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Characterization of some plant extracts by GC–MS

Article  in  Nuclear Instruments and Methods in Physics Research Section B Beam Interactions with Materials and Atoms · January 2009
DOI: 10.1016/j.nimb.2008.10.021

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 Nuclear Instruments and Methods in Physics Research B 267 (2009) 338–342

Contents lists available at ScienceDirect

Nuclear Instruments and Methods in Physics Research B

journal homepage: www.elsevier.com/locate/nimb

Characterization of some plant extracts by GC–MS

A. Iordache *, M. Culea, C. Gherman, O. Cozar
‘‘Babes-Bolyai” University, Str. M. Kogalniceanu, Nr. 1, Cluj-Napoca 400084, Romania

a r t i c l e i n f o a b s t r a c t

Available online 17 October 2008 Different types of herbs often used in pharmaceutical, cosmetic and food industry were extracted and
then analyzed by gas chromatography and gas chromatography–mass spectrometry. The method valida-
PACS: tion parameters showed good linearity, precision and recovery for a standard mixture. Herbs from differ-
80 ent zones of Romania were studied: melissa (Melissa officinalis), nettle (Urtica dioica, Lamium album),
camomile (Matricaria chamomilla). The study was applied for fingerprint chromatograms to characterize
Keywords: the flavors extracted from herb plants of different sources. The identity and quantity of the measured
GC
active compounds was correlated with the expected therapeutic effects. The active principles content
GC-MS
was determined for the same herb, and different amounts of the active principles were determined for
LLE
Herbs
plants of different origin.
Extracts Ó 2008 Elsevier B.V. All rights reserved.
Active principles

1. Introduction officinalis, Urtica dioica, Lamium album from different geographi-

The study of herbs involves the isolation and the structure elu-
cidation of their compounds, for understanding and evaluating
their therapeutic potential. A growing interest for the rapid extrac-
tion from different matrices and for the precise analyses of these
active herb compounds increased the need for optimization of
the experimental design, to obtain better recoveries, low solvent
consumption and reduced extraction times [1–4]. In the latest
years the interest for the study of the organic compounds from
plants and their activity has increased. A lot of extraction methods
and analytical methods as spectrophotometry, high performance
liquid chromatography, capillary electrophoresis, gas chromatog-
raphy (GC) with flame ionization detection (FID), gas chromatogra-
phy–mass spectrometry (GC–MS) are developed for plant active
compounds study. The combination of an ideal separation tech-
nique (GC) with the best identification technique (MS) made GC–
MS an ideal technique for qualitative and quantitative for volatile
and semivolative compounds. In addition, the use of a proper
extraction method is needed.
The aim of the present paper is to develop a rapid method for
the quantitative determination of organic compounds in herbs
used as raw materials in cosmetic, pharmaceutical or food indus-
try. The method was used for a comparison between the bioac-
tive compounds found in Matricaria chamomilla, Melissa
* Corresponding author.
E-mail address: andres_iro2002@yahoo.com (A. Iordache).
cal regions of Romania.
2. Materials and methods
2.1. Apparatus
A Hewlett Packard gas chromatograph 5890 coupled with an EI
mass spectrometer model 5989B was used for the compounds
identification. The GC is equipped with a HP-5MS capillary column
30 m length, 0.25 mm diameter, 0.25 lm film thickness, in the
temperature program: 50 °C for 2 min, then increased to 250 °C
at a rate of 8 °C/min, then with 30 °C/min to 310 °C and kept for
10 min. The helium flow rate was 1 ml/min. A Hewlett Packard
6890 gas chromatograph equipped with a HP-5 fused silica capil-
lary column 30 m length, 0.25 mm diameter, 0.25 lm film thick-
ness and an autosampler was used in the following temperature
program: from 50 °C for 2 min to 250 °C at 8 °C/min with helium
as carrier gas, at 2.8 ml/min flow rate.
2.2. Reagents and chemicals
Solvents (methanol, methylene chloride, hexane, ethyl acetate,
acetone, purchased from Comchim-Bucharest, Romania, were
purified by distillation, when necessary. We have investigated
some standard compounds, known to be usually present in some
herbs: (1) 3-hepten-2-one, used as external standard (ES); (2) 1,8-
cineol (eucalyptol) (synthesized in S.C.Natex s.r.l.); (3) linalool

0168-583X/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.nimb.2008.10.021
 A. Iordache et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 338–342 339

Fig. 1. Separation chromatogram of the standard mixture.

