Fruit and Vegetables - GERSTEL · analytical methods and new instrumentation. Unfortunately, analytical instruments normally do not tolerate introduction of “dirty” samples or

Intelligent Automation for GC/MS and LC/MS

Pesticide Analysis EZFruit and Vegetables

News from GERSTEL GmbH & Co. KG · Eberhard-Gerstel-Platz 1 · D-45473 Mülheim an der Ruhr · Germany · Phone + 49 208 - 76503-0 · Fax + 49 208 - 7650333

SPECIAL

Monitoring of Pesticides

in Fruit and Vegetables

GERSTEL Solutions Worldwide Pesticide Special

■ Automated Liner EXchange for GC Inlets – Handling Dirty Samples

Sample clean-up steps, which are needed in order to prepare for example environmental or food samples for pesticide analysis, are time-consuming and a potential source of errors. Sim-plifi cation - or elimination - of such procedures is often the motivation when developing new analytical methods and new instrumentation. Unfortunately, analytical instruments normally do not tolerate introduction of “dirty” samples or even of extracts that have not been cleaned. For example, extracts containing suspended matter or high molecular weight compounds contaminate a GC inlet within a few injections, causing peak broadening or even loss of sensi-tive compounds. The GERSTEL Automated Liner EXchange enables routine analysis of “dirty” extracts without system down-time since dirty liners are automatically replaced. Page 3.

■ Automated Solid Phase Extraction (SPE)

Performing Solid Phase Extraction (SPE) manually can be time consuming and nerve-racking, especially when recovery and reproducibility are lacking due to sample variability. If SPE can be reliably automated, it becomes a much more effi cient and reproducible process. The GER-STEL SPE provides several benefi ts compared with manual SPE: Improved recovery, precision and reproducibility as well as maximized sample throughput by performing SPE during GC or LC analysis of the preceding sample. In many cases this translates to more than 50 percent time saving for the overall analysis, compared to manual processing. Following the SPE clean-up steps, the MPS can introduce the sample extract directly to the LC/MS or GC/MS system for analysis. A method used for the determination of up to 185 different pesticides using GC/MS and LC/MS is reported. Page 6.

■ Automated Disposable Pipette Extraction (DPX)

Disposable Pipette Extraction (DPX) is a fast and effi cient SPE technique used for a wide range of applications such as pesticide monitoring. Only 200 - 250 µL of sample is needed to reach the required limits of detection using a fully automated process. The extraction step is performed in 30 - 60 seconds and the complete process including elution and rinse steps takes 3 - 6 minutes depending on the application. Additionally, automated DPX is performed during the GC or LC run of the preceding sample ensuring maximum throughput and best possible GC/MS or LC/MS system utilization. Elution requires only a small amount of solvent, which means that DPX effectively provides a concentration step. For many applications, such as pesticides in fruit and vegetables, solvent evaporation is not required - or it can be auto-mated by using Large Volume Injection (LVI) techniques. Page 9.

Published by GERSTEL GmbH & Co. KG Eberhard-Gerstel-Platz 1 45473 Mülheim an der Ruhr Germany

Editorial Director Guido Deußing ScienceCommunicationNeuss, Germany [email protected]

Translation and editingKaj Petersen [email protected]

Scientifi c advisory boardEike Kleine-Benne, Ph.D. [email protected]

Oliver Lerch, Ph.D. [email protected]

Malte Reimold, [email protected]

Contact [email protected]

Design Paura Design, Hagen, Germany www.paura.com 06

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Imprint

One of the most important aspects of controlling pesticide exposure is to monitor pesticide residues in food, water and bever-ages.

Many food samples require extensive sample preparation and sample clean-up in order to reach the required limits of determi-nation and in order to keep the analysis sys-tem suffi ciently stable for a routine analysis environment. Most established analytical methods are based on traditional liquid-liquid extraction, gel permeation chromatog-raphy (GPC) or solid phase extraction (SPE) in combination with GC/MS or LC/MS. The benefi ts of automated sample preparation and automated matrix elimination for the determination of pesticides by GC/MS and LC/MS are demonstrated in the material shown in this GERSTEL Newsletter.

the SBSE technique (GERSTEL Twister®) is a well recognized technique for extracting and concentrating pesticides and other contaminants from aqueous samples. This special issue of the GERSTEL Solutions Worldwide Magazine brings you an overview of GERSTEL technology and applications in the fi eld of pesticide analysis.

Enjoy the magazine!

Yours

Ralf Löscher, Ph.D. International Sales Manager

In this issue

GERSTEL offers a wide range of reliable and effi cient automated sample preparation systems for GC/MS and LC/MS pesticide determination. Our solutions cover the full spectrum from aqueous through non-fat-containing to fat containing samples: The GERSTEL Automated Liner EXchance ALEX technology for GC/MS helps to improve sample throughput in pesticide residue analysis – especially when using the QuE-ChERS method for sample preparation. The GERSTEL SPE provides a fully automated solution including sample preparation and introduction to GC/MS or LC/MS. Dis-posable Pipette Extraction (DPX), a fast dispersive SPE technique that requires very little solvent, enables rapid and effi cient extraction of analytes using only small amounts of solvent and, last, but not least,

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New technology for handling dirty samples

Automated liner exchange for GC injectors

S ample clean-up steps needed to pre-pare environmental or food samples for determination of pesticides are

time-consuming and are potential sourc-es of errors. Simplifi cation or elimination of such procedures is often the motivation for development of new analytical meth-ods and new instrumentation. Unfortu-nately, analytical instruments do not nor-mally tolerate introduction of “dirty” sam-ples or even “dirty” extracts.

