A breath of fresh air!

Transcription

1 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 News from GERSTEL GmbH & Co. KG Eberhard-Gerstel-Platz Mülheim an der Ruhr Germany Phone + 49 (0) gerstel@gerstel.com No. 12 ISSN A breath of fresh air! Solvent-free extraction and µ-scale sample prep for LC/MS and GC/MS... Wine flavor Holding on to fleeting encounters Natural mysteries Ghostbusters in the desert No pain! Efficient extraction and determination of pain killers in biological fluids Sustainable fuel Automated determination of glycerin in biodiesel

2 GERSTEL Solutions worldwide No. 12 In this issue Wine flavor analysis: Holding on to fleeting encounters Solvent-free extraction combined with on-demand multi-dimensional GC/MS and analyte concentration lets you find even the smallest flavor compound peaks in complex matrices...3 Eberhard G. Gerstel Dear reader, In the 12 th issue of GERSTEL Solutions Worldwide magazine, we invite you to go on a voyage to laboratories across the globe that use GERSTEL solutions. We want to bring you a breath of fresh air in a number of ways: GERSTEL technologies such as the Twister, thermal desorption, and DHS are used to extract and concentrate analytes without relying on the use of toxic solvents. Even if you cannot completely eliminate the use of solvents, you can still reduce the amount used: The GERSTEL MPS automates sample preparation and generates calibration standards in the µl-scale. This means that laboratory staff is less exposed to potentially toxic solvents and productivity is improved. The articles in the magazine provide several examples across a range of applications. And how about the air we breathe in homes, offices, and cars? This is where most of us spend % of our lives, increasingly surrounded with materials, such as flooring, that can emit VOCs and SVOCs. Fortunately there are rules and regulations in place that help keep unsafe products out of homes, offices, and automobiles. The GERSTEL TDS and Thermal Extractor are used to test materials to ensure that they can safely be used. The TDS even extracted the facts about what causes the famed fairy circles in the Namib desert as we report in this magazine. Last, but not least, foods, beverages, and consumer products must be safe and should have a pleasant smell. Producers now have new possibilities for extracting, separating and concentrating annoying offflavors even from complex samples to sniff out the source and put a smile on everyone s face again. Enjoy the magazine! Sincerely, E b e r h a r d G. G e r s t e l President / Co-Owner Clincal chemistry: No pain! Pain management drugs: Single step automated liquid extraction and dispersive SPE clean-up enables efficient automated LC-MS/MS determination...6 Water: Easy catch: Pesticides Automated SPE combined with evaporative analyte concentration and LC-MS/MS makes it easy to reach required LODs for pesticides in water...10 Twister extraction: All good things come in threes The usefulness of Twister sorption phases for generating flavor profiles of beverages was tested. And the winner is...12 Natural mysteries: Ghostbusters in the desert South African scientists dig deeper in their search for the causes of the Namib fairy circles...18 A breath of fresh air? When home and office make you suffer Fast and easy determination of VOC- and SVOC emissions from building products used indoors makes producers and occupants breathe a sigh of relief...22 Sustainable fuel: Automated sample preparation and determination of glycerin in biodiesel Fragrance analysis: Fabric softeners caught in a whirl One company s successful search for an alternative to solvent-based extraction of fragrances in household products and detergents...28 News... 17, 31, 32 2 GERSTEL Solutions Worldwide No. 12

3 Wine flavor analysis Holding on to fleeting encounters Companies around the world constantly strive to be a nose ahead in the race to determine flavor and fragrance compounds or to determine which off-flavor compounds are the culprits in customer complaint cases. The scientists who are ahead in the race are not only highly skilled, but also have the right toolkit. Having the ability to combine a variety of extraction techniques with 1- or 2-dimensional separation, mass selective detection, an olfactory detection port (ODP) to determine which compounds are interesting, and even a fraction collector to collect interesting compounds for further analysis this is the toolkit that dreams are made of. When all this is nicely integrated on a single GC/MS system it also becomes very manageable. Japanese scientists from GERSTEL K.K. in Tokyo demonstrate the system that helps sniff out even the smallest peak. In terms of olfactory performance, we humans have nothing like the capabilities of our canine companions. The olfactory center in the brain of a dog is up to 40 times larger than the human olfactory center. A German shepherd has around 220 million olfactory cells; nosy humans have no more than 5 million. Dogs also breathe faster and more intensely, further adding significantly to their olfactory performance. Dogs can sense a significantly larger number of odor-active compounds per unit time than humans. The fact that we humans lead our dogs on a leash and not the other way around bears testament to the fact that olfactory performance may be important, but is not apparently the decisive factor in determining the pecking order on the evolutionary ladder. Dogs have superior olfactory capabilities, Homo sapiens have advanced analytical instrumentation and the skills to decipher the results in order to compensate for olfactory deficiencies. The instrumentation available to us even enables extraction of odor active compounds from complex matrices followed by qualitative or quantitative determination. Gas chromatography (GC), especially in combination with olfactory detection (GC-O), is widely recognized as a highly efficient method. If the column effluent is split at the column outlet, an Olfactory Detection Port (ODP) and a mass selective detector (MSD) can be attached in parallel in order to get both an olfactory signature and a mass spectral identification and quantification of the odor active compound. As an aside, the ODP - MSD combination exposes weaknesses in the technique. You don t always get a signal in the chromatogram even though there is a perceivable smell at the ODP, explains Dr. Nobuo Ochiai, technical director at GERSTEL K.K. in Tokyo, Japan. This proves that the human nose, while not able to compete with its canine counterpart, is still more sensitive than modern analytical equipment when it comes to compounds that have a low odor threshold: Even a few milligrams of methylmercaptan in 100 million cubic meters of air are enough to make people head for the exit. To put that volume into context, it is around 1000 times the volume of the Notre Dame cathedral in Paris. For most compounds, far higher concentrations are required in order to adequately produce an olfactory response. And many times it is necessary to first remove interfering compounds using chromatographic separation in order to get a clear description. Separating olfactory interferences from the analyte can be simple, even for complex samples, if you have the right equipment. GERSTEL Solutions Worldwide No. 12 3

4 A GC/MS system that offers a second dimension of separation with simple, easy to use software control is available from GERSTEL. Called the Selectable 1D/2D- GC-O/MS system, it allows the GC column effluent to be split between the MSD and an Olfactory Detection Port (ODP) or a Preparative Fraction Collector (PFC) to enable parallel MS determination and olfactory monitoring or fraction collection for further analysis. A second dimension at your finger tips Dr. Ochiai and Mr. Sasamoto successfully used heart-cutting from the 1D column in the compact, integrated 1D/2D-GC-O/MS system and used the second dimension to separate and identify flavor and fragrance compounds in foods, beverages and personal care products. The main challenge when analyzing many of these types of samples is to eliminate matrix interference. Coeluting compounds will normally influence both the mass selective detection and the olfactory impression leading to incorrect odor identification and odor intensity readings. If the analytical system does not have sufficient sensitivity to determine the identity or concentration of the odor-active compounds, further automated steps can be taken to concentrate analytes: A) 1D heart-cuts from multiple injections can be taken and transferred to the 2D column for every 2D run. B) Analytes from multiple 2D runs can be collected on a fraction collector connected to the outlet of the 2D column. C) The concentration steps under both A) and B) can be combined. In order to facilitate these steps, the scientists used a single trap PFC. Performance check Two commercially available white wines, a Sauvignon Blanc and a Chardonnay, were used to put the combined 1D/2D-GC-O/ MS-PFC system to the test. Three off-flavor analytes were added to the wine: 1) The classical corkiness culprit trichloroanisol (TCA); 2) 2-isobutyl-3-methoxypyrazine (IBMP), known for its bell pepper flavor; and 3) geosmin ( earth odor in Greek), known for its musty earthy note. Even ultra-trace amounts of these compounds can be detected by the human nose. The IBMP odor threshold is 25 ng/l; TCA starts stinking at 5 ng/l; and geosmin adds a musty note at concentrations as low as 50 ng/l. To test the PFC s performance, standard solutions containing the following 15 compounds at the pg-level were injected directly into the analytical system: Hexanal, 1-hexanol, 3-hexenol, linalool, citrinellol, geraniol, p-cymen-8-ol, phenethyl alcohol, guaiacol, ethylhexanoate, ethyloctanoate, phenethyl acetate, beta-damascenone, gamma-nonalactone and limomene. IBMP, TCA and Geosmin were extracted from spiked wine samples in headspace vials by means of Stir Bar Sorptive Extraction (SBSE) using the GERSTEL Twister. Following the extraction step, the PDMS coated Twisters were removed from the samples, cleaned with DI water, dabbed dry on a Kimwipe, and transferred to individual Thermal Desorption Unit (TDU) liners. The TDU liners were then transferred to the GERSTEL MultiPurpose Sampler (MPS) and placed in individually sealed tray positions from which the MPS transports them to the TDU for analysis. Thermal desorption was performed under the following conditions: TDU initial temperature: 30 C; hold time 0.5 min.; 720 C/min to 200 C; hold time 3 min; Desorb Flow (He) 50 ml/min. The desorbed analytes were cryo-focused in the Cooled Injection System (CIS), PTV-type GC inlet, at 10 C on a CIS liner packed with Tenax TA. CIS analyte desorption was performed by heating the CIS at a rate of 720 C/min to 240 C, with a hold time of 2.0 min. Analytes were transferred to the 1D separation column in splitless mode. The GC/MS system used was a 7890 GC combined with an 5975C MSD (both from Agilent Technologies). The 1D separation was performed on a 30 m long DB-Wax column, 0.25 mm ID, 0.25 µm film thickness 1D/2D-TIC and olfactory traces obtained through SBSE-TD- 1D/2D-GC-O/ MS analysis of wine spiked with 5 50 ng/l off flavor compounds: (a) = 1D/2D-TIC; (b) = 1D/2D olfactory traces. (Agilent Technologies). The second dimension separation was performed using a 10 m long DB-5 column, 0.18 mm ID, 0.40 µm film thickness (Agilent Technologies). The columns were not kept inside the GC oven, but rather placed in individual Low Thermal Mass (LTM) column modules (Agilent Technologies) mounted on the front of the GC. LTM modules can be heated and cooled separately, and, thanks to their low thermal mass, heating and cooling can be performed much faster than traditional air bath GC ovens, resulting in faster separation and shorter analysis cycles. The GC oven was kept at 250 C throughout the analysis cycle, essentially serving as a heated chamber that keeps all transfer capillaries and column connectors at the proper temperature for best system performance. 1D/2D separation- and PFC Concentration Performance The scientists tested the performance of their single trap PFC (GERSTEL) in two steps using an adsorbent trap. Recoveries ranged from 85 to 98 %, with RSDs below 3.2 % (n = 7). Further, compound recoveries over twenty injection cycles were investigated and were found to be in the range from 98 to 116 %. Dr. Ochiai found that the high recoveries achieved using PFC concentration showed that both the analyte transfer in the system and the PFC adsorbent trap performed very well for the analytes at sub-ng levels, whereby useful quantitative and qualitative information could be gained. Following the first test runs, the identification of the spiked offflavor compounds was performed using automated thermal desorption in combination with 1D/2D-GC-O/MS. The 1D column was programmed from 40 C (2 min) at 10 C/min to 240 C. The 2D column was kept at 40 C and was left at this temperature if not used. When a heart-cut was per- 4 GERSTEL Solutions Worldwide No. 12

