1800
Anal. Chem. 1988, 60, 1800-1807
Mass Spectrometry/Mass Spectrometry of Prostaglandins: Daughter Ion Spectra of Derivatized and Isotope-Labeled E and D Prostanoids R. J. Strife* and J. R. Simms Corporate Research Division, Miami Valley Laboratories, The Procter & Gamble Company, P.O. Box 398707, Cincinnati, Ohio 45239-8707
The daughter ion spectra of parent ions produced by electron ionization of four derivatized prostaglandins (PG's) and eight deuteriated analogues have been obtalned on a sector mass spectrometer of BE configuration (reversed-geometry magnetic sector/electrlc sector). Daughter ions were observed under unimoiecuiar and coilisionally actlvated dlssocatfon conditions (6-keV laboratory energy) in the second Heid-free region. The various parent Ions showed two classes of kagmentation: (1) derhrathre-speclfic and (2) backbone-speclflc. The deuterium iabels provided evidence for proposed fragmentation pathways of parent Ions. Isotope labeling also resolved certaln overiapplng daughter Ions obscured by klnetic energy release In the spectra of unlabeled compounds. Use of this information to develop a systematic rationale for choosing parent ion-daughter ion pairs for selected reaction monltoring of PG's in biologlcal fluids by gas chromatographylmass spectrometry/mass spectrometry Is dlscussed.
The trace analysis of arachidonic acid metabolites, namely prostaglandins (PGs),thromboxanes (TXs), and leukotrienes (LT's), continues to be analytically challenging. These assays are important for further understanding the pharmacology of this class of metabolites, as well as for developing new drug candidates to inhibit certain enzymatic reactions in the arachidonic acid cascade. Current methods of analysis rely on extensive sample purification by multiple solid-phase or liquid extractions, high-performance liquid chromatography (HPLC), and even immunoadsorption chromatography (1-5). Subsequent analysis by capillary gas chromatography/mass spectrometry (GC/MS) with selected-ion monitoring (SIM) often relies on the formation of a pentafluorobenzyl ester (PFB) derivative and on using electron-capture negative chemical ionization (EC-NCI) ( 5 , 6 ) . This enhances both the sensitivity and selectivity of detection. Our initial studies of PGEz quantitation in urine, by GC/MS/MS using electron ionization (7)) showed that a small-scale (1-5 mL samples) organic extraction and/or reverse-phase minicolumn (0.5 cm i.d. X 2.5 cm) reduces the residues from biological samples quite adequately for GC/MS analysis. Further reduction of chemical noise was obtained by using MS/MS detection, a well-established technique, in the selected reaction monitoring (SRM) mode (8, 9). The detection was done under unimolecular conditions on an instrument of BE configuration (reversed-geometry magnetic sector/electric sector). Daughter ions were generated in the second field-free region. This approach has also been used for steroids on E B instruments (first field-free region) and referred to as metastable-ion monitoring (10). We have now studied the daughter ion spectra of several ions produced by electron ionization (EI) of four derivatized
* Author to whom correspondence should be addressed.
prostaglandins (E and D type) and their deuteriated analogues in greater detail. The objectives of this study are (1)to defiie more thoroughly the MS/MS fragmentation of E and D prostanoids at high energies (6 keV in the laboratory reference frame) under unimolecular and collisionally activated dissociation (CAD) conditions, (2) to examine the choices available for parent ion to daughter ion reactions, for simplified SRMbased assays of PG's in biological fluids, (3) to begin to investigate how these choices affect the selectivity of SRM-based assays, and (4)ultimately to develop a rationale for choosing which reaction to monitor, given the myriad of possible reactions in any given system. EXPERIMENTAL SECTION Materials and Methods. Prostaglandins El, E2,and Dz were obtained from Upjohn Pharmaceutical (Kalamazoo, MI). The (referred to compound 11-deoxy-13,14-didehydro-15-keto-PGE, below as the PGEl analogue) was synthesized and fully characterized at Procter and Gamble's Miami Valley Laboratories (11). All solvents and derivatization reagents were of HPLC grade or distilled in glass and were obtained from standard suppliers. Prostaglandin standards (50-100 gg) were derivatized to the methyl ester, methoxime, bis(trimethylsily1)ethers by well-known procedures using fresh diazomethane, methoxylamine hydrochloride in dry pyridine, and N,O-bis(trimethylsily1)acetamide/acetonitrile (12,13). An exception was the PGEl analogue, which was only methylated. Deuteriated diazomethane was made by using a kit from Aldrich Chemical (Milwaukee, WI). N,OBis(trimethyl[2H9]silyl)acetamidewas obtained from MSD Isotopes (St. Louis, MO). These reagents were used to synthesize [2H3]methylesters (designated Me-d,) and [2Hg]TMSethers (designated TMS-d9). All products were characterized first by capillary GC/MS using a falling-needle injector, at 315 "C, connected to a 15 m x 0.25 mm i.d. DB-5 column operated isothermally at 270 "C. The column was interfaced directly (transfer line 290 "C) to a Vacuum Generators (VG, Altrincham,England) ZAB-2F mass spectrometer of BE configuration, equipped with a VG 11-2505data system (DS). Typical operating conditions were as follows: source temperature 200 "C, electron ionization at 70 eV, parent ion mass resolution 800 (10% valley),and 6-keV laboratory energy for both unimolecular and CAD experiments. In the case of CAD, the collision region pressure was adjusted to give 50-75% reduction of the parent ion beam. Accurate mass measurements were carried out at a mass resolution of 5000 (10% valley) with probe introduction. For each compound, the four most intense ions in the electron ionization mass spectrum above m / z 200 were chosen as parent ions. Daughter ion spectra were obtained on 500-ng probe samples under unimolecular and multiple-collision CAD conditions. The daughter ions were generated in the second field-free region of the spectrometer. The electric sector was scanned down to 40% of its initial voltage (40% of the parent ion mass). The daughter ion spectra (mass-analyzed ion kinetic energy spectra, referred to below as the MIKE spectra for simplicity) were acquired by using the multichannel analyzer capabilities of the DS at about 2 s per scan (0.1-s interscan delay). In a typical experiment, 30 scans were summed at a daughter ion mass resolution of 200 (10% valley). Probe samples were also used to tune the instrument for SRM.
