Measurements on Natural Specimens. In Table 111, the mean measured plasma oxypurine (hypoxanthine plus xanthine) level for five normal individuals is 0.17 f 0.08 mg/100 ml in excellent agreement with a normal range of 0.1-0.3 mg/100 ml measured by enzymatic procedures (11). The corresponding figure for uric acid of 2.5 f 0.7 mg/100 ml cannot be compared with the normal range of 4-6 mg/ 100 ml. The enzymatic method measures the total uric acid and sodium urate in plasma but a t blood p H (7.4), almost all the uric acid is present as the sodium salt and, hence, undetectable by mass spectrometry. However there is good evidence that certain of the plasma proteins can form loosely bound complexes with uric acid. For normal individuals, the concentration of uric acid bound in this way has recently been reported (13) to be 2.9 =k 0.4 mg/100 ml whicll compares well with the figure obtained by mass spectrometry. Until further work is carried out to elucidate the nature of the interaction between uric acid and plasma protein, however, this apparent agreement should not be overstressed. No alternative methods of measuring oxypurine or uric acid levels in tissue have been reported and there is some evidence ( 2 ) that they are not necessarily related in a simple way to the corresponding levels in plasma. Nonetheless, the data presented in Figure 5 show the expected variation in muscle uric acid concentration between the three groups of in2 dividuals examined. The measured normal range of 26 ppm is increased in gout to between 35 and 150 ppm. Treat-
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(13) J. R. Klinenberg and 1. Kippen, J. Lab. Clin. Med., 7 5 , 503 (1970).
ment with the drug allopurinol which reduces the plasma uric acid concentration and alleviates the clinical manifestation of the disease also causes a marked lowering of the amount of uric acid in the tissue. The present results demonstrate the feasibility of performing multicomponent quantitative analyses. at the parts per million level o n a few milligrams of tissue and plasma by high resolution mass spectrometry without the necessity of tedious and time-consuming extractive procedures. Typically, data from about 15-20 specimens, plus calibrations, can be acquired in a working day. The time taken in processing these data manually is normally about 24 man-hours. With automatic digital computing facilities, this time would be considerably reduced. Applications of this method to other compounds in a variety of tissues are being actively pursued. ACKNOWLEDGMENT
We thank E. F. Scowen for his continuing interest and support, our colleagues of the Dunn Laboratories for the provision of blood specimens, A. Cox of the Hammersmith Hospital for the provision of normal muscle specimens, and R. W. E. Watts of the M R C Clinical Research Centre, Harrow, for the provision of gouty muscle specimens and for many helpful discussions. RECEIVED for review March 15, 1971. Accepted June 25, 1971. Presented in part a t the Trienial Mass Spectrometry Conference, Brussels, 1970. The provision of research grants by the Governors of St. Bartholomew’s Hospital and a grant toward the purchase of the mass spectrometer from the Fleming Memorial Trust are gratefully acknowledged.