For example, extracts containing sus-pended matter or high-molecular-weight compounds contaminate a GC inlet after a few injections, causing peak broadening or even loss of sensitive compounds. Reducing or eliminating clean-up steps will result in dirty extracts and daily – or even hourly – maintenance of the GC system.

System Design for Automated Liner Exchange

A simple and automated liner exchange system is able to overcome most chromato-graphic problems caused by “dirty” samples in GC analysis. A solution is presented that uses a commercially available programma-ble temperature vaporizing (PTV) inlet in combination with an autosampler, which can automatically perform a liner exchange at any time during a sample sequence. Every liner is equipped with a transport adapter, which also allows liquid injection through a septum. Adapters fi tted with liners are transported by means of the autosampler which also performs the liquid injection. The system is based on the Cooled Injec-tion System (CIS 4) inlet and the MultiPur-

Figure 1: Automated Liner Exchange (ALEX) installed on an Agilent 7890 GC equipped with a CIS 4 programmed temperature vaporization (PTV) type inlet. Detailed view of the ALEX System.

17

The GERSTEL Automated Liner EXchange (ALEX) enables routine GC analysis of samples containing large amounts of matrix or other sol-id residue. After several injections, matrix material deposited in the GC inlet leads to adsorption and loss of active analytes, such as pes-ticides. ALEX replaces the GERSTEL CIS inlet liner at user-defi ned in-tervals, eliminating the need for time-consuming clean-up steps dur-ing sample preparation.

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Figure 2 Left: Exploded view of transport adapter for automated liner exchange with liner, adapter, 3 x 5mm septum and septum screw; a hole for carrier gas entry can be seen between the o-rings of the transport adapter. Right: Assembled transport adapter with liner

Figure 3: GC FID chromatogram of a Grob test mixture (1 µL splitless) injected into a CIS 4 equipped with the ALEX system

Figure 4. Screen shot of a control sequence for ALEX embedded in Agilent GC ChemStation and MS ChemStation.

Table 1: QuEChERS method sample preparation steps for multi-residue analysis of pesticides in non-fatty foods such as fruits and vegetables

Weigh 10 g of sample

–> Add 10 ml of Acetonitrile (AcN)

Shake vigorously 1 min

–> Add 4 g MgSO4 and 1 gNaCl

Shake vigorously 1 min

–> Add internal standard solution

Shake 30 sec and centrifuge

Take Aliquot of supernatant

–> Add MgSO4 and sorbent

Shake 30 sec and centrifuge

GC-MS and LC-MS

coupling between the body and the top of the PTV. As a result, no septum bleeding or bleeding of the o-rings of the adapters can be observed.

The body of the injector is identical to the CIS 4 inlet and all commercially available types of liners for this inlet can be used (empty liners or liners fi lled with glass wool or adsorbents). The automated liner exchange head doesn’t affect the ana-lytical performance of the CIS 4 inlet. As an example, Fig. 3 shows a chromatogram of a Grob test mixture with uncompromised peak resolution and peak shapes.

Tests with n-alkane mixtures proved that recovery of high boiling substances is comparable to a normal CIS 4 system. It is clearly seen that this new Automated Liner EXchange (ALEX) system has no infl uence on CIS 4 performance. Methods developed for CIS 4 system can be transferred to the ALEX system without any modifi cations.

A software solution was developed that enables the user to exchange liners at any point in time during the analysis sequence. The software can be operated stand-alone or integrated into the Agilent GC or MS ChemSation. This means that only one se-quence list is needed for the complete sys-

tem. Fig. 4 shows a screen shot of such a sample sequence.

Pesticide analysis of non-fatty foods with reduced sample preparationRecently a new multi-residue method for pesticide analysis in fruits and vegetables was presented (QuEChERS, Quick Easy Cheap Effective Rugged Safe) [1]. Com-pared to previous methods, the QuECh-ERS method enables rapid sample prepa-ration for determination of pesticides such that 8 samples can be prepared in less than 30 minutes. Table 1 summarizes all neces-sary steps of the QuEChERS method.

The main benefi t of this sample prep-aration method is a less time-consuming analysis, which is also less error-prone. Unfortunately, extracts obtained follow-ing this procedure often have a high ma-trix content, which causes chromatograph-ic problems for GC analysis due to residue build-up in the liner.

Fig. 5 shows a picture of a 2 mL vial containing a bell pepper extract and a glass wool packed liner in which 5 µL of this ex-tract has been injected.

pose Sampler (MPS 2) (GERSTEL, Germa-ny). Instead of the septumless head normal-ly used on the CIS 4 for liquid injection, a special support head is mounted. This sup-port head seals the transport adapters, pro-viding an un-compromised carrier gas fl ow throughthe adapter and liner. The support head and the transport adapters are coni-cal. In order to provide a perfect seal, ev-ery transport adapter is fi tted with two o-rings, between which the carrier gas inlet is placed. Such a sealing system has been proven through years of use in other sys-tems where glass tubes are automatically exchanged, such as the GERSTEL Thermal Desorption System (TDS).