5 1D/2D-TIC and Mass Chromatograms (m/z 195 and 197) after PFC-concentration of analytes from 20 injections of spiked wine (Zoomed in 2D-GC-MSanalysis). (1) = IBMP 25 ng/l; (2) = TCA 5 ng/l; (3) = Geosmin 50 ng/l. formed from the 1D separation, the 2D column was programmed from 40 C to 150 C at 5 C/min and then at 20 C/min to 280 C (hold). The column effluent was split between the MS and the ODP with a split ratio of 1:2. The MS was operated in full scan and in SIM mode. The scan range was from 29 to 300 m/z at a scan rate of 2.68 Hz. In SIM mode, nine ions were monitored: m/z 124, 151 and 94 for IBMP; m/z 112, 125 and 182 for Geosmin; and m/z 195, 197 and 210 for TCA. The acquisition rate was 3 Hz for each ion; the ODP was kept at 250 C. Determining off-flavor compounds at ultra-trace levels Just a sneak preview: IBMP, TCA and Geosmin were determined in scan mode using SBSE-TD-1D/2D-GC-O/MS with unequivocal olfactory confirmation. Retention times of the GC-O signals were min (IBMP), min (TCA), and min (Geosmin). But these peaks were completely hidden in the 1D total ion chromatogram (TIC), the scientists stated. Therefore the relevant sections of the 1D chromatogram from to min and from to min were heart-cut to the 2D column. The 2D-GC-O/MS analysis was performed immediately after the 1D separation had been finished using the same GC/MS system and without any system modification. The 2D separation started at a retention time of 17.5 min. The three off-flavor compounds were clearly identified by olfactory detection during the 2D run. As an aside, high-boiling residue was simultaneously back-flushed from the 1D column by increasing the outlet pressure and decreasing the inlet pressure in order to clean up the system for the next run. MS detection in full scan mode unfortunately was not sufficiently sensitive to deliver useful data based on a single injection. The scientists therefore set about concentrating analytes introduced from 20 Twister extractions of the same sample using the integrated single channel PFC. Following the 20-fold concentration step, the Tenax TA trap used in the PFC was desorbed in the TDU and the analytes introduced to the system for 2D-GC-O/ MS determination. No system modification was necessary in order to perform this procedure. The results peak for themselves: IBMP and Geosmin peaks were found in the 2D TIC and the peaks matched the olfactory signals. Even though the TCA peak was almost completely buried in the 2D TIC, the Extracted Ion Chromatogram (EIC) for the TCA ions m/z 195 and 197 clearly displayed a peak matching the olfactory signal for TCA. The mass spectra for all target compounds were compared with data from the Wiley Mass Spectral Library, which is integrated with the Agilent ChemStation. Following library identification, a Sauvignon Blanc wine was analyzed to determine the concentration levels of all three off-flavor compounds using single SBSE- TD-1D/2D-GC-O/MS. The MS was operated in Single Ion Monitoring (SIM) mode. Quantitation was based on four-point standard addition curves, the method resulted in excellent linearity (r ) for all three compounds. Only IBMP was actually detected in the Sauvignon Blanc wine. The determined concentration was 13 ng/l (RSD = 4,4 %, n = 6), which can rightly be described as ultra-trace level. Dr. Nobuo Ochiai: With our integrated system, 1D-GC- O/MS-, 2D-GC-O/MS-, 1D-GC-PFC-, and 2D-GC- PFC analysis can be performed without modifying the system configuration. The Thermal Desorption Unit (TDU) on the system performs splitless introduction and transfer of the trapped and concentrated analytes. We have clearly shown that this enables us to identify off-flavor compounds at ultratrace levels. The 2D-GC-O/ MS system is unique in allowing us to perform the analysis in full scan mode. System performance was shown by determining three off-flavor compounds, TCA, IBMP, and Geosmin spiked into a wine at levels ranging from 5 to 50 ng/l. The combina- Kikuo Sasamoto tion of SBSE and PFC resulted in recoveries between 71 and 78 %. These values clearly show that the SBSE-TD-1D/2D-GC-O- MS technique combined with the single trap PFC is a powerful and versatile tool for the determination of flavor compounds in the ng/l range in real samples. Literature Nobuo Ochiai, Ph.D. Nobuo Ochiai, Kikuo Sasamoto. J. Chromatogr. A, 1218 (2011) Mass spectra of IBMP (a-1), TCA (a-2), and Geosmin (a-3) extracted and concentrated from spiked wine using 20 SBSE extractions combined with PFC concentration and subsequent TD-1D/2D- GC-O/MS determination. Mass spectra from Wiley Mass Spectral Library for IBMP (b-1), TCA (b-2), and Geosmin (b-3). GERSTEL Solutions Worldwide No. 12 5

6 Toxicology laboratories are trying to find ways to minimize sample preparation and enhance productivity. The adaptation of LC-MS/MS instrumentation has become popular due to the technique s high sensitivity and selectivity, low detection limits (e.g. 1 ng/ml), smaller sample volume requirements, and also due to the fact that LC-MS/ MS doesn t require chemical derivatization of analytes. However, LC-MS/MS can require the use of sample clean-up, extraction and concentration steps. These steps have traditionally been performed manually using liquid-liquid or solid-phase extraction (SPE). A different approach is to use SPE to extract the sample matrix. In this case, matrix interferences are bound to the sorbent in order to be removed from the analyte solution. The major advantage of this approach is that no separate wash or elution steps are required, enabling rapid sample preparation while still allowing comprehensive screening of the cleaned sample. Disposable Pipette Extraction (DPX) was developed as an alternative to traditional SPE, combining efficient and rapid extraction with significantly reduced solvent consumption. DPX is a novel dispersive solid-phase extraction technique that uses sorbent loosely contained in a pipette tip enabling highly efficient mixing with the sample solution. Drug Screening No pain! This study focuses on high throughput automated extraction of small volumes of urine samples (< 500 μl) used in the determination of pain management drugs by LC-MS/MS. Disposable pipette extraction (DPX) was used in a novel manner (see figure 2) to extract pain management drugs for comprehensive screening. Extracts were automatically diluted and injected into the LC-MS/MS system. Sample preparation was performed just-in-time, the cycle time averaged 7 min per sample. Validation results show that the automated DPX-LC-MS/MS screening method provides adequate sensitivity for more than 65 analytes and internal standards. Lower limits of quantitation (LLOQ) ranged between ng/ml and % RSDs were below 10 % in most cases. The main advantages of the DPX technology are: rapid extraction, high recoveries, negligible solvent waste generation, and full automation of the extraction combined with sample introduction to the chromatographic system. We have developed a fast automated DPX urine cleanup method using a GER- STEL MultiPurpose Sampler (MPS XL) for comprehensive screening of 49 pain management drugs with LC-MS/MS. The reversed phase sorbent with added salts (DPX-RP- S) used in the method allows the removal of salts and proteins present in urine, resulting in reduced matrix effects. The novel DPX procedure described here combines the advantages of dispersive SPE and liquid extraction in a simple, quick and efficient way. Figure 1: MPS XL MultiPurpose Sampler (dual head version) with GERSTEL DPX option, mounted on top of an Agilent 6460 Triple Quad LC/MS system, for high throughput pain management drug screening. The sorbent is chosen to extract the matrix without binding or absorbing the analytes of interest providing high recoveries. Since the extraction time (3 min) is less than the analytical LC-MS/MS run time (4 min), the extraction of one sample can be performed during the chromatographic analysis of the previous sample, achieving high throughput while processing each sample just in time ensuring that all samples are treated identically. EXPERIMENTAL Materials. Stock solutions for the compounds listed in Table 1 were purchased from Cerilliant. An intermediate analyte stock solution was prepared by combining the analyte stock solutions with acetonitrile, at appropriate concentrations, to evaluate the different drug classes. Deuterated analogues, d3-morphine, d4-buprenorphine, d3-norbuprenorphine, d9-methadone, d3-tramadol, d5-fentanyl, d5-alpha-hydroxy alprazolam, d4-clonazepam, d5-oxazepam, d5-estazolam, d3-cocaine, d5-nordiazepam, d5-propoxyphene, d7-carisoprodol, d5-amphetamine, d4-ketamine, d4-7-aminoclonazepam, and d5-pcp were purchased from Cerilliant. High concentration calibration standard and intermediate QC urine samples were prepared by making 6 GERSTEL Solutions Worldwide No. 12

7 Figure 2: Graphical representation of the automated DPX urine cleanup process. The new extraction procedure described here, combines the advantages of dispersive SPE and liquid-liquid extraction in a simple, quick and efficient manner. Therefore, only one extraction step is needed to eliminate the whole range of potentially interfering matrix compounds. As can be seen, an ACN layer and an aqueous layer are formed thanks to the salts contained in the DPX tip. appropriate dilutions of the combined intermediate analyte stock solution using analyte free urine to give the concentrations listed in Table 1. Calibration standards were then prepared using a dilution ratio strategy from the high concentration sample of 1:2:2:2.5:2. The high, medium and low QC samples were prepared using a dilution ratio strategy from the high concentration sample of 1:1.33:3.33: 8. b-glucuronidase, Type-2, from Helix pomatia was purchased from Sigma-Aldrich. Fresh urine was obtained from a male volunteer. All other reagents and solvents used were reagent grade. Instrumentation. All automated DPX PrepSequences were performed using a MultiPurpose Sampler (MPS XL Dual Tower) with GERSTEL DPX Option as shown in Figure 1. All analyses were performed using an Agilent 1290 Infinity LC with a Zorbax Eclipse Plus C18 column (2.1 x 50 mm, 1.8 μm, 600 bar), an Agilent 6460 Triple Quadrupole Mass Spectrometer with Jet stream electrospray source and GERSTEL MPS XL autosampler configured with an Active Wash Station (AWS). Sample injections were made using a 6 port (0.25mm) Cheminert C2V injection valve fitted with a 2 μl stainless steel sample loop. Sample pretreatment. Hydrolysis of urine was performed by combining 2 ml of urine, 150 μl of the working internal standard solution, 100 μl of b-glucuronidase, and 500 μl of 0.66M acetate buffer, ph 4, vortex mixing for 30 seconds, and then incubating at 55 C for 2 hours. Aliquots of 260 μl of hydrolyzed urine samples were added into clean shell vials for automated cleanup and injection. The automated extraction (DPX Prep Sequence and Clean-up proce- dure) used for this method consisted of the following steps: 1. Aspirate 750 μl of 100 % acetonitrile from the fast solvent delivery station using the 2.5 ml DPX syringe. 2. Pick up a new DPX tip (DPX-RP-S) located within the tray. 3. Add 500 μl of 100 % acetonitrile through the DPX tip, into the urine sample located on the MPS sample tray. 4. Wait for 6 seconds to allow the acetonitrile to completely wet the DPX sorbent. 5. Aspirate the entire sample followed by 1400 μl of air into the DPX tip. 6. After equilibrating for 5 seconds, dispense the contents of the DPX tip, as well as the remaining acetonitrile found within the DPX syringe, back into the original shell vial in the tray. 7. Move the DPX tip to the PipWaste position and dispose of the DPX tip. 8. Transfer 100 μl of the upper liquid layer located within the original shell vial, into a clean, empty, capped autosampler vial with magnetic septum cap located on a VT98 tray. 9. Dilute the extract by adding 900 μl of water into the sample vial. 10. Inject 50 μl of the sample into the HPLC injection valve (2 µl injection loop). A total of 116 MRM transitions (98 Analyte qualifier/quantifier and 18 internal standard transitions) were monitored in a 4 minute analytical window followed by a column regeneration time of 2.5 minutes. A retention time window of 30 seconds was used for each positive ion transition monitored in the dynamic MRM method. Detailed mass spectrometric acquisition parameters are available upon request. Analysis conditions LC Pump: gradient (600 bar), flowrate = 0.5 ml/min Mobile Phase: A - 5 mm ammonium formate, with 0.05 % formic acid B % formic acid in methanol Gradient: Initial 5 % B 0.5 min 5 % B 1.5 min 30 % B 3.5 min 70 % B 4.5 min 95 % B 6.49 min 95 % B 6.5 min 5 % B Run time: 6.5 minutes Injection volume: 2 μl (loop over-fill technique) Column temperature: 55 C Analysis conditions MS Figure 3: Overlaid chromatograms for all 116 dynamic MRM transitions from an extracted urine sample at the MRL. Operation: electrospray positive ion mode Gas temperature: 350 C Gas flow (N2): 12 L/min Nebulizer pressure: 35 psi Capillary voltage: 4400 V GERSTEL Solutions Worldwide No. 12 7

8 1) d3-morphine 6) d3-cistramadol 11) d5-oxazepam 16) d5-amphetamine 2) d4-buprenorphine 7) d5-fentanyl 12) d5-estazolam 17) d5-pcp 3) d3-norbuprenorphine 8) d7-carisoprodol 13) d5-nordiazepam 18) d3-cocaine 4) d9-methadone 9) d4-7-aminoclonazepam 14) d5-α-oh-alprazolam 5) d5-propoxyphene 10) d4-clonazepam 15) d4-ketamine List of internal standards used. RESULTS AND DISCUSSION Figure 3 shows representative dynamic MRM chromatograms for all 49 pain management drugs and internal standards in a hydrolyzed urine sample spiked at the minimum reporting limit (MRL) and cleaned using automated DPX. Table 1. Retention times, high calibration standard concentrations, MRLs and LOQs for all pain management drugs analyzed. (1-18 used internal standard) Figure 4: Representative calibration curves: morphine, flurazepam, cocaine and ketamine. Disclaimer For drug screening only. Not for use in diagnostic procedures. The information provided in this article is intended for reference and research purposes only. GERSTEL offers no guarantee as to the quality and suitability of this data for your specific application. Information, descriptions and specifications in this publication are subject to change without notice. Compound Ret. Time High Cal Std. MRL LOQ [min] [ng/ml] [ng/ml] [ng/ml] 6-MAM Codeine Hydrocodone Hydromorphone Oxycodone Morphine Oxymorphone Meperidine Normeperidine Buprenorphine Norbuprenorphine EDDP Methadone Norpropoxyphene Propoxyphene O-Desmethyl-cis-Tramadol cis-tramadol Fentanyl Norfentanyl Meprobamate Carisoprodol aminoclonazepam Clonazepam Oxazepam Estazolam Alprazolam Diazepam Flunitrazepam Lorazepam Nitrazepam Temazepam α-oh-alprazolam Nordiazepam Bromazepam Clobazam Midazolam Triazolam Flurazepam Ketamine Norketamine Amphetamine MDA MDEA MDMA Methamphetamine Methylphenidate PCP Benzoylecgonine Cocaine GERSTEL Solutions Worldwide No. 12

