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High-performance liquid chromatography of ecdysteroids and ecysteroid-22-phosphates
Journal
of Chromatogruphy,
246 ( 19SZ) 3 17-373
Elsevier Scientific Publishing Company. Amsterdam CHROM.
Printed in The Netherlands
15,031
Note
High-performance liquid steroid-22-phosphates R. ELWYN Deparrnzent
ISAAC,
NICHOLAS
of Biochenristr_v_
chromatography
P. lMILNER
University
and HUW
of Liverpool.
P-0.
of ecdysteroids
and ecdy-
H. REES’ Box 147. Liwrpool
L69 3BX
(Great
Brituirzj
(First received April 29th, 19S7; recked manuscript received May 14th. 1983)
The ecdysteroids (insect moulting hormones) are a group of polyhydroxylated steroids possessing a 5&7ene-6-one ring structure .20-Hydroxyecdysone (II) is generally regarded as the major active hormone’. although ecdysone (I) may also elicit a honnonai response in certain tissues at some stages in development_ 2-DeoxyecdysonezJ and Z-deosy-20-hydrosyecdysone 5*6 have been isolated from the ovaries and eggs of a number of insect species as major ecdysteroid components of largely unidentified highly polar conjugates. These Xdeoxy ecdysteroids may be possible precursors of ecdysteroids I and II. Other ecdysteroids which have been isolated from insects include the 26-hydroxy’-“, 3-epi’“iA, 3-dehydro’s-‘6, S-acetate” and 2% phosphate l8 derivatives. The latter compounds are considered to be inactivation products of the hormones_ Thus, ecdysteroids varying widely in polarities are encountered in insect metabolism_ Complex mixtures of ecdysteroids are amenable to chromatography on reversed-phase high-performance liquid chromatographic (HPLC) systems” and, owing to the wide range of polarities, are best resolved using a methanol-water gradient for elution. In the present paper we describe the chromatography of a wide spectrum of insect ecdysteroids on an efficient, short (15cm) Ultrasphere-ODS (particle size 5 ,urn) column. The data presented (Table I) indicate potential pitfalls in the
0021-9673/S2/~/SO~.7.5
63 1982 Elsevier Scientific Publishing Company
NOTES
identification of some ecdysteroids and metabolites having similar chromatographic properties_ and the value of an efficient reversed-phase column in resolving such steroids. The chromatography of the newly identified ecdysteroid monoacetates and 22-phosphates are aIso reported. Ecdysone- 2%phosphate and 2-deosyecdysone-22phosphate have also been analysed by HPLC using reversed-phase paired-ion and anion exchange (Whatman SAX) chromatography_ ESPERIMEXTAL
The ecdysteroid samples used in the present study were obtained from various souicrSI ecdysone (I) (Simes, Milan, Italy); 30-hydrosyecdysone (II) and &pi-?deosyecd_vsone (XIII) (Dr_ G_ B_ Russell. D.S.LR__ New Zealand); 26-hydrosyecdysone (VII) (Dr. J_ N. Kaplanis_ U.S.D.A.. Beltsville. MD, U.S.A.); 20,26-dihydroayecdysone (VIII). ?-deosy-20-hydrosyecdysone (XII) and 3-epi-?-deosy-20hydrosyecdysone (XIV) (Dr. D. H. S. Horn. C.S.I.R.O., Melbourne. Australia), 2deosyecdysone (XI) (Professor E_ Ohnishi_ Nagoya University_ Nagoya, Japan); poststerone (XVII) (the late Dr. J. B. Siddall, Zoecon Corporation, CA, U.S.A.): inokosterone (XVIII) (Professor T. Takemoto, Tohoku University, Tohoku, Japan); ponasterone A (XIX) (Professor K_ Nakanishi, Columbia University_ New York, NY, USA_): 3-dehydroecdysone (V) and z-dehydro-20-hydrosyecdysone (VI) (prepared by the method of Spindler r& ol_‘O); 3-epiecdysone (III) and 3epi-20h)-dhosyecdysone (IV) (prepared by the method of Dinan and Rees’l), ecdysone-Z(X) and 3-monoacetates (IX) (prepared by the method of GaIbraith and Horn”); ecd}sone-22-phosphate (XV) and %leoqecdysone-22-phosphate (XVI) (isolated
