134
ASSAYS
[16]
Despite these caveats, the platelet serotonin assay for PAF is quick, simple, inexpensive, and has proved very useful in numerous studies of PAF production and its modulation. Acknowledgments This work was carried out in the F. L. Bryant Jr. Research Laboratory and was supported by National Institutes of Health Grant HL 34303.
[16] Q u a n t i t a t i o n o f P l a t e l e t - A c t i v a t i n g F a c t o r b y Gas Chromatography-Mass
Spectrometry
B y K E I T H L . CLAY
Introduction Quantitative analysis of platelet-activating factor (PAF, 1-O-alkyl-2-Oacetylglycero-3-phosphocholine) is complicated by the chemical nature of the molecule and by the necessity to measure it at very low concentrations (picomolar or less) in extremely complex biological mixtures. Measurement of PAF has frequently been accomplished by the use of its biological activities, through which this molecule was initially described. 1 Measurement ofplatelet aggregation 2 or of release ofplatelet serotonin content s has been used to estimate the amount of PAF present in biological mixtures. These procedures can give very valuable information and one should not underestimate the importance of biological assays in working with a substance that was initially described as a biological activity. Since the elucidation of the exact chemical nature of PAF, however, it has become possible to develop specific physical techniques for its measurement. 4 Gas chromatography-mass spectrometry has been used as the primary method of measuring PAF. Procedures have been described for the measurement of this molecule in both the positive-ion electron-impact ionization mode 5 and in the negative-ion chemical ionization mode. 6 These procedures have the advantage over bioassay procedures of better I j. Benveniste, P. M. Henson, and C. G. Cochrane, J. Exp. Med. 136, 1356 (1972). 2 G. Camussi, M. Aglietta, F. Malavasi, C. Tetta, W. Piacibeilo, F. Sanavio, and F. Bussolino, J. Immunol. 131, 2397 (1983). 3 j. M. Lynch, G. Z. Lotner, S. J. Betz, and P. M. Henson, J. Immunol. 123, 1219 (1979). 4 R. C. Murphy and K. L. Clay, Am. Rev. Respir. Dis. 136, 207 (1987). 5 K. Satouchi, M. Oda, K. Yasunaga, and L. Saito, J. Biochem. 94, 2067 (1983). 6 C. S. Ramesha, and W. C. Pickett, Biomed. Environ. Mass Spectrom. 13, 107 (1986).
METHODSIN ENZYMOLOGY,VOL. 187
Copyright© 1990by AcademicPress,Inc. All rightsof reproductionin any formreserved.
[16]
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precision and accuracy and are less subject to false positive values due to cross-reactivity in the biological activities. GC-MS procedures, however, require much more sample preparation and are, therefore, much more labor intensive than the bioassays. A major advantage of GC-MS (or any mass spectrometry-based procedure) is that stable isotopically labeled variants of the analyte of interest can be added to the biological matrix at the earliest time of collection of the sample. These chemically identical, but physically distinguishable, molecules serve as almost ideal internal standards for the quantitation of the endogenous material. It should be noted that a major advantage of the use of stable isotopically labeled variants is that these molecules can serve as positive indicators of the adequacy of each individual analysis: if the internal standard is recovered and gives an adequate signal in the mass spectrometer, the analysis has been successful, even if none of the endogenous material has been detected. With other procedures, it is possible to obtain a false negative result due to inadequate recovery, but the use of the stable isotope-labeled internal standards gives a useful control on each individual sample and corrects for the variability of recoveries to be expected with biological matrixes of differing complexity.
