Table I. Boron Assay Results No. of Assay by Sample determina- isotope No. tions dilution, % 1 2 3
4
6 3 2 2
99.35 97.26 97.23 97.50
Relative standard deviation, %
Assay by titrimetry,
0.05
99.38 97.36 97.20 97.50
0.10 0.08 0.05
%
lo&. This corresponds to errors in the total boron assay of 0.005%and 0.01%, respectively. A weighing error or loss during fusion of 0.02 mg of boron corresponds to 0.02% error in the boron assay.
ACKNOWLEDGMENT We thank V. E. Connolly and E. L. Callis for performing the mass spectrometric analyses. LITERATURE CITED (1) A. R. Eberle, M. W. Lerner, and L. J. Pinto, Anal. Chem., 36, 1282
tervals, especially when a new bottle of reagent is used. For this work the correction factor applied to the mass 89/88 ratio for 10Bmis 0.9994 and that for l0Bois 1.0005.The boron used was more than 90% enriched in boron-10. Boron assay results for four samples are shown in Table I. In each case, there is good agreement between the assay found by the isotope dilution technique and an independently determined chemical titration assay. The largest relative standard deviation found is 0.10%. Standard deviation for duplicate mass spectrometric determinations is 0.003% for l0B, and
(1964). (2) A. R. Eberle and M. W. Lerner, Anal. Chem., 37, 1568 (1965). (3) C. A. Lucchesi and D. D. DeFord, Ana/. Chem., 29, 1169 (1957). (4) A. A. Nemodruk and Z. K. Karolova, "Analytical Chemistry of Boron", E. Seijffers, Ed., Sivan Press, Jerusalem, 1965, p 97. (5) J. C. Landry et al., Mitt. Geb. Lebensmittelunters. Hyg., 65, 65 (1974). (6) E. J. Catanzaro et al., "Boric Acid; Isotopic, and Assay Standard Reference Materials", Nat. Bur. Stand. (US.). Spec. Pub., 260-17, Feb. 1970. (7) M. W. Lerner, "The Analysis of Elemental Boron", USAEC Div. of Tech. Information, TID-25190, Nov. 1970.
RECEIVEDfor review March 23, 1976. Accepted April 29, 1976.
Computer Acquisition and Processing of Metastable Ion Scans in a Double Focusing Mass Spectrometer Lubomir Baczynskyj,* D. J. Duchamp, J. F. Zieserl, M. D. Kenny, and J. B. Aldrich Research Laboratories, The Upjohn Company, Kalamazoo, Mich. 4900 1
A computerized method for automatically acquiring metastable ion data in a double focusing mass spectrometer with reversed geometry is described. Programs have been developed allowing accelerating voltage and electrostatic sector scans. The data acquisition and data reduction are performed in real-time. Time for processing the data of a complete scan is 15-20 s. The accuracy of mass determinations is Increased for weaker metastable peaks and large gains in conveniences are achieved.
