Analysis of the sesquiterpenes in Achillea species usinq liquid chromatography-mass spectrometry with positive ion atmospheric pressure chemical ionisation

The species of the Achillea millefolium group contain different sesquiterpenes which are of chemotaxonomical and pharmacological interest. Therefore the HPLC analysis of these compounds is a fundamental task for the quality control of Achillea samples. As difficulties in identification solely on the basis of retention times may arise, the LC was coupled with atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) allowing a selective and sensitive detection and thus the clear identification of the sesquiterpenes.


Introduction
Several species of the polyploid Achillea millefolium group are characterized by labile proazulenes and other guaianolides, e.g.8a-tigloxy-(I) resp.8aangeloxy-artabsin (2) or achillicin (3) and stable matricarin-derivatives as matricarin Yarrow is widely used in folk medicine against various diseases including internal and external inflammations and according to pharmacological studies some of these sesquiterpenes are responsible for the anthiphlogistic activity [3, 41.In contrast some sesquiterpenes show only little or no activity while others even might trigger the undesirable allergic contact dermatitis [5, 61.Therefore it was an important task to work out a method for the analysis of the sesquiterpenes in Achillea species to check the quality of samples.A high performance liquid chromatographic method on reversed phase was developed allowing the quantitative determination of the relevant sesquiterpenoids using UV-detection [7].
It is obvious that the identification of the sesquiterpenes especially in samples with a complex composition or in samples containing compounds of more than one Achillea species is difficult on the basis of retention times solely.Thus there was the demand for an alternative detection method allowing the unambiguous identification of these compounds.In this paper the coupling of mass spectrometry (MS) with high performance liquid chromatography (HPLC) is presented as additional tool for detection of sesquiterpenoids from Achillea species.Experimental Chemicals HPLC grade water was prepared by distillation of deionised water on a GFL 4020 distillation instrument, methanol (CHROMASOL\$, HPLC grade) was purchased from Riedel-de Haen, dichloromethane (p.a. grade) from J. T. Baker, ammonium acetate (Ammonium aceticum puriss.cryst.) was obtained from Apoka, Vienna, Austria.

Plant material and reference substances
Achillea species were collected in Austria or they were commercial samples.
The plant material was botanically identified at the lnstitute of Pharmacognosy.
Vouchers of the origins are deposited in the Herbarium of the lnstitute of Pharmacognosy, University of Vienna (Austria).

HPLC-MS equipment
The

Sample preparation
100 mg air dried flowerheads were transferred to a 2 ml reacti vial followed by 1 ml dichloromethane.After ultrasonification for 10 min.at room temperature the solution was filtered into a 10 ml round bottom flask, the drug was washed two times with 1 ml dichloromethane respectively, and also filtered.Altogether this procedure was performed four times, the plant extracts were evaporated entirely and redissolved in 500 PI 80% methanol, 20 p1 thereof were analyzed by HPLC-MS.In contrast the labile proazulenes 8a-tigloxy-artabsin (I), MW 346, resp.8aangeloxy-artabsin ( Z ) , showed only some fragments at these conditions and no quasimolecular ions could be found.It is known that the addition of volatile buffers to the mobile phase may enable ionisation and improve the ion abundance.The addition of ammonium acetate resulted in mass spectra of these proazulenes with a prominent peak corresponding to the adduct [M+NH4]' beside a quasimolecular ion

Optimisation of the APCl parameters
[MH]' with low intensity (Fig. 3).Further investigations showed that a minimum concentration of 2 mM ammonium acetate is necessary to obtain the above described mass spectra.
Increasing the concentration to 5 mM buffer leads to moderately increasing peak intensities while the use of higher concentrations up to 10 mM ammonium acetate showed no further advantages.Therefore the addition of 5 mM ammonium acetate to the mobile phase was the best choice.
In contrast to the proazulenes and eudesmanolides the matricarin derivative (5) did not show any adduct with ammonium acetate, the mass spectrum showed same profile as without addition of ammonium acetate (Fig. 2).This fact was confirmed in all following investigations, therefore two types of sesquiterpenes are found in Achillea species: the matricarin type sesquiterpenes giving no adduct with ammonium acetate and showing prominent [MH]' quasimolecular ions (Fig. 2), on the other hand proazulenes and eudesmanolides are present with [M+NH,]' being the base peak and [MH]' with low abundance (Fig. 3 and 4).The latter compounds can easily be recognised by the difference of 17 amu between the ions [M+NH4It and [M+H]'.

Effect of vaporizer temperature and declustering potential
To achieve a reduction of the aerosol droplet size for an improvement of the .
All other parameters (nebulizer gas, curtain gas, needle current, focusing potential, entrance potential and deflector) were investigated in the same manner, but only minor effects on the mass spectra could be observed.

LC-MS detection of sesquiterpenoids from Achillea
Fig. 5 shows the TIC as well as the UV traces from an analysis of Achillea collina.Using LC-APCl MS the sesquiterpenoids can selectively be detected.
The detection limit for these compounds was investigated by injection of various amounts of the reference sesquiterpenes 8a-tigloxy-(1) resp.8a-angeloxyartabsin (2), desacetylmatricarin (5) and santamarin (6).These series of analyses demonstrated that this method is effective for the detection of the sesquiterpenes down to levels of 1 ng in the scan mode (mlz 100mlz 400 1 sec.)and showed that these compounds are detected in the same intensities.Thus, LC-MS offers a much better sensitivity than UV-detection.
The introduced HPLC-APCI MS method allows the selective and sensitive detection of the sesquiterpenes from different species of the Achillea millefolium group.A clear identification of the compounds is possible by the use of the mass spectra even if a complex composition of the sesquiterpenes or a mixture of different species is present.This is an indispensable requirement for the analysis and quality control of medicinally used Achillea samples or extracts.
ionisation efficiency at the given flow rate of the mobile phase of 1 mllmin the vaporizer temperature was increased step by step starting from 200°C.Rising the temperature to 300°C resulted in continous increase of the ion intensities.An increase of the vaporizer temperature to 350°C showed no further effects, whereas temperatures above 350°C produced decreasing ion intensities most likely due to thermal degradation of the compounds.Thus the ideal vaporizer temperature was found to be 300°C.By applying different potential differences ("declustering potential", DP) in the intermediate pressure region of the mass spectrometer the collision induced dissociation (CID) of compounds may be controlled.A series of investigations with the reference compounds showed that a low declustering potential (DP 1) gives mass spectra where only the quasimolecular ions [M+H]' resp.[M+NH,]' can be seen.Increasing the potential to DP 10 leads to more and more fragmentation and higher sensitivity of the total ion current (TIC), whereas values above the ideal DP 10 resulted in too much fragmentation so that only very small or no quasimolecular ions were found.