Development and validation of hplc method for …...The HPLC system, used for method development and method validation was Agilent 1200 Infinity Series, separation module equipped

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Der Pharmacia Lettre, 2015, 7 (1):238-257

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ISSN 0975-5071 USA CODEN: DPLEB4

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Development and validation of hplc method for estimation of tapentadol and its process-related impurities

M. V. Lakshminarayana*, A. Krishnaiah and G. Suneetha

Department of Chemistry, Sri Venkateswara University, Tirupati (A.P.), India

_____________________________________________________________________________________________

ABSTRACT To develop a simple, novel, sensitive, precise and specific HPLC method for the determination of Tapentadol hydrochloride and its process-related impurities in bulk drugs. The chromatographic separation was achieved on Inertsil ODS 3V (250 x 4.6 mm), 5µm column was used with photo diode array detector (PDA) and variable wavelength detector (VWD) as a stationary phase using acetonitrile: ammonium phosphate buffer (pH 6.3 adjusted with tryethylamine) as mobile phase at detection wavelength 219 nm in Gradient mode at a flow rate of 1.0 ml/min. Keywords: Tapentadol hydrochloride, Gradient mode, three impurities, PDA detector. _____________________________________________________________________________________________

INTRODUCTION

Chemically Tapentadol hydrochloride is 3-[(1R, 2R)-3-(dimethyl amino)-1-ethyl-2-methylpropyl] phenol hydrochloride. It is a centrally acting analgesic with dual mode of action as an agonist at the �-opioid receptor and as a norepinephrine reuptake inhibitor [1]. It is used in the treatment of moderate to severe pain. It was reported that tapentadol dosed at 50 mg 3 times daily for 7 days [2]. Analysis plays an important role in the formulation development of any drug molecule. A suitable and validated method has to be available for the analysis of drugs in bulk, in drug delivery systems, in dissolution studies (in vitro), and in biological samples (in vivo). If such a suitable method for a specific need is not available, then it becomes essential to develop a sensitive, accurate, precise, and reproducible method for the estimation of drug samples. An extensive literature survey reveals that tapentadol was analyzed in urine sample by the ultra performance liquid chromatography method [3]. A literature survey further revealed the availability of HPLC methods in canine plasma [4] and pharmaceutical formulations [5, 6]. The computer-assisted literature survey reveals that there is no stability indicating RP-LC method for the determination of process and degradation-related impurities formed under the stress conditions in tapentadol.

In this paper illustrated the development and validation of related substances method for accurate determination of three potential process impurities (fig.-1) in Tapentadol hydrochloride samples as per the International Conference on Harmonization (ICH) recommendations. We present active pharmaceutical ingredient (API) stability test guideline Q1A (R2) issued by ICH suggests that stress studies should be carried out on active pharmaceutical ingredient (API) to establish its inherent stability characteristics, leading to separation of degradation products and hence supporting the suitability of the proposed analytical procedures. It also recommends that the analytical test procedures for stability samples should be stability indicating and should be fully validated. Development of an

M. V. Lakshminarayana et al Der Pharmacia Lettre, 2015, 7 (1):238-257 ______________________________________________________________________________

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accurate and efficient analytical method for determining the quality and evaluating the impurity profile of drug substances provides some of the critical activities carried out during process research and development in order to meet the requirements of various regulatory authorities [7, 8]. The present proposed method deals a simple, rapid, selective and stability-indicating method for determining the process- and degradation-related impurities in Tapentadol bulk drug along with its validation as per the USP and the ICH guidelines [9, 10]. Here describe the investigation in detail. According to literature survey, only two LC–MS methods to detect Tapentadol in biological matrices (urine and oral fluid) [11]. However there have been no studies on HPLC method for detection of Tapentadol in pharmaceutical formulations. To address this shortfall, the aim of the present paper was to develop and validate a new simpler methodology to quantify Tapentadol and impurities in bulk drug using HPLC with diode array detection (HPLC–DAD).

