HPTLC Analysis and Force Degradation Study of Tapentadol Hydrochloride in Bulk and Its Pharmaceutical Formulation
Asmita S.1*, Mahaveer S.1, Sagar W.2, Birendra S.1
1School of Pharmaceutical Sciences, Jaipur National University, Jagatpura, Rajasthan, India.
2JSPM’s Charak College of Pharmacy and Research, Wagholi, Pune.
*Corresponding Author E-mail: asmita.ghemud4@gmail.com
ABSTRACT:
To achieve easy, sensitive reproducible high-performance thin-layer chromatography (HPTLC) densitometric method was developed and validated for the determination of tapentadol HCl and its pharmaceutical formulation. In this method the ethyl acetate: methanol: ammonia (6:4:0.5v/v/v) were used as mobile phase for chromatographic separation of the drug. The method has been performed on precoated silica Tab 60F254 Merck plates and the Rf value was calculated at 0.47 for tapentadol HCL. The calibration curve was plotted in the concentration range of 500-2500ng/ml. The limits of quantitation and limit of detection were found to be 0.07 and 0.23μg/ml respectively. The correlation coefficient (R2) value was obtained at 0.998 for tapentadol HCL. The percent recoveries were obtained between 99.97 to 101.13% for tapentadol HCl. The method has been validated according to ICH guidelines for linearity, precision, accuracy, specificity, and robustness. The degradation behavior was recorded under acidic, basic, neutral, oxidative, photolytic, and thermal stress conditions. The degradation products were well separated from the pure drug under the optimized conditions. As the method could effectively separate the drug from its degradation products, it can be employed as a stability-indicating method for tapentadol HCl.
KEYWORDS: Tapentadol HCl, High-performance thin-layer chromatography, Method validation, Force degradation, etc.
INTRODUCTION:
3-[1-(dimethylamino)-2-methylpentan-3-yl]phenol
Fig. 1: Chemical structure of Tapentadol
It is available in the United States in both immediate and extended-release formulations such as NUCYNTA® and NUCYNTA ER® respectively, from Janssen pharmaceuticals. It appears to be well tolerated in the analgesic dosages 50, 75, and 100 mg recommended for osteoarthritis and lower back pain. In mice, tapentadol has shown more potent action than morphine against heat hyperalgesia. 1-4
A literature review reveals very few methods were developed for the determination of tapentadol hydrochloride such as LC/MS5 UPLC6 Spectrophotometric method 7-10 RP-HPLC methods11-21 HPTLC methods 22-24 and LC/MS/MS 25.
However, very few amounts of information exist in the literature on the force degradation behavior of TAP by the HPTLC-MS method. So our work aimed to develop and validate a rapid, sensitive, selective, and accurate HPTLC method for the determination of TAP and its tablet formulations. The study was further extended for the identification of resulting degradation products by using mass spectroscopy.
MATERIAL AND METHODS:
Chemicals and reagents:
Tapentadol hydrochloride was procured from Precise Chemipharma Pvt. Ltd., Navi Mumbai, India as a gift sample. Vorth TP® was manufactured by Glenmark and labeled to contain 50 mg TAP per tablet purchased from a local pharmacy. Ethyl acetate, methanol, ammonia solution, sodium hydroxide, conc. HCl and 30% H2O2 were of analytical grade.
Instrumentation:
In this study, scanning was carried out by using Camag TLC Densitometric Scanner 3S/N 130319 with Wincats software version 1.4.2 (Camag, Muttenz, Switzerland). A TLC Camag Linomat V autosampler was used, which was equipped with a 100-ml syringe (Camag, Switzerland). Drug degradation was carried out at 277 nm by using an ultraviolet (UV) lamp. Merck HPTLC plates (10 × 10 cm) coated with 60 F254 silica gel (Merck, Darmstadt, Germany) with 0.2 mm thickness were used as a stationary phase.
Selection of Solvents:
Based on the solubility study methanol was selected as the solvent for dissolving TAP.
Preparation of Standard Stock Solutions of TAP:
The standard solution of Tapentadol Hydrochloride was prepared by dissolving 5 mg of the drug in 10 ml of methanol (AR Grade) to obtain a final concentration of 500.0 ug/ml and the applied volume is 1ul-5ul.
