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News from GERSTEL GmbH & Co. KG · Eberhard-Gerstel-Platz 1 · 45473 Mülheim an der Ruhr · Germany · Phone + 49 (0) 2 08 - 7 65 03-0 · [email protected] ww

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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

No. 12

A breath of fresh air!Solvent-free extraction

and µ-scale sample prep for

LC/MS and GC/MS...

Sustainable fuel

Automated determination

of glycerin in biodiesel

No pain!

Efficient extraction and determination

of pain killers in biological fluidsWine flavor

Holding on to fleeting

encounters

Natural mysteries

Ghostbusters in the desert

GERSTEL Solutions worldwide No. 12

Dear reader, In the 12th 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 techno-logies such as the Twister, thermal desorp-tion, and DHS are used to extract and con-centrate 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 auto-mates 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 80-90 % of our lives, increa-singly 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 off-flavors – 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 lPresident / Co-Owner

Eberhard G. Gerstel

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

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 ................... 26

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

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

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.

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 combinat ion 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 us ing 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

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 sep-arate and identify flavor and fragrance com-pounds 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 selec-tive detection and the olfactory impression leading to incorrect odor identification and odor intensity readings.

If the analytical system does not have suf-ficient sensitivity to determine the identity or concentration of the odor-active compounds, further automated steps can be taken to con-centrate analytes: A) 1D heart-cuts from multiple injections can be taken and trans-ferred 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 con-centration 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 ana-lytes were added to the wine: 1) The classical cork-iness culprit trichloroani-

sol (TCA); 2) 2-isobu-tyl-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 com-pounds 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 perfor-mance, standard solu-tions containing the fol lowing 15 com-pounds at the pg-level were injected directly into the analytical sys-tem: Hexanal, 1-hexanol, 3-hexenol, linalool, citrinellol, geraniol, p-cymen-8-ol, phenethyl alcohol, guaiacol, ethylhexanoate, ethyloc-tanoate, phenethyl acetate, beta-damasce-none, 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. Fol-lowing the extraction step, the PDMS coated Twisters were removed from the samples, cleaned with DI water, dabbed dry on a Kim-wipe, 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 trans-ports them to the TDU for analysis. Ther-mal desorption was performed under the following conditions: TDU initial tempera-ture: 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 ana-lyte 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 trans-

ferred to the 1D separa-tion column in splitless mode. The GC/MS sys-tem used was a 7890 GC combined with an 5975C MSD (both from Agi-

lent Technol-og i e s ) . The 1D separation was performed on a 30 m long DB-Wax col-umn, 0.25 mm ID, 0 .25 µm film thickness

(Agilent Technologies). The second dimen-sion 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 con-nectors at the proper temperature for best sys-tem 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 recover-ies 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 informa-tion could be gained. Following the first test runs, the identification of the spiked off- flavor compounds was performed using automated thermal desorption in combina-tion with 1D/2D-GC-O/MS. The 1D col-umn 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 tempera-ture if not used. When a heart-cut was per-

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.


formed from the 1D separation, the 2D col-umn 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. Reten-tion times of the GC-O signals were 12.45 min (IBMP), 16.25 min (TCA), and 16.55 min (Geosmin). “But these peaks were com-pletely hidden in the 1D total ion chromato-gram (TIC)”, the scientists stated. Therefore the relevant sections of the 1D chromatogram from 12.40 to 12.55 min and from 16.10 to 17.00 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 reten-tion time of 17.5 min. The three off-flavor compounds were clearly identified by olfac-tory detection during the 2D run. As an aside, high-boiling residue was simultane-ously back-flushed from the 1D column by increasing the outlet pressure and decreasing the inlet pressure in order to clean up the sys-tem for the next run. MS detection in full scan mode unfortunately was not sufficiently sen-sitive 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 sys-tem for 2D-GC-O/MS determination. No system modifica-tion 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 match-ing the olfactory signal for TCA. The mass spectra for all target compounds were com-pared with data from the Wiley Mass Spec-tral Library, which is integrated with the Agi-lent ChemStation.

Following library identification, a Sau-vignon Blanc wine was analyzed to deter-mine 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 (r2 ≥ 0.9990) 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 ana-lytes. We have clearly shown that this enables us to identify off-flavor compounds at ultra-trace levels. The 2D-GC-O/MS system is unique in allow-ing us to perform the anal-ysis in full scan mode”. Sys-tem performance was shown by determining three off-fla-vor compounds, TCA, IBMP, and Geosmin spiked into a wine at levels ranging from 5 to 50 ng/L. The combina-

1D/2D-TIC and Mass Chromatograms (m/z 195 and 197) after PFC-concen-tration of analytes from 20 injections of spiked wine (Zoomed in 2D-GC-MS-analysis). (1) = IBMP 25 ng/L; (2) = TCA 5 ng/L; (3) = Geosmin 50 ng/L.

tion of SBSE and PFC resulted in recover-ies 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.

Mass spectra of IBMP (a-1), TCA (a-2), and Geosmin (a-3) extrac-ted 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).

Kikuo Sasamoto Nobuo Ochiai, Ph.D.

Literature

Nobuo Ochiai, Kikuo Sasamoto. J. Chromatogr. A, 1218 (2011) 3180-3185.


Drug Screening

No pain!

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 sensitiv-ity and selectivity, low detection limits (e.g. 1 ng/mL), smaller sample volume require-ments, and also due to the fact that LC-MS/ MS doesn’t require chemical derivatization of analytes. However, LC-MS/MS can re-quire 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 sep-arate wash or elution steps are required, enabling rapid sam-ple preparation while still allow-ing comprehensive screening of the cleaned sample. Disposable Pipette Extraction (DPX) was developed as an alternative to traditional SPE, combining effi-cient and rapid extraction with significantly reduced solvent consumption. DPX is a novel dispersive solid-phase extrac-tion technique that uses sorbent loosely contained in a pipette tip enabling highly efficient mix-ing with the sample solution.

The main advantages of the DPX technol-ogy 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 manage-ment 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 pro-cedure described here combines the advan-tages of dispersive SPE and liquid extraction in a simple, quick and efficient way.

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 analyt-ical LC-MS/MS run time (4 min), the extrac-tion of one sample can be performed during the chromatographic analysis of the previous sample, achieving high throughput while pro-cessing each sample “just in time” ensuring that all samples are treated identically.

EXPERIMENTAL

Materials. Stock solutions for the com-pounds 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 con-centrations, to evaluate the differ-ent drug classes.

D e u t e r a t e d a n a l o g u e s , d3-morphine, d4-buprenorphine, d3-norbuprenorphine, d9-meth-adone, d3-tramadol, d5-fentanyl, d5-alpha-hydroxy alprazolam, d4-clonazepam, d5-oxazepam, d5-estazolam, d3-cocaine, d5-nor-diazepam, d5-propoxyphene, d7-carisoprodol, d5-amphet-amine, d4-ketamine, d4-7-ami-noclonazepam, and d5-PCP were purchased from Cerilliant. High concentration calibration stan-dard and intermediate QC urine samples were prepared by making

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.

