Determination of Volatile Constituents of Human Blood and Tissue Specimens by Quantitative High Resolution Mass Spectrometry Walter Snedden and Robin B. Parker
Medical Professorial Unit, St. Bartholomew’s Hospital, London, E.C.1, England A technique utilizing multiple peak integrated ion current mass spectrometry at high resolution has been designed to enable the simultaneous determination of up to five compounds present in human blood plasma and muscle tissue. Apart from simple desiccation, no chemical extraction of the specimen or isolation of the compounds to be determined is required. The compounds are determined at the 10-100 ppm level to an accuracy of &fOoJo, with a consumption of only 5 mg of desiccated material for a duplicate analysis. The method i s illustrated by comparative estimations of purine metabolites in normal and gouty individuals. Where comparable data are available from alternative sources, the agreement with the mass spectrometric method is good.
THEQUANTITATIVE determination in blood and tissue of many substances of biological importance is hampered frequently by their insolubility or instability toward extractive procedures. Furthermore, since the amount of specimen available for analysis from human sources must be quite small (e.g., 10 mg of tissue or 0.5 ml of plasma), the problem of accurate determination of several compounds simultaneously at concentrations between 10 and 100 ppm becomes formidable. In an attempt to overcome these difficulties, high resolution mass spectrometry has been used to detect and measure small amounts of compounds directly in desiccated tissue and blood plasma. The considerations which determined the choice of a mass spectrometric method for this purpose were as follows: The technique was applicable to the determination of any compound in any substrate subject to the limitations below: the compounds should vaporize at not more than 350 O C ; the compounds should give characteristic ions which could be resolved from the background ions of the substrate; and the substrate should be capable of being freeze-dried without loss of the compounds of interest. Qualitative identification and quantitative determination of a number of substances could be performed simultaneously without the need to extract them from the substrate or purify them by any chromatographic procedure with consequent reduction in sample loss. A high speed of analysis and sample throughput could be achieved after the mass spectrometer had been adjusted and calibrated for a given series of compounds. In principle, one or more ion peaks which are characteristic only of each compound to be determined are selected from the total high resolving power spectrum of all the materials evaporated from the tissue or plasma. The intensities of these ions are then integrated over the lifetime of their parent compounds in the mass spectrometer inlet. Each integrated ion intensity then may be related to the absolute amount of the corresponding parent compound admitted to. the mass spectrometer. In this way, the high sensitivity of the direct insertion probe inlet may be fully utilized for quantitative measurements without complications due to sample fractionation. At the same time, the power of the high resolution
mass spectrometer to separate ions of a given atomic composition effectively isolates each substance of interest from the complex mixture of material evaporated from the specimen. Thus laborious and time consuming extractive and purification procedures are rendered unnecessary. Preliminary accounts of the application of this technique to the measurement of hypoxanthine and xanthine in skeletal muscle have been published already (I, 2). In this paper, experimental details for estimating up to five compounds simultaneously are presented together with a discussion of the accuracy attainable with particular reference to mixtures of the purines hypoxanthine I, xanthine 11, uric acid 111, and their isomers allopurinol IV and oxipurinol V. R
0
0
VI
The simultaneous determination of these five compounds is of importance in the study of gout and its treatment (3). Microscopic examination of skeletal muscle sections taken from gouty individuals has indicated ( 4 ) the presence of crystalline material attributable to certain of these substances. A discussion of the clinical aspects of these measurements will be published elsewhere. Quantitative mass spectral measurements on whole human tissue do not appear to have been reported previously by (1) R. B. Parker, W. Snedden, and R. W. E. Watts, Biochem. J., 115, 103 (1969). (2) Zbid., 116, 317 (1970). (3) “The Metabolic Basis of Inherited Disease,” J. B. Stanbury, J. B. Wyngaarden, D. S. Fredrickson, Ed. 2nd ed., McGrawHill, New York, N.Y., 1966, p 667. (4) R. W. E. Watts, J. T. Scott, R. A. Chalmers, L. Bitensky, and J. Chayen, Quart. J. Med., N.S., 40, 1 (1971).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
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J
i
(01
25
66
29
20
100
90
(L)
27
66
22
I9
IO0
94
Figure 1. Partial high resolution mass spectrum of substances evaporated from human muscle, showing peaks attributable to uric acid Relative intensities of uric acid peaks from muscle b. Relative intensities of same peaks from pure uric acid c. Nominal mass of multiplet a.