9 Table 2. Extracted QC samples % accuracies and % RSDs. Table 1 lists the column retention times, concentrations for the highest calibration standard, MRLs and LLOQs for the 49 analytes in this screening assay. LLOQ concentrations are higher (5 fold factor increase) in comparison to those listed in our previous work performed with an automated concentration step using a solvent evaporation station [1]. However, the LLOQs of this modified cleanup method are still below the original MRLs and cycle times are considerably shorter. Representative calibration curves are shown in Figure 4. Regression analysis for all pain management drugs analyzed within this method resulted in R 2 values of 0.99 or greater. Compound QCL Avg. Accuracy [%] QCH Avg. Accuracy [%] [ng/ml] [n = 4] % RSD [ng/ml] [n = 4] % RSD 6-MAM Codeine Hydrocodone Hydromorphone Oxycodone Morphine Oxymorphone Meperidine Normeperidine Buprenorphine Norbuprenorphine EDDP Methadone Norpropoxyphene Propoxyphene o-desmethyl-cis-tramadol Tramadol Fentanyl Norfentanyl Meprobamate Carisoprodol aminoclonazepam Clonazepam Oxazepam Estazolam Alprazolam Diazepam Flunitrazepam Lorazepam Nitrazepam Temazepam α-oh-alprazolam Nordiazepam Bromazepam Clobazam Midazolam Triazolam Flurazepam Ketamine Norketamine Amphetamine MDA MDEA MDMA Methamphetamine Methylphenidate PCP Benzoylecgonine Cocaine The DPX automated sample cleanup time was reduced from 7 to 3 min/sample; the total cycle time per sample for the extraction process and injection was reduced from 13 to 7 min/sample, fitting with the just in time sample preparation strategy available using the MAESTRO software and increasing throughput. Using this automated procedure for extraction and analysis over 200 samples can be processed per day. The accuracy and precision of the method was measured for all pain management drugs analyzed by extracting replicate QC samples (n=4) at high and low concentrations. Table 2 shows the resulting accuracy and precision data for all pain management drugs. Accuracy data averaged 98.0 % (range: %) and precision data (% RSD) averaged 4.2 % (range: %) for all pain management drugs determined. CONCLUSIONS As a result of this study, we were able to show: The automated DPX cleanup method using the GERSTEL MPS XL Dual Tower robotic sampler for pain management drug screenings in urine provided cycle times of approximately 7 min/sample allowing throughput of over 200 samples per day. 49 pain management drugs can be rapidly and reproducibly isolated from hydrolyzed urine samples using an automated DPX cleanup procedure coupled to LC- MS/MS analysis using the Agilent 6460 Triple Quadrapole Mass Spectrometer. Linear calibration curves resulting in R 2 values 0.99 or greater were achieved with LOQs lower than the minimum reporting limits for the majority of pain management drugs analyzed. The DPX-LC-MS/MS method provided good accuracy and precision averaging 98.0 % (range %) accuracy with 4.2 % RSD (range: %) for all pain management drugs analyzed. References [1] Determination of Pain Management Drugs using Automated Disposable Pipette Extraction and LC-MS/MS, GERSTEL AppNote AN Authors Oscar G. Cabrices, Fred D. Foster, John R. Stuff, Edward A. Pfannkoch, GERSTEL, Inc., 701 Digital Dr. Suite J, Linthicum, MD 21090, USA William E. Brewer Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street, Columbia, SC 29208, USA GERSTEL Solutions Worldwide No. 12 9

10 Water Easy catch: pesticides Pesticides in water are easy to catch i.e. extract, but often an additional analyte concentration step is required in order to meet the limits of detection (LODs) specified in rules and regulations. A complete automated sample preparation system consisting of a GERSTEL MultiPurpose Sampler (MPS) with SPE option and a solvent evaporation station was put to the test in the work described here. System performance, including automated extraction, was tested for 18 pesticides at the 10 ng/l level in groundwater. The analytes were extracted, concentrated, and determined using LC-MS/MS. Performance criteria were ruggedness, recovery of analytes, method linearity and sample to sample reproducibility. The pesticides appear to have taken the bait. Havingto reach ever lower limits of detection (LODs)is a daily challenge in modern laboratories. In order to succeed in obtaining sufficiently sensitive analysis methods, sample preparation techniques such as Solid Phase Extraction (SPE) or Liquid-Liquid Extraction (LLE) are often used as combined extraction and concentration steps. The concentration factor achieved in these cases depends on the ratio between the extracted sample volume and the amount of solvent used for analyte elution from the SPE cartridge or for liquid extraction. Following the extraction, a further evaporative concentration step can be performed, significantly improving the limits of detection for the overall analytical method. For example, an extra concentration step can help to meet the requirements of the water EU framework directive 2008/105/EC for pesticides in ground and drinking water. For the evaporative concentration step, semi-automated solutions are widely used, in which a number of samples are concentrated under a flow of nitrogen at a moderately increased temperature. In such systems, sample transfer is handled manually. The GERSTEL MultiPosition Evaporation Station (mvap) in combination with the GERSTEL MultiPurpose Sampler (MPS) now offers fully automated concentration of sample extracts. The system enables complete automation of all sample preparation steps including introduction to the LC- or GC-system. The evaporation is controlled by controlling the applied vacuum, tem- Manual Sample Preparation - Sample filtration - Transfer of filtrate to 25 ml Vial SPE Process - 20 ml sample/filtrate added to cartridge - SPE Cartridges: M & N C18ec - Elution with 2 ml MeOH (Concentration 10:1) mvap Evaporation - Evaporation time: 30 min - Temperature: 60 º C - Pressure: 200 mbar - Agitation: 600 rpm - Evaporation to dryness mvap Reconstitution - Addition of 0.5 ml H 2 O - Incubation time 5 min - Shaking: 600 rpm Transfer to LC-MS/MS - Transfer of concentrate to 1.5 ml vials - Analysis with LC-MS/MS Automated sample preparation process used for the analysis work performed. The mvap is a six-position evaporation station for the GERSTEL MultiPurpose Sampler (MPS). Samples are concentrated at slightly elevated temperatures under moderate vacuum, enabling significantly improved limits of detection. GERSTEL Multi Purpose Sampler MPS with automated Solid Phase Extraction (SPE) and evaporation module mvap. 10 GERSTEL Solutions Worldwide No. 12

11 Hexazinon Diuron Isoproturon Desethylatrazin Metribuzin Cyanazin Overlay chromatogramm of quantifier MRMs for six selected pesticides extracted from a spiked sample at 10 ppb. Matrix calibration curves for six selected analytes extracted from spiked groundwater sample. perature, and agitation leading to reproducible results independent of the solvent used. The user can also benefit from a real increase in laboratory efficiency, since batches of samples can be processed automatically overnight. In this work, the performance of the complete system including the mvap is demonstrated. Experimental SPE extraction and clean-up was performed using a GERSTEL MultiPurpose Sampler (MPS) fitted with SPE option. Eluate concentration was performed using the GERSTEL MultiPosition evaporation station (mvap) under MAESTRO software control. LC-MS/MS analysis work was performed using an Agilent 1290 LC system coupled to an Agilent 6490 Triple Quadrupole Mass Spectrometer. In the mvap, samples were concentrated at slightly elevated temperatures under moderate vacuum, enabling significantly improved limits of detection. Method parameters are listed in the table to the left. Spiked groundwater samples were used for matrix calibration and analysis. Conclusions A completely automated process involving SPE and an evaporation step was tested for ruggedness, recovery of analytes, method linearity and sample to sample reproducibility. Data obtained using the mvap to concentrate extracts containing 18 selected pesticides from groundwater samples clearly demonstrates that the system delivers the required analytical performance while requiring almost no manual handling steps. Calibration curves, sample to sample reproducibility, and recovery rates for the analytes selected, demonstrate the applicability for routine laboratory work. The GERSTEL MPS with SPE and mvap in combination with an LC-MS/MS system is a powerful tool for determining pesticides in water at low concentration levels. Hexazinon RSD 2.8 % Isoproturon RSD 6.5 % Desethylatrazin rel. STD 9.6 % Metribuzin RSD 9,2 % Cyanazin RSD 7,8 % Diuron RSD 9,5% response number of repetitions Signal responses and standard deviations for selected analytes from twelve consecutive extractions and analyte determinations. Recovery of determined pesticides spiked at 10 ng/l References Directive 2008/105/EC of the European Parliament and of the Council of 16 December 2008 on environmental quality standards in the field of water Policy, amending and subsequently repealing Council Directives 82/176/EEC, 83/513/EEC, 84/156/EEC, 84/491/EEC and 86/280/ EWG, and ammending Directive 2000/60/EC,of the European Parliament and the Council. Authors Meike Holtmann, TeLA GmbH, Fischkai 1, D Bremerhaven, Germany. Susanne Sperling, GERSTEL GmbH & Co. KG, Eberhard-Gerstel- Platz 1, D Mülheim an der Ruhr, Germany. Concentration in ng/l (average of 12 individually prepared samples) The determined 18 pesticides with recoveries. GERSTEL Solutions Worldwide No

12 Twister extraction All good things come in threes A novel Ethylene Glycol- (EG) and Silicone based combined sorbent phase has been developed for stir bar sorptive extraction (SBSE) using the GERSTEL Twister with the aim of improving recovery of analytes across a wide polarity range. In this article, the performance of EG-Silicone, polyacrylate (PA), and polydimethylsiloxane (PDMS) Twisters is investigated to determine their usefulness in generating qualitative flavor profiles of beverages such as whisky, white wine, and multivitamin juice. Stir bar sorptive extraction (SBSE) is based on principles similar to solid phase micro-extraction (SPME). Both techniques rely on partitioning of analytes between a sorbent phase and a liquid sample phase, resulting in extraction and concentration of the analytes in the sorbent phase depending on the partitioning coefficient. Following sample extraction, the coated stir-bar is thermally desorbed in a flow of carrier gas, releasing and transferring the analytes to a GC/MS system. The most widely used Twister phase is polydimethylsiloxane (PDMS), which is non-polar. It has been reported that the extraction efficiency of the PDMS based Twister can be up to 250 times higher than for PDMS based SPME fibers [1] due to the much larger sorbent phase volume, improved phase ratio and improved phase contact during extraction, all of which enable more efficient extraction and extraction of larger volumes. Successful applications of SBSE include extraction and analysis of VOCs, SVOCs, PAHs, pesticides, and offodors in water; drugs of abuse such as Tetrahydrocannabinol (THC), barbiturates and benzodiazepines; phthalates and various metabolites in biological fluids; flavor compounds, preservatives, thrichloroanisole, pesticides, and fungicides in food and beverages [2,3]. For polar compounds with an octanol-water partition coefficient (Ko/w) lower than 10,000 (logko/w) < 4), it has been found that recoveries gradually decrease with decreasing Ko/w when using PDMS based Twisters. Among the more hydrophilic solutes are, for example, polar pesticides, alcohols, esters, and phenolic compounds. Although recoveries could successfully be improved for many polar pesticides by adding 30 % NaCl (w/w) into the water sample, the salting-out technique does not necessarily help for all polar compounds and there has increasingly been demand for a Twister with a more polar phase. Two new Twisters with more polar phases are now available from GERSTEL: The Polyacrylate (PA) Twister and the Ethylene Glycol (EG) Silicone Twister. These new Twisters extract several classes of polar compounds more efficiently than the PDMS Twister due to their polar nature. In addition, the EG-Silicone Twister, since it is silicone based, will also efficiently extract non-polar compounds. Experimental Samples: Scotch whisky (40 % EtOH v/v); white wine, sauvignon blanc (13 % EtOH v/v) and multivitamin juice. Instrumentation. The TD-GC/MS analysis was performed using a Thermal Desorption Unit (TDU) combined with a MultiPurpose Sampler (MPS) and a Cooled Injection System (CIS 4) programmed temperature vaporization (PTV) type inlet (all GERSTEL). An Agilent 6890N gas chromatograph with a 5975B inert XL (triple axis) mass selective detector (MSD) was used. The entire analysis system was operated under MAESTRO software control integrated with Agilent ChemStation software using one integrated method and one integrated sequence table. The more polar PA- and EG-Silicone Twisters do retain some water during extraction of aqueous samples, but excess water can be eliminated prior to GC/MS analysis by operating the TDU in solvent vent mode. In this mode, water is evaporated at low initial temperature, for example at C and ambient pressure (0 kpa) for a pre-determined time before the temperature ramp for the thermal desorption starts. As a result, the introduction of water into the GC/ MS system is avoided or significantly reduced. An alternative way to reduce water background is to leave the Twisters exposed to a dry atmosphere for approximately 15 minutes. In this work, we used the TDU solvent vent mode for water removal since it is an automated process, which delivers more reproducible and reliable results. Extraction of aqueous samples using an EG-Silicone or a PA Twister is performed in exactly the same way as with a PDMS Twister. Aqueous sample was transferred into a 10 ml headspace vial. The Twister was added and the vial was sealed with a screw cap. The extraction was performed at room temperature for 60 min while stirring at 1000 rpm on a multiple position magnetic stirrer. After the extraction had been completed, the Twister was removed from the sample with a magnetic rod and briefly rinsed with HPLC grade water. After carefully drying it with a lint-free tis- 12 GERSTEL Solutions Worldwide No. 12

