GERSTEL Solutions No. 6.pdf

News from GERSTEL GmbH & Co. KG · Aktienstraße 232 – 234 · D-45473 Mülheim an der Ruhr · Germany · Phone +49 (0) 208 - 76503-0 · Fax +49 (0) 208 - 7650333ww

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No. 6 April 2006

ISS

N 1

619

-007

6

G L O B A L A N A L Y T I C A L S O L U T I O N S

GERSTEL MPS with new option for standard cartridges

Automated Solid Phase Extraction (SPE)

SBSE tracks down odors

Twister sniffs out aftertaste

New: Dual SBSE Fast GC/MS

Fast screening ofpesticide multi-residues

New: GERSTEL MAESTRO Software

MAESTRO conductssymphony of solutions

A s a family business, we have always enjoyed freedom from short or medium term interests

of investors, allowing us to pursue promising long-term strategies and projects. The GERSTEL tradition is to supply a steady stream of innovative solutions, providing reliable growth and dependable support for our customers over the years.

We are in the fortunate position to have sufficient resources to keep and extend our strong position as a global provider of GC solutions while adding LC and LC/MS solutions to the offering. Since 1998, GERSTEL has had double-digit annual growth; our organization has grown by more than 100 % over the past 5 years.

When GERSTEL started offering automated sample preparation for Liquid Chromatography and LC/MS, we initially focused on the German market, working with experienced local partners to build our expertise and our customer base. We are now expanding our scope, offering LC and LC/MS solutions world-wide through our network of subsidiaries and distributors. Many of the techniques and technologies used for GC sample prep can be applied to LC. A series of products and solutions have been developed around the GERSTEL MultiPurpose Sampler (MPS), a combined autosampler and liquid handling robot. This year at PittCon,

Customer focused Solutions for LC and LC/MS

GERSTEL expands its offering

GERSTEL is introducing automated on-line SPE for the MPS.

GERSTEL solutions can significantly increase productivity and throughput of LC and LC/MS labs, improving Return On Investment (ROI) for the instrumentation. In many cases, added benefits are provided, such as improved precision and significantly reduced solvent consumption with associated savings and improvements of the laboratory environment.

Our integrated solutions and inte-grated software helps control every step of the process, from automated Sample Prep through Sample Introduction to control of the entire GC, GC/MS, LC or LC/MS system. Just one method and one sequence table controls the entire process providing simple and efficient operation with less risk of error.

By offering both GC and LC solutions, we strive to more completely cover the needs of our chromatography laboratory customers. Labs mostly have both techniques in-house, struggling with different sample prep hardware and different software for the two. By relying on solutions from one vendor, the complexity is reduced significantly.

Yours sincerely

For almost 40 years, GERSTEL has built a strong reputation providing accessories and system solutions for gas chromatography, focusing mainly on automated sample preparation and sample introduction. Our solutions are based on instruments from Agilent Technologies making the combined solutions unbeatable in terms of quality, performance and overall value. GERSTEL is the largest partner of Agilent Technologies world-wide for customer oriented analytical solutions.

In this issueCompany profile■GERSTEL, Inc. USA: Join the „Gerstelization“

3

Title

■NEW: Automated

SPE option for MPS

4Application ■Improved determination of toxins in mussels 6■6 times faster screening of pesticide multi-residues 17

Software ■New MAESTRO software offers enhanced productivity 10

Research ■The Twister sniffs out the aftertaste 12■Drug testing: reliable determination of THC in saliva 15■GERSTEL ODP sniffs out insect communication 20

Distributor ■SRA Instruments, Italia 16

News ■Analytical Supplies 2006/2007 9■New EU regulation on organic chemicals in toys 24

Book tip ■»Bugs in the system« 23

Imprint 24

Eberhard G. Gerstel

Holger Gerstel

Eberhard G. Gerstel and

Holger Gerstel

3GERSTEL solutions worldwide – April 2006

GERSTEL solutions worldwide Editorial

A cornerstone of the GERSTEL philosophy is exceptional customer

support. This philosophy has helped GERSTEL become the leading Agilent Technologies Premier Solutions Partner worldwide.

The GERSTEL U.S. Headquarters in Baltimore boasts a well-equipped applications lab. Our chemists have many years of experience developing analytical methods and associated automated sample preparation procedures. Our resources are available to you as a customer; we want to meet your requirements with a GERSTEL solution. To further expand our support capabilities in the US, GERSTEL recently established a partnership with Ray Marsili, a recognized authority with many years of experience in food and flavor analysis. Ray and his group are experts in Olfactory Detection and Chemometric techniques. The Marsili Consulting Group maintains a contract lab in Rockford, Illinois, equipped with instrumentation from GERSTEL, Agilent Technologies, and LECO. This lab will serve as a demonstration and application development facility, focused primarily on Food, Flavor, and Fragrance applications, key GERSTEL areas of expertise.

To most efficiently help new users become productive, our training facility in Baltimore offers multi-day training courses throughout the year. Courses cover setup, maintenance and trouble-shooting of complete systems as well

as special techniques and accessories: Our TDS courses cover adsorbent tube air sampling and direct thermal extraction of solids using the Thermal Desorption System (TDS). Our courses on the MultiPurpose Sampler (MPS 2) robotic sampler cover special accessories such as the Twister Desorption Unit (TDU) and Automated Liner Exchange (ALEX). Various sample introduction techniques are emphasized, that can be automated using the MPS 2 in combination with a CIS 4 inlet. These include Large Volume Liquid Injection (LVI), Headspace GC analysis, automated SPME and automated Sample Preparation Functions in combination with the GERSTEL MAESTRO software. To put information at your finger tips at all times, a comprehensive set of more than 80 video clips is available on CD covering all aspects of instrument maintenance.

Both the MPS 2 and TDS instrument platforms are compatible with the revolutionary GERSTEL Twister, based on the Stir Bar Sorptive Extraction (SBSE) technique. A half-day Twister® method development course illustrated with a series of application examples is offered in conjunction with the MPS 2 and TDS courses. Please refer to the GERSTEL U.S. web site www.gerstelus.com for details on upcoming courses!

A key focus for GERSTEL is to help our customers meet constant demands for higher productivity and improved performance. This past year, GERSTEL

successfully launched the Modular Accelerated Column Heater (MACH) Fast GC accessory. MACH has helped many laboratories push back the boundaries of performance and productivity, combining conventional column separations with faster heat-up and cool-down in a standard GC 6890. The MACH system has been successfully implemented for environmental applications such as TPH, pesticides and PCB’s and also for traditional forensic applications such as analysis of drugs of abuse and arson accelerants.

In addition to Fast GC applications, MACH enables optimized dual column chromatography. Separate temperature programs can be set up for the columns used, ensuring optimal performance for each column. For more complex separations, MACH can be coupled with GERSTEL‘s Multi-Column Switching (MCS) multidimensional GC system to produce a highly efficient single-GC solution with independently controlled columns. MACH-based multidimensional systems are used for complex samples in Food, Flavor and Fragrance and other applications.

Join the many who have found “GERSTELIZATION” to be the cornerstone to building more effective analytical capabilities.

Call us to find out how GERSTEL technology can benefit you!

The GERSTEL, Inc. applications laboratory

in Baltimore. Our chemists have many

years of experience developing analytical

methods and associated automated sample

preparation procedures.

GERSTEL, Inc., located in Baltimore, MD, USA has just finished a banner year measured by sales success as well as by expansion of Customer oriented capabilities. The North American GERSTEL field support team continues to grow with additions in 2005 to Service, Applications, and Technical Support.

Join the “Gerstelization”


GERSTEL solutions worldwide Company profile

GERSTEL solutions worldwide Title

I n recent years, food inspectors have increasingly found residues of restricted or outlawed antibiotics

in food products of animal origin such as meat and fish, that are imported to Europe. When the active compound chloramphenicol (CAP) was found it caused real upheaval. CAP is a known human carcinogenic. It is suspected of causing genetic damage in human cells as well as irreversible damage to the blood-forming cells of the bone marrow.

The determination of CAP is usually performed by LC/MS. The sensitivity of the method depends greatly on sample preparation. Norbert Helle, Ph.D. is an applications expert in the field of food safety and owner of TeLA GmbH, a contract laboratory in Bremerhaven, Germany. According to Dr. Helle, a

high matrix load can result in incorrect quantification of CAP even when highly selective LC-MS/MS methods are used. The same is the case for many other analytes in the areas of food, pharmaceutical or environmental analysis. Solid Phase Extraction (SPE) is the sample preparation technique of choice for many such samples enabling analysts to separate analytes from matrix prior to LC or GC analysis. Over the past 25 years, SPE has become one of the most effective and most widely used procedures for sample clean-up.

According to Dr. Helle, manual SPE methods have serious drawbacks. Not only is a lot of time and patience needed to perform SPE manually: Recovery and reproducibility can be subject to extreme deviations. This is largely dependent on the experience of the user and on how

meticulously each step is performed. If the SPE process and all associated liquid handling steps are automated, the process becomes much more reliable and efficient.