Interactions between Rock and Organic Matter Esterification and Transesterification Induced in Sediments by Methanol and Ethanol Patrick Arpino a n d G u y Ourisson Laboratory Associated with the CNRS-lnstitut de Chimie, Esplanade, 67-Strasbourg, France Transesterification and esterification of waxes and mono- and dicarboxylic acids contained in clay sediments have been observed after extraction with solvent mixtures containing methanol or ethanol; thls leads to changes in the quantitative analysis of the alcohols and the free acids. These modifications are due to the catalytic activity of clays, especially montmorillonite. Particular attention must therefore be paid to extraction methods employed in organic geochemistry for the study of alcohols, esters, and fatty acids in sediments. THEORGANIC MATTER found in sediments, which is considered to be more abundant than that present above the surface of the Earth ( I ) , contains the residues of biological activity trapped in the mineral sediments. The evolution of this sedimentary organic material, its “diagenesis,” can be due to the action of microorganisms in the first stages of burial. However, it is (1) J. M. Hunt, Proceedings of the International Oil Conference, Budapest, Hungary, 1962. 1656
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entirely a n abiotic process during the very long subsequent periods. At the chemical level, this diagenesis can imply various reactions, for example reductive hydrogenolysis (2), dehydroall of which have genation (3), oxidation (4), and cracking (3, been encountered in examples studied in this laboratory. These diagenetic reactions can sometimes be simulated by replacing time with a n increase in temperature, i.e., by heating the rock prior to extraction (5). I n every case, interpretation of the results requires strict control over extraction methods. Some observations on the (2) H. Knoche, P. Albrecht, and G. Ourisson, Angew. Chem., 80, 666 (1968); Angew. Chem. Znt. Ed. Eng., 7, 631 (1968). (3) P. Albrecht, Thkse: “Constituants Organiques de Roches Sdirnentaires,” Strasbourg (1969) and P. Albrecht and G. Ourisson, Angew. Chem., 83, 221 (1971); Angew. Chem. Int. E d . Eng. 10, 209 (1971). (4) G . Mattern, P. Albrecht, and G. Ourisson, Chem. Commun. 1970, 1570. ( 5 ) P. Albrecht and G. Ourisson, unpublished work, 1971.
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
intervention of a clay, acting as a catalyst during mild extraction processes, are described below. Esterification and Transesterification Catalyzed by Montmorillonite. A lacustrine Eocene sediment (50 million years) from Bouxwiller (Alsace, France), was extracted to study its soluble organic compounds (6). The rock contained 25% of total organic carbon and the argillaceous fraction consisted of 60 % montmorillonite. Different solvents were used during the various extractions of the same rock, using either a soxhlet for 72 hours, or sonication (3 X 20 mn at 4OOC) followed by centrifugation and decantation. In all cases, the total extract obtained was fracticnated on a silica gel column, using solvents of increasing polarity. The fractions isolated were analyzed by IR, MS, a n d G L C . Many compounds were extracted in this way, especially hydrocarbons (6) and pentacyclic triterpanes (7). However, various modifications affecting esters and fatty acids can be attributed to the catalytic role of the rock clays. (1) When chloroform and ultra-sonics were employed, waxes and esters of alcohols and aliphatic acids having 22 t o 34 carbon atoms were isolated. Free acids and alcohols, in the same proportions as those contained in the waxes, and dicarboxylic acids were also obtained from the total extract. (2) When a benzene--methanol mixture 3/1 and ultrasonics were used, more fractions of methyl monoesters and methyl diesters were isolated. However, less wax and more alcohols were found than before. (3). When a benzene-ethanol mixture 3/1 was used, methyl esters were no longer found, but a n equivalent quantity of ethyl esters was isolated. (4). Sonication does not play a role in these modifications, although it can cause dissociation of organic solvent molecules (8). Experiment 2 was repeated in a soxhlet with a benzene-methanol mixture (3/1) for 72 hours. After separation, it was found that the waxes had been quantitatively transesterified to give methyl esters and alcohols. ( 5 ) . These reactions do not proceed in the absence of rock. Thus, 200 mg of a mixture of waxes and fatty acids, isolated during Experiment 1, were dissolved in 50 ml of benzene-methanol mixture (3/1). After the application of ultra-sonics for 45 min at 40°C, no methyl esters were detected. DISCUSSION
Transesterification reactions have been carried out using molecular sieves, which are synthetic alumino-silicates. These reactions proceed with displacement of the equilibrium by the retention of the alcohol, capable of reversing the reaction, in the molecular sieves (9). Montmorillonite, a very widespread clay in many recent sediments (IO), can include various organic compounds such as amines ( I I ) , alcohols (12), or aliphatic acids (13). In our case, the waxes could have been hydrolyzed; this would have given fragments capable of being retained either through their functional group or by inclusion of their aliphatic chain. (6) P. Arpino, P. Albrecht, and G. Ourisson, C. R. Acad. Sci. Paris, 270, 1763 (1970). (7) P. Arpino, unpublished work, 1971. (8) A. Weissler, I. Pecht, and M. Anbar, Science, 150, 1288 (1965). (9) D. P. Roelofsen, J. W. M. de Graaf, J. A. Hagendoorn, H. M.