13 Analysis conditions TDU: 40 ml/min solvent vent (0.5 min) EG-Silicone and PA Twister: 40 C (0.5 min); 120 C/min; 220 C (5 min) PDMS Twister: 40 C (0.5 min); 120 C/min; 270 C (5 min) PTV: split 1: C (0.5 min); 12 C/s; 300 C (5 min) Polar separation Column: 15 m ZB-FFAP (Phenomenex) di = 0.25 mm df = 0.25 μm Pneumatics: He, constant flow = 1.4 ml/min Oven: 50 C (2 min); 5 C/min; 60 C; 10 C/min; 165 C; 20 C/min, 250 C (5 min) Non-polar separation Column: 30 m ZB-5 (Phenomenex) di = 0.25 mm df = 0.25 μm Pneumatics: He, constant flow = 1.2 ml/min Oven: 60 C (2 min); 5 C/min; 200 C; 10 C/min; 300 C (5 min) sue, the Twister was stored in a 1.5 ml vial. The Twister was finally placed in a TDU glass liner and the liner stored on an MPS sample tray for GC/MS analysis. Scotch Whisky The EG-Silicone Twister is especially well suited for extraction of polar compounds which form hydrogen bonds as hydrogen donors, for example, phenols and similar substances. In figure 1, a comparison of three chromatograms from three extractions of a whisky using different Twisters is shown. The EG-Silicone Twister extraction provided the best recovery for phenols, ethyl esters and fatty acids from whisky. It is clearly seen that the EG-Silicone Twister extracts more compounds, and in greater amount. In table 1, peak areas are listed for the annotated compounds shown in the chromatograms. The peak areas that result from the EG-Silicone Twister extraction are an order of magnitude higher than the compound peaks obtained using the PA or PDMS Twisters for almost all compounds. Due to its polydimethylsiloxane basis, the EG-Silicone Twister also has high affinity for nonpolar analytes like long carbon-chain ethyl esters and acids. When comparing the chromatograms from the EG-Silicone- and PDMS Twister extractions, it becomes clear that the EG-Silicone Twister extraction (top chromatogram) results in the same number of peaks in the region after 25 minutes, but the peaks are significantly larger and recoveries significantly better. Table 2 shows the extraction efficiency (recovery in %) for selected whisky components: phenol, o-cresol, ciswhisky lactone and eugenol, obtained with three types of Twisters from spiked water samples. The highest recovery for phenol and o-cresol was obtained using an EG-Silicone Twister: 5.7 % and 9.8 %, respectively. The PDMS Twister gave high extraction efficiency for non-polar compounds like lactone and eugenol: 24.3 % and 32.9 %, respectively. In direct comparison with the PA Twister, the EG-Silicone Twister provided higher sensitivity for whisky lactone (6.1 %) and eugenol (29.5 %). These results prove that the EG-Silicone Twister extracts phenolic substances very efficiently and that it is also highly suitable for many non-polar compounds. As is clearly seen in table 3, a significantly larger number of phenols and aromatic compounds were extracted from the whisky sample with the EG-Silicone Twister than with the PDMS Twister. The total number of extracted compounds is 126 for Figure 1. Whisky extraction chromatograms obtained using EG-Silicone, Acrylate, and PDMS Twisters, non-polar column separation. 5 ml whisky sample (20 % EtOH (v/v), 1:1 dilution with water), 1000 rpm for 1 hour at room temperature. Peak identification: 1. Phenol; 2. Ethyl hexanoate; 3. o-cresol; 4. p-cresol; 5. Phenethyl alcohol; 6. o-ethylphenol; 7. 2,4- Xylenol; 8. Ethyl octanoate; 9. Octanoic acid; 10. Ethyl decanoate; 11. Decanoic acid; 12. Ethyl dodecanoate; 13. Dodecanoic acid. Table 1. Peak Areas of marked peaks obtained from extraction using three different Twister types. Peak Compounds Extracted Ion Peak Areas No. [m/z] EG-Silicone PA PDMS 1 Phenol E E E+04 2 Ethyl hexanoate E E E+06 3 o-cresol E E E+05 4 p-cresol E E E+05 5 Phenethyl alcohol E E E+06 6 o-ethylphenol E E E ,4-Xylenol E E E+05 8 Ethyl octanoate E E E Ethyl decanoate E E E Ethyl dodecanoate E E E+07 GERSTEL Solutions Worldwide No

14 Table2.Recovery in % for selected whisky standard substances obtained with three types of Twisters. Whisky Log EG- PA PDMS standards Ko/w Silicone Phenol o-cresol cis-whisky lactone Eugenol Table 3. Whisky compounds extracted by SBSE using the PDMS and EG-Silicone Twister respectively. Compound class PDMS EG- Silicone Phenols and aromatic compounds Fusel alcohols Fatty acids Aliphatic acid ethyl esters Other esters Lactones 1 2 Acrolein derivates 7 7 Terpenes and norisoprenoids 6 7 Miscellaneous 6 12 Total the EG-Silicone Twister and 92 for the PDMS Twister. For the other compounds classes, both Twisters extract a similar number of compounds, but the EG- Silicone Twister generally gives better recovery. In order to achieve better separation of polar compounds from the whisky sample, a ZB- FFAP column was subsequently used. The resulting chromatogram is shown in figure 2, the whisky profile was obtained based on extraction with an EG- Silicone Twister. Table 4 lists the proposed compound names identified with the mass spectral database (Wiley 6N). All identified peaks have a hit quality higher than 80. The plausibility of the identification was checked against literature to ensure that the reported compounds were known to be present in whisky. Using the polar column, the peaks for acids, phenols and other polar compounds show a better peak shape. Many important whisky compounds (vanillin, ethyl vanillate, etc.), which were covered by broad co-eluting acid peaks when using the ZB-5 non-polar column, were now well separated and could easily be identified. Multivitamin Juice Extraction of multivitamin juice or of other fruit juices is often negatively influenced by fruit pulp, which blocks analyte access to the extraction phase and/or hinders phase separation following the extraction. In contrast, the presence of fruit pulp has no effect on the SBSE extraction process for multivitamin juice. A 10 ml sample was directly dispensed into a 10 ml vial, the Twister was added and the sample stirred for 1 hour at 1000 rpm. Both EG-Silicone- and PDMS Twisters were used for the extraction. As can be seen in the chromatograms in figure 3, the EG-Silicone Twister extracts more compounds than the PDMS Twister and with better recovery. The peaks obtained using the EG-Silicone Twister are significantly larger. In the chromatogram obtained with EG-Silicone Twister, 39 peaks were clearly identified. Nine compounds were not at all found or identified using the PDMS Twister: formic acid, acetic acid, furfural, furfural alcohol, 2-hydroxycyclopent-2-enone, 3-methyl- 2,5-furandione, 5-methyl-2-furfural, 2,3-dihydro-3,5-dihydroxy-6- methyl-4h-pyran-4-one and Hydroxymethylfurfurole (HMF). Most of these compounds are furfurals and derivatives of furan. Moreover, the peaks for these nine compounds were very large using EG-Silicone Twister extraction, for example furfural (No. 3) and HMF (No. 21). Some important terpenes in the multivitamin juice were extracted by both Twisters, these are listed in table 6. Eight terpenes were selected and their peaks integrated based on extracted ion chromatograms (EICs). The EIC masses used and the resulting peak areas are also listed in table 6. It can be seen that EG-Silicone- and PDMS Twisters have similar extraction efficies for the terpenes judging by the very similar peak areas obtained using the two Twisters. For more polar alcohol-terpenes linalool, 4-terpineol, alpha-terpineol, and nerolidol, the EG-Sil- icone Twister does provide better recovery than the PDMS Twister; conversely, for monoterpenes like alpha-pinene, betamyrcene, delta-3- carene and d-limonene the PDMS Twister gives better recovery. White Wine (Sauvignon Blanc) EG-Silicone-, PA- and PDMS Twisters were used to extract a broad range of volatile compounds and generate a flavor profile of the white wine. Subsequently, the extraction results for the three Twister types were compared. PA- and PDMS Twisters can be added directly to the wine sample without modifying the sample. Prior to extraction with EG-Silicone Twister, the wine sample needed to be neutralized to ph 3.6 in order to avoid break-down of the Twister phase. Chromatograms were obtained using both ZB-5 (non-polar) and ZB-FFAP (polar) columns, the tentatively identified wine compounds are listed in Table 7. Except for the oven programs and column flow rates used, all conditions for TDU, CIS, and MSD were the same for all analyses performed. A stacked view comparison of chromatograms from extractions using different Twisters is shown in figure 4. It can be seen that the Table 4. Tentatively identified compounds found in Scotch whisky by Twister extraction and GC/MS analysis using a ZB-FFAP Column. Peak Proposed Peak Proposed Peak Proposed No. Identity No. Identity No. Identity 1 Ethyl octanoate 9 trans Whisky lactone 17 Capric acid 2 Ethyl nonanoate 10 o-cresol 18 Farnesol 3 Ethyl decanoate 11 p-ethylguaiacol 19 Lauric acid 4 1-Decanol 12 d-nerolidol 20 Vanillin 5 Phenethyl acetate 13 Octanoic acid 21 Ethyl vanillate 6 Ethyl dodecanoate 14 o-ethylphenol 22 Myristic acid 7 Guaiacol 15 2,4-Xylenol 8 Phenethyl alcohol 16 p-ethylphenol Figure 2. SBSE-TD-GC/MS chromatogram, polar column separation, resulting from an EG-Silicone Twister extraction of a 5 ml whisky sample diluted 1:1 with water (20 % EtOH v/v). Sample extracted for one hour at room temperature and 1000 rpm. 14 GERSTEL Solutions Worldwide No. 12

15 EG-Silicone Twister extracts a larger number of individual substances (30 tentatively identified peaks) from wine than the PDMS and PA Twisters. Substances like furfural, cis- and trans-4-hydroxymethyl-2-methyl- 1,3-dioxolane, glycerin, malic acid, methyl 2,3-dihydroxybenzoate are only found in the EG-Silicone- and PA Twister based chromatograms. Furthermore, most peaks are much larger in the EG- Silicone Twister-based chromatogram than in the PA Twister-based chromatogram. The EG-Silicone Twister extracts acids much more efficiently from wine than the PDMS Twister, see peaks No. 11, 17, 21, 22, and 24 as well as alcohols like 2,3-butanediol (No. 3), 1-hexanol (No. 6), and Phenethyl alcohol (No.15). PDMS Twisters, conversely, extract larger amounts of esters compared to EG-Silicone Twister (see No. 4, 7, 12, 13, 18, 25). To achieve better resolution and separation of polar compounds extracted from the wine, a polar column was also used. As can be seen in figure 5, the polar column produced sharp acid peaks and enabled Table 5. Identified compounds found in multivitamin juice by Twister extraction and GC/MS analysis using a ZB-5 Column. Peak Proposed Identity Peak Proposed Identity Peak Proposed Identity No. No. No. 1 Formic acid 14 gamma-terpinene 27 Nerolidol 2 Acetic acid 15 alpha-terpinolene 28 Methoxyeugenol 3 Furfural 16 Linalool 29 alpha-cubebene 4 Furfural alcohol 17 Apple oil 30 Myristic acid 5 Isoamyl acetate 18 2,3-Dihydro-3,5-31 Nootkatone dihydroxy-6-methyl- 4H-pyran-4-one 6 2-Hydroxycyclopent Terpineol 32 8-Hydroxy-6-2-en-one methoxy 7 alpha-pinene 20 alpha-terpineol 33 9-Hexadecenoic acid 8 3-Methyl-2,5-21 Hydroxymethyl- 34 Palmitic acid Furandione furfurole (HMF) 9 5-Methyl-2-furfural 22 Eugenol 35 Limetin 10 beta-myrcene 23 trans-caryophyllene 36 Xanthotoxin 11 delta-3-carene 24 alpha-humulene 37 Linoleic acid 12 D-Limonene 25 Valencene 38 Isopimpinellin 13 Isoamylbutyrate 26 Elemicin 39 Squalene the separation of several key polar compounds that were covered by big co-eluting ester peaks in the chromatogram produced on the non-polar column. Although a different column was used, the quantitative results and determined compound identities obtained from EG-Silicone- and PDMS Twister extractions were in good agreement. Some polar acids, alcohols, as well as other polar compounds could be extracted only using the EG-Silicone Twister. Additionally, 5-methyl-2-furfural (No. 11) and Hydroxymethylfurfurole (HMF) (No. 24 ), p-hydroxyphenethyl Table 6. Peak area responses of Terpenes resulting from Twister extractions. Peak No. Compounds Extracted Ion Peak Areas [m/z] EG-Silicone PDMS 7 alpha-pinene E E beta-myrcene E E delta-3-carene E E D-Limonene E E Linalool E E Terpineol E E alpha-terpineol E E Nerolidol E E+05 Figure 3. Multivitamin juice chromatogram obtained from EG-Siliconeand PDMS Twisters, non-polar column separation. 10 ml sample, 1000 rpm, 1 hour, room temperature. Figure 4. Sauvignon Blanc chromatogram profiles obtained from EG-Silicone-, PDMS- and PA Twister extractions of 5 ml samples for one hour at 1000 rpm, non-polar column separation. GERSTEL Solutions Worldwide No

