1

On-line Solid-Phase Extraction for Liquid Chromatography-Mass Spectrometry 1

Analysis of Pesticides 2

Paolo Luccia and Oscar Núñez

b,* 3

a Department of Nutrition and Biochemistry, Faculty of Sciences, Pontificia Universidad 4

Javeriana, Bogotà, D.C., Colombia. 5

b Department of Analytical Chemistry, University of Barcelona. Martí i Franquès 1-11, 6

E08028 Barcelona, Spain 7

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* Corresponding author: Oscar Núñez 9

Department of Analytical Chemistry, University of Barcelona 10

Martí i Franquès 1-11, E-08028, Barcelona, Spain. 11

Phone: 34-93-403-3706 12

Fax: 34-93-402-1233 13

e-mail: oscar.nunez@ub.edu 14

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KEYWORDS: Pesticides; Preconcentration; Liquid chromatography; Mass 16

spectrometry; Water analysis 17

Abstract 18

Public concern about pesticides in food and water has increased dramatically in 19

the last two decades. In order to guarantee consumers’ health and safety, analytical 20 methods that could provide fast and reliable answers without compromising accuracy 21 and precision are required. 22

Sample treatment is probably the most tedious and time-consuming step in many 23 analytical procedures and, despite the significant advances in chromatographic 24

separations and mass spectrometry techniques, sample treatment is still one of the most 25

important parts of the analytical process for achieving good analytical results. 26

Therefore, over the last years, considerable efforts have been made to simplify the stage 27 and to develop fast, accurate and robust methods which allow the determination of a 28 wide range of pesticides without compromising the integrity of the extraction process. 29

This review article intends to give a short overview of recently developed on-30 line solid-phase extraction, pre-concentration and clean-up procedures for the 31

determination of pesticides in complex matrices by liquid chromatography-mass 32 spectrometry techniques. 33

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1. Introduction 36

Pesticides are substances employed in agriculture because of their capacity to 37

protect crops against a wide range of threats from weeds, fungal diseases and insect 38

pests, avoiding economic losses and increasing agricultural productivity and 39

performance. However, in spite of these advantages, they must be carefully used 40

because they can cause serious environmental pollution and serious health effects such 41

as diseases of the nervous system, reproductive disorders, cancer or even genetic 42

problems [1]. Because of the huge volumes of production of these compounds and their 43

continuous worldwide use, some pesticides have become relatively persistent substances 44

in the environment. Thus, residues of these compounds and/or their transformation 45

products may easily leach from pesticide application point sources on soil surfaces or 46

infiltrate from environmental waters (lakes or rivers) that were polluted as a result of 47

direct application and/or runoff, and finally reach aquifers and alter ground-water 48

quality which often are used as sources for drinking water supply [2,3]. Consequently, 49

pesticides must undergo extensive efficacy, environmental and toxicological testing to 50

be registered by governments for legal use in specified applications. The monitoring of 51

pesticide residues in both food and water is nowadays a priority objective in order to get 52

extensive evaluation of food and water quality and to avoid possible risks to human 53

health. For instance, very low pesticide residue limits (10 g/kg) in fruits and vegetables 54

intended for baby food production have been established by the European Union [4]. 55

Regarding pesticide pollution in environmental waters, the United States Environmental 56

Protection Agency (US-EPA) has established maximum concentration levels (MCLs) 57

for some pesticides in drinking water, such as atrazine (3.0 mg/L) 58

(http://www.epa.gov/oppsrrd1/reregistration/atrazine/atrazine_update.htm). European 59

legislation is more restrictive and the Council Directive 80/778/EEC [5] has set limits 60

for pesticides at 0.1 g/L for individual pesticides and 0.5 g/L for the sum of all 61

pesticides in water used for human consumption, while Directive 2008/105/EC [6] set 62

environmental quality standards for priority substances and certain pollutants in surface 63

waters, such as atrazine (0.6 g/L) and diuron (0.2 g/L). 64

Traditionally, gas chromatography-mass spectrometry (GC-MS) have been 65

proposed for the analysis of pesticides [7-11], and frequently by using well established 66

library searching routines. But today, liquid chromatography coupled with tandem mass 67

spectrometry (LC-MS/MS) is becoming a powerful tool for pesticide residue analysis in 68

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complex matrices [12-17] due to its selectivity and sensitivity, a substantial reduction of 69

sample treatment steps compared with other methodologies such as GC-MS(/MS), and 70

its reliable quantification and confirmation at the low concentration levels required by 71

legislation. Moreover, the demands of high sample throughput in short time frames have 72

given rise to high efficiency and fast or even ultra-fast LC methods, which are 73

becoming also very popular for the analysis of pesticides. Among the several modern 74

approaches in HPLC methods that enable the reduction of the analysis time without 75

compromising resolution and separation efficiency, UHPLC methods either using sub-76

2m totally porous particle-packed columns or partially porous core-shell columns 77

