords Forced degradation, ICH, Linezolid, LOD, LOQ, RP-HPLC

INTRODUCTION

The oxazolidinones represent the first truly new class of antibacterialagents to reach the marketplace in several decades.[1] Linezolid, (s)-N-[[3-[3-fluoro-4(4-morpholinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl] aceta-mide (Figure 1) was the first oxazolidinone to be developed and approvedfor clinical use. Linezolid is a synthetic antibiotic used for the treatment ofserious infections caused by Gram-positive bacteria that are resistant to

Address correspondence to Sharmistha Mohapatra, Department of Pharmaceutical Analysis &Quality Assurance, Roland Institute of Pharmaceutical Sciences, Berhampur, Ganjam, Orissa-760010,India. E-mail: sharmistha.mhptr@gmail.com

Journal of Liquid Chromatography & Related Technologies, 34:2185–2195, 2011Copyright # Taylor & Francis Group, LLCISSN: 1082-6076 print/1520-572X onlineDOI: 10.1080/10826076.2011.585548

Journal of Liquid Chromatography & Related Technologies, 34:2185–2195, 2011Copyright # Taylor & Francis Group, LLCISSN: 1082-6076 print/1520-572X onlineDOI: 10.1080/10826076.2011.585548

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several other antibiotics.[2,3] Linezolid is active against most Gram-positivebacteria that cause diseases including streptococci, vancomycin-resistantenterococci (VRE), and methicillin-resistant Staphylococcus aureus(MRSA).[4–7] The main indication of linezolid is the treatment of severeinfections caused by Gram-positive bacteria that are resistant to other anti-biotics; it should not be used against bacteria that are sensitive to drugs witha narrower spectrum of activity, such as penicillins and cephalosporins. Inboth the popular press and the scientific literature, linezolid has beencalled a ‘‘reserve antibiotic’’—one that should be used sparingly so that itwill remain effective as a drug of last resort against potentially intractableinfections.[8] A literature review revealed that the HPLC method has beenthe technique of choice for the separation and determination of linezolidin biological fluids. Thus far, several HPLC methods have been described toanalyze linezolid in various body fluids including LC=MS=MS, microboreLC=ESI-MS=MS, HPLC with electrospray tandem mass spectrometry, LCmethods using ultraviolet (UV) detection,[9–15] and fluorescence detec-tion.[16] For the assays in the pharmaceutical dosage forms, the methodsreported in literature are HPLC,[17–20] capillary electrophoresis,[21] TLCfollowed by densitometric and first derivate spectrophotometry,[22] andHPTLC by densitometric analysis.[23] However, any generally recom-mended or rapid stability indicating analytical method for the determi-nation of linezolid in pharmaceutical dosage form has not yet beendescribed in any pharmacopoeia or literature. As a result, in this study anovel rapid, selective, linear, precise, and sensitive stability indicatingRP-HPLC method with the UV detection for the estimation of linezolidin pharmaceutical dosage form was developed and validated as per theICH guideline.

EXPERIMENTAL

Chemicals and Reagents

Linezolid (amorphous form) was obtained from Glenmark Pharmaceu-tical, tablets of Linezolid (600mg of linezolid per tablet) were obtainedfrom Glenmark (Zyvox) and Microcryson (Lizoforce), and HPLC grademethanol was purchased from the Merck.

FIGURE 1 Structure of Linezolid.

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Instrumentation

The chromatographic system consisted of a pump, Model LC-Class-Vpversion 6.12 SPI Shimadzu, equipped with UV-VIS detector Model SPD-10A,Shimadzu, integrator Model Hypersil ODS C-18 (250� 4.6mm, packed with5 m) column Shimadzu. Injections were carried out using a 20mL loop atroom temperature.

Analytical Procedure

Stock solution of linezolid (1mg=mL) was prepared in methanol whichcould be stored at 268K for over 1 month with no evidence of decompo-sition. Standards solutions of linezolid were prepared with methanol inthe range of 0.01mg=mL to 34mg=mL by diluting with methanol:water(50:50 v=v). Each solution was then injected into the column and the chro-matograms were recorded. The calibration curve for the HPLC analysis wasconstructed by plotting the peak area of normalization of linezolid againstconcentration.

Analysis of Tablets

Ten tablets were weighed to obtain the average weight and thenpowdered. The fine powder, equivalent to 10mg of linezolid, was weighedand transferred into a 25-mL calibrated volumetric flask and dissolvedusing methanol. This mixture was sonicated (30min) and then filteredthrough a 0.45 mm membrane filter. After filtration, the appropriate vol-ume (1.0mL) was taken into a 10-mL flask and diluted up to the mark withsame mobile phase. All determinations were conducted in triplicate. Theamount of linezolid was calculated from the related linear regressionequations.