For many years, the study of metastable ions had been confined mainly to physical chemistry. In recent years, however, the usefulness of metastable ions in the analysis of mass spectra of organic molecules has been clearly demonstrated ( I ) . Rapid progress in this field has been achieved due to the pioneering work of Beynon, McLafferty, and others. The recent progress in instrumentation has made it possible for many laboratories, whose principal interest lies in the area of organic mass spectrometry, to become involved in this field. Recently we acquired a double focusing mass spectrometer with reversed geometry, the Varian MAT CH5 DF. The particular feature of this geometry is the arrangement of the analyzing sectors. The ion source is followed by the magnetic sector which is followed by the electrostatic sector analyzer and terminated by the detector. The advantages of such an arrangement have been discussed in the literature (2-4). T o detect metastable ions, two types of experiments can be performed. The accelerating voltage can be scanned (for the CH5 DF, from 1 to 3 kV or from 2 to 3 kV) while maintaining the magnetic and electrostatic sectors constant ( 5 ) .Such scans yield information about the precursors of a given fragment ion. Scanning of the electrostatic sector (ESA), at constant magnetic field and accelerating voltage, gives rise to a spectrum 1358
ANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976
of metastable peaks, which correlates a given ion with its daughter ions. This last technique has been called DAD1 ( 4 ) (direct analysis of daughter ions) or MIKES ( 2 ) (mass-analyzed ion kinetic energy spectrometry). Using the manual mode, we recently studied the fragmentations of some prostaglandin molecules by both defocusing techniques (6). Although the information obtained from these experiments was very useful, the process of obtaining such results was tedious. T o increase the accuracy of the measurements, four determinations of the tops of each metastable peak were made. Considering, that in general there are several metastable peaks in each ESA scan of a given ion, the acquisition of these data manually was very time-consuming. Another disadvantage of this manual procedure on our mass spectrometer is that, in the decoupled mode, the accelerating voltage or the ESA voltage can slowly drift during the measurements. Such measurements can last from a few minutes to an hour per scan depending on the number of peaks measured. This small drift can lead to a decrease in accuracy. Furthermore, the electron multiplier is normally operated a t high gains because of the low intensity of some metastable peaks. This introduces noise on top of the peaks and makes the determinations more difficult. The shape of the metastable peaks is normally Gaussian; however, i t is well known that not all metastable peaks have a Gaussian profile ( I ) . For "flat" or "dish" top metastable peaks, it is important to determine the center rather than the high point of the metastable peak. For the above reasons, we decided to computerize the scanning and processing of metastable ion data. Appropriate programs were written to allow rapid scanning of the ESA or the accelerating voltage, acquiring the data automatically, and processing and presenting the final results in an appropriate format. This project became part of a larger programming effort in which three mass spectrometers may scan simultaneously in different modes. Data are acquired in real time and
Table I. A. C o m m a n d f o r S c a n n i n g the E l e c t r o s t a t i c S e c t o r Voltage. B. C o m m a n d f o r Scanning the
CH5
8/30/75 SEO NO 18 I O TETRACOSANE
ESA SCAN
I O N SCANNED
338,391
A c c e l e r a t i n g Voltage A ‘“ENTER
MANUAL DATA
OP
L.B. I D TETRACOSANE ES 3 , 5 0 6 . 4 0 , 3 3 8 . 3 9 1 2
‘4
5
**MANUAL DATA SENT
07
05
04
03
02
01
-
0.0
Figure 1. Spectrum of metastable ions produced by an ESA scan of the M.+ of tetracosane. This is an average of 4 scans followed by smoothing
DATE: 5 / 9 / 7 5 T I M E : 1 9 . 3 7 USER: L . B I, D . : TETRACOSANE E . S . A . SCAN.
CH5
MASS OF ION SCANNED: 3 3 8 . 3 9 1 2 I N I T I A L VOLTAGE: 506.4000 SCAN RATE: 3 DURATION OF SCAN: 7 9 SEC. SET SAMPLING RATE TO
06
625
CH5
8130175 SEQ N O
8 I O TETRACOSANE
HV S C A N .
I O N SCANNED
57.070
100
B **ENTER
MANUAL DATA
50
OP
L.B. I D TETRACOSANE AS 1 , 3 , 3 . 1 9 8 1 , 5 7 . 0 7 0 4 $ **MANUAL DATA SENT
CH5 DATE: 8 / 3 0 / 7 5 TIME: 1 5 . 5 8 USER: L . 6 I. D . : TETRACOSANE ACCELERATING VOLTAGE SCAN. MASS OF ION SCANNED: 5 7 . C 7 0 4 I N I T I A L VOLTAGE: SCAN RATE: 3 DURATION OF SCAN: 68 SEC. SET SAMPLING RATE TO
E/EO=
3.1981
625
processed using a time-shared computer (IBM-1800). A brief account of the total system was presented a t the 23d Annual Conference on Mass Spectrometry and Allied Topics (7).
