Pesticides Analysis Quantitative Analysis of Pesticides in Dietary Supplement by GC/MS/MS
Aihua Liu, Ph.D. Director, R&D
DYAD LABS | dyadlabs.com 2 Outline
Background
Method Development
Method Validation
Proficiency Tests
Conclusions
DYAD LABS | dyadlabs.com 3 Background
Accurate analysis of pesticide in dietary supplement sample becomes more and more important.
The compendium pesticide methods can’t cover all dietary supplements which have various and complex matrices.
Develop a fast, simple, specific, sensitive and comprehensive method to quantify 112 pesticides in botanical and non- botanical dietary supplement.
DYAD LABS | dyadlabs.com 4 Method Development
Sample Preparation
Sensitivity Issue
Peak Tailing Issue
Matrix Effect Issue
DYAD LABS | dyadlabs.com 5 Sample Preparation----Extraction Solvent
Adopt QuEChERS (Quick, Easy, Recovery (%) Analyte Cheap, Effective, Rugged and Safe) AOAC Method Dyad Method technique. Azinphos-ethyl 25.84 95.00 Fenchlorphos oxon 27.35 84.75 Fenitrothion 60.65 98.25 Optimize extraction solvent Heptachlor 66.25 91.50 (1) AOAC method solvent------Malaoxon 23.85 115.25 (2) USP method solvent----- Methidathion 28.40 96.75 (3) Dyad method solvent------Paraoxon-methyl 15.65 74.50
DYAD LABS | dyadlabs.com 6 Sample Preparation----dSPE
Dispersive solid phase extraction (dSPE) is a simple, low cost, fast and sorbent-based technique to clean up sample.
Different dSPEs were compared, and Agilent 5982-5122 dSPE was selected.
Result (ng/mL) Nominal Conc. Analyte dSPE1 dSPE2 (ng/mL) (Agilent 5982-5211) (Agilent 5982-5122) Chlorpyrifos (Chlorpyrifos-ethyl) 150 85.7 147 Dimethoate 150 49.0 151 Fenitrothion 150 119 128
DYAD LABS | dyadlabs.com 7 Sample Preparation----Low Recovery
Some pesticides have low recovery since they stick to the surface of containers during extraction.
The analyte protectant (AP) can prevent this low recovery issue by occupying the container surface.
Result (ng/mL) Analyte Nominal Conc. (ng/mL) Without AP With AP Malathion 150 115 167 Pendimethalin 150 67.5 136
DYAD LABS | dyadlabs.com 8 Sample Preparation Procedure
GC/MS/MS
Add water, AP and 0.1% Add ceramic 1. Centrifuge + Dry down sample FA in MeCN +homogenizer 2. dSPE under N2
LC/MS/MS
DYAD LABS | dyadlabs.com 9 Sensitivity Issue---dMRM and RTL
Too many Multiple Reaction Monitoring (MRM) transitions cause low sensitivity issue.
Dynamic MRM(dMRM) mode can keep sensitivity by setting up different time segments for many analytes.
dMRM requires consistent retention times
Retention Time Locking (RTL) ensures the consistency of retention time by adjusting as flow rate.
DYAD LABS | dyadlabs.com 10 Peak Tailing Issue---AP with Sandwich Injection Mode
Some pesticides have peak tailing issue, which is caused by stickiness to the GC system.
AP with hydroxyl groups can block active sites in GC system to prevent peak tailing issue.
Traditional Injection Mode without AP Sandwich Injection Mode with AP
DYAD LABS | dyadlabs.com 11 Matrix Effect—Internal Standard (IS) Selection
USP 561 uses only one IS. Method IS USP 561 Carbophenothion AOAC 2007.01 uses two IS. AOAC 2007.01 Parathion-d10, HCH-d6 Atrazine-d5, Carbophenothion, Diazion-d10, Dyad Labs uses five IS Dyad Labs Dimethoate-d6, Linuron-d6
Analyte: Lindane Analyte: Lindane IS: Atrazine-d5 IS: Carbophenothion R2=0.9998 R2=0.9917
DYAD LABS | dyadlabs.com 12 Matrix Effect—MRM Transition Selection
Each pesticide has three MRM transitions.
Choose the transition with most sensitive and least interference to minimize matrix effect
A: Transition has interference B: Transition has no interference
A B
DYAD LABS | dyadlabs.com 13 Method Validation
Specificity
Linearity and Sensitivity
Accuracy and Range
Precision
Stability
Batch Size
DYAD LABS | dyadlabs.com 14 Selected Matrix for Validation
Matrix Type Matrix No. Description 1 Psyllium Seed Powder 2 Garlic Bulb Powder 3 Broccoli Sprout Powder 4Red Wine Botanical Mixed 5Mango Powder Matrix 6 Fenugreek Seed Powder 7 Blackcurrant Fruit Powder 8 Mushroom Powder 9 Ginseng Panax Root Powder Non‐Botanical 10 Sport Supplement 1 Mixed Matrix 11 Sport Supplement 2 12 Sport Supplement 3
DYAD LABS | dyadlabs.com 15 Specificity
Method specificity is evaluated by analyzing blank diluent, blank matrix extract, analyte only and IS only.
No significant contribution/interference peaks were observed in these samples.
Blank Diluent Blank Non-Botanical Matrix Blank Botanical Matrix
Analyte Transition Analyte Transition Analyte Transition
IS Transition IS Transition IS Transition
DYAD LABS | dyadlabs.com 16 Linearity
Each pesticide has its own limit. Five different curve ranges were designed to cover all 112 pesticides using 9 standard curve points.
The curve regression type is quadratic with weigh of 1/x and R2 must be ≥0.995.
Conc. Curve Type Level (ng/mL) Type I Type II Type III Type IV Type V Std 1 2.00 x Std 2 5.00 x x Std 3 10.0 x x x Std 4 20.0 x x x x x Std 5 50.0 x x x x x Std 6 100 x x x x x Std 7 200 x x x x x Std 8 500 x Std 9 1,000 x QC 100 x x x x x
DYAD LABS | dyadlabs.com 17 Proficiency Test
Assigned Range Dyad Result Proficiency Test Sample Analyte (µg/kg) (µg/kg) Malathion 42.7-109.7 75.8 Brown Rice (T09116QC) Pendimethalin 46.8-120.5 99.3
Wheat Flour (T09117QCsale) Fenitrothion 56.0-145 88.1
Animal Feed (T09118QC) Dimethoate 121-286 170
DYAD LABS | dyadlabs.com 18 Conclusions
This pesticide method is simple, fast, specific, sensitive and comprehensive.
This pesticide method has been successfully validated in both botanical and non-botanical dietary supplement.
This pesticide method has been successfully applied for proficiency test samples.
DYAD LABS | dyadlabs.com 19 Thank You.