Assay Procedure
Reagents Acetyl-d3 chloride, (Acetic anhydride)-d6, Acetic acid-d4, Hydrofluoric acid, 48%, Acetic acid (Aldrich, Milwaukee, WI) Bis(trimethylsilyl)trifluoroacetamide (BSTFA), Silicic acid solid-phase extractor cartridges (Supelco, Bellefonte, PA) Bis(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) (Pierce, Rockford, IL) Silica gel G thin-layer chromatography plates (Analtech, Newark, DE) Octadecylsilyl solid-phase extractor cartridges (Analytichem, Harbor City, CA) L-Lysophosphatidylcholine-palmitoyl, stearoyl, and oleoyl (acyllysoGPC). Phospholipase C(Type XIII, from Bacillas cereus), l-OHexadecyl-2-O-acetylglycero-3-phosphocholine (hexadecyl-PAF) (Sigma, St. Louis, MO) 1-O-Octadecyl-2-O-acetylglycero-3-phosphocholine (Octadecyl-PAF); Hexadecyllysoglycero-3-phosphocholine and octadecyllysoglycero3-phosphocholine (lyso-PAF) Bachem, Bubendorf, Switzerland) Chloroform, dichloromethane, methanol, diethyl ether, hexane, perchloric acid, 70% (Fisher, Fair Lawn, N J)
136
ASSAYS
[16]
Internal Standards
Stable isotopically labeled variants of PAF and similar molecules are not commercially available, but preparation of excellent internal standards is quite simple and inexpensive. In essence, acetylation of the appropriate lysophospholipid with deuterated acetylating reagents will give the acetylated phosphatidylcholine with deuterium atoms in the acetyl moiety of the sn-2 position. The preparation of approximately I0 mg of internal standard by two different methods will be described. Method 1
To 200/xl of perdeuteroacetic acid (acetic acid-d4), are added 10 mg of lyso-PAF (or acyllyso-GPC) followed by 50/zl of acetyl-d3 chloride. The solution is mixed by gentle agitation and left at room temperature for 30 min. A small amount of hydrogen chloride gas will be produced in the reaction, so it should be kept in a fume hood. At the end of 30 min, 2 ml of methanol are cautiously added, followed by 4 ml dichloromethane. Two phases are then produced by the addition of 2 ml water. The mixture should be gently shaken for several minutes, centrifuged to separate the phases, and the water (top) layer removed. The water layer should then be shaken with a fresh 2 ml portion of dichloromethane and the dichloromethane (bottom) layer combined with the initial extract. The dichloromethane phase will contain essentially all of the deuterated phosphatidylcholine and should be taken to dryness for further purification. Method 2
To 200 /xl of dichloromethane are added 10 mg of lyso-PAF (or acyllyso-GPC) and the solution sonicated to form a dispersion of the phospholipid. To this suspension is added 50/~1 of (acetic anhydride)-d6 and the suspension mixed. Perchloric acid (70%, 5/xl), is then added and the suspension mixed rapidly for 15 sec. Immediately on addition of the perchloric acid, the suspension should become clear as the reaction proceeds and the phospholipid goes into solution. After mixing for 15 sec, an additional 4 ml of dichloromethane is added, followed immediately by the addition of 2 ml of methanol and 2 ml water. The phases are mixed, separated, washed, and taken to dryness as described in method 1. It is critical to the success of this method that the material not be allowed to stand longer than a few seconds in an attempt to force completion. The reaction is essentially complete as soon as the reagents are mixed; any further delay in isolation of the products will result in oxidation and production of dark-colored products, and decreased yields. Both of these procedures result in rapid and essentially complete reaction of the lysophospholipid with the acetylating reagent. For further
[16]
GC-MS or PAF
137
purification, it is necessary only to separate the unreacted lysophospholipid from the acetylated product, although even that minor purification will not be necessary for some applications. Application of the dichioromethane layer as a streak on a preparative silica gel G TLC plate and development in chloroform/methanol/water (50:30:5, v/v/v) will result in clean separation of the acetylated from the nonacetylated lysophospholipid. Scraping and extraction of the appropriate area of the TLC plate will yield a solution suitable for use as internal standards for subsequent G C - M S analysis. It should be noted that the exact amount of trideuterated-PAF in this solution must be independently quantitated. Principle of Method PAF is too involatile to be directly analyzed by GC-MS. It must, therefore, be converted to a molecule that is amenable to gas phase analysis. The following steps are designed to: (1) isolate PAF from the biological mixture in a purity sufficient for subsequent G C - M S analysis; (2) hydrolyze the polar phosphocholine moiety to yield a diglyceride which can be derivatized to give a molecule with favorable properties for G C - M S ; (3) prepare a derivative for positive-ion electron impact ionization-mass spectrometry for either estimation of the molecular species composition of the sample or for quantitative analysis of PAF and related molecules. The following listing presents two parallel schemes, the individual steps of which are interchangeable, depending on the required analysis. 1. Add deuterated internal standards to biological sample. 2. Lipid extraction (Bligh and Dyer solvent extraction or solid-phase ODS cartridge). 3. Isolation of PAF-enriched fraction (TLC, silica gel G or solid-phase silica cartridge). 4. Hydrolysis of phosphate ester (phospholipase C or hydrofluoric acid). 5. Isolation of diglyceride (TLC or solvent extraction). 6. Chemical derivatization (trimethylsilyl or tert-butyldimethylsilyl). 7. Gas chromatography-mass spectrometric analysis (selected-ion monitoring or full scan mass spectra). Assay in Biological Fluids The two parallel methods outlined above will be described, but it should be borne in mind that each of the steps of one method can be substituted for the analogous step in the other method. The choice of the