3-[(1R,2R)-3-(Diethylamino)-1-ethyl-2-methylpropyl] phenol N-oxide

HON+

O-

3-[(1R,2R)-3-(methylamino)-1-ethyl-2-methylpropyl] phenol

CH3

NHH3C

HO

HON

CH3

CH3CH3

H3C HCl

3-((2R,3R)-1-(dimethylamino)-2-methylpentan-3-yl)phenol hydrochloride

(Tapentadol hydrochloride)

(2R,3R)-3-(3-methoxy phenyl)-N,N,2-trimethylphentan-1-amine hydrochloride

CH3

NCH3

CH3

OH3C

Impurity-A Impurity-B Impurity-C

HCl

Fig.-1: Structures of Tapentadol hydrochloride and their impurities

MATERIALS AND METHODS 2.1. Chemicals and Reagents Tapentadol hydrochoride and related impurities received as complementary gift from Alchem Laboratories, Mumbai, India. HPLC grade acetonitrile, Methanol and Isopropyl alcohol. Potassium di hydrogen phosphate, orthophospharic acid, 1-Octane sulphonic acid sodium salt, Triethyl amine and Ammonium phosphate were purchased from Merck. High pure water was prepared by using Millipore Milli Q plus water purification system. 2.2. Instrumentation The HPLC system, used for method development and method validation was Agilent 1200 Infinity Series, separation module equipped with auto sampler, comprising of four pumps, column oven and PDA Detector. Data acquisition was done by using Chromeleon software 2.3. Chromatographic conditions Chromatographic separation was achieved gradient elution of the mobile phase A: buffer (0.01 M Potassium dihydrogen phosphate and 1.0g 1-Octane sulfonic acid at pH 3.2), Methanol and isopropyl alcohol in the ratio of 75:5:20 v/v and mobile phase B: acetonitrile and water in the ratio of 90: 10 v/v. Mobile phase composition was maintained as (T/%B): 0/7, 15/10, 25/20, 40/25, 60/30, 61/7 and 70/7 with flow rate of the mobile phase 1.0


240


ml/min. Separation was performed on Inertsil ODS 3V (250 x 4.6 mm), 5µm column was used with photo diode array detector (PDA) and detector wavelength was kept at 219 nm for monitoring the separation. Injection volume - 20µl, column oven temperature is 10°C and run time is 70 minutes. 2.4 Preparation of diluent Mix pH 6.3 buffer (5.7 g of ammonium phosphate transferred into 1000 ml of water and adjusted pH to 6.3 with Triethylamine) and methanol in the ratio of (48:52). Mixed well and degas. 2.5 (a) Preparation of standard stock solution Weighed and transferred accurately 25 mg of Tapentadol hydrochloride working standard into 100ml of volumetric flask, added about 70ml of diluent, sonicate to dissolve and dilute to the volume with diluent and mix. 2.5 (b) Preparation of standard solution: Diluted 5ml of standard stock solution to 50ml with diluent. Further diluted 4 ml of this solution to 50 ml with diluent. 2.6 Preparation of sample solution Weighed and transferred accurately about 100 mg of sample into 100 ml of volumetric flask. Added about 30 ml of diluent, sonicated to dissolve and made up to volume with diluent then mixed well.

RESULTS AND DISCUSSION 3.1 Method development 3.1.1 Selection of wavelength: The ultraviolet (UV) absorption spectra of Tapentadol and its impurities were confirmed by using photo diode array detection (PDA). Tapentadol and its impurities were found to have varying absorption maxima over a rage of wavelength. But it was found that at 219 nm. Tapentadol and its impurities were found to have optimum UV absorption. Therefore, 219 nm was selected for the study and quantification of Tapentadol impurities (See Spiked sample Chromatogram and Peak purity) 3.1.2 Method optimization In pursuit of symmetric peak shapes and shorter run time, feasibility of various mixture (s) of solvents and columns were used such as Waters Symmetry-C18, 250x4.6 mm, 5µm column. Solution -A (0.1% Ortho Phosphoric Acid) and solution – B (Acetonitrile). The gradient program was set as (time (min)/% solution B): 0/10, 15/30, 40/30, 60/30, 61/7, 70/7 at flow rate of 1.0 mL/min. The diluent was considered as buffer (0.5M ammonium Phosphate to pH 6.5 with Triethylamine) and Methanol in the ratio of 48:52 v/v. In this case longer retention times and poor peak shape of Tapentadol Hydrochloride was observed. In second time used different columns such as YMC Pack ODS AM, Hypersil BDS, Inertsil ODS 3V and different buffers such as potassium dihydrogen phosphate, Trifluoroacetic acid were also tried with different gradient methods to achieve the best chromatographic separation. In this case found poor resolution between Tapentadol hydrochloride, Tapentadol Diastereomer, impurity-A and impurity-B and also poor peak shapes. To improve resolution of impurities and peak shapes of analytes changed by pH from 2.5 to 7.0 with phosphate buffer. Particularly at pH 3.2 the peaks were strongly retained Tapentadol and its Diastereomeric isomer and also Tapentadol and impurity - A peaks were co eluted. To avoid this co eluted peaks, added ion pair reagent (sodium 1-octane sulfonicacid) to the mobile phase (pH 3.2) on a C18 column. In this case peak shapes were significantly improved but Tapentadol hydrochloride and Tapentadol Diastereomer were co-eluted.