Automated SPE option for the MultiPurpose Sampler (MPS) based on standard cartridges

At PittCon 2006 in Orlando and at Analytica 2006 in Munich, GERSTEL is introducing a newly developed automated SPE Option for the GERSTEL MultiPurpose Sampler (MPS). The SPE Option is based on newly developed technology incorporated into an SPE station. Standard cartridges are used, enabling simple transfer of existing methods. The cartridges are fitted with disposable needles for liquid transfer, completely eliminating the risk of carry over between samples.

According to Ralf Bremer, Managing Director of GERSTEL, the SPE station

Thousands500

400

300

200 MPS

Recovery: 92 %

Relative Standard Deviation: 2.0 %

Manual

Relative Standard Deviation: 2.2 %

Recovery: 90 %

CAP manual SPE

CAP MPS

1 2 3 4 5 6 7 8

Recovery and reproducibility of chloramphenicol

determination in prawn meat with manual and

automated sample preparation, respectively.

Transport sample vial into the SPE vial position,Transport cartridge to the SPE waste position

Condition cartridge with 4 mL MeOH and 4 mL H2O,Flow: 50 µL/s

MOVE

ADD 5+6

ADD 7

ADD 8+9

SPE -> SHIFT

ADD 10

WAIT

MOVE

SPE -> SHIFT

MOVE

Add 4 mL sample to cartridge, Flow: 50 µL/s

Rinse cartridge with 1 mL H2O and 2 mL MeOH/H2O(1:10), flow: 50 µL/s

Transport SPE carriage with the cartridge from SPEwaste to SPE vial position

Elution of CAP with 3 mL MeOH/H2O (1:1),Flow: 30 µL/s

Wait 30 seconds for eluent to transfer completely

Move SPE carriage to waste vial position

Discharge cartridge to the waste vial position

Transport sample vial back to the tray

Prep method for automated

SPE using the MPS to

analyse chloramphenicol

in food products of animal

origin like meat and fish.

All steps are selected by

mouse-click from a menu

and added to the list.

New: Automated SPE option for the MultiPurpose Sampler (MPS) based on standard SPE cartridges

Automated Solid Phase Extraction

GERSTEL solutions worldwide – April 2006

4 5GERSTEL solutions worldwide – April 2006

As many users will tell you, performing manual Solid Phase Extraction (SPE) requires a lot of time - and strong nerves when insufficient recovery and bad reproducibility are experienced. It can be hard to discern what went wrong: Was conditioning adequate? Did the cartridge run dry in an unguarded moment? Was the eluent flow rate too high? Did cartridges get mixed up? If only cartridges could speak to us. If the SPE process can be reliably automated, such questions will not arise in the first place. GERSTEL has now introduced an Automated SPE Option for the GERSTEL MultiPurpose Sampler (MPS). It is based on standard cartridges making it easy to transfer and automate existing methods. Fully automated liquid handling and exact timing of all processes make SPE a more relaxing and much more efficient activity.

Fragmentation spectrum of CAP

257.0

fragment

parent321.0

Prawn sample,spiked with 0.01 µg/kg CAP

1 pg CAP

2 4 6 8 10 min

Chloramphenicol

NO2 CH CH OHCH2

OH NH CO CHCI2

GERSTEL solutions worldwide Title

simplifies SPE, saving a significant amount of time for the user. Not only is the process fully automated: All steps in the process are performed during the chromatographic run of the preceding sample. Overlapping sample prep and analysis ensures best possible productivity. Following the automated SPE step, the prepared sample is automatically introduced to the GC, GC/MS, LC or LC/MS system by the MPS. Alternatively, depending on the needs of the user, the MPS can be used as a stand-alone sample preparation robot, independent of the chromatographic system.

Automated liquid handling in the MPS provides the user with the capability to deliver defined, reproducible volumes of liquid for conditioning, extraction and elution of SPE cartridges. Flow rates are kept constant and the transfer of defined volumes performed in a reliable manner. This helps to produce better and more reliable analytical results. Similarly, standard addition and sample introduction is performed automatically in a reliable and reproducible manner for best possible analytical results. If automated drying steps are required, the MPS, fitted with a gas purge option, can perform this step automatically as well.

Cartridge elution is performed under positive pressure in a sealed system and liquids are transferred from the cartridge into a closed vial using disposable needles. This ensures that the SPE process is performed without introduction of contaminants, without analyte carry-over and without loss of analytes for improved results. For every sample a new cartridge is used.

Chromatogram of transfer 321.0 – 257.0; detection of a CAP residue of 0.01 µg/kg in a prawn sample

following automated SPE on the GERSTEL MultiPurpose Sampler MPS 2.

LC/MS. In his experience, manual SPE using standard cartridges can provide good results under tightly controlled conditions, but the manual process is of course very time consuming. MPS with the Automated SPE Option provides better results than Dr. Helle expected. For CAP determinations using the MPS, standard deviations were 2.0 % compared with 2.2 % for manual SPE. The recovery was 92 % on the MPS compared with 90 % for manual SPE. The MPS provided slight, but clear improvements over the best achievable manual results, and, more importantly, a big improvement in productivity. Even complex samples can be conveniently and reliably processed in around half the time it normally takes – and everything is done automatically by the MPS.

Bremer: “Since the SPE station of the MPS is designed for standard SPE cartridges, existing validated manual methods can be directly transferred to the MPS and automated”. The complete process is controlled separetely by the MAESTRO Software or through integrated control from the Agilent Technologies ChemStation Software. Integrated control means that everything from the SPE process through dilution, standard addition, derivatization, mixing and sample introduction to the GC/MS or LC/MS is controlled from one method and one sequence table.

Norbert Helle has been using a GERSTEL MPS with Automated SPE Option successfully for the deter-mination of chloramphenicol (CAP) in food products of animal origin by

Ralf Bremer

Dr. Norbert Helle



Health-wiseMussels can be lethal – especially if consumed in the wrong season. The danger to our health is posed by algae toxins, accumulated in mussel tissue in high concentrations during periods of warm weather and strong algae growth. In order to better protect consumers, the European Union (EU) requires that such seafood products be tested on lab mice. The chemist Norbert Helle, Ph.D. and a group of experts have shown a better way: A method for the determinination of marine biotoxins such as the highly Paralytic Shell Fish Poisoning (PSP) toxin has been accepted into German federal regulations. The method is based on liquid chromatography (LC) with fluorescence detection.

The authorDr. Norbert Helle

TeLA GmbH

Fischkai 1, 27572 Bremerhaven

Germany

Improved determination of toxins in mussels




GERSTEL solutions worldwide Application

Mussels have their traditional place on European menus in fall and winter. Popular advice

says to eat mussels only in months that have an ‘r‘ in their names, that is, mainly during the cold wet season of the year. Those who disregard the rule and eat mussels harvested in the summer risk poisoning by marine biotoxins. These toxins cause diarrhea and vomiting, interfere with the autonomic nervous system, produce palsy symptoms and in serious cases even cause death.

Marine biotoxins are accumulated from algae which the mussels feed on. Algae can produce a variety of toxins, which accumulate in the mussel tissue. The risk of poisoning rises during the summer months because algae growth increases dramatically with rising ocean temperatures. As the food supply for the mussels increases, so does their uptake of toxins.

Classes of marine biotoxins

Various organic compound classes are counted among marine biotoxins. Paralytic Shell Fish Poisoning (PSP) toxins are highly polar tetrahydropurines. A relatively large number of different PSP toxins are known. Only a few of these are formed directly in toxic algae, for example in the Alexandrium types, most are produced by the mussels as metabolites of algae toxins.

PSP toxins are among the most potent biotoxins known. The consumption of PSP impacted mussels can affect the nervous system, causing apnea and even death. Diarrhetic Shellfish Poisoning (DSP) toxins, also called dinophysis toxins, are compounds with several cyclic ether groups. The most widely encountered toxin of this class is Okadaic acid.

Poisoning with DSP toxins can cause nausea, vomiting and diarrhea. Algae that produce DSP toxin, like Dinophysis acuminata and Dinophysis acuta, can be found in the North Sea and Baltic Sea.

The active ingredient in Amnesic Shellfish Poisoning (ASP) toxins is Domoic acid, which impairs the central nervous system and can cause loss of short-term memory (amnesia). This of course has the unfortunate effect that people can no longer remember what they have eaten, complicating both diagnosis and treatment.

7

GERSTEL solutions worldwide ApplicationBesides the three main groups of

marine biotoxins, additional toxins from algae or mussels have been isolated and categorized. These include yessotoxins, pectenotoxins and the azaspiro acids.

EU-Guidelines, Focusing on Consumer Protection

As toxic algae spread in the oceans, and the number of illnesses caused by mussel consumption increased, the call for improved consumer protection was heard worldwide. The European Union (EU) has established guidelines for the control of algae toxins in mussels. The guidelines are now to be adapted into national law by the Member States.