Conditions of Forced Degradation Studies

Conditions of forced degradation studies were in compliance with rec-ommendations of the International Commission of Harmonization.[24,25]

Samples of linezolid (10.0mg) were accurately weighed into 5-mL vials.Different vials with linezolid were placed in water bath maintained at60� 2�C containing aqueous saturated solutions of sodium hydroxide(0.02M NaOH), diluted hydrochloric acid (0.5N HCl), and hydrogen per-oxide (6%H2O2), respectively, and another vial with sample was placed in aheat chamber, set at 80� 2�C=0.5 hr for thermal degradation. UV degra-dation was performed by exposing the sample in a watch glass to UV lamp

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of 365 nm for 48 hr. After definite time intervals, determined by rate ofdegradation, the respective vials were taken out of the respective chambers,cooled to room temperature, and the contents dissolved in methanol. Theso obtained solution was quantitatively transferred into a volumetric flaskand made up to total volume of 10.0mL with methanol. From this,1.0mL of the solution was taken and diluted to 10mL with the mobilephase (methanol:water 50:50 v=v) to get a sample solution of 100 mg=mL.The chromatograms were interpreted using the following dependence ofpure chromatogram of linezolid.

RESULTS AND DISCUSSION

In the initial trials the following mobile phases were used: acetonitrileand water (20:80 v=v) (mobile phase 1) and acetonitrile and water(50:50 v=v) (mobile phase 2) as the mobile phases. Mobile phase 1 has beenrejected due to a lack of linezolid signal on chromatogram. When samplesof linezolid were analyzed using LiChrospher ODS column and a mobilephase 2, peaks shape were not good and retention time was �12min; there-fore, organic modifier concentration was changed from 50% to 70%, but noimprovement was observed. Subsequent attempts were made by lowering ofpH of the mobile phase (using 80% ortho-phosphoric acid) and replace-ment of acetonitrile by methanol. In both cases, marked improvementwas observed. Eventually, a mobile phase composed of water:methanol(50:50 v=v) gave the best results. During these studies injection volumewas 20 mL and the mobile phase flow rate was constant at 1.0mL=min.The analytical wavelength was 254 nm.

Validation of the Method

Validation of HPLC method was in compliance with recommendationsof the ICH guidelines.[22,23]

Selectivitity

This method was selective for the linezolid (Retention time about5.117min), in the presence of degradation product. The selectivity ofHPLC method is illustrated in Figure 2. The typical excipients includedin the drug formulation do not interfere with selectivity of the method(Figure 2a and 2b). The analysis of the chromatogram of linezolid, itsdegradation product, revealed the following efficiencies of the column:for linezolid N¼ 2900, degradation product N¼ 4968, (where N representstheoretical plate number). The separation factors between linezolid and

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product of degradation¼ 3.6. The resultant chromatograms of acidic, alka-line, oxidative, thermal, and photolytic degradation are shown in Figures 3–7,respectively.

FIGURE 3 Resultant chromatogram of acidic degradation (0.5N HCl). (Color figure available online.)

FIGURE 2 Representative chromatogram of Linezolid (100mg=mL). a) Representative chromatogramof Linezolid (100mg=mL) Formulation (Zyvox1 600mg); and b) Representative chromatogram ofLinezolid (100mg=mL) Formulation (Lizoforce1 600mg). (Color figure available online.)

Stability Indicating RP-HPLC for Linezolid in Dosage Form 2189

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Precision and Accuracy

The repeatability of the method was examined by injecting the solutionconsisting of linezolid (0.08mg=mL, 0.10mg=mL, and 0.12mg=mL) intothe HPLC system for three consecutive days. The results are given inTable 1. The repeatability of the HPLC method was good and precise,and the RSD values were obtained between 0.710% and 1.016%.

Linearity

The linearity of the method was determined in terms of the correlationcoefficient between concentration of linezolid and the peak normalizationof linezolid. The calibration curve of linezolid was given in the Figure 8.The calibration range was between 0.001mg=mL to 3.4mg=mL presentedwith the equation of:

y ¼ ac þ b ¼ ð51134� 0:0029Þ � c þ ð12348� 0:010Þ

FIGURE 4 Resultant chromatogram of alkaline degradation (0.02M NaOH). (Color figure availableonline.)

FIGURE 5 Resultant chromatogram of oxidative degradation (6% H2O2). (Color figure availableonline.)

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The correlation coefficient (r2¼ 0.999) closed to unity. The valuesobtained showed good linearity. The intra-day and inter-day accuracy ofmethod was also examined. The evaluated data are given in Table 2.

Sensitivity

Limit of quantitation (LOQ¼ 0.0663) and limit of detection (LOD¼0.01983) having signal-to-noise ratio of 10 and 3, respectively, were calcu-lated by using the following formula:-

LOD ¼ 3 Sa=b and LOQ ¼ 10 Sa=b

where Sa¼ standard deviation of intercept of standard curve; b¼ slope ofstandard curve.

The results of LOD and LOQ are illustrated in Table 2.

FIGURE 6 Resultant chromatogram of thermal degradation (80� 2�C). (Color figure availableonline.)

FIGURE 7 Resultant chromatogram of photolytic (UV) degradation (365nm). (Color figure availableonline.)