EXPERIMENTAL Interface Hardware. The same hardware that is used to interface three mass spectrometers (CH5 DF, CH7, and LKB 9000) to the IBM 1800 computer for data acquisition of normal spectra is also used for the data acquisition of,metastable scans on the CH5 DF. An extra switch on the interface is provided on this mass spectrometer for selection of metastable vs. normal scan modes. The interfacing hardware was designed and constructed in our electronics laboratory. Scanning is initiated by the computer either automatically according to a time scheme, or in response to an operator button. Minor modifications of the scanning circuit of the CH5 DF were made to allow the computer to start and stop the scanning of the magnet, the accelerating voltage, pr the electrostatic sector analyzer. The intensity signal is fed through a set of autoranging amplifiers into a 12 bit A/D convertor. The digitized intensities are sent to the computer a t an operator selected rate under the control of a real-time clock in the interface. A detailed description of the total system will be published a t a later date. Description of t h e Computerized Method. Initiation of Erperiment. In Table IA, a typical setup of an electrostatic sector (ESA) scan is shown. A series of commands are entered using the Texas Instrument Silent 700 teletype writer. The ES command sets up for scanning the electrostatic analyzer. This command is followed by three input parameters separated by commas: the first number (3) is the scan speed, the second (506.40) is the initial voltage Eo of the ESA, and the third (338.3912) is the exact mass of the ion being scanned. The computer prints a summary of the experiment and indicates to the operator the sampling rate to be selected. A similar setup for scanning the accelerating voltage is shown in Table IB. The parameters in the AS command are: 1 or 2 depending on the selected range (1 to 3 kV or 2 to 3 kV), scan speed, initial accelerating voltage, and mass. The values of initial voltages (Eo) are read from a digital voltmeter. Data Acquisition. A “SCAN” button a t the instrument is used to request the computer to start a scan. Low sampling rates of the intensity signal from the electron multiplier are required because of the characteristics of the scanning circuitry of the CH5 D F (slowest scanning rate -310 s, fastest scanning rate ~7 s). To reduce noise, it is advantageous to sample the signal faster than required and average the readings. Knowing the scan rate and duration of the scan, the computer averages every 2“ points where n is such that the total number of points after averaging is less or equal to 3000. Thus for scan
12
14
16
18
2 0
22
24
26
2 8
30
Figure 2. Spectrum of metastable ions (HV Scan) showing the origins of the ion occurring at m/e 57 in the mass spectrum of tetracosane
speed 3, duration of scan -79 s, and sampling rate 625 Hz, every 32 data points are averaged, yielding 1541 points for a complete scan. Results of the data acquisition step are accumulated in a “RAW DATA FILE” on disk. As soon as the data acquisition part is completed, the data processing programs are executed automatically. Data Processing. The processing of the data consists of determining the positions of the peaks as a function of time and converting these measurements to voltages and to masses. The relationship between voltage and time is determined for each scan rate by using a calibration compound. In the initial processing step, each scan is reformated and transferred to a “TEMPORARY FILE” on disk, which can hold 40 scans. These data may be reprocessed with different options, averaged, printed, and plotted. Figure 1shows an ESA spectrum of the molecular ion of tetracosane (m/e 338). This spectrum was obtained by averaging four scans and smoothing the data points. Considerable noise reduction results from the averaging process. Further noise reduction is obtained by smoothing the data. A seven-point