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News from GERSTEL GmbH & Co. KG · Eberhard-Gerstel-Platz 1 · D-45473 Mülheim an der Ruhr · Germany · Phone + 49 2 08 - 7 65 03-0 · Fax + 49 2 08 - 7 65 03 33 Forensic toxicology Drugs of abuseExtraction in Blood alcohol analysisAutomated Static Headspace as the method of choice ForgeryHot in pursuit Forensic Sciences and Toxicology Drugs of abuse: Extraction in seconds Blood alcohol I: Automated Headspace Analysis – the method of choice Blood alcohol II: Ethanol determination in biological samples by dual rail MPS 7 Extraction in seconds SPE: Poison Analysis EZ DPX: Doing Drugs? Forgery: Hot in pursuit of forgers Automated Sample Preparation – In Forensic Science and in Toxicology, body fluids are regularly analyzed for residues of drugs of abuse, therapeutic drugs and of their metaboli- Automated Sample Preparation II – 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. GERSTEL online Information on liam (Bil ) E. Brewer, Ph.D. from the Uni- products, applications, events and Body fluids represent a complex and downloads, general information heterogeneous matrix. Accurate versity of Southern Carolina. Professor about GERSTEL and customer focused determination of drugs, pharmaceuti- Brewer is the Owner-President of DPX solutions: cals and metabolites in blood and urine Labs ( requires both a suitable chromatographic The DPX technique has now been auto- system and adequate sample preparation. mated by GERSTEL, a leader in automa- Sometimes more than one extraction tion of sample preparation and sample int- technique is needed for a successful result. roduction for GC/MS and LC/MS. Solid Phase Extraction (SPE) is among "The reactions we got from the foren- the most widely used sample clean-up and sic scientists at the SOFT 2008 meeting", analyte extraction techniques in forensic says Ro bert J. Collins, Ph.D., President of GERSTEL GmbH & Co. KG and toxicology laboratories. GERSTEL, Inc. in Baltimore, MD, "lead Eberhard-Gerstel-Platz 1 Traditional y, SPE requires the use of us to believe that automated DPX is seen 45473 Mülheim an der Ruhr, Germany significant quantities of solvent, some of by experts as a very promising alternative Editorial Director which are toxic. Fol owing several labor to standard extraction techniques". intensive steps, many methods require In order to provide an efficient solu- that the solvent be evaporated in order to tion for the determination of drugs and concentrate the analytes of interest and metabolites in blood in a routine labora- achieve the necessary detection limits. tory environment, GERSTEL and DPX Translation and editing Depending on the chemical properties Labs LLC col aborated on automating of the analytes, a chromatographic deter- the DPX technique. "DPX immedia- mination may also require further sample tely struck us as the right solution", says Scientific advisory board preparation steps such as derivatization. Bob Collins. Furthermore, for this appli- Eike Kleine-Benne, Ph.D. The sum total of sample preparation steps cation, samples should be prepared and can amount to a significant bottleneck for derivatized just prior to analysis. Just in Oliver Lerch, Ph.D. laboratory productivity and a risk to occu- time sample preparation eliminates ana- pational health unless adequate and costly lyte degradation and under-reporting of Malte Reimold, Ph.D.
safety precautions are taken. concentration levels since no sample is If the tedious and labor intensive steps waiting for an extended period of time can be eliminated, the overal task can in the autosampler before being analyzed. be performed more efficiently and faster Also, in order to optimize GC/MS sys- while producing accurate results and using tem utilization and sample throughput, a Paura Design, Hagen, Germany 1619-0076 · 10 / 2009 only a fraction of the amount of solvent sample should be ready for introduction normally used. All this is possible thanks every time the GC/MS finishes its run to Disposable Pipette Extraction (DPX), and becomes ready for the next sample. a technique developed by Professor Wil- In summary, performing GC/MS analy- GERSTEL Solutions worldwide Forensic Special ture was centrifuged and the supernatant Automated Disposable Pipette Ext- transferred to a clean label ed test tube raction (DPX) is subsequently perfor- containing 0.1 mL of 0.1 M HCl. med on the prepared samples using the Preparing urine samples for the MultiPurpose Sampler (MPS) equipped determination of benzodiazepines: with 1 mL CX tips from DPX Labs (www.
In order to determine the total As the name suggests, CX level of free, bound and meta- tips contain a novel and unique cation bolized residues of drugs and exchange material with additional slightly pharmaceuticals in urine, apolar cha racteristics. The DPX process hydrolysis must be per- is completely automated: 250 µL of a 30 formed of the respective % solution of acetonitrile in water is dis- conjugates, such as for pensed onto the DPX sorbent inside the example glucuronides of tip for conditioning. The conditioning sol- benzodiazepines, which vent is subsequently discarded to waste. are metabolites of the The DPX tip is then immersed into the drugs that are formed to sample and a defined volume is aspirated facilitate excretion of the into the tip. Air is then aspirated into the substances from the body. tip, causing turbulent mixing and efficient The hydrolysis reaction extraction of analytes into the sorbent. Fol- is started by add ing 10 µL lowing a 30 second equilibration time, in of a solution of the enzyme which the sorbent is allowed to settle, the b-glucuronidase and 50 µL extracted sample is discarded to waste. The of a 0.1 M sodium phosphate sorbent is rinsed twice, first with 0.5 mL buffer with pH 4 to a 0.2 mL of a 30 % solution of Acetonitrile in water sample of urine. The mixture is and then with 0.5 mL acetonitrile. The ex- kept at 55 °C for two hours before tracted analytes are eluted using 0.7 mL being al owed to cool to room tempera- of a solution consisting of 2 % concen- ture. Acetonitrile (0.25 mL) is then added trated ammonia, 78 % CH2Cl2 and 20 % in order to preci pitate the enzyme. Fol- lowing centrifugation, the supernatant is The eluate was dispensed directly into transferred to a clean, label ed test tube an autosampler vial. The total amount of and 200 µL of 0.1M HCl is added. The time required for extraction and liquid prepared samples are then placed in the handling was less than 6 minutes per MPS sample tray. sis and sample preparation carefully syn- chronized and in paral el benefits both the quality of results and the throughput. The Automated DPX process 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 All steps are performed automati- cally by the MPS.
