Adulteration Screening Analysis Guides
Execution-ready operating procedures covering sample preparation, extraction protocols, chromatographic & spectrometric methods, SST verification criteria, batch sequences, and official reporting.
• Buffer 25 mM Na₂HPO₄ pH 7.6: Dissolve 3.55 g of Anhydrous di-Sodium Hydrogen Phosphate in 1000 mL Ultrapure water. Adjust pH to 7.6 using dilute Orthophosphoric acid (H₃PO₄). Filter through 0.45 µm nylon membrane filter and degas for 20 min.
• Primary Stock Standard (1.0 mg/mL): Accurately weigh 10.0 mg of Omeprazole and Lansoprazole RS into separate 10 mL volumetric flasks. Dissolve and make up to volume with Methanol.
• Mixed Working Standard (0.05 mg/mL each): Pipette 0.5 mL of Omeprazole and 0.5 mL of Lansoprazole stock solutions into a single 10 mL volumetric flask. Make up to volume with Methanol.
• LOD Solution (0.003 mg/mL): Pipette 0.6 mL of Mixed Working Standard into a 10 mL flask and make up with Methanol.
• Tablets / Capsules: Finely pulverize sample. Weigh 1.0 g of sample into a 50 mL centrifuge tube.
• Extraction Solvent: Pipette 20.0 mL of Methanol into the tube. (*Note: For softgels, use 20.0 mL of Chloroform as soft capsule matrices do not dissolve in Methanol).
• Extraction: Sonicate in ultrasonic bath for 15 minutes. Centrifuge at 3000 rpm for 10 minutes.
• Filtration: Filter supernatant through 0.45 µm PTFE / Nylon syringe filter into HPLC vial.
Vial 1: Blank (Methanol) [1x] ➔ Vials 2–7: Working Standard 0.05 mg/mL [6x for SST] ➔ Vial 8: LOD Solution [1x] ➔ Vial 9: Blank [1x] ➔ Vials 10–11: Sample 1 (Inj A & B) [2x] ➔ Vials 12–19: Samples 2 to 5 with Blanks ➔ Vial 20: LOD Solution [1x IQC Bracket] ➔ End: LOD Solution [1x].
• SST: Retention Time %RSD ≤ 2.0%, Tailing Factor T ≤ 2.0, Theoretical Plates N ≥ 2000.
• Identification: Positive if Sample RT matches Standard RT (±2%) AND PDA UV spectrum (190–400 nm) similarity index > 0.999.
• Confirmation: Fresh re-extraction by Second Analyst on separate date + GCMS confirmation (PKKK/300/UP/004 or 017).
• Buffer 20 mM KH₂PO₄ pH 3.0: Dissolve 2.72 g of Potassium Dihydrogen Phosphate in 1000 mL Ultrapure water. Adjust pH to 3.0 with Orthophosphoric acid. Filter (0.45 µm) and degas for 20 min.
• Domperidone Stock (1.0 mg/mL): Accurately weigh 10.0 mg Domperidone RS into 10 mL volumetric flask. Dissolve and dilute with Methanol.
• Working Standard (0.05 mg/mL): Dilute 0.5 mL stock to 10 mL with Methanol.
• LOD Solution (0.003 mg/mL): Dilute 0.6 mL working standard to 10 mL with Methanol.
• Weigh 1.0 g of tablet/capsule/liquid sample into 50 mL centrifuge tube.
• Add 20.0 mL Methanol (or Chloroform for softgels). Sonicate for 15 minutes, centrifuge at 3000 rpm for 10 minutes. Filter through 0.45 µm Nylon filter.
| Time (min) | Buffer pH 3.0 (%) | Acetonitrile (%) | Flow |
|---|---|---|---|
| 0.01 – 1.99 | 90 % | 10 % | 1.0 mL/min |
| 2.00 – 3.99 | 70 % | 30 % | 1.0 mL/min |
| 4.00 – 7.99 | 50 % | 50 % | 1.0 mL/min |
| 8.00 – 11.99 | 40 % | 60 % | 1.0 mL/min |
| 12.00 – 14.09 | 30 % | 70 % | 1.0 mL/min |
| 14.10 – 20.00 | 90 % | 10 % (Re-equilibration) | 1.0 mL/min |
• SST: Retention Time %RSD ≤ 2.0%, Tailing T ≤ 2.0, N ≥ 2000.
• Positive Match: Sample RT within ±2% of Domperidone RS & UV Spectrum similarity > 0.999.
