Kikuo Sasamoto

Nobuo Ochiai, Ph.D. 


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

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.
1D/2D-TIC and Mass Chromatograms (m/z 195 and 197) after PFC-concentration of analytes from 20 injections of spiked wine (Zoomed in 2D-GC-MSanalysis). (1) = IBMP 25 ng/L; (2) = TCA 5 ng/L; (3) = Geosmin 50 ng/L
Mass spectra of IBMP (a-1), TCA (a-2), and Geosmin (a-3) extracted and concentrated from spiked wine using 20 SBSE extractions combined with PFC concentration and subsequent TD-1D/2D-GC- O/MS determination. Mass spectra from Wiley Mass Spectral Library for IBMP (b-1), TCA (b-2), and Geosmin (b-3).

Wine flavor analysis

Holding on to fleeting encounters

Companies around the world constantly strive to be a nose ahead in the race to determine flavor and fragrance compounds – or to determine which off-flavor compounds are the culprits in customer complaint cases. The scientists who are ahead in the race are not only highly skilled, but also have the right toolkit. Having the ability to combine a variety of extraction techniques with 1- or 2-dimensional separation, mass selective detection, an olfactory detection port (ODP) to determine which compounds are interesting, and even a fraction collector to collect interesting compounds for further analysis – this is the toolkit that dreams are made of. When all this is nicely integrated on a single GC/MS system it also becomes very manageable. Japanese scientists from GERSTEL K.K. in Tokyo demonstrate the system that helps sniff out even the smallest peak.

In terms of olfactory performance, we humans have nothing like the capabilities of our canine companions. The olfactory center in the brain of a dog is up to 40 times larger than the human olfactory center. A German shepherd has around 220 million olfactory cells; nosy humans have no more than 5 million. Dogs also breathe faster and more intensely, further adding significantly to their olfactory performance. Dogs can sense a significantly larger number of odor-active compounds per unit time than humans. The fact that we humans lead our dogs on a leash and not the other way around bears testament to the fact that olfactory performance may be important, but is not apparently the decisive factor in determining the pecking order on the evolutionary ladder. Dogs have superior olfactory capabilities, Homo sapiens have advanced analytical instrumentation and the skills to decipher the results in order to compensate for olfactory deficiencies. The instrumentation available to us even enables extraction of odor active compounds from complex matrices followed by qualitative or quantitative determination. Gas chromatography (GC), especially in combination with olfactory detection (GC-O), is widely recognized as a highly efficient method. If the column effluent is split at the column outlet, an Olfactory Detection Port (ODP) and a mass selective detector (MSD) can be attached in parallel in order to get both an olfactory signature and a mass spectral identification and quantification of the odor active compound. As an aside, the ODP - MSD combination exposes weaknesses in the technique. “You don’t always get a signal in the chromatogram even though there is a perceivable smell at the ODP”, explains Dr. Nobuo Ochiai, technical director at GERSTEL K.K. in Tokyo, Japan. This proves that the human nose, while not able to compete with its canine counterpart, is still more sensitive than modern analytical equipment when it comes to compounds that have a low odor threshold: Even a few milligrams of methylmercaptan in 100 million cubic meters of air are enough to make people head for the exit. To put that volume into context, it is around 1000 times the volume of the Notre Dame cathedral in Paris. For most compounds, far higher concentrations are required in order to adequately produce an olfactory response. And many times it is necessary to first remove interfering compounds using chromatographic separation in order to get a clear description.
Separating olfactory interferences from the analyte can be simple, even for complex samples, if you have the right equipment. A GC/MS system that offers a second dimension of separation with simple, easy to use software control is available from GERSTEL. Called the Selectable 1D/2D-GC-O/MS system, it allows the GC column effluent to be split between the MSD and an Olfactory Detection Port (ODP) or a Preparative Fraction Collector (PFC) to enable parallel MS determination and olfactory monitoring or fraction collection for further analysis.

