PKKK/300/UP/051 Determination of Hydroquinone by HPLC

High Pressure Liquid Chromatography with photo diode array detector

Whatman PTFE disposable syringe filter, 13 mm diameter, 0.45 µm pore size or equivalent

Nylon filter, 47 mm diameter, 0.45 µm pore size or equivalent

Ultrasonic bath

Volumetric flasks

Methanol (HPLC grade)

Potassium Dihydrogen Phosphate (KH2PO4)

Water(Ultrapure water, 18 mega ohm)

Orthophosphoric acid 85% (H3PO4)

0.05M Phosphate Buffer Solution adjusted with orthophosphoric acid to pH 2.5

Diluent: 0.05M Phosphate Buffer Solution pH 2.5

Reference material: Hydroquinone Reference Standard

Test Method

Reference Standard Preparation (Reference Standard Stock Solution)

~ 20.0 mg of hydroquinone reference standard x Purity

100

Reference Standard Solution

Pipette separately, 1 mL, 2 mL, 3 mL, 4 mL and 5 mL of reference standard stock solution into a 10 mL of volumetric flask and make up to volume with diluent. Filter each of the solution before injecting into the HPLC column. (0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL and 0.5 mg/mL of Hydroquinone respectively)

Limit of Quantitation (LOQ) Solution

Pipette 100 µL of reference standard stock solution into a 100 mL volumetric flask and make up to volume with diluent (0.001mg/mL of hydroquinone)

Internal Quality Control (IQC Solution)

Pipette 0.02 mL (20 µL) of standard stock solution from part 6.1 and spiked in 0.5 g of blank sample. Dilute both standard and blank sample with 20mL diluent. (0.004% w/w of Hydroquinone).

Sample Preparation

Perform sampling procedure according to PKKK/300/KOS/001 Proses Persampelan Untuk Analisis Sampel Unit Kosmetik

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

System suitability test

Sequence of injection

Calculation

Determine the amount of hydroquinone in the portion of sample taken by using the following formula and the amount of hydroquinone presence in the sample will be reported as percentage of weight per sample weight (% w/w):-

Determine the concentration of IQC solution from the calibration curve as constructed in 6.2. Plot the recovery value (%) of hydroquinone obtained on the IQC chart. (Currently using limit Recovery 80-120% for IQC > 10µg/kg, based on Official Journal of European Communities 2002/657/EC)

Results Interpretation

Five different peak areas/heights from chromatogram of reference standard solutions represent five different concentration levels of standard solutions. The peak area of the sample solution that obtained from the standard calibration curve is directly proportional to the concentration of hydroquinone in the sample.

A dilution process shall be carried out if in any case, the peak area/ height of sample solution are more than the area/ height of the highest standard solution concentration (0.5mg/mL), so that the peak area/height falls within the standard calibration range.