In order to grip and transport the adapters, the autosampler has been mod-ifi ed slightly and fi tted with an electrical gripper. Up to 97 conditioned liners are stored in a special tray; the transport adapt-ers provide a gas-tight seal for contamina-tion-free storage.

For liquid injections, every transport adapter is equipped with a 5 x 3 mm sep-tum that is commercially available and is, for example, used in Agilent’s cool on-col-umn inlet. The top of the injector, and the transport adapter in particular, remain cool during the analysis due to effective heat de-


184

Figure 5: 2 mL Vial containing a QuEChERS method bell pepper extract and a liner packed with glass wool in which 5 µL of this extract has been injected.

Figure 6: Peak area trend (GC / TOF-MS) for 5 µL injections of a standard solution in matrix (bell pepper) into an empty, deactivated baffl ed liner

Liner Empty baffl ed liner (deactivated)

Injection Volume 5 µL (AcN solutions)

Injection Speed 10 µL/sec

Injection Mode Solvent vent for 15 sec (50 mL/min; 8.2 psi),

Splitless sample transfer, Purge Flow 50 mL/min @ 150 sec

CIS 4 Temp. Program 50°C (0.25 min) - 12°C/sec – 280°C (30 min)

Table 3: Injection conditions for Pesticide Analysis after QuEChERS sample extraction method, GERSTEL MPS 2 with ALEX system, GERSTEL CIS 4, Agilent 6890 GC, Varian FactorFour XMS column (30 m, 0.25 mm ID, 0.25 µm fi lm), Leco Pegasus 3 TOF-MS; Liner: Empty baffl ed liner, deactivated.

Residue build-up in the GC liner very quickly affects the analysis of many pesti-cides, as can be seen in Fig. 6. A 5 µL stan-dard solution made up in a bell pepper ma-trix was injected 20 times into a deactivat-ed baffl ed liner. Peak area trends for three different pesticides are presented. For endo-sulfane sulphate and chlorothalonil, peak areas decrease over the course of the 20 in-jections. This can be explained with in-creasing matrix contamination of the lin-er, leading to loss of analytes. For dichloro-phos the situation is different; the peak ar-eas increase. This effect is described in the literature as “matrix-induced chromato-graphic response enhancement” [2]. This means that matrix components cover re-maining active sites in the chromatographic system leading to higher response for sen-sitive analytes.

Fig. 6 shows clearly that when analyz-ing extracts obtained with the QuEChERS method, a liner exchange is required af-ter 10 or at least 15 runs for vegetables like bell peppers. In order to implement the QuEChERS method in a laboratory for a routine automated analysis, it is absolutely necessary to have the capability of exchang-ing liners automatically as is provided by the new GERSTEL ALEX system.

The following is an example of a sequence for routine analysis:

1 Liner Exchange2 Standard Injections for recalibration (3 or 5 concentration levels)3 Sample Injections (7 up to 10 runs)4 Liner Exchange5 ..Steps 2-4 are repeated..

ConclusionsThe system described herein for auto-

matic exchange of PTV inlet liners enables automated GC analysis of samples or ex-tracts with a high content of high boiling substances or suspended matter.

The chosen application, determining pesticides in fruits and vegetables with QuEChERS sample preparation, demon-strates that a reduction of sample prepa-ration steps combined with a GC system which tolerates injection of solutions with a high matrix content is a powerful solution.

Compound sd Compound sd

1 Cyhalothrin 9.5% Imazalil 7.2%

2 Cyhalothrin 6.7% Cresoxim-methyl 6.7%

Atrazine 9.0% Methamidophos 6.5%

Azoxystrobin 5.9% Permethrin 7.0%

Bifenthrin 6.8% Permethrin 2 6.8%

Carbaril 12.9% Procymidone 4.7%

Chloropyriphos-methyl 6.9% Tebuconazol 6.8%

Chlorpyrifos-ethyl 8.5% Thiabendazole 6.8%

Chlorthalonil 29.3% Tolylfl uanide 8.2%

Cyprodinil 7.7% Trifl uraline 6.9%

Dichlorvos 12.0% Tritane 3.5%

Endosulfan sulphate 12.7% o-Phenylphenol 6.8%

Ethion 8.0% p,p’-DDD 6.1%

Table 2 Standard deviations for different pesticides achieved under optimized injection conditions (see Table 3) for 10 injections in one liner. Even though only 10 injections per liner are performed, standard deviations are still high for several pesticides. Apart from the specifi c chemistry of some pesticides, this is due to the fact that 5 µL of the acetonitrile extract had to be injected into an empty liner. Acetonitrile is known not to be a suitable solvent for GC analysis. Normally for such a solution, and such an injection volume, a liner with glass wool should be used. Unfortunately, some substances are very sensitive and show discrimination on glass wool liners. On the other hand a 5 µL injection volume is necessary in order to meet required detection limits of 0.01 mg/kg.

