Computer Evaluation of Gas Chromatographic-Mass Spectrometric Analyses of Steroids from Biological Materials Robert Reimendal and Jan B. Sjovall Department of Chemistry, Karolinska lnstitutet, Stockholm, Sweden
Procedures are described for computer analysis of GC-MS data obtained by repetitive scanning and recorded on magnetic tape in analyses of TMS and MO-TMS derivatives of steroids. Completely or incompletely separated compounds are first located by a search for peaks in individual fragment ion current chromatograms. A search for molecular ions is then made based on fragment losses typical of TMS and MO-TMS derivatives. Potential molecular ions fulfilling given criteria are suggested in terms of steroid structure. The search is repeated with the peak-front and peak-tail difference spectra to permit resolution of mixtures. The computer evaluation is automatic or is carried out under supervision using a CRT display. The procedures are designed as aids in the rapid evaluation of large numbers of routine analyses. Examples are given of analyses of steroids in bovine corpus luteum, in human, urine, and in plasma of a patient with choriocarcinoma.
Several computer systems have been described which permit rapid acqusition of data in gas chromatographicmass spectrometric (GC-MS) analyses [for references, see review by Burlingame and Johanson ( I ) ] . This makes it possible to use repetitive scanning to obtain a maximum amount of information in analyses of complex mixtures (2, 3). However, evaluation of results becomes time consuming, and an efficient use of gas chromatography-mass spectrometry in qualitative and quantitative routine analyses of complex mixtures of steroids in blood, tissues, and urine requires computer-aided evaluation of data. This paper describes procedures which have been found useful in analyses of samples from patients and in studies of steroid metabolism.
EXPERIMENTAL Instruments. An LKB 9000 gas chromatograph-mass spectrometer equipped with a scan interval programmer and an acceleration voltage scan unit was used (4). Spectra were recorded on magnetic tape using the interface for an incremental or synchronous tape recorder described by Jansson et al. ( 5 ) . The tape was processed in an IBM 1800 computer with 24-K core storage, two 500-K disk storages, two 9 track magnetic tape units, a card read punch, a line printer, a plotter, and a CRT display. Analytical Procedures. Mono- and disulfates of steroids in plasma were isolated, solvolyzed, and converted into trimethylsilyl ( T M S ) ethers as described previously (6). Urinary steroids were extracted with Amberlite XAD-2 (7) and were subjected to (1) A . L. Burlingame and G . A. Johanson, Anal. C h e m . , 44, 337R (1972) (2) R. A . Hites and K. Biemann. Anal. Chem., 40, 1217 (1968). (3) R. A. Hites and K . Biemann, Anal. C h e m . , 42, 855 (1970). (4) R. Reimendal and J . Sjovall,Anal. Chem., 44, 21 (1972). (5) P.-A. Jansson, S. Melkersson, R. Ryhage and S. Wikstrom, Arkiv Kemi. 31, 565 (1970) (6) 0.Janne, R. Vihko, J . Sjovall, and K. Sjovall. Clin. Chim. Acta, 23, 405 (1969). (7) H . Bradlow, Steroids, 11, 265 (1968).
enzymatic hydrolysis and solvolysis (8). They were analyzed as 0-methyloxime ( M 0 ) - T M S derivatives, prepared as described by Thenot and Horning (9). Columns, 2-3 m X 4 mm, containing 1.5% SE-30 on Chromosorb W H.P., 80-100 mesh, were used. Temperatures of column, molecule separator, and ion source were about 230, 250, and 290 “C, respectively. To emphasize diagnostically important ions of high mass, the electron energy was kept a t 22.5 eV. Following injection of the sample, magnet scans covering the desired m / e range ( m / e 0-550 or 0-820) were initiated automatically after a selected delay and with selected constant or increasing intervals (4). S e a r c h for Compound Zones on t h e Tape. This is carried out using a peak locating program, PLOCP. The spectra are read from the tape into core and a matched background is subtracted ( 4 ) . A sequence of three spectra are always simultaneously in core. The changes with time of the intensities of all fragment ion currents (except for those given by major background ions) are followed. When a predetermined number of increasing values followed by a n equal or decreasing value have been detected in a fragment ion current, the peak maximum value and the retention time of the scan are registered. The number of fragment ion current chromatograms showing a peak a t this scan is registered. Before the start of the search, the program is given values for five variables to define a “peak of a compound of interest”: 1. minimum number of increasing intensity values preceding a decreasing one. The setting of this value depends on the frequency of the scanning and on a threshold set for the noise level; 2. minimum number of fragment ion current chromatograms showing simultaneous appearance of a peak; 3. minimum intensity of the highest of these peaks; 4. minimum intensity at this peak scan of any fragment ion current showing a higher or equal value compared to that of the previous scan; 5 . minimum value of the result obtained by multiplying the highest peak intensity with the number of chromatograms showing a peak. If this value is exceeded, it is taken as a n indication of the presence of a peak of a compound also when the minimum number of simultaneously appearing peaks is not reached. In addition to the vafues of the five variables, retention time limits are given so that only the interesting part of the chromatogram is searched. The values of the variables will determine the limit of sensitivity in detection of peaks. The choice of values is determined by the noise level, electronic or chemical, in the analysis. If only major compounds are of interest the minimum values may be set high. Common values for intensities correspond to 