quadratic smoothing function is used for this purpose. Figure 2 shows an accelerating voltage scan of the ion at m/e 57 of tetracosane. This spectrum is a single scan without smoothing. In determining peak centers, the processing program first skips over the tail of the main beam ion. Since the intensities of the metastable peaks are much smaller than the intensity of the main beam ion, they would become insignificant after normalization. For this reason, the data points (intensities) corresponding to the main beam ion are ignored. The program then subtracts the offset which is the lowest point in the scan. A threshold is arbitrarily set to 5% of the highest point in the scan (excluding the main beam ion). Only peaks above this threshold are considered. The program then determines the high point (position of the maximum) and the center at 3/4height for each metastable peak id terms of point number (time). The intensity, the high point, the center at % height and the width a t 3/4 height are computed for the 20 most intense metastable peaks of each scan. The final part of processing the data consists of correlating the centers of the peaks with voltages and masses. I t is assumed that the scan of the electrostatic sector, or accelerating voltage, is linear with time. Thus, from the peak position and the slope and the intercept of the line, the voltage E corresponding to each metastable peak is calculated. Using this voltage, the mass of the main beam ion ( m l )and the initial voltage Eo, the mass of the daughter or precursor ion (mz) can be computed for each metastable peak from the well known equation
E Eo’m1
mz = -
A printed report, such as shown in Table 111, is automatically obtained for each scan and is also produced when a scan is reprocessed or when an averaged spectrum is computed. For each metastable peak, ANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976
1359
Table 11. A. Calibration for the ESA Mode. B. Calibration Report for the HV Mode A
386
LINEAR REGRESSION RESULTS-BEST F I T OF 14 PTS SLOPE = 0.00947 Y INTERCEPT = -9.16587 VARIATION = 0.00000 STD DEVIATION = 0,00000 CORRELATION COEFF = 1 .00000 360
50
50
200
150
100
250
350
300
EO-€(THEOR.)
450
400
Figure 3. Mass spectrum of cholesterol CH5
9 /6 /7 5 SE(1 NO
9
I D CHOLESTEROL
ESA S C A N .
I O N SCANNED
386.354
EO-E(CALC.)
43.481637 64.468257 85.454324 106.440929 127.426980 148.41 3631 169.399667 190.386287 211.372323 232,358943 253.34501 0 274.331138 295.317728 316.30431 7
POINT NBR.
43.534825 64.475716 85.4771 77 106.418060 127.389240 148.390717 169.392254 190.363404 211.395207 232.305763 253.337536 274.369339 295.279957 316.372292
MASS (THEOR.)
5564.8 7776.0 9993.6 12204.8 14419.2 16636.8 18854.4 21068,8 23289.6 25497.6 27718.4 29939.2 321 47.2 34374.4
MASS(CALC.
309.3521 295.3365 281.3208 267.3052 253.2895 239.2739 225.2582 211.2426 197.2269 183.21 13 169.1956 155,1800 141.1643 127.1487
309.3166 295.3313 281.3055 267.3203 253.3147 239.2890 225.2631 211.2576 197.2116 183.2465 169.2006 155.1546 141 .1895 127.1030
B LINEAR REGRESSION RESULTS-BEST F I T OF 4 PTS SLOPE = -0.00016 Y INTERCEPT = 0.14953 VARIATION = 0.00000 STO DEVIATION = 0,00000 CORRELATION COEFF = 1.00000 EO-E(THE0R.)
EO-EICALC.)
-1.567964 -2.351 942 -3.135926 -3.919907
E/EO=
0.7
08
0.9
0.6
05
04
03
02
01
POINT NBR.
-1.567935 -2.352022 -3.135847 -3.919934
MASS (THEOR.)
10454.4 16227.2 19998.4 24771.2
MASS(CALC.)
85.1017 99.1173 113,1330 127.1486
85.101 2 99.1187 113.1315 127.1490
0.0
Flgure 4. Spectrum of metastable ions (ESA Scan) produced by the M.+ ( d e 386) of cholesterol. This is an average of 8 scans with smoothing
Table 111. Report of Metastable Ion Scan (ESA Scan) Produced by Maf of Cholesterol
VITAMIN 03 CH5 DATE: 9/6/75 TINE: 12.49 USER: L.O 1.0.: CHOLESTEROL SEQU. NO. 9 AVERAGE OF 8 METASTABLE SCANS
I:, I 'i"
1
loo
-I
METASTABLE SCAN REPROCESSED METASTABLE SCAN HAS BEEN SMOOTHED
ll
I
I I 158 I I I
i
I
50
LbO
IS0
HIGH POINT
CENTER AT 314 H.
