1130 computer, input and checking of a structure takes about 1 second. By our experience, the coding system described here seems to represent an optimum procedure with respect to short coding time, required previous knowledge, ease of training, punched card economy, computer time, and error detecting capability. The coding, however, is based upon existing graphical structure formulas. Producing a graphical picture of the structure from the official chemical names as the only basis, however, requires a highly skilled chemist with special knowledge and takes longer by far than the coding itself. The principle of the procedure presented here is by no means new. However, this special composition of different steps which leads to simple and time-saving operation may find some interest.
The input and checking programs are written for an IBM 1130 computer. Most of the programming was done in FORTRAN IV, some input and output routines, however, in 1130-ASSEMBLER to achieve overlapped execution. Program listings may be obtained upon request. Condensed Storage of Connection Tables. To reduce the need for total storage capacity, the connection tables are compressed to about 6 bytes per non-hydrogen atom, i.e., 80-120 bytes are necessary for storage of each structure. During the reading of these short-format connection tables from external storage devices, the connection table is simultaneously filled up to its full format. PRESENT EXPERIENCE AND ASSESSMENT The coding method described here was applied to the collection of mass spectra of the Mass Spectrometry Data Centre (MSDC), Aldermaston. About 8000 structures have been coded. After a few hours of training, a coding speed of about 50 structures per hour was achieved. Using a n IBM
RECEIVED for review July 13, 1972. Accepted October 19, 1972.
Computerized Quantitation of Drugs by Gas Chromatography-Mass Spectrometry Lubomir Baczynskyj, David J. Duchamp, John F. Zieserl, Jr., and Udo Axen Phj.sicul and Analytical Chemistry Research, The Upjohn Company, Kalamazoo, Mich. 49001 A fully computerized method for quantitative determination of picogram amounts of drugs using a GC-MScomputer system is described. In this method, the magnetic field is scanned repetitively Over a mass range and the intensities of selected ions of a drug and its deuterated analog are measured by the comPuter. In the final report, the ratio of Protium to deuterium form of the drug is obtained. The lower limit of detection of the drugs investigated so far i s 200-300 picograms. The reproducibility of the individual measurements is good, as shown by the reported results.
using an accelerating voltage alternator (AVA). The heights of the two traces are measured, and their ratio yields the composition of the mixture. This technique applied to isotopically labeled mixtures such as glucose and gluCose-d7 ( 2 ) gave isotopic abundances on aslittle as 0.55 pg with an average deviation of 5.5z. More recent applications of this method to such drugs as chlorpromazine and its metabolites (3), nortriptyline (4), and prostaglandin El (PGE1) ( 5 ) have proved its usefulness and general applicability to the field of drug analysis. The extension of this approach to PGEz and PGFzcy has allowed the determination of picomole quantities of these prostaglandins by using the corresponding (3,3,4,4-d4) compounds as carriers (6). Contrary to the AVA approach (2), in our method the accelerating voltage is maintained constant, while the magnetic field is repetitively scanned at a slow rate over a narrow mass range (20-30 amu). This is similar to the system recently described by Hites and Biemann (7) in which the whole mass range is continuously scanned during a G C run and certain ions diagnostic of particular structural features are plotted as a function of the scan number to yield a “mass chromatogram.” Such an approach gives a very useful qualitative information, but previously has not been widely used for quantitative measurements.
THE COMBINATION of gas chromatography (GC) with mass spectrometry (MS) has given the analyst an extremely versatile and sensitive tool. Although the use of the mass spectrometer for quantitative determinations of components of mixtures has been known for many years in the petroleum industry ( I ) , the recent emphasis on the qualitative information contained in a mass spectrum has overshadowed its quantitative aspect. In the present communication, we report the use of a computerized GC-MS system for quantitative determinations of very small amounts of drugs. It had been shown previously ( 2 ) that it is possible to determine the composition of an unresolved mixture in the gas chromatographic effluents by utilizing the mass spectrometer as the detector for the gas chromatograph. In this method, the intensities of two preselected ions, each characteristic of one component of the mixture, are continuously recorded by ( I ) J. H. Beynon. “Mass Spectrometry and Its Applications to Organic Chemistry,” Elsevier Publishing Co., Amsterdam 1960, p
424. (2) C. C. Sweeley. W. H. Elliott, I. Fries, and R. Ryhage, ANAL. CHEM., 38,1549 (1966).