In order to test the MPS-DPX-CIS-GC/ MS system in practice, the scientists ana- If needed, the sorbent is conditioned lyzed blood and urine samples that had with solvent prior to the extraction been spiked with different drugs and pharmaceuticals. Compounds determi- ned included amphetamines, benzodiaze- 1 Sample is drawn into the pipette tip for direct contact pines, cocaine and methadone as wel as with the solid phase sorbent. tetrahydrocannabinol (THC) and meta- There is no contact between bolites. For details, please see the graphic the sample and the syringe representations on this page. The analy- used to aspirate the sample and therefore no risk of cross 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 2 Air is drawn into the pipette tip from below through the frit. ppm) and d5-OH-Alprazolam (0.2 ppm) Turbulent air bubble mixing were used. For the opiates, equivalent deu- creates a suspension of sor- 4 Extracted analytes are eluted using a suitable bent in the sample, ensuring solvent, which is added from above for most terated compounds were used (each at a optimal contact, highly efficient efficient elution. The eluate is collected in a vial level of 0.1 ppm).
extraction, and high recovery.
for subsequent sample introduction to LC/MS Required manual sample The extracted sample is preparation steps discharged, typically after 30 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- Preparing blood samples: 0.5 mL of ace- If needed, the sorbent can be nation can be performed in parallel for best possible tonitrile was added to a 0.25 mL sample washed to remove unwanted residue.
throughput and system utilization.
of whole blood followed by mixing to pre- cipitate proteins in the sample. The mix- GERSTEL Solutions worldwide Forensic Special 50 µL of ethyl acetate were added and the mixture kept at 70 °C for The analysis was performed on a 20 minutes. When the extract 6890N/5975 (inert XL) GC/MS had cooled off, 50 µL of the solu- system from Agilent Technolo- tion was introduced to the CIS gies. The GC was fitted with a inlet using the Large Volume Cooled Injection System (CIS 4) Injection (LVI) technique. PTV-type inlet. An MPS Prep- Station with DPX option and The conclusion reached by THC and metabolites in blood: Total ion chromatogram of 10 ng/mL THC and metabolites extracted from 0.5 mL whole blood fol owing MAESTRO Software control protein precipitation, centrifugation and DPX-RP. Derivatization was used for automated sample was performed in the CIS inlet by injecting 50 µL of DPX eluent preparation and sample intro- "As we had expected, the ana- together with 20 µL of 50/50 BSTFA/acetonitrile. No additional duction. The complete system lysis based on automated DPX solvent evaporation or derivatization step was performed. Analytes: including GC/MS was operated delivers excel ent results", said 1) THC-TMS, 2) OH-THC-TMS, 3) COOH-THC-2TMS using one integrated method Bob Collins. Even though all and one sequence table directly analyses were performed on from the Agilent Technologies very smal sample volumes (250 ChemStation Software, operated µL blood or 200 µL urine), the integrated with the MAESTRO resulting peak intensities were highly satisfactory – even in full scan mode. The MultiPurpose Sampler (MPS) with automa- ted DPX enables fast sample Some analytes must be deriva- preparation of difficult samples tized to enable GC/MS deter- while delivering high sensitivity mination. "The Cooled Injection and accurate results. The additi- System (CIS) inlet offers an inert onal liquid handling capabilities and temperature programmable of the dual rail MPS PrepSta- environment", says Prof. Brewer, tion enabled ful automation of "which is highly suited to evapo- al the required liquid handling rating and purging excess solvent steps such as derivatization and while simultaneously, or at least addition of an internal standard. Benzodiazepines in urine: Total ion chromatogram of 0.2 ppm sequential y, performing deriva- This added level of automation benzodiazepines in 0.2 mL urine fol owing enzymatic hydrolysis tization of analytes." provided best possible produc- and DPX. Derivatization was performed in the CIS inlet by inject- For the derivatization of tivity and throughput. The ins- ing 50 µL of DPX eluent together with 20 µL of 50/50 MTBSTFA/ benzodiaze pines, 20 µL of 50 trument combination used pro- acetonitrile. No separate solvent evaporation step was performed. % N- (t-butyldimethylsilyl)- ved to be especially useful for Increasing the sample volume to 0.5 mL and performing multiple the determination of basic drugs DPX extractions would increase the sensitivity. 1) Diazepam, 2) (MTBSTFA) in acetonitrile such as cocaine, methadone, PCP, Nordiazepam-d5-TBDMS, 3) Nordiazepam-TBDMS, 4) Fluni- was aspirated into the autosam- TCAs and Meperidine. trazepam, 5) 7-aminoflunitrazepam, 6) Oxazepam-2TBDMS, 7) pler syringe fol owed by 20 µL Most benzodiazepines Temazepam-TBDMS, 8) Nitrazepam, 9) Lorazepam-2TBDMS, 10) of air and 50 µL of the DPX were easy to determine using Clonazepam-TBDMS, 11) Alprazolam, 12) a-OH-Alprazolam-d5- eluate. "The resulting ‘Sand- MTBSTFA derivatization in TBDMS, 13) a-OH-Alprazolam-TBDMS wich' injection was performed the GC inlet. The following were slowly, using a programmed stop determined: Diazepam, Nordia- flow method to ensure that the zepam, Oxazepam, Temazepam, solvent was completely remo- Alprazolam and a-OH-alpra- ved through the split vent prior zolam. "DPX combined with to the derivatization step", the GC/MS determination provi- application specialist explains. ded excel ent results for the 11 The CIS temperature quickly listed benzodiazepines in urine", ramped to 300 °C, which star- Bob Col ins notes, "plus we got ted the derivatization process good recovery and great sensi- Methamphetamine 14.4 % and helped transfer the deriva- tivity for the opiates. For most tized analytes to the GC column opiates, we achieved limits of in splitless mode for highest pos- determination under 1 ng/mL sible recovery and lowest limits of in whole blood". Ralf Bremer, General Manager for produc- "Automated analyte deriva- tion and R&D, is thril ed about tization in the GC inlet proved the use of the CIS 4, PTV-type to be both simple and highly inlet for evaporative concentra- practical", said Prof. Brewer. "The tion and analyte derivatization: Drugs of abuse in blood: Total ion chromatogram of a DPX extract "This year, we are celebrating the of 250 µL whole blood spiked at 0.5 ppm with drugs of abuse method was successful y applied using d5-PCP as internal standard. Chromatograms from the 5th to the determination of benzo- 25th anniversary of the introduc- and 20th injections are shown alongside each other to demonstrate diazepines in blood. Compounds tion of the first GERSTEL CIS. the ruggedness of the analysis. The insert shows the extracted that were not successfully deriva- It is very reassuring to see that the ion chromatogram (EIC) from the 5th injection. The sample was tized in this way were derivatized improvements we have regularly protein precipitated with 0.5 mL acetonitrile, and the supernatant directly in the sample vial". For engineered into the CIS over the was transferred to clean tubes. After adding 0.1 mL of 0.1 M HCl, this approach, the DPX eluate years enable us to stay well ahead automated DPX was performed. 1) Meperidine, 2) d5-PCP (ion was evaporated to dryness under of the competition". 205), 3) PCP, 4) Methadone, 5) Methaqualone, 6) Amitriptyline, a flow of nitrogen in the sample 7) Cocaine, 8) cis-Doxepin, 9) Imipramine, 10) trans-Doxepin, 11) Desipramine, 12) Pentazocine, 13) Codeine (Septum bleed from vial. 50 µL of MTBSTFA and vial cap after repeat injections from the same vial).
