WO2013024398A2

WO2013024398A2 - An improved method for the quantitative determination of linezolid

Info

Publication number: WO2013024398A2
Application number: PCT/IB2012/054017
Authority: WO
Inventors: Venkatraman JAYARAMAN; Sundara Kalyana BALAJI; Sanjiv DIXIT; Jagadish KADIA
Original assignee: Alembic Pharmaceuticals Limited
Priority date: 2011-08-12
Filing date: 2012-08-07
Publication date: 2013-02-21
Also published as: WO2013024398A3

Abstract

The present invention relates to an improved reversed-phase liquid chromatographic (RP-LC) method for the quantitative determination of Linezolid. The present invention further provides a stability indicating analytical method using the samples generated from forced degradation studies.

Description

AN IMPROVED METHOD FOR THE QUANTITATIVE DETERMINATION OF LINEZOLID

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Linezolid is chemically known as N-[[(5S)-3-[3-Fluoro-4-(4-morpholinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl] acetamide and marketed by Pfizer in US under brand name Zyvox. Linezolid is a synthetic antibacterial agent of the oxazolidinone class. It is used for the treatment of infections caused by multi-resistant bacteria including streptococci and methicillin-resistant Staphylococcus aureus.The structural formula is as shown beloow.

The product mixture of a reaction rarely is a single compound pure enough to comply with pharmaceutical standards. Side products and byproducts of the reaction and adjunct reagents used in the reaction will, in most cases, be present. At certain stages during processing of the Linezolid contained in the product mixture into an active pharmaceutical ingredient ('API'), the Linezolid must be analyzed for purity, typically by HPLC or GC analysis, to determine if it is suitable for continued processing or ultimately for use in a pharmaceutical product.

The U.S. Food and Drug Administration's Center for Drug Evaluation and Research (CDER) has promulgated guidelines recommending that drug applicants identify organic impurities of 0.1% or greater in the active ingredient. 'Guideline on Impurities in New Drug Substances,' 61 Fed. Reg. 371 (1996); 'Guidance for Industry ANDAs: Impurities in Drug Substances,' 64 Fed. Reg. 67917 (1999). Unless an impurity has been tested for safety, is in a composition proven to be safe in clinical trials, or is a human metabolite, the CDER further recommends that the drug applicant reduce the amount of the impurity in the active ingredient to below 0.1%. In order to obtain marketing approval for a new drug product, manufacturers must submit to the regulatory authority evidence that the product is acceptable for administration to humans. Such a submission must include, among other things, analytical data showing the impurity profile of the product to demonstrate that the impurities are either absent, or present in a negligible amount. Therefore, there is a need for analytical methods to detect impurities to identify and assay those impurities.

Generally, impurities (side products, byproducts, and adjunct reagents) are identified spectroscopically and by other physical methods and then the impurities are associated with a peak position in a chromatogram (or a spot on a TLC plate). Thereafter, the impurity can be identified by its position in the chromatogram, which is conventionally measured in minutes between injection of the sample on the column and elution of the particular component through the detector, known as the 'retention time' ('Rt'). This time period varies daily based upon the condition of the instrumentation and many other factors. To mitigate the effect that such variations have upon accurate identification of an impurity, practitioners use 'relative retention time' ('RRt') to identify impurities.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a reversed-phase liquid chromatographic (RP-LC) method for the quantitative determination of Linezolid.

In another aspect, the present invention provides an HPLC method for Linezolid containing less than about 5% area by HPLC, preferably less than about 3% area by HPLC, more preferably less than 1% area by HPLC, of total impurities.

In another aspect, the present invention further provides a stability indicating analytical method using the samples generated from forced degradation studies.

In yet another aspect, the present invention provides a simple, accurate and well-defined stability indicating an Ultra performance liquid chromatography (UPLC) method for the determination of Linezolid in the presence of degradation products.