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The % of isopropyl alcohol and acetonitrile played a key role in the retention time and resolution between the impurities. After many logical trials, chromatographic condition was achieved by using above combination of solvents. In the presence of Inertsil ODS 3V, 250x 4.6, 5 um column using the mobile phase contains Solution-A and solution-B. Solution-A contains buffer (0.01 M Potassium dihydrogen phosphate and 1.0g 1-octane sulfonicacid at pH 3.2), methanol and isopropyl alcohol in the ratio of 75:5:20 v/v and solution-B contains acetonitrile and water in the ratio of 90: 10 v/v. Mobile phase composition was maintained as (T/%B): 0/7, 15/10, 25/20, 40/25, 60/30, 61/7 and 70/7 with flow rate of the mobile phase 1.0 ml/min. In this case good resolution and satisfactory peak shapes were observed between Tapentadol hydrochloride and its impurities. 3.2 Method Validation The developed method was validated for Related Substances of Tapentadol hydrochloride in accordance with ICH guidelines [12]. 3.2.1 System precision The system precision was evaluated by six replicate analyses of a Tapentadol hydrochloride standard solution. The retention time of Tapentadol hydrochlorides peak was about 15.1 minutes and the mean, standard deviation (SD) and % relative standard deviation for the peak areas of Tapentadol hydrochloride were calculated. The results are tabulated in table-1. 3.2.2 Method precision Prepared six samples of Tapentadol hydrochloride of single batch spiking with known impurities at specification level and analysed as per the test method. The % of individual impurities and total impurities were quantified and the results are tabulated in table -2. 3.2.3 Ruggedness (Intermediate precision): Ruggedness of the method has been verified along with method precision by analysing the six samples, of same batch used for method precision, as per test method by different analyst on a different day using different instrument and different column. Calculated the % RSD for individuals and total impurities of six results and compared with the method precision results. The overall %RSD was not more than 10. 3.2.4 Specificity Blank, standard and sample solution spiked with known impurities at specification limit and individual impurity solution were injected into the HPLC system. There was no interference from the blank at the retention time of Tapentadol hydrochloride and known impurity peaks. The chromatogram of spiked sample and peak purity reveals that the impurities were well separated and there were no co-eluting peaks and no interference of impurities at the retention time of analyte peak. This method is specific for the determination of related substance in Tapentadol hydrochloride. Sample spiked with known impurities and individual standard solutions of retention time (RT), Relative retention time (RRT) and purity data were tabulated in table 3a and 3b respectively. Refer fig-2 for the chromatogram of blank, fig-3 for the standard chromatogram, fig-4 for the chromatogram of sample spiked with known impurities and fig-5, 6 & 7 for individual impurity-A, impurity-B &and impurity-C respectively. 3.2.5 Determination of LOD and LOQ The limit of detection (LOD) and limit of quantification (LOQ) were predicted for the impurity-A, impurity-B, impurity-C and Tapentadol hydrochloride from the linearity using the following formula. LOD = 3.3x Residual standard deviation/slope LOQ = 10 x Residual standard deviation/slope Predicted LOD value was very low hence lowest detected values consider as LOD and these LOD & LOQ values was verified by giving six replicate injections of solution containing Impurity-A, Impurity-B, Impurity-C and


