Analysis of Steroids by Off-Line Computerized Gas Chromatography-Mass Spectrometry Robert Reimendal and Jan Sjovall Department of Chemistry, Karolinska Instifutet, Stockholm, Sweden Procedures are described for handling of data recorded on magnetic tape in gas chromatographic-mass spectrometric analyses of steroids from biological materials. The methods are intended for use in biochemical and clinical studies where large numbers of samples consisting of complex mixtures are analyzed. The aim has been to achieve high capacity and speed in the processing of data. In isothermal analyses, spectra are taken automatically with a continuously increasing interval between scans, Thus, the number of spectra is reduced markedly compared to that obtained by repetitive scanning with constant intervals. Short processing times are achieved by limiting the preliminary evaluation of spectra to 100120 selected ions, the intensities of which are listed in retention time sequence. Plots of fragment ion current chromatograms, and computer search for appearance of gas chromatographic peaks may also be used in the evaluation. Sensitivity limits in applications of fragment ion current plots to quantitative work are discussed. A computer search for possible molecular ions has been used as an aid in the interpretation of the normalized spectra which are often due to mixtures of compounds.
USEOF GAS chromatography-mass spectrometry as a routine method in qualitative and quantitative analysis necessitates computer handling of data. Different on-line and off-line systems for data acquisition and evaluation have been described (see 1-6). Gas chromatography-mass spectrometry has been used in this laboratory in studies of bile acid and steroid metabolism, and all analyses during the last three years have been carried out using the interface for off-line data acquisition described by Ryhage's group (2, 3). Since a large number of analyses are made by different groups, high capacity and simplicity in data processing have been attempted. This paper describes methods which are presently in routine use.
(1.0-9.9 sec); and a factor for increase of this interval with time after sample injection according to the formula: I t = k ' t + Io
where I , is the interval between scans after t minutes, and l o is the initial interval a t the time of sample injection. The factor, k , can be set a t 0.01, 0.02, or 0.03 corresponding to an increase of the interval with 0.6, 1.2, or 1.8 seconds per minute retention time. The instrument has also been equipped with a unit for acceleration voltage scanning. The voltage is increased linearly by 10% (or less) in 2 o r 5 sec, and the scan is repetitive. The interface for incremental or continuous (15 kHz) recording of mass spectra o n magnetic tape is that described by Jansson et al. ( 2 , 3). An incremental (PEC 1800-9) o r a synchronous (PEC 3800-9) tape recorder is used. The magnetic tape is processed in a n IBM 1800 computer which has a 24-K core storage of 16-bit words, two 500-K disk storages, a 60-kHz nine-channel magnetic tape unit, a line printer (80 lines per min), a card read punch, and a plotter. Gas Chromatography-Mass Spectrometry. Steroid fractions were obtained from biological materials by methods described previously (7-9). Samples were analyzed as trimethylsilyl (TMS) ethers prepared with hexamethyldisilazane and trimethylchlorosilane in pyridine (IO), or as O-methyloxime trimethylsilyl ether (MO-TMS) derivatives (11). Usually a 3-m x 4-mm column of 1.5% SE-30 (GC-grade) on Chromosorb W H.P., 80-100 mesh, was used a t 230 "C. The temperatures of the molecule separator and the ion source were about 250 and 290 "C, respectively. The energy of the bombarding electrons was 22.5 eV. RESULTS AND DISCUSSION
Instruments. An LKB 9000 gas chromatograph-mass spectrometer with mass marker is used. The standard scan stop has been replaced with a scan stop unit based o n the mass marker. This permits simpler setting of the mass a t which scanning should be interrupted. The preamplifier following the electron multiplier has been equipped with several input resistors so that a n optimal gain can be selected for different types of analyses. The instrument has been equipped with a unit for automatic scan start. This permits variation of three time functions: the time between sample injection and initiation of first scan (1-60 min); the time of a constant interval, IO,between scans
Incremental US. Continuous Recording of Spectra. During the past two years about 60,000 spectra, taken during analyses of biological materials, have been processed by the computer. When the automatic scan is used, many more spectra are recorded than those finally normalized and plotted. When such large numbers of analyses are made, the incremental mode of operation (see Ref. 3) is most suitable; it gives short records o n the tape and short processing times in the computer. There are three drawbacks with this method: loss of information on metastable ions, a larger variability in peak height reading than with continuous sampling, and difficulties with mass marker adjustment due to differing hydrogen content of the ions (3). These drawbacks are of little practical importance in biological studies of a group of compounds with similar elemental composition. The mass marker is adjusted for hydrocarbon type ions,
(1) R . A. Hites and K. Biemann, ANAL. CHEM., 40, 1217 (1968). (2) B. Hedfjall, P.-A. Jansson, Y.Mbrde, R. Ryhage, and S. Wikstrom, J. Sci. Instrum., 2, 1031 (1969). (3) P.-A. Jansson, S. Melkersson, R. Ryhage, and S. Wikstrorn, Arkiv Kemi, 31, 565 (1970). (4) L. Bergstedt and G. Widmark, Chromatographia, 2, 529 (1969). (5) C. C. Sweeley, D. B. Ray, W. I. Wood, J. F. Holland, and M. I. Kritchevsky, ANAL. CHEM., 42, 1505 (1970). (6) R. A. Hites and K. Biemann, ibid., p 855 (1970).