(with sub-3 m superficially porous particles) are among the most popular in the 78

analysis of pesticides [18-20]. 79

Nevertheless, due to the increased number of pesticides used worldwide and the 80

variety and complexity of food and environmental matrices, the use of ultra-fast 81

separations is not enough to develop fast analytical methods for the analysis of pesticide 82

residues. Besides, multi-residue screening methods able to analyze not only target but 83

non-target pesticide residues or even unknown compounds are demanded. Nonetheless, 84

despite the advances in chromatographic separations and mass spectrometry techniques, 85

sample treatment is still one of the most important parts of the analytical process and 86

effective sample preparation is essential for achieving good analytical results [21]. Ideal 87

sample preparation methods should be fast, accurate, precise and must keep sample 88

integrity. Therefore, over the last years, considerable efforts have been made to develop 89

modern approaches in sample treatment techniques for the analysis of pesticide residues 90

without compromising the integrity of the extraction process. 91

In 2003, Anastassiades et al. [22] developed an ideal sample extraction and 92

clean-up procedure named QuEChERS, acronym of “Quick, Easy, Cheap, Effective, 93

Rugged, and Safe” which has become particularly popular to determine moderately 94

polar pesticides residues in various food matrices [23,24]. However, sample 95

manipulation is still required when employing QuEChERS. The use of on-line solid-96

phase extraction (SPE), which minimizes sample manipulation and provides both high 97

preconcentration factors and recoveries, is becoming very popular in the analysis of 98

pesticide residues in environmental water samples [25-27]. It is an increasingly 99

powerful and rapid technique used to improve the sample throughput and overcome 100

many of the limitations associated with the classical off-line SPE procedure. Other more 101

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SPE-based selective approaches such as the use of molecularly imprinted polymers 102

(MIPs) have also been proposed for the analysis of pesticides [25]. 103

In this manuscript, the state-of-the-art of on-line SPE methodologies for the LC-104

MS analysis of pesticide residues in complex matrices such as food and environmental 105

water samples will be reviewed. We are aiming to give a short overview of recently 106

developed on-line SPE extraction, pre-concentration and clean-up procedures, so the 107

advantages and disadvantages of on-line SPE versus off-line SPE procedures, the most 108

frequently used SPE sorbents for the clean-up and preconcentration of pesticides, and 109

applications of on-line SPE to pesticide residue analysis will be described by means of 110

relevant application examples. 111

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2. On-line SPE versus off-line SPE 113

The choice of the proper sample treatment methodology in pesticide residue 114

analysis depends on several aspects, such as the sample matrix composition and the 115

physical-chemical properties of the target pesticides. Taking into consideration that 116

these compounds are usually found at very low concentration levels (ng/L-µg/L), 117

sample treatment undoubtedly represents one of the most challenging parts of the 118

overall analytical method for obtaining adequate preconcentration and clean-up 119

efficiencies [17]. 120

SPE is probably still the most frequently used procedure in sample preparation 121

for pesticide, veterinary and other residues in food and environmental samples since it 122

offers many benefits and advantages over other sample preparation methods. In fact, 123

SPE is fast, robust and highly versatile, offering users a reliable tool to successfully 124

perform different analytical tasks, such as purification, trace enrichment, desalting, 125

derivatization, solvent exchange and class fractionation. In principle, SPE is analogous 126

to liquid-liquid extraction (LLE) since it involves partitioning, but in this case between a 127

liquid (sample matrix or solvent with analytes) and a solid sorbent phase with 128

compounds that are extracted from the sample and adsorbed onto the sorbent material, 129

thus providing concentrated sample extracts that are almost free of interfering matrix 130

components [24]. 131

Two main different SPE approaches are currently available: off-line and on-line 132

procedures. Off-line SPE is a very simple technique to use that employs economical and 133

uncomplicated equipment formed by disposable extraction columns, microplates and 134

cartridges available in a wide range of reservoir volumes, formats and sorbents. Figure 1 135

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shows the general SPE process which basically consists in four different steps: a) the 136

solid sorbent is wetted by an appropriate solvent in order to activate the functional 137

groups on its surface (conditioning step); b) sample is percolated through the sorbent 138

(loading step); c) clean-up by washing the sorbent with a solvent of low elution strength 139

to eliminate matrix components that have been retained on the solid sorbent (washing 140

step); and d) elution of analytes of interest by employing an appropriate solvent with a 141

higher elution strength (elution step). Off-line SPE methodology has been extensively 142

used to extract pesticides and, despite the fact that today there is a general trend towards 143

on-line methods, it is probably still the most widely used configuration in sample 144

preparation for environmental and food analysis in general [28-30]. Off-line SPE 145

approach remains a useful technique for analyzing pesticides in complex matrices, 146

because of its greater flexibility and whenever elution solvent is not compatible with the 147

subsequent method of analysis [24]. For instance, a simple and sensitive method for the 148

simultaneous determination of 103 pesticide residues in tea by using LC-MS/MS was 149

recently developed and validated by Huang et al. [31]. The residual pesticides were 150

extracted from spiked and real tea samples with ACN and then purified using Carb-NH2 151