0003-2700/88/0360-1 800$01.50/0 0 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60,NO. 17, SEPTEMBER 1, 1988 R> COOCH,
OTMS
R2
R,
Mol Wt
PGEI
-CH*CH2- = N - O C h
.--OTMS
541
PGE2
>=C;H
=N-OCH,
--OTMS
539
---0TMS
=N-OCH,
539
P ~ D z
;,--.c; H
1801
H
11 deoxy, 13.14-didehydro 15 keto PGE, (methyl ester)
348
Structures and numbering scheme for the derivatized prostaglandins (PG’s). C,-C, and C13-C20 are referred to as the C, and Cy side chains, respectively. Flgure 1.
100.1
73 I
Figure 4. Unimolecuhr (A), CAD (B), and isotope-shifted (TMS-d,) CAD (CFMIKE spectra of m l z 295 ( m l z 304 in the deuteriated derivative) of PGE2-MeMox(TMS),. Asterisks denote the shifted peaks. The peak
height percentage is relative to the parent ion intensity. v .......” o
c
H
3
366
OTMS
Electron ionization mass spectrum of PGE, as the methyl ester 0-methoxime bis(TMS) ether derivative (PGE,-MeMox(TMS),). Asterisks denote parent ions selected for MSlMS.
Flgure 2.
and
CHsO-N
FC
TMSO
A
J
II
100
I
150
’
200
m/z
Unimolecular (A) and CAD (B)-MIKE spectra of m l z 225 of PGE,-MeMox(TMS),. The peak height percentage is relative to the parent ion intensity. Figure 3.
RESULTS AND DISCUSSION The structures of the derivatized PG’s and the skeletal numbering scheme are shown in Figure 1. GC/MS analyses were consistent with the literature (13). The electron ionization mass spectrum for the PGEz methyl ester methoxime bis(TMS) ether (major isomer, designated as the MeMox(TMS)zderivative) obtained in this study is shown in Figure 2 as a point of reference. The structures of three of the parent ions ( m / z 225,295,366)and the corresponding MIKE spectra are shown in Figures 3,4,and 5,respectively. These MIKE spectra are also listed in Table I, together with data from the other PG‘s examined. The data set is restricted to daughter ions of at least 0.1% intensity, by height, of the parent ion beam to facilitate comparison. The MIKE spectra of isotope-labeled compounds are not listed. Rather, the mass shifts observed for parent and daughter ions are given in the second and last columns of the table, respectively. Accurate mass results for the PGE, analogue are also listed in the table. The proposed structures of the selected parent ions in MeMoxTMSz derivatives of PG’s are derived from extensive high-resolution data and isotope-labeling studies reported in the literature (13-15). This information was helpful in interpreting the MIKE spectra, since the relative abundance of a CAD fragment formed via a high-energy process is determined by the structure of the parent ion. In this case, the internal energy of the precursor ion before collision is the
I
,
2M)
250
’ 300
350
m/z
Unimolecular (A) and CAD (B)-MIKE spectra of m l z 366 of Pa,-MeMox(TMS),. The peak height percentage is relative to the parent ion intensity. Flgure 5.