NOTES
319
from eggs of Schistocerca gregaria”). The mixture of ecdysteroids used in the separation shown in Fig. 1 was obtained from ecdysteroid conjugates, isolated from developing eggs of Schistocerca gregaria, by hydrolysis with a crude enzyme preparation from Helix pomatia*. HPLC grade methanol was purchased from Rathburn Chemicals (Walkerbum, Great Britain) and tetra-fz-butylammonium hydroxide solution (40 y0 in water) and AnalaR-grade orthophosphoric acid were purchased from BDH (Poole, Great Britain). High-performance liquid chonzatograplzy HPLC was carried out using a Model ALC/lOO Iiquid chromatograph (Waters Assoc., Northwich, Great Britain) linked to a Perkin-Elmer LC-55 variable wavelength detector set at 250 nm. Linear solvent gradients were formed with the aid of a Model 660 solvent programmer (Waiers Assoc.) and samples, dissolved in methanol, were injected via a Model U6K injector (Waters Assoc.). An Altex Ultrasphere-ODS column (15 cm x 4.6 mm, particle size 5 pm) (Anachem, Luton. Great Britain) was used for reversed-phase chromatogaphy. Some reversed-phase separations of ecdysteroid phosphates involved addition of tetra-n-butylammonium hydroside (BDH) adjusted to pH 7.5 with orthophosphoric acid. to the aqueous methanol soIvent to make a 0.6 7; solution. RESULTS
AND
DISCUSSION
Reversed-phase HPLC using a methanol-water gradient on an UltrasphereODS column allowed the separation of ecdysteroids of widely differing polarities in a single run (Table I). The application of such a system to the analysis of biological extracts is illustrated in Fi_g. I_ The chromatogram shows the separation of a series of ecdysteroids isolated from developing eggs of 27.gregaria. These ecdysteroids probably form part of a metabolic scheme for the biosynthesis and inactivation of 20hydroxyecdysone (II)_ It is apparent from Table I that several of the ecdysteroids are either not resolved or have very similar chromatographic properties. Thus, potential pitfalls in identification of radioactive ecdysteroid metabolites are clear. However, use of such a reversed-phase column in conjunction with an adsorption-type column, such as APSHypersil (aminopropyl) or silica. obviates such problems_ The system described in this paper allows good resolution of four ecdysteroids and their 3-epimers on this short column (Figs 1 and 2). Although separation of ecdysteroids from their 3epimers has been achieved on some other types of reversed-phase columns (PBondapak CISt 30 cm’“), this was not always the casez3. On reversed-phase. the 3epiecdysteroids have very similar retention volumes to the corresponding 3-dehydro compounds (Table I), but these pairs can be easily separated by chromatography on an APS-Hypersil column 23. It is interesing that the order of elution of some plant ecdysteroids during isocratic reversed-phase HPLC was changed when different organic solvents in admixture with water were used as mobile phases’“. It is important to note that whereas on silica thin-layer chromatography (TLC) on a reversedecdysone-‘>- and 3-monoacetates co-migrate with 3dehydroecdysone”, phase system these three ecdysone derivatives are easily resolved. Therefore, the
NOTES
L
20
10 !-cc?
0
Fig_ l_ Separation ofecdysteroids on an Ultrasphere-ODS column (15 cm x 3.6 mm I.D.). Mobile phase: linear _gadient of 40 p,Omethanol in water increkng to SO“/‘, methanol in water over 10 min: flow-rate, 1 mi.:min_ Detection: UV absorbance at 150 run. Peaks: II = 20-hydroxyecdysone: V!I = 26-hydroxyecdjxone; I = ecdysone; III = 5-epiccd~sone; XII = I-deoxy-20-hydroxgecdcdysone; IX = ecdysone-3acetate; XI = Z-deoxyecdysone; XIII = j-epi-Z-deoqecdysone.
