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Method for quantitative analysis of 16,16-dimethyl-prostaglandin E2 from plasma using deuterated carrier and gas chromatography-mass spectrometry
ANALYTICAL
BIOCHEMISTRY
Method
100,
109- 117 (1979)
for Quantitative Analysis of 16,16-Dimethyl-prostaglandin from Plasma Using Deuterated Carrier and Gas Chromatography-Mass Spectrometry
E,
S.~TEFFENRUD Department
of Chemistry,
Karolinska
Institutet,
S-104
01 Stockholm,
Sweden*
AND
F. H. LINCOLN Experimental
Chemistry
Research,
The Upjohn
Company,
Kalamazoo,
Michigan
49001
Received June 1, 1979 The preparation of 3,3,4,4-2H,16,16,-dimethyl-prostaglandin EZ (PGE2), 16,16-dimethyl5,6-didehydro-PGE,, and 5,6-3H,16,16-dimethyl-PGE, is described. These compounds have been used for development of a gas-liquid chromatography-mass spectrometry method for quantitative determination of corresponding nondeuterated prostaglandin. The method is based on addition of a known amount of carrier to the sample and after purification and derivation the ratio between the protium and deuterium form is measured in the mass spectrometer. With this technique 40 pg of 16,16-dimethyl-PGE, can be determined with a precision of ?2.6%.
trimester pregnancy with 16,16-dimethylPGE, have shown few gastrointestinal side effects, neither anti-emetic nor anti-diarrheic medication was needed (2-4). For a detailed study of the pharmacokinetics of 16,16-dimethyl-PGE, it was necessary to prepare the tritium-labeled and the deuterated analog and to develop a sensitive and accurate analytical method. The synthesis of tritium-labeled 16,16dimethyl-PGE,, deuterated 16,16-dimethylPGEz, and the development of an isotopedilution technique for gas-liquid chromatographic-mass spectrometric quantitation of 16,16-dimethyl-PGE, is described in this report.
A number of prostaglandin analogs have been synthesized during recent years. Among those which have been studied with regard to uterotonic potency, 16,16-dimethyl-prostaglandin E2 (PGE# was found to be the most potent analog when compared with six other prostaglandins and prostaglandin analogs as described earlier (1). Induction of abortion during first and second 1 Abbreviations used: PGE,, prostaglandin E2, 1la, 15 -dihydroxy -9 -keto -prost -5 -cis , 13 -trans -dienoic acid; PGE,, prostaglandin E,, 1la,15 dihydroxy-9keto-prost-13-trans-enoic acid; r-BDMS, tertbutyldimethyl silyl; AVA, accelerating voltage alternator; F1,, 9a,lla,l5 trihydroxyPGF,,, prostaglandin prost-13-trans-enoic acid; PGF,,, prostaglandin F,a, 9a,lla,15 dihydroxy - prost - 5- cis,l3 - trans - dienoic acid, PGB,, prostaglandin B,, 15-hydroxy-9-ketoprost-5-cis,8(12),13-trans-trienoic acid; BSA, N,O-bis(trimethylsilyl)acetamide,THP,l5-tetrahydropyranyl; TMS, trimethylsilyl ether; tic, thin-layer chromatography; gc-ms, gas chromatograph-mass spectrometry. * Present address: Department of Clinical Chemistry, Karolinska Sjukhuset. S-10401 Stockholm, Sweden.
MATERIALS
AND METHODS
Preparation of 3,3,4,4-2H,16,16,-DimethylPGE,
The synthesis is outlined in Fig. 1. Lactol I (5), 0.70 g was condensed with the yield 109
0003-2697/79/170109-09$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
110
STEFFENRUD
&zCOOH
~THP
AND
.
ZTHP
FIG.
~2COOH
ZH
III
LINCOLN
:H
IV 1. Synthesis
of 16,16-di[3,3,4,4-2H,]methyl-PGE,.
prepared from 1 g of 2,2,3,3-2H,4-carboxybutyl-triphenyl-phosphonium bromide (6). Chromatography over acid-washed silica gel using 30% ethyl acetate-70% hexane elution afforded 290 mg of II as an oil. Oxidation of 190 mg of this material by 0.2 ml of Jones’ reagent in acetone solution at - 15°C followed by THP ether hydrolysis with acetic acid-water at 40°C and chromatography over 15 g of acid-washed silica gel (75% ethyl acetate-25% hexane elution) gave 58 mg of product IV as a colorless oil. The NMR spectrum (CDC&) showed a sharp singlet at 6 2.3 for the protons attached to carbon 2. Otherwise the spectrum was similar to nondeuterated 16,16-dimethyl-PGE, (5). For the mass spectrometric determination of the deuterium content the O-methyloxime acetyl derivative of the methyl ester was prepared. Repetitive electric scanning over a narrow mass range around the molecular ion gave
The synthesis is outlined in Fig. 2. Crystalline 16,16-dimethyl-PGF,a V (mp 8283°C from acetone-hexane, [alo + 40”, c = 1.0736 in 95% EtOH) (5) was protected as the tribenzoate methyl ester and con-
vetted via bromination, dehydrobromination, and deprotection to the corresponding 5-yne compound. VI (7); [a], + 24”, c = 0.8465 in 95% EtOH; NMR (d6 acetone 6) 5.56 (m, 2H, C,,,,, vinyl), 5.1 (s, 4H, 3XOH + COOH), 3.65-4.4 (m, 3H, C,C,, and C,,H), 1.28 (s), 0.88 (s), and 0.82 (s); mass spectrum (tetra TMS) M+ (668) not observed, 653 (M+-15), 569 (M+-C,H,,), 479 (569+-TMSOH); infrared (thin film cm-‘) 3380, 2960, 2920, 2860, 2640, 2220 (W, C=C), 1710, 1240, 1160, 1110, 1085, 1055, 1020, 995, and 975. The lj-tetrahydropyranyl ether was prepared after protection of the 9- and 1I-hydroxy groups as a boronate ester (8), the protecting group removed and the 11-hydroxy group converted to the t-butyl dimethylsilyl derivative VII (7). Oxidation with Jones’ reagent and hydrolysis of the protecting groups afforded 16,16-dimethyl-5,6-didehydro-PGE, VIII as a pale yellow oil slightly more polar on tic (A-IX, Rf 0.37) than 16,16-dimethyl-PGE,; NMR (CDCl, 6) 5.97 (s, 3H, 2XOH + COOH), 5.7 Cm, 2H9 G14 vinyl), 3.7-4.3 (m, 2H, C,, and C,, H), 1.28 (s), 0.9 (9H). Mass spectrum (tri TMS) weak M+ 594, M+CH, = 579.3336. Calcd for C3,,H5&05 = 579.3357. Also 495 (M+-C,H&, 489 (579+-TMSOH), 405 (495+-TMSOH), 351 (495+-o=cOTMS), 261 (351+TMSOH), 117 (CO,TMS+); infrared (thin
QUANTITATIVE
ANALYSIS
OF
16,16-DIMETHYL-PROSTAGLANDIN
film)
2. Synthesis
111
of 16,l~dimethyl-S,~didehydro-ME*.
