电脑技术英语作文

method was studied for the determination of artemisinin from Artemisia annua L. extracts. The technique does not

require any kind of derivatisation prior to the analysis. All samples were simply dissolved in methanol and

injected into the mobile phase. Detection was achieved by using mass spectrometry with atmospheric pressure

chemical ionisation. The ionisation technique is relatively soft and provides protonated molecular ion and

informative structural fragmentation for the compound. Benzophenone was used as a chromatographic standard for

the determination of the analytical reproducibility. The supercritical carbon dioxide mobile phase used in the

system was modified by 10% methanol. The average absolute retention time was 3. min with a standard

deviation of 0.017 min and a relative standard deviation of 0.4% with respect to benzophenone for the procedure.

The correlation coefficient was 0.998 and detection limit 370 pg on column.

Introduction

Artemisinin (qinghaosu) is a sesquiterpene lactone with an endoperoxide oxygen bridge across the seven member rings, Fig. 1. It is the clinically active antimalarial constituent isolated from the traditional Chinese herb Artemisia annuaL. Several thousand cases of malaria in China have been successfully treated with artemisinin. Total chemical synthesis of artemisi-nin is complex (including 11 steps) and gives a low yield(~30%). Therefore, the main source for artemisinin in pharmaceutical science is from plant extraction. The plant, Artemisia annua L., contains most of the artemisinin (% of the total artemisinin) in the leaves at an average concentration less than 0.1% calculated by dry weight. Elsewhere an average artemisinin content of 0.4% was also reported. The content of artemisinin varies according to its origin and growing conditions, such as temperature, humidity, light and nutrition. Interest in the antimalarial potential of artemisinin as well as its derivatives is currently the subject of numerous investiga-tions.

Several analytical techniques have been reported for the qualitative and quantitative determination of artemisinin in

biological and crude plant extracts. Methods such as thin layer chromatography (TLC), HPLC with electrochemical detec-tion,

ultraviolet detection, polarographic detec-tion, thermospray and electrospray mass spectrometric

detection, evaporative light scattering detection (ELSD), chemiluminescent detection (CL), and gas chromatography

with mass spectrometric detection, enzyme-linked im-munosorbant assay (ELISA), and electrophoresis with UVdetection are the most commonly used tech

niques. Super-critical fluid chromatography with FIDand ELSD has also been reported.

All these techniques have been well adapted for the determination of artemisinin in a wide range of samples, however they have some limitations related to the chromatog- raphy and detection in terms of resolution, being time consuming, requiring derivatisation, etc. ELISA requires a three-step derivatisation to modify artemisinin into ELISA active dihydroartemisinin carboxymethylether. However, the technique gives the impression of being too complicated (three- step derivatisation) for routine analysis requirements. Due to the absence of appropriate UV absorption, UV detection is ineffective for quantitation of artemisinin. A derivatisation is needed to convert artemisinin into a UV active compound that absorbs with a large extinction coefficient at longer wave-lengths (the procedure mainly consists of hydrolysis of artemisinin). ELSD requires a warming-up stage prior to each run, approximately 20–30 min. This is a very significant disadvantage of such a technique. Chemiluminescent detection is based on a secondary formation of a chemiluminescent reagent like aminophthalate. It is not a direct measurement, so the selectivity and sensitivity of the system are also dependent on the other chemical used. The resolution is low and also many compounds in biological or crude plant extracts could provide the oxidation conditions.

In consideration of the chromatographic techniques, GC is a temperature programming technique, thus limiting the tech- nique for thermally labile compounds, including artemisinin (which decomposes at a temperature above 150 °C27) and has a long analysis time.28 Although, as mentioned above the determination of artemisinin by HPLC-MS does provide satisfactory results for routine screening of artemisinin, the analysis technique requires time for sample preparation and clean-up steps (long analysis time), uses a mobile phase that consists of quite a large amount of environmentally unfriendly organic solvents and can give low peak resolution.

It would preferable to have, in addition, a more selective and sensitive technique. Also from an environmental point of view, it is important to minimise the use of undesirable solvents in analysis. In these respects, supercritical fluid chromatography is an alternative technique to GC and HPLC. It offers faster analysis time with good resolution, analysis of thermally labile compounds without derivatisation and it can be interfaced to a wide range of GC- and HPLC-like detectors. Although, an organic modifier is common in supercritical mobile phase, the amount of organic solvent used is very much less (10% MeOH in supercritical CO2) than for HPLC. Another feature of the technique used is the APCI ionisation technique which has not been encountered as an analysis technique for artemisinin interfaced with SFC in literature. In this paper SFC interfaced to APCI-MS using environmental friendly supercritical CO2 as the mobile phase for determination of artemisinin is discussed.