(Fluka, Sweden); (4) geraniol (Fluka, Sweden); (5) alpha-terpinyl-
acetate (Fluka, Sweden); (6) geranyl acetate (Fluka, Sweden); (7)
amyl salicylate (synthesized in S.C. Natex s.r.l); (8) methyl myris-
tate (C14:0 diluted 10% in ethylbenzene and acetophenone; Poly-
science Corporation, Evantson, Illinois, USA); (9) methyl palmitate
(C16:0 diluted 10% in ethylbenzene and acetophenone; Poly-
science Corporation, Evantson, Illinois, USA) and (10) methyl stea-
rate (C18:0 diluted 10% in ethylbenzene and acetophenone;
Polyscience Corporation, Evantson, Illinois, USA). The standard
and solvents purity was tested by GC and GC/MS. An aliquot of
100 ll of each standard compounds formed the standard mixture
(a total of 900 ll, compounds 2–10), 3-hepten-2-one was added
separately, after extraction, as external standard for the recovery
study.
2.3. Plant material
Herb samples, dried flowers and leaves, M. chamomilla, M.
officinalis, U. dioica and L. album were harvested in four different
geographical zones (Petrosani, Catalina (Cluj), Baia Mare and Va-
tra Dornei) of Romania, purchased from market. The control
samples were harvested at The Agronomic Institute Cluj-
Napoca.
2.4.1. Standard solutions
A stock solution was obtained by mixing 100 ll of each stan-
dard compound in ethylbenzene and acetophenone (v/v). The
working standard mixture was prepared by diluting the stock solu-
tion to obtain a concentration of 2.7% (v:v).
2.4.2. Method validation
The linearity of the GC–MS signal was studied in the range of
0–100 lg. The regression curves obtained for the standards gave
good correlation coefficients, over 0.995, as reported in previous
work. Relative standard deviations (RSD) were lower than 3%.
The average values were obtained from the results of four extrac-
tion procedures and two injections of each extract. The recovery
for LLE procedure was 97% (n = 4) for the standard mixture. The
sensitivity was lower than 10 lg at a ratio S/N = 10 and the
L.O.D. was 10 ng, S/N = 10 for each standard. The validation meth-
od was tested on a hydro alcoholic herbs extract, kept seven days
at the room temperature. This herb extract was prepared for a
vermouth aroma composition. Good precision values were ob-
tained from this vermouth hydro alcoholic extract. The relative
standard deviation mean value was lower than 5%, when four
extractions were analyzed twice.
3. Results and discussion

2.4. LLE extraction procedure The separation chromatogram of the standards mixture is pre-
sented in Fig. 1. The peaks at 7.5 min and 10.1 min are the solvents
For the method validation study, a mixture of three solvents
(solvent A), was prepared in the ratio 5:1:1, v/v/v: ethyl ace-
tate:hexane:methylene chloride. The LLE extraction procedure Table 1
for validation was: 30 ll standard mixture in 0.9 ml solution Precision and recovery study for the extraction procedure (n = 4).
distilled water:ethanol (1:1, v/v), 0.9 ml distilled water and Compounds Mean SD RSD Standard Standard Recovery
0.3 ml solvent A, (3:3:1 v/v/v) were mixed with 0.5 g NaCl for (n = 4) (%) extract mixture (%)
5 min and then centrifuged for 2 min. One microliter of 3-hep- 1. 3-Hepten-2-one (ES) 8.50 0.23 2.74 1.00 1.00
ten-2-one was added to the supernatant and then 1 ll of the 2. 1,8-Cineol 8.10 0.07 0.81 0.95 0.96 98.97
resulting solution was injected twice, by using the autosampler (eucalyptol)
3. Linalool 10.16 0.02 0.16 1.20 1.24 96.67
injector.
4. Geraniol 8.46 0.10 1.24 0.99 1.05 94.31
The extraction procedure from herbs: 1 g of each dried and 5. Alpha-terpinyl 3.68 0.01 0.40 0.43 0.44 97.57
crushed plant sort was mixed with 10 ml ethanol and 10 ml dis- acetate
tilled water and warmed at the temperature of 50 °C for 30 min, 6. Geranyl acetate 8.50 0.05 0.62 1.00 1.04 96.28
agitating from time to time. The extract was filtrated and 3 ml of 7. Amyl salicylate 9.01 0.04 0.43 1.06 1.09 97.23
8. Methyl myristate 9.22 0.07 0.77 1.08 1.08 100.82
hydroalcoholic extract were mixed with 3 ml distilled water,
(C14:0)
1 ml solvent A, 5 g NaCl and 0.3 ll methyl myristate, added as 9. Methyl palmitate 9.85 0.17 1.71 1.16 1.19 97.14
internal standard for the quantitative determination of the or- C16:0)
ganic compounds. The new mixture was agitated for 5 min and 10. Methyl stearate 9.01 0.22 2.47 1.06 1.09 96.93
(C18:0)
then centrifuged. The supernatant was separated and concen-
Mean values 1.13 97.32
trated, and then 4 ll were injected twice (n = 2) into the GC
and GC/MS. tR = retention time; SD = standard deviation, RSD = relative standard deviation.
340 A. Iordache et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 338–342

