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PART - A

SECTION-I DEVELOPMENT AND

VALIDATION OF A STABILITY INDICATING HPLC ASSAY

METHOD FOR DETERMINATION OF TAPENTADOL

HYDROCHLORIDE IN TABLET FORMULATION

Tapentadol… Part-A (Section-I)

39

SECTION I:

DEVELOPMENT AND VALIDATION OF A STABILITY INDICATING HPLC

ASSAY METHOD FOR DETERMINATION OF TAPENTADOL

HYDROCHLORIDE IN TABLET FORMULATION

1. INTRODUCTION OF TAPENTADOL HYDROCHLORIDE

Tapentadol is a novel centrally acting analgesic that was approved for use by the

Food and Drug Administration. It has structural similarities to tramadol. It provides

analgesia at similar levels of more potent narcotic analgesics such as hydrocodone,

oxycodone and morphine, but with a more tolerable side effect profile. Tapentadol has

been approved for use as immediate release oral tablets in dosage forms of 50 mg, 75 mg

and 100 mg. Tapentadol has been placed in schedule II by the FDA, as having a high

potential for abuse [1].

1.1 Description

Tapentadol is chemically (-)-(1R,2R)-3-(3-Dimethylamino-1-ethyl-2-methyl-

propyl)-phenol hydrochloride (Figure 1).Its molecular formula is C15H15NO2S.HCl having

molecular weight 257.80 gm/mole. Tapentadol is a µ-opioid receptor agonist and as a

norepinephrine reuptake inhibitor [2].

HO

N

Figure: 1 (-)-(1R,2R)-3-(3-dimethylamino-1-ethyl-2-methyl-propyl)-phenol

1.2 Pharmacodynamics

Tapentadol was characterized as an µ-opioid receptor agonist and a

norepinephrine transporter inhibitor in receptor binding assays and in functional µ-opioid

receptor and norepinephrine synaptosomal reuptake assays. Norepinephrine and µ-opioid

receptor reuptake inhibitors have analgesic effects, although the pain conditions in which

http://edocket.access.gpo.gov/2009/pdf/E9-11933.pdf�


40

these two drug classes are most efficacious may be different. For example, it appears that

µ-opioid receptor agonists are mostly effective against acute moderate-to-severe pain,

whereas norepinephrine reuptake inhibitors are particularly effect against chronic pain.

This implies that a medication that combines both mechanisms of action may be effective

a broad spectrum of pain conditions. The exact mechanism of action is unknown [3].

Tapentadol is a centrally acting oral analgesic with a dual mechanism of action,

combining mu-opioid receptor agonist and norepinephrine reuptake inhibition in a single

molecule. Norepinephrine plays a role in the endogenous descending pain inhibitory

system, and the analgesic efficacy of norepinephrine reuptake inhibitors has been shown

in neuropathic pain.

Analgesic effect of tapentadol has been demonstrated in a wide range of animal

models of pain with nociceptive and neuropathic components, and development of

tolerance to its analgesic effect was twice as slow as that of morphine. Although

Tapentadol has a 50-fold lower binding affinity to mu-opioid receptor, its analgesic

potency is only 2 to 3 times lower than that of morphine, indicating that the dual mode of

action may result in an opiates paring effect [4].

1.3 Pharmacokinetics

Absorption of Tapentadol is rapid with the Cmax occurring in the serum at between

1.25 -1.5 hours post dose. Tapentadol is primarily detected as conjugated metabolites in

the serum; the Cmax of the conjugates is between 1.25 - 2 hours post dose.

Approximately 99% of a dose is accounted for in the urine and 1% in the feces. More

than 50% of the dose is excreted after 4 hours (t1/2 = 3.93 hours) and over 95% within 24

hours of dosing [5].

Tapentadol has no pharmacologically active metabolites. The primary metabolic

pathway is via glucuronidation, with sulfation of the phenolic hydroxyl group occurring

to a minor extent. Minor phase-I bio transformations include hydroxylation of the

aromatic ring, as well as demethylation and subsequent conjugation. The results of in

vitro studies show that these metabolites are either unable to bind to, or have a low

affinity for the µ-opioid receptor, and therefore are not likely to contribute to any

analgesic activity.


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2. LITERATURE REVIEW

The literature reviews regarding Tapentadol hydrochloride suggest that various

analytical methods were reported for its determination as drug, in pharmaceutical

formulation and in various biological fluids. The literature reviews for analysis of

Tapentadol hydrochloride are as under:

1. Sherikar, Omkar D.; Mehta, Priti J. have developed and validated three

methods for determination of Tapentadol hydrochloride in bulk and laboratory tablet

sample. In RP-HPLC method, elution was achieved in isocratic mode using combination

of 50 mM phosphate buffer pH 3.62 and acetonitrile in ratio of 70:30 (% v/v) with 0.1%

triethylamine and using HiQSil C8 column having specification, 250 × 4.6 mm and 5 mm

particle size. The flow rate was 1 ml/min and detection was done at 285 nm. UV-

spectrophotometric determination of Tapentadol was carried out at 272 nm. Third method

consists of quantification of Tapentadol using Folin-Ciocalteu reagent in presence of 20%

sodium carbonate solution. The blue color chromogen formed is measured at wavelength

of maximum absorption 750 nm for Tapentadol against reagent blank. All 3 developed

methods were validated according to ICH guidelines [7].

