ANALYTICAL CHEMISTRY, VOL. 50, NC). 1, JANUARY 1978
nium(V1) were present. Some uranium(V1) is not able to drift out of the zone of weak adsorption and is eluted as a separate peak in the zirconium fraction. Table IV shows that for large amounts of uranium(V1) losses in this separate peak can be as high as about 10% of the total uranium(V1) present. For smaller amounts of uranium the losses become less prominent, while they become negligable for trace amounts. In order to avoid the observed losses of uranium the fast movement of uranium(I1) with its labile zone had to be retarded. This was attained by inserting a buffer of 30 mL of 2 M hydrochloric acid containing 75% methanol between the sulfuric acid and hydrochloric acid eluting agents. The lower concentration of hydrochloric acid leads to a lower peak of sulfuric acid preceding it, as is shown in Figure 3. It has been shown that the presence of methanol or other organic solvents increases the anion adsorption of uranium substantially in both hydrochloric and sulfuric acid solutions ( 1 0 ) . No information seems to be available about mixtures of both acids containing organic solvents as well as some pure acid-organic solvent mixtures of relevant concentrations. Some values for distribution coefficients of such mixtures are therefore presented in Table I1 (methanol) and I11 (acetone). The tables show that the coefficients increase substantially with methanol or acetone as well as with hydrochloric acid concentration when the sulfuric acid concentration is constant. They increase with increasing solvent concentration and slowly decrease with increasing sulfuric acid concentration when the hydrochloric acid is constant. Generally acetone causes a larger increase in pure or predominantly sulfuric acid solutions a n d methanol in pure or predominantly hydrochloric acid solutions. Table I shows that in about 2.5 M aqueous sulfuric acid, the concentration in the wave preceding the hydrochloric acid eluting agent in Figure 2, the distribution coefficient of uranium(V1) is about 4. Uranium(V1) therefore moves relatively fast down the column and has not enough time to escape from the small region of low distribution coefficients completely. When the buffer is inserted the sulfuric acid concentration
47
at its peak will be about 1.8 M. I n addition a methanol concentration of about ’75% will be present. This will increase the distribution coefficient to a value of about. 23. A much faster and complete escape of the uranium from the region of weak adsorption does result. No extra uranium peak occurs and recoveries are quantitative, as is shown in Table IV. About 60 ng of uranium found in th.e “zirconium fraction” when 10 kg uranium were separated are rather uncertain because of a variable blank of between 200 a r d 250 ng. The phenomena described from which the losses of uranium result are of general importance and do apply also to other elements. They may occur fairly often and therefore become important when two different inorganic acids or other eluting agents are used successively for removal of two different groups of interfering elements from an anion exchange column, while the element to be analyzed fo-r is still retained. The approach to use a small amount of solution containing an organic solvent as “buffer” in order t o increase distribution coefficients in the critical border region between the eluting agents also seems to be of fairly generid validity and probably could find wide application when separation of more than two components or groups of component!; on an anion exchange column is planned.
LITERATURE CITED (1) 9. L. Jangida, N. Krishnamachari,M. S. Varde, and V. Venkatasubramanian, Anal. Chim. Acta, 32, 91 (1965). (2) F. W. E. Strelow and C. R. van Zyl, J . S. A f r . Chem. Irist., 20, 1 (1967). (3) S. Fisher and R . Kunin, Anal. Chem., 29, 400 (1957). (4) T. Kirijama and R. Kuroda, Anal. Chim. Acta, 71, 375 (1974). (5) K. A. Kraus and F. Nelson, Proc. Int. C o d , Peaceful Uses At. Energy Geneva, 1955, 7, 113 (1956). (6) J. Korkisch, A. Farag, and F. Hecht, 2 . Anal. Chem., 161, 92 (1958). (7) F. W. E. Strelow, M. L. Kokot, T. N. van der Walt. and M. Bhaga, J . S. Afr. Chem. Inst., 29, 97 (1976). (8) T. Baltakmens, Anal. Chem., 47, 1147 (1975). (9) F. W . E. Strelow and P. F. S. Jackson, A,wl. Chem., ,26. 1481 (1974). (10) J. Korkisch. Mikrochlm. Acta, 816 (196.4).
