PKKK/300/UP/056 Identification of Steroids in Cosmetic Products by HPLC (ASEAN Harmonised Method ACM 007)

High Performance Liquid Chromatography with PDA

Ultrasonic bath

Glass conical flasks with screw cap

Volumetric flask (10 mL, 10mL, 5 mL)

Shaker

PVDF syringe membrane filter, 0.45 µm pore size or equivalent

Autosampler vial

Acetonitrile HPLC grade

Methanol HPLC grade

Ultrapure water, 18 ohm

Prednisolone, Prednisone, Cortisone, Betamethasone, Dexamethasone, Hydrocortisone Acetate, Triamcinolone Acetonide, Betamethasone 17-Valerate and Mometasone Furoate Reference Standard

Chromatographic Parameters

Column : Kinetex Phenyl-Hexyl 100A 4.6mm x 150mm x 2.6µm

Gradient System :

Mobile Phase A : Water

Mobile Phase B : Acetonitrile

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Injection volume : 10 L

Column temperature : 40 ◦C

Flow Rate : 0.7 mL/min

Wavelength : 240 nm

Test Method

Standard Stock Solution Preparation (0.5 mg/mL)

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

Pipette 1 mL of each reference standard stock solutions. Mix it in 10 mL volumetric flask and make up to volume with methanol. Filter the resulting solution with 0.45 µm syringe membrane filter.

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

System Suitability Test (SST)

6.4.1.2 Resolution between adjacent peaks of reference materials should be more than 1.0.

6.4.1.3 The Tailing factor of peaks must be less than 2.0.

6.4.1.4 The Theoretical plate must be more than 1500.

Internal Quality Control (IQC)

6.4.3.1 Inject 10 µL of mix standard solution after every 10 samples & at the end of analysis.

6.4.3.2 Acceptance criteria for Internal Quality Check (IQC) :

The percentage difference of retention time for mix standard solution should not be more than 3 % for every batch of 10 samples.

Interpretation

Compare the retention time (RT) obtained from sample solution and reference standard solutions.

A steroid compound in the sample solution is positively identified when:

6.5.2.1 The retention time is similar to retention time obtained from reference standard solution.

6.5.2.2 Peak area of the compound is above than peak area of the LOD concentration (0.05 mg/mL).

Peak purity index close to 1.0.

Note: The similarity of spectrum maybe reviewed as part of interpretation of result. The spectrum of compound of interest in sample solution is identical to the spectrum of working standard with similarity of more than 0.95 to the spectrum of standard.