The structures of three HETEs, two DHETs and three THETAs are shown in . Mass spectra of these eicosanoids exhibited carboxylate molecular ions [M-H]− with m/z 319 for HETEs, m/z 337 for DHETs, and m/z 353 for THETAs as the most abundant ions. For MS/MS experiments, the molecular ions were isolated, accelerated toward the ICR cell and dissociated. Some similar fragmentation patterns of HETEs, DHETs and THETAs were observed, indicating the common backbone structures among these compounds. However, there were unique fragmentations reflecting the structural characteristics that can be used to identify these compounds.
Structures of HETEs (11-HETE, 12-HETE and 15-HETE), DHETs (11,12-DHET and 14,15-DHET) and THETAs (11,12,15-THETA, 11,14,15- THETA and 13,14,15- THETA).
Common fragmentation pathways for HETEs, DHETs and THETAs
MS/MS spectra of all compounds indicated losses of H2O and CO2 to form a series of ions. For HETEs ( and ), which consist of one hydroxyl group, m/z 301 corresponding to [M-H-H2O]−, was observed in all SORI-CID and IRMPD spectra. The m/z 257 was formed by a loss of CO2 from [M-H-H2O]−. DHETs with two hydroxyl groups, as shown in and , the molecular ions (m/z 337) lost two H2O molecules one after another, to form m/z 319 and 301, assigned as [M-H-H2O]− and [M-H-2H2O]−, respectively. These ions could further lose CO2 to form [M-H-H2O-CO2]− (m/z 275) and [M-H-2H2O-CO2]− (m/z 257), respectively. The loss of H2O and CO2 are the major fragmentation pathways for HETEs and DHETs. In the SORI-CID and IRMPD spectra of m/z 353 of THETAs ( and –), which have three hydroxyl groups, the [M-H]− ions easily lost one to three H2O molecules to give [M-H-H2O]− (m/z 335), [M-H-2H2O]− (m/z 317) and [M-H-3H2O]− (m/z 299). After the losses of 2 or 3 H2O, [M-H-2H2O]− and [M-H-3H2O]− could further dissociate to form [M-H-2H2O-CO2]− (m/z 273) and [M-H-3H2O-CO2]− (m/z 255), respectively.
Figure 2 MS/MS spectra of [M-H]−, m/z 319 for HETEs (100 pg) obtained from negative ion ESI SORI-CID and IRMPD FTICR. (A) MS/MS spectrum of 11-HETE by SORI-CID; (B) MS/MS spectrum of 11-HETE by IRMPD; (C) MS/MS spectrum of 12-HETE by SORI-CID; (D) MS/MS (more ...)
High-resolution accurate mass measurements by SORI-CID FTICR for HETEs.
Figure 3 MS/MS spectra of [M-H]−, m/z 337 for DHETs obtained from negative ion ESI SORI-CID and IRMPD FTICR. (A) MS/MS spectrum of 11,12-DHET by SORI-CID; (B) MS/MS spectrum of 11,12-DHET by IRMPD; (C) MS/MS spectrum of 14,15-DHET by SORI-CID; (D) MS/MS (more ...)
High-resolution accurate mass measurements by SORI-CID FTICR for DHETs.
Figure 4 MS/MS spectra of [M-H]−, m/z 353 for THETAs obtained from negative ion ESI SORI-CID and IRMPD FTICR. (A) MS/MS spectrum of 11,12,15-THETA by SORI-CID; (B) MS/MS spectrum of 11,12,15-THETA by IRMPD; (C) MS/MS spectrum of 11,14,15-THETA by SORI-CID; (more ...)
High-resolution accurate mass measurements by SORI-CID FTICR for 11,12,15-THETA.
High-resolution accurate mass measurements by SORI-CID FTICR for 13,14,15-THETA.
Compared with HETEs and DHETs, the relative intensities of these ions (loss of H2O and CO2) in THETAs were lower than in HETEs and DHETs. The results suggest that the carbon-carbon bond ruptures became the major fragments of THETAs. In comparison of three THETAs, the relative intensities of ions corresponding to the losses of H2O and CO2 for 13,14,15-THETA were slightly higher than for 11,14,15- and 11,12,15-THETA, suggesting that the positions of hydroxyl groups affected the losses of H2O and CO2 from the molecular ions. When three hydroxyl groups were adjacent, as with 13,14,15-THETA, it was easier to lose H2O or CO2.
