New stability indicating liquid chromatographic method for the determination of Pterostilbene in capsules

 

Gujju. Hima Bindu*, Mukthinuthalapati Mathrusri Annapurna

Department of Pharmaceutical Analysis & Quality Assurance, GITAM Institute of Pharmacy,

GITAM University, Visakhapatnam-530045, India

 

ABSTRACT:

Pterostilbene is used for the treatment of cancer, diabetes, anti-hyperlipidemic and fungal infections.A new RP-UFLC method has been developed for the determination of Pterostilbene. Shimadzu Model UFLC system, equipped with PDA detector and SunfireC18 column was used for the present chromatographic study. Mobile phase consisting of a mixture of tetra butyl ammonium hydrogen sulphate andacetonitrile (32:68, v/v)with flow rate 1.0 mL/min was chosen for the determination of Pterostilbene. Linearity was observed over the concentration range 0.1–100 μg/ml (R² = 0.9999) with linear regression equation y = 161775.7385x - 15849.1207. The LOD and LOQ were found to be 0.01642 μg/ml and 0.05421 μg/ml respectively. Solutions of Pterostilbenewere subjected to stress conditions such as acidic, alkaline, oxidation and thermal degradations. The method was validatedas per ICH guidelines. This method is quite suitable for the determination of Pterostilbene in pharmaceutical formulations.

 

 

 

INTRODUCTION:

Pterostilbene (Figure 1) belongs to the group of phytoalexinsobtained from the plant region¹. It is chemically known as trans 3, 5-dimethoxy-4’-hydroxy-trans-stilbene. It is a dimethylated analogue of resveratrol, C₁₆H₁₆O₃  having molecular weight 256.296 g/mol. Usually it is found ingrapes, blueberries, and in age-old darakchasava, an ayurvedic medicine from India²and in the tree species Pterocarpus marsupium and Guibourtia tessmanii^3-8. It exhibits anti-cancer^9-11anti-diabetic, antioxidant^12-15 and anti-fungal¹⁶ activities. Only three liquid chromatographic methods have beendeveloped for the determination of Pterostilbenewith fluorescence detection¹⁷, in rat plasma¹⁸ and dragon blood¹⁹ and no stability indicating

 

 

liquid chromatographic method has been developed for the assay of pharmaceutical formulations. Currently the authors have proposed a very simple, rapid, precise and robust validated stability indicating reverse phase UFLC method for the determination of Pterostilbene in capsules.

 

Figure 1:Chemical structure of Pterostilbene

 

EXPERIMENTAL:

Chemicals and Reagents

Pterostilbene standard (>99.0% purity) was obtainedfrom Oxford laboratory, India.  Acetonitrile (HPLC grade), _{tetra butyl ammonium hydrogen sulphate,}sodium hydroxide, methanol, hydrochloric acid, hydrogen peroxide, disodium hydrogen phosphate, potassium dihydrogen phosphate and glacial acetic acid were obtained from Merck (India). All chemicals were of analytical grade and used as received.  Pterostilbene is available as capsules with brand names such as Pterostilbene (Source Naturals Inc. (Canada); Label claim: 50 mg), Pterostilbene (Absorb Health (North Carolina); Label claim: 100 mg). Pterostilbene is procured from Booyahchicago , Delhi, India.

 

Preparation of _{Tetra butyl ammonium hydrogen sulphate solution} (TBHS)

_{10 mMTetra butyl ammonium hydrogen sulphate solutionwas prepared by dissolving 3.3954 g of TBHS in HPLC grade water in a 1000mL volumetric flask, sonicated for half an hour and filtered.}

 

Preparation of Pterostilbene stock solution (1 mg/ml)

Stock solution (1000 μg/ml) was prepared by dissolving 25 mg of Pterostilbene in mobile phase in a 25 mL volumetric flask. Working solutions were prepared from the stock solution with mobile phase and all the solutions were filtered through 0.45 μm membrane.

 

Instrumentation

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 tetra butyl ammonium hydrogen sulphate and acetonitrile (38:62, v/v) was used as the mobile phase. The flow rate was 1.0 mL/min and 20 µl of each sample was injected into the UFLC system.

