Anal. Chem. 1993, 85, 2400-2402
2400
TECHNICAL NOTES
Synthesis of Pentafiuorobenzoic Anhydride: A Superior Derivatizing Agent for Lipids Susan T. Weintraub;lt Rajiv K. Satsangi,?Anne Marie Simmons,$Robert F. Williams,t and R. Neal Pinckardt Departments of Pathology and RadiologylResearch Imaging Center, The University of Texas Health Science Center at Sun Antonio, Sun Antonio, Texas 78284
INTRODUCTION
Instrumentation. Mass spectra were acquired on a FinniganMAT Model 4615 quadrupole mass spectrometer in combination with an INCOSdata system. Sample introduction was by means The use of mass spectrometry for identification and of either a gas chromatograph or direct insertion probe. For GC quantitative measurement of extremely low levels of endogseparations, the column was connected directly to the mass enous biological substances has been advanced to a significant spectrometer via a deactivated fused-silica transfer line that was degree by development of derivatives suitable for electron maintained at 250 "C. For electron capture mass spectrometry capture ionization under chemical ionization conditions. This under chemicalionizationconditions with negativeion detection, technique, commonly termed negative ion chemical ionization methane was used as the reagent gas at a pressure of approxi(NICI),generally exhibits detection limits in the subpicogram mately 0.5 Torr. The ion source temperature was 100O C and the range, thereby permitting analysis of highly active substances electron energy was 70 eV. Electron impact ionization spectra were obtained at 70 eV with an ion source temperature of 160 such as prostaglandins and platelet-activating factor (PAF) O C . l3C NMR spectra were recorded on a General Electric Model from a variety of biological matrices. The derivatives most QE 300-MHz spectrometer, with CDCl3as solvent and reference. often used are pentafluorobenzyl esters of acids such as A Perkin-Elmer Model 283B infrared spectrometer was employed prostaglandins and pentafluorobenzoyl esters for PAF and to obtain IR spectra of PFB-anh, prepared as a Nujol mull. alcohols. Prepafation of pentafluorobenzoyl esters has rouHexafluoroacetone Method. PFB-anh was synthesized by tinely been accomplished by reaction with pentafluorobenzoyl a modification of the methods of Abdel-Baky and Giese3 and chloride (PFB-Cl). However, complications such as exchange Crabtree et a1.2 Hexafluoroacetone (2 mmol, 230 pL) containing of deuterium from stable isotope-labeled internal standards1 1.6 equiv of DzO was transferred by means of a syringe into a and degradation of sensitive unsaturated analytes can arise septum-capped flask that was maintained under N2. Benzene as a result of the HC1 which is produced during the reaction. (2 mL; distilled from sodium/benzophenone)was added, followed In order to avoid these problems, Crabtree and co-workers2 by pentafluorobenzoyl chloride (5.2 mmol, 750 pL from a freshly opened ampule). Next, pyridine (5.2 mmol, 420 pL, previously modified the method of Abdel-Baky and Gieses and synthedried over CaH2and stored over 4-A molecular sieves) was added sized pentafluorobenzoic anhydride (PFB-anh), which they via syringe over a period of 5 min, and then the reaction was have successfully used for derivatization of alcohols, hydroxy allowed to stir for 30 min at room temperature. After the addition fatty acid methyl esters and hydroxyeicosatetraenoic acid of 10 mL of ethyl acetate and 3 mL of 5% HC1, the organic layer methyl esters.21~However, the coupling agent used for the was removed and washed with 7 mL of HzO. The ethyl acetate synthesis, trichlorotrifluoroacetone, is no longer commercially was dried briefly over sodium sulfate, filtered, and concentrated, available. In the present report we describe two alternative yielding 1.1g of a crystallizing oil. After addition of 5 mL of methods for preparation of PFB-anh and present preliminary hexane, the mixture was allowed to stand overnight at -20 O C . results on the use of this reagent for direct derivatization of The white crystals which were recovered after decantation of the supernatant were washed two times with 2 mL of hexane and the potent phospholipid autacoid, PAF. dried overnight under high vacuum. A 90% yield was obtained. Dicyclohexylcarbodiimide Method. Pentafluorobenzoic EXPERIMENTAL SECTION acid (210 mg, 