The packed-column SFC coupled with APCI-MS studied to analyse artemisinin was a SF3 model Gilson packed column SFC system (Anachem, Luton, UK). The system consists of a microprocessor-controlled Gilson model 308 master pump and a Gilson model 306 slave pump. The 308 master pump was fitted with a chiller unit (Anachem, Luton, UK) to cool the pump head to 210 °C to produce liquid CO2 and used to deliver SFC grade CO2 mobile phase modified with 10% methanol at a flow rate of 2 ml min21 to produce a pressure of 200 bar at the column inlet. The 306 slave pump was used to deliver the programmed addition of organic modifier into the mobile phase. The two Gilson pumps were connected to a Gilson model 811C dynamic mixer to produce a homogeneous mobile phase. Finally the mobile phase was passed through a Gilson

821pressure regulator. Samples were injected via a Rheodyne 7125 injector valvefitted with a 10 µl injection loop (Anachem, Luton, UK). Separation was achieved on a 25 cm 3 4.6 mm id CN column, packed with 5 µm packing material of cyanopropyl (Spher-isorph, Fison Chromatography, Loughborough, UK).

Detection was performed using a Trio 2000 single quadru-pole mass spectrometer (VG

Biotech, Altrincham, UK). The packed column SFC was interfaced with APCI-MS through a drawn 75 µm id fused-silica restrictor. The restrictor tip was drawn in a bunsen flame and cut to give an inlet pressure of approximately 200 bar. The restrictor was inserted into the APCI probe, which has a heated tip at the end, and positioned at the APCI source. The total SFC column effluent was delivered into the atmospheric pressure ion source through the heated probe (300 °C) using nitrogen boil-off gas from a liquid nitrogen dewar as bath gas at a flow-rate of 200 l h21. The source temperature was maintained at 120 °C. Ionisation was achieved via a 3 kV discharge at the corona pin, generating proton donor methanol reagent ions, ([MeOH2]+) and/or methanol clusters ([MeOH)nH]+), which transfer protons to the analytes. To obtain mass spectra containing several fragment ions, the cone voltage was varied between 30 and 90 V and MS data were collected in the full-scan mode from 100–350 u in 1 s. Single ion monitoring (SIM) was used for quantification of the artemisinin. The position of the restrictor, probe, bath gas flow rate, probe temperature and source temperature have been optimised, for details see ref. 29 and the values were re-checked. The mass spectrometer was operated in positive ion mode for both full scan and single ion mode for the qualitative and quantitative analysis of artemisinin.

Chemicals

Artemisinin was obtained from Aldrich (Gillingham, Dorset, UK) and used without

further purification. Fluorescence grade methanol (99.99%) was obtained from Fisher Scientific Int. (Loughborough, Leicestershire, UK) and benzophenone from Lancaster (Lancaster, UK). High purity grade CO2, SFC grade was obtained from BOC Ltd (Surrey, UK). A standard artemisinin stock solution was prepared in methanol to give a concentration of 0.5 mg ml21. All solutions used were prepared from this stock solution in methanol.

SFC-APCI-MS conditions for the chromatographic elution were initially optimised.

Different concentrations of MeOH modifier (v/v) were applied to achieve a complete elution and a good peak shape. The best concentration of MeOH in the mobile phase was found to be 10%. All the optimum conditions used for the analysis of artemisinin are given in Table 1. Although, a high APCI probe temperature is applied to the probe, most of the heat is used to overcome the Joule–Thomson effect, to complete vaporisation of the mobile phase and to heat the bath gas. However, thermal decomposition of artemisinin at the APCI probe is insignificant.

Fig. 2 shows the TIC chromatogram for artemisinin standard under the optimum

conditions. The chromatogram shows a good peak shape with an analysis time of less than 4 min. This retention time is approximately 2 or 3 times less than those in literature for HPLC analysis.1,10,13,16,20,23 Elution of artemisinin under the optimum condition was straightforward. The Gauss-ian like peak shape of the chromatogram demonstrates the absence of hydrogen bonding interactions between the com-pound and the stationary phase. The corresponding APCI mass spectrum at a cone voltage of 30 V gives the protonated molecule ion at m/z = 283 u in approximately 10% relative intensity.