identification of 3dehydroecdysone and ecdysone monoacetates by cochromatography with authentic compounds must be carried out on both adsorption and reversedphase systems. Reversed-phase columns are unsuitable for the chromatography of the highly polar ecdysteroid phosphates unless the anionic group is first neutral&d. We have added tetra-n-butylammonium hydro.xide to the eluting solvent as the anion pairing agent”, which resuk in greater retention of the phosphate conjugates on the reversed-phase column (Table I, solvent system B). The reproducible results obtained by this method should facilitate the identification of radiolabelled ecdysteroid metabelites by co-chromatography with authentic steroid conjugate. We have used chromatography on a strong anion exchange column as a major step in the purification of ecdysone-22-phosphate and 2_deosyecdysone-22phosphate from the eggs of Schistocerca gregaria Is. For routine analytical studies, phosphate buffer (0.02 AZ) pH 5.0 was employed as the mobile phase, but when it was necessary to isolate ecdysteroid conjugates free of phosphate salts, this buffer was replaced with ammonium acetate (O-1 M). These systems separate ecdysone-22phosphate, Z-deosyecdysone-22-phosphate (Table II) and a highIy polar 20-hydrosyecdysone conjugate of, as yet, unknown structure (data not presented). In conclusion, complex mixtures of ecdysteroids can be resolved on an Ultrasphere reversed-phase column using a methanol-water gradient (total elution volume, 20 ml)_ Small elution vohunes and low solvent flow-rates result in greater efficiency in the detection of UV absorbing compounds and in the monitoring of radiolabekd components either by liquid scintillation counting of fractions or by the use of an on-line radioactivity scintillation monitor_ Ecdysteroid phosphates are
321
NOTES TABLE
I
RmENTION
VOLUMES
FOR ECDYSTEROIDS
ON ULTRASPHERE-ODS
Colurrm: 15 cm x 66 mm LD_ Modile phases: A, linear gradient of 40 y0 methanol in water increasing to 80 % methanol in water over 20 min; B, 46 y0 methanol in water containing 0.6 % tetra-n-butylammonium hydroxide; flow-rate, 1 ml/min.
.-I I
11 III LV V VI VII VIII IX X XI XII XIII XIV xv XVI XVII XVII1 XIX
Ecdysone IO-Hydrosyecdysone 3-Epiecdysone 3-Epi-20-hydroxyecdysone 3-Dehydroecdysone 3-Dehydro-20-hydroxyecdysone 26-Hydroxyccdysone 20,26_Dihydrosyecdysone Ecdysone-3-acetate Ecdysone-2-acetate 2-Deoxyecdysone 2-Deoxv-20-hydroxyecdysone 3-Epi-2-deosyecdysone 3-Epi-2-deosy-20-hydroxyecdysone Ecdysone-22-phosphate ZDeoxyecdysone-22-phosphate Poststerone Inokosterone Ponasterone A
13-I 9.4 13.6 9.9 13.5 9.9 9.7 4.9 16.0 15.3 18.6 14.5 20.0 16.6 1.3 2.0 9.6 9.9 17.2
B
8.5 21.5
I
20
10 Mm
0
Fig. 2. Separation of ecdysone (I), 20-hydroxyecdysone (II), j-epiecdysone (III) and j-epi-2Ohydrosyecclysone (IV) on an Ultrasphere-ODS column (15 cm x 4.6 mm I.D.). Chromato_mphic conditions \*ere the same as those described for Fis 1.
NOTES
32
TABLE
Ii
RETEhTf09 PHOSPHATE
VOLUME FOR OX PARTISIL-SAX
ECDYSONE-2%PHOSPHATE
AND
‘-DEOXYECDYSONE-Z-
Column: 50 cm x 9-d mm I.D. Mobile phases: C. 0.02 M phosphate bufier, pH 5.0; D, 3.1 Ammonium ;fcztxS; flow-rate_ 5 ml;min. Ecd:srrroid
Rerention volume (ml)
J lobile pilase
Ecdysone-X-phosphate l?-Deoxvccdyone-22-p-nosphaie
C
D
91 110
105
go
amenabIe to both anion eschanse and paired-ion reversed-phase aIthou$ peak shape is much better in the latter method.