(7) 0. Janne, R. Vihko, J. Sjovall, and K. Sjovall, CIL7. Chim. Acta, 23, 405 (1969). (8) B. E. Gustafsson, J.-A. Gustafsson, and J. Sjovall, Eur. J. Biochem. 4, 568 (1968). (9) J.-A. Gustafsson, C. Shackleton, and J. Sjovall, ibid., 10, 302 (1969). (10) M. Makita and W. W. Wells, A w l . Biochem., 5 , 523 (1963). (11) W. L. Gardiner and E. C . Homing, Biochim. Biophys. Acta, 115, 524 (1966).
EXPERIMENTAL
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
21
I I.:.,,
I':lll
STEROIDS,INFANT FAECES, 1,5% SE-30,230' MO-TMS
SPECTR MINUTES
30
40
50
0
10
20
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STEROIDS,INFANT FAECES, 1,5% SE-30,230' TMS
SPECTRUM NO 160 MINUTES
I
50
t. i.-n
I
1
120
40
100
30
60 20
(0 10
2'0 0
Figure 1. Total ion current recordings in the gas chromatographic-mass spectrometric analysis of TMS and MO-TMS derivatives of steroids in the monosulfate fraction from faeces of a 15-day old infant given ACTH which means that the reading for background silicone peaks is taken somewhat too late. This results in a falsely high intensity value for the mass preceding a background peak at masses above 500 (Le., the reading for m/e 502 is taken when the peak at mje 503 is just starting to rise). However, the stability of the mass marker is such that the error is constant and thus is eliminated after background subtraction. The variability of the peak height reading (only the highest value between two mass marker pulses is written on the tape) is of the order *lox. This is satisfactory in all qualitative work, and is improved if multiple spectra are taken during the elution of a compound. Thus, the incremental mode of operation is used in all routine analyses. If higher accuracy is required-e.g., for isotope determinations-multiple spectra are taken. The continuous mode of operation is used only for special applications where particularly high sensitivity and accuracy is required (see below). Processing of Data. The gas chromatograph-mass spectrometer is used by several people in the different groups of the laboratory and the individual investigator uses his own magnetic tapes. When the scan was initiated manually, usually during the appearance of a peak, all spectra were transferred from the individual tapes to a common tape and were given identification letters and numbers. Matrix lists of spectra were then printed overnight from this tape. Be22
0
cause of the time required for printing of a matrix list (mje 10-SOO), this method obviously cannot be applied when automatic scanning is used. The large number of spectra obtained during automatic scanning, and the time needed t o evaluate the data were reduced in two ways : By continuously increasing the interval between scans so that a n unnecessarily large number of spectra would not be taken during isothermal gas chromatography; and by considering the intensities of a limited number of ions instead of the whole spectrum in the preliminary evaluation of data. Examples of analyses performed with a n interval increase of 0.6 sec X min-' are shown in Figure 1. The initial interval (lo,at injection of sample) was 6 sec, and no scans were initiated during the first 3 min following sample injection. In this way about 160 spectra were obtained compared to about 550 if a n interval of 6 sec had been kept throughout the analysis. Temperature programming will also reduce the number of spectra, but the changing relative intensities of the background peaks make background correction difficult with the present program. I n this, the intensities of prominent background peaks in a n average of 10 background spectra are compared with those in the sample spectrum. A background correction factor is then calculated. The possibility that the sample gives an ion with the same mass as any of those used for the comparison is also considered. The subtraction of a
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
STEROIOS IN
ION CURRENT
INFANT FAECES (TMS)
A
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20
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30 40 50 Figure 2. Computer plots of fragment ion currents from the analysis shown in Figure 1. The m/e values of the ion currents plotted are shown a t the right end of the chromatograms. The spectrum numbers correspond to those given in the lower chromatogram of Figure 1
10
