Instruments
The ultra-violet spectrophotometric instrument is T80 + UV/V Spectrophotometer Instrument Ltd (UK). It is connected to a computer. The cells used are 1-cm width quartz cells. The weighing device is an analytical balance (Sartorius, model 2474, Germany). Other devices and instruments used to achieve the work are an ultrasonic bath (Power sonic, model 405, Korea), a centrifuge device (90-1 Centrifuge, Shanghai Surgical Instruments Factory, China), a porcelain mortar, volumetric dark flasks, and several scales of glass pipettes.
Solvents and chemicals
Standard active pharmaceutical ingredients are nicotinamide powder 99% (BDH Laboratory Supplies, England), tretinoin powder ≥ 98% gifted by Rama Pharma Co, Aleppo, and methanol of analytical grade (Merck, Germany).
Preparation of standard solutions
Standard solution of nicotinamide
First, 20 mg of NCT was weighed. Then it was transferred into a 20-mL flask and diluted with methanol to the mark to obtain a standard stock solution of NCT with a concentration of 1000 µg/mL. Then, six quantities of 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL were pipetted out to 10-mL flasks and diluted with methanol to prepare a series of standard solutions of NCT with concentrations of 20, 40, 60, 80, 100, and 120 µg/mL.
Standard solution of tretinoin
After weighing 20 mg of TRT, it was transferred into a 20-mL flask and diluted with methanol to obtain a standard stock solution of TRT with a concentration of 1000 µg/mL. Then 0.5 mL was pipetted out to a 20-mL flask to obtain a stock solution of TRT with a concentration of 25 µg/mL. Then, ten quantities from 0.2 to 2.0 mL were pipetted out to 10-mL flasks and diluted with methanol to prepare stock solutions of TRT with concentrations of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 µg/mL.
Preparation of standard mixture of nicotinamide and tretinoin
The synthetic mixture of NCT and TRT was made in the ratio of 1:40 (TRT: NCT). The same procedure was followed in Sects. 2.3.1 and 2.3.2. Except that the same final 20-mL flask was used for both NCT and TRT with concentrations of 1000 µg/mL and 25 µg/mL respectively.
Preparation of synthetic mixture from pharmaceutical formulas
Solution formula
Locacid®, a dermal solution formula which is produced by Universal Pharma Co, Damascus. It is labeled to contains 1000 µg/mL of TRT (0.1% w/v%). The volume of the container is 30 mL. Two quantities of 0.5 mL of the dermal solution were pipetted. Each one was transferred to a 20-mL flask. A quantity of 20 mg of NCT was weighed and transferred to one of the previous flasks. Both flasks were diluted with methanol to obtain two work solutions. The first one (A) contains a synthetic mixture of; the dermal solution formula, which includes excipients and TRT, plus NCT with a ratio of 1:40 (TRT: NCT) (the same ratio as if the formula also contains 4% of NCT). The second one (B) only contains the formula of the dermal solution which includes excipients and TRT. This procedure was done to study the absorbance of all ingredients of the formula and compare it to the absorbance of the standard solution of TRT. Next, two quantities of 0.4 mL were pipetted from A and B. Each of them was transferred to 4 of 10-mL flasks to obtain 8 solutions. Four of them (A series) contain TRT, excipients, and NCT, while the others (B series) contain only TRT and excipients. Standard additions were added before diluting with methanol. Standard additions of 0%, 50%, 100%, and 150% of NCT and TRT were added to the A series to obtain final concentrations of 40, 60, 80, and 100 µg/mL and 1.0, 1.5, 2.0, and 2.5 µg/mL of NCT and TRT respectively. Standard additions of 0%, 50%, 100%, and 150% of TRT were added to the B series to obtain final concentrations of 1.0, 1.5, 2.0, and 2.5 µg/mL of TRT. The flask with a concentration of 1.0 µg/mL of TRT which has no standard addition was given according to the labeled 0.1% concentration of the formula.
Cream formula
Retinoram®, a dermal cream formula which is produced by Rama Pharma Co, Aleppo. It is labeled to contain 1000 µg/g of TRT (0.1% w/w%). The net weight of the cream is 30 g. Two quantities of 500 mg of the cream were weighed. Each one was transferred to a mortar with 10 mL of methanol for trituration then transferred to a 20-mL flask. Next, each of them was sonicated for 10 min to ensure the maximum disintegration of the cream and full dissolution of TRT. After sonication, a quantity of 20 mg of NCT was weighed and added to one of the previous flasks. Both were diluted with methanol. Then, each of them was centrifuged to obtain two work solutions. The first one (A) contains a synthetic mixture of; the cream formula, which includes excipients and TRT, plus NCT with a ratio of 1:40 (TRT: NCT) (the same ratio as if the formula also contains 4% of NCT). The second one (B) only contains the formula of the cream which includes excipients and TRT. Next, two quantities of 0.4 mL were pipetted from A and B. Each of them was transferred to 4 of 10-mL flasks to obtain 8 solutions. Four of them (A series) contain TRT, excipients, and NCT, while the others (B series) contain only TRT and excipients. Standard additions were added before diluting with methanol. Standard additions of 0%, 50%, 100%, and 150% of NCT and TRT were added to the A series to obtain final concentrations of 40, 60, 80, and 100 µg/mL and 1.0, 1.5, 2.0, and 2.5 µg/mL of NCT and TRT respectively. Standard additions of 0%, 50%, 100%, and 150% of TRT were added to the B series to obtain final concentrations of 1.0, 1.5, 2.0, and 2.5 µg/mL of TRT. The flask with a concentration of 1.0 µg/mL of TRT which has no standard addition was given according to the labeled 0.1% concentration of the formula.