chromatography,
_ACKXOWLEDGEXfE;r;TS
We thank the Agricultural Research Council and the Science and Engineering Research Council for financial support and Professor T. W. Goodwin for his continued interest and encouragement. Gifts of ecdysteroids are gratefully acknowledged_ REFEREXCES 1 L_ I. Gilbert and D. S. King in hf. Roc_kstcin (Editor), The Ph_nioIog_~of Insecta, Vol. 1. Academic Press. New York. 2nd ed_. 1973. p_ 119. C. Hen-u. _M_ Lagueux. B. Lu and J. A. Hoffman. Life Sci.. 22 (1978) 21-11. E. Ohnishi. hl. Takashi. F. Chatani. S. lkekawa and S. Sakuni, Scie~rcr. 197 (1977) 66. L. K. Dinan and H. H. Rm. J. fnsecr Plr~sid.. 17 (1951) 51. E. Ohnishi and T. htizuno. Insrcr Bioclzenr., 11 (1951) 155. R. E. Isaac. H. H. Rees and T_ W_ Goodsin. J. Chem. Sot_, C/rem. Common.. (19Sl) 41s. J. Clrem. Ser.. Chem. AI. J_ Thompson_ J. X;. Kaplanis. \V. E. Robbins and R. T. Yamamoto, S 9 10 11 12 I3 11 15 16 17 18 19
Conzmun., (1967) 650. J. N_ Kaplanis. W. E. Robbins, M. J. Thompson and S. R. Dutky. Scier;ce, 180 (1973) 307. J. S_ Kaplanis. 51. J. Thompson. S. R. Dutky and W_ E. Robbins. Srero&. 3-l (i979) 333_ D. R. Greenwood and G. B. Russell. Experienria. 34 (1978) 687. J. Xoolnan. L. Reum and P. Karlson. Hoppe-Se&r-s Z. Phniol. Chem.. 360 (1979) 1351. H_ N. Xige J_ A_ Stoboda. M. J_ Thompson_ J. N. Kaplanis, S. R. Dutky and W. E. Robbins, Lipids. 9 (1971) 971. &ci_J. Thompson, J. X. Kaplanis. W_ E. Robbins. S. R. Dutky and H. N. Nigg. Sreroids. 24 (1974) 359_ R. T_ Mayer. J. L. Durrant, G. M. Holman. G. F. Weirich and J. A. Sloboda. Sreroids, 34 (1979) 555. P- Karlson_ H. Bugany. H. Dopp and G.-X- Hoyer. Hoppe-Se&v‘s Z_ PlJuiol_ C~J~JIJ..353 (1971) i610. J. Koolman and K.-D. Spindler. Hoppe-Sevler’s Z. Physiol. Chem.. 3% (1977) 1339. R. E_ Isaac, H. H. Rees and T. W. Good&. 1. Chem. Sot.. Chem. Commun.. (19Sl) 594. R. E. Isaac, M. E. Rose. H. H. Rm and T. W. Goodwin, J. Chem. Sot., Chem. Conmzun.. (19X+2)249. R. Lafcnt. P. Beydon, B. AIauchamp, G. Somme-Martin, M. Andrianjafintrimo and P. Krien, in F. S&r& A. Zabza. J. J. Menu and B. Cymborowski (Editors), Regulation of Insecx Developmenr and Beltnviour, Part I_ Wroclow Technical University Press, Wroclow, 19&l, p_ 125. K.-D. Spindler. J. Koolman. F. Mosora and H. Emmerich, 1. Insect Ph_llrioI..23 (19771441. L. N. Dinan and H. H. Rees, Sreroids_ 31(197S) 629. Xl. N_ Galbraith and D_ H. S. Horn_ dusr. J_ Cjzem_, 22 (1969) 1045. L. Xi. Dinan, P. L. Doncahey. H_ H. R= and T_ W. Goodwin, J. Chronmrogr.. 205 (1981) 139. I_ D. Wifso~. C. R. Bidbp and E. D. Morgan. J. Chromafogr., 238 (1982) 97. E_ Tomlinson, T. M. Jefferies and C. Xl. Riley. J_ Chromarogr., 159 (1975) 315.
of Chromatogruphy,
246 ( 19SZ) 3 17-373
Elsevier Scientific Publishing Company. Amsterdam CHROM.
Printed in The Netherlands
15,031
Note
High-performance liquid steroid-22-phosphates R. ELWYN Deparrnzent
ISAAC,
NICHOLAS
of Biochenristr_v_
chromatography
P. lMILNER
University
and HUW
of Liverpool.