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 0.5 – 50 ng/mL and % RSDs were below 10 % in most cases.


appropriate dilutions of the combined inter-mediate analyte stock solution using analyte free urine to give the concentrations listed in Table 1. Calibration standards were then pre-pared 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 pre-pared using a dilution ratio strategy from the high concentration sample of 1:1.33:3.33: 8. b-Glucuronidase, Type-2, from Helix poma-tia 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 Mul-tiPurpose 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 Quad-rupole 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 stain-less 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, vor-tex mixing for 30 seconds, and then incu-bating 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-

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.

Figure 3: Overlaid chromatograms for all 116 dynamic MRM transitions from an extracted urine sample at the MRL.

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 acetoni-trile 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 posi-tion 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 Ana-lyte qualifier/quantifier and 18 internal stan-dard transitions) were monitored in a 4 min-ute analytical window followed by a column regeneration time of 2.5 minutes. A reten-tion time window of 30 seconds was used for each positive ion transition monitored in the dynamic MRM method. Detailed mass spec-trometric acquisition parameters are available upon request.

Analysis conditions LC

Pump: gradient (600 bar), flowrate = 0.5 mL/minMobile Phase: A - 5 mM ammonium formate, with 0.05 % formic acid B - 0.05 % formic acid in methanolGradient: 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 % BRun time: 6.5 minutesInjection volume: 2 µL (loop over-fill technique)Column temperature: 55°C

Analysis conditions MS

Operation: electrospray positive ion modeGas temperature: 350°CGas flow (N2): 12 L/minNebulizer pressure: 35 psiCapillary voltage: 4400 V


Figure 4: Representative calibration curves: morphine, flurazepam, cocaine and ketamine.

RESULTS AND DISCUSSION

Figure 3 shows representative dynamic MRM chromatograms for all 49 pain management drugs and internal standards in a hydro-lyzed 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)

Compound Ret. Time High Cal Std. MRL LOQ [min] [ng/mL] [ng/mL] [ng/mL] 6-MAM1 1.60 100 10 5 Codeine1 1.43 500 50 25 Hydrocodone1 1.56 500 50 25 Hydromorphone1 1.01 500 5 25 Oxycodone1 1.51 500 50 25 Morphine1 0.71 500 50 25 Oxymorphone1 0.83 500 50 25 Meperidine1 2.27 500 50 25 Normeperidine1 2.33 500 50 25 Buprenorphine2 3.02 100 10 5 Norbuprenorphine3 2.60 100 10 5 EDDP4 2.76 500 50 25 Methadone4 3.22 500 50 25 Norpropoxyphene5 2.98 1000 100 50 Propoxyphene5 3.16 1000 100 50 O-Desmethyl-cis-Tramadol6 1.71 25 25 12.5 cis-Tramadol6 2.13 250 25 12.5 Fentanyl7 2.64 10 1 0.5 Norfentanyl7 2.04 10 1 0.5 Meprobamate8 2.58 500 50 25 Carisoprodol8 3.38 500 50 25 7-aminoclonazepam9 2.12 400 40 20 Clonazepam10 3.17 400 40 20 Oxazepam11 3.38 400 40 20 Estazolam12 3.30 400 40 20 Alprazolam13 3.42 400 40 20 Diazepam13 3.75 400 40 20 Flunitrazepam13 3.23 400 40 20 Lorazepam12 3.39 400 40 20 Nitrazepam13 3.15 400 40 20 Temazepam13 3.50 400 40 20 α-OH-alprazolam14 3.29 400 40 20 Nordiazepam13 3.63 400 40 20 Bromazepam12 3.05 400 40 20 Clobazam13 3.34 400 40 20 Midazolam13 3.08 400 40 20 Triazolam13 3.41 400 40 20 Flurazepam13 2.79 400 40 20 Ketamine15 2.01 1000 100 50 Norketamine15 2.01 1000 100 50 Amphetamine16 1.60 1000 100 50 MDA16 1.64 1000 100 50 MDEA16 1.82 1000 100 50 MDMA16 1.69 1000 100 50 Methamphetamine16 1.66 1000 100 50 Methylphenidate16 2.16 1000 100 50 PCP17 2.54 50 5 2.5 Benzoylecgonine18 1.99 250 25 12.5 Cocaine18 2.13 250 25 12.5

Disclaimer

For drug screening only. Not for use in diagnostic proce-

dures. 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. Infor-

mation, descriptions and specifications in this publication

are subject to change without notice.

List of internal standards used.

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


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 extract-ing replicate QC samples (n=4) at high and low concentrations. Table 2 shows the result-ing accuracy and precision data for all pain management drugs. Accuracy data averaged 98.0 % (range: 77 - 110 %) and precision data (% RSD) averaged 4.2 % (range: 0.9 -12.8 %) 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 manage-ment drug screenings in urine provided cycle times of approximately 7 min/sam-ple allowing throughput of over 200 sam-ples per day.

49 pain management drugs can be rapidly and reproducibly isolated from hydro-lyzed 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 R2 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 77 - 110 %) accuracy with 4.2 % RSD (range: 0.9 -12.8 %) for all pain management drugs analyzed.

Table 2. Extracted QC samples % accuracies and % RSDs.

Compound QCL Avg. Accuracy [%] QCH Avg. Accuracy [%] [ng/mL] [n = 4] % RSD [ng/mL] [n = 4] % RSD