SPE cartridges. Good recoveries ranging from 65 to 114% with intra-day precisions 152

lower than 19.6% were obtained. An off-line SPE gas chromatography–mass 153

spectrometry was also proposed by Ma et al. [32] for the determination of 154

organophosphorus pesticides (dichlorovos, methyl parathion, malathion, and parathion) 155

in underground water. The method showed limits of detection for spiked water samples 156

in the range of 4–10 ng/L together with satisfactory recoveries for all analytes (59.5-157

94.6%). Another example highlighting the versatility and usefulness of off-line SPE 158

procedure has been reported by Kouzayha et al. [33]. In this study, the authors 159

developed a new multi-residue method based on off-line SPE modality for 160

determination and quantification of 67 pesticides in water samples. The use of a 161

centrifugation technique in both the drying and elution steps allowed good recoveries 162

(higher than 65-68% for the 67 analyzed pesticides) with relative standard deviations 163

lower than 12.3%. However, off-line SPE procedure also presents several weaknesses 164

which include: time consuming and labor-intensive steps, requirement of large amount 165

of organic solvents, and possible loss of analytes during the evaporation steps or sample 166

manipulation that may lead to sample contamination, and less accuracy and precision 167

with respect to the on-line mode. These aspects, together with the demands of high 168

sample throughput in short time frames, have given rise to high efficiency and fast or 169

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even ultra-fast analytical methods that employ automated and/or on-line SPE procedures 170

[34]. In this context, on-line SPE modality has become very popular over the last few 171

years for the analysis of pesticides in complex samples [25]. In fact, on-line SPE has 172

several advantages over off-line methods such as high sample throughput, reduced 173

sample manipulation, improved precision, and low reagent consumption. Furthermore, 174

on-line systems are especially beneficial when the amount of sample is limited, or when 175

very high sensitivity is required because of the low concentration of target molecules in 176

the sample. Although SPE technique can be easily coupled on-line to liquid 177

chromatography (LC) and gas-chromatography (GC) systems, analytical methods which 178

combine SPE with LC are the most frequently used approach. Different systems and 179

configurations are available [24]. As an example, Figure 2 shows the most common on-180

line SPE configuration which normally involves the implementation of a small SPE 181

column within the injection loop of a six-port rotary valve. The SPE column should 182

usually be as small as possible (i.e., 30 x 2 mm i.d. or 8 x 3 mm i.d.) in order to prevent 183

band broadening leading to poor resolution and chromatographic performance. 184

Furthermore, SPE column sorbent must be obviously compatible with the one of the 185

analytical column. As an example, Viglino et al. [35] used an automated on-line SPE 186

method combined with LC-electrospray-tandem mass spectrometry (LC-ESI-MS/MS) 187

with positive electrospray ionization for the simultaneous analysis of pharmaceuticals, 188

pesticides and some metabolites in drinking, surface and wastewater samples. The total 189

analysis time, including the period required to flush the C18 SPE cartridge with organic 190

solvent and reconditioning the LC column, was only 20 min, thus overcoming some of 191

the limitations associated with the classical off-line SPE (i.e., tedious and time 192

consuming sample procedure) and making possible the development of a faster method 193

together with high preconcentration factors and recoveries. On the other hand, Díaz-194

Plaza et al. [36] reported an on-line coupling reversed-phase liquid chromatography–gas 195

chromatography method for organophosphorus, organochlorine and triazine pesticides 196

using the through oven transfer adsorption desorption (TOTAD) interface with 197

subsequent simultaneous electron-capture and nitrogen–phosphorus detection by post-198

column splitter. The use of fully automated on-line RP LC/GC has also been reported 199

for the determination of four organophosphorus pesticides (dimethoate, methidathion, 200

chlorpyriphos and fenitrothion) in olive oil [37,38]. However, the majority of reports on 201

the application of on-line SPE describe pesticides monitoring in aqueous environmental 202

samples with only a few that have been applied to food analysis. Examples of the latter 203

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include analysis of mepiquat and chlormequat in pears, tomatoes, and wheat flour [39], 204

and N-methylcarbamates (oxamyl, dioxacarb, metolcarb, carbofuran, carbaryl and 205

isoprocarb) and their metabolites in apple, pear and cucumber samples [40]. 206

If on-line SPE mode has several advantages over traditional off-line SPE, it also 207

has limitations and drawbacks. For instance, certain drawbacks of the on-line approach 208

are the possibility of memory effects and progressive deterioration of the column 209

sorbent when the SPE column is re-used. The latter may lead to important changes in 210

the SPE selectivity and capacity. Furthermore, on-line SPE approach also possesses less 211

flexibility regarding the choice of eluting solvents since usually it must be the same as 212

the mobile phase used in the analytical column. Finally, in most cases, even though the 213

use of an automated on-line instrument is quite straightforward, experienced personnel 214

are required for method development and eventual trouble-shooting [41]. 215

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3. Sorbents used for on-line SPE 217

