PKKK/300/UP/030 Identifikasi dan Kandungan Menthol, Camphor, Methyl Salicylate dan Thymol dalam produk tradisional menggunakan GCMS

Instrument: Gas Chromatography Mass Spectrometer (GCMS)

Column: BP-624, 6% Cyanopropylphenyl 94% Dimethylpolysiloxane, with dimension of 30 m x 0.25 mm and particle size of 1.4 µm or equivalent.

Analytical balance and microbalance

Sonicator

Reference standard: Primary or secondary RS of equivalent.

Menthol

Camphor

Methyl Salicylate

Thymol

Eugenol

Chemical:

Methanol

Glassware and consumables:

Conical flask

Amber volumetric flask

Disposable syringe without needle, 5 ml

Nylon solvent filter, 0.45µm

Amber glass screw cap vial, 2 ml

Amber glass screw cap vial, 50 ml

Liquid form: Pipette 10 µl of eugenol reference standard into a 25 ml amber volumetric flask. Dissolve and dilute to volume with methanol. Filter the solution through a 0.45 µm nylon syringe filter into an amber vial.

Powder form: Weigh 10 mg of eugenol reference standard into a 25 ml amber volumetric flask. Dissolve and dilute to volume with methanol. Filter the solution through a 0.45 µm nylon syringe filter into an amber vial.

For methyl salicylate: Pipette 7 µl of methyl salicylate into a 20 mL amber volumetric flask and repeat steps ii and iii.

Preparation of Mixed Working Standard Solution (0.1 mg/ml or 100 µg/ml).

Pipette 12.5 ml of each standard stock solution into an amber 50 ml vial.

Refer to the table below for preparation details:

Table 1: Preparation of mix working standard solution for calibration curve.

Option to prepare mix working standard solution for calibration in 5 mL volumetric flask. The preparation as in the table below:

Table 2: Preparation of mix working standard solution for calibration curve.

For system suitability test (SST), use the Level 3 concentration (20 µg/ml of each compound) as the system suitability solution.

Pipette 50 µl of mixed working standard solution into 5 ml volumetric flask and add 1000 µl of internal standard (IS) solution.

Use the Level 1 concentration (3 µg/ml of each compound) as the LOQ solution.

Patch / Medicated Plaster

Cut the sample into small pieces.

Cool the solution to room temperature.

Sampling the sample according to PKKK/300/UP/001.

Cool the solution to room temperature.

Perform appropriate dilutions if the sample concentration exceeds the linear range of the calibration curve.

Document the step-by-step dilution preparation in the worksheet.

The GCMS system used for detection of menthol, camphor, methyl salicylate and thymol in traditional product are given in table 2 below.

Table 3: GCMS system for identification and assay of menthol, camphor, methyl salicylate and thymol

Table 4: Oven temperature program for identification and assay of menthol, camphor, methyl salicylate and thymol

Table 5: SIM mode for identification and assay of menthol, camphor, methyl salicylate, and thymol

(*) Remarks: The data acquisition time windows will be adjusted according to the observed retention times of target analytes to ensure comprehensive peak capture and optimal instrumental efficiency.

For each injection, a volume of 0.5 µl of the solution will be administered. The sequence of injection is shown in the table below.

Table 6: Sequence of injection for identification and assay of menthol, camphor, methyl salicylate and thymol

The acceptance criteria for SST, LOD and LOQ are shown in the table below.

Table 7: Requirement for SST, LOD and LOQ of identification and assay of menthol, camphor, methyl salicylate and thymol

Standard injection and calibration curve

Generate a calibration curve by plotting concentration against area, and determine the linearity equation along with the correlation coefficient (R2).

This step (ii) can be performed using the instrument’s software (GCMSsolution).

Ensure that the correlation coefficient (R²) is equal to or greater than 0.995. If the correlation does not meet this requirement, re-inject the working standard solution until the desired criteria are met.

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.

Identification

The identification of the target compound is considered positive when the following criteria are met:

Retention time

The retention time of the target compound in the sample chromatogram corresponds to the retention time in the chromatogram of the working standard.

Mass Spectrum Confirmation

Confirmation is based on the detection of the target ion and reference ions.

Reference ions are monitored as relative ion intensity, with the maximum permissible tolerance defined in Table 8:

Table 8: Tolerance for reference ion intensity for identification of menthol, camphor, methyl salicylate and thymol

Reporting Results

NOT DETECTED: The compound is reported as NOT DETECTED when the peak area ratio of the compound to the internal standard (IS) in the sample solution is below the peak area ratio of the standard solution at the limit of detection (LOD, 1 µg/mL).

BELOW QUANTITATION LIMIT: The compound is reported as BELOW QUANTITATION LIMIT (3 µg/mL) when the peak area ratio of the compound to IS in the sample solution is below the peak area ratio of the standard solution at the limit of quantitation (LOQ, 3 µg/mL)

Quantifiable Amounts: If the peak area ratio of the compound to IS in the sample solution exceeds the peak area ratio of the standard solution at the LOQ level, the compound's concentration must be calculated using the calibration curve extrapolation as outlined in section B. Assay.

Assay

The amounts of menthol, camphor, methyl salicylate, and thymol are determined using the extrapolation of the calibration curve.

Therefore, the final amount of each compound in the sample is calculated and reported as percent weight / weight (% w/w).

This calculation in step (ii), is performed using the following formula:

Equation 1: Calculation for the amount of menthol, camphor, methyl salicylate and thymol

Limits or specifications

Menthol and Camphor

The limits for menthol and camphor are defined according to the Drug Registration Guidance Document (DRGD), Third Edition, Eight Revision, July 2024.

The limits are outlined in Table 9:

Table 9: Limits or specifications for the assay of menthol and camphor

Methyl Salicylate and Thymol

The DRGD does not specify limits or guidelines for methyl salicylate and thymol.

If the amounts of these compounds need to be reported, they will be presented as the measured values.

Each sample must have a dedicated analysis report, which includes the following details:

Sample Details

Sample name

Sample number

Batch number

Analysis Date

Equipment Parameters

Equipment name

Gas chromatographic (GC) conditions

Mass spectrometry (MS) conditions.

Reference Standard (RS)

List of RS used in the analysis

Preparation procedure for RS, including printed RS weight

Sample Preparation

Sample preparation procedure

Printed sample weight (duplicate weighing)

Interpretation of Results

Detailed interpretation based on criteria outlined in the SOP

Evidence of Analysis

Evidence must be attached to the analysis report, including:

Compilation of chromatograms for calibration curve solutions

Chromatogram for LOD and LOQ solutions

Chromatogram of working standard (first injection of SST)

Compilation of chromatograms for SST

Chromatogram for blank solution prior to and after the sample solution injection

Chromatograms for samples A and B

IQC table