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In the Service of Science and Justice

Forensic toxicologyDrugs of abuse

Extraction in seconds

Blood alcohol analysis

Automated Static Headspace as the method of choice

Forgery

Hot in pursuit of forgers

Forensic Sciences and Toxicology

Drugs of abuse: Extraction in seconds

In Forensic Science and in Toxicology, body fluids are regularly analyzed for residues of drugs of abuse, therapeutic drugs and of their metaboli-tes. In general this type of analysis requires extensive sample prepara-tion. GERSTEL has introduced automated Disposable Pipette Extraction (DPX). DPX is a fast and efficient dispersive SPE technique used for a wi-de range of applications such as drugs of abuse, therapeutic drug moni-toring, comprehensive screening, pharmacology studies (NNK), as well as pesticides in fruit and vegetables. DPX is based on unique and pa-tented dispersive SPE devices: Pipette tips that incorporate loosely con-tained sorbent material, which is mixed with the sample solution. Tur-bulent air bubble mixing creates a suspension of sorbent in the sample ensuring optimal contact and highly efficient extraction. The extraction is performed much faster than with traditional SPE techniques.

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Published by GERSTEL GmbH & Co. KG Eberhard-Gerstel-Platz 1 45473 Mülheim an der Ruhr, GermanyEditorial Director Guido Deußing ScienceCommunicationNeuss, Germany [email protected] and editingKaj Petersen [email protected] advisory boardEike Kleine-Benne, Ph.D. [email protected] Lerch, Ph.D. [email protected] Reimold, [email protected] [email protected] Paura Design, Hagen, Germany www.paura.de

Drugs of abuse: Extraction in seconds 2

Blood alcohol I: Automated Headspace Analysis – the method of choice 5

Blood alcohol II: Ethanol determination in biological samples by dual rail MPS 7

SPE: Poison Analysis EZ 8

DPX: Doing Drugs? 11

Forgery: Hot in pursuit of forgers 14

Automated Sample Preparation – Overview I 15

Automated Sample Preparation II – Overview II 16

B ody fluids represent a complex and heterogeneous matrix. Accurate

determination of drugs, pharmaceuti-cals and metabolites in blood and urine requires both a suitable chromatographic system and adequate sample preparation. Sometimes more than one extraction technique is needed for a successful result. Solid Phase Extraction (SPE) is among the most widely used sample clean-up and analyte extraction techniques in forensic and toxicology laboratories.

Traditionally, SPE requires the use of significant quantities of solvent, some of which are toxic. Following several labor intensive steps, many methods require that the solvent be evaporated in order to concentrate the analytes of interest and achieve the necessary detection limits. Depending on the chemical properties of the analytes, a chromatographic deter-mination may also require further sample preparation steps such as derivatization. The sum total of sample preparation steps can amount to a significant bottleneck for laboratory productivity and a risk to occu-pational health unless adequate and costly safety precautions are taken.

If the tedious and labor intensive steps can be eliminated, the overall task can be performed more efficiently and faster while producing accurate results and using only a fraction of the amount of solvent normally used. All this is possible thanks to Disposable Pipette Extraction (DPX), a technique developed by Professor Wil-

liam (Bill) E. Brewer, Ph.D. from the Uni-versity of Southern Carolina. Professor Brewer is the Owner-President of DPX Labs (www.dpxlabs.com).

The DPX technique has now been auto-mated by GERSTEL, a leader in automa-tion of sample preparation and sample int-roduction for GC/MS and LC/MS.

“The reactions we got from the foren-sic scientists at the SOFT 2008 meeting”, says Ro bert J. Collins, Ph.D., President of GERSTEL, Inc. in Baltimore, MD, “lead us to believe that automated DPX is seen by experts as a very promising alternative to standard extraction techniques”.

In order to provide an efficient solu-tion for the determination of drugs and metabolites in blood in a routine labora-tory environment, GERSTEL and DPX Labs LLC collaborated on automating the DPX technique. “DPX immedia-tely struck us as the right solution”, says Bob Collins. Furthermore, for this appli-cation, samples should be prepared and derivatized just prior to analysis. Just in time sample preparation eliminates ana-lyte degradation and under-reporting of concentration levels since no sample is waiting for an extended period of time in the autosampler before being analyzed. Also, in order to optimize GC/MS sys-tem utilization and sample throughput, a sample should be ready for introduction every time the GC/MS finishes its run and becomes ready for the next sample. In summary, performing GC/MS analy-

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2GERSTEL Solutions worldwide Forensic Special

All steps are performed automati-cally 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 sor-bent in the sample, ensuring optimal contact, highly efficient 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 efficient 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 determi-nation can be performed in parallel for best possible throughput and system utilization.

Automated DPX processsis and sample preparation carefully syn-chronized and in parallel benefits both the quality of results and the throughput. The MAESTRO software with its PrepAhead and Scheduler functions makes it extre-mely easy for the analyst to plan, optimize, and set up the whole process.

From theory to practice

In order to test the MPS-DPX-CIS-GC/MS system in practice, the scientists ana-lyzed blood and urine samples that had been spiked with different drugs and pharmaceuticals. Compounds determi-ned included amphetamines, benzodiaze-pines, cocaine and methadone as well as tetrahydrocannabinol (THC) and meta-bolites. For details, please see the graphic representations on this page. The analy-sis was performed using deuterated inter-nal standards: For blood samples, d5-PCP (0.2 ppm) was used. For the determination of benzodiazepines, d5-Nordiazepam (0.2 ppm) and d5-OH-Alprazolam (0.2 ppm) were used. For the opiates, equivalent deu-terated compounds were used (each at a level of 0.1 ppm).

Required manual sample preparation steps

Preparing blood samples: 0.5 mL of ace-tonitrile was added to a 0.25 mL sample of whole blood followed by mixing to pre-cipitate proteins in the sample. The mix-

ture was centrifuged and the supernatant transferred to a clean labelled test tube containing 0.1 mL of 0.1 M HCl.

Preparing urine samples for the determination of benzodiazepines:

In order to determine the total level of free, bound and meta-

bolized residues of drugs and pharmaceuticals in urine, hydrolysis must be per-formed of the respective conjugates, such as for example glucuronides of benzodiazepines, which are metabolites of the drugs that are formed to facilitate excretion of the substances from the body.

The hydrolysis reaction is started by add ing 10 µL

of a solution of the enzyme b-glucuronidase and 50 µL

of a 0.1 M sodium phosphate buffer with pH 4 to a 0.2 mL

sample of urine. The mixture is kept at 55 °C for two hours before

being allowed to cool to room tempera-ture. Acetonitrile (0.25 mL) is then added in order to preci pitate the enzyme. Fol-lowing centrifugation, the supernatant is transferred to a clean, labelled test tube and 200 µL of 0.1M HCl is added. The prepared samples are then placed in the MPS sample tray.