background matched to the sample in this way is satisfactory to correct for differing amounts of effluent directed into the ion source and for the decrease in background intensity that may occur when a compound zone passes through the ion source. However, it does not correct for the changes in relative intensities of peaks in the background spectrum which occur during temperature programming to high temperatures. At present, no stationary phase is available that will not give a background spectrum at the temperatures and sensitivity levels required for analysis of steroids from biological extracts. The number and mass of ions to be considered in the preliminary evaluation of data are selected by the individual investigator. Hites and Biemann ( 6 ) have discussed methods for objective selection of mje values to be plotted as “mass chromatograms,” but their method makes use of normalized spectra and requires more computer capacity and time than is available to us. Furthermore, when a known group of compounds is being studied, a subjective choice of ions may be preferable. In studies of TMS ethers of steroids, 100-120 mje values are selected. These values are given to the computer via the card reader together with sample identification numbers, settings for the automatic scan unit (initial interval, interval increase, and delay time), and the retention time of a suitable reference compound. The uncorrected intensities of the mje values selected are taken from the magnetic tape and are printed (in blocks of 20 m/e values per page) in the sequence in which the spectra were taken (one spectrum per line). The retention time when the spectrum was taken (in minutes, and relative to the standard) is printed, as well as the number of the spectrum in the individual analysis, and the number of the tape sector where it is stored. Because of external noise, 0.5-1 % of the tape sectors contain values which do not represent mass spectra. To be accepted as a spectrum (and printed), intensities must be recorded at mje 18,207, and 503 (when silicone columns are used). This simple criterion reduces the number of erroneous spectra to 0.1-0.2%. The remaining errors are probably due to external noise appearing in the middle of a scan, and a short sequence of intense peaks is usually seen. It has not been necessary to eliminate these spectra by programming. The computer time required to follow 20 mje values is about 1 sec per spectrum and the rate is limited by the printer.
When 100 m/e values are monitored, each spectrum takes about 5.5 sec. This rate is sufficiently high for most routine purposes. The list of intensities permits the individual investigator to read the changes in fragment ion current and to correlate retention times with the appearance of specific peaks. On the basis of this information, compounds which are already well known can be identified and these spectra require no further treatment. Regions of the chromatogram which are of particular interest may be further studied in different ways. Spectra may be normalized and plotted after subtraction of a matched background, or chromatograms may be plotted of unnormalized or normalized intensities of a sequence of mje values or of single m/e values. The procedures which have been used are similar to those described by Hites and Biemann (6, 12) and some of them have been reported (13). Some examples of analyses of biological samples will be given. Figure 1 shows the total ion current recording in the analysis of TMS and MO-TMS derivatives of steroids in the monosulfate fraction from faeces of a 15-day infant receiving ACTH (this sample was kindly supplied by Drs. P. Eneroth and J.-A. Gustafsson). Several peaks with retention times of about 45-55 min are seen. Bile acids and hydroxylated cholesterol derivatives are often present in Cip and CB1steroid fractions obtained from faeces, and a number of mje values typical for these classes of compounds were selected, and the ion currents were plotted. Figure 2 shows the different chromatograms obtained. In each chromatogram the lowest intensity value found in any spectrum is subtracted as a background. The appearance of peaks with appropriate mje values and retention times indicates the presence of TMS derivatives of the following compounds (interpretations based on comparisons with spectra of authentic compounds) : 22E-hydroxycholesterol [spectrum No. 128, mje 173 (side chain)], chenodeoxycholic acid [No. 142, m/e 593 (M - 15), 518 (M - 90), 173 (side chain)], cholic acid [No. 147, m / e 606 (M - 90), 516 (M - 2 X go), 501 (M - (2 X 90 15)), 173 (side chain)],
+
(12) R. A. Hites and K. Biemann in “Advances in Mass Spectrometry,” Vol. 4,E. Kendrick, Ed., The Institute of Petroleum,
London, 1968, p 37. (13) R. Reimendal and J. Sjovall in “Proceedings of the 3rd International Congress on Hormonal Steroids,” Hamburg, 1970, Excerpta Medica, Amsterdam, 1971, in press. ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
23
CORPUS LUTEUM STEROIOS (MO-TMS)
nn
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00
-
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1
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SPECTRUM NO.
