Chemicals and reagents
All the solvents were of analytical grade and purchased from Qualigens (Thermo Fisher Scientific). Purified water was purchased from JK labs (Mumbai). The tetrahydrocurcumin (Sabiwhite) was obtained from Sami Labs, Bengaluru, Karnataka, India.
Method development
Preparation of standard solution
Ten milligrams of tetrahydrocurcumin was accurately weighed and transferred to a 50 ml volumetric flask. Thirty-five milliliters of methanol was added and with the aid of sonication, it was dissolved completely. Volume was made up and solution was mixed well. Ten milliliters of aliquot was withdrawn and diluted to 50 ml with diluent.
Preparation of sample solution
Five hundred milligrams of the 1% w/w tetrahydrocurcumin gel was weighed on a butter paper and transferred the gel along with butter paper to a 50 ml beaker containing 25 ml of diluent. The sample was sonicated for 5 min to form clear solution and the contents of the beaker were transferred to a 50 ml volumetric flask. The beaker was rinsed 2 times by 10 ml of methanol and transferred the same to the volumetric flask. The volume was made up to the mark with diluent and mixed well. The solution was filtered through a 0.45 μm Nylon syringe filter discarding 3-5 ml of sample. Ten milliliters aliquot was further diluted with methanol to 25 ml.
Chromatographic conditions
The system comprised of a Jasco PU-2089 HPLC Pump equipped with Jasco-2070 UV/Vis detector. The Mobile phase composed of acetonitrile: methanol: water (40:23:37% V/V); adjusted to a pH of 3.0 ± 0.05 was used for the study. The mobile phase was filtered through a 0.45 μm pore size membrane PVDF filter and degassed ultrasonically after mixing. Twenty microliters of the solution was injected. The flow rate was maintained at 1 ml/min. The column used for the study was Hypersil BDS, C18 column, 250 mm × 4.6 mm, 5 μm. The column oven temp was maintained at 25 °C. The detection was carried out at wavelength 280 nm. The run time for the analysis was 10 min.
Validation of method: quantitation of THC in bulk and pharmaceutical dosage form
The developed method was validated for system suitability, accuracy, precision, linearity and robustness in accordance with ICH guideline for validation of analytical procedures Q2(R1).
System suitability
System suitability testing helps to decide whether the developed chromatographic system is suitable for the analysis. %RSD of retention times, asymmetry factors and theoretical plates were the parameters for the study. Six replicate samples containing tetrahydocurcumin were analyzed using the developed method.
Filter study is carried out to ensure that no drug is retained by the filter during sample preparation. The solution was collected after passing through a 0.45 μm PVDF filter and a 0.45 μm Nylon filter, by discarding 5 ml of solution. The results were compared to unfiltered centrifuged sample solution.
Specificity
The mobile phase or the excipients in the formulation should not interfere with the analysis. The developed method needs to be specific. The blank, placebo solution, THC solution and gel solution were injected and peak purity was determined.
Linearity
THC solutions at 5 levels were prepared from 10 to 150% of working concentration. At each level, analysis was carried out in triplicate. The peak areas versus concentrations data was evaluated by linear regression analysis. Three data set corresponding to triplicate analysis were constructed. This data was analyzed by the test one-way ANOVA with post test Tukey using a significant level of α = 0.05 (95% of confidence interval).
Accuracy
An accuracy study was performed by adding known amounts of THC to the placebo preparation. The actual and measured concentrations were compared and recovery was calculated. Recovery of the method was evaluated at three different concentration levels (corresponding to 50, 100 and 150% of test preparation concentration). For each concentration level, three sets were prepared and measured.
Precision
The precision of the assay method was evaluated in terms of repeatability by performing six independent assays (intra-day) of THC. Under the same experimental conditions, intermediate precision of the method was checked by another person performing the same procedure on a different day (inter-day). %RSD not more than 2 was taken as the limit.
Robustness
The factors chosen for this study were the change in wavelength (+, −3 nm), flow rate (+,−10%) and column temperature (+,−2 °C). The appropriate amounts of THC, bulk drug and formulation were weighed and diluted with methanol. The effect of changed parameters on analysis of THC in bulk drug was evaluated in terms of RT, asymmetry factor, theoretical plates and assay.