Automated Disposable Pipette Ext-raction (DPX) is subsequently perfor-med on the prepared samples using the MultiPurpose Sampler (MPS) equipped with 1 mL CX tips from DPX Labs (www.dpxlabs.com). As the name suggests, CX tips contain a novel and unique cation exchange material with additional slightly apolar cha racteristics. The DPX process is completely automated: 250 µL of a 30 % solution of acetonitrile in water is dis-pensed onto the DPX sorbent inside the tip for conditioning. The conditioning sol-vent is subsequently discarded to waste. The DPX tip is then immersed into the sample and a defined volume is aspirated into the tip. Air is then aspirated into the tip, causing turbulent mixing and efficient extraction of analytes into the sorbent. Fol-lowing a 30 second equilibration time, in which the sorbent is allowed to settle, the extracted sample is discarded to waste. The sorbent is rinsed twice, first with 0.5 mL of a 30 % solution of Acetonitrile in water and then with 0.5 mL acetonitrile. The ex-tracted analytes are eluted using 0.7 mL of a solution consisting of 2 % concen-trated ammonia, 78 % CH2Cl2 and 20 % isopropanol.

The eluate was dispensed directly into an autosampler vial. The total amount of time required for extraction and liquid handling was less than 6 minutes per sample.

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Benzodiazepines in urine: Total ion chromatogram of 0.2 ppm benzodiazepines in 0.2 mL urine following enzymatic hydrolysis and DPX. Derivatization was performed in the CIS inlet by inject-ing 50 µL of DPX eluent together with 20 µL of 50/50 MTBSTFA/acetonitrile. No separate solvent evaporation step was performed. Increasing the sample volume to 0.5 mL and performing multiple DPX extractions would increase the sensitivity. 1) Diazepam, 2) Nordiazepam-d5-TBDMS, 3) Nordiazepam-TBDMS, 4) Fluni-trazepam, 5) 7-aminoflunitrazepam, 6) Oxazepam-2TBDMS, 7) Temazepam-TBDMS, 8) Nitrazepam, 9) Lorazepam-2TBDMS, 10) Clonazepam-TBDMS, 11) Alprazolam, 12) a-OH-Alprazolam-d5-TBDMS, 13) a-OH-Alprazolam-TBDMS

THC and metabolites in blood: Total ion chromatogram of 10 ng/mL THC and metabolites extracted from 0.5 mL whole blood following protein precipitation, centrifugation and DPX-RP. Derivatization was performed in the CIS inlet by injecting 50 µL of DPX eluent together with 20 µL of 50/50 BSTFA/acetonitrile. No additional solvent evaporation or derivatization step was performed. Analytes: 1) THC-TMS, 2) OH-THC-TMS, 3) COOH-THC-2TMS

Drugs of abuse in blood: Total ion chromatogram of a DPX extract of 250 µL whole blood spiked at 0.5 ppm with drugs of abuse using d5-PCP as internal standard. Chromatograms from the 5th and 20th injections are shown alongside each other to demonstrate the ruggedness of the analysis. The insert shows the extracted ion chromatogram (EIC) from the 5th injection. The sample was protein precipitated with 0.5 mL acetonitrile, and the supernatant was transferred to clean tubes. After adding 0.1 mL of 0.1 M HCl, automated DPX was performed. 1) Meperidine, 2) d5-PCP (ion 205), 3) PCP, 4) Methadone, 5) Methaqualone, 6) Amitriptyline, 7) Cocaine, 8) cis-Doxepin, 9) Imipramine, 10) trans-Doxepin, 11) Desipramine, 12) Pentazocine, 13) Codeine (Septum bleed from vial cap after repeat injections from the same vial).

Instrumentation

The analysis was performed on a 6890N/5975 (inert XL) GC/MS system from Agilent Technolo-gies. The GC was fitted with a Cooled Injection System (CIS 4) PTV-type inlet. An MPS Prep-Station with DPX option and MAESTRO Software control was used for automated sample preparation and sample intro-duction. The complete system including GC/MS was operated using one integrated method and one sequence table directly from the Agilent Technologies ChemStation Software, operated integrated with the MAESTRO software.

Derivatization

Some analytes must be deriva-tized to enable GC/MS deter-mination. “The Cooled Injection System (CIS) inlet offers an inert and temperature programmable environment”, says Prof. Brewer, “which is highly suited to evapo-rating and purging excess solvent while simultaneously, or at least sequentially, performing deriva-tization of analytes.”

For the derivatization of benzodiaze pines, 20 µL of 50 % N- (t-butyldimethylsilyl)-N-methyltrifluoracetamide (MTBSTFA) in acetonitrile was aspirated into the autosam-pler syringe followed by 20 µL of air and 50 µL of the DPX eluate. “The resulting ‘Sand-wich’ injection was performed slowly, using a programmed stop flow method to ensure that the solvent was completely remo-ved through the split vent prior to the derivatization step”, the application specialist explains. The CIS temperature quickly ramped to 300 °C, which star-ted the derivatization process and helped transfer the deriva-tized analytes to the GC column in splitless mode for highest pos-sible recovery and lowest limits of determination.

“Automated analyte deriva-tization in the GC inlet proved to be both simple and highly practical”, said Prof. Brewer. “The method was successfully applied to the determination of benzo-diazepines in blood. Compounds that were not successfully deriva-tized in this way were derivatized directly in the sample vial”. For this approach, the DPX eluate was evaporated to dryness under a flow of nitrogen in the sample vial. 50 µL of MTBSTFA and

50 µL of ethyl acetate were added and the mixture kept at 70 °C for 20 minutes. When the extract had cooled off, 50 µL of the solu-tion was introduced to the CIS inlet using the Large Volume Injection (LVI) technique.

The conclusion reached by the experts

“As we had expected, the ana-lysis based on automated DPX delivers excellent results”, said Bob Collins. Even though all analyses were performed on very small sample volumes (250 µL blood or 200 µL urine), the resulting peak intensities were highly satisfactory – even in full scan mode. The MultiPurpose Sampler (MPS) with automa-ted DPX enables fast sample preparation of difficult samples while delivering high sensitivity and accurate results. The additi-onal liquid handling capabilities of the dual rail MPS PrepSta-tion enabled full automation of all the required liquid handling steps such as derivatization and addition of an internal standard. This added level of automation provided best possible produc-tivity and throughput. The ins-trument combination used pro-ved to be especially useful for the determination of basic drugs such as cocaine, methadone, PCP, TCAs and Meperidine.