2. Dousa, Michal; Lehnert, Petr; Adamusova, Hana; Bosakova, Zuzana have

developed and validated a sensitive and specific high performance liquid

chromatographic method for the seperation and determination of tapentadol enantiomers.

Ten different chiral columns were tested in a normal phase system. Excellent enantio-

separation with the resolution more than 2.5 for all enantiomers was achieved on

Chiralpak AD-H using mixture of heptanepropan-2-ol-diethylamine (980:20:1, v/v/v).

The detection was carried out using fluorescence detector at excitation wavelength of 295

nm and emission wavelength of 273 nm. The influence of mobile phase composition,

mainly organic modifiers, additives, aliphatic alkanes and water content in mobile phase,

on retention and enantio-separation was studied. This method is suitable for routine

determination of chiral purity of (R,R)-Tapentadol in enantiopure active pharmaceutical

ingredient [8].

3. Kathirvel, Singaram; Satyanarayana, SuggalaVenkata; Devalarao,

Garikapati have documented simple, rapid, selective, and isocratic RP-LC method for

the quantitation and determination of tapentadol and its related substances in bulk

samples and pharmaceutical dosage forms in the presence of its 2 process-related

impurities. Chromatographic seperation was achieved on the reversed phase, Enable

column (C18 (5-mm, 250 × 4.6 mm, i.d.)) at ambient temperature using a mobile phase


42

consisting of 0.02 M potassium dihydrogen orthophosphate (adjusted to pH 6 with 1 M

KOH) and acetonitrile (80:20, v/v). Flow rate was 1 ml/min with UV detection at 215 nm

[9].

4. Goud, EdigaSasiKiran; Reddy, V. Krishna have determined sensitive, specific,

precise, and linear reverse phase HPLC method for the analysis of related substances in

Tapentadol in bulk and pharmaceutical dosage form. The known related substances are

methoxy impurity [(2R,3R)-3-(3-methoxyphenyl)-N,N,2-trimethylpentanamine] and

alcoholic impurity [(2S)-1-dimethylamino-3-(3-methoxyphenyl)-2-methylpentan-3-ol

hydrochloride]. The method was carried out on a Zodiac C18 column (250 mm × 4.6 mm;

5 µm) using a mobile phase mixture of phosphate buffer pH 7.0, acetonitrile and MeOH

in a gradient elution at a flow rate of 1.0 ml/min at wavelength of 220 nm. The method

can be used for the detection and quantitative estimation of known and unknown

impurities in drug and pharmaceutical dosage form [10].

5. Marin, Stephanie J.; Hughes, John M.; Lawlor, Bryan G.; Clark, Chantry J.;

McMillin, Gwendolyn A. have developed a fast (7.5 min) liquid chromatography-time-

of-flight mass spectrometry (LC-TOF-MS) method in which sixty-seven drugs and

metabolites were separated in serum or plasma. This method was developed as a blood

drug screen, with emphasis on the detection of common drugs of abuse and drugs used to

manage chronic pain. Compound identification is based on chromatographic retention

time, mass, isotope spacing and isotope abundance. Data analysis software (Agilent)

generates a compound score based on how well these observed criteria matched

theoretical and empirical values. The method was validated using fortified samples and

299 residual patient specimens. The accuracy of positive results was >90% for drugs

and/or metabolites[11].

6. Bhatasana, Purvi T.; Parmar, Ashok R. have investigated reversed phase high-

performance liquid chromatography method for the quantification of Tapentadol

hydrochloride in tablet dosage form. The mobile phase consisting of solvent A MeOH:

Solvent B Acidic water (pH 3.8 adjusted by triethylamine and o-phosphoric acid) in ratio

of (58:42% v/v) was delivered at the flow rate of 1.2 ml/min and UV detection was

carried out at 271 nm. The validation of method carried out as per ICH guidelines. As per

validation data it was found that method is specific, robust, and precise within the

described concentration range [12].

7. Jiang, Xue; Jin, Yi; Xu, Haiyan; Xu, Pingwei; Zhai, Nannan; Yuan, Bo have

derived a method to identify the metabolites of Tapentadol in rat urine, the Tapentadol


43

and its metabolites in the rat urine were identified or confirmed through MRM and full

scan MS2 by liquid chromatography tandem mass spectrometry. The parent drug and its

fifteen kinds of metabolites were found in the urine. Among the metabolites, dehydro-

Tapentadol and dehydro-tapentado-O-glucuronide were firstly discovered. The LC-

MS/MS method is a simple and fast way for the analysis of the metabolites of Tapentadol

in the rat urine [13].

