Standard Operating Procedure (SOP)

1.0 Scope

This SOP applies to the detection and quantification of pesticide residues in fruits, vegetables, and plant-based matrices, following AOAC 970.52, 985.22, and 970.53.

2.0 Principle

Pesticides are extracted using solvents, purified by Florisil/silica cleanup, concentrated, and quantified via Gas Chromatography (GC) with selective detectors:

ECD (Electron Capture Detector): Organochlorines

NPD (Nitrogen Phosphorus Detector): Organophosphates and Organonitrogen

FPD (Flame Photometric Detector): Organophosphates

3.0 Responsibilities

Analyst: Conducts the procedure and maintains records.

Supervisor: Reviews QC, validates results.

QA Officer: Ensures compliance with accreditation standards.

4.0 Safety and Precautions

Checklist before starting work:
☐ Lab coat, nitrile gloves, goggles
☐ Certified fume hood operational
☐ MSDS for solvents available
☐ Waste containers labeled (halogenated vs non-halogenated)

⚠️ Hazards:

Solvents: Flammable and toxic → avoid ignition sources.

Standards: Extremely toxic → handle only with calibrated pipettes.

Florisil dust: Respiratory hazard → wear mask if handling dry powder.

5.0 Equipment and Apparatus

Gas Chromatograph with ECD, NPD, FPD

Capillary columns DB-5 or DB-1701 (30 m x 0.25 mm x 0.25 μm)

Homogenizer (stainless steel jar)

Analytical balance (±0.1 mg)

Rotary evaporator with water bath ≤40 °C

Kuderna-Danish (K-D) concentrator tubes with Snyder column

Florisil glass columns with Teflon stopcock

Volumetric flasks, pipettes, centrifuge (optional)

6.0 Reagents and Standards

Solvents (residue grade): Acetonitrile, Petroleum Ether, Hexane, Acetone, Dichloromethane, Ethyl Acetate

Sodium Sulfate (anhydrous, pre-baked 600 °C, stored sealed)

Florisil (60–100 mesh, activated 675 °C, deactivated 1–2% water)

Certified pesticide standards (DDT, Malathion, Lindane, Carbaryl, etc.)

Calibration standards (multi-level: 0.01–0.5 µg/mL)

7.0 Procedure

7.1 Sample Preparation

Collect representative sample (50–100 g).

Homogenize using blender/homogenizer until uniform.

Record sample weight in logbook.

7.2 Extraction

AOAC 970.52 & 970.53 (Organochlorines, Organophosphates):

Add 200 mL acetonitrile to 50 g sample.

Blend 2–3 min.

Filter homogenate into suction flask.

Transfer 100 mL extract to separatory funnel.

Add 100 mL petroleum ether → shake.

Add 500 mL sodium sulfate solution (10%) → shake.

Discard aqueous layer, retain petroleum ether layer.

Wash extract twice with 100 mL sodium sulfate solution.

Pass through sodium sulfate drying bed.

AOAC 985.22 (Organonitrogen pesticides):

        Extract with dichloromethane.

        Partition into acetonitrile-saturated hexane (removes fats).

        Back-extract into dichloromethane.

7.3 Cleanup (Florisil Column Chromatography)

Pack glass column:

        Glass wool → 2 cm Na₂SO₄ → 10–15 g Florisil → 2 cm Na₂SO₄

Condition with 50 mL petroleum ether.

Load concentrated extract.

Elute sequentially:

        Fraction 1: 200 mL (6% ether/petroleum ether) → HCB, Lindane

        Fraction 2: 200 mL (15%) → Aldrin, Dieldrin

        Fraction 3: 200 mL (50%) → Malathion, Parathion

7.4 Concentration

K-D concentrator + Snyder column in 40 °C bath.

Reduce volume to 5–10 mL.

Final concentration under N₂ → 1–2 mL (hexane).

7.5 GC Analysis

Carrier gas: Helium/Hydrogen.

Injection: 0.5–1 µL, splitless.

Injector temp: 250 °C.

Oven: 80 °C (1 min) → ramp 10–15 °C/min → 280 °C (hold 10 min).

Detector temps: ECD 320 °C; NPD 280 °C; FPD 250 °C.

Run calibration standards and samples.

Confirm retention times vs standards.

8.0 Quality Control (QC)

9.0 Calculations

Residue (mg/kg or ppm) = (C × V) / (W × R)

Where:

        C = Analyte concentration in extract (µg/mL)

        V = Final extract volume (mL)

        W = Sample weight equivalent (g)

        R = Recovery correction (if applied, else R=1)

10.0 Records

Sample logbook entries

Extraction sheets

GC run sheets with chromatograms

QC summary report

11.0 Method Limitations

Historical AOAC methods → limited for modern pesticides.

Not confirmatory (requires GC-MS/MS).

Possible matrix effects.

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