C HROMATOGRAPHY Chromatographic selectivity is a prerequisite for most applications of LC-MS, as

3.4.2 C HROMATOGRAPHY Chromatographic selectivity is a prerequisite for most applications of LC-MS, as

mass selectivity does not completely eliminate isobaric interferences and matrix effects that may affect the relative response of analytes. Although separation of all analytes is not considered necessary for detection of pesticide residues due to the high selectivity provided by MS=MS, ion suppression is observed either from coelution with other analytes or, more likely, coeluting matrix components. The most important and widely used separation technique for pesticides is LC on reversed-phase (RP) columns. Separation is based on differences in hydrophobicity by partitioning between an apolar stationary phase and a polar mobile phase. Together with appropriate control of operational parameters such as solvent com- position, pH, temperature, and flow rate, reversed phase can enable separations of many pesticides with a wide range of polarities and molecular weight. Columns with

C 8 and C 18 stationary phases on high purity silica are the most widely used.

3.4.2.1 Mobile Phases The selection of mobile-phase constituents for LC-MS using RP is not an easy task,

and the conditions described in the literature are clearly rarely optimized. The selec- tion of mobile phase is important to obtain a good chromatographic separation, but it

76 Analysis of Pesticides in Food and Environmental Samples also affects the analyte ionization and the sensitivity of the mass spectrometer. 89 For

example, analyte charge should be suppressed by manipulation of the mobile-phase pH for optimum retention but this can have a detrimental affect on MS response. 90 Contrary to conditions for RP LC retention, for optimized electrospray (ES) ioniza- tion, the pH should be adjusted to promote the charged state of the analyte over its neutral species as ionization takes place in the liquid phase. In contrast, for optimized atmospheric pressure chemical ionization (APCI), in which ionization takes place in the gas phase, formation of the neutral species is favored due to the higher volatility of

the neutral versus charged species and hence better vaporization. 91 A compromise can

be sought by experimentation but this is particularly difficult when a wide range of pesticides, with differing properties, have to be analyzed. The situation is further complicated if polarity switching is employed so that positive and negative ions are periodically sampled throughout the analytical run. Alternatively, the target pesticides are divided into anionic and cationic groups and analysis performed separately.

For routine application, even designs of orthogonal nebulizers for atmospheric pressure ionization interfaces are still restricted to the use of volatile buffers. The concentration of the buffer, or acid or base used to adjust=control the pH, should be as low as possible for ES. If not, competition between analyte and electrolyte ions for conversion to gas-phase ions decreases the analyte response. If a species is in large excess, it will cover the droplet surface and prevent other ions to access the surface, and thus to evaporate. A species in large excess will also catch all charges available and prevent the ionization of other molecules present at much lower concentration. Ammo- nium acetate and ammonium formate are generally applicable at pH 7 but concentra- tions should be kept to a minimum. RP LC separations are sometimes improved at acidic pH, using acetic acid or formic acid, as such or in combination with ammonium acetate or ammonium formate. The addition of reagents postcolumn can be used to generate pH conditions optimum for ionization without changing chromatographic separation but this approach is rarely implemented for routine analyses.

Methanol and=or acetonitrile are used as organic modifiers. Low surface tension and a low dielectric constant of the solvent promote ion evaporation, which favor the ionization process. The gas-phase basicity (proton affinity) and gas-phase acidity (electron affinity) are also important solvent properties in the positive and negative ionization modes, respectively. Those features encourage the use of methanol versus acetonitrile. Methanol is also preferred over acetonitrile when MS with ES is coupled to gradient LC because the lower eluotropic strength of methanol causes compounds to elute at a higher percentage of organic solvent, where ES sensitivity is increased. In most cases, more pesticides appear to elute in the middle of the analytical run. Given the extra demand on MS acquisition in terms of obtaining sufficient data points across a peak and a long enough dwell time for sensitivity, it is surprising that few authors have reported efforts to optimize the gradient conditions so that pesti- cides exhibit a wider elution profile. 92

3.4.2.2 Ion Pair, Hydrophilic Interaction, and Ion Chromatography Volatile ion pair reagents (e.g., heptafluorobutyric acid and tetrabutyl ammonium)

have been added either to the sample vial 93 or to the mobile phase to improve the

Analysis of Pesticides by Chromatographic Techniques 77 chromatography of ionic species, such as paraquat and diquat. 94 The introduction of

an ion pair reagent into the mobile phase increases the interactions between the quaternary ammonium compounds and the C 18 stationary phase, providing the necessary retention and resolution. 95 The type and quantity of ion pair reagent added has to be a compromise between improvement in separation and retention and minimizing the suppression observed in ES. Hydrophilic interaction chromatog-

raphy (HILIC) 96 has been explored for the determination of paraquat and diquat by MS=MS without the need for ion-pairing reagents. HILIC separates compounds by passing a hydrophobic or mostly organic mobile phase across a neutral hydrophilic stationary phase, causing solutes to elute in order of increasing hydrophilicity. Although the chromatography behavior on HILIC is not as good as that observed using the ion pair systems, the MS sensitivity using the HILIC mobile phase was

claimed to be significantly greater. 97 Although ion chromatography (IC) allows separation of ionic compounds that

have no retention on conventional reversed-phase LC columns, it is rarely used for the determination of pesticides by LC-MS due to the difficulties encountered by spraying the nonvolatile salts used in high ionic strength eluents. Exceptions include

chlormequat, 99 as elution is possible with volatile buffers, and glyphosate, when a suppressor is used between IC system and MS=MS to remove salts from the eluent to

make coupling with the ES source possible.

3.4.2.3 Fast Liquid Chromatography One of the primary parameters that influence LC separation is the particle size of the

packing materials used to effect the separation. There has been a long trend of reducing particle size in LC (e.g., 10, 5, and 3 mm). Initially, the smallest particles were used in short columns, leading to fast analysis times but relatively modest gains in resolving power. Flow rates and column length were restricted by the back pressure generated. By using smaller particles, increases in speed of analysis, improved resolution, and sensitivity are possible. 100 According to the van Deemter equation, which describes the relationship between linear velocity (flow rate) and plate height (HETP or column efficiency), when the particle size decreases to <2.5 mm not only is there a significant gain in efficiency, but the efficiency does not diminish at increased flow rates or linear velocities. In order to take advantage of such small particle sizes, instrumentation capable of high-pressure operation with low system and dead volume is required; so, in 2004, Swartz and Murphy introduced

the first LC system capable of operation up to 15,000 psi. 101 These systems have been termed ultraperformance liquid chromatography (UPLC) or ultra-HPLC

(UHPLC) to differentiate them from HPLC. 102 With sub 2 mm particles, half-height peak widths of <1 s can be obtained, posing significant challenges for the MS.

In order to accurately and reproducibly integrate an analyte peak, the MS must have a sufficient acquisition rate to capture enough data points across the peak (>10 points=peak), requiring very short dwell times and interchannel delays on a

triple quadrupole instrument or the fast spectral acquisition of the TOF analyzer and the software tools to handle the increased number of results. Improved detection limits for LC-MS are achieved by narrower chromatographic peaks effectively

78 Analysis of Pesticides in Food and Environmental Samples increasing the concentration of analytes entering the MS source increasing signal

intensity and the improved resolution may reduce ion suppression by separating species that may coelute in conventional LC. 103 In addition, shorter analytical run times are possible without compromising chromatographic resolution leading to an increase in sample throughput. This technology offers considerable benefits over conventional LC-MS and its application to the determination of pesticide res- idues 104,105 is growing rapidly.