RECEIVED for review August 25, 1977. Accepted September 26. 1977.
Separation of Prostaglandins and Thromboxanes by Gas Chromatography with Glass Capillary Columns F. A. Fitzpatrick Research Laboratories, The Upjohn Company, Kalamazoo, Michigan 4900 1
Prostaglandin and thromboxane methyl ester oxime trimethylsllyl ethers were efflciently separated by gas chromatography using a commercial open-tubular, glass capillary column coated with OV-101. Separation and detection of all currently known primary transformation products of the arachidonic acid cascade was achieved in 30 min in a single, isothermal gas chromatographic run. Kovats indices for 24 compounds were determined. The method is reliable and it evades some dlfficultles which have plagued more sophisticated glass capillary chromatographic procedures.
T h e discovery of the transient compounds: prostaglandin endoperoxides (1-4), thromboxane A2 (5, 6), and prostacyclin (7), has complicated the determination of the classical E, F, 0003-2700/78/0350-0047$01 OO/O
and D series prostaglandins (PG). The concentration of these stable prostaglandins, however, reflects the concentration of their unstable precursors indirectly. .411 prostaglandin elements derive ultimately from Czopolyunsaturated fatty acids stored in cells (8). Since arachidonic acid predominates in most cells, the sequential formation of the prostaglandins, thromboxane, and prostacyclin is designated the arachidonic acid cascade (Figure 1). Arachidonic acid, immobilized as a phospholipid is transformed by a cyclooxygenase enzyme complex into the prostaglandin endolperoxides, 9,ll-epoxy15-hydroperoxyprosta-5,13-dienoicacid (PGGJ and 9 , l l epoxy-15-hydroxyprosta-5,13-dienoic acid (PGH2). Upon formation, P G H 2 rapidly converts, both enzymatically and chemically, into PGE2, PGD2,and PGFZa,and into two labile compounds: thromboxane A2 (TxA,) and prostacyclin (PGI2). TxA2 and PGIz, in turn, convert rapidly into their stable C 1977 American Chemical Society
48
ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY 1978
COOH
ARACHIDONIC A C I D
I
fatty acid cyclo-oxygenase
6OH PGG2
I
peroxidase
--
~ 1 1 1 1
OH
1
PGH2 prostacyclin syntheta/
/thromboxane synthetase
PGD2
isomerase
\
&
I/
!\
0 - ,
\\“
0
I
mild acid
OH 9-keto 1 reductase
-
-
OH PROSTACYCLIN
0
OH
0
OH
OH
PGD2
PGE2
PGF2a
TXA2
\
hydrolysis
OH 6-KETO-PG F1a
TXB2
Flgure 1. Currently accepted biosynthetic and chemical transformations in the arachidonic acid cascade
products: thromboxane B2 (TxBz)and 6-keto-PGF1, (9, IO), respectively. Subsequent metabolism may produce the 15ketoprostaglandins (11-13); the 13,14-dihydro-15-ketoprostaglandins a n d the di-, tetra-, or hexanorcarboxylic acid products from the p oxidation of the 13,14-dihydro-15ketoprostaglandins. Prostaglandins with @-keto1functionality (E or D series) dehydrate in the presence of acids or bases, so degradation products associated solely with isolation or derivatization steps are common. Equivalent transformation of homo-y-linolenic acid (Cz0:3) into the “1” series prosta-
glandins (prosta-13-enoic acids) occurs, and transformations of other Czopolyunsaturated acids are plausible. T h e precursor fatty acid and the enzymatic composition of each cell govern the nature and degree of these transformations. Sample complexity is thus a major deterrent to the clarification of the physiological roles of prostaglandins and thromboxanes. Radioimmunoassay (RIA) of prostaglandins and thromboxane B2is sensitive and potentially selective (14, 15) but analysis of prostaglandin mixtures requires an appropriate
ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY 1978
