Gas Phase Microanalysis of Zooecdysones Hiroshi Miyazaki, Masataka Ishibashi, and Chiyoko Mori Research Laboratories, Pharmaceutical Division, Nippon Kayaku Co., Simo, Kita-ku, Tokyo, Japan
Nobuo lkekawa Laboratory of Chemistry for Natural Products. Tokyo lnstitute of Technology. Ohokayama. Meguro-ku. Tokyo. Japan
Gas phase microanalysis of the insect moulting hormones and their extraction and clean-up method were investigated. The extraction with a modified Soxhlet apparatus using tetrahydrofuran as solvent was found the most effective by a test using 3H-ecdysterone. After adsorption of the extract on Carplex, the major part of the lipid could be removed by Soxhlet extraction with n-hexane, benzene, and then ether. Further extraction with tetrahydrofuran recovered ecdysones quantitatively. Previously, we found that HFB-TMS derivatives prepared by an exchange reaction of OTMS into OHFB are suitable for the microanalysis of phytoecdysones. By the present GC-MS analysis, the exchange reaction has been shown to occur at C-2 equatorial OTMS on phytoecdysones. While in the case of a-ecdysone, di-HFB-tri-TMS derivative was obtained. Picogram levels of HFB-TMS derivatives of zooecdysones in insects were detected by electron capture detector. For the more specific analysis of zooecdysones, mass fragmentography of TMS derivatives was developed. Quantatative estimation of lo-'' g level of a-ecdysone and ecdysterone can be obtained by focusing at m / e 564 and m / e 561, respectively. By this technique, 53 ng of a-ecdysone and 78 ng of ecdysterone were estimated in a pupa ( 2 day-old) of silkworm.
T h e microdetermination of the insect moulting hormones in biological systems has until now been carried out by bioassays with Calliphora uincia ( I ) , M u s c a d o m e s t i c a (2), C h i l o suppressalis (3), and other insects. However, more sensitive and more specific determination of ecdysones is necessary for the studies on the excretion (4, 5), metabolism (S), a n d mode of action of the moulting hormones. In a previous report (7), we described the gas chromatographic separation of phytoecdysones, using trimethylsilyl ether (TMS) and heptafluorobutyrate ( H F B ) , which can be detected at the subnanogram level by electron capture technique, and also their application to the analysis of ecdysone homologs in plants. This method could not be applied to the analysis of zooecdysones because of the interference with large amounts of lipids in insects. We have explored a better method of extraction and clean-up of zooecdysones in a biological system. Then ( 1 ) P. Karlson, Vitam. H o r m . , (New Y o r k i . 14, 227(1956); Angew, Chem., 2, 175(1963). (2) J. N. Kaplanis, L. A. Tabor, M . J. Thompson, W. E. Robbins, and T . J. Shortino, Steroids, 8, 625(1966). (3) Y . Sato, M . Sakai, S. Imai, and S. Fujioka, Appl. €ntomoi. ZOO/., 3 , 49( 1968). ( 4 ) S. 8.Weir, Nature. 228, 580(1970). (5) A . Willing, H . H. R e e s , and T. W . Goodwin, J insect P h y s f o l . . 1 7 , 281 71 1971 1 . (6) K. Nakanishi, "Chemistry of Natural Products," Vol. 7, Butterworths, London, 1970, p 167. ( 7 ) N. Ikekawa, F. Hattori. J. Rubio-Lightbourn, H . Miyazaki. M . Ishibashi, and C. Mori, J. Chromatogr. S c i , 10, 233(1972).
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 7, JUNE 1973
electron capture technique was applied to the purified ecdysones using H F B derivatives, whose structures were elucidated by mass spectrometry. Furthermore, for the purpose of getting more specific detection and more precise estimation, mass fragmentography (8) was applied to the analysis of ecdysones in insects as their TMS derivatives.