8. Ramanaiah, Ganji; Ramachandran, D.; Srinivas, G.; Jayapal, G.; Rao,

Purnachanda; Srilakshmi, V. have documented simple, rapid, selective, precise, and

accurate isocratic reverse phase high performance liquid Chromatography assay method

for simultaneous estimation of Tapentadol and Paracetamol in tablet formulations. The

seperation was achieved by C18 column (Hypersil BDS, 150 × 4.6 mm i.d.); in mobile

phase pH 6.8 Phosphate Buffer and MeOH in the ratio of 700:300 vol./vol. The flow rate

was 1.0 ml/min and the sepd. drugs were detected using UV detector at the wavelength of

215 nm. The method was validated as per ICH guidelines [14].

9. Jin, Yi; Jiang, Xue; Zhai, Nannan; Xu, Pingwei; Yuan, Bo; Xu, Haiyan have

derived sensitive and rapid LC-MS/MS method to determine the concentration of

Tapentadol in rat plasma. After the extraction from plasma by protein precipitation,

analytes and internal standards were separated by a Diamonsil C18 column. Methanol-5

mmol/L ammonium acetate-acetic acid (58:42:0.5, v:v:v) were used as the mobile phase.

The multiple reaction monitoring was used for quantitative. determination in positive

mode. The transitions were m/z: 222.2 for Tapentadol, m/z: 307.1 for fluconazol. No

significant interferences for the detection of Tapentadol and fluconazol from endogenous

substances in plasma were observed in the present study [15].

10. Giorgi, M.; Meizler, A.; Mills, P. C. have developed and validated a simple

HPLC-FLOURECENCE based method to quantify TAP in plasma. Several parameters

both in the extraction and detection method were evaluated. The applicability of the

method was determined by administering TAP orally to two dogs; the protocol yielded

the expected pharmacokinetic results and plasma collected by jugular venipuncture at

regular intervals. The mobile phase consisted of acetonitrile (A):acetic acid (B) (33 mM),

delivered in gradient mode (5-95% B [0-20 min], 95-5% B [20-25 min] and finally 5% B

isocratically [25-32 min]) with a flow rate of 1 ml/ min. Excitation and emission

wavelengths were of 273 and 298 nm, respectively. TAP was extracted from the plasma

using a mixture of Et2O:CH2Cl2 (7:3, v/v) [16].


44

3. AIM OF PRESENT WORK

As per discussion in the literature review UV, LC-MS and HPLC methods for the

determination of Tapentadol hydrochloride in pharmaceutical dosage forms or in

metabolite and plasma are reported. HPLC is the most commonly used method for

analysis of Tapentadol hydrochloride. An extensive literature survey reveals few HPLC

methods for estimation of Tapentadol hydrochloride in pharmaceutical dosage forms as

well as biological fluids; however, not all of these are stability indicating. Most of the

reported methods either do not include stress degradation studies or are not completely

optimized and validated, and they are cumbersome, time-consuming and expensive.

Method validation is an essential step in drug analysis. The process confirms that the

analytical procedure employed for the analysis is suitable for its intended use and shows

reliability of the results produced by any method. The primary objective of the present

work was thus to develop and validate a stability indicating HPLC method for the assay

of Tapentadol hydrochloride from its dosage form (tablets). This work also deals with the

forced degradation of Tapentadol hydrochloride under stress condition like acid

hydrolysis, base hydrolysis, and oxidation, thermal and photolytic stress. Hence, the

method is useful for routine quality control analysis and also for determination of

stability.

The aim and scope of the proposed work are as under:

To develop suitable HPLC method for Tapentadol hydrochloride.

Forced degradation study of Tapentadol hydrochloride under stress condition.

To resolve all major impurities generated during the force degradation studies of

Tapentadol hydrochloride.

Perform the validation for the developed method.


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4. EXPERIMENTAL

4.1 Materials

Tapentadol hydrochloride standard was provided by Ami Life sciences

Laboratories Ltd., Baroda (India). Tapentadol tablets containing 50 mg Tapentadol

hydrochloride and the inactive ingredient used in drug matrix were obtained from market.

HPLC grade methanol was purchased from Spectrochem Pvt. Ltd., Mumbai (India).

HPLC grade water was produced in-house by Milli Q (Millipore, Millford, USA) system.

Membrane filters of 0.45µm (Millipore) were used. Analytical grade ortho-phosphoric

acid, hydrochloric acid, sodium hydroxide pellets and 30% v/v hydrogen peroxide

solution were obtained from Ranbaxy Fine Chemicals, New Delhi (India).

