chapter-7 determination of metaxalone and its impurities...

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Chapter-7

Determination of Metaxalone and its Impurities

In Solid Oral Dosage Form

Overview

Rapid determination of Metaxalone and its related substances in solid oral dosage form using the

developed and validated, stability indicating, RP-UPLC method is presented in this chapter.

7.1 LITERATURE REVIEW

UPLC is a new category of separation technique based upon well-established principles of liquid

chromatography, which utilizes sub-2 µm particles for the stationary phase. These particles operate

at elevated mobile phase linear velocities to affect dramatic increases in resolution, sensitivity and

speed of analysis. Owing to its speed and sensitivity, this technique has gained considerable

attention in recent years for pharmaceuticals and biomedical analysis [1-3]. In the present work, this

technology has been applied to the method development and method validation study of related

substances and the assay determination of Metaxalone in pharmaceutical dosage forms.

Very few methods have appeared in the literature for the assay determination of META in bulk and

pharmaceutical dosage forms by high-performance liquid chromatography (HPLC) [4-6].

Narasimha et al. [7], described method validation of META by using UV spectroscopy. Nirogi RV

et al. [8], described a method for quantification of META in human plasma by HPLC coupled to

tandem mass spectroscopy. META is not official as drug substance as well as drug product in the

European Pharmacopoeia (Ph. Eur.) and United State Pharmacopoeia (USP). To the best of our

knowledge, none of the currently available analytical methods can separate and quantify all the

known related compounds and degradation impurities of META API and dosage forms.

Determination of Metaxalone and its Impurities In Solid Oral Dosage Form

247

Furthermore, there is no stability-indicating HPLC/UPLC method reported in the literature that can

adequately separate all impurities and accurately quantify META API and dosage forms. It is,

therefore, felt necessary to develop a new rapid, stability-indicating method for the related

substance determination and quantitative determination of META.

Therefore, a reproducible stability-indicating RP-UPLC method is developed for the quantitative

determination of META and its two known impurities (Imp-A and Imp-B), this developed proposed

method is also able to separate two unknown degradation products from interested compounds

within 6.0 min. This method is successfully validated according to the ICH guidelines (Validation

of Analytical Procedures: Test and Methodology Q2) [9]. Chemical structures, UV spectra and

IUPAC name of META, Imp-A and Imp-B are presented in Figure 7.1.

________________________________________________________________________ Metaxalone (META): 5-[(3,5-dimethylphenoxy)methyl]-1,3-oxazolidin-2-one

Impurity-A (Imp-A): 3-(3,5-dimethylphenoxy)propane-1,2-diol

Impurity-B (Imp-B): 3,5-dimethylphenol

[Figure 7.1 Chemical structures, UV spectra and IUPAC name of META, Imp-A and

Imp-B]

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248

7.2 THE SCOPE AND OBJECTIVES OF PRESENT STUDY

There is no stability-indicating HPLC/UPLC method reported in the literature that can adequately

separate all impurities and accurately quantify META API and dosage forms. It is, therefore, felt

necessary to develop a new rapid, stability-indicating method for the related substance

determination and quantitative determination of META. We intend to opt for a faster

chromatographic technique UPLC, for the said study. An attempt is made to determine whether

UPLC can reduce analysis times without compromising the resolution and sensitivity.

The objectives of the present work are as follow:

To developed a rapid, stability indicating RP-UPLC method for determination of

metaxalone and its related substances in solid oral dosage form.

Forced degradation study.

To separates metaxalone from its all impurities (A, B) known impurities/ degradation

products and any unknown degradation product generated during forced degradation study.

Perform analytical method validation for the proposed method as per ICH guideline.

7.3 METAXALONE

Metaxalone, which is 5-[(3,5-dimethylphenoxy)methyl]-1,3-oxazolidin-2-one [Figure 7.2], was first

synthesized by Lunsford, et al. [10]. The chemistry of the metabolic products was proposed by

Bruce et al. [11]. Metaxalone (marketed by King Pharmaceutical under the brand name

SKELAXIN®, approved by the US FDA in 1962 [12]) is a muscle relaxant and useful to relieve the

pain caused by strains, sprains, and other musculoskeletal conditions. Its exact mechanism of action

is not known, but it may be due to general central nervous system depression. It is considered to be

a moderately strong muscle relaxant, with relatively low incidence of side effects, low toxicity [13]

and no reports of major safety issues [14]. In addition, because of new indications it seems that

interest would be continued for this drug product [15-17]. Metaxalone exhibits increased

bioavailability when taken with food [18]. Metaxalone molecular weight is 221.252 g/mole with


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molecular formula C12H15NO3. Its melting point is 122°C [12]. Metaxalone is a white to almost

white, odorless crystalline powder freely soluble in chloroform, soluble in methanol and in 96 %

ethanol but practically insoluble in ether or water [12].