EXPERIMENTAL G a s Chromatography. A Shimadzu Seisakusho Model GC5AP equipped with electron capture detector (Model ECD-5, Ni63) and hydrogen flame ionization detector was employed; pulsed voltage was 48 V; pulse interval, 75 psec; pulse width. 8 psec. The column packings were 1.5% OV-101 and 1.5% OV-1 on Chromosorb W H P , 80-100 mesh. The column temperature was 260 "C, and the flow rate of carrier gas (nitrogen) was 60 ml/min. The injection port and the detector temperature were at 280 "C. M a s s Spectrometry a n d M a s s Fragmentography. An LKB9000s GC-MS system equipped with a multiple ion detector (MID) was employed. T h e column was 100 cm X 4-mm i.d., glass coil with 1.5% OV-101 on Chromosorb W H P , 80-100 mesh (Applied Science Lab.). The column temperature was 270 "C; the ionization current was 60 pA; the voltages were 20 eV and 70 eV; the accelerating voltage were 1.75 KV and 3.5 KV; the ion source temperature was 290 "C. S a m p l e s a n d Reagents. T h e ecdysone samples in this study were m-ecdysone, 3H-ecdysterone, 2-deoxyecdysone; ecdysterone ($-ecdysone), ponasterone, inokosterone, and cyasterone. As a model compound, 3,22-di-hydroxycholest-5-ene was also used. Trimethylsilylimidazole ( T S I M ) , heptafluorobutyryl imidazole (HFBI), heptafluorobutyric acid (HFBA) were obtained from Pierce Chemical Co., and Tokyo Kasei Kogyo Co.. Ltd. All reagents were used after distillation. The silkworm, Bombyx mori, pupae of F1 hybrid between two races, J 131 and C 131, were used. Heptafluorobutyryl a n d Trimethylsilyl Derivatization. Ecdysterone, 0.5 mg, was dissolved in 20 p1 of TSIM in a small Teflon (Du Pont) capped tube and the solution was heated at 100 "C for 1 hr. T o this solution, 20 p1 of HFBI and 2 p1 od HFBA were added and the mixture was heated at 50 "C for 2 hr. Benzene (0.2 ml) was added and washed with ice cold 5% KaHCO3 and then with water. After drying over NaZS04, the solvent was evaporated. Ecdysterone mono-HFB-penta-TMS; Anal: Calcd for C&8&8F&5, F, 12.8270.Found 13.35%. Extraction a n d Purification of Zooecdysones. T e n pupae were p u t into a 100-ml beaker, and 20 ml of acetone and dry ice were added. The beaker was covered with aluminium foil. The pupae were dried completely under reduced pressure and temperature below 50 "C. T o the obtained residue, 5 pg of cyasterone and 15 grams of sea sand were added, and ground to fine powder in mortar. T h e powder was p u t into a paper filter thimble and extracted with 100 ml of tetrahydrofuran ( T H F ) in a modified Soxhlet apparatus for 24 hr. This apparatus was designed in such a way t h a t a glass container for the filter paper thimble is heated by the vapor of the solvent. The T H F extract was concentrated to half volume and 3 grams of Carplex No. 80 (silicic acid) was added. Then t h e T H F was completely eliminated by rotary evaporator. The residue was p u t into a filter paper thimble and extracted in the Soxhlet succesively for 1 hr with 50 ml of each of the following solvents, n-hexane. benzene, ether, and T H F . The T H F extract was passed through 3 grams of silica gel (Merck, Silica gel 60 for chromatography) column prepared in a glass filter (15 AG-3). Then the column was washed with T H F . The el(8) C. G . Hammar, B. Holmstedt, and R . Ryhage, A n a / Bfochem , 25,
532 (1968)
68 5
Mt-(90x2+1 5 )
M' - ( 93tl5 )
331
841 890
600
700
I
800
I
I
Mt-90 94 6
n
900
1021
$ 1
MI-15
h
1100
1000
Figure 1. Mass spectrum of ecdysterone-mono-HFB-penta-TMS
uate and washing were combined a n d T H F was evaporated in vacuum. T h e residue was purified by preparative TLC (silica gel, plate 20 X 20 cm, thickness 500 p ) using CHCIS-EtOH ( 2 : l ) a s the developing solvent system. A wide band containing compounds with R f values from 0.45 to 0.9 was extracted with T H F using a n ultrasonic generator (over all yield of recovery was 7 5 7 0 ) , the T H F was evaporated, t h e residue dissolved in 100 p1 of T S I M , a n d t h e solution was heated a t 100 "C for 30 min. This solution was analyzed by mass fragmentography. In the case of GLC analysis using ECD, 100 pl of HFBI and 10 p1 of HFBA were added t o the T M S solution, a n d t h e reaction mixture was heated a t 50 "C for 2 hr. Benzene (0.2 ml) was added and t h e mixture was poured into ice-water. T h e organic layer was washed with a n ice cold 5% NaHCOa solution and dried over NaZS04. T h e product was purified by preparative T L C using benzene as developing solvent. T h e region corresponding to ecdysones was extracted with benzene and analyzed by GLC.