antibody population and radiolabeled tracer for each component. T h e production and evaluation of different antibodies is time consuming, and the radiolabeled tracers are commercially available for few prostaglandins, so tedious biosynthetic preparations are necessary, or else [1251]histamide derivatives must be used (16). Immunological methods, despite their potential selectivity, often suffer in practice from nonspecific interferences unless antibodies of high affinity ( K , > lo9), and specificity are obtained. For prostaglandin analysis, particularly, t h e possible presence of unknown cross-reacting compounds looms alarmingly since t h e recent discovery of new enzymatic pathways (1-7), and t h e implicit understanding that undiscovered pathways may yet exist. Gas chromatography combined with mass spectroscopy (GC/MS) a n d deuterated carrier internal standards is, conditionally, t h e most accurate method for prostaglandin analysis ( 17-19), b u t multicomponent analyses are hindered by t h e limited availability of deuterated internal standards, and by the chromatographic resolution between the individual components. In practice, derivatives are often selected for their favorable mass spectral properties rather t h a n their favorable chromatographic properties, and the chromatograph itself is often operated at suboptimal conditions in order t o achieve maximum compatibility with the mass spectrometer. G C / M S and RIA are not suitable for profile studies on samples containing many prostaglandins, although their value for discrete analysis is undisputed. High performance gas chromatography (HPGC) using wall coated open tubular columns (WCOT),or support coated open tubular columns (SCOT), despite its unparalleled chromatographic efficiency, has been used infrequently, in the routine sense, for biochemical separation a n d analysis (20-27). Schomberg (28) has summarized t h e technical complexities causing this reluctant acceptance of glass capillary HPGC. These include: (1)T h e preparation of thermostable, glass, open tubular gas chromatographic columns with reproducible characteristics. (2) Adaptation of low volume capillary columns t o instruments designed to accommodate packed columns. ( 3 ) Accurate and precise injection of samples into low volume capillary columns without sample degradation, and without exposure of the column to excessive amounts of heated solvent vapors and derivatizing agents. Specialists have addressed these problems individually; however, their solutions have not coalesced into a routine tool for t h e analytical nonspecialist. By combining commercial glass, open tubular capillary columns with t h e novel injection system of Van den Berg and Cox (29),a practical, reliable system for high performance gas chromatography of prostaglandins has been developed. Access t o this powerful, analytical technique is now permitted without t h e frustrating apprenticeship which previously limited its application. Separation and detection of all of the currently known primary transformation products of t h e arachidonic acid cascade has been achieved for t h e first time in a single isothermal gas chromatographic run. EXPERIMENTAL Apparatus. A Perkin-Elmer Model 3920 gas chromatograph with a heated injector block, dual flame ionization detectors, and a temperature programmer was used. Chromatograms were recorded on a Varian A-25 single pen recorder, 0-1 mV full-scale. An all-glass, solventless injector, constructed according to Van den Berg and Cox (29),was mounted horizontally in the heated injector block of the chromatograph. This device is related to one described earlier by Ros (30) and to a system used by Siggia et al. for reaction gas chromatography (31). A needle valve, instead of a capillary restrictor, regulated the column/vent gas flow ratio in our system.