20
40
80
60
120
100
- 7
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Figure 4. Computer plots of fragment ion currents from the analysis shown in Figure 3 CORPUS LUTEUM STEROIDS (MO-TMSI GO
3
MINUTES
10
EO/
20
100
I
ION
CURRENT
125
30
Figure 3. Computer recording of partial ion currents in the analysis of free steroids from bovine corpus luteum The sums of intensities of ions between nije 34-500 (upper curve), 200-500 (middle curve), and 273-500 were plotted 24E-hydroxycholesterol [No. 148, m/e 503 (M - 43), 413 43)), 145 and 159 (side chain)] and 20,225(M - (90 dihydroxycholesterol [No. 153, m/e 389 (M - 173), 299 (M - (90 173)) 173 (side chain)]. The computer time taken to obtain this information is about of that required to plot the sequence of spectra taken during the elution of these compounds. Another example of the use of fragment ion current recordings is given in Figures 3-5. Figure 3 shows a computer plot of partial ion currents in a n analysis of MO-TMS derivatives of steroids in bovine corpus luteum (a detailed report of this study will be published by B. Gustafsson, G. Schumacher, and J. Sjovall). The sum of all intensities between m/e 34 and 500, 200 and 500, and 273 and 500 have been plotted after subtraction of the lowest sum found in any spectrum. The chromatogram is normalized so that the highest value gives full scale deflection. The sample was very impure and from Figure 3, it is evident that the impurities give their most intense ion(s) between m/e 200 and 273. Some compound(s) (spectra No. 40-60) give major ion(s) between mje 34 and 200. Each partial ion current plot is completed in about 30 sec and these chromatograms give information about the m/e range in which abundant ions occur. In the same analysis, a search was then made for the presence of MO-TMS derivatives of 20-keto-21-deoxy steroids (typical ions at m / e 70, 87, loo), 20-hydroxy-21-deoxy steroids (mje 117), 3-ketod4 steroids ( m / e 125, 137, 153), progesterone [m/e 372 (M)], pregnenolones [m/e 417 (M), 402 (M - 15), 386 (M - 31), 312 (M - go)], pregnanolones (m/e 419, 404,
+ +
24
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
462
372
SPECTRUM NO. 20
3
60
40
10
80
20
100 120 30
MINUTES
Figure 5. Computer plots of fragment ion currents from the analysis shown in Figure 3
388) and pregnenediols [m/e462 (M)]. In addition, l-monoglycerides may contaminate steroid fractions from tissues and some typical ions were monitored [m/e 399 (CN), 397 (CE.,,),395 (CI8:*), and 371 (CE)] (14). The results are shown in Figures 4 and 5 . The distribution of the peaks indicates the presence of two pregnenolones, one with a 20-keto group (spectrum No. 47) and one with a 20-hydroxy group and 3k e t o d 4 structure (spectrum No. 59). A pregnenediol is indicated in spectrum No. 50, followed by a pregnanediol (m/e 117 peak extends much beyond mje 462 peak). All the peaks of diagnostic significance for a 3-hydroxypregnan-20one MO-TMS derivative are found in spectrum 48. The ~~
(14) C. B. Johnson and R. T. Holman, Lipids,1 , 3 7 1 (1966).