[Figure 7.2 Chemical structure of Metaxalone (META)]

Indications

For the treatment of painful peripheral musculoskeletal conditions and spasticity from upper motor

neuron syndromes.

Pharmacodynamics

Metaxalone is a skeletal muscle relaxant indicated as an adjunct to rest, physical therapy, and other

measures for the relief of discomforts associated with acute, painful musculoskeletal conditions.

The mode of action of this drug has not been clearly identified, but may be related to its sedative

properties. Metaxalone does not directly relax tense skeletal muscles in man [12].

Mechanism of action

The mechanism of action of metaxalone in humans has not been established, but may be due to

general central nervous system depression.

Absorption

The absolute bioavailability of metaxalone from Skelaxin tablets is not known.

Protein binding

Not available.

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250

Metabolism

Probably hepatic.

Route of elimination

Metaxalone is metabolized by the liver and excreted in the urine as unidentified metabolites.

Half life

9.2 (+/- 4.8) hours.

Toxicity

LD50=775mg/kg (Rat, oral); LD50=1690 mg/kg (Mouse, oral). When determining the LD50 in rats

and mice, progressive sedation, hypnosis and finally respiratory failure were noted as the dosage

increased. In dogs, no LD50 could be determined as the higher doses produced an emetic action in

15 to 30 minutes [12].

Affected organisms

Humans and other mammals.

7.4 EXPERIMENTAL

7.4.1 Materials and reagents

Metaxalone (99.9% w/w) working standard, Impurity-A (99.8% w/w) reference standard, Impurity-

B (99.7% w/w) reference standard, placebo (Batch No. F027P) and Metaxalone tablets (Batch No.

F027) are provided by Dr. Reddy’s laboratories Ltd., Hyderabad, India. HPLC grade acetonitrile

and methanol are obtained from J.T.Baker (NJ, USA). GR grade orthophosphoric acid and

triethylamine are obtained from Merck Ltd. (Mumbai, India). 0.22 µm nylon membrane filter and

nylon syringe filters are purchased from Pall life science limited (India). 0.22 µm PVDF syringe

filters are purchased from Millipore (India). High purity water is generated using Milli-Q Plus


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water purification system (Millipore®, Milford, MA, USA). All other chemicals used are of

analytical grade.

7.4.2 Equipments

Cintex digital water bath (Mumbai, India) is used for specificity study. Photo stability studies are

carried out in a photo-stability chamber (SUNTEST XLS+, ATLAS, Germany). Thermal stability

studies are performed in a dry air oven (Cintex, Mumbai, India).

7.4.3 Preparation of mobile phase

The mobile phase is consisted a mixture of water, acetonitrile, methanol and triethylamine in the ratio

of 50:25:25:0.1 (v/v/v/v), pH is adjusted to 6.3 with orthophosphoric acid and filtered through 0.22 µm

nylon membrane filter.

7.4.4 Diluent preparation

Mobile phase is used as diluents.

7.4.5 System suitability solution preparation

Suitability solution is prepared by dissolving standard substances in diluent to obtain solution

containing 1000 µg/mL of Metaxalone, 1 µg/mL of Imp-A and 1 µg/mL of Imp-B.

7.4.6 Standard solution preparation

Standard solution are prepared by dissolving META working standard in diluent to obtain solution

containing 1000 µg/mL for assay and 1µg/mL for related substances.

7.4.7 Sample solution preparation

Sample solution is prepared by dissolving sample (twenty tablets are crushed to fine powder by

mortar and pestle) in diluent to obtain a solution containing 1000 µg/mL of Metaxalone (for assay

and related substances). It is then filtered through 0.22 µm PVDF syringe filter and the filtrate is

collected after discarding first few milliliters.

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252

7.4.8 Placebo solution preparation

Twenty tablets of placebo are crushed to fine powder. An accurately weighed portion of the powder

equivalent to 25 mg of META is taken into 25 ml volumetric flask. About 20 ml of diluent is added

to this volumetric flask and sonicated in an ultrasonic bath for 10 minutes. Dilute volume up to the

mark and mixed well. It is then filtered through 0.22 µm PVDF syringe filter and the filtrate is

collected after discarding first few milliliters.

7.4.9 Chromatographic conditions

Analysis is performed on an Acquity UPLCTM

system (Waters, Milford, USA), consisting of a

binary solvent manager, sample manager, and PDA (photo diode array) detector. System control,

data collection and data processing are accomplished using Waters EmpowerTM

-2 chromatography

data software. The chromatographic condition is optimized using an Acquity® HSS-T3 (100 mm x

2.1 mm, 1.8 µm) column. The mobile phase is consisted a mixture of water, acetonitrile, methanol

and triethylamine in the ratio of 50:25:25:0.1 (v/v/v/v), pH is adjusted to 6.3 with orthophosphoric

acid and filtered through 0.22 µm nylon membrane filter. Mobile phase is used as a diluent. The

finally selected and optimized conditions are as follows: injection volume 1 µL, isocratic elution,

flow rate 0.3 mL/min, column oven temperature at 45°C, detection wavelength 230 nm for assay

and 205 nm for related substances determination. Under these conditions, the backpressure in the

system is about 6,000 psi.

