Investigations on the use of analyte protectants for multiresidue GC analysis

Michelangelo Anastassiades, Bünyamin Tasdelen, Ellen Scherbaum

Chemisches und Veterinäruntersuchungsamt Stuttgart, Schaflandstr. 3/2, 70736 Fellbach, Germany

References and Acknowledgement

[1] M. Anastassiades and S.J. Lehotay, in A. Di Muccio (Editor), European Pesticide Residues Workshop, EPRW, Rome, 2002.

[2] M. Anastassiades, K. Maštovska and S.J. Lehotay, J Chromatogr A 1015 (2003) 163.

[3] K. Maštovska, S.J. Lehotay and M. Anastassiades, Anal Chem 77 (2005) 8129

The project has been financially supported by the Landesstiftung Baden-Württemberg GbmH in Germany.

Summary

Experiments

Results

55 potential APs have been individually tested for their ability to protect a range of (67) pesticides representing various classes:

Carbaryl

Ditalim

phosParathion-mD

icofolC

aptanFolpetC

hlorothalonilD

iclofluanidTolyfluanidC

lozolinateTPPProcym

idon

pH 4pH 4.5

pH 5pH 5.5

pH 60

1

relative respons

Malic Acid

Introduction

Matrix-induced enhancement effects are well-known to seriously affect measurement accuracy in GC applications, mostly leading to overestimated results, when using the convenient matrix free calibration standards. There are various approaches on how to deal with matrix effects: Intensive cleanup of the extracts, instrument maintenance measures including the use of inert materials may reduce but not eliminate this effect. The use of matrix-matched standards and the standard additions approach can effectively overcome this problem, but they are often troublesome. The use of isotopically labelled ISTDs is the best approach, but it is currently not feasible in multiresidue analysis due to the high price and limited availability of the standards.The use of “analyte protectants” (APs) was first introduced 4 years ago, at the EPRW in Rome, as a concept to deal with the notorious problem of matrix effects [1-3]. APs are chemicals, that are added to sample extracts and calibration solutions (e.g. prepared in neat solvent) to improve chromatographic behaviour of pesticides and to equalize the matrix effects. The function of APsis to interfere with the interactions that pesticides undergo with active sites on the GC-inlet and column surfaces and that typically lead to degradation and/or peak broadening in GC separations. The most effective APs are polar compounds with the ability to undergo H-bonds such as sugars and their derivatives. APs are added, individually or in mixtures, to extracts at concentrations much higher than those of the pesticides to be protected. In the initial study some representative pesticides were tested [2,3]. In this study the influence of APs on a greater number of GC-amenable pesticides and their applicability in routine multiresidue analysis is being investigated.

PSA cleanup

Addition of „Analyte

Protectants“(AP)

„ Deg

ree

of an

alyt

epro

tect

ion”

Raw Extract

Cleaned up Extract

Cleaned-up Extract + AP

Standard + AP

Standard in Pure Solvent

APs reduce analyte interactions with active

sites and thus errors related to matrix-induced

peak enhancement phenomena

Analyte Protectants- Principle

PSA cleanup

PSA cleanup

Addition of „Analyte

Protectants“(AP)

Addition of „Analyte

Protectants“(AP)

„ Deg

ree

of an

alyt

epro

tect

ion”

Raw Extract

Raw Extract

Cleaned up Extract

Cleaned up Extract

Cleaned-up Extract + AP

Standard + AP

Cleaned-up Extract + AP

Standard + AP

Standard in Pure Solvent

Standard in Pure Solvent

APs reduce analyte interactions with active

sites and thus errors related to matrix-induced

peak enhancement phenomena

Analyte Protectants- Principle

In order to determine the overall most promising APs, a valuation system was employed, where peak height, peak area as well as the ratio thereof (as a measure of peak shape) were taken into account. The overall ranking of the AP-candidates (over the entire pesticide range) is shown in the table to the left. The APs were added at 1 mg/mL in most cases (ethylglycerol 10 mg/mL, shikimic acid 0.5 mg/mL). As in the previous study sugar derivatives were the overall most efficient APs. Acidic APs were found to effectively protect base-sensitive compounds such as captan, folpet and chlorothalonil. However they were also shown to negatively affect basic pesticides. A negative impact of AP-candidates on certain pesticides was observed in several cases. A differentiated evaluation of the various APs is thus necessary. The general correlation bet-ween the volatilities of the APs and the pesticides, as regards the protection potential, observed in the previous studies could be confirmed. So that early eluting APs better protected early eluting pesticides.