1mmol) and dicyclohexylcarbodiimide(DCC;100 mg, 0.5 mmol) were placed in a dry 5-mL round-bottom flask. Materials. Pentsfluorobenzoic acid, pentafluorobenzoylchloDry toluene (1mL) was added, the atmosphere over the solution ride, dicyclohexylcarbodiimide,and dry toluene were purchased was flushed with dry nitrogen, and the flask was then capped from Aldrich Chemical Co. (Milwaukee, WI). The octadecyl homolog of PAF, 1-O-octadecyl-2-acetyl-sn-glycero-3-phospho- with a glass stopper. The reaction mixture was maintained at room temperature overnight, with stirring by means of a small choline (18O-AGEPC),was obtained from Bachem Bioscience magnetic stir bar. The dicyclohexylurea (DCU)which precip(Philadelphia, PA). Hexafluoroacetone containing 1.6 equiv of itated was removed by filtration, and 500 MLof deionized water DzO was obtained from Cambridge Isotope Laboratories (Wobum, was added to the filtrate. The mixture was stirred for 15 min MA). RadiolabeledPAF homologswere synthesizedas previously to convert any remaining DCC to DCU. The organic layer was described.6 All other reagents were of the highest purity separated and dried over anhydrous sodium sulfate and the commercially available. solvent removed by a stream of dry nitrogen at 45 O C . The solid residue was recrystallized as flakes from n-heptane. The yield t Department of Pathology. of product was 180 mg (88%). f Radiology/Research Imaging Center. Reaction of PAF with PFB-anh. Aliquota of radiolabeled (1)Haroldsen, P. E.; Gaskell, S. J.; Weintraub, S. T.;Pinckard, R. N. J. Lipid Res. 1991,32, 723-729. PAF (l-0-octadecyl-2-[~~]acetyl-sn-glycero-3-phosphochol~e, (2) Crabtree, D. V.; Adler, A. J.; Handelman, G. J. J. Chromatogr. 1.75nmol,3.96 mCi/mmol)in chloroform/methanolsolution were 1989,466,251-270. to glass tubes fitted with Teflon-lined caps, and the transferred (3) Abdel-Baky, S.; Giese, R. W. J. Org. Chem. 1986,51, 339Q-3391. solvent was removed by a stream of nitrogen. PFB-anh (550 pg) (4) Crabtree, D. V.;Adler, A. J. J. Chromotogr. 1991, 543, 405-411. was added in toluene, and the solvent was evaporated. The tubes (6)Weintraub, S. T.; Lear, C.; Pinckard, R. N., submitted to Biol. were capped and heated under a variety of conditions, as noted Mass Spectrom. 0003-2700/93/0385-2400$04.00/0
0 1993 Amerlcan Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 65, NO. 17, SEPTEMBER 1, lQQ3 2401 100
50
I
7
RFMF
FMFR
3 100
80
1
I
120
140
Table I. Derivatization of PAF with Pentafluorobenzoic Anhydride time, recovery of [W-aeetatelAGEPC/PFBO h 80 "C loo O C 120 oc
I
167
117
m/z 60
1Y3
I
160
180
200
220
240
r 191488
100 1
50
i
3616 (74.3) 3730 (76.6)
2935 (60.3) 2746 (56.4)
The values represent the amount of radioactivity in dpm (from of the sample)migrating at Rf0.85 after TLC fractionation of the PFB derivative of the octadecyl homolog of PAF. The numbers in parentheses are the percent recoveries calculated for the complete l/g
406 I
rxl0 m/z
737 (15.1) 837 (17.2)
1 2
260
280
300
320
340
360
380
400
420
derivatizationprocedure. The derivativewas produced by reacting 1.75 nmol of [1~-acetatel-180-AGEPC with 650 pg of PFB-anh for the designated time and temperature. An Rfvalue of 0.85 is similarly exhibited by the derivative produced by phospholipaae C treatment and reaction with PFB-Cl.
Flgurr 1. Electron impact ionization mass spectrum of pentafiuo-
robenzoic anhydride.
1
loo
for each experiment. After heating, the residue in each tube was dissolved in 400pL of hexane and extracted three times with 400 pL of water. The hexane layer was evaporated by a stream of dry nitrogen, and the residue was dissolved in a small volume of hexane prior to TLC fractionation and determination of radioactivity or mass spectral analysis. TLC Purification of PFB Derivatives. Glass microfiber plates impregnatedwith silicic acid (Toxi-GramsBlank B-Plus19, Toxi-Lab, Inc., Irvine CA) were used in a modification of the method of Christman and Blah6 The plates were developed for 10 cm with hexane, air-dried, and developed a second time for 10 cm with hexane/ethyl acetate (91, v/v). After drying in air, each lane was cut into fractionswhich were extractedwith hexane/ ethyl acetate (91). A portion of the combined organic phase was removed for determination of radioactivity, and the remainder was concentratedunder nitrogen prior to mass spectral analysis.
