76
ASSAYS
[9]
Tetradeuterated (5S,6S)-LTA4 methyl ester is accessible via this twostep procedure. The yield of each step is modest but reproducible according to the known chemical instability of these epoxidic compounds. Nevertheless, when stored under specific conditions, no decomposition of this compound has been found to occur over 1 year. Acknowledgment The authors wish to thank Mrs. Th. Beaucourt and F. Toupet for their assistance in the preparation of this manuscript.
[9] Q u a n t i t a t i v e G a s C h r o m a t o g r a p h y - M a s s S p e c t r o m e t r y A n a l y s i s o f L e u k o t r i e n e B4
By W. RODNEY MATHEWS Introduction L e u k o t r i e n e B4, (5S,12R)-5,12-dihydroxy-(6Z,8E,lOE,14Z) eicosatetraenoic acid, a metabolite of arachidonic acid, is a mediator in a variety of inflammatory disorders.l As leukotriene B4 (LTB4) is an extremely potent compound and there are many structurally similar rnetabolites of arachidonic acid, sensitive and selective assays are required in order to establish its role in inflammation. Assays utilizing GC-MS have been used successfully to analyze a variety of eicosanoids and the technique of negative-ion chemical ionization (NCI)-mass spectrometry provides an especially sensitive method for the analysis of eicosanoids. 2 This chapter describes a GC-NCI-MS assay for LTB4 employing [2H4]LTB4 as an internal standard which is both sensitive and selective for quantifying LTB4 in biological matrixes) In order to give LTB4 suitable electron capture and chromatographic properties necessary for GC-NCIMS, the pentafluorobenzyl (PFB) ester of the carboxylic acid and the trimethylsilyl (TMS) ethers of the alcoholic hydroxyl groups of LTB4 are formed.
I M. K. Bach, Annu. Rev. Microbiol. 36, 371 (1982). 2 R. C. Murphy, Progr. Biochem. Pharmacol. 20, 84 (1985). 3 W. R. Mathews, G. L. Bundy, M. A. Wynalda, D. M. Guido, W. P. Schneider, and F. A. Fitzpatrick, Anal. Chem. 60, 348 (1988).
METHODS IN ENZYMOLOGY, VOL. 187
Copyrigh'~ © 1990 by Academic Press, Inc. All fights of reproduction in any form reserved.
[9]
GAS C H R O M A T O G R A P H Y - - M A S S S P E C T R O M E T R Y O F
LTB4
77
Assay Method
Principle. The G C - M S assay for LTB4 described here is based on G C - N C I - M S of the P F B , T M S derivative of LTB4 with selected-ion monitoring using a stable isotope analog of LTB4 as an internal standard. The general steps are as follows: (1) Addition of the [2H4]LTB4 internal standard to the sample; (2) partialpurificationof the sample; (3) formation of the PFB ester of the carboxylic acid group of LTB4 ; (4) purificationby silicic acid chromatography; (5) silylation of the alcohol hydroxyls of LTB4; (6) determination of the ratio of unlabeled to labeled LTB4 internal standard by selected-ion monitoring G C - M S . Reagents
[ZH4]LTB4 (I0.0 tzg/ml ethanol) or other stable isotope-labeled internal standard LTB4 (Cayman Chemical, Ann Arbor, MI) Pentafluorobenzyl Bromide (PFBBr, Pierce, Rockford, IL) N,N'-Diisopropylethylamine, sequanal-grade (Pierce) Bis(trimethylsilyl)trifluoroacetamide (BSTFA, Pierce) 10% BSTFA in hexane Silicic Acid (Silicar CC-4, Mallinckrodt) Octadecyl columns (Baker-I0 SPE, J. T. Baker Chemical Co, Phillipsburg, NJ), 1.0 ml DB-I fused silica capillary column, 12 m (J&W Scientific, Folsom, CA) Internal S t a n d a r d
At the present time, stable isotopically labeled leukotriene B4 is not commercially available, although [2H4]LTB 4 should be available soon from Cayman Chemical. In the mean time, a suitable isotopically labeled standard can be prepared. Several options for preparation of an internal standard are available using either 2H or tsO. [2H4]LTB4 can be chemically synthesized. A method starting with 2,5-undecadiynol has been described.3 Alternatively, [2Ha]LTB4 can be generated biosynthetically from commercially available [2HaJarachidonic acid. 4 A biosynthetic method for converting LTA4 to LTB4 is available 5 and [2H4]LTA4 can be purchased from Cayman Chemical or synthesized. 6 An alternative to deuteriumlabeled LTB4 is [taO2]LTB4. This can be easily prepared from LTB4 by 180 4 M. Dawson, J. H. Vine, M. J. Forest, C. M. McGee, P. M. Brooks, and T. R. Watson, J. Label. Comp. Radiopharmacol. 3, 291 (1987). 5 A. L. Maycock, M. S. Anderson, D. M. DeSousa, and F. A. Kuehl, Jr., J. Biol. Chem. 257, 13711(1982). J. P. Lellouche, F. Aubert, and J. P. Beaucourt, Tetrahedron Lett. 29, 3069(1988).
