Electrospray tandem mass spectrometry of ecdysteroid moulting hormones

Electrospray mass spectrometIy has aided the structural characterization of ecdysteroid moulting hormones as a means of understanding factors controlling the moulting cycle in crabs. Possible fragmentation routes were frrst obtained by controlled collision-activated dissociation (CAD) initiated by cone voltage fragmentation. Low energy CAD MSIMS analyses of the protonated molecules [M+H]+ confrrmed the characteristic fmgerprint patterns obtained by cone voltage fragmentation. MSIMS analyses of selected intermediate fragments provided additional structural data and established the fragmentation routes.


Introduction
Over one hundred different steroids, with varying degrees of hydroxylation, containing the basic 5f3, 7-en-6-one moiety of ecdysone have been isolated from plants, insects and crustaceans.Ecdysteroids in invertebrates have been related to the animals' life cycle and especially moulting [1].Research has generally pointed to the Y organs from crustaceans and the prothoracic gland from insects as the primary secretory glands.However, auxiliary sources of ecdysteroids, in both animal groups, have also been proposed [2].
Insects and crustaceans do not biosynthesize ecdysteroids de novo, but biotransform dietary cholesterol or C-24 alkylsterols into ecdysone derivatives [3,4].As well, a wider variety of ecdysteroids have been found in the blood of crustaceans than in most insects.In plants, there are indications that ecdysteroids playa role in defence against insects, but their function is not as well defmed as in animals.
The earlier structural work reported in the literature was carried out on compounds isolated from plant sources.Larger quantities of material facilitated the full spectroscopic characterization and chemical degradation needed for the Wlambiguous identification of the molecules.Later, advances in chromatography, immunoassays and spectroscopy enabled full or partial characterization of ecdysteroids in a variety of species where a-ecdysone 1 and J3-ecdysone 2 are major components (Figure 1).
The present study was originally initiated to elucidate the structure of the moulting hormones of snow crabs as a means of understanding the moulting cycle in crabs.The knowledge of the identity of the moulting hormones might, perhaps, enable us to determine what factors, particularly environmental, might interfere with the efficacy of moulting hormones and cause massive moult failures within snow crab populations.Such a phenomenum was observed off the Avalon Peninsula in the early 1980's with devastating consequences to the snow crab industry.
Mass spectrometry is the technique of choice for structural elucidation studies of these ecdysteroids.GC-MS and LC-MS have been used with strong and mild ionization techniques.Because ecdysteroids are polar molecules, they need derivatization prior to volatilization for GC related analyses.Due to the lack of reactivity of the hindered hydroxy positions, this necessity has led to the development of alternate MS approaches [5].
While electron impact (EI) was the only available MS technique in the late sixties and early seventies, a plethora of different MS techniques, covering a wide range of ionization modes, emerged during the last decade.A short summary ofMS techniques used in the literature to investigate the structure of ecdysteroids is presented in Table 1.More information on techniques used in the analysis of ecdysteroids and MS techniques in general, can be found in recent reviews [13,14].
Electrospray ionization is well established as a robust LC-MS technique that allows rapid, accurate and sensitive analysis of a wide range of analytes from low molecular weight polar compounds (less than 200Da) to biopolymers larger than 100KDa.Although ESMS has been distinguished for its ability to produce intact multiple-charged ions from proteins and other biopolymers [15,16], compounds of less than lKDa produce singlecharged molecules.Under appropriate experimental conditions, gas-phase fragmentations are minimized and the subsequent ions, which possess low internal energy, are sufficiently stable to pass from the ion source to the detector without dissociation.This is common for ions produced under "very soft" ionization processes.
Dissociations may be induced or activated by collision.In this process, a portion of the kinetic energy of the ion is converted to internal energy by colliding with a neutral gas phase species, usually in the pressurized collision cell of tandem MSIMS instruments [17,18].Ions that have undergone this collisional excitation process may subsequently fragment.Thus, collision-activated dissociation CAD MSIMS, also known as low-energy CAD MSIMS, is a valuable method for generating structural information if the primary ionization process does not impart enough internal energy for spontaneous fragmentation to occur [19].
Another method of generating structural information, known as cone voltage fragmentation, is by dissociation of the protonated molecular ion [M+H]+ by controlled adjustment of the accelerating voltage (0±250V) applied to the sampling cone or focus voltage of the electrospray source.This procedure is also known as CAD in the atmospheric pressure/vacuum interface region, under mild conditions [20].Unlike CAD experiments performed using tandem mass spectrometers, no mass filtering precedes ion-neutral collisions.Mass analysis of surviving precursor ions, plus the decomposition products generated by the subtotal of all dissociations, from all precursors, contribute to the ions observed in the CAD spectrum.
The present study relates to the structural characterization and differentiation of aecdysone and J)-ecdysone using electrospray MS in the positive and negative ion modes.Evidence of the possible fragmentation routes was frrst obtained by cone voltage fragmentation.Structural information was also derived from low energy tandem mass spectral analysis (MSIMS) of the protonated molecules [M+H]+.Rationalization of the fragmentation routes was made by obtaining the product and precursor ion spectra of the various intermediate ions.
Table 1 Examples of the analysis of ecdysteroids using various mass spectroscopy techniques