3420, 2960, 2940, 2860, 2660, 2240
(CsC, weak), 1745, 1715 sh, 1245, 1160, 1080, 1015, 1000, and 975 cm-‘. Preparation PGEz
PLASMA
VIII
VII FIG.
Ez FROM
of5,6-3H216,16-Dimethyl-
5,6-3H~16,16-Dimethyl-PGE~ was prepared by catalytic hydrogenation of 16,16dimethyl-5,6-did~hydro-PGE~ using tritium gas. One milligram of 16,lQdimethyl-5,6didehydro-PGE, was dissolved in 0.5 ml of methanol and 1 mg of Lindlars catalyst was added. The solution was stirred under tritium atmosphere for 15 min as described earlier (9) and finally filtered and left to stand in room temperature for about 60 min. The methanol was evaporated and about 200 pg of unlabeled 16,16-dimethyl-PGE~ was added. The material was purified by reversed phase pa~ition chromatographies using system C-50. The yield was about 2% (based on added tritium gas). About 7.5 x 10 dpm of this material was added to 25 pg of 16,16-dimethyl-PGE,. The O-methyloxime acetyl derivative of the methyl ester was prepared as described before (10) and subjected to oxidative ozonolysis as described earlier (11). After treatment of the reaction products with diazomethane, gas chromatography with simultaneous detection of radioactivity was performed. When the sample was injected on a 1% SE-30
column at 170 and 23O”C, all radioactivity seen was eluted with the solvent peak. At the lower temperature an unlabeled compound with ac - value of 16.1 appeared. The mass spectrum of the material in this peak gave an ion at m/e 301 (141) and prominent ions at m/e 270 (M-31), m/e 241 (M-60), mle 239 (M-2 x 31), m/e 227 (M-74), m/e 183 (M-2 x 59), m/e 167 (M-(74 + 60)), and m/e 149 (M-(59 + 3 x 31) base peak) indicating that this material was due to compound B in Fig. 3. If any of the double bonds in A5 or Al3 positions in the tritium-labeled 16,16dimethyl-PGE, structure would have been reduced with tritium gas any of compounds C, D, and E, should have been formed. The calculated c - values of those are 21,2 1, and 26, respectively. Since no radioactive product of these c- values was found the product of this reduction must be 16,16di[5,6-3H~]methyl-PGE~. Preparation of [3,3,#,4JHJ[5,C3H,]Labeled 16,16-Dimethyl-PGE,
5,6-3H,16,16-Dimethyl-PGEB was added to 600 pg of 3,3,4,4-2H,16,16-dimethylPGE, to give a specific activity of 116.5 @/mol. Thin-layer chromatography of this compound showed an& = 0.46. No radioactive impurities could be detected upon radio-gas
112
STEFFENRUD
AcO
H
AND LINCOLN
OAc
0
E
3. 16,16-Dimethyl-FGE, (A) and compounds formed by oxidative ozonolysis of the possible products after the catalytic hydrogenation of 16,16-dimethyl-5,6-didehydro-PGE,. FIG.
chromatography of the 0-methyloxime trimethyl&y1 ether derivative of the methyl ester (1% SE-30 on Chromosorb W, 230°C). The radioactive purity was determined to be greater than 98%. Chromatographic Methods
Reversed phase partition chromatography was performed as described earlier (12), using system C-48 (moving phase, methanol:water 144: 156 (v/v); stationary phase, chloroform:isooctanol, 15: 15 (v/v)). Silicic acid chromatography was carried out on Silic AR CC4, 100-200 mesh, Mallinckrodt analytical reagent. The columns (0.25 g) were eluted with mixtures of ethyl acetate in benzene (1:9,2:8, and 4:6) or ether in hexane (0:lO and 1:9) followed by methanol. Five milliliters of each solvent mixture was used. Thin-layer chromatography was run on silica gel-coated glass plates as described before (13). The solvent system was ethyl acetate:2,2,4-trimethylpentane:acetic acid: water, 110:20:30: 100 (v/v/v/v). Gas-liquid chromatography with simultaneous registration of mass and radioactivity was performed on a Barber-Colman gas chromatograph Model 5000. The column was a 2 m x 5 mm i.d. silanized glass tubing packed with 1% SE-30 on Chromosorb W, HP 100- 120 mesh (Varian Aerograph, Walnut Creek, Calif.).
