Development of Simple and Sensitive HPLC Method for
Determination of Glyphosate Residues in Soybean
Om Prakash Sharma
National Forensic Science Laboratory
Introduction
Literature review
Methodology
Results and discussion
Conclusions/Reccomendations
2
Outlines
3
Insecticides Organochlorines Organophosphates Carbamates Pyrethroides
Herbicides Bipyridyls Chlorophenoxy Acetanilides Glyphosate Triazines
Fungicides Thiocarbamates Dithiocarbamates Cupric Salts Tiabendazoles Triazoles Dicarboximides Dinitrophenols Organotin compounds
Rodenticides Warfarines Indanodiones
Fumigants Aluminium and Zinc phosphides Methyl bromode Ethylene dibromide
Pesticides - Classification by Use and Chemical
Structure
The annual import of pesticides in Nepal is about 345T a.i. (2011/12)
55.85% insecticides, 27.70% fungicides, 10.92% herbicides and 4.81% others. (2011/12)
Average pesticides use in Nepal is 142 g a.i./ha, which is very low as compared to other Asian counties.
Cotton (2560g/h), Tea (2500g/h) and Vegetables (14oog/h) (Thapa 2002)
4
Use of Pesticides in Nepal
Country A,I,/Ha (KG)
Taiwan 17
Japan 12
USA 7
Europe 2.5
Pakistan 1.3
Thailand 1
India 0.5
5
Pesticide Consumption Trend in Different
Countries
Country A.I./Ha(Kg)
Bangaladesh 0.4
Bhutan 0.1
Nepal 0.142
S.Korea 6.6
Risks Toxic to humans Impact on environment and ecosystems Benefits Crop protection Food preservation Material preservation Disease co
6
Pesticides – Risks and Benefits
It is estimated that there are 3 million cases of pesticide poisoning each year and up to 220,000 deaths, primarily in developing countries. (WHO, 2011)
Children are particularly vulnerable to the harmful effects of pesticides even at very low levels of exposure
7
Global Health Impact of Pesticides
Air
Soil
Water
Food
8
Pesticides in Different Media
Crops Routinely Sprayed (Fruits, Vegetables, Grains)
Bioaccumulation in animals and products (Fish, Eggs, Meat, Dairy)
Many food products have detectable levels of pesticides
Guideline levels of pesticides in food (MRL)
Guidelines to limit the population exposure: Acceptable daily intake (ADI)
9
Food Residues
10
Major routes of absorption Dermal Ocular Ingestion Inhalation
Important to consider
Biotransformation Into Inactive or more active metabolites
Elimination Urinary excretion Biliary/Fecal excretion Excretion in milk
Distribution and storage Fat soluble pesticides are stored in adipose tissues Others
Toxicokinetic Varies for Different Pesticides
Post-emergent, Non-selective systemic
and foliar applied herbicide
Commercialized to variety of formulations
High efficacy, low toxicity, low cost
Came to market in 1974
Blocks shikimate pathway - Biosynthesis of aromatic amino acids (trp, phe, tyr)
Occupies >30% of the herbicide sale volume around the world
Recently, growth rate of market demand is 15-20% (USAD)
11
(Roundup, Touchdown, Vision
etc.)
What is Glyphosate ?
Source USDA AER 786
12
2012 2006 1998 2012 2006 1998
Concern : Herbicide Use Will Go Up
Environmental contamination
-Threats on aquatic organisms
-Threats on terrestrial organisms
Food contamination
-Threats on human health
Analytical problem
-Highly water soluble
-Low mass, low volatility, highly polar
-Lack of chromophore and fluorophore group
13
Problems
To explore the simple and effective GP extraction method from protein rich crop matrix (soybean)
To develop the sensitive and reliable HPLC method for determination of glyphosate in soybean matrix
To determine the residues of glyphosate in soybeans
14
Objectives
Structure and properties
Metabolism
Environmental fate
Toxicity
Residues in food
15
Brief About Literature
16
•Tendency to form dipolar molecule (zwitterions)
•C2-N bond is more available for microbial cleavage
General structure pKa=0.8
pKa=2.3
pKa=6.0
pKa=11
•Exists in different ionic species
•Weak organic acid, polar and highly water soluble
Source: Piriyapittaya et al.,2008
Structure/Properties
17
•Considered as a stable herbicide in plant tissues •Little is known about metabolism and enzymes involved in plants
Metabolism of glyphosate
Microbial degradation in soil and water
Glyphosate has strong adsorption capacity with soil particles
Low potential to contaminate water
Adsorption affinity depends on:
-Presence of phosphorus and metal ions such as
Fe, Cu, and Al-oxides in soil
-Ca and Mg ions in water
Low tendency of accumulation in animal tissues
Movement through volatilization is negligible
18
Environmental Fate
LD50 4,230 mg/kg (WHO) 5,600 mg/kg (Monsanto).
