PKKK/300/UP/023 Identifikasi Antifungal dalam produk tradisional menggunakan LCMS

Material and Equipment

Instrument:

Triple Quadrupole LCMS – 8045 (High Performance Liquid Chromatography Mass Spectrometer Liquid Chromatography with mass spectrometry) and heated ESI as interface.

Analytical balance & microbalance

Column: Phenomenex Kinetex XB-C18 1.7µm, 100 x 2.1 mm or equivalent

Reference standards:

Fluconazole

Ketonazole

Clotrimazole

Nystatin

Griseofulvin

Econazole

Miconazole

Itraconazole

Chemicals:

Methanol (LCMS grade)

Acetonitrile with 0.1% Formic Acid (LCMS grade)

Deionised water with 0.1% Formic Acid (LCMS Grade)

Deionized water

Formic acid

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

Measure 900 ml of acetonitrile and transfer in solvent bottle.

Repeat step 6.2(i) and 6.2(ii) for deionized water.

Degassed filtered mobile phase at least for 30 minutes to ensure no bubble is present.

Or used ready-made mobile phase of deionised water with 0.1% Formic Acid and acetonitrile with 0.1% Formic Acid, transfer into solvent bottle and proceed to step v.

Preparation of Reference Standard

Individual Stock Standard (250 µg/ml)

Mixed Stock A (10 µg/ml)

Pipette 200 µl of each stock standard into 5 ml amber volumetric flask. Make up to mark with methanol and vortex to dissolve. Final concentration for mix stock A is 10 µg/ml.

Mixed Stock B (1.0 µg/ml)

Pipette 500 µl of mixed stock A into 5 ml amber volumetric flask. Make up to mark with methanol and vortex to dissolve. Final concentration for mix stock B is 1.0 µg/ml.

Mixed Working Standard (WS, 100 ng/ml)

Pipette 150 µl of mixed stock B and 1350 µl of methanol into a 2 ml amber vial. Vortex to dissolve. Final concentration of mixed working standard is 100 ng/ml.

Limit of detection (LOD, 50 ng/ml)

Pipette 75 µl of mixed stock B and 1425 µl of methanol into a 2 ml amber vial. Vortex to dissolve. Final concentration of LOD is 50 ng/ml.

Single Standard Stock Solution

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 Ujian Penyaringan).

Sample preparation for capsule

By using disposable pipette, withdraw about 5 – 10 ml of clear supernatant layer to a syringe fitted with a 0.22 µm Nylon filter.

Pipette 0.1 ml of the above solution into 2 ml amber vial and dilute to 1 ml with methanol. Vortex to dissolve.

Warm it until the sample disperses in a 60°C water bath for 10 minutes.

By using a disposable pipette, withdraw about 5-10 ml of the sample solution to a syringe fitted with a 0.22 µm Nylon filter.

Pipette 0.5 ml of the above solution into a 2 ml amber vial and dilute to 1 ml with methanol. Vortex to dissolve.

Experimental Method

Table 1: Liquid chromatography (LC) parameters used for detection of Anti-fungal

Mobile Phase Method

Gradient program for mobile phase use for identification of anti-fungal in traditional products shown as below:

Table 2: Gradient program for mobile phase used for identification of anti-fungal in traditional products.

Mass spectrometry (MS) parameter used for identification of anti-fungal in traditional product are given in table below

Table 3: Mass spectrometry (MS) parameter used for identification of anti-fungal.

MRM parameter used for detection of anti-fungal in traditional product are given in table below.

Table 4: MRM parameter for identification of anti-fungal.

Sequence of Injection

Table 5: Sequence of injection for identification of anti-fungal.

System Suitability Test (SST) Requirement

6.5.6.1 System suitability test need to be performed for each batch run to ensure the entire system is working properly. Perform at least 6 replicate injection of working standard solution (100 ng/ml).

Acceptance criteria:

Tailing factor: Not more than, NMT 2.0.

Theoretical plate (N): Not less than, NLT 2000.

%RSD (retention time) for 6 injections of mix working standard solution: %RSD NMT 1.0%

S/N ratio of LOD solution: S/N > 3

Internal Quality Control (IQC)

Acceptance criteria for Internal Quality Control Check (IQC):

The percentage difference of retention time for LOD solution should not be more than 3 % for every batch of 5 samples.

Result Interpretation

Identification of target analyte is positive when.

The retention time of target compound(s) in the sample is similar to the working standard solution.

Confirmation of mass spectrum based on detection of the target (MRM 1) and reference ions (MRM 2). Reference ions are monitored as relative intensity is as the following:

Relative Ion Intensity (% of base peak) > 50: Tolerance ± 20%.

Relative Ion Intensity (% of base peak) 20 - 50: Tolerance ± 25%.

Testing result for each analyte is reported as NOT DETECTED in the sample when the peak area of a specific analyte in a sample solution is below the peak area of the standard solution at LOD level, 50 ng/ml (equivalent to 10 ppm in 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 LCMS system.

List and preparation procedures of RS use in analysis.

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

Interpretation of result (Result: Detected/Not Detected)

Evidence of analysis must be attached together in analysis report:

Chromatogram of System Suitability (SST) on each compound and results.

Chromatogram of LOD on each compound and results.

Chromatogram of 1st and 2nd injection of sample (with minimum information of sample name, sample number, analysis method, date and time of analysis, confirmation of target ion (MRM1) and reference ion (MRM2).

IQC table