6-MAM 12.5 101 12.7 75.0 107 4.9 Codeine 62.5 98 3.0 375.0 105 2.3 Hydrocodone 62.5 96 3.8 375.0 104 2.7 Hydromorphone 62.5 99 2.3 375.0 102 1.6 Oxycodone 62.5 98 2.9 375.0 103 2.8 Morphine 62.5 97 6.5 375.0 102 1.5 Oxymorphone 62.5 100 2.6 375.0 101 3.0 Meperidine 62.5 97 3.2 375.0 104 4.5 Normeperidine 62.5 98 2.6 375.0 103 4.0 Buprenorphine 12.5 103 10.8 75.0 106 4.2 Norbuprenorphine 12.5 91 9.3 75.0 97 7.7 EDDP 62.5 106 1.5 375.0 107 1.7 Methadone 62.5 107 3.0 375.0 108 2.2 Norpropoxyphene 125.0 96 1.9 750.0 97 1.5 Propoxyphene 125.0 96 2.2 750.0 97 1.0 o-Desmethyl-cis-Tramadol 31.3 94 2.1 187.5 93 1.3 Tramadol 31.3 94 2.6 187.5 94 0.9 Fentanyl 1.3 109 7.0 7.5 102 4.0 Norfentanyl 1.3 99 12.1 7.5 98 2.8 Meprobamate 62.5 87 3.3 375.0 88 1.1 Carisoprodol 62.5 90 2.8 375.0 84 1.1 7-aminoclonazepam 50.0 90 10.1 300.0 92 6.2 Clonazepam 50.0 89 9.1 300.0 101 2.6 Oxazepam 50.0 84 12.9 300.0 97 5.9 Estazolam 50.0 87 3.7 300.0 90 3.2 Alprazolam 50.0 100 3.3 300.0 104 5.5 Diazepam 50.0 98 3.8 300.0 98. 4.5 Flunitrazepam 50.0 109 6.9 300.0 94 4.3 Lorazepam 50.0 92 6.7 300.0 97 8.7 Nitrazepam 50.0 97 12.6 300.0 97 5.0 Temazepam 50.0 95 7.8 300.0 98 2.2 α-OH-alprazolam 50.0 77 4.1 300.0 79. 6.2 Nordiazepam 50.0 110 10.6 300.0 96 5.8 Bromazepam 50.0 104 12.0 300.0 90 2.3 Clobazam 50.0 102 5.3 300.0 97 4.4 Midazolam 50.0 102 5.3 300.0 97 4.4 Triazolam 50.0 100 4.2 300.0 104 6.3 Flurazepam 50.0 101 3.2 300.0 98 4.2 Ketamine 125.0 88 1.4 750.0 89 1.0 Norketamine 125.0 91 2.1 750.0 88 1.4 Amphetamine 125.0 100 2.4 750.0 102 1.5 MDA 125.0 99 2.5 750.0 103 1.4 MDEA 125.0 102 2.5 750.0 103 1.7 MDMA 125.0 101 2.4 750.0 101. 1.4 Methamphetamine 125.0 101 2.6 750.0 103 1.4 Methylphenidate 125.0 99 2.3 750.0 103 2.0 PCP 6.3 107 7.3 37.5 105 3.0 Benzoylecgonine 31.3 97 3.4 187.5 98 2.5 Cocaine 31.25 98 3.3 187.5 100 1.2

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, Univer-sity of South Carolina, 631 Sumter Street, Columbia, SC 29208, USA

References

[1] Determination of Pain Manage-ment Drugs using Automated Disposa-ble Pipette Extraction and LC-MS/MS, GERSTEL AppNote AN-2011-06

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 in-crease) in comparison to those listed in our previous work performed with an automated concentration step using a solvent evapora-

tion station [1]. However, the LLOQs of this modified cleanup method are still below the original MRLs and cycle times are consider-ably shorter. Representative calibration curves are shown in Figure 4. Regression analysis for all pain management drugs analyzed within this method resulted in R2 values of 0.99 or greater.


Water

Easy catch: pesticides

Havingto reach ever lower limits of detec-tion (LODs)is a daily challenge in mod-

ern laboratories. In order to succeed in obtain-ing sufficiently sensitive analysis methods, sample preparation techniques such as Solid Phase Extraction (SPE) or Liquid-Liquid Extraction (LLE) are often used as com-bined 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 extrac-tion, 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

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 tothe 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.

Automated sample preparation process used for the analysis work performed.

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 H2O - 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

GERSTEL Multi Purpose Sampler MPS with auto-mated Solid Phase Extraction (SPE) and evapora-tion module mVap.

The mVAP is a six-position evaporation station for the GERSTEL MultiPurpose Sampler (MPS). Samples are con-centrated at slightly elevated temperatures under moderate vacuum, enabling significantly improved limits of detection.

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 concen-trated under a flow of nitrogen at a mod-erately increased temperature. In such sys-tems, 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 automa-tion of all sample preparation steps including introduction to the LC- or GC-system. The

evaporation is controlled by con-trolling the applied vacuum, tem-


1 2 3 4 5 6 7 8 9 10 11 12number of repetitions

response

HexazinonRSD 2.8 %

Desethylatrazinrel. STD 9.6 %

CyanazinRSD 7,8 %

MetribuzinRSD 9,2 %

DiuronRSD 9,5%

IsoproturonRSD 6.5 %

Signal responses and standard deviations for selected analytes from twelve consecutive extractions and analyte determinations.

Recovery of determined pesticides spiked at 10 ng/L

Concentration in ng/L (average of 12 individually prepared samples)

The determined 18 pesticides with recoveries.

Matrix calibration curves for six selected analytes extracted from spiked groundwater sample.

Desethylatrazin

Diuron

Hexazinon

Isoproturon

Metribuzin

Cyanazin

Overlay chromatogramm of quantifier MRMs for six selected pesticides extracted from a spiked sample at 10 ppb.

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 pro-cessed automatically overnight. In this work, the performance of the complete system includ-ing 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 us-ing the GERSTEL MultiPosi-tion 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 Quad-rupole 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.

Authors

Meike Holtmann, TeLA GmbH, Fischkai 1, D-27572 Bremerhaven, Ger-many. Susanne Sperling, GERSTEL GmbH & Co. KG, Eberhard-Gerstel-Platz 1, D-45473 Mülheim an der Ruhr, Germany.

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 Parlia-ment and the Council.

Spiked groundwater samples were used for matrix calibration and analysis.

Conclusions

A completely automated process involving SPE and an evapora-tion step was tested for rug-gedness, recovery of analytes, method linearity and sample to sample reproducibility. Data obtained using the mVAP to concentrate extracts contain-ing 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 ap-plicability 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 con-centration levels.


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 sor-bent 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 off-odors in water; drugs of abuse such as Tetrahydrocannabinol (THC), barbiturates and benzodiazepines; phthalates and various metabolites in biological fluids; flavor compounds, preservatives, thrichloroan-isole, 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 gradu-ally decrease with decreasing Ko/w when using PDMS based Twist-ers. Among the more hydrophilic solutes are, for example, polar pes-ticides, 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 increas-ingly 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) Sili-cone Twister. These new Twisters extract several classes of polar com-pounds 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 Agi-lent 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 sol-vent vent mode. In this mode, water is evaporated at low initial tem-perature, for example at 30-40 °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 repro-ducible 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 mag-netic 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-


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 compari-son of three chromatograms from three extractions of a whisky using different Twisters is shown. The EG-Silicone Twister extraction pro-vided 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 polydimethyl-siloxane basis, the EG-Silicone Twister also has high affinity for non-polar 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, cis-whisky 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 com-pounds like lactone and eugenol: 24.3 % and 32.9 %, respectively. In direct comparison with the PA Twister, the EG-Silicone Twister pro-vided 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, Acry-late, 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 94 1.1E+07 2.3E+06 6.0E+04 2 Ethyl hexanoate 88 1.8E+06 2.8E+05 7.6E+06 3 o-Cresol 108 1.5E+07 1.7E+06 1.8E+05 4 p-Cresol 108 1.1E+07 1.4E+06 1.6E+05 5 Phenethyl alcohol 91 2.4E+07 7.9E+06 1.6E+06 6 o-Ethylphenol 107 9.3E+06 6.1E+05 2.1E+05 7 2,4-Xylenol 107 1.7E+07 1.3E+06 4.3E+05 8 Ethyl octanoate 88 1.5E+08 4.0E+06 1.4E+08 10 Ethyl decanoate 88 2.3E+08 1.2E+07 2.3E+08 12 Ethyl dodecanoate 88 1.1E+08 5.0E+06 7.0E+07

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 1:10 -100°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 µmPneumatics: He, constant flow = 1.4 mL/minOven: 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 µmPneumatics: He, constant flow = 1.2 mL/minOven: 60°C (2 min); 5°C/min; 200°C; 10°C/min; 300°C (5 min)


the EG-Silicone Twister and 92 for the PDMS Twister. For the other compounds classes, both Twisters extract a similar num-ber 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 chromato-gram 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 iden-tified peaks have a hit quality higher than 80.