4.2 Instrumentation

The chromatographic system used to perform development and validation of this

assay method was comprised of a LC-10ATvp binary pump, a SPD-M10Avp photodiode-

array detector and a rheodyne manual injector model 7725i with 20μl loop (Shimadzu,

Kyoto, Japan) connected to a multi-instrument data acquisition and data processing

system (Class-VP 6.13 SP2, Shimadzu).

4.3 Mobile phase preparation

The mobile phase consisted of methanol – 0.002 M potassium dihydrogen

phosphate buffer pH 3.0(60: 40 v/v). To prepare the buffer solution, 0.2722 g potassium

dihydrogen phosphate were weighed and dissolved in 1000 ml HPLC grade water and

then adjusted to pH 3.0 with ortho-phosphoric acid. Mobile phase was filtered through a

0.45 μm nylon membrane (Millipore Pvt. Ltd. Bangalore, India) and degassed in an

ultrasonic bath (Spincotech Pvt. Ltd., Mumbai).

4.4 Diluent Preparation

HPLC grade water was used as diluent.

4.5 Standard Preparation

Tapentadol hydrochloride standard stock solution containing 500µg/ml was

prepared in a 100 ml volumetric flask by dissolving 50.00mg of Tapentadol and then

diluted to volume with water as a diluent. Further take 10 ml of this stock solution in 50

ml volumetric flask and make up to mark with diluents. The concentration obtained was

100 µg/ml of Tapentadol hydrochloride.


46

4.6 Test Preparation

Twenty tablets were weighed and the average weight of tablet was determined.

From these, five tablets were weighed and transfer into a 500 ml volumetric flask. About

50 ml of diluent was added and sonicated for a minimum 30 min. with intermittent

shaking. Then content was brought back to room temperature and diluted to volume with

diluent. The sample was filtered through 0.45µm nylon syringe filter. Further take 10 ml

of this stock solution in 50 ml of volumetric flask and make up to mark with diluent. The

concentration obtained was 100 µg/ml of Tapentadol hydrochloride. The degradation

samples were prepared by transferring powdered tablets, equivalent to 50 mg of

Tapentadol hydrochloride into a 250 ml round bottom flask. Then prepared samples were

subjected to acidic, alkaline and oxidant media and also for thermal and photolytic

conditions. After completing the degradation treatments, the stress content solutions were

allowed to equilibrate to room temperature and diluted with mobile phase to attain 100

µg/ml concentrations of Tapentadol hydrochloride. Specific conditions were described as

follows.

4.7 Chromatographic Conditions

Chromatographic analysis was performed on a Phenomenex Luna C8 (150mm ×

4.6mm i.d., 5μm particle size) column applying an isocratic elution using methanol –

0.002 M potassium dihydrogen phosphate buffer pH 3.0 (60: 40 v/v) as a mobile phase.

The mobile phase was filtered through 0.45μm membrane filter and degassed for 30

minute in an ultrasonic bath prior to its use. Flow rate of mobile phase was adjusted to

1.00 ml/min and injection volume was 20 μL. The chromatographic experiment was

performed at ambient temperature and detection was carried out at 272 nm. The

chromatographic run time was up to 10 minutes. In degradation study chromatography

was done upto 30 minutes to observe whether any degradation product was eluted after

specified run time (10 minutes) or not.


47

5. RESULT AND DISCUSSION

5.1 Development and Optimization of the HPLC Method

Proper selection of the method depends upon the nature of the sample (ionic or

ionisable or neutral molecule), its molecular weight and solubility. Tapentadol

hydrochloride can be dissolved in polar solvent hence RP-HPLC was selected to estimate

them. To develop a rugged and suitable HPLC method for the quantitative determination

of Tapentadol hydrochloride, the analytical conditions were selected after testing the

different parameters such as diluents, buffer, buffer concentration, organic solvents for

mobile phase and mobile phase composition and other chromatographic conditions. Our

preliminary trials using different composition of mobile phases consisting of water with

methanol or acetonitrile, did not give good peak shape.

The mobile phase consisted of methanol – 0.002 M potassium dihydrogen

phosphate buffer pH 3.0 (60: 40 v/v). To prepare the buffer solution, 0.2722 g ammonium

potassium dihydrogen phosphate was weighed and dissolved in 1000 ml HPLC grade

water. Mobile phase was filtered through 0.45 μm nylon membrane (Millipore Pvt. Ltd.

Bangalore, India) and degassed in an ultrasonic bath (Spincotech Pvt. Ltd., Mumbai).

By using 0.002 M potassium dihydrogen phosphate buffer in 1000 ml of HPLC

water and keeping mobile phase composition as methanol – 0.002 M potassium

dihydrogen phosphate buffer (60: 40, v/v), best peak shape was obtained. For the

selection of organic constituent of mobile phase, methanol was chosen to resolve

degradation peaks of drug from drug peak properly and to attain good peak shape. Figure

2 represents UV spectrum for wavelength selection. Figure 3 and Figure 4 represent the

chromatograms of standard and test preparation respectively.