parameter of least effect on the daughter ion spectrum. We arbitrarily decided to select the four most abundant ions above mlz 200 as parent ions. This limitation was an attempt to balance issues regarding instrumental response and selectivity during SRM, since this study was to provide the basis for subsequent analysis of biological samples. Also, the most significant daughter ions were observed in the energy range above 40% of the parent ion energy. MIKE Spectra of PGEz-MeMox(TMS)z.The parent ion of m/z 225 carries 6.7% of the total ion current (TIC, summed from m/z 50-550). Its structure, shown in Figure 3,is thought to be a highly conjugated, even-electron oxonium ion (13,16). It is not surprising that very few daughter ions are observed under unimolecular conditions (MIKE spectrum, Figure 3A) given the stability of such an ion. An alternate structure for mlz 225, proposed by Hartzell and Andersen, (15) is not consistent with Hamberg and Samuelsson’s spectrum (14) for 5,6,8@,11@,12a,14,15-[2H],-PGEz-MeMox(TMS)z, unless a very specific deuterium migration occurs from CI5. Collisionally activated dissociation (Figure 3B) produced remote-site fragmentations ( I 7) involving elimination of alkanes at various positions along the carbon chain from Cz0
1802
ANALYTICAL CHEMISTRY, VOL. 60,
NO. 17, SEPTEMBER
1, 1988
Table I. Summary of Daugher Ion Spectra for PG’s under CAD Conditions
Compound
Parent Ion ( X TIC), Mass Shift d,-methyl, Mass Shift dg-
PGE2-MeMoxTMS,
2 2 5 ( 6 . 7 ) , + O , +9
Daughter Ions
(0) 195 181 167 155 153 135 73
COOCH,
+OTMS
3 6 6 ( 1 . 0 ) , + 3 , +9
OTMS
COOCH 3
263 235 221 207 205 193 190 179 173
(.1) (.2) (.3) (.2) (.3)
Mass Loss ‘ 2
H6
4
3 Ha
‘ 4
Hl 0
-C,H,o -‘SHl
2
(-1)
-C,H,,OSi
(.7)
l‘-
(.6)
(.1) (.1) (.1) (1.1) (.3) (.3)
(.1) (.3)
338 335 324 295 276 265 263 251 237 225
(.2) (1.3) (.3) (1.8)
493 479 477 450 436 435 418 392 335 328
Mass Shifts !,-methyl, dg%
OH, 6’
-CH,OH -C2H,O2 -C3H602
-‘4
-C,
,
‘2
H, OSi
-C5H1002
-(C,H1,OSi+CH,) -‘6
H l 2 ‘2
-‘2
H4
-(C,Hl0OSi+CH,OH)
-0CH3 -C3H6
-C5H1 1
H, ,OSi
(1.1)
-C3
(.7) (.7) (1.8) (.5) (1.6)
-Cs H9 02 - ( C , H , 1 +CH, OH)
(.5) (-2) (-4)
-CH,
(.2)
‘ 4 Hl 0 -C5H12 3‘H5°2
-c6 H l 1’2
-C7H1 3’2 -‘BH1 3’2
+o, +9 +o, +9 +o, +9 +o, +9 +o, +9 +o, +o +o, +o +o, +9 +o, +9 +o, +9 +o, +9 + 3 , +9 +o, +9 +o, +o +o, +9 +o, +o +3, +3, +3, +3, +3,
+o, +o, +o, +o, +o,
+9 +9 +9 +9
+o +9 +9 +9 +9 +9
TMSO’
5 0 8 ( . 9 ) , + 3 , +18
COOCH)
btMS
PGE,-MeMoxTMS,
~TMS
(.3) (.1) (3.9) (.4) (.2) (.2)
-C2H5
-OCH3
-C,H, OSi - ( C H OSi +CN ) -(C6H120SiC3Hg1 - ( 2 ~C,H,,OSl)
, ,,
+3, +3, +O, +3, +3, +0, +3, +3,
+3,
+18 +18 +18 +18 +18 +18 +9 +9
+o
+ 3 , +9
225 ( 1 . 7 ) - Identical to PGE2-MeMoxTMS,
297 ( 6 . 1 ) , + 3 , +9
+o,
-OCH,
266 231 207 193 181 175 167
(.1) (.1) (.3) (1.0) (.3) (.3)
- ( C, H,
353 337 326 312 297 278 255 253 239 225
(.3) (.1) (1.4) (1.4) (3.4) (.7)
-CH, -CH,O
+9 +3, +9 + 3 , +o
ccooc +OTMS
368 ( 2 . 9 ) , + 3 , +9 COOCH,
OTMS
(.2)
(.7) (.7) (.7)
?
-C,H, ,OSi -(C,H9SiOCH,)
6‘-
,OSi+CH,OH)
H l 2 ‘2
-C7H1 4’2
-C3H6
-C4Ha -C5H1 1
-C,H,,OSi -C,H13 O*
+o, +o +o, +9 +o, +o +o, +9
+3, +3, +3, +3, +3, +3, +3,
+9 +9 +9 +9 +9
+o +9 +9 +9 +9
ccoo
CH,O-N
TMSO
(5.5)
-‘6
‘ 7
-‘EH,
H l 1’2 H l 3 ‘2 5’2
+o, +o, +o.
ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988
Table I (Continued)
Parent Ion (% TIC), Mass Shift d,-methyl, Mass Shift d, -TMS
Compound
4 2 0 ( . 3 ) , + 3 , +9
M+
-(cH,o.+
TMSOH)
Daughter Ions (%P) 404 390 389 362 360 332 330 320 318 304
(.3) (.3) (.7) (.4) (.4) (.3) (1.2) (1.2) (.6) (.4)
Mass Loss
Mass Shifts $,-methyl, d,*
-CH, -C2H6
-CH, 0 -c,Hl 0 -‘2 ‘ 4
H4 ‘2 H8 ‘2
-C,HloOSi -C, HI 2 O* -C5H1002 ‘ 6 Hl 2 ’;
+ 3 , +9 + 3 , +9 +o, +9 + 3 , +9 +o, +9 +o, +9 + 3 , +o + 3 , +9 +o, +9 +o, +9
*(TMS migration, loss of C15-C20) PGD2-MeMoxTMS,
378 ( 2 . 3 1 , + 3 , +9
~t
+ TMSOH)
-(C,H11*
4 1 8 ( . 7 ) , + 3 , +9
M+ -(cH,o.