GERSTEL Solutions worldwide Forensic Special Automated Headspace Analysis – the method of choice Forensic laboratories face the need to analyze a large number of samples Edward A. Pfannkoch, of human blood and body fluids for alcohol content. When faced with and Jacqueline A. Whitecavagethis challenge factors that need to be considered are sample throughput, Gerstel, Inc., 701 Digital Drive, Suite J, resolution, and carryover. Linthicum, MD 21090, USA A successful method for these analyses that meets the specifications set by the Preparation of standards should be fast, precise, and accurate. California Department of Justice Blood • Secondary standard (SS). 0.25 mL Current methods used in these analyses Alcohol Operating Procedures (Title of absolute (200 proof) ethanol and use a gas chromatograp coupled to a static 17) [2]. We also configured and tested a 0.125 mL of n-propanol pipetted into headspace sampler and flame ionization separate dual-column/dual-FID system a 100mL volumetric flask and diluted detector (FID). The x, y, z robotic auto- that adds confirmation because of the with bottled water.
sampler used in this study has a capacity different elution order of ethanol on the • Quality control standard (QC). 0.15 of up to 128 headspace samples, which is two columns.
mL of absolute (200 proof) ethanol and a distinct advantage compared to other 0.125 mL of n-propanol pipetted into samplers commercially available Results Experimental a 100 mL volumetric flask and diluted obtained with the instrument and me- with bottled water.
thodology described in this report meet Instrumentation • Resolution standard (RS). 0.25 mL of the specifications set by the California Analyses were performed on a 6890 GC absolute (200 proof) ethanol, 0.1 mL Department of Justice Blood Alcohol equipped with single or dual FID (Agi- methanol, 0.1 mL isopropanol, 0.01 Operating Procedures (Title 17). A dual- lent Technologies), and a GERSTEL mL acetone and 0.125 mL n-propanol column, dual-FID blood alcohol analysis MPS 2 MultiPurpose sampler configured pipetted into a 100 mL volumetric flask system that can be used for confirmation for static headspace injection.
and diluted with bottled water.
of ethanol peaks was also tested and pro- • Blank standard. 0.125 mL of n-propa- duced results with good precision (below Reagents nol pipetted into a 100 mL volumet- • Ethyl alcohol, absolute, 200 proof, ric flask and diluted with bottled water. 99.5%, A.C.S. reagent grade All standards above were diluted 1:6 in • Methyl alcohol, 99.8%, A.C.S. reagent bottled water prior to use. 500 µL of standard was then pipetted into a 20 Headspace gas chromatography (HS- • Acetone 99.5%, A.C.S. reagent grade mL headspace vial. 1 mL of 1000 µg/ GC) for determination of ethanol content • n-Propanol (1-propanol) 99.5% A.C.S. mL internal standard (n-propanol) and of blood is widely used by forensic labs reagentgrade (IS) 1 mL of the blood alcohol mix resolu- to test automobile drivers charged with • Isopropanol (2-propanol), 99.5%, tion control standard (Restek, # 36256, DUI (driving under the influence). The A.C.S. reagent grade Lot# A034323) was diluted in 18 mL method originates from 1964 when G. • Blood alcohol mix resolution control bottled water. 4 mL of standard was Machata [1] published the first use of standard (Restek, # 36256). 0.100 g/dL then pipetted into a 20 mL headspace HS-GC for quantitative analysis. The in water of 8 compounds: acetaldehyde, vial. All vials were crimp-capped using method includes the use of an internal acetone, acetonitrile, ethanol, ethyl ace- blue silicone/PTFE septa.
standard (IS) compound. Tert-butanol tate, isopropanol, methanol and methyl or n-propanol may be used as internal ethyl ketone (MEK). 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 GERSTEL Solutions worldwide Forensic Special GC 6890 (Agilent Technologies) Split/splitless, 100°C 30 m DB-ALC2 (Agilent) di = 0,53 mm, df = 2,0 µm MultiPurpose Sampler (MPS) 1 mL (500 µL/s) Table 1. Method parameters for California com- Table 2. Example of calculation and check of K factor. GC 6890 (Agilent Technologies) Average peak area Standard deviation Split/splitless, 150 °C 30 m DB-ALC1 (Agilent) di = 0,32 mm, df = 1,8 µm MultiPurpose Sampler (MPS) 1 mL (500 µL/s) Table 3. Method parameters for dual-column Table 4. Precision of 12 replicas using dual-column configuration.