In one aspect, the UPLC method described in the present invention has the following advantages when compared with prior art methods for determining the Linezolid and its related impurities:

All the impurities were well separated with a minimum resolution 6.0 (limit: Not less than 2.0);
Gradient profile to elute all related impurities and organic phase is 80% which ensure the elution and detection of non polar impurities forming during the process or stress study;
the present method mobile phase pH is about 4.5 which is more stable in all normal C18 columns;
consistency in specificity, precision & reproducibility with good peak shape; and
the degradation impurities from stress studies are well separated from the known impurities.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 (A-G) illustrates the UPLC chromatograms of blank, sample and spiked (Imp-D, Imp-B, Imp-II, Imp-I and Imp-C spiked in Linezolid) samples.

Fig. 1-A: Blank
Fig. 1-B: Test Sample chromatogram
Fig. 1-C: Spiked Sample Chromatogram (System Suitability)
Fig. 1-D: Spiked Sample Chromatogram (Specificity)

Fig. 2 (A-G) illustrates the typical HPLC chromatograms of stressed test samples of Linezolid.

Fig. 2-A: Blank (degradation)
Fig. 2-B: Test Sample (Used for degradation)
Fig. 2-C: Photolytic degradation
Fig. 2-D: Thermal degradation
Fig. 2-E: Acid Degradation
Fig. 2-F: Base Degradation
Fig. 2-G: Peroxide degradation

DETAILED DESCRIPTION OF THE INVENTION

As used herein, 'limit of detection (LOD)' refers to the lowest concentration of analyte that can be clearly detected above the base line signal, is estimated is three times the signal to noise ratio.

As used herein, 'limit of quantization (LOQ)' refers to the lowest concentration of analyte that can be quantified with suitable precision and accuracy, is estimated as ten times the signal to noise ratio.

As used herein, 'gradient elution' refers to the change in the composition of the gradient eluent over a fixed period of time, stepwise or at a constant rate of change, as the percentage of the first eluent is decreased while the percentage of the second eluent is increased.

As used herein, 'gradient eluent' refers to an eluent composed of varying concentrations of first and second eluents.

The five main known impurities of Linezolid are:

Impurity-B: N-[[(5S)-3-[4-(4-morpholinyl) phenyl]-2-oxo-5-oxazolidinyl] methyl] acetamide (Desfluoro), having following structure:

The impurity B is detected and resolved from Linezolid by UPLC with an relative retention time (hereafter referred as rrt) of 0.67.

Impurity-C: Bis {N-[[(5S)-3-[3-fluoro-4-(4-morpholinyl) phenyl]-2-oxo-5-oxazolidinyl] methyl]}acetamide (Bis Linezolid), having following structure:
The impurity C is detected and resolved from Linezolid by UPLC with an rrt of 1.71.
Impurity-D:(5S)-5-(amino methyl)-4-(3-fluoro-4-morpholin-4-ylphenyl)-1,3- oxazolidin-2-one (Amine), having following structure:
The impurity D is detected and resolved from Linezolid by UPLC with an rrt of 0.34.
Impurity-I: (R)-[N-3-(3-Fluoro-4-morpholinylphenyl)-2-oxo--5-oxazolidinyl]methyl azide, having following structure:
The impurity I is detected and resolved from Linezolid by HPLC with an rrt of 1.64.
Impurity-II: (R)-[N-3-(3-Fluoro-4-morpholinylphenyl)-2-oxo--5-oxazolidinyl]methyl methanesulfonate , having following structure:
The impurity II is detected and resolved from Linezolid by UPLC with an RRt of 1.14.

According to one aspect of the present invention, there is provided a reversed-phase liquid chromatographic (RP-LC) method for quantifying, by area percent, the amounts of Linezolid and all impurities, preferably, imp-B, imp-C, imp-D, Imp-I and Imp-II present in a sample of Linezolid.

According to another aspect of the present invention , there is provided a stability indicating analytical method using the samples generated from forced degradation studies.

According to another aspect of the present invention , there is provided an accurate and well-defined stability indicating an HPLC method for the determination of Linezolid in the presence of degradation products.

