S OLID -P HASE E XTRACTION SPE is a cleanup technique where HPLC sorbents are used to separate analytes from

6.3.2 S OLID -P HASE E XTRACTION SPE is a cleanup technique where HPLC sorbents are used to separate analytes from

the sample matrix. While the sorbents used for SPE are in many cases chemically similar to those used for HPLC, the particle size is much larger. SPE differs from the HPLC process, in that the analyte is eluted stepwise, rather than a gradient increase (or decrease) of the solvent strength in the mobile phase used in gradient HPLC. While the resolution of HPLC columns is typically measured as thousands of theoretical plates, SPE columns are typically in the 10 –50 theoretical plate range [109]. The SPE process is usually conducted by eluting sample extracts through SPE columns (open syringe barrels or cartridges) or disks. Recently, dispersive SPE has been introduced, which involves mixing the sample extracts directly with SPE sorbents [12]. The most common types of sorbents commonly used for SPE cleanup

include: C 18 , styrene –divinyl benzene (SDVB), aminopropyl (NH 2 ), primary secondary amine (PSA), trimethyl ammonium strong anion exchange (SAX), graphitized carbon black (GCB), Florisil, silica, and alumina.

Two strategies have been used for SPE cleanup of sample extracts, which can

be referred to as analyte isolation and matrix isolation. Analyte isolation typically entails analytes that are adsorbed from an organic solvent –water mixture onto

a nonpolar SPE sorbent. The SPE column may be rinsed with an aqueous solution to remove interfering coextractives, followed by elution of the adsorbed analytes with an organic solvent. Matrix isolation entails using the SPE columns which act as ‘‘chemical filters’’ and trapping matrix components while the pesticides are eluted through the

column. Analyte isolation using nonpolar reversed phase SPE columns (C 18 or SDVB) works very well with nonpolar pesticides such as the OCs and the synthetic pyrethroids

Determination of Pesticides in Food of Vegetal Origin 159 but not with polar, water-soluble pesticides, as they may not be consistently adsorbed

onto nonpolar reversed phase SPE sorbents from an organic solvent –water mixture. Therefore, few pesticide multiclass MRMs use analyte isolation [110 –114].

Matrix isolation is more widely used in pesticide residue cleanup. Generally, matrix isolation SPE cleanup procedures are devised so that both polar and nonpolar pesticides will be eluted through the SPE column(s) while certain matrix components will be retained. The following section provides examples of how various SPE sorbents have been used for matrix isolation SPE cleanup. Further information on matrix isolation cleanups of sample extracts is provided in Table 6.1.

TABLE 6.1 SPE Column and Dispersive SPE Cleanups Used for the Analysis of Pesticides in Produce Samples

SPE Sorbent(s) a Pesticides b Elution Solvent c References SPE Columns

C 18 Multiclass

Acetone –water (7:3)

Acetonitrile –water (7:3)

Acetone –hexane (1:7)

[39] OH

EtAc –hexane (1:9)

[119] OH, PYR

Ethyl ether –PE (3:7)

[120] OH, OP, PYR

EtAc –hexane (3:7)

[108] Florisil=silica

EtAc –hexane (1:1)

[121] GCB

ON, OP

Acetone –PE (3:7)

Multiclass

Acetone –EtAc (1:1)

[118] OP

Acetonitrile –toluene (3:1)

[78] GCB=Florisil

Acetonitrile –toluene (3:1)

OH, PYR Hexane –DCM–acetone (10:60:30) [79] GCB=NH2

Multiclass

[27,116,117,124] OH, OP

Acetonitrile –toluene (3:1)

[125] GCB=PSA

Acetone –toluene (3:1)

Multiclass

Acetonitrile –toluene (3:1)

[126] OH, OP

Hexane –acetone (1:1)

[127] OP

Acetone –toluene (3:1)

[128] GCB=SAX=PSA

Acetone –toluene (3:1)

Multiclass

[28] NH 2 Multiclass

Acetonitrile –toluene (3:1)

[129 –131] NMC

Acetone

Methanol –DCM (1:99 or 2:98) [25,132 –135]

Methanol –acetone (3:7)

[63] SAX=PSA

[115] OH, ON

PE –acetone (2:1)

[3,4] OH, PYR

PE –acetone (2:1)

Acetone –hexane (3:7)

[137] (continued )

160 Analysis of Pesticides in Food and Environmental Samples

TABLE 6.1 (continued) SPE Column and Dispersive SPE Cleanups Used for the Analysis of Pesticides in Produce Samples

SPE Sorbent(s) a Pesticides b Elution Solvent c References Silica

[138] OH, OP, NMC

Acetone –DCM (1:1)

[39] Dispersive SPE

EtAc –hexane (3:7)

PSA Multiclass

[12,13,15,16,17,139,140] Fungicides

Acetonitrile

[18] OH, OP

[8] NMC, OP

[14] GCB=PSA

[141] a C 18 , Octadecyl-derivatized silica; GCB, graphitized carbon black; NH 2 , aminopropyl; PSA, primary secondary amine; SAX, strong anion exchanger.