~~
~
Table I. Masses, Atomic Compositions, and Relative Intensities of the Characteristic Ions Used for Quantitative Analysis of Compounds I-V Atom mle comp. RI(1) RI(I1) RI(II1) RI(IV) RI(V) 52.01875 CaHzN . . . . . . . . . 14.6 30.7 54.0218" GH2Nn 13.8 4 3 . 2 73.5 . . . .. . 136.0385* CsHaNaO 100 ... . . . 100 ... 152.0334b CsHiNaOn . . . 100 . . . . . . 100 168 .0293b CsH4N403 . . . . . . 100 ... ... Internal standard m/e 5 5 from perfluorokerosine. Internal standard m/e 142 from 1-methylnaphthalene.
other workers. However Majer and Boulton (5) have published recently some preliminary estimations of tyramine in extracts of rat brain by an integrated ion-current technique. EXPERIMENTAL Materials. Specimens of normal skeletal muscle were obtained from patients undergoing minor surgical operations. None of these subjects had suffered previously from any disease of purine metabolism. Specimens from untreated and allopurinol-treated gout patients were the same as those obtained earlier for microscopic investigation (4). The purpose of these biopsies was explained fully to each patient and his free consent to the procedure was obtained. Tenmilliliter samples of blood were withdrawn from healthy volunteers. Each specimen was immediately transferred to a heparinized container, centrifuged at 4 "Cand the plasma layer separated for investigation. The specimens were dehydrated and 2-3 mg portions admitted to the mass spectrometer (Varian MAT SM1) as already described (I). ( 5 ) J. R. Majer and A. A. Boulton, Nature, 225,658 (1970). 1652
'
Qualitative Observations. Before attempting quantitative measurements, it was necessary to ensure that the characteristic ions, on which subsequent quantitative analyses of I-V were based (See Table I), were completely separated from background ions of the same nominal mass arising from the fragmentation of other constituents of the specimen. In Figure 1, the relative intensities of the ions having the correct atomic composition to be derived from uric acid are compared, at a resolving power of 20,000 (10% valley definition), with a standard mass spectrum. With the exception of mle 69 which was not used for quantitative measurements, the agreement was satisfactory. This indicated further that interference from unresolved ions of almost the same precise mass but derived from sources other than uric acid was negligible. Similar conclusions could be drawn about the characteristic ions of the other four compounds in this study. Quantitative Measurements. To obtain abundance data of high sensitivity, independent of vaporization temperature and inlet fractionation, for a series of ions from the same sample, a technique involving multiple peak selection and ion current integration was devised. The latter part is a development of the methods used at low resolving power for the measurement of small amounts of organometallic compounds (6, 7) and for the determination of the absolute sensitivity of the mass spectrometer (8). Instrumentation. The first requirement was fulfilled by a five-channel peak preselector which allowed the ion accelerating voltage to be cycled between five different values selected with reference to a standard. Figure 2 shows a simplified diagram of the arrangement which consists essentially of five peak-matching circuits linked to a multiway rotary switch SI. Adjustment of VR1-VR6 allowed any mass within 30% (6) A. E. Jenkins and J. R. Majer, Tulanta, 14, 777 (1967). (7) B. R. Kowalski, T. L. Isenhour, and R. E. Sievers, ANAL. CHEM., 41, 998 (1969). (8) K. E. Habfast and K. H. Maurer, ASTM E-14, 14th Conference on Mass Spectrometry and Allied Topics, Dallas, Texas, 1966.