242


Tapentadol hydrochloride. These LOD and LOQ verified values were shown in table -4 and the corresponding chromatogram of LOQ was shown in fig-8. The LOD and LOQ values are summarized in table - 5 and 6. The Precision at LOQ values were shown in table -7. 3.2.6 Linearity Linearity for impurity-A, impurity-B and impurity-C was performed from about LOQ to 200 % of specification limit with respect to test concentration [Concentration depicted was based on taking 0.1% for each of impurity-A, impurity-B and impurity-C of test concentration (1000 µg/ml) as 100%]. A graph was plotted with concentration on X axis and peak area on Y-axis for impurities and correlation coefficient was determined and the Relative response factor (RRF) values for known impurities were calculated. The results are tabulated in table-8 and represented graphically in fig.9, 10, 11 & 12. Moreover, Correlation coefficient (r) is not less than 0.99 for impurities. 3.2.7 Accuracy Known amount of impurity-A, impurity-B and impurity-C spiked in the sample solution at about 40 %, 100 % and 150 % of specification limit with respect to test concentration and sample solution was analyzed. Total amount found in the spiked sample was calculated. The amount recovered is calculated by subtracting the amount already present in the sample from amount found. From amount recovered and amount added, % recovery was calculated. The results are tabulated in table-9a, 9b and 9c. 3.2.8 Robustness The Robustness of the method was evaluated by changing the flow rate (± 10%), Column oven temperature (±5°C), pH of buffer (± 0.2unit) and organic content (± 10 %) in the method and injected the sample solution spiked with known impurities and verified the retention time of impurities. RRTs for the known impurities are comparable. The results are tabulated in table – 10a & 10b. 3.2.9 Stability in analytical solution: Standard solution and sample spiked with the known impurities at specification level were injected into HPLC system initially and different time intervals up to 24 hours at 10°C and 25°C and determined the cumulative % RSD for the peak areas. Finally observed that, the cumulative %RSD for the peak area of Diastereomer isomer, impurity-A, impurity-B and impurity-C, highest unspecified impurity, total impurities and Tapentadol hydrochloride were not more than 10 hence it was concluded that the Standard solution and spiked sample solution are stable for about 23 hours at 10°C and 13 hours at 25°C. The results are tabulated in table –11a, 11b & 11c. 3.2.10 Forced degradation: Forced degradation study was carried out by treating the sample under the following different conditions in the presence of like acid, alkali, peroxide, photolytic, thermal, hydrolytic and humidity. The forced degradation studies and peak purity results were tabulated in table-10. Control sample (Unstressed sample) is shown in fig.-13. 3.2.10.1 Acid degradation The Tapentadol hydrochloride sample was treated with 10 ml of 5N HCl by heating at 90°C for 37 hours and neutralized the solution with 5N NaOH. Treated sample solution was analyzed as per the test method. In this case there was no significant degradation in presence of acid medium and the drug was most stable in acid. The acid degraded sample chromatogram was shown in fig.-14. 3.2.10.2 Alkali degradation The Tapentadol hydrochloride sample was treated with 10 ml of 5N NaOH solution by heating at 90°C for 37 hours and neutralized the solution with 5N HCl and filtered 0.45µ membrane filter. The treated sample solution was analyzed as per the test method. In this case there was no significant degradation in presence of alkali medium and the drug was most stable in base. The base degraded sample chromatogram was shown in fig.-15.


243


Table –1: System Precision data of Tapentadol hydrochloride

Sr. No. Area of Tapentadol peak 1 60723 2 60265 3 59231 4 59803 5 58872 6 59706

Mean 59767 SD 671.6

% RSD 1.12

Table-2: Method precision of Tapentadol hydrochloride with known impurities

S. No. Diastereomer isomer (% w/w)

Impurity-A (% w/w)

Impurity-B (% w/w)

Impurity-C (% w/w)

Highest Unspecified Impurity (%w/w) Total impurities

(% w/w) 1 0.013 0.091 0.096 0.109 0.024 0.342 2 0.013 0.094 0.101 0.110 0.026 0.364 3 0.012 0.090 0.097 0.109 0.027 0.346 4 0.012 0.095 0.094 0.108 0.027 0.374 5 0.013 0.095 0.098 0.106 0.026 0.362 6 0.013 0.091 0.099 0.109 0.026 0.410

Mean 0.013 0.093 0.098 0.109 0.026 0.366 SD 0.0005 0.0023 0.0024 0.0014 0.0011 0.0245

%RSD 3.85 2.47 2.45 1.28 4.23 6.69

Table- 3a: Sample spiked with known impurities

Name of the peak RT (min) RRT Peak Purity (Match factor) Diastereomer isomer 13.561 0.90 981.695 Impurity-A 17.180 1.14 999.866 Impurity-B 19.250 1.27 999.585 Impurity-C 25.191 1.67 999.858 Tapentadol HCl 15.106 1.00 991.955

RRT calculated with respect to retention time of Tapentadol peak.