49
Thermostable, open tubular glass capillary columns, 25 or 50 m X 0.25 mm i.d. coated with OV-17 or OV-101 (Perkin-Elmer, Norwalk, Conn.) were mounted in an aluminum mandril (Alltech Associates, Arlington Heights, Ill.). Solid vespel polyamide ferrulds ('/le in.) were bored to accommodate the outside diameter of the capillary, and the capillary was connected to a low volume Swagelok union. Glass lined stainless steel tubing, '/I6 in. X 0.01 in. i.d. (Alltech Associates), was connected to the all-glass injector with the aid of vespel reducing ferrules, and the low volume union was then attached to the glass lined tubing. The capillary outlet was connected directly to the flame ionization jet with the minimum length of glass lined stainless steel tubing necessary. An assortment of vespel ferrules, vespel reducing ferrules, brass unions, brass reducing unions, glass lined stainless steel tubing ( l / 1 6 in. o.d. X 0.01 in. i.d.1 and a precision needle valve (Nupro) are the only components needed to mount the capillary column to this injector in most chromatographs. The dimensions of the chromatograph dictate the sizes required. We have mounted the columns and injector vertically, in a Hewlett-Packard Model 5713 chromatograph with equal facility. Chromatographic Conditions. Helium carrier gas, hydrogen, and compressed air were purified over a desiccant and a 4-A molecular sieve (Applied Science Laboratories, State College, Pa.). An inlet pressure of 50 psig produced a linear carrier gas velocity of 38 em-s-', or approximately 1-2 mL/min. The gas inlet pressure was adjusted to obtain maximum column efficiency with a standard alkane mixture. The heated injector block and detector oven were at 250 and 300 "C. respectively. The Kovats retention index for the prostaglandins was determined with the column operated a t 245 "C isothermally according to Schomburg and Dielmann (32). Reagents. Hexane, methanol, and methylene chloride, distilled in glass (Burdick and Jackson, Muskegon, Mich.); anhydrous ether (Mallinckrodt. St. Louis, Mo); bis(trimethylsily1)acetamide(BSA), o-methylhydroxylamine hydrochloride, o-butylhydroxylamine hydrochloride (Applied Science Laboratories, State College, Pa.); n-methyl-n-nitroso-n-nitroguanidine, o-ethylhydroxylamine hydrochloride (Eastman, Rochester, N.Y.); potassium hydroxide solution, 4 5 7 ~wjv, and potassium hydroxide pellets (Baker, Phillipsburg, N.J.) were used as received. Silylation grade pyridine was stored over 4-?i molecular sieves during use. All prostaglandins and thromboxanes were supplied by the Experimental Chemistry Laboratories of The Upjohn Company. Other chemicals cited were the highest puriry available. Esterification. Prostaglandins and thromboxanes dissolved in methanol (0.1 mL) were treated with excess ethereal diazomethane (1.0 mL) for 5 min at 25 "C. The diazomethane solution was prepared immediately before use. Excess reagent was evaporated with a nitrogen stream a t 25 "C. Oximation. Prostaglandin methyl esters containing carbonyl groups were converted to alkyl or aryl (oximes. Fifty nanograms to 50 pg of prostaglandin or thromboxane methyl esters were treated with a solution (0.2 mL) of o-ethyl-, o-methyl-, o-butyl-, or o-benzylhydroxylamine hydrochloride in anhydrous pyridine (1.0 mg/mL). The mixture was warmed at 40 "C for 2 h or allowed to stand overnight at 25 "C.The pyridine was evaporated under a nitrogen stream and the residue was reconstitut.ed in 20 pL of methanol. One mL of 0.9% w/v sodium chloride and 5 pL of 1 M citric acid were added and the prostaglandin oxime methyl esters were extracted with 3 X 2 mL of hexane. The combined hexane extracts were dried over anhydrous sodium sulfate, concentrated to 0.5 mL, and quantitatively transferred to a 1.0-mL glass-stoppered volumetric flask. The hexane was evaporated and the prostaglandin methyl ester oximes were concentrated into the tube bottom. This residue was heated with 50 pL of BSA for 1-2 h a t 40 "C.The bulk of the BSA was evaporated under a dry nitrogen stream a t 25 "C and the contents of the tube were then diluted with 0.1 mL of hexane. Sample Injection. A Hamilton 701 syringe was inserted through the septum of the inlet port and the sample (1-10 pL) was quantitatively deposited onto the tip of the glass probe within the injection system (Figure 2). The temperature a t the probe tip, directly below the sample inlet port, was 40-45 "C. In this position, solvent and volatiles exited through the vent, but the prostaglandin and thromboxane derivatives remained on the probe
50
ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY 1978
I I
C H R O M A T O G R A P H OVEN
I
I
1
1 1
I
I
I
MAGNET
CARRIER GAS INLET OXYGEN
TRAP
GLASS L I N E D STAINLESS STEEL T U B I N G
MOLECULAR SIEVE
DETECTOR
Figure 2. Schematic representation of chromatographic system with Van den Berg injector. A block. C = Chromatograph oven
= Carrier gas inlet junction. B = Heated injector
Table I. Kovats Indexa of F Series Prostaglandins
Compound
1,
13,14-dihydro-15-keto-PGF,, PGF,, 13,14-dihydro-l5-keto-PGF,, 15-keto-PGF,, 15-keto-PGF;; a
Conditions were identical to those listed in Figure
3.