Non-carcinogenic (USEPA) and without mutagenic effect to humans (IPCS)
Effects on non-target organisms and overall environmental impact are not fully understood
19
Toxicity associated with glyphosate
Monsanto advertises that Roundup is less toxic
than table salt (which has an LD50 of3000
mg/kg).
This is misleading because this LD50 is only a
measure for acute oral toxicity.
20
Roundup – Less toxic Than Table Salt ?
21
More Toxic Than We Think
Modern advances in research indicates that glyphosate based herbicides have association with
-Reproductive toxicity,
-Developmental toxicity,
-Endocrine disruption activity
- Carcinogenic activity
Exhibits synergistic effect with inactive ingredients
e.g. (POEA)
Sequence of Human Development
1 2 3 4 5 6 7 8 12 16 20 38
Implantation
Prenatal
Death
Emryonic period
Major Morphological abnormalities
Fetal Period
Physiological and Functional
Defects
Central Nervous System
Heart
Ears
Eyes
Limbs
Palate
External Genetalia
Developmental Toxicity
Infertility
Spontaneous abortion
Altered length of pregnancy
Low sperm count and low sperm motility
Birth defects
Epididymal and testicular abnormalities
Altered sexual behavior, altered puberty onset,
lactation problems, altered menopause onset.
23
Reproductive Toxicity Via Endocrine
Disruption
Surveys of causes of mortality/morbidity by occupation showed farmers have increased risk of certain (mostly rare) cancers:
Non-Hodgkin’s lymphoma, Leukemia, Multiple myeloma
Soft-tissue sarcoma
Breast cancer
Brain tumor
Prostate cancer
Ovarian cancer
24
Carcinogenic Activity
Glyphosate contamination in drinking water is not
considered as serious threat so far
Residues may results in crops, vegetables, fruits and
animal tissues.
Major source of exposure to human is expected
through food
Low levels detected in secondary food commodities:
milk, meat, and eggs
In some GM crops, more glyphosate residues have been reported
25
Glyphosate Residues in Soybean, Other Crops
and Food Commodities
Country Wheat
grain
Bareley Maize Soybean Soybean
(transgenic)
USA - - 3 0.45-17 0.27-23
France 9.5 - - - -
Belzium 1.2 20 - -
UK 1 1.4 -11 - -
26
Application pattern: Repeated and preharvest
Source:Pesticide residues in food-2011
Maximum residues found in different cropps (mg/kg) (From WHO/FAO supervised trials)
Rate of application: 0.84 kg ae/ha
Glyphosate Residues Contd.
MRLs of glyphosate in dry soybean: 20mg/kg (Codex
Alimentarius Commission)
ADI: 0.3mg/kg (Codex Alimentarius Commission)
EU maximum threshold limit in drinking water 0.1mg/l
27
Glyphosate Residues Contd.
Gas chromatography
-Derivatized to a less polar, volatile and thermally
stable derivative
-GC-ECD, GC-NPD, GC-MS, GC/MS/MS)
Capillary electrophoresis
-Suffered with low sensitivity owing to the limited
sample injection volume
-MS, UV and indirect fluorescence detection
-Provides high resolutions and efficiency with MS
28
Mostly Used Analytical Methods to Determine
Glyphosate Residues
Ion exchange chromatography -Used to detect ionic analytes including glyphosate -IC-ICP-MS, IC-ESI-MS. (IEC)
ELISA -High sensitivity and selectivity -High cost, difficulty to get commrecial test kit -Mostly used to detect glyphosate in water
NMR technique -Mostly used to analyze glyphosate in biological samples
29
Analytical Methods Contd.
HPLC
-Derivatized to add fluorophore or chromophore
groups
-Post or precolumn derivatization
-MS,UV,FLD detection system
-FMOC-Cl, TsCl, O-pthalaldehyde, CNBF, N, N-
dimethyl-2-mercaptoethylamine hydrochloride and
mixture of o-phthalaldehyde and 2- mercaptoethanol
30
Analytical Methods Contd.