Mos t benz od iaz ep ine s were easy to determine using MTBSTFA derivatization in the GC inlet. The following were determined: Diazepam, Nordia-zepam, Oxazepam, Temazepam, Alprazolam and a-OH-alpra-zolam. “DPX combined with GC/MS determination provi-ded excellent results for the 11 listed benzodiazepines in urine”, Bob Collins notes, “plus we got good recovery and great sensi-tivity for the opiates. For most opiates, we achieved limits of determination under 1 ng/mL in whole blood”. Ralf Bremer, General Manager for produc-tion and R&D, is thrilled about the use of the CIS 4, PTV-type inlet for evaporative concentra-tion and analyte derivatization: “This year, we are celebrating the 25th anniversary of the introduc-tion of the first GERSTEL CIS. It is very reassuring to see that the improvements we have regularly engineered into the CIS over the years enable us to stay well ahead of the competition”.

EIC

TIC

Drug Average RSDAmphetamine 13.9 %Methamphetamine 14.4 %Meperidine 5.8 %PCP 2.2 %Methadone 3.3 %Methaqualone 3.6 %Amitriptyline 3.1 %Cocaine 3.8 %Cis Doxepin 2.8 %Imipramine 3.3 %Trans Doxepin 3.2 %Pentazocine 5.3 %Codeine 4.2 %Desipramine 6.4 %


Blood alcohol I

Automated Headspace Analysis – the method of choice

Edward A. Pfannkoch, and Jacqueline A. WhitecavageGerstel, Inc., 701 Digital Drive, Suite J,Linthicum, MD 21090, USA

Forensic laboratories face the need to analyze a large number of samples of human blood and body fluids for alcohol content. When faced with this challenge factors that need to be considered are sample throughput, resolution, and carryover.

A successful method for these analyses should be fast, precise, and accurate. Current methods used in these analyses use a gas chromatograp coupled to a static headspace sampler and flame ionization detector (FID). The x, y, z robotic auto-sampler used in this study has a capacity of up to 128 headspace samples, which is a distinct advantage compared to other samplers commercially available Results obtained with the instrument and me-thodology described in this report meet the specifications set by the California Department of Justice Blood Alcohol Operating Procedures (Title 17). A dual-column, dual-FID blood alcohol analysis system that can be used for confirmation of ethanol peaks was also tested and pro-duced results with good precision (below 5 % RSD).

Introdution

Headspace gas chromatography (HS-GC) for determination of ethanol content of blood is widely used by forensic labs to test automobile drivers charged with DUI (driving under the influence). The method originates from 1964 when G. Machata [1] published the first use of HS-GC for quantitative analysis. The method includes the use of an internal standard (IS) compound. Tert-butanol or n-propanol may be used as internal standard for the determination of alcohol in blood. The choice of which internal standard to use depends on the type of column utilized in the GC instrument. Blood is a very complex matrix that varies depending on the individual. The salt or lipid content may be different and headspace analysis with the use of an IS provides fast measurements that can be automated. In this study, a GERSTEL MultiPurpose Sampler (MPS) robotic autosampler with a headspace gas-tight syringe was used to analyze ethanol solutions in different concentrations. In this study, we developed a method

that meets the specifications set by the California Department of Justice Blood Alcohol Operating Procedures (Title 17) [2]. We also configured and tested a separate dual-column/dual-FID system that adds confirmation because of the different elution order of ethanol on the two columns.

Experimental

InstrumentationAnalyses were performed on a 6890 GC equipped with single or dual FID (Agi-lent Technologies), and a GERSTEL MPS 2 MultiPurpose sampler configured for static headspace injection.

Reagents• Ethyl alcohol, absolute, 200 proof,

99.5%, A.C.S. reagent grade• Methyl alcohol, 99.8%, A.C.S. reagent

grade• Acetone 99.5%, A.C.S. reagent grade• n-Propanol (1-propanol) 99.5% A.C.S.

reagentgrade (IS)• Isopropanol (2-propanol), 99.5%,

A.C.S. reagent grade• Blood alcohol mix resolution control

standard (Restek, # 36256). 0.100 g/dL in water of 8 compounds: acetaldehyde, acetone, acetonitrile, ethanol, ethyl ace-tate, isopropanol, methanol and methyl ethyl ketone (MEK).

Preparation of standards • Secondary standard (SS). 0.25 mL

of absolute (200 proof ) ethanol and 0.125 mL of n-propanol pipetted into a 100mL volumetric flask and diluted with bottled water.

• Quality control standard (QC). 0.15 mL of absolute (200 proof ) ethanol and 0.125 mL of n-propanol pipetted into a 100 mL volumetric flask and diluted with bottled water.

• Resolution standard (RS). 0.25 mL of absolute (200 proof ) ethanol, 0.1 mL methanol, 0.1 mL isopropanol, 0.01 mL acetone and 0.125 mL n-propanol pipetted into a 100 mL volumetric flask and diluted with bottled water.

• Blank standard. 0.125 mL of n-propa-nol pipetted into a 100 mL volumet-ric flask and diluted with bottled water. All standards above were diluted 1:6 in bottled water prior to use. 500 µL of standard was then pipetted into a 20 mL headspace vial. 1 mL of 1000 µg/mL internal standard (n-propanol) and 1 mL of the blood alcohol mix resolu-tion control standard (Restek, # 36256, Lot# A034323) was diluted in 18 mL bottled water. 4 mL of standard was then pipetted into a 20 mL headspace vial. All vials were crimp-capped using blue silicone/PTFE septa.


Figure 2. FID overlay of Blank/Internal Standard (IS), Resolution Standard and Secondary Standard (SS) for California Compliance.

Quality control criteria for California compliance

• Calibration runs consist of 6 secondary standards followed by a resolution stan-dard. The calibration constant K is then calculated for each of the 6 secondary standards and the mean is calculated. The value of the K constant for each of the six determinations must fall within ± 1.5 % of the mean value.

• The results for the resolution standard must show a resolution of 0.01 % ace-tone in the presence of 0.20 % ethanol.

• Analysis runs consist of a Blank (water, no IS) and standards (SS, QC and RS) followed by the sample set (2 replicas per sample) followed by two additional standards (QC and SS).

• The result of the blank sample should be less than 0.01 %.