The basis is the Commission Decision 2002/225/EC of March 15, 2002 which specifies maximum allowable concentrations of individual toxin groups and lays out accepted analysis methods. According to regulation (EC) No. 854/2004 (EU Hygiene Package), the guidelines have to be implemented by January 1, 2006. In Germany, the maximum allowable concentrations of ASP toxins (20 mg Domoic acid/kg mussel meat), DSP toxins (400 µg/kg mussel hepatopancreas), and PSP toxins (800 µg/kg mussel meat) are specified by the Fish Hygiene Regulation. In the

implementation of European regulations, Germany has deviated from the analysis guidelines.

The EU guidelines require that tests for marine biotoxins be performed on a routine basis using bioassays. In the case of mussel toxins, this means performing the test on animals: In order to determine if marine biotoxins are present in a sample, an extract of the mussel tissue to be examined is injected into the abdominal cavity of a mouse. If the mouse subsequently dies, it is considered proof that an excess concentration of biotoxins was present.

The use of animals for routine testing for algae toxins has met with severe criticism. Germany mainly relies on chemical analysis methods; animal experiments are used only in cases where inconclusive results have been obtained. The chemical analysis approach is justified based on a federally ensured right to use scientifically verifiable methods. Additionally, the German Animal Rights Law stipulates that animal testing should only be conducted if no scientifically sound practical alternative is available. The analysis technique mainly used is Liquid Chromatography (LC), in combination with highly sensitive fluorescence detection.

Pre column methodLAWRENCE 1991

Post column methodTHIELERT 1991, OSHIMA 1989,

FRANCO 1993

25 g mussel sample

Homogenate

Supernatant

Cleaned extract

Derivatized extract

Derivatized solutionto be analyzed

LC Separation

Chromatogram

Add 25 mL HCI 0.1 M,mix using an Ultraturrax (2 min)

centrifugation

Adjust pH to 3-4 , clean upsupernatantconditioned with methanol/waterand 0.1 M HCI

Add 0.5 mL supernatant, elute with2 mL water, mix eluates

Adjust 1 mL to pH = 8 (NaOH),oxidize 100 µL with 275 µLperiodate solution, approximately2 min reaction time

Add 20 µL concentratedacetic acid

inject 20 µL, separate on C-18LC columEluent A: ammonium formiate 0.1 MEluent B: eluent A + 5 % acetonitrile

Fluorescence detection:Ex.: 335 nm, Em.: 400 nm

inject 20 µL ,separate on C-18RPLC columnbinary gradient:ammonium phosphate+ sodium octane sulfonate+ acetonitril

filtration

centrifugation

Add 25 mL HCI 0.1 M, mixusing an Ultraturrax (2 min)

Online-derivatizationwith:ammonia +periodic acid +sulfuric acid10 m reaction loop at 47 °C

Fluorescence detection:Ex.: 335 nm, Em.: 400 nm

Der

ivat

izat

ion

Det

erm

inat

ion

Sam

ple

Pre

para

tion

Ext

ract

ion

Der

ivat

izat

ion/

Det

erm

inat

ion

Ext

ract

ion

Chromatogram

Separated analytes

solution to be analyzed

25 g mussel sample

Homogenate

Supernatant

Schematic

comparison of

pre-column

derivatization and

online post-column

derivatization as

described by

Lawrence et al. [1]




LC – the method of choice for detection of PSP toxins

The chemical structures of PSP toxins are based on a tetrahydropurine ring, substituted at several positions. The derivatized analytes are highly polar, i.e. very water soluble, and do not have adequate chromophores for UV detection.

In order to achieve the required sensitivity for detection of PSP toxins, methods based on fluorescence detection [1] were developed as far back as the 1980’s, relying on the formation of a fluorescing aromatic structure formed through oxidation of the toxin molecules. Various oxidants were used, among them hydrogen peroxide and perchloric acid.

While the pre-column derivatization described by Lawrence et al. [1] offers the advantage of a simpler instrument set-up and lower reagent consumption, the advantage of on-line post column derivatization is that it requires less manual sample preparation and generally performs better with less standard deviation.

In Germany, the set of laws regulating Foods, Tobacco products, cosmetic products, and other related consumer products is called the LMBG. The § 35 LMBG working group deals specifically

with mussel toxins. It has been working for several years on the development of a standardized procedure to detect PSP toxins. Following preliminary round robin tests involving a number of laboratories running both methods, a final comparative study was performed in 2004 with a procedure based on the method of Lawrence et al., which was modified by Walther [2]. The results of the study, in which nine laboratories participated, were so good, that the proposed method was accepted in the collection of official methods according to § 35 LMBG.The samples for the round robin test were based on an extract of the toxic algae Alexandrium fundyense, containing GTX-2, GTX-3, GTX-5 and saxitoxine. The toxic extract was added to the unexposed meat of blue mussels. Extraction with acetic acid followed by oxidation using hydrogen peroxide yielded fluorescing derivatization products. These were separated on a reversed phase column with highly sensitive fluorescence detection at the extinction wave length of 330 nm and emission wave length of 390 nm.

Depending on the detector used, the detection limit of the method lies between 5 µg/kg and 50 µg/kg mussel meat, clearly meeting the requirements of EU regulations. The upper limit specified by

tests conducted to check sample homogeneity and stability revealed a problem at the participating laboratories: the fluorescing derivatization products of PSP toxins are thermally labile. The detector response clearly showed analyte degradation after only two hours at room temperature.

After 24 hours the signal was reduced by as much as 40 percent. Additionally, the stability of individual derivatized PSP toxins varied greatly. The GTX-5 derivatization product, for example, was decomposed significantly faster than the saxitoxine derivatization product. This could lead to significant error in results, especially when longer sample series are run over night or during the weekend. The effect can be reduced by using cooled sample trays. Ideally, though, every sample should receive

mAU

140

120

100

80

60

40

20

10 20 30 40 50 min

GTX-2 and GTX-3

GTX-5

saxitoxine

Sample chromatogram ofthe round robin test extracts: Separation following pre-column derivatization of GTX-2/3, GTX-5 and saxitoxine.

Comparison data for manual and automated sample preparation. Analyte degradation is clearly observed in samples that have been prepared manually and left on the autosampler for more than two hours prior to sample introduction and analysis. The MPS prepares every sample at the exact same point in time prior to analysis, providing significantly better results.

Program for derivatization of PSP toxins with

the GERSTEL MultiPurpose

Sampler (MPS).

Absorption [%]120

100

80

60

40

20

00 5 10 15 20

time [h]

MPSmanual sample preparation

Vial transport from 2 mL tray to the Agitator

Add 50 µL H2O2 to sample, start derivatization

Mix sample in the Agitator at 500 rpm for 1.5 minutes,20 seconds on time, 2 seconds off time, alternating direction

Add 30 µL glacial acetic acid to stop derivatization

Mix sample in the Agitator at 500 rpm for 1.5 minutes,20 seconds on time, 2 seconds off time, alternating direction

Vial transport from Agitator back to 2 mL tray

Inject 5 µL of sample into LC column for separation andsubsequent fluorescence detection

MOVE

ADD 1

MIX 1

ADD 2

MIX 2

MOVE

INJECT

the EU is 800 µg/kg. Please note: GTX-2 and GTX-3 are not separated when using pre-column derivatization since they form the same product. A post column derivatization method is required in order to determine these compounds individually. This would be relevant for research purposes only.

Automated sample prep provides improved results

The results of the final round robin test were highly satisfactory. Initially though,




equal treatment, being derivatized at exactly the same point in time prior to analysis.

The goal of on-time sample preparation can be accomplished if an intelligent LC autosampler is used. Ideally, an autosampler should provide high sample throughput combined with simple programming of sample preparation steps such as adding standards or reagents, mixing, heating or even diluting. All these requirements are met by the GERSTEL MultiPurpose Sampler (MPS).

MPS 3, the sampler of choice

The MPS is easily controlled and operated through the GERSTEL MASter and the new MAESTRO Software, which can both be integrated in the ChemStation from Agilent Technologies. The required sample prep steps are selected by mouse-click from a drop-down menu list. Examples are: MOVE, ADD or MIX. The derivatization method for PSP toxins on the MPS 3 includes the following steps: A vial with sample extract, to which NaOH 1M has been added, is placed into the heated sample agitator of the MPS 3 [MOVE]. The agitator has a built-in orbital shaker for mixing. As a second step, derivatization reagent (H2O2 10 %) is added [ADD 1]. The sample is mixed for 1.5 minutes [MIX 1], then concentrated acetic acid is added [ADD 2], and then mixed again for 1.5 minutes [MIX 2]. The oxidation reaction stops as soon as the pH-value is lowered. The robotic arm puts the vial back into its original tray position, where it remains until sample introduction. Exactly five minutes after the derivatization started, the sample is injected into the LC.

Interlaboratory round robin test on algae toxins in mussel meat and mussel products. Number of participants: 9, overall mean value toxin: x; repetitive standard deviation: Sr; relative standard deviation: SR; Repeatability limit per lab: r; Interlaboratory Repeatability limit: R

Results

The exact and reproducible timing of all sample prep steps in the MPS 3 brought significant improvements in correlation and standard deviation when compared with manual sample preparation. Ten analyses were performed in sequence on an Agilent Technologies LC 1100 system with the following result: If sample preparation was performed manually and the samples subsequently left in the autosampler until analyzed, a significant decrease in signal intensity was found due to decomposition of the oxidation product. As an example, a 30% drop in the concentration of the gonyautoxin GTX-5 is observed after 12 hours at 25 °C.