78
ASSAYS
[9]
exchange,7 although care must be taken to avoid hydrolysis and los s of the label during the sample workup. After a stable isotopic analog of LTB4 is obtained, it should be examined for isotopic distribution by derivatization and mass spectrometry as described below. The internal standard should be completely free of nonlabeled LTB4 in order to obtain a linear standard curve. Deuterated LTB4 can be stored as an ethanolic solution under argon at - 7 0 ° for several months without noticeable degradation. We use a solution containing 10/zg/ml [ZH4]LTB4 in ethanol which can be accurately pipetted.
Sample Preparation Prior to the GC-MS analysis of LTB4, a certain amount of sample preparation is required. The amount and type of sample preparation necessary depends on the nature of the sample matrix, and ranges from relatively minimal preparation when determining the amount of LTB4 produced by isolated cell suspensions to a more difficult and extensive one when LTB4 levels in more complex matrixes such as urine and blood are the goal. A method that has been successful for the analysis of LTB4 production by isolated neutrophils is described here. This method has also been used to determine LTB4 levels in lung lavageates from guinea pigs. When analyzing more complex samples, preparation methods employing solid-phase extraction techniques 8 and immunoaffinity columns 9 should be considered.
Neutrophils Following stimulation of neutrophils, the cells are removed by centrifugation and 100/zl [EH4]LTB4 (10 ng) is added to an aliquot (0.5 ml) of the supernatant. The supernatant is adjusted to pH 3 with HCI and extracted twice with ethyl acetate (1.0 ml). The ethyl acetate layers are pooled and transferred to a 13 × 100 mm culture tube. The ethyl acetate is removed under a stream of nitrogen. The residue is reconstituted in 1.0 ml of methanol/water (10:90, v/v) and applied to a conditioned 1.0 ml Baker-10 SPE column. These columns are conditioned by washing with 5 ml water followed by 5 ml methanol and finally 5 ml water. After the sample is loaded, the columns are washed with 2.0 ml of methanol/ water (20:80, v/v) and then eluted with 2.0 ml of methanol/water (80 : 20, v/v). The 80% methanol eluant is transferred to a screw-capped 7 R. C. Murphy and K. L. Clay, this series, Vol. 86, p. 547. 8 H. Salad and S. Steffenrud, J. Chromatogr. 378, 35 (1986). 9 j. j. Vrbanac, J. W. Cox, T. D. Eller, and D. R. Knapp, this volume [7].
19]
GAS CHROMATOGRAPHY--MASS SPECTROMETRY OF L T B 4
79
13 x 100 mm culture tube topped with a Teflon-lined cap for derivatization.