P-0.
of ecdysteroids
and ecdy-
H. REES’ Box 147. Liwrpool
L69 3BX
(Great
Brituirzj
(First received April 29th, 19S7; recked manuscript received May 14th. 1983)
The ecdysteroids (insect moulting hormones) are a group of polyhydroxylated steroids possessing a 5&7ene-6-one ring structure .20-Hydroxyecdysone (II) is generally regarded as the major active hormone’. although ecdysone (I) may also elicit a honnonai response in certain tissues at some stages in development_ 2-DeoxyecdysonezJ and Z-deosy-20-hydrosyecdysone 5*6 have been isolated from the ovaries and eggs of a number of insect species as major ecdysteroid components of largely unidentified highly polar conjugates. These Xdeoxy ecdysteroids may be possible precursors of ecdysteroids I and II. Other ecdysteroids which have been isolated from insects include the 26-hydroxy’-“, 3-epi’“iA, 3-dehydro’s-‘6, S-acetate” and 2% phosphate l8 derivatives. The latter compounds are considered to be inactivation products of the hormones_ Thus, ecdysteroids varying widely in polarities are encountered in insect metabolism_ Complex mixtures of ecdysteroids are amenable to chromatography on reversed-phase high-performance liquid chromatographic (HPLC) systems” and, owing to the wide range of polarities, are best resolved using a methanol-water gradient for elution. In the present paper we describe the chromatography of a wide spectrum of insect ecdysteroids on an efficient, short (15cm) Ultrasphere-ODS (particle size 5 ,urn) column. The data presented (Table I) indicate potential pitfalls in the
0021-9673/S2/~/SO~.7.5
63 1982 Elsevier Scientific Publishing Company
NOTES
identification of some ecdysteroids and metabolites having similar chromatographic properties_ and the value of an efficient reversed-phase column in resolving such steroids. The chromatography of the newly identified ecdysteroid monoacetates and 22-phosphates are aIso reported. Ecdysone- 2%phosphate and 2-deosyecdysone-22phosphate have also been analysed by HPLC using reversed-phase paired-ion and anion exchange (Whatman SAX) chromatography_ ESPERIMEXTAL
The ecdysteroid samples used in the present study were obtained from various souicrSI ecdysone (I) (Simes, Milan, Italy); 30-hydrosyecdysone (II) and &pi-?deosyecd_vsone (XIII) (Dr_ G_ B_ Russell. D.S.LR__ New Zealand); 26-hydrosyecdysone (VII) (Dr. J_ N. Kaplanis_ U.S.D.A.. Beltsville. MD, U.S.A.); 20,26-dihydroayecdysone (VIII). ?-deosy-20-hydrosyecdysone (XII) and 3-epi-?-deosy-20hydrosyecdysone (XIV) (Dr. D. H. S. Horn. C.S.I.R.O., Melbourne. Australia), 2deosyecdysone (XI) (Professor E_ Ohnishi_ Nagoya University_ Nagoya, Japan); poststerone (XVII) (the late Dr. J. B. Siddall, Zoecon Corporation, CA, U.S.A.): inokosterone (XVIII) (Professor T. Takemoto, Tohoku University, Tohoku, Japan); ponasterone A (XIX) (Professor K_ Nakanishi, Columbia University_ New York, NY, USA_): 3-dehydroecdysone (V) and z-dehydro-20-hydrosyecdysone (VI) (prepared by the method of Spindler r& ol_‘O); 3-epiecdysone (III) and 3epi-20h)-dhosyecdysone (IV) (prepared by the method of Dinan and Rees’l), ecdysone-Z(X) and 3-monoacetates (IX) (prepared by the method of GaIbraith and Horn”); ecd}sone-22-phosphate (XV) and %leoqecdysone-22-phosphate (XVI) (isolated
NOTES
319
from eggs of Schistocerca gregaria”). The mixture of ecdysteroids used in the separation shown in Fig. 1 was obtained from ecdysteroid conjugates, isolated from developing eggs of Schistocerca gregaria, by hydrolysis with a crude enzyme preparation from Helix pomatia*. HPLC grade methanol was purchased from Rathburn Chemicals (Walkerbum, Great Britain) and tetra-fz-butylammonium hydroxide solution (40 y0 in water) and AnalaR-grade orthophosphoric acid were purchased from BDH (Poole, Great Britain). High-performance liquid chonzatograplzy HPLC was carried out using a Model ALC/lOO Iiquid chromatograph (Waters Assoc., Northwich, Great Britain) linked to a Perkin-Elmer LC-55 variable wavelength detector set at 250 nm. Linear solvent gradients were formed with the aid of a Model 660 solvent programmer (Waiers Assoc.) and samples, dissolved in methanol, were injected via a Model U6K injector (Waters Assoc.). An Altex Ultrasphere-ODS column (15 cm x 4.6 mm, particle size 5 pm) (Anachem, Luton. Great Britain) was used for reversed-phase chromatogaphy. Some reversed-phase separations of ecdysteroid phosphates involved addition of tetra-n-butylammonium hydroside (BDH) adjusted to pH 7.5 with orthophosphoric acid. to the aqueous methanol soIvent to make a 0.6 7; solution. RESULTS
