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

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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

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Global Health Impact of Pesticides

Air

Soil

Water

Food

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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)

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Food Residues

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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

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Objectives

Structure and properties

Metabolism

Environmental fate

Toxicity

Residues in food

15

Brief About Literature

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•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

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•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

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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

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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.

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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.

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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

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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

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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 - -

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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

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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

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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

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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

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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

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Sample Collection

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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

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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)

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Instrumentation and Chromatographic

Separation

37

Agilent 1100 Series HPLC Equipment

Instrumentation

LOD and LOQ:

Recovery:

Precision:

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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

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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

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Selection of Extracting Solvents

Water +

methanol

Water +

dichoromethane

Water Glyphosate

Glyphosate

Glyphosate

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•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

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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

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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.

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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.

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y = 3508.35x - 7.48

(A) Solvent standard

y = 2458.90 x- 1.85

(B) matrix-matched standard

Calibration Curves

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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

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•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)

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Residues of GP in Soybeans

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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

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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.

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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.

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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.

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Recomendations

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