138
ASSAYS
[16]
optimum method will be dictated by the particular question and by the resources available in the laboratory. The step common to both procedures, and the one that is critical for achievement of good accuracy and precision is addition of the deuterated internal standards. In general, it is desirable to add an amount of internal standard that is similar to the amount expected in the sample. Accuracy of addition of internal standards is essential for reliable quantitative results. Solutions of deuterated PAF and analogous compounds should be kept in ethanol solution, rather than chloroform or dichloromethane because of the difficulty of accurately pipetting the latter solvents. To ensure that the internal standard adequately corrects for losses or degradation which may occur in any isolation scheme, the internal standards should be added to the biological sample at the earliest possible time permitted by the experiment. Method A
Lipids are extracted by the method of Bligh and Dyer. 7 One volume of the aqueous sample is mixed with equal volumes of methanol and chloroform. After thorough mixing, the solution is induced to separate into two phases by the addition of one volume each of water and chloroform. After mixing and low-speed centrifugation, the chloroform phase of this extraction is then taken to dryness and redissolved in a small amount of chloroform/methanol (1 : 1, v/v) for application to a silica gel G TLC plate. On a separate portionof the TLC plate, a standard solution of tritiated PAF should be applied to allow detection of the appropriate region of the plate by scanning with a radioactive TLC scanner (Berthold) after development. After drying, the plate is developed in a solvent system of chloroform/ methanol/water (50 : 30 : 5, v/v/v). PAF will move to a n R f of approximately 0.3, but that value can change slightly with each analysis, and the exact position should be located by the use of radioactive standards. For extraction of this PAF fraction from the TLC plate, the silica gel scraped from the plate is shaken with 2 ml methanol/water (9 : 1, v/v). The silica gel is removed from the solution by filtration or centrifugation and the solution taken to dryness. When dry, the sample is subjected to phosphate ester hydrolysis with phospholipase C. Water (1 ml), diethyl ether (1 ml), and 5 units of phospholipase C are added to the sample and the suspension mixed by continuous rotation of the reaction tube for 2 hr. At the end of 2 hr, I ml hexane is added, the mixture shaken thoroughly, and then centrifuged to effect good phase separation. The upper organic layer is
E. G. Bligh, and W. J. Dyer, Can. J. Biochem, Physiol. 37, 911 (1959).
[16]
GC-MS or PAF
139
removed, taken to dryness with a gentle stream of nitrogen, and the diglyceride is purified by TLC on silica gel G with development in choloroform/methanol/acetic acid (98 : 2 : 1, v/v/v). Prior to analysis as the trimethylsilyl derivative, it is necessary to effect intramolecular rearrangement of the sn-2-acetyl group to the sn-3 position, since the naturaly occurring 1,2-isomer does not give useful spectra as the trimethylsilyl derivative. Isomerization is conveniently effected by simply allowing the diglyceride to remain on the TLC plate overnight, after which it is located by scanning with the radioactive TLC scanner, scraped, and extracted with diethyl ether containing 10% methanol. This ethereal solution is taken to dryness and the trimethylsilyl derivative prepared by heating for 15 min at 60 ° in a closed tube with 50/~1 of BSTFA. The trimethylsilyl derivative of the 1,3-isomer of the diglyceride derived from hexadecyl-PAF gives the mass spectrum illustrated in Fig. 1. For quantitative analysis, the ion at m/z 175 and its deuterated analog at m/z 178 are alternately measured by the G C - M S , and the ratio of the ion currents of these two ions used to measure the amount of PAF in the original sample. Figure 2 illustrates the chromatograms obtained when the analysis described was applied to measurement of PAF in the stimulated human neutrophil.
100
1'75
80
6o E ._> n20
(M- I 5) 415 I,l,l,l,T,l,l,l,l,], 50
100
j,l~ 150
,,1',1,i, 1, l, l T ~ r ~ r T r - ~ T q - T ~ r - i 200
250
300
350
~ r 400
m/z FIG. 1. Electron-impact ionization mass spectrum of the trimethylsilyl derivative of the 1,3-isomer of the hexadecylacetyl diglyceride derived from hexadecyl-PAF.
140 ~ i225t
, ~
"~, 5oo-[ o,
4,'
£
[16]
ASSAYS
.........
m/z 175 ~ , ~ ,
.....
,
. . . . . . . .
~
,
. . . . . . .
,
I . . . . . . . .
I .
.
, ., , .
.
. . . .. ~ .
.
.....
~ ....