Preferably, the method for determining the amount of impurities in a Linezolid sample comprises the steps of:

combining a Linezolid sample with a mixture of Water and acetonitrile in the ratio of 60:40 (v/v) to obtain a solution;
injecting the sample solution into a 100mm×2.1mm column with 1.7μm Acquity UPLC BEH C18 column;
gradient eluting the sample with a mixture of A Eluent and B Eluent in the ratio of 75:25 (v/v) initial and progressively increased to 20:80(v/v) in 9.5 minutes 40 minutes time;
Preparing Eluent A by adding Potassium dihydrogen ortho phosphate in water and the pH observed was about 4.5 (A Eluent) and a B Eluent with methanol: 75:25 (v/v); and
Measuring of the amounts of Linezolid and each impurity at 250nm wavelength with a UV detector (having an appropriate recording device).

The buffer in step-(d) may be prepared by mixing about 1L water with about 2.72 g potassium dihydrogen ortho phosphate.

Preferably, the ratio of mobile phase buffer and solvent in step-(d) may be continued at the same ratio for 2 minutes then changed linearly to 65:35 (v/v) within 6 minutes followed by subsequent ratio change of 20:80 (v/v) is within 1.5 minutes. After 5.5 minutes the initial gradient of 80:20 is for 3.0 minutes to be conditioned for every analysis. The column temperature may be maintained at about 40⁰C.

The LOD /LOQ values of Linezolid e and its related impurities, Imp-I, Imp-II, Imp-B, Imp-C and Imp-D are summarized in Table 1.

Sr. No	Components	LOD (%)	LOQ ( %)
1	Imp-I	0.0032	0.0095
2	Imp-II	0.0055	0.0169
3	Imp-B	0.0059	0.0180
4	Imp-C	0.0054	0.0165
5	Imp-D	0.0056	0.0169
6	Linezolid	0.0045	0.0139

Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities and degradation products. The specificity of the LC method for Linezolid Intentional degradation was attempted to stress conditions of photolytic degradation (as per ICH recommended condition), acid hydrolysis (using 1.0M HCl), base hydrolysis (using 1M NaOH), and oxidative degradation (using 3.0% H₂O₂) to evaluate the ability of the proposed method to separate Linezolid from its degradation products. To check and ensure the homogeneity and purity of Linezolid peak in the stressed sample solutions, PDA-UV detector was employed. Assessment of mass balance in the degraded samples was checked by comparing with control standard sample using PDA detector. Related substances studies were carried out on the stressed samples against Linezolid standard sample and the mass balance (Area of the Linezolid + sum of the area of all impurities + sum of area all degradants) was tabulated.

Preferably, the limit of detection (LOD) and limit of quantification (LOQ) were estimated to be 0.0056 % and 0.0169 % for imp-D, 0.0059 % and 0.0180 for imp-B, 0.0045 % and 0.0139 % for Linezolid , 0.0055 %, 0.0169 %l for imp-II,0.0031 % ,0.0095% and 0.0054%,0.0165% for Imp-C were estimated at a signal-to-noise ratio of 3:1 and 10:1 by LOQ precision, respectively by injecting a series of diluted solutions with known concentration.

The accuracy of the related substances method with the spiked impurities was evaluated in duplicate at four different concentration levels, i.e. LOQ (0.0171% for imp-D, 0.0173 % for imp-B ,0.0167% for Imp-II,0.0096% for Imp-I and 0.0165 for imp-C), 50 % level (0.074% for imp-D, 0.075 % for imp-B ,0.075% for Imp-II,0.076% for Imp-I and 0.074 for imp-C), 100% level (0.149% for imp-D, 0.151 % for imp-B ,0.152% for Imp-II,0.152% for Imp-I and 0.149 for imp-C) and 150% level (0.224% for imp-D, 0.227 % for imp-B ,0.228% for Imp-II,0.229 % for Imp-I and 0.225 for imp-C). 100% level (1.988µg/ml for imp-2, 2.008µg/ml for imp-1 and 2.002µg/mL for imp-3) and 50 % level in bulk drug sample. The % recoveries for Imp-1, Imp-2 and Imp-3 were calculated from the standard solution.

