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Mass spectrometric quantitation and analysis of leukotrienes and other 5-lipoxygenase metabolites
PROSTAGLANDINS
MASS SPECTROHBTRIC QUANTITATION AND ANALYSIS OF LEUROTRIENRS
AND OTHER 5-LIPOXYGENASE MNTABOLITES
Robert C. Murphy Department of Pharmacology University of Colorado Health Sciences Center Denver, CO 80262 USA Abstract Detailed studies of the 5-lipoxygenase pathway of arachidonic acid metabolism in vivo is a difficult challenge, but nonetheless, an important pursuit. The leukotrienes are perplexing compounds to quantitate due, in part, to their production in very small quantities by only certain cells, as well as to their chemical/biochemical instability. Several mass spectrometric techniques have been developed to quantitate 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4). The mass spectral properties of terbutyldimethylsilyl derivatives of LTB4 are reported here which are quite favorable for electron impact ionization. Catalytic reduction of LTB4 prior to derivatization greatly improved capillary gas chromatographic behavior as well as electron impact mass spectral properties. Subpicomole quantities could be readily detected by selected ion recording of the M-57 ion, which is the most abundant ion in the mass spectrum. Lipoxygenase products labeled with oxygen-18 at the carboxyl moiety are uniquely stable to catalytic reduction and, thus, may serve as useful internal standards. Introduction Considerable effort in the past few years has been placed in the development of analytical methodology for quantitation of metabolites of arachidonic acid in physiological fluids. Aside from bioassay, methods based on physical chemical properties have emerged using high pressure liquid chromatography with ultraviolet absorption (1) or radioimmunoassay detection (2-4) as well as mass spectrometry (5-8). This latter instrumental method enjoys distinct distinct advantages over other techniques when used with the ancillary procedures of combined gas chromatography, selected ion recording and stable isotope dilution. Advantages include unique molecular specificity, picomolar sensitivity and excellent precision and accuracy. We have recently described the use of negative ion chemical ionization (NICI) and pentafluorobenzyl ester, trimethylsilyl ether derivative of 5-hydroxyeicosatetraenoic acid (7, 8). Since
NOVEMBER
1984VOL. 28N0.5
597
PROSTAGLANDINS the majority of the ion current is carried by the carboxylate anion, subpicomole amounts of 5-HETE can be detected (9) and quantitated (10). However, the gas chromatographic properties of such polyunsaturatedmolecules as 5-HETE and LTB4 are not ideal with substantial thermal instability noted by many investigators. Furthermore, NICI sensitivity is affected by poorly understood variables. Therefore, we have investigated alternative derivatives for the analysis of LTB4 by mass spectrometetric techniques. Methods Positive ion electron impact (7OeV) ionization was carried out on a VG 7070H mass spectrometer using 200 uA emission. A capillary gas chromatographic column (10 meters, DB-1, J & W Scientific Co., Ranch0 Cordova, CA, USA) was threaded directly into the ion source. A splitless on-column injector (J & W Scientific)was employed with an isothermal column oven temperature of 255O and transfer lines of 260'. The carrier gas was helium at a flow rate approximately l.ZmL/min. Catalytic reduction of LTB4 was carried out using 5% Rh on alumina (100 ug per sample) suspended in methanol and bubbled with hydrogen for 15 min at 0'. This catalyst was used to reduce hydrogenolysis as a significant side reaction. The t-butyldimethylsilyl derivative was made by treating the free carbolic acid with 10% MTBSTFA (Regis Chemical Co., Morton Grove, IL, USA) and 2% Corey's reagent (t-butyldimethylsilylchloride (1 M) plus imidazole (2.5 M) in DMF) in acetonitrile for 1 hr at 60'. After derivatization, solvent and excess reagent was evaporated under vacuum and the derivatized dissolved in hexane. Results and Discussion The EI mass spectra of the trF(t-butyldimethylsilyl) derivative (TBDMS) and the methyl ester, di(t-butyldimethylsilyl) ether derivative of LTB4 are shown in Figure 1. The mass shifts for the dioxygen-18 labeled carboxyl isotopimers are indicated above the major nominal masses. The mass spectral fragmentations are rather straightforward as indicated in the structural insets. Even though the tri-TBDMS derivative is 100 daltons heavier, the fragmentation of this derivative is more favorable for quantitative analysis due to the abundance of the ion at m/z 435 (which is derived from ion & following loss of t-butyldimethylsilanol). An even more favorable TBDMS derivative investigated was the saturated LTB seen in Figure 2. The base peak corresponded to loss of t-buty'iradical (M-57) at m/z 629. The ion at m/z 497 corresponds to the loss of t-butyldimethylsilanolfrom this abundant ion. Even electron ions at m/z 257, 345, 485 and 573 correspond to cleavage of those bonds adjacent to the ether oxygen atoms with charge retention in the ether fragment.