Different types of sorbents are currently available to carry out SPE of pesticides. 218

To achieve optimal SPE extraction performances, one of the key factors is therefore the 219

choice of the proper type of sorbent since its nature strongly controls parameters of 220

primary importance such as selectivity, affinity and capacity [42]. This choice obviously 221

depends on the physical-chemical properties of the target pesticides as well as the on the 222

nature of the sample matrix. In fact, the SPE extraction performances are determined by 223

the interaction of pesticides with the chosen sorbent as well as of the interaction of 224

sample matrix with both sorbent and pesticides [24]. Several sorbents have so far been 225

employed for SPE extraction of pesticides, including alumina, silica gel, Florisil, ion-226

exchange resins, octadecyl-, octyl-, phenyl-silica based sorbents and graphitized black 227

carbon (GBC) [43]. Schenck et al. [44] have recently evaluated the clean-up and 228

extraction efficiencies of pesticide residues in fresh fruits and vegetables using GBC, 229

octadecylsilyl, strong anion exchange, aminopropyl, and primary secondary amine 230

(PSA) SPE columns. As a result, aminopropyl and PSA sorbents were found to provide 231

the most effective clean-up, removing the greatest number of sample matrix 232

interferences whereas GBC removed most of the visible plant pigment in the extracts 233

but also showed a lower ability to remove fatty acid matrix interferences. Furthermore, 234

the authors revealed that using an acetone extraction followed by a PSA clean-up, both 235

polar and non-polar pesticides present in samples at 1.0 ng/g could be recovered. 236

However, in general, polar-SPE (silica, alumina, Florisil) is more suitable for the clean-237

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up procedure of most apolar pesticides (i.e., organochlorine and some 238

organophosphorus compounds) from organic extracts. On the other hand, octadecyl-239

bonded silica (C18) allows retention of a wide variety of analytes with different 240

polarities. In the last few years, other novel material technology, such as monoliths [45], 241

molecularly imprinted polymers (MIP) [45,46], immunoaffinity columns (IAC), and 242

hydrophilic polymeric sorbents have also been introduced as SPE sorbents. For 243

instance, MIPs are synthetic and intelligent materials with an artificially generated 244

three-dimensional network that is able to specifically rebind a target molecule [45-49]. 245

MIP has the advantages to be cost-effective, and chemically and thermally stable 246

without storage limitations and stability problems regarding organic solvents [50,51]. 247

Recently, magnetic or magnetically modified adsorbents materials [52] as SPE sorbents 248

have been shown to allow high degree of clean up and enrichment factors in the analysis 249

of pesticide residues in environmental samples. The most widely used magnetic 250

nanoparticle (NP) for residue analysis is probably C18 fabricated Fe3O4 core-shell NPs 251

that have proved to be suitable for the preconcentration or cleaning up of both non polar 252

and moderately polar pesticides [53]. Graphene-based SPE has also been proposed for 253

the determination of organophosphorus pesticide residues in apple juices, showing 254

average recoveries of the analytes at two spiked levels (5 and 20 ng/mL) for real-sample 255

analysis of 69.8-106.2% [30]. However, only few of these SPE sorbents have been 256

applied in the on-line mode. The fact that many solid phases, such as GBC, are not 257

pressure resistant is one possible reason. Hypercrosslinked sorbents, which can be 258

effectively packed in precolumns, allows to overcome this problem thus offering an 259

alternative for the on-line enrichment of polar pollutants from aqueous samples [54]. 260

Among all the possible sorbents for the SPE extraction, on-line SPE using hydrophilic 261

lipophilic balanced (HLB) sorbent is probably one of the most popular analytical 262

choices. For instance, the direct coupling of an on-line Oasis HLB SPE cartridge to LC–263

MS/MS has been developed by Stoob et al. [55] for the analysis of neutral (triazines, 264

phenylureas, amides, chloracetanilides) and acidic (phenoxyacetic acids and triketones) 265

pesticides in natural water. Absolute extraction recoveries from 85 to 112% were 266

obtained for the different analytes. Detection limits for environmental samples between 267

0.5 and 5 ng/L were obtained, and matrix induced ion suppression smaller than 25% 268

was also observed. Recently Togola et al. [56] highlighted the advances of on-line SPE 269

coupled with ultra-high performance liquid chromatography (UHPLC)-MS/MS for 270

determining the fate of 18 pesticides and their degradates in aquatic organisms exposed 271