Results and Discussion

California DOJ Blood Alcohol Opera-ting Procedure (Title 17). The instru-ment used in this study was a GERS-TEL MPS autosampler that can be pro-grammed to be used with headspace sy-ringes. For this study we used a 2.5 mL syringe that can also be programmed to inject different volumes (recommen ded volumes from 0.25 mL up to 2.5 mL). The headspace syringe adaptor is heated and can be controlled to optimize the syrin-ge temperature. This results in great time savings and excellent sample throughput. For this analysis we selected an incuba-tion temperature of 65 ºC and a syringe temperature of 70 ºC. It is recommended to use a slightly higher syringe tempe-rature to avoid condensation. Secondary and resolution standards were analyzed.

An example chromatogram is displayed in Fi-gure 2. It can be seen that there is no ethanol carry-over in the blank and the IS repro-duces well. Using the gas chromato-graph in the iso-thermal mode, we were able to sepa-rate the alcohols present in the SS and also the com-pounds present in the resolution standard in ap-proximately 3.5

minutes (Figure 3). The updated chro-matographic conditions include the use of a capillary GC column instead of the packed column currently used for these analyses in California.

Response

Blood Alcohol Dual-Column Con-firmation Method. We configured a system with dual complimentary alcohol columns from a single inlet and dual FIDs for blood alcohol analysis [3]. The dual system has an advantage since the order of elution is different for each column, enabling confirmation of the peak iden-tification. In order to verify the precision of the splitter (Figure 4) we installed two identical columns and checked the response of the secondary standard on both columns.

Conclusions

• The GERSTEL MPS 2 robotic auto-sampler is capable of delivering perfor-mance for blood alcohol analysis that meets or exceeds the California Title 17 Forensic Alcohol Analysis and Breath Alcohol Analysis performance criteria.

• Testing during a 3-month period show-ed good robustness and reproducibility.

• This instrumentation provides increased sample throughput by accommodating up to 128 samples and by using the “prep ahead” function to equilibrate multiple samples simultaneously.

• The GERSTEL MPS 2 autosampler performed well when used with a dual column blood alcohol confirmation method.

Compound Average peak area Standard deviation RSD [%] ALC1 ALC2 ALC1 ALC2 ALC1 ALC2Methanol 2321294 2628655 81681 93324 3,52 3,55Acetaldehyde 12326272 13373526 534287 578415 4,33 4,33Ethanol 4958271 5519370 180264 202302 3,64 3,67Isopropanol 9719454 10594110 379814 418120 3,91 3,95Acetone 31021477* 21991434 1350778* 962275 4,35* 4,38Acetonitrile 11314551 481138 4,25n-Propanol 9320684 9919804 344806 370832 3,70 3,74MEK 39378050 42850828 1711203 1844065 4,35 4,30Ethyl acetate 52394805 55868378 1904410 2026058 3,63 3,63

Table 4. Precision of 12 replicas using dual-column configuration.

GC 6890 (Agilent Technologies)Inlet: Split/splitless, 150 °C Split 1:5Column: 30 m DB-ALC1 (Agilent) di = 0,32 mm, df = 1,8 µmOven: isotherm, 35 °C

MultiPurpose Sampler (MPS)Incubation: 65 °C (15 min)Syringe: 2,5 mL, 70 °CInjektion: 1 mL (500 µL/s)

Table 3. Method parameters for dual-column dual-FID system.

EtOH n-Propanol (IS)File name Peak Area Peak Area K-factor -1,5% / +1,5%7230002 113206882 106875141 0,1794 Pass / Pass7230003 116621856 109083941 0,1777 Pass / Pass7230004 114897369 107497317 0,1778 Pass / Pass7230005 111194673 104605854 0,1787 Pass / Pass7230006 115948256 109036988 0,1787 Pass / Pass7230007 114896941 107920646 0,1785 Pass / PassAverage 114460996 107503314 0,1785 0,176 / 0,181StD 1975014 1662204 0,0006 % RSD 1,73 1,55

Table 2. Example of calculation and check of K factor.

GC 6890 (Agilent Technologies)Inlet: Split/splitless, 100°C Split 1:5Column: 30 m DB-ALC2 (Agilent) di = 0,53 mm, df = 2,0 µm Oven: isotherm, 40 °C

MultiPurpose Sampler (MPS)Incubation: 65 °C (15 min)Syringe: 2,5 mL, 70 °CInjektion: 1 mL (500 µL/s)

Table 1. Method parameters for California com-pliance.


Figure 5. Dual-FID traces of Restek (#36256) Resolution Control Standard and Internal Standard (IS).

Figure 4. Dual-FID traces of Secondary Standard (SS) using two identical columns DB-ALC 2.

Blood alcohol analysis using the

Dual Rail MPS PrepStation

Scientists from the Federal Bureau of Investigation Laboratory in Quantico, VA have used a GERSTEL MPS Prep-Station to combine automated liquid sample preparation such as addition of standards with automated head-space analysis.

Detection, identification, and quantitation of etha-nol and other low molecular weight volatile com-pounds in liquid matrices by headspace gaschro-matography–flame ionization detection (HS–GC–FID) and headspace gas chromatography–mass spectrometry (HS–GC–MS) are becoming com-monly used practices in forensic laboratories. Al-though it is one of the most frequently utilized procedures, sample preparation is usually done manually. Implementing the use of a dual-rail, pro-grammable autosampler can minimize many of the manual steps in sample preparation.

The autosampler is configured so that one rail is used for sample preparation and the other rail is used as a headspace autosampler for sample int-roduction into the gas chromatograph inlet. The sample preparation rail draws up and sequenti-ally adds a saturated sodium chloride solution and internal standard (0.08%, w/v acetonitrile) to a headspace vial containing a biological sam-ple, a calibrator, or a control. Then, the analyti-cal rail moves the sample to the agitator for incu-bation, followed by sampling of the headspace for analysis. Using DB-624 capillary columns, the method was validated on a GC–FID and confirmed with a GC–MS. The analytes (ethanol, acetonitrile) and possible interferences (acetaldehyde, metha-nol, pentane, diethyl ether, acetone, isopropanol, methylene chloride, n-propanol, and isovaleralde-hyde) were baseline resolved for both the GC–FID and GC–MS methods. This method demons-trated acceptable linearity from 0 to 1500 mg/dL. The lower limit of quantitation (LOQ) was deter-mined to be 17 mg/dL and the limit of detection was 5 mg/dL.J. Chromatogr. B 850 (2007) 230–235

Figure 3. FID trace of Resolution Standard for California Compliance.

Blood alcohol II


Poison analysis EZ

Application specialists from TeLA GmbH have developed a new me-thod that dramatically simplifies LC/MS determination of pesticide le-vels, providing high-quality results independent of the sample mat-rix type and complexity.