0.4-1% of the dynamic range of the amplifier, and usually 4-10 simultaneously appearing peaks are required to signify a peak of a compound. The use of the fifth variable above makes it possible to detect also those compounds which give rise to only one major fragment ion. The search for compound zones requires about 1.5 sec per spectrum. When a “peak of a compound of interest” has been detected, the retention time, relative retention time, number of chromatograms showing a peak, percentage contribution of these peaks to the total ionization, m / e and intensity values for variables 3 and 4, percentage contribution of the fragment ion in variable 4 to the total ionization, number of the scan and number of the tape sector containing the spectrum are printed on one line by the line printer. The direction of further data treatment is set with the console data switches. If the CRT display is used for rapid inspection of data, the total ion current chromatogram ( u p (8) H. Eriksson and J.-A. Gustafsson, Eur. J. Eiochem., 16, 268 (1970). (9) J.-P. Thenotand E. C. Horning, Anal. Lett.. 5 , 21 (1972).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 7, JUNE 1973
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to the retention time of the peak found) appears on the display and the background-corrected, normalized spectrum is then shown. Since the spectra immed,iately preceding and following the “peak maximum spectrum” are in core, these spectra may be displayed, and any difference spectrum between the three spectra may also be calculated and displayed. In all cases, plotted spectra may be directly obtained. A search for potential molecular ions in the original spectrum and in the difference spectra may be made automatically. In the most rapid routine procedure, the printed information on located peaks is the only output. The total ion current chromatogram is plotted at the end of the search. At any stage, the search procedure can be interrupted uia the teletype and any selected spectrum or difference spectrum in any selected analysis may be displayed.
Search for Potential Molecular Ions. The program used in previous studies (4) has been simplified to give a higher search rate and better ability to detect molecular ions of steroids with 4-6 substituents. The background-corrected, normalized spectrum is searched for molecular ions and fragment ions typical of TMS and MO-TMS derivatives of steroids. The search starts at m / e 820 and continues through lower m / e values within a range set at the start of the search. All peaks are subjected to an approximative correction for isotope peak contribution. The intensity, I p , of a peak, m / e = P, is compared with the intensity, I p - l , of the peak at m / e = P - 1. When IP equals I p - 1 or exceeds the value (P/800) x IP-1. P is considered not to be an isotope peak. This method intentionally overcorrects both for P + 1 and P + 2 peaks. The factor Pi800 is also used for correction of intensities of possible fragment ions from P. Depending on their intensities, fragment ions are divided into ten groups scoring 0-9 points. Relative intensities of 0-0.31% give no points; 0.32-0.63 = 1; 0.64-1.27 = 2; 1.28-2.55 = 3; 2.56-5.11 = 4; 5.12-10.23 = 5; 10.24-20.47 = 6; 20.48-40.95 = 7; 40.96-81.91 = 8; >81.92 = 9 points. Depending on their possible origin, fragment ions are then classified into five groups: M and M - 15 is group PM; M - 90, M - 103, and M - 105 is TMS1; M - 180 is TMSP; M - 31 and M - 121 is MO; M - 18 and M - 33 is OH. When a potential molecular ion is above m / e 550, M - 117 is included in T M S l and M - 205 in TMS2. Before the start of the search, the program is given structural limits for steroids. This information is divided into parent hydrocarbon structure, (e.g., estrane, androstane, pregnane, cholane, and cholestane), and number and nature of substituents, (e.g., double bonds, trimethylsiloxy groups, 0-methyloxime groups, and keto groups). The search procedure is divided into three test phases: 1. tests whether an ion should be regarded as a potential M + ; 2. tests whether criteria for an M + are fulfilled; 3. tests whether the mass of a suggested M + may be the molecular weight of one or several of the permitted steroid structures. The assignment of numerical values to the relative intensities and scores required in test phases 1 and 2 is empirical and is based on studies of spectra of mixtures of reference compounds and steroids of biological origin. The aim has been to obtain maximal sensitivity in detection of potential molecular ions with a minimum of irrelevant information, N o attempt has been made to use computer methods to optimize the values. 1. When a peak, P, with a relative intensity, IP, exceeding 0.31% (after correction for isotope contribution) is found, P is considered as a potential M + and the tests of stage 2 are made. If I p < 0.3270, the possibility is considered that the intensity of the molecular ion is below the limit of detection. A search is then made for P - 15, and if I P - 1 5 is 0.31, if either of IP-31 or ( I p - 9 0 I p - 1 0 3 ) score 5 points or more. 2. When a potential M - has been found by the criteria above, tests are made for presence and intensities of certain fragment ions. If any of the following six criteria is fulfilled, the tests of stage 3 are performed. a ) I p - 1 5 > 4 points; b) T M S l > 7 points; c) I p - 1 5 > 2 points and TMSP > 4 points; d) P M > 4 points and T M S l > 2 points; e) I p - 1 5 > 2 points and OH > 2 points; f ) (PM MO) > 5 points and MO > 2 points. Two additional criteria are used for potential molecular ions at m / e values above 550; a ) PM > 2 points and T M S l > 3 points or TMS2 > 4 points; b) TMS2 > 7 points. As mentioned above, scores for Ip-117 and I p - 2 0 5 are included in T M S l and TMS2, respectively, when P > m / e 550.