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in mesocosms. Limits of quantification from 15 to 25 ng/L for pesticides and 272

metabolites have been obtained, with linearity range up to 1 μg/L. On the other hand, 273

on-line Hypersil GOLD C18 column has also been showed to achieve detection limits in 274

the range of 2 to 24 ng/L for the compounds of interest by using only 1 mL of filtered 275

water sample. Good recoveries from 87 to 110% in surface as well as wastewater 276

samples were obtained whereas matrix effects observed for some compounds was lower 277

than 25% [35]. 278

Finally, it is apparent that the development of new selective materials for a given 279

application as well as of universal sorbent suitable for every purpose will continue to be 280

a major part of the scientific research and technological innovation in the field of SPE, 281

with special attention to the development of high pressure resistant sorbents able to be 282

coupled on-line with fast or even ultra-fast analytical methods (i.e., UHPLC). 283

284

4. Applications of on-line SPE to pesticide residue analysis 285

On-line SPE coupled to LC-MS(/MS) methods are becoming very popular for 286

the preconcentration and analysis of pesticide residues especially in water samples, and 287

many examples of their application varying in number of pesticides and the type of SPE 288

sorbent used can be found in the literature. In Table 1, a selection of some of these 289

examples published in the last years is presented [35,57-63]. Columns and SPE 290

cartridges based on C18 can be found among the most commonly used sorbents for the 291

on-line SPE preconcentration of pesticides in water samples [35,63-65]. For instance, 292

Hernández et al. [65] reported the use of an C18 SPE cartridge (10x2 mm) for the on-293

line SPE-LC-MS/MS analysis of 29 pesticides (1 fungicide, 16 insecticides, 10 294

herbicides and 2 acaricides) and 6 metabolites in environmental water samples by the 295

direct injection of only 1.3 mL of filtered water sample, with a total analysis time of 18 296

min. Figure 3 shows, as an example, the chromatograms of two positive water samples. 297

Recently, Sun et al. [63] proposed the use of a C18 SPE column (7.5x4.6 mm, 10 µm 298

particle size) for the on-line SPE LC-MS/MS analysis of 9 emerging pesticides in lake 299

water and seawater samples at trace level. To enhance method sensitivity, the authors 300

proposed the use of a large enrichment water sample volume (50 mL), allowing the 301

detection of these compounds at 1-10 ng/L. 302

Polymeric sorbents are also commonly employed for the on-line SPE 303

preconcentration of pesticides, such as PLPR-s (macroporous spherical particles of 304

polystyrene and divinylbenzene), which is usually employed for the analysis of medium 305

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to highly polar pesticides [58,60-62,66-69], or Strata-X SPE (polymeric sorbent with N-306

vinylpyrrolidine functional groups) [59]. For example, Köck-Schulmeyer at al. have 307

been using PLPR-s sorbents for the on-line SPE LC-MS/MS monitoring pesticides in 308

wastewater treatment plants, river waters and groundwaters from Catalonia (Spain) 309

[61,62,69]. With the proposed method, and using a quadrupole-linear ion trap MS 310

instrument, limits of detection down to 5 ng/L (groundwater samples) with good 311

accuracies and precisions were achieved. 312

An interesting application is the one recently reported by Huntscha et al. [57] 313

using a home-made single mixed-bed multilayer cartridge with 4 extraction materials 314

for the on-line SPE LC-MS/MS multi-residue analysis of 88 polar organic 315

micropollutants with a broad range of chemical properties (pesticides, biocides, 316

pharmaceuticals, corrosion inhibitors, and many of their transformation products) in 317

ground, surface and wastewater samples. The SPE cartridge was prepared in-house by 318

filling an empty stainless steel cartridge with 10 mg Oasis HLB (hydrophilic-lipophilic 319

balance, Waters) as the first material in the enrichment flow direction. As second 320

material, 10 mg of a mixture of Strata X-AW, Strata X-CW (both from Phenomenex) 321

and Isolute ENV+ (Biotage) in a ratio of 1:1:1.5 (X-AW:X-CW:ENV+) was used. 322

Although hydrophobicity is, in general, the driving force for the enrichment of 323

compounds on typical reversed-phase materials such as alkyl-modified silica or 324

poly(styrene-divinylbenzene) polymers, the introduction of new polymeric sorbents 325

with novel functional groups in the polymeric structure extended the applicability of 326

SPE to more hydrophilic compounds. For instance, the use of Oasis HLB proposed by 327

Huntscha et al. [57], which provides lipophilic (divinylbenzene-rings) and hydrophilic 328

(N-vinyl-pyrrolidine) groups, allows to achieve retention of both non-polar and polar 329

compounds. For most of the compounds evaluated, limits of quantification were below 330

10 ng/L in groundwater (n=71) and surface water (n=70) and below 100 ng/L in 331

wastewater (n=70). 332

As previously commented, novel material technology such as molecularly 333

imprinted polymers are beginning to be used in on-line SPE procedures for sample 334

preparation [51], and some examples describing its application to the analysis of 335

pesticides can be found in the literature. For instance, Koeber et al. [70] proposed a 336

novel and highly selective on-line sample clean-up procedure based on the use of MIPs 337

as SPE material for the analysis of triazines in river water samples. The method 338

comprises the combination of a restricted access material (RAM) and a MIP allowing a 339