References[1] M. Anastassiades, S. Lehotay, D. Stajnbaher

and F. Schenck: Fast and easy multiresidue method employing acetonitrile extraction/partitioning and “dispersive solid-phase extraction” for the determination of pesticide residues in produce. J AOAC Int 86(2) (2003) 412-31.

[2] M. Anastassiades, K. Mastovska, S.J. Lehotay: Evaluation of analyte protectants to improve gas chromatographic analysis of pesticides. J. Chromatogr. A 1015 (2003) 163-184

Laboratory time for sample preparation is reduced dramatically and at the same time a high sample throughput for the analyti-cal instrument is ensured.


5


Fully automated Sample clean-up and Pesticide Screening with Agilent 6410 LC/MS QQQ online SPE System, Agilent ordering Number: 5990-3866EN

Pesticide analysis EZWhen the sample matrix no longer matters

Application specialists from TeLA GmbH have developed a new method that dramatically simplifi es LC/MS determination of pesticide levels, providing high-quality results independent of the sample matrix type and complexity.

Norbert Helle, Ph.D. and Meike Baden, TeLa GmbH, Fischkai 1, 27572 Bremerhaven, Germany; Phone: +49 471 / 4832 430; Fax: +49 471 / 4832 438; E-mail: [email protected]

Fully automated Sample clean-up and Pesticide

When the sample matrix no longer matters

6


P esticides, fungicides and herbicides are needed in order to provide an adequate supply of food to the ever-growing human population across the

world. The other side of the coin is that residues of these types of compounds in foods cannot be allowed to endanger or affect the health of the consumer. To ensure that foods do not endanger us, maximum ac-ceptable levels, sometimes referred to as tolerated lev-els, have been established for individual compounds according to the current state of scientifi c knowledge. If these levels are exceeded, it would be illegal to mar-ket the contaminated product in Europe. Correspond-ing laws were established by the EU. This legal basis must be, or, in some cases, already has been, adapted into National law by EU member states.

In Germany, the details on maximum acceptable levels of residues can be found in the German LF-GB, acronym for the compendium of laws govern-ing Food, Feed and various consumer products. As an aside, the term “consumer products” in this context spans a great variety of products ranging from pack-aging that comes into contact with food, feed or per-sonal care products to personal care products them-selves, such as cosmetics, tooth paste or shampoo or other personal care items that make more than brief contact with skin or mucous membranes.

Rules, unless properly enforced, are of course worth less than the proverbial paper they were print-ed on. In other words trust is fi ne, but we should verify and if needed take corrective action to ensure compli-ance and best possible consumer safety. This requires a network of reliable laboratories, which is not a trivial matter, as can be seen a bit later in this text.

World-wide, around 700 pesticides are in use, very few of which can be legally used throughout Europe. Various compounds classes have been established, but even these can cover a wide range of polarities, mak-ing it diffi cult to develop a fast all encompassing ana-lysis method.

Still, effective multi-residue methods are in use for the determination of pesticides, helping to en-sure food safety. When fruits and vegetables are ana-lyzed for pesticide residues, often several pesticides are found. The effects on human health have only been documented for very few of these compounds or com-pound groups.

Tracking down pesticides using GC/MS and LC/MSClassical pesticide analysis relied on gas chromatog-raphy (GC) using an electron capture detector (ECD) or a nitrogen phosphorous detector (NPD). The most widely used detector today is the mass selective de-tector (MSD).

In Germany, the analytes that are mainly in fo-cus are those listed in the DFG S19 method, a multi-residue method for the determination of pesticides in food, which enjoys Europe-wide recognition. The analysis of the 270 compounds listed in the S19 meth-od does, however, require signifi cant sample prepara-tion including a gel chromatography clean-up step to separate analytes from the matrix.

Different analysis techniques are used for differ-ent types of pesticides. Liquid chromatography (LC) combined with a mass selective detector (MS) is used to determine polar to moderately apolar compounds. Gas chromatography (GC), most often in combination

with a mass selective detector (MSD) covers apolar to mod-erately polar compounds. As can be seen from this de-scription, there is some over-lap between the techniques. Recently a new multi-resi-due method for the deter-mination of pesticide lev-els in fruits and vegetables was presented (QuECh-ERS: Quick, Easy, Cheap, Ef-fective, Rugged & Safe) [*]. Compared to previous meth-ods, the QuEChERS sample preparation steps are much less time-consuming, en-abling the preparation of 8 samples in less than 30 min-utes. QuEChERS is a sam-ple preparation method well suited for both GC, GC/MS and LC/MS analysis. The QuEChERS sample prepa-ration steps are listed below.