Preparation for a pharmaceutical dosage form solution:
Twenty tablets were crushed and triturated well in a mortar with a pestle. The average tablet weight was determined and a powder sample equivalent to 13.4 mg of Tapentadol HCl was transferred into a 10 ml volumetric flask. About 10 ml methanol was added, and the flask was sonicated for 15 min. and solution was filtered through Whatman paper no. 41. The final volume was made up to the mark to obtain a tablet stock solution of 500.0 ug/ml
Determination of λ Max for TAP:
After chromatographic development, standard stock solution bands of different concentrations were scanned over a range of 200-400 nm. A standard stock solution was applied to the TLC plate with the help of the Camag Linomat-V automatic sample applicator, the plate was chromatographed in a twin-through glass chamber saturated with methanol for 30 min. After chromatographic development, the plate was removed and air-dried. The separated bands on the TLC plate were scanned over the wavelength range of 200-400 nm. It was observed that the drug showed absorbance at 277 nm. The spectrum obtained which is shown in Fig. 2
Fig. 2: Spectrodensitogram of Tapentadol Hydrochloride (wavelength 277 nm)
Chromatographic conditions:
Samples were applied in the form of bands of 6 mm width with a 100 μl sample syringe on aluminum plates precoated with silica gel G60F254(20×20cm), using the autosampler. An application volume rate of 4 μl was used, and the space between bands was 14mm. The slit dimension was 5 × 0.045 mm, and the scanning speed was 20 mm/s. Selected mobile phase i.e. ethyl acetate: methanol: ammonia (6:4:0.5v/v/v) was assembled of linear ascending development which was carried out in a glass chamber saturated with the mobile phase. Development of the plates was left till the mobile phase migrates 8 cm. Following the development, the plates were air-dried; bands were visualized under a UV lamp at 277 nm and densitometric scanning was performed using a CAMAG TLC scanner in the reflectance–absorbance mode at 277 nm and operated by WINCATS software. The radiation source was a deuterium lamp. The densitogram of TAP HCl is shown in Fig.No.3. The chromatographic method was validated concerning linearity, precision, accuracy, and specificity as summarized in ICH guidelines 26
METHOD VALIDATION:
Several experimental parameters, such as mobile phase composition, activation time, saturation time, scan modes, and detection wavelength were optimized during method development. Promising results were obtained by using mobile phase ethyl acetate: methanol: ammonia (6: 4: 0.5: v/v/v) and the maximum resolution was found with Rf value 0.47 at concentration 2000 ng/ml by applying volume 1-5μl having an optimum wavelength for detection was 277 nm at which good detection for tapentadol hydrochloride was obtained.
Fig. 3 Typical densitogram of Tapentadol hydrochloride
Linearity of the calibration curve:
The stock solution was applied in the range of 500-2500g/spot for tapentadol hydrochloride to evaluate the linearity. The plates were developed in a 10×10 cm twin trough chamber using a freshly prepared developing phase (5ml) i.e. ethyl acetate: methanol: ammonia (6:4:0.5v/v/v) and scanned at 277 nm. Peaks were integrated. The Response area was plotted against the corresponding concentrations to obtain the calibration curve. Linear correlation was obtained between peak area and concentration of tapentadol hydrochloride in the range 500-2500 ng/spot with correlation coefficient R2=0.998. The linear regression equation was found to be Y = 252.5+2171xX. The calibration curve for TAP depicted in Fig. 4
Fig. 4 Calibration curve for Tapentadol hydrochloride at 277mm
Accuracy (Recovery Study):
Accuracy means a measure of the exactness of an analytical method. Accuracy was checked by the standard addition method. The accuracy of the proposed method was ascertained based on the recovery study. Recovery studies were carried out by the addition of standard working solutions to pre-analyzed tablet solutions at three different levels, 80%, 100%, and 120%. At each level of the amount, three determinations were performed which were shown in Table 1.