Alternatively, if sample preparation was performed on-time, automatically by the MPS 3, the determined concentrations remained constant over a long period. The MPS 3 can prepare a sample during analysis of the preceding sample, saving time and optimizing

system utilization while delivering significantly improved results.

Conclusion

The analysis results reported in this article clearly demonstrate that chemical analysis using HPLC and fluorescence detection is superior to bioassays (animal testing) for the determination of algae toxins in mussel meat. In addition, automated, on-time sample preparation and sample introduction has been shown to provide a significant improvement in the quality of results.

Literature[1] Lawrence, J.F. and Menard, C. (1991),

„Liquid Chromatography determination of

paralytic shellfish poisons in shellfish after

prechromatographic oxidation“, J. Assoc.

Off. Anal. Chem., Vol. 74, No. 6, 1006-1012

[2] Personal message by Dr. Lutz Walther,

Bavarian State Office for Health and Food Safety

The updated GERSTEL consumables catalogue

Analytical Supplies 2006/2007 provides an extensive

list of consumables, accessories and spare parts

that are essential for the smooth operation of your

GERSTEL system. Our solutions include automated

sample preparation, analyte enrichment and sample

introduction devices for GC, GC/MS, LC and LC/MS.

Additional GC techniques are available such as Fast GC,

Headspace, SPME, Thermal Desorption, Stir Bar Sorptive

Extraction, Multi-Dimensional GC, Olfactory Detection

and Preparative Fraction Collection. To request a

catalogue, please contact: [email protected].

Download: www.gerstel.com/GERSTEL_Supplies_

2006_2007_en.pdf

Analytical Supplies 2006/2007




There were several good reasons to move on from the MASter Software”,

says Fred Schwarzer Ph.D., software development manager at GERSTEL. “Primarily, we needed a new program architecture to handle the growing number of accessories that are often working alongside each other for sample preparation and sample introduction.” The original MASter software was never intended to accommodate such a large number of modules. The range of GERSTEL modules has grown to include not only GC inlets and multidimensional switching for GC, but also complete autosamplers that perform automated

GERSTEL solutions worldwide Software

MAESTRO software conducts GERSTEL symphony of solutions

New control software offers users enhanced productivity and simplified operation

sample preparation, sometimes even combined with a Fast GC accessory or a temperature programmable column oven for fast LC analysis. We needed a modern, modular and easily expandable software platform to support our ever-increasing number of new accessories and systems, in short: software that connects all parts and turns them into an integrated GERSTEL solution.

MAESTRO offers GERSTEL users a new control software with a simplified user interface combined with significantly expanded capabilities – especially for the widely used MultiPurpose Sampler MPS.

Plugin Modules

MAESTRO is designed for plug-in modules. Software control modules for accessories and instruments are not programmed into the core program. Rather, these building blocks are added as independent modular plug-ins, making it easier to add new accessories and modules as they are developed, without interfering with existing parts of the program.

“As soon as the instrument is plugged in it is recognized by the controller. The relevant program modules are then automatically loaded and activated”, says Fred Schwarzer. Apart from this, MAESTRO has been prepared to support the widely used COM-Model, making it easier to integrate it with other software platforms.

Increased productivity and flexibility for GC/MS and LC/MS laboratories

To meet ever increasing demands for higher performance and higher productivity, labs often use combinations of systems and accessories from different instrument vendors. The MASter software already provides an integrated

solution for operating every available GERSTEL accessory and instrument in combination with instruments from Agilent Technologies. The complete system is conveniently operated through the ChemStation software using just one method and one sequence table.

MAESTRO provides additional functionalities, more operational flexibility and easier set-up of automated sample preparation functions, mainly used for GC/MS and LC/MS. All MAESTRO functions are available during stand-alone operation as well.

Whenever priority samples arrive in the lab, the software enables flexible addition to, and rearrangement of, the scheduled samples in the autosampler. Samples can be added even when the active sequence incorporates overlapping sample prep and analysis, a key MAESTRO feature for improved productivity.

MAESTRO includes several new tools to make your life as a user as easy and productive as possible: To support new users and to provide help whenever questions arise, a context sensitive on-line help function is right at your finger tips. The on-line help functions include explanation of parameters as well as ranges and other relevant information.

MAESTRO has integrated maintenance scheduling to ensure that preventive maintenance is performed after a user-defined number of injections. This maintenance scheduling function gives users the added comfort of

MPS with automated liner exchange (ALEX).



knowing that they will be prompted to perform scheduled maintenance such as a liner or septum exchange before any problems arise due to wear or build-up of contamination in the system. The system is designed to avoid unforeseen down-time and keep productivity high.

Should any unforeseen problems arise during the analysis sequence, the sequence is halted and the user can be notified by email. This enables the user to take quick action, ensuring that the system is not left idle on the bench .

Planning your work is made easier with the MAESTRO software. During set-up of an analysis sequence, the software shows the time required to run the scheduled samples. During the analysis run, the remaining time for the scheduled sequence and the time to next injection is always shown to enable changes to the sequence and planning of further analysis work.

Optimized liquid sample preparation and sample introduction

The MPS samplers can handle even the most complex sample matrices. Sample clean-up and preparation can be performed automatically. Automated sample prep techniques cover the complete range of sample types and analytes. The following liquid

With 15 years of experience from the MASter Software under our belt, and based on feed-back from our many users worldwide, GERSTEL has developed a new control software. The new MAESTRO software is designed to integrate our growing range of

accessories for GC/MS and LC/MS. MAESTRO has a modern graphical user

interface, designed for intuitive operation. In stand-alone operation or fully integrated with the ChemStation Software, MAESTRO puts reliable productivity at your finger tips.

GERSTEL solutions worldwide Software

Dr. Fred Schwarzer

Novel automated techniques for eliminating difficult sample matrices. Automated Liner Exchange ALEX (photo on left hand side), Automated TDU-liner EXchange ATEX, and TDU liquid option for thermal extraction of liquid samples.

■ MAESTRO, in combination with GERSTEL Sample Prep technology, can simplify and automate the handling of samples with a heavy matrix load. Depending on the amount and type of matrix and the sample volume to be introduced, one of the following options would best meet the challenge: Automated Liner EXchange (ALEX) system, Automated TDU-liner EXchange (ATEX) system or Twister Desorption Unit (TDU) liners with liquid sample cups. The MPS can inject samples directly into either of these systems.

Automated SPE option for MPS

■With the MAESTRO Software, the SPE process and chromatography analysis can be performed using the Prep Ahead feature, that is in parallel, ensuring that no time is wasted waiting for sample preparation. Defined volumes of sample, conditioning solvents and elution solvents are added in a reproducible manner, all controlled from the MAESTRO Software PrepBuilder. In addition, sample prep steps such as standard addition or derivatization can be performed on the eluted analytes prior to GC/MS or LC/MS analysis.

MAESTRO sequence table.

One method and one sequence

table controls the complete process

from sample preparation and

introduction to GC/MS or LC/MS

analysis.

Planning work is made easier

with the MAESTRO software.

During set-up of an analysis

sequence, the software shows

the time required to run the

scheduled samples. Sample

Prep and chromatography are

performed in parallel using the

Prep Ahead function to ensure

best productivity.

sample prep techniques are performed automatically: Solid Phase Extraction (SPE), Standard Addition, Dilution, Extraction, Derivatization, Mixing, Heating, Twister Back Extraction and Membrane Extraction.

For GC, a wide range of sample introduction techniques are available: Liquid, Large Volume Injection (LVI), Headspace, Solid Phase Micro Extraction

(SPME), Twister Desorption and Thermal Desorption. In the MAESTRO software, liquid “sandwich injections” have been added to the list. In this type of injections, the sample is surrounded by solvent and/or air inside the syringe. Whenever the sample is introduced to the GC, the extra air or solvent ensures complete transfer of analytes from the syringe to the GC System.



Using a method based on Stir Bar Sorptive Extraction (SBSE), the German Research Laboratory

for Food Chemistry has succeeded for the first time in determining minute amounts of odor-causing substances in the mouth at defined time intervals after the consumption of food. The method can be used to determine aftertaste and it has helped explain how aftertaste develops.