dual-FID system. Quality control criteria Results and Discussion minutes (Figure 3). The updated chro- for California compliance matographic conditions include the use California DOJ Blood Alcohol Opera- of a capil ary GC column instead of the • Calibration runs consist of 6 secondary ting Procedure (Title 17). The instru- packed column currently used for these standards fol owed by a resolution stan- ment used in this study was a GERS- analyses in California.
dard. The calibration constant K is then TEL MPS autosampler that can be pro- calculated for each of the 6 secondary grammed to be used with headspace sy- standards and the mean is calculated. ringes. For this study we used a 2.5 mL The value of the K constant for each of syringe that can also be programmed to Blood Alcohol Dual-Column Con- the six determinations must fal within inject different volumes (recommen ded firmation Method. We configured a ± 1.5 % of the mean value.
volumes from 0.25 mL up to 2.5 mL). The system with dual complimentary alcohol • The results for the resolution standard headspace syringe adaptor is heated and columns from a single inlet and dual FIDs must show a resolution of 0.01 % ace- can be controlled to optimize the syrin- for blood alcohol analysis [3]. The dual tone in the presence of 0.20 % ethanol. ge temperature. This results in great time system has an advantage since the order • Analysis runs consist of a Blank (water, savings and excellent sample throughput. of elution is different for each column, no IS) and standards (SS, QC and RS) For this analysis we selected an incuba- enabling confirmation of the peak iden- fol owed by the sample set (2 replicas tion temperature of 65 ºC and a syringe tification. In order to verify the precision per sample) fol owed by two additional temperature of 70 ºC. It is recommended of the splitter (Figure 4) we instal ed standards (QC and SS).
to use a slightly higher syringe tempe- two identical columns and checked the • The result of the blank sample should be rature to avoid condensation. Secondary response of the secondary standard on less than 0.01 %.
and resolution standards were analyzed.
both columns.
chromatogram is Conclusions gure 2. It can be • The GERSTEL MPS 2 robotic auto- seen that there is sampler is capable of delivering perfor- no ethanol carry- mance for blood alcohol analysis that over in the blank meets or exceeds the California Title 17 and the IS repro- Forensic Alcohol Analysis and Breath duces wel . Using Alcohol Analysis performance criteria.
the gas chromato- • Testing during a 3-month period show- graph in the iso- ed good robustness and reproducibility.
thermal mode, we • This instrumentation provides increased were able to sepa- sample throughput by accommodating rate the alcohols up to 128 samples and by using the present in the SS "prep ahead" function to equilibrate and also the com- multiple samples simultaneously.
pounds present • The GERSTEL MPS 2 autosampler in the resolution performed well when used with a dual column blood alcohol confirmation Figure 2. FID overlay of Blank/Internal Standard (IS), Resolution Standard and Secondary Standard (SS) for California Compliance.
GERSTEL Solutions worldwide Forensic Special analysis using the Scientists from the Federal Bureau of Figure 3. FID trace of Resolution Standard for California Compliance.
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 Figure 4. Dual-FID traces of Secondary Standard (SS) using two identical is used for sample preparation and the other rail is columns DB-ALC 2.
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.
Figure 5. Dual-FID traces of Restek (#36256) Resolution Control Standard J. Chromatogr. B 850 (2007) 230–235 and Internal Standard (IS).
GERSTEL Solutions worldwide Forensic Special When the sample matrix no longer matters .
Poison analysis EZ Application specialists from TeLA GmbH have developed a new me-thod that dramatically simplifies LC/MS determination of pesticide le- Fully automated Sample clean-up and vels, providing high-quality results independent of the sample mat- Pesticide Screening with Agilent 6410 rix type and complexity. LC/MS QQQ online SPE System, Agi- lent ordering Number: 5990-3866EN Norbert Hel e, Ph.D. and Meike Baden, TeLa GmbH Bremen, GermanyPesticides, fungicides and herbici- seen from this description, there is some ble to those reached using the S19 method. des are needed in order to provide overlap between the techniques. Recently The QuEChERS method is much faster, an adequate supply of food to the a new multi-residue method for the deter- requires much less sample preparation, ever-growing human population across mination of pesticide levels in fruits and covers a wider range of analytes and is the world. The other side of the coin is vegetables was presented (QuEChERS: more readily automated. In addition, much that residues of these types of compounds Quick, Easy, Cheap, Effective, Rugged & smal er volumes of partly toxic organic sol- in foods cannot be allowed to endanger or Safe) [*]. Compared to previous methods, vents are required, compared with other affect consumer health. the QuEChERS sample preparation steps currently used methods for determining World-wide, around 700 pestici- are much less time-consuming, enabling pesticides in fruits and vegetables. In addi- des are in use, very few of which can be the preparation of 8 samples in less than tion to the financial benefits of a much legal y used throughout Europe. Various 30 minutes. QuEChERS is a sample pre- higher laboratory throughput, the cost of compounds classes have been established, paration method well suited for both GC, materials at around one Euro per sample but even these can cover a wide range of GC/MS and LC/MS analysis. The QuE- is relatively low.
polarities, making it difficult to develop ChERS sample preparation steps are lis- The limits of QuEChERS are encoun- a fast all encompassing ana lysis method. ted below. tered whenever samples with more com- Stil , effective multi-residue methods The main benefit of this sample prepa- plex matrices need to be analyzed, such as are in use for the determination of pesti- ration method is that the overall analysis is garlic, onion, artichoke or avocado with cides, helping to ensure food safety or to less time-consuming and less error-prone much higher fat content. This can lead determination the cause in case pesticides than more traditional approaches. Unfor- to problems with interferences, than can have been used as poison. tunately, extracts obtained following this especially influence quantification unless procedure often have a high matrix con- further clean-up steps are performed.