+
+
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 7 , JUNE 1973
3. When an M + has been defined by the tests of stages 1 and 2, all molecular weights of permitted combinations of steroid skeletons and substituents are compared with the m / e value of this M+. If matching values are found, the scores for appropriate fragment ion groups are added. The P M score is always included in the total score, the T M S l score is included if the molecular weight requires the presence of a TMS group, the TMSP score if two T M S groups are required, the MO score if an 0-methyloxime is required, and the OH score if a free hydroxyl group is required. If the total score is higher than 7, the name of the steroid structure is translated from the mass contributions of the skeleton and the substituents and is printed together with the molecular weight. The latter also appears in the spectrum on the CRT display. The suggested molecular weights are listed in order of decreasing m / e values. When several potential molecular ions of different mass are detected, only those structures are printed which give more than half of the highest score of a preceding structure with higher molecular weight. When a potential molecular ion can be explained as the result of losses of fragments used in defining a molecular ion of higher mass, the name is printed together with the possible relationship to the previously defined M+. When SE-30 columns are used, the search for an M + is interrupted 181 mass units below the m / e value of the highest M + found to fulfill the criteria for a molecular ion of a steroid. With the steroid structure limits commonly used, the search is rapid and the line printer is rate limiting (80 lines/min). The TMS or MO-TMS derivatives of some steroid structures give too few or too small peaks in the high mass range to permit recognition of a molecular weight. These steroids often give a few ions of high intensity at lower mass, and special pattern recognition programs may be added to the search. Thus, a base peak at m / e 117 representing more than 40% of the total ionization is strongly indicative of a 2-trimethylsiloxyethyl side chain, and the computer output is “pregnanediol side chain.” When an intense peak at m / e 253 is present together with peaks a t m / e 343 and 423 the computer output is “ABCD-rings with 3 double bonds.” Commonly occurring contaminants, e.g., dioctyl phthalate, are also recognized and their names are printed. Additional pattern recognitions may be added to the program depending on the origin and nature of the steroid sample analyzed. The program searching for molecular ions operates either with spectra selected by the biochemist or with spectra selected by the peak locating program. In the latter case, the search occurs automatically when a peak of a compound has been located. When the spectrum at the peak maximum has been searched for molecular ions, the preceding spectrum is subtracted and the resulting difference spectrum is searched. The search is then repeated with the “peak-tail” difference spectrum. If requested a t the start of the program, the search of difference spectra may be omitted but this search often yields important information. Comparison with Spectra of Authentic Steroid Derivatives. A file of reference spectra has been established, and programs similar to those described by Hertz et al. (IO) have been written which may be used for identification of spectra of single components. However, since spectra are often due to mixtures, and all steroid derivatives are not in the file, it is more valuable to use the collection of reference spectra in searches for correlations between steroid structure and occurrence of specific fragment ions and losses of specific fragments. In the method used, the computer is given the masses of 1-5 ions and/or fragment losses, the minimum relative intensities that they must have, and whether or not they must all be present. The search rate depends on the number of spectra found to fulfill the criteria and is limited by the line printer. When no spectra are found, the rate is about 20 spectra per second. This could be increased by having the library of spectra on a magnetic disk.