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selective sample preparation to be achieved in an on-line mode. The RAM column 340

combines size exclusion and adsorption chromatography, reducing the concentration of 341

matrix molecules present in the river water by a cutoff of 15 kDa, while the MIP 342

column selectively retains the triazine analytes whereas the residual matrix is not 343

retained and separated completely, removing then all matrix and non-target compounds. 344

As an example, Figure 4a shows the chromatograms obtained for a solution of a humic 345

acid (150 mg/L) after being analyzed by LC-UV without sample clean-up (1), after an 346

on-line RAM SPE clean-up procedure (2), and after the proposed on-line RAM-MIP 347

SPE clean-up procedure (3), showing the strong capacity to reduce matrix components 348

of this method. In Figure 4b the selectivity of the multidimensional on-line RAM-MIP 349

SPE clean-up platform is demonstrated, showing that only molecules with a triazine 350

structure are recognized and enriched. Total analysis time, including on-line sample 351

clean-up, was lower than 15 min, achieving limits of detection in the range 10-50 ng/L 352

(1.5 mL of sample analyzed) with acceptable recoveries (51-102%). In addition, the 353

performance of the MIP material did not change even after more than 300 enrichment 354

and desorption cycles. 355

A new on-line SPE sample treatment system, turbulent flow chromatography 356

(TFC), which combines high-throughput and high reproducibility by means of 357

separating analytes from various matrices with reduced sample handling [21], has also 358

been proposed for the analysis of pesticides [71-74]. Within these systems, the sample 359

can be injected directly onto a narrow diameter column (0.5 or 1.0 mm) packed with 360

large particles (30-60 µm) at a high flow rate (higher than 1 mL/min) helping creating a 361

very high linear velocity inside the turbulent flow column. Under turbulent flow 362

conditions the improved mass transfer across the bulk mobile phase allows for all 363

molecules to improve their radial distribution, however, under these conditions a 364

laminar zone around the stationary phase particles still exists, where diffusional forces 365

still dominate the mass transfer process. Molecules with low molecular weight diffuse 366

faster than molecules with a high molecular weight, forcing large molecules to quickly 367

flow to waste while retaining the small analytes [21]. Asperger et al. [72] proposed the 368

use of an on-line SPE TFC-LC-MS/MS system for the rapid analysis of eleven trace 369

level priority pesticides in surface and drinking water. The use of TFC columns (50x1 370

mm, 30-50 µm particle size) enables fast on-line SPE at high sampling flow-rates (5 371

mL/min). Both polymeric (Oasis HLB) and carbon based (Hypercarb) TFC columns 372

allow complete extraction of pesticides with good recoveries from water volumes up to 373

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50 mL. Figure 5 shows the chromatogram obtained for the analysis of 10 mL sample of 374

river water by on-line TFC SPE-LC-APCI-MS/MS. Afterwards, the same group 375

proposed the combination of two different TFC columns (polymer based column 376

followed by a carbon based column) for the rapid extraction of pesticides from small 377

water sample volumes (10 mL) at high flow rates (5 mL/min) [71]. Analytes are eluted 378

with purely organic eluent from the TFC column, and the effluent is re-mixed with 379

water previous to LC column to provide efficient analyte focusing. A short monolithic 380

LC column is then used for the chromatographic separation to achieve a fast on-line 381

TFC-LC-APCI-MS/MS analysis. This approach allowed quantitative results for nearly 382

30 pesticide species (triazines, phenyl ureas and organophosphorous pesticides among 383

others) in less than 14 minutes, with a method well reproducible, robust and extremely 384

sensitive achieving LODs between 0.1 and 1 ng/L. Recently, Quinete et al. [74] also 385

described the application of an on-line TFC SPE-UHPLC-APCI-MS/MS method for the 386

analysis of chlorinated pesticides, such as endosulfans, at part per trillion levels, by 387

injecting 20 mL water samples into a Turboflow HTLC C18 XL column. 388

On-line SPE-LC-MS(/MS) approaches have also been described for the clean-up 389

and preconcentration of pesticides from food products after an extraction step from the 390

solid sample matrix and reconstitution in an adequate solvent [39,73,75,76]. For 391

instance, chlormequat and mepiquat residues have been analyzed in pear, tomato and 392

wheat flour samples by direct injection of the food extract onto an on-line SPE 393

(Prospekt) coupled with LC-ESI-MS/MS using a strong cation-exchange resin as SPE 394

sorbent [39]. Surrogate standards (d9-chlormequat, d6-mepiquat) were employed to 395

compensate for recovery losses and potential MS/MS signal suppression. A limit of 396

quantification for both cationic analytes at, or below, 5 µg/kg was obtained, with good 397

intra- and inter-assay precisions with mean variability values lower than 7%. In another 398

work, Vichapong et al. [76] analyzed carbamate residues in food and environmental 399

samples by on-line SPE-LC in a reversed-phase C18 bead (25-40 µm). Fruit and 400

vegetable samples were extracted with acetonitrile, and after solvent removal in a rotary 401

evaporator, the residue was dissolved with water and analyzed by the proposed method. 402