Derivat&ation The PFB ester, TMS ether derivative of LTB4 is formed as follows. The sample is first dried under a stream of nitrogen and then any residual moisture is removed by adding dichloromethane, 0.25 ml, to the tube and drying under a stream of nitrogen. The residue is dissolved in 50 /xl acetonitrile and 20 txl N,N'-diisopropylethylamine and l0/zl PFBBr are added. The tube is capped and the reaction allowed to proceed for l0 min at room temperature. Excess reagent is removed with a stream of nitrogen and the residue dissolved in 100 tzl ethyl acetate. A small Silicar CC-4 column (ca. 0.5 × 1.0 cm) is prepared in a Pasteur pipette by slurry packing the silicic acid in ethyl acetate. The sample is applied to the column which is washed with 2.0 ml ethyl acetate and then the PFB-LTB4 is eluted with 2.0 ml dichloromethane/ethyl acetate ( l : l , v/v). The dichloromethane/ethyl acetate is concentrated with a stream of nitrogen and transferred to a clean 1.0-ml Reacti-vial. The remainder of the solvent is removed and the TMS derivative formed. BSTFA, 50 tzl, and acetonitrile, 50/zl, are added to the Reacti-vial which is closed with a Teflon-lined cap and heated for 30 min at 60 ° in a heating block. After cooling, the excess reagent is removed under nitrogen and the residue dissolved in 50/xl hexane/BSTFA (90 : 10, v/v) for GC-MS. At this point, the sample may be stored under argon at - 7 0 ° and is stable for several weeks. G C - M S Analysis
General The NC1 mass spectrum of PFB,TMS-LTB4 is quite simple, consisting of the base peak at m/z 479 corresponding to the loss of the PFB moiety (C6F6CH2) and ions at m/z 389 and m/z 299 corresponding to successive losses of trimethylsilyl alcohol [(CH3)3SiOH]. This can be seen in the spectrum presented elsewhere in this volume by Ian Blair. 1° LTB4 is quantified by monitoring the (M-PFB) ion at m/z 479 for LTB4 and the corresponding ion at m/z 483 for the [2I-I4]LTB4 internal standard using the selected-ion-monitoring scan mode. A standard curve is constructed by adding a constant amount of [2Hs]LTB4 (10 ng;2.9 pmol) to 0-100 ng lo I. A. Blair, this volume [2].
80
ASSAYS
[9]
(0-298 pmol) LTB4 followed by derivatization as described above. The areas of the m/z 479 and m/z 483 peaks are determined and a plot of the amount of LTB4 vs the peak area ratio (m/z 389)/(m/z 483) constructed. An example of a standard curve is shown in Fig. 1.
Standard Conditions GC-NCI-MS is carried out using a VG 70SE mass spectrometer equipped with a HP 5890 GC and a VG 11-250J data system. The GC column is a 12 m DB-1 (0.25 mm id, 0.25/zm film thickness, J&W Scientific) fused silica capillary column. The column is equipped with a dedicated HP on-column injector and the column is introduced directly into the ion source of the mass spectrometer. Samples (1.0/zl) are injected with the oven temperature at 50°. After 1 min the temperature is increased to 265 ° at 20°/min and then programmed to 290 ° at 5°/min. Under these conditions LTB4 elutes at approximately 9.3 min. The heated transfer line to the ion source is maintained at 270 ° and the ion source itself is heated to 180°. Methane is used as the CI reagent gas at a pressure of 1 x 10-5 torr measured at the source housing. The mass spectrometer operating conditions are typically: accelerating voltage, 8 kV; ionization energy, 80 eV;
pMOLESLTB4= 16.9 /RATIO M/ZM/4793/483.3 E - 0.006 65r,,,.
oD
N
N
432-
.
!
e
30
150
300
pMOLESLT84 FIc. I. Typical standard c u r v e for L T B 4 . T h e equation o f the regression line is indicated.
[9]
GAS CHROMATOGRAPHY-MASS SPECTROMETRY OF LTB4
8!
emission current, 1 mA; resolution, 1000. The instrument is operated in the selected-ion-monitoring mode under the control of the data system and peak areas for m/z 479.3 and 483.3 are calculated by the data system.