7.4.10 Method Validation

The method described herein has been validated for simultaneous determination of Assay and its

related substances by RP-UPLC.

7.4.10.1 Specificity

Forced degradation studies are performed to demonstrate selectivity and stability-indicating

capability of the proposed method. The sample are exposed to acid hydrolysis [1 N HCl (3 mL),


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60°C, 1h], base hydrolysis [1N NaOH (3 mL), 60°C, 30 min], oxidative [30 % H2O2 (3 mL), 60°C,

1h], thermal [105°C, 6h], water hydrolysis [at 60 °C for 2h], and photolytic degradation [1.2

million Lux hours]. All exposed samples are than analysed by the proposed method.

7.4.10.2 System suitability

System suitability parameters are measured so as to verify the system performance. System

precision is determined on six replicate injections of standard preparation. All important

characteristics including % RSD, resolution (between META and Imp-B), tailing factor and

theoretical plate number are measured.

7.4.10.3 Precision

The precision of the system is determined using the sample preparation procedure described above

for six real samples of formulation and analysis using the same proposed method. Intermediate

precision is studied by other scientist, using different columns, different UPLC, and is performed

on different days.

7.4.10.4 Accuracy

To confirm the accuracy of the proposed method, recovery experiments are carried out by the

standard addition technique for assay and impurities addition technique for related substances. The

accuracy of the assay method for META is evaluated in triplicate (n=3) at the five concentrations of

500, 750, 1000, 1250 and 1500 µg/mL (50, 75, 100, 125 and 150 %) of drug product, and the

recovery is calculated for each added (externally spiked) concentration. The mean of percentage

recoveries (n=15) and the relative standard deviation are calculated. For all impurities, the recovery

is determined in triplicate (n=3) for 0.5, 0.75, 1.0, 1.25 and 1.5 µg/mL (50, 75, 100, 125 and 150 %)

of the analyte concentration (1000 µg/mL) of the drug product, and the recovery of the impurities is

calculated. The mean of percentage recoveries (n=15) and the relative standard deviation are also

calculated for related substances.

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254

7.4.10.5 Linearity

For related substances test, linearity is demonstrated from LOQ (0.1 µg/mL) to 2.0 µg/mL of

standard concentration of Impurities using a minimum of nine calibration levels (LOQ, 0.2, 0.4,

0.6, 0.8, 1.0, 1.2, 1.5 and 2.0 µg/mL) for Imp-A and Imp-B. For assay test, linearity is demonstrated

from 50% to 150% of standard concentration using a minimum of seven calibration levels (500,

700, 800, 1000, 1200, 1400 and 1500 µg/mL) for META. The method of linear regression is used

for data evaluation. The peak areas of the standard compounds are plotted against the respective

META, Imp-A and Imp-B concentrations. Linearity is described by the linearity equation,

correlation coefficient and Y-intercept bias is also determined.

7.4.10.6 Robustness

The robustness is a measure of the capacity of a method to remain unaffected by small but

deliberate changes in flow rate (± 0.05 mL/min), change in column oven temperature (± 5 °C) and

change in organic solvent ratio (± 10%). All important characteristic including % assay, resolution

(between META and Imp-B), tailing factor, theoretical plates number and the retention behaviour

of interested compound are evaluated.

7.4.10.7 Solution stability

The stability of the sample solution is established by storage of the sample solution at ambient

temperature for 24h. The sample solution is re-analyzed after 12 and 24h, and the results of the

analysis are compared with the results of the fresh sample. The stability of standard solution is

established by the storage of the standard solution at ambient temperature for 24h. The standard

solution is re-injected after 12 and 24h, and % RSD is calculated.

7.4.10.8 Filter compatibility

Filter compatibility is performed for nylon 0.22 μm syringe filter (Pall Life sciences) and PVDF

0.22 μm syringe filter (Millipore). To confirm the filter compatibility in proposed analytical


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method, filtration recovery experiment is carried out by sample filtration technique. Sample is

filtered through both syringe filters and percentage assay is determined and compared against

centrifuged sample.

7.5 RESULTS AND DISCUSSION

7.5.1 Method Development and Optimization

The main objective of the RP-UPLC method development is to rapid assay and related substances

determination of Metaxalone in pharmaceutical formulation are: the method should be able to

determine assay (AS) and related substances (RS) in single run and should be accurate,

reproducible, robust, stability indicating, filter compatible, linear, free of interference from blank /

placebo / impurities / degradation products and straightforward enough for routine use in quality

control laboratory.