Turbulent flow chromatography coupled to LC-MS/MS has also been recently 403

proposed for the analysis of pesticide residues in grapes, baby food and wheat flour 404

matrices [73]. Sample extracts were injected into the on-line TFC SPE-LC-MS/MS 405

system consisting of a polymeric based cyclone MCX-2 (50x0.5 mm) TurboFlow 406

column for sample preparation and a C18 Hypersil Gold (150x4.6 mm, 5µm) column 407

13

for analytical separation. Limits of detection between 0.8 and 6.0 ng/g for baby food 408

and 0.8-10.3 ng/g for the other matrices, with acceptable precisions (RSDs lower than 409

22%) and good sample recoveries (67-124%), were obtained within a total analysis time 410

of 13 min. 411

412

5. Concluding remarks and future perspectives 413

The use of on-line SPE preconcentration approaches are becoming very popular 414

for the analysis of pesticides in complex matrices, especially when dealing with 415

environmental water samples, because of their many benefits and advantages over other 416

sample treatment procedures such as high sample throughput, reduced sample 417

manipulation, improved precision and low reagent consumption. It is also very 418

advantageous when a limited amount of sample is available or high sensitivity is 419

required. However, when developing on-line SPE LC-MS/MS methodologies for the 420

analysis of pesticides some limitations and drawbacks must also be considered. The 421

most important one relies in the re-utilization of the on-line SPE sorbents, so it is 422

important to evaluate the possibility of memory effects and, especially, the deterioration 423

of the SPE sorbent because of matrix components, in particular when complex matrices 424

are analyzed. Despite all this, many on-line SPE sorbents commercially available today 425

are very stable and, together with adequate washing programs in the SPE procedure, 426

many SPE cycles can be carried-out without observing any problem. Another 427

consideration to take into account when developing on-line SPE preconcentration is that 428

elution solvents must be compatible with the LC-MS/MS method, being in general the 429

same mobile phase used for the chromatographic separation. 430

Several SPE sorbents have been evaluated for the on-line SPE LC-MS/MS 431

analysis of pesticides. However the increased necessity in the multi-residue analysis of 432

pesticides with a wide range of physicochemical properties is making necessary the 433

simultaneous combination of sorbents with different stationary phases in order to 434

achieve simultaneously good recoveries for different pesticide families, being this one 435

of the main topics that may be exploited in the future. 436

Novel on-line SPE strategies such as the use of restricted access materials and 437

molecularly imprinted polymer sorbents, as well as turbulent flow chromatography 438

approaches are beginning to become popular when dealing with some particularly 439

difficult applications in the analysis of pesticides. MIPs are cost-effective, stable 440

regarding presence of organic solvents and very selective to the target pesticides to be 441

14

analyzed, which would make them appropriate for some specific applications, in 442

particular for those families of compounds not correctly preconcentrated when dealing 443

with multi-residue analysis. As regards TFC, this novel strategy has a powerful capacity 444

to remove matrix interferences while keeping the advantages of on-line approaches such 445

as reproducibility, robustness and sensitivity. All this novel on-line SPE strategies will 446

be sure exploited in the near future for the analysis of pesticides. 447

Most of the on-line SPE LC-MS/MS applications dealing with the analysis of 448

pesticides are focused in environmental water analysis because of the simplicity of 449

sample manipulation. However, several interesting applications of these on-line 450

approaches to the analysis of pesticides in food samples are also reported in the 451

literature. 452

Finally, the new advances in manufacturing SPE sorbents more pressure 453

resistant will facilitate the coupling of on-line SPE approaches to UHPLC-MS/MS 454

methodologies, making more attractive the application of these methodologies for the 455

analysis of pesticides in both environmental waters and food samples. 456

457

Conflict of interest statement 458 459

The authors have declared no conflict of interests. 460

15

461

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607

608 609

610 611 612

613

20

Figure captions 614 615

Figure 1. Scheme of a general SPE process. 616

617

Figure 2. Scheme of a common on-line SPE configuration with a small SPE column 618

within the injection loop of a six-port rotary valve. 619

620

Figure 3. On-line SPE-LC-ESI-MS/MS chromatograms from positive water samples. 621

(a) ground water containing 9 ng/L of pyrimicarb (273>72), 650 ng/L of bromacil 622

(259>203), 95 ng/L of terbutryne (242>186), 13 ng/L of diuron (233>72) and 10 ng/L 623

of terbuthylazine (230>174), and (b) surface water containing 520 ng/L of carbendazim 624

(192>160), 150 ng/L of dimethoate (230>199), 27 ng/L of terbumeton (226>170), 170 625

ng/L of terbacil (215>159) and 25 ng/L of methidathion (303>145). Reprinted with 626

permission from ref. [65]. Copyright 2001 Elsevier. 627

628

Figure 4. (a) Reduction of interfering matrix components. A solution of a humic acid 629