PGD,-MeMoxTMS,
+ TMSOH)
468 ( 4 . 5 ) , + 3 , +18
TMSQ
% -COOCH,
+ OI \ T M S
H,CO-N
508 (1.4), + 3 , +18
M+
- CH,O.
347 332 288 274 262 257 245 231
(1.8) (.6) (.6)
402 390 386 374 345 330 328 316 302 277
(.2) (.2) (.7)
(.3) (.2) (2.6) (.3) (.4) (.6)
437 422 378 346 325 296
(1.2) (-3) (1.6) (.7) (.2) (2.7)
(.1) (.2) (.9) (.3) (.2)
(.1)
418 ( 6 . 6 ) 366 ( . 3 ) 328 ( - 3 )
-OCH, -(OCH,+CH,) -C,H, OSi -(OCH, +C,H,Oz ) - (C2H3 OTMS) -(C3H1,0Si+CH30) -(CH3OH+C5H9O2) -(CH,OH+C,H1102)
+ 3 , +9 + 3 , +6 + 3 , +o +9 + 3 , +o + 3 , +O + O , +9 +O, +9
-CH4
+ 3 , +9 + 3 , +9 +o, +9 + 3 , +9 + 3 , +9 +o, +9 + 3 , +o + 3 , +o +o, +9 +o, +9
-C2H4
-CH,OH -C,H, ?
-C,H,O2 -C,HloOSi -‘SH1
0’2 -C6H1 2’2
-C,H1302 -0CH3 -(OCH-,+CH,) -C,H,,OSi -(C,H,,OSi+CH,OH) ?
-(C8H,3O,+OCH3)
-C,H, ,OSi -(C8H,402) -(2x C,H,,OSi)
+o,
+3, +3, +3, +3, +0, +0,
+18 +15 +9 +9 +18 +18
+3,
+o +18 +o
+O,
+3,
(Other remote site fragments
-
0BS. 217.1219 CALC. 217.1228
a
=
3 PPM M?
20, OBS. 234.1619 CALC. 234.1620 A = . 5 PPM C15H2
C, 5H2 1 0 3
f)Bs. 749.1506 (‘AIL. 249.1491 A = 6 PPM
- (C, H , , C O . +
CH3OH)
234 (2.7)) +0, ”-’+
249 ( 1 . 3 ) , +3,
199 192 189 175 173 171 161 157 147 132
(3.5) (1.9) (1.0) (1.0) (.4) (.4) (.6) (-7) (.2)
219 216 205 191 178 163 135
(.1) (.2)
231 217 199 189 175
(.1) (5.0) (.6) (.6)
(.3)
Mass Shifts Mass Loss
d,-methyl, d,=
-H20 -C2 H
-co
-C2 H 2 0 -(C2H,+HZO) - (H, OtCO ) -C2 H4 CO - ( C, H20+H20) -C, H, 0 -C5H,O
to, to, +o,
(.3) (.6)
(4.8) (3.2) (.9)
(-5) 161 ( . 3 ) 147 ( . 3 )
- -
-
.
to C15. We have previously referred to this as backbonespecific fragmentation for prostaglandins (7). These ions are shifted by 9 u in the MeMox(TMS-d9j2derivative (parent ion m / z 234), consistent with the proposed fragmentation. Formation of the trimethylsilyl cation (and presumably the neutral trienone) gives the most intense daughter ion at m / z 73 and is a derivatiue-specific fragment. The observed CAD efficiency (18) was less than 1%.Any particular daughter ion was less than 0.8% by height of the parent ion beam. Peak heights relative to the unattenuated parent ion beam (e.g., before CAD) are a factor of 2-3 lower. The ion of m / z 295 is also thought to be an even-electron, conjugated oxonium ion. The unimolecular decomposition of m / z 295,carrying 4.3% of the TIC, is shown in Figure 4A. The daughter ions are 20 times more intense than in the unimolecular MIKE spectrum of m / z 225. The most intense daughter ions involve loss of methanol or TMSOH, both derivative-specific fragmentations, but with charged retained on the PG backbone, not the derivative group. These cleavages are confirmed by loss of methanol-d,,and TMSOH-d, in the deuteriated derivatives. The loss of TMSOH is particularly interesting given the proposed structure of the oxonium ion. We suspect that a protonated ion, shown in Scheme IA, is formed by successive hydrogen transfers. This ion can easily lose TMSOH, forming a resonance-stabilized carbonium ion. Synthesis of other labeled compounds would be necessary to test various mechanisms that could lead to this ion, but it is beyond the scope of our study. ‘I’he daughter ions at m / t 190 and 173 involve combination losses of methyl or methanol from the ester, and TMSOH, again confirmed by deuterium labeling. The fragmentation is therefure derivative-specific, but doubly so. Two different derivative groups froin separate sites a w lost.
