PKKK/300/UP/032 Identifikasi Dopamine dalam produk tradisional menggunakan HPLC

Materials and Equipment

Instrument:

High Performance Liquid Chromatography with photodiode array (PDA) detector

Analytical & microbalance

Column: ZORBAX Eclipse Plus C18 or equivalent

Reference standards: Primary or secondary RS or equivalent

Dopamine

Chemicals:

Methanol

Potassium dihydrogen phosphate

Phosphoric acid

Deionized water

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

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

Preparation of Standard Solution, Working Standard and LOD solution

Standard solution

Pipette 60 µL of standard stock solution into 1.5 mL vial and add 1440 µL of water. Mix well. Concentration of working standard solution is 0.02 mg/ml (20 ppm).

Pipette 12 µL of standard stock solution into 1.5 mL vial and add 1488 µL of deionized water. Mix well. Concentration of LOD solution is 0.004 mg/ml (4 ppm).

Use diluent, deionized water as blank.

Preparation of sample

Sample handling (sampling)

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

Sample preparation for analysis

For solid sample: Weigh 1 gm of the sample in a beaker and dissolve with 20 mL of mobile phase.

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 dopamine.

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.

Working standard solution 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 (for SST) and 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 3 for an example of the sequence table:

Table 3: Sequence injection for identification of dopamine in HPLC

LOD solution must be injected at the end of the analysis.

System suitability test requirement

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

Tailing factor not more than (NMT) 2.0.

Theoretical plate (N) > 2000.

Internal Quality Control (IQC)

The percentage difference of retention time for working 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 parameter 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 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