16 alcohol (No. 27) and ethyl 3-(4-hydroxyphenyl)- propenoate (Z or E) (No. 29) were found only when combining EG-Silicone Twister extraction with separation on a polar column (Table 8). CONCLUSION The novel EG-Silicone Twisters and PA Twisters presented in this work enable higher extraction efficiency than traditional PDMS Twisters for polar compounds in samples like whisky, multivitamin juice and white wine. For compounds like phenols, furans, alcohols, and acids, use of EG-Silicone Twister results in the best extraction efficiency and shows better performance than the PA Twister. For non-polar compounds such as terpenes and ethyl esters etc., EG-Silicone Twisters, due to their dimethylsiloxane base, provide extraction efficiencies similar to those achieved using PDMS Twisters. By using both the EG-Silicone- and the PDMS Twister in a sequential SBSE process, an overall analyte profile of non-polar and polar organic compounds in a sample can be obtained. The ph value of the sample is a critical point for the EG-Silicone Twister. In water-based standards, the optimum ph range was found to be from 3.5 to 10.0, for wine samples from 3.6 to 7.0. Like the PDMS Twister, the extraction using the EG-Silicone Twister is easy to perform. Only a few instrumental parameters have to be adjusted. Additionally operating the TDU in solvent vent mode is important when desorbing EG-Silicone- and PA Twisters in order to remove excess water that is retained due to their polar nature. This is needed to eliminate water from the GC/MS system. Further information - Applications: AppNote 3/2011 ( Table 7. Tentatively identified compounds extracted from white wine using different Twisters and separated on a ZB-5 GC Column. Peak Proposed Identity Peak Proposed Identity Peak Proposed Identity No. No. No. 1 2-Methyl-butanol 11 Hexanoic acid 21 Nonanoic acid 2 3-Methyl-butanol 12 Ethyl hexanoate 22 Malic acid 3 2,3-Butanediol 13 1-Hexyl acetate 23 Methyl 2,3-dihydroxy- 4 Ethyl butanoate 14 Glycerine 24 Capric acid 5 Furfural 15 Phenethyl alcohol 25 Ethyl decanoate 6 1-Hexanol 16 2,3-Dihydro-3,5-26 p-hydroxyphenethyl dihydroxy-6-methyl- alcohol 4H-pyran-4-one 7 Isoamyl acetate 17 Octanoic acid 27 2,4-Di-tert-butylphenol 8 trans-4-hydroxymethyl- 18 Ethyl octanoate 28 Methyl 2,5-dihydroxy 2-methyl-1,3-dioxolane benzoate 9 cis-4-hydroxymethyl- 19 Phenethyl acetate 29 Lauric acid 2-methyl-1,3-dioxolane 10 Citraconic anhydride 20 Ethyl dl-malate 30 Ethyl laurate Table 8. Tentatively identified compounds extracted from white wine using different Twisters and separated on a ZB-FFAP Column. Peak Proposed Identity Peak Proposed Identity Peak Proposed Identity No. No. No. 1 1-Hexyl acetate 11 5-Methyl-2-furfural 21 Glycerine 2 1-Hexanol 12 Phenethyl acetate 22 2,3-Dihydrobenzofuran 3 Ethyl octanoate 13 Hexanoic acid 23 Lauric acid 4 Acetic acid 14 Phenethyl alcohol 24 Hydroxymethylfurfurole (HMF) 5 Furfural 15 Ethyl dl-malate 25 Malic acid 6 trans-4-hydroxy- 16 Octanoic acid 26 Myristic acid methyl-2-methyl- 1,3-dioxolane 7 2,3-Butanediol 17 Nonanoic acid 27 p-hydroxyphenethyl alcohol 8 Ethyl decanoate 18 2,3-Dihydro-3,5-28 Palmitic acid dihydroxy-6-methyl- 4H-pyran-4-one 9 cis-4-hydroxymethyl- 19 Decanoic acid 29 Ethyl 3-(4-hydroxy 2-methyl-1,3-dioxolane phenyl)-propenoate (Z or E) 10 Clorius 20 2,4-Di-tert-butylphenol Acknowledgment The authors would like to thank Dr. Kevin MacNamara, Irish Distillers, Pernod-Ricard for his kind support. References 1. Frank David, Bart Tienpont,Pat Sandra, Stir-bar sorptive extraction of trace organic compounds from aqueous matrices, LCGC North America, 21: (2003) 2. Kevin Mac Namara, Michelle Lee, Albert Robbat Jr., J.Chromatogr. A 1217 (2010) Kevin Mac Namara, Dagamara Dabrowska, Meike Baden, Norbert Helle, LC/GC Chromatography, Sep Authors Figure 5. Sauvignon blanc chromatogram profiles obtained from EG-Siliconeand PDMS Twister extractions of 5 ml samples for one hour at 1000 rpm, polar column separation. Yunyun Nie, Eike Kleine-Benne GERSTEL GmbH & Co. KG, Eberhard- Gerstel-Platz 1, D Mülheim an der Ruhr, Germany 16 GERSTEL Solutions Worldwide No. 12

17 Literature Mass spectrometry in food safety safe and plentiful food supply is a A global concern and laboratories worldwide are working hard to ensure that contaminated food doesn t reach consumers. In Mass Spectrometry in Food Safety: Methods and Protocols, experts in the field provide both context and detailed information. Chapters in the book cover topics such as regulations in various countries as well as stateof-the art methods and instrumentation. Key topics in food safety are covered including the determination of low levels of pesticides, mycotoxins, veterinary drugs and chemical contaminants from packaging materials. One chapter, written by the recognized food analysis expert Dr. Norbert Helle and his team, specifically addresses automated solid phase extraction for preparation and clean-up of food samples in combination with LC-MS/MS. Method chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Dr. Norbert Helle More Information Mass Spectrometry in Food Safety Methods and Protocols Series: Methods in Molecular Biology, Vol. 747 Zweigenbaum, Jerry (Ed.) 1 st Edition., 2011, XII, 416 p. 104 illus. Humana Press, ISBN Innovation Multi-Position Evaporation Station (mvap) Olfactometry More than 1,000 GERSTEL ODPs sold Sniffing out odors A six-position evaporation station (mvap) is available for the GERSTEL MultiPurpose Sampler (MPS). Samples are concentrated under moderate heat and vacuum, enabling significantly improved limits of detection. Solvent exchange to an HPLC- or GC compatible solvent can be performed for improved chromatography. The mvap can be used in combination with SPE, Dispersive SPE (DPX) or liquid/liquid extraction to evaporate solvent from extracts combined with injection to GC/MS or LC/MS. Every step is controlled by mouse-click using the MAESTRO PrepBuilder. Just one method and one sequence table is needed for the entire process including GC/MS or LC/ MS analysis. When you need to pin down odor causing compounds, standard analysis methods quickly reach their limits. Only the parallel use of analytical instrumentation and the human olfactory senses provides real answers. The GERSTEL Olfactory Detection Port (ODP) makes it happen! More than one thousand users have opted for the ODP connected to their GC/MS system not even counting the more recently introduced ODP 3 with heated mixing chamber. The ODP enables sensory detection of odors by the human nose simultaneously with analytical detection by any GC detector, including MSD, FID, and FPD. Voice recognition software allows the sensory analyst to describe odors and fragrances in real time these voice descriptors are recorded and converted to editable text files. For each GC/MS run a complete report is generated, including a chromatogram superimposed with an annotated olfactogram. Text of the descriptors spoken by the analyst is placed above each olfactogram peak. The analyst can assign any of four intensity levels to each eluting component, and the olfactogram is recorded with this intensity information. The ODP is an effective tool for obtaining simultaneous sensory and analytical information to determine flavors and odors in foods, beverages, fragrances, and other complex samples and to help identify sources of odors. GERSTEL Solutions Worldwide No

18 Natural mysteries Ghostbusters in the desert According to the Himba people, the large circular patches with reduced vegetation on the floor of the Namib Desert are a supernatural phenomenon of mystical origin. The fairy circles appear similar to crop circles, a phenomenon known for centuries and by some attributed by some to be signs of extraterrestrial visitors. South African scientists have worked to unearth the secret of the mysterious circles. In their view, the circles are created not by extra-terrestrial forces, but rather by subterranean phenomena. Young Himba girl. by Guido Deußing Sand samples for Naudé et al., 2011 were collected from sampling location 7: NamibRand Nature Reserve. Fairy circles occur in a broken belt in the pro-namib zone of the west coast of southern Africa, extending from southern Angola through Namibia to just south of the Orange River in South Africa. Most of the localities where fairy circles occur lie between the 50 and 100mm isohyet, with some of them extending between the 100 and 150mm isohyet. Map with courtesy from: Van Rooyen M.W., Theron G.K., Van Rooyen N., Jankowitz W.J., Matthews W.S., Mysterious circles in the Namib Desert: review of hypotheses on their origin. Journal of Arid Environments 57, It gets hot, really hot, in the Namib Desert; and really cold as well. In some places temperatures hover above the 50 C mark during the day and descend below the freezing point at night, it is an incredibly dry, arid, but in places grass-covered desert. When the sun rises it not only heats up the desert, it also exposes a spectacular sight: Thousands of mysterious circles cover the vast expanse of the Namib, side by side, like pockmarks on the desert s sand-colored face. The so-called fairy circles are surrounded by tales and myths. Their uniform appearance, sheer number, and distribution have always sparked human imagination. Natural displays of such regularity are typically awarded a deeper meaning or attributed to higher powers and tales are spun. The tales of the Himba, a semi-nomad people in Northern Namibia, relate that a dragon lives underneath the desert sand and breathes fire. The fire rises to the surface in hot bubbles singeing the ground in circular shapes the birth of a fairy circle. Of course the description is naïve like most folklore; however, at a second glance it appears closer to the truth than the explanations offered by scientists in the 1970s and 1980s, which many still cling to. According to those theories, the fairy circles are remnants of termite activity: In their search for food, termites supposedly devoured the thin grass cover in a circular area, presumably never venturing more than a certain distance from their mound. Other experts suspect that ants, clearing the ground of grass seeds, have caused the circular patches with no or dying vegetation. (Basic and Applied Dryland Research 1, 2 (2007), ). This is not the case ; scientists of the University of Pretoria in South Africa now discard previous theories as well as oral tradition. Instead of relying on speculation, chemists Yvette Naudé and Egmont Rohwer from the Department of Chemistry and their colleague Gretel van Rooven from the Department of Botany had gone into the Namib Desert to investigate the matter based on scientific facts using chemical analysis instrumentation and on-site observation of insect activity. Their investigative research allowed the scientists to draw a scientifically based conclusion proving natural geochemically based causes (Journal of Arid Environments 75, 5 (2011) ). The ground is breathing Naudé and her colleagues first went on location in the desert for a visual examination, similar to what police detectives do at a crime scene. For the record: fairy circles are circular areas on the ground, which are free of vegetation or covered by still live (yellowing) or dead (gray) vegetation. They are typically surrounded by comparatively lush vegetation. None of the dead grass in the circle showed marks caused by termite mandibles, Yvette Naudé explains. According to the scientist, the fact that the inner circle contained dead as well as live vegetation indicated that this was a fairy circle in the making. Thus the theory of hungry termites and grass seed-collecting ants could be investigated on site dur- The Namib desert is a cool coastal desert extending for 1,200 miles (1,900 km) along the Atlantic coast from Namibe (formerly Moçâmedes) in Angola southward across Namibia to the Olifants River in the Western Cape province of South Africa. It reaches inland 80 to 100 miles (130 to 160 km) to the foot of the Great Escarpment. The southern portion merges with the Kalahari on the plateau atop the escarpment. Its name is derived from the Nama language, implying an area where there is nothing. The Namib is arid and is almost totally uninhabited (Source: Encyclopedia Brittanica ). 18 GERSTEL Solutions Worldwide No. 12

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20 Fairy circles cover the vast expanse of the Namib like pockmarks on the desert s sand-colored face. ing the formation of a circle and could not be confirmed. Another observation was made and yet another theory formed and tested: the partly lush vegetation at the edge of the fairy circles could indicate the involvement of allelopathic compounds. Some plants release these in order to harm other forms of vegetation that are trying to encroach on their territory. Are plants on the vegetation-rich edge of the fairy circle able to do so? At first glance, the idea seemed both possible and plausible, according to Yvette Naudé. However, potting trials proved that allelopathy did not play a role. Additionally, fairy circles also develop in vegetation-free, sandy areas: The sandy ground in these fairy circles looks shaken and churned up, the researchers describe, similar to craters found on the ocean floor, which are caused by gas bubbles percolating out of the ground. Based on this observation, Yvette Naudé and colleagues developed the hypothesis that gases and fluids of geological origin play a role in the formation of the fairy circles. The scientists theory: Gases and fluids seep in specific migration pathways. Once the gases The tales of the Himba, a semi-nomad people in Northern Namibia, relate that a dragon lives underneath the desert sand and breathes fire. reach the soil surface they start to disperse forming a circle. There are plenty of potential gas sources in Namibia, suggests Yvette Naudé. These include both big oil and natural gas reservoirs and the country s geothermal activity visible at the hot springs in Namibia s spa towns. The scientists started their investigations by determining the soil gas composition in selected fairy circles as well as in the matrix, areas between the circles where there are no geobotanical anomalies. Gas samples were collected by inserting suitable collection funnels into the ground. Several times throughout the day the levels of carbon monoxide (CO), carbon dioxide (CO 2 ), oxygen (O 2 ), hydrogen sulfide (H2S) and nitrogen dioxide (NO 2 ) were determined using a portable gas analyzer. The gas analysis allowed a number of conclusions to be drawn concerning the soil chemistry, reports Yvette Naudé. CO, for example, allowed conclusions about the presence of natural gas. Natural gas is not considered toxic for plants, but it turns out to be an important stress factor for the vegetation. The presence of hydrocarbons was shown to lead to an increase in the activity of oxidizing and reducing (e.g. sulfur-reducing) bacteria, which again lowers the level of oxygen in the soil. This may have farreaching, almost cascading effects for the soil chemistry, the scientists explain: Apart from the oxygen decreasing periodically in the soil of the fairy circles, as we have found during our measurements, an upwelling of gases can lead to increased formation of organic acids, which influence the ph value of the soil and therefore the availability of important minerals for plant development. Plants grown in soil collected from fairy circles, the scientists report further, did not flourish, in contrast to plants that were planted in soil from the vegetation-rich edges of the fairy circles and in the matrix. To develop a clearer picture as to the presence and distribution of hydrocarbon compounds in the soil of the fairy circles, the scientists turned to thermal desorption (GERSTEL TDS) in combination with gas chromatography and mass-selective detection. The advantages of the thermal desorption technique: It is cost effective, simple A different theory was formed and tested by Yvette Naudé and colleagues of the University of Pretoria in South Africa. One of the scientists initial theories: the partly lush vegetati circles could indicate the involvement of allelopathic compo 20 GERSTEL Solutions Worldwide No. 12