Verschoor, and H. Van Bekham, Rec. Trav. Chim. Pays Bas, 88, 193 (1970). (10) G. Millot, “G6ologie des argiles,” Mason, Paris, 1%3, p 378. (11) A. Weiss, “Organic Geochemistry,” G . Eglinton and M. T. J. Murphy, Ed., Springer-Verlag, New York, N. Y.,1969, p 737. (12) A . K. Galwey, Chem. Commun., 1969, 577. (13) G. W. Brindley and W . F. Moll Jr., Amer. Mineral., 50, 1355 (1965).
However, the inclusion could not have made possible the fatty acid-methyl ester transformation, contrary t o the above observations. Alternatively, montmorillonite could play the role of a Lewis acid in these organic reactions (14). Excess methanol or ethanol used in the solvent could displace the equilibrium (acid-catalyzed by the clay), in favor of a n esterified or transesterified product. From the organic geochemical point of view, it must be considered that the use of solvents containing alcohols may lead to modified substances, according to the degree of catalytic activity in the rock studied. In a study of alcohols of geological origin, Sever and Parker (15) obtained, from sediments, fatty alcohols in abnormally high quantities, considering their content in the biosphere. The chain lengths were comparable to those of the acids present in the same sediments. Extraction using methanol with the application of ultra-sonics did not change the extract in their case and did not produce the alcohols isolated, from other organic compounds. In this case, therefore, the use of methanol seemed permissible. Waxes, which are very widespread in the plant kingdom in the epicuticular layer of leaves (16), are probably stable in the geological conditions present in many sediments. Since they are not changed during extraction, they are difficult to detect by GLC, for they have from 40 to 70 carbon atoms. In IR, the carbonyl group of the ester at 1740 cm-l is poorly visible, also because of the length of the chain. They may therefore be easily missed. In our laboratory, the use of methanol for extraction of argillaceous sediments always increases the amount of free alcohols, in direct relation to the disappearance of the waxes. Precautions must be taken for the study of traces of organic material in rocks poor in carbon. H a n et a/. observed, amongst the contaminants included in the external layers of a meteorite (17), the presence of methyl palmitate in the fraction eluted with benzene and of palmitic acid among the free acids. They had extracted the meteorite with a mixture of benzene-methanol (3/1). The methyl palmitate isolated was doubtless not initially present. For quantitative analysis of fatty acids in sediments, methanol is generally used for the extraction. The acids are either retained o n a column of silica impregnated with potassium hydroxide (18) or eluted from a silica column with a polar solvent. There is nevertheless a risk of losing a portion of these acids if they have been previously methylated during the extraction, because the two methods only allow separation of acidic compounds. Our lack of knowledge about the types of interactions between rock and organic matter explains the unexpected difficulties often encountered in organic geochemistry. This same ignorance regarding the precise conditions experienced by buried organic material, for periods of millions of years, must warn against a noncritical transposition of analytical methods and mechanisms to geochemistry, even though they are accepted in classical organic chemistry. RECEIVED for review March 19, 1971. Accepted May 25, 1971.
D. H. Solomon, Clays Clay Minerals, 16, 31 (1968). J. Sever and P. L. Parker, Science, 164, 1052 (1969). G. Eglinton and R. S. Hamilton, ibid., 156, 1322 (1967). J. Han, B. R. Sirnoneit, A. L. Burlingame, and M. Calvin, Nature, 222, 364 (1969). (18) W. Van Hoeven, Ph.D. Thesis, “Organic Geochemistry,” University of California, Berkeley, Calif., 1969.
(14) (15) (16) (17)
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