Quantitation and optimization of β-carotene and α-tocopherol in emulsion prototype with reversed-phase chromatography

Emulsion products that are rich in pro-vitamin A, vitamin E and antioxidants have been successfully formulated from red palm oil (FO-MSM), pumpkin (SLK), and dragon fruit (SBN). This study was aimed to determine the concentrations of β-carotene and αtocopherol in the prototype products of FO-MSM, SLK, and SBN by reverse phase chromatography and to determine the optimal deodorization time for FO-MSM on βcarotene and α-tocopherol levels. Optimal conditions for analysis of samples was with the mobile phase methyl dichloride: methanol: acetonitrile (2: 1: 3). The injection volume was 10 μL for β-carotene and 20 μL for α-tocopherol. Isocratic elution was carried out at a flow rate of 1.0 mL/min at room temperature (25±2°C). The mobile phase was first eliminated for 45 mins. The β-carotene and α-tocopherol peaks were identified by matching the peak retention times (Rt) of β-carotene and α-tocopherol in the sample with the standard Rt of β-carotene and α-tocopherol. The reverse-phase chromatography successfully determined β-carotene and α-tocopherol in prototype products of FO-MSM, SLK, and SBN. The developed emulsion prototype had 2044.5±196.1 ppm of β-carotene and 38.8±0.4 ppm of α-tocopherol. The content of β-carotene in the developed emulsion prototype was more than tenfold increase from the original prototype without sacrificing the product's taste. β-carotene showed no significant difference in reducing the deodorization time from 5 hrs to 1 hr, while the 2.5-fold increase of the α-tocopherol content was observed. Therefore, the optimal deodorization time for FO-MSM was determined based on the value of α-tocopherol and not by β-carotene. RP-18 was successfully used to determine β-carotene and α-tocopherol in FO-MSM, SLK, and SBN emulsion prototype products

Development and Validation of a Method for Ionic Dyes Determination in Guava Fibrous Matrix Using Reverse Phase Chromatography

In this work, a method for food dyes determination in fibrous fruit pulps was developed. The analytes Tartrazine, Twilight Yellow FCF, Erythrosine, Orange G and Allura Red were analyzed by reverse phase chromatography with diode array (DAD) detection. The sample preparation consisted of a simple procedure, based on initial pH adjustment of the sample (pH = 10.0), followed by an extraction with acetonitrile (5.0 mL and agitation for 30 s), ultrasonic (5 min) and centrifugation (4000 rpm, 20 ºC, for 20 min). The supernatant had its pH adjusted to 3.0, followed by filtration. The method was validated, with the analytical curves prepared in a matrix, in the range of 50 to 150 mg kg-1 for all the analytes studied (determination coefficients (R2) > 0.99). The method veracity was evaluated in terms of recovery, obtaining recoveries of 93-110%. The repeatability was less than 13.7%, while the intermediate precision was less than 19.0%. The limits of detection and quantification were determined, being 15 and 30 mg kg-1, respectively. The combined relative uncertainties were less than 13.8%. Therefore, it can be concluded that a simple method was developed, suitable for monitoring the presence of dyes in fruit pulps in routine analyzes.

Method Development, Validation and Stability Indicating Studies for Simultaneous Estimation of Anti-Hypertensive Drugs from Pharmaceutical Formulation by RP-HPLC

Objective: Method development, validation & stability indicating studies for simultaneous estimation of Anti-Hypertensive drugs, Benidepine (BEN) and Metoprolol (MET) from pharmaceutical formulation by RP-HPLC. Methods: For present work, reverse phase chromatography was selected as its suggested use for ionic and moderate to non-polar compounds. Reverse phase chromatography is simple, suitable, better regarding efficiency, stability, and reproducibility. C18 packed column, a 100 X 2.1mm. ID column of 5.0 μm particle packing, was selected for separation of BEN and MET. Different solvent systems were tried and optimized in combinations as mobile phase. BEN (4 μg/ml) and MET (50 μg/ml) in 15mM ammonium formate-Methanol (15:85 v/v) was developed as it was showing good peak shapes and a significant amount of resolution. The mobile phase was flowed at 1.2 ml/min with detection of BEN analytes at 236 nm and MET analytes at 225 nm respectively. Result: Method development was done. Specificity, linearity, accuracy, precision, robustness, limit of detection and limit of quantitation were used to accomplish validation. The method was found linear from 32.5 – 500 µg.ml-1 for both BEN and MET individually. The percentage recovery of BEN when placed for period of 12 hours was found to 100% in 0.1N/M NaOH at 60˚C and Thermal (60˚C); 12 % degradation in 0.1N/M HCl at 60˚C; Oxidation (3-6% H2O2) at room temperature whereas for MET was 100 % in 0.1N/M NaOH, 0.1N/M HCl at 60˚C, at thermal (60˚C) as well as oxidation by 3-6% H2O2 at room temperature. Conclusion: Developed analytical method for the simultaneous estimation of Benidipine (BED) and Metoprolol (MET) in both bulk and tablet formulation has obliged the ICH guidelines including, tailing factor (T), separation factors (α), theoretical plates (N), capacity factor (k’), resolution (R) and RSD (%). The validated stress degradation studies under thermal, oxidative, alkali and acid ascertained few degradation products for Benidipine whereas the Metoprolol was unaffected with forced degradation studies. Keywords: Benidipine, Metoprolol, Reverse-Phase High Performance Liquid Chromatography, Stability indicating method.