675 2364 4371 302 302
620 950 1060 2170 2040 3390 3970 5180 5740 6350 6990 8070 9300
649 955 1082 2162 2037 3378 3969 5175 5727 6340 6994 7810 9293
231
y' 200
Figure 5. Mass spectrum of vitamin
250
300
350
384
400
2872 912 370 655 911 279 405
WIDTH AT 314 H. 92 61 42 74 100 187 64 76 117 98 90 81 3 125
E/EO 0.97978 0.96048 0.95289 0.88832 0.84797 0,81563 0.78030 0,70820 0.67520 0.63855 0.59945 0.55067 0.46201
MASS 378.157 371.089 368.155 3k3.210 327.619 31 5.123 301.472 273.617 260.867 246,708 231,602 212.754 176.500
TRANSITION FROM 186 TO: 378 371 368 343 327 315 301 273 261 247 231 213 178
t
8
+
18 43 59 71 85 113 125 139
+ 15
+ t t
+
t
+ +
+ 155
t 173 t
206
D3
the report lists intensity (in arbitrary units), two measures of peak location (high point and position of peak center at 3/4 height) and peak width at 3/4 height (in units of point number X 10). Two measures of peak position are helpful in detecting abnormal peak shapes. The report also lists the calculated EIEo, the calculated mass, the nominal mass of the precursor (HV) or daughter (ESA) ion, and the nominal mass of the neutral fragment. The elapsed time from scan completion to the printing of the report is about 20 s. Calibration. Even though the circuitry of the CH5 DF mass spectrometer is designed for linear voltage scans, it is not obvious that a linear voltage-to-time calibration would work for the accuracy required. A number of experiments have convinced us that a straight line with a non-zero intercept would be sufficiently accudrate. Different values of slope and intercept are required for each scan speed of each scan type-ESA, HV (1to 3 kV) and HV (2 to 3 kV). Accurate slope and intercept values for correlating peak positions to voltage are determined by use of a calibration compound. The calibration is performed by the computer using the molecular ion of tetracosane (mle 338) for ESA mode (see Figure 1).In the HV mode, the ion a t mle 57 of tetracosane is used (see Figure 2). In the calibration step, the computer matches the experimental peak positions to the known peaks of the calibration spectrum by correlating an approximate calculated mass to the known mass. Slope and intercept are determined by a linear regression calculation using (Eo - E ) theoretical vs. point number (based on sampling rate). A typical report of the calibration program for the ESA mode is shown in Table IIA. The fit to a straight line is essentially perfect. The intercept is quite significant; this is due to the delay between scan start and actual beginning of voltage sweep. The agreement between the calculated and theoretical (Eo - E ) and mass are excellent. A similar report for the HV 1360
INTENSITY
ANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976
mode is shown in Table IIB. Each slope and intercept from calibration is automatically stored for use in subsequent experiments.
RESULTS AND DISCUSSION The information obtained from metastable ion experiments, mainly used for theoretical studies, can be quite valuable in the solution of structure problems. We are often involved in structural elucidation of organic compounds by mass spectrometry. To solve these problems, we must sometimes make a detailed study of the processes occurring in the mass spectrometer. The speed and convenience of the system described here makes i t possible for us to apply metastable ion studies to many more problems than we could by the manual method. Metastable ions are useful in studying fragmentation pathways because a metastable peak generally implies a one-step process between the precursor and product ions. However, the absence of a metastable does not guarantee that this process does not occur in the mass spectrometer.Also, the presence of a metastable peak could arise from a two-step reaction-a fast reaction followed by a slow one. But it is generally accepted that such processes occur only rarely and that the presence of a metastable peak links two ions as precursor and product in a given fragmentation scheme (8). A study of the fragmentation of cholesterol (I) provides an example of the use of our computerized technique. Figure 3 shows the low resolution spectrum of cholesterol.
