Article pubs.acs.org/JAFC
Development of an Automated Column Solid-Phase Extraction Cleanup of QuEChERS Extracts, Using a Zirconia-Based Sorbent, for Pesticide Residue Analyses by LC-MS/MS Bruce D. Morris* and Richard B. Schriner Food and Bioanalytical Division, R. J. Hill Laboratories, Private Bag 3205, Hamilton East, New Zealand S Supporting Information *
ABSTRACT: A new, automated, high-throughput, mini-column solid-phase extraction (c-SPE) cleanup method for QuEChERS extracts was developed, using a robotic X-Y-Z instrument autosampler, for analysis of pesticide residues in fruits and vegetables by LC-MS/MS. Removal of avocado matrix and recoveries of 263 pesticides and metabolites were studied, using various stationary phase mixtures, including zirconia-based sorbents, and elution with acetonitrile. These experiments allowed selection of a sorbent mixture consisting of zirconia, C18, and carbon-coated silica, that effectively retained avocado matrix but also retained 53 pesticides with 70% recoveries of all 263 pesticides. Analysis of avocado extracts by LC-Q-Orbitrap-MS showed that the method developed was removing >90% of di- and triacylglycerols. The method was validated for 269 pesticides (including homologues and metabolites) in avocado and citrus. Spike recoveries were within 70−120% and 20% RSD for 243 of these analytes in avocado and 254 in citrus, when calibrated against solvent-only standards, indicating effective matrix removal and minimal electrospray ionization suppression. KEYWORDS: QuEChERS, SPE, LC-MS/MS, ITSP, Z-Sep, zirconia, pesticide, multiresidue, avocado, citrus
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INTRODUCTION The “quick, easy, cheap, effective, rugged, and safe” (QuEChERS) method for the analysis of multiclass pesticide residues in fruits and vegetables introduced the use of dispersive solid-phase extraction (d-SPE) cleanup, to reduce the amounts of coextracted matrix in extracts, before instrumental analysis, using a mixture of MgSO4 and PSA sorbents, with C18 and graphitized carbon black (GCB) added if required to improve removal of nonpolar matrix and chlorophyll.1−4 In the original QuEChERS method, d-SPE was used instead of column SPE (c-SPE) to provide a quicker and cheaper cleanup.1 Recently the zirconia-based sorbent HybridSPE, in well-plates or columns, has been utilized for the removal of phopholipids from plasma5−7 and eggs.8 The zirconia materials Z-Sep and Z-Sep+ have been evaluated for d-SPE cleanup of QuEChERS extracts for analysis of environmental pollutants and pesticides in fish and shrimp9−11 and pesticides from oily fruits or vegetable oils,12−16 due to their abilities to remove the lipophilic matrix. However, in our experience, used routinely, Z-Sep d-SPE can result in the transfer of solid phase into analysis vials and subsequently into the HPLC, building up over time to cause retention of some analytes and poor peak shapes or carry-over. Consequently, we investigated the development of an automated c-SPE cleanup, based on zirconia-coated silica, using Instrument Top Sample Preparation (ITSP) mini-cartridges, on a robotic X-Y-Z instrument autosampler. This could be as quick and cheap as d-SPE, as many instruments are already equipped with robotic autosamplers; however, it could also give the improved matrix removal that is possible with c-SPE17 and avoid zirconia transfer to the LC-MS/MS. © XXXX American Chemical Society