Tracking down pesticides using tent, which causes chromatographic prob- To enable reliable and rugged analysis lems for GC analysis due to residue build- independent of the sample matrix, we up in the liner unless an automated liner looked for a similarly effective alterna- Classical pesticide analysis relied on gas exchange system such as the GERSTEL tive sample preparation procedure. We chromatography (GC) using an electron ALEX is used. (Cf.: GERSTEL Solutions found that automated solid phase extrac- capture detector (ECD) or a nitrogen Worldwide Magazine No. 5 p. 18) (http:// tion (SPE) based on the GERSTEL Mul- phosphorous detector (NPD). The most tiPurpose Sampler (MPS) provided an widely used detector today is the mass excellent solution. The GERSTEL SPE, selective detector (MSD). we have previously used successfully for a In Germany, the analytes that are number of applications, including aflato- mainly in focus are those listed in the DFG xins, chloramphenicol and malachite green S19 method, a multi-residue method for in foods. In summary, we can report that the determination of pesticides in food, Weigh 10 g of sample
our automated SPE-LC-MS/MS-ESI which enjoys Europe-wide recognition. multi-residue method reduces the num- The analysis of the 270 compounds listed –> Add 10 ml of Acetonitrile (AcN) ber of manual steps required to a mini- in the S19 method does, however, require Shake vigorously 1 min
mum while increasing laboratory through- significant sample preparation including a –> Add 4 g MgSO4 and 1 gNaCl put. The results are solid and reproducible gel chromatography clean-up step to sepa- Shake vigorously 1 min
combined with high sensitivity and good rate analytes from the matrix. –> Add internal standard solution limits of determination. Different analysis techniques are used Shake 30 sec and centrifuge
for different types of pesticides. Liquid –> Take Aliquot of supernatant Intrumental requirements chromatography (LC) combined with –> Add MgSO4 and sorbent Shake 30 sec and centrifuge
a mass selective detector (MS) is used The GERSTEL SPE was fitted with an to determine polar to moderately apolar –> Take Aliquot of supernatant –> inject to GC-MS and LC-MS injection valve; sample introduction to the compounds. Gas chromatography (GC), Agilent LC 1200 was performed directly most often in combination with a mass by the SPE system; detection was perfor- selective detector (MSD) covers apolar to The results obtained using QuE- med using an Agilent 6410 MS/MS Triple moderately polar compounds. As can be ChERS sample preparation are compara- Quad instrument. GERSTEL Solutions worldwide Forensic Special Sample Preparation: 15 mL of an aceto-
The settings for the ion source were opti- Raw sample extracts were automati- nitrile/water mixture (80:20) was added mized for the flow and eluent used. The cally loaded onto standard SPE cartridges to a five gram sample of fruit or vegetable following parameters were used: N2 tem- and cleaned. A new cartridge was used for for extraction. The SPE cartridge (M&N perature: 340 °C; carrier gas flow (N2): every sample to eliminate cross-contami- C-18ec, 6 mL, 1 g) was conditioned using 9 L/min; nebulizer pressure: 30 psi. The nation. Macherey-Nagel cartridges con- 10 mL methanol (MeOH) and 10 mL triple quadrupole instrument was opera- taining C18 reversed phase material were water. All steps in the sample preparation ted in MRM mode, with 5 different time found to produce excel ent, reliable results. procedure, including sample introduction segments, monitoring two transitions for Automated SPE clean-up as described were fully automated. each pesticide. In each segment 40 to 50 in this article took around 20 minutes to 5 mL sample was added to the cart- analytes were monitored. complete. Apart from the first sample, the ridge, which was subsequently rinsed with SPE process was performed during LC/ 5 mL water. Analytes were then eluted The proof of the pudding MS or GC/MS analysis of the preceding using an acetonitrile/water mixture added sample, ensuring that the SPE step was at a flow rate of 600µL/min. In contrast to When using the QuEChERS method, it is performed without increasing the overall most manual SPE methods, the liquid is necessary to adapt the clean-up steps to the analysis time. Once the first sample had not aspirated through the cartridge under sample at hand. It has been clearly shown been prepared for analysis, the LC/MS or vacuum, rather it is added under positive that for "uncomplicated" matrices, such as GC/MS system never had to wait idly for pressure using a syringe. This means that lettuce or cucumber, additional clean-up the next sample. flows, and therefore also the elution speed, steps are not required following the aceto- An LC 1200 Rapid Resolution HPLC are accurately controlled and results more nitrile/water extraction. For complex mat- system from Agilent Technologies was reproducible. This holds true even when rices that contain fat and other chal enging used for the analysis. In order to achieve sample matrix changes the restriction matrix components, further clean-up steps good separation combined with method across the cartridge. The eluate was con- are of course needed. For this purpose we ruggedness, the conscious decision was centrated for six minutes at 50 °C and the used the GERSTEL SPE system. made to only seek a moderate reduction 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 fol owing 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 Calibration curves for that the Multimode source provided sig- nine pesticides, deter- nificantly lower detection limits for some mined using the TeLA pesticides than the ESI source. For other GmbH SPE-LC-MS/MS compounds, however, a lower response was pesticide multi-residue obtained than with the ESI ion source. GERSTEL Solutions worldwide Forensic Special Overlay medium polarity sections of 8 different chromatograms: 8 sepa- rate sample preparations and injections of a bell of the analysis time. The total analysis time pepper sample spiked required to determine around 140 com- with a standard mixture pounds was in the order of 35 minutes. of pesticides, 100 ng/mL each. The peaks shown This time period was more than sufficient are for the pesticides to prepare the following sample for just- Terbutylazin, Cyprodinil, in-time sample introduction to the LC/ Prochloraz, Flusilazol and Fenoxycarb, all showing Sample clean-up using SPE contri- good reproducibility. butes not only to the ruggedness of the method, it also improves reproducibility and linearity, among other things. To il us- trate this, a bell pepper sample was spiked Determination of polar and apolar pesticides with a pesticide mixture and analyzed. Fol- respectively in orange oil. lowing SPE clean-up, retention times and Overlay chromatograms peak areas of the analytes showed excel ent covering 9 different con- reproducibility. The linearity was excel ent, centrations are shown.
both for polar compounds like Carbenda- zim and Thiabendazole as well as for apo- lar pesticides like Diazinon and Pirimi- Orange oil samples were cleaned up using a slightly modified SPE method. The efficiency of SPE clean-up is il us- trated by the fact that the intense yel ow 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.