I, = 1OQ( [log R, - log R,
100,.
0
10
20
30
MINUTES
Flgure 3. Separation of F series prostaglandins as methyl ester TMS ethers. Column: 40 m OV-101, 245 OC isothermal. Carrier gas: He, 50 psig, 38 c m = s-'; Hydrogen: 20 psig; air: 50 psig. FID 32X Chart speed 10 min/in. Approximately 500 ng injected. (1) PGFz0, (2) PGF,d, (3) PGF,, (4) 13,14-dihydr0-15-keto-PGF,~, (5) PGF,,, (6) 13,14-dihydro-1 5-keto-PGF,,, (7) 15-keto-PGF,,
tip. Solvent was entirely vented after 3 min. The probe was then magnetically guided past the carrier gas inlet junction until the tip was in the heated zone (250 "C) of the injector block. Prostaglandin and thromboxane derivatives vaporize at this temperature and the redirected gas flow now sweeps them onto the chromatographic column. Complete transfer from the probe tip to the column took 4 min at 250 "C. With the capillary column at 145 "C initially, the vaporized compounds condensed at its head. The column temperature was then adjusted for isothermal or programmed operation. Overall memory effects were less than 2%.
RESULTS A commercial, glass, open tubular OV-101 capillary column separated structurally similar prostaglandins and thromboxanes efficiently. Figure 3 shows the separation of several F series prostaglandins as their methyl ester trimethylsilyl ethers. Table I lists the corresponding Kovats retention indices. F prostaglandins with keto groups at the '2-15 position could be chromatographed without formation of a carbonyl derivative; however, prostaglandins with P-keto1 functionality and thromboxanes, with acetal functionality, had t o be converted to oximes for proper gas chromatography. Figure
2650 2675 2699 2705 2717 2739 2771 2803 2756
40
t
I
I)/( [log R, + i - log R, 1) +
2
1
I
I
0
10
20
I 30
MINUTES
Figure 4. Separation of prostaglandin methyl ester methoxime TMS 1) PGA, (syn-, anti-), (2', 2) PGD2 ethers. Conditions as in Figure 3. (i', Syn(syn-, anti-), (3', 3) PGE, (syn-, anti-), (4) TxB,, (5)6-keto-PGF,,. and anti- refer only to the resolution of two geometrical isomers of the oxime, and do not imply any assignment of conformation
4 shows the separation of a standard mixture of the primary transformation products of the arachidonic acid cascade as their methyl ester methoxime trimethylsilyl ethers. There was no apparent loss of trimethylsilanol during the separation of the prostaglandin oximes. Formation of methyl esters first simplifies the removal of excess derivatizing agent in the subsequent oximation reaction. I t is conceivable that trimethylsilyl esters could also be suitable derivatives if they were
ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY 1978 50
r
I
Table 11. Kovats Index of Prostaglandin and Thromboxane Oximes‘
2 I 3
Compound PGA, EA,
PGBl PGB, PGD, ED,
PGEl PGE, 0
10
20 MINUTES
51
30
Figure 5. Separation of prostaglandin methyl ester butyloxime TMS 2) PGD, ethers Conditions as in Figure 3. (l’, 1) PGAp (syn-, anti-), (2’, (syn-, anti-), (3’,3) PGEp (syn-, anti-), (4’, 4) = 6-keto-PGF,, (syn-, anti-), (5’,5) = TxB, (syn-, anti-)
