PKKK/300/UP/026 Identifikasi Anti-hipertensi dalam produk tradisional menggunakan HPLC

Materials and Equipments

Instrument:

High Performance Liquid Chromatography with photodiode array (PDA) detector

Analytical & microbalance

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

Reference standards: Primary or secondary RS or equivalent

Captopril

Methyldopa

Atenolol

Metoprolol

Labetalol

Nifedipine

Prazosin

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)

Amber Volumetric Flasks (Grade A)

Nylon syringe filter 0.45 µm

Disposable syringe without needle (5 ml)

2 ml amber glass screw cap vial

Preparation of Mobile Phase

Buffer preparation:

To prepare sodium dihydrogen orthophosphate dihydrate (25mM) buffer pH 3.0, dissolve 3.9 g of sodium dihydrogen orthophosphate dihydrate in 1L of deionized water.

Measure 1 L of acetonitrile and transfer in 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

Combine mix working standard solution by transfer 0.8 ml of captopril stock solution and 0.4 ml of each of the other reference standard solutions into one amber volumetric flask (10 ml) and make up to volume with methanol. Mix well.

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 0.5 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-hypertensives are given in Table 1 below:

Table 1: HPLC system used for identification of anti-hypertensive

Mobile Phase Method

Solvent gradient program for mobile phase is use in identification of anti-hypertensives 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 anti-hypertensive in HPLC

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

System Suitability Test (SST) requirement

System suitability test needs 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.

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-hypertensives 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 RS

IQC table