Ion --
I D V I T A M I N 03
ESA SCAN
I O N SCANNED
384,339
10 Scans Each t h e Average o f 9 or 10 Scans
36 Scans Reprocessed WISmoothing
36 Scans
9/6/75 SED NO 4
CH5
Table IV. Statistical Analysis of Selected Ions in ESA Scans of the Molecular Ion of Tetracosane
281.32 Mean c s.0. Std. Dev. Range
281.24 .05 ,113
t
211.37 .06 .28
t
.01
281.23 i .01 .04 .14
281.23 .04 .10
t
.01
211.37 t .01 .05 .20
211.40 .04 .13
?
169.36 t .01 .05 .16
169.39 t .01 .04 .13
112.89 .08 .36
112.94 .05 .13
Figure 6. Spectrum of metastable ions (ESA Scan) produced by the Ma+ (m/e 384) of vitamin DJ. This is an average of 3 scans with smooth-
211.24 Mean i S.D. Std. Oev. Range
.01
.01
ing CH5
9 / 5 / 7 5 SEO NO
7
ID VITAMIN 03
HV SCAN.
I O N SCANNED
158.100
169.19 Mean ?- S.D. Std. Oev. Range
169.36 t .01 .07
.32
113.13 Mean t S.D. S t d . Dev. Ranae
112.95 .18 .78
?
.03
5
.01
t
.01
Having obtained such a spectrum, one normally has to guess which fragment ions originate directly from the molecular ion. In the system described here, it takes only 3 or 4 min to switch to the metastable mode, to focus on the molecular ion at m/e 386, and to obtain the results shown in Table 111. The plot of an averaged spectrum resulting from such scans is shown in Figure 4. These results show, for example, that the fragment ions at m/e 371,368,301 come directly from the molecular ion; the ions a t 353 and 275 probably do not. The usefulness of such information in the interpretation of mass spectra of compounds of unknown structure is well established.
(11)
Metastable scans can sometimes suggest structural similarities not obvious in normal mass spectra. The low resolution mass spectrum of vitamin D3 (11)is shown in Figure 5. At first glance this spectrum looks quite different from that of cholesterol (Figure 3). When one compares the ESA scan of the molecular ion of vitamin D3 (shown in Figure 6) to that of the molecular ion of cholesterol (Figure 4), similarities in fragmentation are quite apparent. Both molecular ions lose 18 (HzO), 59, 85, 113, 139, and 208 indicating close structural similarities. However vitamin D3 has a pronounced metastable peak corresponding to the loss of 248, which is cleavage across the (7-8) double bond. This cleavage occurs a t a point in D3 which is structurally dissimilar to cholesterol and leads to the dominant ions (136, 118) in the spectrum of vitamin D3. An example of the use of our computerized system for studying precursor ions is given by the HV scan of the ion a t m/e 158 in the spectrum of vitamin Ds. This scan (Figure 7) shows that this ion, the third strongest ion in the spectrum, arises from m/e 176 (loss of HzO). This therefore indicates that ion 176 contains oxygen and that both 176 and 158 probably arise from the lower part of vitamin D3. A statistical analysis of the precision of mass measurements obtained by using our computerized system is shown in Table
& E/EO=
,
7
12
14
16
18
20
22
24
26
28
30
Figure 7. Spectrum of metastable ions (HV Scan) obtained from ion at m/e 158 of vitamin D3
IV. Using results from repeated runs, statistics were computed on mass determinations on four metastable peaks of different intensities. They were extracted from ESA scans of the molecular ion of tetracosane (see labeled peaks in Figure l). Column 1gives the statistics-mean ( h t d error of mean), standard deviation of the sample, and range (maximum mass - minimum mass)-for masses obtained by processing these scans without use of the smoothing and averaging options. Excellent precision of the mass measurements is shown for the more intense peaks as evidenced by the low standard deviation and narrow range. The mass of the 113 ion, with a very small metastable peak, is measured with much less precision. The signal for this peak is not much above the electronic noise. Column 2 gives similar statistics on masses obtained by reprocessing these data as individual scans with smoothing. The precision for the most intense peak is essentially unchanged. However, a considerable improvement in precision is obtained for the less intense peaks. Statistics showing the effect of the averaging option (Column 3) were computed on masses from 10 spectra, each the average of 9 or 10 individual scans. By use of this option, masses from small peaks may be measured with a precision comparable to masses from large peaks. This