The main benefi t of this sample preparation meth-od is that the overall analysis is less time-consuming and less error-prone than more traditional approach-es. Unfortunately, extracts obtained following this pro-cedure often have a high matrix content, which causes chromatographic problems for GC analysis due to res-idue build-up in the liner unless an automated liner exchange system such as the GERSTEL ALEX is used. (Cf.: GERSTEL Solutions Worldwide Magazine No. 5 p. 18) (http://www.gerstel.com/solutions_no5.htm)

QuEChERS method:

Weigh 10 g of sample –> Add 10 ml of Acetonitrile (AcN)Shake vigorously 1 min –> Add 4 g MgSO4 and 1 gNaClShake vigorously 1 min –> Add internal standard solutionShake 30 sec and centrifuge –> Take Aliquot of supernatant –> Add MgSO4 and sorbentShake 30 sec and centrifuge –> Take Aliquot of supernatant –> inject to GC-MS and LC-MS

The results obtained using QuEChERS sample preparation are comparable to those reached using the S19 method. The QuEChERS method is much faster, requires much less sample preparation, covers a wider range of analytes and is more readily auto-mated. In addition, much smaller volumes of part-ly toxic organic solvents are required, compared with other currently used methods for determining pesti-cides in fruits and vegetables. In addition to the fi nan-cial benefi ts of a much higher laboratory throughput, the cost of materials at around one Euro per sample is relatively low.

The limits of QuEChERS are encountered when-ever samples with more complex matrices need to be analyzed, such as garlic, onion, artichoke or avocado

Calibration curves for nine pesticides, determined using the TeLA GmbH SPE-LC-MS/MS pesticide multi-residue method.

[*] M. Anastassiades, S. Lehotay, D. Stajnbaher and F. Schenck: Fas t and easy multiresidue method employing acetonitrile extraction/partitioning and “dispersive solid-phase extraction” for the determination of pesticide residues in produce. J AOAC Int 86 (2) (2003) 412-31.

7

Norbert Helle and Meike Baden in front of the SPE LC-MS/MS system used for the pesticide multi-residue method: Agilent Series LC 1200 and a GERSTEL SPE system mounted across an Agilent 6410 MS/MS Triple Quad.

with much higher fat content. This can lead to prob-lems with interferences, than can especially infl uence quantifi cation unless further clean-up steps are per-formed.To enable reliable and rugged analysis independent of the sample matrix, we looked for a similarly effective alternative sample preparation procedure. We found that automated solid phase extraction (SPE) based on the GERSTEL MultiPurpose Sampler (MPS) provided an excellent solution. The GERSTEL SPE, we have pre-viously used successfully for a number of applications, including afl atoxins, chloramphenicol and malachite green in foods. In summary, we can report that our automated SPE-LC-MS/MS-ESI multi-residue meth-od reduces the number of manual steps required to a minimum while increasing laboratory throughput. The results are solid and reproducible combined with high sensitivity and good limits of determination.

Instrumental requirements The GERSTEL SPE was fi tted with an injection valve; sample introduction to the Agilent LC 1200 was per-formed directly by the SPE system; detection was per-formed using an Agilent 6410 MS/MS Triple Quad in-strument.

Sample Preparation: 15 mL of an acetonitrile/wa-ter mixture (80:20) was added to a fi ve gram sample of fruit or vegetable for extraction. The SPE cartridge (M&N C-18ec, 6 mL, 1 g) was conditioned using 10 mL methanol (MeOH) and 10 mL water. All steps in the sample preparation procedure, including sample introduction were fully automated.

5 mL sample was added to the cartridge, which was subsequently rinsed with 5 mL water. Analytes were then eluted using an acetonitrile/water mixture added at a fl ow rate of 600µL/min. In contrast to most man-ual SPE methods, the liquid is not aspirated through the cartridge under vacuum, rather it is added un-der positive pressure using a syringe. This means that fl ows, and therefore also the elution speed, are accu-rately controlled and results more reproducible. This holds true even when sample matrix changes the re-striction across the cartridge. The eluate was concen-trated for six minutes at 50 °C and the residual ana-lytes taken up in 5 mL of a acetonitrile/formic acid mixture (30:70).

Sample introduction and analyte separation: 20 µL of the cleaned-up extract was introduced di-rectly to the LC/MS-MS System. The temperature of the column (ZorbaxXDB C-18 100x2.1 mm, 1.8 µm rapid resolution) was set to 50 °C; fl ow rate: 0.5 mL/min resulting in a column head pressure of approxi-mately 420 bar. A solvent mixture of 5mM formic ac-id (A) and acetonitrile (B) was used as mobile phase based on the following gradient programming: 0 min (20 % B); 5 min (20 % B), 30 min (90 % B).

Detection: Analytes were detected with positive Electron Spray Ionization (ESI) using the electron spray ion source or, alternatively, the Agilent Multi-mode ion source. Our experiments clearly showed that the Multimode source provided signifi cantly lower de-tection limits for some pesticides than the ESI source. For other compounds, however, a lower response was obtained than with the ESI ion source. The settings for the ion source were optimized for the fl ow and eluent used. The following parameters were used: N2 temper-ature: 340 °C; carrier gas fl ow (N2): 9 L/min; nebuliz-er pressure: 30 psi. The triple quadrupole instrument was operated in MRM mode, with 5 different time seg-ments, monitoring two transitions for each pesticide. In each segment 40 to 50 analytes were monitored.

The proof of the puddingWhen using the QuEChERS method, it is necessary to adapt the clean-up steps to the sample at hand. It has been clearly shown that for “uncomplicated” ma-trices, such as lettuce or cucumber, additional clean-up steps are not required following the acetonitrile/water extraction.

For complex matrices that contain fat and other chal-lenging matrix components, further clean-up steps are of course needed. For this purpose we used theGERSTEL SPE system.

