Preparation of topical formulations containing different clay minerals and vitamin C derivative (Ascorbyl glucoside)

Four O/W emulsions (F-SP, F-SW, F-SGY, and F-W4) (Table 1) were prepared. The aqueous phase (Phase B) was pre-mixed using a magnetic bar for 10 minutes to achieve complete dispersion of the kaolin, then the oily phase (Phase A) and the aqueous phase (Phase B) were heated to 85 ± 2 ^oC separately. The aqueous phase was poured into the oily phase with agitation speed of 1500 RPM provided by an mechanical stirrer until cooling to below 40 ^oC. The ascorbyl glucoside, at 5.0 %, was solubilized separately in the citrate buffer (Phase C) and the pH of this solution was corrected into the range of 5.5 and 6.0 with sodium hydroxide aqueous solution at 10% according to Nagase recommendation. This precedent was used in all formulations. Sodium hydroxide aqueous solution and preservative (Phase D) were then poured into the fresh emulsion below 35 ^oC and homogenized using a Dremel Moto-Toll model 395 homogenizer at ~ 24000 RPM for 3 minutes, which was

considered the ideal homogenization time for the emulsions to achieve complete homogeneity.

Table 1. Qualitative and quantitative (%w/w) composition of emulsions Base, SP, SW, F-SGY, and F-W4

*For each formulation was formulated the blank formulation correspondent named active-free (without the active ascorbyl glucoside).

2.7.1 Emulsion centrifuge test and pH evaluation of the formulations

Formulations were evaluated initially by centrifugation of the samples 24 h after the preparation of the emulsions. The formulations were centrifuged for 30 minutes at a speed of 3000 RPM using an Allegra 6R centrifuge(ANVISA Cosmetics Products Stability Guide, 2004; Colipa/CTFA Guidelines on Stability Testing of Cosmetic Products, 2004).

The pH of each formulation was measured in triplicate by means of a laboratory bench peagometer at 25.0 °C. The peagometer was previously calibrated

with pH 4.0 and 7.0 solutions. The samples were diluted in (w/w), weighing 0.5 g of each formulation and adding 5 mL of distilled water. The electrode was inserted into the aqueous preparation(Davis, 1997). The results were generated from the mean values (accepted significance values 5.0-10.0%).

2.8 ASCORBYL GLUCOSIDE HPLC METHOD VALIDATION 2.8.1. Equipment, standards and reagents

Samples was quantified using an Agilent 1100 series HPLC (Agilent Technologies, CA, USA) coupled with UV (254 nm) and a diode array detector (DAD). A mobile phase of 85% orthophosphoric acid HPLC grade at 0.1% (pH 3.8) was pumped at a flow rate of 1.0 mL/min through a Vydac® TM Reverse Phase C18 / 250 mm x 4.6 mm (ambient temperature) and the volume for each injection was 20 μL. A HP Chemstation software V. 32 was used for data acquisition.

The ascorbyl glucoside (AA2G) (Hayashibara, Okayama, Japan) was purchased from Nagase America Corporation (New York, NY) with purity of 100.0%.

The water and 85% orthophosphoric acid HPLC grade from Fisher Scientific were used to prepare the mobile phase. All chemicals and reagents were used without any further purification.

2.8.2 Calibration curve - Linearity

Aliquots of stock solution of ascorbyl glucoside (purity 100.0%) were diluted by adding the de-ionized water as needed to prepare concentrations ranging from 180 to 5 µg mL⁻¹. Three samples of each concentration were prepared. The least-squares fit method was employed to evaluate statistically the results for linearity by regression line and coefficient of linear correlation (r²) (HUANG et al., 2004; LIN et al., 2007).

2.8.3 Specificity

a) Preparation of Stock matrix formulation Solution (active free)

Analysis of active-free formulations without ascorbyl glucoside. About 20 mg

of each o/w emulsion were weighed and dispersed, separately, in 10 mL of de-ionized water. Samples were mixed using a mixer model M16715 (Thermolyne, Columbia, Maryland, USA) for 1 minute, then centrifuged at 3000 RPM for 3 minutes. The supernatant was discarded and the remaining liquid reserved (HUANG et al., 2004; LIN et al., 2007).

b) Sample Preparation containing Matrix formulation only

The aliquot of 960 µl of the previously reserved stock solution of the matrix was added into a HPLC vial and the volume was filled up to 1 ml with deionized water (40 µl added volume). It was mixed for 1 minute, filtered through 0.45 μm microfilter and injected with HPLC-UV.

c) Sample containing matrix formulation solution added with AA2G (active)

The same volume of each matrix formulation stock solution (960 µL), a aliquot of 40 µl of AA2G stock solution at concentration of 1000 µg / mL were added into a HPLC vial, thus completing the volume to 1 mL. The final solution was mixed for 1 minute, filtered through 0.45 μm microfilter and injected in HPLC-UV.

2.8.4 Limits of detection (LD) and quantification (LQ)

The LD and LoQ were based on Standard Deviation of response and slope of calibration curve:

Equation I: LD= 3σ/s Equation II: LQ=10σ/s

Wherein,

s – Slope of calibration curve

σ – Standard deviation of the y-intercept of the regression line

2.8.5 Precision

Stock solution was quantified precisely and diluted with de-ionized water to three different concentrations; 80, 100 and 120 µg/mL. Samples were injected into HPLC for analysis in triplicates on the same day and the analysis was continued for two consecutive days. The standard deviation (S.D.) and relative standard deviation (R.S.D.) were then calculated.

2.8.6 Accuracy/Recovery

In order to measure accuracy/recovery, known aliquots of each formulation were added with known concentrations of ascorbyl glucoside (AA2G) and diluted to final concentrations of 80, 100 and 120 µg/mL of active with de-ionized water. The recovery (%) was calculated using equation III (Lin et al. 2007).

Equation III: %R = [(C1-C2)/C3] x100 Wherein,

C1 – Final concentration of the formulation aliquot solution added with known concentrations of the active solution. The C1 was calculated from average peak area ratio of each sample to ascorbyl glucoside (AA2G) by the obtained linear equation.

C2 – Known concentration of the active contained into the formulation aliquot solution C3 − Known concentration of the active added to the formulation aliquot solution

2.8.7 Robustness

The robustness was evaluated to certify the ability of an analytical method to remain unaffected by small variations in method parameters. The variations applied in the method were; change in the mobile phase, change in the temperature during the HPLC analyze, change in the flow rate at HPLC. The standard deviation (S.D.) and relative standard deviation (R.S.D.) were then calculated.