Unique fragmentation pathways for HETEs, DHETs and THETAs
Besides the similarities of fragmentation, there were significant differences in fragmentations among HETEs, DHETs and THETAs obtained from SORI-CID and IRMPD that indicated the characteristics of the molecular structures.
(a) MS/MS characteristics of HETEs by SORI-CID and IRMPD (1) SORI-CID of HETEs
, and E show the MS/MS spectra obtained from SORI-CID FTICR for 11-, 12-, and 15-HETE, respectively. In the SORI-CID spectrum of [M-H]−
for 11-HETE, the major characteristic product ions were m/z
167 and 149. The characteristic m/z
167 is similar to that reported fragmentation by triple quadrupole and ion trap mass spectrometers [33
]. The MS/MS spectra for 11-HETE by a triple quadrupole mass spectrometer had the dominant ion of m/z
] while spectra from FTICR and ion trap [39
] contained other characteristic ions. The mechanism for formation of m/z
167 was previously described [41
] as the charge-remote formation of the aldehyde by proton transfer and the cleavage of the C10–C11 bond. Another characteristic ion of the m/z
149 was observed and proposed as a cleavage of the C11–C12 bond and a loss of CO2
. Another particular ion with high intensity was observed at m/z
275. It was formed by a direct loss of CO2
from the [M-H]−
ion. The losses of CO2
O by [M-H]−
ion formed the highest abundant m/z
257, although these two ions were not detected in the previous studies [33
Proposed major fragmentation pathways of 11-, 12- and 15-HETE by negative ion ESI-FTICR. (A) 11-HETE; (B) 12-HETE; (C) 15-HETE.
For 12-HETE, the major characteristic ions were m/z
179, 163 and 135 and were similar to the results from the ion trap mass spectrometer [39
]. Again, the m/z
257 is the highest abundant ion. The mechanism for the m/z
179 formation was previously described [41
]. We proposed that the negative charge was located at the carboxylate because the presence of the m/z
135 ion, corresponding to a loss of CO2
from the m/z
179. The m/z
163 was presumably the result of carbon-carbon bond cleavage from C12–C13 and loss of CO2
The characteristic product ions of 15-HETE were m/z
219, 175 and 113 and were similar to previous studies [33
]. The m/z
219 was the product ion formed by the identical mechanism as previously described for the m/z
179 of 12-HETE [41
]. The m/z
175 was the subsequent loss of CO2
from the m/z
219 (). The elemental composition of the m/z
113 indicated the fragment of C7
O-1, suggesting the double bond conjugation and cleavage of C13–C14 bond. The abundance of this ion increased in IRMPD (see below).
(2) IRMPD of HETEs
The major product ions generated from IRMPD were similar to those generated from SORI-CID, but the relative intensities of these ions were different. In IRMPD spectra of [M-H]−
for all three HETEs (), the relative intensity of m/z
257 decreased, while this ion was the highest abundant ion in SORI-CID spectra of HETEs. It indicated that the loss of CO2
from the [M-H-H2
ion for HETEs became less effective with IRMPD. For 12-HETE (), m/z
179 was the most abundant ion, indicating that the break of C11–C12 bond was the major pathway [41
]. For 15-HETE (), m/z
219 and 113 became the major ions. The m/z
219 was formed by the break of C14–C15 bond and the charge was located on the carboxyl group. The formation of m/z
113 was the result of double bond conjugation and the rupture of C13–C14 bond as in SORI-CID.
(b) MS/MS characteristics of DHETs by SORI-CID and IRMPD (1) SORI-CID of DHETs
In the SORI-CID spectrum of [M-H]−
337, for 11,12-DHET is shown in and the ions are shown in . The prominent m/z
167, 163, 135, 179, 197, 153 and 169 were observed and were similar to the results from ion trap mass spectrometer [40
]. Formation of the highest abundant m/z
167 was similar to 11-HETE [41
]. If the charge was relocated on the hydroxyl oxygen at C12, the m/z
169 could be formed by the same fragmentation mechanism. The m/z
197 was a result of the cleavage of C11–C12 bond [41
]. Both m/z
179 and 153 corresponded to a loss of H2
O and CO2
from the m/z
197, respectively. The m/z
135 was formed by a loss of CO2
from the m/z
179 or a loss of H2
O from the m/z
153, respectively. The m/z
163 was derived from the cleavage of C12–C13 and the losses of H2
O and CO2
. The major fragmentation pathways of 11,12-DHET were proposed in .
