Methoxypyrazine standards

Unlabelled 2-methoxy-3-alkylpyrazines are commercially available (Pyrazine Specialities, Inc., Atlanta, GA, USA). They can also be synthesized by the method of Murray and Whitfield (1975). The chloro compounds (8) and (9), prepared by the method of Murray and Whitfield (1975), are converted to trideuterated methoxypyrazines (6) and (7) by the action of sodium methoxide- Hj (Fig. 2 Harris et d. 1987 Lacey et d. 1991). 

To determine whether the response of the mass spectrometer is linear, it is calibrated by andysis of mixtures of (1) and (6), and mixtures of (4) and (7), at a minimum of six mole ratios covering the useful concentration range, preparing each cdibration mixture in duplicate. The instrumentd response (i.e. weighted linear regression of Hq/ H3 peak area ratio on mole ratio) is calculated 

Appropriate quantities of the trideuterated methoxypyrazines (6) and (7) in purified dichloromethane are added to samples (typically 240 ml) and allowed to stand for several hours. After adjustment to pH 5, the sample is distilled at atmospheric pressure. Methoxypyrazines in the distillate (50 ml) are trapped with a strongly acidic ion-exchange resin (AG50WX4,200 mg) then, after filtration and washing of the resin, are removed by water adjusted to pH 10 (1 ml) which in turn is extracted with dichloromethane (2 X 0.5 ml). The dichloromethane extracts are slowly concentrated under nitrogen in the dark at room temperature over 6-7 h to ca. 20 il in tapered micro-vials, then transferred to glass capillaries that are sealed and stored in the dark at — 20°C imtil mass spectrometry. 

Day-to-day variation of instrumental performance can be expected. It is not practical to perform a full instrumental calibration on each day of analysis, but it is necessary to compare the instrumental response with the sample to that with a standard (e.g. 1 jtl of a dichloromethane solution of (1), (4), (6) and (7) at 200 pg/1, and (3) at 20 pg/1) on the day of analysis after, as required, mass calibration and optimization of source parameters. Quantitative analysis should be attempted only if the response to the standard is within 10% of the mean of previous satisfactory determinations. 

Methoxypyrazines can contribute to wine flavour even at low parts per trillion levels. Sensory detection thresholds in water have been determined as 2 ng/1 (parts 

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Methoxypyrazines are compounds with very low detection thresholds which must be determined at very low levels. For these compounds, different selective isolation methods have been proposed (Allen et al. 1994 Sala et al. 2002). Some authors use a simple extraction (Kotseridis et al. 1999 Falcao et al. 2007) or an optimized headspace SPME procedure (Chapman et al. 2004 Prouteau et al. 2004) using in most cases isotopically-labelled internal standards to compensate for matrix effects. In spite of the claims of the authors, all these methods present some difficulties to accurately determine the compounds at the lowest levels at which they can be found. A recent report has presented an advanced method combining the preconcentration ability of headspace SPME with the selectivity of comprehensive GC (Ryan et al. 2005). 

In studies of methoxypyrazines, a stable isotope labeled internal standard has not always been used. Calo et al. (6) used 5ec-butylmethoxypyrazine as an internal standard to quantify isobutylmethoxypyrazine in a comparison of grape varieties. Recently, Hashizume and Umeda have used 2-methyl-3-Ai-propylpyrazine as an internal standard to quantify methoxypyiazines in Japanese red wine and grape samples (7). However, the increased potential for lack of precision and accuracy needs to be recognized, and the natural occurrence of 5gc-butylmethoxypyrazine is a drawback to its use as an internal standard. 

Table 1. Coefficient of variation of replicate mass spectrometry determinations of methoxypyrazines (1), (2) and (3) using trideuterated isobutylmethoxypyrazine (5) as internal standard...

Althou the amino aclci/sugar solutions were buffered in an effort to maintoin pH, a drop in pH was encountered with Increasing reaction times. Therefore, after heat treatment, each sample was adjusted to pH 9.0 with O.IN NaCH. One ml of a solution containing 2-methoxypyrazine in distilled water (2 ppm) was added as an internal standard. Firml sample volume was 15 ml. lyrazines were then isolated, s >arated and quantified using an... 

Figure 4.10. Extracted ion chromatograms of GC/IT-MS/MS analysis of a 1 2.5 diluted wine spiked with four methoxypyrazines at 0.25 ng/L and two ethoxypyrazines at 45 ng/L as internal standards. The chromatographic conditions are described in Table 4.3. (49) 3-Ethyl-2-methoxypyrazine (m/z 119) (50) 3-isopropyl-2-methoxypyrazine (IPMP) (m/z 109) (51) 3-ethyl-2-ethoxypyrazine (internal standard 1) (m/z 124) (52) 3-isopropyl-2-ethoxypyrazine (I.S. 2) (m/z 123) (53) 3-seobutyl-2-methoxypyrazine (SBMP) (m/z 81) (54) 3-isobutyl-2-methoxypyrazine (IBMP) (m/z 109).

Kotseridis, Y.S., Spink, M., Brindle, I.D., Blake, A.J., Sears, M., Chen, X., Soleas, G., Inglis, D., and Pickering, G.J. (2008). Quantitative analysis of 3-alkyl-2-methoxypyrazines in juice and wine using stable isotope labelled internal standard assay, / Chromatogr. A, 1190,294-301. 

Fig. 2. Trideuterated methoxypyrazines, used as internal standards in quantitative wine analysis by isotope-dilution mass spectrometry, and the corresponding chloropyrazines used in their synthesis...

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