Raw sample extracts were automatically loaded onto standard SPE cartridges and cleaned. A new car-tridge was used for every sample to eliminate cross-contamination. Macherey-Nagel cartridges contain-ing C18 reversed phase material were found to pro-duce excellent, reliable results.

Automated SPE clean-up as described in this arti-cle took around 20 minutes to complete. Apart from the fi rst sample, the SPE process was performed dur

Norbert Helle and Meike Baden in front of the SPE LC-MS/MS system used for the pesticide multi-residue method: Agilent Series LC 1200 and a GERSTEL SPE system mounted across


8

ing LC/MS or GC/MS analysis of the preceding sample, ensuring that the SPE step was performed without in-creasing the overall analysis time. Once the fi rst sample had been prepared for analysis, the LC/MS or GC/MS system never had to wait idly for the next sample.

An LC 1200 Rapid Resolution HPLC system from Agilent Technologies was used for the analysis. In or-der to achieve good separation combined with meth-od ruggedness, the conscious decision was made to only seek a moderate reduction of the analysis time. The total analysis time required to determine around 140 compounds was in the order of 35 minutes. This time period was more than suffi cient to prepare the following sample for just-in-time sample introduc-tion to the LC/MS system.

Sample clean-up using SPE contributes not on-ly to the ruggedness of the method, it also improves reproducibility and linearity, among other things. To illustrate this, a bell pepper sample was spiked with a pesticide mixture and analyzed.

Following SPE clean-up, retention times and peak areas of the analytes showed excellent reproducibility. The linearity was excellent, both for polar compounds like Carbendazim und Thiabendazole as well as for ap-olar pesticides like Diazinon und Pirimiphosmethyl.

Orange oil samples were cleaned up using a slight-ly modifi ed SPE method. The effi ciency of SPE clean-up is illustrated by the fact that the intense yellow col-or of the sample was transferred to the cartridge while the resulting extract was a clear and colorless liquid. Recovery for the various compounds in this diffi cult matrix ranged from 70 to 90 % while recoveries from fruit and vegetable samples were mainly in the range from 80 to 100 %. It is worth noting that the Zorbax SB-C18 Rapid Resolution columns used provided ex-cellent peak symmetry.

One fi nal comment: Every method must prove its worth in practice. The test, as always, is in the analy-sis of real world samples. To prove the validity of our

Overlay medium polarity sections of 8 different chromatograms: 8 separate sample preparations and injections of a bell pepper sample spiked with a standard mixture of pesticides, 100 ng/mL each. The peaks shown are for the pesticides Terbutylazin, Cyprodinil, Prochloraz, Flusilazol and Fenoxycarb, all showing good reproducibility.

Carbendazim

Thiabendazol

Determination of polar and apolar pesticides respectively in orange oil. Overlay chromatograms covering 9 dif ferent concentrat ions are shown.

method, we took part in a Europe-wide round robin with 46 participating laboratories. A vegetable sample (zucchini) had to be analyzed for 185 different pes-ticide residues. Out of 46 laboratories, TeLA GmbH was among the 12 that managed to correctly identi-fy and quantify the analytes thus meeting the round robin requirements and passing the test.

128 of the 185 pesticides were determined using our SPE-LC-MS/MS pesticide multi-residue method. 90 of the 185 pesticides were determined using a GC/MS system (GC 6890 / MSD 5973, both from Agi-lent Technologies) in combination with the GERSTEL MultiPurpose Sampler (MPS) using a Retention Time Locking (RTL) method.


9

All steps are performed automatically by the MPS.If needed, the sorbent is conditioned with solvent prior to the extraction process.

1 Sample is drawn into the pipette tip for direct contact with the solid phase sorbent. There is no contact between the sample and the syringe used to aspirate the sample and therefore no risk of cross contamination.

2 Air is drawn into the pipette tip from below through the frit. Turbulent air bubble mixing creates a suspension of sorbent in the sample, ensuring optimal contact, highly effi cient extraction, and high recovery.

3 The extracted sample is discharged, typically after 30 seconds.

If needed, the sorbent can be washed to remove unwanted residue.

4 Extracted analytes are eluted using a suitable solvent, which is added from above for most effi cient elution. The eluate is collected in a vial for subsequent sample introduction to LC/MS or GC/MS.

The total time required for extraction in the examples shown in this article was always less than 6 minutes. Sample preparation and GC/MS or LC/MS determination can be performed in parallel for best possible throughput and system utilization.

