A new Validated Stability Indicating RP-UFLC Method for the Estimation of Pterostilbene
Kalyani Lingamaneni*, Mukthinuthalapati Mathrusri Annapurna
Department of Pharmaceutical Analysis and Quality Assurance
*Corresponding Author E-mail: kalyani.lingamaneni@gmail.com
ABSTRACT:
Pterostilbene belongs to phytoalexins group and it is chemically trans 3, 5-dimethoxy-4’-hydroxy-trans-stilbene. Pterostilbene is used for the treatment of diabetes, cancer and fungal infections. A new stability indicating chromatographic method has been developed for the quantification of Pterostilbene and the method was validated. The chromatographic study was studied using Shimadzu Model CBM-20A/20 Alite UFLC system with Sunfire C18 column and PDA detector. A mixture consisting of 0.1M ammonium formate and methanol (15:85, v/v) with flow rate 0.8 mL/min. Linearity was observed over the concentration range 0.05–150 μg/mL (R2 = 0.9999) with linear regression equation y = 207961.521x-16083.7544. The LOD and LOQ were obtained to be 0.01569 μg/mL and 0.04782 μg/mL respectively. Pterostilbene drug solutions were subjected to stress conditions such as acidic, alkaline, oxidation and thermal degradations. The method was validated and can be applied for the quantification of Pterostilbene pharmaceutical formulations.
KEYWORDS: Pterostilbene, RP-UFLC, Stability-indicating, Validation, ICH guidelines.
INTRODUCTION:
Pterostilbene (PS) (Figure 1) is a dimethylated analogue of resveratrol with molecular formula C16H16O3 and molecular weight 256.296 g/mol. Pterostilbene is found in grapes, blueberries1 and in age-old darakchasava, an ayurvedic medicine from India2 and in the tree species Pterocarpus marsupium3. Pterostilbene is supposed to show anti-cancer activity4, anti-diabetic5 and anti-fungal activity6. .Liquid chromatographic methods were developed for the estimation of Pterostilbene in rat plasma7, dragon blood8 and in biological fluids9 and HPLC10-11 methods. Yu Jeong12 et al and Loïc Becker13 et al studied Pterostilbene and its derivatives using LC-MS and Mathrusri14-15 et al developed spectrophotometric techniques for the quantification of Pterostilbene in pharmaceutical formulations. A new validated stability indicating RP-UFLC method was developed for the quantification of Pterostilbene in capsules.
Figure 1. Chemical structure of Pterostilbene (PS)
MATERIALS AND METHODS:
Chemicals and Reagents:
Pterostilbene standard (API) was procured as a gift sample from Maithili Life Sciences Private Limited (India) (>99.0% purity). Ammonium formate (HPLC grade) and methanol (HPLC grade) were obtained from Merck India. All other chemicals such as sodium hydroxide, hydrochloric acid, hydrogen peroxide were of analytical grade and obtained from Qualigens Fine Chemicals (India).
Pterostilbene is available with brand names such as Pterostilbene (Absorb Health); Label claim: 100 mg) and Pterostilbene (Source Naturals Inc.; Label claim: 50 mg) as capsules.
Preparation of 0.1M Ammonium formate:
6.3 gm of ammonium formate was accurately weighed and dissolved in water in a 1000 ml volumetric flask to attain 0.1M ammonium formate solution.
Preparation of Pterostilbene stock standard solution (1 mg/mL):
Pterostilbene stock solution (1000 μg/mL) was prepared by dissolving 25 mg of PS in methanol in a 25 mL volumetric flask. Working standard solutions were prepared from the stock solution using the mobile phase and all the solutions were filtered through 0.22 μm membrane after sonication.
Instrumentation and chromatographic conditions:
Chromatographic separation was achieved by using a Shimadzu Model CBM-20A/20 Alite UFLC system, equipped with SPD M20A prominence PDA detector with Sunfire C18 (150 mm × 4.6 mm i.d., 5 µm particle size) column. A mixture of 0.1M ammonium formate and methanol (15:85, v/v) was used as the mobile phase with flow rate 0.8 mL/min for the chromatographic study and 20 µl of each sample was injected into the UFLC system.