21 According to the Himba people, the firy breath of the dragon rises to the surface in hot bubbles singeing the ground in circular shapes the birth of a fairy circle. and it doesn t rely on toxic organic solvents, explains Yvette Naudé. Solvent-free extraction brings several benefits: Cost savings; a cleaner base-line and lower detection limits; a clean chromatogram without solvent peak masking of analytes; higher sensitivity since there is no analyte dilution; and last, but not least, cleaner laboratory air. Thermal desorption GC/MS also requires much less sample than, for example, Soxhlet extraction, which is commonly used for isolating and analyzing hydrocarbons in soil. The scientists took soil samples in carefully selected locations as follows: A 40 gram sample of soil was placed in a prepared glass vial, a polydimethylsiloxane-coated GERSTEL Twister was added and the vial was sealed. The Twister has been used successfully in many applications to extract organic compounds from solid, liquid, and gaseous samples. PDMS is used as sorption phase to concentrate extracted analytes from the sample. Back to the soil samples: The sealed vial was heated to 50 C for 50 minutes. When the scientists then removed the twister from the sample, magnetic particles were clearly visible at its end, another clue confirming the theory of a micro-seepage of hydrocarbon compounds in the ground, which, according to Yvette Naudé, is known to induce microbial changes of magnetic and iron-containing minerals. In order to rule out analytical bias caused by magnetic particles, Yvette Naudé and her colleagues subsequently replaced the Twisters with 10 cm long pieces of PDMS tubing as extraction medium using the same extraction method as described above. The PDMS tubing was subsequently removed from the sample and transferred to a TDS glass tube for thermal desorption in the TDS. The thermally desorbed organic analytes were then cryo-focused in the GERSTEL Cooled Injection System (CIS) GC inlet and transferred to the GC column for separation and mass selective detection. The analysis results support the theory of the scientists: We detected alkenes and microbiological metabolites of alkanes. Higher alkene concentrations were found in soil from the center of the fairy circle and lower concentrations in the matrix, the area without geobotanic anomaly. This suggests that there is strong microbial activity in the soil inside the fairy circles. Additionally, the alkane-alkene concentration ratio found allowed the scientists to draw conclusions as to the activity of a particular fairy circle: higher ratios suggested a newly active circle. Our observations and our results do not support the involvement of termites or ants, concludes Yvette Naudé. However, one thing remained to be explained why are fairy circles round? Yvette Naudé: The overlying sand causes dispersion of rising gases from a seep, creating a funnellike formation which is seen at the surface as a circle. The circles are not all the same size, and can vary dramatically depending on seep rates, sand conditions, and the underlying geology. Another complicated case solved with the help of GERSTEL technology. Acknowledgements Special thanks go to Dr. Yvette Naudé and colleagues as well as to Mr. Marc Springer, Namibia Allgemeine Zeitung, for supplying the photographs used in the article. on at the edge of the fairy unds. Yvette Naudé with Pieter Stoutjesdijk, GERSTEL Regional Sales Manager Europe, Middle East, Africa and India. The scientist solved the puzzle using the GERSTEL TDS and the GERSTEL Twister. GERSTEL Solutions Worldwide No

22 A breath of fresh air When home and office make you suffer Building products used indoors in homes and offices can have significant impact on indoor air quality (IAQ) through emission of volatile- or semi-volatile organic compounds (VOCs/SVOCs). In order to protect the health and well-being of occupants in homes and company buildings from potentially toxic emissions, EU and national regulations require that products used indoors be tested following clearly defined methods. In Germany, and increasingly throughout Europe, the AgBB evaluation scheme is used. Material emission testing relies mainly on environmental test chambers combined with sampling of chamber air onto adsorbent tubes and Thermal Desorption GC/MS analysis. Tests generally take 28 days, but thermal extraction offers an easier and less expensive way of getting reliable information about product emissions, for R&D purposes or for quality control of existing approved products. PVC, linoleum, carpeting, laminate, parquet, and cork the choice of floor covering for homes and offices seems almost endless - and once you have chosen the type, a similarly endless choice of producers and quality levels can cause headaches even before the flooring has been installed. Unfortunately, a successful installation may not quite signal the end of your headaches. If the carefully chosen flooring or the glue used to install it - emits VOCs/SVOCs, these could contaminate the indoor air and even cause irritation and negative health effects. And to top it off, reactions to contaminants in air are highly individual, varying significantly from person to person. Hardened breathers of inner city air and perpet- Used for the determination of material emissions: The GERSTEL TDS/TDS A2 mounted on a GC/MS System. ually recycled indoor atmospheres in modern energy-efficient buildings may feel nothing. Others may be in for constant suffering while in the building. And the list of real or perceived symptoms is endless. If headaches, mucus membrane irritation, fatigue, allergic reactions, immune system deficiency, frequent infections, deterioration of pre-existing asthmatic conditions, depressions, or simply a sudden general lack of well-being occurs after moving into a new building or after a building has been renovated or redecorated, the informed physician should not exclude a case of sick building syndrome (SBS). According to the US EPA, indicators of SBS include: Building occupants complain of symptoms associated with acute discomfort, e.g., headache; eye, nose, or throat irritation; dry cough; dry or itchy skin; dizziness and nausea; difficulty in concentrating; fatigue; and sensitivity to odors. The cause of the symptoms is not known. Most of the complainants report relief soon after leaving the building. We humans in modern society spend most of our lives indoors, depending on the season up to % of every day. This means that IAQ in homes and offices has significant and decisive influence on our health and well-being. Temperature and relative humidity (RH) are also critical factors. In addition, VOC- (C 6 -C 16 ) and Text: Guido Deußing 22 GERSTEL Solutions Worldwide No. 12

23 SVOC (>C 16 -C 22 ) contamination plays a role that is increasingly in focus of regulating government agencies. Many construction products used in buildings are potential sources of VOC- or SVOC emissions. Apart from the flooring materials and the glues used to install them, some of the culprits may be paints, lacquers, varnishes, coatings, wood preservation products, wall paper, caulks and sealants, cement, prefabricated bricks, and concrete. We are surrounded by a huge range of industrially produced materials that contain a long list of ingredients and additives to make them easy to use, low cost and durable. The European Union is recognizing the importance of this area and is moving towards regulation of emission of chemicals into indoor air. In the Proposal for a Regulation of the European Parliament and of the Council laying down harmonized conditions for the marketing of the construction products the following is stated: Annex I, Part 3. Hygiene, health and the environment: The construction works must be designed and built in such a way that they will not be a threat neither to the hygiene nor health of the occupants and neighbors, nor exert a exceedingly high impact over their entire life cycle to the environmental quality nor to the climate, during their construction, use and demolition, in particular as a result of any of the following: (a) the giving-off of toxic gas; (b) the emissions of dangerous substances, Fig.: AgBB scheme for health-related evaluation of construction products. Gerd Bittner: A product that meets the AgBB criteria is well suited for use indoors in buildings. * VOC: Retention time range equal to C 6 -C 16, SVOC: Retention time range equal to > C 16 -C 22, ** LCI: Lowest Concentration of Interest. volatile organic compounds (VOC), greenhouse gases or dangerous particles into indoor or out door air; Etc. The European Union states that it takes into account the extraordinary importance of the European Construction Products Directive for the well-being of building occupants. The German Federal Environmental Agency (Umweltbundesamt UBA) states: Building products used to construct a building or incorporated into a building must in particular fulfill these requirements that no chemical, physical, or biological influences pose any danger or give rise to inappropriate inconvenience ( 16 MBO). Uniform assessment protocol So far, so good. But, to paraphrase a popular saying, good intentions don t always pave the way to paradise. True to the old credo: Trust, but verify. Construction products should be checked in a standardized way in order to even the playing field for producers by applying the same rules to everyone while allowing the consumer to win by being allowed to live, work, and play in a healthy indoor environment. The Committee for Healthrelated Evaluation of Building Products produced the AgBB evaluation scheme, which is used in Germany and increasingly throughout Europe. The process enables a clear and uniform assessment of emissions of VOCs and SVOCs under standard conditions. These test conditions for flooring have given us, for the first time, a set of stan- Causes of Sick Building Syndrome: According to the US EPA, the following have been cited as causes of or contributing factors to sick building syndrome: Inadequate ventilation Chemical contaminants from indoor sources: For example: adhesives, carpeting, upholstery, manufactured wood products, copy machines, pesticides, and cleaning agents may emit volatile organic compounds (VOCs), including formaldehyde) Chemical contaminants from outdoor sources: For example, pollutants from motor vehicle exhausts; plumbing vents, and building exhausts (e.g., bathrooms and kitchens) can enter the building through poorly located air intake vents, windows, and other openings. In addition, combustion products can enter a building from a nearby garage) Biological contaminants: Bacteria, molds, pollen, and viruses are types of biological contaminants. These contaminants may breed in stagnant water that has accumulated in ducts, humidifiers and drain pans, or where water has collected on ceiling tiles, carpeting, or insulation. These elements may act in combination, and may supplement other complaints such as inadequate temperature, humidity, or lighting. Even after a building investigation, however, the specific causes of the complaints may remain unknown. Source: USEPA - GERSTEL Solutions Worldwide No

24 LCI values LCI is an acronym for Lowest Concentrations of Interest, i.e. the lowest concentration of toxicological relevance for a particular compound in indoor air in residential and office buildings. LCI values are not equivalent to or related to Occupational Exposure Limits (OELs) or Recommended Exposure Levels (RELs) as specified for occupational safety. OEL values OEL is an acronym for Occupational Exposure Limits, By definition, the occupational exposure limit is the contaminant level to which you can be exposed continually, day after day during your whole working life without experiencing any negative health effects as a result. AgBB Committee for health related assessment of construction products (German: AgBB) was formed in 1997 by a Working Group for Environmental Health Protection brought together under a cooperation between the Health Authorities of the German States. Among the members of the AgBB are State Health Authorities, the German Federal Environmental Protection Agency (UBA), the German Institute for Construction Technology (DIBt), The federal Institute for Material Research (BAM), and various other regional and federal State Agencies. Some test methods for Emission testing of flooring materials DIN EN ISO Emission chamber test method. DIN EN ISO Sampling, storage, and preparation of samples. DIN ISO Determination of VOCs in indoor air and in Environmental test chambers. Sampling on TenaxTA followed by Thermal Desorption GC/MS. DIN ISO Determination of formaldehyde and other carbonyl compounds; Sampling Evaluation scheme for health related assessment of emissions from construction products (AgBB) German Institute for Construction Technology (DIBt) Product Approval details More Information Gerd Bittner, Textiles & Flooring Institute (TFI), Charlottenburger Allee 41, Aachen, Germany, Phone dardized test conditions for approval of flooring materials that are used for an annual verification check of the emission properties of approved products, states Gerd Bittner of the Textiles & Flooring Institute (TFI) in Aachen, Germany. Testing of flooring materials and flooring systems (i.e. including the glue used to install the flooring) is performed at the TFI using environmental test chambers based on the DIN EN ISO , DIN EN ISO , and DIN ISO standards for indoor air. These standards specify conditions for all aspects of testing various flooring materials in environmental test chambers as well as the analytical determination of identity and concentration of emitted organic compounds (VOCs/SVOCs). Chamber air is collected using active pumped sampling onto a suitable adsorbent tube after three and 28 days. The tubes are typically filled with Tenax TA and the analysis, as specified in the AgBB scheme is performed by Thermal Desorption - Gas Chromatography combined with Mass Spectrometry Detection (GC/MS) of the analytes. A non-polar separation column is used, which means that individual analytes can be assigned to a boiling point range or retention time range C 6 -C 16 (VOC) or >C 16 - C 22 (SVOC) as specified in the AgBB scheme for health-related evaluation of construction products rev The term individual analytes refers to both identified and non-identified compounds. The AgBB scheme requires a limit of detection of 1 μg/m 3 for each compound in order to comprehensively cover and describe the material emissions. Depending on the specific requirements, quantitative information on individual compounds must be obtained. Whenever individual compound concentrations exceed 5 μg/m 3, they must be quantified both individually and in summation as part of the relevant group. Exceptions are EU category 1 and 2 carcinogens. For identified carcinogens and compounds that have an LCI value, compound specific quantification must be performed. Unidentified compounds as well as compounds to which no LCI value is assigned are quantified as toluene equivalents. GERSTEL TE: Thanks to the large thermal extraction tube, a range of different sample types and amounts can be analyzed based on thermal extraction in the TE. Thermal extraction as a highly suitable rapid test method The test over 28 days, as required in the AgBB scheme, results in a comprehensive and standardized emission profile, according to Gerd Bittner. Typical peak patterns can be observed and compared during data analysis and key analytes are therefore easily found. Emissions from different materials are easily compared both quantitatively and qualitatively, and quantitation using internal standard, typically expressed as an equivalent toluene concentration, is easily performed as specified for the unknown minor compounds and those without LCI values. However, emission chamber test results take almost a month to produce and they are highly labor- and cost intensive. This poses a serious problem for the industry, especially during product development: Test cycles of a month can cause significant project delays with serious consequences, for example, in terms of development cost and loss of competitiveness. A clear indication of the emission profile of a product in every development stage, or during trouble shooting following customer complaints, can save companies both lots of time and pots of money. For these reasons, the TFI has for many years offered their customers accelerated emission tests based on thermal extraction, a dynamic headspace technique based on trapping on a standard adsorbent tube. Industry clients come to the TFI for emission tests during product development; for regular Quality Control of product batches; for trouble shooting following customer complaints; as well as for sample identity verification. Testing is often performed using the GERSTEL Thermal Extractor (TE). The large extraction tube of the TE (ID 14 mm, length of heated zone 75 mm) can be loaded with much larger and more representative samples than regular thermal desorption tubes. We use the Thermal Extractor to test textiles, elastic flooring material, multi-layer systems, as well as glues used to install flooring material, reports Mr. Bittner. The samples are heated in a flow of inert gas and the extracted analytes are purged onto the adsorbent tube and concentrated on TenaxTA. Thermal Desorption (TD)-GC/MS analysis is subsequently performed following the AgBB guidelines. By adapting the Thermal Extraction methods used to the corresponding emission chamber methods, we have achieved good qualitative correlation between thermal extraction and emission chamber tests for various materials; in other words, we find the same typical peak patterns, making it easy to compare results, says Gerd Bittner, before concluding: In our experience, the accelerated thermal extraction tests give us the ability to quickly establish the emission potential of flooring materials and their associated adhesive systems, as well as to compare emissions from different combinations. Thermal extraction is a valuable and efficient complement to standard emission chamber tests. 24 GERSTEL Solutions Worldwide No. 12

