744
Anal. Chem. 1987, 59, 744-748
erties of such polymers to further enhance the detection capabilities.
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (8) (9) (10)
Habert, M. K.; Baldwin, R. P. J . Chromatogr. 1985, 345, 43. Wang, J.: Freiha, B. Anal. Chem. 1984, 56, 2266. Santos, L. M.; Baldwin, R. P. Anal. Chem. 1986, 58, 846. Marko-Varga, G.: Appelquist. R.: Gorton, L. Anal. Chim. Acta 1988, 179, 371. Ikariyama, Y.; Heineman. W. R. Anal. Chem. 1986, 5 8 , 1803. Sittampalam, G.,Wilson, G. S. Anal. Chem. 1983, 55, 1608. Wang, J.; Hutchins, L. D. Anal. Chem. 1985, 5 7 , 1536. Wang, J.; Tuzhi, P.; Golden, T. Anal. Chim. Acta. in press. Oyama. N.: Anson, F. C. J . Am. Chem. SOC.1979, 101, 3450. Cox, J. A.: Kulesza, P. J. Anal. Chlm. Acta 1983, 154, 71.
(11) Facci, J.; Murray, R. W. Anal. Chem. 1982, 5 4 , 772. (12) Geno, P. W.: Ravichandran, K.; Baldwin, R. P. J . Nectroanal. Chem. 1985, 183, 155. (13) Oyama, N.; Anson, F. C. Anal. Chem. 1980, 52, 1192. (14) Rubinstein,'I.; Bard, A. J. J . Am. Chem. SOC. 1981, 103, 5007. (15) Moore, R. B.; Martin, C. R. Anal. Chem. 1966, 58, 2569.
RECEIVED for review August 21, 1986. Accepted November 11,1986. This work was supported by the National Institutes of Health (Grant No. GM 30913-03) and the American Heart Association. T.G. acknowledges the support of Battelle Pacific Northwest Laboratory for the award of a Summer Fellowship during the course of this work.
Determination of Volatile Constituents of Chinese Medicinal Herbs by Direct Vaporization Capillary Gas ChromatographyIMass Spectrometry Chen Yaozu,* Li Zhaolin, Xue Dunyuan, and Qi Limin
Department of Chemistry, Lanzhou University, Lanzhou, People's Republic of China
Analyses of the volatile constituents of Chinese medlclnal herbs have been accomplished via direct vaporiratlon GC/MS using sample slres of 3-15 mg of pulverlred herb samples subjected to heatlng at 250 OC for 60 s In a specially constructed vaporlror. By this new technique, three Chinese medicinal herbs have been analyzed and the results compared with those obtalned by tradltlonai steam distillatlon extractlon methods. Good agreement between the two techniques has been observed. Direct vaporitation, however, offered several distinct advantages. The method was rapid wlth a rnlnlmal sample preparatlon step, employed a small sample slre, and avoided the potential Interferences from lmpurltles derived from the solvents during extractlon and distillation.
Many Chinese medicinal herbs contain volatile essential oils as their effective pharmacological ingredients. In conventional methods of analyzing for essential oils, steam distillation and/or extraction with various organic solvents is often employed prior to the determination step. These methods involve extensive analytical manpower and are time-consuming. Besides, during such chemical treatments some constituents may be lost and the sample may suffer from potential contamination by impurities from the solvents employed. More recently, a method developed for the volatile monomers and additives of rubberlike polymeric samples involved a technique described by H u ( I , 2) as chromatopyrography. In this basic principle, a vaporization chamber has been specially constructed in this laboratory to permit direct analysis by GC/MS. In this paper, we report the analysis of micrcquantities of three major Chinese medicinal herb samples by this technique of direct vaporization and compare the results with those obtained by the conventional steam distillation extraction methods.
