Evaluation of Zirconia Adsorbents in QuEChERS Cleanup for Hazelnut, Avocado and Soybeans

Hans Ragnar Norli, Gunvor Viki Seneset , Nina Svendsen, Agnethe Christiansen and Sven

Roar Odenmarck Bioforsk Plant Health and Plant Protection

Høgskoleveien 7N-1430 Ås, Norway

• Primarily establish a QuECheRS cleanup method for analysis of nuts including other fatty matrices

• Develop a simpler and faster in house cleanup method for fatty matrices without a freeze out step and CaCl2 treatment

• Investigate the new zirconia materials from Supelco

Background

Outline• Matrices:

– Hazelnut (60-70% fat)– Avocado (10-15% fat)– Soybean (≈ 40 % fat)

• Pesticides:– Total: 333– 122: GC-MS/MS– 211: LC-MS/MS

• Cleanup:– In house – Z-sep– Z-sep+

Instruments

Agilent 7890 A GC, 7000B GC/MS-MS (2012) Multimode inlet (Cooled by

Pressurized Air) Backflush at column midpoint Rt-lock (ditalimfos)

Agilent 1200 HPLC, 6410 Triple Quad LC/MS (2009) ESI+ Dynamic MRM Programmed injection (sample dilution)

QuEChERS Sample PreparationIn house methodAvocadoHazelnut Soybean

With Z-sepAvocadoHazelnut Soybean

With Z-sep+AvocadoHazelnut Soybean

Amount (g) 5 5 5

Water (mL) 10 (0 avocado) 10 (0 avocado) 10 (0 avocado)

Spiking mg/kg 0.1 0.1 0.1

ACN (mL) 10 10 10

Phase separation

Supel QuE Citrate (EN) tube

Supel QuE Citrate (EN) tube

Supel QuE Citrate (EN) tube

Cleanup 1 Freezing out -20°C 1.5 h* Supel QuE Z-sep(EN) tube

Supel QuE Z-sep+ (EN) tube

Cleanup 2 0.5 g CaCl2

Cleanup 3Only for GC(Omitted for LC)

Supel QuE PSA/C18 (EN) tube

*Hans Ragnar Norli, Agnethe Christiansen, Ermias Deribe Journal of Chromatography A, 1218 (2011) 7234– 7241

Removal of lipids with freezing

Hans Ragnar Norli, Agnethe Christiansen, Ermias Deribe Journal of Chromatography A, 1218 (2011) 7234– 7241

Gravimetric Studies

No Cleanup In House Z-sep Z-sep+0

0.5

1

1.5

2

2.5

3

3.5

Avocado

Hazelnut

Soybean

mg/

mL R

esid

ue

Visual Inspection of Residues

No cleanup In house Z-sep Z-sep+

Percent residue removed

Avocado 0 89 59 27

Hazelnut 0 89 57 61

Soybean 0 94 57 51

No Cleanup In-House Z-sep Z-sep+

Hazelnut

Chromatograms (LC-MS/MS) Hazelnut extracts

Red= Z-sepGreen= Z-sep+Black= In-House

Full Scan: m/z 100-1000

Recovery and Precision

Hazelnut Avocado SoybeansRecovery %

In-House

Z-sep Z-sep+ In-House Z-sep Z-sep+ In-House

Z-sep Z-sep+

<60 8 6 10 7 6 13 8 5 11

60-69 4 2 5 2 1 4 1 0 1

70-120 84 89 76 87 89 68 77 90 63

121-140 1 1 4 12 2 10 10 2 12

>140 3 3 4 2 2 5 3 3 12

Precision%

< =20 93 98 98 91 96 99 94 96 63

> 20 6 2 2 9 4 1 5 4 37

Percent of pesticides of 333 within different recovery categories

Pesticides giving recoveries < 60 % in all three matrices after In house and Z-sep+ cleanup

Pesticide Recovery range (%) Pesticide Recovery range (%)

In-House Z-sep+ Pesticide In-House Z-sep+

Acephate 52-56 Propamocarb 50-54

4-Bromophenylurea 52-55 Prothiconazole- desthio

11-27

Aldicarb-sulfoxide 42-51 Tebuconazole 29-49

Chinomethionate 5-10 Thiabendazole 40-46

Diclofluanide 13-33 Tolylfluanid 12-36

Diniconazole 21-41 Trihclorfon 29-52

Fenamiphos 47-54 Tricyclazole 46-56

Fenamiphos-sulfoxide Triticonazole 21-40

Fenhexamid 35-56

Fluazinam 20-40

Hexaconazole 14-32

Metconazole 21-40

Omethoate 43-49

Pinoxaden 28-33

Prochloraz 56-58

Combination of matrix and pesticide with recoveries < 60 % in all cleanup methods

Pesticide Matrix Recovery range

Fenbutadin oxide Hazelnut 35-56

Carboxin Avocado 16-47

Benfuracarb Hazelnut 0-31

Diafenthiuron Avocado 13-27

Diafenthiuron Soybean 23-37

Recoveries < 60 % for all 9 combinations of matrices and cleanup methods

Pesticide Recovery range

Pymetrozine 5-59

Spirotetramat-enol 0-12

Cyromazine 20-55

Matrix Effects

• Normally related to quantification of pesticides using calibration standards in pure solvent