A second dimension at your finger tips

Dr. Ochiai and Mr. Sasamoto successfully used heart-cutting from the 1D column in the compact, integrated 1D/2D-GC-O/MS system and used the second dimension to separate and identify flavor and fragrance compounds in foods, beverages and personal care products. The main challenge when analyzing many of these types of samples is to eliminate matrix interference. Coeluting compounds will normally influence both the mass selective detection and the olfactory impression leading to incorrect odor identification and odor intensity readings.
If the analytical system does not have sufficient sensitivity to determine the identity or concentration of the odor-active compounds, further automated steps can be taken to concentrate analytes: A) 1D heart-cuts from multiple injections can be taken and transferred to the 2D column for every 2D run. B) Analytes from multiple 2D runs can be collected on a fraction collector connected to the outlet of the 2D column. C) The concentration steps under both A) and B) can be combined. In order to facilitate these steps, the scientists used a single trap PFC.

Performance check

Two commercially available white wines, a Sauvignon Blanc and a Chardonnay, were used to put the combined 1D/2D-GC-O/ MS-PFC system to the test. Three off-flavor analytes were added to the wine: 1) The classical corkiness culprit trichloroanisol (TCA); 2) 2-isobutyl- 3-methoxypyrazine (IBMP), known for its bell pepper flavor; and 3) geosmin (“earth odor” in Greek), known for its musty earthy note. Even ultra-trace amounts of these compounds can be detected by the human nose. The IBMP odor threshold is 25 ng/L; TCA starts stinking at 5 ng/L; and geosmin adds a musty note at concentrations as low as 50 ng/L. To test the PFC’s performance, standard solutions containing the following 15 compounds at the pg-level were injected directly into the analytical system: Hexanal, 1-hexanol, 3-hexenol, linalool, citrinellol, geraniol, p-cymen-8-ol, phenethyl alcohol, guaiacol, ethylhexanoate, ethyloctanoate, phenethyl acetate, beta-damascenone, gamma-nonalactone and limomene. IBMP, TCA and Geosmin were extracted from spiked wine samples in headspace vials by means of Stir Bar Sorptive Extraction (SBSE) using the GERSTEL Twister. Following the extraction step, the PDMS coated Twisters were removed from the samples, cleaned with DI water, dabbed dry on a Kimwipe, and transferred to individual Thermal Desorption Unit (TDU) liners.
The TDU liners were then transferred to the GERSTEL MultiPurpose Sampler (MPS) and placed in individually sealed tray positions from which the MPS transports them to the TDU for analysis. Thermal desorption was performed under the following conditions: TDU initial temperature: 30 °C; hold time 0.5 min.; 720 °C/min to 200 °C; hold time 3 min; Desorb Flow (He) 50 mL/min. The desorbed analytes were cryo-focused in the Cooled Injection System (CIS), PTV-type GC inlet, at 10 °C on a CIS liner packed with Tenax TA. CIS analyte desorption was performed by heating the CIS at a rate of 720 °C/min to 240 °C, with a hold time of 2.0 min. Analytes were transferred to the 1D separation column in splitless mode. The GC/MS system used was a 7890 GC combined with an 5975C MSD (both from Agilent Technologies). The 1D separation was performed on a 30 m long DB-Wax column, 0.25 mm ID, 0.25 μm film thickness (Agilent Technologies). The second dimension separation was performed using a 10 m long DB-5 column, 0.18 mm ID, 0.40 μm film thickness (Agilent Technologies). The columns were not kept inside the GC oven, but rather placed in individual Low Thermal Mass (LTM) column modules (Agilent® Technologies) mounted on the front of the GC. LTM modules can be heated and cooled separately, and, thanks to their low thermal mass, heating and cooling can be performed much faster than traditional air bath GC ovens, resulting in faster separation and shorter analysis cycles. The GC oven was kept at 250 °C throughout the analysis cycle, essentially serving as a heated chamber that keeps all transfer capillaries and column connectors at the proper temperature for best system performance.