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
of the references to be preselected at resolving powers of up to 20,000 without loss of ion focusing. Setting Up Procedure. After the mass spectrometer resolving power was adjusted to some suitable value between 15,000 and 20,000, a mixture of the substances to be determined was admitted. Each channel of the peak selector was then adjusted to display on the mass spectrometer oscilloscope one of the ion beams to be measured (See Table I). The ion accelerating voltage sweep was then decreased to exclude all multiplet peaks except that of interest. At a sweep speed of 2 cmlsec, this display could be recorded on a fast response pen recorder. Measurement Procedure. After admission of the sample, the vaporization temperature was raised until the most volatile component (hypoxanthine at 230 "C)began to appear. Recording was then initiated, each channel being selected in turn by manual adjustment of S1 during the oscilloscope fly-back. T o avoid excessive tailing of the less volatile uric acid, the vaporization temperature was increased during the run as shown in Figure 3 to a maximum of 300 "C. Recording was continued until all the material of interest had evaporated. Typical evaporation profiles for the molecule ions of hypoxanthine, xanthine and uric acid evaporated from normal muscle are shown in Figure 3. The areas of each profile could then be related to the absolute concentration of its precursor molecule (2). Allopurinol and oxipurinol were distinguished from their equally volatile structural isomers hypoxanthine and xanthine, respectively, on the basis of the differing relative intensities of m/e 52.0187 and 54.0218 for the two pairs of compounds (9). For specimens containing all five components, profiles of these fragments were combined with those of the molecule ions above to yield a set of linear simultaneous Equations 1-5 from which the concentrations of I-V were calculated. c1w3 = AIM (1) c2w2 c3wS = A152 (2) C4wl CSW4 = A136 (3) Cswi C7W2 Caw3 = As4 (4)
+ + + + Cow4 + Ciows
= As2
(5)
(9) R. A. Chalmers, R. B. Parker, H. A. Simmonds, W. Snedden, and R. W. E. Watts, Biochem. J., 112,527 (1969).
SI
E.S.A.(+)
,
I.A.V. RS.U. (REFERENCE VOLTAGE)
E.S.A. (ZERO)
POWER SUPPLY
11 11
Figure 2. Simplifide diagram of multichannel peak preselector Where Wl-Wr are the weights of I-V evaporated from a known weight of the specimen; A52-A168 are the measured profile areas for the appropriate components of m/e 52-168 in the spectra of the unknown samples, and CI-Cla are the coefficients obtained from the standard mass spectra and calibration plots for each of the pure compounds I-V. In each case the profiles for m/e 52 and m/e 54 had to be obtained from a separate sample of the same specimen. Calibrations. The mass spectrometer was calibrated for each experiment by recording the evaporation profiles for two sets of mixtures of known compositions, one set consisting of compounds 1-111 only, the other comprising 1V and V. In order to assess the effect of the carrier media on the precision of the analysis, separate mixtures of 1-111 were diluted with deionized water, anhydrous sodium sulfate, and desiccated muscle from which all endogenous purine had been evaporated. The concentrations of these mixtures ranged from 10-1OOO ng/mg. Because of the very low solubility in water of compounds IV and V, sodium sulfate only was used as their diluent. Calibration mixtures containing similar concentrations of caffeine (1 :3 : 5 tri-N-methyl xanthine VI) in both methanol and sodium sulfate, were used to compare th'e precision of determination and detection limits of a relatively nonpolar purine with those obtainable for the more polar compounds 1-111.
URIC A C I D
Figure 3. Evaporation profiles of hypoxanthine, xanthine, and uric acid Ordinates denote the instantaneous molecular-ion intensities measured sequentially on three channels at the probe temperatures indicated PROBE TEMPERATURE
2
PROGRAM I
TIME (MIN.)
0
I
2
3
4
5
b
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
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4%
ERROR
241
20
e.
1-u(AQ.