Optimization of Sensitivity Although the spectra of LTB4 is not complex and the derivatization procedure is relatively straightforward, obtaining optimal sensitivity can be difficult. In order to obtain optimal sensitivity in the G C - M S analysis of LTB4, care must be taken to ensure that both the chromatograph and mass spectrometer are being operated efficiently. A few general comments may help to obtain the necessary sensitivity. Relatively few problems have been encountered in the derivatization of LTB4 with the exception of the hydrolysis of the TMS ethers. This can be minimized by using a solution of hexane containing BSTFA instead of hexane to dissolve the samples for injection. Since derivatized LTB4 is relatively stable, it is useful to generate a stock of derivatized LTB4 (I mg/ml) as well as serial dilutions of this standard. The overall G C - M S sensitivity should be checked with these standards. The chromatography of PFB,TMS-LTB4 can be a problem especially at low levels. The use of a new, short GC column (12 m) coupled with the use of an on-column injector and a direct interface to the MS ion source will usually be successful. The performance of a used GC column can often be recovered by removing the first meter. Since even 6-m columns can provide sufficient resolution for LTB4 analysis, 3 this can be repeated several times. If poor chromatography is suspected it may be useful to prepare a PFB ester derivative of a saturated long-chain fatty acid (e.g., stearic acid). The PFB fatty acids are less sensitive to active sites on the column and can help distinguish GC from MS problems. A clean ion source is essential for optimal sensitivity in the NCI mode. The use of fluorinated hydrocarbons for tuning and calibration can rapidly contaminate the ion source and should be kept to a minimum. A clean source can be used for approximately 1 week before loss of sensitivity becomes a problem. Exact MS operating conditions will vary from instrument to instrument, however, the general discussion of negative-ion chemical ionization-mass spectrometry found in the review by Oehme I1 discusses several important factors and is quite useful. ~ M. Oehme, in "Mass Spectrometry of Large Molecules" (S. Facchetti, ed.), p. 233. Elsevier, Amsterdam, 1985.
ASSAYS
[9]
Tetradeuterated (5S,6S)-LTA4 methyl ester is accessible via this twostep procedure. The yield of each step is modest but reproducible according to the known chemical instability of these epoxidic compounds. Nevertheless, when stored under specific conditions, no decomposition of this compound has been found to occur over 1 year. Acknowledgment The authors wish to thank Mrs. Th. Beaucourt and F. Toupet for their assistance in the preparation of this manuscript.
[9] Q u a n t i t a t i v e G a s C h r o m a t o g r a p h y - M a s s S p e c t r o m e t r y A n a l y s i s o f L e u k o t r i e n e B4
By W. RODNEY MATHEWS Introduction L e u k o t r i e n e B4, (5S,12R)-5,12-dihydroxy-(6Z,8E,lOE,14Z) eicosatetraenoic acid, a metabolite of arachidonic acid, is a mediator in a variety of inflammatory disorders.l As leukotriene B4 (LTB4) is an extremely potent compound and there are many structurally similar rnetabolites of arachidonic acid, sensitive and selective assays are required in order to establish its role in inflammation. Assays utilizing GC-MS have been used successfully to analyze a variety of eicosanoids and the technique of negative-ion chemical ionization (NCI)-mass spectrometry provides an especially sensitive method for the analysis of eicosanoids. 2 This chapter describes a GC-NCI-MS assay for LTB4 employing [2H4]LTB4 as an internal standard which is both sensitive and selective for quantifying LTB4 in biological matrixes) In order to give LTB4 suitable electron capture and chromatographic properties necessary for GC-NCIMS, the pentafluorobenzyl (PFB) ester of the carboxylic acid and the trimethylsilyl (TMS) ethers of the alcoholic hydroxyl groups of LTB4 are formed.
I M. K. Bach, Annu. Rev. Microbiol. 36, 371 (1982). 2 R. C. Murphy, Progr. Biochem. Pharmacol. 20, 84 (1985). 3 W. R. Mathews, G. L. Bundy, M. A. Wynalda, D. M. Guido, W. P. Schneider, and F. A. Fitzpatrick, Anal. Chem. 60, 348 (1988).