EXPERIMENTAL SECTION Samples. The Chinese medicinal herbs Atractylodes macrocephala koidz (I), Zingiber officimleRoscoe (11),and Amomum globosum Lour (111) were all purchased from local markets and properly identified by Zhang Guoliang of the Department of Biology, Lanzhou University. Herb samples for direct vaporization were first pulverized to pass through a 60-mesh screen. Essential oil extracts for the same herbs were also obtained by steam distillation followed by extraction with ether. Gas Chromatography. Analyses were performed on a Shimadzu Model 9A gas chromatograph equipped with a flame ionization detector (FID) at a operating temperature of 260 "C and with nitrogen as carrier gas at a flow rate of 60 mL/min; hydrogen and air flow rates were 40 and 400 mL/min, respectively, with nitrogen being used as a makeup gas at a flow rate of 40 mL/min. Columns employed for the analysis of herb samples are as follows: for samples I and 111, a 30-m SE-54 capillary column; for sample 11, a 35-m PEG-2OM capillary column. Operating conditions for analysis are as follows: for herb sample I, from 120 to 140 "C at a rate of 1.5 OC/min, then to 230 O C at a rate of 2 OC/min; for herb sample 11, from 70 to 200 OC at a rate of 3 "C/min; for herb sample 111,from 65 to 160 "C at a rate of 2 OC/min, then to 230 "C at a rate of 10 OC/min. Mass Spectrometry. Mass spectra were obtained on a Finnigan MAT 312/S188 magnetic scanning mass spectrometer equipped with an electron impact (EI) source. Instrument parameters used were as follows: ionizing voltage, 70 eV; source temperature, 220 "C; emission current, 300 wA; scanning speed, 1 s/decade. Vaporization Chamber. The specially constructed device is illustrated in Figure 1 and the schematic shown in Figure 2 illustrates the overall instrumental layout. The herb powder (3-15 mg) in a platinum boat is placed onto the metal spoon (Figure 1,item 26) at the end of the operation rod which is then inserted into the front of the vaporization chamber and locked into place. When the temperature of the furnace and block heater have reached 250 O C , the ball valve is opened slowly and the operation rod is pushed forward to allow the sample boat to lie just outside the quartz heating tube. After a short time the sample boat on the spoon is then pushed into the center of the quartz tube. The volatile constituents of the sample then vaporize instantly and
0003-2700/87/0359-0744$01.50/0 1987 American Chemical Society
745
ANALYTICAL CHEMISTRY, VOL. 59, NO. 5, MARCH 1, 1987 Table I. Volatile Constituents of Atractylodes macrocephala Koidz
peak ID (Figure 3) 1 2
3 5 6 7 8 9 10 11
16 20 23
compounds
formula
0-maaliene eremophila-l(lO),ll-diene cyperene trans-caryophyllene y-elemene cu-humulene acoradiene y-patchoulene aromadendrene y-cadinene atractylon 2,5-diacetylbenzofuran butenolide A
relative content," 70
mol wt
DV/GC/MS
steam distillation
204 204 204 204 204 204 204 204 204 204 216 202 232
0.34 0.80 1.56 1.26 2.50 0.69 0.89 7.19 1.06 13.57 45.58 0.92 3.65
0.17 0.39 0.29 1.12
7.09 0.49 0.29 2.36 0.67 6.32 63.03 1.55 1.05
Values are the arithmetic mean of five measurements.
\ ?
11
16
8
21
16
I1
h
Flgwe 1. Schematic diagram of the direct vaporization device: 1, nut: 2, silicon rubber septum; 3, ball valve (stainless steel); 4, carrler gas inlet; 5, silicon rubber septum: 6, nut: 7, quartz tube (110 mm X 6 mm i.d.): 8, furnace (74 mm X 55 mm 0.d. with 10 mm i.d.); 9, asbestos; 10, silica wool: 11, nut: 12, silicon rubber septum: 13, silicon rubber septum; 14, nut; 15, block heater: 16, silicon rubber septum; 17, nut: 18, injection port of GC: 19, syringe (60 mm); 20, knurled screw; 21, fixing screw: 22, fixing plate; 23, thermocouple for temperature control; 24, fixing screw: 25, bearing of quartz tube; 26, metal spoon: 27, platinum boat; 28, operation rod; 29, silicon rubber septum: 30, sliding block: 31, silicon rubber septum: 32, knob of operation rod. I
IO
20
I
30
I
40
,
50
I
60:flW)
Flgure 3. Chromatogram of volatile constituents of Atracfylodes macrocephala Koidz (I): (a) by direct vaporization: (b) by steam distillation-extraction method.
are swept into the chromatographic column by the carrier gas. The sample spoon is withdrawn just outside the quartz tube after 60 s until the analysis is completed.
RESULTS AND DISCUSSION
I_
- - - - - - - - - - - - - - - - - - - - - - - - - -/ -I - - - __ ,
Flgure 2. Schematic flow diagram of the direct vaporizatlon/capillary gas chromatography/mass spectrometry instrumentation: (A) cylinder of carrier gas; (Bo, B, B,, B,, B3) pressure gauge: (C, C,, C, C,) pressure regulator: (D,, D), rotameter: (E,, E, E3) filter; (F,, F2)buffer tube: (G) vaporization device; (H) injection port: ( I ) capillary column: (J) ion source of mass spectrometer: (K) FID; (L) splitter.