AND
DISCUSSION
Reversed-phase HPLC using a methanol-water gradient on an UltrasphereODS column allowed the separation of ecdysteroids of widely differing polarities in a single run (Table I). The application of such a system to the analysis of biological extracts is illustrated in Fi_g. I_ The chromatogram shows the separation of a series of ecdysteroids isolated from developing eggs of 27.gregaria. These ecdysteroids probably form part of a metabolic scheme for the biosynthesis and inactivation of 20hydroxyecdysone (II)_ It is apparent from Table I that several of the ecdysteroids are either not resolved or have very similar chromatographic properties. Thus, potential pitfalls in identification of radioactive ecdysteroid metabolites are clear. However, use of such a reversed-phase column in conjunction with an adsorption-type column, such as APSHypersil (aminopropyl) or silica. obviates such problems_ The system described in this paper allows good resolution of four ecdysteroids and their 3-epimers on this short column (Figs 1 and 2). Although separation of ecdysteroids from their 3epimers has been achieved on some other types of reversed-phase columns (PBondapak CISt 30 cm’“), this was not always the casez3. On reversed-phase. the 3epiecdysteroids have very similar retention volumes to the corresponding 3-dehydro compounds (Table I), but these pairs can be easily separated by chromatography on an APS-Hypersil column 23. It is interesing that the order of elution of some plant ecdysteroids during isocratic reversed-phase HPLC was changed when different organic solvents in admixture with water were used as mobile phases’“. It is important to note that whereas on silica thin-layer chromatography (TLC) on a reversedecdysone-‘>- and 3-monoacetates co-migrate with 3dehydroecdysone”, phase system these three ecdysone derivatives are easily resolved. Therefore, the
NOTES
L
20
10 !-cc?
0
Fig_ l_ Separation ofecdysteroids on an Ultrasphere-ODS column (15 cm x 3.6 mm I.D.). Mobile phase: linear _gadient of 40 p,Omethanol in water increkng to SO“/‘, methanol in water over 10 min: flow-rate, 1 mi.:min_ Detection: UV absorbance at 150 run. Peaks: II = 20-hydroxyecdysone: V!I = 26-hydroxyecdjxone; I = ecdysone; III = 5-epiccd~sone; XII = I-deoxy-20-hydroxgecdcdysone; IX = ecdysone-3acetate; XI = Z-deoxyecdysone; XIII = j-epi-Z-deoqecdysone.
identification of 3dehydroecdysone and ecdysone monoacetates by cochromatography with authentic compounds must be carried out on both adsorption and reversedphase systems. Reversed-phase columns are unsuitable for the chromatography of the highly polar ecdysteroid phosphates unless the anionic group is first neutral&d. We have added tetra-n-butylammonium hydro.xide to the eluting solvent as the anion pairing agent”, which resuk in greater retention of the phosphate conjugates on the reversed-phase column (Table I, solvent system B). The reproducible results obtained by this method should facilitate the identification of radiolabelled ecdysteroid metabelites by co-chromatography with authentic steroid conjugate. We have used chromatography on a strong anion exchange column as a major step in the purification of ecdysone-22-phosphate and 2_deosyecdysone-22phosphate from the eggs of Schistocerca gregaria Is. For routine analytical studies, phosphate buffer (0.02 AZ) pH 5.0 was employed as the mobile phase, but when it was necessary to isolate ecdysteroid conjugates free of phosphate salts, this buffer was replaced with ammonium acetate (O-1 M). These systems separate ecdysone-22phosphate, Z-deosyecdysone-22-phosphate (Table II) and a highIy polar 20-hydrosyecdysone conjugate of, as yet, unknown structure (data not presented). In conclusion, complex mixtures of ecdysteroids can be resolved on an Ultrasphere reversed-phase column using a methanol-water gradient (total elution volume, 20 ml)_ Small elution vohunes and low solvent flow-rates result in greater efficiency in the detection of UV absorbing compounds and in the monitoring of radiolabekd components either by liquid scintillation counting of fractions or by the use of an on-line radioactivity scintillation monitor_ Ecdysteroid phosphates are
321
NOTES TABLE
I
RmENTION
VOLUMES
FOR ECDYSTEROIDS
ON ULTRASPHERE-ODS
Colurrm: 15 cm x 66 mm LD_ Modile phases: A, linear gradient of 40 y0 methanol in water increasing to 80 % methanol in water over 20 min; B, 46 y0 methanol in water containing 0.6 % tetra-n-butylammonium hydroxide; flow-rate, 1 ml/min.