____
I
-
-
-
-
-
-
+Q, -
-H20 -CH, OH -(cH, O H + H ~ O ) -C, H, 0 , -C, H6 0 , -C,H,O* -C5Hi 0 0 2
k-coocH3 a+
to, +o, to, to, to, to, +o, to, +o, +o,
___
+o, +o, +o,
-
+3,
-
+o, tu, to, to, +u, +o,
-
-
-
-
___
-
Scheme I. (A) Loss of TMSOH from m / z 295 in PGEz-MeMox(TMS)2,(B) Origin of m / z 296 from m / z 468 in PGDz-MeMox(TMS),, and (C) Loss of Water from m / z 217 in PGE, Analogue Methyl Ester
CAD (MIKE spectrum shown in Figure 4B)did not increase the absolute intensities of the original daughter ions of m / z 295 ( e g , unimolecular conditions) but did cause new daughter ions to appear. These new daughters involved successive cleavages of the general formula C,H2,0 along CI&, perhaps also remote-site in nature. As expected, these ions were not shifted in the deuteriated methyl esters’ spectra and were shifted by 9 u in the spectra of deuteriated TMS ethers. Another advantage of comparing the MIKE spectra of native and deuteriated derivatives was the resolution of peaks obscured by kinetic energy release in the spectra of the native
ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988
compounds. The kinetic energy release limits the obtainable resolution in the daughter ion spectrum. The daughter ions a t m / z 193 and 207 were resolved in the MIKE spectrum of the deuteriated TMS ether (parent ion m / z 304)) shifting to m/z 202 and 216, respectively (Figure 4C). The peaks formerly overlapping them in the MIKE spectrum, m / z 190 and 205, involve loss of labeled groups and are not shifted. Observation of reactions in the fist field-free region also improves daughter ion mass resolution, but a t the expense of parent ion mass resolution. This may have implications for SRM selectivity in biological samples. The unimolecular and CAD-MIKE spectra of m / z 366 (1% of the TIC) are shown in plots A and B of Figure 5, respectively. This parent ion has at least two contributing structures (13-15), one of which is an odd-electron radical species. The variety of ions in the unimolecular spectrum is much larger than that observed for the even-electron oxonium ions. The most abundant ion is formed via a backbone-specific fragmentation giving a loss of 71 daltons (Da) by cleavage of c16-c2@This produces the even-electron oxonium daughter ion at m / z 295. Derivative-specific losses of CH30 ( m / z 335) and TMSOH (mlz 276) and a backbone-specific cleavage between C8 and Cg,yielding m / z 225, give the next three most abundant daughter ions. Once again, the analysis of deuterium-labeled species supports these fragmentation hypotheses (see Table I). It also shows that the methanol loss occurs from the parent ion structure containing the methoxime. CAD enhanced the C5-Cs cleavage (hydrogen rearrangement is necessary) so that m / z 251 was of comparable intensity to loss of TMSOH. Other changes were minor. Finally, in this study the parent ion at m / z 508 (0.9% of the TIC) originates from the molecular ion by loss of OCH, from the methoxime (13). The unimolecular MIKE spectrum of m / z 508 shows one of the most intense daughter ions of any of the parent ions investigated. Loss of TMSOH gives m / z 418, about 4% of the height of the parent ion beam. CAD causes relatively weak daughter ions to appear from cleavages along the backbone. O t h e r Observations from CAD of PGE, PGD2, a n d PGEl Analogue. These three prostanoids show similar patterns in their CAD-MIKE spectra, including generally more intense losses of TMSO(H) and MeO(H), as well as the sequential cleavages along the C, (Cl-C7) and Cy (C13-C20)side chains. PGEl-MeMox(TMS)2 lackes the unsaturation at the 5,6 position on the carbon backbone, compared to that of PGE2. However, the ion at m / z 225 has a structure and daughter ion spectrum identical with the PGE2 derivative. A unique feature of the m / z 297 daughter ion spectrum (analogous to m / z 295 for PGE2) was that the most intense peak appeared a t m / z 193 (loss of 104 u, 2.7% of the parent intensity). Isotope labeling showed that the product ion was formed by loss of the TMS group a t C-15 and the ester methyl group a t C-1. This implies the total composition of the neutral or neutrals lost is C4HI20Si, with the origin of the oxygen atom in question. The possibility of migration of the CI5-TMSto the C1-carboxymethyl group oxygen, followed by a one-step loss of methyl trimethylsilyl ether, is suggested. Migration of 'I'MS groups is a well-known phenomenon, even over relatively long distances (16,19). The specific hypothesis given here could be investigated by selective oxygen-18 labeling of the carboxymethyl group (20). In the E1 mass spectrum of PGD,-MeMox('l'MS),, the structure of m / z 296 is reportedly unknown (15). This ion is the most abundant daughter ion (2.7%)of m / z 468. This latter ion is the most abundant ion in the E1 mass spectrum above m J z 200. Since the reaction of m / z 468 to m J z 296 is a likely choice for SRM of PGD2,it is important to understand
1805
the origin of the daughter ion. The parent ion, m / z 468, is formed by P-cleavage of C5H11.