Table 1: Accuracy (Recovery study) for Tapentadol Hydrochloride
Sr. No. |
Level of Recovery |
% Recovery |
1 |
80% |
99.97 |
2 |
100% |
101.13 |
3 |
120% |
99.99 |
Precision:
The precision of the proposed method was determined by the repeatability of the system and the method for standard and tablet was determined by analyzing three replicates of different concentrations of standard tapentadol hydrochloride (500 ng/spot, 1000 ng/spot, and 1500 ng/spot) Results expressed in terms of %RSD which was determined found to be 1.2 for system precision and 0.7 for method precision which was less than 2%. So the developed method was precise. Data are given in Table 2
Table 2: Results for Linearity and Precision
Sr. No. |
Parameter |
Values |
1 |
Detection wavelength (nm) Beer’s law limit (ng/spot) |
277 nm 500-2500(ng/spot) |
2 |
Linearity range |
1-5ul |
3 |
Regression Equation |
y = 252.5+2171xX |
4 |
Slope (m) |
252.5 |
5 |
Intercept (c) |
2171 |
6 |
Correlation coefficient (r) |
0.998 |
7 |
Precision Intraday Interday |
1.48 1.47 |
8 |
Limit of detection (LOD) |
0.07 |
9 |
Limit of quantitation (LOQ) |
0.23 |
LOD and LOQ:
To determine detection and quantification following equations are used.
Limit of detection (LOD) = 3.3 ×N/B and
Limit of quantitation (LOQ) = 10 × N/B
Where ‘N’ is the standard deviation of the response and B is the slope of the corresponding calibration curve, were used.
The LOD and LOQ were 0.07 and 0.23, respectively, indicating the high sensitivity of the method (Table 2).
Analysis of Vorth TP® tablets:
The developed method was successfully applied the for determination of tapentadol hydrochloride in commercial tablet dosage form with a label claim of 50mg/tablet. The amount of tapentadol hydrochloride estimated was found to be 49.50 mg/tablet with a % label claim of 101.13 % and the Rf value was in the same range i.e. 0.47 (Table 3)
Table 3: Determination of TAP in Vorth TP® Tablets by the Proposed HPTLC Method (ASSAY)
Pharmaceutical formulation |
Amt of the drug in formulation (mg/tablet) |
Amt of drug applied(ng/spot) |
Amt of the area find |
% label claim |
SD |
RSD |
Vorth TP® |
50 |
2000 |
4820 |
101.13 |
1.4 |
0.029 |
Table 4: Robustness data for Tapentadol hydrochloride in terms of Rf value and peak area
Sr. No. |
Parameter |
Rf |
Area |
Mean |
SD |
%RSD |
A I 1 2 3 |
Robustness Saturation time Duration of chamber saturation (5mins.) Change in plate activation Time (100ºC for 10 mins.) Change in plate activation time (120ºC for 15 mins.) |
0.44 0.47 0.50 |
4698 4766 4762 |
4742 |
38.15 |
0.80 |
II
1 2 3 |
Total Mobile phase variation EA:MeOH:AMM 6:4:0.5 6:3:0.5 7:4:0.5 |
0.47 0.46 0.52 |
4766 4765 4812 |
4781 |
26.85 |
0.56 |
III 1 2 3 |
Mobile phase variation 6:4:0.5 5:9:4.1:0.5 6.1:3.9:0.5 |
0.47 0.49 0.51 |
4766 4769 4854 |
4796 |
49.9 |
1.04 |
IV 1 2 3 |
Development to scan 0 to 30min 45min 1hr |
0.47 0.48 0.50 |
4766 4767 4799 |
4777 |
18.7 |
0.39 |
Robustness:
Robustness is the capacity of the method to remain unaffected by small deliberate variations.
This study was done by applying small changes in developing system composition like small changes in plate activation time and varying the saturation time (±5 min). In each case, the retardation factor (Rf) was determined. The results are shown in Table 4.
Specificity
By analyzing the standard drug and test samples specificity was determined. The band of TAP HCl in the sample should be matched with the Rƒ and spectrum of standard drug. There was no interference from the blank at the analytical peaks. Peak purity reveals the homogeneity of peaks and there was no interference between the standard and sample peaks.