GERSTEL LC/MS Effluent Optimizer (LEO) One final comment: Every method must prove its worth in practice. The test, as Optimized LC separation and MS detection – always, is in the analysis of real world sam- ples. To prove the validity of our method, get the best of both worlds we took part in a Europe-wide round robin with 46 participating laboratories. A vege- In LC/MS, we work towards the mu- table sample (zucchini) had to be analyzed mizer (LEO) Optimized LC separation for 185 different pesticide residues. Out of tually irreconcilable goals of achieving and MS detection – get the best of both 46 laboratories, TeLA GmbH was among the perfect LC separation and com- worlds to the LC effluent and/or chan- the 12 that managed to correctly identify bining it with the most efficient ioniza- ging its pH. The LEO module is quickly and quantify the analytes thus meeting tion and lowest achievable MS detec- and easily instal ed in your LC/MS sys- the round robin requirements and pas- tion limits for our analytes. The LC sepa- tem. A solvent mixture, buffer solution sing the test.
ration may require a certain pH and or reagent is then easily added to the 128 of the 185 pesticides were deter- polarity range of the eluent, while ana- effluent ensuring that the LC separa- mined using our SPE-LC-MS/MS pesti- cide multi-residue method. 90 of the 185 lyte ionization in the LC/MS ionization tion can be performed under optimal pesticides were determined using a GC/ source requires yet another pH, a dif- conditions while also enabling maxi- MS system (GC 6890 / MSD 5973, both ferent buffer – or even derivatization mum yield in the MS ionization process. from Agilent Technologies) in combina- of the analyte for best possible effici- Whether you are looking to perform pH tion with the GERSTEL MultiPurpose ency or optimized spectral information. adjustment or post-column derivatiza- Sampler (MPS) using a Retention Time How to optimize both? Wel the logical tion, for method development or rou- Locking (RTL) method.
answer is to take the effluent from the tine analysis, when you use LEO and the perfect LC separation and then optimize GERSTEL MAESTRO software you can *] M. Anastassiades, S. Lehotay, D. Stajnbaher and F. it for MS analysis. This task is easily pos- easily and efficiently control all para- Schenck: Fast and easy multiresidue method emplo- sible when you add the GERSTEL LC/ meters as part of the overall method. ying acetonitrile extraction/partitioning and "disper- sive solid-phase extraction" for the determination of MS Effluent Optimizer (LEO) module Just one sequence table controls the pesticide residues in produce. J AOAC Int 86 (2) to your LC/MS/MS system. Application entire system from sample preparation (2003) 412-31.
examples show sensitivity gains of up through LC separation and effluent opti- to a factor of 40 by simply adding a salt mization to MS analysis. It is al done at solution GERSTEL LC/MS Effluent Opti- the click of a mouse.
GERSTEL Solutions worldwide Forensic Special 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.
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 LC
Mobile Phase: A - 4.5 mM
Ammonium acetate 2.1 mm x 30 mm, 3.5 µm, Eclipse XDB C18 (Agilent) Inj. volume: 10 µL Automated Sample Preparation Analysis conditions MS
Positive ion mode, Single reaction
monitoringRun time: Comprehensive Analysis of Drugs of Abuse in Blood and Urine with Desolvation Temp.: 391 °C Automated Disposable Pipette Extraction and HPLC/MS/MS IIn order to analyze biological spe- rapid, minimal solvent waste is genera- Compound
M + H Dwell Cone
cimens for drugs and their metabolites, ted, and the entire process can be ful y Time Voltage
it is necessary to perform sample prepa- automated including introduction of the [m/z] [ms]
ration to eliminate matrix interference. extract to the chromatographic system. Solid-phase extraction is general y the The GERSTEL MPS autosampler per- d3-Oxymorphone 305 preferred sample preparation technique, forms DPX extractions in approximately Oxymorphone 302 in this study Disposable Pipette Extrac- 5 minutes using reversed phase (DPX- tion (DPX) was utilized. DPX is a novel RP) or cation exchange (DPX-CX) sor- Hydromorphone 286 dispersive solid-phase extraction tech- bent material. For chemical analysis of d3-Oxycodone 319 nique that uses loosely contained sorbent target drugs, GC/MS or HPLC/MS/MS Oxycodone in a disposable pipette tip. The sample is are general y the preferred techniques. 6-MAM aspirated into the tip where it is actively The advantage of LC/MS/MS is that mixed with the sorbent and forms a sus- chemical derivatization of the analytes is pension. The main advantages of the DPX not required, making sample preparation Hydrocodone technology are that the extraction is very simpler and less time consuming. In addi- GERSTEL Solutions worldwide Forensic Special The supernatant was decanted into a 30 s equilibration time to allow analyte a clean labeled sample tube. 100 binding, the resulting solution was dis- µL of 0.1 M HCl was added to pensed to waste. To wash off excess mat- the solution, and the sample rix, a 500 µL wash of 10% acetonitrile/ tube was placed on the MPS water was added to the sorbent material 2 sample tray for automated through the top of the DPX tip and dis- pensed to waste followed by an additional wash using 500 µL of acetone. For elution Urine sample preparation. of the analytes, 700 µL of 78/20/2 (v/v) of 50 µL of 0.6 M sodium ace- tate buffer (pH = 4) and 10 nium hydroxide was added to the sorbent µL of ß-glucuronidase was material through the top of the DPX tip added to a 200 µL sample of and dispensed directly into a clean HPLC urine. The solution was thermo- vial. All eluents were dried and reconsti- stated at 70°C for 2 hours, and tuted with 100 µL of methanol and 400 then cooled to room temperature. µL of 4.5mM ammonium acetate before To precipitate proteins, 250 µL of injection. acetonitrile was added to the hydro- lyzed urine and the sample was vortex Results and Discussion mixed and centrifuged. The supernatant was decanted into a clean labeled sample The DPX-CX extractions were readily tube. 200 µL of 0.1 M HCl was added to performed using the GERSTEL MPS Sample preparation
