🇲🇾 NPRA — PKKK/300/UP/021 · PKKK/300/UP/011 · PKKK/300/UP/014

◈ Seksyen Pengujian Produk & Kosmetik · Unit Penyaringan

Steroid HPLC Screening Step-by-Step Guide

Complete Operational Workflow for Shimadzu Prominence-i (LabSolutions)

Comprehensive, execution-ready practical guide for screening 8 steroid adulterants (Dexamethasone, Betamethasone, Prednisone, Prednisolone, Triamcinolone acetonide, Hydrocortisone acetate, Cortisone acetate, Beta-17-valerate) in traditional medicines.

Instrument Shimadzu Prominence-i (LC-2030C 3D)

Column Kinetex Phenyl-Hexyl 100Å (150×4.6mm, 2.6µm)

Flow & Temp 0.7 mL/min @ 40°C

PDA Detection 240 nm (Scan 190–400 nm)

Extraction LLE with pH 7.0 adjustment

⚙️ Panduan Pengendalian Alat & Radas Yang Digunakan (Instrument References):

PKKK/300/UP/011 · HPLC 3 & 4

Shimadzu Prominence-i Operating Guide

Buka Panduan Alat →

PKKK/300/UP/003 · RRLC HP1200

Agilent 1200 Series RRLC Guide

Buka Panduan Alat →

PKKK/300/UP/014 · Ekstraksi

Pengekstrakan Cecair-Cecair (LLE pH 7.0)

Buka SOP Pengekstrakan →

PKKK/300/UP/005 · Neraca Analitikal

Alat Timbang Precisa XT 120A

Buka SOP Alat Timbang →

PKKK/300/UP/019 · Penyejatan

Water bath Memmert WB 45

Buka SOP Waterbath →

PKKK/300/UP/058 · Sonikasi

Ultrasonic Bath Branson 8210

Buka SOP Sonikator →

PHASE 1

Reagent Preparation & Standard Solutions

Ref: PKKK/300/UP/021 Para 6.1 & 6.4

1.1 Mobile Phase Preparation:

Prepare Mobile Phase A: Ultrapure Water (18.2 MΩ·cm) and Mobile Phase B: HPLC Grade Acetonitrile (ACN). Sonicate and degas both solvents for 15 minutes in ultrasonic bath before placing onto LC reservoir tray.

1.2 Primary Stock Standard Solutions (1.0 mg/mL):

Weigh accurately 10.0 mg of each steroid reference standard into separate 10 mL volumetric flasks. Dissolve and make up to volume with HPLC grade Methanol.

Prednisone Prednisolone Betamethasone Dexamethasone Triamcinolone acetonide Hydrocortisone acetate Cortisone acetate Betamethasone-17-valerate

1.3 Mixed Working Standard Solution (0.05 mg/mL):

Pipette 0.5 mL from each of the 8 individual stock solutions into a single 10 mL volumetric flask. Dilute to volume with Methanol (Final concentration = 0.05 mg/mL each).

1.4 Diluent & Blank:

Prepare Methanol : Water (1:1 v/v) as the blank and diluent.

PHASE 2

Liquid-Liquid Extraction (LLE) Protocol

Ref: PKKK/300/UP/014 Para 6.3 & PKKK/300/UP/021 Para 6.3

⚠️ CRITICAL REQUIREMENT: Per SOP 021 Para 6.3, the sample aqueous solution MUST BE ADJUSTED TO pH 7.0 before organic extraction.

2.1 Sample Weighing & Dissolution:

Weigh 1.5 g (or more) of pulverized traditional product sample into a 100 mL beaker. Add 10–50 mL of deionized water.

2.2 Sonication:

Sonicate the mixture in an ultrasonic bath for 10–15 minutes to ensure complete dispersion of the matrix.

2.3 pH Adjustment to pH 7.0:

Check pH using pH indicator paper. Add dilute Ammonia solution (if acidic) or dilute HCl (if basic) dropwise until the solution reaches exactly pH 7.0.

2.4 Extraction with Organic Solvent (2× Extractions):

Transfer solution into a 250 mL separating funnel. Add 50 mL of Chloroform (or Ethyl Acetate).
*Pengekstrakan dilakukan menggunakan kaedah lenggang secara perlahan bagi memastikan perpindahan analit ke fasa organik yang maksimum serta mengelakkan pembentukan emulsi. Repeat extraction with another 50 mL organic solvent.

2.5 Phase Separation & Evaporation:

For Chloroform: collect bottom organic layer; filter through filter paper into evaporating dish. Discard top aqueous layer. Evaporate organic extract in evaporating dish until completely dry on a water bath (Memmert WB 45).

2.6 Reconstitution & Filtration:

Reconstitute dry residue with 3.0 mL of Ethanol. Filter through 0.45 µm Nylon / PTFE syringe filter into standard 2 mL HPLC autosampler vial.

PHASE 3

Shimadzu Prominence-i & LabSolutions Setup

Ref: PKKK/300/UP/011 & PKKK/300/UP/021 Table 1 & 2

3.1 Power On & Software Login:

Switch on main power & HPLC machine (password: 00000). Open LabSolutions software ➔ Login (User ID: Admin, Password blank) ➔ Click Instrument ➔ LC-2030C 3D.

