PKKK/300/UP/027 Identifikasi Domperidone dalam produk tradisional menggunakan HPLC

Materials and Equipments

Instrument:

High Performance Liquid Chromatography with photodiode array (PDA) detector

Analytical & microbalance

Column: Zorbax Eclipse Plus C18 5µm, 150 mm x 4.6 mm or equivalent

Reference standards: Primary or secondary RS or equivalent

Domperidone

Chemicals:

Methanol

Acetonitrile

Deionized water

Sodium dihydrogen orthophosphate

Glasswares and consumables:

Beakers (50 ml)

Solvent filter system

Nylon solvent filter 0.45 µm

Solvent bottle (1 L)

Volumetric Flasks (Grade A)

Nylon syringe filter 0.45 µm

Disposable syringe without needle (5 ml)

2 ml glass screw cap vial

Preparation of Mobile Phase

Buffer preparation:

To prepare sodium dihydrogen phosphate buffer pH 3.2, dissolve 3.9 g of sodium dihydrogen orthophosphate dihydrate in 1L of deionized water.

Measure 1 L of acetonitrile and transfer into another solvent bottle.

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

Preparation of Reference Standard

Individual standard stock solution

Working standard solution (0.015 mg/ml)

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 methanol.

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

Experimental Method

The HPLC system used for identification of domperidone are given in Table 1 below:

Table 1: HPLC system used for identification of domperidone

Mobile Phase Method

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

Table 2: Gradient program for mobile phase

Refer to document PKKK/300/UP/004 (RRLC 1200 Series), PKKK/300/UP/012 (HPLC Prominence-i) and PKKK/300/UP/017 (HPLC Flexar) on how to set-up method and HPLC system.

Standard Injection

Working standard solution must be injected at the beginning of analysis for six times to obtain system suitability test (SST). Repeat the injection or prepare new mix standard solution if the chromatogram of RS 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).

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: Injection sequence for identification of anti-hypertensive in HPLC

LOD 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 mix working standard solution (0.015 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 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 parameter including equipment name, mobile phase program and HPLC system

List and preparation procedures of RS use in analysis

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

Interpretation of result (Detected, Not Detected or suspected of specific Domperidone).

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 RS

IQC table