• Mixed Standard Stock (EG ~222.7 µg/mL & DEG ~223.7 µg/mL): Prepared in Methanol.
• 5-Level Calibration Curve:
- Level 1 (LOQ): 6.01 µg/mL EG / 6.04 µg/mL DEG (135 µL stock ➔ 5 mL)
- Level 2: 8.01 µg/mL EG / 8.05 µg/mL DEG (180 µL stock ➔ 5 mL)
- Level 3 (SST): 10.02 µg/mL EG / 10.06 µg/mL DEG (225 µL stock ➔ 5 mL)
- Level 4: 12.02 µg/mL EG / 12.07 µg/mL DEG (270 µL stock ➔ 5 mL)
- Level 5: 14.02 µg/mL EG / 14.09 µg/mL DEG (315 µL stock ➔ 5 mL)
- LOD Solution: 2.0 µg/mL (45 µL stock ➔ 5 mL)
• SIM Ions:
Ethylene Glycol (EG) [RT 4.5–11.0 min]: Target Quant: m/z 31; Qualifiers: m/z 33, m/z 62.
Diethylene Glycol (DEG) [RT 11.0–20.0 min]: Target Quant: m/z 45; Qualifiers: m/z 75, m/z 31.
1. Accurately pipette 100 µL of medicinal syrup sample into a 10 mL volumetric flask.
2. Add HPLC grade Methanol, dissolve, and make up to the 10.0 mL mark.
3. Vortex thoroughly for 30 seconds. Transfer into GC autosampler vial for injection.
• Initial: 100 °C (hold 1.0 min)
• Ramp 1: 10.0 °C/min to 130 °C (hold 7.0 min)
• Ramp 2: 20.0 °C/min to 240 °C (hold 3.0 min)
• Post-run: 20.0 °C/min to 250 °C (hold 3.0 min). Total Time: 23.0 min.
• SST (6 Injections of Level 3 Standard): Peak Area %RSD ≤ 10.0%, Tailing Factor Tf ≤ 2.5, Theoretical Plates N ≥ 2000. LOD S/N ≥ 3, LOQ S/N ≥ 10.
• Compliance Limit: EG ≤ 0.10% v/v and DEG ≤ 0.10% v/v. If > 0.10% v/v ➔ FAIL / OOS.
• Weigh 1.5 g sample ➔ dissolve in 10–50 mL water ➔ sonicate 10–15 min ➔ adjust pH to exactly 7.0 with Ammonia/HCl ➔ extract 2× with 50 mL Chloroform (gentle rocking) ➔ evaporate bottom layer to dryness ➔ reconstitute in 3.0 mL Ethanol ➔ filter 0.45 µm.
0.01–1.99 min (80% A : 20% B) ➔ 2.00–13.99 min (75% A : 25% B) ➔ 14.00–17.99 min (70% A : 30% B) ➔ 18.00–19.99 min (60% A : 40% B) ➔ 20.00–21.99 min (50% A : 50% B) ➔ 22.00–29.99 min (30% A : 70% B) ➔ 30.00–35.00 min (80% A : 20% B re-equilibration).
• Positive match: Sample RT within ±2% of RS AND UV spectrum overlay similarity > 0.999.
• Mandatory 2nd Analyst Protocol: Second analyst extracts new sample on different date at pH 7.0 ➔ Confirmation on GCMS (PKKK/300/UP/004 / 017).
• Accurately weigh 1.0 g of cosmetic cream into a 50 mL centrifuge tube.
• Add 20.0 mL of Methanol. Sonicate at 40 °C for 15 minutes to melt cream matrices. Cool to room temperature.
• Centrifuge at 3500 rpm for 10 minutes. Filter supernatant through 0.45 µm PTFE syringe filter into HPLC vial.
• Retention Time: Hydroquinone elutes at ~5.5 – 6.5 minutes.
• SST: %RSD of 6 standard injections ≤ 2.0%, Tailing factor T ≤ 1.8.
• Identification: Sample RT matches Hydroquinone RS (±2%) with UV spectral purity similarity > 0.999.
• Stock Standard (100 µg/mL): Accurately weigh 10.0 mg Lovastatin RS in 100 mL volumetric flask with ACN.
• Working Standard (20 µg/mL): Dilute 5.0 mL stock to 25 mL with Acetonitrile.
• Sample Prep: Weigh accurately 20 pulverized tablets/capsules. Weigh portion equivalent to ~20 mg Lovastatin into 100 mL flask. Sonicate 20 min in ACN, filter 0.45 µm.