Proposed major fragmentation pathways of 11,12-DHET by negative ion ESI FTICR.
The CID spectrum of [M-H]−
337, for 14,15-DHET had several characteristic ions () that were similar to the results from the ion trap mass spectrometer [39
] with the most abundant fragments of m/z
207 and 257 (). The m/z
257 was the result of the loss of 2H2
O and CO2
from the [M-H]−
ion. The m/z
207 was derived from the cleavage of the C13–C14 bond as previously described [39
]. In addition to m/z
207, other major ions were observed at m/z
129, 163, 175, and 219. The m/z
163 was from a loss of CO2
from the m/z
207. If the charge was relocated on the hydroxyl oxygen at C15, m/z
129 can be formed at high abundance by the same dissociation as m/z
207. The m/z
219 resulted from a loss of H2
O and the charge-driven cleavage of the C14–C15 bond from the molecular ion as described for 15-HETE above. The subsequent loss of CO2
219 formed m/z
175 with high abundance. Some other low abundant ions were also observed in the spectra, such as m/z
127 derived from cleavage at the C7–C8 bond, m/z
167 derived from the cleavage at the C10–C11 bond. illustrates the proposed dissociation reactions of 14,15-DHET.
Proposed major fragmentation pathways of 14,15-DHET by negative ion ESI FTICR.
(2) IRMPD of DHETs
The IRMPD spectra of [M-H]− for DHETs were similar to those formed by SORI-CID, indicating the same major fragmentation patterns. In the IRMPD spectra of [M-H]−for 11,12- and 14,15-DHETs (), the intensity of m/z 257 also decreased similar to HETEs as described above. The intensities of m/z 319 and 301, which were the results of the loss of one and two H2O from the molecular ions, became higher, indicating easy losses of H2O to produce [M-H-H2O]− and [M-H-2H2O]−. Interestingly, the m/z 163 instead of m/z 167 was observed as the high abundant ion for 11,12-DHET () similar to 12-HETE. The results suggested that 11,12-DHET possibly lost H2O at the C11 position and fragmented with the mechanism similar to 12-HETE. More low abundant ions were observed in the low m/z range of the IRMPD spectrum of 11,12-DHET, such as m/z 58, 123, 113, 177, and 139, indicating further fragmentation from excessive energy. For 14,15-DHET, the intensities of m/z 175, 163, and 129 became lower in the IRMPD spectrum with the m/z 207 as the highest abundance ion (). This suggested the dominant C13–C14 cleavage.
In general, the major product ions in the CID spectra of HETEs and DHETs obtained from FTICR were similar to ions obtained from ion trap and triple quadrupole mass spectrometers [34
]. However, more intense and more characteristic fragmentation ions were obtained from FTICR similar to ion trap than triple quadrupole mass spectrometry. Losses of H2
O and CO2
to form characteristic ions are more favorable in FTICR. Furthermore, the structures of these fragments can be ascertained based on the corresponding high-resolution and accurate mass data from FTICR. For example, the m/z
163 for 12-HETE and 11,12-DHET has the masses of 163.11239 and 163.11235, respectively. This ion has the elemental composition of C11
O-1 ( and ). However, the m/z
163 for 14,15-DHET has the mass of 163.14947 with the elemental composition of C12
-1 (). These results indicated that the fragmentation mechanisms of the m/z
163 for 11-HETE/11,12-DHET and 14,15-DHET are different as shown in , and .
(c) MS/MS characteristics of THETAs by SORI-CID and IRMPD (1) SORI-CID of THETAs
All THETAs exhibited identical [M-H]−, m/z 353. show the MS/MS spectra obtained from SORI-CID FTICR of 11,12,15-, 11,14,15-, and 13,14,15-THETA, respectively. Their high-resolution and accurate mass spectrometric data and corresponding predicted elemental composition of their fragments are shown in , and , respectively. With three hydroxyl groups, the spectra of THETAs were more complex than spectra of HETEs and DHETs.
High-resolution accurate mass measurements by SORI-CID FTICR for 11,14,15-THETA.