Results and Discussion
The electrospray mass spectrum (positive ion mode) of a-ecdysone 1 was recorded with a low focus voltage (30Volts) and gave the protonated molecular ion [M+H]+ at mlz 465, in addition to the adducts [M+~]+ and [M+H+MeOH]+ at mlz 482 and 497, respectively (Figure 2a).However, since this simple ESMS lacked substantial structural information, a higher focus voltage was employed to induce fragmentation of the protonated [M+H]+ molecule.Such a technique can provide structurally useful infonnation.Thus, the ESMS of a-ecdysone 1 were recorded with focus voltages of 50 and 75 volts as shown in Figures 2b and 2c.When higher focus voltages were used, the adduct with sodiwn [M+Na]+ at mlz 487 was observed in all the ESMS .The fragmentation route of the protonated molecular ion of this steroid was rationalized to occur by simple losses of either one, two or three molecules of water, to afford the [M+H-H 2 0]+, [M+H-2H 2 0]+ and [M+H-3H 2 0]+ fragment ions at mlz 447, 429 and 411, respectively.The elimination of two or three molecules of water can occur by a concerted single step or, more likely, by a multistep process involving the loss of molecules of water from the [M+H-H 2 0]+ or [M+H-2H 2 0]+ fragment ions.
Low energy tandem MS analyses were conducted to rationalize the fragmentation pathways leading to the various fragmentations obtained in the conventional ESMS.The product ion spectrum, also called daughter ion spectrum, arising from the fragmentation in the RF only hexapole collision cell of the quadrupole-hexapole-quadrupole instrument was obtained.The [M+H]+ ion at mlz 465 was selected for the recording of the tmimolecular mass-analyzed ion kinetic energy (MIKE) and collisional activated dissociation (CAD) MS/MS.One of the main advantages of the MS/MS technique is that the origin of fragment ions can be ascertained.The CAD MS/MS of the [M+H]+ ion of a-ecdysone 1 suggested the fonnation of the following product ions at mlz 447, 429, 411, 331, 109 and 98 (Figure 3a).Second generation product ions of the intennediate fragm~nt ions [M+H -H 2 0]+ at mlz 447 were generated in an MS/MS experiment and afforded the product ions at mlz 429, 411, 331, 109 and 98, as shown in Figure 3b.Third generation product ions of the intennediate fragment ion [M+H -2H 2 0]+ at mlz 429, were generated in a different MS/MS experiment and afforded the product ions at mlz 411, 331, 109 and 98 (Figure 3c).
The Schematic representation of the applicable scan mode is conveniently represented on all the MS/MS figures with symbols (filled circle indicates a flXed or preselected mass, open circle indicates a scanned or variable mass), as described by Wysocki [19] and originally introduced by Cooks and co-workers [21].
In a different set of experiments, the precursors of the [M+H-2H 2 0]+ fragment ion at mlz 427 were sought using the precursor ion scan technique (parent ion scan).It was established that this ion originated from either the intennediate ion [M+H-H 2 0]+ at mlz 447 or the protonated molecule [M+H]+ at mlz 465 (Figure 5).Similarly, it was established that the [M+H-H20-HO-CH=CH-CH2-C(Me)20H]+ ion at mlz 331, originated from either the intennediate fragment ion [M+H -H 2 0]+ at mlz 447, by loss of the 116 a.m.u.side chain, or the protonated molecule [M+H]+ at mlz 465, by the concerted loss of a molecule of water and the 116 a.m.u.side chain (Figure 6).The ESMS of a-ecdysone 1, in the negative ion mode, afforded a major peak at mlz 499, assigned to [M-H+2H 2 0l, resulting from the deprotonated molecular ion adduct with two molecules of water.
The ESMS of J3-ecdysone 2, in the positive ion mode, was recorded with a focus voltage of 30 Volts and showed the protonated molecule [M+H]+ at mlz 481 in addition to the adduct [M+H+MeOH]+ at m/z 513 (Figure 7a).However, as this mass spectrum did not provide any substantial structural infonnation, ESMS were recorded with higher focus voltages to induce fragmentation of the molecular ion [M+H]+.The ESMS of J3-ecdysone 2, recorded with focus voltages of 50 and 75 Volts, are shown in Figure 7b and 7c respectively.In these conditions, fragment ions originating from the loss of one, two or three molecules of water were observed.These fragment ions were assigned as [M+H-H 2 0]+, [M+H-2H 2 0]+ and [M+H-3H 2 0]+ at mlz 463, 445 and 427, respectively.As in the case of a-ecdysone 1, a fragment ion, resulting either from the loss of the HO-CH==CH-CH2-C(Me)20H (116 a.m.u.) side chain from the intermediate fragment ion [M+H -H 2 0]+ at mlz 463 or from the loss of the HO-CH==CH-CH==C(Me)2 (98 a.m.u.) side chain from the intennediate fragment ion [M+H-2H 2 0]+ at mlz 445, was observed at mlz 347.This fragment ion may lose a molecule of water to afford the fragment ion [M+H-2H 2 0- 116]+ at mlz 329.
CAD MSIMS of the protonated molecular ion [M+H]+ at mlz 481, afforded a series of product ions at mlz 463, 445, 427, and 371, as shown in Figure 8.The proposed fragmentation route of the protonated molecule [M+H]+ of f3-ecdysone 2, as determined by ESMS and CAD MSIMS, is shown in Figure 9.As in the case of 1, the formation of the fragment ion [M+H-2H 2 0]+ at mlz 445, may originate either by the concerted losses of two molecules of water from the protonated molecular ion [M+H]+ or by a two-step process involving the loss of a molecule of water from the intermediate fragment ion [M+H-H 2 0]+ at mlz 463.The product fragment ion at mlz 371, has been assigned as [M+H-3H 2 0- CH 2 ==C(Me)2]+ and originated from the loss of the CH 2 ==C(Me)2 (56 a.m.u.) side chain from the [M+H-3H 2 0]+ product ion at mlz 427.
The ESMS of f3-ecdysone 2, in the negative ion mode, afforded only a major peak at mlz 515, which was assigned to the molecular deprotonated adduct with two molecules of water, namely [M-H+2H 2 0l.
Preliminary investigation on hemolymph obtained from snow crabs seems to indicate the presence of these two moulting hormones in small amounts [22].
ESMS was performed either by direct injection into the electrospray source through the Rheodyne injector with a 20 ~ loop or through the Hewlett Packard 10 HPLC system containing the VYDAC column.Obviously the ESMS spectra were identical in each case.
The ESMS (positive and negative ion modes) were measured with a Fisons VG-Quattro quadrupole-hexapole-quadrupole mass spectrometer, equipped with an electrospray ionization source, capable of analyzing ions up to mlz 4000.A 486, 66MHz personal computer equipped with Fisons MASSL YNX Mass Spectrometer Data System Software, was used for data acquisition and processing.
The temperature of the ES ionization source was maintained at 70°C.The voltage of the ES capillary was 3.4 KV and the HV lens was at 0.4 KV throughout the whole operation.ESMS were recorded with a focus setting varying from 30 to 75 volts.Generally, higher voltage induced fragmentation of lower molecular weight samples.Conventional ESMS were obtained by scanning in the Multichannel Analysis mode (MCA) with a scan dwell time of Is/250 a.m.u .. Spectra are an average of 3-4 scans.The mass scale was calibrated using a polyethylene glycol mixture (PEG 300/600/1000) in the positive ion mode and a sugar mixture (com syrup, raffmose, maltose and maltotetraose) in the negative