00
1 . (SOLID ~ MIXTURE)
xx
CAFFEINE (SOLID OR SOLNJ
SOLUTION)
-.-.HANDLING
ERRORS
Figure 4. Dependence of analytical precision on concentration and diluent for caffeine, hypoxanthine, xanthine, and uric acid in solution and solid mixtures
lb
20
IO0 CONCENTRATION
Table 11. Analysis of Test Mixtures Mixture A HX X UA Alp Found, ng/mg 45 77 51 77 Theory, ng/mg 54 58 56 68 +17 Error, % +9 -13 -33 Mixture B Found, ng/mg Theory, ng/mg Error, % Mixture C Found, ng/mg Theory, ng/mg Error, Mixture D Fouhd, ng/mg Theory, ng/mg Error,
Oxp 70 70 0
140 147 +5
175 164 -7
149 167 +11
148 137 -8
178 169 -5
94 76
97 94 +3
119 125
80 89 -10
93 106 -12
161 156 $3
170 184 -7
161 169 -5
225 207 +9
+23
132 131 +I
-5
At the beginning and end of each set of profiles, the intensity of an internal standard (See Table I) was recorded t o check the constancy of the mass spectrometer sensitivity. These reference materials, which were admitted simultaneously with each sample at constant pressure from reservoir inlets, were also used as mass references to preset the peak selector. The overall accuracy of the method was checked by analyzing a series of mixtures containing known concentrations of all five components together. RESULTS
Instrument Performance. To achieve successful measurements of integrated abundance for a number of ion beams simultaneously and at high resolution, the outputs of the analyzer power supplies and ion current amplifier had to remain constant to a high degree over a prolonged period of time. With the present arrangement, the stability of the analyzer, including the peak preselector, was sufficient to maintain a peak in the center of the display oscilloscope to within a distance equivalent to a + 5 ppm mass difference for a period of about 8 hours without intermediate adjustment. Over the same period of time, changes in mass spectrometer sensitivity remained less than 5 % as measured by the constancy of the ion currents derived from the internal 1654
200 (nQ/nq)
standards. A prerequisite for this performance was the control of the ambient temperature to within *2 "C. The routine examination of biological samples caused only a slight (about 20 %) deterioration in sensitivity, at constant 20,000 resolving power, over a period of six months. Throughout this time, the ion source was baked for about six hours each night. Since the spectrometer was calibrated on a day-today basis, the analytical accuracy was not affected. Calibrations. Linear relationships were found between the areas of the molecular-ion profiles of compounds I-V and the corresponding weights of material evaporated over the concentration range invesligated. In Figure 4 the mean percentage deviations of the experimental points from the appropriate regression lines are plotted against concentration expressed as ng/mg of sample (ppm) of caffeine, hypoxanthine, xanthine, and uric acid both in solution and in solid mixtures with sodium sulfate. The use of purine-free muscle as a diluent gave results indistinguishable from those obtained with sodium sulfate. Part of the total observed error is attributable to inaccuracies in the preparation and admission to the mass spectrometer of the standard solutions and mixtures. The maximum estimated errors from these sources are also shown in Figure 4 (dashed curve). Analyses. The analyses of some typical mixtures of the 5 compounds in known concentrations are presented in Table 11. With very few exceptions, the overall error for concentrations above 100 ppm was within *lOP7, and +20P7, for concentrations less than 100 ppm. The concentrations of hypoxanthine, xanthine, and uric acid in the plasma of a series of normal subjects are shown in Table 111. Also the mean measured oxypurine (hypoxanthine plus xanthine) and uric acid levels are compared with those obtained by enzymatic spectrophotometric methods (10, 11). In Figure 5 the concentrations of uric acid in the muscle of gout patients before and during allopurinol treatment are compared with those in normal muscle. DISCUSSION
Precision of Measurement. An examination of Figure 4 shows that for a relatively nonpolar material such as caffeine,
-
(10) L. Liddle, J. E. Seegmiller, and L. Laster, J . Lab. Clin. Med., 54, 903 (1959). (11) S. Jorgensen and H. E. Poulsen, Acta Pharrnacol. Toxicol., 11, 223 (1955).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