For 11,12,15-THETA, ( and ), the major ions were m/z
197, 167, 157, 139 and 127. The most abundant product ion was m/z
197. This ion was the result of the charge-driven cleavage of C11–C12 by the loss of neutral aldehyde from the enolate anion in which the charge was relocated on the hydroxyl oxygen at C12. If the charge was relocated on the hydroxyl oxygen at C11, the m/z
157 was formed by the similar fragmentation mechanism as shown in . The m/z
157 then lost H2
O to form m/z
139. The mechanism for formation of m/z
167 was the same as the ion of 11-HETE [41
]. If the charge was relocated on the hydroxyl oxygen at C-15, the m/z
235 was likely formed by the losses of H2
O and hexyl aldehyde from the cleavage of C14–C15 bond. illustrates the proposed fragmentation pathways for 11,12,15-THETA.
Proposed mechanism of formation of the m/z 197 and m/z 157 in the negative ion SORI-CID spectrum of [M-H]− for 11,12,15-THETA.
Proposed fragmentation pathways of 11,12,15-THETA by negative ion ESI FTICR.
The SORI-CID spectrum of [M-H]−
for 11,14,15-THETA ( and ) was dominated by the m/z
167. The formation of the m/z
167 was the result of the double bond conjugation and then, the break of C10–C11 bond as previously described for 11-HETE [41
]. In addition, the unique characteristic m/z
85 was observed for 11,14,15-THETA. The elemental composition of this ion was assigned as C4
. The proposed mechanism for its formation was the cleavages of C10–C11 bond and C14–C15 bond. Besides the m/z
167 and 85, the m/z
129, 205, 223, and 235 were also observed for 11,14,15-THETA. The proposed fragmentation pathways for these ions were similar to DHETs as shown in .
Proposed fragmentation pathways of 11,14,15-THETA by negative ion ESI FTICR.
When 11,12,15- and 11,14,15-THETA were compared, 11,12,15-THETA needs more collisional energy to fragment than 11,14,15-THETA. The different positions of the second hydroxyl group and the third double bond in the structures of 11,12,15-THETA and 11,14,15-THETA resulted in a remarkable and diagnostically useful difference in their major fragmentation pathways. The m/z 197, 207, 157 and 139 were the characteristic product ions with m/z 197 as the highest abundant ion for 11,12,15-THETA. The m/z 167, 205 and 85 were the characteristic product ions with m/z 167 as the most abundant ion for 11,14,15-THETA.
and show the SORI CID spectrum and fragments of [M-H]−
for 13,14,15-THETA. The m/z
193 was observed as the most abundant ion, which was proposed to form by the double bond conjugation and the break of C12 –C13 bond similar to the formation of m/z
179 for 12-HETE [41
]. This ion then lost CO2
to form the m/z
149. Other product ions were m/z
253, 235, 223, 217, 205, 173, 161, and 129. If the charge was relocated at C15 oxygen group, the m/z
253 was formed by the charge-driven loss of neutral aldehyde from C14–C15 bond. The m/z
235, 217 and 173 were formed by the losses of H2
O or CO2
253. The m/z
223 and 129 were formed by breaking of the C13–C14 bond. The former ion lost H2
O to form the m/z
205, and then lost CO2
to produce the m/z
161. The unique characteristic m/z 59 was observed for 13,14,15-THETA. The proposed formation of this ion was the cleavage of C12–C13 bond and C14–C15 bond similar to the m/z
85 for 11,14,15-THETA. The proposed fragmentation pathways for 13,14,15-THETA are shown in .
Proposed fragmentation pathway of 13,14,15-THETA by negative ion ESI FTICR.
(2) IRMPD of THETAs
show the product ion spectra obtained from IRMPD of 11,12,15-, 11,14,15-, and 13,14,15-THETA, respectively. The major product ions obtained from IRMPD were similar to SORI-CID, suggesting similar major fragmentation pathways of THETAs. However, there were some differences between SORI-CID and IRMPD spectra. For 11,14,15-THETA (), the intensities of the m/z 205 in the IRMPD spectrum increased two-fold, and the abundance of m/z 85 decreased, compared with SORI-CID. IRMPD favored the m/z 205 by the cleavage of neutral aldehyde from C13–14 bond and the subsequent loss of H2O. This may be due to the product ion obtaining more energy from the laser, and then fragmention to generate higher abundant m/z 205. For 11,12,15-THETA, the relative abundance of the m/z 207 increased with IRMPD. However, the abundance of the other product ions, such as m/z 157, 167, 177, 139 and 127, decreased. For 13,14,15-THETA, there were obvious differences between IRMPD and SORI-CID. The abundance of the m/z 59 and 173 decreased ten-fold in the IRMPD spectrum, and the other ions such as m/z 205, 149, 129, 217, 235 and 253 also decreased. These two techniques complemented each other, providing more useful structural information.