Automated DPX process

I n this study, we present a novel solid-phase extraction technique called dis-posable pipette extraction (DPX). The

solid-phase sorbent contained in the DPX tip is loose, which AN/2009/1 - 2 permits mixing of solutions to provide unsurpassed extraction effi ciency and short equilibra-tion times. DPX extractions are automated using the GERSTEL MultiPurpose Sampler (MPS), enabling effi cient, high-throughput sample preparation. The GERSTEL DPX-Q and the DPX-Qg with graphitized car-bon black, represent the only commercial-ly available automated QuEChERS applica-tion for multi-residue analysis of pesticides. DPX is a fast and efficient solid phase ex-traction technique used for a wide range of applications such as drugs of abuse, thera-peutic drug monitoring, comprehensive screening, pharmacology studies, as well as pesticides in fruits and vegetables. The DPX process is shown schematically in Fig-ure 1. If needed, the sorbent is conditioned

Disposable Pipette Extraction (DPX)

Automated multi-residue Pesticide Analysisin Fruits and Vegetables by DPX-QuECHERS

One of the most important aspects of reducing pesticide exposure is monitoring of pesticide residues in foods. A number of analytical methods have been developed, many of them based on traditional liquid-liquid extraction, gelpermeation or solid phase extraction in combination with GCMS or LC-MS. Recently, the QuEChERS (quick, easy, cheap, effective, rugged and safe) sample preparation methods have been developed to help monitor pesticides in a range of food samples. These methods, however, still require many manual steps, such as centrifugation, leading to increased total analysis time. There is a need for a simple, reliable and readily automated technique to clean up QuEChERS type extracts in order to improve laboratory productivity for monitoring pesticide residues in foods.

with solvent prior to extraction. The sam-ple is then drawn into the pipette tip for di-rect contact with the solid phase sorbent. Turbulent air mixing creates a suspension of the sorbent in the sample ensuring opti-mal contact and highly efficient extraction. The extracted sample is discharged, typical-ly after 30 seconds. If needed, the sorbent can be washed to remove unwanted resi-due. The extract is then eluted into a vial for subsequent LC or GC analysis.

For sample cleanup methods, such as QuEChERS which focuses on removing fatty acids and water, the DPX method simply incorporates the steps of aspirat-ing the sample solution, mixing with the sorbent, and dispensing the solution into the vial for analysis. There are no wash- or elution steps, the extractions can there-fore take place in less than 1 minute. The GERSTEL MPS 2 with MAESTRO software control automates the entire process including sample introduction.

In the following study, DPX-Qg tips are used to remove sample interferences from QuEChERS type extracts prior to GC/MS analysis.


Figure 2. Picture of extracts before and after clean-up.

ExperimentalInstrumentation: Analyses were per-formed on a 7890 GC equipped with a 5975C MSD with triple axis detector (Agilent Technolo-gies), PTV inlet (CIS 4, GERSTEL) and MPS 2 robotic sam-pler with 10 µL sy-ringe (GERSTEL).

Standard preparation: A composite stan-dard of organochlorine and organophos-phate pesticides was prepared at a concen-tration of 1000 µg/L in acetonitrile. The standard was diluted to 20, 50, 100 and

10

250 µg/L. Twe nt y - f ive micro-liters of a matrix matching solution were added to the standards.

Sample preparation: Processed fruit and vegetable extracts were provided by the University of South Carolina. The pesti-cide standard was diluted to obtain con-centrations of 20 and 200 µg/L in 500 µL of the extracts. DPX extraction: 1 mL QuEChERS DPX tips were provided by DPX Labs, LLC. 500 µL of vegetable extract was manually transferred into a test tube. The extracts, 0.5 mL each, were drawn through the DPX tips 3 times. This process was automated using the GERSTEL MPS 2 autosampler with 2.5 mL syringe. The extract was then transferred into a 2 mL autosampler vial. 1 µL of eluent was injected into the GC.

Results and DiscussionThe spinach extract was cleaned using a DPX Qg-1TA tip and the orange extract was cleaned using both the Q-1TA and Qg-1TA tips. The Qg-1TA tips contain graphitized carbon black which is more effective in removing chlorophyll from the extracts. Figure 2 shows a photograph of the spinach extracts before and after processing with the DPX tips. The green color is effectively removed from the spin-ach sample. The orange sample still had a mild orange tint when the Q-1TA tip was used. The Qg-1TA tip effectively removed all color and was used for all subsequent experiments. Figures 3 show chromato-graphically the effectiveness of the cleanup of the samples using DPX for the spinach sample, respectively. The DPX Qg-1TA tips effectively remove interferents, espe-cially free acids in the 15-22 minute reten-tion time window. Each extract type was spiked at 20 and 200 ppb in triplicate and cleaned up using DPX. An example chro-matogram for a spinach extract spiked at 200 ppb is shown in Figure 4. Table 1 shows the results for the DPX cleanup.

The recoveries were calculated from an external six point calibration plot for each analyte. The external standards


were matrix matched. The results show good recover-ies for the mix of OC and OP pesticides used in this study. The average % RSDs (n=3) are less than 10 % for

both matrices at both 20 and 200 ppb spike lev-els. The recoveries at the 200 ppb level range from 68-179 % with an average value of 119 % for the orange ex-tracts and range from

34-124 % and average 91 % for the spinach ex-

tracts. These values could be improved with further optimization of the DPX automation method (num-ber of extracts and aspira-tion speeds) and the use of internal standards in the GC/MS method. The re-coveries with and without DPX cleanup are shown in Table 2 for the spinach and orange extracts, clearly showing the elimination of matrix interferences with the DPX Qg-1TA tip.