25 Thermal extraction using the GERSTEL TE: an efficient alternative to environmental chamber testing practical examples from the TFI Thermal extraction of a carpet tile Thermal extraction of a raw material 1 Ethylbenzene 2+3 Total xylenes 4 Butylglycol 15 Butyldiglycol 16 2-ethyl-hexanoic acid Optimization of carpet tiles I: A carpet tile was taken from a production facility for accelerated VOC/ SVOC emission testing. The result: Large compound peaks were found in the retention time range from min. To determine the source(s) of the emissions, all raw products used in the production of the carpet tile were individually tested for emissions using thermal extraction. The TFI quickly identified the source and was able to propose a clear strategy for optimizing the carpet tile production to ensure lower material emissions. Thermal extraction of carpet tile before change Thermal extraction of carpet tile after optimization AgBB test result summary after three days. Requirement TVOC (C 6-C 16) 599 µg/m³ 300 µm/m³ R TVOC (without LCI) 223 µg/m³ 100 µg/m³ AgBB test result summary after three days. Requirement TVOC (C 6-C 16) µg/m³ 300 µm/m³ R TVOC (without LCI) µg/m³ Optimization of carpet tiles II: Thermal Desorption GC/MS chromatogram of analytes extracted from a carpet tile sample using thermal extraction. Major compound peaks were found in the retention time range from 8 14 min (upper chromatogram). The source of the emissions was identified and substituted. The change was successful as can be seen in the lower chromatogram, showing the emission test of the optimized product. Environmental test chamber Thermal extraction Useful tool and great complement: Comparison of emission tests performed after three days in an environmental test chamber (above) and accelerated emission testing using thermal extraction (below). Good correlation is seen between the peak patterns in the two chromatograms, both obtained by Thermal Desorption-GC/ MS. Thermal extraction results in a more efficient recovery of higher boiling compounds as can be seen. Thermal Extraction is an efficient tool for evaluating intermediate or final products either in the product development stage; for trouble shooting following customer complaints; or for production control. GERSTEL Solutions Worldwide No

26 Automated determination of glycerin in biodiesel Sustainable fuel When Biodiesel is produced, glycerin is generated as a by-product, and it must be removed since it can cause damage to diesel engines. EU- and U.S. guidelines specify the maximum allowable concentrations of free and total glycerin in Biodiesel. A standard method based on GC/FID is available, but it is relatively labor intensive. If the right autosampler and sample preparation robot is used, the entire process can be automated. Biodiesel is in many ways comparable to mineral oil based diesel fuel. However, unlike conventional diesel fuel, biodiesel is not obtained from crude oil but from renewable raw materials: In the U.S. mainly from soybean oil, in Europe often from rapeseed oil. When the books are balanced as to how environmentally friendly we are, Biodiesel is counted as renewable energy and is regarded as a sustainable fuel if certain criteria are met. Chemically speaking, Biodiesel consists of fatty acid methyl esters (FAMEs). Depending on which raw material was used to produce the fuel, the FAMEs are classified as either soy methyl esters (SMEs) or rape methyl esters (RMEs). Regardless of type and origin of the basic biogenic raw material, FAMEs are produced by trans-esterification of fats and oils (triglycerides). In the process of the alkaline or acidic catalyzed reaction, the trivalent alcohol glycerin is substituted by methanol in order to ensure adequate viscosity of the resulting fuel at a wide temperature range. In the U.S., Biodiesel for use in Diesel engines must conform to ASTM D6751 and the amount of Glycerin is determined using ASTM Method D Standard Test Method for the Determination of Free and Total Glycerin in B-100 Biodiesel Methyl Esters by Gas Chromatography. Glycerin, an undesirable travel companion Apart from FAMEs or SMEs and RMEs, substandard glycerin (SSG) is generated during the Biodiesel production process and a residue is formed consisting of glycerin, water, catalyst, excess methanol and free fatty acids. The SSG by-product is toxic and flammable, but unsuitable as a fuel and generally undesirable, since it forms solid sediment, which can block the fuel filter. When separated from biodiesel, SSG can be purified and re-introduced into the production stream. Additionally, SSG is an important raw material in the production of pharmaceutical and industrial glycerin. Improved analytical efficiency with automation Pure Biodiesel is referred to as B 100 diesel. Some engines can operate on B 100 diesel, but in most cases a mixture of the biogenic fuel and mineral oil based diesel is used. In Germany, the Biofuel Quota Act was enacted in 2007, making it mandatory to add as much as five percent biodiesel to conventional diesel (B 5). If you are not sure whether your vehicle should be operated on pure biodiesel or on a mixture such as B 5, you should contact the vehicle manufacturer for advice. Determining whether biodiesel is free from glycerin requires a suitable analysis method. For the determination of the amount of free and total glycerin and of mono-, di- and triglycerides, European standard EN and its American counterpart ASTM Method D6584 prescribe the use of gas chromatography (GC) with flame ionization detection (FID). The analytes must first be transferred into a form that is suitable for GC and this is done through derivatization, which is normally a tedious, labor intensive and time-consuming task, Dr. John R. Stuff explains. Dr. Stuff and Jacqueline A. Whitecavage, experienced application chemists from GERSTEL, Inc. in Baltimore, MD set out to automate the method from A to Z, reducing the workload while maximizing sample throughput. The sample preparation steps were transferred to the autosampler and fully synchronized with the GC run to ensure that the GC never has to wait for the next sample to be ready. Dual syringe system for liquid handling For the analysis, Stuff and Whitecavage used a GC 6890 from Agilent Technologies with a GERSTEL Cooled Injection System (CIS 4) and FID. Automated sample preparation and sample introduction to the GC was performed using the Dual Rail version of the GERSTEL MultiPurpose Sampler (MPS). The MPS was equipped with two different syringe sizes, a 10 μl on-column syringe and an 80 μl sideport syringe with dilutor module. Bio- 26 GERSTEL Solutions Worldwide No. 12

27 GC/FID system with GERSTEL Dual Rail MPS used to automate ASTM Method D : Standard Test Method for the Determination of Free and Total Glycerin in B-100 Biodiesel Methyl Esters by Gas Chromatography. Samples are prepared during GC analysis of the preceding sample. Whenever the GC becomes ready, the next sample is ready to be injected ensuring best possible system utilization. The screenshot (MAESTRO Sequence Scheduler) illustrates the high efficiency of the biodiesel analysis using MPS GC/FID. Chromatogram of a biodiesel standard. diesel standards containing glycerin, monoolein, diolein, triolein, butanetriol, and tricaprin, all in pyridine, as specified in ASTM D were purchased. Biodiesel B-100 was purchased locally. The glycerin, mono-, di- and triolein standards as well as butanetriol, tricaprin and MSTFA were placed in separate vials in the MPS for further processing. Biodiesel B-100 samples were weighed directly into 10 ml screw cap vials and placed on the MPS tray. The samples and standards were prepared by the MPS based on a GERSTEL MAESTRO Prep- Sequence. ASTM D specifies preparation of a five point calibration curve for glycerin, mono-, di-, and triolein from stock standards. Heptane was used for rinsing and dilution. Derivatization was performed using N-methyl-N-trimethylsilyl trifluoracetamide (MSTFA). To reduce the required number of manual steps, the GERSTEL, Inc. scientists set up the MultiPurpose Sampler (MPS) for automated sample preparation. The necessary instructions: Add, Move, Mix, Dilute, Wait, and Inject are selected via mouse click in the menu of the MAESTRO control software PrepBuilder and added to the individual prep method. MAESTRO operates fully integrated into the ChemStation and GC MassHunter software (Agilent Technologies). The manual effort is reduced to the weighing of 100 mg of the sample into 10 ml headspace vials and placing them in Automated process steps with the MPS the MPS sample tray, explains Jacqueline Whitecavage. Standards were prepared in empty 10 ml vials placed on the autosampler. All further steps are fully automated and synchronized for best possible throughput: Standard preparation, adding internal standards and derivatization reagent, mixing, incubating, rinsing, and introducing the sample into the CIS. The automated steps of the procedure are performed by the MPS. Processing a sample in the MPS requires approximately 27 minutes. The GC run takes a total of 38 minutes including a seven-minute cool-down phase. We optimized the method to ensure maximum throughput, said John R. Stuff. In other words, the MPS prepares the calibration standards at the required concentration levels, after which the first sample is derivatized and injected into the CIS. The next sample is always prepared just-in-time for when the GC is ready for the next run. The scientists are satisfied with their results: The results show good linearity for the standards and a good reproducibility (RSDs range from 2.1 to 2.5 %) for the biodiesel sample. Automating the sample preparation process means that valuable resources can be used more efficiently and laboratory staff is less exposed to potentially toxic solvents and reagents. Furthermore, system productivity can be maintained overnight and throughout the weekend meaning that valuable instrumentation is used much more efficiently. Analysis conditions Chromatogram of a real biodiesel sample analyzed using the MPS-GC/FID system. Diolein calibration curve. Reference J. R. Stuff, J. A. Whitecavage. Full Automation of ASTM Method D Standard Test Method for the Determination of Free and Total Glycerin in B-100 Biodiesel Methyl Esters by Gas Chromatography using a GERSTEL Dual Rail PrepStation. GERSTEL AppNote 1/2010 ( Text: Guido Deußing 1. Addition of 100 µl butanetriol solution as internal standard 1 2. Addition of 100 µl tricaprine solution as internal standard 2 3. Addition of 100 µl derivatization reagent 4. Mix (1 min) 5. Wait (15 min) 6. Dilution with 8 ml heptane 7. Mix (1 min) 8. Sample injection 1 µl on-column CIS: Column: On-column; 60 C (0.05 min); 0.2 C/s to 230 C (2 min); 0.5 C/s to 380 C (10 min) 10 m Rtx-Biodiesel TG (Restek), ID = 0.32 mm, df = 0.1 µm Carrier gas: Helium, 3 ml/min (constant flow) GC Oven: 50 C (1 min); 15 C/min to 180 C; 7 C/min to 230 C; 30 C/min to 380 C (10 min) FID: 380 C GERSTEL Solutions Worldwide No

28 Fragrance Analysis Fabric softeners caught in a whirl To determine fragrances used in household products and detergents, liquid-liquid extraction (LLE) combined with GC/MS is often used. Scientists in the Firmenich Research and Development department in Singapore have now turned to Stir Bar Sorptive Extraction (SBSE) in their search for an alternative extraction method with promising results. Fabric softeners are not too important for the washing process itself, but they offer many positive effects that are appreciated at the ironing board at the very latest. Additionally, fabric softeners endow textiles and clothes with a distinct fragrance, helping consumers to form lasting bonds with the product. What we perceive as a single scent or fragrance is typically a complex mixture of individual fragrance compounds. These interact to form the complete olfactory impression that distinguishes the consumer product. It is the task of the quality control department to ensure batch to batch uniformity of the olfactory impression or fingerprint. How efficiently this is achieved depends to a large extent on the extraction and sample preparation techniques used. To isolate volatile fragrance compounds from soaps, detergents, or fabric softeners, the chemist traditionally reaches for the liquid/liquid extraction (LLE) technique. LLE is based on differences in analyte solubility in two immiscible solvents. These are typically a hydrophilic (aqueous) phase on the one side and a hydrophobic organic solvent on the other side of the phase divide. According to Khim Hui, scientist at Firmenich Asia Private Ltd. in Singapore, this is where the drawbacks of the LLE technique are seen: The LLE technique is able to selectively pick out interesting analytes from complex matrices, but overall, this sample preparation technique is very labor intensive and cumbersome. Getting good phase separation is often difficult when the sample to be extracted contains surfactants. Furthermore, large quantities of costly solvent are required; solvents must be discharged in a responsible manner after use, which is both expensive and time consuming. And, last but not least, solvents can have a negative impact on the laboratory work environment. The scientists Khim Hui and Diana Koh started searching for an attractive alternative to the LLE technique: We looked for a technique that would minimize or even eliminate solvent use while extracting fragrance compounds with good recovery and delivering first rate validation data, Diana Koh states. After a comprehensive search, the scientists decided to pursue Stir Bar Sorptive Extraction (SBSE) using the GERSTEL Twister [1]. Over the past decade since its introduc- Analysis conditions TDU : Initial temperature: 50 C; delay time: 0.50 min; initial time: 0.00 min; ramp at 80 C/ min; end temperature: 150 C; hold: 1.00 min; Desorption mode: splitless CIS 4 : Initial temperature: 10 C; equilibration time: 0.50 min; initial time: 0.00 min; ramp at 12 C/s; end temperature: 280 C; hold: min; CIS 4 liner Tenax TA; Vent time: 0.00 min; Purge time: 0.00 min; Purge flow: 80 ml/min Agilent GC/MS 6890/5973N GC oven: 50 C (3.00 min); 3 C/min; 260 C 20 C/min; 300 C (5.00 min) Column: HP-5MS, 30m x 0.25mm x 0.25μm Constant flow rate: 1.2 ml/min, helium MSD: Mass range: , Quad: 150 C, Source: 230 C 28 GERSTEL Solutions Worldwide No. 12