 

Method validation²⁰

Linearity

A series of Pterostilbenesolutions (0.1-100 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 concentration of the Pterostilbene 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 International Conference on Harmonization guidelines Q2 (R1).²⁰

 

Method precision, accuracy and robustness           

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 (309 and 313 nm), composition of mobile phase (66 and 70% of acetonitrile) and flow rate (0.9 and 1.1 mL/min). Robustness of the method was studied using at a concentration level 20 μg/mlPterostilbene.

 

Forced degradation studies²¹

Acid and base degradation

Acidic and alkaline degradations were performed by treating the Pterostilbene solution (1 mg/mL) with 0.1 N hydrochloric acid and 0.1 N sodium hydroxide respectively. The solutions were refluxed for 1 hour at 80 ºC, cooled, neutralized and diluted with mobile phase as per the requirement.

 

Oxidation degradation

Oxidation degradation was performed by treating the Pterostilbene solution (1 mg/mL) with 30% hydrogen peroxide. The solution was refluxed for 1 hour at 80 ºC, cooled and diluted with mobile phase as per the requirement.

 

Thermal degradation

For thermal stress testing, 1 mg/mlPterostilbenesolution was heated in thermostat at 80 ºC for 1 hour, cooled, filtered and diluted as per the requirement before use.

 

Applicability of proposed method to Capsules

To perform the assay of QC samples of Pterostilbene twenty capsules were procured from thelocal pharmacy store and the contents were finely powdered. Powder equivalent to 100 mg Pterostilbene 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.

 

_{Stability of mobile phase and drug solutions}

Pterostilbene drug solutions as well as the mobile phase were tested for 48 hours for its stability. The assay of drug solutions was monitored for a period of 48 hours to determine the solution stability and the retention time was observed for the stability testing of mobile phase.

 

RESULTS AND DISCUSSION:

A newstability indicating liquid chromatographic method hasbeen developed for the determination of Pterostilbene.The previously published methods were compared with the present proposedmethods in detail in Table 1.

 

 

 

 

Table. 1. Literature survey of Pterostilbene

 

Optimisation of _{high performance liquid chromatographic method}

Trials were made initially on Shimadzu Model CBM-20A/20 Alite UFLC system, equipped with SPD M20A prominence PDA detector with different columns using different mobile phases in various compositions for the development of liquid chromatographic method and finally Sunfire C18 (150 mm × 4.6 mm i.d., 5 µm particle size) column was chosen and the work was proceeded. Blunt peaks were observed when formic acid: acetonitrile (40: 60, v/v) was tried at a flow rate of 1 ml/min and further changes in the mobile phase composition didn’t give any fruitful results and therefore the authors have finally attempted a mobile phase consisting of a mixture of tetra butyl ammonium hydrogen sulphate and acetonitrile (32:68, v/v) with flow rate 1.0 mL/min. in which Pterostilbene was eluted as a sharp peak at 3.3 ± 0.08 mins (UV detection 311 nm) and therefore the above mobile phase was selected.

 

Method validation

Linearity

Pterostilbene has shown linearity 0.1-100 μg/ml (R² = 0.9999) with linear regression equation y = 161775.7385x - 15849.1207(Figure 2) and the LOD and LOQ were found to be 0.01642 μg/ml and 0.05421 μg/ml respectively (Table 2).The complete separation of the analysis has taken less than 10 min and the method can be applied successfully for performing long-term stability studies of Pterostilbeneformulations. The typical chromatograms obtained for blank and Pterostilbene were shown in Figure 3A and 3B.In all the UFLC runs the theoretical plates were more than 2000, capacity factor was more than 2 and the tailing factor was less than 2.