Table –3b: Individual solution solutions

Name of the peak RT (min) Peak Purity (Match factor) Impurity-A 17.226 999.907 Impurity-B 19.304 999.716 Impurity-C 25.204 999.896 Tapentadol HCl 15.099 999.919

Table – 4: Verification at LOD

Sample No. Peak area

Impurity-A Impurity-B Impurity-C Tapentadol HCl 1 1767 1097 1405 1461 2 1860 848 1486 1797 3 1596 1202 1538 1586 4 1717 975 1555 1702 5 2861 1806 1295 2814 6 1657 962 1078 2012

Mean 1910 1148 1393 1895 SD 474.8 344.4 181.6 487.6

%RSD 24.86 30.00 13.04 25.73

3.2.10.3 Peroxide degradation The sample was treated with 5 ml of hydrogen peroxide solution (30%) by heating at 90°C for 1 hour. The treated sample solution was analyzed as per the test method. In this case the sample was more sensitive. The peroxide degraded sample chromatogram was shown in fig-16. 3.2.10.4 Photolytic degradation


244


Table -5: Precision at LOQ

Sample No. Peak area

Impurity-A Impurity-B Impurity-C Tapentadol 1 4447 3855 4196 5129 2 4593 3725 3718 5800 3 4336 3466 4096 5227 4 4226 3547 4356 5107 5 3930 3721 4182 5435 6 4889 3946 3586 4894

Mean 4404 3710 4022 5265 SD 326.7 180.6 301.8 315.5

%RSD 7.42 4.87 7.50 5.99

Table – 6: Summary of LOD values

Compound Name LOD (in µg/ml) LOD (in %w/w) Impurity-A 0.050 0.005 Impurity-B 0.072 0.007 Impurity-C 0.051 0.005

Tapentadol HCl 0.062 0.006

Table –7: Summary of LOQ Values

Compound Name LOQ (in µg/ml) LOQ (in %w/w) Impurity-A 0.149 0.015 Impurity-B 0.216 0.022 Impurity-C 0.152 0.015

Tapentadol HCl 0.187 0.019

Table-8: Linearity of Tapentadol and their impuriti es

% Lavel Impurity-A Impurity-B Impurity-C Tapentadol Conc. (µg/ml) Area Conc. (µg/ml) Area Conc. (µg/ml) Area Conc. (µg/ml) Area 3 0.029898 0 0.027163 0 0.030465 0 0.062461 2065 5 0.049830 1604 0.045272 921 0.050775 1116 0.104102 3020 8 0.079728 1787 0.072435 1582 0.081240 2050 0.166563 5149 10 0.099660 3696 0.090543 1805 0.101550 3606 0.208204 5790 30 0.298979 10325 0.271630 4800 0.304649 9792 0.624611 16730 50 0.498299 16668 0.452717 8503 0.507749 15543 1.041018 27761 80 0.797278 27618 0.724347 14897 0.812398 23260 1.665628 44902 100 0.996598 33216 0.905434 16522 1.015497 29853 2.082035 56559 120 1.195917 39134 1.086520 21413 1.218596 34955 2.498442 67497 150 1.494896 50495 1.358150 26488 1.523246 44140 3.123053 85704 200 1.993195 65947 1.810867 33694 2.030994 60032 4.164070 112171 CC 0.9996 0.9986 0.9996 0.9999

Slope 33285.506 18945.761 29167.998 27015.256 Intercept 70.292 145.441 130.829 192.987

RSS 3316314.8 3382708.7 3139709.1 2366891.1 RRF 1.23 0.70 1.08 1.00

CC= Correlation Coefficient RSS= Residual Sum of Squares

The sample was exposed to UV light of 200 Watt-hrs/sq-mtr and fluorescent light of 1200 KLux-hrs. The treated sample was analysed as per the test method. In this case there was no significant degradation in presence of photolytic and the drug was most stable in UV light. The UV light degraded sample chromatogram was shown in fig-17. 3.2.10.5 Thermal degradation The sample was kept in oven at 105°C for about 87 hours. The treated sample was analyzed as per the test method. In this case there was no significant degradation in presence of heat and the drug was most stable in thermal condition. The thermal degraded sample chromatogram was shown in fig-18.