The plausibility of the iden-tification 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 eas-ily be identified.

Multivitamin Juice

Extraction of multivitamin juice or of other fruit juices is often neg-atively influenced by fruit pulp, which blocks analyte access to the extraction phase and/or hinders phase separation following the extrac-tion. 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 Twist-ers 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-ter-penes linalool, 4-terpineol, alpha-terpineol, and nerolidol, the EG-Sil-

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

Table 4. Tentatively identified compounds found in Scotch whisky by Twister extraction and GC/MS analysis using a ZB-FFAP Column.

Table2.Recovery in % for selected whisky standard substances obtained with three types of Twisters.

Whisky Log EG- PA PDMS standards Ko/w Silicone Phenol 1.46 5.7 4.2 3.0 o-Cresol 1.95 9.8 5.2 1.5 cis-Whisky 2.00 6.1 1.7 24.3 lactone Eugenol 2.27 29.5 3.3 32.9

Table 3. Whisky compounds extracted by SBSE using the PDMS and EG-Silicone Twister respectively. Compound class PDMS EG- Silicone Phenols and aromatic compounds 14 40 Fusel alcohols 10 10 Fatty acids 11 11 Aliphatic acid ethyl esters 15 15 Other esters 22 22 Lactones 1 2 Acrolein derivates 7 7 Terpenes and norisoprenoids 6 7 Miscellaneous 6 12

Total 92 126

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 tempera-ture and 1000 rpm.

icone Twister does provide bet-ter recovery than the PDMS Twister; conversely, for monoterpenes like alpha-pinene, beta-myrcene, 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 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 93 1.7E+05 3.9E+05 10 beta-Myrcene 93 1.4E+06 2.6E+06 11 delta-3-Carene 93 2.8E+06 4.7E+06 12 D-Limonene 68 1.3E+07 1.8E+07 16 Linalool 71 1.5E+06 4.3E+05 19 4-Terpineol 71 4.3E+05 2.7E+05 20 alpha-Terpineol 59 1.5E+06 4.3E+05 27 Nerolidol 69 5.2E+05 4.8E+05

Figure 3. Multivitamin juice chromatogram obtained from EG-Silicone- and PDMS Twisters, non-polar column separation. 10 mL sample, 1000 rpm, 1 hour, room temperature.

Table 5. Identified compounds found in multivitamin juice by Twister extraction and GC/MS analysis using a ZB-5 Column.

EG-Silicone Twister extracts a larger number of indi-vidual 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 chro-matograms. 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

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

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.

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- 19 4-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


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 Hydroxymethyl- furfurole (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

Table 8. Tentatively identified compounds extracted from white wine using different Twisters and separated on a ZB-FFAP Column.

alcohol (No. 27) and ethyl 3-(4-hydroxyphenyl)-propenoate (Z or E) (No. 29) were found only when combining EG-Silicone Twister extraction with sepa-ration on a polar col-umn (Table 8).

CONCLUSION

The novel EG-Silicone Twisters and PA Twisters presented in this work enable higher extraction efficiency than traditional PDMS Twisters for po-

lar compounds in samples like whisky, multivitamin juice and white wine. For compounds like phe-nols, 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 op-erating 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.

Authors

Yunyun Nie, Eike Kleine-Benne GERSTEL GmbH & Co. KG, Eberhard-Gerstel-Platz 1, D-45473 Mülheim an der Ruhr, Germany

Figure 5. Sauvignon blanc chromatogram profiles obtained from EG-Silicone- and PDMS Twister extractions of 5 mL samples for one hour at 1000 rpm, polar column separation.

References

1. Frank David, Bart Tienpont,Pat Sandra, Stir-bar sorptive extraction of trace organic compounds from aqueous matrices, LCGC North America, 21: 21-27 (2003)2. Kevin Mac Namara, Michelle Lee, Albert Robbat Jr., J.Chromatogr. A 1217 (2010) 1363. Kevin Mac Namara, Dagamara Dabrowska, Meike Baden, Norbert Helle, LC/GC Chromatography, Sep. 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

Acknowledgment

The authors would like to thank Dr. Kevin MacNamara, Irish Distillers, Pernod-Ricard for his kind support.

Further information

www.gerstel.com - Applications: AppNote 3/2011 (http://www.gerstel.com/pdf/p-gc-an-2011-03.pdf)


A six-position e v ap o r a t i o n 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 per-formed for improved chromatog-raphy. The mVAP can be used in

Innovation

Multi-Position Evaporation Station (mVAP)

Olfactometry

More than 1,000 GERSTEL ODPs sold – Sniffing out odors

combinat ion with SPE, Dis-

persive SPE (DPX) or liquid/liquid extrac-

tion 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 intro-duced ODP 3 with heated mix-ing 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 soft-ware allows the sensory analyst to describe odors and fragrances in real time – these voice descrip-tors are recorded and converted to editable text files. For each

GC/MS run a complete report is generated, including a chro-matogram 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 sen-sory and analytical information to determine flavors and odors in foods, beverages, fragrances, and other complex samples and to help identify sources of odors.

A safe and plentiful food supply is a global concern and laboratories world-

wide are working hard to ensure that contaminated food doesn’t reach con-sumers. 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 state-of-the art methods and instrumen-tation. Key topics in food safety are covered including the determination

Literature

Mass spectrometry in food safety

Dr. Norbert Helle

More Information

Mass Spectrometry in Food Safety Methods and ProtocolsSeries: Methods in Molecular Biology, Vol. 747 Zweigenbaum, Jerry (Ed.)1st Edition., 2011, XII, 416 p. 104 illus.Humana Press, ISBN 978-1-61779-135-2

of low levels of pesticides, mycotoxins, veter-inary drugs and chemical contaminants from packaging materials. One chapter, written by the recognized food analysis expert Dr. Nor-bert Helle and his team, specifically addresses automated solid phase extraction for prepara-tion and clean-up of food samples in combina-tion with LC-MS/MS. Method chapters con-tain 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.


It gets hot, really hot, in the Namib Desert; and really cold as well. In some places tem-

peratures hover above the 50 °C mark dur-ing 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 sur-rounded by tales and myths. Their uniform appearance, sheer number, and distribution have always sparked human imagination. Natural displays of such regularity are typ-ically awarded a deeper meaning or attrib-uted 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 explana-tions 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 ven-turing more than a certain distance from their mound. Other experts suspect that ants, clear-ing the ground of grass seeds, have caused the circular patches with no or dying vegetation.