GLC 1 JONTP
34-500
PREGNANCY7 PRURITUS
-
PLASMA DISULFATE5
-
AS-DIOL-17A
434 419 344 305 329 254 239 129
INTENSITY OF ALL IONS MULTIPLIED BY 4 254
A5-OIOL-17A
7
344
9
AS-DIOL-17%
10
11
MINUTES
Figure 6. “Fragment ion map” of the TMS ethers of 5-androstene-3p,l7-diol isomers from the steroid disulfate fraction in plasma of a pregnant woman with pruritus in the third trimester The upper curve is the computer plot of partial ion current (m/e 34-500). The lower curves are expanded plots of m / e values typical for 5. androstene-3,17-diol TMS ethers with retention times between 7-11 min. The amount of each androstenediol was about 30 ng different monoglycerides are also present (spectra No. 20, 39, 40, and 43). This search of the magnetic tape was completed in about 8 min. At the bottom line of Figures 4 and 5, vertical lines are computer indications of the presence of peaks in any cf the fragment ion current chromatograms. These lines consist of two parts separated by a horizontal dash. The height of the lower part is proportional to the number of chromatograms in which a peak is found (two or more increasing followed by one decreasing intensity value). The height of the upper part is proportional to the sum of intensities of all the peaks found, full scale being reached at 400 units (empirically chosen, representing 10% of the dynamic range of the analog to digital converter if present in a single peak). These lines can be drawn without fragment ion current plots and be used as a n aid in the choice of spectra to be normalized and plotted. The lines are plotted very rapidly and 200 fragment ion current chromatograms (in blocks of 20) can be searched in less than 10 min. However, fragment ions with low mass may give rise to many nonspecific peaks of high intensity (as seen in Figures 4 and 5). In these searches, fragment ions with high mass, preferably above mJe200, should be chosen. In some applications-e.g. determination of the homogeneity of a G L C peak with respect to specific ions-a “threedimensional” mode of plotting ion intensities is more clear than the mode shown in Figures 2,4, and 5. This is exempli-
fied in Figure 6. The “three-dimensional” plots (which may be called fragment ion maps) are mostly used in deuterium determinations when spectra are taken over the GLC peak with shortest possible intervals and average spectra are calculated. It is then necessary to ascertain the absence of interfering compounds in all parts of the GLC peak (15). Whenever ratios between fragment ion intensities are to be determined, corrected backgrounds must be subtracted from all spectra. Chromatograms of four m/e values can then be plotted as shown in Figure 7. The highest intensity for any of the m/e values is used to set full scale deflection and other intensities are normalized relative to this value. The apparent tailing of the m/e 284 chromatogram at a retention time of about 14.3 min is due to the presence of a pregnane-3,20adiol with 3a,5P configuration whereas the major component has a 3 a , h configuration. The ratios between the intensities of the four ions chosen (m/e 374, 345,284, and 269) are different in different epimers as is seen by comparison with the peak of the 3@,5a:epimer appearing at 17.2-17.3 min. The computer will also calculate and print the ratios between the intensities of any of the ions selected. Search for Molecular Ion in the Normalized Spectra. When spectra from a n analysis of a crude steroid fraction are
(15) T. Cronholm and J.
Sjovall, Eur. J . Biochern., 13, 124 (1970).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
25
,
G L C 1 J D N T P 34-500
PREGNANCY,
7
PRURITUS
PLASMA D I S U L F A T E 5
G L C 1 F O L T P 284 269 374 345
FRAGMENT I O N CURRENT 269
P-OIOL3A35A p, 269
P - 3 9 20 9 2 1 - O L
345 (ISOTOPE)
‘9
8: F O L T P 239 304 329 462
FRAGMENT
ION CURRENT
AS-DIOL
I4!