is due to the well-known reduction of random noise achieved by averaging. Since the intense peak (281) is less affected by random noise, averaging gives only a small increase in precision. Similarly, increasing the number of scans in the average much beyond 10 gives only marginal precision gains. A measure of the accuracy of mass measurements is given by comparing theoretical masses with the calculated means shown in Table IV. Mass values are generally within 0.2 amu. Calculated masses in the center of the spectrum show a small positive bias; those at the ends show a small negative one. This results from the small nonlinearity of the scanning circuitry of our mass spectrometer and our assumption of a linear calibration curve. Using a higher order calibration curve would remove most of this bias. However, for practical purposes, the accuracy achieved here is quite sufficient. Our short experience with this system indicates that the weak point is our mass spectrometer. Increased stability of the accelerating voltage and electrostatic sector voltage should increase the accuracy of the measurements. The amount of sample consumed in these experiments is substantial. RouANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976
1361
tinely we use 10 to 100 times as much sample as for a normal low resolution scan. Two major advantages of the computerized over the manual method of acquiring metastable data are evident from our experience. First, an order of magnitude in convenience is gained by using the computer. Second, masses are determined with considerably higher accuracy. In a practical sense, the experiments involving smoothing and averaging cannot be run without a computer. The determination of the masses of weak metastable peaks cannot be performed manually on our mass spectrometer to better than fl amu; whereas, with the computerized system, much better determinations are routinely achieved. A fully automated system for processing metastable ion data produced by the Barber-Elliot-Major defocusing technique has been described in the literature (IO). In that system, all the metastable transitions for all ions in a spectrum can be detected. For our system, it would have been quite simple from the programming point of view to let the computer find the top of each normal ion, from Ma+ downwards, and to take an ESA scan on each of them. However, the management of such large sets of data presents some problems and tends to dilute the attention of the mass spectrometrist. In our system, we have taken the approach that only ions of interest, selected by the operator, will be scanned. Each ion of interest is rapidly brought in focus using the magnetic field and the digital mass marker display. The total time for scanning and processing
each spectrum is usually less than 2 min. It was pointed out to us by Prof. Beynon that, as with low or high resolution mass spectra, the mass spectrometrist is usually selective with the choice of ions that he considers. These are either intense ions or structurally significant ions of lower abundance. The same should be true for metastable peaks if we want to use them on a routine basis. LITERATURE CITED (1) R. G. Cooks, J. H. Beynon, R. M. Caprioli, and G. R. Lester, “Metastable
Ions”, Elsevier Scientific Publishing Co., Amsterdam, 1973. (2) J. H. Beynon,,R. G. Cooks, J. W. Amy, W. E. Baitinger, and T. Y . Ridley, Anal. Chem., 45, 1023A (1973). (3) T. Wachs, P. F. Bente 111, and F. W. McLafferty, lnt. J. Mass Specfrom. /on Phys.,9, 333 (1972). (4) K. H. Maurer, C. Brunnee, G. Kappus, K. Habfast, U. Schroder, and P. Schulze, 19th Annual Conference on Mass Spectrometry, ASTM Committee E-14, Atlanta, Ga., May 1971. (5) M. Barber and R. M. Elliott, 12th Annual Conference on Mass spectrometry, ASTM Committee E-14, Montreal, Canada, June 1964. (6) L. Baczynskyj and R. J. Wnuk, 22d Annual Conference on Mass Spectrometry and Allied Topics, Philadelphia, Pa., May 1974. (7) L. Baczynskyj, J. F. Zieserl, M. D. Kenny, J. B. Aldrich, and D. J. Duchamp, 23d Annual Conference on Mass Spectrometry and Allied Topics, Houston, Texas, May 1975. ( 8 ) See Chapter 2 of Reference 1. (9) F. W. Mclafferty, “interpretation of Mass Spectra”, 2d ed.,W. A. Benjamin, Inc., Reading, Mass., 1973, Chapter 7. (IO) J. E. Coutant and F. W. McLafferty, lnt. J. Mass Spectrom. /on Phys., 8, 323 (1972).