A
.
i
go61 5001
m/z 178
10 Time, minutes 12 F[o. 2. Selected-ion monitoring chromatograms for measurement of PAF from human PMNs by GC-MS as the trimethylsilyl derivative. The top trace (m/z 175) is a measure of the amount of PAF in the sample; the bottom trace (m/z 178) is the ion current derived from the added deuterated internal standards, [2H3]hexadecyl-PAF and [2H3]octadecyl PAF.
Method B
In this procedure, the biological sample is mixed with 4 volumes of absolute ethanol to stop enzymatic activity and to extract PAF from the mixture. This ethanolic mixture is placed on ice for 1 hr to allow for good ~00908070¢-
_=
(M-57)
60-
415
50-
> k¢, 40n" 30-
355
20i
~.0~ e, 50
~.00
~50
200
250
300
350
400
450
500
m/z FIG. 3. Electron-impact ionization mass spectrum of tert-butyldimethylsilyl derivative of the diglyceride derived from hexadecyl-PAF.
141
GC-MS OF PAF
[16]
protein precipitation and then centrifuged to separate the ethanolic solution from the precipitate. The ethanol is then diluted to approximately 50% by the addition of 3 volumes of water. This solution is then applied directly to a reversed-phase extractor column (100 mg silica-ODS, Bond-Elut, Analytichem) which has been activated with 5 ml ethanol and then washed with 5 ml water. The sample application can be conveniently performed either with the use of a vacuum manifold or with positive pressure from a syringe. After passing the 50% ethanolic solution through the ODS packing, the column is washed with 5 ml water followed by 5 ml 50% aqueous
m/z 415
s°~a4t
'~°°I
A
II /I
16
5354 /
/~
o '°°°1
//
,~
t6 B686
i7
tO
t7
J.8
i7
i8
17
tB
m/z 418
m/z 429
u} t6
m/z 432
t6
Time. minutes Fro. 4. Selected-ion monitoring chromato~'ams from an analysis of PAF derived from stimulated routine mast cells, The top two chromatograms (m/z 4]5 and m/z 418) are from hexadecyl-PAF and [2H3]hexadecyl-PAF. The bottom two traces are from the cellular hexadecanoylacetylphosphocholine (m/z 429) and its added internal standard [2H3]hexadecanoy 1 acetyl phosphocholine (m/z 432).
142
ASSAYS
[17]
ethanol. A silicic acid solid-phase extractor cartridge (500 mg silica, Supelco) which has been washed with 5 ml ethanol is then connected directly to the end of the ODS cartridge with an adapter (Analytichem) and the PAF is eluted directly from the ODS cartridge onto the silica with 5 ml ethanol. The cartridges are uncoupled and the silica is washed with an additional 5 ml ethanol. The PAF fraction is then eluted into a polyethylene tube with 4 ml of methanol/water (3 : 1). This solution is then taken to dryness on a centrifugal vacuum dryer (Savant). The phosphate ester is cleaved with hydrofluoric acid (48%, 0.5 ml) which is added to the polyethylene tube containing the dried extract and left at room temperature for 4 hr. At the end of this time the diglycerides produced are extracted with 2 ml hexane. The hexane is washed once with water and then taken to dryness with a gentle stream of nitrogen. The dried extracts are then prepared for G C - M S analysis as the TBDMS derivative by reaction with 50/zl MTBSTFA for 15 rain at 60 ° The TBDMS derivative of PAF gives the mass spectrum illustrated in Fig. 3. This derivative gives an intense M - 5 7 ion (m/z 415 for the hexadecylacetyl diglyceride from PAF) for each of the acetyl-containing diglycerides related to PAF. The combination of GC retention time and ion at M - 5 7 gives good identification of the molecular species of compounds related to PAF. By alternately measuring the M - 5 7 ion and its deuterated analog 3 mass units higher, accurate measurement of the amount of PAF can be made. Figure 4 illustrates measurement of hexadecyl-PAF (m/z = 415 and 418) and the palmitoylacetyl analog (m/z = 429 and 432) produced in response to calcium ionophore stimulus of murine mast cells. The two peaks for PAF and the three peaks observed for the palmitoyl analog are from all of the possible positional isomers of the acyl groups produced by intramolecular isomerization.
[ 17] Q u a n t i t a t i v e A n a l y s i s o f P l a t e l e t - A c t i v a t i n g F a c t o r b y Gas Chromatography-Negative-Ion Chemical Ionization Mass Spectrometry
By WALTER C. PICKETT and CHAKKODABYLUS. RAMESHA Mass spectrometric analysis of the platelet-activating factor (PAF) has been severely limited due to the inherent involatility of this phospholipid. Although valuable structural information has been gathered with respect to the intact molecule utilizing fast atom bombardment mass METHODS IN ENZYMOLOGY, VOL, 187
Copyright © 1990 by Academic Press, inc. All rights of reproduction in any form reserved.