The c - values were calculated in relation to normal fatty acid methyl esters. Gas -liquid chromatography -mass spectrometry. The mass spectrometric quantita-
tions were performed with a combined gas-liquid chromatograph-mass spectrometer, LKB 9000A. The gas-liquid chromatographic column (1.5 m x 2 mm, glass) was packed with 1% Dexsil 300 on Chromosorb W DMCS, 80- 100 mesh (Analabs., North Haven, Conn.). The conditions were: column temperature: 26O”C, carrier gas flow about 20 ml/min, electron energy: 25 eV, and trap current: 120 PA. An LKB AVA-unit was used. The two ions to be measured were intermittently focused every second. As a control of the analytical procedure, a standard was injected every fourth run in order to check the focusing (6). Preparation of Derivatives for Gas -Liquid Chromatography and Mass Spectrometry
The methods used for preparation of methyl esters 0-methyloxime trimethylsilyl ether and acetyl derivatives have been described earlier (10). Ethyl esters were prepared by dissolving the compounds in 0.5 ml of ethanol and treatment with diazoethane in ether. After evaporation, the ethyl ester was dissolved in 100 ~1 of t-butyldimethylchlorosilane/ imidazole/dimethylformamide (1 mM/2.5
QUALITATIVE
ANALYSIS
OF 16, I~DIMETHYL-PROSTAGLANDIN
& FROM PLASMA
f 13
mM/l ml) Applied Science Laboratories The methanol fractions that contained radioactivity were evaporated and subInc., State College, Pa.) and heated at 100°C jected to reversed-phase partition chromafor 1 h as described before (14). This treatment converted the ethyl ester of 16,16- tography using 4.5-g columns and system dimethyl-PGE, to the ethyl ester t-butyl C-48. 16,16-Dimethyl-PGE~ appeared at V, dimethylsilyl ether of l&16-dimethyl-PGB,. = 150 ml. The fractions containing radioAfter cooling, the reaction mixture was activity were combined and after evaporaextracted three times with 0.5 ml of hexane. tion, the residue was dissolved in ethanol The extracts were combined, reduced to a and treated with diazoethane. Further purismall volume, and passed through a silicic fication was obtained by silicic acid chromaacid column (0.25 g) and the column was tography using stepwise increasing concenwashed with hexane to a total volume of trations of ethyl acetate in benzene. After 5 ml. The l&16-dimethyl-PGB* derivative evaporation of the solvent, the residue was was eluted from the column with 5 ml of treated with t-butyldimethylchlorosilanel imidazole/dimethylform~ide and purified ether in hexane (1:9). as described above. The derivatized comSolvents pound was dissolved in a small volume of hexane before analysis in the mass specEther, methanol, chloroform (all analytitrometer. The overall recovery of the purical grade, Merck) ethylacetate, and hexane fication and derivatization procedures was (pesticide grade, Fisher) were used without about 40%. further puri~cation. The benzene (analytical grade, Merck) RESULTS was glass distilled. Quuntitative Analysis of 16,16-DimethylRecovery from Plasma PGE, For practical reasons, titrated blood bank plasma was used in the recovery tests. One plasma pool of 150 ml was divided into three equal parts. One was used as the blank. To the other two parts [5,6-3H2] 16,16-dimethylPGE, (1.3 ,&i/pmol) was added to give concentrations of 104 and 520 pg/ml, respectively (see Table 2) and equilibrated for 5 min. Each of the three parts was further divided into 5 x lo-ml samples and transferred into tubes containing 1000 ng [3,3,4,43HZ]- [5,6--“H&labeled 16, l~dirne~yl-~E~. The plasma was equilibrated with the carrier for 10 min. The plasma was acidified to pH 4 with 2 M HCl and poured through an XAD-2 column (1.5 x 15 cm). The column was washed with water until neutral effhrent and eluted with 4 x 15ml of methanol. The yield of radioactivity in the methanol fractions was 90-95% of added amount.
The mass spectrum of the t-BDMS derivative of the ethyl ester of [3,3,4,4-2H,]-[5,63H,]-labeled 16,16-dimethyl-PGE, is shown in Fig. 4. The ion of m/e 409 is due to elimination of 99 mass units from the molecular ion, probably by cleavage between carbon 15 and 16. Since this ion is of high intensity and the ion at m/e 405 where the corresponding ion from the protium form appears, is of low intensity these ions were selected for measurements during the analysis. The ratio between the recorded ion intensities at m/e 405 and 409 was 12.3 2 0.201 1000 (n = 5) when the t-BDMS derivative of the ethyl ester of [3,3,4,4-2H,]-[5,6-3H,]labeled-16,16-dimethyl-PGE, was analyzed using the AVA equipment. 16,16-Dimethyl”PGE~ was added to [3, 3,4,~zH~]-[5,6-~H~]-labeled-16,16-dimethylPGE, in ratios l:lOOO, 2:1000, 4: 1000, 8:1000, and 16:lOOO and the mixtures were derivatized as described above.
STEFFENRUD
AND LINCOLN
r
M-9! LO9
Y 10
M-57
100
150
200
250
300
350
LOO
1 LSf 1 ,L.,.,.,l. 450
5oM [I ,.,., 500
I
,
“le
FIG. 4. Mass spectrum of the tert-butyl dimethylsilyl ether derivative of the ethyl ester of 16,16di[3,3,4,4-eH,]methyl-PGE~. Conditions: electron energy: 22.5 eV, trap current: 120 PA.
Aliquots of about 20 ng of the different mixtures were analyzed in the mass spectrometer equipped with the AVA unit, and the ion intensities at mle 405 and 409 were recorded. The slope of the standard curve after injection of 20 ng from each of the mixtures was close to 45” (Fig. 6). The precision and accuracy of the method determined by repetitive injections of 20 ng of the different standard mixtures is shown in Table 1. Since the precision at 1: 1000 is 18.9% and 20 ng of l&16-dimethyl-PGE, of this mixture was injected each time, the limit of detection with the equipment used is lower than 20 pg (0.05 pmol).
rier, the samples were worked up as described under Material and Methods. The analytical data and recoveries of added 16,16-dimethyl-PGEB are given in Table 2. The level of added 16,16-dimethyl-PGE, (column 2, Table 2) in each plasma sample was determined by one single injection of 20 ng of the isolated compound (carrier plus protium form) and was not calculated from mean values after several determinations. This presentation was chosen in order to give a true picture of the precision obtained. A typical AVA run of a plasma sample is shown in Fig. 5, containing 100 pg/ml 16,16dimethyl-PGE,.
