Current Spectroscopy and Chromatography - Volume 10, Issue 1, 2024

Developed and validated a new reverse phase high-performance liquid chromatographic (RP-HPLC) method, and it is prompt, precise, sensitive and robust for the estimation of eugenol in seed powder extract of Myristica fragrans.

The chemometric approach was utilized to obtain a rugged and definitive chromatographic method for the purpose.

Method variables such as acetonitrile (%) and flow rate were investigated for robustness and optimization by using a face-centered cubic design (FCCD). The Design Expert 12.0.1.0 software has been employed for this optimization. Further, the effects of factors were monitored on the concentrations of eugenol recovered from seed powder extract. Chromatograms have been developed by using an optimized mobile phase mixture containing methanol-water-acetonitrile (10:40:50, v/v/v) and Symmetry^® C18 column (5 μm, 3.9 ×150 mm). The mobile phase was derived at a flow rate of 1 mL/min, and estimation of eugenol was performed at ʎmax 272 nm.

Validation of the method has been carried out to reveal its selectivity, linearity, precision, accuracy, LOD, and LOQ. Linear calibration plot for eugenol was held over the concentration across 6.25 and 100 μg/mL (R² = 0.999). The coefficient of variation was less than 1%, and accurate recovery of eugenol was observed between 96.80 and 99.56%. The LOD and LOQ have been established to be 1.97 and 6.25 μg/mL, respectively. Intraday and Inter-day coefficients of variation have 1.81-1.91 and 1.92-1.57, respectively. Antioxidant activity (AA) by DPPH assay of seed powder extract in five different solvents was performed, and % AA activity was calculated against ascorbic acid.

The validated method has founded to be highly robust and will be applied for the analysis of eugenol's formulation. The highest % AA has reported in hexane solvent.

There are very few methods for simultaneously determining a combined dose of SAL and KET.

The current study aims to explore accurate, precise, simple, and cost-effective HPLC and HPTLC techniques for the simultaneous assessment of Salbutamol (SAL) and Ketotifen (KET).

The determination of Salbutamol and Ketotifen was performed by HPLC and HPTLC methods using 280 nm and 258 nm as the determination wavelength, respectively. Methanol was used to dissolve the drug for estimation in HPLC using mobile phase methanol: 10mM di-Potassium hydrogen orthophosphate in the ratio of 55:45 v/v of pH 4 at a flow rate of 1mL/min and in chloroform: toluene: methanol (7: 2: 3 v/v/v) for the estimation in HPTLC. Moreover, a statistical comparison was made between the results obtained through HPLC and HPTLC of Salbutamol (SAL) and Ketotifen (KET) using the Student’s t-test and F-test.

A linear response was observed in the range of 4-24 µg/mL and 2-12 µg/mL, respectively, for SAL and KET for HPLC. R² was found to be 0.9998 and 0.9999, respectively. For HPTLC, the linear response was observed in the concentration range of 20-120 ng/ spot and 10 - 60 ng/ spot for SAL and KET, respectively. R² was found to be 0.9988 and 0.9998, respectively. The limit of detection (LOD) for HPLC was estimated as 0.34 µg/ml and 0.10 µg/ml for SAL and KET, respectively, and for the HPTLC method, the LOD was estimated as 4.8 µg/ml and 1.5 µg/ml, respectively. Analysing the marketed formulation by using both methods, SAL and KET within the range of 100 ± 2% were recovered. The results obtained after the estimation of the Mastifen S tablet by applying both methods were according to nominal content. Degradation studies were performed using both methods. It was found that Salbutamol was unstable in hydrolytic, oxidative and thermal degradation, whereas stable in photolytic conditions. Ketotifen was found to be stable in thermal and photolytic conditions and unstable in hydrolytic and oxidative conditions.

The proposed stability indicating HPLC and HPTLC methods for SAL and KET was found to be simple, accurate, and reproducible for quantitative estimation in pharmaceutical dosage form, without interference from the excipients or degradation products from the main drug component.

Standardization of Triphala Juice was performed by using the WHO Guidelines. The Parameters included Preliminary Analysis, Phytochemical Identification, Heavy Metal Estimation, etc. A new simple, specific, precise and accurate UV Spectrophotometric, High-Performance Liquid.

Chromatography and High-Performance Thin Layer Chromatography method has been developed for the Estimation of Gallic Acid in pure form.

The UV- Spectrophotometric method was developed using Schimadzu 1800 UV - Visible spectrophotometer using methanol as a solvent. The method was shown to be linear, with a detection wavelength of 273 nm for Gallic Acid.

The separation was achieved on the Schimadzu Prominence-I RP-HPLC and the column used was C18 column using mobile phase consisting of mixture of Methanol: 0.1% OPA (50:50). The detection was carried out at 280 nm with a flow rate of 0.7 ml/min. The retention time for Gallic Acid was found 3.89 minutes. The calibration curve was found linear (r² = 0.999) for RP- HPLC method.

The HPTLC method was developed using Aetron Sprayline instrument, Methanol as solvent and mobile phase consisting of Toluene: Ethyl Acetate: Formic Acid: Methanol (3:3:0.9:0.2). The method was found linear and the wavelength of detection for Gallic Acid was 254 nm, respectively.

The percentage recoveries for both methods were found in the 98.0- 102.0% range.

The methods were validated in accordance with International Conference on harmonization acceptance criteria for specificity, linearity, precision, accuracy, robustness and system suitability. The excipients did not interfere in the determination of Gallic acid in Triphala Juice.

The suggested approach was effectively implemented for the quantitative determination of gallic acid in Triphala juice, which would aid in quality control.