formed after oximation. Figure 5 shows the separation of these same compounds as their methyl ester butyloxime trimethylsilyl ethers. Table I1 lists the Kovats retention indices for these and 18 other prostaglandin or thromboxane methyl ester oxime trimethylsilyl ethers. Multiple values for single, authentic prostaglandins indicate the resolution of the syna n d anti-isomer of the alkyl oxime. A 40-m OV-101 column retained the prostaglandin benzyloxime derivatives excessively (12C-180 min) under the conditions shown in Figures 3-5. The composition of the sample under study will dictate the choice of oxime derivative; however, the butyloxime is generally useful. T h e butyloximes of PGA,, PGD2, PGE2, TxB, and 6-keto-PGF,, are resolved in 40 min a n d their 15-keto metabolites all elute beyond 47 min. The methoximes of PGA2, PGD,, PGE2, TxB2, and 6-keto-PGF,, are resolved in 30 min b u t their 15-keto metabolites, especially 15-keto-PGE2and 13,14-dihydro-15-keto-PGE2,have similar Kovats indices. The increased retention of the butyloximes allows a longer clearance time between injection and elution of the prostaglandins, for the elution of other compounds isolated from biological matrices. Also, after derivatization, the butyloximes can be removed from excess derivatizing agent by partitioning between hexane and saline more efficiently t h a n the corresponding methoximes. The chromatographic conditions cited represented a compromise between an acceptable resolution a n d short analysis times. Operation a t reduced linear gas velocities would produce optimized separations from a theoretical viewpoint, b u t a t the expense of longer chromatographic intervals. The column temperature limit was only 250 “C; consequently the increased analysis time could not be compensated for by elevating the temperature. Figure 6 shows a useful application of this methodology. A potent, prostaglandin synthetase preparation, the sheep seminal vesicle acetone powder (33)was incubated with arachidonic acid as previously described ( 3 4 ) . It is known that this system produces, mainly, PGE,; in the presence of the cofactor, reduced glutathione (GSH). Upon reevaluation; however, capillary gas chromatography revealed that 6keto-PGF,, formation dominated when cofactor was absent. Chang and Murota (35)have recently reported a similar result for bovine seminal vesicles using radiometric thin-layer chromatography. Since all of the currently known transformation products of arachidonic acid are detected in a single, isothermal run, the glass capillary method is a qualitative improvement over a previously reported HPLC method ( 3 4 ) .
13,14-dihydroPGE, 13,14-dihydroPGE,
Methoximes, Ix
Butyloximes, Ix
Ethyloximes, y!
2542 2604 2534 2589 2696 2719 2717 2761 2676 27 1 2 2703 2759 2689 2748 2721
2768 2845 2745 2826 2943 2956 2930 2960 2870 2916 2904 297 1 2887 2954 29 19
2595 2652 2573 2631 2766 2776 2766 2800 2731 2762 2751 2810 2726 2790 2776
2765 2706
2968 2897
28 14 2752
2752 2948 27 95 2708 3048, 3151 279.5, 2810 3118, 3144 2842, 2852 2717 2761 13,14-dihydro-l5- 2704, 2750 3070, 3096 2 7 8 6 , 2803 keto-PGE , 2717, 2761 3118, 3144 2825, 2832 15-keto-PGF1, 2744 29 26 2780 2956 2795 15-keto-PGF:, 2736 2890 2747 2925 2762 27 50 13-14-dihydro-152922 2756 keto-PGF, 2937 2799 13,14-dihydro-l52890 2732 2700 keto-PGF,, 27 1 2 2906 2755 15(S)-15-methyl 2538 2762 2597 PGA, 2604 2845 2661 15(S)-15-methy1 2964 2757 2758 PGB, 15(S)-15-methyl 2913 2741 2691 PGD, 2772 2717 3000 2849 2796 -B, 3013 3215 29 14 13,14-dihydro-152819 keto-TxB, 3227 3240 6-keto-PGFI, 2823 2988 2845 2995 2856 13,14-dihydro-6,2849 3155, 3173 2884, 2896, 16-diketo-PGF,, 2905 2858 3178. 3192 6,15-dike to-PG F , a 2836 3105; 3170 2884, 2899, 2908 3178, 3209 15-keto-PGE,
a
The conditions were identical to those in Figures 4 and
5.