Avocado extracts were selected as a matrix with high oil content,18,19 and experiments were carried out to evaluate the weight of matrix removed after acetonitrile (MeCN) elution through ITSP c-SPE cartridges with six different stationary phases. Recoveries of 263 pesticides and metabolites spiked on avocado were determined through five of these sorbents and, along with matrix weight-removal results, allowed selection of a Z-Sep/C18/CarbonX mixture for further method development. Investigation of the effect of different elution solvents (MeCN, MeCN/MeOH (1:1), MeOH, coextracted citrate in a CEN (European Committee for Standardization method,20), QuEChERS extract, and formate buffer at three concentrations in MeCN/MeOH (1:1), on pesticide recoveries through Z-Sep/C18/CarbonX, resulted in a method using elution of CEN QuEChERS extracts with 100 mM formate buffer in MeCN/MeOH (1:1). To the best of our knowledge, this study is the first to use ITSP mini-cartridges for cleanup of QuEChERS extracts and zirconia solid phase in an SPE column, rather than used dispersively, for pesticide residue analysis. Removal of avocado di- and triacylglycerols by Z-Sep, monitored by LC-QOrbitrap-MS, is also presented. The method was validated for the analysis of 269 pesticides, including homologues and metabolites, in avocado and citrus, to give spike recovery and reproducibility data. Special Issue: 51st North American Chemical Residue Workshop Received: November 17, 2014 Revised: February 7, 2015 Accepted: February 9, 2015
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DOI: 10.1021/jf505539e J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
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Automated Mini-Column SPE and Dispersive SPE. Miniaturized c-SPE cartridges, for use on X-Y-Z autosamplers, were supplied by ITSP Solutions (Hartwell, GA, USA), with 30 mg of Z-Sep/C18/ CarbonX (2:5:0.24), 45 mg of PSA/C 18 /CarbonX/MgSO 4 (3:3:0.25:5), and 10 mg of Z-Sep or 30 mg of Z-Sep+ sorbents, the first two as homogeneous mixtures. Cartridges with 20 mg of HLB/ Z-Sep/CarbonX (1:1:0.22) were handmade in the laboratory. ITSP c-SPE cartridges were 3.5 cm long, including a 0.6 cm tip, with a sorbent bed 6 mm in diameter × 2.5 mm in height (0.5 mm bed height for 10 mg of Z-Sep). Sorbent weights for each mixture were amounts that could fit into the fixed cartridge volume. The upper section of the cartridge comprised a syringe needle guide with a sealing septum on top (Figure 1). Robotic c-SPE used a PAL HTS-xt, X-Y-Z
MATERIALS AND METHODS
Reagents and Consumables. MeCN and MeOH (Lichrosolv grade), formic acid (98−100%), NaCl (99.5%), trisodium citrate dihydrate, disodium hydrogen citrate sesquihydrate, and ammonium formate were purchased from Merck (Darmstadt, Germany). MgSO4 (anhydrous) used for QuEChERS extractions was from Scharlau (Sentmenat, Spain). Water was purified using a Sartorius (Goettingen, Germany) Arium 611UV unit. Formate buffer (100 mM) for c-SPE was prepared in MeOH/MeCN (1:1) by dissolving ammonium formate (70 mM) and adding 30 mM formic acid. Lower concentrations of formate buffer (50 and 10 mM) were prepared by dilution of the 100 mM solution with MeOH/MeCN (1:1). Two zirconia-coated silica sorbents, Supel Que Z-Sep and Z-Sep+, were obtained from Supelco (Bellefonte, PA, USA), as was Supel-Select HLB SPE. Z-Sep+ is a zirconia-coated silica, with octadecyl derivatization of silanol groups. Silanols are underivatized in Z-Sep material. PSA and C18-functionalized silicas, and anhydrous MgSO4 used for SPE experiments were from UCT (Bristol, PA, USA). The carbon-coated silica, CarbonX was supplied by United Science Corp (Center City, MN, USA). Ceramic grinding rods (3/8 in. × 7/8 in.) to aid QuEChERS extractions were sourced from SPEX (Metuchen, NJ, USA). Analytical Standards. High-purity analytical standards were obtained from Dr. Ehrenstorfer (LGC Standards, Middlesex, UK), Sigma-Aldrich (St. Louis, MO, USA), Wako (Osaka, Japan), Accustandard (New Haven, CT, USA), and Chemservice (West Chester, PA, USA). Penthiopyrad was purchased from Kanto Chemical Co. (Tokyo, Japan). Atrazine-d5, used as an internal standard, and triphenylphosphate, added