The APCI-MS mass spectrum of artemisinin at 30 V cone voltage shows high progressive

fragmentation, this case was similar even when a lower cone voltage was applied. This mass

spectra indicates that artemisinin under the APCI-MS condi- tions used is not stable enough to give the protonated molecule ion as most abundant ion. The most abundant ion (100% relative intensity) under the analysis conditions was the fragment ion of artemisinin at m/z = 251 which was the monitored and used ion in SIM mode to quantitate artemisinin in samples. Since the fragmentation pattern is specific for each compound, identifica-tion of the artemisinin is unambiguous. Increasing the cone voltage to 50 V and then to 90 V, leads to almost complete fragmentation of the structure due to collision-induced dissocia-tion reaction. Fig. 3 shows the mass spectra of artemisinin standard at different cone voltages applied. The most abundant ion at m/z = 251 u which is the artemisinin specific ion and is possibly produced by losing an oxygen molecule from the protonated molecule ion. Further fragmentation occurred by breaking the aromatic rings until reaching the stable ion at m/z = 105 u at high cone voltage.

The analyte ions introduced in the ionisation source undergo a series of reactions with the

reagent ion, e.g. proton transfer, charge transfer, cluster or addition reactions. Hence the most common reaction in the APCI ionisation is the proton transfer reaction,31 it is clear that the other reactions are also possible for artemisinin. The ions above the protonated molecular ion at m/z = 328 and m/z = 298 u on the mass spectrum (mass spectrum at 10 and 30 cone voltage) are the adducted ions of clustered methanol and methyl groups to the artemisinin, [M + C2H5O + H]+ and [M + CH3 + H]+ respectively.

The fragment ions below the protonated molecular ion occurring corresponding to the

following species; m/z = 283 u [M + H]+, m/z = 277 u [M 2H5]+, m/z = 251 u [M 2O2 + H]+, m/z = 219 u [M 2 O4 + H]+, m/z = 191 u [M 2 O4C2H4 + H]+, m/z = 178 u [M 2 O4C3H5+H]+, m/z = 162 u [M 2 O5C3H5 + H]+, m/z = 151 u [M 2 O5C4H4 + H]+, m/z = 133 u [M 2 O5C5H10 + H]+ and the lowest fragment ion in the scanned range was at m/z = 105 u [M 2 O5C7H14 + H]+. Fig. 4 shows the proposed fragmentation pattern for artemisinin, in which the ions were chosen in respect to their high intensity at 50 and 90 V. This fragmentation must be regarded as provisional. Since the nature of ionisation techniques used in MS are different from each other, to compare fragmentation of artemisinin in Fig. 4 with literature was unworkable.

Reproducibility of the system was determined in full scan mode

using retention times. Benzophenone was used as a chromatographic standard compound and the relative retention times were calculated from the ratio of absolute retention times of artemisinin to those of benzophenone. For this investigation a concentration of 250 ng on column containing benzophenone was injected several times to achieve sufficient data to carry out a statistical calculation. Table 2 shows the reproducibility data for artemisinin. Both SD and RSD within day and restrictor are quite low, showing good reproducibility. The restrictor is one of the most important and the same time most fragile part of the interface since it often breaks down. The restrictor is a home made version, prepared by drawing the 75 µm id fused silica tubing in a flame, and cutting the end to give more or less the required pressure. SFC pressure is controlled by the restrictor. Consequently replacing the restrictor causes different effects on the chromatographic retention times and it is difficult to maintain the previous pressure even at the same flow rate. However to minimise this effect the use of relative retention times were preferred which are more convenient rather than absolute retention time in reproducibility studies. During the reproducibility course three different restrictors were used and for each restrictor a minimum 10 injections were made. The total number of the restrictors used overall was 5. Reproducibil- ity between days and restrictors for the system is well satisfactory, related results have been published

else- where.