245


Table-9a: Recovery of Impurity-A (Relative response factor =1.23)

Level Sample

No. Amount actual

(µg/ml) Amount added

(µg/ml) Amount found

(µg/ml) Amount recovered

(µg/ml) %

Recovery Mean

% RSD

%Recovery Min Max

40% (Level-

1)

sample-1 0.000 0.408

0.405 0.405 99.3 99.1 0.53 98.5 99.5 sample-2 0.402 0.402 98.5

sample-3 0.406 0.406 99.5 100%

(Level-2)

sample-1 0.000 1.019

0.970 0.970 95.2 94.9 2.54 92.4 97.2 sample-2 0.990 0.990 97.2

sample-3 0.942 0.942 92.4 150%

(Level-3)

sample-1 0.000 1.529

1.560 1.560 102.0 99.1 5.07 93.3 102.0 sample-2 1.559 1.559 102.0

sample-3 1.426 1.426 93.3 Overall 97.7 3.57 92.4 102.0

Table -9b: Recovery of Impurity-B (Relative response factor =0.70)

Level Sample

No. Amount actual

(µg/ml) Amountadded

(µg/ml) Amount found

(µg/ml)

Amount recovered (µg/ml)

% Recovery

Mean%

RSD

%Recovery

Min Max

40% (Level-

1)

sample-10.266 0.372

0.675 0.409 109.9 111.5 2.31 109.9 114.5 sample-2 0.676 0.410 110.2

sample-3 0.692 0.426 114.5 100%

(Level-2)

sample-10.266 0.930

1.248 0.982 105.6 103.7 1.61 102.4 105.6 sample-2 1.226 0.960 103.2

sample-3 1.218 0.952 102.4 150%

(Level-3)

sample-10.266 1.395

1.616 1.350 96.8 103.2 8.88 96.8 113.7 sample-2 1.852 1.586 113.7

sample-3 1.649 1.383 99.1 Overall 106.2 5.93 96.8 114.5

Table-9c: Recovery of Impurity-C (Relative response factor=1.08)

Level Sample

No. Amount actual (µg/ml)

Amount added (µg/ml)

Amount found (µg/ml)

Amount recovered (µg/ml)

% Recovery

Mean %

RSD

%Recovery Min Max

40% (Level-

1)

sample-1 0.000 0.429

0.468 0.468 109.1 106.5 2.15 104.9 109.1 sample-2 0.450 0.450 104.9

sample-3 0.452 0.452 105.4 100%

(Level-2)

sample-1 0.000 1.072

1.123 1.123 104.8 107.0 3.53 104.8 111.4 sample-2 1.194 1.194 111.4

sample-3 1.124 1.124 104.9 150%

(Level-3)

sample-1 0.000 1.608

1.709 1.709 106.3 104.6 1.44 103.7 106.3 sample-2 1.667 1.667 103.7

sample-3 1.668 1.668 103.7 Overall 106.0 2.44 103.7 111.4

3.2.10.6 Hydrolytic degradation The sample was treated with 10 ml of water and kept in water bath at 90°C for 41 hours. The treated sample was analyzed as per the test method. In this case there was no significant degradation in presence of water and the drug was most stable in water. The water degraded sample chromatogram was shown in fig-19. 3.2.10.7 Humidity degradation The sample was kept in humidity chamber at 25°C/90% RH for 127 hours. The treated sample was analyzed as per the test method. In this case there was no significant degradation in presence of humidity and the drug was most stable in humidity. The humidity degraded sample chromatogram was shown in fig-20. In the above all degradation studies the peak purity data was satisfactory and the final results are compiled in table-12.