29 0.25 R e 0.2 R s a 0.15 p o n t i o 0.1 s e min 1 hour 2 hour 3 hour 4 hour Time (t) Graph 1 and 2 (left to right): Response Ratio versus different extraction times R e s p o n s e R a t i o Response Ratio vs Extraction Time Response Ratio vs Stirring Speed Stirring Speed (rpm) EUCALYPTOL RM 1 ZESTOVER RM 2 CAMPHOR RM 3 HEXYLCINNAM RM 9 IC ALDEHYDE DECAL RM 5 EUCALYPTOL RM 1 ZESTOVER RM 2 CAMPHOR RM 3 HEXYLCINNAMIC RM 9 ALDEHYDE DECAL RM 5 Graph 3 and 4 (left to right): Response Ratio versus different stirring speeds tion, the Twister has proven its worth by efficiently extracting volatile- and semivolatile organic chemical compounds (VOCs and SVOCs) from a wide range of complex matrices. SBSE not only requires little, if any, solvent, says Khim Hui, it is also surprisingly simple to perform. SBSE is performed using a glass encased magnetic stir bar with a relatively large volume of sorbent phase coated on the outside. While the Twister stirs the sample, analytes are efficiently extracted into the sorbent phase, in this case PDMS, recovery rates are generally much higher than those obtained with SPME. A large number of samples can be extracted simultaneously and the subsequent Twister Desorption and analyte determination is fully automated making the overall process highly efficient. A GERSTEL Thermal Desorption Unit (TDU) mounted on a GERSTEL Cooled Injection System (CIS 4) PTV-type GC inlet and a GC 6890 /5973N MSD from Agilent Technologies were used. R e s p o n s e R a t i o min 1 hour 2 hour 3 hour 4 hour Time (t) R e R s a p t o i n o s e Response Ratio vs Extraction Time Response Ratio vs Stirring Speed Stirring Speed (rpm) VERDOX RM 4 LILIAL RM 6 LILIAL RM 6 AMYLCINNAMIC RM 7 ALDEHYDE HEXYL RM 8 SALICYLATE GALAXOLIDE RM MIP AMYLCINN RM 7 ALDEHYD HEXYL RM 8 SA VERDOX RM 4 GALAXOL RM 10 As an aside, SBSE is based on the partition of analytes between PDMS and the sample. The Twister stirs the sample and nonpolar or moderately polar compounds are extracted and concentrated. The partition coefficient of a compound between PDMS and water is very close to its K O/W value (octanol/water partition coefficient). K O/W is a physical chemical parameter used to describe hydrophilic or hydrophobic properties of a compound [2]. K O/W has been used, among other things, to describe whether an environmental pollutant such as a pesticide was likely to accumulate in fat tissue. A high Log K O/W value is characteristic of a hydrophobic compound that would be extracted with high recovery using PDMS. Theory guides experiment decides To test the SBSE technique for their applications, the scientists analyzed fabric softener samples spiked with the following flavor and fragrance compounds: 1,8-cineol (RM 1), Zestover (RM 2), camphor (RM 3), Verdox (RM 4), Decal (RM 5), Lilial (RM 6), amylcinnamaldehyde (RM 7), hexylsalicylate (RM 8), hexyl-cinnamaldehyde (RM 9) as well as Galaxolid 70 MIP (RM 10). An internal standard (IS) was added to the Twister stir bar prior to the extraction by letting it stir an IS solution, which was generated by adding 100 μl IS stock solution (150 mg/l of 1,4-dibromobenzol in acetonitrile) to 20 ml deionized (DI) water and letting the Twister stir the IS solution for an hour. The kinetics of the extraction depend on the analyte migration rate to and into the PDMS phase of the Twister, which among other things, depend COMPOUNDS Mean STDEV % RSD Eucalyptol Zestover Camphor Verdox Decal Lilial Amylcinnamic Aldehyde Hexyl Salicylate Hexylcinnamic Aldehyde Galaxolide 70 MIP Table 2: Calculated %RSD for Reproducibility (Mean of 50 measurements) on diffusion rates, stirring conditions, and the sample volume. Diana Koh states: The peak area depends on the extraction time. In their study, the scientists determined the optimal extraction time to be one hour at room temperature while stirring the Twister at 800 rpm. To get reliable data, Diana Koh performed the analysis over five different days, achieving good results with good reproducibility and RSDs of less than 16 percent across the board. From the 50 determinations per compound performed over five days, Diana Koh determined the reproducibility to be less than 12.5 %. Three-point calibration curves Repeatability Day 1 Day 2 Day 3 Day 4 Day 5 COMPOUNDS Mean STDEV RSD % Mean STDEV RSD % Mean STDEV RSD % Mean STDEV RSD % Mean STDEV RSD % Eucalyptol Zestover Camphor Verdox Decal Lilial Amylcinnamic Aldehyde Hexyl Salicylate Hexylcinnamic Aldehyde Galaxolide 70 MIP Table 1: % RSDs calculated based on 10 individual sample extractions for every listed result. GERSTEL Solutions Worldwide No

30 Response Ratio EUCALYPTOL RM 1 Response Ratio ZESTOVER RM 2 Response Ratio CAMPHOR RM 3 Response Ratio VERDOX RM 4 Response Ratio RM e e e-002 y=5.409e -002 R 2 = e-003 y=6.715e -002 R 2 = e-002 y=8.646e -002 R 2 = y=6.097e -001 R 2 = e-002 y=2.176e -001 R 2 = Concentration Ratio Concentration Ratio Concentration Ratio Concentration Ratio Concentration Ratio Response Ratio RM 6 Response Ratio RM 7 Response Ratio RM 8 Response Ratio RM 9 Response Ratio RM y=7.397e -001 R 2 = y=2.111e -001 R 2 = y=2.541e -001 R 2 = y=7.646e -002 R 2 = y=8.549e -002 R 2 = Concentration Ratio Concentration Ratio Concentration Ratio Concentration Ratio Concentration Ratio Graph 5: Twister calibration curve of each compound Method Comparision Method % Dosage found % Leakage (Corrected) LLE SBSE Table 3: Correlation of determination (R 2 ) were established for each fragrance compound added resulting in good linearity with R 2 - values higher than for all compounds. Comparison of SBSE and LLE Chromatogram 1: Monitored perfume raw materials in Liquid Softener using SBSE technique. Comparing LLE & SBSE method at same dosage (with Encapsulation Technology in softener application) Chromatogram 2: Profile from Liquid-liquid Extraction. As observed, there is little/no signal detected for monitored compounds (10 RMs). But how did SBSE stack up to the LLE technique, which has been tested and validated extensively throughout the company and proven for real life samples over a long period of time? Khim Hui: In order to compare the techniques, reference samples were prepared at different concentration levels: (0.30 %; 0.50 % and 0.75 %). It was shown that the analysis results obtained using SBSE were quite close to the results obtained with the traditional LLE technique. Further, the comparison showed that SBSE resulted in much higher sensitivity than LLE. The final conclusion: SBSE is a fast, sensitive, and highly reproducible alternative to standard sample preparation techniques such as LLE for the determination of fragrance compounds in fabric softener. In addition, SBSE can be performed using much smaller sample sizes while still allowing quantitative determination at lower concentration levels. Last, but not least, SBSE allows us to reduce use of organic solvents dramatically, this is a very positive result, the scientist says. References 1. C. Franc, F. David, G. de Revel. J. Chromatogr. A 1216 (2009) M.W. Maylan, P.H. Howard; J. Pharm. Sci. 1995,84, Chromatogram 3: Profile from Stir Bar Sorptive Extraction. Sensitivity is enhanced, thus allows detection of raw materials leakage in trace analysis. More information Diana Koh Guat Fen, Khim Hui Ng, Firmenich Asia Private Limited, 10 Tuas West Road, Singapore Text: Guido Deußing 30 GERSTEL Solutions Worldwide No. 12

31 New GERSTEL K.K. offices in Tokyo Customer focused solutions with a difference Growing demand for GERSTEL Solutions over a number of years has also resulted in a steady growth in GERSTEL K.K. team members. In November 2011 GERSTEL K.K. became the fourth GERSTEL organization, after Germany, the U.S., and Switzerland, to move to new and larger offices. The benefit: significantly more room, better facilities, and a central location to provide the best possible support for our customers in terms of stock availability and customer focused solutions with advanced application support, a hallmark of the GERSTEL K.K. organization. In 2004, six years after Dr. Manfred Schwarzer first moved to Tokyo to support the Japanese distributor Yokogawa Analytical Systems Inc., GERSTEL K.K. was founded. At that time, Dr. Schwarzer moved back to Germany to take up the key position of Software Development Manager, and Hirooki Kanda joined New and larger: the GERSTEL K.K. offices are centrally located in Tokyo, Japan. The GERSTEL K.K. team. GERSTEL K.K. as General Manager. Mr. Kanda and his team have further developed GERSTEL K.K. into an accepted brand and house-hold name in Japan for automated sample preparation and sample introduction. Based on a strong focus on solutions and applications, GERSTEL K.K. has produced solid and consistent year-on-year growth since it was founded. Among the customers are major global players in fields such as Automotive, Semiconductors, Flavor and Fragrance, Food and Beverage producers, as well as Pharmaceutical companies and water suppliers. The customers are supported by an internationally renowned team of application scientists under the leadership of Dr. Nobuo Ochiai. Thanks to close ties and strong cooperation with international partners and scientists, all signs point to further growth for the GERSTEL organization. GLOBAL ANALYTICAL SOLUTIONS GERSTEL and Agilent - 25 years of partnership On November 30, 2011, GERSTEL management joined Agilent Technologies European Management to celebrate 25 years of successful global partnership. GERSTEL Presidents/Owners Eberhard G. Gerstel and Holger Gerstel along with Managing Director Ralf Bremer and representatives from the GERSTEL sales, service, and marketing organizations attended the ceremony at the Agilent facilities in Waldbronn, Germany. Danilo Cazzola, Vice President EMEA Sales, Marketing and Service, Maurizio Rosati, Senior Director of Sales EMEAI, and Fred Strohmeier, Vice President and General Manager, Agilent Deutschland GmbH congratulated GERSTEL, citing multiple highlights of the cooperation on both sides over the years. Most recently, a customer survey returned a highly favorable customer satisfaction rating for combined GERSTEL- Agilent customers. Eberhard G. Gerstel received an engraved anniversary trophy to add to the long line of annual prizes awarded to GERSTEL for top performance. GERSTEL is ranked as Premier Solution Partner, Platinum Level, the only Agilent Solution Partner world-wide to have reached this level. In the mid-eighties, Fred Strohmeier Eberhard G. Gerstel Danilo Cazzola both companies recognized their high synergypotential and the opportunity to do more for customers. GERSTEL sample preparation and sample introduction technologies are integrated with Agilent GC/MS and LC/MS instrumentation under unified software control and the complete solution is supported by GERSTEL. The potential was first recognized 25 year ago and, a great deal has been accomplished since then. Both parties agreed that this was only the beginning of the story a partnership worth celebrating. GERSTEL Solutions Worldwide No

32 Literature Flavor, Fragrance, and Odor Analysis, Second Edition There are many advantages to stir bar sorptive extraction (SBSE) for isolating and concentrating flavor active chemicals from foods. These include simplicity, wide application range, efficient analyte concentration, and generally the absence of masking solvent peaks. Written from a practical, problem-solving perspective, the second edition of Flavor, Fragrance, and Odor Analysis highlights this powerful technique and emphasizes the range of applications available. Topics discussed include: Sequential SBSE, a novel extraction procedure A simplified method for switching from one-dimensional to two-dimensional GC/ MS How to improve analytical sensitivity and recovery of phenolic compounds with aqueous acylation prior to SBSE GC-MS Analyzing and combating off-flavors caused by metabolites from microorganisms A technique for measuring synergy effects between odorants The identification of the characterizing aroma-active compounds of tropical fruits with high economic potential The parameters utilized during the production of aqueous formulations rich in pyrazines How spectral deconvolution can be used to speciate the subtle differences in essential oil content and track key ingredients through the manufacturing process The final chapter summarizes chemical identities of characterizing aroma chemicals in fruits, vegetables, nuts, herbs and spices, and savory and dairy flavors. It also provides a brief compendium of the characterization of off-flavors and taints that are reported in foods. With contributions from a distinguished panel of international experts, this volume provides chemists and researchers with the latest techniques for analyzing and enhancing food flavor and fragrance. More Information Ray Marsili (Edi.), Flavor, Fragrance, and Odor Analysis, Second Edition, 280 Pages, ISBN-10: and ISBN-13: GERSTEL online: Information on products, applications, events and downloads, as well as general information about GERSTEL and our customer focused solutions: and Imprint Published by GERSTEL GmbH & Co. KG Eberhard-Gerstel-Platz Mülheim an der Ruhr, Germany Editorial director Guido Deussing Uhlandstrasse Neuss, Germany guido.deussing@pressetextkom.de Scientific advisory board Dr. Eike Kleine-Benne eike_kleine-benne@gerstel.de Dr. Oliver Lerch oliver_lerch@gerstel.de Dr. Malte Reimold malte_reimold@gerstel.de Translation and editing Kaj Petersen kaj_petersen@gerstel.com Design Paura Design, Hagen, Germany ISSN / G L O B A L A N A L Y T I C A L S O L U T I O N S GERSTEL, Inc., USA sales@gerstelus.com GERSTEL BRASIL gerstel-brasil@gerstel.com GERSTEL GmbH & Co. KG, Germany gerstel@gerstel.com GERSTEL AG, Switzerland gerstel@ch.gerstel.com GERSTEL K.K., Japan info@gerstel.co.jp GERSTEL LLP, Singapore sea@gerstel.com Subject to change. GERSTEL, GRAPHPACK and TWISTER are registered trademarks of GERSTEL GmbH & Co. KG. Printed in Germany 0312 Copyright by GERSTEL GmbH & Co. KG