RESULTS AND DISCUSSION The aim of the present study was to find methods for rapid evaluation of large numbers of gas chromatographicmass spectrometric analyses of steroids in blood, urine, tissues, and feces from animals and humans. In these analyses, spectra are taken continuously to avoid loss of important information and to permit quantitative determinations. The most comprehensive evaluation of data (10) H . S . Hertz, R. A. (1971).
Hites, and K . Biemann, Anal. Chem.. 43, 681
Table I. Computer Evaluation of Spectra Obtained in the Analysis of TMS Ethers of Steroids in the Disulfate Fraction from Plasma of a Patient with Choriocarcinoma Scan
Noa
Retention time Min Reib
No of peaksC
M / e of most intense iond Peak Max
Per cent of total ionization ine Peaks Max
27
9.6
0.54
58
21 5
215
83.4
8.2
28
9.8
0.55
7
372
371
17.4
25.8
33
10.8
0.61
9
345
344
5.9
7.4
34
11.0
0.62
75
21 5
215
87.3
6.9
52 56 69 70 71
15.2 16.2 19.9 20.2 20.5
0.85 0.91 1.12 1.14 1.15
21 7 8
361 117 129 118 117
361 117 117 117 117
78.4 56.7 14.7 16.5 58.5
34.3 43.6 44.4 65.1 55.0
5 5
Selected by the computer as representing a spectrum at the maximum Relative to cholestane. Number of fragment ion current chromatograms showing a peak with the retention time at which the spectrum was taken. M/e values of fragment ion currents a
of a peak of a compound.
can obviously be obtained if all spectra and all fragment ion current chromatograms [mass chromatograms (3)]are made available to the biochemist. A microfilm system for this purpose has been described [see ( 1 2 ) ] . However, the time required to study all these spectra and chromatograms is too long if gas chromatography-mass spectrometry is to be used in routine analyses of steroid samples from patients. The process of evaluation of a GC-MS analysis may be divided into three parts: 1. location of compounds, whether eluted singly or in mixtures, and determination of the retention times, 2. interpretation of the mass spectra taken a t the located peak maxima of the individual compounds, and 3. quantitative determination of identified steroids. The latter is based on peak areas in appropriate fragment ion current chromatograms and is not described in this paper. Peak Locating Program. This is based on a search for peaks in all individual fragment ion current chromatograms [cf. ( 3 ) ] .Thus, it is possible to detect overlapping compounds provided that their peak maxima differ by a t least one scan and that their mass spectra are different. Thm cannot be achieved if the total ion current serves as the basis for the search (12). Furthermore, the base line of fragment ion current chromatograms can usually be well defined, whereas the base line of the total ion current chromatogram may be difficult to define when complex mixtures are analyzed. The detection limit is also lower when fragment ion rather than total ion current serves as the basis for the search. However, our peak locating program fails to detect overlapping isomeric compounds if the isomers give spectra with similar relative intensities. It may also fail to detect the appearance of small amounts of a saturated steroid overlapping with a peak due mainly to the unsaturated analog. However, overlapping compounds of the latter type are usually detected in the subsequent computer search for molecular ions, particularly in the search of the difference spectra. Depending on the distribution of scans over the apex of the peak, one or two indications of a peak maximum may (11) K Biemann in The Applications of Computer Techniques in Chemical Research,’ P. Hepple, Ed., Institute of Petroleum, London, 1972, D 5 D. Henneberg, K. Casper, E. Ziegler, and B. Weimann, Angew. Chem., 84, 381 (1972).
Potential molecular ions (score) 434(33) 4 74 ( 13) 434(27) 474(24) 524( 17) 434(37) 524(22) 434(37) 524(25) 464 (12)
P-diol 462(13)
P-diol
P-diol
General structure of steroid/
Androstenediol Contaminant g Androstenediol Contaminant g Not present Androstenediol Not present Androstenediol Not present Contaminant Pregnanediol Pregnenediol Pregnanediol Pregnanediol
giving the largest peak (peak) and the highest intensity (maxi at the scan indicated. e Percentage of total ion current given by fragment ion currents showing a peak (peaks) or having the highest intensity (max). Supported by inspection of spectrum. g 1-Monopalmitin. See text.