Following recent discoveries (2-7), attention in prostaglandin research has now turned toward the relative amounts of classical prostaglandins, thromboxane and 6-keto-PGF1, produced by different organs, and how “shunting” between the three metabolic pathways regulates the physiology of cells. The glass capillary gas chromatographic method should prove useful since it can separate all of the compounds of interest. T h e applications of this method have been qualitative, b u t the quantitative aspects are also promising. Table I11 lists injection reproducibility for sub-microgram quantities of prostaglandin or thromboxane methyl ester butyloxime tri-
52
ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY 1978
and mass spectrometric analysis. T h e columns prepared by Maclouf et al. (27) and Rigaud et al. (36) were undoubtedly superior to the commercial columns used here, b u t with this simple, reliable system difficult separations have been achieved routinely. 60
50
-
ACKNOWLEDGMENT
-
Jacques Maclouf and Michel Rigaud of the Laboratoire de Biochimie, CHU Dupuytren, Limoges, France, contributed vital suggestions on the injection system and the general problem of capillary gas chromatographic analysis of prostaglandins. These contributions are gratefully acknowledged.
Y
VI 2 L 0
a
40-
LITERATURE CITED
s 30 3' 13
0
10
30
20
MINUTES
Figure 6. Capillary chromatographic profile of prostaglandin products from seminal vesicle synthetase enzyme. Conditions as in Figure 5. (l', 1) PGE2, (2) PGD2, (3', 3)6-keto-PGF,,
Table 111. Injection Reproducibility Compound PGD, PGE, 6-keto-PGF,, TxB,
Micrograms injec te da 0.227 0.568 1.136 0.188 0.47 0
0.940 0.159 0.398 0.796 0.264 0.660 1.320
Peak height, Re1 std mm dev, n = 5 31.6 2 66.8 2 154.0 c 25.0 r 50.7 i 116.0 2 16.3 2 36.4 c 84.0 ? 18.5 c 46.2 + 110.0
2
3.2 6.4
310.0%
7.2
29.6% 24.7%
2.2 5.7 8.0 2.6 3.5 5.0 2.0 4.1 6.2
211.2% 26.8% +15.9% +9.5% 25.9% 210.8% 28.9% 25.6%
+8.8%
Compounds were chromatographed as methyl ester butyloxime TMS ethers using the conditions of Figure 5. The micrograms listed were deposited on the glass probe in 3 p L of hexane. The relative standard deviation of 5 injections includes the error of syringe sampling plus the error of probe manipulation. The peak height is the sum of both syn- and anti-isomers. a
methylsilyl ethers with flame ionization detection. T h e minimum, on-column, quantity of compound which can be quantitated (signal/noise = 5/1 at attenuation 8 X ) is approximately 20 f 5 ng. T h e potential of glass capillary chromatography for certain biochemical problems has been reported (20-27). Maclouf et al. (27) first reported its utility for prostaglandin separation
