PKKK/300/UP/021 Identifikasi Steroid dalam Produk tradisional menggunakan HPLC

Materials and Equipment

Instrument:

High Performance Liquid Chromatography with photodiode array (PDA) detector

Analytical & microbalance

Column: Kinetex Phenyl-Hexyl 2.6µm, 150 x 4.6 mm or equivalent

Reference standards: Primary or secondary RS or equivalent

Prednisolone

Prednisone

Betamethasone

Dexamethasone

Triamcinolone acetonide

Hydrocortisone acetate

Cortisone acetate

Betamethasone-17-valerate

Chemicals:

Ethanol

Acetonitrile

Deionized water

Ammonia

Methanol

Glassware and consumables:

Beakers (50 mL)

Solvent filter system

Nylon solvent filter 0.45 µm

Solvent bottle (1 L)

Nylon syringe filter 0.45 µm

Volumetric Flasks (Grade A)

Disposable syringe without needle (5 mL)

2 mL glass screw cap vial

Preparation of Mobile Phase

Measure 1 L of water and transfer in a solvent bottle.

Repeat step i. for acetonitrile.

Degassed filtered mobile phase at least for 20 minutes to ensure no bubble present.

Preparation of Reference Standard

Individual standard stock solution

6.3.3.1 Prepare a single standard stock solution following the same procedure outlined in 6.3.1 to 6.3.2, but using a single standard instead of multiple standards.

6.3.3.2 This procedure is applicable for screening selected compounds only

Preparation of sample

Sample handling (sampling)

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

Sample preparation for analysis

Sample preparation differ between dosage forms. Please refer to document PKKK/300/UP/013 (Pengekstrakan Fasa Pepejal Bagi Produk Tradisional, Suplemen Kesihatan dan Kosmetik) and PKKK/300/UP/014 (Pengekstrakan Fasa Cecair Bagi Produk Tradisional, Suplemen Kesihatan dan Kosmetik). Use LLE only if SPE is not feasible.

Sample pH is adjusted to pH7 before the extraction process is performed.

Experimental Method

Table 1: HPLC system used for identification of steroids.

Mobile Phase Method

Solvent gradient program for mobile phase is use in identification of steroids shown as below:

Table 2: Solvent gradient program for identification of steroids.

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

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 a mix working standard solution six times (SST) before injecting the sample.

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

A blank or diluent must be injected before the next sample injection. Refer to Table 3 for an example of the sequence table:

Table 3: Sequence injection for identification of steroid in HPLC

System suitability test requirement

System suitability tests need to be performed for each batch run to ensure the entire system is working properly. Perform at least six replicate injections of mix working standard solution (0.05 mg/mL).

Acceptance criteria:

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

Tailing factor not more than (NMT) 2.0.

Theoretical plate not less than (NLT) 2000.

Internal Quality Control (IQC)

The percentage difference of retention time for a mixed standard 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 steroid 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 the sample is sequenced at 6 or more, a chromatogram of standard before sample must be presented.

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

IQC table