3.2 Install Analytical Column:

Connect Phenomenex Kinetex Phenyl-Hexyl 100Å (150 × 4.6 mm, 2.6 µm) in the direction of flow. Set Column Oven temperature to 40 °C.

3.3 Method Parameters & Gradient Time Program:

In Method Settings, configure:

Time (min)	Module / Command	Water / Line A (%)	ACN / Line B (%)	Flow Rate
0.01 – 1.99	Pump B Conc	80 %	20 %	0.7 mL/min
2.00 – 13.99	Pump B Conc	75 %	25 %	0.7 mL/min
14.00 – 17.99	Pump B Conc	70 %	30 %	0.7 mL/min
18.00 – 19.99	Pump B Conc	60 %	40 %	0.7 mL/min
20.00 – 21.99	Pump B Conc	50 %	50 %	0.7 mL/min
22.00 – 29.99	Pump B Conc	30 %	70 %	0.7 mL/min
30.00 – 35.00	Pump B Conc	80 %	20 % (Re-equilibration)	0.7 mL/min

PDA Detector: 240 nm (Scan range: 190–400 nm), Cell Temp: 40 °C, Injection Volume: 10 µL, Stop Time: 35.0 min.

3.4 Auto Purge & Baseline Stabilization:

Execute Auto Purge on Lines A & B. Download Method File ➔ Click Instrument ON/OFF to start flow. Click Plot button in RealTime window. Observe baseline and pressure stability (~120–180 bar) for at least 15 minutes before starting injections.

PHASE 4

Real-Time Batch Sequence Table

Ref: PKKK/300/UP/021 Table 3 & Para 6.6

Load autosampler rack into Tray 1 and populate Real Time Batch table:

Vial / Row	Sample / Standard Name	Vial Type	Inj Count	Purpose / Rule
1	Blank (Methanol : Water 1:1)	Diluent	1	System equilibrium & baseline blank
2–7	Mixed Working Standard (0.05 mg/mL)	Standard	6	System Suitability Test (SST)
8–9	Sample 1 (Duplicate: Inj 1 & Inj 2)	Sample	2	Sample 1 Adulterant Screening
10	Blank (Methanol : Water 1:1)	Diluent	1	Carryover elimination
11–12	Sample 2 (Duplicate: Inj 1 & Inj 2)	Sample	2	Sample 2 Adulterant Screening
13	Blank (Methanol : Water 1:1)	Diluent	1	Carryover elimination
14–19	Samples 3 to 5 (Duplicate each)	Sample	6	Screening batch
20	Mixed Working Standard (0.05 mg/mL)	IQC Check	1	IQC Bracket (every 5 samples)
21	Blank (Methanol : Water 1:1)	Diluent	1	Wash
22–23	Sample 6 (Duplicate)	Sample	2	Subsequent samples
End	Mixed Working Standard (0.05 mg/mL)	End Std	1	Final Bracket Check

PHASE 5

Data Evaluation, SST Criteria & Confirmation Protocol

Ref: PKKK/300/UP/021 Para 6.6, 6.7, 6.8 & Borang UP/005

1. System Suitability Test (SST) Criteria (n = 6)

Retention Time %RSD: Must be ≤ 2.0% across all 6 replicate standard injections.
Peak Tailing Factor (T): T ≤ 2.0.
Theoretical Plates (N): N ≥ 2000.
Peak Resolution: All 8 steroid peaks must be well separated (except Betamethasone & Dexamethasone critical pair).

2. Internal Quality Control (IQC) Acceptance

Retention Time Drift: Percentage difference in retention time for mixed standard across the batch must be ≤ 3.0%.

3. Positive Identification Criteria (Sample Chromatogram vs Standard)

A steroid compound is declared POSITIVE (DIKESAN) if and only if:

Retention Time Match: Retention time of sample peak matches the working standard peak within ±2%.
UV Spectral Match: PDA UV absorption spectrum (190–400 nm) overlay shows a spectral similarity index > 0.999 compared to reference standard.

🔒 MANDATORY 2ND ANALYST CONFIRMATION PROTOCOL (Para 6.8.2 – 6.8.4):
If a sample tests POSITIVE for any steroid:

A Second Analyst must independently prepare a fresh sample extraction on a different date per `PKKK/300/UP/014` (adjusted to pH 7.0).
The sample extract must be subjected to confirmatory analysis using GCMS (`PKKK/300/UP/004` Agilent 7890/5975C or `PKKK/300/UP/017` Agilent 8890/5977B) before issuing official failed report.

PHASE 6

Post-Analysis Column Wash & System Shutdown

Ref: PKKK/300/UP/011 Para 6.5

6.1 Organic Flush:

Pump 100% Acetonitrile (or Methanol) at 0.7 mL/min for 30 minutes to flush all matrix residues from the column.

6.2 Storage Flush:

Pump 70% Acetonitrile : 30% Ultrapure Water for 15 minutes as the column storage solvent.

6.3 Instrument Shutdown:

In LabSolutions RealTime window, click Instrument ON/OFF to turn off pumps, oven, and PDA lamp. Close software and log the run in the instrument logbook (Rekod Penyelenggaraan Alat Shimadzu Prominence-i).