Recovery of 16,16-Dimethyl-PGE, from Plasma
DISCUSSION
Unlabeled 16,16-dimethyl-PGE, was added to titrated plasma and after addition of carTABLE PRECISION
Compound 16,16-Dimethyl-PGE,
DETERMINED
BY REPETETIVE
Prostaglandins of the PGE type possess a /3-ketol system which make them suscepti1 ANALYSIS
OF STANDARD
Injected deuterated carrier Ox)
Total H-form injected (PP)
H-Form injected m
20 20 20 20 20 20
0 20 40 80 160 320
0 0.10 0.20 0.40 0.80 1.60
MIXTURES
Percentage H-form found (mean r SD) (N = 5) 0.106 0.233 0.403 0.846 1.612
0 lr + -t ” +
18.9 2.6 2.2 2.5 1.4
QUANTITATIVE
ANALYSIS
OF 16,16-DIMETHYL-PROSTAGLANDIN TABLE
E2 FROM PLASMA
115
2
RECOVERY OF ADDED 16, 16-DIMETHYL-PGEz H/D (%I
Plasma f&&N
0.066 0.007 -0.039 0.064 -0.023 0.233 0.212 0.175 0.225 0.209 1.083 1.065 0.949 1.048 1.00
33 4 -20 32 -12 118 108 88 114 106 548 538 480 530 506
Mean 2 SD (pgiml)
7 k 24
0
107 2 12
104
520 + 27
520
3
i
1 RETENTION
113 104 85 110 102 105 103 92 102 97
doned due to either unsuitable fragmentation in the mass spectrometer or difficulties with the derivatization prior to the gc-ms analysis. Many other derivatives were tried until it was found that the t-BDMS ether derivative of 16,16-dimethyl-PGB, showed a simple mass spectrum where the base peak represented a high percentage of the total
ble to degradation under gas chromatographic conditions. This degradation can be prevented by conversion to a O-methyloxime derivative (10). Therefore the U-methyloxime trimethylsibyl ether derivative and the O-methyloxime acetates were tried for quantitative analysis of l&16-PGEz. Both were aban-
2
Recovery I6,16-dimethyl-PGE, (%I
Added amount (ppfml)
I
5
TIME
6
t
7
MINUTES
FIG. 5. Recording of ion intensities at m/e 405 and m/e 409 after injection of about 20 ng of 16,16di[3,3,4,~*H~methyl-~E~ from a plasma sample. The lower tracings are from the least sensitive galvanometer, while the tracings from the middle and the upper galvanometer represent 10 and 100 times amplification.
116
STEFFENRUD
5 NANOG/
10 pG OLCARRIER
15
FIG. 6. Ratios of peak heights of 16,16-dimethylPGEl and 16,16-di[3,3,4,4-2H,]methyl-PGE, derivative (each injection 20 ng totally) versus composition of injected material.
ionization (9%). The usefulness of t-BDMS ether derivatives for gc-ms have also been described before (14- 19). It has also been shown that dehydration of E-prostaglandins to A-prostaglandins could occur during formation of trimethylsilyl ether derivatives with BSA-pyridine 1:l (21). In the case of 16,16-dimethyl-PGE, it was found advantageous to make t-BDMS derivatives and to use conditions that also caused dehydration into 16,16-dimethylPGB,. The mass spectrum of 16,16-dimethylPGBz ethyl ester, t-BDMS ether (Fig. 4) shows that the strong peak at m/e 409 represents as much as 9% of the total ionization. Together with excellent gas chromatographic properties this derivative gives a very high sensitivity with good precision for AVA analyses (Table 1). The precision showed at low levels is considerably better than that reported for other prostaglandins or prostaglandin analogs (6,2 1,22). Another advantage of preparing ?-BDMS derivatives of the E-prostaglandins is the short time needed for dehydration and derivatization, e.g., 1 h at 100°C. This should be compared to 12 h at room temperature for preparation O-methyloximes and another
AND LINCOLN
1 or 12 h for silylation and acetylation, respectively. Thus it should be possible to successfully employ t-BDMS ether derivatives for quantitative determination of other prostaglandins or prostaglandin analogs as shown for the major urinary metabolites of PGF,, and PGFza (17,18). The technique described has been successfully used for the quantitative determination of plasma levels of 16,16-dimethylPGEz in the human female during induction of abortion with vaginal suppositories containing this drug (S. Steffenrud, unpublished data, 1978). ACKNOWLEDGMENTS This study has been supported by research grants from The Expanded Programme of Research Development and Research Training in Human Reproduction of the World Health Organization. The skillful technical assistance of Eva Ohlsson is gratefully acknowledged. I am greatly indebted to Dr. Krister Green for valuable discussions and constructive criticism.
REFERENCES 1. Wiqvist, N., Martin, J. N., Bygdeman, M., and Green, K. (1975) Prostaglandins 9, 255. 2. Lundstrom, V., Bygdeman, M., Fotiov, S., Green, K., and Kinoshita, K. (1976) Prostagfandins 16, 167. 3. Martin, J. N., Bygdeman, M., Ramadan, M., Green, K., Leader, A., Lundstrom, V., and Wiqvist, N. (1976) Prosraglandins 11, 123. 4. Leader, A., Bygdeman, M., Gr6en, K., Martin, J. N., and Wiqvist, N. (1975) Prostaglandins 10, 357. 5. Magerlein, B. J., DuCharme, D. W., Magee, W. E., Miller, W. L., Robert, A., and Weeks, J. R. (1973) Prosraglandins 4, 143. In this reference 16,16-dimethyl-PGF,o is described as an oil. 6. Gr6en, K., Granstriim, E., Samuelsson, B., and Axen, U. (1973) Anal. Biochem. 54,434. 7. Analogous reactions for the PGFZa series have been described by Lin, C. H., Stein, S. J., and Pike, J. E. (1976) Prostaglandins 11, 377. 8. Nishizawa, E. E., Miller, W. L., Gorman, R. R., Bundy, G. L., Swensson, J., and Hamberg, M. (1975) Prostaglandins 9, 109. 9. Samuelsson, B. (1964)J. Biol. Gem. 239,409l.
QUANTITATIVE
ANALYSIS
OF 16,16-DIMETHYL-PROSTAGLANDIN
10. Green, K. (1969) Chem. Phys. Lipids 3, 254. 11. Green, K. (1971) Biochemistry 10, 1072. 12. Bergstram, S., and Sjdvall, J. (1951) Acta Chem. Stand. 5, 1267. 13. Green, K., and Samuelsson, B. (1%4) J. Lipid Res. 5, 117. 14. Corey, E. J., and Venkateswadu, A. (1972) .I. Amer. Chem. Sot. 94,619O. 15. Watson, J. T., and Sweetman, B. J. (1974) Org. Mass Speclrom. 9, 39. 16. Kelly, R. W. and Taylor, P. L. (1976) Anat. Chem. 48,465.
E2 FROM PLASMA
I17
17. Brash, A. R., DraBan, G. H., Glare, R. A. and Baille, T. A. (1976)Biochem. Sot. Truns. 4,706. 18. Brash, A. R., Baille, T. A., Glare, R. A. and Draffan, G. H. (1976) Biochem. Med. 16,77. 19. Quillam, M. A. and Westmore, J. B. (1977) Anal. Chem. SO, 59. 20. Sweetman, B. J., Friiiich, J. C. and Watson, J. T. (1973) Prostaglandins 3, 75. 21. Lincoln, F. H., Axen, U., Green, K., Ohlsson, H. andSamuelsson, B. (1976)AnaI. Left. 9,187. 22. Green, K. and Steffenrud, S. (1976) Anal. Biochem. 76,606.