Table. 2. Linearity of Pterostilbene

*Mean of three replicates

 

 

Figure 2:Linearity graph of Pterostilbene

 

Method precision, accuracy and robustness           

The method precision was performed by assaying the drug samples (n=3) of each at three different concentration levels (10, 50 and 100 μg/ml) on the same day (intra-day precision) and on three different days (inter-day precision). The % RSD range was found to be0.13-0.78 and 1.12-1.45for intra-day and inter-day precision studies respectively (Table 3) which is less than 2.0 indicating that the method is precise. Recovery studies were performed by spiking pure drug solutions with Pterostilbene formulation extracted solution (10 μg/ml) so as to yield a total concentration 9, 10 and 11 μg/ml. The % RSD was found to be 0.39-0.53 which is less than 2.0 % indicating that the method is accurate. The percentage recovery observed was 99.60-100.93 % (Table 3). The method was tested for its robustness by inducing small deliberate changes in the method parameters such as mobile phase composition, flowrate, detection wavelength, temperature etc. followed by evaluation of the assay results. The mobile phase composition (TBHS buffer: acetonitrile) was slightly varied as30:70 and 34:66 (± 2 %, v/v, established method is 32:68 v/v) whereas the flow rate was varied as 0.9and 1.1 mL/min (± 0.1 mL/min) with detection wavelength at 309 and 313 nm (± 2 nm). The results shown in Table 4 indicates that the % RSD (0.33-1.40) of the assay determined under robustness conditions was less than 2.0% indicating that the established method is robust.

 

Table. 3. Precision and accuracy studies of Pterostilbene

*Mean of three replicates

 

Table.4.Robustness study of Pterostilbene

*Mean of three replicates

 

Applicability of developed methods to Capsules

The proposed analytical technique was applied for the assay of Pterostilbene in capsules (PTEROSTILBENE^® Source Naturals Inc.; Label claim: 50 mg and PTEROSTILBENE^® Absorb Health.; Label claim: 100 mg) and the percentage of recovery is found to be 98.88- 99.02 (Table 5). The overlay chromatogram obtained for the marketed formulations were shown in Figure 3C.

 

Table. 5. Assay of Pterostilbene capsules

*Mean of three replicates

 

Forced degradation behaviour

Pterostilbene standard and capsule powder were exposed to different stress conditions and a slight decomposition was observed on exposure of Pterostilbenedrug solution to acidic (5.84 %), alkaline (0.41 %), oxidation (-0.03 %) and thermal (1.75 %) degradations indicating that the drug is highly resistant (< 6% degradation) towards the all the degradations (Table 6). The phenolic group present in the Pterostilbene chemicalstructure may be responsible for the alkaline degradation for which the extra peak might have eluted at 2.375 min.Also it was observed that Pterostilbene lost its peak shape slightly showing the tailing in the respective chromatogram leading to decrease in the theoretical plates. The typical chromatograms obtained during the assay of stressed samples were shown in Figure 4 (A-F).

 

 

Figure 3: Typical chromatograms of Pterostilbene [A] Blank [B] Pterostilbenepure standard [C] VitaMonk (Label claim: 50 mg)

Table. 6. Forced degradation studies of Pterostilbene

 

 

 

Figure 4: Typical chromatograms of Pterostilbene [A] Blank [B] Pterostilbene standard [C] Acidic degradation [D] Oxidative degradation [E] Alkaline degradation [F] Thermal degradation

 

 

_{Stability of solutions and mobile phase}

The drug solutions as well as the mobile phase were proved to be quite stable during a study of 48 hours as the % RSD was found to be 0.0808 and0.0960 which is less than 2.0 for the solution and mobile phase stability studies (Table 7).

 

Table.7. Study of Pterostilbene solution stability and mobile phase stability

Solution stability			Mobile phase stability
Time (h)	*Assay (%)	Statistical analysis	Retention time (min)	Statistical analysis
Initial	98.72	Mean = 98.85 SD = 0.079917 % RSD =0.0808	3.361	Mean = 3.358 SD = 0.003225 % RSD =0.0960
6	98.80		3.355
12	98.93		3.362
18	98.92		3.359
24	98.88		3.357
48	98.87		3.354

*Mean of three replicates

 

CONCLUSION:

The proposed three methods were validated as per ICH guidelines and can be applied for the determination of Pterostilbene incapsules. The three methods were found to be robust, accurate, precise and specific.

 

ACKNOWLEDGEMENTS:

The authors are grateful to Oxford Laboratories, India for providing the gift samples of Pterostilbene.There is no conflict of interest.

 

 

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Accepted on 20.09.2018          © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(9): 3851-3856.