1D/2D separation- and PFC Concentration Performance

The scientists tested the performance of their single trap PFC (GERSTEL) in two steps using an adsorbent trap. Recoveries ranged from 85 to 98 %, with RSDs below 3.2 % (n = 7). Further, compound recoveries over twenty injection cycles were investigated and were found to be in the range from 98 to 116 %. Dr. Ochiai found that the high recoveries achieved using PFC concentration showed that both the analyte transfer in the system and the PFC adsorbent trap performed very well for the analytes at sub-ng levels, whereby useful quantitative and qualitative information could be gained. Following the first test runs, the identification of the spiked offflavor compounds was performed using automated thermal desorption in combination with 1D/2D-GC-O/MS. The 1D column was programmed from 40 °C (2 min) at 10 °C/min to 240 °C. The 2D column was kept at 40 °C and was left at this temperature if not used. When a heart-cut was performed from the 1D separation, the 2D column was programmed from 40 °C to 150 °C at 5 °C/min and then at 20 °C/min to 280 °C (hold). The column effluent was split between the MS and the ODP with a split ratio of 1:2. The MS was operated in full scan and in SIM mode. The scan range was from 29 to 300 m/z at a scan rate of 2.68 Hz. In SIM mode, nine ions were monitored: m/z 124, 151 and 94 for IBMP; m/z 112, 125 and 182 for Geosmin; and m/z 195, 197 and 210 for TCA. The acquisition rate was 3 Hz for each ion; the ODP was kept at 250 °C.

Determining off-flavor compounds at ultra-trace levels

Just a sneak preview: IBMP, TCA and Geosmin were determined in scan mode using SBSE-TD-1D/2D-GC-O/MS with unequivocal olfactory confirmation. Retention times of the GC-O signals were 12.45 min (IBMP), 16.25 min (TCA), and 16.55 min (Geosmin). “But these peaks were completely hidden in the 1D total ion chromatogram (TIC)”, the scientists stated. Therefore the relevant sections of the 1D chromatogram from 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 retention time of 17.5 min. The three off-flavor compounds were clearly identified by olfactory detection during the 2D run. As an aside, high-boiling residue was simultaneously back-flushed from the 1D column by increasing the outlet pressure and decreasing the inlet pressure in order to clean up the system for the next run. MS detection in full scan mode unfortunately was not sufficiently sensitive to deliver useful data based on a single injection. The scientists therefore set about concentrating analytes introduced from 20 Twister extractions of the same sample using the integrated single channel PFC. Following the 20-fold concentration step, the Tenax TA trap used in the PFC was desorbed in the TDU and the analytes introduced to the system for 2D-GC-O/ MS determination. No system modification was necessary in order to perform this procedure. The results peak for themselves: IBMP and Geosmin peaks were found in the 2D TIC and the peaks matched the olfactory signals. Even though the TCA peak was almost completely buried in the 2D TIC, the Extracted Ion Chromatogram (EIC) for the TCA ions m/z 195 and 197 clearly displayed a peak matching the olfactory signal for TCA. The mass spectra for all target compounds were compared with data from the Wiley Mass Spectral Library, which is integrated with the Agilent ChemStation.
Following library identification, a Sauvignon Blanc wine was analyzed to determine the concentration levels of all three off-flavor compounds using single SBSE-TD- 1D/2D-GC-O/MS. The MS was operated in Single Ion Monitoring (SIM) mode. Quantitation was based on four-point standard addition curves, the method resulted in excellent linearity (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-GCO/ MS-, 2D-GC-O/MS-, 1D-GC-PFC-, and 2D-GCPFC analysis can be performed without modifying the system configuration. The Thermal Desorption Unit (TDU) on the system performs splitless introduction and transfer of the trapped and concentrated analytes. We have clearly shown that this enables us to identify off-flavor compounds at ultratrace levels. The 2D-GC-O/ MS system is unique in allowing us to perform the analysis in full scan mode”. System performance was shown by determining three off-flavor compounds, TCA, IBMP, and Geosmin spiked into a wine at levels ranging from 5 to 50 ng/L. The combination of SBSE and PFC resulted in recoveries between 71 and 78 %. These values clearly show that the SBSE-TD-1D/2D-GC-OMS 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.