BIOCHEMISTRY
Method
100,
109- 117 (1979)
for Quantitative Analysis of 16,16-Dimethyl-prostaglandin from Plasma Using Deuterated Carrier and Gas Chromatography-Mass Spectrometry
E,
S.~TEFFENRUD Department
of Chemistry,
Karolinska
Institutet,
S-104
01 Stockholm,
Sweden*
AND
F. H. LINCOLN Experimental
Chemistry
Research,
The Upjohn
Company,
Kalamazoo,
Michigan
49001
Received June 1, 1979 The preparation of 3,3,4,4-2H,16,16,-dimethyl-prostaglandin EZ (PGE2), 16,16-dimethyl5,6-didehydro-PGE,, and 5,6-3H,16,16-dimethyl-PGE, is described. These compounds have been used for development of a gas-liquid chromatography-mass spectrometry method for quantitative determination of corresponding nondeuterated prostaglandin. The method is based on addition of a known amount of carrier to the sample and after purification and derivation the ratio between the protium and deuterium form is measured in the mass spectrometer. With this technique 40 pg of 16,16-dimethyl-PGE, can be determined with a precision of ?2.6%.
trimester pregnancy with 16,16-dimethylPGE, have shown few gastrointestinal side effects, neither anti-emetic nor anti-diarrheic medication was needed (2-4). For a detailed study of the pharmacokinetics of 16,16-dimethyl-PGE, it was necessary to prepare the tritium-labeled and the deuterated analog and to develop a sensitive and accurate analytical method. The synthesis of tritium-labeled 16,16dimethyl-PGE,, deuterated 16,16-dimethylPGEz, and the development of an isotopedilution technique for gas-liquid chromatographic-mass spectrometric quantitation of 16,16-dimethyl-PGE, is described in this report.
A number of prostaglandin analogs have been synthesized during recent years. Among those which have been studied with regard to uterotonic potency, 16,16-dimethyl-prostaglandin E2 (PGE# was found to be the most potent analog when compared with six other prostaglandins and prostaglandin analogs as described earlier (1). Induction of abortion during first and second 1 Abbreviations used: PGE,, prostaglandin E2, 1la, 15 -dihydroxy -9 -keto -prost -5 -cis , 13 -trans -dienoic acid; PGE,, prostaglandin E,, 1la,15 dihydroxy-9keto-prost-13-trans-enoic acid; r-BDMS, tertbutyldimethyl silyl; AVA, accelerating voltage alternator; F1,, 9a,lla,l5 trihydroxyPGF,,, prostaglandin prost-13-trans-enoic acid; PGF,,, prostaglandin F,a, 9a,lla,15 dihydroxy - prost - 5- cis,l3 - trans - dienoic acid, PGB,, prostaglandin B,, 15-hydroxy-9-ketoprost-5-cis,8(12),13-trans-trienoic acid; BSA, N,O-bis(trimethylsilyl)acetamide,THP,l5-tetrahydropyranyl; TMS, trimethylsilyl ether; tic, thin-layer chromatography; gc-ms, gas chromatograph-mass spectrometry. * Present address: Department of Clinical Chemistry, Karolinska Sjukhuset. S-10401 Stockholm, Sweden.
MATERIALS
AND METHODS
Preparation of 3,3,4,4-2H,16,16,-DimethylPGE,
The synthesis is outlined in Fig. 1. Lactol I (5), 0.70 g was condensed with the yield 109
0003-2697/79/170109-09$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
110
STEFFENRUD
&zCOOH
~THP
AND
.
ZTHP
FIG.
~2COOH
ZH
III
LINCOLN
:H
IV 1. Synthesis
of 16,16-di[3,3,4,4-2H,]methyl-PGE,.
prepared from 1 g of 2,2,3,3-2H,4-carboxybutyl-triphenyl-phosphonium bromide (6). Chromatography over acid-washed silica gel using 30% ethyl acetate-70% hexane elution afforded 290 mg of II as an oil. Oxidation of 190 mg of this material by 0.2 ml of Jones’ reagent in acetone solution at - 15°C followed by THP ether hydrolysis with acetic acid-water at 40°C and chromatography over 15 g of acid-washed silica gel (75% ethyl acetate-25% hexane elution) gave 58 mg of product IV as a colorless oil. The NMR spectrum (CDC&) showed a sharp singlet at 6 2.3 for the protons attached to carbon 2. Otherwise the spectrum was similar to nondeuterated 16,16-dimethyl-PGE, (5). For the mass spectrometric determination of the deuterium content the O-methyloxime acetyl derivative of the methyl ester was prepared. Repetitive electric scanning over a narrow mass range around the molecular ion gave
The synthesis is outlined in Fig. 2. Crystalline 16,16-dimethyl-PGF,a V (mp 8283°C from acetone-hexane, [alo + 40”, c = 1.0736 in 95% EtOH) (5) was protected as the tribenzoate methyl ester and con-
vetted via bromination, dehydrobromination, and deprotection to the corresponding 5-yne compound. VI (7); [a], + 24”, c = 0.8465 in 95% EtOH; NMR (d6 acetone 6) 5.56 (m, 2H, C,,,,, vinyl), 5.1 (s, 4H, 3XOH + COOH), 3.65-4.4 (m, 3H, C,C,, and C,,H), 1.28 (s), 0.88 (s), and 0.82 (s); mass spectrum (tetra TMS) M+ (668) not observed, 653 (M+-15), 569 (M+-C,H,,), 479 (569+-TMSOH); infrared (thin film cm-‘) 3380, 2960, 2920, 2860, 2640, 2220 (W, C=C), 1710, 1240, 1160, 1110, 1085, 1055, 1020, 995, and 975. The lj-tetrahydropyranyl ether was prepared after protection of the 9- and 1I-hydroxy groups as a boronate ester (8), the protecting group removed and the 11-hydroxy group converted to the t-butyl dimethylsilyl derivative VII (7). Oxidation with Jones’ reagent and hydrolysis of the protecting groups afforded 16,16-dimethyl-5,6-didehydro-PGE, VIII as a pale yellow oil slightly more polar on tic (A-IX, Rf 0.37) than 16,16-dimethyl-PGE,; NMR (CDCl, 6) 5.97 (s, 3H, 2XOH + COOH), 5.7 Cm, 2H9 G14 vinyl), 3.7-4.3 (m, 2H, C,, and C,, H), 1.28 (s), 0.9 (9H). Mass spectrum (tri TMS) weak M+ 594, M+CH, = 579.3336. Calcd for C3,,H5&05 = 579.3357. Also 495 (M+-C,H&, 489 (579+-TMSOH), 405 (495+-TMSOH), 351 (495+-o=cOTMS), 261 (351+TMSOH), 117 (CO,TMS+); infrared (thin
QUANTITATIVE
ANALYSIS
OF
16,16-DIMETHYL-PROSTAGLANDIN
film)
2. Synthesis
111
of 16,l~dimethyl-S,~didehydro-ME*.