According to another aspect of the present invention, there is provided a chromatographic method to get the separation of all impurities and stress studies degradants from analyte peak. Satisfactory chromatographic separation was achieved using the mobile phase consists of phosphate buffer (2.72 g potassium dihydrogen ortho phosphate in 1000ml of HPLC water, and solvent methanol.

In the optimized conditions the Linezolid , Imp-I, Imp-II , Imp-B, Imp-C and Imp-D were well separated with a resolution of greater than 6 and the typical retention times (RT) of Imp-D, Imp-B , Linezolid , Imp-II,Imp-I and Imp-C were about 2.04, 4.04, 5.99 , 6.81,9.800 and 10.27 minutes, and typically shown in Figure 1. The system suitability results and the developed LC method were found to be specific for Linezolid and its five impurities, namely Imp-1, Imp-2 and Imp-3.

The system suitability values and mass numbers of Linezolid and its impurities were summarized in Table 2.

Compound (n=1)	Rt	Rs	N	T	(m/z)
Imp-D	2.046		5652	1.31	295.30
Imp-B	4.040	16.40	15146	1.12	319.35
Linezolid	5.991	14.53	32559	1.14	337.35
Imp-II	6.810	6.22	47132	1.21	374.38
Imp-I	9.800	35.95	1076188	1.03	321.30
Imp-C	10.275	12.42	1184484	1.15	615.62

*n=1: determination, Rt: retention time, Rs: USP resolution, N: number of theoretical plates (USP tangent method), T: USP tailing factor, m/z: mass number.

No considerable degradation observed in Linezolid bulk samples, under stress conditions such as photolytic stress and thermal stress,. Some level of degradation in test solution was achieved using 3% hydrogen peroxide at RT for 24 hours, 1M sodium hydroxide at RT for 10 minutes and 1M HCL at 60°C for 4 hours . Impurities observed in stress condition using PDA detector, typically shown in Figure. 2. The peak test results obtained from PDA & LC-MS/MS confirm that the Linezolid peak is homogeneous and pure in all analyzed stress samples. The mass balance of stressed samples was close to 98.0% indicates that no co-eluting impurities in the main peak. The mass balance of Linezolid is unaffected in the presence of Imp-B, Imp-C, Imp-D, Imp-I and Imp-II, which confirms the stability indicating method developed.

The forced degradation study results were summarized in Table 3.

Stress Condition	Impurity- D (%)	Impurity- B (%)	Impurity- II (%)	Impurity- I (%)	Impurity- C (%)	Major unknown impurity (%)	% Degrad-ation
Sample	ND	ND	ND	ND	ND	0.03
Sample (Heat at 90°C 5 days)-1	ND	ND	ND	ND	ND	0.03	----
Sample (photo deg. 1.2 lux hrs)-1	ND	ND	ND	ND	ND	0.03	----
Sample, 1M NaOH, 10 min RT	ND	5.36	ND	ND	ND	ND	5.30
Sample, 1M HCl 5min	ND	ND	ND	ND	ND	0.03	----
Sample, 1M HCl 4 hrs at 60°C	3.25	ND	ND	ND	ND	0.45	3.91
Sample, 1M HCl 24 hrs at RT	0.27	ND	ND	ND	ND	0.10	0.31
Sample, 3%H2O2, 5 min RT	ND	ND	ND	ND	ND	0.62	1.13
Sample, 3%H2O2, 4 hrs at 60°C	ND	ND	ND	ND	ND	56.10	99.48
Sample, 3%H2O2, 24 hrs at RT	ND	ND	ND	ND	ND	9.17	18.27

The percentage recovery of Linezolid of its impurities in bulk drug samples was ranged from LOQ to 150.0%. The percentage recovery of Imp-B, Imp-C, Imp-D, Imp-I and Imp-II in bulk drugs samples was ranged from 99.93 to 115.98. HPLC chromatograms of blank, pure sample and all three impurities spiked in Linezolid bulk drug sample were shown in Table 4.