ASSAYS
[16]
Despite these caveats, the platelet serotonin assay for PAF is quick, simple, inexpensive, and has proved very useful in numerous studies of PAF production and its modulation. Acknowledgments This work was carried out in the F. L. Bryant Jr. Research Laboratory and was supported by National Institutes of Health Grant HL 34303.
[16] Q u a n t i t a t i o n o f P l a t e l e t - A c t i v a t i n g F a c t o r b y Gas Chromatography-Mass
Spectrometry
B y K E I T H L . CLAY
Introduction Quantitative analysis of platelet-activating factor (PAF, 1-O-alkyl-2-Oacetylglycero-3-phosphocholine) is complicated by the chemical nature of the molecule and by the necessity to measure it at very low concentrations (picomolar or less) in extremely complex biological mixtures. Measurement of PAF has frequently been accomplished by the use of its biological activities, through which this molecule was initially described. 1 Measurement ofplatelet aggregation 2 or of release ofplatelet serotonin content s has been used to estimate the amount of PAF present in biological mixtures. These procedures can give very valuable information and one should not underestimate the importance of biological assays in working with a substance that was initially described as a biological activity. Since the elucidation of the exact chemical nature of PAF, however, it has become possible to develop specific physical techniques for its measurement. 4 Gas chromatography-mass spectrometry has been used as the primary method of measuring PAF. Procedures have been described for the measurement of this molecule in both the positive-ion electron-impact ionization mode 5 and in the negative-ion chemical ionization mode. 6 These procedures have the advantage over bioassay procedures of better I j. Benveniste, P. M. Henson, and C. G. Cochrane, J. Exp. Med. 136, 1356 (1972). 2 G. Camussi, M. Aglietta, F. Malavasi, C. Tetta, W. Piacibeilo, F. Sanavio, and F. Bussolino, J. Immunol. 131, 2397 (1983). 3 j. M. Lynch, G. Z. Lotner, S. J. Betz, and P. M. Henson, J. Immunol. 123, 1219 (1979). 4 R. C. Murphy and K. L. Clay, Am. Rev. Respir. Dis. 136, 207 (1987). 5 K. Satouchi, M. Oda, K. Yasunaga, and L. Saito, J. Biochem. 94, 2067 (1983). 6 C. S. Ramesha, and W. C. Pickett, Biomed. Environ. Mass Spectrom. 13, 107 (1986).
METHODSIN ENZYMOLOGY,VOL. 187
Copyright© 1990by AcademicPress,Inc. All rightsof reproductionin any formreserved.
[16]
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135
precision and accuracy and are less subject to false positive values due to cross-reactivity in the biological activities. GC-MS procedures, however, require much more sample preparation and are, therefore, much more labor intensive than the bioassays. A major advantage of GC-MS (or any mass spectrometry-based procedure) is that stable isotopically labeled variants of the analyte of interest can be added to the biological matrix at the earliest time of collection of the sample. These chemically identical, but physically distinguishable, molecules serve as almost ideal internal standards for the quantitation of the endogenous material. It should be noted that a major advantage of the use of stable isotopically labeled variants is that these molecules can serve as positive indicators of the adequacy of each individual analysis: if the internal standard is recovered and gives an adequate signal in the mass spectrometer, the analysis has been successful, even if none of the endogenous material has been detected. With other procedures, it is possible to obtain a false negative result due to inadequate recovery, but the use of the stable isotope-labeled internal standards gives a useful control on each individual sample and corrects for the variability of recoveries to be expected with biological matrixes of differing complexity.