3420, 2960, 2940, 2860, 2660, 2240
(CsC, weak), 1745, 1715 sh, 1245, 1160, 1080, 1015, 1000, and 975 cm-‘. Preparation PGEz
PLASMA
VIII
VII FIG.
Ez FROM
of5,6-3H216,16-Dimethyl-
5,6-3H~16,16-Dimethyl-PGE~ was prepared by catalytic hydrogenation of 16,16dimethyl-5,6-did~hydro-PGE~ using tritium gas. One milligram of 16,lQdimethyl-5,6didehydro-PGE, was dissolved in 0.5 ml of methanol and 1 mg of Lindlars catalyst was added. The solution was stirred under tritium atmosphere for 15 min as described earlier (9) and finally filtered and left to stand in room temperature for about 60 min. The methanol was evaporated and about 200 pg of unlabeled 16,16-dimethyl-PGE~ was added. The material was purified by reversed phase pa~ition chromatographies using system C-50. The yield was about 2% (based on added tritium gas). About 7.5 x 10 dpm of this material was added to 25 pg of 16,16-dimethyl-PGE,. The O-methyloxime acetyl derivative of the methyl ester was prepared as described before (10) and subjected to oxidative ozonolysis as described earlier (11). After treatment of the reaction products with diazomethane, gas chromatography with simultaneous detection of radioactivity was performed. When the sample was injected on a 1% SE-30
column at 170 and 23O”C, all radioactivity seen was eluted with the solvent peak. At the lower temperature an unlabeled compound with ac - value of 16.1 appeared. The mass spectrum of the material in this peak gave an ion at m/e 301 (141) and prominent ions at m/e 270 (M-31), m/e 241 (M-60), mle 239 (M-2 x 31), m/e 227 (M-74), m/e 183 (M-2 x 59), m/e 167 (M-(74 + 60)), and m/e 149 (M-(59 + 3 x 31) base peak) indicating that this material was due to compound B in Fig. 3. If any of the double bonds in A5 or Al3 positions in the tritium-labeled 16,16dimethyl-PGE, structure would have been reduced with tritium gas any of compounds C, D, and E, should have been formed. The calculated c - values of those are 21,2 1, and 26, respectively. Since no radioactive product of these c- values was found the product of this reduction must be 16,16di[5,6-3H~]methyl-PGE~. Preparation of [3,3,#,4JHJ[5,C3H,]Labeled 16,16-Dimethyl-PGE,
5,6-3H,16,16-Dimethyl-PGEB was added to 600 pg of 3,3,4,4-2H,16,16-dimethylPGE, to give a specific activity of 116.5 @/mol. Thin-layer chromatography of this compound showed an& = 0.46. No radioactive impurities could be detected upon radio-gas
112
STEFFENRUD
AcO
H
AND LINCOLN
OAc
0
E
3. 16,16-Dimethyl-FGE, (A) and compounds formed by oxidative ozonolysis of the possible products after the catalytic hydrogenation of 16,16-dimethyl-5,6-didehydro-PGE,. FIG.
chromatography of the 0-methyloxime trimethyl&y1 ether derivative of the methyl ester (1% SE-30 on Chromosorb W, 230°C). The radioactive purity was determined to be greater than 98%. Chromatographic Methods
Reversed phase partition chromatography was performed as described earlier (12), using system C-48 (moving phase, methanol:water 144: 156 (v/v); stationary phase, chloroform:isooctanol, 15: 15 (v/v)). Silicic acid chromatography was carried out on Silic AR CC4, 100-200 mesh, Mallinckrodt analytical reagent. The columns (0.25 g) were eluted with mixtures of ethyl acetate in benzene (1:9,2:8, and 4:6) or ether in hexane (0:lO and 1:9) followed by methanol. Five milliliters of each solvent mixture was used. Thin-layer chromatography was run on silica gel-coated glass plates as described before (13). The solvent system was ethyl acetate:2,2,4-trimethylpentane:acetic acid: water, 110:20:30: 100 (v/v/v/v). Gas-liquid chromatography with simultaneous registration of mass and radioactivity was performed on a Barber-Colman gas chromatograph Model 5000. The column was a 2 m x 5 mm i.d. silanized glass tubing packed with 1% SE-30 on Chromosorb W, HP 100- 120 mesh (Varian Aerograph, Walnut Creek, Calif.).