Figure 2: Wavelength selection of standard preparation


48

Figure 3: Chromatogram of standard preparation

Figure 4: Chromatogram of test preparation

5.2 Degradation Study

The degradation samples were prepared by transferring powdered tablets,

equivalent to 50.0 mg Tapentadol hydrochloride into a 250 ml round bottomed flask.

Then drug content were employed for acidic, alkaline and oxidant media and also for

thermal and photolytic stress conditions. After the degradation treatments were

completed, the stress content solutions were allowed to equilibrate to room temperature

and diluted with diluent to attain 100 µg/ ml Tapentadol hydrochloride concentrations.

Specific degradation conditions were described as follows.


49

5.2.1 Acidic condition

Acidic degradation study was performed by heating the drug content in 0.1 N HCl

at 60° C for 30 min and mixture was neutralized. In acidic degradation, it was found that

around 7% of the drug degraded. (Figure 5)

Figure 5: Chromatogram of acidic forced degradation study

5.2.2 Alkaline condition

Alkaline degradation study was performed by ambient temperature in 0.05N

NaOH for 30 min and mixture was neutralized. In alkali degradation, it was found that

around 22 % of the drug degraded. (Figure 6)

Figure 6: Chromatogram of alkali forced degradation study

5.2.3 Oxidative condition

Oxidation degradation study was performed by heating the drug content in 30%

v/v H2O2 at 80° C for 45 min. Major degradation was found in oxidative condition that

product was degraded up to 12 %. (Figure 7)


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Figure 7: Chromatogram of oxidative forced degradation study

5.2.4 Thermal condition

Thermal degradation was performed by exposing solid drug to dry heat at 80˚ C in

a conventional oven for 72 hr. In thermal degradation, it was found that around 0.45 % of

the drug degraded. (Figure 8)

Figure 8: Chromatogram of thermal degradation study

5.2.5 Photolytic condition

Photolytic degradation study was performed by exposing the drug content in UV-

light for 72 hours. In photolytic degradation, it was found that around 0.26% of the drug

degraded. (Figure 9)


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Figure 9: Chromatogram of UV-light degradation study

5.3 Method Validation

5.3.1 Specificity

The specificity of the method was determined by checking the interference of

placebo with analyte and the proposed method was eluted by checking the peak purity of

Tapentadol hydrochloride during the force degradation study. The peak purity of the

Tapentadol hydrochloride peak was found satisfactory (0.999) under different stress

condition. There was no interference of any peak of degradation product with drug peak.

5.3.2 Linearity

Seven points calibration curve were obtained in a concentration range from 40-

160μg/ml for Tapentadol hydrochloride. The response of the drug was found to be linear

in the investigation concentration range and the linear regression equation was y =

3E+07X + 24079 with correlation coefficient 0.999. (Figure 10) Chromatograms obtained

during linearity study were shown in figure 11-17.

Figure 10: Linearity curve for Tapentadol hydrochloride


52

Figure 11: Linearity study chromatogram of level-1 (40%)




53





54


5.3.3 LOD and LOQ

The limit of detection and limit of quantification were evaluated by serial dilutions

of Tapentadol hydrochloride stock solution in order to obtain signal to noise ratio of 3:1

for LOD and 10:1 for LOQ. The LOD value for Tapentadol was found to be 0.6 ppm and

the LOQ value 0.2 ppm. Chromatograms of LOD and LOQ study were shown in Figure

18-19.

Figure 18: Chromatogram of LOD Study of Tapentadol hydrochloride


55

Figure 19: Chromatogram of LOQ study of Tapentadol hydrochloride

5.3.4 Precision

The result of repeatability and intermediate precision study are shown in Table 1.

The developed method was found to be precise as the %RSD values for the repeatability

and intermediate precision studies were < 0.50 % and < 0.94 %, respectively, which

confirm that method was precise.

Table 1: Evaluation data of precision study

Set Intraday (n = 6) Interday (n = 6)

1 100.2 100.2

2 100.0 100.1

3 100.1 101.3

4 100.9 99.58

5 101.2 98.44

6 100.9 100.2

Mean 100.5 100.0

Standard deviation 0.500 0.932

% RSD 0.497 0.932

5.3.5 Accuracy

The HPLC area responses for accuracy determination are depicted in Table 2. The

result shows that excellent recoveries (99.67 - 100.15 %) of the spiked drug were


56

obtained at each added concentration, indicating that the method was accurate.

Chromatograms obtained during accuracy study were shown in Figure 20-22.