PS-DIOL
329 239
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462
P - 3 9 16 9 2 0 - O L
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15
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Figure 7. Computer plot of fragment ion currents ( m / e 269,284,345,374 middle curves; m/e 239,329,304,462 lower curves) after subtraction of corrected backgrounds The analysis is the same as that shown in Figure 6 and the partial ion current plot is shown for comparison
inspected, it is difficult to see the various possible steroid structures that may be represented, since spectra are frequently due to mixtures. It is therefore of considerable help t o have a computer search which will suggest all the molecular ions which may be present, with a large freedom of choice among steroids that may occur in the sample. Permitting molecular weights given by androstane and pregnane derivatives containing any substituent combination with a maximum of 2 double bonds, 5 keto groups, 1 hydroxyl group, and 5 trimethylsiloxy groups, a search was made for molecular ions in the analysis shown in Figure 1. The search was started at m/e 909 and each peak found (excluding isotope peaks) was considered as a possible molecular ion. To be accepted as such, typical fragment losses had to be present, and the relative intensities of the parent and fragment ion peaks had to exceed certain values. Ten sets of criteria were used which were based on the presence of the following ions (the minimum relative intensity required is given in parentheses): M (0.2), M 15 (5); M (l), M - 15 (l), M - 90 (1); M (l), M - 15 (l), 26
M - 105 (1); M (l), M - 15 (l), M - 180 (5); M (0.2), M 15 (l), M - 90 (0.2), M - 105 (0.2); M (3,M - 90 ( 5 ) ; M (absent), M - 90 (9,M - 103 ( 5 ) ; M (absent), M - 15 (l), M - 90 (1); M (l), M - 18 (1); M (l), M - 33 (1) (to be accepted, the latter two alternatives require the presence of free hydroxyl or keto group in the structure calculated from the molecular ion). When M was absent, it was calculated from the fragment ions indicated. The mass of the molecular ion was translated by the computer to a steroid name using the terms androst, pregn, ene, 01, one, and TMS for the masses with which the skeleton and substituents contribute to the molecular weight. Each set of criteria above was given a n identification letter (S if requiring a TMS group, F if requiring a free hydroxy or keto group, C if M was calculated) and a number. The search was done according to all ten sets of criteria and the identification letter and number of the set(s) according t o which an M was defined were printed after the name of the steroid. In most cases the main steroid gave the longest list of sets. The result of the search is shown in Table I. In
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
Table I. Computer Calculation of Molecular Ions in Spectra from Analysis Shown in Figure 1 (TMS Derivatives) No. of remaining possible structures No. of Not No. of No. of failures No. of M+ structures not highly Supported by supported by unlikely inspection of fragmentation to detect Spectrum calcd compatible Compounds identifiedb No. by computer with ret. time structures mass spectrum pattern“ correct M+ ... A6-3P-ol-17-one 5 1 1 2 13 9 A6-3P-ol-17-one 14 6 3 ... 1 2 1 2 ... A6-3P,17a-01 16 9 4 2 1 1 ... A6-3/3,17a-01 17 8 4 2 21 2 1 ... 1 ... ... A6-3/3,17P-01 22 8 6 ... 1 1 ... A5-3P,17P-0l 2 3 ... A-diolones 2 4 24c 11 29 5 2 2 1 ... ... P6z1e-3p-ol-20-one 33 9 2 5 2d ... P5-3P-ol-20-one 35 15 1 9 2 2 18 P6-3P-ol-20-one; A-triol 37 12 4 3 4d 1 A-3, 11-01-17-one; P-3, 17-01-20-one 8 2 1 1 le P-diolone; P-3,2040 13 diol 46 13 8 1 2J 2 ... P6-3P,17a-ol-20-one P6-3p,20a-ol 49 9 4 3 1 1 ... PS-3/3,20a-ol; As50 13 8 ... 2 3 ... 3P,16a,17P-01 55 10 6 ... 2f 2 ... P6-3P,16a-ol-20-one 56 13 7 2 2J 2 ... P6-3P,16a-ol-20-one 62 12 7 ... 1 3 le P-3,17,20-01: P-3. 2i-oi-20-one 1 2 63 5 2 P-3.17.20-01 , 72 13 9 ... 1J 3 1J Ps-3P,17~,20~-ol e 73 10 8 ... ... 1 le P6-3P,17a,20a-~l a In most cases, one of these had the same molecular ion as the correct choice. In the other cases M+ was absent and was calculated from other fragments. * A = androstane, P = pregnane; greek letters denote configuration of hydroxyl groups, superscript indicates position of double bond. The compounds were identified on the basis of their spectra and retention times. Low intensity spectrum with many peaks. Possibilities which could not be excluded. e M - 90 or M - 18 was chosen as M+. One major compound. I
.
.
I
.
.
I
.
.