METHODS IN ENZYMOLOGY, VOL. 187
Copyrigh'~ © 1990 by Academic Press, Inc. All fights of reproduction in any form reserved.
[9]
GAS C H R O M A T O G R A P H Y - - M A S S S P E C T R O M E T R Y O F
LTB4
77
Assay Method
Principle. The G C - M S assay for LTB4 described here is based on G C - N C I - M S of the P F B , T M S derivative of LTB4 with selected-ion monitoring using a stable isotope analog of LTB4 as an internal standard. The general steps are as follows: (1) Addition of the [2H4]LTB4 internal standard to the sample; (2) partialpurificationof the sample; (3) formation of the PFB ester of the carboxylic acid group of LTB4 ; (4) purificationby silicic acid chromatography; (5) silylation of the alcohol hydroxyls of LTB4; (6) determination of the ratio of unlabeled to labeled LTB4 internal standard by selected-ion monitoring G C - M S . Reagents
[ZH4]LTB4 (I0.0 tzg/ml ethanol) or other stable isotope-labeled internal standard LTB4 (Cayman Chemical, Ann Arbor, MI) Pentafluorobenzyl Bromide (PFBBr, Pierce, Rockford, IL) N,N'-Diisopropylethylamine, sequanal-grade (Pierce) Bis(trimethylsilyl)trifluoroacetamide (BSTFA, Pierce) 10% BSTFA in hexane Silicic Acid (Silicar CC-4, Mallinckrodt) Octadecyl columns (Baker-I0 SPE, J. T. Baker Chemical Co, Phillipsburg, NJ), 1.0 ml DB-I fused silica capillary column, 12 m (J&W Scientific, Folsom, CA) Internal S t a n d a r d
At the present time, stable isotopically labeled leukotriene B4 is not commercially available, although [2H4]LTB 4 should be available soon from Cayman Chemical. In the mean time, a suitable isotopically labeled standard can be prepared. Several options for preparation of an internal standard are available using either 2H or tsO. [2H4]LTB4 can be chemically synthesized. A method starting with 2,5-undecadiynol has been described.3 Alternatively, [2Ha]LTB4 can be generated biosynthetically from commercially available [2HaJarachidonic acid. 4 A biosynthetic method for converting LTA4 to LTB4 is available 5 and [2H4]LTA4 can be purchased from Cayman Chemical or synthesized. 6 An alternative to deuteriumlabeled LTB4 is [taO2]LTB4. This can be easily prepared from LTB4 by 180 4 M. Dawson, J. H. Vine, M. J. Forest, C. M. McGee, P. M. Brooks, and T. R. Watson, J. Label. Comp. Radiopharmacol. 3, 291 (1987). 5 A. L. Maycock, M. S. Anderson, D. M. DeSousa, and F. A. Kuehl, Jr., J. Biol. Chem. 257, 13711(1982). J. P. Lellouche, F. Aubert, and J. P. Beaucourt, Tetrahedron Lett. 29, 3069(1988).
78
ASSAYS
[9]
exchange,7 although care must be taken to avoid hydrolysis and los s of the label during the sample workup. After a stable isotopic analog of LTB4 is obtained, it should be examined for isotopic distribution by derivatization and mass spectrometry as described below. The internal standard should be completely free of nonlabeled LTB4 in order to obtain a linear standard curve. Deuterated LTB4 can be stored as an ethanolic solution under argon at - 7 0 ° for several months without noticeable degradation. We use a solution containing 10/zg/ml [ZH4]LTB4 in ethanol which can be accurately pipetted.