There are different ways to approach a good wine. Connoisseurs always swear by their own personal method. Certain principles and details, however, simply follow from instinct and from human

anatomy. Minor tricks of the trade and some experience can then help even amateurs to a more qualified assessment and maybe to more enjoyment. To sum it up: wine tasting is not just for specialists. Even the ancient Romans knew that a wine should be judged not only by its taste, but by its color and aroma as well. Sounds simple, and it actually is – at least the part about the taste. The flavor evaluation, the palate test, still holds many secrets. Simple descriptors such as sweet sour, salty bitter, creamy, spicy or toasty cannot fully describe the complex interactions that provide the

Stir Bar Sorptive Extraction (SBSE) helps determine odor causing substances

The Twister sniffs out the aftertaste

What do wine and mouthwash have in common? Both leave an aftertaste. How aftertaste develops has not yet been clearly determined, but it is well known that flavors and odors play an essential part; in fact, a more correct term would be “afterodor”. A scientist from the German Research Laboratoy for Food Chemistry sheds some light on the secrets of how we taste.

full experience of tasting food and drink. Whether food or drink has a short or long lasting taste, and whether it turns out to be enjoyable, depends mainly on to what extent and for how long characteristic flavors find their way from the oral cavity via the throat to the olfactory sensors in the nose. This process is referred to as retronasal odor perception. In contrast, when flavors are sensed in combination with inhalation through the nose, it is called orthonasal odor perception.

But how do the flavor compounds that are released from foods or beverages in our mouth reach their destination,



GERSTEL solutions worldwide Research


the olfactory sensors? To adequately answer this question it is not enough to watch a connoisseur during wine tasting. You can reach some conclusions about the person from the smacking, slurping and guttural sounds emanating during the process of expert tasting. Still it remains unclear exactly how the odorous substances reach the olfactory sensing apparatus. If modern imaging tools are used, however, it becomes obvious that this is a precisely controlled process, which permits a flavor transfer only at certain times.

Learning from Mother Nature: The way of human tasting

If there is food in the oral cavity and lips and jaws are closed, the palate and tongue base form a barrier that actually seals the oral cavity and prevents us from unintentionally swallowing food or literally getting it down the wrong pipe. A small experiment demonstrates the incredible efficiency of this natural lock inside the mouth. Take a small sip of coffee or wine and leave it in your mouth. Now close your lips. In doing so, keep your lower jaw completely still. What is your perception? Now start to chew. Does anything change in your perception? And then swallow. What is your perception now?

„As a general rule, taste impressions like sour, bitter, sweet or salty can be detected during the first part of this experiment, but no typical flavor notes like toasty, fruity or flowery“, explains Dr. Andrea Büttner, a scientist at the German Research Laboratory for Food Chemistry. During chewing, namely the second part of the experiment, the natural barrier in the oral cavity opens

Stir Bar Sorptive Extraction (SBSE) helps determine odor causing substances

for a short time and small amounts of aroma components reach the olfactory bulb via the throat.

“The real perception of the wine or coffee aroma does not occur until immediately after swallowing, with the first breath.”

An aroma impulse, by the way, can also be registered when we place food into our mouth. Andrea Büttner: „This is an important protective mechanism provided by Mother Nature. Through it we receive a first impression on whether the substance in our mouth is fit to eat or not.” The phenomenon has physiological reasons: as soon as we open the jaw, the tongue-palate barrier opens up for a short time. After closing mouth and jaw again, excess air is released from the mouth cavity into the nose via the throat cavity; flavor substances from the food are thus transferred to the olfactory sensors in the nose, where a discernable sensory impulse is released through the sensory bulb to our brains. Proof that this is how the process works was provided by real-time breath analysis using Proton Transfer Reaction Mass Spectrometry (PTR-MS).

After taking a sip of wine, a test person exhales through the nose and then closes the lips and tongue-palate barrier. In the on-line PTR-MS measurement, a distinct initial peak is seen. The signal is due to the presence of ethyl acetate formed by reaction between ethanol and acetic acid. This highly volatile compound is easily sensed at relatively low concentrations and often identified as a nail polish smell. In wine, ethyl acetate occurs naturally. In this experiment, ethyl acetate is used as a marker to detect the transfer of volatile wine flavor compounds from the oral cavity to the nose cavity. The initial ethyl acetate peak during wine intake lasts only 2.5 seconds. Even though the test person keeps the wine in the mouth for some time, only isoprene, a metabolite which is released into the atmosphere via our breath, is detected, but no ethyl acetate.

You can’t smell with your mouth full

Though the experienced connoisseur seldom swallows during wine tasting, he or she is able to give a comprehensive retronasal evaluation of a wine. Using a few tricks of the trade, mother nature and

her physiological miracle of sensory perception is outwitted:

By skillfully opening and closing the mouth and the palate barrier, and by combining these motions with a special breathing technique, flavors are pumped from the oral cavity through the throat cavity and on into the nose. This process is often accompanied by sounds that may seem less than appetizing to the uninitiated. The process can be examined and traced via PTR-MS. Incidentally, experienced wine tasters never bite off more than Open palate while food is inserted into the mouth

with a spoon (video-fluoroscopic snap-shot)

GC-System with

MPS 2 and TDU

for fully automated

analysis of up to 196

GERSTEL Twisters

they can chew, that is, they never take too much wine into their mouth when tasting. Apart from the risk of choking, this would lead to a reduction of the vapor phase volume in the mouth, effectively reducing both the transfer of flavor compounds into the vapor phase and the subsequent transfer of the vapor phase to the nose cavity. If, on the other hand, the amount of wine taken into the oral cavity is too small, dilution of the sample by saliva and a too large volume of vapor phase can significantly impact the sensory perception. The volume needs to be just right.

Back to the experiments: As soon as the test person has swallowed the wine and taken a breath, there is another





To avoid contact, and thus a direct extraction of

substances from the oral mucosa, a special sampling

vessel for the Twister has been developed. BOSS

provides unobstructed access of saliva and gas

phase to the Twister, thereby ensuring an efficient

extraction of flavor compounds.

0 1 2 3 4 5 6

The Twister sniffs out the aftertaste

clear ethyl acetate signal. Even after the wine has been swallowed, volatile flavor compounds from the wine are detected in the breath. Even though there is no more wine in the mouth, volatile compounds, that are typical of the aroma for the tasted wine, are released and can be determined through sensory perception or by using analytical instrumentation such as gas chromatography (GC) with olfactory detection or MS detection. The duration of the “aftertaste” depends on several factors, among them the extent to which flavor compounds are metabolized by the saliva, resulting in reduced sensory perception and a displacement of the aroma profile. Thioles, such as 2-furfurylthiol, that adds a typical roasted coffee flavor, or 4-mercapto-4-methyl-2-pentanone, characteristic for Sauvignon-Blanc wines, are efficiently metabolized by saliva. Earthy pea or bell pepper type notes, however, are saliva persistent.

Flavor persistence was determined based on sensory evaluation in parallel with direct saliva analysis using the Buccal Odor Screening System (BOSS). BOSS is a novel analysis method based

on Stir Bar Sorptive Extraction (SBSE) using the GERSTEL Twister. The Twister is a glass-coated magnetic stir bar with an outer coating of polydimethylsiloxane (PDMS) that can extract organic compounds such as flavors or off-odors from aqueous and other liquid samples. Direct contact between the Twister and the oral mucous membranes could lead to extraction of adsorbed compounds that do not contribute to the sensory perception. The Twister is kept isolated from the membrane surfaces by placing it inside a small, perforated glass vial. The vial is sealed with a glass stopper and placed inside the oral cavity of the test-person for a defined period of time. The perforated vial walls ensure unobstructed contact of the Twister with saliva and with the oral cavity gas phase. The test person moves saliva around the vial during the extraction phase.

Sampling using the BOSS procedure is performed as follows: a subject tastes a food sample and subsequently places the perforated glass vial with the Twister into his or her mouth. The vial is moved around in the saliva inside

the oral cavity. The lips are kept sealed and breathing only takes place via the nose. After a specified period of time, the glass capsule is removed from the mouth and the twister taken out and dabbed dry on a lint-free paper. The extracted compounds in the PDMS are determined by thermal desorption in combination with GC separation and olfactory detection. In her work, Dr. Andrea Büttner established a clear correlation between the concentrations of extracted flavor compounds and the flavor perception of the subject. With BOSS it was possible for the first time to detect even small amounts of flavor compounds in the oral cavity at any given point in time after consumption of a beverage or food sample.

In other words, for quite a while after a food has been swallowed, traces of flavor compounds can be detected that have been absorbed by oral mucous. These can produce a flavor impression over a period of time, known as the aftertaste. This finding is especially of interest to food manufacturers. Customer acceptance and the success of any given product in the marketplace depends heavily on whether consumers enjoy the taste and aftertaste. In addition, Dr. Büttner considers the BOSS method a promising, extremely helpful instrument for diagnosing halitosis, as well as for examining the effectiveness of toothpastes and mouthwashes.

It can be safely said that the BOSS system can neither replace wine tasting nor can it provide specific conclusions about individual properties of wines and other products. It is simply a tool to describe the aftertaste of a given product. Dr. Büttner adds: „Wine tasting is and will remain a subjective matter, incidentally, one that is more strongly influenced by the mood and the environment in which it is performed than most people realize.“

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Real-time PTR-MS

monitoring of volatile

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pass the olfactory

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nose when drinking

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Drug testing

Reliable determination of THC in salivaI n the U.S., drug testing for federal

employees is required by law and tests are performed according to the

guidelines of the „Federal Drug Testing Program“. Since 1988, only laboratories certified by the US-Department of Health and Human Services (DHHS) are authorized to perform such tests. The DHHS has since proposed to incorporate oral fluid testing to supplement or replace other techniques. This would allow less invasive sample collection and could help ensure that samples are not tampered with or substituted, protecting those providing samples from incorrect test results.