The spiked solution of META (1000 µg/mL), IMP-A (1 µg/mL) and IMP-B (1 µg/mL) is subjected

to separation by RP-UPLC. Initially the separation of all compounds is studied using water as a

mobile phase-A (MP-A) and acetonitrile (ACN) as a mobile phase-B (MP-B) on a UPLC column

(Eclipse Plus C18, RRHD, 50 x 2.1mm; 1.8µm) using a Waters (UPLC) system with the linear

gradient program. The flow rate of 0.3 mL/min is selected with regards to the backpressure and

analysis time as well. Various types of MP-A and MP-B are studied to optimize the method, which

are summarized in Table 7.1 with the associated observations.

Based on mobile phase selection experimental study, the optimized UPLC parameters are; flow rate

0.3 mL/min; column oven temperature 45°C; mixture of water, ACN, MeOH and TEA in the ratio

of 50:25:25:0.1 v/v/v/v respectively (pH adjusted 6.3 with OPA). In order to achieve symmetrical

peak shape of all substances and more resolution between META and Imp-B different stationary

phases are explored. Peak merging (Imp-B and META) and broad peak shape of META is observed

with an Acquity BEH C8 (100 x 2.1 mm, 1.7µm) column. Poor resolution (Imp-B and META) and

broad peak shape of META is observed with an Acquity BEH C18 (100 x 2.1 mm, 1.7µm) column.

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256

Finally the desired separation with symmetrical peaks is obtained using Acquity HSS-T3 (100 x

2.1mm, 1.8µm) column. Column oven temperature is also studied and found that 45°C is more

appropriate with respect to separation and peak shape. Based on compounds UV response, 230nm

(for assay) and 205nm (for related substances) is found more appropriate for determination of

META and its impurities from single run. META, Imp-A, and Imp-B are well resolved from each

other and there is no chromatographic interference observed due to blank and placebo in a

reasonable time of 6.0 minutes, which are presented in Figure 7.3 and 7.4. Overlaid specimen

chromatograms of assay study are presented in Figure 7.5.

Table 7.1 Summary of mobile phase optimization

MP-A MP-B Observation

Water CAN Co-eluting peak of Imp-B and META

0.1% aqueous TEA CAN Co-eluting peak of Imp-B and META

0.1% aqueous TEA ACN:MeOH

(50:50 v/v)

Poor resolution (Imp-B and META), USP

tailing more than 2.0 for META peak

Mixture of water, ACN, MeOH

and TEA [50:25:25:0.1 v/v/v/v]

N.A. Poor resolution (Imp-B and META), USP

tailing more than 3.0 for META peak

Mixture of water, ACN, MeOH

and TEA [50:25:25:0.1 v/v/v/v],

pH adjusted 6.3 with OPA

N.A. Poor resolution (Imp-B and META) and

broad peak shape of META.

USP = United state pharmacopoeia; MeOH=Methanol; TEA=Triethylamine;

OPA= Orthophosphoric acid; ACN= Acetonitrile

[Figure 7.3 Expanded overlaid specimen chromatograms of blank, placebo and

sample (at 230nm)]


257

[Figure 7.4 Overlaid specimen chromatograms of blank, placebo, diluted standard

and spiked impurities along with analyte (at 205nm)]

[Figure 7.5 Overlaid specimen chromatograms of assay study (at 230nm)]

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258

7.5.2 Analytical Parameters and Validation

After satisfactory development of the method it is subjected to method validation as per ICH

guideline [9]. The method is validated to demonstrate that it is suitable for its intended purpose by

the standard procedure to evaluate adequate validation characteristics (system suitability, accuracy,

precision, linearity, robustness, solution stability, filter compatibility and stability indicating

capability).

7.5.2.1 Specificity

Specificity is the ability of the method to measure the analyte response in the presence of its

potential impurities. Forced degradation studies are performed to demonstrate selectivity and

stability indicating capability of the proposed RP-UPLC method. Figure 7.3 and 7.4 are show that

there is no any interferences at the RT (retention time) of META due to blank, placebo and

impurities. Stress studies are performed at concentration of 1000 µg/mL of META to provide the

stability indicating property and specificity of the proposed method. Spectral purity of META, Imp-

A and Imp-B are presented in Figure 7.6.

Forced degradation studies are performed by the stress conditions acid hydrolysis [1 N HCl (3 mL),

60°C, 1h, Figure 7.7], base hydrolysis [1N NaOH (3 mL), 60°C, 30 min, Figure 7.8], oxidative [30

% H2O2 (3 mL), 60°C, 1h, Figure 7.9], thermal [105°C, 6h, Figure 7.10], water hydrolysis [at 60 °C

for 2h, Figure 7.11], and photolytic degradation [1.2 million Lux hours, Figure 7.12] to evaluate the

ability of the proposed method to separate META from its degradation products. Minor degradation

products are observed when META is subjected to acid, heat, photolytic and hydrolytic conditions.