Method Validation16:
Linearity:
A series of Pterostilbene solutions (0.05–150 μg/mL) were prepared from the stock, diluted with mobile phase and 20 µL was injected in to the UFLC system. The peak area of each chromatogram was noted and a calibration curve was plotted by taking the concentration of PS standard solutions on the x-axis and the corresponding peak area on the y-axis. The limit of quantification (LOQ) and limit of detection (LOD) were based on the standard deviation of the response and the slope of the constructed calibration curve (n=3), as described in ICH guidelines Q2 (R1) 16.
Precision and accuracy and robustness studies:
The intra-day and inter-day precision studies of the three methods were performed at three different concentration levels (10, 50 and 100 µg/mL) and on three different days respectively and the %RSD was calculated. The accuracy of the assay method was calculated at three different levels (80%, 100% and 120%) by the standard addition method and the percentage recoveries were calculated. The robustness of the assay method was performed by introducing deliberate changes in the chromatographic conditions that includes detection wavelength (305 and 315 nm), composition of mobile phase (13 and 17% of ammonium formate) and flow rate (0.7 and 0.9 mL/min). Robustness of the method was studied using at a concentration level 20 μg/mL PS.
Stress degradation studies17:
Acid and base induced degradation
Acidic and alkaline degradations were performed by treating the drug solution (1 mg/mL) with 1 ml of 0.1 N hydrochloric acid and 0.1 N sodium hydroxide respectively. The solutions were refluxed for 1 hour at 80 ºC, cooled, neutralized with 0.1 N sodium hydroxide and 0.1 N hydrochloric acid respectively and diluted with mobile phase as per the requirement before injecting in to the HPLC system.
Oxidation induced degradation:
Oxidation degradation was performed by treating the drug solution (1 mg/mL) with 1 ml of 30% hydrogen peroxide. The solution was refluxed for 1 hour at 80 ºC, cooled and diluted with mobile phase as per the requirement.
Thermal induced degradation:
For thermal stress testing, 1 mg/mL drug solution was heated in thermostat at 80 ºC for 1 hour, cooled, filtered and diluted as per the requirement before use.
Applicability of developed method to the marketed formulations (Capsules):
To perform the assay twenty capsules of Pterostilbene were procured from the local pharmacy store and the contents inside the capsule were finely powdered. Powder equivalent to 100 mg PS was accurately weighed and dissolved in mobile phase in a 100 ml volumetric flask and the contents of the flask were sonicated for 30 min and filtered through 0.45 μm membrane filter before injection in to the UFLC system.
RESULTS AND DISCUSSION:
A new stability indicating RP-UFLC method has been developed for the estimation of Pterostilbene in pharmaceutical formulations. A review of the previously published liquid chromatographic methods was summarized in Table 1 and the present proposed method was compared with it.