To quantitate the technique, a series of artemisinin standard solutions of 1 ng, 10 ng, 50

ng, 100 ng and 250 ng on column, was prepared to study linearity of response (LOR) and limit of detection (LOD) of the system. Both parameters were deter-mined in selected ion mode (SIM) and the monitored ions were the specific fragmented ion of artemisinin at m/z = 251 u and the protonated molecule ion at m/z = 283 u as confirmation ion. The peak areas were calculated and used to plot a calibration graph against the amount injected on the column at a cone voltage of 30 V. The points represents the response of the detector at each concentration and shows an excellent straight line fit producing a correlation coefficient (R2) of 0.998 and the related equation y = 29390x + 61980, which was used to quantitate the amount of the artemisinin in the Artimisia annua L. extracts. The above equation was also used to work out the analytical reproducibility (precision) and accuracy of the technique. Artemisinin concentration of 1 ng and 50 ng on column was injected several time in the system then related peak areas were calculated by monitoring the ion m/z = 251 u in SIM. The result obtained showed a analytical reproducibility (precision) of 2.5% RSD using the peak areas. Accuracy was calculated by applying the above equation and the peak areas for 1 ng and 50 ng concentration. Average relative error was below 8%.

The theoretical limit of detection for the mass was deter-mined for a signal to noise

ratio of 3:1 using the 1 ng concentration of artemisinin standard solution. The theoretical limit of detection for artemisinin was estimate to be 370 pg on column using signal to noise ratio of 3:1.

A large number of alcoholic extract samples (ca. 150 alcoholic extracts) containing

artemisinin have been successfully ana-lysed using the SFC-APCI-MS technique. Fig. 5 shows the chromatogram of one of the artemisinin extracts from Artemisia annua L. Identification of artemisinin peaks was based on the retention time, mass spectrum and fragmentation pattern, with the peak at 3.53 min corresponding to the artemisinin in the extract. Quantification of artemisinin in samples was based on the calibration graph which was generated for each batch of extracts.

The chromatogram also contains other peaks, which are associated with the

co-extracted compounds. The identification of the co-extracted compounds was attempted using the corresponding mass spectra and the possible existence of other products in the plant extracts. As mentioned above, artemisinin does not produce a protonated ion at 100% relative intensity under the conditions applied even at low cone voltage, therefore its analogues were also expected to behave similarly. However, the mass spectra of the co-extracted compounds did not show certain specific m/z values to be expected. Most importantly a lack of the standard compounds for all natural products, makes the mass spectra comparison impossible, and hence no identification can reasonably be attempted. Fig. 6 shows the mass spectra of the co-extracted compounds.

Packed column SFC in combination with APCI-MS method offers a definitive

technique for the both qualitative and quantitative analysis of artemisinin samples. The technique does not require any derivatisation prior to the injection and offers excellent analytical reproducibility. The SFC-APCI-MS methods gave a Gaussian shape peak for artemisinin and the retention time was decreased approximately 2 or 3 times. Controlled fragmentation was possible through adjustment of the cone voltage for providing structural information for artemisinin. The detector response for the APCI-MS was found to be linear (R2 = 0.998) over a large concentration range from 1 ng to 250 ng on column and the detection limit was 370 pg on column (S/N = 3:1). The system was also found to be reproducible in retention times (Table 1). Analytical reproduci-bility (precision) was calculated as 2.5% RSD, and 8% relative error for accuracy.

The authors are grateful to Celal Bayar University, Manisa, Turkey and the

European Union (FAIR CT96 2003) for financial support, and to William Ransom and Son Ltd. (Hitchin, England) and General Extract GmbH (Flensbury, Germany) for supplying the artemisinin extracts.

1 J. F. S. Ferreira and J. Janick, Phytochemistry, 1996, 41, 97–104. 2 B. A. Avery, K. K. Venkatesh and M. A. Avery, J. Chromatogr., B., 1999, 730, 71–80.

3 I. S. Lee, H. Elsohly and C. D. Hufford, Pharm. Res., 1991, 7, 199. 4 A. Brossi, B. Venugopalan, L. D. Gerpe, H. J. C. Yeh, J. L. Flippen- Anderson, P. Buchs, X. D. Luo, W. Milhous and W. Peters, J. Med. Chem., 1988, 31, 8.

5 G. Schmid and W. Hofheinz, J. Am. Chem. Soc., 1983, 105, 624–625.

6 T. Ravindranathan, M. A. Kumar, R. B. Menon and S. V. Hiremath, Tetrahedron Lett., 1990, 31, 755–758.

7 D. L. Klayman, A. J. Lin, N. Acton, J. P. Scovill, J. M. Hoch, W. K. Milhous and A. D. Theoharides, J. Nat. Prod., 1984, 47, 715–717. 8 D. L. Klayman, Science, 1985, 228, 1049–1055.