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 phe-nomenon 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.

by Guido Deußing

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®).

Sand samples for Naudé et al., 2011 were col-lected 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 iso-hyet, 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., 2004. Mysterious circles in the Namib Desert: review of hypotheses on their origin. Journal of Arid Environments 57, 467-485.

(Basic and Applied Dryland Research 1, 2 (2007), 121-137).

„This is not the case“; scientists of the University of Pretoria in South Africa now discard previous theories as well as oral tradi-tion. Instead of relying on speculation, chem-ists Yvette Naudé and Egmont Rohwer from the Department of Chemistry and their col-league Gretel van Rooven from the Depart-ment of Botany had gone into the Namib Desert to investigate the matter based on sci-entific facts using chemical analysis instru-mentation 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) 446-456).

The ground is breathing

Naudé and her colleagues first went on loca-tion 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 vege-tation or covered by still live (yellowing) or dead (gray) vegetation. They are typically sur-rounded 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 the-ory of hungry termites and grass seed-collect-ing ants could be investigated on site dur-

Young Himba girl.



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 cir-cles could indicate the involvement of allelo-pathic 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, accord-ing to Yvette Naudé. However, potting trials proved that allelopathy did not play a role. Additionally, fairy circles also develop in veg-etation-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

reach the soil surface they start to disperse forming a circle.” There are plenty of poten-tial gas sources in Namibia, suggests Yvette Naudé. These include both big oil and natu-ral gas reservoirs and the country’s geothermal activity visible at the hot springs in Namib-ia’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 fun-nels into the ground. Several times throughout the day the levels of carbon monoxide (CO), carbon dioxide (CO2), oxygen (O2), hydro-gen sulfide (H2S) and nitrogen dioxide (NO2) were determined using a portable gas analyzer. The gas analysis allowed a number of conclu-sions to be drawn concerning the soil chem-istry, 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 hydrocar-

bons was shown to lead to an increase in the activity of oxidizing and reducing (e.g. sul-fur-reducing) bacteria, which again lowers the level of oxygen in the soil. This may have far-reaching, 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 min-erals for plant development. ” Plants grown in soil collected from fairy circles”, the scien-tists report further, “did not flourish, in con-trast 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 detec-tion. The advantages of the thermal desorp-tion technique: “It is cost effective, simple

„Fairy circles“ cover the vast expanse of the Namib like pockmarks on the desert’s sand-colored face.

The tales of the Himba, a semi-nomad people in Northern Namibia, relate that a dragon lives under-neath the desert sand and breathes fire.

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 vegetation at the edge of the fairy circles could indicate the involvement of allelopathic compounds.


and it doesn’t rely on toxic organic solvents,” explains Yvette Naudé. Solvent-free extrac-tion 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 desorp-tion 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 care-fully 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 success-fully 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 sam-ple. 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 vis-

ible 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 micro-bial changes of magnetic and iron-contain-ing minerals.

In order to rule out analytical bias caused by magnetic particles, Yvette Naudé and her colleagues subsequently replaced the Twist-ers with 10 cm long pieces of PDMS tub-ing 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 trans-ferred to the GC column for separation and mass selective detection.

The analysis results support the the-ory 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 sup-port the involvement of termites or ants”, con-cludes 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 funnel-like 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.

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.

One of the scientists‘ initial theories: the partly lush vegetation at the edge of the fairy circles could indicate the involvement of allelopathic compounds.

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.

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.


PVC, linoleum, carpeting, laminate, par-quet, and cork – the choice of floor cov-

ering 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 con-taminate the indoor air and even cause irri-tation 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-

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.

Used for the determination of material emissions: The GERSTEL TDS/TDS A2 mounted on a GC/MS System.

ually recycled indoor atmospheres in mod-ern energy-efficient buildings may feel noth-ing. 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, aller-gic reactions, immune system deficiency, fre-quent 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 mod-

ern society spend most of our lives indoors, depend-ing on the season up to 80 – 90 % 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 addi-tion, VOC- (C6-C16) and Te

xt: G

uido

Deu

ßing


Fig.: AgBB scheme for health-related evaluation of con-struction products. Gerd Bittner: „A product that meets the AgBB criteria is well suited for use indoors in buildings.“ * VOC: Retention time range equal to C6-C16, SVOC: Retention time range equal to > C16-C22, ** LCI: Lowest Concentra-tion of Interest.

SVOC (>C16-C22) contamination plays a role that is increasingly in focus of regulating gov-ernment agencies. Many construction prod-ucts 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, lac-quers, varnishes, coatings, wood preservation products, wall paper, caulks and sealants, cement, prefabricated bricks, and concrete. We are surrounded by a huge range of indus-trially produced materials that contain a long list of ingredients and additives to make them easy to use, low cost and durable. The Euro-pean 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 lay-ing down harmonized conditions for the mar-keting of the construction products” the fol-lowing 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 exceed-ingly 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,

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 Direc-tive 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 apply-ing the same rules to everyone while allow-ing the consumer to win by being allowed to live, work, and play in a healthy indoor environment. The Committee for Health-related 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 con-ditions. “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 fac-tors 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 com-pounds (VOCs), including formaldehyde)

Chemical contaminants from out-door 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 con-taminants 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 combina-tion, and may supplement other complaints such as inadequate temperature, humidity, or lighting. Even after a building investiga-tion, however, the specific causes of the complaints may remain unknown.

Source:

USEPA - www.epa.gov/iaq/pubs/sbs.html


dardized test conditions for approval of floor-ing materials that are used for an annual ver-ification 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. includ-ing the glue used to install the flooring) is performed at the TFI using environmental test chambers based on the DIN EN ISO 16000-11, DIN EN ISO 16000-9, and DIN ISO 16000-6 standards for indoor air. These standards specify conditions for all aspects of testing various flooring materials in envi-ronmental test chambers as well as the ana-lytical determination of identity and con-centration of emitted organic compounds (VOCs/SVOCs). Chamber air is collected using active pumped sampling onto a suit-able 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 Desorp-tion - 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 C6-C16 (VOC) or >C16-C22 (SVOC) as specified in the AgBB scheme for health-related evaluation of construction products rev. 2010.

The term “individual analytes” refers to both identified and non-identified com-pounds. The AgBB scheme requires a limit of detection of 1 µg/m3 for each compound in order to comprehensively cover and describe the material emissions. Depending on the specific requirements, quantitative infor-mation on individual compounds must be obtained. Whenever individual compound concentrations exceed 5 µg/m3, they must be quantified both individually and in summa-tion as part of the relevant group.

Exceptions are EU category 1 and 2 car-cinogens. For identified carcinogens and compounds that have an LCI value, com-pound specific quantification must be per-formed. Unidentified compounds as well as compounds to which no LCI value is assigned are quantified as toluene equivalents.