Sample collection
Sample preparation
Derivatization
Chromatographic separation
validation
31
Methodology
Total 51 soybean samples collected from local markets of Bangkok and Kathmandu
Thailand : 44 samples with 24 different
brand names
Nepal: 7 samples with 5 different brand name
Kept in freezer at 4oC until experimentation
Organic soybean was used as blank samples
32
Sample Collection
33
1gm blended soybean in 50 ml PP tube
Extracted with water-methanol (4:1, v/v) using circular tube rotator for 1hrs
Supernatant
Protein precipitation with equal volume of methanol (Vortex mixing)
Centrifuged at 4000 rpm for 30 min
Centrifuged at 400 rpm for 15 min
Sample Preparation
34 HPLC analysis
Derivatization with FMOC-Cl
Filtration through PVDf membrane(0.5µ)
Vortex mixing
5 fold dilution with water
Supernatant
35
Addition of 0.25 ml of 50 mM borate buffer (pH 2.5) (Vortex mixing)
Addition of 2g/L FMOC-Cl in CAN (0.3 ml)
Allowed to react for over night at 40
Quenching the reaction ml) with 2%H3PO4 (0.3ml)
(Vortex mixing)
1 ml soybean extract in 15 ml PP tube
HPLC analysis
Derivatization Procedure
Equipment: -Agilent 1100 series HPLC system comprised with quaternary pump, degasser,
auto autosampler, thermosttated column oven and fluorescence and diode array detectors was utilized
HPLC column: -Reversed phase C18 columns (ACE 5 m 4.6 250 mm, Hichrome)
Mobile phase:
-Phosphate buffer (A) and acetonitrile (B)
36
Instrumentation and Chromatographic
Separation
37
Agilent 1100 Series HPLC Equipment
Instrumentation
LOD and LOQ:
Recovery:
Precision:
38
Six replicates for each concentration levels
Calculated from post-extraction spiked solution, at 0.05, 0.1 and 0.5 µg/ml
Six multiple injection for each concentration at same day for repeatability and three different days for intermediate precision
Calculated from matrix matched standard solution at concentration of 0.005 µg/ml
From pre-extraction spiked solution at 0.05, 0.1 and 0.05 µg/ml
Validation Methods
Solvent selection
Derivatization
HPLC conditions and analysis
Calibration
Recovery and precision
Glyphosate residues in soybean
39
Results and Discussion
Solvents used in extraction
-Water
- mixture of water and dichloromethane
- mixture of water and methanol
Water-methanol solvent system was selected as most effective for extraction
40
Selection of Extracting Solvents
Water +
methanol
Water +
dichoromethane
Water Glyphosate
Glyphosate
Glyphosate
41
42
•FMOC-OH leads Interference.
•Studied for different time course ( 15, 30, 45, 60 min, 2hrs and overnights),pH of borate bufrfer (pH 8.5, 9 and 9.5), concentration of FMOC-Cl (1,2 and 5 g/L) and temperature RT and 40 o C.
Derivatization
0
20
40
60
80
100
120
140
160
1 2 3
FMOC-Cl cocentration (g/L)
Det
ecto
r re
spon
se (
LU
xS
)
43
•Derivatization optimized at 5g/L FMOC-Cl, over-night time course, and 40 o C reaction temperature.
Effect of FMOC-Cl Concentration on
Derivatization
44
G
G
G
(a) FMOC-Cl 1 mg/ml
(b) FMOC-Cl 2 mg/ml
(c) FMOC-Cl 5 mg/ml
0
20
40
60
80
100
120
8.5 9.0 9.5
pH
Det
ecto
r re
spon
se (
LU
xS
)
45
Borate buffer with pH 9.5 is suitable for the complete reaction between glyphosate and FMOC-Cl to give stable derivative.
Effect of pH of Borate Buffer on
Derivatization Reaction
46
(a)40 o C
(b)Room
temperature
G
G
Effect of Temperature on Derivatization
C18 reverse column (ACE 5 m 4.6 250 mm, Hichrome, UK), Gradient elution
Mobile phase: 50 mM phosphate buffer (pH2.5)and ACN( 65:35, v/v)
To eliminate unreacted reagent and side reactants in the column, acetonitrile ratio was increased
Other optimized conditions: Flow rate-0.8ml/min; Injection volume-20µl; Colume compartment temperature: 40 o C
47
HPLC Conditions and Separations
48
G
G
•phosphate buffer with pH 5.75 gave less retention time and may co-eluted or interfered with other unretained compounds.