RESULTS AND DISCUSSION E x t r a c t i o n a n d Clean-up Methods. We encountered many more difficulties in t h e extraction a n d clean-up procedure for the zooecdysones t h a n for the phytoecdysones due to t h e presence of very small amounts of ecdysones and large amounts of lipids which were extracted together. T h e clean-up method of phytoecdysones was applied to the zooecdysones without satisfactory results. By using 3H-labeled ecdysterone, we established a more suitable condition for extraction and clean-up. For t h e extraction of zooecdysones, a modified Soxhlet apparatus was used. In this apparatus, the extraction was carried out a t the boiling point of t h e solvent, rendering a more effective extraction. T h e solvent should be as effective as possible for the extraction of ecdysones, with a minimum contamination by other lipids. After testing several kinds of solvents, tetrahydrofuran (THF) was found best. THF proved to be a better solvent t h a n methyl ethyl ketone for extraction of phytoecdysones, because it led to obtaining a small amount of residue, slightly colored, and without significant loss of ecdysones. When a known amount of 3H-ecdysterone was added to the silkworm pupa, t h e recovery by extraction was nearly quantitative (98.5%). For the clean-up method, the adsorption on Carplex No. 80 (silicic acid) and reelution procedure, which was used for phytoecdysones, was also effective for zooecdysones. Although alumina was a good adsorbant of lipids, the recovery of 3H-ecdysterone was only 30%. T h e THF extract (1.6 grams from 10 pupa) was a d sorbed on Carplex and extracted successively with n-hexane, benzene, a n d ether. By this procedure t h e major parts of the lipids (95%) were removed a n d the ecdysones were not extracted. T h e extraction of ecdysones from Carplex was carried out with THF. This THF solution was passed through a short column of silica gel a n d the col-
u m n was eluted with T H F . T h e eluate, without further purification, can be analyzed by mass fragmentography using a GC-MS system. However, for t h e use of ECD technique a further clean-up was necessary, because the peak of the ecdysones were hidden by the solvent peak. Therefore, the extract was purified by preparative TLC, converted to H F B via T M S derivative and again purified by TLC. T h e sample t h u s obtained was analyzed by GLC using electron capture detector. S t r u c t u r e of the H F B - T M S Derivatives. In the previous paper (7), it was reported that the hydroxy groups of ecdysones are partially silylated with trimethylsilylimidazole (TSIM) a t room temperature and completely silylated a t 100 "C. Both derivatives, which show single sharp peaks on GLC, can be converted into H F B derivatives with heptafluorobutyrylimidazole (HFBI) and heptafluorobutyric acid (HFBA), as a catalyst, without removal of T M S reagent. Although the H F B derivatives showed a single sharp peak, their structures remained to be clarified. Now those structures were elucidated by mass spectrometry. T h e mass spectrum of ecdysterone HFB-TMS derivative obtained by the exchange reaction is shown in Figure 1. T h e base peak of m / e 685 and the elemental analysis suggesting mono-HFB-penta-TMS derivative indicated t h a t one of the O T M S in the steroidal skeleton was exchanged to a OHFB group. A base peak of hexa-TMS derivative of ecdysterone appeared a t m / e 561 a n d was assigned to fragment I (7, 9). T h e corresponding fragment I1 of mono-HFB-penta-TMS derivative should give the ion mle 685. When 2-deoxyecdysone which has no equatorial hydroxy group, was treated in the same manner, the retention time of the GLC peak did not shift, but a small peak having a shorter retention time t h a n t h e T M S derivative appeared. This peak may correspond to t h e monoHFB-tri-TMS derivative from peaks of m / e 46'7 (base CH
*C'
CH3'CH,
TMSO
I
TMSO
R=TMS
mle.561
'
1:
ill We.564
U R = H F B mle.685
I
r l l l m c n t Inns
1
(9) M. Koreeda. K . Nakanishi, S. Imai, T. Tsuchiya. and N. Wasada. Mass Spectrosc . 17. 669(1969).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 7, JUNE 1973
1165
CH2
685
Mt-(214t73x2+l 5)tZ
/
I
XlOO
ago
Mt-90x3
y+O W
CH3
@
OTMS 7
600
700
809
Mi-1 5
1
800
L L L1 0 7 0 Mt-90
1000
900
1100
1200
Figure 2. Mass spectrum of inokosterone-di-HFB-tetra-TMS
peak) and m l e 845 ( M + - 15). From these data, equatorial C-2 O T M S of ecdysterone seemed to be converted into H F B ester during the reaction. T h e same derivatization of inokosterone gave a di-HFB-tetra-TMS compound as shown in Figure 2 and the position of the H F B groups may be located at C-2 and C-26. Ponasterone and cyasterone gave mono-HFB derivatives. Furthermore, the generalization t h a t the equatorial O T M S group is converted to the OHFB group faster t h a n the axial one, was proved by the reaction of several bile acids and steroidal hormones (10).