Identification of THETAs as arachidonic acid metabolites in rabbit aorta samples
The THETA fraction in rabbit aorta samples was analyzed by negative ion LC-FTICR to obtain structural information for identification of THETA isomers. The mass spectrum of the samples showed the most abundant ion of m/z 353 ([M-H]−). The molecular weight was determined as 354 Da with the elemental composition of C20H34O5.
(top panel) shows the total ion chromatogram of the MS/MS of m/z 353 by IRMPD. show the product ions of m/z 353 obtained from different retention times by IRMPD. The m/z 335, 317, and 299 were observed, suggesting the subsequent losses of one to three H2O from [M-H]−. These ions indicated three OH groups were present in the structures. The low abundance of these ions also suggested that the positions of three OH groups were not adjacent. The changes in spectral patterns at different retention times (from A to D) suggested that the peak contained more than one compound. A complete separation of THETA regioisomers on a reverse phase LC column was not obtained.
Figure 5 Total ion chromatogram (top panel) and product ion mass spectra of THETAs isolated from rabbit aorta by negative ion LC-ESI-FTICR with IRMPD. MS/MS spectrum of m/z 353 obtained from negative ion ESI FTICR at detection time of (A) 8.93 min; (B) 9.28 min; (more ...)
These mass spectra revealed the presence of characteristic products, m/z 85, 167, 205, 197, 157, 139 and 207, which belonged to THETAs. The m/z 167, 205 and 85 were the characteristics of 11,14,15-THETA, and the m/z 197, 157, 139 and 207 were the characteristics of 11,12,15-THETA. As shown in , the characteristics of 11,14,15-THETA, such as m/z 167, 205 and 85, were observed, but no m/z 197, 157, 139 and 207 were observed, suggesting that 11,14,15-THETA eluted first. With a longer eluting time (as shown in ), the m/z 197, 157, 139 and 207 appeared and the intensities of these ions increased, suggesting that 11,12,15-THETA eluted after 11,14,15-THETA.
shows the total ion chromatograms (top panel by IRMPD) and the selected ion chromatograms of m/z 85, 139, 157, 167, 197, 205, and 207 by both SORI-CID and IRMPD. The m/z 167, the major ion for 11,14,15-THETA and the minor ion for 11,12,15-THETA, exhibited a broad peak. According to the different retention times of these ions (run time of 25.00 min + detection time), the ions could be divided into two groups. A group of m/z 167, 205 and 85 () with identical detection times (9.44 min), suggesting that they were generated from one compound, 11,14,15-THETA. Another group of m/z 197,157, 139 and 207 () with identical detection times (9.64 min), suggesting that they were from 11,12,15-THETA (see also ). The m/z 205 also exhibited a small peak at a longer detection time (10.60 min). This ion is present in the spectra of both 11,14,15- and 13,14,15-THETA. However, the peak of the biological sample at 10.60 min did not have m/z 193 and the retention time did not match 13,14,15-THETA, suggesting the possibility of another THETA being present at very low concentration.
Figure 6 Total ion chromatograms and selected ion chromatograms of THETA fraction (top panels) isolated from rabbit aorta by negative ion LC-ESI-FTICR with SORI-CID and IRMPD. Total ion chromatograms by SORI-CID (A) and IRMPD (B); Selected ion chromatogram of (more ...)
To further confirm the identity of these THETAs, a mixture of 11,12,15-THETA and 11,14,15-THETA was analyzed by LC-FTICR using the same conditions as the biological sample. The retention times and MS/MS spectra of biological THETAs were identical to the THETA standards. Comparing to the abundance of THETA standards, the concentration of THETAs in biological samples was approximately 800 pg on the column. The results indicated that both 11,12,15- and 11,14,15-THETA were produced in the rabbit aorta with 11,14,15-THETA being the major isomer.