Sample Preparation Prior to the GC-MS analysis of LTB4, a certain amount of sample preparation is required. The amount and type of sample preparation necessary depends on the nature of the sample matrix, and ranges from relatively minimal preparation when determining the amount of LTB4 produced by isolated cell suspensions to a more difficult and extensive one when LTB4 levels in more complex matrixes such as urine and blood are the goal. A method that has been successful for the analysis of LTB4 production by isolated neutrophils is described here. This method has also been used to determine LTB4 levels in lung lavageates from guinea pigs. When analyzing more complex samples, preparation methods employing solid-phase extraction techniques 8 and immunoaffinity columns 9 should be considered.
Neutrophils Following stimulation of neutrophils, the cells are removed by centrifugation and 100/zl [EH4]LTB4 (10 ng) is added to an aliquot (0.5 ml) of the supernatant. The supernatant is adjusted to pH 3 with HCI and extracted twice with ethyl acetate (1.0 ml). The ethyl acetate layers are pooled and transferred to a 13 × 100 mm culture tube. The ethyl acetate is removed under a stream of nitrogen. The residue is reconstituted in 1.0 ml of methanol/water (10:90, v/v) and applied to a conditioned 1.0 ml Baker-10 SPE column. These columns are conditioned by washing with 5 ml water followed by 5 ml methanol and finally 5 ml water. After the sample is loaded, the columns are washed with 2.0 ml of methanol/ water (20:80, v/v) and then eluted with 2.0 ml of methanol/water (80 : 20, v/v). The 80% methanol eluant is transferred to a screw-capped 7 R. C. Murphy and K. L. Clay, this series, Vol. 86, p. 547. 8 H. Salad and S. Steffenrud, J. Chromatogr. 378, 35 (1986). 9 j. j. Vrbanac, J. W. Cox, T. D. Eller, and D. R. Knapp, this volume [7].
19]
GAS CHROMATOGRAPHY--MASS SPECTROMETRY OF L T B 4
79
13 x 100 mm culture tube topped with a Teflon-lined cap for derivatization.
Derivat&ation The PFB ester, TMS ether derivative of LTB4 is formed as follows. The sample is first dried under a stream of nitrogen and then any residual moisture is removed by adding dichloromethane, 0.25 ml, to the tube and drying under a stream of nitrogen. The residue is dissolved in 50 /xl acetonitrile and 20 txl N,N'-diisopropylethylamine and l0/zl PFBBr are added. The tube is capped and the reaction allowed to proceed for l0 min at room temperature. Excess reagent is removed with a stream of nitrogen and the residue dissolved in 100 tzl ethyl acetate. A small Silicar CC-4 column (ca. 0.5 × 1.0 cm) is prepared in a Pasteur pipette by slurry packing the silicic acid in ethyl acetate. The sample is applied to the column which is washed with 2.0 ml ethyl acetate and then the PFB-LTB4 is eluted with 2.0 ml dichloromethane/ethyl acetate ( l : l , v/v). The dichloromethane/ethyl acetate is concentrated with a stream of nitrogen and transferred to a clean 1.0-ml Reacti-vial. The remainder of the solvent is removed and the TMS derivative formed. BSTFA, 50 tzl, and acetonitrile, 50/zl, are added to the Reacti-vial which is closed with a Teflon-lined cap and heated for 30 min at 60 ° in a heating block. After cooling, the excess reagent is removed under nitrogen and the residue dissolved in 50/xl hexane/BSTFA (90 : 10, v/v) for GC-MS. At this point, the sample may be stored under argon at - 7 0 ° and is stable for several weeks. G C - M S Analysis
General The NC1 mass spectrum of PFB,TMS-LTB4 is quite simple, consisting of the base peak at m/z 479 corresponding to the loss of the PFB moiety (C6F6CH2) and ions at m/z 389 and m/z 299 corresponding to successive losses of trimethylsilyl alcohol [(CH3)3SiOH]. This can be seen in the spectrum presented elsewhere in this volume by Ian Blair. 1° LTB4 is quantified by monitoring the (M-PFB) ion at m/z 479 for LTB4 and the corresponding ion at m/z 483 for the [2I-I4]LTB4 internal standard using the selected-ion-monitoring scan mode. A standard curve is constructed by adding a constant amount of [2Hs]LTB4 (10 ng;2.9 pmol) to 0-100 ng lo I. A. Blair, this volume [2].