Significant degradation is observed when the drug product is subjected to base hydrolysis, and

slight degradation is observed when the drug product is subjected to oxidative hydrolysis. The

purity of the peaks obtained from the stressed sample is verified using the PDA detector. The

obtained purity angle is less than purity threshold for all the stressed samples. An assay of samples

is performed by comparison with reference standards, and the mass balance [% assay + % known


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impurities + area % unknown impurities, at 205nm] for each of the stressed samples is calculated.

The results from forced degradation study are given in Table 7.2. Peak purity plots of all stress

condition samples are presented in Figure 7.13.

[Figure 7.6 Spectral purity of META, Imp-A and Imp-B]

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260

[Figure 7.7 Overlaid chromatograms of acid hydrolysis study]

[Figure 7.8 Overlaid chromatograms of base hydrolysis study]

[Figure 7.9 Overlaid chromatograms of peroxide degradation study]


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[Figure 7.10 Overlaid chromatograms of heat degradation study]

[Figure 7.11 Overlaid chromatograms of hydrolytic degradation study]

[Figure 7.12 Overlaid chromatograms of photolytic degradation study]

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262

Table 7.2 Summary of forced degradation results

Degradation

condition

Mass

balance#

Purity Observation

Angle Threshold

Control sample 99.9 0.055 0.261 N.A.

Acidic hydrolysis 99.2 0.095 0.285 No degradation observed

Alkaline hydrolysis 98.8 0.131 0.333 Significant degradation observed

Oxidation 98.5 0.075 0.284 No significant degradation observed

Water hydrolysis 99.5 0.055 0.258 No significant degradation observed

Thermal (solid) 100.1 0.055 0.261 No significant degradation observed

Photolytic 100.3 0.055 0.259 No significant degradation observed

N.A. Not applicable; # = % assay + % known impurities + area % unknown impurities

Base hydrolysis Water hydrolysis

Oxidative Acid hydrolysis


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Thermal Photolytic

[Figure 7.13 Obtained spectral purity plot of META in forced degradation study]

7.5.2.2 System suitability

The percentage RSD of area counts of six replicate injections is below 1.0 % [Table 7.3]. The

parameters all complied with the acceptance criteria and system suitability is established. As seen

from this data, the acceptable system suitability parameters would be: resolution between Imp-B

and META is not less than 1.5, theoretical plates is not less than 5000, tailing factor for META is

not more than 2.0. Specimen chromatogram of system suitability solution and diluted standard are

presented in Figure 7.14 and 7.15 respectively.

Table 7.3 System suitability results (system precision, method precision and

intermediate precision)

Test Parameters Imp-A

(1µg/mL)

Imp-B

(1µg/mL)

META

(1µg/mL)

META

(1000µg/mL)

System Precision Area % RSD 0.8 0.9 0.9 0.3

Precision (n=6) USP resolution N.A. N.A. N.A. 2.34

USP tailing 1.34 1.52 1.12 1.19

USP plate count 8831 7219 11975 11994

Intermediate precision

(n=6)

USP resolution N.A. N.A. N.A. 2.32

USP tailing 1.32 1.52 1.13 1.15

USP plate count 8906 6570 11848 12162

N.A.= Not applicable

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264

[Figure 7.14 Specimen chromatogram of system suitability solution]

[Figure 7.15 Specimen chromatogram of diluted standard solution]

7.5.2.3 Precision

The system precision of the related substance (at 205 nm) method is verified by injecting six

replicate injections of a standard solution contains META (1 µg/mL) and its two impurities of 1

µg/mL of each. The RSD (%) of the peak areas is calculated for each compound (system precision).

Method precision experiments are conducted in six individual preparations of META (1000 µg/mL)

spiked with 1 µg/mL each of the impurities and the RSD (%) for area percentage of each impurities

is calculated. Precision of assay (at 230 nm) method is evaluated by performing six (n=6)

independent assays of META tablet at 1000 mg/mL level against a qualified working standard. The

RSD (%) of the six results is calculated. The intermediate precision of the assay and RS method is

evaluated by different analyst, instrument and day. The RSD (%) of peak area of META, Imp-A

and Imp-B in system precision is within 1.0% [Table 7.3]. The RSD (%) results of META and its

impurities for precision and intermediate precision are presented in [Table 7.4]. These results


265

confirmed the high precision of the method. Overlaid chromatogram for the assay standard

preparation (at 230 nm), diluted standard preparation (at 205 nm), assay precision study and related

substances precision study are presented in Figure 7.16, 7.17, 7.18 and 7.19 respectively.