Norbert Helle, Ph.D. and Meike Baden, TeLa GmbH Bremen, Germany

P esticides, fungicides and herbici-des 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 consumer health.

World-wide, around 700 pestici-des 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, making it difficult to develop a fast all encompassing ana lysis method.

Still, effective multi-residue methods are in use for the determination of pesti-cides, helping to ensure food safety or to determination the cause in case pesticides have been used as poison.

Tracking down pesticides using GC/MS and LC/MS

Classical pesticide analysis relied on gas chromatography (GC) using an electron capture detector (ECD) or a nitrogen phosphorous detector (NPD). The most widely used detector today is the mass selective detector (MSD).

In Germany, the analytes that are mainly in focus 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 method does, however, require significant sample preparation including a gel chromatography clean-up step to sepa-rate analytes from the matrix.

Different analysis techniques are used for different 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 moderately polar compounds. As can be

seen from this description, there is some overlap between the techniques. Recently a new multi-residue method for the deter-mination of pesticide levels in fruits and vegetables was presented (QuEChERS: Quick, Easy, Cheap, Effective, Rugged & Safe) [*]. Compared to previous methods, the QuEChERS sample preparation steps are much less time-consuming, enabling the preparation of 8 samples in less than 30 minutes. QuEChERS is a sample pre-paration method well suited for both GC, GC/MS and LC/MS analysis. The QuE-ChERS sample preparation steps are lis-ted below.

The main benefit of this sample prepa-ration method is that the overall analysis is less time-consuming and less error-prone than more traditional approaches. Unfor-tunately, extracts obtained following this procedure often have a high matrix con-tent, which causes chromatographic prob-lems for GC analysis due to residue 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 QuE-ChERS sample preparation are compara-

ble 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 automated. In addition, much smaller volumes of partly toxic organic sol-vents are required, compared with other currently used methods for determining pesticides in fruits and vegetables. In addi-tion to the financial benefits of a much higher laboratory throughput, the cost of materials at around one Euro per sample is relatively low.

The limits of QuEChERS are encoun-tered whenever samples with more com-plex matrices need to be analyzed, such as garlic, onion, artichoke or avocado with much higher fat content. This can lead to problems with interferences, than can especially influence quantification unless further clean-up steps are performed.To enable reliable and rugged analysis independent of the sample matrix, we looked for a similarly effective alterna-tive sample preparation procedure. We found that automated solid phase extrac-tion (SPE) based on the GERSTEL Mul-tiPurpose Sampler (MPS) provided an excellent solution. The GERSTEL SPE, we have previously used successfully for a number of applications, including aflato-xins, chloramphenicol and malachite green in foods. In summary, we can report that our automated SPE-LC-MS/MS-ESI multi-residue method reduces the num-ber of manual steps required to a mini-mum while increasing laboratory through-put. The results are solid and reproducible combined with high sensitivity and good limits of determination.

Intrumental requirements

The GERSTEL SPE was fitted with an injection valve; sample introduction to the Agilent LC 1200 was performed directly by the SPE system; detection was perfor-med using an Agilent 6410 MS/MS Triple Quad instrument.

When the sample matrix no longer matters ...

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


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

Sample Preparation: 15 mL of an aceto-nitrile/water mixture (80:20) was added to a five 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 cart-ridge, which was subsequently rinsed with 5 mL water. Analytes were then eluted using an acetonitrile/water mixture added at a flow rate of 600µL/min. In contrast to most manual SPE methods, the liquid is not aspirated through the cartridge under vacuum, rather it is added under positive pressure using a syringe. This means that flows, and therefore also the elution speed, are accurately controlled and results more reproducible. This holds true even when sample matrix changes the restriction across the cartridge. The eluate was con-centrated for six minutes at 50 °C and the residual analytes taken up in 5 mL of a acetonitrile/formic acid mixture (30:70).Sample introduction and analyte separa-tion: 20 µL of the cleaned-up extract was introduced directly 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; flow rate: 0.5 mL/min resulting in a column head pressure of approximately 420 bar. A solvent mixture of 5mM formic acid (A) and acetonitrile (B) was used as mobile phase based on the following gradient pro-gramming: 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 Multimode ion source. Our experiments clearly showed that the Multimode source provided sig-nificantly lower detection 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 opti-mized for the flow and eluent used. The following parameters were used: N2 tem-perature: 340 °C; carrier gas flow (N2): 9 L/min; nebulizer pressure: 30 psi. The triple quadrupole instrument was opera-ted in MRM mode, with 5 different time segments, monitoring two transitions for each pesticide. In each segment 40 to 50 analytes were monitored.

The proof of the pudding

When 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” matrices, such as lettuce or cucumber, additional clean-up steps are not required following the aceto-nitrile/water extraction. For complex mat-rices that contain fat and other challenging matrix components, further clean-up steps are of course needed. For this purpose we used the GERSTEL SPE system.

Raw sample extracts were automati-cally loaded onto standard SPE cartridges and cleaned. A new cartridge was used for every sample to eliminate cross-contami-nation. Macherey-Nagel cartridges con-taining C18 reversed phase material were found to produce excellent, reliable results.

Automated SPE clean-up as described in this article took around 20 minutes to complete. Apart from the first sample, the SPE process was performed during LC/MS or GC/MS analysis of the preceding sample, ensuring that the SPE step was performed without increasing the overall analysis time. Once the first 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 order to achieve good separation combined with method ruggedness, the conscious decision was made to only seek a moderate reduction


Carbendazim

Thiabendazol

of the analysis time. The total analysis time required to determine around 140 com-pounds was in the order of 35 minutes. This time period was more than sufficient to prepare the following sample for just-in-time sample introduction to the LC/MS system.

Sample clean-up using SPE contri-butes not only to the ruggedness of the method, it also improves reproducibility and linearity, among other things. To illus-trate this, a bell pepper sample was spiked with a pesticide mixture and analyzed. Fol-lowing SPE clean-up, retention times and peak areas of the analytes showed excellent reproducibility. The linearity was excellent, both for polar compounds like Carbenda-zim and Thiabendazole as well as for apo-lar pesticides like Diazinon and Pirimi-phosmethyl.

Orange oil samples were cleaned up using a slightly modified SPE method. The efficiency of SPE clean-up is illus-trated by the fact that the intense yellow color 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 difficult mat-rix ranged from 70 to 90 % while recove-ries 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 excellent peak symmetry.