(1) M. Hamberg, J. Svensson, T. Wakabayashi, and B. Samuelsson, Proc. Natl. Acad. Sci. U . S . A . ,71, 345-349 (1974). (2) M. Hamberg and B. Samuelsson, Proc. Natl. Acad. Sci. U . S . A . ,71, 3400-3404 (1974). (3) C.Malmsten, M. Hamberg, J. Svensson, and 8. Samuelsson, Proc. Natl. Acad. Sci. U . S . A . ,72, 1446-1450 (1975). (4) D. Nugteren and E. Hazelhof, Biochim. Siophys. Acta., 326, 448-461 (1973). (5) M. Hamberg. J. Svensson, and B. Samuelsson. Proc. Natl. Acad. Sci. U . S . A . , 72,2994-2998 (1975). (6) P. Needleman, S.Moncada, S. Bunting, J. Vane, M. Hamberg. and B. Samuelsson, Nature (London), 261, 550-560 (1976). (7) S.Moncada, R. Gryglewski, S. Bunting, and J. Vane, Nature (London). 263, 663-665 (1976). (8) R. Gorrnan, "Prostaglandins, Thromboxanes, and Prostacyclin" in "Biochemistry and Mode of Action of Hormones", Series 11, H. V. Rickenberg, Ed., 1977, in press. (9) C. Pace-Asciak, J . A m . Chem. Soc., 98, 2348-2349 (1976). (10) R. Johnson et al., Prostaglandins, 12, 915-928 (1976). (11) H. Vonkeman, D. Nugteren, and D. vanDorp, Biochim. Biophys. Acta, 187, 581-583 (1969). (12) J. Jarabak, Proc. NaN. Acad. Sci. U . S . A . ,69, 533-534 (1972). (13) S.C. Lee and L. Levine, J . Biol. Chem., 250, 548-552 (1975). (14) B. Samuelsson, E. Granstrom, K . Green, M. Hamberg, and S. Hammarstrom, Ann. Rev. Biochem.. 44, 669,(1975). (15) L. Levine, R. GcMiozCernosek, and H. VanVunakis. "Specific Antibodies: Reagents for Quantitative Analysis of Prostaglandins" in Adv. Biosci. 9, 71-83 (1973). (16) J. Maclouf, N. Pradel, P. Pradelles. and F. Dray, Biochim. Biophys. Acta.. 431, 139-146 (1976). (17) K. Green, M. Hamberg, and B. Samuelsson, "Quantitative Analysis of Prostaglandins and Thromboxanes by Mass Spectrometric Methods." in Adv. Prostaglandin Thromboxane Res., 1, 47-59 (1976). (18) A. Lawson, Clin. Chem. (Winston-Salem. N . C . ) ,21, 803-824 (1975). (19) R. W. Kelly and P. L. Taylor, Anal. Chem., 48, 465-467 (1976). (20) K. Grob, Helv. Chim. Acta. 51, 718 (1968). (21) A. L. German, C. Pfaffenberger, J. P. Thenot, H. Horing, and E. C. Horning, Anal. Chem.. 45, 930-935 (1973). (22) A. L. German and E. C. Horning. J . Chromatogr. Sci., 11, 76-82 (1973). (23) J. A . Vollmin, Chromatographia, 3 , 233-241 (1970). (24) A. Ros and I. Sommerville, J. Obstet. Gynaecol., 78, 1096-1 107 (1971). (25) M. Novotny and A. Zlatkis, J . Chromatogr. Sci., 8 , 346-350 (1970). (26) R. F. Adams, F. L. Vandemark, and G. Schmidt, J . Chromatogr. Sci., 13, 63-68 (1977). (27) J. Maclouf. M. Riaaud. J. Durand. and P. Chebroux. Prostadandins, 11. 999-1018 (i976j (28) G Schomburg, R Dielmann, H Husmann, and F Weeks, J Chromatcgr , 122 .- -, 55-72 .. . - (19761 , - -, (29) P. M. J. Van den Berg and T. P. Cox, Chromatcgraphia,5 , 301-305 (1972). (30) A. Ros, J . Gas Chromatogr., 3 , 252 (1965). (31) S.Siggia, L. Whitlock, and J. Tao, Anal. Chem., 41, 1387-1392 (1969). (32) G. Schomburg and G. Dielmann, J. Chromatcgr., Sci., 11, 151-159 (1973). (33) D. Walhch and E. Daniels, Biochim. Sbphys. Acta, 231, 445-457 (1971). (34) F. A. Fitzpatrick, Anal. Chem., 48, 499-502 (1976). (35) W. C.Chang and S. Mwota, Biochim. Biophys. Acta, 486, 136-144 (1977). (36) M.R i g a d , P. Chebrow, J. Durand, J. Maclouf, and C. Mandani, Tetrahedron Lett., 44, 3935-3938 (1976).
RECEIVED for review August 25, 1977. Accepted October 25, 1977.