RESULTS Synthesis of Pentafluorobenzoic Anhydride. Both the hexafluoroacetone and the DCC methods yielded the same product, pentafluorobenzoic anhydride, as confirmed by a variety of physicochemicalmeasurements. The melting point of the product was 66-68 "C, in agreement with the literature. In the electron impact ionization mass spectrum, shown in Figure 1, a molecular ion a t mlz 406 was observed, along with a base peak at mlz 195, representative of the pentafluorobenzoyl moiety. The infrared spectrum revealed the presence of a doublet (1742 and 1805 cm-1) characteristic for carbonyl groups of anhydrides in agreement with the results of Crabtree and associates.2 Furthermore, the 13C NMR spectrum of the product was consistent with the one described by Crabtree. In our first synthesis of PFB-anh by the hexafluoroacetone method, we utilized toluene as the solvent. For subsequent reactions, we chose to use benzene instead of toluene because of the ease in removing water from benzene, in addition to its greater volatility. In instances where there are concerns about the safety of benzene, substitution with toluene can be made without loss of product yield. Use of PFB-anh for Derivatization of PAF. In these preliminary studies, we have found that in the absence of solvent, PFB-anh is capable of formingthe pentafluorobenzoyl derivative of PAF in a manner analogous to our direct derivatization procedure, which utilizes heptafluorobutyric anhydride (HFB-anh).'S8 The results for recovery of radio(6) Christman, B. W.; Blair, I. A. Biomed. Environ. Mass Spectrom.
1989,18, 258-264.
(7)Sataangi, R. K.; Ludwig, J. C.; Weintraub, S.T.; Pinckard, R. N. J. Lapad Res. 1989,30,929-937. (8)Weintraub, S. T.; Lear, C. L.; Pinckard, R. N. J.Lipid Res. 1990, 31,719-725.
I
Time (rnin)
I1
60
65
70
75
Flgure 2. Selected ion retrieval trace for QC electron capture mass spectral analysis of 18:O-AGEPC/PFB formed by direct reaction with PFB-anh. GC conditions: column, 15 m X 0.32 mm D E 1 (J&W Scientific):heilum linear velocity, 80 cm/s: initial column temperature of 170 O C held for 1 min, followed by an increase to 280 O C at a rate
of 30 OC/min.
labeled derivatized PAF are shown in Table I. A selected ion retrieval trace for GC electron capture mass spectral analysis of the PFB derivative of 18:O-AGEPC is shown in Figure 2. The electron capture mass spectrum (not shown)was identical to those of PAF derivatives formed either by our direct derivatization method or by phospholipase C treatment and esterification by PFB-C1, with an intense ion a t mlz 580 representative of [MI- predominating.
DISCUSSION In previous reports, we described the use of direct derivatization techniques for mass spectral analysisof PAF homolags and anal0gs.~1~ In those studies we showed that the heptafluorobutyryl esters yieldedvaluable structural information, and in agreement with others? the pentafluorobenzoyl esters provided exceptionaldetection limits for quantitative analysis. But derivatization using PFB-C1 has been a source of some concern related to deleterious consequences from the generation of HC1 during the reaction. Since we produce HFB derivatives by using HFB-anh, we were interested in developing an analogous procedure employing PFB-anh. Unfortunately, PFB-anh is not commerciallyavailable. Crabtree's method for synthesis of the anhydride appeared to be quite straightforward, but our efforts were complicated by the fact that the coupling agent that Crabtree used, trichlorotrifluoroacetone, waa no longer available from commercial sources. We felt that a logical alternative to trichlorotrifluoroacetone (9) Ramesha, C. S.; Pickett, W. C. Biomed. Mass.Spectrom. 1986,13, 107-1 11.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 17, SEPTEMBER 1, 1993
would be hexafluoroacetone. Since hexafluoroacetone is a gas at room temperature, for convenience it is generally supplied in a hydrated form-usually the trihydrate. However, in Crabtree’s method, only 1 equiv of water is needed for anhydride formation. To avoid the necessity of compensating for an excess of 2 equiv of water, we utilized a commercialpreparation of hexafluoroacetonewhich contained 1.6 equiv of D2O and readily obtained our desired product. From repeated preparation of PFB-anh via hexafluoroacetone/DzO we found that the easiest way to be assured of obtaining maximum product yield was to use a fresh bottle of reagent each time. We, furthermore, investigated a different approach to the synthesis-coupling of pentafluorobenzoic acid by means of DCC. This reaction proved highly successful when carried out in dry toluene. In view of the fact that the reagents for the DCC method are somewhat less
costly and are available from a wide variety of commercial sources, we feel that this latter method will be more practical for large-scale production of PFB-anh. Our future studies will focus on optimization of conditions for direct reaction of PFB-anh with PAF isolated from a wide variety of biological sources.
ACKNOWLEDGMENT This work was supported by NIH Grant HL-22555. We are grateful for the expert technical assistance of Cynthia Lear.
RECEIVED for review February 15,1993. Accepted on May 6, 1993.