One final comment: Every method must prove its worth in practice. The test, as always, is in the analysis of real world sam-ples. To prove the validity of our method, we took part in a Europe-wide round robin with 46 participating laboratories. A vege-table sample (zucchini) had to be analyzed for 185 different pesticide residues. Out of 46 laboratories, TeLA GmbH was among the 12 that managed to correctly identify and quantify the analytes thus meeting the round robin requirements and pas-sing the test.

128 of the 185 pesticides were deter-mined using our SPE-LC-MS/MS pesti-cide multi-residue method. 90 of the 185 pesticides were determined using a GC/MS system (GC 6890 / MSD 5973, both from Agilent Technologies) in combina-tion with the GERSTEL MultiPurpose Sampler (MPS) using a Retention Time Locking (RTL) method.

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

Overlay medium polarity sections of 8 different chromatograms: 8 sepa-rate 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.

Determination of polar and apolar pesticides respectively in orange oil. Overlay chromatograms covering 9 different con-centrations are shown.

In LC/MS, we work towards the mu-tually irreconcilable goals of achieving the perfect LC separation and com-bining it with the most efficient ioniza-tion and lowest achievable MS detec-tion limits for our analytes. The LC sepa-ration may require a certain pH and polarity range of the eluent, while ana-lyte ionization in the LC/MS ionization source requires yet another pH, a dif-ferent buffer – or even derivatization of the analyte for best possible effici-ency or optimized spectral information. How to optimize both? Well the logical answer is to take the effluent from the perfect LC separation and then optimize it for MS analysis. This task is easily pos-sible when you add the GERSTEL LC/MS Effluent Optimizer (LEO) module to your LC/MS/MS system. Application examples show sensitivity gains of up to a factor of 40 by simply adding a salt solution GERSTEL LC/MS Effluent Opti-

mizer (LEO) Optimized LC separation and MS detection – get the best of both worlds to the LC effluent and/or chan-ging its pH. The LEO module is quickly and easily installed in your LC/MS sys-tem. A solvent mixture, buffer solution or reagent is then easily added to the effluent ensuring that the LC separa-tion can be performed under optimal conditions while also enabling maxi-mum yield in the MS ionization process. Whether you are looking to perform pH adjustment or post-column derivatiza-tion, for method development or rou-tine analysis, when you use LEO and the GERSTEL MAESTRO software you can easily and efficiently control all para-meters as part of the overall method. Just one sequence table controls the entire system from sample preparation through LC separation and effluent opti-mization to MS analysis. It is all done at the click of a mouse.

GERSTEL LC/MS Effluent Optimizer (LEO)

Optimized LC separation and MS detection –get the best of both worlds NEW


Automated Sample Preparation

Doing Drugs ?Comprehensive Analysis of Drugs of Abuse in Blood and Urine with

Automated Disposable Pipette Extraction and HPLC/MS/MS

I In order to analyze biological spe-cimens for drugs and their metabolites, it is necessary to perform sample prepa-ration to eliminate matrix interference. Solid-phase extraction is generally the preferred sample preparation technique, in this study Disposable Pipette Extrac-tion (DPX) was utilized. DPX is a novel dispersive solid-phase extraction tech-nique that uses loosely contained sorbent in a disposable pipette tip. The sample is aspirated into the tip where it is actively mixed with the sorbent and forms a sus-pension. The main advantages of the DPX technology are that the extraction is very

rapid, minimal solvent waste is genera-ted, and the entire process can be fully automated including introduction of the extract to the chromatographic system. The GERSTEL MPS autosampler per-forms DPX extractions in approximately 5 minutes using reversed phase (DPX-RP) or cation exchange (DPX-CX) sor-bent material. For chemical analysis of target drugs, GC/MS or HPLC/MS/MS are generally the preferred techniques. The advantage of LC/MS/MS is that chemical derivatization of the analytes is not required, making sample preparation simpler and less time consuming. In addi-

tion, highly efficient ionization, in com-bination with tandem mass spectrometry results in high sensitivity and selectivity. This study focused on performing auto-mated extraction of reduced sample volu-mes coupled with LC/MS/MS to provide high throughput analysis “one sample at a time”. The sample preparation time was decreased sufficiently to allow the extrac-tion of a sample during the chromato-graphic analysis of the previous sample in the sequence.

Experimental

Instrumentation. Sample extrac-tion and introduction in the LC/MS/MS system was automated using a GERSTEL MPS dual rail PrepStation with DPX option.

Analysis conditions LCMobile Phase: A - 4.5 mM Ammonium acetate B - MethanolGradient: Initial 90 % A / 10 % B 2 min 85 % A / 15 % B 3 min 65 % A / 35 % B 4 min 50 % A / 50 % B 6 min 35 % A / 65 % B 8 min 90 % A / 10 % B (4 min)Flowrate: 350 µL/minColumn: 2.1 mm x 30 mm, 3.5 µm, Eclipse XDB C18 (Agilent)Inj. volume: 10 µL

Analysis conditions MSPositive ion mode, Single reaction monitoringRun time: 10 minCapillary: 3 kVExtractor: 2.81 VSource Temp.: 130 °CDesolvation Temp.: 391 °C

Compound M + H Dwell Cone Time Voltage [m/z] [ms] [V]

d3-Oxymorphone 305 50 30Oxymorphone 302 50 30Morphine 286 50 30Hydromorphone 286 50 30d3-Oxycodone 319 50 30Oxycodone 316 50 306-MAM 328 50 30Codeine 300 50 30Hydrocodone 300 50 30


The supernatant was decanted into a clean labeled sample tube. 100 µL of 0.1 M HCl was added to the solution, and the sample tube was placed on the MPS 2 sample tray for automated DPX extraction.

Urine sample preparation. 50 µL of 0.6 M sodium ace-tate buffer (pH = 4) and 10 µL of ß-glucuronidase was added to a 200 µL sample of

urine. The solution was thermo-stated at 70°C for 2 hours, and

then cooled to room temperature. To precipitate proteins, 250 µL of

acetonitrile was added to the hydro-lyzed urine and the sample was vortex

mixed and centrifuged. The supernatant was decanted into a clean labeled sample tube. 200 µL of 0.1 M HCl was added to the sample solution, and the sample tube was placed on the MPS sample tray for automated DPX extraction.

Extraction

A GERSTEL MPS dual rail PrepSta-tion was set up with 1 mL DPX-CX tips (DPX Labs, LLC, Columbia, SC) for ext-raction of drugs from blood and hydro-lyzed urine. The following automation method was used: 250 µL of 30% aceto-nitrile/water was slowly added through the top of the DPX tip at a rate of 50 µL/s to wet the sorbent. The sample was then aspirated into the DPX tip at a rate of 90 µL/s and mixed with the sorbent by dra-wing in an additional 2 mL of air. After

Sample preparation

All opiate standards were obtained from Cerilliant (Round Rock, TX). A 10 ppm stock solution was prepared in methanol for all sample fortifications. Two inter-nal standards were used, d3-Oxymor-phone for quantitation of Oxymorphone and d3-Oxycodone for all other opiates. All solvents used were of HPLC grade or higher.