Assay Procedure
Reagents Acetyl-d3 chloride, (Acetic anhydride)-d6, Acetic acid-d4, Hydrofluoric acid, 48%, Acetic acid (Aldrich, Milwaukee, WI) Bis(trimethylsilyl)trifluoroacetamide (BSTFA), Silicic acid solid-phase extractor cartridges (Supelco, Bellefonte, PA) Bis(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) (Pierce, Rockford, IL) Silica gel G thin-layer chromatography plates (Analtech, Newark, DE) Octadecylsilyl solid-phase extractor cartridges (Analytichem, Harbor City, CA) L-Lysophosphatidylcholine-palmitoyl, stearoyl, and oleoyl (acyllysoGPC). Phospholipase C(Type XIII, from Bacillas cereus), l-OHexadecyl-2-O-acetylglycero-3-phosphocholine (hexadecyl-PAF) (Sigma, St. Louis, MO) 1-O-Octadecyl-2-O-acetylglycero-3-phosphocholine (Octadecyl-PAF); Hexadecyllysoglycero-3-phosphocholine and octadecyllysoglycero3-phosphocholine (lyso-PAF) Bachem, Bubendorf, Switzerland) Chloroform, dichloromethane, methanol, diethyl ether, hexane, perchloric acid, 70% (Fisher, Fair Lawn, N J)
136
ASSAYS
[16]
Internal Standards
Stable isotopically labeled variants of PAF and similar molecules are not commercially available, but preparation of excellent internal standards is quite simple and inexpensive. In essence, acetylation of the appropriate lysophospholipid with deuterated acetylating reagents will give the acetylated phosphatidylcholine with deuterium atoms in the acetyl moiety of the sn-2 position. The preparation of approximately I0 mg of internal standard by two different methods will be described. Method 1
To 200/xl of perdeuteroacetic acid (acetic acid-d4), are added 10 mg of lyso-PAF (or acyllyso-GPC) followed by 50/zl of acetyl-d3 chloride. The solution is mixed by gentle agitation and left at room temperature for 30 min. A small amount of hydrogen chloride gas will be produced in the reaction, so it should be kept in a fume hood. At the end of 30 min, 2 ml of methanol are cautiously added, followed by 4 ml dichloromethane. Two phases are then produced by the addition of 2 ml water. The mixture should be gently shaken for several minutes, centrifuged to separate the phases, and the water (top) layer removed. The water layer should then be shaken with a fresh 2 ml portion of dichloromethane and the dichloromethane (bottom) layer combined with the initial extract. The dichloromethane phase will contain essentially all of the deuterated phosphatidylcholine and should be taken to dryness for further purification. Method 2
To 200 /xl of dichloromethane are added 10 mg of lyso-PAF (or acyllyso-GPC) and the solution sonicated to form a dispersion of the phospholipid. To this suspension is added 50/~1 of (acetic anhydride)-d6 and the suspension mixed. Perchloric acid (70%, 5/xl), is then added and the suspension mixed rapidly for 15 sec. Immediately on addition of the perchloric acid, the suspension should become clear as the reaction proceeds and the phospholipid goes into solution. After mixing for 15 sec, an additional 4 ml of dichloromethane is added, followed immediately by the addition of 2 ml of methanol and 2 ml water. The phases are mixed, separated, washed, and taken to dryness as described in method 1. It is critical to the success of this method that the material not be allowed to stand longer than a few seconds in an attempt to force completion. The reaction is essentially complete as soon as the reagents are mixed; any further delay in isolation of the products will result in oxidation and production of dark-colored products, and decreased yields. Both of these procedures result in rapid and essentially complete reaction of the lysophospholipid with the acetylating reagent. For further
[16]
GC-MS or PAF
137
purification, it is necessary only to separate the unreacted lysophospholipid from the acetylated product, although even that minor purification will not be necessary for some applications. Application of the dichioromethane layer as a streak on a preparative silica gel G TLC plate and development in chloroform/methanol/water (50:30:5, v/v/v) will result in clean separation of the acetylated from the nonacetylated lysophospholipid. Scraping and extraction of the appropriate area of the TLC plate will yield a solution suitable for use as internal standards for subsequent G C - M S analysis. It should be noted that the exact amount of trideuterated-PAF in this solution must be independently quantitated. Principle of Method PAF is too involatile to be directly analyzed by GC-MS. It must, therefore, be converted to a molecule that is amenable to gas phase analysis. The following steps are designed to: (1) isolate PAF from the biological mixture in a purity sufficient for subsequent G C - M S analysis; (2) hydrolyze the polar phosphocholine moiety to yield a diglyceride which can be derivatized to give a molecule with favorable properties for G C - M S ; (3) prepare a derivative for positive-ion electron impact ionization-mass spectrometry for either estimation of the molecular species composition of the sample or for quantitative analysis of PAF and related molecules. The following listing presents two parallel schemes, the individual steps of which are interchangeable, depending on the required analysis. 1. Add deuterated internal standards to biological sample. 2. Lipid extraction (Bligh and Dyer solvent extraction or solid-phase ODS cartridge). 3. Isolation of PAF-enriched fraction (TLC, silica gel G or solid-phase silica cartridge). 4. Hydrolysis of phosphate ester (phospholipase C or hydrofluoric acid). 5. Isolation of diglyceride (TLC or solvent extraction). 6. Chemical derivatization (trimethylsilyl or tert-butyldimethylsilyl). 7. Gas chromatography-mass spectrometric analysis (selected-ion monitoring or full scan mass spectra). Assay in Biological Fluids The two parallel methods outlined above will be described, but it should be borne in mind that each of the steps of one method can be substituted for the analogous step in the other method. The choice of the
