PKKK/300/UP/029 Identifikasi PDE-5 dalam produk tradisional menggunakan LCMS-MS

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

Micropipette 100-1000 µl

Column: Phenomenex Kinetex XB-C18 1.7µm, 150 x 2.1 mm or equivalent

Reference standards:

Aminotadalafil

Desmethylcarbodenafil

Dimethylsildenafil

Homosildenafil

Hydroxythiohomosildenafil

N-Desmethyl Acetildenafil

Noracetildenafil

Norneosildenafil

Sildenafil Citrate

Sildenafil N-Oxide

Tadalafil

Thiodimethylsildenafil

Thiohomosildenafil

Thiosildenafil

Vardenafil

Yohimbine

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)

Amber Volumetric Flasks (Grade A)

Pipette tips

Disposable pipette

Spatula

Nylon syringe filter 0.22 µm

Disposable syringe without needle (5 ml)

Preparation of Mobile Phase

Measure 900 ml of acetonitrile and transfer in solvent bottle.

Mobile Phase B

Measure 900 ml of deionized water and transfer in solvent bottle.

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

If ready-made mobile phase of deionised water with 0.1% Formic Acid and acetonitrile with 0.1% Formic Acid are available, skip steps 6.2.1 to 6.2.3. Transfer ready solvent into solvent bottle and proceed to step 6.2.4.

Preparation of Reference Standard

Multiple Standard Stock Solution

Individual Stock Standard (250 µg/ml)

Mixed Stock A (10 µg/ml)

Pipette 800 µl of each stock standard into 20 ml amber volumetric flask. Make up to mark with methanol and vortex to dissolve. Final concentration for mix stock A is 10 µg/ml (10ppm).

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 (1ppm).

Mixed Working Standard (WS, 500 ng/ml)

Pipette 750 µl of Mixed Stock B and 750 µl of methanol into a 1.5 ml amber vial. Vortex to dissolve. Final concentration of mixed working standard is 500 ng/ml (500ppb).

Limit of detection (LOD, 150 ng/ml)

Pipette 225 µl of Mixed Stock B and 1275 µl of methanol into a 1.5 ml amber vial. Vortex to dissolve. Final concentration of LOD is 150 ng/ml (150ppb).

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).

Sample preparation for Capsule, Tablet, Liquid, Powder

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

If further dilution of the sample is necessary, refer to step 6.4.4 iii b).

Pipette 10 ml of sample into a 50 ml beaker and add accurately 10 ml of methanol. Vortex to mix the solution thoroughly.

If the analysis result indicates oversaturation of the sample, dilution is required.

The extend of dilution depends on the sample’s initial results. Prepare multiple dilutions to determine the most suitable concentration for analysis.

Example of 1:100 dilution procedure:

Pipette 1000 µl of prepared sample solution from beaker into a 10 ml volumetric flask (Volumetric Flask A). Mark up with methanol and sonicate for 15 minutes (1:10 dilution).

Pipette 1000 µl of the solution from Volumetric Flask A into another 10 ml volumetric flask (Volumetric Flask B). Mark up with methanol and sonicate for another 15 minutes (1:100 dilution).

Experimental Method

To optimize the usage of reference standards, the analyst shall perform an initial LC/MS-MS trial run of the sample without injecting the 17 PDE-5 reference standards. This preliminary screening is intended only to provide an early indication of the possible adulterant(s) present. Based on the observed compound(s), only the relevant PDE-5 reference standard(s) will be prepared as described in Paragraph 6.3.2 and injected during the actual analysis.

In the event that the preliminary screening does not indicate the presence of any of the 17 PDE-5 compounds, the analyst shall prepare only the mixed standard solution containing the main PDE-5 compounds (namely sildenafil, tadalafil and vardenafil), or prepare the appropriate standard(s) based on the client’s request, together with the sample for the actual analysis.

The chromatogram generated from this trial run is for internal screening purposes only and will not be included in the analysis report.

Table 1: Liquid chromatography (LC) parameters used for detection of PDE-5 inhibitors

Mobile Phase Method

Gradient program for mobile phase use for identification of phosphodiesterase-5 inhibitors (PDE-5) in traditional products shown as below:

Table 2: Gradient program for mobile phase used for identification of PDE-5 inhibitors in traditional products

Mass spectrometry (MS) parameter used for identification of phosphodiesterase-5 inhibitors (PDE-5) in traditional product are given in table below:

Table 3: Mass spectrometry (MS) parameter used for identification of PDE-5 inhibitors

MRM parameter used for detection of PDE-5 inhibitor in traditional products are given in table below.

Table 4: MRM parameter for identification of PDE-5 inhibitors

Sequence of Injection

Table 5: Sequence of injection for identification of PDE-5 inhibitors

System Suitability Test (SST) Requirement

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 (500 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 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 is based on the detection of target and reference ions (refer to m/z setting under Table 4). Reference ions are monitored as relative intensity. The maximum permitted tolerance for reference ion intensity is as follow:

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

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

C. Relative Ion Intensity (% of base peak) 10-20: Tolerance ± 30%

The test result is reported as NOT DETECTED in sample when the peak area in the sample solution is below the peak area of the standard solution at the LOD level of 150 ng/ml (equivalent to 30 ppm in sample).

Analysis report

Each sample will have one analysis report. The report must include the following details:

Sample details (Sample name, sample number and batch number)

Analysis date

Equipment parameters including equipment name, mobile phase program and LCMS system.

List and preparation procedures for the reference standard (RS) used in analysis.

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

Interpretation of result (Result: Detected / Not Detected)

Evidence of analysis must be attached to the report, including:

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

Chromatogram of LOD on each standard compound and its results.

Chromatogram of the 1st and 2nd injection of the sample (with information on sample name, sample number, analysis method, and the date and time of analysis) overlaid with individual standards and LOD.

Relative intensity of individual compounds in a sample.

IQC table