Table 2: Evaluation data of accuracy study

Level

(%)

Amount added concentration a

(mg/ml)

Amount found

concentration a

(mg/ml)

%

Recovery % RSD

50 0.04993 0.05001 100.15 0.429

100 0.10053 0.10059 100.06 0.614

150 0.15053 0.15004 99.67 0.662

a Each value corresponds to the mean of three determinations

Figure 20: Accuracy study chromatogram of level-1 (50%)



57


5.3.6 Solution stability study

Table 3 shows the results obtain in the solution stability study at different time

intervals for test preparation. It was concluded that the test preparation solution was found

stable up to 48 h at 2 - 8˚ C and ambient temperature, as during this time the result was

not decreased below the minimum percentage.

Table 3: Evaluation data of solution stability study

Intervals

% Assay for test

preparation solution

stored at 2-5 ˚C

% Assay for test

preparation solution

stored at ambient

temperature

Initial 100.28 100.03

12 h 100.23 99.74

24 h 100.04 99.45

36 h 99.80 99.35

48 h 99.62 99.22


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5.3.7 Robustness

The result of robustness study of the developed assay method was established in

Table 4. The result shown that during all variance conditions, assay value of the test

preparation solution was not affected and it was in accordance with that of actual. System

suitability parameters were also found satisfactory; hence the analytical method would be

concluded as robust. Chromatograms obtained during robustness study were shown in

figure 23-27.

Table 4: Evaluation data of robustness study

Robust conditions % Assay

System suitability

parameters

Theoretical

plates Asymmetry

Flow 0.9 ml/min 100.56 4235 1.66

Flow 1.1 ml/min 100.84 4430 1.41

Buffer pH 2.8 99.75 4316 1.28

Buffer pH 3.2 100.45 4615 1.36

Methanol-Buffer (58: 42,v/v) 100.22 4218 1.29

Methanol-Buffer (62: 38,v/v) 99.80 4306 1.17

Column change 99.82 4416 1.12

Figure 23: Standard chromatogram (0.9 ml/min flow rate)


59

Figure 24: Standard chromatogram (1.1 ml/min flow rate)

Figure 25: Standard chromatogram (Methanol-Buffer (62: 38, v/v))

Figure 26: Standard chromatogram (Methanol-Buffer (58: 42, v/v))


60

Figure 27: Standard chromatogram (Column change)

5.3.8 System suitability

A system suitability test of the chromatographic system was performed before

each validation run. Five replicate injections of standard preparation were injected and

asymmetry, theoretical plate and % RSD of peak area were determined for same.

Acceptance criteria for system suitability, asymmetry not more than 2.0, theoretical plate

not less than 4000 and % RSD of peak area not more than 2.0 were fulfilled during all

validation parameter.


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6. CALCULATIONS AND DATA

Calculation formula used

1. Calculation formula for % assay of Tapentadol hydrochloride

WeightTestWeightStandard

AreaStandardMeanAreaTestMean

Assay 5005010

100% ×××=

StandardofPotencyClaimLable

WeightTestMean×××

1050

2. Relative standard deviation

100% ×=sMeasurmentofValueMean

sMeasurmentofDeviationStandardRSD

3. Recovery

100×=AddedAmountfoundAmount

Recovery%

4. Amount found

ionConcentratStandardAreaStandardMean

AreaTestMeanmlmgFoundAmount ×=)/(

5. Amount added

VolumeWeight(mg/ml)AddedAmount =


62

Specificity Study for Analytical Method Validation of Tapentadol hydrochloride

Tablets

Standard weight (mg) 50.0

Standard dilution 100 10 50

Standard potency 100%

Standard concentration (mg/ml) 0.100

Replicate 1 2 3 4 5

Standard area 2635243 2640284 2610340 2666187 2650326

Mean standard

area 2640476

Standard deviation 20577.16

%RSD 0.779

Replicate Test

area

1 2642536

2 2639482

Mean test area 2641009

Test weight (mg) 1060.4

Label claim (mg) 50.0

Mean test weight

(mg) 212.1

% Assay 100.12

Calculation:

Prototype calculation for one set:

4.1060500

5010

10050

26404762641009% ×××=Assay 100

501.212

1050

×××

= 100.12 %


63

Linearity Study for Analytical Method Validation of Tapentadol hydrochloride

Tablets





Concentration of linearity stock

sol. 0.500

Replicate 1 2 3 4 5

Standard area 2636824 2632522 2649920 2664215 2632256

Mean standard area 2643147


%RSD 0.522

Concentration

level %

Volume of linearity stock

solution taken (ml)

Diluted to

(ml)

Final

concentration

(mg/ml)

Mean

area

40 4.0 50 0.04 1059324

60 6.0 50 0.06 1594467

80 8.0 50 0.08 2182468

100 10.0 50 0.1 2645621

120 12.0 50 0.12 3206743

140 14.0 50 0.14 3698564

160 16.0 50 0.16 4236551

Correlation co-

efficient 0.999

Slope 3.0×107

Intercept 24079


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Precision Study for Analytical Method Validation of Tapentadol hydrochloride