9 D. J. Charles, J. E. Simon, K. V. Wood and P. Heinstein, J. Nat. Prod., 1990, 53, 157–160.

10 N. Pras, J. F. Visser, S. Batterman, H. J. Woerdenbag, T. M. Malingre and C. B. Lugt, Phytochem. Anal., 1991, 2, 80–383.

11 M. M. Eldomiaty, I. A. Almeshal and F. S. Elferaly, J. Liq. Chromatogr., 1991, 14, 2317–2330.

12 N. Acton and D. L. Klayman, Planta Med., 1985, 441–442.

13 K. L. Chan, K. H. Yuen, S. Jinadasa, K. K. Peh and W. T. Toh, Planta Med., 1997, 63, 66–69.

14 J. F. S. Ferreira, D. J. Charles, K. Wood, J. Janick and J. E. Simon, Phytochem. Anal., 1994, 5, 116–120.

15 D. Ortelli, S. Rudaz, E. Cognard and J. L. Veuthey, Chromatogra- phia, 2000, 52, 445–450.

16 H. N. Elsohly, E. M. Croom and M. A. Elsohly, Pharm. Res., 1987, 4, 258–260.

17 C. G. Thomas, S. A. Ward and G. Edwards, J. Chromatogr., B, 1992, 583, 131–136.

18 Z. M. Zhou, Y. X. Huang, G. H. Xie, X. M. Sun, Y. L. Wang, L. C. Fu, H. X. Jian, X. B. Guo and G. Q. Li, J. Liq. Chromatogr., 1988, 11, 1117–1137.

19 M. P. Maillard, J. L. Wolfender and K. Hostettmann, J. Chromatogr., A, 1993, 7, 147–1.

20 P. Sahai, R. A. Vishwakarma, S. Bharel, A. Gulati, M. Z. Abdin, P. S. Srivastava and S. K. Jain, Anal. Chem., 1998, 70, 3084–3087.

21 H. T. Chi, K. Ramu, J. K. Baker, C. D. Hufford, I. S. Lee, Y. L. Zeng and J. D. McChesney, Biol. Mass Spectrom., 1991, 20, 609–628.

22 P. Christen and J. L. Veuthey, Curr. Med. Chem., 2001, 8, 1827–1839.

23 M. D. Green, D. L. Mount, G. D. Todd and A. C. Capomacchia, J. Chromatogr., A, 1995, 695, 237–242.

24 H. J. Woerdenbag, N. Pras, R. Bos, J. F. Visser, H. Hendriks and T. M. Malingre, Phytochem. Anal., 1991, 2, 215–219.

25 M. Jaziri, K. Shimomura, K. Yoshimatsu, M. L. Fauconnier, M. Marlier and J. Homes, J. Plant Physiol., 1995, 145, 175–177. 26 H. L. Chen, K. T. Wang, Q. S. Pu, X. G. Chen and Z. D. Hu, Electrophoresis, 2002, 23, 2865–2871.

27 M. Kohler, W. Haerdi, P. Christen and J. L. Veuthey, Phytochem. Anal., 1997, 8, 223–227.

28 S. S. Mohamed, S. A. Khalid, S. A. Ward, T. S. M. Wan, H. P. O. Tang, M. Zheng, R. K. Haynes and G. Edwards, J. Chromatogr., B, 1999, 731, 251–260.

29 K. Dost, PhD Thesis, University of Nottingham, Nottingham, 1999. 30 A. E. Ashcroft, Ionisation Methods in Organic Mass Spectrometry, The Royal Society of Chemistry, Cambridge, 1997.

31 R. K. Mitchum, W. A. Korfmacher, G. F. Moler and D. L. Stalling, Anal. Chem., 1982, , 719–722.

32 Y. McAvoy, K. Dost, D. C. Jones, M. D. Cole, M. W. George and G. Davidson, Forensic Sci. Int., 1999, 99, 123–141.

33 K. Dost and G. Davidson, J. Biochem. Biophys. Meth., 2000, 43, 125–134.

34 D. C. Jones, K. Dost, G. Davidson and M. W. George, Analyst, 1999, 124, 827–831.