(b) Phosphate buffer pH 2.5
(a)Phosphate buffer pH 5.75
Effect of the pH of the Phosphate Buffer on
Retention Time
Calibration performed in both solvent standard and
matrix-matched standard in 0.005-1µg/ml range
Sensitivity of the method for matrix-matched standard was 30% less than in solvent standard.
Linearity was approximately similar.
49
Calibration
50
G
G
(a) Solvent standard
(b) matrix-matched standard
•Matrix- matched standards contained more interferrents from sample which canreact with derivatizing reagent and produce interference during analysis.
51
y = 3508.35x - 7.48
(A) Solvent standard
y = 2458.90 x- 1.85
(B) matrix-matched standard
Calibration Curves
52
Analyte spiked level
(µg/ml)
(n=5)
% Recovery Average
Recovery
0.05 100.9
95.2 0.1
92.47
0.5 92.23
% Recovery was determined from pre-extraction spikes
Recoveries
Analyte
Concentration (µg/ml)
Intra-day (%RSD) Inter-day (%RSD)
PA
(LUxS) RT(min) PA
(LUxS) RT(min)
0.05 1.29 0.44 2.42 0.68
0.1 0.84 0.18 1.37 0.29
0.5 0.15 0.30 0.46 0.46
53
•Determined on the basis of Retention time and Peak area
•Evaluated from matrix matched standards at 0.05, 0.1 and 0.5 µg/ml
Repetability and Intermediate Precision
Residues ranges from 0.23 – 4.61 mg/kg
50% samples have nearly consistent residues of GP from both lots. But, in other 50% it was not consistent in both lots.
Variable residues may be due to different pattern of storage, different GP treatment pattern, temperature etc.
Residues determined by applying proposed method are quite below the MRL set by Codex Alimentarius Commission
(20mg/Kg for soybean)
54
Residues of GP in Soybeans
55
Soybean sample ID
GP residue (µg/g)±SD(n=3)
Lot.No. 1 Lot. No. 2
SB-01 0.47±0.09 0.27±0.03
SB-02 0.23±0 -
SB-03 0.60±0.02 1.34±0.06
SB-04 0.31±0.02 -
SB-05 0.53±0.03 1.05±0.08
SB-06 0.26±0.01 2.4±0.4
SB-07 0.89±0.0 0.93±0.009
SB-08 0.97±0.03 0.86±0.01
SB-09 0.45±0.02 0.31±0.09
SB-10 2.88±0.02 2.99±4.01
SB-11 0.67±0.08 0.68±0.08
SB-12 0.24±0.02 0.39±0.01
SB-13 4.25±0.16 4.61±0.79
SB-14 1.47±0.09 1.65±0.00
56
SB-16 1.09±0.05 0.73±0.02
SB-17 0.73±0.04 0.50±0.04
SB-18 0.58±0.00 0.92±0.30
SB-19 2.05±0.14 1.09±0.08
SB-21 0.35±0.01 0.93±0.02
SB-22 1.79±0.10 2.05±0.06
SB-23 1.89±0.08 1.70±0.13
SB-24 1.24±0.13 1.0±0.02
SB-25 0.25±0.01 -
SB-26 0.38±0.01 -
SB-27 0.57±0.03 0.99±0.03
SB-28 0.53±0.01 -
SB-29 0.46±0.01 0.63±0.06
Effective extraction method to remove protein
FMOC-Cl appears to be suitable derivatizing agent in protein rich sample matrix
Very good sensitivity with LOD and LOQ 0.125 and 0.25 mg/kg, respectively; correlation 0.99929
Recovery ranges from 92-101%
Excellent precision with standard deviation ranges from 0.15-1.29% (repeatability) on the basis of PA
However, matrix effect was not completely removed.
57
Conclusions
Further study is required to find more efficient protein precipitating agent
It is required to investigate the methods to reduce interferences caused by excess amount of FMOC-Cl
Effect of derivatization by-products such as FMOC-OH and other co-extracted fluorogenic and polar compounds should be explored to develop the suitable HPLC method.
To study the effects of aqueous-organic reagent ratio in derivatization reaction mixture.
58
Recommendations
Further validation is required by applying the proposed method on other protein rich and complex sample matrix such as corn, wheat and soybean derived food.
From this study, the residues determined were quite below the maximum residue levels (MRLs) of glyphosate set my international authority. Therefore, taking soybeans and food derived from these soybeans is recommendable.
59
Recomendations
60