• Analyte signal interferences; either suppression or enhancement caused by co-eluting sample components

• Quantification is either over- or under-estimated

The nature of Matrix effects*

• In LC-MS/MS with electrospray signal suppression is most predominant – Caused by competition between matrix

components and analytes to obtain available protons

• Signal enhancement may be caused by co -elution of isobaric ions

*Journal of Chromatography A, 1304(2013)109-120

The nature of Matrix effects #

• In GC-MS/MS signal enhancement is caused by matrix components blocking active sites:– more analyte molecules can reach the detector

• Signal suppression can be observed when non-volatile compounds accumulate in the GC-system creating new active sites

# Talanta 140(2015)81-87

Matrix effects

Type Supression or Enhancement

Soft 0-20 %

Medium 20 -50 %

Strong > 50 %

Medium or strong matrix effects are overcome by matrix matched calibration standards, analyte protectants (GC) or dilution of sample

Slope Solvent > Slope matrix

OK ! !

Slope Solvent

Slope Matrix

Too Low!

Response

Concentration

𝑴𝑬 (% )=(( 𝑺𝒍𝒐𝒑𝒆𝒐𝒇 𝒄𝒂𝒍𝒊𝒃𝒓𝒂𝒕𝒊𝒐𝒏𝒄𝒖𝒓𝒗𝒆 𝒊𝒏𝒎𝒂𝒕𝒓𝒊𝒙𝑺𝒍𝒐𝒑𝒆𝒐𝒇 𝒄𝒂𝒍𝒊𝒃𝒓𝒂𝒕𝒊𝒐𝒏𝒄𝒖𝒓𝒗𝒆𝒊𝒏 𝒔𝒐𝒍𝒗𝒆𝒏𝒕 )−𝟏)𝒙𝟏𝟎𝟎

Slope Solvent < Slope matrix

Too High !

Slope Matrix

Slope Solvent

OK !

Response

Concentration

𝑴𝑬 (% )=(( 𝑺𝒍𝒐𝒑𝒆𝒐𝒇 𝒄𝒂𝒍𝒊𝒃𝒓𝒂𝒕𝒊𝒐𝒏𝒄𝒖𝒓𝒗𝒆 𝒊𝒏𝒎𝒂𝒕𝒓𝒊𝒙𝑺𝒍𝒐𝒑𝒆𝒐𝒇 𝒄𝒂𝒍𝒊𝒃𝒓𝒂𝒕𝒊𝒐𝒏𝒄𝒖𝒓𝒗𝒆𝒊𝒏 𝒔𝒐𝒍𝒗𝒆𝒏𝒕 )−𝟏)𝒙𝟏𝟎𝟎

Percent of pesticides in hazelnut within different matrix effect categories

Soft 0-20 (%) Medium 20-50 (%) Strong > 50 (%)

LC GC LC GC LC GC

In house 94 75 5 14 1 11

Z-Sep 94 78 3 17 3 5

Z-sep+ 96 86 4 10 0 4

Distribution of matrix effects GC-MS/MS (Hazelnut)

-60

-40

-20

0

20

40

60

80

100

Series1; 2992.45742092457In House

𝑀𝐸(%

)

-100

-80

-60

-40

-20

0

20

40

60

80

100

Series1; 138.454011741683

Z-Sep

𝑀𝐸(%

)

-100

-80

-60

-40

-20

0

20

40

60

80

100

Z-sep+ 𝑀𝐸(%

)

Distribution of matrix effects LC-MS/MS (Hazelnut)

-100

-80

-60

-40

-20

0

20

40

60

80

100

Series1; 818.586003789824

In-House

ME

(%)

-100

-80

-60

-40

-20

0

20

40

60

80

100

Series1; 844.43462195558

Z-sep

ME

(%)

-80

-60

-40

-20

0

20

40

60

80

Z sep+M

E (%

)

Distribution of matrix effects > 50 for HazelnutPesticide Instrument In-House Z-sep Z-sep+ Comments4-Chloroaniline GC -76 -78 Metabolite of diflubenzuronePhtalimide GC 69 Degradation product of folpetEPN GC 59Parathion methyl GC 59Dicofol, p,p'- GC 63 134Deltamethrin isomer # 1 GC 177 -52 -79 Always difficult on GC (Move to LC)Deltamethrin isomer # 2 GC 65 Always difficult on GC (Move to LC)Dichlofluanid GC 71Fluazinam GC 71 138 55Paraoxon GC 72Tolylfluanid GC 76Endosulfan sulfate GC 103Chlorothalonil GC 146Paraoxon-methyl GC 833Folpet GC 2992 -61Phenyl phenol -orto GC 91

Propyzamide GC -58Amitraz LC -89 -85 -86 Degrades!Bromukonazole isomer #2 LC 7723-OH-Carbofurane LC 817 UnstableCyanazine LC 8196Cl-4OH-Phenylpyridazine LC -657 Metabolite of pyridateAtrazine desisopropyl LC 783Flonicamid LC 844Phosmet oxon LC 789

Thanks to:

Gunvor: Sample prepartation and photograhps

Nina: LC-MS/MS results Agnethe: Experimental design and discussion of results

Sven: LC-MS/MS-Chromatograms