ConclusionsThis study demonstrates

the feasibility of using the DPX Qg-1TA for cleanup of QuEChERS type extracts

CompoundSpinach

No DPX DPXDichlorvos 120 51Mevinphos 145 34Phorate 170 93

-BHC 150 76-BHC 170 51

Diazinon 160 96Methyl Parathion 300 54

Ronnel 195 63Aldrin 210 124Trichloronate 245 87Heptachlor Epoxide 155 102

t-chlordane 175 116Prothiofos 265 104Dieldrin 260 123Endrin 195 118ß-Endosulfan 180 102Fensulfothion 165 88Sulprofos 250 122DDT 195 117Endrin Ketone 155 97Average 193 91

AnalyteSpinach Extracts

% Recovery % RSD20 ppb 200 ppb 20 ppb 200 ppb

Dichlorvos 92 51 8.3 15Mevinphos 60 34 8.3 15Phorate 16 93 32 5.2

-BHC 47 76 16 6.2-BHC 105 51 17 24

Diazinon 117 96 2.5 3.9Methyl Parathion 165 54 20 14Ronnel 95 63 11 9.8Aldrin 148 124 1.9 3.3Trichloronate 152 87 8.3 6.8Heptachlor Epoxide 120 102 4.2 3.4t-chlordane 135 116 3.7 3.5Prothiofos 162 104 4.7 6.3Dieldrin 128 123 9.0 3.2Endrin 142 118 5.4 3.9ß-Endosulfan 138 102 12 7.4Fensulfothion 63 88 4.6 11Sulprofos 200 122 4.3 6.4DDT 208 117 6.9 7.3Endrin Ketone 138 97 2.1 7.7Average 122 91 9.1 8.2

Table 1. Pesticide recoveries and % RSD. Table 2. Pesticide recoveries with and without DPX clean-up; spike level = 200 ppb

B

A

Time-->

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Abundance

Abundance

1000000

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2000000

1500000

10.00 15.00 20.00 25.00

10.00 15.00 20.00 25.00

1000000

500000

2000000

1500000

2500000

2500000

Figure 3. Chromatograms of spinach extract before (A) and after (B) clean-up.

Abundance

2000

10000

Time--> 10.00 15.00

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20.00

4000

8000

14000

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Figure 4. Chromatogram of spinach extract, spiked with 200 ppb, after DPX clean-up.

prior to GC/MS analysis. The DPX tips re-move matrix interferents, leading to better analyte recovery and reducing the need for maintenance since there is less build-

up of non-volatile material in the GC in-let. Full automation of the sample cleanup and injection is accomplished using the GERSTEL MPS 2.

11

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[email protected]

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Subject to change. GERSTEL®, GRAPHPACK® and TWISTER® are registered trademarks of GERSTEL GmbH & Co. KG.Printed in Germany · 0208b · © Copyright by GERSTEL GmbH & Co. KG

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[email protected]

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About GERSTELGERSTEL develops, produces and supports solutions that include auto-mated sample preparation and sample introduction for GC/MS and LC/MS. The available techniques include au-tomated Solid Phase Extraction (SPE), which can be performed in combinati-on with Standard Addition, Derivatiza-tion, and Eluate Concentration with or without Keeper Solvent. Additionally, in combination with a GC or GC/MS system, Automated Liner Exchange (ALEX) enables automated matrix eli-mination in the GC, allowing the user to dramatically reduce the amount of effort going into sample prepara-tion, for example by using the QuE-ChERS sample preparation method for the determination of pesticides in non-fatty fruits and vegetables.

Sample preparation is performed simultaneously with the LC/MS or GC/MS run of the preceding sample enabling highest possible productivity and system utilization. The system is controlled through the GERSTEL MA-ESTRO software in stand-alone mode or fully integrated with the Agilent Technologies GC/MS or LC/MS soft-ware. One method and one sequence table controls the complete system.

A method for fast screening of pesticide

multi residues in aqueous samples using

dual stir bar sorptive extrac-

tion (dual SBSE) - thermal

desorption (TD) – fast GC/MS

has been developed. Reco-

very of 82 pesticides – orga-

nochlorine, carbamate, orga-

nophosphorous, pyrethroid

and others – for the SBSE

was evaluated as a function

of octanol water distribution

coeffi cients (log Ko/w: 1.7-

8.35), sample volume (2-20

mL), salt addition (0-30 %

NaCl), and methanol addition

(0-20 %). The optimized method consists

of a dual SBSE performed simultaneously

on respectively a 20 mL sample containing

30 % NaCl and a 20 mL sample without

modifi er (100 % sample solution). One ex-

traction with 30 % NaCl is mainly targeting

solutes with low Ko/w (log Ko/w < 3.5) and

Pesticides in Wateranother extraction with unmodifi ed sample

solution is targeting solutes with medium

and high Ko/w (log Ko/w >

3.5). After extraction, the

two stir bars were placed in

a single glass desorption li-

ner and were simultaneous-

ly desorbed. The desorbed

compounds were analyzed

by fast GC/MS using a mo-

dular accelerated column

heater (MACH). The method

showed good linearity (r2 >

0.9900) and high sensitivi-

ty (limit of detection: < 10

ng/L) for most of the target

pesticides. The method was applied to the

determination of pesticides at ng/L levels in

river water and brewed green tea. n

Further informationAppNote 12/2008 (http://www.gerstel.com/pdf/p-gc-an-2008-12corrected.pdf))

Documents

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