Table 7.4 Precision (n=6) and Intermediate precision (n=6) results

Substance Precision Intermediate precision

Mean % % RSD Mean % % RSD

META (1000µg/mL) 99.3 0.9 99.5 1.1

Imp-A (1µg/mL) 0.098 2.4 0.101 2.3

Imp-B (1µg/mL) 0.103 2.7 0.097 2.6

[Figure 7.16 Overlaid chromatograms of replicate standard injection (at 230 nm)]

[Figure 7.17 Overlaid chromatograms of diluted standard solution (at 205 nm)]

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266

[Figure 7.18 Overlaid chromatograms of assay precision study (at 230 nm)]

[Figure 7.19 Overlaid chromatograms of related substance precision study (at

205 nm)]

7.5.2.4 Accuracy

The accuracy of an analytical procedure expresses the closeness of agreement between the true

value and the observed value. The accuracy of the assay method for META is evaluated in triplicate

(n=3) at the five concentrations of 500, 750, 1000, 1250 and 1500 µg/mL (50, 75, 100, 125 and

150%) of drug product, and the recovery is calculated for each added (externally spiked)

concentration. For all impurities, the recovery is determined in triplicate (n=3) for 0.5, 0.75, 1.0,


267

1.25 and 1.5 µg/mL (50, 75, 100, 125 and 150%) of the analyte concentration (1000 µg/mL) of the

drug product, and the recovery of the impurities is calculated. The amount recovered is within ± 1.5

% (for assay) and ± 5.0 % (for related substances) of amount added, which indicates that there is no

interference due to excipients present in pharmaceutical dosage forms. It is confirmed from results

that the method is highly accurate [Table 7.5]. Overlaid specimen chromatograms of assay and

related substances accuracy are presented in Figure 7.20 and 7.21 respectively.

Table 7.5 Accuracy results

Substance

At 50%

(n=3)

At 75%

(n=3)

At 100%

(n=3)

At 125%

(n=3)

At 150%

(n=3)

%

Rec.$

%

RSD

%

Rec.$

%

RSD

%

Rec.$

%

RSD

%

Rec.$

%

RSD

%

Rec.$

%

RSD

META

(1000µg/mL) 100.7 0.9 100.2 0.5 99.5 1.2 99.8 0.8 99.1 1.3

META

(1µg/mL) 95.7 3.1 97.3 2.0 97.9 3.7 96.7 2.4 98.9 3.4

Imp-A

(1µg/mL) 95.2 4.8 98.2 2.9 103.1 3.6 98.8 3.2 99.4 4.1

Imp-B

(1µg/mL) 102.6 3.6 101.1 3.4 104.4 4.3 101.4 2.8 97.6 3.9

$= Recovery

[Figure 7.20 Overlaid specimen chromatograms of assay accuracy study (at 230 nm)]

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268

[Figure 7.21 Overlaid specimen chromatograms of accuracy study (related substances at 205 nm)]

7.5.2.5 Linearity

The linearity of an analytical method is its ability to elicit test results that are directly proportional,

or by a well-defined mathematical transformation to the concentration of analyte in a sample within

a given range. The detector response linearity for Imp-A, Imp-B and META are assessed by

injecting nine separately prepared solutions covering the range of LOQ (0.1 µg/mL) to 2.0 µg/mL

(LOQ, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.5 and 2.0 µg/mL) of the normal analyte concentration (1

µg/mL). For META assay the response function is determined by preparing standard solutions at

seven different concentration levels ranging from 500 to 1500 µg/mL (500, 700, 800, 1000, 1200,

1400 and 1500 µg/mL). The correlation coefficients, slopes and y-intercepts of the calibration curve

are determined [Table 7.6]. The correlation coefficient obtained is greater than 0.999 in both cases

[Table 7.6]. Overlaid specimen chromatograms of linearity study of assay and related substances

are presented in Figure 7.22 and 7.23 respectively. Linearity graph of Imp-A, Imp-B, META (at

205nm) and META (at 230 nm) are presented in Figure 7.24 to 7.27 respectively.


269

Table 7.6 Regression statistics

Compound Linearity

range (µg/mL)

Correlation

coefficient (r2)

Linearity (Equation) Y- intercept

bias at 100%

META 500 to 1500 0.9998 y =3423.3(x) + 50372 1.451

META 0.1 to 2.0 0.9998 y =28098(x) + 102.38 0.368

Imp-A 0.1 to 2.0 0.9998 y =33041(x) – 208.53 -0.634

Imp-B 0.1 to 2.0 0.9994 y =42025(x) + 813.06 1.887

[Figure 7.22 Overlaid specimen chromatograms of linearity study (Assay)]

[Figure 7.23 Overlaid specimen chromatograms of linearity study (related

substances)]

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270

[Figure 7.24 Linearity curve of impurity-A]

[Figure 7.25 Linearity curve of impurity-B]

[Figure 7.26 Linearity curve of META at 205 nm]