246


Table-10a: Robustness RRT comparison

Name of the peak I II II IV V VI VII VIII IX X XI X II XIII Diasteriomer isomer ND ND ND ND 0.90 ND 0.90 0.90 0.90 0.91 0.90 0.90 0.90

Impurity-A 1.12 1.12 1.11 1.13 1.12 1.12 1.13 1.13 1.13 1.12 1.12 1.14 1.14 Impurity-B 1.26 1.25 1.22 1.27 1.25 1.26 1.27 1.27 1.26 1.23 1.26 1.28 1.27 Impurity-C 1.69 1.62 1.68 1.62 1.66 1.67 1.67 1.66 1.68 1.48 1.66 1.65 1.67 Tapentadol 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

Table-10b: Robustness Parameters details

Sr. No Experiment (Actual values)

I Plus Flow (1.1 ml) II Minus Flow (0.9 ml) III Plus column oven temperature (40°C) IV Minus column oven temperature (30°C) V Plus pH (3.4) VI Minus pH (3.0) VII Plus Organic mobile phase-A (Methanol) VIII Minus Organic mobile phase-A (Methanol) IX Plus Organic mobile phase-A (IPA) X Minus Organic mobile phase-A (IPA) XI Plus Organic mobile phase-B XII Minus Organic mobile phase-B XIII Specificity

Table-11a: Solution stability for Standard solution

IntervalHours

Tapentadol at 10°C Tapentadol at 25°C Peak area Cumulative %RSD Peak area Cumulative %RSD

Initial 52836 NA 51449 NA 3 53191 0.47 51948 0.68 7 53214 0.40 50800 1.12 13 51432 1.60 53162 1.93 16 52419 1.40 NA NA 20 52167 1.30 NA NA 23 52506 1.19 NA NA

Table-11b: Solution stability for spiked sample solution at 10°C

Interval Hours

Diastereomer isomer

Impurity-A

Impurity-B

Impurity-C

Highest Unknown Impurity

Total Impurities

Peak Area

Cum. %RSD

Peak Area

Cum. %RSD

Peak Area

Cum. %RSD

Peak Area

Cum. %RSD

Peak Area Cum. %RSD Peak Area

Cum. %RSD

Initial 3734 NA 31492 NA 17482 NA 32909 NA 6591 NA 94705 NA 3 3557 3.43 31305 0.42 19690 8.40 34265 2.85 7144 5.69 95961 0.93 7 3911 4.74 31611 0.49 19296 6.26 32067 3.35 7752 8.11 97310 1.36 13 3245 7.86 30666 1.35 18638 5.14 32803 2.77 8084 8.94 96161 1.11 16 3565 6.85 31634 1.27 17706 5.19 33341 2.44 7916 8.25 99426 1.84 20 2996 9.47 32344 1.72 17666 5.09 33076 2.18 7264 7.53 95986 1.67 23 3349 8.86 30830 1.78 19734 5.32 32272 2.20 6932 7.45 95733 1.57


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Table-11c: Solution stability for spiked sample solution at 25°C

Interval Hours

Diastereomer isomer

Impurity-A

Impurity-B

Impurity-C

Highest Unknown Impurity

Total impurities

Peak Area

Cum. %RSD

Peak Area

Cum. %RSD

Peak Area

Cum. %RSD

Peak Area

Cum. %RSD

Peak Area Cum. %RSD Peak Area

Cum. %RSD

Initial 2205 NA 32370 NA 17945 NA 33488 NA 6643 NA 95099 NA 3 2173 1.03 31167 2.68 19549 6.05 33411 0.16 6563 0.86 95530 0.32 7 2262 2.04 33849 4.14 17263 6.43 33053 0.70 7123 4.47 96657 0.84 13 2182 1.81 32716 3.39 17619 5.58 33389 0.58 7987 9.24 96710 0.84

Table-12: Forced Degradation Results

S. No.

Condition

Diastereomer isomer (%w/w)

Imp-A (%

w/w)

Imp-B (%

w/w)

Imp-C (%

w/w)

Highest unspecified

impurity (% w/w)

Total impurities (% w/w)

Difference in total impurity

Peak Purity

1 Control Sample (unstressed Sample)

0.012 ND ND ND 0.018 0.044 NA 992.105

2 Acid stressed (10ml, 5N HCl,37 hour at 90°C)

ND ND ND ND 0.035 0.093 0.049 986.052

3 Base stressed (10ml, 5N NaOH, 37 hours at 90°C)

0.051 1.062 0.043 ND 0.947 3.929 3.885 987.300

4 Peroxide stressed (30% H2O2, 5ml, 1 hour at 90°C)

0.051 ND ND ND 5.098 14.072 14.028 986.863

5 Light stressed sample 0.010 ND ND ND 0.026 0.046 Nil 985.492

6 Heat stressed (95 hours at 105°C)

ND ND ND ND 0.024 0.042 Nil 985.534

7 Humidity stressed (127 hours at 25°C/90%RH)

ND ND ND 0.023 0.010 0.033 Nil 986.068

8 Hydrolytic stressed (41 hours at 90°C)

ND 0.043 0.017 ND 0.022 0.557 0.513 985.579

Fig-2: Chromatogram of blank


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Fig-3: Chromatogram of standard solution