’
be obtained for a single component. If the spectrum is taken near the true maximum, most fragment ion current chromatograms will show a peak maximum a t this scan. If two spectra are symmetrically taken on both sides of the maximum, some fragment ion current chromatograms will show the peak maximum in one scan and some in the other. This results in storage of duplicate spectra. Search for Potential Molecular Ions of Steroid Derivatives. When the criteria for a peak maximum of a compound are fulfilled, the normalized spectrum is searched for potentia1 molecular ions of steroid derivatives. The search is based on information about the nature of the sample and the derivatization procedure used. This information is given to the program in the form of limitations of structures to be searched for. Thus, if in vitro studies are made with a CIS steroid precursor, it would usually be unnecessary to search for Czl and C 2 7 steroids among the products. Analyses of steroids in urine and blood may be limited to a certain class of steroids. The conditions of derivative formation are important in determining whether complete or partial protection of polar groups is obtained. The standard procedure used for formation of T M S ethers leaves hindered hydroxyl groups unreacted, whereas a,P-unsaturated keto groups may give rise to enol ethers. The method for formation of MO-TMS derivatives described by Thenot and Horning (9) is very suitable since it leaves only one keto group free ( a t C - l l ) , and no unreacted hydroxyl groups. The empirical criteria for a molecular ion were chosen to permit detection of all possible molecular ions in spectra of mixtures. When the program was tested on 240 reference spectra of T M S ethers, allowing the presence of androstanes and pregnanes with 0-3 double bonds, 0-6 T M S groups, 0-1 free hydroxyl groups, 0-3 keto groups and no MO groups, M + was found in 232 cases (96.7%). M - 90 was defined as M + in 6 cases (2.5%) and no M + was found in 2 cases (0.8%). One of the latter steroids was pregnanediol T M S ether and the computer output was “pregnanediol side chain.” When tested on 77 spectra of partial MO-TMS derivatives, allowing the presence of androstanes and pregnanes with 0-3 double bonds, 0-6 T M S groups, 0-1 free hydroxyl groups, 0-3 MO groups and 0-1 keto groupqthe correct M + was found in 75 cases (97.5%). The wrong molecular weight was given in 2 cases. ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 7, J U N E 1973
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Table II. Computer Evaluation of Spectra Obtained in the Analysis of MO-TMS Derivatives of Steroids in Corpus Luteum of a Pregnant Cowa Scan No
Retention time Min Re1
M l e of most intense ion Peak Max
No of
peaks
31 38 46 47
10.8 11.9 13.7 14.0
0.61 0.67 0.77 0.79
41 115 54 39
103 205 143 100
103 205 100 100
48
14.2
0.80
11
388
388
50
14.7
0.83
15
267
117
51 56 60 61
15.0 16.2 17.3 17.6
0.84 0.91 0.97 0.99
114 17 45 67
117 116 386 81d
117 116 100 100
62
17.9
1 .oo
127
100
100
66
19.0
1.07
7
117
117
70
20.2
1.14
71
70
372
71
20.5
1.15
115
372
372
75
21.8
1.22
83
117
117
117 22.0 1.24 62 153 76 149 23.4 1.31 10 71 80 2.27 52 129 121 40.4 129 a For explanation of Table headings, see footnotes in Table I. M-31 of a pregnanolone MO-TMS derivative present in small amounts. The molecular ion of this compound was found by the computer in the difference spectrum scan 47 - scan 46 where it gave the highest score. Ions of this mass were not present but were calculated from fragments
*
Per cent of total ionization in Peaks Max
Potential molecular ions (score)
General structure of steroid
-
None No steroid None N o steroid Pregnenolone 41 7 ( 3 7 ) 417(32) Pregnenolone . . .6 388(16) 45.0 16.6 Pregnanolone 419(23) Pregnenolone 41 7 (22) 346( 14 ) c Not present 31 3 (12) Not present 20.1 5.5 Pregnenediol 462(18) 388 (9) 21.6 92.2 Pregnenediol 462 (18) 42.1 No steroid 8.8 None Pregnenolone 27.4 6.4 417(41) Pregnenolone 33.5 9.0 417(31) Pregnanolone 419( 19) 346( 16 ) c Not present 11.3 Pregnanolone 419 (28) 87.8 Pregnenolone 417(23) Not present 346( 19) P-diole 69.8 Pregnanediol 82.0 462( 1 9 ) f Pregnenediolf Pregnenedione 372(25) 9.5 19.3 Not present 403(19)' Not present 376 (1 5 ) C 10.9 372(25) Pregnenedione 80.3 387( 18 ) c Not present 299 (14) Not present 24.7 Pregnenolone 58.1 417(36) 301 (18) Not present Pregnenolone 54.2 37.0 417(35) 16.4 25.9 Not present 301(11) 7.2 Cholesterol 63.4 458(28) of the steroid derivative. The mass spectra did not support the presence of a steroid with this molecular weight. The most intense peak ion m/e 81. is probably due to contamination with squalene. e See text. Detected in the difference SDectrum 66-67. 65.3 79.6 46.1 59.5
12.0 20.7 7.6 18.1
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