RECEIVEDfor review December 4,1975. Accepted April 26, 1976.
Probability Based Matching System Using a Large Collection of Reference Mass Spectra Gail M. Pesyna,’ Rengachari Venkataraghavan, Henry E. Dayringer, and F. W. McLafferty * Department of Chemistry, Cornel1 University, Ithaca, N.Y. 14853
The Probability Based Matching (PBM) system, which utlllres a reverse searching procedure and weightlng of mass and abundance data, has been modifledto permlt matching of an unknown spectrum against a large data base not restricted to spectra taken under the same experimental condltions. The performance has been evaluated quantitatively using recall/ reliability plots following procedures developed for informatlon retrleval systems. Sensltlvity to lmpuritlesand other errors in the reference spectra have been decreased by “flagging” up to four peaks to Ignore them In the calculation. PBM has been tested with four classes of criteria for structural matchlng, showing that the great majority of incorrect matches are of structurally related compounds. The utlllty of PBM Is partlcularly striking for giving useful performance in the identification of components in as low as 10% concentration.
Research in the area of document and information retrieval has firmly established that system efficiency is increased by the proper weighting of the relative importance of items used for identifying each member of a library ( I ) . Similar conclusions have been reached by those studying mass spectral retrieval systems (2-6): for example, Crawford and Morrison ( 3 )showed that using a logarithmic scale of peak abundances gave a substantial improvement in retrieval performance, in Current address, Science and Technology Committee, U.S. House of Representatives, 2321 Rayburn Building, Washington, D.C. 20515.
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ANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976
part by reducing the importance of the base peak in the spectrum. A “Probability Based Matching” (PBM) system has recently been proposed ( 5 ) which also weights the mle values of the peaks according to their “uniqueness”, applying this on a small reference library (less than 100 spectra) from one instrument using a reverse search. This paper describes the application of the PBM concept to a large data base (23 879 spectra) (7) and testing of the resulting system using a statistically large sample of unknown spectra. This system was designed in particular to emphasize high reliability in retrieval, as those unknowns for which only low confidence matches can be achieved usually must also be examined by a mass spectrometrist or an interpretive algorithm such as the “Self-Training Interpretive and Retrieval System” (STIRS) (8).The results show that weighting of the mass as well as the abundance values improves retrieval performance, and confirms the value of “reverse searching” for the spectra of mixtures (5, 6). “Probability Based Matching” is based upon the “General Rule of Multiplication” of probability theory (9) which states that if n independent events occur with probabilities p1, p z , . . . ,pn then the probability of all n of these events occurring is given by Equation 1. Thus if peaks with
n pi n
overall probability =
i=l
(1)
masses rnl and r n z having intensities i l and iz occur in mass spectra with probabilities p 1 and pz, the probability that both occur at random in an unknown spectrum is p 1 X p2. If this