(ethyl benzene and acetophenone) for the methyl esters used in Table 4
the standard mixture. Absolute recovery values were determined Organic compounds found in Urtica dioica.

by using external calibration with the standard 3-hepten-2-one. Compounds tR 1 2 3

The recovery and precision experimental data are presented in Ta- lg/g lg/g lg/g
ble 1. The mean values are results from four extraction procedures
1. Butoxy propanol 10.2 0 0 0
and two injections of each extract. The relative standard deviation 2. 4-Vinyl phenol M = 120 11.8 0 0 0
3. Carvone 12.2 0 5.6 0
4. Dihydroactinidiolide M = 180 16.8 0 5.6 206.6
5. 11 Methyl myristate (SI = 2 mg/g) 19.5 2000 2000 2000
Table 2
6. Loliolide M = 196 20.3 0 0 942.5
Organic compounds found in Matricaria chamomilla.
7. Hexahydrofarnisyl acetone 21 0 5.6 0
Compounds tR 1 2 3 4 8. Neophytadiene M = 278 21.2 0 80.5 0
9. Benzyl salicylate M = 228 21.5 0 5.6 0
(min) lg/g lg/g lg/g lg/g 10. Methyl palmitate M = 270 22 0 5.6 0
1. 1,3 Butandiol 4.2 85.6 61.9 0 40.9 11. Ethyl palmitate M = 284 22.8 0 5.6 0
2. 4-Vinyl phenol 11.8 9.2 6.9 0 0 12. Phytol 24.3 0 226.4 69.9
3. 2-Methoxy-6-vinyl phenol 14.8 178.6 103.1 0 0 13. Gamma-sitosterol 32.8 0 5.6 0
4. Trans-beta-farnesene 15.6 0 68.7 61 0 Total value 0 346.1 1219
5. Spathulenol M = 220 17.6 70.9 74.2 0 0
tR(GC) = retention time.
6. Alpha-bisabolol oxide B M = 238 18.6 160.2 230.9 216.8 55.1
1. Urtica dioica harvested from Baia Mare region.
7. Alpha-bisabolol M = 222 18.9 485.3 0 72.1 312.3
2. Urtica dioica harvested from Petrosani region.
8. Bisabolone oxide M = 236 19.1 0 233.7 100 0
3. Urtica dioica harvested from Catalina region (Cluj-Napoca).
9. Methyl myristate (SI = 2 mg/g) 19.5 2000 2000 2000 2000
10. Herniarin M = 178 19.7 893.2 378 48.5 50.5
11. Chamazulene M = 184 19.7 46 82.5 97.1 49.0
12. Bisabolol oxide A M = 238 20 1798 1093 1050 682.2
13. En-in-dicycloether M = 200 21.7 1647 941.6 563.7 1292.1
14. En-in-dicycloether M = 200 21.8 582 277.7 132 215.2
15. Methyl palmitate M = 270 22 181.4 68 0 0
16. Palmitic acid M = 256 22.6 179.6 433 0 0 Table 5
17. Ethyl palmitate M = 284 22.8 0 0 46.9 29.3 Organic compounds found in L. album.
18. 9-Octadecenoic acid 23 0 34.4 0 0
Compounds tR 1 2 3 4
19. 9,12-Octadecadienoic acid 24.8 0 41.2 0 0
20. Methyl linoleate M = 294 24.8 0 0 54 0 lg/g lg/g lg/g lg/g
21. Ethyl oleate M = 310 24.8 0 0 53.4 0
1. Iso-butyl formiate 3.9 6.9 43.4 0 0
22. Achilin M = 246 25.1 18.4 20.6 27 0
2. Butoxy propanol 10.2 0 4.3 0 71.8
23. Methyl heritol M = 258 25.2 0 0 27 35.9
3. M = 120 11.8 0 4.3 0 160.4
24. M = 288 25.9 0 0 101.9 27.8
4. Carvone 12.2 0 0 0 21
25. M = 288 26.4 0 68 148.3 20.2
5. Propyl benzene 12.3 6.9 0 0 0
26. Glyceryl oleate diacetate 27.6 240 96 129 27.8
6. tert-Butyl phenol 13.2 0 0 0 21
M = 440
7. 4-Vinyl-2-methoxy phenol 13.4 0 4.3 0 89.9
27. Stearic acid M = 270 28 0 24.7 0 0
8. Dihydroactinidiolide M = 180 16.8 4.6 0 0 142.7
Total value 8575.2 4338.1 2928.7 2838.3
9. Methyl myristate (SI = 2mg/g) 19.5 2000 2000 2000 2000
tR = retention time. 10. Loliolide M = 196 20.3 0 0 0 87.4
1. Matricaria chamomilla harvested from Baia Mare region. 11. Hexahydrofarnisyl acetone 21 4.6 0 0 0
2. Matricaria chamomilla harvested from Petrosani region. 12. Methyl palmitate M = 270 22 4.6 0 16.8 0
3. Matricaria chamomilla harvested from Catalina region (Cluj-Napoca). 13. Palmitic acid M = 256 22.6 0 0 0 256.6
4. Matricaria chamomilla harvested from Vatra Dornei region. 14. Phytol 24.3 122.3 0 71.1 40.9
15. Gamma-sitosterol 32.8 0 0 12.3 0
Total value 149.9 56.3 100.2 891.7

tR(GC) = retention time.