(C16-C20)from the molecular ion, giving an oxonium ion. In the MIKE spectrum of m / z 468, there is an intense loss of CH30 (greater than 1%))generally characteristic of ions carrying the methoxime functionality (at Cll for PGD2) in this study. Furthermore, the methoxy loss is not from the ester functionality as demonstrated by the shift of this daughter ion by 3 u in the spectrum of the methyl-d, compound. In contrast, the daughter ion a t m / z 296 is not shifted in the methyl-d3 ester's spectrum and is shifted by 18 u in the case of the TMS-dgcompound. This implies that both C15 and Cg are present. It is suggested that the m / z 296 daughter ion may be generated by loss of the methoxy radical and then the C, side chain (Scheme IB). The lack of an ion for only loss of the C, side chain suggests the process may be activated by loss of methoxy, and the second step, forming the more stable even-electron oxonium ion, is rapid under multiple-collision conditions (Scheme IB). The structure proposed for m/z 296 is further supported by its CAD-MIKE spectrum, showing loss of 26 u ( C N ) as the third most abundant daughter ion. The PGEl analogue requires only methylation to pass through the GC. This simplified derivatization is an advantage for the analysis of biological samples. The E1 spectrum of the methyl ester is characterized by much fragmentation. The base peak in the spectrum occurs at m / z 99, presumably the acylium ion C5H11CO+. Elemental compositions of the parent ions at m / z 206, 217,234, and 249 (carrying 1.8, 1.3,2.7, and 1.3% of the TIC, respectively) were deduced by accurate mass measurement for oxygen and unsaturation content, as well as by making the methyl-d3 ester. For example, the ion a t m / z 249 is shifted to m / z 252 in the methyl-d, ester and has an observed mass of 249.1506, C16H2103 (calculated 249.1491). The proposed parent ion structures are summarized in Table I. These parent ions fragment like other PG's along the carbon backbone, with some exceptions. The parent ion at m/z 249 showed a loss of methanol from the ester that was 5 times more intense than the other PG's studied. The parent ion a t m / z 234 showed the most intense backbone-specific fragmentation that we have observed, losing C,H8, perhaps from the C y chain (C16-c20) in a McLafferty rearrangement. When m/z 217 was chosen as the parent ion, significant losses of CO a n d CH2C0 were observed. Also, this ion lost water to yield an abundant ion (3.5%)at m / z 199. It is suggested that enolization, followed by rearrangement to a conjugated allylic alcohol, precedes loss of H 2 0 (Scheme IC). Appearance energy measurements on simpler ketones have shown that enol ions in the gas phase are thermodynamically more stable than their keto isomers by 58-130 kJ/mol (21). Furthermore, extensive hydrogen rearrangement occurs in PG's, as in the isomerization of TMS-oxonium ions. Loss of TMSOH often gives rise to the most intense daughter ion in the MIKE spectrum. Finally, an interesting loss of a C2H fragment from the 217 parent ion (1.9%)was observed. Very little kinetic energy was released coinpared to that of the other fragmentations. Considerations for Selected Reaction Monitoring. The choices for SRM are guided by a number of considerations including parent ion intensity (percentage of TIC carried by the parent ion), individual daughter ion intensity, parent ion m/z, daughter ion m l z , and class of fragmentation (vide infra). The first two choices listed are of fundamental importance to achieving the required instrumental response but affect selectivity as well. The latter three have a larger bearing on selectivity and reduction of chemical noise in biological samples. P a r e n t and D a u g h t e r Ion Intensities. Some general observations can be drawn from these results, with the caution
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that other PG's need to be studied further. Parent ion intensities parallel stability so that a relatively intense parent ion may have poor daughter ion intensities, as with m / z 225 in PGE,. CAD must be carried out under multiple-collision conditions to fragment this ion. Monitoring the reaction of m / z 225 to 73 (designated 225' 73') in the GC/MS/MS mode, we found reasonable chromatograms for nanogram quantities of standards (signal/noise was 1O:l) injected on column. In our judgment, this level of sensitivity was not adequate for the simplified assays we wish to design for biological samples. In contrast, the much less abundant parent ion at m / z 366, an odd-electron radical cation, shows many daughter ions, even under unimolecular conditions. However, the observed instrumental sensitivity is only slightly better, because of the low percentage of the total ion current carried by m / z 366. Therefore, on the BE instrument, selection of parent-daughter ion pairs to obtain the required response is somewhat of a paradox. An ion like m / z 295 in PGEz represents a compromise. Even under unimolecular (UM) conditions, its daughters were 20 times more intense than those of m / z 225. 173', a double derivaInitial GC/MS/MS trials (295' tive-specific fragmentation) showed that 50-100 pg quantities were detectable in standards with a s / n ratio of 1O:l. In other cases, the SRM choice is more obvious. For PGDz there is an intense ion a t m / z 468 in the E1 spectrum that clearly stands out from other fragment ions in the 200-550 u range. This same ion fragments with relative good efficiency in a backbone-specific fashion to m/z 296 (2.7% of the parent) and is a clear-cut choice for sensitivity and selectivity in SRM. Parent Ion and D a u g h t e r Ion m / z . It is a generally accepted principle in ion monitoring of biological samples that a favorable increase in signal/chemical noise may often be realized by monitoring less intense ions a t higher mlz. However, intense lower m / z ions should not be ruled out as potential parent ions, if they show good CAD efficiencies, since SRM is a more selective method of detection. A potential problem with several of the higher m / z parent ions in these derivatives is that their MIKE spectra tend to become dominated by loss of the derivative groups. Thus m / z 508, commonly chosen for selected ion monitoring of PGE2, fragments to lose mainly TMSOH. Derivative-specific fragmentations for SRM can show limited enhancement of selectivity relative to SIM ( 7 ) . Little is known about the influence of daughter ion mass on selectivity of MS/MS assays. An opportunity to investigate this occurs for m / z 366 of PGE2. A homologous series of alkyl-type fragments of increasing mass, along Cl-C6 of the upper side chain, are