598
NOVEMBER
1984 VOL. 28 NO. 5
PROSTAGLANDINS
Figure 1. Electron impact mass spectra (70eV) of (A) tri (TBDMS) derivative of LTB4 and (B) methyl ester, di (TBDMS) derivative of LTB4. When carboxyl oxygen atoms ( l ) are labeled with oxygen-18, observed masses are indicated by small numbers above the major ions. b
I
.57
Figure 2. Electron impact mass spectrum (70 eV> of the tri (TBDMS) derivative of 5,12 dihydroxyeicosanoic acid obtained catalytic hydrogenation of LTB4.
NOVEMBER
1984VOL.28NO.S
by 599
PROSTAGLANDINS
The relative abundance of the ion at m/z 629 compares favorably to the relative abundance of the carboxylate anion from the pentafluorobenzyl ester, trimethylsilyl ether derivative of LTB,, using NICI conditions. Furthermore, the gas chromatographic behavior of the saturated derivative shown in Figure 2 is substantially improved. There was no indication that thermal degradation seen for LTB,+ or 5-HETE (11) during gas chromatography was taking place. While catalytic reduction of eicosanoids prior to derivatization for GC-MS analysis has not been widely employed, this procedure is quite rapid. The hydrogenolysis of vinylic hydroxyl moieties can be a serious side rection, but careful choice of the metal catalyst can minimize this reaction (12). One unique feature of the oxygen-18 isotopic variants, whose use we have advocated for internal standards, is that they are stable to catalytic hydrogenation as seen in Figure 3. This is not true for deuterium-labeled eicosanoids which would readily exchange the deuterium atoms with protium atoms on the catalyst surface and, thus, be lost in this process. Overall, the saturated LTB4 as the tri-TBDMS derivative displays considerable promise as a tool for the quantitative analysis of LTB& in biological fluids.
l-
5
-67
Figure 3. Electron impact mas?'ipectrum ot dioxygen-18 labelled LTB& following catalytic reduction and TBDMS derivatization. Acknowledgements The author wishes to acknowledge the technical contribution of Forrest S. Anderson to this work. LTB4 was kindly supplied by Dr. J. Rokach, Merck-Frosst, Canada. This work was supported, in part, by grant HL 25785 from the National Institutes of Health.
600
NOVEMBER
1984 VOL. 28 NO. 5
PROSTAGLANDINS
References 1.