Conclusion
Mass spectral analyses of ecdysones have been facilitated using electrospray ionization.AbWldant signals, corresponding to the protonated molecules, were observed in each case, using this ionization technique.Collision induced dissociations in the atmospheric pressure/vacuwn interface were promoted by increasing the accelerating voltages and have provided additional structw"al information.MSIMS spectra of the moulting hormones, obtained using low energy collisional activation, permitted the rationalization of the fragmentation pathways.Furthermore, daughter and parent ion series and MSIMS spectra of selected intermediate ions, formed during the cone voltage fragmentation of the ionized species, permitted rationalization of the fragmentation behaviour.
proposed fragmentation route of the protonated molecule [M+H]+ of ecdysone, as determined by ESMS and CAD MS/MS, is shown in Figure 4.The product ion at mlz 331 has been tentatively assigned as originating either from the [M+H-H 2 0]+ fragment ion at mlz 447 by the loss of the HO-CH=CH-CH2-C(Me)20H (116 a.m.u.) side chain or from the [M+H-2H 2 0]+ fragment ion at mlz 429 by the loss of the HO-CH=CH-CH=C(Me)2 (98 a.m.u.) side chain.The product ions at mlz 109 and 98 are produced, from the side chain, by elimination and were attributed to [CH 2 =CH-CH=CH-CH=C(Me)2+H]+ and [HO-CH=CH-CH=C(Me )2+H]+, respectively.

Figure 9 .
Figure 9. Major fragmentation routes of the [M+H]+ ion of r>-ecdysone 2 observed by ESMS and CAD MSIMS.