cleaved with similar intensities. However, the low percentage of the TIC carried by m / z 366 makes such a SRM study difficult on the BE instrument. Class of Fragmentation. Our initial studies on biological samples lead us to believe that the backbone-specific fragmentations confer superior selectivity over derivative-specific fragmentations in the analysis of biological samples (7). Stated another way, when SRM is based on a derivative-specific fragmentation, any component in a SIM chromatogram that is derivatized has an enhanced probability of appearing in a SRM chromatogram. I t is another paradox that in prostaglandin analysis, derivatization is required for GC introduction and that the most intense ions are often from derivativespecific fragmentations. As an example, Figure 6 shows the SIM and SRM chromatograms of the PGE, analogue in a single 2-mL ether extract of a 1-mL plasma sample. The drug was spiked at 5 ng/mL. The ether was evaporated, the residue was methylated, and the sample was analyzed by SIM (mlt 234, the most significant ion above m / z 200 in this derivative) and
B
1
A
11
Plasma SIM m/z234
Plasma SRM 234+- i7a+
I
-
-
Blank
Spiked 5.4 ng/ml
L
5
10
15
0 Time (Min )
l
-
i
'
1
,
5
Figure 6. SIM ( m / z 234) and SRM (234' 178') chromatograms of plasma extracts of the PEE, analogue methyl ester. The traces are from an analogue recorder and have not been digitally smoothed. The standard compound shows an identical chromatographic profile due to the presence of two isomers in the synthetic sample.
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SRM (234' 178+), a backbone-specific fragmentation). About 400 pg of the analogue was injected onto the column. Not surprisingly, selected-ion monitoring is a failure, and components in the extract are detected at retention times long beyond that of the PG. Selected-reaction monitoring shows a large increase in selectivity, with a total analysis time of 5 min. In a slightly different approach, a second step of derivatization may be added to form the methyl ester bis(methoxime) derivative. Its mass spectral properties are superior, showing a higher m / z (264 u) and more intense (9.7% of the TIC) parent ion, from a McLafferty rearrangement cleaving C1-C7. However, the daughter ion spectrum of m / z 264 is dominated by derivative-specific loss of CH,O to produce m/z 233. Monitoring the reaction 264' 233' led to little improvement in selectivity compared to monitoring the ion m / z 264 in the same plasma extracts. Also, our initial results on PGE2 in urine show reaction monitoring of loss of TMSOH 205') does improve selectivity compared to that of (295' SIM, but less so than other more selective fragmentations like 295' 173' ( 7 ) . It should be noted that Gaskell et al. have successfully used derivative-specific fragmentations in GC/MS/MS assays of TBDMS steroids for a number of years (IO,22). Derivatization of phenols also provided analytically useful derivative-specific fragmentations (23). The underivatized phenols showed little fragmentation to daughter ions so, in this case, derivatization provided a useful solution. The end result depends on the interferences in the matrix and the sample preparation scheme, so the derivative-specific approach should not be immediately ruled out. Subsequent to our first report ( 7 ) ,Schweer et al. reported on the GC/MS/MS of several PG's on a triple-quadrupole instrument, using E1 of the MeMoxTMS derivatives (24,25). Nonphysiologic levels of deuteriated PG's were spiked into various matrices, and the 1% levels of the do (unlabeled) PG present in these materials were studied. The well-recognized advantages of MS/MS, namely the increase in signal/chemical noise and greater selectivity of SRM versus SIM, were demonstrated for several PG's. Comparative determinations of the same PG under different reaction monitoring schemes were not investigated. Also, the sample preparations involved multiple steps and in one case provided reasonably good detection by SIM. Further simplification of the sample preparation schemes was not addressed. However, sensitivity was increased compared to that of our work because of higher apparent CAD efficiencies on the triple-quadrupole instrument. Vrbanac has taken an approach similar to Gaskell's steroids work using E1 of TBDMS derivatives (26). However,
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988
the sample preparations involved very specific immunoadsorption chromatography. In the plasma determination cited here, sample preparation was minimized. The masses of extracts (n = 8) found by careful weighing of extracts were 50 clg (s = 10 Mg). The physical state of the sample was therefore satisfactory for injection onto the capillary column. More recent SRM approaches have used EC-NCI to gain selectivity and intensity advantages in forming parent ion beams of RCOO- from pentafluorobenzyl esters (27, 28). Derivative-specific losses (TMSOH) are dominant in the daughter ion spectra, but loss of C 0 2 can be induced. However, use of the PFB derivative necessitates a chromatography step to remove reagents prior to sample analysis. It is interesting that in a report dealing with both EC-NCI and positive ion E1 methods Frohlich (27) showed the determination of PGD2 in urine using the lower-boiling MeMox296'). (TMS)2 derivative (468' We are continuing to use this derivative to further probe effects on selectivity of analysis, discussed above. However, the analyses are being carried out on the ion trap mass spectrometer (ITMS) (29, 30). We have observed CAD efficiencies of 50% for the parent ions of the PGE2 derivative discussed here. Excellent sensitivity is observed for less than 75-pg quantities (on column) in initial trials. We are proceeding to study selectivity effects in real biological samples with optimized sample preparation schemes.