2-Deoxy-D-ribose, 4,6-O-Ethylidene-a-D-glucose, 4-Hydroxybenzoic acid, Adipic acid, Allylacetic acid, Formic acid, Succinic acid, Pyruvic acid, Brij92 (Diethylene glycol oleyl ether), Butyric acid, Citric acid, Caffeine, D-(+)-Galactose, D-(+)-Gluconic acid-δ-lactone, D-(+)-Glucose, D-(+)-Ribonic acid-γ-lactone, D-(+)-Xylose, D-Fructose, Diethylene glycol, DL-Malic acid, D-Mannite, D-Sorbitol, Acetic acid, Ethylene glycerol, Fumaric acid, Polyethylene glycols (mixture of), Gallic acid, Glutaric acid, Glycolic acid, Glycyl-glycine, Hexaethylene glycol, Caffeic acid, L-(+)-Arabinose, L-(+), Gulonic acid-γ-lactone, Lactose, L-Sorbose, Maleic acid, meso-Erythitol, Lactic acid, myo-Inositol, Oxalic acid, Pentaethylene glycol, Pimelic acid, Propyl gallate, Adonitol, Sucrose, Shikimic acid, Sorbic acid, Tetraethylene glycol, Triethylene glycol, Triglycerol, Valeric acid, Vanillic acid, Tataric acid, Xylitol

(E)

OH

OH

OH

O

OH

OH OOH

OH OH

OH

OHOH

OHOO

OH

OH

OH

HO

AP 1-Mix

AP 2-Mix

δ-Gluconolactone

Shikimic acid

Sorbitol

Ethyl glycerol

applies to the impact of acidic APs on base-sensitive pestici-des. In this case, a „measured pH“ of 4 corresponded to an amount of 0.35 mg malic acid per mL acetonitrile (with 2 % water). Pesticides known to make problems in GC-analysis have been shown to be effectively protected during GC-Injection upon addition of acids. Among the compounds protected are also some compounds that are also problematic in LC-MS/MS analysis (captan, folpet, chloro-thalonil, dicofol), so that GC analysis still remains essential.

The protective potential of APs towards susceptible analytes depends not only on its type (chemical structure) but also on the amount added to the extract. As shown in the figure below, this concentration-dependence also

Based on the results of the ranking study, the initially suggested AP-mixture con-sisting of Sorbitol and Ethyl-glycerol (AP1) was extended by δ-Gluconolactone to better protect some intermediate eluting pesticides as well as Shikimic acid to protect base sensitive compounds (AP2).

The figure below shows how the addition of the APs to the solvent-free standard solutions used for calibration as well as to the matrix extracts (QuEChERS) does not only significantly improve the peak shapes and resolution but it also helps to significantly reduce quantification errors of “difficult” GC-compounds.

The concept of using „Analyte Protectants“ in order to improve the GC analysis of problematic compounds has been more thoroughly studied. Two new APs (δ-Gluconolactone and Shikimic acid) were added to the generic-AP mixture in order to better protect base-sensitive pesticides.The usefulness of the AP-concept to deal with errors related to matrix-induced enhancement effects in a convenient way (without the need of matrix matching) could be confirmed.

Ranking Analyte Protectant Score 1 D-(+)-Gluconic acid-δ-lactone 2.06 2 4,6-O-Ethylidene-α-D-glucose 1.77 3 L-(+)-Arabinose 1.76 4 Triglycerol 1.72 5 D-Fructose 1.72 6 D-Sorbitol 1.72 7 Brij ' 92 1.60 8 D-(+)-Glucose 1.59 9 2-Deoxy-D-ribose 1.57 10 Adonitol 1.56 11 Xylitol 1.56 12 Shikimic acid 1.55 13 D-(+)-Xylose 1.55 14 D-(+)-Ribonic acid-γ-lactone 1.42 15 Polyethylene glycols (mix of) 1.37 16 AP Mix 1 1.36 17 L-Sorbose 1.36 18 L-(+)-Gulonic acid-γ-lactone 1.25 19 D-Mannite 1.24 20 Ethyl glycerol. 1.21 21 meso-Erythitol 1.08 22 Vanillic acid 1.06 23 Citric acid 1.01 24 Gallic acid 0.96 25 Caffeic acid 0.83

40 DL-Malic acid 0.25

0

20

40

60

80

100

120

o-Phenylphenol Captan Folpet Chlorothalonil Carbaryl

in pure solvent Cucumber extract Solvent+AP2 Cucumber + AP2

1 % 1 % 7 % 10 % 10 %

Bias

22 % 160 % 60 % 26 % 58 %

Enhancement factor 2,1 1,61,81,41,1

Carbarylpure solvent

AP2: Sorbitol (0.15 mg/mL)

Ethylglycerol (3 mg/mL)Gluconolacton (1 mg/mL)Shikimic acid (0.5 mg/mL)

cucumber

solvent + AP2

cucumber + AP2

relative respons

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