Mathews, W.R.,J.Rokach and R.C. Murphy. Analysis of leukotrienes by high pressure liquid chromatography. Anal. Biochem. 118: 96. 1981. 2. Lewis, R.A., C.W. Lee, L. Levine, R.A. Morgan, Z.J.W. Weiss, J.W. Drazen, H. Oh, D. Hoover, E.J. Corey and K.F. Austen. Biology of the c-6-sulfidopeptide leukotrienes. In Adv. Pros.Thrombox.Leukotr. Res., Vol. 11 (eds. B. Samuelsson, R. Paoletti and P. Ramwell) pp. 15-26, 1981. 3. Hayes, E.C.,P. Lombardo, Y. Girard, A.L. Maycock J. Rokach, A.S. Rosenthal, R. Young, R.W. Egan and H.J. Zweerink. Measuring leukotrienes of slow reacting substance of anaphylaxis: Development of a specific radioimmunoassay. J. Immunol. 131: 429. 1983. Salmon, J.A., P.M. Simmons and R.M.J. Palmer. A 4. radioimmunoassay for leukotriene B4. Prostaglandins-24: 225-234. 1982. 5. Blair, I.A., S.E. Barrow, K.A. Wadel, P.J. Lewis and C.T. Dollery. Prostacyclin is not a circulating hormone in man. Prostaglandins 23: 579. 1983. 6. Hubbard, W.C., Mx. Phillips and D.F. Taber. Selective synthesis of octadeuterated 5-HETE for use in GC-MS quantitation of 5-HETE. Prostaglandins 23: 61-66. 1982. Strife, R.J. and R.C.Murphy. Preparation of 7. pentafluorobenzyl esters of arachidonic acid lipoxygenase metabolites: Analysis by gas chromatography and negative ion chemical ionization mass spectrometry. J. Chromatog. 305: 3-12. 1984. Strife, R.J.and R.C. Murphy. Stable isotope labeled 58. lipoxygenase metabolites of arachidonic acid: Analysis by negative ion chemical ionization mass spectrometry. Prostagl. Leukotr. Med. 13: l-8. 1984. 9. Burghuber, O.C., R. Strife, J. Zirrolli, M.M. Mathias, J.T. Reeves, R.C. Murphy and N.F. Voelkel. Leukotriene inhibitors attenuate H202-induced rat lung injury. Submitted. 10. Strife, R.J., N.F.Voelkel and R.C. Murphy. 5-HETE in biological fluids by NICI. Biomed. Mass Spectrom. In press. 11. Boeynaems, J.M., A.R. Brash, J.A. Oates and W.C. Hubbard. Preparation and assay of monohydroxy-eicosatetraenoicacids. Anal. Biochem. 104: 259-267. 1980. 12. House, H.O. In: Modern Synthetic Reactions, 2nd Edition. (Benjamin Inc., Philippines), p. 13, 1972.
NOVEMBER
1984 VOL. 28 NO. 5
601
MASS SPECTROHBTRIC QUANTITATION AND ANALYSIS OF LEUROTRIENRS
AND OTHER 5-LIPOXYGENASE MNTABOLITES
Robert C. Murphy Department of Pharmacology University of Colorado Health Sciences Center Denver, CO 80262 USA Abstract Detailed studies of the 5-lipoxygenase pathway of arachidonic acid metabolism in vivo is a difficult challenge, but nonetheless, an important pursuit. The leukotrienes are perplexing compounds to quantitate due, in part, to their production in very small quantities by only certain cells, as well as to their chemical/biochemical instability. Several mass spectrometric techniques have been developed to quantitate 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4). The mass spectral properties of terbutyldimethylsilyl derivatives of LTB4 are reported here which are quite favorable for electron impact ionization. Catalytic reduction of LTB4 prior to derivatization greatly improved capillary gas chromatographic behavior as well as electron impact mass spectral properties. Subpicomole quantities could be readily detected by selected ion recording of the M-57 ion, which is the most abundant ion in the mass spectrum. Lipoxygenase products labeled with oxygen-18 at the carboxyl moiety are uniquely stable to catalytic reduction and, thus, may serve as useful internal standards. Introduction Considerable effort in the past few years has been placed in the development of analytical methodology for quantitation of metabolites of arachidonic acid in physiological fluids. Aside from bioassay, methods based on physical chemical properties have emerged using high pressure liquid chromatography with ultraviolet absorption (1) or radioimmunoassay detection (2-4) as well as mass spectrometry (5-8). This latter instrumental method enjoys distinct distinct advantages over other techniques when used with the ancillary procedures of combined gas chromatography, selected ion recording and stable isotope dilution. Advantages include unique molecular specificity, picomolar sensitivity and excellent precision and accuracy. We have recently described the use of negative ion chemical ionization (NICI) and pentafluorobenzyl ester, trimethylsilyl ether derivative of 5-hydroxyeicosatetraenoic acid (7, 8). Since
NOVEMBER
1984VOL. 28N0.5
597