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 stan-dardized 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 concentra-tion, is easily performed as specified for the unknown minor compounds and those with-out 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 exam-ple, in terms of development cost and loss of competitiveness. A clear indication of the emission profile of a product in every devel-opment stage, or during trouble shooting fol-lowing customer complaints, can save com-panies 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 emis-sion tests during product development; for regular Quality Control of product batches; for trouble shooting following customer com-plaints; as well as for sample identity veri-fication. 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 representa-tive 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 floor-ing material”, reports Mr. Bittner. The sam-ples are heated in a flow of inert gas and the extracted analytes are purged onto the adsor-bent tube and concentrated on TenaxTA. Thermal Desorption (TD)-GC/MS analy-sis is subsequently performed following the AgBB guidelines.

“By adapting the Thermal Extraction methods used to the corresponding emis-sion chamber methods, we have achieved good qualitative correlation between ther-mal 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 acceler-ated thermal extraction tests give us the abil-ity to quickly establish the emission poten-tial of flooring materials and their associated adhesive systems, as well as to compare emis-sions from different combinations. Thermal extraction is a valuable and efficient comple-ment to standard emission chamber tests.“

Gerd Bittner, Textiles & Flooring Institute (TFI), Charlottenburger Allee 41, 52068 Aachen, Germany, Phone +49 241 96790-0 www.tfi-online.de

More Information

GERSTEL TE: Thanks to the large thermal extrac-tion tube, a range of different sample types and amounts can be analyzed based on thermal extraction in the TE.

LCI values

LCI is an acronym for “Lowest Concent-rations of Interest”, i.e. the lowest con-centration of toxicological relevance for a particular compound in indoor air in resi-dential 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 Expo-sure 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 16000-9 Emission chamber test method.

• DIN EN ISO 16000-11 Sampling, sto-rage, and preparation of samples.

• DIN ISO 16000-6 Determination of VOCs in indoor air and in Environmental test chambers. Sampling on TenaxTA follo-wed by Thermal Desorption – GC/MS.

• DIN ISO 16000-3 Determination of formaldehyde and other carbonyl com-pounds; Sampling

• Evaluation scheme for health related assessment of emissions from construc-tion products (AgBB)

• German Institute for Construction Techno-logy (DIBt) Product Approval details


Optimization of carpet tiles I: A carpet tile was taken from a pro-duction facility for accelerated VOC/SVOC emission testing. The result: Large compound peaks were found in the retention time range from 10 – 14 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.

Optimization of carpet tiles II:Thermal Desorption GC/MS chro-matogram 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.

Useful tool and great complement: Comparison of emission tests performed after three days in an environmental test cham-ber (above) and accelerated emission testing using thermal extraction (below). Good cor-relation is seen between the peak patterns in the two chro-matograms, 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.

Thermal extraction using the GERSTEL TE: an efficient alternative to environmental chamber testing – practical examples from the TFI

1 Ethylbenzene

2+3 Total xylenes

4 Butylglycol

15 Butyldiglycol

16 2-ethyl-hexanoic acid

Thermal extraction of a carpet tile

Thermal extraction of a raw material

Thermal extraction of carpet tile before change

Thermal extraction of carpet tile after optimization

AgBB test result summary after three days.

Requirement

TVOC (C6-C16) 599 µg/m³ 300 µm/m³

R 0.169 1

TVOC (without LCI) 223 µg/m³ 100 µg/m³

AgBB test result summary after three days.

Requirement

TVOC (C6-C16) 206.7 µg/m³ 300 µm/m³

R 0.061 1

TVOC (without LCI) 93.2 100 µg/m³

Environmental test chamber

Thermal extraction


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 renew-able 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 con-sists 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 viscos-ity of the resulting fuel at a wide temperature range. In the U.S., Biodiesel for use in Die-sel engines must conform to ASTM D6751 and the amount of Glycerin is determined using ASTM Method D6584-07 “Standard Test Method for the Determination of Free and Total Glycerin in B-100 Biodiesel Methyl Esters by Gas Chromatog-raphy”.

Glycerin, an un-desirable travel companion

Apart from FAMEs or SMEs and RMEs, sub-standard glycerin (SSG) is generated during the Biodiesel production process and a residue is formed consisting of glyc-

erin, water, catalyst, excess methanol and free fatty acids. The SSG by-product is toxic and flammable, but unsuitable as a fuel and gen-erally undesirable, since it forms solid sedi-ment, 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 mate-rial 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 Ger-many, 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 vehi-cle 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 analy-sis method. For the determination of the amount of free and total glycerin and of mono-, di- and triglycerides, European stan-dard EN 14105 and its American counterpart ASTM Method D6584 prescribe the use of gas chromatography (GC) with flame ion-ization detection (FID). “The analytes must first be transferred into a form that is suitable for GC and this is done through derivatiza-tion, which is normally a tedious, labor inten-sive and time-consuming task”, Dr. John R. Stuff explains. Dr. Stuff and Jacqueline A. Whitecavage, experienced application chem-ists 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 White-cavage used a GC 6890 from

Agilent® Technologies with a GERSTEL Cooled Injec-tion System (CIS 4) and FID. Automated sample prepara-tion and sample introduction to the GC was performed using the Dual Rail version of the GERSTEL Mult iPurpose Sampler (MPS). The MPS

was equipped with two differ-ent syringe sizes, a 10 µL on-col-

umn syringe and an 80 µL side-port syringe with dilutor module. Bio-

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.


Analysis conditions

CIS: 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)

Column: 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

Chromatogram of a biodiesel standard.

Chromatogram of a real biodiesel sample analy-zed using the MPS-GC/FID system.

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

Reference

J. R. Stuff, J. A. Whitecavage. Full Automa-tion of ASTM Method D6584-07 “Stan-dard Test Method for the Determination of Free and Total Glycerin in B-100 Bio-diesel Methyl Esters by Gas Chromatogra-phy” using a GERSTEL Dual Rail PrepStation. G E R S T E L A p p N o t e 1 / 2 0 1 0 (www.gerstel.com/pdf/p-gc-an-2010-01.pdf)

diesel standards containing glycerin, mono-olein, diolein, triolein, butanetriol, and tri-caprin, all in pyridine, as specified in ASTM D6584-07 were purchased. Biodiesel B-100 was purchased locally. The glycerin, mono-, di- and triolein standards as well as butane-triol, tricaprin and MSTFA were placed in separate vials in the MPS for further pro-cessing. 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 D6584-7 specifies prep-aration 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. scien-tists 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 indi-vidual prep method. MAESTRO operates fully integrated into the ChemStation® and GC MassHunter® software (Agilent Tech-nologies). “The manual effort is reduced to the weighing of 100 mg of the sample into 10 mL headspace vials and placing them in

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 syn-chronized for best possible throughput: Stan-dard preparation, adding internal standards and derivatization reagent, mixing, incubat-ing, rinsing, and introducing the sample into the CIS. The automated steps of the proce-dure 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 lev-els, 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.” Auto-mating the sample preparation process means that valuable resources can be used more effi-ciently and laboratory staff is less exposed to potentially toxic solvents and reagents. Fur-thermore, system productivity can be main-tained overnight and throughout the week-end meaning that valuable instrumentation is used much more efficiently.