(150 mg/L) injected into different LC system configurations and the extract analyzed 630

using UV/visible detection (λ=220 nm): (1) LC column only, (2) on-line RAM-LC 631

coupling, and (3) on-line RAM-MIP-LC coupling. (b) Selectivity of the 632

multidimensional on-line RAM-MIP SPE clean-up platform. The upper chromatogram 633

was obtained by injecting a standard mixture of different pesticides (LC-MS system). 634

The lower chromatogram was obtained after enrichment by RAM-MIP. Reprinted with 635

permission from ref. [70]. Copyright 2001 American Chemical Society. 636

637

Figure 5. Total ion chromatogram and extracted MRM traces of eight pesticides 638

obtained from on-line TFC SPE-LC-APCI-MS/MS analysis of a 10 mL river water 639

sample (River Parthe, Leipzig, Germany). Reprinted with permission from ref. [72]. 640

Copyright 2002 Elsevier. 641

21

Table 1. Selection of on-line SPE methods for the analysis of pesticide residues in water samples by LC-MS

Compounds Sample On-line SPE sorbent Sample volume Recoveries LC conditions MS conditions LOQs Ref.

88 polar organic

micropollutants

including pesticides

Ground, surface

and wastewater

Home-made single mixed-bed

multilayer cartridge with 4

extraction materials: 10 mg Oasis

HLB, 10 mg of Strata X-AW, Strata

X-CW and Isolute ENV+ in a ratio

1/1/1.5

50 mL

(adjusted to pH 7)

80-120% Atlantis T3 column (150 x 3.0 mm

I.D., 3µm particle size)

Gradient elution

A) 5 mM ammonium acetate in water

B) methanol with 0.1% formic acid

Flow-rate: 300 µL/min

Triple quadrupole instrument

ESI in polarity switching

mode

SRM acquisition mode

0.1-200

ng/L

[57]

37 pesticides Surface water Macroporous spherical particles of

polystyrene and divinylbenzene

(PLRP-s) with dimensions 10 x 2

mm

1500 µL

(two injections of

750 µL)

60-129% Poroshell 120 EC-C18 column (50 x

4.6 mm I.D., 2.7 µm particle size)

Gradient elution

A) 5 mM ammonium acetate and

0.01% formic acid in water

B) Acetonitrile

Flow-rate: 350 µL/min

Triple quadrupole instrument

ESI (positive ionization)

SRM acquisition mode

0.3-33

ng/L

[58]

20 biocides and

pesticides

Surface and

wastewater

Strata-X extraction cartridge

(polymeric sorbent with N-

vinylpyrrolidine functional groups),

20 x 2.1 mm, 33 µm particle size

20 mL 65-90% Xbridge-C18 column (50 x 2 mm

I.D.)

Gradient elution

A) water with 0.1% formic acid

B) methanol with 0.1% formic acid

Flow-rate: 300 µL/min

Triple quadrupole instrument

ESI (positive and negative

ionization mode)

SRM acquisition mode

3-100

ng/L

[59]

22 medium to highly

polar pesticides

Groundwater

Wastewater

PLRP-s cartridge (10 x 2 mm), and

HySphere Resin GP

(polydivinilbenzene, 10-12 µm

particle size).

5 mL 75-178% Purospher Star RP-18 end-capped

column (125 x 2.0 mm I.D., 5 µm

particle size)

Gradient elution

A) water

B) acetonitrile

Flow-rate: 300 µL/min

Quadrupole-linear ion trap

instrument

ESI (positive and negative

ionization mode)

SRM acquisition mode

0.02-3.91

ng/L a

[60,61]

Groundwater

[62]

Wastewater

9 pesticides Sea and lake

water

C18 SPE column (7.5 x 4.6 mm

I.D., 10 µm particle size)

50 mL 47-92% Shimadzu VP-ODS column (250 x

4.6 mm I.D., 5µm particle size)

Gradient elution

A) water with 0.1% formic acid and

5 mM ammonium formate

B) acetonitrile with 0.1% formic acid

Flow-rate: 1 mL/min

Ion-trap mass analyzer

instrument

ESI (positive ionization

mode)

SRM acquisition mode (only 1

transition)

2-10

µg/L a

[63]

Pesticides and

pharmaceuticals

Drinking,

surface and

wastewater

C18 Hypersil GOLD column (20 x

2.1 mm I.D., 12 µm particle size)

1.5 mL 87-110% C18 Hypersil Gold column (50 x 2.1

mm I.D., 3 µm particle size)

Gradient elution

A) water with 0.1% formic acid

B) methanol

Flow-rate: 200 µL/min

Triple quadrupole instrument

ESI (positive ionization mode)

SRM acquisition mode

2-24 ng/L [35]

a LODs

22

Figure 1

23

Figure 2

24

Figure 3

25

Figure 4

26

Figure 5