~
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Table 11. Computer Calculation of Molecular Ions in Spectra from the Analysis of a Steroid Fraction from Bovine Corpus Luteum Shown in Figures 3-5 (MO-TMS Derivatives Were Analyzed) No. of remaining possible structures No. of No. of Not No. of No. of M+ structures highly Supported by supported by failures Spectrum calcd not compatible unlikely inspection of fragmentation to detect No. by computer with ret. time structuresa mass spectrum patternb correct M+ Compounds identifiedc 46 8 4 2 1 1 ... P6-3P-ol-20-one 8 4 2 1 1 Id P6-3P-ol-20-one 47 P-3P-ol-fO-one 48 11 7 2 1 1 1 P6-3P-ol-20-one P-Sp-ol-2O-one 49 11 4 6 1 ... ... P-3P-ol-20-one 50 11 ... 8 2 1 ... P-3@-01-20-one P5-3P,2OP-0l 51 9 ... 5 2 2 ... P5-3P,2Op-0l P-3P,20p-Ol 54 11 7 3 1 ... ... P4-3,20-one 59 10 5 3 1 1 P4-20P-ol-3-one a The mass of M+required simultaneous presence of two underivatized substituents and two double bonds. M+ had the same mass as that of the identified compound or was obtained by calculation from other fragments. Tentative identification by mass spectrum and retention time. P = pregnane, greek letters denote orientation of hydroxyl groups, superscript indicates position of double bond. IZ M+ of the pregnanolone was considered as an isotope peak from M+ of the pregnenolone. t . .
spite of the complex nature of many spectra, the “chemical” background of the sample being intense, surprisingly few steroid structures were suggested and many could be eliminated o n the basis of retention time for which n o limits had been set.
A similar search was made for the analysis shown in Figures 3-5. Molecular weights of steroids containing any combination of a maximum of 2 double bonds, 1 keto group, 2 hydroxyl groups, 5 trimethylsiloxy groups and 5 0-methyloxime groups were permitted. In addition to the criteria listed ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
27
CONT.
C O N T - SCAN 117
P-OIOL
A
SCAN 117
P-DIOL
r -
9 CONT-
10
11
12
13
14
A;= fl. C O N T - SCAN 117
S C A N 117
P-DIOL 300 P G
MINUTES
Figure 8. Computer plots of fragment ion current, m/e 117, obtained by scanning m/e 110-120 every other second (30 ng, 1.5 ng, and 300 pg) or every 3rd second (75 pg) in four analyses of different amounts of the sample used in the analysis shown in Figures 6 and 7. The sample was analyzed at a higher temperature, and the peaks correspond to the pregnanediol TMS ethers at 14.3 and 17.3 min in Figures 6 and 7. The continuous mode for recording of spectra was used (see text) above, molecular weights of 0-methyloximes were considered in the following cases: M ( l x ) , M - 15 (l), M - 31 (1); M (l), M - 31 ( 5 ) ; M (absent), M - 15 (l), M - 31 (1). The result of the search is shown in Table 11. The criteria for M were set empirically after tests using reference spectra of about 200 steroid derivatives. With the reference compounds, M was found in about 8 5 % of the cases. The complexity of the mixtures from biological material, however, makes the search procedure less suitable for steroids with more than three oxygen-containing substituents. The spectra of about 500 steroid derivatives are presently being studied in collaboration with D r . J.-A. Gustafsson in order to establish empirical criteria for search of steroids in analysis of samples from patients. Search programs based on the use of abbreviated spectra with the 3 most intense ions in each 20 mass unit interval have been tested but are still too time consuming since presearch criteria based on relative intensity (16) are difficult to use when the spectra are due to several compounds in differing concentrations. These search methods have been extensively studied and recently discussed by Biemann et a/. (16). Quantitative Applications. The fragment ion current chromatograms can be used for quantitative estimations in the same way as “mass fragmentography” has been used (1719). Since long time constants can be used, the latter method is more sensitive than when entire spectra are taken. However, selection of m/e values must precede the analysis (6) which is a disadvantage in analyses of naturally occurring unknown steroid mixtures. (16) H. S. Hertz, R. A. Hites, and K. Biemann, ANAL. CHEM., 43, 691 (1971). (17) C. C. Sweeley, W. H. Elliott, J. Fries and R. Ryhage, ibid., 38, 1549 (1966). (18) C.-G. Hammar, B. Holmstedt, and R. Ryhage, A n d . Biochem., 25, 532, (1968). (19) R. W. Kelly, J . Chromatogr. 54, 345 (1971).