Compound (n=1)	Recovery at LOQ level	Recovery at 100% level
Imp-D	115.98	101.29
Imp-B	104.01	102.39
Linezolid
Imp-II	96.26	103.67
Imp-I	105.76	100.80
Imp-C	100.70	100.05

*n= determination

In deliberate varied chromatographic conditions (pH and column) the resolution between all the components is not less than 6.0, illustrating the robustness of the method.

Experimental

The LC system, used for method development and forced degradation studies and method validation was Waters-Acquity (manufactured by Waters India Ltd) LC system with a photo diode detector. The out put signal was monitored and processed using Empower 2 software system (designed by Waters India) on IBM computer (Digital Equipment Co).

The chromatographic column used was a Waters Acquity BEH C18 100mm ×2.1 mm column with 1.7 μm particles. The mobile phase consists of 0.02M potassium dihydrogen ortho phosphate buffer (2.72g of potassium dihydrogen ortho phosphate in 1000ml of HPLC water), and solvent is methanol. The flow rate of the mobile phase was kept at 0.3 ml/min.Beginning with the gradient ratio of mobile phase buffer and solvent 75:25(v/v), system was continued at the same ratio for 2 minutes. The ratio was changed linearly 65:35(v/v) within 6 minutes and again the ratio was changed linearly 20:80(v/v) within 1.5 minutes the system was continued at the same ratio for 5.5 minutes. After 0.5 minutes the initial gradient of 75:25 is for 3 minutes to be conditioned for every analysis. The column temperature was maintained at 40⁰C and the wavelength was monitored at a wavelength of 250 nm. The injection volume was 2 μL for related substances and assay determination. Mixture of Water and acetonitrile in the ratio of 50:50 (v/v) was used as diluent during the standard and test samples preparation.

Preparation of reference solution:

7.5 mg of each Imp-B,Imp-C,Imp-D,Imp-I,Imp-II and Linezolid were accurately weighed and transferred to the 100mL volumetric flask(BOROSIL-Class-A) , separately; 20ml of diluent was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with diluent. Pipette out 1.0mL from solution and transferred in to a 100mL volumetric flask (BOROSIL-Class-A), and made up to mark with diluent.

A working solution of 500μg/ml was prepared for related substances determination analysis.

Claims

A HPLC method for analyzing Linezolid, wherein the mobile phase comprises two or more liquids, including a first eluent A and a second eluent B, and the relative concentration of the liquids is varied to a predetermined gradient.
A HPLC method according to claim 1, wherein the first eluent A is buffer.
A HPLC method according to claim 1, wherein the first eluent B is methanol.
A HPLC method according to claim 1, wherein gradient of A eluent and B eluent in the ratio of 75:25 (v/v) initial and progressively increased to 20:80(v/v) in 9.5 minutes and 40 minutes time.
A HPLC method according to claim 2, wherein buffer is about 2.72 g potassium dihydrogen ortho phosphate in 1 liter of water and pH about 4.5.
A HPLC method for Linezolid containing less than about 5% area by HPLC, preferably less than about 3% area by HPLC, more preferably less than 1% area by HPLC, of total impurities.
A HPLC method determining the amount of impurities in Linezolid sample comprises the steps of:

a) combining a Linezolid sample with a mixture of Water and acetonitrile in the ratio of 60:40 (v/v) to obtain a solution;

b) injecting the sample solution into a 100mm×2.1mm column with 1.7μm UPLC BEH C18 column;

c) gradient eluting the sample with a mixture of A eluent and B eluent in the ratio of 75:25 (v/v) initial and progressively increased to 20:80(v/v) in 9.5 minutes and 40 minutes time.

d) Preparing eluent A by adding Potassium dihydrogen ortho phosphate in water and the pH observed was about 4.5 (A eluent);

e) Preparing B eluent is methanol; and

f) Measuring of the amounts of Linezolid and each impurity at 250nm wavelength with a UV detector (having an appropriate recording device).