THCStructure of Δ9-tetrahydrocannabinol (THC) the main active compound in cannabis.

O

OH

Saliva samples are highly suited to test for the presence of controlled substances, such as THC, that have been taken through the mouth. Swallowing cannabis or smoking - and inhaling - it are natural respiration or ingestion processes that leave behind residues and traces in the mouth and respiratory tract. Saliva analysis can provide additional information as to the timing of any drug intake. As for the details of obtaining a sample, it is often considered less invasive to provide a saliva sample than to provide a urine sample under supervision – let alone to have a blood sample taken.

Twister extraction time study for THC spiked into blank oral fluid

shows almost quantitative extraction after 90 minutes.

Until now, standard GC/MS-methods that are used for determination of THC-metabolites in urine have not been able to reliably determine the active agent of Cannabis, Δ9-tetrahydrocannabinol (THC) in saliva at the required levels. New methods being explored include GC-MS/MS and LC-MS/MS which require purchase of expensive, specialized instrumentation to achieve the necessary detection limits.

Stir Bar Sorptive Extraction (SBSE) using the GERSTEL-Twister has now been shown to provide an answer to the challenge, while reducing the amount of sample preparation and clean-up to an absolute minimum: GERSTEL scientists, in cooperation with Greg Grinstead, Ph.D. of Marshfield Laboratories (Marshfield, Wisconsin, U.S.A.), have shown that SBSE combined with thermal desorption and GC/MS (EI) with selected ion monitoring can reliably extract and quantify THC in oral fluid at concentrations comfortably below the cutoff levels proposed for confirmatory testing of alternative specimens in federal workplace drug testing programs. The confirmatory testing parameters (chromatography; resolution; three selected ions with acceptable ion abundance ratios; QC requirements; LOD/LOQ etc.) that have been applied successfully for confirmatory testing of drugs in urine by the National Laboratory Certification Program for almost 20 years could be extended to the more demanding analysis of THC in oral fluid. For more detail please see GERSTEL application note 6/2005. www.gerstel.com/application.htm




Since March 2005, SRA Instruments Italia has been the official GERSTEL distributor for Italy

L ocated in Cernusco sul Naviglio near Milan, SRA has a network of

14 specialized and trained sales and service agents throughout Italy ensuring efficient and competent local support for all customers.

SRA Instruments Italia S.r.l.

Viale Assunta, 101

I-20063 Cernusco sul Naviglio (MI)

Tel. +39-02-9214.3258

Fax +39-02-9247.0901

[email protected]

www.srainstruments.com

SRA Instruments is well-known as an Analytical Solutions Provider in the Italian market. “Our mission”, says Armando Miliazza, Managing Director of SRA, “is to provide complete customer solutions. The partnership with A.I. Tech and GERSTEL allows us to more completely cover the needs of our customers. Our highly specialized staff understands the analytical needs of modern labs providing each customer with the solution that meets her or his requirements. We provide installation, training, analytical support - including method development - as well as service support”.

SRA Instruments Italia obtained ISO 9001:2000 certification in February 2005.

SRA Instruments, GERSTEL distributor in Bella Italia

Gigi Cobelli, SRA Technical Support Manager instal-

ling a new system. SRA offers complete solutions

including service and application support.

From left to right: Roberto Gaita, Gianluca Stani and

Armando Miliazza.

The SRA Booth at the MAC 2005 exhibition in Milano.

SRA Instruments Italia is made up of 18 dedicated professionals with many years of experience in the analytical instrument market. Previously, the main focus has been on providing analyzers and complete solutions for the environmental, industrial and petrochemical markets based on Agilent Technologies instrumentation. In March 2005, the then GERSTEL distributors in Italy, Roberto Gaita and Sabrina Brait and their company A.I.Tech formed a partnership with SRA. On that occasion, Roberto Gaita became a member of the board of directors of SRA. The partnership has enabled SRA to provide solutions from GERSTEL to the environmental market while extending their offering into the Food and Beverage market. Through the partnership, GERSTEL customers in Italy have gained access to the extended sales, support and service network of SRA providing efficient local support throughout the entire country.

GERSTEL solutions worldwide Distributor



6 times faster screening of pesticide multi-residues in aqueous samples

Take Two!

SBSE can be performed in parallel on two aliquots of the same sample under different extraction

conditions using two Twisters. Using this dual Twister approach, the two extractions can be optimized individually: For aqueous samples [6], one extraction can be optimized for hydrophilic solutes with salt addition [7, 8] and the other extraction can be optimized for more hydrophobic solutes with addition of an organic solvent such as methanol [9]. After extraction, the two Twisters can be simultaneously desorbed using a thermal desorption system. Dual SBSE has previously been proposed for multiresidue screening of pesticides in food samples such as vegetables, fruits and green tea [5].

SBSE methods involve batch sample preparation (parallel extraction of up to 60 samples on one stir-plate, typically for 1 hour) and sequential analysis (separation and detection); sample throughput is mainly determined by the separation step. SBSE methods can therefore

Over the past few years, stir bar sorptive extraction (SBSE) [1] using stir bars coated with polydimethylsiloxane (PDMS), has been successfully applied to trace analysis of organic compounds in various sample matrices such as water, soil, food, beverages and biological fluids [2-4]. The PDMS coated stir bars are commercially available from GERSTEL under the name Twister®. Several authors have indicated that SBSE provides high recovery and extremely low limits of detection (LOD) at the sub-ng/L level, particularly for hydrophobic solutes [2-4].

provide high sample throughput when combined with Fast GC.

A Fast GC system with direct resistive heating is commercially available in which a capillary column is enclosed in a resistively heated toroid-formed assembly (Low thermal mass GC [10]) The system is available under the name of Modular Accelerated Column Heater (MACH). The MACH system provides fast temperature programming rates combined with rapid cool-down and short equilibration times for shortest possible analytical cycle times. Using the MACH system, a maximal heating rate of 30 ºC/s can be achieved, cool-down from 400 ºC to 100 ºC typically takes less than one minute [10]. The MACH system can be directly integrated with Agilent GC instruments to allow full use of conventional injectors, detectors, sampling systems, and software. We recently optimized and validated a dual

The AuthorNobuo Ochiai, Ph.D.

GERSTEL K.K., 2-13-18 Nakane, Meguro-ku, Tokyo, 152-0031 Japan

[email protected]

Twister method for both polar pesticides with low Ko/w, and apolar pesticides with high Ko/w, at very low levels (sub-µg/L) in aqueous samples. Dual Twister analysis was performed in combination with a MACH Fast GC/MS system and a quadrupole MS providing maximum

sample throughput. The principle and potential of Dual Twister-Fast GC/MS is demonstrated in the following, using the determination of pesticides in river water and brewed green tea as examples.

Experimental

The dual Twister procedure works as follows: Two 20 mL aliquots of a sample, containing 30 % NaCl or 0-20 % methanol




Figure 1: Total ion chromatogram obtained by

Dual SBSE-TD-RTL GC/MS of a river water sample

spiked with 82 pesticides at 500 ng/L level.

Figure 2: Total ion chromatogram and some representative mass

chromatograms obtained by Dual SBSE-TD-LTM GC/MS

of a river water sample spiked with 82 pesticides at 500 ng/L level.


respectively, were transferred to two 20 ml vials. A Twister was added to each vial and stirred for 60 min. The two Twisters were then simultaneously desorbed using a GERSTEL TDU thermal desorption system, programmed from 40 ºC (1 min) at 720 ºC/min to 280 ºC (5 min) in splitless mode. The desorbed compounds were cryo-focused in a GERSTEL CIS 4 PTV inlet at -100 ºC. The CIS 4 inlet, operated in splitless mode, was then programmed at 720 ºC/min to 280 ºC (5 min), thus transferring the analytes to the capillary column. The separation was performed on a DB-5 column (10 m x 0.18 mm i.d., 0.18 µm film thickness, Agilent Technologies), which was coiled and packaged tightly together with a heating wire, a sensor, and ceramic fibers in the MACH column module (wide format, 5 inch module). The column was connected to the GC inlet with a 1 m long, 0.32 mm i.d. fused deactivated silica capillary and to the MS detector with a 1m long, 0.18 mm i.d. fused deactivated silica capillary, using



Literature

[1] E. Baltussen, P. Sandra, F. David, C. A. Cramers, J. Microcol. Sep., 11 (1999) 737.

[2] E. Baltussen, C. A. Cramers, P. J. F. Sand-ra, Anal. Bioanal. Chem., 373 (2002) 3.

[3] F. David, B. Tienpont, P. Sandra, LC GC N AM, 21 (2003) 108.