Tablets



Standard potency 100

Label claim (mg) 50

Mean test weight(mg) 212.1


Replicate 1 2 3 4 5

Standard area 2650482 2642783 2662431 2659700 2661483



%RSD 0.320

Description Mean area Test weight (mg) % Assay

Set 1 2654802 1060.5 100.22

Set 2 2598553 1040.8 99.95

Set 3 2626458 1050.4 100.10

Set 4 2690485 1068 100.85

Set 5 2704605 1070.2 101.17

Set 6 2659400 1055.5 100.87

Mean 100.53


%RSD 0.497

Calculation:


1060.5500

5010

1002.50

26553762654802% ×××=Assay 100

501.212

1050

×××

= 100.22 %


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Intermediate precision study for Analytical Method Validation of Tapentadol

hydrochloride Tablets




Mean test weight(mg) 212.1


Replicate 1 2 3 4 5

Standard area 2656894 2612843 2622852 2645264 2654220



%RSD 0.742

Description Mean area Test weight (mg) % Assay

Set 1 2685413 1072.4 100.25

Set 2 2564282 1025.4 100.12

Set 3 2712485 1072.2 101.28

Set 4 2732412 1098.5 99.58

Set 5 2694528 1095.8 98.44

Set 6 2678845 1070.6 100.17

Mean 99.97


%RSD 0.851

Calculation:


4.1072500

5010

1008.49

26384152685413% ×××=Assay 100

501.212

1050

×××

= 100.25 %


66

Comparison for Precision and Intermediate Precision Study for Analytical Method

Validation for Tapentadol hydrochloride Tablets

Set %Assay

Precision study

1 100.22

2 99.95

3 100.10

4 100.85

5 101.17

6 100.87

Intermediate precision study

1 100.25

2 100.12

3 101.28

4 99.58

5 98.44

6 100.17

Mean 100.22

Standard

deviation 0.770

%RSD 0.767


67

Accuracy study for Analytical Method Validation of Tapentadol hydrochloride

tablets



Standard potency 100.00%


Replicate 1 2 3 4 5

Standard area 2661342 2654813 2645290 2632167 2640524



% RSD 0.436


68

Recovery Level Mean area Weight

(mg) Volume

(ml)

Amount added

concentration (mg/ml)

Amount Found

concentration (mg/ml)

% Recovery

Mean %

Recovery *S.D. %

RSD

50% set-1 1325614 25.1 500 0.0502 0.050083 99.77

100.15 0.4294 0.429 set-2 1320890 24.8 500 0.0496 0.049905 100.61 set-3 1324312 25.0 500 0.0500 0.050034 100.07

100% set-1 2646503 50.2 500 0.1004 0.099988 99.59

100.06 0.6140 0.614 set-2 2642261 50.0 500 0.1000 0.099827 99.83 set-3 2698725 50.6 500 0.1012 0.101961 100.75

150% set-1 3931521 75.0 500 0.1500 0.148537 99.02

99.67 0.6601 0.662 set-2 3982364 75.5 500 0.1510 0.150458 99.64 set-3 3999836 75.3 500 0.1506 0.151118 100.34

* S.D.= Standard deviation

Calculation:

Prototy pe calculation for one set:

0.10026468261325614)/( ×=mlmgFoundAmount = 0.050083 mg/ml

50025.1)/( =mlmgAddedAmount = 0.0502 mg/ml

1000.050200.050083

×=Recovery% = 99.77 %


69

Robustness Study for Analytical Method Validation of Tapentadol hydrochloride Tablets

Flow Rate at

0.9ml/min Flow Rate at

1.1ml/min Buffer

pH =2.8 Buffer

pH=3.2 Methanol-Buffer

62: 38 Methanol-Buffer

58: 42 Column Change

Replicate Standard Area Standard Area Standard Area Standard Area Standard Area Standard Area Standard Area

1 2666590 2645162 2634542 2630493 2660292 2649524 2656437 2 2638472 2626450 2601867 2602137 2637264 2699645 2683452 3 2659164 2663600 2672804 2656329 2690154 2603452 2701568 4 2614598 2634612 2649138 2680164 2613467 2646853 2623864 5 2629345 2615483 2622963 2634762 2619400 2616437 2605483

Mean 2641633.8 2637061.4 2636262.8 2640777 2644115.4 2643182.2 2654160.8 S.D. 21342.832 18399.360 26757.929 29275.378 31537.090 37200.590 39993.820

% RSD 0.808 0.698 1.015 1.109 1.193 1.407 1.507 Replicate Test Area Test Area Test Area Test Area Test Area Test Area Test Area

1 2688437 2625482 2645761 2637628 2635861 2659486 2679483 2 2625483 2694154 2614383 2668539 2664831 2639462 2620134

