1. Some rectifications on the composition of costus root oil (Saussurea costus (Falc.) Lipsch) and absolute and GC-MS-FID analyses thereof Juergen Wanner a, Alin Bosilcov b juergen.wanner@kurtkitzing.de a Kurt Kitzing Co., 86757 Wallerstein, GERMANY b Brueder Unterweger GmbH, Thal-Aue 13, 9911 Assling, AUSTRIA MATERIALS Essential oil (EO) of costus (Saussurea costus) is a commercial oil from Brüder Unterweger, Assling, Austria, product code 43639; Indian costus oil & absolute were from Essential Nature, Stuttgart, Germany and another Indian oil from Adrian Essential Oils Ltd., UK. LITERATURE [1] Chang K-M & Kim G-H (2008). Comparison of Volatile Aroma Components from Saussurea lappa C.B. Clarke Root Oils. Journal of Food Science and Nutrition, 13, 128–133. [2] Akakabe Y, Washizu K, Matsui K & Kajiwara T (2005). Concise synthesis of (8Z,11Z,14Z)-8,11,14-heptadecatrienal, (7Z,10Z,13Z)-7,10,13-hexadecatrienal, and (8Z,11Z)-8,11-heptadecadienal, components of the essential oil of marine green alga Ulva pertusa. Bioscience, Biotechnology, and Biochemistry, 69(7), 1348–1352. [3] Zahara K, Tabassum S, Sabir S, Arshad M, Qureshi R, Amjad MS & Chaudhari SK (2014). A review of therapeutic potential of Saussurea lappa-An endangered plant from Himalaya. Asian Pacific Journal of Tropical Medicine, 7(Suppl 1), S60–S69. [4] Pandey MM, Rastogi S & Rawat AKS (2007). Saussurea costus: Botanical, chemical and pharmacological review of an ayurvedic medicinal plant. Journal of Ethnopharmacology, 110, 379–390. [5] Gwari G, Bhandari U, Andola HC, Lohani H & Chauhan N (2013). Volatile constituents of Saussurea costus roots cultivated in Uttarakhand Himalayas, India. Pharmacognosy Research, 5(3), 179–82. [6] Thömel E and Klein F (1976). Über neue Inhaltsstoffe von Costuswurzelöl. Tetrahedron, 32, 163–165. [7] Grieder B, Maurer A (1977). Sesquiterpenoids from Costus oil (Saussurea lappa CLARKE). Helv. Chim. Acta, 60(7), 2177–2194. [8] Steglich W, Fugmann B, Lang-Fugmann S (2000). Roempp Encyclopedia Natural Products. Georg Thieme Verlag, p. 258, Stuttgart, Germany, ISBN 0-86577-988-0 Costus root oils and an absolute were analyzed by GC-MS-FID for the sake of a rather comprehensive chemical analysis which was hard to find in the literature. To our surprise hexadecatrienal was described in one paper as the main component [1] which we could not believe just for olfactory reason since unsaturated aldehydes exhibit a very strong and penetrating odor [2]. Since costus root has a millennia-long history for its aromatic, spicy and medicinal values there is a realm of literature [3,4] but only incomplete or sometimes questionable data about the complete composition of the volatiles [1,5-7]. Even though there is nowadays only limited use of costus root oil due to allergenic properties [8] there is still some interest in the authenticity and chemical constitution of this oil. About 90% of the complete oil or absolute could be identified with aplotaxene (heptadeca-1,8,11,14-tetraene) being the major component followed by the lactones dehydrocostunolide, dihydrodehydrocostus lactone and the costols while in the case of the absolute the lactones were the main ingredients. To identify doubtful substances it may, in some cases, be helpful to take the oil apart through simple separation techniques or perform a derivatization reaction. CONCLUSION Mass spectra alone are often not sufficient for identifying compounds in essential oil analyses, even though that approach can be found in some publications. Even comparing retention indices may not suffice especially for sesquiterpenoids which can have very similar spectra. To narrow down multiple options it can be useful to perform simple operations with the aim to assign questionable compounds to a certain chemical substance group or exclude some options. Microscale separation of EOs on silica or alumina gel into a apolar fraction, which contains all hydrocarbons (and partially the ethers, epoxides) and a polar fraction, which includes all terpenoids can be done in a very short time. The separation provides additional information and can in certain cases uncover coelutions. Another method to gain more information is the practice of derivatization. Here care must be taken to avoid isomerisation of the EO components, which are prone to acidic catalyzed rearrangements. Only gentle methods can be used like thermochemolysis with trimethyl sulfonium hydroxide to form methyl derivatives of H-acidic substances, base-catalyzed acylation of alcohols, phenols, amines or silylation. Aldehydes can be disclosed by reaction with  -aminocapronic acid to form water soluble imine adducts. Some methods described above were used to identify and confirm heptadeca-1,8,11,14-tetraene (aplotaxene) as the major constituent in costus root oil by moving it in the apolar fraction. Elema-1,3,11(13)-trien-12-ol was firstly identified as an alcohol by acetylation and secondly by the mass spectrum in paper form since there was no entry in the used MS libraries. It should be noted that the composition of costus root oil probably depends on distillation time since the high boiling lactones are the major volatiles in the rhizome and make up half of the costus absolute and can only be recovered completely by prolonged distillation time. CHEMICAL ANALYSES (GC-MS-FID) 7820A Agilent dual channel GC-FID; HP-1 60m x 0.32 mm x 0.25  m and HP-Innowax 60m x 0.32 mm x 0.5  m 6890N-5973 Agilent GC-MSD; HP-1 60m x 0.32 mm x 0.25  m Thermo Scientific Trace GC Ultra with 2 split/splitless injectors and a FID detector Thermo Scientific ISQ Single quadrupole mass spectrometer Thermo Scientific TriPlus RSH autosampler GC column: 50 m x 0,25 mm x 1.0  m SE-52 FID-MS-splitter consisting of a quartz X connector, incoming: 1) the end of the GC column; 2) a short 0.25 mm transfer line from the second injector as an auxilary gas outgoing: 1) a short transfer line (1 m x 0.25 mm) to the FID; 2) a short restriction line (0.3 m x 0.1 mm) to the MS ion source Temp.prg.: 60 ° C 1Min. @ 3°C/Min. to 230°C 13 Min. Carrier gas He 5.0 @ 1,5 ml/min. const. flow; injector 230°C, FID 250°C, MS interface 250°C, ion source 150°C, EI 70 eV, scan 40- 500 amu 0.1  L EO samples were injected neat at a split ratio of 1:100 or 1.0  L of a dilution 1:10 in an appropriate solvent Analysis software: Xcalibur v2.2 Identification: MS-Libraries & retention indices from NIST08, Wiley8th ed., Identification of Essential Oil Components by GC/MS 4th ed. by Adams, Allured Publishing, MassFinder terpenoids library Quantification: FID area percent normalization EXPERIMENTAL 100  l oil on top of a silica gel (10 g) chromatography column, elution with pentane (30 ml) = apolar fraction followed by elution with CH 2 Cl 2 (30 ml) = polar fraction Fig.1: separation of the EO into an apolar (mainly terpene hydrocarbons) and a polar fraction (mainly terpenoids) Fig.2: original EO versus acetylated EO (the corresponding acetates of the alcohols are shifted towards higher retention) EXPERIMENTAL 31  L acetic anhydride, 69  L triethylamine, 4 mg p-dimethyl- aminopyridine and 10  L oil in a micro vial were heated 1 h @ 60° C. Neutralization in 10 mL 1N KH 2 PO 4, extraction with 1 mL pentane