1. Lamium album harvested from Baia Mare region.
Table 3 2. Lamium album harvested from Petrosani region.
Organic compounds found in Melissa officinalis. 3. Lamium album harvested from Cluj-Napoca region (The Agronomic Institute).
4. Lamium album harvested from Catalina region (Cluj-Napoca).
Compounds tR 1 2 3
lg/g lg/g lg/g
1. Linalool 9.4 0 18.4 0
2. Citronellal 10.4 0 0 197.2
3. Z-citral 12 0 0 300.3 values were below 3% for the extraction procedure. The recovery
4. Methyl citronellal 12.3 0 0 24.2 gave 97% for the standard mixture by LLE extraction (3:3:1,v/v/v),
5. E-citral 12.6 0 0 592 very close to other extraction procedures. The compounds were
6. Geranyl acetate 2,3-epoxy- 12.8 0 0 20.1
identified by GC–MS. The active principles content in the herbs
7. Geranyl acetate 12.8 0 0 14.3
8. trans-Caryophyllene 15.1 0 0 11.3 harvested from different geographical regions are presented in
9. Caryophyllene oxide 17.6 0 0 14.7 Tables 2–5.
10. Methyl myristate (2mg/g) 19.5 2000 2000 2000 The ion chromatograms of M. chamomilla, M. officinalis and L.
11. Loliolide 20.2 23.6 23.1 6.8
album herbs are presented in Fig. 2. The qualitative and quantita-
12. Palmitic acid 21.30 0.3 0.1 9.8
13. Methyl palmitate 22 31.4 25.9 11.6
tive differences are due to the different climatic regions, specific
14. Gamma-sitosterol M = 414 32.8 21.9 0 6.8 soil quality and air temperature.
Total value 77.2 67.5 1209.1 Matricaria chamomilla shows high quantity of bioactive com-
tR = retention time.
pounds. Alpha-bisabolol, the important active compounds respon-
1. Melissa officinalis harvested from Catalina region (Cluj-Napoca). sible for anti-inflammatory effect is diminished or oxidized. There
2. Melissa officinalis harvested from Baia Mare region. are samples where the organic compounds are lost probably be-
3. Melissa officinalis harvested from Cluj-Napoca region (Agronomic Institute). cause of the geographical conditions or the drying processes.
 A. Iordache et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 338–342 341

Fig. 2. Active compounds separation of Matricaria chamomilla, Melissa officinalis, Lamium album herbs.

Fig. 3 presents the quantitative variation in active principles in
herbs from different regions from Romania. M. officinalis herb
showed low content in active principles. These results could be
due to the differences between the soil types, fertilizers, slopes,
sunshine, location and climate. The highest quantity of bioactive
compounds was found in Cluj-Napoca species, Baia Mare and Cat-
alina regions. The L. album and U. dioica samples are characterized
(Fig. 3) by very small amounts of active principles.
Our rapid and simple method outlined the active principles in
the studied herbs which are responsible for some therapeutic
and aromatic effects. The important qualitative and quantitative
differences among herbs require their surveillance after harvest-
ing and drying, to be then used for medicinal or pharmaceutical
purposes. The extraction method presented is simple, rapid and
inexpensive, with reduced solvent consumption. GC–MS method
used for the analysis of the obtained extracts can be an interest-
342 A. Iordache et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 338–342

Fig. 3. Comparative variation of the total active principles in herbs from different geographical regions; the missing results are due to very low quantity of the compounds
extracted from herbs in similar conditions.

ing tool for testing the amount of some active principles in herbs References
used in cosmetic, drugs, pharmaceutical or food industry.
[1] C. Gherman, M. Culea, O. Cozar, Talanta 53 (2000) 253.
[2] N. Carro, C.M. Garcia, R. Cela, Analyst 122 (1997) 325.
Acknowledgement [3] K. Robards, M. Antolovich, Analyst 122 (1997) 11R.
[4] P. Pallado, G. Tassinato, M. D’Alpaos, P. Traldi, Rapid Commun. Mass Spectrom.
This work has been supported by the Romanian Research Foun- 11 (1997) 1335.
dation (Grant ID 501).

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