Abstract
A targeted search for heliotropin (or piperonal) in the flower fragrance of heliotrope and in cured vanilla beans of Tahitian origin is reported. Heliotropin is not detected in the fragrance emitted by heliotrope, using dynamic headspace SBSE sampling, followed by thermal desorption and analysis by conventional GC-MS. Main constituents are anisaldehyde and benzaldehyde, which confirms previous findings. Less than one part per million of heliotropin is accurately detected in authentic Tahitian vanilla bean, in using both GC-MS and LC-MS/MS. This confirms previous observations, which had shown that 4-methoxyaromatic compounds are the characteristic elements in the flavor of Tahitian vanilla, with p-anisaldehyde as the the main aroma donator. We suggest that heliotropin was named after the discovery of piperonal by degradation of piperine, the odor of which was reminiscent of the heliotrope flower. Later, its identification in vanilla extracts was the result of either a wrong interpretation of analytical data, or, much more probably, adulterations of these extracts. As a result, heliotropin should not be cited any longer as a characteristic consituent of Tahitian vanilla.

"It remains doubtful whether this aromatic aldehyde occurs in the beans (fruit) of various vanilla species, also whether the odor of heliotrope flowers is due to piperonal." Since its first publication in 1949 [1] , this statement has not stimulated any complete investigation, and, conversely, there has been a continuing controversy on the alleged occurence of heliotropin (or piperonal) in cured vanilla beans from Tahiti [2] , even until recently [3] . Such confusing allegations may still generate problems in food labelling, despite this heliotropin has been used for a long time as an additive in artificial vanilla blends, or even as an adulterant in natural vanilla flavors [4] . We report herein, a targeted search for heliotropin in the fragrance of heliotrope flowers and in cured vanilla beans of Tahitian origin.
Heliotrope flowers
The analysis of fragrance given off by heliotrope flowers (Heliotropum spp.) has been reported in several recents papers [5] [6] [7] . It is noteworthy, none of these publications mentioned heliotropin, and in one of them, dealing with H. peruvianum [6] , it was even stated that "heliotropin does not exist in heliotrope".
Heliotrope flowers
In 1984, we had initiated a research program on the systematic analysis of the volatile compounds (so-called headspace) emitted by fragrant flowers, including that of heliotrope. In 1991, using our standard procedure [8] , we had identified 66 volatile constituents in common heliotrope flowers Heliotropum arborescens (Tables 1 and 2). Among the main components listed in Table 1 , C-6 aliphatic alcohols and aldehydes play an important role in the immediate fresh and green impression of the fragrance. This is rapidly dominated, however, by the characteristic almond-like odor provided by benzaldehyde and p-anisaldehyde, which confirms recent findings [7] .
Heliotrope flowers
Fifteen years later, we were able to retrieve the GC-MS acquisition data file, and carry out a selective search for heliotropin in using the ion extraction technique with ions 121, 149 and 150 within the appropriate GC retention window. This proved to be negative. More recently (May 2006), a new investigation was carried out with living heliotrope flowers, using sorptive dynamic headspace extraction with a Twister TM coated with PDMS polymer as the trapping device. After thermal desorption and cryofocussing, the volatile fraction was analyzed by GC-MS. The major components were found to be this time benzaldehyde, p-anisaldehyde and 2-phenylethanol, in decreasing order of abundance. Again, no heliotropin could be detected, although no specific work was undertaken to determine its limit of detection under these conditions.
Tahitian vanilla bean
A comprehensive analysis of the volatile fraction of cured vanilla beans cultivated in Tahiti (Vanilla tahitensis Moore) has been published recently [9] . Although the authors claim that they could identify 276 constituents, data on the identity and concentration of only 20 of them was disclosed. In particular, no information was provided on the presence of heliotropin.
Tahitian vanilla bean
With no intention of carrying out such a comprehensive investigation, we have focussed our analysis on the possible presence of heliotropin in an industrial ethanolic oleoresin which had been prepared in 1977, a sample of which (ca. 200 g) had been used at that time for a similar purpose [10] , and carefully stored since then. Using direct injection GC-MS and the ion extraction technique within a selected retention window, we were unable to detect any heliotropin. This enabled us to conclude that, if heliotropin is present in the extract, its concentration should be in the low ppm range. This was confirmed upon GC-MS in SIM mode (using ions 149 and 150), which showed that the lower limit of detection is about 1 ppm.
Tahitian vanilla bean
Authentic Tahitian vanilla beans were thoroughly extracted with diethylether in August 2006. GC-MS analysis of the crude extract by static headspace SPME showed the presence of high levels of anis alcohol, anisaldehyde and a relatively low level of vanillin. This was considered to be sufficient to confirm that the plant species is V. tahitensis.
Tahitian vanilla bean
The crude extract was then submitted to a more sensitive analysis by LC-MS/MS with positive electrospray ionization (ESI), with a view to detecting and quantifying the heliotropin, if present. For the construction of the calibration curve (R 2 >0.99), the ion at m/z 93 was used. It corresponds to a grand daughter ion (C 6 H 4 O+H) + generated by the successive losses of CH 2 O (30) and CO (28) from the parent ion at m/z 151. Under these conditions, the concentration of heliotropin was found to be 0.67 ppm. A similar measurement was performed with the 1977 extract, which was found to contain 2.3 ppm heliotropin. It should be noted, however, that this quantitative determination does not comply strictly with the recommended guidelines of the European Commission [11] , and would therefore justify further testing by LC-MS/MS with at least two distinct transition ions. Additional data on standard deviation, recovery rate in a spiked sample etc. would also be desirable, although they are outside the remit of this paper. Whatsoever, these results confirm that heliotropin is only a trace constituent (less than 10 ppb) in this sample of cured beans of V. tahitensis. Previous well supported observations [10, 12] had shown that heliotropin is not detected in extracts of authentic Tahitian vanilla beans, using either GC-MS [10] or HPLC-UV [12] . Whereas no lower limit of detection (LOD) was given by the former authors, the latter estimated it to be less than 10 ppm.
Tahitian vanilla bean
Consequently, heliotropin cannot be considered any longer as a characteristic constituent of V. tahitensis fruit, on both organoleptic and compositional standpoints. Even as a trace element, heliotropin is not specific either of V. tahitensis, because it had been detected at a similar concentration level (<1 ppm) in a methanol extract of V. planifolia [13] .
Tahitian vanilla bean
In conclusion, this paper should put an end to an exceptionally long controversy [12] , based largely on misunderstanding and involvement of adulterated vanilla extracts [14] . It certainly started ca. 140 years ago when pure piperonal was first prepared from natural piperin by German chemists, who assigned to this new compound the odor of heliotrope flower, thus the name of "heliotropin". The characteristic odor of heliotrope flower is mainly due to panisaldehyde. This element accounts in greatest part for the specific aroma of cured Tahitian vanilla bean, a feature that had already been highlited one century ago by Walbaum [15] .
Tahitian vanilla bean
to SPME using the standard device (Supelco) fitted with a PA fiber (polyacrylate). As a result, acids (such as anisic acid) and phenols (such as vanillin, 4-hydroxybenzaldehyde) were not recovered, while neutral components (anis alcohol, anisaldehyde, anisylethyl ether etc.) were selectively concentrated and much better detected by GC and GC-MS .

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