GC/FID system with GERSTEL Dual Rail MPS used to automate ASTM Method D6584-07: “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) illus-trates the high efficiency of the biodiesel analysis using MPS GC/FID.

Automated process steps with the MPS

Diolein calibration curve.

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Fragrance Analysis

Fabric softeners caught in a whirl…

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. Addi-tionally, 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 inter-act to form the complete olfactory impres-sion that distinguishes the consumer prod-uct. It is the task of the quality control depart-ment 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 prepara-tion techniques used.

To isolate volatile fragrance compounds from soaps, detergents, or fabric softeners, the chemist traditionally reaches for the liq-uid/liquid extraction (LLE) technique. LLE is based on differences in analyte solubility in two immiscible solvents. These are typ-ically a hydrophilic (aqueous) phase on the one side and a hydrophobic organic solvent on the other side of the phase divide. Accord-ing 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 cumber-some.” Getting good phase separation is often difficult when the sample to be extracted con-tains surfactants. Furthermore, large quanti-ties 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 tech-nique that would minimize or even eliminate solvent use while extracting fragrance com-pounds with good recovery and delivering first rate validation data”, Diana Koh states. After a comprehensive search, the scientists decided to pursue Stir Bar Sorptive Extrac-tion (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: 10.00 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: 29.0 – 400.0, Quad: 150 °C, Source: 230 °C

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.


Fragrance Analysis

Fabric softeners caught in a whirl…

tion, the Twister has proven its worth by effi-ciently extracting volatile- and semivolatile organic chemical compounds (VOCs and SVOCs) from a wide range of complex matri-ces. “SBSE not only requires little, if any, sol-vent”, says Khim Hui, “it is also surprisingly simple to perform“. SBSE is performed using a glass encased magnetic stir bar with a rela-tively large volume of sorbent phase coated on the outside. While the Twister stirs the sam-ple, 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 ana-lyte determination is fully automated mak-ing 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.

As an aside, SBSE is based on the parti-tion of analytes between PDMS and the sam-ple. The Twister stirs the sample and non-polar or moderately polar compounds are extracted and concentrated. The partition coefficient of a compound between PDMS and water is very close to its KO/W value (octa-nol/water partition coefficient).

KO/W is a physical chemical parameter used to describe hydrophilic or hydrophobic properties of a compound [2]. KO/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 KO/W value is characteristic of a hydro-phobic compound that would be extracted with high recovery using PDMS.

Theory guides – experiment decides

To test the SBSE technique for their appli-cations, the scientists analyzed fabric softener

Graph 3 and 4 (left to right): Response Ratio versus different stirring speeds

0.000000

0.100000

0.200000

0.300000

0.400000

0.500000

0.600000

0.700000

700 800 900

Response

Ratio

Stirring Speed (rpm)

Response Ratio vs Stirring Speed

VERDOX

LILIAL

AMYLCINNAMIC ALDEHYDE

HEXYL SALICYLATE

GALAXOLIDE 70 MIP

RM 10

RM 8

RM 7

RM 4

RM 6

0.000000

0.010000

0.020000

0.030000

0.040000

0.050000

0.060000

0.070000

0.080000

700 800 900

Response

Ratio

Stirring Speed (rpm)

Response Ratio vs Stirring Speed

EUCALYPTOL

ZESTOVER

CAMPHOR

HEXYLCINNAMIC ALDEHYDE

DECAL

RM 1

RM 2

RM 3

RM 9

RM 5

Graph 1 and 2 (left to right): Response Ratio versus different extraction times

0

0.05

0.1

0.15

0.2

0.25

30 min 1 hour 2 hour 3 hour 4 hour

Response

Ratio

Time (t)

Response Ratio vs Extraction TimeEUCALYPTOL

ZESTOVER

CAMPHOR

HEXYLCINNAMIC ALDEHYDE

DECAL0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

30 min 1 hour 2 hour 3 hour 4 hour

Response

Ratio

Time (t)

Response Ratio vs Extraction TimeLILIAL

AMYLCINNAMIC ALDEHYDE

HEXYL SALICYLATE

VERDOX

GALAXOLIDE 70 MIP

RM 1

RM 2

RM 3

RM 9

RM 5

RM 6

RM 7

RM 8

RM 4

RM 10

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), amyl-cinnamaldehyde (RM 7), hexylsalicylate (RM 8), hexyl-cinnamaldehyde (RM 9) as well as Galaxolid 70 MIP (RM 10). An internal stan-dard (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-dibro-mobenzol in acetonitrile) to 20 mL deion-ized (DI) water and letting the Twister stir the IS solution for an hour. The kinetics of the extraction depend on the analyte migra-tion rate to and into the PDMS phase of the Twister, which among other things, depend

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 tem-perature while stirring the Twister at 800 rpm. To get reliable data, Diana Koh performed the analysis over five different days, achiev-ing “good results with good reproducibil-ity 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 5COMPOUNDS Mean STDEV RSD % Mean STDEV RSD % Mean STDEV RSD % Mean STDEV RSD % Mean STDEV RSD %

Eucalyptol 0.011391 0.000569 5.0 0.012481 0.001269 10.2 0.012263 0.000979 8.0 0.012726 0.001675 13.2 0.012806 0.001285 10.0Zestover 0.004632 0.000450 9.7 0.004311 0.000264 6.1 0.004320 0.000135 3.1 0.004182 0.000423 10.1 0.004503 0.000408 9.1Camphor 0.016166 0.000759 4.7 0.016907 0.001131 6.7 0.016440 0.000769 4.7 0.016788 0.002001 11.9 0.016717 0.001316 7.9Verdox 0.915239 0.018752 2.0 0.972578 0.033094 3.4 0.866965 0.046048 5.3 0.861180 0.067641 7.9 0.891986 0.042382 4.8Decal 0.014214 0.001540 10.8 0.013926 0.001113 8.0 0.012846 0.000584 4.5 0.011892 0.001824 15.3 0.011710 0.000817 7.0Lilial 0.543458 0.019006 3.5 0.578859 0.027545 4.8 0.545785 0.035792 6.6 0.541996 0.053128 9.8 0.538627 0.025521 4.7Amylcinnamic Aldehyde 0.157428 0.006136 3.9 0.171888 0.012369 7.2 0.155054 0.011036 7.1 0.156900 0.017694 11.3 0.159980 0.008911 5.6Hexyl Salicylate 0.166794 0.015632 9.4 0.194849 0.020899 10.7 0.172095 0.019607 11.4 0.170137 0.026629 15.7 0.174985 0.013832 7.9Hexylcinnamic Aldehyde 0.082476 0.006007 7.3 0.093632 0.009226 9.9 0.081535 0.008477 10.4 0.082533 0.012220 14.8 0.084395 0.005973 7.1Galaxolide 70 MIP 0.058829 0.004466 7.6 0.067701 0.006918 10.2 0.058252 0.007216 12.4 0.056675 0.008331 14.7 0.058113 0.004954 8.5

Table 1: % RSDs calculated based on 10 individual sample extractions for every listed result.