[Figure 7.27 Linearity curve of META at 230 nm]

y = 33041x - 208.53 R² = 0.9998

0

10000

20000

30000

40000

50000

60000

70000

0.0 0.5 1.0 1.5 2.0 2.5

Are

a

Con. (In µg/mL)

Linearity of Impurity-A

y = 42025x + 813.06 R² = 0.9994

0

20000

40000

60000

80000

100000

0.0 0.5 1.0 1.5 2.0 2.5

Are

a

Con. (In µg/mL)

Linearity of Impurity-B

y = 28098x + 102.38 R² = 0.9998

0

10000

20000

30000

40000

50000

60000

0.0 0.5 1.0 1.5 2.0 2.5

Are

a

Con. (In µg/mL)

Linearity of META for RS

y = 3423.3x + 50372 R² = 0.9998

0

1000000

2000000

3000000

4000000

5000000

6000000

0 200 400 600 800 1000 1200 1400 1600

Are

a

Con. (In µg/mL)

Linearity of META for Assay


271

7.5.2.6 Limit of detection (LOD) and limit of quantification (LOQ)

The LOD and LOQ for META and its impurities are determined at a signal to noise ratio of 3:1 and

10:1, respectively, by injecting a series of dilute solutions with known concentrations. Precision

study is also carried out at the LOQ level by injecting six (n=6) individual preparation and

calculating the % RSD of the area for each impurity and for META. The determined limit of

detection, limit of quantification and precision at LOQ levels for META, Imp-A and Imp-B are

presented in Table 7.7. Specimen chromatogram of LOQ study is presented in Figure 7.28.

Overlaid chromatograms of LOQ precision study is presented in Figure 7.29.

[Figure 7.28 Specimen chromatogram of LOQ for Imp-A, Imp-B and META]

[Figure 7.29 Overlaid chromatograms of LOQ precision study]

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272

Table 7.7 Results of LOD, LOQ and LOQ precision (n=6)

7.5.2.7 Robustness

To determine the robustness of the method, the experimental conditions are deliberately changed.

The resolution of META and imp-B is evaluated. The effect of change in flow rate ± 0.05mL/min

(0.25 and 0.35 mL/min), column oven temperature ± 5°C (40 and 50°C), mobile phase pH ± 0.2

units (6.1 and 6.5 pH) and mobile phase composition ± 10% (for acetonitrile) are studied. During

study other chromatographic conditions are kept same as per the experimental section.

In all the deliberately varied chromatographic conditions, all of the analytes are adequately

resolved, and the order of elution remained unchanged. Robustness study obtained results are

presented in Table 7.8. Specimen chromatograms of robustness study are presented in Figure 7.30,

7.31, 7.32 and 7.33. Figure 7.30 indicate that USP resolution 1.56 is also adequate for the peak

separation in developed method.

Table 7.8 Robustness study results

Condition Parameters Imp-A Imp-B META

Normal methodology USP resolution -- -- 2.34

Retention time in min 2.350 2.827 3.159

USP tailing 1.34 1.52 1.12

USP plate count 8831 7219 11975

Assay % w/w 0.098 0.103 99.3

At flow rate 0.25 mL/min USP resolution -- -- 2.31


USP tailing 1.31 1.48 1.16


Assay % w/w 0.097 0.102 99.1

At flow rate 0.35 mL/min USP resolution -- -- 2.51


USP tailing 1.29 1.53 1.12


Assay % w/w 0.098 0.102 99.4

META Imp-A Imp-B

LOD (µg/mL) 0.03 0.04 0.04

LOQ (µg/mL) 0.1 0.1 0.1

LOQ precision (% RSD) 4.5 4.9 5.7


273

At 40°C column oven temp. USP resolution -- -- 2.77


USP tailing 1.3 1.41 1.18


Assay % w/w 0.099 0.104 99.7

At 50°C column oven temp. USP resolution -- -- 2.07


USP tailing 1.22 1.35 1.23


Assay % w/w 0.098 0.103 98.9

At mobile phase pH 6.1 USP resolution -- -- 1.70


USP tailing 1.31 1.36 1.13


Assay % w/w 0.097 0.104 99.6

At mobile phase pH 6.5 USP resolution -- -- 2.36


USP tailing 1.27 1.33 1.17


Assay % w/w 0.098 0.102 99.0

Mobile phase [-10% ACN] USP resolution -- -- 2.77


USP tailing 1.12 1.44 1.1


Assay % w/w 0.098 0.103 99.8

Mobile phase [+10% ACN] USP resolution -- -- 1.56


USP tailing 1.38 1.52 1.36


Assay % w/w 0.099 0.102 99.8

[Figure 7.30 Specimen chromatograms of robustness study (change in organic

solvent ratio, ± 10%)]

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274

[Figure 7.31 Specimen chromatograms of robustness study (change in mobile phase pH)]

[Figure 7.32 Specimen chromatograms of robustness study (column oven

temperature)]


