PKKK/300/UP/024 Identifikasi Diuretik dalam produk tradisional menggunakan HPLC

Materials and Equipments

High Performance Liquid Chromatography with photodiode array (PDA) detector

pH Meter

Reference standards: Primary or secondary RS or equivalent

Spironolactone

Furosemide

Hydrochlorothiazide

Amiloride Hydrochloride

Sodium Dihydrogen Phosphate

Deionized water

Phosphoric Acid

Acetonitrile

Methanol

Beakers (50 ml)

Spatula

Solvent filter system

Nylon solvent filter 0.45 µm

Solvent bottle (2 L and 1L)

Amber Volumetric Flasks (20 ml)

Disposable syringe without needle (5 ml)

2 ml glass screw cap vial

Preparation of Mobile Phase

Measure 1 L of acetonitrile.

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

Preparation of Reference Standard

Individual standard stock solution

Concentration of each standard stock: 0.5 mg/ml (500ppm)

Pipette 0.8 ml of each standard stock solution into 20 ml amber volumetric flask. Dilute to volume with methanol and mix well.

Concentration of each standard in mixed solution: 0.02 mg/ml (20 ppm)

Pipette 1.25 ml of mix working standard solution into 5 ml amber volumetric flask. Dilute to volume with Methanol 0.005 mg/ml (5 ppm).

6.3.4.1 Prepare a single standard stock solution following the same procedure outlined in 6.3.1 to 6.3.3, but using a single standard instead of multiple standards.

6.3.4.2 This procedure is applicable for screening selected compounds only

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 10 ml of the sample in a beaker and dissolve with 10 ml methanol.

Experimental Method

The HPLC system used for identification of anti-diuretic are given in Table 1 below.

Table 1: HPLC system used for identification of diuretic

Mobile Phase Method

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

Table 2: Gradient program for mobile phase

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

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 mix 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 diuretics 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 (0.02 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 anti-diabetic 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)

If sample sequence is more than 5 samples, a standard chromatogram will be injected again after 5 sample injections set and presented along with report with the nearest standard chromatogram injection.

Overlay spectrum of sample that have similar retention time with RS.

IQC table