Mean 2656960 2659818 2630072 2653083.5 2650346 2649474 2649808.5 Standard weight

(mg) 50.1 50.1 50.1 50.1 50.1 50.1 50.1

Test weight (mg) 1061.2 1061.2 1061.2 1061.2 1061.2 1061.2 1061.2 Label claim

(mg) 50 50 50 50 50 50 50

Mean test weight (mg) 212.2 212.2 212.2 212.2 212.2 212.2 212.2

% Assay 100.56 100.84 99.75 100.45 100.22 99.80 99.82


70

Solution Stability Study for Analytical Method Validation of Tapentadol

hydrochloride Tablets

System suitability of standard preparation for solution stability

Initial

After 12

hours

After 24

hours

After 36

hours After 48 hours

Standard Standard Standard Standard Standard

Replicate Peak area Peak area Peak area Peak area Peak area

1 2671428 2669432 2668568 2664348 2666284

2 2638876 2634164 2632942 2627427 2630422

3 2644247 2641965 2643986 2639612 2642674

4 2637271 2636568 2633260 2630157 2632133

5 2657610 2655617 2652591 2646244 2650319

Mean 2649886.4 2647549.2 2646269.4 2641557.6 2644366.4

S.D. 14455.748 14789.185 14910.300 14788.489 14686.569

%RSD 0.546 0.559 0.563 0.560 0.555

Solution stability for standard preparation at 2 -8°C

After 12 hours After 24 hours After 36

hours

After 48

hours

Standard Standard Standard Standard

Replicate Peak area Peak area Peak area Peak area 1 2668522 2644623 2606498 2611867 2 2617094 2619700 2662493 2645962 3 2675643 2677601 2670682 2682654 4 2637430 2630962 2632920 2616492 5 2650492 2652943 2605671 2621507 1 2622983 2638634 2645936 2647964 2 2648708 2626490 2607929 2614104

Mean 2645838.9 2641564.7 2633161.3 2634364.3 S.D. 21802.376 19424.005 27491.574 25989.393

%RSD 0.824 0.735 1.044 0.987


71

Solution stability for standard preparation at room temperature

After 12 hours After 24 hours After 36 hours After 48

hours

Standard Standard Standard Standard

Replicate Peak area Peak area Peak area Peak area

1 2671428 2669432 2668568 2664348

2 2638876 2634164 2632942 2627427

3 2644247 2641965 2643986 2639612

4 2637271 2636568 2633260 2630157

5 2657610 2655617 2652591 2646244

1 2594672 2612846 2627142 2647318

2 2645914 2659449 2600796 2662873

Mean 2641431.1 2644291.6 2637040.7 2645425.6

S.D. 23804.218 18914.334 21318.127 14477.555

%RSD 0.901 0.715 0.808 0.547

Solution stability for test preparation at 2 -8°C

Initial After 12

After 24 hours After 36

After 48 hours Standard Standard Standard Standard Standard

Replicate Peak area Peak area Peak area Peak area Peak area 1 2671428 2669432 2668568 2664348 2666284 2 2638876 2634164 2632942 2627427 2630422 3 2644247 2641965 2643986 2639612 2642674 4 2637271 2636568 2633260 2630157 2632133 5 2657610 2655617 2652591 2646244 2650319

Replicate Test Area Test Area Test Area Test Area Test Area 1 2646872 2654737 2649438 2638006 2637472 2 2658168 2647691 2643104 2641755 2632564

Mean 2652520 2651214 2646271 2639880.5 2635018 % Assay 100.28 100.23 100.04 99.80 99.62 Standard

weight (mg) 50.1 50.1 50.1 50.1 50.1

Test weight

1061.2 1061.2 1061.2 1061.2 1061.2 % Difference

0.05 0.24 0.48 0.66


72

Solution stability for test preparation at room temperature

Initial After 12 hours After 24 hours

After 36 hours After 48 hours

Standard Standard Standard Standard Standard Replicate Peak area Peak area Peak area Peak area Peak area

1 2671428 2669432 2668568 2666284 2664348 2 2638876 2634164 2632942 2630422 2627427 3 2644247 2641965 2643986 2642674 2639612 4 2637271 2636568 2633260 2632133 2630157 5 2657610 2655617 2652591 2650319 2646244

Replicate Test Area Test Area Test Area Test Area Test Area 1 2646832 2637774 2631067 2628132 2624515 2 2644988 2638451 2629975 2627782 2624274

Mean 2645910 2638112.5 2630521 2627957 2624394.5 % Assay 100.0308 99.736009 99.449006 99.35207 99.21738863

Standard weight (mg) 50.1 50.1 50.1 50.1 50.1

Test weight (mg) 1061.2 1061.2 1061.2 1061.2 1061.2

% Difference compared to that of Initial 0.2947909 0.5817938 0.678728 0.813411141

Calculation:


1061.2500

5010

1001.50

26498862652520% ×××=Assay 100

502.212

1050

×××

= 100.28 %


73

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