Blood sample preparation. A 250 µL blood sample was spiked at the specified concentration with the stock opiates mix. To precipitate proteins, 500 µL of acetonitrile was added and the solu-tion was vortex mixed and centrifuged.

a 30 s equilibration time to allow analyte binding, the resulting solution was dis-pensed to waste. To wash off excess mat-rix, a 500 µL wash of 10% acetonitrile/water was added to the sorbent material through the top of the DPX tip and dis-pensed to waste followed by an additional wash using 500 µL of acetone. For elution of the analytes, 700 µL of 78/20/2 (v/v) of methylene chloride/isopropanol/ammo-nium hydroxide was added to the sorbent material through the top of the DPX tip and dispensed directly into a clean HPLC vial. All eluents were dried and reconsti-tuted with 100 µL of methanol and 400 µL of 4.5mM ammonium acetate before injection.

Results and Discussion

The DPX-CX extractions were readily performed using the GERSTEL MPS dual rail PrepStation. These DPX tips are ideal for basic drugs due to their mixed-mode cation exchange and reversed phase characteristics. The entire extraction pro-cess took approximately 5 minutes per sample. Because a basic eluent is used with the cation exchange sorbent, the eluents had to be solvent exchanged into the HPLC mobile phase. The extract was dried in about 4 minutes using low heat and nitrogen gas flow. All HPLC/MS spectra were collected using single reac-tion monitoring (SRM) because under the HPLC conditions used we were un-able to generate quality daughter ions for the opiate drugs using multiple reaction monitoring (MRM). Although SRM MS analysis may not provide the best sensiti-


vity for the analysis of these drugs at low concentrations, this study focused on the automated DPX extraction and the uti-lity of this automated sample prepara-tion for HPLC/MS analysis of opiates. A rapid resolution HPLC column was chosen to generate chromatographic data in less than 10 minutes.

Conclusion

Automated DPX extraction of opiates from biological specimens can be perfor-med successfully using the GERSTEL MPS dual rail PrepStation. In the work presented here, the total extraction time was 5 minutes, additionally 4 minutes were required for evaporation and solvent exchange. The total sample preparation

Extracted ion chromatogram of a DPX extract of whole blood spiked with 100 ppb of the opiate mix and with 400 ppb of the internal stan-dards (d3-oxymorphone and d3-oxycodone) in whole blood. Even when performing SRM MS analysis, the sensitivity is more than sufficient, demonstrating the high ionization efficiency of the electrospray system. Most importantly, no matrix effect or ion suppression was observed, showing that sample extraction and cleanup with DPX is well suited for the analysis. (1) d3-oxymorphone, (2) oxymorphone, (3) morphine, (4) hydromorphone, (5) d3-oxycodone, (6) oxycodone, (7) 6-MAM, (8) codeine, and (9) hydrocodone.

Overlay total ion LC/MS chromatograms of DPX extracts of a blank blood sample and of a matrix matched sample, both spiked at 0.5 ppm. The overlay shows that interferences are negligible

Recovery and % RSD for opiates (400 ppb) extracted from whole blood.

Extracted ion chromatogram of a DPX extract of whole blood spiked with 400 ppb of opiates. The chromatogram is free from interferences, the opi-ates were extracted reproducibly and with high recoveries. It is notewor-thy that this blood sample was only 0.25 mL. (1) d3-oxymorphone, (2) oxymorphone, (3) morphine, (4) hydromorphone, (5) d3-oxycodone, (6) oxycodone, (7) 6-MAM, (8) codeine, and (9) hydrocodone.

Recovery and %RSD for opiates (500 ppb) extracted from urine.

Analysis of urine spiked with 500 ppb of opiates. Again, extracts were free from interferences. No matrix effect or ion suppression for opiates was seen. (1) d3-oxymorphone, (2) oxymorphone, (3) morphine, (4) hydro-morphone, (5) d3- oxycodone, (6) oxycodone, (7) 6-MAM, (8) codeine, and (9) hydrocodone.

time was less than the chromatographic run time, which means that the next sam-ple can be prepared while separation of the current sample is in progress. When- ever the LC/MS/MS system has fi-nished a run, the next sample is ready to be introduced ensuring the highest pos-sible throughput. Additionally, “just in time” sample preparation helps to ensure that the prepared sample is not kept in the autosampler for a long time prior to being analyzed, reducing the risk of ana-lyte degradation and helping to maintain sample integrity. The DPX-CX tips work very well for extraction of opiates, reco-veries were in the range from 60 to 85 % with RSD’s below 6 %. Future work will focus on determining the lower limits of detection and quantitation using tandem

mass spectrometry with multiple reaction monitoring. Also, automated DPX com-bined with HPLC/MS/MS will be opti-mized for other drugs and metabolites.

Further information: GERSTEL AppNote 7/2009

Sparkle T. Ellison, William E. Brewer, Stephen L. MorganDepartment of Chemistry and Biochemistry, University of SouthCarolina, 631 Sumter Street, Columbia, SC 29208, USA

Fred D. FosterGERSTEL, Inc., 701 Digital Dr. Suite J,Linthicum, MD 21090, USA


Forensic science has contributed greatly to the exposure of forgeries: “Today we

are in a position to determine if handwrit-ten or printed text, numbers, or signatu-res are original or if they have been mani-pulated, if documents have been altered or if they have been forged entirely”, says Dr. Andreas Rippert of the Department of Forensic Sciences of the Zurich Can-tonal Police. The forensic chemist adds: “We can now also pinpoint the time when a document was forged, which could help us solve open cases.”

Pyrolysis GC/MS complicates the picture

Pyrolysis GC/MS is often used to exa-mine paper and documents. The sample is pyrolyzed under anaerobic conditions, i.e. under a flow of oxygen-free inert gas. While Pyrolysis GC/MS provides a lot of information, the technique does pose a problem, according to Dr. Rippert: “At-tempts to reveal the document’s mate-rial composition using pyrolysis GC/MS result in a large number of peaks, which could originate either from the ink or from the paper. In addition, the high temperatures used give rise to decompo-sition products that further complicate data interpretation.”

To expose forgeries, the Document Laboratory of the Zurich Cantonal Police successfully applies thermal desorption coupled with GC/MS.