(3) C. G. Hammar, B. Holmstedt, and R. Ryhage, A/ru/. Biochem., 25, 532 (1968). (4) T. E. Gaffney, C. G . Hammar, B. Holmstedt, and R. E. McMahon, ANAL.CHEM., 43,307 (1971). (5) B. Samuelsson, M. Hamberg, and C. C. Sweeley, A n d . Biochem., 38, 301 (1970). (6) U. Axen, K . Green, D. Horlin; and B. Samuelsson, Biochem. Biophys. Res. Commun., 45, 519 (1971). (7) R. A. Hites and K. Biemann, ANAL.CHEM.,42,855 (1970).
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Figure 2. Mass spectrum of the major GC peak of the benzyloxime-methyl ester, di-TMS ether of PGE2(I)
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EXPERIMENTAL
Apparatus. The GC-MS system used in this study is the LKB-9000 interfaced to a n IBM-1800 computer (8). The scanning circuit of the magnet was slightly modified to allow short magnetic scans to be taken. The G C column used throughout this study was a 4-ft 1% SE-30 o n Gas Chrom Q 80jlOO mesh. Reagents. The syntheses of [3,3,4,4-d4]-prostaglandin EZand [3,3,4,4-d4]-prostaglandin FZCY have been reported previously (6). Since one of the compounds was a viscous liquid, retaining a certain amount of solvent, and since both compounds contain some isotopic impurities, a n exact weight of each of these deuterated prostaglandins was calibrated (using the present method) against an exact weight of crystalline PGE, and PGF2wTHAM, respectively. In this way, the actual weight of the tetradeuteroprostaglandins could be determined in each sample. The [17,17,17-d31-7P,17a-dimethyltestosterone was prepared from 7P-methylandrost-4(8) M. F. Grostic, J. F. Zieserl, Jr., D. A. Griffith, M. D. Kenny, L. Baczynskyj, and D. J. Duchamp, the Upjohn Company, Kalamazoo, Mich., unpublished work, 1972. 480
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ene-3,17-dione, by protecting the 3-ketone as the pyrrolidyl enamine and reacting the 17-ketone with CD8MgI followed by hydrolysis of the enamine (9). The final product was a crystalline material of high isotopic purity. In the case of the prostaglandins, suitable derivatives for GC were prepared by reacting PGE, with benzylhydroxylamine hydrochloride (IO), followed by methylation of the carboxylic acid function with diazomethane and then blocking the free hydroxyl groups as trimethylsilyl ethers (TMS). In a similar manner, PGF2a was first esterified using diazomethane and then silylated. The steroid was amenable to G C without further derivatization. Procedure. In a typical experiment, a known amount of a deuterium labeled drug, used as carrier, is added to the sample which contains the same drug in its protium form. From a low resolution mass spectrum of the carrier, the operator selects an intense ion characteristic of the drug. He enters this mass number (R) and its protium equivalent along with five or more words into the computer using toggle switches at the interface. Whenever a GC peak of interest begins to emerge from the GC, the operator pushes the interface SCAN button, and the computer scans the requested number of times (variable from 1 to 40) over a preselected mass range. The number of scans and the mass range are selected in such a way, that the first scan is taken at the very beginning of the G C peak and the last scan at the very end. The computer processes the data and immediately prints a final report on a typewriter a t the mass spectrometer. The operator may run more GC runs without reentering his manual data words. Computer processing of the data occurs in three stages. First, during data acquisition the intensity-mass pairs are sampled at 10 Kc. As it is received by the computer, the intensity information is averaged to reduce noise. In practice each intensity value, stored for further processing, is the average of 32 or 64 measured intensities. In the second stage, following data acquisition, each mass spectral scan is processed separately to yield intensity values for up to six selected ions. The data are lightly smoothed and corrected (9) M. E. Herr, J. A. Hogg, and R. H. Levin, J. Amer. Chem. Soc., 78, SO0 (1956). (10) P.G. Devaux, M. G. Horning, R. M. Hill. and E. C. Horning, A w l . Biorhrm.. 41,70 (1971).