[4] M. Kawaguchi, R. Ito, K. Saito, H. Nakaza-wa, J. Pharm. Biomed. Anal., in press

[5] N. Ochiai, K. Sasamoto, H. Kanda, T. Ya-magami, F. David, B. Tienpont, P. Sandra, J. Sep. Sci., 28 (2005) 1083.

[6] N. Ochiai, K. Sasamoto, H. Kanda, S. Nakamura, in preparation.

[7] V. M. Leon, B. Alvarez, M. A. Cobollo, S. Munoz, I. Valor, J. Chromatogr. A, 999 (2003) 91.

[8] S. Nakamura, S. Daishima, Anal. Bioanal. Chem., 382 (2005) 99.

[9] T. Benijts, J. Vercammen, R. Dams, H. P. Tuan, W. Lambert, P. Sandra, J. Chromatogr. B, 755 (2001) 137.

[10] J. C. Luong, R. L. Gras, H. J. Cortes, R. M. Mustacich, in: Proceedings of 27th ISCC,

Riva der Garda, Italy, 2004, I.O.P.M.S., Kor-trijk, Belgium, 2004, CD-ROM paper PL11.


press-fit connectors on both sides. The column temperature was programmed from 40 ºC (2 min) at 75 ºC/min to 300 ºC (2 min). Helium was used as carrier gas. The carrier gas pressure was programmed from 82 kPa (2 min) at 36 kPa/min to 207 kPa (held for 2 min), keeping the carrier gas approximately at a constant flow rate of 1.1 mL/min. The host GC oven (Agilent 6890 GC) and the MSD interface were kept at a constant temperature of 250 ºC. The mass spectrometer (Agilent 5975 MSD) was operated in scan mode using electron-impact ionization (electron-accelerating voltage: 70V). The scan range was set from m/z 58 to 510 and sampling rate of zero, resulting in scan rate of 10.83 scan/s.

Results and Discussion

Figure 2 shows the total ion chromatogram (TIC) and some representative mass chromatograms obtained by Dual Twister-Fast GC/MS of spiked river water samples. 82 pesticides – organochlorine,

carbamate, organophosphorous, pyre-throid and others – spiked at 500 ng/L could be clearly determined. The method showed good linearity over the range 25-1000 ng/L (r2 > 0.9900) and low LOD (< 10 ng/L; signal-to-noise ratio of 3:1) for most of the target pesticides. Using this method, the first and last compounds to elute were dichlorvos with a retention time of 3.722 min and imibenconazole with a retention time of 6.519 min, respectively. For comparison, the same compounds have retention times in conventional GC-MS analysis with retention time locking (RTL) of 5.829 min and 35.930 min, respectively, a factor of 5.5 longer for imibenconazole using the conventional RTL method, as seen in figure 1.

For river water samples, although fenobucarb (Ko/w = 2.79) and diazinon (Ko/w = 3.86) were present at ultra trace levels (16 and 4.9 ng/L, respectively), well defined mass chromatograms were obtained without interference from matrix compounds at acceptable precision (RSD

< 12 %, n = 5). The compounds could be determined using a standard addition calibration method. A brewed green tea sample was found to contain pirimiphos-methyl (Ko/w = 4.20; 10 ng/L, RSD 7.8 %) and tebuconazole (Ko/w = 3.89; 240 ng/L, RSD 8.1 %).

Conclusion

Dual Twister Fast GC/MS was successfully applied to fast screening of pesticide multiresidues in aqueous samples. The proposed method has many practical advantages, for example high sensitivity, good linearity, remarkable precision and high sample throughput. Also, the method allowed the determination of ng/L levels of pesticides in river water and brewed green tea samples with very low Relative Standard Deviation in the range from 7.8 to 12 %.

Acknowledgements

The author thanks Dr. Frank David of the Research Institute for Chromatography for his helpful advice. My coworkers and coauthors of the related article [6], Kikuo Sasamoto, Hirooki Kanda, Chiaki Nagamori and Yuki Ishizuka of GERSTEL K.K. as well as Dr. Sadao Nakamura of Yokogawa Analytical Systems are also thanked for their contribution.



C ountless species have fallen victim to environmental changes throughout the ages, but insects

have withstood even the most adverse conditions. Many people would rather have seen insects die out with the dinosaurs, since most are considered nuisances or are simply considered frightening. The desire to control bugs, or rather to wipe them out, comes from more than just annoyance or fear: Insects transmit major diseases such as malaria, yellow fever and typhus and are one of the main sources of crop failure which can result in financial losses or even famine.

What evolution could not accomplish over millions of years, man is trying to master himself. Reaching for the fly swatter is a natural reflex, the ultimate fly swatter of course being large amounts of pesticides applied to fields and crops to deal with insects that are out to eat

Following nature’s scent

A German forestry zoologist uses gas chromatography (GC) and a modified Olfactory Detector Port (ODP) to „eavesdrop“ on insect communication. His goal is to snoop out information on how insects communicate in order to develop new plant protection chemicals and maybe provide a new generation of biosensors that can detect forest fires in the early stages.

our lunch. But this is not necessarily ultimately in our interest since such chemicals could end up harming the very humans that they should help provide with safe, adequate and ample food supplies.

Often the great significance of the tiny creatures in regard to life and its diversity is too easily ignored, according to the American insect researcher, May R. Berenbaum: “insects are our most important partners in creating life on earth, because they often take the lead in the formation of terrestrial ecosystems.”

But how could we keep insects from destroying complete harvests and crops without resorting to dangerous chemicals? “In order to provide a solution, it is helpful to know what makes a plant attractive to insects“, explains Prof. Stefan Schütz, Manager of the Institute for Forest Zoology and Forest Protection at the University of Göttingen, Germany.

Just keep those antennae out

To warn members of their species or to find a mate, insects use pheromones, volatile airborne messengers, that are emitted by the insects. Pheromones are in turn detected by other insects using highly sensitive smell receptors, located on their antennae. Insects also perform olfactory detection of emissions from plants. Volatile organic compounds (VOCs) are emitted from plants for various purposes, sometimes intentionally, sometimes involuntarily, for

instance if plant tissue is damaged and the plant injured. The potato beetle is specialized in detecting and finding its host plant, the potato, by the smell. In doing so, the insect, which belongs to the family of leaf beetles, prefers fully-grown and damaged plants. “Young plants, on the other hand, are not attractive to the potato beetle“, explains Schütz.

The transmission of olfactory stimuli determines the behavior of the potato beetle. Thus, it is clear that the smell patterns of young and fully-grown potato plants are different. To verify this hypothesis, Schütz developed a special apparatus using a gas chromatograph (GC) in combination with a Electro-Antennographic Detector (EAD). The EAD is based on GERSTEL Olfactory Detector Port (ODP), that has been modified and equipped with a special detection cell.

From the left: Prof. Stefan Schütz during an interview

with GERSTEL representative Detlef Bergemann and

GERSTEL Solutions Editor Guido Deußing.




The central element of the detection cell is an antenna, which has been gently removed from an insect. The antenna generates a detectable electric potential when stimulated with specific substances. Dr. Bernhard Weißbecker, an institute staff member, explains: “If the base and top of the antenna are mounted with electrodes, an Electro Antennogram (EAG) can be recorded, that provides information on the volatile compounds detected by the donor insect.“

The house longhorn beetle

The functional test of the apparatus was performed by Schütz using a house longhorn beetle, which is found in Europe and in countries within the temperate climate zones. The house longhorn beetle deposits its eggs in aged wood, the larvae remain in the wood for two to eight years, feeding on the wood. This can cause severe structural damage, for example, to wooden houses. Schütz examined the emissions of pine wood and found that the house longhorn beetle detects substance classes, which largely fall outside the typical odor pattern of softwoods detected by the human nose.

Schütz used the EAD to detect odors and odor patterns that are perceived by insects. In parallel, he used mass spectrometry (MS), to identify the chemical compounds found in the sample. „If two different detection systems are used, however, comparison and correlation of the chromatograms can prove very complex”, says Schütz.

EAD in useThe EAD is based on a modified GERSTEL ODP 2, equipped with a special detection cell (diagram to the left). The central element of the detection cell (pictured above) is an insect antenna, which has been gently amputated from the insect. The antenna generates a detectable electric potential when stimulated with specific volatile substances to which the receptors react, explains Bernhard Weißbecker, Ph.D. (pictured above).


GERSTEL Olfactory Detector Port (ODP)When individual odor compounds or complex odor patterns need to be characterized, the GERSTEL ODP 2 provides a useful interface between the GC and the human nose, the ultimate arbiter of smell. GC ODP is a proven and reliable technique for characterization of odors and odor causing compounds from various sources. The ODP 2 works in combination with all standard GCs.

The Treasury of Life

About a million different species of insects are known today and have

been scientifically determined. Experts suspect that this is only a

fraction of the species that exist*, mainly in tropical rain forests.




used airtight emission chambers made of steel and Teflon foil, which he attached directly to the trunks. Sampling was performed by closed loop stripping using activated charcoal as adsorbent. The odor analytes were eluted from the activated charcoal, separated by gas chromatography and detected by MSD and EAD-FID. Significant differences in the odor composition were found. „The EAD trace from jewel beetle antennae allowed us to identify potential marker substances, through which the beetle is able to spot attractive trees”, says Schütz.