Analysis of Zooecdysones by Electron Capture Technique. Ecdysones in ten pupae (older stage) of silkworm, Bombix mori, were extracted, after addition of cyasterone as a n internal standard, and purified as described above. HFB-TMS derivatives of the extracted ecdysones were analyzed by GLC witt ECD (Figure 5). T h e GLC peaks show t h a t the TLC separation was effective and the extraction period of 12 hr was insufficient. T h e major moulting hormone in this stage was shown to be ecdysterone, whose amount can be estimated from the
noTM HFBoF HFB OHFB
or-Ecdysone
Ponasterone
( M*=1072 )
TMSO
TMSO
( M+=948)
"'""e HFBoe
TMSO
( fl.1036)
TMSO
( M+=1005)
I1
H O
0
Inokosterone OHFB HFBO
I
IIrVCtUlP
0'
Tfli-YiB
OPTlldfllCI
1
TMSO
GLC analysis of the H F B derivative of a-ecdysone showed a prominent peak, accompanied with a small peak shown in Figure 3. Those peaks could be assigned to diHFB-tri-TMS and mono-HFB-tetra-TMS compounds, respectively, by mass spectrometry. Relative retention times to cholesteryl butyrate were 1.07 (di-HFB) and 1.43 (mono-HFB), respectively. One H F B could be located a t equatorial (2-2. In contrast to ecdysterone, C-22-OTMS may also be susceptible to exchange with HFB, presumably due to the absence of '2-20 oxygen substituent. Indeed, when the model compound 3,22-dihydroxycholest5-ene was treated under the same conditions, a fairly large amount of the 3,22-di-HFB derivative was obtained, as shown in Figure 4. From the patterns shown in Figures 3 and 4, it may de deduced t h a t the exchange reaction rate is influenced by the functional groups on the steroidal skeleton. (10) H. Miyazaki, M . Ishibashi, C. Mori, and N. ikekawa, 92nd Annuai Meeting of the Pharmaceutical Society of Japan. 1972.
