PKKK/300/UP/035 Identifikasi Tretinoin dalam produk tradisional menggunakan HPLC

Materials and Equipment

Instrument:

High Performance Liquid Chromatography with photodiode array (PDA) detector

Analytical & microbalance

Column: Zorbax SB-C18 5 µm, 250 x 4.6 mm or equivalent

Reference standards: Primary or secondary RS or equivalent

Tretinoin

Chemicals:

Methanol (HPLC Grade)

Glacial acetic acid

Deionized water

Glassware and consumables:

Erlenmeyer flask (50 mL)

Amber coloured volumetric flask (20 mL)

Amber coloured vial (2 mL)

Solvent filter system

Solvent bottle (1 L)

Disposable syringe without needle (5 mL)

Nylon solvent filter 0.45 µm

Preparation of Mobile Phase

Accurately measure 846 mL of methanol and transfer into a clean 1 L solvent bottle.

Degas the filtered mobile phase at least for 20 minutes to ensure the removal of air bubbles.

Preparation of Standard Solution and Working Standard (WS) Solution

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.

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.

6.3.3. LOD solution preparation: (0.002 mg/ml) of tretinoin in methanol.

Sample handling (sampling)

Sampling processes differ between different types of dosage form. Please refer to the document PKKK/300/UP/001 (Proses Persampelan).

Sample preparation for analysis

For solid sample: Weigh about 0.5 g of sample into a 50 mL Erlenmeyer flask. Add 20 mL of methanol to the flask.

For liquid sample: Measure 10 mL of the sample in a beaker and dissolve with 10 mL methanol.

Experimental Method

Table 1: HPLC system used for identification of Tretinoin.

Refer to document PKKK/300/UP/003 (RRLC 1200 Series), PKKK/300/UP/011 (HPLC Prominence-i) and PKKK/300/UP/016 (HPLC Flexar) on how to set-up method and HPLC system.

Standard Injection and System Suitability

Working standard solution must be injected at the beginning of analysis (six injections). Repeat the injection or prepare new working standard solution if the chromatogram of reference standard is not well separated or peak is not sharp.

Sample Injection

Retention time and UV spectrum observation

To observe and check retention time and UV spectrum of the sample and RS, refer to document PKKK/300/UP/003 (RRLC 1200 Series), PKKK/300/UP/011 (HPLC Prominence-i) and PKKK/300/UP/016 (HPLC Flexar).

Injection Sequence

First, inject a blank or diluent, then inject working standard solution six times (SST). Inject the LOD solution before injecting the sample.

If more than five samples are injected in a single analysis, the LOD solution must be injected once every five samples.

A blank or diluent must be injected before the next sample injection. Refer to Table 2 for an example of the sequence table:

Table 2: Sequence injection for identification of Tretinoin in HPLC

Working standard solution must be injected at the end of the analysis.

System suitability test requirement

System suitability test need to be performed for each batch run to ensure the entire system is working properly. Perform at least six replicate injections of working standard solution (0.005 mg/mL).

Acceptance criteria:

%RSD for retention time: Not more than, NMT 2.0%

Tailing factor not more than (NMT) 2.0.

Internal Quality Control (IQC)

The percentage difference of retention time for LOD solution should not be more than 3 % for every batch of 5 samples.

Acceptance criteria for test sample

Analysis report

Each sample will have one analysis report. Report must consist of detail as below:

Sample details (Sample name, sample number and batch number)

Analysis date

Equipment parameters including equipment name, mobile phase program and HPLC system.

List and preparation procedures of reference solution use in analysis.

Sample preparation procedure and printed sample weight (for solid sample only)

Interpretation of result (Detected, Not Detected or suspected of specific tretinoin compound)

Evidence of analysis must be attached together in analysis report:

Chromatogram of 1st and 2nd injection of sample (with minimum information of compound name, peak area and retention time).

Chromatogram of 1st and 2nd injection of standard (with minimum information of compound name, peak area and retention time). If sample is sequence at 6 or more, chromatogram of standard before sample must be presented.

Overlay spectrum of sample that have similar retention time with reference standard.

IQC table