T h e chromatograms obtained for the three Chinese medicinal herbs studied by direct vaporization and extraction by steam distillation are illustrated in Figures 3, 4, and 5. Atractylodes macrocephala koidz (I). In the case of herb sample I, 23 constituents were separated. It is clear from the resultant chromatograms (Figure 1) that the present approach of direct vaporization is qualitatively equal t o that of steam distillation. There are a few very minor differences in the relative responses obtained by the two different analytical approaches. Of these 23 constituents, the major 13 compounds were subsequently identified as CI5 terpenoids by mass spectrometry. Quantitation was then performed by both techniques and the results are listed in Table I.
746
ANALYTICAL CHEMISTRY, VOL. 59, NO. 5, MARCH 1, 1987
Table 11. Volatile Constituents of Zingiber officinale Roscoe peak ID (Figure 4)
formula
wt
a-pinene camphene @-pinene a-phellandrene myrcene @-phellandrene a-limonene sabinene A3-carene p-cymene a-terpinene 6-methylhept-5-en-2-one 2,3,6-Trimethylanisole linalool bornyl acetate terpinen-4-01 @-citronellol citral b borneol geraniol a-bergamotene or a-zingiberene or unknown 3-bisabolene 2-P-farnesene nerolidol eugenol
2
3 4
5 6 7 8 9 10 11
12 14 16 23 26 27 36 37 38 39 40 45 46 49 53
relative content: % DV/GC/MS steam distillation
mol
compounds
136 136 136 136 136 136 136 136 136 134 136 126 150 154 196 154 156 152 154 154 204 204 204 222 164
0.86 2.58 0.38 0.05 0.44 0.22 0.93 6.38 0.07
1.34 4.12
0.08 0.06 0.65 0.30 1.24 8.80 0.07 0.09 0.07 0.17 0.14 0.41
0.08
0.06 0.23 0.07 0.24 0.22 0.75 0.26 0.48 0.19
0.09
0.50 0.12 0.34 0.35 0.99 28.96 16.45 0.79 0.57 0.29
1.12
27.70 21.67 0.70
0.57 0.22
a Values are the arithmetic mean of five measurements. -
51
22
I
19
M
I
9
43
I
3
I
I
I
1
20
30
40
50
1
I
I
I
1
lo
20
30
40
50
io
1
60
tmrrli
Flgure 4. Chromatogram of volatile constituents of Zingiber officinale Roscoe (11): (a) by direct vaporization: (b) by steam distillation-extraction method.
Zingiber officinale Roscoe (11). In the case of herb sample 11, 54 constituents were observed by gas chromatography (Figure 4), of which 25 were identified for quantification (Table 11). These results were consistent with previously reported results (3-6).
58
cmin)
Ij
‘mrn)
Flgure 5. Chromatogram of Amomun globosum Lour (111): (a) by direct vaporization: (b) by steam distillation-extraction.
Amomum globosum Lour (111). In this particular case, 59 constituents were separated by gas chromatography (Figure 5) of which 47 were identified by mass spectrometry (Table 111). Lawrence (7) has previously identified 15 of these
ANALYTICAL CHEMISTRY, VOL. 59, NO. 5, MARCH 1, 1987
747
Table 111. Volatile Constituents of Amomum globosum Lour
1 2
3" 4 50 6 7" 8 9 10 11"
12" 13" 14 15" 16" 17 19 20 2 1"
22" 23" 24" 25" 26" 27" 28" 29" 30" 31" 32 33" 35" 36" 39" 40a 41a 43" 44" 45 46" 47" 48 51" 53" 58 59"
relative content: % DV/GC/MS steam distillation
mol
peak ID (Figure 5)
compounds
wt
a-pinene camphene benzaldehyde &pinene myrcene a-phellandrene a-terpinene p-cymene a-limonen l,8-cineol A3-carene sabinene fenchone linalool sabinene hydrate y-terpinene camphor terpinen-4-01 a-terpineol 3-phenyl-2-propen-1-01 4-phenylbutan-2-one cinnamic aldehyde anethol thymol maltol 4-phenyl-3-buten-2-one y-cadiene 0-patchoulene methyl cinnamate l-methyl-2-(2-propenyl)benzene a-begamotene caryophyllene 2-fi-farnesene a-humulene P-cubebene a-muurolene a-gurjunene p-bisabolene y-muurolene 6-cadinene copaene P-guaiene nerolidol carotol a-cubebene farnesol all-trans-farnesylacetate