Table 1: Review of liquid chromatographic methods
|
Mobile phase (v/v) |
λ (nm) |
Method |
Comments |
Reference |
|
Acetonitrile: 0.1% formic acid |
320 |
HPLC |
Gradient mode Rat plasma |
7 |
|
Acetonitrile: 1% acetic acid (41: 59) |
310 |
HPLC |
Dragon blood |
8 |
|
Acetonitrile: water (50∶50) |
Excitation 330 Emission 374 |
HPLC |
Fluorescence detection Rat serum |
9 |
|
Tetra butyl ammonium hydrogen sulphate: Acetonitrile (32:68) |
311 |
UFLC |
Isocratic mode |
10 |
|
0.1% Tri fluoro acetic acid: Acetonitrile (10:90) |
219 |
HPLC |
Isocratic mode |
11 |
|
Ammonia formate: Methanol (15:85) |
311 |
UFLC |
Isocratic mode |
Present work |
Method optimisation:
Initially trials were using different mobile phases in various compositions and with Phenomenex column. Mobile phase consisting of formic acid and acetonitrile (40: 60) was used with a flow rate of 0.8 ml/min but a blunt peak was observed and even after increasing the flow rate to 1 ml/min there was no improvement in the peak shape. Then mobile phase consisting of ammonium formate and methanol (50: 50) was tried with flow rate 1 ml/min and no peak was observed and therefore the same mobile phase with 15: 85 v/v ratio was used with flow rate 1.2 ml/min but peak splitting was observed. The previous column was replaced with Sunfire C18 column keeping the above chromatographic conditions same and due to which Pterostilbene was eluted at 1.948 min (flow rate 1.0 ml/min) the drug peak was eluted at 2.313 min with tailing greater than 2. Finally the chromatographic conditions such as mobile phase ammonium formate: methanol (15: 85), flow rate 0.8 ml/min with UV detection 310 nm were established where Pterostilbene was eluted as a sharp peak at 2.759 ± 0.03 mins (UV detection 310 nm) with acceptable system suitability parameters.
Method validation:
Linearity:
Pterostilbene has shown linearity 0.05-150 μg/mL (R2 = 0.9999) (Table 2) with linear regression equation y = 207961.521x-16083.7544 (Figure 2). The LOD and LOQ were found to be 0.01569 μg/mL and 0.04782 μg/mL respectively. The run time was 10 min and the method can be applied for performing long-term stability studies of PS in pharmaceutical formulations. The chromatograms obtained during the study were shown in Figure 3. The theoretical plates were more than 2000 and the tailing factor was less than 2.
Table 2: Linearity
|
Conc. (μg/ml) |
*Mean Peak Area ± SD |
RSD (%) |
|
0.05 |
10633.00 ± 77.66 |
0.73 |
|
0.1 |
20620.67 ± 151.80 |
.0.74 |
|
0.5 |
106622.33± 1007.56 |
0.94 |
|
1 |
204699.33±930.40 |
0.45 |
|
5 |
1066179.00±10020.40 |
0.94 |
|
10 |
2086538.00±9999.52 |
0.48 |
|
20 |
4076829.33±10188.91 |
0.25 |
|
50 |
10442841.00±59154.23 |
0.57 |
|
80 |
16291838.00±200000.33 |
1.23 |
|
100 |
20864776.67±84261.88 |
0.40 |
|
150 |
31282585.33±75886.96 |
0.24 |
*Mean of three replicates
Precision, Accuracy and Robustness study of Pterostilbene:
The proposed liquid chromatographic method for the estimation of Pterostilbene is found to be precise and accurate (Table 3) as the % RSD was found to be less than 2.0. The method was also found to be robust as the % RSD was less than 2.0 (Table 4).
|
|
|
|
Figure 2. Calibration curve of Pterostilbene
|
Figure 3.Typical chromatograms of A) Placebo B) Pterostilbene API (Rt 2.769 min) C) Pterostilbene capsule (Label claim: 50 mg) (Rt 2.780 min) |
Table 3: Precision and accuracy studies
|
Conc. (µg/ml) |
Intra-day precision |
Inter-day precision |
|
|
*Mean peak area ± SD (%RSD) |
*Mean peak area ± SD (%RSD) |
||
|
10 |
2086538.00 ± 9999.52 (0.48) |
2127812.33 ± 20723.69 (1.28) |
|
|
50 |