Fig-4: Chromatogram of sample spiked with known impurities


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Fig-5: Chromatogram of impurity-A

fig-6: Chromatogram of impurity-B


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fig-7: Chromatogram of impurity-C

fig-8: Chromatogram of LOQ


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fig-9: Linearity graph for impurity-A

fig-10: Linearity graph for impurity-B


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fig-11: Linearity graph for impurity-C

fig-12: Linearity graph for Tapentadol HCl


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fig-13: Control sample chromatogram

fig-14: Acid stressed sample chromatogram


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fig-15: Base stressed sample chromatogram

fig 16: HPLC chromatogram of peroxide stressed sample


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fig-17: HPLC Chromatogram of UV stressed sample

fig 18: HPLC chromatogram of heat stressed sample


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fig 19: HPLC chromatogram of hydrolytic stressed sample

fig- 20: HPLC chromatogram of humidity stressed sample


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CONCLUSION

A rapid, precise, user friendly and reproducible HPLC method for estimation of Tapentadol hydrochloride related three impurities in bulk form was developed and validated as per ICH Guidelines. The LOD and LOQ measurements are also established for the further scope of utilizing this method. Because of its wide range of linearity, use of readily available mobile phase and RSD values for all parameters were found to be less than 10, which indicates the validity of method and results obtained by this method fairly reliable. This method can be used by the industries and academic institutions for the estimation of Tapentadol Hydrochloride. Moreover, successfully performed degradation studies on impurity-A, impurity- B, impurity- C and Tapentadol hydrochloride. Acknowledgement: The authors are highly indebted to the authorities of Sri Venkateswara University, for providing laboratory facilities and Ph.D registration.

REFERENCES

[1] T.M. Tzschentke, T. Christoph, B. Kogel, J. Pharmacol. Exp. Ther., 2007, 323, 265. [2] S.E. Daniels, D. Upmalis, A. Okamoto, C. Lange, J. Häeussler, Curr.Med.Res. Opin., 2009, 25, 765. [3] J.A. Bourland, A.A. Collins, S.A. Chester, S. Ramachandran, R.C. Backer, J. Anal. Toxicol., 2010, 34, 458. [4] M. Giorgi, A. Meizler, P. C. Mills, J.Pharm.Biomed. Anal., 2012, 68, 148. [5] R. P. Kanzariya, T.P. Patel, K.G. Kapuriya, S.D. Faldu, Method development and validation of tapentadol hydrochloride by RP-HPLC method, Inventi Impact: Pharm Analysis and Quality Assurance. [6] J. Gandhi, N.J. Shah, A.N. Lumbhani, Simple, Pharma Science Monitor, 2012, 4, 2440. [7] International Federation of Pharmaceutical Manufacturers and Associations (IFPMA), Impurities in new drug substances,” in Proceedings of the International Conference on Harmonization (ICH ’06), Methodology Q3A(R2), Geneva, Switzerland, 2006. [8] International Federation of Pharmaceutical Manufacturers and Associations (IFPMA), Good manufacturing practice guide for active pharmaceutical ingredients, in Proceedings of the International Conference on Harmonization (ICH ’05), Methodology Q7A, Geneva, Switzerland, 2005. [9] The United States Pharmacopeia, Validation of Compendial Methods, United States Pharmacopeia, 32nd edition, 2009. [10] International Federation of Pharmaceutical Manufacturers and Associations (IFPMA), Validation of analytical procedures, in Proceedings of the International Conference on Harmonization (ICH ’96), Methodology Q2 (R1), Geneva, Switzerland, 1996. [11] C. Coulter, M. Taruc, J. Tuyay, C. Moore, J.Anal. Toxicol, 2010, 34, 458. [12] Validation of analytical procedures: text and methodology”; International Conference on Harmonization, draft revised guidance on Q2(R1) Fed Regist. 1995, 60.

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Development and validation of hplc method for …...The HPLC system, used for method development and method validation was Agilent 1200 Infinity Series, separation module equipped