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Drug Stability in Solution

The stability of standard and sample preparations was determined over48 hr. Standard and sample preparations were stored at ambient tempera-ture under laboratory light conditions. Solutions were analyzed at 0, 24,and 48hr. The results were evaluated for the percentage difference fromtime zero. Less than 1.0% difference was observed, which demonstrates thatthe standard and sample preparations were stable for up to 48 hr, whenstored at ambient temperature under laboratory light conditions.

FIGURE 8 Calibration curve of Linezolid. (Color figure available online.)

TABLE 1 Accuracy and Precision for Linezolid Assay

Intra-Day

ParameterDay 1

(n¼ 10)Day 2

(n¼ 10)Day 3

(n¼ 10)Inter-Day forAll 30 Days

Added concentration 0.080mg=mLMeasured concentration(mg=mL)

0.08005� 0.0008 0.08006� 0.0007 0.08008� 0.0007 0.08006� 0.0007

Recovery (%) 97.45� 0.85 99.73� 0.25 99.92� 0.51 99.03� 0.54SD 1.015 1.009 1.001 1.008RSD (%) 1.04 1.01 1.00 1.016

Added concentration 0.100mg=mLMeasured concentration(mg=mL)

0.10003� 0.0008 0.10006� 0.0006 0.10004� 0.0007 0.10005� 0.0007

Recovery (%) 99.94� 0.59 100.09� 0.32 100.02� 0.41 100.01� 0.44SD 0.915 0.909 0.921 0.915RSD (%) 0.915 0.908 0.920 0.914

Added concentration 0.120mg=mLMeasured concentration(mg=mL)

0.12003� 0.0009 0.12005� 0.0007 0.12004� 0.0002 0.12003� 0.0005

Recovery (%) 99.98� 0.25 101.38%� 0.54 100.37%� 0.51 100.57� 0.43SD 0.726 0.734 0.712 0.724RSD (%) 0.728 0.725 0.710 0.721

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Application to Tablets

The present method was applied to the analysis of linezolid in threebatches of tablets of Lizoforce and Zyvox. The results presented inTable 3 are in good agreement with the labeled content. All data representthe average of three determinations.

Application to Stability Study

The proposed chromatographic method was successfully applied to theevaluation stability of linezolid in the solid phase. From the forced degra-dation study the drug linezolid was found to be more susceptible to oxidat-ive, alkaline, thermal, and photolytic as compared to acidic degradation(Table 4). The possible degraded product are may be due to presence ofacetamide substituent at the S-methyl position of oxazolidone derivative,which was found to be more reactive toward acid, alkali, and hydrogen per-oxide by giving different degradation products by breaking of the methyllinkage. The UV light and elevated temperature can cause changes inthe morpholine ring attached to the oxazolidone ring due to presence ofloan pair of electron on Nitrogen atom giving rise to degradedproducts.[26,27]

TABLE 2 The Intra-Day and Inter-Day Accuracy and Linearity of the Proposed Method

Intra-Day

Parameter Day 1 (n¼ 10) Day 2 (n¼ 10) Day 3 (n¼ 10)Inter-Day forAll 30 Days

Slope (a�Da) 5.1169� 0.003 5.1096� 0.0012 5.1138� 0.0047 5.1134� 0.0029Intercept (b�Db) 1.238� 0.54 1.312� 0.41 1.292� 0.23 1.280� 0.39Correlation coefficient (r2) 0.9999 0.9999 0.9998 0.9999RSD of slope a 0.768 0.792 0.771 0.777RSD of intercept b 0.0817 0.0923 0.0859 0.0866Frequency (n) 10 10 10 10LOD mg=ml 0.01970 0.01984 0.01995 0.01983LOQ mg=ml 0.0659 0.0647 0.0683 0.0663

RSD: Relative standard deviation; LOD: limit of detection; LOQ: limit of quantitation.

TABLE 3 Results Obtained in Determination of Linezolid in Tablets(Lizoforce and Zyvox)

Formulation Mean� (mg)� SD % Recovery

Lizoforce 599.94� 0.081 99.99Zyvox 601.05� 0.026 100.17

�Each value is average of 3 batches of tablets.

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CONCLUSION

The developed method for the determination of linezolid based on theuse of RP-HPLC with UV detection was reliable, simple, robust, sensitive,and precise. Furthermore the method is rapid; therefore, it is less timeconsuming and economical. The purposed method is stability indicating;therefore, it is suitable for estimation of linezolid in the presence ofdegraded products. In addition it is also suitable for estimation of drugin commercial tablets. Due to this, it can also be applied in the qualitycontrol of linezolid tablets.

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TABLE 4 Results Obtained in Forced Degradation Studies of Linezolid

Conditions AppliedPeak Area Drug andMajor Degradants

Rt of Drug and MajorDegradants (min)

% DrugRecovered

% DrugDecomposed

Standard drug (SD) 5168936 5.117 100 0SDþ 2ml of 0.5NHCl (1=2hr)

476108591873452

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SD solution underthermostat at80� 2�C (1=2hr)

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SD solution under UVat 365 nm (48hr)

443145910102557

5.5502.4333.108

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