.-I I
11 III LV V VI VII VIII IX X XI XII XIII XIV xv XVI XVII XVII1 XIX
Ecdysone IO-Hydrosyecdysone 3-Epiecdysone 3-Epi-20-hydroxyecdysone 3-Dehydroecdysone 3-Dehydro-20-hydroxyecdysone 26-Hydroxyccdysone 20,26_Dihydrosyecdysone Ecdysone-3-acetate Ecdysone-2-acetate 2-Deoxyecdysone 2-Deoxv-20-hydroxyecdysone 3-Epi-2-deosyecdysone 3-Epi-2-deosy-20-hydroxyecdysone Ecdysone-22-phosphate ZDeoxyecdysone-22-phosphate Poststerone Inokosterone Ponasterone A
13-I 9.4 13.6 9.9 13.5 9.9 9.7 4.9 16.0 15.3 18.6 14.5 20.0 16.6 1.3 2.0 9.6 9.9 17.2
B
8.5 21.5
I
20
10 Mm
0
Fig. 2. Separation of ecdysone (I), 20-hydroxyecdysone (II), j-epiecdysone (III) and j-epi-2Ohydrosyecclysone (IV) on an Ultrasphere-ODS column (15 cm x 4.6 mm I.D.). Chromato_mphic conditions \*ere the same as those described for Fis 1.
NOTES
32
TABLE
Ii
RETEhTf09 PHOSPHATE
VOLUME FOR OX PARTISIL-SAX
ECDYSONE-2%PHOSPHATE
AND
‘-DEOXYECDYSONE-Z-
Column: 50 cm x 9-d mm I.D. Mobile phases: C. 0.02 M phosphate bufier, pH 5.0; D, 3.1 Ammonium ;fcztxS; flow-rate_ 5 ml;min. Ecd:srrroid
Rerention volume (ml)
J lobile pilase
Ecdysone-X-phosphate l?-Deoxvccdyone-22-p-nosphaie
C
D
91 110
105
go
amenabIe to both anion eschanse and paired-ion reversed-phase aIthou$ peak shape is much better in the latter method.
chromatography,
_ACKXOWLEDGEXfE;r;TS
We thank the Agricultural Research Council and the Science and Engineering Research Council for financial support and Professor T. W. Goodwin for his continued interest and encouragement. Gifts of ecdysteroids are gratefully acknowledged_ REFEREXCES 1 L_ I. Gilbert and D. S. King in hf. Roc_kstcin (Editor), The Ph_nioIog_~of Insecta, Vol. 1. Academic Press. New York. 2nd ed_. 1973. p_ 119. C. Hen-u. _M_ Lagueux. B. Lu and J. A. Hoffman. Life Sci.. 22 (1978) 21-11. E. Ohnishi. hl. Takashi. F. Chatani. S. lkekawa and S. Sakuni, Scie~rcr. 197 (1977) 66. L. K. Dinan and H. H. Rm. J. fnsecr Plr~sid.. 17 (1951) 51. E. Ohnishi and T. htizuno. Insrcr Bioclzenr., 11 (1951) 155. R. E. Isaac. H. H. Rees and T_ W_ Goodsin. J. Chem. Sot_, C/rem. Common.. (19Sl) 41s. J. Clrem. Ser.. Chem. AI. J_ Thompson_ J. X;. Kaplanis. \V. E. Robbins and R. T. Yamamoto, S 9 10 11 12 I3 11 15 16 17 18 19
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