The document laboratory of the Zurich Cantonal Police uses a GERSTEL Thermal Desorption System (TDS) in combination with a GERSTEL Cooled Injection System (CIS) and an Agilent Technologies GC 6890 / 5973 MSD. The system is used to differentiate between ink samples.

Using a TDS/CIS-GC/MS system, the document laboratory of the Zurich Cantonal Police is able to identify ink from ballpoint pens from different manufacturers. It can also be determined when a particular text has been written. Cut-outs of less than 5 mm diameter from the document are sufficient for analysis and conclusive findings.

During thermal desorption, the carrier gas sweeps across the paper sample at successively increased temperatures. During this process, all relevant volatile and semi-volatile compounds are desorbed and determined.

Thermal Desorption – the method of choice

“To reach a clear conclusion about the authenticity of a document, a GC intro-duction method is needed that provides the possibility of varying, i.e. program-ming the temperature over the course of the thermal desorption / thermal extrac-tion step. Organic compounds are extrac-ted from the sample in successive steps at different temperatures”, says Dr. Rolf Hofer of the Department of Forensic Sci-ences of the Zurich Cantonal Police. Dr. Hofer and his forensic expert colleagues used the GERSTEL Thermal Desorp-tion System (TDS) to develop this ana-lysis method. “As carrier gas sweeps across the paper sample at increasing tem-peratures, the relevant analytes ran-ging from volatile to semi-volatile, are successively desorbed and cryo-focused prior to introduction to the GC/MS system.”

Analysis and results Dr. Andreas Rippert: “At tempera-tures below 100 °C, volatiles are ex-tracted, especially phenol and ben-zene derivatives, as well as hydrocar-bons up to heptadecane.”

At temperatures above 100 °C less volatile compounds such as fatty

Hot in pursuit of forgers

Forgery

acids, phthalates, and higher-boiling hyd-rocarbons are extracted. The scientist explains: “If ink has been applied to the document during the past weeks, i.e. in case of ‘fresh tracks’, hydrocarbons are emitted in clearly detectable quantities, as are semi-volatile compounds like phen-oxyethanol and phenoxyethoxyethanol. At 210 °C, final residues of volatile sub-stances are desorbed even from older ink samples.” Dr. Rolf Hofer adds: “While the complete range of detected substan-ces is required for conclusive classifica-tion and differentiation of written mate-rial, phenoxyethanol and phenoxyethoxy-ethanol are the main indicators when it comes to age determination.”


GERSTEL MAESTRO software

• Stand-AloneoperationorintegratedintheAgilentChemStationorMassHunterSoftware

• OnesequencetableoperatestheentiresystemincludingLC/MSorGC/MS

• SamplePrepbyMouse-ClickusingthePrepBuilderfunctions

• Schedulerforeasyplanning

• PrepAhead/MultipleSampleOverlap:Automatedoverlappingofsamplepreparationandanalysisformaximumthroughput

• Prioritysamplescanbeaddedtothesystematanypointintheanalysissequence

• LOGfileandServiceLOGfilefunctionsensuretraceability

• AutomatedE-mailnotificationifthesequenceisstopped

• Controlofupto4systemsfromonePC

• Real-timemonitoringofallmodulesandparameters

• Interactiveon-linehelpfunction

Sample Prep by Mouse-Click

TheMultiPurposeSampler(MPS)isanautosamplerandsam-plepreparationrobotforGCandLC.Samplepreparationstepsareperformedduringtheanalysisoftheprecedingsampleforbestpossiblesystemutilizationandhighestsamplethrough-put.Samplepreparationstepsareperformed inacontrolledandhighlyaccurateandreproduciblemannerforbestpossibleresults.Everystepisselectedbymouse-clickfromapull-downmenuintheMAESTROsoftwareandaddedtotheoverallGC/MSorLC/MSmethod.Availablesamplepreptechniquesare:

• SolidPhaseExtraction(SPE)• DisposablePipetteExtraction(DPX)• Internalstandardaddition• Weighing,Sonication,Centrifugation• Derivatization• Extractionanddilution• Heating,conditioningandmixing• TwisterBackExtraction(TBE)• AutomatedLinerEXchange(ALEX)• AutomatedTwisterdesorptionandanalysis(SBSE)• SolidPhaseMicroExtraction(SPME)• SPMEMultiFibreExchanger(MFX)• ThermalDesorptionandThermalExtraction(TDS/TDU)• DynamicHeadspace(DHS)

• MultiColumnSwitching(MCS)

MAESTRO Software enables Sample Prep by Mouse-Click. All sample preparation steps are con-veniently and easily selected from a drop down menu and added to the method. Example:

ADD

Add solvent, internal standard or reagent

MOVEMove the vial or cartridge

MIXAgitate or stir and incubate the sample at a set temperature

INJECTIntroduce an aliquot of the sample to the GC or LC system

Automated Sample Preparation by Mouse-Click (Part I)

Next generation software for automated sample preparation and sample introduction. MAESTRO optimizes performance and throughput of GERSTEL modules

and systems.

GERSTEL GmbH & Co. KGEberhard-Gerstel-Platz 145473 Mülheim an der RuhrGermany

+49 208 - 7 65 03-0+49 208 - 7 65 03 33

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GERSTEL GmbH & Co. KGEberhard-Gerstel-Platz 145473 Mülheim an der RuhrGermany

+49 208 - 7 65 03-0+49 208 - 7 65 03 33

[email protected]

GERSTEL, Inc.Caton Research Center1510 Caton Center Drive,Suite HBaltimore, MD 21227USA

+1 410 - 247 5885+1 410 - 247 5887

[email protected]

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

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

GERSTEL K.K.2-13-18 Nakane, Meguro-ku152-0031 TokyoDai-Hyaku Seimei ToritsudaiEkimae Bldg 2FJapan

+81 3 57 31 53 21+81 3 57 31 53 22

[email protected]

GERSTEL AGEnterpriseSurentalstrasse 106210 SurseeSwitzerland

+41 41 - 9 21 97 23+41 41 - 9 21 97 25

[email protected]

As us how GERSTEL technology can benefit you

Ultrasonic Bath improved sample preparation

Bar Code Reader tracking and ID verification of samples and extracts

Heated Agitator highly controlled reactions such as analyte derivatization

Weighing Option automated weighing of liquids and liquid additions

Centrifuge separation of matrix and extract And more … You name it!

Intelligent Automated Sample Preparation for

LC/MS and GC/MS (Part II)

www.gerstel.com

Additional techniques, now available from the leader in automated sample preparation:

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