10442841.00 ± 59154.23 (0.57) |
11051642.67 ± 118030.86 (1.34) |
|
|
100 |
20864776.67 ± 84261.88 (0.40) |
2138561.33 ± 186396.94 (1.43) |
|
|
Accuracy |
|||
|
Conc. (µg/ml) |
*Mean peak area ± SD (% RSD) |
Drug Found (µg/ml) |
*Recovery (%) |
|
9 |
1882494.00 ± 6248.05 (0.33) |
8.97 |
99.72 |
|
10 |
2066511.33 ± 9949.28 (0.48) |
10.01 |
100.14 |
|
11 |
2294863.67 ± 9387.84 (0.41) |
10.96 |
99.62 |
*Mean of three replicates
Table 4: Robustness study of Pterostilbene (20 µg/ml)
|
Parameter |
Condition |
*Mean peak area |
*Mean peak area ± SD (% RSD) |
|
Flow rate (± 0.1 ml/min) |
0.7 |
4075319 |
4072011 ± 6822.278 (0.16) |
|
0.8 |
4076548 |
||
|
0.9 |
4064165 |
||
|
Detection wavelength (± 5 nm) |
305 |
4076846 |
10130.96±10130.96 (0.25) |
|
310 |
4076118 |
||
|
315 |
4058946 |
||
|
Mobile phase composition (± 2 %) |
17:83 |
4068461 |
4071037 ± 4457.722 (0.11)
|
|
15:85 |
4068465 |
||
|
13:87 |
4076184 |
Mean of three replicates
Stress degradation studies:
Pterostilbene was exposed to different stress conditions and the percentage of drug decomposition during acidic hydrolysis was 36.49 % and 62.07 % during oxidation. A very slight drug decomposition i.e. less than 5 % was observed during alkaline (1.30 %) and thermal (2.30 %) degradations indicating that the drug is highly resistant towards the alkaline and thermal degradations (Table 5). The typical chromatograms obtained during the stress degradation studies were shown in Figure 4. The system suitability parameters such as the theoretical plates (>2000), tailing factor (<1.5) were shown in Table 5 and are within the acceptable criteria.
Assay of Pterostilbene capsules:
The proposed analytical technique was applied for the assay of Pterostilbene in capsules (Label claim: 100 mg) and the percentage of recovery was found to be 99.76- 99.84 (Table 6). The chromatogram obtained during the assay of Pterostilbene capsules was shown in Figure 3C.
Table 5: Forced Degradation Studies of Pterostilbene (50 µg/ml)
|
Stress Conditions |
Rt (min) |
*Mean peak area |
*Drug recovered (%) |
*Drug decomposed (%) |
Theoretical plates |
Tailing factor |
|
Standard drug |
2.770 |
10442841 |
100 |
- |
3447.612 |
1.336 |
|
Acidic degradation |
2.779 |
6632339 |
63.51 |
36.49 |
4043.015 |
1.385 |
|
Alkaline degradation |
2.645 |
10306564 |
98.70 |
1.30 |
3475.534 |
1.349 |
|
Oxidative degradation |
2.792 |
3961239 |
37.93 |
62.07 |
3890.851 |
1.354 |
|
Thermal degradation |
2.764 |
10202155 |
97.70 |
2.30 |
3441.224 |
1.335 |
Table 6: Assay of Pterostilbene
|
Formulation |
*Labelled amount (mg) |
*Amount found (mg) |
* % Assay |
|
Brand I |
50 |
49.92 |
99.84 |
|
Brand II |
100 |
99.76 |
99.76 |
*Mean of three replicates
Figure 6.Typical Chromatograms of Pterostilbene [A] Blank [B] Pterostilbene pure drug sample [C] acidic degradation [D] thermal degradation [E] Oxidative degradation [F] alkaline degradation
CONCLUSION:
The proposed stability indicating RP-UFLC method for the estimation of Pterostilbene capsules was validated as per ICH guidelines and it can be applied not only for the determination of Pterostilbene but also for the kinetic studies. The method was found to be robust, accurate, precise and specific.
ACKNOWLEDGEMENT:
The authors are grateful to Maithili Life Sciences Private Limited (India) for providing the gift samples of Pterostilbene and there is no conflict of interest.
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Received on 10.11.2020 Modified on 19.12.2020
Accepted on 21.01.2021 © RJPT All right reserved
Research J. Pharm. and Tech 2021; 14(3):1284-1288.
DOI: 10.5958/0974-360X.2021.00227.4