275

[Figure 7.33 Specimen chromatograms of robustness study (flow rate variation)]

7.5.2.8 Solution stability

Drug stability in pharmaceutical formulations is a function of storage conditions and chemical

properties of the drug and its impurities. Conditions used in stability experiments should reflect

situations likely to be encountered during actual sample handling and analysis. Stability data is

required to show that the concentration and purity of the analyte in the sample at the time of

analysis corresponds to the concentration and purity of the analyte at the time of sampling. META

(1000 µg/mL) spiked solution (with 1 µg/mL of each impurity) is prepared in the diluent by leaving

the test solutions at room temperature. The spiked solution is re-analyzed at 12h and 24h time

intervals, assay and related substances are determinate for the compounds and compared against

fresh sample. The sample solution does not show any appreciable change in assay and related

substances value when stored at ambient temperature up to 24h, data are presented in Table 7.9.

The results from solution stability experiments confirmed that sample solution is stable for up to

24h during assay and related substances determination. Standard solution is re-injected after 12 and

24h time intervals and % RSD of all injected standard injections are calculated. Standard solution

does not show any appreciable change in % RSD (RSD for META is less than 1.0%) value when

stored at ambient temperature up to 24h. Overlaid specimen chromatograms for sample solution

stability of assay and related substances are presented in Figure 7.34 and 7.35 respectively.

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276

[Figure 7.34 Specimen chromatograms of assay sample solution stability]

[Figure 7.35 Overlaid specimen chromatograms of related substance sample

solution stability]

Table 7.9 Solution stability results

Compound 0h 12h 24h

META (1000µg/mL) 1005 1004 1003

Imp-A (1µg/mL) 1.003 1.007 1.008

Imp-B (1µg/mL) 0.975 0.967 0.978


277

7.5.2.9 Filter compatibility

Filter compatibility is performed for PVDF 0.22 µm syringe filter (Millipore) and nylon 0.22 µm

syringe filter (Pall Life sciences). To confirm the filter compatibility in proposed analytical method,

filtration recovery experiment is carried out by sample filtration technique. Sample is filtered

through both syringe filters. Assay and related substances is determined (in µg/mL) and compared

against centrifuged sample. The sample solution does not show any significant changes in assay

and related substances with respect to centrifuged sample. Filter compatibility results are presented

in Table 7.10, which indicates that both syringe filters are compatible with sample solution.

Overlaid specimen chromatograms of assay and related substance test (filter compatibility) are

presented in Figure 7.36 and 7.37 respectively.

Table 7.10 Filter compatibility results

Compound Centrifuged PVDF filter 0.22µm Nylon filter 0.22µm

META (1000µg/mL) 1005 1004 1003

Imp-A (1µg/mL) 0.951 0.961 0.964

Imp-B (1µg/mL) 0.985 0.972 0.981

[Figure 7.36 Overlaid specimen chromatograms of assay filter compatibility study]

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278

[Figure 7.37 Overlaid specimen chromatograms of related substance filter compatibility study]

7.6 CALCULATION FORMULA

7.6.1 Assay (% w/w)

Calculated the quantity, in mg, of META in the portion of solid oral pharmaceutical formulation

using the following formula:

Where,

Cstd = Concentration of standard solution in mg/mL

Cs = Concentration of sample solution in mg/mL

Rs = Compound peak response obtained from the sample preparation

Rstd = Compound peak response (mean peak area) obtained from the standard preparation


279

7.6.2 Impurity (% w/w)

Calculate % impurity present in the finished product formulation using the following formula:

Where,

Cstd = Concentration of standard solution in mg/mL

Cs = Concentration of sample solution in mg/mL

Rs = Compound peak response obtained from the sample preparation

Rstd = Compound peak response (mean peak area) obtained from the standard preparation

RRF= Compound related response factor

7.6.3 Relative standard deviation (% RSD)

It is expressed by the following formula and calculated using Microsoft excel program in a

computer.

Where,

SD= Standard deviation of measurements

= Mean value of measurements

7.6.4 Accuracy (% Recovery)

It is calculated using the following equation:

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280

7.7 CONCLUSION

The rapid isocratic RP-UPLC method is developed for quantitative and related substances analysis

of Metaxalone in pharmaceutical formulation. Satisfactory results are obtained from validation of

the method. The run time (6 min) enabled rapid determination of META. This method exhibited an

excellent performance in terms of sensitivity and speed. This stability-indicating method can be

applied for the routine analysis of production samples and to check the stability of Metaxalone in

bulk drug and formulation. Moreover, it can be applied for determination of assay, blend

uniformity, content uniformity and in vitro dissolutions of pharmaceutical products, where sample

load is higher and high throughput is essential for faster delivery of results.

Note: Intellectual Property Management (IPM) clearance number for the present research

work is: PUB 00143-11.


281

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