136 136 106 136 136 136 136 134 136 154 136 136 152 154 154 136 152 154 154 134 148 132 148 150 126 146 204 204 162 132 204 204 204 204 204 204 204 204 204 204 204 204 222 222 204 222 264
0.12 0.05 0.12 0.34 0.09 1.58 0.06 1.06 0.43 0.66 0.05 0.07 0.06 1.27 0.06 0.44 0.66 0.39 1.17 0.52 5.87 0.44 0.67 0.33 0 2.13 2.42 5.51
0.10 0.06 0.07 0.20 0.09 1.11
0.07 1.08 0.71 1.90 0.05 0.11 0.23 1.83 0.08 0.07 0.21 0.45 1.03 0.76 3.58 1.02 0.41 0.21 0.49 0.24 2.63 6.11 1.42 0.25 0.22 5.05 0.57 12.15 1.58 0.41 0.52 1.01 0.62 2.56 0.40 0.41 0.42 4.03 0.56 30.00 0.68
2.11
0.32 0.21 4.80 0.43 12.17 1.50 0.17 0.18 0.93 0.51 2.49 0.33 0.39 0.45 5.82 0.55 26.63 1.09
" Compounds are not found in this herb previously. *Values are the arithmetic mean of five measurements. ~
Table IV. Reproducibility of Relative Retention Values (rn)of Selected Volatile Constituents of Amomum globosum Lour yrt at the following peak no. (Figure 5)
expt. no.
10
22
29
33
36
45
51
58
2 3 4 5
1.144 1.143 1.127 1.145 1.144
2.456 2.469 2.479 2.472 2.463
3.342 3.364 3.341 3.368 3.359
3.602 3.631 3.605 3.636 3.625
3.830 3.853 3.859 3.859 3.851
4.234 4.273 4.303 4.280 4.265
4.679 4.712 4.750 4.720 4.700
5.093 5.155 5.220 5.167 5.118
av std dev re1 dev, %
1.141 0.0076 0.66
2.468 0.0088 0.36
3.355 0.013 0.39
3.620 0.015 0.41
3.850 0.012 0.31
4.271 0.025 0.59
4.712 0.026 0.55
5.151 0.049 0.95
1
constituents. The remaining 32 identified constituents in this work are therefore the first reported findings of these terpenoids in this Chinese medicinal herb. Comparison of Techniques. While there are a few minor differences in the chromatograms obtained by steam distillation vs. the direct vaporization approach, the principal constituents of these herbs are clearly observed by both techniques. With regards to quantitation by the two methods,
it is observed that the direct vaporization method is generally more efficient a t extracting the volatile constituents than the steam distillation ether extraction method. There are only a few instances where the amount extracted by steam distillation exceeds that observed by direct vaporization. Effects of T e m p e r a t u r e a n d Duration of Heating. It was observed experimentally that different durations of heating (15-120 s) and different temperatures of the quartz
748
ANALYTICAL CHEMISTRY, VOL. 59, NO. 5, MARCH 1, 1987
Table V. Reproducibility of Relative Percentage Content of Selected Volatile Constituents of Amomurn globosum Lour 9i content at following peak no. (Figure 5)
expt no.
10
22
29
33
36
45
51
58
2 3 4 5
1.95 1.85 1.98 1.85 1.86
3.63 3.48 3.60 3.53 3.65
6.16 6.03 6.16 6.14 6.04
5.20 4.94 5.00 5.08 5.02
11.94 11.99 11.93 12.24 12.66
2.52 2.53 2.58 2.68 2.56
3.93 3.99
30.28
4.12
4.04 4.06
29.83 30.04 30.09
av std dev re1 dev, %
1.90 0.062 .32
3.58 0.071
6.11 0.065 1.1
5.05 0.099 2.0
12.15 0.31
2.57 0.064 2.5
4.03 0.072 1.8
1
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Flgwe 6. Dependence of the absolute yield of volatile constituents of Amomun globosum Lour (111) on the temperature and duration of heating during vaporization: (a) peak no. 33; (b) peak no. 36.
