PKKK/300/UP/018 Pengesanan Bahan Kawalan dan Terlarang dalam produk Tradisional menggunakan GCMS

Materials and Equipments

Instrument:

Gas Chromatography with single quadrupole Mass Spectrometry (EI)

Column:

HP 5MS 0.25µ, 30 m x 0.25 mm or equivalent

Reference standards:

Primary or secondary RS or equivalent (Refer Appendix A)

Chemical:

Ethanol

Glasswares and consumables:

Beakers

Volumetric Flasks (Grade A)

Nylon syringe filter 0.45 µm

Disposable syringe without needle (5 mL)

2 mL glass screw cap vial

Working standard solution

Sample handling (sampling)

Sampling processes differ between different types of dosage form. Please refer to the document PKKK/300/UP/001 (Proses Persampelan).

Sample preparation for analysis

The sample preparation process varies depending on the compound of interest. Three main methods are applied: Solid-Phase Extraction (SPE), Liquid-Liquid Extraction (LLE), and direct dissolved with ethanol.

When samples require pH adjustment prior to analysis, either Solid-Phase Extraction (SPE) or Liquid-Liquid Extraction (LLE) may be employed. Detailed procedures for both techniques can be found in documents PKKK/300/UP/013 and PKKK/300/UP/014.

For preparing samples by direct dissolving in Ethanol, the procedure is as follows:

Weight 1 gram of the sample (for dosage form such as capsule, powder, pill and cream) or measure 10 mL of the sample (for liquid dosage forms) into a beaker.

Experimental Method

The GCMS system used for detection of controlled and prohibited compounds in traditional products are given in Table 1 below.

Table 1: GCMS system for detection of controlled and prohibited compound

Column Temperature Program

Method apply column temperature program for detection of prohibited and restricted compound in traditional product:

Table 2: Column Temperature Program

Refer to document PKKK/300/UP/004 (GCMS Agilent 7890/5975C), PKKK/300/UP/010 (GCMS Shimadzu QP2010 Ultra), PKKK/300/UP/017 (GCMS Agilent 8890/5977B) on how to set-up temperature program and GCMS system.

System Suitability Test by Tuning Evaluation and System Verification

Conduct daily tuning using the system's built-in software to ensure the GCMS is performing optimally and capable of delivering accurate and reliable results.

The generated tuning report must meet predefined criteria for the GCMS model before proceeding with sample analysis.

Table 3: Tuning Parameters and Acceptance Criteria for GCMS Systems (Agilent).

Table 4: Tuning Parameters and Acceptance Criteria for GCMS Systems (Shimadzu)

For qualitative screening with Agilent GCMS Systems, configure the instrument using Standard Spectra Tune rather than Autotune. This preserves standard ion-ratio targets, ensuring consistent fragmentation patterns that are crucial for reliable spectral library matching.

For routine system performance verification, the Tune Evaluation function shall be performed daily (or as specified). This function is accessed in the instrument software by selecting Tune > Tune Evaluation.

Tune Evaluation automatically turns on the MS and collects the required data to generate a detailed verification report.

The report provides an automatic review and evaluation of key tune parameters, including: Mass Axis Calibration, Isotope Ratios, Precursor’s Presence, Air and Water Levels, and Multiplier Voltage. The acceptance criteria for evaluation remain consistent, irrespective of whether the system is evaluating a Standard Spectra Tune or an Autotune.

Standard Injection

Method verification is required when a compound of interest has not been tested (e.g. in a new instrument). Verification is performed by injecting a working standard solution once as assurance that the method is able to detect compounds with minimum library match at 80% in similarity. Repeat the process if library matching is less than minimum requirement or prepare a new working standard (by altering working standard concentration depending on peak intensity) if the problem is not resolved.

In the confirmation test (2nd analysis), a working standard solution is injected together during sample analysis (to ensure the system is at optimal condition with minimum library matching at 80%).

Sample Injection

The sample contains compounds if the retention time matched with reference standard and mass spectrum of the sample has minimum similarity matching with the library at 80%. No carry over from the previous sample is ensured by checking the chromatogram of diluent before sample injection (absent of compound detected).

Identification of Steroids by GCMS

Identification of Steroids by GCMS in Multi-Component Samples

Individual Analysis of Sample

If a received sample consists of different components or colours (e.g. a green capsule and a red capsule) and is requested for analysis using this GCMS screening method, each component shall be analysed individually. Sample extraction may utilize Solid-Phase Extraction (SPE) at varying pH levels (pH 3, 7, or 11), or by Direct Dissolve Ethanol, depending on the target analyte profile (e.g., SPE pH 3 for initial NSAID screening).

Re-analysis for Positive Screening Results

If one of the analysed sample components (e.g. the green capsule) detects the presence of a steroid compound (e.g. Dexamethasone) via the GCMS screening method, the second analyst is mandated to repeat the confirmatory analysis for that specific component. The repeat analysis must use the exact same extraction technique (e.g. SPE pH3) and test method as utilized by the first analyst.

Follow-up Action for Negative Screening Results

For any sample component that does not detect the target compound(s) via the initial GCMS screening method (e.g., the red capsule), or where the detected compound (e.g., Dexamethasone detected in SPE pH 3) is known to yield a LODcleaner, more definitive result using a different established extraction (e.g., SPE pH 7), that component shall then proceed to analysis using the specific steroid HPLC method (Method Reference: PKKK/300/UP/047), utilizing the SPE pH 7 extraction procedure.

This step is to ensure comprehensive screening, as the GCMS method may miss polar steroids or those less efficiently extracted at the initial screening pH (e.g., pH 3, optimized for NSAIDs). Switching to the HPLC method with the optimized extraction (SPE pH 7) provides the necessary sensitivity and selectivity for conclusive steroid detection.

Determination of Limit of Detection (LOD)

Limit of Detection (LOD) for a compound is determined using peak-to-peak (PtP) Signal-to-Noise Ratio (S/N) analysis.

Analyze the prepared solution to generate a PtP profile of the compound.

Calculate the Signal-to-Noise Ratio (S/N) from the PtP data.

LOD Calculation

The LOD concentration is calculated to achieve a minimum PtP S/N ratio of 3:1 using the formula:

LOD Concentration =

Example: For a 1 mg/mL (equivalent to 1000 ppm) Chlorpheniramine standard with a PtP S/N of 3924.65:

LOD Concentration =

= 0.76 ppm

Verification of LOD

If the PtP S/N ratio is less than 3, prepare a higher concentration and repeat the injection until the PtP S/N is at least 3.

This procedure provides a temporary LOD concentration for use in the confirmation test (2nd analysis) until method validation is completed.

Upon completion of method validation, the validated LOD will replace the temporary LOD and a new procedure will be developed accordingly.

Mass spectrum observation

To observe and check mass spectrum of the sample and reference standard, refer to document PKKK/300/UP/004 (GCMS Agilent 7890/5975C), PKKK/300/UP/010 (GCMS Shimadzu QP2010 Ultra), PKKK/300/UP/017 (GCMS Agilent 8890/5977B).

Injection Sequence

Injection of diluent (ethanol) must be performed before and after sample injection. Refer to Table 5 for example of sequence table:

Table 5: Injection sequence for GCMS analysis

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, GCMS system and name of method file.

List and preparation procedures of reference standard use in analysis.

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

Interpretation of result (Detected, Not Detected or suspected of specific compound),

utilizing the initial sample analysis as the primary basis for reporting.

Evidence of analysis must be attached together in analysis report:

Tuning 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).

Library matching for mass spectrum of sample that have similar retention time with RS (contain information of similarity index and comparison of mass spectrum with RS)