PKKK/300/UP/060 Screening of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) in Cosmetic Products Using HPLC

High Pressure Liquid Chromatograph with photo diode array detector

Ultrasonic bath

Zorbax Eclipse Plus C18;150 mm x 4.6 mm i.d., 5 µm

Micropipette

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

10 mL, 50 mL Volumetric flasks

50 mL glass stoppered conical flask

Acetonitrile (HPLC grade)

Methanol (HPLC grade)

Sodium Dihydrogen Phosphate Dihydrate (NaH2PO4.2H2O)

Water (Ultrapure water, 18 mega ohm)

Orthophosphoric acid 85% (H3PO4)

Reference material: Aspirin, Ibuprofen, Diclofenac Sodium, Mefenamic Acid, Naproxen, Paracetamol, Piroxicam, Sulindac, Ketoprofen, Indomethacin, Phenylbutazone

Chromatographic Parameters

Test Method

Mobile Phase Preparation

6.3.1.1Mobile Phase A (Sodium Dihydrogen Phosphate Buffer): Dissolve 3.9 g NaH2PO4.2H2O in 1000 mL of water. Mix well and adjust to pH 3.2 with Orthophosphoric acid. Filter and degassed before use.

6.3.1.2 Mobile Phase B: Filter and degassed 1000 mL Acetonitrile.

Reference Standard Stock Solution Preparation

6.3.2.1 Weigh separately 2 mg of Aspirin, Ibuprofen, Diclofenac Sodium, Mefenamic Acid, Naproxen, Paracetamol, Piroxicam, Sulindac, Ketoprofen, Indomethacin, Phenylbutazone Reference Standard into 10 mL volumetric flask. Add 5 mL of Methanol and sonicate for 20 minutes. Make up to volume with Methanol (0.2 mg/mL / 200 ppm).

Pipette 3.75mL of Indomethacin standard stock solution and Naproxen standard stock solution into a 50 mL volumetric flask and make up to volume with Methanol. (0.015 mg/mL / 15 ppm).

Pipette 3.75 mL of the remaining Reference Standard Stock Solution (except for Indomethacin and Naproxen) into a 50 mL flask and make up to volume with Methanol (0.015 mg/mL / 15 ppm).

Limit of Detection (LOD) Solution Preparation

Limit of Detection (LOD) Solution Preparation (LOD-Mix Standard A2)

Pipette 300 µL of Mix Standard A1 solution into a vial and add 1200 µL of Methanol (0.003 mg/mL / 3 ppm).

6.3.4.2 Limit of Detection (LOD) Solution Preparation (LOD-Mix Standard B2)

Pipette 300 µL of Mix Standard B1 Solution into a vial and add 1200 µL of Methanol. (0.003mg/mL / 3ppm).

Note:

The weight of the reference standard can be modified to minimize the usage of the reference standard.

Sample Preparation

6.3.5.1 Weigh approximately 0.5 g of sample into a 50 mL glass stoppered conical flask. Add 20 mL of Methanol. Sonicate for 10 minutes. Filter the solution with a 0.45 µm nylon filter prior to injecting into the HPLC.

Note: The weight of the sample can be modified due to the availability and amount of the sample received.

System Suitability Test (SST)

ii) Theoretical plates NLT 1500

iii) Resolution NLT 1.5

Injection Sequence

Results Interpretation

Aspirin, Ibuprofen, Diclofenac Sodium, Mefenamic Acid & Naproxen are analysed and interpreted at 220 nm.

Paracetamol, Piroxicam, Sulindac, Ketoprofen, Indomethacin and Phenylbutazone are analysed and interpreted at 254 nm.

Compare the Retention Time, Area and Spectrum obtained from the sample chromatogram against the LOD-Mix Standard Solution (A2/B2) chromatogram. A compound is reported as detected in the sample if:

Retention time corresponds to standard with difference less than +/- 1%

The sample peak area is more than LOD-Mix Standard (A2/B2)

The spectrum of the sample with a similarity of more than 0.999 to the spectrum of standard

The analysis shall be repeated by a second analyst using the same method when the sample was detected with NSAIDs in the first analysis.

Result documented in Certificate of Analysis:

If NSAIDs not detected : ‘Tidak dikesan’

If NSAIDs detected : ‘Dikesan’