as a system monitoring compound (SMC), were from Sigma-Aldrich. A 20 mg/L solution of triphenylphosphate was prepared in MeOH and a 2 mg/L solution of atrazine-d5 in MeCN. Pesticides were separated into 10 mixes, prepared in MeCN with 0.05% formic acid (10 mg/L). These were used for spiking experiments and diluted in 10 mM formate buffer (aqueous, pH 4)/MeOH/MeCN (13:2:1) to give calibration standards (0.02, 0.05, 0.1, 0.2, 0.4, 1, 2, 5, 10, 20, 40 μg/L). Samples and Extraction. Samples, for analyte recovery experiments and method validation, of avocado (cv. Hass), citrus (orange, mandarin, and lemon) and buttercup squash were obtained from New Zealand growers. Avocados were mature, but unripe, and citrus fruits were without wax coating. Real samples of avocado, navel orange, and mandarin were purchased from local markets in Hamilton, New Zealand, and used to demonstrate the application of the validated method for recovery of incurred residues. Extraction was based on the citrate-buffered CEN QuEChERS method.4,20 Frozen samples were homogenized using a Blixer 6 blender (Robot Coupe, Vincennes, France). Subsamples (5 g for avocado and citrus, 20 g for squash) were weighed into 50 mL polypropylene tubes containing two ceramic rods. SMC (100 μL of 20 mg/L) was added to each. Spikes (50 μL of each 10 mg/L mix) were split between two samples, with approximately 50% (five mixes) of the pesticides in each, which separated parents from breakdown products (e.g., acephate from methamidophos, naled from dichlorvos, furathiocarb from carbofuran). Water (10 mL) and MeCN (9.65 mL, to give 10 mL total volume including spikes, or 10 mL for unspiked blanks), were added, and tubes were shaken by hand for 1 min. After the addition of salt mixture (13 g of MgSO4/ NaCl/trisodium citrate dihydrate/disodium hydrogen citrate sesquihydrate (8:2:2:1)), tubes were shaken on a horizontal shaker for 15 min. For citrus an additional 4 g of trisodium citrate was added along with the salt mixture, to compensate for the citric acid in samples and bring the pH back into the buffer region. Tubes were centrifuged at 2000 rpm, for 5 min, and then 1 mL of the MeCN layer was transferred into autosampler vials, to which 100 μL of atrazine-d5 (2 mg/L) was added. For validation, three samples of avocado, and samples of lemon, orange, or mandarin, were extracted without spiking, plus in triplicate or quadruplicate with spikes, using different technicians, in three batches, over a more than 2 week period, to give a total of 10 replicates. Lemons, oranges, and mandarins were extracted in separate batches.
Figure 1. Automated ITSP c-SPE cartridge. autosampler (CTC Analytics, Zwingen, Switzerland), with HW-54P and HW-96P aluminum trays (ITSP Solutions) and a 250 μL syringe (CTC Analytics). c-SPE cartridges were conditioned (with 150 μL of elution solvent) in the HW-96P tray, then transferred, on the end of the syringe needle, to the HW-54P tray, where they were inserted into 2 mL amber glass vials with split-septa clip caps. Extract (150 μL) was then loaded onto the cartridge followed by elution solvent (150 μL), with the combined eluent collected. For cartridges with 30 mg of sorbent (Z-Sep/C18/CarbonX and Z-Sep+) or 45 mg of PSA/C18/ CarbonX/MgSO4, approximately 75 μL of solvent was retained; therefore, 150 μL was 2 column void volumes. Elution rate was 2 μL/s. Cleaned up extracts were diluted 5-fold for analysis, with 10 mM formate buffer (aqueous, pH 4)/MeOH/MeCN (13:2:1). D-SPE used 30 mg of Z-Sep/C18/CarbonX (2:5:0.24) in a 2 mL glass vial. Two-fold diluted avocado extract (150 μL) was added, plus 150 μL of MeCN, to match the dilution on c-SPE. The vial was vortexed for 1 min, the solid-phase was allowed to settle for 5 min, and then 200 μL of extract was transferred to a second vial for 5-fold