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ACKNOWLEDGMENT We thank Peggy Meyer and Eileen Fletcher for help in the preparation of this manuscript. LITERATURE CITED (1) Balazy, Michael; Murphy, Robert C. Anal. Chem. 1088, 5 8 , 1098-1 101. (2) Westcott, Jay Y.; Stenmark, Kurt R.; Murphy, R. C. Prostaglandins 1986, 31, 227-37. (3) Fischer, C.; Meese, C. 0. Biomed. Mass Spectrom. 1085, 12, 399-404. (4) Zipser, Robert D.; Morrison, Aubrey; Laffi, Giacomo; Duke, Robert J. Chromatogr. 1085, 339, 1-9. (5) Hubbard, H. Lge; Eller, Thomas D.; Mais, Dale E.; Halushka, Perry V.; Baker, Renee H.; Blair, Ian A., Vrbanac, J. James; Knapp, Daniel R. Prostaglandins 1087, 33, 149-161. (6) Min, 8 . H.; Pao, J.; Garland, W. A,; diSilva, J. A. F.; Parsonnet, M. J. Chromatogr. 1080, 183, 411-419.
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(7) Strlfe, Robert J. Presented at the 33rd Annual Conference on Mass Spectrometry and Allied Topics; San Dlego, CA, May 28-31, 1985. (8) Johnson, Jodie V.; Yost, Richard A. Anal. Chem. 1985, 57, 758788A. (9) Kondrat, R. W.; Cooks, R. G. Anal. Chem. 1078, 50, 81-92A. (10) Gaskell, Simon J.; Mlllington. David S. Biomed. Mass Spectrom. 1078, 5 , 557-558. (11) Oliver, J. D.; Strickland, L. C. Acta Crystallogr., Sect. C : Cryst. Struct. Commun. 1085, C41, 1477-1480. (12) Hubbard, Walter C. I n Methods in €nzymology; Lands, William E. M., Smith, William L., Eds.; Academic: New York, 1982; Vol. 86,section 5. (13) Green, Krister. Chem. Phys. Lipids; 1060, 3 , 254-272. (14) Hamberg, Mats; Samuelsson, Bengt J. Biol. Chem. 1071, 246, 1073- 1077. (15) Hartzell, C. J.; Andersen, N. H. Biomed. Mass Spectrom. 1085, 12. 303-309. (16) Middleditch, Brian S.; Desiderio, Dominic M. J. Org. Chem. 1073, 38, 2204-2209. (17) Tomer, Kenneth B.; Crow, Frank W.; Gross, Michael L. J. Am. Chem. SOC. 1983, 15, 5487-5488. (18)\Todd, Peter J.; McLafferty, F. W. I n Tandem Mass Spectrometry; McLafferty, F. W., Ed.; Wiley: New York, 1983; Chapter 7. (19) Gaskell, Simon J.; Smith, Andrew G.; Brooks, Charles J. W. Biomed. Mass Spectrom. 1975, 2 , 148-155. (20) Murphy, R. C.; Clay, K. L. I n Methods in Enzymology; Lands, William E. M., Smith, William L., Eds.; Academic: New York, 1982; Vol. 86, Section 5, pp 547-551. (21) Holmes, J. L.; Lossing, F. P. J. Am. Chem. SOC. 1080, 102, 1591-1595. (22) Gaskell, S. J.; Porter, C. J.; Green, B. N. BlOmed. Mass Spectrom. 1085, 12, 139-141. (23) Hunt, D. F.; Shabanowitz, J.; Harvey, M. T.; Coates, M. Anal. Chem. 1085, 57, 527-537. (24) Schweer, Horst; Seyberth, Hannsjorg W.; Schubert, Rolf. Biomed Mass Spectrom. 1086, 73,611-819. (25) Schweer, H.; Soeding, K.; Kammer, J.; Seyberth, H. W. I n Advances in Prostaglandin, Thromboxane, and Leukotriene Research ; Samuelsson, B., Paoletti, R., Ramwell, P. W., Eds.; Raven: New York, 1987; Vol. 17, pp 622-626. (26) Vrbanac, J. J.; Knapp, D. R. Presented at the 35th Annual Conference on Mass Spectrometry and Allied Topics; Denver, CO, May 24-29, 1987. (27) Frohlich, J. C.; Sawada, M.; Bochmann, G.; Oelz, 0. I n Advances in Prostaglandin, Thromboxane, and Leukotriene Research; Zor, U., Naor, Zvi, Kohen, Fortune, Eds.; Raven: New York, 1987; Vol. 16, pp 363-372. (28) Schweer, H.; Meese, C. 0.; Furst, 0.; Kuhl, P. Gonna; Seyberth, H. W. Anal. Biochem. 1087, 164, 156-163. (29) Louris, John N.; Cooks, R. Graham; Syka, John E. P.; Keliey, P. E.; Stafford, George C., Jr.; Todd, John F. J. Anal. Chem. 1987, 59, 1677-1685. (30) Strife, Robert J.; Kelley, P. E.; Weber-Grabau, M. Presented at the 35th Annual Conference on Mass Spectrometry and Allied Topics, Denver, CO, May 24-29, 1987.
RECEIVED for review December 14,1987. Accepted April 16, 1988.