138
ASSAYS
[16]
optimum method will be dictated by the particular question and by the resources available in the laboratory. The step common to both procedures, and the one that is critical for achievement of good accuracy and precision is addition of the deuterated internal standards. In general, it is desirable to add an amount of internal standard that is similar to the amount expected in the sample. Accuracy of addition of internal standards is essential for reliable quantitative results. Solutions of deuterated PAF and analogous compounds should be kept in ethanol solution, rather than chloroform or dichloromethane because of the difficulty of accurately pipetting the latter solvents. To ensure that the internal standard adequately corrects for losses or degradation which may occur in any isolation scheme, the internal standards should be added to the biological sample at the earliest possible time permitted by the experiment. Method A
Lipids are extracted by the method of Bligh and Dyer. 7 One volume of the aqueous sample is mixed with equal volumes of methanol and chloroform. After thorough mixing, the solution is induced to separate into two phases by the addition of one volume each of water and chloroform. After mixing and low-speed centrifugation, the chloroform phase of this extraction is then taken to dryness and redissolved in a small amount of chloroform/methanol (1 : 1, v/v) for application to a silica gel G TLC plate. On a separate portionof the TLC plate, a standard solution of tritiated PAF should be applied to allow detection of the appropriate region of the plate by scanning with a radioactive TLC scanner (Berthold) after development. After drying, the plate is developed in a solvent system of chloroform/ methanol/water (50 : 30 : 5, v/v/v). PAF will move to a n R f of approximately 0.3, but that value can change slightly with each analysis, and the exact position should be located by the use of radioactive standards. For extraction of this PAF fraction from the TLC plate, the silica gel scraped from the plate is shaken with 2 ml methanol/water (9 : 1, v/v). The silica gel is removed from the solution by filtration or centrifugation and the solution taken to dryness. When dry, the sample is subjected to phosphate ester hydrolysis with phospholipase C. Water (1 ml), diethyl ether (1 ml), and 5 units of phospholipase C are added to the sample and the suspension mixed by continuous rotation of the reaction tube for 2 hr. At the end of 2 hr, I ml hexane is added, the mixture shaken thoroughly, and then centrifuged to effect good phase separation. The upper organic layer is
E. G. Bligh, and W. J. Dyer, Can. J. Biochem, Physiol. 37, 911 (1959).
[16]
GC-MS or PAF
139
removed, taken to dryness with a gentle stream of nitrogen, and the diglyceride is purified by TLC on silica gel G with development in choloroform/methanol/acetic acid (98 : 2 : 1, v/v/v). Prior to analysis as the trimethylsilyl derivative, it is necessary to effect intramolecular rearrangement of the sn-2-acetyl group to the sn-3 position, since the naturaly occurring 1,2-isomer does not give useful spectra as the trimethylsilyl derivative. Isomerization is conveniently effected by simply allowing the diglyceride to remain on the TLC plate overnight, after which it is located by scanning with the radioactive TLC scanner, scraped, and extracted with diethyl ether containing 10% methanol. This ethereal solution is taken to dryness and the trimethylsilyl derivative prepared by heating for 15 min at 60 ° in a closed tube with 50/~1 of BSTFA. The trimethylsilyl derivative of the 1,3-isomer of the diglyceride derived from hexadecyl-PAF gives the mass spectrum illustrated in Fig. 1. For quantitative analysis, the ion at m/z 175 and its deuterated analog at m/z 178 are alternately measured by the G C - M S , and the ratio of the ion currents of these two ions used to measure the amount of PAF in the original sample. Figure 2 illustrates the chromatograms obtained when the analysis described was applied to measurement of PAF in the stimulated human neutrophil.
100
1'75
80
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(M- I 5) 415 I,l,l,l,T,l,l,l,l,], 50
100
j,l~ 150
,,1',1,i, 1, l, l T ~ r ~ r T r - ~ T q - T ~ r - i 200
250
300
350
~ r 400
m/z FIG. 1. Electron-impact ionization mass spectrum of the trimethylsilyl derivative of the 1,3-isomer of the hexadecylacetyl diglyceride derived from hexadecyl-PAF.
140 ~ i225t
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[16]
ASSAYS
.........
m/z 175 ~ , ~ ,
.....
,
. . . . . . . .