for base-line drift. Next the reference peak (R) is located by finding the largest intensity in the mass range R - 0.5 to R 3.0. To compensate for magnet drift, all other peaks are located relative to the reference peak (R). Maximum peak height, optionally corrected for background, is used as a measure of ion intensity. The operator may elect to plot one of the scans to check the computer processing. In the third stage, ion intensities from all the scans are processed to give a h a 1 report. The area under the intensity cs. time curve for each ion is computed. The time between scans varies somewhat, but is accurately measured by computer clock during data acquisition. The area of each ion is then divided by the area of the reference ion. These area ratios are used for quantification. A typical output of a run is shown in Figure 1. At the head of the report, total areas (absolute and relative) are listed for ions of interest. A scan by scan listing follows. The first column (N) corresponds to scan number. D (T) refers to the time between scan starts. Columns 3 and 5 list the intensities of the ions rnje 528 and 524, respectively, and columns 4 and 6 correspond to the areas for each scan for the same two ions. These areas are obtained by multiplying the sum of two consecutive D (T)’s by the intensities of the two ions.
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RESULTS AND DISCUSSION
Various techniques for monitoring particular ions in a GC effluent are possible and have been described. Recently the well-known AVA method has been computerized (11, 12). Two variations of the modified hardware for multiple ion detection have been reported (13, 14). Another approach, somewhat closer to the method described in this paper, is to maintain the magnetic field constant and to scan the accelerating voltage, respectively, over a limited mass range. A recent application of such a method to a drug problem has been reported (15). Also in quadrupole mass spectrometers, one can use an automatic peak selector to monitor one or more (up to eight) ions and measure the peak heights of the ions of interest (16). All these approaches seem to yield satisfactory results. Our choice of scanning the magnet repetitively was based on the following reasons. First, we had a GC-MS System (LKB-9000) interfaced to an IBM-1800 computer, which could scan the magnetic field repetitively at regular intervals. Implementation of quantitative measurements of ion intensities required no new interface hardware. Only the additional necessary software had to be developed. Second, one of the problems encountered with the AVA technique is the exact reproduction of the two (or three) accelerating voltages originating at the alternator. Furthermore, during an experiment in which ions of mass 350 or higher are monitored, the magnet heats up and tends to drift. Thus computerized AVA requires laborious refocusing during the experiment to obtain high precision. By maintaining the accelerating voltage constant and by scanning the magnetic field, the drift of the mag(11) J. F. Holland, C. C. Sweeley, R. E. Thrush, R. E. Teets. and M. A. Bieber, ANAL.CHEM., 45,308 (1973). (12) J. T. Watson, D. Pelster, B. J. Sweetman, and J. C. Frolich, Twentieth Annual Conference on Mass Spectrometry and Allied Topics, Dallas, Texas, June 4-9, 1972. (13) C. G. Hammar and R . Hessling, ANAL.CHEM.,43,298 (1971) (14) P. D. Klein, J. R . Haurnann, and W. J. Eisler, ibid., 44, 490 ( 1972).
(15) B. H. Albrecht, J. R. Plattner, D. Flagerman, S. Markey, and R. C. Murphy: Twentieth Annual Conference on Mass Spectrom-
etry and Allied Topics, Dallas, Texas, June 4-9, 1972. (16) N. H. Andersen, i n “Prostaglandins,” Auu. N . Y. Acud. Sci., 180, 196 (1971).
