Sophisticated Online Techniques in the Authenticity Assessment of

Chapter 4

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Sophisticated Online Techniques in the Authenticity Assessment of Natural Flavors Armin Mosandl, Mirjam Kreck, Jochen Jung and Sabine Sewenig Institut für Lebensmittelchemie, Johann Wolfgang Goethe-Universität Frankfurt/Main, Marie-Curie-Strasse 9, D-60439 Frankfurt/Main, Germany

The acceptance of food strongly depends on their flavor and aroma impressions. Consequently, authentication of genuine flavors is an important topic in view of quality assurance in the food industry and in consumer protection. Both phenomena, enantioselectivity as well as isotope discrimination during biosynthesis, may serve as inherent parameters in the authenticity control of natural flavor compounds, provided that suitable methods and comprehensive data from authentic sources are available. Besides site specific natural isotope fractionation, measured by NMR-spectroscopy (SNIF-NMR), enantio-selective capillary gas chromatography (enantio-cGC) and comparative isotope ratio mass spectrometry (IRMS), have proved to be highly efficient tools in the origin specific analysis. Nevertheless, analytical authentication of genuine food constituents is a permanent challenge, due to the complexity of food matrices. So far, enantioselective and/or IRMS online coupling techniques are the methods of choice in order to determine the authenticity of flavors. The benefits of stir bar sorptive extraction (SBSE)-enantio-MDGC/MS and multielement GC-IRMS techniques are outlined.

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© 2007 American Chemical Society Ebeler et al.; Authentication of Food and Wine ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

53


Stir Bar Sorptive Extraction ( SBSE )-enantio-MDGC-MS A novel, solventless, and simple technique for extraction of organic analytes from aqueous samples [stir bar sorptive extraction (SBSE)], has been recently introduced (/). SBSE takes advantage of the high enrichment factors of sorptive beds, but with the application range and simplicity of SPME (2-5). The stir bar is coated with a thick film of polydimethylsiloxane (PDMS), in which the aqueous sample extraction takes place during stirring for a predetermined time. After that time it is removed and placed into a glass tube, which is transferred into a thermal desorption system (TDS) where the analytes are thermally recovered, cryofocused and evaluated on-line with a capillary MDGC-MS system (Figure 1). In addition to the extraction of organic analytes from aqueous samples, the PDMS stir bars are also suitable for headspace and in vivo headspace sampling. Headspace sampling is a technique widely used to characterize the volatile fraction of several matrices, particularly aromatic and medicinal plants. SBSE has also been shown to be a successful technique for headspace sampling, because the PDMS stir bars enrich higher amounts of trapping material than SPME and, therefore, exhibit better extraction efficiency for analyzing minor components (6). Enantioselective analysis has been widely applied in quality assurance of natural flavors and essential oils, and the knowledge of enantiomeric ratios of characteristic constituents can be used as a potent genus chemitaxonomic marker. The enantioselective analysis of chiral compounds in complex natural materials by multidimensional gas chromatography (MDGC) has been shown to be an efficient and selective tool for this purpose (7-P) This connection allows the combination of the high extraction efficiency of the stir bar with the high selectivity of the enantio-MDGC-MS system. Thus, it is possible to determine the exact enantiomeric ratios of minor components in complex natural materials such as food flavors or essential oils.

SBSE Sampling A stir bar consisting of a magnetic core sealed inside a glass tube with a length of 1.2 cm, an outer diameter (o.d.) of 1.2 mm and coated with 55 PDMS was used. Stir bars are manufactured and offered by Gerstel (Muhlheim/Ruhr, Germany) under the name Twister®. The stir bar is conditioned in a desorption tube (178 mm length, 6 mm o.d., 4 mm i.d. glass tube) of a thermal desorption unit (Gerstel TDS-2) at 300 °C for 2 h.

Ebeler et al.; Authentication of Food and Wine ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

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ί i Downloaded by UNIV OF SOUTHERN CALIFORNIA on March 26, 2017 | http://pubs.acs.org Publication Date: December 28, 2006 | doi: 10.1021/bk-2007-0952.ch004

TDS-system

ί

C I S

-

3 P

T

V [

non chiral pre column

I lllve-T^

chiral main column ITD

I

detector main column OFEIMI OFENII FD detector pre column

"press-fit" connector

Figure 1. Schematic diagram ofSBSE-enantio-MDGC-MS (Reproducedfrom reference 10.)

Essential Oil Sampling Procedure The conditioned stir bar is placed in a 10 mL water sample with an essential oil concentration of 1 ppm and stirred at 1200 rpm for 20 min at ambient temperature. The stir bar is removed and dried, using a clean paper cloth. After positioning the stir bar in the middle of the heating zone of the desorption tube, the desorption program is started.