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,
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,
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m/z 178
10 Time, minutes 12 F[o. 2. Selected-ion monitoring chromatograms for measurement of PAF from human PMNs by GC-MS as the trimethylsilyl derivative. The top trace (m/z 175) is a measure of the amount of PAF in the sample; the bottom trace (m/z 178) is the ion current derived from the added deuterated internal standards, [2H3]hexadecyl-PAF and [2H3]octadecyl PAF.
Method B
In this procedure, the biological sample is mixed with 4 volumes of absolute ethanol to stop enzymatic activity and to extract PAF from the mixture. This ethanolic mixture is placed on ice for 1 hr to allow for good ~00908070¢-
_=
(M-57)
60-
415
50-
> k¢, 40n" 30-
355
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~.00
~50
200
250
300
350
400
450
500
m/z FIG. 3. Electron-impact ionization mass spectrum of tert-butyldimethylsilyl derivative of the diglyceride derived from hexadecyl-PAF.
141
GC-MS OF PAF
[16]
protein precipitation and then centrifuged to separate the ethanolic solution from the precipitate. The ethanol is then diluted to approximately 50% by the addition of 3 volumes of water. This solution is then applied directly to a reversed-phase extractor column (100 mg silica-ODS, Bond-Elut, Analytichem) which has been activated with 5 ml ethanol and then washed with 5 ml water. The sample application can be conveniently performed either with the use of a vacuum manifold or with positive pressure from a syringe. After passing the 50% ethanolic solution through the ODS packing, the column is washed with 5 ml water followed by 5 ml 50% aqueous
m/z 415
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m/z 418
m/z 429
u} t6
m/z 432
t6
Time. minutes Fro. 4. Selected-ion monitoring chromato~'ams from an analysis of PAF derived from stimulated routine mast cells, The top two chromatograms (m/z 4]5 and m/z 418) are from hexadecyl-PAF and [2H3]hexadecyl-PAF. The bottom two traces are from the cellular hexadecanoylacetylphosphocholine (m/z 429) and its added internal standard [2H3]hexadecanoy 1 acetyl phosphocholine (m/z 432).
142
ASSAYS
[17]
ethanol. A silicic acid solid-phase extractor cartridge (500 mg silica, Supelco) which has been washed with 5 ml ethanol is then connected directly to the end of the ODS cartridge with an adapter (Analytichem) and the PAF is eluted directly from the ODS cartridge onto the silica with 5 ml ethanol. The cartridges are uncoupled and the silica is washed with an additional 5 ml ethanol. The PAF fraction is then eluted into a polyethylene tube with 4 ml of methanol/water (3 : 1). This solution is then taken to dryness on a centrifugal vacuum dryer (Savant). The phosphate ester is cleaved with hydrofluoric acid (48%, 0.5 ml) which is added to the polyethylene tube containing the dried extract and left at room temperature for 4 hr. At the end of this time the diglycerides produced are extracted with 2 ml hexane. The hexane is washed once with water and then taken to dryness with a gentle stream of nitrogen. The dried extracts are then prepared for G C - M S analysis as the TBDMS derivative by reaction with 50/zl MTBSTFA for 15 rain at 60 ° The TBDMS derivative of PAF gives the mass spectrum illustrated in Fig. 3. This derivative gives an intense M - 5 7 ion (m/z 415 for the hexadecylacetyl diglyceride from PAF) for each of the acetyl-containing diglycerides related to PAF. The combination of GC retention time and ion at M - 5 7 gives good identification of the molecular species of compounds related to PAF. By alternately measuring the M - 5 7 ion and its deuterated analog 3 mass units higher, accurate measurement of the amount of PAF can be made. Figure 4 illustrates measurement of hexadecyl-PAF (m/z = 415 and 418) and the palmitoylacetyl analog (m/z = 429 and 432) produced in response to calcium ionophore stimulus of murine mast cells. The two peaks for PAF and the three peaks observed for the palmitoyl analog are from all of the possible positional isomers of the acyl groups produced by intramolecular isomerization.
[ 17] Q u a n t i t a t i v e A n a l y s i s o f P l a t e l e t - A c t i v a t i n g F a c t o r b y Gas Chromatography-Negative-Ion Chemical Ionization Mass Spectrometry
By WALTER C. PICKETT and CHAKKODABYLUS. RAMESHA Mass spectrometric analysis of the platelet-activating factor (PAF) has been severely limited due to the inherent involatility of this phospholipid. Although valuable structural information has been gathered with respect to the intact molecule utilizing fast atom bombardment mass METHODS IN ENZYMOLOGY, VOL, 187
Copyright © 1990 by Academic Press, inc. All rights of reproduction in any form reserved.