Table 111. Purine Concentration (mg/100 ml) Subject HXa X (Hx X) UA
+
A
B C
D E Mean measured Normal range
0.12 0.11 0.18 0.13 0.15
0.13 0 0 0.03 0.02
0.25 0.11 0.18 0.13 0.15 0.17 0.1-0.3
0 Concentrations of hypoxanthine (Hx), xanthine acid (UA) in normal plasma. Sodium urate. c Protein bound.
... 2.27 3.19 2.24 2.36 2.52 4.P-6.0’ 2.‘ f 0 . 4 e (>.),
and uric
there is no essential difference in the precision of measurement of a solution or a solid mixture of the same concentration provided the latter is thoroughly mixed. The minimum detectable concentration of caffeine in each case was about 0.5 ppm under the present conditions of measurements. It was possible, of course, to increase the sensitivity considerably by simply increasing the mass spectrometer gain. It was not possible, however, to introduce subnanogram quantities of material into the mass spectrometer in a sufficiently reproducible manner for proper calibration. Consequently the precision of measurements under such conditions was very poor and any figure quoted for a minimum detectable concentration became questionable. A more useful concept, therefore, for quantitative purposes is the smallest measurable concentration ( 2 ppm in the case of caffeine) which could be measured with a precision of + l o % or better. At least half the observed error could be accounted for by handling inaccuracies. The remainder was most probably a combination of variation in mass spectrometer sensitivity (contributing not more than f5 for concentrations of caffeine less than 20 ppm) and errors in the integration procedure. Several methods of integration of the evaporation profile were attempted (e.g., weighing a cut-out of the profile shape and numerical integration by Simpson’s rule), but the most convenient was to determine simply the sum of the recorded ordinates. Provided at least ten ordinates were obtained per profile, this method was as satisfactory as the others. In the case of the more polar purines 1-111, the precision of measurement was less and showed marked dependence on the method of introduction. When aqueous solutions were used, the minimum measurable concentration was as high as 30 ppm while the error increased much more rapidly for smaller amounts than was observed for solutions of caffeine. In addition the solid residue from solutions 1-111 usually required excessively high temperatures (>350 “C)for vaporization and gave profiles with prolonged tailing. This appeared to be due to adsorption of the polar purines on the surface of the gold sample container of the inlet. These gold containers were normally regarded as having negligible surface activity but especially after prolonged cleaning the surface activity was very pronounced. Surface deactivation took place after a container has been used for 3-4 samples, provided it was not heated to more than 300 “C in between. Consistent evaporation profiles usually could then be obtained but the mechanism of the deactivation process remains unclear. Containers made from borosilicate glass proved equally prone to surface activity and were not improved by standard silanization procedures (12). (12) H. H. Wotiz
and S. J. Clark, “Gas Chromatography of Steroid Hormones,” Plenum Press, New York, N. Y.,1966, p 75.
kT*::::S
I
GOUT BEFORE TREATMENT
I
GOUT
DURING
TREATMENT
Figure 5. Comparison of muscle uric acid levels (vertical lines) of 6 normal subjects, 5 gout patients before having allopurinol treatment, and 6 gout patients during allopurinol therapy
By contrast, the profiles of 1-111 from muscle, plasma, or sodium sulfate exhibited much less tailing, were of a more consistent area, and required lower vaporization temperatures than did those from aqueous solutions. This was taken as evidence that essentially complete evaporation of the purines took place and only negligible amounts were retained by the tissue and plasma proteins. This was further indicated in Figure 4, where the minimum measurable concentration of 1-111 in a solid mixture was now 10 ppm. Their error curve was much more closely parallel to that of caffeine indicating a much reduced adsorption of the polar purines on the surface of the sample container. Some adsorption on the sodium sulfate undoubtedly took place, however, accounting for the decreased measurement precision compared with caffeine under the same conditions. This was particularly noticeable if the solid mixture was allowed to stand for more than 24 hours. Best results were obtained from mixtures used immediately after preparation. The error curves of IV and V in sodium sulfate mixtures were closely similar to those of 1-111 and the same considerations apply. Overall Accuracy. Contributions to the total overall error (Table 11) in the simultaneous determination of compounds I-V probably arose from two main sources besides the calibration errors referred to above: the arithmetical operations necessary to solve Equations 1-5 tended to allow some error accumulation and all five profiles were not measured from the same sample. Nevertheless, the results shown in Table I1 indicated that with very few exceptions, the total error in the analysis of the test mixtures at concentrations down to 50 ppm was approximately twice the calibration error for a given component. Consequently the error in measuring such five-component mixtures at the 10-ppm level was probably no more than +20z. When compared with the natural variation of purine levels within a group of normal individuals (Table 111 and Figure 5 ) , this error is not considered to be excessive.
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
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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 pH (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-
*
(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 on 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 MRC 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 and Guy 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
e
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). In 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