Headspace Stir Bar Sorptive Extraction (HSSE) Essential oil vapors of 0.3 g carefully dried plant material of Melaleuca alternifolia (C), Eucalyptus globulus (L.) and Thymus vulgaris (L.) are extracted by a Twister® coated with 55 film of polydimethylsiloxane, which is held at one end of magnetic wire fastened at the lid of a 20 mL vial. After an equilibration time of 15 min the stir bar is removed and positioned in the middle of the heating zone of the desorption tube and the desorption program is started. The headspace sampling device is outlined in Figure 2.


55 In K/V0-HSSE A stir bar was placed in a 25 mL flask, which was fixed to a holder near the living plants and contained two or three branches. The opening of the flask was sealed carefully with Parafilm® and, after an equilibration time of 15 min for Melaleuca alternifolia (C), 30 min for Eucalyptus globulus (L.) and Thymus


vulgaris (L.), the stir bar was removed and the analytes were desorped as described before. The in vivo headspace sampling device is outlined in Figure 2.

Thermal Desorption—Capillary M D G C - M S The TD-MDGC-MS consists of a Gerstel TDS thermal desorption system mounted on a Siemens Sichromat 2, with two independent column oven programs and a live T-switching device, coupled to the transfer line of a Finnigan MAT ITD 800, using an open split interface. For thermal desorption conditions,cf. ref. (10).

Results and Discussion In order to obtain accurate information about the enantiomeric purity of chiral compounds in essential oils, analytical procedures of highest selectivity are mandatory. Principal limitations of enantioselective analyses are known, such as racemates of natural origin, generated by non-enzymatic reactions (autoxidation, photooxidation, etc.), racemization during processing or storage (if structural features of chiral compounds are sensitive) and blending of natural and synthetic chiral compounds (9). A typical well-known example is the acid-catalyzed racemization of linalool during distillation (11-13). The pH value of plant material and distillation time are critical parameters in the chirality evaluation of pH-sensitive compounds. One possibility for obtaining accurate information about the native enantiomeric purity of labile compounds is to avoid the racemization or formation of artifacts during processing by using the headspace analysis of plant material. HSSEenantio-MDGC-MS is a new method for the reliable determination of genuine enantiomeric ratios, as well as the in vivo HSSE technique. The enantiomeric purities of chiral monoterpenes in the gaseous emission of the living plants Melaleuca alternifolia (C), Eucalyptus globulus (L.) and Thymus vulgaris (L.)

are determined and compared with the results of the headspace sampling and essential oil analysis of authentic materials.


56 Β

A

Holder

Stir bar Magnetic wire Stir bar


Plant material Flask, closed with parafilm

Figure 2. Scheme of the headspace sampling (A), in vivo headspace sampling device (B). (Reproducedfrom reference 10.)

Figure 3 shows the simultaneous enantioselective analysis of the standard compounds ct-pinene (1), β-pinene (3), a-phellandrene (4), linalool (6), terpinen-4-ol (7), ot-terpineol (9), borneol (8), sabinene (2) and limonene (5), experimental data see (70).

Conclusion The combination of SBSE, thermal desorption and enantio MDGC-MS allows the determination of the enantiomeric purity of chiral flavor compounds in essential oils and fruits, avoiding the usual expensive and time consuming isolation steps for complex matrices. The presented sampling methods are suitable for establishing characteristic authenticity profiles of fruit flavors by enantioselective capillary gas chromatography and may be utilized in quality assurance of processed fruits ( Table I ).

Isotope Discrimination The natural cycles of the bioelements carbon, oxygen, hydrogen (nitrogen, sulfur) are subjected to various discrimination effects, such as thermodynamic isotope effects during water evaporation and condensation or isotope equilibration between water and C 0 . On the other hand the processes of photosynthesis and secondary plant metabolism are characterized by kinetic isotope effects, caused by defined enzyme catalyzed reactions (75). 2


57

A


SIM:m/z93

OH ft.R

Β SIM:m/z7/0

c

> k

-OH

TIC 5,51

~ι 30:01

•IL

u

\—K. 1

1

1

1

31:41

33:21

35;OI

36:41

r 38.21

45:01

5tf;tfl

55:01

mm

Figure 3. SBSE-enantio-MDGC analysis of the standard compounds a-pinene (1), sabinene (2), β-pinene (3),a-phellandrene (4), limonene (5), linalool (6), terpinen~4-ol (7), borneol (8), a-terpineol (9), main column; Λ and B, temperature program 1; C, temperature program 2. (Reproducedfrom reference 10.)


58 Table I. Enantiomeric purity [%] of fresh Andalusian strawberries and products, using SBSE-enantio-MDGCMS

^sjA ^ 0


pulp

S:>99 R:99 R:l,4 in fruit S:98,6

^ Ύ ^ ο ^

R:l,5 S:98,5 R:99 R:99

^γ^οΗ

R:n.d. S: n.d. R: n.d. S: n.d. R: n.d. S: n.d.

o^^cfr

^^CT^

R: >99 S: 99 S: 99 S: 99 S: 99 S: 99

S: n.d.

S: n.d.

R: >99

S: 99.9 >99.9 52.9 55.7 51.8 52.0 53.3

Sophisticated Online Techniques in the Authenticity Assessment of

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