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Figure 4.
Mass spectrum of 7p,l7~-dirnethyltestosterone (111)
net is easily taken into account when the peaks of interest are identified by software. Third, at present the accelerating voltage alternator, that is commercially available, allows only two or three ions to be monitored. In our system, six ions can be monitored simultaneously, and this number can be increased by a simple software change. Fourth, the acquisition and storage of the data in the computer allow certain digital manipulations to be performed for each scan. The raw data can be smoothed to reduce the error due to noise on top of the peaks. For low intensity ions, correction may be made for sloping base line due to collision processes taking place in the analyzer. We have applied this method to the following drugs: PGE2, PGF2cr, and 7fl,17cr-dimethyltestosterone. P G F 2 aand PGE? were labeled at the 3,3,4,4-positions with deuterium ( 6 ) , whereas the steroid contained a CD, group at the 17cr position. The benzyloxime-methyl ester di-TMS derivative of PGE? (I) and the methyl ester tri-TMS derivative of PGFzcr (11) were injected in the gas chromatograph. The 7@,17crdimethyltestosterone (111) was run as such. C H , G
1 -
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C-OCH,
OSi(CHJ), OSi(CH,), C34H5TN05Si2:mo1 wt 615 I
os
C,,Hs,O,Si,~mol wt 584 I1 R=HorD
C,,H3,0,:mol wt 316 I11
The mass spectra of each of these compounds ( R = H ) are shown in Figures 2, 3, and 4. In the case of I , the mass spectrum of the major G C peak is shown. The ions of interest used in the multiple scanning experiments are marked by arrows. ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
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Table I. 528 :524 Ions of Benzyloxime-Methyl Ester-DiTMSEther of PGEz (I) Amount H (as Amount H PGE,) measured, ng Sample Measured H :D injected, (assuming D:H ( X 1000) ng linearity) Blank 0.27, 0.25, 0.31 1OOO:l 0.79,0.72, 0.70 1.0 0.6, 0.5, 0.5 1.53, 1.49, 1.52 2.0 1.4, 1.4, 1.4 1000:2 1000:5 4.06, 3.97, 3.98 5.1 4.3, 4.2, 4.2 9.11, 9.16, 9.28 10.2a 10.1,10.2*, 10.3 1000: 10 1000:20 18.96, 18.20, 18.47 20.4 21.4,20.5, 30.8 1000:50 43.96,44.61,43.70 50.9 50.0, 50.7, 49.6 1OOO:lOO 85.21,84.73,83.85 101.8 97.1, 96.5, 95.5 This value was used for standardization. 5
Table 11. 427:423 Ions of Methyl Ester-Tri-TMS Ether of PGF,a(II) Estimated std dev of Amount H Average single Amount measured, Pg meamea- H (as (assuming sured sured injected, linearity)* Sample H : D H : D ( X PGFpcu) AverD: H N ( X 1000) 1000) pg age Rangeb Blank 15 2.65 0.25 1OOO:l 5 4.9 0.3 200 265 240-336 1000:3 5 8.0 0.3 600 616 579-668 1000 :5 5 11.0 0.2 1000 970 947-1005 1000:7.5 5 15.6 0.6 1500 1502 1436-1598 1000:10 0.3 5 19.9 2000’ 2000‘ 1946-2031 1000:15 5 27.5 0.5 2990 2878 2812-2930 1000:20 5 37.8 0.2 3990 4067 4037-4090 a This value was used for standardization Calculated from average response by subtracting blank and multiplying by factor calculated from standard. Table I shows the analysis of the M+ -91 ion of the PGE2 derivative shown above (I). All samples contained 1 pg of the deuterated material per pl. Injections were all one microliter. The protium amounts injected varied from 1 to 100 ng. As can be seen from the “Measured H: D” column of Table I, the reproducibility, and therefore the precision, of the method is quite good for all ratios over this wide range. In the last column, we show each measurement reduced to ng by subtracting blank values and multiplying the difference by a factor obtained from one of the 1000 :10 measurements (equivalent to a linear standard curve through the origin). The measurements are quite linear for the higher ratios; however, for best results a