dilution with 10 mM formate buffer (aqueous, pH 4)/MeOH/MeCN (13:2:1) for analysis. Gravimetric Recovery Experiments. Glass 2 mL vial inserts (350 μL flat-bottomed) were weighed on a five-figure analytical balance (Sartorius CPA225D), then either 150 μL of 2-fold-diluted crude avocado extract or the eluent from 150 μL of the same extract passed through automated ITSP c-SPE was collected in the preweighed inserts. Solvent was evaporated at 40 °C under a gentle flow of nitrogen, and the inserts were allowed to re-equilibrate to room temperature (15 min) before reweighing to determine the dry weight of avocado matrix recovered. LC-MS/MS Parameters. LC-MS/MS was carried out on Triple Quad 5500 and 6500 triple-quadrupole instruments (AB Sciex, Toronto, ON, Canada), coupled to 1200 HPLC systems (Agilent, Palo Alto, CA, USA) with PAL HTS-xt autosamplers and DLW syringes (CTC Analytics). The MS/MSs were operated in positive electrospray ionization (ESI) mode, with source temperatures of 350 °C (Triple Quad 5500) B
DOI: 10.1021/jf505539e J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
extracts were finally diluted 10-fold with MeCN, to match the dilution of crude extract, before injection. Data analysis used Thermo Xcalibur Qual Browser. Extracted ion chromatograms were constructed by entering the accurate masses of each TAG or DAG peak, with a 10 ppm range. To identify TAGs and DAGs, accurate masses were entered into ChemSpider Advanced Search.21
and 285 °C (Triple Quad 6500), and an ionspray voltage of 5500 V. Multiple reaction monitoring (MRM) transitions are given in Supporting Information Table SI-B and were optimized by undertaking multiple injections with differing declustering potential (DP) and collision energy (CE) parameters. HPLC used Ascentis Express C18 columns (10 cm × 2.1 mm, 2.7 μm particle size) from Supelco at a flow rate of 0.4 mL/min and a column oven temperature of 50 °C. Mobile phase A was 10 mM ammonium formate/formic acid buffer (pH 4) in H2O/MeOH (9:1), and mobile phase B was MeOH. Gradient elution started at 12.5% B (held for 0.5 min) and ramped to 40% B over 1.5 min, then to 62.7% B over 6 min, followed by a final ramp to 95% B over 6 min (held at 95% for 2 min), to give an even spread of peaks across the chromatogram. Injection volume was 10 μL. Validated Method. The final method validated used QuEChERS extraction of 5 g subsamples of homogenized fruit, with citrate buffering, as outlined under Samples and Extraction. Automated c-SPE was carried out with ITSP cartridges containing 30 mg of Z-Sep/C18/ CarbonX (2:5:0.24). Extracts (150 μL) were loaded onto cartridges and eluted with 150 μL of 100 mM formate buffer in MeOH/MeCN (1:1), with loading and elution volumes collected. After 5-fold dilution with 10 mM formate buffer (aqueous, pH 4)/MeOH/MeCN (13:2:1), extracts were analyzed by LC-MS/MS. Details are provided in the relevant c-SPE and LC-MS/MS sections. LC-Q-Orbitrap-MS Analysis. The LC consisted of an Ultimate 3000 UHPLC pump, autosampler, degasser, and column compartment and was coupled to a Q-Exactive quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) operated in positive ESI mode. A Kinetex C18, 10 cm × 2.1 mm, 2.6 μm particle size, HPLC column (Phenomenex, Torrance, CA, USA) was used. Mobile phase A was 10 mM ammonium formate/formic acid buffer (pH 4) in water/MeOH (9:1), and mobile phase B was 10 mM ammonium formate/0.015% formic acid in MeOH/isopropanol (1:1). The flow rate was 0.4 mL/min, and the column oven held at 50 °C. The injection volume was 5 μL. The elution gradient started at 30% B (held for 0.5 min) with a ramp to 60% B at 2 min, followed by a second ramp to 98% B at 10 min (held for 8 min before returning to 30% B for re-equilibration). The Q-Exactive was tuned using LTQ ESI Positive Ion Calibration Solution