The c - values were calculated in relation to normal fatty acid methyl esters. Gas -liquid chromatography -mass spectrometry. The mass spectrometric quantita-
tions were performed with a combined gas-liquid chromatograph-mass spectrometer, LKB 9000A. The gas-liquid chromatographic column (1.5 m x 2 mm, glass) was packed with 1% Dexsil 300 on Chromosorb W DMCS, 80- 100 mesh (Analabs., North Haven, Conn.). The conditions were: column temperature: 26O”C, carrier gas flow about 20 ml/min, electron energy: 25 eV, and trap current: 120 PA. An LKB AVA-unit was used. The two ions to be measured were intermittently focused every second. As a control of the analytical procedure, a standard was injected every fourth run in order to check the focusing (6). Preparation of Derivatives for Gas -Liquid Chromatography and Mass Spectrometry
The methods used for preparation of methyl esters 0-methyloxime trimethylsilyl ether and acetyl derivatives have been described earlier (10). Ethyl esters were prepared by dissolving the compounds in 0.5 ml of ethanol and treatment with diazoethane in ether. After evaporation, the ethyl ester was dissolved in 100 ~1 of t-butyldimethylchlorosilane/ imidazole/dimethylformamide (1 mM/2.5
QUALITATIVE
ANALYSIS
OF 16, I~DIMETHYL-PROSTAGLANDIN
& FROM PLASMA
f 13
mM/l ml) Applied Science Laboratories The methanol fractions that contained radioactivity were evaporated and subInc., State College, Pa.) and heated at 100°C jected to reversed-phase partition chromafor 1 h as described before (14). This treatment converted the ethyl ester of 16,16- tography using 4.5-g columns and system dimethyl-PGE, to the ethyl ester t-butyl C-48. 16,16-Dimethyl-PGE~ appeared at V, dimethylsilyl ether of l&16-dimethyl-PGB,. = 150 ml. The fractions containing radioAfter cooling, the reaction mixture was activity were combined and after evaporaextracted three times with 0.5 ml of hexane. tion, the residue was dissolved in ethanol The extracts were combined, reduced to a and treated with diazoethane. Further purismall volume, and passed through a silicic fication was obtained by silicic acid chromaacid column (0.25 g) and the column was tography using stepwise increasing concenwashed with hexane to a total volume of trations of ethyl acetate in benzene. After 5 ml. The l&16-dimethyl-PGB* derivative evaporation of the solvent, the residue was was eluted from the column with 5 ml of treated with t-butyldimethylchlorosilanel imidazole/dimethylform~ide and purified ether in hexane (1:9). as described above. The derivatized comSolvents pound was dissolved in a small volume of hexane before analysis in the mass specEther, methanol, chloroform (all analytitrometer. The overall recovery of the purical grade, Merck) ethylacetate, and hexane fication and derivatization procedures was (pesticide grade, Fisher) were used without about 40%. further puri~cation. The benzene (analytical grade, Merck) RESULTS was glass distilled. Quuntitative Analysis of 16,16-DimethylRecovery from Plasma PGE, For practical reasons, titrated blood bank plasma was used in the recovery tests. One plasma pool of 150 ml was divided into three equal parts. One was used as the blank. To the other two parts [5,6-3H2] 16,16-dimethylPGE, (1.3 ,&i/pmol) was added to give concentrations of 104 and 520 pg/ml, respectively (see Table 2) and equilibrated for 5 min. Each of the three parts was further divided into 5 x lo-ml samples and transferred into tubes containing 1000 ng [3,3,4,43HZ]- [5,6--“H&labeled 16, l~dirne~yl-~E~. The plasma was equilibrated with the carrier for 10 min. The plasma was acidified to pH 4 with 2 M HCl and poured through an XAD-2 column (1.5 x 15 cm). The column was washed with water until neutral effhrent and eluted with 4 x 15ml of methanol. The yield of radioactivity in the methanol fractions was 90-95% of added amount.
The mass spectrum of the t-BDMS derivative of the ethyl ester of [3,3,4,4-2H,]-[5,63H,]-labeled 16,16-dimethyl-PGE, is shown in Fig. 4. The ion of m/e 409 is due to elimination of 99 mass units from the molecular ion, probably by cleavage between carbon 15 and 16. Since this ion is of high intensity and the ion at m/e 405 where the corresponding ion from the protium form appears, is of low intensity these ions were selected for measurements during the analysis. The ratio between the recorded ion intensities at m/e 405 and 409 was 12.3 2 0.201 1000 (n = 5) when the t-BDMS derivative of the ethyl ester of [3,3,4,4-2H,]-[5,6-3H,]labeled-16,16-dimethyl-PGE, was analyzed using the AVA equipment. 16,16-Dimethyl”PGE~ was added to [3, 3,4,~zH~]-[5,6-~H~]-labeled-16,16-dimethylPGE, in ratios l:lOOO, 2:1000, 4: 1000, 8:1000, and 16:lOOO and the mixtures were derivatized as described above.
STEFFENRUD
AND LINCOLN
r
M-9! LO9
Y 10
M-57
100
150
200
250
300
350
LOO
1 LSf 1 ,L.,.,.,l. 450
5oM [I ,.,., 500
I
,
“le
FIG. 4. Mass spectrum of the tert-butyl dimethylsilyl ether derivative of the ethyl ester of 16,16di[3,3,4,4-eH,]methyl-PGE~. Conditions: electron energy: 22.5 eV, trap current: 120 PA.
Aliquots of about 20 ng of the different mixtures were analyzed in the mass spectrometer equipped with the AVA unit, and the ion intensities at mle 405 and 409 were recorded. The slope of the standard curve after injection of 20 ng from each of the mixtures was close to 45” (Fig. 6). The precision and accuracy of the method determined by repetitive injections of 20 ng of the different standard mixtures is shown in Table 1. Since the precision at 1: 1000 is 18.9% and 20 ng of l&16-dimethyl-PGE, of this mixture was injected each time, the limit of detection with the equipment used is lower than 20 pg (0.05 pmol).
rier, the samples were worked up as described under Material and Methods. The analytical data and recoveries of added 16,16-dimethyl-PGEB are given in Table 2. The level of added 16,16-dimethyl-PGE, (column 2, Table 2) in each plasma sample was determined by one single injection of 20 ng of the isolated compound (carrier plus protium form) and was not calculated from mean values after several determinations. This presentation was chosen in order to give a true picture of the precision obtained. A typical AVA run of a plasma sample is shown in Fig. 5, containing 100 pg/ml 16,16dimethyl-PGE,.
Recovery of 16,16-Dimethyl-PGE, from Plasma
DISCUSSION
Unlabeled 16,16-dimethyl-PGE, was added to titrated plasma and after addition of carTABLE PRECISION
Compound 16,16-Dimethyl-PGE,
DETERMINED
BY REPETETIVE
Prostaglandins of the PGE type possess a /3-ketol system which make them suscepti1 ANALYSIS
OF STANDARD
Injected deuterated carrier Ox)
Total H-form injected (PP)
H-Form injected m
20 20 20 20 20 20
0 20 40 80 160 320
0 0.10 0.20 0.40 0.80 1.60
MIXTURES
Percentage H-form found (mean r SD) (N = 5) 0.106 0.233 0.403 0.846 1.612
0 lr + -t ” +
18.9 2.6 2.2 2.5 1.4
QUANTITATIVE
ANALYSIS
OF 16,16-DIMETHYL-PROSTAGLANDIN TABLE
E2 FROM PLASMA
115
2
RECOVERY OF ADDED 16, 16-DIMETHYL-PGEz H/D (%I
Plasma f&&N
0.066 0.007 -0.039 0.064 -0.023 0.233 0.212 0.175 0.225 0.209 1.083 1.065 0.949 1.048 1.00
33 4 -20 32 -12 118 108 88 114 106 548 538 480 530 506
Mean 2 SD (pgiml)
7 k 24
0
107 2 12
104
520 + 27
520
3
i
1 RETENTION
113 104 85 110 102 105 103 92 102 97
doned due to either unsuitable fragmentation in the mass spectrometer or difficulties with the derivatization prior to the gc-ms analysis. Many other derivatives were tried until it was found that the t-BDMS ether derivative of 16,16-dimethyl-PGB, showed a simple mass spectrum where the base peak represented a high percentage of the total
ble to degradation under gas chromatographic conditions. This degradation can be prevented by conversion to a O-methyloxime derivative (10). Therefore the U-methyloxime trimethylsibyl ether derivative and the O-methyloxime acetates were tried for quantitative analysis of l&16-PGEz. Both were aban-
2
Recovery I6,16-dimethyl-PGE, (%I
Added amount (ppfml)
I
5
TIME
6
t
7
MINUTES
FIG. 5. Recording of ion intensities at m/e 405 and m/e 409 after injection of about 20 ng of 16,16di[3,3,4,~*H~methyl-~E~ from a plasma sample. The lower tracings are from the least sensitive galvanometer, while the tracings from the middle and the upper galvanometer represent 10 and 100 times amplification.
