PKKK/300/UP/053 Identification and Quantitation of Tretinoin (Retinoic Acid) in Cosmetic Products by HPLC

All reagents must be of analytical quality or HPLC grade.

Water used must be Ultrapure water, 18 mega ohm

Methanol (HPLC) Grade

Glacial acetic acid

Mobile phase : mixture of methanol,water and acetic acid 85:15:0.5 (v/v/v)

Reference material: Tretinoin, secondary or primary standard. Protect it from light

Light Resistant glassware

Erlenmeyer flask

Amber coloured volumetric flasks

Amber coloured autosampler vials

High Pressure Liquid Chromatography

Analytical column: Hypersil Gold C18, 5 µm, 250mm x 4.6mm or equivalent

UV Detector, with detection capability at 353nm

Standard stock solution: (0.1 mg/mL) of tretinoin in methanol.

Accurately weigh 2.00 mg of tretinoin into a 20 mL amber volumetric flask. Dissolve in 10 mL methanol, and if necessary sonicate for 1-2 minutes. Make up to volume with methanol.

Standard working solution: (0.005 mg/mL) of tretinoin in methanol.

Pipette 1mL of standard stock solution into a 20 mL amber volumetric flask. Make up to volume with methanol. Filter through a 0.45 µm syringe filter into an amber autosampler vial.

Note : The standard stock & working solution is stable for 2 months when kept in temperature between 2-8 °C

6.3.4.1 Conditions:

Column oven temperature : 30oC

Flow rate : 1.4 mL/min

UV detector wavelength : 353 nm

Injection volume : 20 µL

Instrument run time : 20 min

6.3.4.2 Inject blank/diluent, standard and sample solutions into the HPLC

6.3.4.3 Compare the retention time and spectrum obtained for the sample solution with that of the tretinoin standard solution.

Tailing factor should be NMT 2.0

Theoretical plates should be NLT 1500

Internal Quality Control (IQC)

LOD solution prepared in 6.3.3 will be injected for every 5 sample injection. The sequences for IQC are as follows:

LOD solution will be injected for each matrix (maximum five samples per matrix) in a single run.

Calculate the % difference of retention time for LOD solutions injected for every batch of 5 samples.

% difference of retention time for LOD solution injection should not be more than 2.0%.

Results Interpretation

Compare the retention time, peak purity and spectrum obtained from the sample chromatogram with that of tretinoin standard solution chromatogram.

The identity of tretinoin is positive if the retention time and the UV spectrum of sample is similar to that obtained from the standard solution and the peak area of sample is more than the peak area of LOD solution.

The acceptance criteria for peak purity : Impurity in the sample solution is not detected when peak purity index is close to 1.0.

Test Method (Quantitation)

The reagents and HPLC condition are the same as in identification procedure

Standard preparation

Standard stock solution: Accurately weigh 2.5 mg of tretinoin in a 25 mL amber volumetric flask. Dissolve in 10 mL methanol, and if necessary sonicate for 1-2 minutes. Make up to volume with methanol (0.10 mg/mL of tretinoin).

Standard solution: Pipette separately, 1 mL, 2 mL, 3 mL, 4 mL and 5 mL of standard stock solution into a 20 mL amber volumetric flask and make up to volume with methanol. Filter each of the solution into an amber autosampler vial before injecting into the HPLC column. (5 mcg/mL, 10 mcg/mL, 15 mcg/mL, 20 mcg/mL and 25 mcg/mL of Tretinoin respectively).

Sample preparation

Note: The amount of sample used can be adjusted depending on the availability and condition of sample received.

System suitability test (SST)

Sequence of injection

Determine the amount of tretinoin in the portion of sample taken by using the following formula. The amount of tretinoin present in the sample will be reported as percentage of weight per sample weight (% w/w).

Results Interpretation

Five different peak areas from chromatogram of standard solutions representing five different concentration levels of standard solutions. The peak area of the sample solution obtained from the standard calibration curve is directly proportional to the concentration of tretinoin in the sample.

The peak area of sample solutions must fall within the standard calibration range. Therefore, a dilution process shall be carried out if the peak area of sample solutions is more than the peak area of the highest standard solution concentration (25 mcg/mL).