Fig 5: Densitograph of Specificity for TAP HCl
Forced degradation studies:
Forced degradation studies were carried out by ICH guidelines Q1A (R2) 27. TAP was exposed to different stress conditions. TAP is freely soluble in water so all degradation samples were prepared directly under respective stressors. TAP stock standard solution (500ug/ml) was used during forced degradation studies. Furthermore, a concentration of 5μg/band of each degraded sample was then applied on HPTLC plates in replicates. The procedure under chromatographic conditions was then followed. From the relative peak area of TAP in each chromatographed sample, TAP % degradation was then calculated which is shown in Table 5
Hydrolytic degradation:
Acidic hydrolysis was carried out at 80°C by using solutions of 0.1 N HCl for half an hour, while basic hydrolysis was performed at room temperature using 0.1 N NaOH for 45min. For neutral hydrolysis, distilled water was used at 80°C for half an hour. Separate 5 mg of TAP was transferred to four separate 10-ml volumetric flasks and then mixed with 3-ml of 0.1 N HCl, 0.1N NaOH, and distilled water. The prepared solutions were kept away from light to exclude the possible photodegradation at 80°C except for 0.1 N NaOH, which was kept at room temperature. Then apply the sample to a plate for development in the mobile phase. Spot of HCl, sample, and NaOH on 2 TLC plates after heating then observe under UV for detection wavelength of 254nm, mark the band with a pencil, and then scratch with a spatula. Take scratched product in a volumetric flask, add methanol to it then check mass. The degraded product is shown in Fig.5
Fig.5 Typical densitogram of tapentadol hydrochloride and its degradation products in the acid medium
Oxidative degradation:
In oxidative degradation, 3%H2O2 was used. By mixing 5 mg of Tapentadol with 3ml of either 3% H2O2 in separate 10 ml volumetric flasks, the solutions were kept at 80°C for half an hour away from light to prohibit the possible effect of light. Samples were then cooled and evaporated on a water bath to remove the remaining H2O2, and then the volume was adjusted using methanol.
Photolytic degradation:
The effect of light on Tapentadol HCl solid and liquid samples was studied. Then, 5mg of Tapentadol HCl were transferred separately into two 10ml volumetric flasks. Samples were subjected to UV light for 1hr (liquid sample) or half an hour (for solid sample). 5ml of methanol was added to each flask, and the volume was then adjusted with methanol.
Thermal degradation:
Tapentadol HCl 5 mg was stored at 80°C for 1 hr in an oven. The powder was transferred to a 10 ml volumetric flask, dissolving in methanol, and then the volume was completed with methanol.
Table 5: Data for degradation study
Sr. No. |
Stress conditions |
% Drug degradation |
1 |
0.1 N NaOH 1hr heating |
15% |
2 |
0.1 N HCL |
18% |
3 |
3% H2O2 1 hr heating |
11% |
4 |
Distilled water |
13% |
5 |
Thermal 60℃ 1 hrs heat |
08% |
6 |
UV at 277 nm 24 HRS |
09% |
RESULT:
During the optimization of the method many trials were taken, initially, various mobile phases were used starting from ethyl acetate: methanol: triethylamine, followed by the addition of glacial acetic acid, formic acid, and ammonia to get a good peak shape and RF value. Ammonia was added in different concentrations with ethyl acetate and methanol which resulted in good separation. The final mobile phase selected was ethyl acetate: methanol: ammonia (6:4:0.5) resulted in a sharp peak and satisfactory Rf value. After method optimization, the method was validated as per ICH guidelines. All validated parameters were found satisfactory in the standard limits. The method was applied for degraded samples to verify its capability of usage in the shelf-life period (stability-indicating nature) determination. The method is also capable to detect successfully all the degradation products.
CONCLUSION:
The developed method may help to reduce the amount of organic phase, which makes it more economic and safe for industrial use. Good linearity was found for the drug between concentrations ranging from 1-6 ul. The developed method is precise and reproducible as indicated by low % RSD (<2%). The drug was well separated from impurities and the Rf value was found to be 0.47. Correlation coefficients were found in prescribed limits (R2 = 0.990) and accuracy studies resulted in mean recoveries between 98-104%. The method was found to be robust with the mobile phase variations. The degradation behavior of Tapentadol was studied under acidic, basic, oxidative, neutral, photolytic, and thermal stress. The developed method can separate the degradation products and drugs with good resolution under the optimized condition. The degradation study proves that the method is stability-indicating. From the mass study, we can conclude that the highest degradation was found in HCl with a base peak of maximum intensity at 58.9 m/z. I conclude that the stability-indicating HPTLC-MS method was developed and validated as per ICH guidelines in terms of linearity, accuracy, precision, recovery, ruggedness, and robustness for the quantitation of Tapentadol HCl in tablets.
ACKNOWLEDGEMENT:
The authors are thankful to Precise Chemipharma Pvt. Ltd., Navi Mumbai, India for providing a gift sample of Tapentadol Hydrochloride and Diya lab, Mumbai for elucidation of the mass of the degraded products.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 11.12.2020 Modified on 26.10.2021
Accepted on 25.04.2022 © RJPT All right reserved
Research J. Pharm. and Tech. 2022; 15(6):2765-2770.
DOI: 10.52711/0974-360X.2022.00463