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 7, JUNE 1973
peak area. T o obtain a more accurate content of ecdysterone in the insects, we applied the orthogonal polynomial equation (11) with addition of a known amount of sample. T o the five groups of pupae, known proportional amounts of ecdysterone were added and the amount of the ecdysterone in each group was determined by GLC. From Figure 6, the exact amount of ecdysterone was calculated to be 85 ng in one pupa. On the other hand, the peak corresponding with n-ecdysone could not be recognized by this method. Analysis by Mass Fragmentography. For more precise and specific determination of zooecdysones, the mass fragmentographic technique was applied to the completely silylated derivatives, which are more stable t h a n partial T M S or HFB-TMS derivatives. As shown in the mass spectra of TMS derivatives of phytoecdysones. the compounds having O T M S groups a t (11) G. Taguchi. "Statistical Anaiysis, ' Maruzen. Tokyo, 1972, p 197
2
6
4
8
10 rnin
Figure 3. Gas chromatographic separation of a-ecdysone HFBTMS derivative ( I ) Di-HFB-tri-TMS derivative ( I I ) Mono-HFB-tetra-TMS derivative column conditions 1 5 % OV-101 on Chromosorb W HP. 100 X 4-mm I d , 260 "C 60 ml min Flame ionization detector
0
10
rnin
t
20
Ecaysterone
Figure 5. Gas chromatographic separation of ecdysones in silkworm pupae (more developed stage) _ _ Extraction with THF for 24 hrs; --- Extraction with THF for 12 hrs:
_._._.The
extract was derivatized to HFB-TMS without separation by TLC. Column conditions, 1.5% 101 on Chromosorb W HP, 100 X 4-mm i.d.. 260 " C . 60 ml. Electron capture detector
10 rnin
5
Figure 4. Gas chromatographic separation of 3,22-dihydroxycholest-5-ene HFB-TMS derivatives obtained by exchange reaction 1
( I ) DI-HFB der vative ( I I ) Mono-HFB-mono-TMS derivative ( I l l ) DITMS derivative Column conditlons 1 5% OV-101 on Chromosorb W HP 100 cm X 4-mm I d 230 "C 60 ml min Flame ionization detector
C-20 and C-22 exhibited base peak of mle 561 which can be assigned to fragment I. Therefore, this key fragment ion was chosen for the analysis of ecdysterone. In the case of a-ecdysone (Figure 7 ) which does not give t h e peak a t m / e 561, the strong peak m / e 564 (fragment 111) was selected. As a n internal standard of this estimation, cyasterone which gave the same fragment ion mle 561 was used. T h e calibration curves of the mle 564 for a-ecdysone a n d mle 561 for ecdysterone are shown in Figure 8. The mass fragmentogram of 2 day-old 10 pupae focusing a t mle 561 and mle 564 is shown in Figure 9. T h e amounts of a-ecdysone and ecdysterone were calculated to be 53 ng and 78 ng in one pupae, respectively. T h e mass fragmentogram of older pupae is shown in Figure 10. Focusing a t mle 564 did not give the peak cor-
I
x
x+a x*2a x+4a x.80 a:0., "9
Weight Figure 6. Estimation of ecdysterone in pupae.of silkworm by additions of known proportional amounts of ecdysterone for using orthogonal polynomial equation
responding to a-ecdysone. Monitoring a t mle 561 gave clear evidence for the presence of ecdysterone a n d a n unknown compound having a shorter retention time t h a n ecdysterone which was also detected in 2-day-old pupae. When the retention time of this compound was compared with t h a t of phytoecdysones reported in the previous paper (7), it did not correspond to any of them. Furthermore, monitoring at' mle 685 (fragment 11) by mass fragmentography of its HFB-TMS derivative gave a new peak having a slightly shorter retention time than ecdysterone. Therefore, this unknown compound seems to contain a n
425
t
474
400 400
I
500
J-824
600
700
N'
800
Figure 7. Mass spectrum of a-ecdysone-penta-TMS ANALYTICAL CHEMISTRY, VOL. 45, NO. 7, J U N E 1973
1167
Cyastcrone
Unknown
1‘
i
A A-
-
h
I\
Ecdysterone
J_v_I
J b %564
5
IO
15
20 min.
Figure 10. Mass fragmentogram of ecdysones from silkworm pupae of more developed stage
I
2
1
3
4 x10-”og
Figure 8. Calibration curve of (ai a-ecdysone(rn/e 564) and f b i ecdysterone(rn/e 561)
oxygen function a t C-20 and the same steroidal skeleton as the ecdysones. T h e determination of the complete structure is under investigation in our laboratory.
ACKNOWLEDGMENT 1
Cyasterone 1.S)
1c
aEcdysone-
5
IO
I5
20 rnin.
Figure 9. Mass fragmentogram of ecdysones from 2 day-old
silkworm pupae
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ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 7, JUNE 1973
The authors are grateful to W. Tanaka, Nippon Kayaku Co., and M. Kobayashi, T h e Sericultural Experiment Station, for their encouragements and valuable suggestions through this work, to T. Takemoto and H. Hikino, Tohoku University, for the samples of a-ecdysone, 3Hecdysterone, inokosterone, and cyasterone and to D. H. S. Horn, C.S.I.R.O., Melbourne, for the sample of deoxyecdysone. Received for review November 30, 1972. Accepted January 17, 1973. This research was supported by a research grant from T h e Ministery of Education.