PROSTAGLANDINS the majority of the ion current is carried by the carboxylate anion, subpicomole amounts of 5-HETE can be detected (9) and quantitated (10). However, the gas chromatographic properties of such polyunsaturatedmolecules as 5-HETE and LTB4 are not ideal with substantial thermal instability noted by many investigators. Furthermore, NICI sensitivity is affected by poorly understood variables. Therefore, we have investigated alternative derivatives for the analysis of LTB4 by mass spectrometetric techniques. Methods Positive ion electron impact (7OeV) ionization was carried out on a VG 7070H mass spectrometer using 200 uA emission. A capillary gas chromatographic column (10 meters, DB-1, J & W Scientific Co., Ranch0 Cordova, CA, USA) was threaded directly into the ion source. A splitless on-column injector (J & W Scientific)was employed with an isothermal column oven temperature of 255O and transfer lines of 260'. The carrier gas was helium at a flow rate approximately l.ZmL/min. Catalytic reduction of LTB4 was carried out using 5% Rh on alumina (100 ug per sample) suspended in methanol and bubbled with hydrogen for 15 min at 0'. This catalyst was used to reduce hydrogenolysis as a significant side reaction. The t-butyldimethylsilyl derivative was made by treating the free carbolic acid with 10% MTBSTFA (Regis Chemical Co., Morton Grove, IL, USA) and 2% Corey's reagent (t-butyldimethylsilylchloride (1 M) plus imidazole (2.5 M) in DMF) in acetonitrile for 1 hr at 60'. After derivatization, solvent and excess reagent was evaporated under vacuum and the derivatized dissolved in hexane. Results and Discussion The EI mass spectra of the trF(t-butyldimethylsilyl) derivative (TBDMS) and the methyl ester, di(t-butyldimethylsilyl) ether derivative of LTB4 are shown in Figure 1. The mass shifts for the dioxygen-18 labeled carboxyl isotopimers are indicated above the major nominal masses. The mass spectral fragmentations are rather straightforward as indicated in the structural insets. Even though the tri-TBDMS derivative is 100 daltons heavier, the fragmentation of this derivative is more favorable for quantitative analysis due to the abundance of the ion at m/z 435 (which is derived from ion & following loss of t-butyldimethylsilanol). An even more favorable TBDMS derivative investigated was the saturated LTB seen in Figure 2. The base peak corresponded to loss of t-buty'iradical (M-57) at m/z 629. The ion at m/z 497 corresponds to the loss of t-butyldimethylsilanolfrom this abundant ion. Even electron ions at m/z 257, 345, 485 and 573 correspond to cleavage of those bonds adjacent to the ether oxygen atoms with charge retention in the ether fragment.
598
NOVEMBER
1984 VOL. 28 NO. 5
PROSTAGLANDINS
Figure 1. Electron impact mass spectra (70eV) of (A) tri (TBDMS) derivative of LTB4 and (B) methyl ester, di (TBDMS) derivative of LTB4. When carboxyl oxygen atoms ( l ) are labeled with oxygen-18, observed masses are indicated by small numbers above the major ions. b
I
.57
Figure 2. Electron impact mass spectrum (70 eV> of the tri (TBDMS) derivative of 5,12 dihydroxyeicosanoic acid obtained catalytic hydrogenation of LTB4.
NOVEMBER
1984VOL.28NO.S
by 599
PROSTAGLANDINS
The relative abundance of the ion at m/z 629 compares favorably to the relative abundance of the carboxylate anion from the pentafluorobenzyl ester, trimethylsilyl ether derivative of LTB,, using NICI conditions. Furthermore, the gas chromatographic behavior of the saturated derivative shown in Figure 2 is substantially improved. There was no indication that thermal degradation seen for LTB,+ or 5-HETE (11) during gas chromatography was taking place. While catalytic reduction of eicosanoids prior to derivatization for GC-MS analysis has not been widely employed, this procedure is quite rapid. The hydrogenolysis of vinylic hydroxyl moieties can be a serious side rection, but careful choice of the metal catalyst can minimize this reaction (12). One unique feature of the oxygen-18 isotopic variants, whose use we have advocated for internal standards, is that they are stable to catalytic hydrogenation as seen in Figure 3. This is not true for deuterium-labeled eicosanoids which would readily exchange the deuterium atoms with protium atoms on the catalyst surface and, thus, be lost in this process. Overall, the saturated LTB4 as the tri-TBDMS derivative displays considerable promise as a tool for the quantitative analysis of LTB& in biological fluids.