tube had little if any effect upon the resultant chromatograms. Therefore, under different vaporization conditions, it can be concluded that no decomposition of the constituents had occurred. However, the temperature and duration of vaporization did have a marked effect on the quantitative results. For example, the quantitation of peaks 33 and 36 in herb sample I11 (Figure 5a) clearly illustrated t h a t optimum vaC in 60 s (Figure 6) and porization conditions were a t 250 ' that too low a temperature or too short a duration could result in poor yields. This temperature dependence could result in poor sensitivity and reproducibility and stresses the need to select the proper vaporizer temperature and duration of va-
2.6
29.74
30.00 0.21 0.70
porization. Too long a duration of vaporization may result in widening the chromatographic elution profile and reducing efficiency of resolution and quantitation. Reproducibility. The relative retention values and relative percentage amounts of eight selected principal constituents of herb sample I11 are listed in Tables IV and V. Under optimum experimental conditions, the average relative deviations of retention values and relative percentage content were calculated to be less than 0.95% and 3.3470, respectively. It can be concluded, therefore, that the reproducibility of the direct vaporization method is satisfactory.
Registry No. P-Maaliene, 489-29-2; eremophiula-l(10),11diene, 10219-75-7; cyperene, 2387-78-2; trans-caryophyllene, 87-44-5; y-elemene, 29873-99-2;a-humulene, 6753-98-6;acoradiene, 24048-44-0; y-patchoulene, 508-55-4; aromadendrene, 489-39-4; y-cadinene, 39029-41-9; atractylon, 6989-21-5;2,5-diacetylbenzofuran, 28221-81-0;butenolide A, 73069-14-4; a-pinene, 80-56-8; camphene, 79-92-5;P-pinene, 127-91-3;a-phellandrene, 99-83-2; myrcene, 123-35-3;@-phellandrene,555-10-2; a-limonene, 138-86-3; sabinene, 3387-41-5; A3-carene, 13466-78-9;p-cymene, 99-87-6;a-terpinene, 99-86-5;6-methylhept-kn-2-one, 110-93-0 2,3,6-trimethylanisole, 21573-36-4;linalool, 78-70-6; bornyl acetate, 76-49-3; terpinen-4-01, 562-74-3; P-citronellol, 106-22-9; citral b, 106-26-3;borneol, 507-70-0; geraniol, 106-24-1; a-bergamotene, 17699-05-7;a-zingiberene, 495-60-3; P-bisabolene, 495-61-4; Zp-farnesene, 28973-97-9; nerolidol, 142-50-7; eugenol, 97-53-0; benzaldenhyde, 100-52-7;l,&ineol, 470-82-6; fenchone, 119579-5; sabinene hydrate, 546-79-2; y-terpinene, 99-85-4; camphor, 7622-2; a-terpineol, 98-55-5; 3-phenyl-2-propen-1-01,104-54-1;4phenylbutan-2-one, 2550-26-7;cinnamaldehyde, 104-55-2;anethol, 104-46-1;thymol, 89-83-8; maltol, 118-71-8;4-phenyl-3-buten2-one, 122-57-6; P-patchoulene, 514-51-2; methyl cinnamate, 103-26-4; l-methyl-2-(2-propenyl)benzene, 1587-04-8; caryophyllene, 87-44-5; P-cubebene, 13744-15-5; a-muurolene, 10208-80-7; a-gurjunene, 489-40-7; y-muurolene, 30021-74-0; &cadinene, 483-76-1; copaene, 3856-25-5; P-guaiene, 88-84-6; carotol, 465-28-1; a-cubebene, 17699-14-8; farnesol, 4602-84-0; all-trans-farnesylacetate, 4128-17-0.
LITERATURE CITED (1) Hu, J. C. A. Anal. Chem. 1981, 5 3 , 311A. (2) Hu, J. C. A. J . Chromatogr. Sci. 1981, 19, 634. (3) Yosioka, I.; Nishio, T.; Tani, T.; Kitagaw. I. J . Pharm. Soc. Jpn. 1976, 96, 1229. (4) Fu Shun-mo; Fang Hong-ju; Lin Gou-sheng; Xiao Pei-gen Acta Phytotaxon. Sin. 1981, 19. 196. (5) Kami, T.; Nakayama, M.; Hayashi, S. Phytochem. Rep. 1972, 1 1 ,
3377. (6) Masada, Y. Analysis of Essential Oils by Gas Chromatography and Mass Spectromehy; Hirokawa Publishing: Tokyo, 1975; p 251. (7) Lawrence, B. M.; Hogg, J. W.; Terhune, S.J. Phyfochem. Rep. 1972, 1 1 . 1534.
RECEIVED for review April 21, 1986. Accepted October 27, 1986.