116
STEFFENRUD
5 NANOG/
10 pG OLCARRIER
15
FIG. 6. Ratios of peak heights of 16,16-dimethylPGEl and 16,16-di[3,3,4,4-2H,]methyl-PGE, derivative (each injection 20 ng totally) versus composition of injected material.
ionization (9%). The usefulness of t-BDMS ether derivatives for gc-ms have also been described before (14- 19). It has also been shown that dehydration of E-prostaglandins to A-prostaglandins could occur during formation of trimethylsilyl ether derivatives with BSA-pyridine 1:l (21). In the case of 16,16-dimethyl-PGE, it was found advantageous to make t-BDMS derivatives and to use conditions that also caused dehydration into 16,16-dimethylPGB,. The mass spectrum of 16,16-dimethylPGBz ethyl ester, t-BDMS ether (Fig. 4) shows that the strong peak at m/e 409 represents as much as 9% of the total ionization. Together with excellent gas chromatographic properties this derivative gives a very high sensitivity with good precision for AVA analyses (Table 1). The precision showed at low levels is considerably better than that reported for other prostaglandins or prostaglandin analogs (6,2 1,22). Another advantage of preparing ?-BDMS derivatives of the E-prostaglandins is the short time needed for dehydration and derivatization, e.g., 1 h at 100°C. This should be compared to 12 h at room temperature for preparation O-methyloximes and another
AND LINCOLN
1 or 12 h for silylation and acetylation, respectively. Thus it should be possible to successfully employ t-BDMS ether derivatives for quantitative determination of other prostaglandins or prostaglandin analogs as shown for the major urinary metabolites of PGF,, and PGFza (17,18). The technique described has been successfully used for the quantitative determination of plasma levels of 16,16-dimethylPGEz in the human female during induction of abortion with vaginal suppositories containing this drug (S. Steffenrud, unpublished data, 1978). ACKNOWLEDGMENTS This study has been supported by research grants from The Expanded Programme of Research Development and Research Training in Human Reproduction of the World Health Organization. The skillful technical assistance of Eva Ohlsson is gratefully acknowledged. I am greatly indebted to Dr. Krister Green for valuable discussions and constructive criticism.
REFERENCES 1. Wiqvist, N., Martin, J. N., Bygdeman, M., and Green, K. (1975) Prostaglandins 9, 255. 2. Lundstrom, V., Bygdeman, M., Fotiov, S., Green, K., and Kinoshita, K. (1976) Prostagfandins 16, 167. 3. Martin, J. N., Bygdeman, M., Ramadan, M., Green, K., Leader, A., Lundstrom, V., and Wiqvist, N. (1976) Prosraglandins 11, 123. 4. Leader, A., Bygdeman, M., Gr6en, K., Martin, J. N., and Wiqvist, N. (1975) Prostaglandins 10, 357. 5. Magerlein, B. J., DuCharme, D. W., Magee, W. E., Miller, W. L., Robert, A., and Weeks, J. R. (1973) Prosraglandins 4, 143. In this reference 16,16-dimethyl-PGF,o is described as an oil. 6. Gr6en, K., Granstriim, E., Samuelsson, B., and Axen, U. (1973) Anal. Biochem. 54,434. 7. Analogous reactions for the PGFZa series have been described by Lin, C. H., Stein, S. J., and Pike, J. E. (1976) Prostaglandins 11, 377. 8. Nishizawa, E. E., Miller, W. L., Gorman, R. R., Bundy, G. L., Swensson, J., and Hamberg, M. (1975) Prostaglandins 9, 109. 9. Samuelsson, B. (1964)J. Biol. Gem. 239,409l.
QUANTITATIVE
ANALYSIS
OF 16,16-DIMETHYL-PROSTAGLANDIN
10. Green, K. (1969) Chem. Phys. Lipids 3, 254. 11. Green, K. (1971) Biochemistry 10, 1072. 12. Bergstram, S., and Sjdvall, J. (1951) Acta Chem. Stand. 5, 1267. 13. Green, K., and Samuelsson, B. (1%4) J. Lipid Res. 5, 117. 14. Corey, E. J., and Venkateswadu, A. (1972) .I. Amer. Chem. Sot. 94,619O. 15. Watson, J. T., and Sweetman, B. J. (1974) Org. Mass Speclrom. 9, 39. 16. Kelly, R. W. and Taylor, P. L. (1976) Anat. Chem. 48,465.
E2 FROM PLASMA
I17
17. Brash, A. R., DraBan, G. H., Glare, R. A. and Baille, T. A. (1976)Biochem. Sot. Truns. 4,706. 18. Brash, A. R., Baille, T. A., Glare, R. A. and Draffan, G. H. (1976) Biochem. Med. 16,77. 19. Quillam, M. A. and Westmore, J. B. (1977) Anal. Chem. SO, 59. 20. Sweetman, B. J., Friiiich, J. C. and Watson, J. T. (1973) Prostaglandins 3, 75. 21. Lincoln, F. H., Axen, U., Green, K., Ohlsson, H. andSamuelsson, B. (1976)AnaI. Left. 9,187. 22. Green, K. and Steffenrud, S. (1976) Anal. Biochem. 76,606.