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ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
The sensitivity of the method utilizing incremental recording of spectra was evaluated with a mixture of cholestane and progesterone bis-0-methyloxime. These compounds have the same molecular weight and similar retention times o n SE-30 columns (separation factor 1.13). Between 2 and 200 ng were injected at 230 “C column temperature, giving a retention time of about 10 min. Two ng of cholestane was easily detected from mje 372, 217, and 218 whereas 10 ng of the progesterone derivative could be just detected from the mje 372, 341, 273, 153, 125, and 100 chromatograms. The peak heights for cholestane were linearly proportional to the amount injected. In the case of the progesterone derivative, the relationship was nonlinear. Comparisons using either a flame ionization detector o r the mass spectrometer as detector with the same column, showed that the nonlinearity was more pronounced with the mass spectrometer where it was partly related to the temperature of the molecule separator. A similar behavior was shown by MO-TMS derivatives-e.g., of testosterone and 17a-hydroxyprogesterone-whereas the TMS ether of 5-androstene-3,l3,17P-diol behaved as cholestane. At present, the only method available which will counteract the loss in sensitivity due t o adsorption and thermal lability is the addition as internal standard of a large excess of the compound labeled with a stable isotope (20-22). Using the incremental mode to record spectra, the sensitivity limit for common steroids which do not suffer extensive loss in the column-separator system, and which have retention times between 5 and 15 min, is about 1 ng (signal to noise ratio about 3). To obtain a higher sensitivity the continuous mode of recording must be utilized. A slow repetitive scan (20) B. Samuelsson, M. Hamberg, and C. C . Sweeley, Anal. Biochem., 38, 301 (1970). (21) L. Siekmann, H.-0. Hoppen, and H. Breuer, Z . Anal. Clzern., 252, 294 (1970). (22) T. E. Gaffney, C.-G. Hammar, B. Holmstedt, and R. E. McMahon, ANAL. CHEM.,43, 307 (1971).
(1-5 sec) is made over a selected range of m/e values (10% or less of the mass of the ion focused with the magnet field at the start of the scan). This can be done with the magnet or the accelerating voltage. The electron multiplier voltage is set at 3.1-3.7 kV and a high input resistance is used in the electrometer amplifier (50,000 MO). The values recorded on the tape are read into the computer which finds the mass spectrometric peaks and calculates the area under each peak. When magnet scanning is used, the beginning and end of the peaks can be determined using the mass marker pulses simultaneously recorded o n the tape. The areas obtained for the mass(es) of interest are used to construct the gas chromatogram. A five-point smoothing of the GLC curve is first made and the lowest value is subtracted. The gas chromatogram is drawn by the plotter setting the highest value found t o full scale deflection. Four recordings obtained in this way in analyses of different amounts of the same sample as that shown in Figures 6 and 7 are shown in Figure 8. M / e 117 was scanned repetitively during the elution of pregnanediol TMS ethers. The ratio between the peak areas shows that there is a relatively larger loss of the pregnanediol eluted last when 75-pg amounts are analyzed.
When the continuous mode of recording is used as described, the sensitivity limit (signal/noise ratio about 3) for compounds such as cholestane and TMS ethers of pregnanediols and 5-androstene-3@,17@-diol is of the order of 20-50 pg. A major factor determining this value is a low frequency fluctuation of the intensity of the background peaks. The detection limit for substances lost in the column-detector system is much higher. Thus, progesterone bis-0-methyloxime could not be detected when less than 500 pg was injected. ACKNOWLEDGMENT
The electronic designs of the automatic scan start unit and the accelerating voltage scan unit were made by research engineer S. Melkersson and Miss A. Oberg. RECEIVED for review July 13, 1971. Accepted August 11, 1971. Work supported by grants from the Swedish Medical Research Council (Grant No. 13)3-2189), the Wallenberg Foundation, and the Bank of Sweden Tercentenary Fund.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
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