the sample solution, and the sample tube dual rail PrepStation. These DPX tips are was placed on the MPS sample tray for ideal for basic drugs due to their mixed- All opiate standards were obtained from automated DPX extraction. mode cation exchange and reversed phase Cerilliant (Round Rock, TX). A 10 ppm characteristics. The entire extraction pro- stock solution was prepared in methanol Extraction
cess took approximately 5 minutes per for al sample fortifications. Two inter- sample. Because a basic eluent is used nal standards were used, d3-Oxymor- A GERSTEL MPS dual rail PrepSta- with the cation exchange sorbent, the phone for quantitation of Oxymorphone tion was set up with 1 mL DPX-CX tips eluents had to be solvent exchanged into and d3-Oxycodone for all other opiates. (DPX Labs, LLC, Columbia, SC) for ext- the HPLC mobile phase. The extract was Al solvents used were of HPLC grade raction of drugs from blood and hydro- dried in about 4 minutes using low heat lyzed urine. The fol owing automation and nitrogen gas flow. Al HPLC/MS method was used: 250 µL of 30% aceto- spectra were col ected using single reac- Blood sample preparation. nitrile/water was slowly added through tion monitoring (SRM) because under A 250 µL blood sample was spiked at the top of the DPX tip at a rate of 50 µL/s the HPLC conditions used we were un- the specified concentration with the stock to wet the sorbent. The sample was then able to generate quality daughter ions for opiates mix. To precipitate proteins, 500 aspirated into the DPX tip at a rate of 90 the opiate drugs using multiple reaction µL of acetonitrile was added and the solu- µL/s and mixed with the sorbent by dra- monitoring (MRM). Although SRM MS tion was vortex mixed and centrifuged. wing in an additional 2 mL of air. After analysis may not provide the best sensiti- GERSTEL Solutions worldwide Forensic Special Recovery and % RSD for opiates (400 ppb) extracted from whole Overlay total ion LC/MS chromatograms of DPX extracts of a blank blood Extracted ion chromatogram of a DPX extract of whole blood spiked with sample and of a matrix matched sample, both spiked at 0.5 ppm. The 400 ppb of opiates. The chromatogram is free from interferences, the opi- overlay shows that interferences are negligible 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.
Extracted ion chromatogram of a DPX extract of whole blood spiked Analysis of urine spiked with 500 ppb of opiates. Again, extracts were free with 100 ppb of the opiate mix and with 400 ppb of the internal stan- from interferences. No matrix effect or ion suppression for opiates was dards (d3-oxymorphone and d3-oxycodone) in whole blood. Even when seen. (1) d3-oxymorphone, (2) oxymorphone, (3) morphine, (4) hydro- performing SRM MS analysis, the sensitivity is more than sufficient, morphone, (5) d3- oxycodone, (6) oxycodone, (7) 6-MAM, (8) codeine, demonstrating the high ionization efficiency of the electrospray system. and (9) hydrocodone.
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.
vity for the analysis of these drugs at low time was less than the chromatographic mass spectrometry with multiple reaction concentrations, this study focused on the run time, which means that the next sam- monitoring. Also, automated DPX com- automated DPX extraction and the uti- ple can be prepared while separation of bined with HPLC/MS/MS will be opti- lity of this automated sample prepara- the current sample is in progress. When- mized for other drugs and metabolites.
tion for HPLC/MS analysis of opiates. ever the LC/MS/MS system has fi- A rapid resolution HPLC column was nished a run, the next sample is ready to Further information: chosen to generate chromatographic data be introduced ensuring the highest pos- GERSTEL AppNote 7/2009 in less than 10 minutes. sible throughput. Additional y, "just in time" sample preparation helps to ensure that the prepared sample is not kept in Sparkle T. El ison, Wil iam E. Brewer, the autosampler for a long time prior to Stephen L. Morgan Automated DPX extraction of opiates being analyzed, reducing the risk of ana- Department of Chemistry and Biochemistry, from biological specimens can be perfor- lyte degradation and helping to maintain University of South med successful y using the GERSTEL sample integrity. The DPX-CX tips work Carolina, 631 Sumter Street, Columbia, MPS dual rail PrepStation. In the work very wel for extraction of opiates, reco- SC 29208, USA presented here, the total extraction time veries were in the range from 60 to 85 % was 5 minutes, additional y 4 minutes with RSD's below 6 %. Future work will Fred D. Foster were required for evaporation and solvent focus on determining the lower limits of GERSTEL, Inc., 701 Digital Dr. Suite J, exchange. The total sample preparation detection and quantitation using tandem Linthicum, MD 21090, USA GERSTEL Solutions worldwide Forensic Special Hot in pursuit of forgers To expose forgeries, the Document Laboratory of the Zurich Cantonal Police successfully applies thermal desorption coupled with GC/MS.