80
ASSAYS
[9]
(0-298 pmol) LTB4 followed by derivatization as described above. The areas of the m/z 479 and m/z 483 peaks are determined and a plot of the amount of LTB4 vs the peak area ratio (m/z 389)/(m/z 483) constructed. An example of a standard curve is shown in Fig. 1.
Standard Conditions GC-NCI-MS is carried out using a VG 70SE mass spectrometer equipped with a HP 5890 GC and a VG 11-250J data system. The GC column is a 12 m DB-1 (0.25 mm id, 0.25/zm film thickness, J&W Scientific) fused silica capillary column. The column is equipped with a dedicated HP on-column injector and the column is introduced directly into the ion source of the mass spectrometer. Samples (1.0/zl) are injected with the oven temperature at 50°. After 1 min the temperature is increased to 265 ° at 20°/min and then programmed to 290 ° at 5°/min. Under these conditions LTB4 elutes at approximately 9.3 min. The heated transfer line to the ion source is maintained at 270 ° and the ion source itself is heated to 180°. Methane is used as the CI reagent gas at a pressure of 1 x 10-5 torr measured at the source housing. The mass spectrometer operating conditions are typically: accelerating voltage, 8 kV; ionization energy, 80 eV;
pMOLESLTB4= 16.9 /RATIO M/ZM/4793/483.3 E - 0.006 65r,,,.
oD
N
N
432-
.
!
e
30
150
300
pMOLESLT84 FIc. I. Typical standard c u r v e for L T B 4 . T h e equation o f the regression line is indicated.
[9]
GAS CHROMATOGRAPHY-MASS SPECTROMETRY OF LTB4
8!
emission current, 1 mA; resolution, 1000. The instrument is operated in the selected-ion-monitoring mode under the control of the data system and peak areas for m/z 479.3 and 483.3 are calculated by the data system.
Optimization of Sensitivity Although the spectra of LTB4 is not complex and the derivatization procedure is relatively straightforward, obtaining optimal sensitivity can be difficult. In order to obtain optimal sensitivity in the G C - M S analysis of LTB4, care must be taken to ensure that both the chromatograph and mass spectrometer are being operated efficiently. A few general comments may help to obtain the necessary sensitivity. Relatively few problems have been encountered in the derivatization of LTB4 with the exception of the hydrolysis of the TMS ethers. This can be minimized by using a solution of hexane containing BSTFA instead of hexane to dissolve the samples for injection. Since derivatized LTB4 is relatively stable, it is useful to generate a stock of derivatized LTB4 (I mg/ml) as well as serial dilutions of this standard. The overall G C - M S sensitivity should be checked with these standards. The chromatography of PFB,TMS-LTB4 can be a problem especially at low levels. The use of a new, short GC column (12 m) coupled with the use of an on-column injector and a direct interface to the MS ion source will usually be successful. The performance of a used GC column can often be recovered by removing the first meter. Since even 6-m columns can provide sufficient resolution for LTB4 analysis, 3 this can be repeated several times. If poor chromatography is suspected it may be useful to prepare a PFB ester derivative of a saturated long-chain fatty acid (e.g., stearic acid). The PFB fatty acids are less sensitive to active sites on the column and can help distinguish GC from MS problems. A clean ion source is essential for optimal sensitivity in the NCI mode. The use of fluorinated hydrocarbons for tuning and calibration can rapidly contaminate the ion source and should be kept to a minimum. A clean source can be used for approximately 1 week before loss of sensitivity becomes a problem. Exact MS operating conditions will vary from instrument to instrument, however, the general discussion of negative-ion chemical ionization-mass spectrometry found in the review by Oehme I1 discusses several important factors and is quite useful. ~ M. Oehme, in "Mass Spectrometry of Large Molecules" (S. Facchetti, ed.), p. 233. Elsevier, Amsterdam, 1985.