nonlinear standard curve obviously is needed for the lower ratios. With mixtures of pure H:D samples, the reproducibility was within 10% on 1-ng samples injected on column. Tables 11 and I11 show similar measurements obtained on I1 and 111. Smaller amounts of the deuterated materials were used in these experiments, 200 ng and 300 ng for I1 and 111, respectively. To better evaluate the precision of the method, all samples were run at least 5 times. The fourth column of each table shows the standard deviation to be expected in a single measurement of the ratio. For all ratios this standard deviation is well below l o x , and for the larger ratios is around 2 %. As in Table I, here also the lower ratios will require use of nonlinear standard curve for most accurate results. These data show that by use of this method, samples of PGF2a as 482
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Table 111. 319:316 Ions (M+) of 7p,17aDimethyltestosterone (111) Estimated std dev Amount H Average of measured, pg measingle Amount (assuming sured measured Hinlinearity) Sample H : D ( X H : D ( X jected, AverD: H N 1000) 1000) pg age Range Blank 10 2.2 0.17 1000:1 10 3.51 0.19 299 324 253-388 1000:2 10 4.69 0.23 598 614 506-715 1000:3 5 5.95 0.2 897 923 875-993 0.2 1495 1428 1395-1467 1000:5 5 8.01 1000:7.5 5 10.80 0.1 2242 2114 2027-2177 1000: 10 5 14.37 0.3 2990a 2990a 2909-3091 1000:20 5 24.96 0.3 5980 5590 5467-5695 This value was used for standardization. Q
small as 200 pg may be measured to an accuracy of better than lox, provided that a nonlinear standard curve is used and blanks are determined accurately. Extensive experience with the method has led us to the following observations. The sensitivity of this method is a function of the performance of the mass spectrometer. This varies from day to day because of the extensive use of our GC-MS system. (No special precautions were taken to obtain the data in Tables I, 11, and 111.) The value of the blank varies with column bleed, source contamination, etc. To maintain adequate sensitivity of the mass spectrometer, the source and electron multiplier slits must be fairly wide open. This results in considerable overlap of adjacent peaks at higher masses. High pressures in the ion source and analyzer result in lower precision. Improvements in the pumping system of the mass spectrometer should increase precision and sensitivity. The method has been extensively tested and is available for routine use by investigators in our laboratories. It has been used to measure a number of samples of PGEz and PGFpa of biological origin (17, 18),and has been extended to differentiate between endogenous and administered prostaglandins (19). The applicability of this method to the analysis of other compounds of biological interest is obvious, provided the deuterated carrier is available, the compound is amenable to gas chromatographic analysis, and a suitable ion below rn/e 800 can be found in the mass spectrum. ACKNOWLEDGMENT
We thank J. C. Babcock and J. A. Campbell for providing 7/3,17a-dimethyltestosterone and its deuterated analog used in this study, and our electronics group for the outstanding support with the instrumentation aspect of this work. RECEIVED for review September 11, 1972. Accepted November 1,1972. (17) U. Axen, L. Baczynskyj, D. J. Ducharnp, and J. F. Zierserl, Jr., in “The Prostaglandins, Clinical Applications in Human Reproduction,” E. M. Southern, Ed., Futura Publishing Co., Mount Kisco, N.Y., 1972. p 279. (18) U. Axen. L. Baczynskyj, D. J. Duchamp, and J. F. Zieserl, Jr., J. Reprod. Med., in press. (19) U. Axen, L. Baczynskyj, D. J. Duchamp, K . T. Kirton, and J. F. Zieserl, Jr.. “Advancesin the Biosciences,” Vol. 9, Pergamon Press/Vieweg, Braunschweig, in press.