Forensic science has contributed greatly Thermal Desorption – acids, phthalates, and higher-boiling hyd- to the exposure of forgeries: "Today we the method of choice rocarbons are extracted. The scientist ex- are in a position to determine if handwrit- plains: "If ink has been applied to the ten or printed text, numbers, or signatu- "To reach a clear conclusion about the document during the past weeks, i.e. in res are original or if they have been mani- authenticity of a document, a GC intro- case of ‘fresh tracks', hydrocarbons are pulated, if documents have been altered duction method is needed that provides emitted in clearly detectable quantities, or if they have been forged entirely", says the possibility of varying, i.e. program- as are semi-volatile compounds like phen- Dr. Andreas Rippert of the Department ming the temperature over the course of oxyethanol and phenoxyethoxyethanol. of Forensic Sciences of the Zurich Can- the thermal desorption / thermal extrac- At 210 °C, final residues of volatile sub- tion step. Organic compounds are extrac- tonal Police. The forensic chemist adds: stances are desorbed even from older ink ted from the sample in successive steps samples." Dr. Rolf Hofer adds: "While "We can now also pinpoint the time when at different temperatures", says Dr. Rolf the complete range of detected substan- a document was forged, which could help Hofer of the Department of Forensic Sci- ces is required for conclusive classifica- us solve open cases." ences of the Zurich Cantonal Police. Dr. tion and differentiation of written mate- Hofer and his forensic expert col eagues rial, phenoxyethanol and phenoxyethoxy- used the GERSTEL Thermal Desorp- ethanol are the main indicators when it complicates the picture tion System (TDS) to develop this ana- comes to age determination." Pyrolysis GC/MS is often used to exa- lysis method. "As carrier gas sweeps across mine paper and documents. The sample the paper sample at increasing tem- is pyrolyzed under anaerobic conditions, peratures, the relevant analytes ran- i.e. under a flow of oxygen-free inert gas. ging from volatile to semi-volatile, While Pyrolysis GC/MS provides a lot are successively desorbed and cryo- focused prior to introduction to the of information, the technique does pose GC/MS system." a problem, according to Dr. Rippert: "At- tempts to reveal the document's mate- rial composition using pyrolysis GC/MS Analysis and results result in a large number of peaks, which Dr. Andreas Rippert: "At tempera- could originate either from the ink or tures below 100 °C, volatiles are ex- from the paper. In addition, the high tracted, especial y phenol and ben- temperatures used give rise to decompo- zene derivatives, as well as hydrocar- sition products that further complicate During thermal desorption, the carrier gas bons up to heptadecane." sweeps across the paper sample at successively data interpretation." At temperatures above 100 °C increased temperatures. During this process, all relevant volatile and semi-volatile compounds less volatile compounds such as fatty are desorbed and determined. 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 The document laboratory of the Zurich Cantonal Police uses a GERSTEL Thermal Desorption a particular text has been written. Cut-outs of System (TDS) in combination with a GERSTEL Cooled Injection System (CIS) and an Agilent less than 5 mm diameter from the document are Technologies GC 6890 / 5973 MSD. The system is used to differentiate between ink samples. sufficient for analysis and conclusive findings. GERSTEL Solutions worldwide Forensic Special GERSTEL MAESTRO software
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.
• Stand-Alone operation or integrated in the Agilent ChemStation or MassHunter Software • One sequence table operates the entire system including • Sample Prep by Mouse-Click using the PrepBuilder functions• Scheduler for easy planning• PrepAhead / Multiple Sample Overlap: Automated overlapping of sample preparation and analysis for maximum throughput • Priority samples can be added to the system at any point in the analysis sequence • LOG file and Service LOG file functions ensure traceability• Automated E-mail notification if the sequence is stopped• Control of up to 4 systems from one PC Sample Prep by Mouse-Click • Real-time monitoring of all modules and parameters • Interactive on-line help function The MultiPurpose Sampler (MPS) is an autosampler and sam-ple preparation robot for GC and LC. Sample preparation steps are performed during the analysis of the preceding sample for best possible system utilization and highest sample through-put. Sample preparation steps are performed in a controlled and highly accurate and reproducible manner for best possible results. Every step is selected by mouse-click from a pull-down menu in the MAESTRO software and added to the overall GC/ MS or LC/MS method. Available sample prep techniques are: • Solid Phase Extraction (SPE) MAESTRO Software enables Sample Prep by • Disposable Pipette Extraction (DPX) Mouse-Click. All sample preparation steps are con- • Internal standard addition veniently and easily selected from a drop down • Weighing, Sonication, Centrifugation menu and added to the method. Example: • Derivatization • Extraction and dilution • Heating, conditioning and mixing Add solvent, internal standard or reagent • Twister Back Extraction (TBE) • Automated Liner EXchange (ALEX) Move the vial or cartridge • Automated Twister desorption and analysis (SBSE) Agitate or stir and incubate the sample • Solid Phase Micro Extraction (SPME) at a set temperature • SPME Multi Fibre Exchanger (MFX) • Thermal Desorption and Thermal Extraction (TDS/TDU) Introduce an aliquot of the sample • Dynamic Headspace (DHS) to the GC or LC system • Multi Column Switching (MCS) Intel igent Automated Sample Preparation for LC/MS and GC/MS (Part I )
Additional techniques, now available from the leader in automated sample preparation: improved sample preparation Bar Code Reader tracking and ID verification As us how GERSTEL technology of samples and extracts highly controlled reactions such as analyte derivatization automated weighing of liquids and liquid additions separation of matrix and extract GERSTEL GmbH & Co. KG GERSTEL, Inc.
Eberhard-Gerstel-Platz 2-13-18 Nakane, Meguro-ku 2-13-18 Nakane, Megur 45473 Mülheim an der Ruhr 1510 Caton Center Drive, Surentalstrasse 1 Linthicum, MD 21090 Dai-Hyaku Seimei T Dai-Hyaku Seimei oritsudai Baltimore, MD 21227 +49 208 - 7 65 03-0 +1 410 - 247 5885 +81 3 57 31 53 21 +1 410 - 247 5887 +81 3 57 31 53 22 Subject to change. GERSTEL® Subject to change. GERSTEL , GRAPHP , GRAPHPACK and TWISTER ed trademarks of GERSTEL GmbH & Co. KG.
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C2chi#080035 1.4

J_ID: CHI Customer A_ID: 08-0027 Cadmus Art: CHI20564 Date: 23-MAY-08 Stage: I CHIRALITY 00:000–000 (2008) Use of Large-Scale Chromatography in the Preparation of Armodafinil WILLY HAUCK,1 PHILIPPE ADAM,2 CHRISTELLE BOBIER,2* AND NELSON LANDMESSER3 1Novasep Inc., Boothwyn, Pennsylvania 2Novasep SAS, Pompey, France 3Cephalon Inc., West Chester, Pennsylvania Armodafinil, the (R)-enantiomer of modafinil, is a medication used to

Pharmacuetical form: solution for intramuscular, intravenous or Active ingredients: Trimethoprim, Sulfadoxine Target species: Horse, cattle, pig, goat, cat, guinea pig Name and address of the marketing authorisation holder:bela-pharm GmbH & Co.KG, Lohner Str. 19, 49377 Vechta - GermanyComposition:1.0 ml solution contains:Pharmacological active substance: Trimethoprim