The trees that were classified as attractive by the scientists clearly suffered increased infestation by jewel beetles. The remaining pines were not intested within the observation period or in the following year.

“This means that with the help of our GC-MS/EAD system, we are able to find weakened trees”, says Schütz. These findings would enable us to target odor-based pest reduction treatments specifically to the trees at risk or simply to remove these to reduce the spread of pests and the damage caused by them, while avoiding the use of pesticides.

Early detection of smoldering fires

The sensory abilities of insects can also be used for early detection of smoldering fires, for example forest fires, helping to

Pine wood scentOf the terpenes (indicated by peaks in the green chromatogram at bottom), only α-pinene caused a measurable response on the anten-nae of the house longhorn beetle. 3-carene, p-cymene and α-terpineol were not detected. Apart from α-pinene, the house longhorn beetle reacts extremely sensitively to certain saturated aldehydes from hexanal to decanal as well as to certain unsaturated aldehydes and alcohols.

Smoke stack gas analysisThe antennae of the jewel beetle can detect guaiacol in smoke stack gases with high sensitivity (as low as 1 pg/ml). FID: Flame Ionisation DetectorEAD: Electro Antennographic Detector(Antenna of the jewel beetle) 1: α-pinene2: 3-carene3: 2-methoxy-phenol (guaiacol) 4: 2-methoxy-guaiacol 5: 4-acetyl-guaiacol6: 4-formyl-guaiacol

22

When sampling procedures such as solid phase micro extraction (SPME) or sampling onto thermal desorption tubes (TDS) are used for analyte enrichment, the aim is to get the maximum possible amount of analytes onto your GC column. The sample is quantitatively desorbed onto the GC column in splitless mode for highest sensitivity. In order to have both ODP and MS detection, the column effluent is split between the ODP and MSD.

The EAD operates under atmospheric pressure and the MSD under vacuum. This can make it a challenge to set up the flows to the two detectors. Schütz used a GERSTEL GRAPHPACK cross-piece and suitable restriction capillaries.

“We proved that our GC/MS/EAD system can examine the interaction between insects and their habitat”, said Schütz. At the same time, it is being investigated whether house longhorn beetles react negatively to certain odors enabling the scientists to devise environmentally friendly wood preservation measures.

Protecting pines from the pests

Among the forest pests is the beautiful steel-blue jewel beetle that causes considerable damage to pine forests during its very active reproduction phase. During gestation, it eats its way through the pine tree crown, while the larvae feed underneath the bark. For egg deposition, the jewel beetle prefers weakened trees. Healthy trees can destroy the offspring by sap flow or by accelerated cell growth in the affected area, also referred to as callusing. Schütz performed an olfactory analysis of odor emissions from a pine trunk. For this purpose the scientist

Prof. Stefan Schütz, Head of the Institute for Forestry Zoology and Forest Protection at the University of Göttingen.

MothsMoths, butterflies and beetles can be attrac-ted and captured using pheromones.

Silk mothsSilk moth caterpillar: The moth responds to the phero-mone bombykol in a concentration of 1000 molecules per cubic meter air. “The sensitivity is comparable to a dog’s nose“, explains the physicist and forestry scientist, Bernhard Weißbecker, Ph.D., a member of Prof. Schütz’s team.




An ambivalent relationship: Man and insect

It is estimated that we share this earth with up to 10 trillion insects! That would mean two billion bugs per human being. The relationship between man and bug is not always positive: We sometimes compete for the same resources and some insects have the irritating habit of feeding on our blood. In many parts of the world, insects cause transmission of serious and debilitating disease. On the positive side, insects play a vital role, helping to feed billions of humans by enabling agricultural production and by sustaining ecosystems, from which we obtain our foods. Overall, insects are often vilified, as observed by the American entomologist Prof. May R. Berenbaum in her book: „Bugs in the System: Insects and Their Impact on Human Affairs“ (Helix Books). Professor Berenbaum’s book is highly informative and entertaining.

She helps greatly in battling common misconceptions and prejudices. The diverse relationships and dependencies between man and bug are described, from ancient times to present day. Berenbaum shows the influence insects have had on the Arts, History and Literature and delivers a systematic presentation of physiology and survival strategies. When reading this book it soon becomes obvious how ignorant we humans are regarding insects. The author presents complicated interactions between individual species and the ecosystem – as fascinating as the relationship between man and insect.

GERSTEL solutions worldwide »book tip«

House longhorn beetleUp to 20 mm long, house longhorn beetle larvae eat their way through wooden beams, furniture as well as door and window molding, leaving behind distinctive flight holes and a weakened structure.

Infested and uninfested Pine trees by GC/FID/EAD

The EAD trace from the chromatogram represents the electro-physiological response of the steel-blue jewel beetle antenna to the emitted compounds. It is shown here in direct comparison with the GC/FID chromatogram. The arrows indicate the differences in signal between an attractive and an unattractive pine.

fight these more efficiently. Quite the opposite of the usual animal instinct to flee a fire, jewel beetles – in Canada also known as black fire beetles - head for forest fires in droves, sometimes from as far away as 80 km (50 miles).

They come for a reason: Jewel beetles need the wood of freshly burnt trees as nourishment for their larvae. The beetles are not threatened by predators, these have mostly left the area, and the trees can no longer defend themselves against the larvae.

To determine which compounds emitted by burning pine wood attract the beetles, samples were collected on activated charcoal and analyzed in the laboratory using GC/MS/EAD and GC/FID/EAD. „Parallel detection by FID and EAD enables quantitative determination of all the organic compounds that exist in the smoke gases“, explains Prof. Schütz, „while the specific reaction of the beetle antenna to certain substances in the EAD allows us to pin-point the active compounds in question”. The identification of those active compounds is performed using MS detection.

Schütz was able to demonstrate the following: The antennae of black fire beetles are especially sensitive to guaiacol compounds, detecting these at concentrations as low as 1 pg/mL. Based on his calculations just one single pine trunk, charred over a length of two meters, has black fire beetles seek out the place from distances up to one kilometer away, even under light wind conditions.

During field trials with a portable EAD-device this estimate was confirmed, indicating that an early forest fire warning system could be developed based on biosensors.

Infested an uninfested pine trees by GC/EAD/MSD

The main emissions from pine trees are terpenes, especially α-pinene and 3-carene. These substances are emitted both from trees that are attractive to jewel beetles and from trees that are not. The trees that attract jewel beetles, however, emit a much higher amount per unit time. Attractive pines also release the following compounds among others:1: 1,7,7-trimethyl-bicyclo [2,2,1] hept-2-en2: decahydro-methanoazulene 3: dimethyl-Undecadienone




GERSTEL TDS 3 / TDS A Thermal Desorption

System in combination with an Agilent Technologies

GC/MS system combined with a GERSTEL Thermal

Extractor TE 2. Such a system was used to develop

the analytical method listed in EN 71-11. Appendix

A provides detailed information on the analytical

method for determination of VOCs in toys.

In 2005, the European Commission tightened requirements for the safety

of toys. The European Standard, EN 71 Safety of Toys, was expanded to cover organic chemical compounds in toys. The newly approved EN 71 Part 10 covers sample preparation and extraction and Part 11 covers methods of analysis. Producers of toys must meet the new specifications to obtain the CE mark required for selling products in

New EU regulation

on organic chemicals in toys

Safety of toys

the EU. GERSTEL Thermal Extraction and GERSTEL Thermal Desorption technology was used for determination of low levels of VOC emissions as described in EN 71- Part 11.

Some details: A list of the VOCs that must be monitored in toys according to EN 71 can be found in appendix A of Part 11 of the method. An initial screening using Headspace GC is performed to determine whether VOCs are being emitted from the toy material. If significant VOC emissions are found, a more detailed determination must be performed. For this, thermal extraction in a GERSTEL Thermal Extractor (TE 2) is performed. VOCs emitted from the toy sample are concentrated onto a TDS tube, packed with Tenax TA. The TDS tube is subsequently thermally desorbed in a GERSTEL Thermal Desorption System (TDS 3) fitted with a GERSTEL TDS A autosampler. Analytes are automatically refocused in a GERSTEL CIS 4 inlet before being transferred to the GC/MS system for determination. For calibration, TDS tubes are spiked with standard solutions using a GERSTEL Tube Standard Preparation System (TSPS). The TDS tubes filled with Tenax TA are conditioned using a GERSTEL Tube Conditioner (TC 1).

Published by

GERSTEL GmbH & Co. KG,Aktienstrasse 232 – 23445473 Mülheim an der Ruhr, Germany

Editor

Guido Deußing,ScienceCommunication, Neuss, Germany [email protected]

Scientific advisory board

Ulrike Grüter, Ph.D. [email protected]

Eike Kleine-Benne, Ph.D. [email protected]

Contact

[email protected]

Design

Paura Design, Hagen, Germany www.paura.com

Print

BasseDruck, Hagen, Germany

Imprint

www.gerstel.com

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