l-
5
-67
Figure 3. Electron impact mas?'ipectrum ot dioxygen-18 labelled LTB& following catalytic reduction and TBDMS derivatization. Acknowledgements The author wishes to acknowledge the technical contribution of Forrest S. Anderson to this work. LTB4 was kindly supplied by Dr. J. Rokach, Merck-Frosst, Canada. This work was supported, in part, by grant HL 25785 from the National Institutes of Health.
600
NOVEMBER
1984 VOL. 28 NO. 5
PROSTAGLANDINS
References 1.
Mathews, W.R.,J.Rokach and R.C. Murphy. Analysis of leukotrienes by high pressure liquid chromatography. Anal. Biochem. 118: 96. 1981. 2. Lewis, R.A., C.W. Lee, L. Levine, R.A. Morgan, Z.J.W. Weiss, J.W. Drazen, H. Oh, D. Hoover, E.J. Corey and K.F. Austen. Biology of the c-6-sulfidopeptide leukotrienes. In Adv. Pros.Thrombox.Leukotr. Res., Vol. 11 (eds. B. Samuelsson, R. Paoletti and P. Ramwell) pp. 15-26, 1981. 3. Hayes, E.C.,P. Lombardo, Y. Girard, A.L. Maycock J. Rokach, A.S. Rosenthal, R. Young, R.W. Egan and H.J. Zweerink. Measuring leukotrienes of slow reacting substance of anaphylaxis: Development of a specific radioimmunoassay. J. Immunol. 131: 429. 1983. Salmon, J.A., P.M. Simmons and R.M.J. Palmer. A 4. radioimmunoassay for leukotriene B4. Prostaglandins-24: 225-234. 1982. 5. Blair, I.A., S.E. Barrow, K.A. Wadel, P.J. Lewis and C.T. Dollery. Prostacyclin is not a circulating hormone in man. Prostaglandins 23: 579. 1983. 6. Hubbard, W.C., Mx. Phillips and D.F. Taber. Selective synthesis of octadeuterated 5-HETE for use in GC-MS quantitation of 5-HETE. Prostaglandins 23: 61-66. 1982. Strife, R.J. and R.C.Murphy. Preparation of 7. pentafluorobenzyl esters of arachidonic acid lipoxygenase metabolites: Analysis by gas chromatography and negative ion chemical ionization mass spectrometry. J. Chromatog. 305: 3-12. 1984. Strife, R.J.and R.C. Murphy. Stable isotope labeled 58. lipoxygenase metabolites of arachidonic acid: Analysis by negative ion chemical ionization mass spectrometry. Prostagl. Leukotr. Med. 13: l-8. 1984. 9. Burghuber, O.C., R. Strife, J. Zirrolli, M.M. Mathias, J.T. Reeves, R.C. Murphy and N.F. Voelkel. Leukotriene inhibitors attenuate H202-induced rat lung injury. Submitted. 10. Strife, R.J., N.F.Voelkel and R.C. Murphy. 5-HETE in biological fluids by NICI. Biomed. Mass Spectrom. In press. 11. Boeynaems, J.M., A.R. Brash, J.A. Oates and W.C. Hubbard. Preparation and assay of monohydroxy-eicosatetraenoicacids. Anal. Biochem. 104: 259-267. 1980. 12. House, H.O. In: Modern Synthetic Reactions, 2nd Edition. (Benjamin Inc., Philippines), p. 13, 1972.
NOVEMBER
1984 VOL. 28 NO. 5
601