OH

Acetonitrile –toluene (3:1)

b OH, Organohalogens; ON, organonitrogens; OP, organophosphates; PYR, synthetic pyrethroids; NMC, N -methylcarbamates.

c DCM, dichloromethane; EtAc, ethyl acetate; PE, petroleum ether.

6.3.2.1 C 18 SPE Cleanup Octadecyl (C 18 ) SPE columns have been used to filter out planar nonpolar sample

matrix components, primarily pigments and waxes, from sample extracts. When acetonitrile –water [11,25], acetone–water [4,5,115], or acetonitrile [37,116] extracts

are eluted through C 18 SPE columns, nonpolar pigments and lipids will be retained in the column, whereas both nonpolar and polar pesticides will be eluted through the column. Dispersive SPE with C 18 may also be used for the cleanup of plant pigments in acetone –water produce sample extracts (F.J. Schenck, unpublished data, 2006).

6.3.2.2 NH 2 and PSA SPE Cleanup The most effective SPE sorbents for the matrix isolation cleanup of plant extracts

are aminopropyl (NH 2 ) and ethylenediamine-N-propyl (PSA) (see Figure 6.1) [12,21,126,142]. These two sorbents are weak anion exchangers and will interact strongly with acidic matrix components, such as fatty acids, to the extent that strong organic solvents, such as acetone and acetonitrile, are incapable of desorbing them. This is important since high concentrations of fatty acids (e.g., oleic, linoleic, and palmitic acids) in food extracts will produce ions that can interfere with the GC-MS selected ion monitoring (SIM) determination of coeluting pesticides [126,129].

NH 2 and PSA are the most effective sorbents for reducing the effects of sample matrix enhancement [143]. Dispersive SPE with PSA sorbent, which greatly reduces the time and effort required for an SPE cleanup, has been widely used (see Table 6.1). Dispersive SPE cleanup with PSA is not as effective as PSA SPE columns [129], especially for the HPLC-postcolumn derivatization fluorescence determination of

Determination of Pesticides in Food of Vegetal Origin 161

No cleanup

C 18 + GCB + SAX

PSA

FIGURE 6.1 GC=ECD chromatograms before and after SPE cleanup of sample extracts from a blank asparagus sample. Asparagus was extracted with acetone using the method of Luke et al. [35].

(c) 0 5 10 15 20 25 30 35 min FIGURE 6.2 LC-FLD chromatograms of (a) mixed carbamate standard 1.0 ng=mL in

methanol (peak identities are aldicarb sulfoxide, aldicarb sulfone, oxamyl, methomyl, 3-OH carbofuran, aldicarb, propoxur, carbofuran, carbaryl, isoprocarb, BDMC, methiocarb); (b) blank green bean-QuEChERS acetonitrile extraction with PSA-dispersive SPE cleanup; and (c) blank green bean-QuEChERS acetonitrile extraction with PSA SPE column cleanup.

162 Analysis of Pesticides in Food and Environmental Samples

6.3.2.3 Tandem GCB=NH 2 , GCB=PSA, and GCB=SAX=PSA SPE Column Cleanup

GCB adsorbs planar compounds, and has been widely used for the removal planar molecules like pigments and sterols from produce sample extracts. GCB will not remove fatty acids and other polar compounds, thus it is usually used in combination

with NH 2 or PSA SPE sorbents. These two sorbents when used together provide an excellent cleanup. The problem with GCB is that it may retain planar aromatic pesticides such as hexachlorobenzene (HCB) [12,16,27]. Thus, solvent mixtures containing toluene are commonly used to elute pesticides through GCB columns. Acetonitrile –toluene (3:1) mixtures have been used for the multiclass MRM elution

of pesticides through tandem GCB-NH 2 [27,116,124], GCB-PSA [21], and GCB-SAX-PSA [28]. Schenck and Howard-King [125,127] found acetone –toluene (3:1) to be a better elution solvent than acetonitrile –toluene (3:1) since much smaller

volumes of solvent were needed to elute HCB through tandem GCB-NH 2 or GCB-PSA SPE columns.

6.3.2.4 Alumina, Florisil, and Silica SPE Column Cleanup Alumina (Al 2 O 3 ), silica (SiO 2 ), and Florisil are very polar sorbents that have

been used for the cleanup of sample extracts for many years. While these three sorbents provide a very effective cleanup, they will oxidize, decompose, or adsorb polar OPs pesticides [40]. They are suitable for the cleanup of nonpolar analytes such as OCs and pyrethroids. Some multiclass MRMs entail splitting the sample extract, cleaning up part of the extract on Florisil for OH analysis, and using the part that had not been cleaned up on Florisil for OP analysis [11].