Table 2: Calculated %RSD for Reproducibility (Mean of 50 measurements)

COMPOUNDS Mean STDEV % RSD

Eucalyptol 0.012334 0.001270 10.3Zestover 0.004390 0.000377 8.6Camphor 0.016604 0.001255 7.6Verdox 0.901589 0.059065 6.6Decal 0.012918 0.001589 12.3Lilial 0.549745 0.036067 6.6Amylcinnamic Aldehyde 0.160250 0.012906 8.1Hexyl Salicylate 0.175772 0.021475 12.2Hexylcinnamic Aldehyde 0.084914 0.009471 11.2Galaxolide 70 MIP 0.059914 0.007433 12.4


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).

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, Sin-gapore 638377

References

1. C. Franc, F. David, G. de Revel. J. Chromatogr. A 1216 (2009) 3318-33272. M.W. Maylan, P.H. Howard; J. Pharm. Sci. 1995,84, 83-92

y=5.409e -002

R2=0.9989

0

1.00e-002

2.00e-002

0 0.2 0.4

EUCALYPTOLResponse Ratio

Concentration Ratio

RM 1

0

0.05

0.1

0 0.5 1 1.5

RM 10Response Ratio

Concentration Ratio

y=8.549e -002

R2=0.9986

0

5.00e-003

1.00e-002

0 0.05 0.1 0.15

ZESTOVERResponse Ratio

Concentration Ratio

RM 2

y=6.715e -002

R2=0.9969

0

2.00e-002

0 0.2 0.4

CAMPHORResponse Ratio

Concentration Ratio

RM 3

y=8.646e -002

R2=0.9984

0

1

0 1 2 3

VERDOXResponse Ratio

Concentration Ratio

RM 4

y=6.097e -001

R2=0.9999

0

2.00e-002

0 0.05 0.1 0.15

RM 5Response Ratio

Concentration Ratio

y=2.176e -001

R2=0.9977

0

0.5

1

0 0.5 1 1.5

RM 6Response Ratio

Concentration Ratio

y=7.397e -001

R2=0.9998

0

0.2

0 0.5 1 1.5

RM 7Response Ratio

Concentration Ratio

y=2.111e -001

R2=0.9999

0

0.2

0.4

0 0.5 1 1.5

RM 8Response Ratio

Concentration Ratio

y=2.541e -001

R2=0.9962

0

0.1

0 0.5 1 1.5 2

RM 9Response Ratio

Concentration Ratio

y=7.646e -002

R2=0.9989

were established for each fragrance compound added resulting in good linearity with R2- val-ues higher than 0.996 for all compounds.

Comparison of SBSE and LLE

But how did SBSE stack up to the LLE tech-nique, 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 com-parison showed that SBSE resulted in much higher sensitivity than LLE”. The final con-clusion: “SBSE is a fast, sensitive, and highly reproducible alternative to standard sam-ple 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 determina-tion 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.

Graph 5: Twister calibration curve of each compound

Table 3: Correlation of determination (R2)

Method Comparision

Method % Dosage found % Leakage (Corrected)LLE 0.53 0.1SBSE 0.51 0.5

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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 ben-efit: significantly more room, better facil-ities, and a central location to provide the best possible support for our customers in terms of stock availability and cus-tomer 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 sup-port the Japanese distributor Yokogawa Analytical Systems Inc., GERSTEL K.K. was founded. At that time, Dr. Schwar-zer moved back to Germany to take up the key position of Software Develop-ment 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 fur-

ther developed GERSTEL K.K. into an accepted brand and house-hold name in Japan for automated sample prepara-tion and sample introduction. Based on a strong focus on solutions and appli-cations, GERSTEL K.K. has produced solid and consistent year-on-year growth since it was founded. Among the custom-ers are major global players in fields such as Automotive, Semiconductors, Flavor and Fragrance, Food and Beverage pro-ducers, as well as Pharmaceutical compa-nies and water suppliers.

The customers are supported by an internationally renowned team of application scientists under the leader-ship 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.

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 , Market ing and Ser vice, Maurizio Rosati, Senior Director of Sales EMEAI, and Fred Strohmeier, Vice President and General Manager, A g i l e n t D e u t s c h l a n d G m b H 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-Agi l en t cu s tomer s . Eberha rd G. Gerstel received an engraved anniversary trophy to add to the long

GLOBAL ANALYTICAL SOLUTIONS

GERSTEL and Agilent - 25 years of partnership

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

On November 30, 2011, GERSTEL management joined Agilent Technologies‘ European Management to celebrate 25 years of successful global partnership.

both companies recognized their h i g h s y n e r g y -potential and the opportunity to do

m o r e f o r customers. GERSTEL s a m p l e 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.


www.gerstel.com

GERSTEL, Inc., USA +1 410 - 247 5885 [email protected]

GERSTEL K.K., Japan +81 3 57 31 53 21 [email protected]

GERSTEL LLP, Singapore +65 6622 5486 [email protected]

GERSTEL GmbH & Co. KG,Germany +49 208 - 7 65 03-0 [email protected]

GERSTEL AG, Switzerland +41 41 - 9 21 97 23 [email protected]


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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

GERSTEL BRASIL +55 11 5665 8931 [email protected]

More Information

Ray Marsili (Edi.), Flavor, Fragrance, and Odor Analysis, Second Edition, 280 Pages, ISBN-10: 1439846731 and ISBN-13: 978-1439846735.

www.gerstel.com

GERSTEL online: Information on products, applications, events and downloads, as well as general information about GERSTEL and our customer focused solutions: www.gerstel.com and www.gerstelus.com.

Imprint

ISS

N 1

619-

0076

· 03

/ 201

2

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

Editorial director Guido Deussing Uhlandstrasse 16 41464 Neuss, Germany [email protected]

Scientific advisory board Dr. Eike Kleine-Benne [email protected] Dr. Oliver Lerch [email protected] Dr. Malte Reimold [email protected]

Translation and editing Kaj Petersen [email protected]

Design Paura Design, Hagen, Germany www.paura.de

There are many advantages to stir bar sorptive extraction (SBSE) for isolat-

ing and concentrating flavor active chem-icals 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 pow-erful technique and emphasizes the range of applications available. Topics discussed include:

Flavor, Fragrance, and Odor Analysis, Second Edition

• Sequential SBSE, a novel extraction pro-cedure

• 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 microorgan-isms

• 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 pro-duction of aqueous formulations rich in pyrazines

• How spectral deconvolution can be used to speciate the subtle differences in essential oil content and track key ingre-dients through the manufacturing process

The final chapter summarizes chemical identities of char-acterizing aroma chemicals in fruits, vegeta-bles, nuts, herbs and spices, and savory and dairy flavors. It also provides a brief com-pendium 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 tech-niques for analyzing and enhancing food fla-vor and fragrance.

Literature

Documents

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