Anal. Chem. 1980, 52, 379-384
Association from the National Institute of Dental Research and is part of the dental research program conducted by the National Bureau of Standards in cooperation with the American Dental Association Health Foundation. Certain commercial materials and equipment are identified in this paper
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in order to specify adequately the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the material or equipment identified is necessarily the best available for this purpose.
Microcomputer-Assisted Mass Spectral Data Acquisition System Utilizing a Mass Marker Brian D. Andresen,”‘ William A. Wagner, and Frank T. Davis University of Florida, College of Pharmacy, Gainesville, Florida 326 10
Computer-assisted mass spectral data acquisition has achieved significant advances in the past decade. Through the use of large-to-medium computers with sufficient memory capacity, speed, and appropriate programs, low resolution mass spectral data can now be acquired, digitized, stored, and manipulated most easily (1-5). I t has now become essential for most applications that a computer control all aspects of the mass spectral data gathering. This would include continuous scanning of the mass spectrometer during any gas chromatographic-mass spectrometric (GC-MS) analyses of complex mixtures, acquisition of the data from the instrument, the processing of the data, and orderly arrangement of the results for rapid human interpretation. The favorable capabilities of a computer-assisted mass spectral data acquisition system would allow larger volumes of data to be acquired and processed, faster data access for interpretation, and permanent data storage. As a further extension, the interpretation of mass spectral data can be left completely under the control of the data system which accurately searches known library compilations for the nearest spectral match (6-16). In this manner, complex mixtures can be completely characterized without any human interpretation in the shortest possible time (6). Investigations into the availability of commercial computers, programmed for mass spectrometry data acquisition, demonstrated that most were relatively expensive. Complete GC-MS-computer systems could be obtained for $50 000$60 000 that possessed dedicated programs for mass spectral data acquisition and display. However, these systems were considered to be too costly and, in addition to duplicating existing analytical equipment, they could not easily be interfaced to another mass spectrometer. Many mass spectrometry research centers have indeed successfully interfaced rather large and sophisticated computers to their own instruments. However, these complex and refined data systems have been developed over a period of many years and are difficult to achieve and nearly impossible to duplicate. Recent developments in the field of microcomputer technology appeared to contribute all aspects of the much larger systems (i.e., speed and calculating power), yet were very cost effective if applied to mass spectral data acquisition only. Current literature has revealed only a limited number of microcomputer data systems which have been applied to an analytical task. These include automated titrations (17-19), a low-pass filter to smooth analog data (20),a microcomputer controlled dual wavelength controller (21), photoacoustic measurements using a microcomputer (22), as well as the commercial gas and high pressure liquid chromatographs. In these applications the microcomputer contributed significantly to the overall effectiveness of the analytical technique. In addition to these favorable reports, very recent developments in mass storage systems have become available. New “floppy ‘Present address and reprints: Department of Pharmacology,The Ohio State University, 333 West Tenth Ave., Columbus, Ohio 43210. 0003-2700/80/0352-0379$01 .OO/O
disk” storage systems could be interfaced to the microprocessor allowing large blocks of data to be stored and retrieved rapidly. This new technology seemed ideally suited for cost-effectively storing large blocks of data common to GC-MS runs and mass spectral libraries, which are often an integral part of normal mass spectral activities. All of these preliminary findings and unique features appeared most favorable for allowing a microcomputer to be interfaced to a mass spectrometer. A recent report has indicated that this approach is feasible with quadrupole mass analyzers (23, 24). The objective of the work reported here was to design a user-oriented GC-MS-microcomputer (GCMS-MC) system that could be applied easily to a DuPont 491 magnetic sector mass spectrometer. Many new microprocessors were currently available to accomplish the data acquisition, however, a Z-80-based microcomputer system was chosen because of its versatile and large (158) machine code instruction set incorporating blockdata-transfer capabilities. This function appeared essential when moving a whole mass spectrum in memory with the fewest machine code statements in the shortest period of time. A variety of designs was attempted, utilizing different combinations of hardware and software which would be most amenable to continuously acquire, process, store, and display mass spectal data. A practical design was eventually developed which was not simply a controller, but a user-oriented data system capable of meeting the goals outlined above, and possessing the capabilities to expand both the hardware and software when more complex tasks are required. The primary goal was simply to utilize a microcomputer system to aid in the rapid analysis of drugs and metabolites. Mass spectral data were to be acquired in a digitized form and then compared to a limited library of compounds. However, once the system was implemented, the emphasis was to develop additional programs to more fully utilize the data that had been stored by the microprocessor. The completed data system could acquire data and search a library file of’ known compounds as well as present hardcopy data of the results. We report here how this was accomplished and in addition describe the automated analysis of drugs from the body fluids of overdose victims.
INSTRUMENTATION The microcomputer system interfaced to a DuPont 491, double-focusing magnetic sector mass spectrometer is shown schematically in Figure 1. The computer system was assembled from commercially available components (Digitrl Group, Denver, Colo. 80206) and operated at 2.5 MHz using an 8-bit word, 2-80 microprocessor (Zilog,Inc., Los Altos, Calif‘. 94022), 32K of random access memory (RAM, TMS 4044) (Exatron, Sunnyvale, Calif. 94086), two SA801 shugart disk drives and controller (Digital Group), and an input/output interface (Digital Group) coupled to an audiocassette deck (Wevcor Mer. Co.), television (Sanyo VM-4209, Compton, Calif. 90220), Diablo printer (Data Dimension, Inc., Greenwich, Conn. 06830), and keyboard (Digital 0 1980 American Chemical Society
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Figure 2. A 10-bit A / D converter circuit configured for continuous data acquisition
Group). In addition the computer system was designed with 8 parallel input/output ports. A data bus connected all parts of the system to the 2-80 microprocessor. Commands and programs were entered through the keyboard, while assembled programs and data were entered through the disk system or from audiotapes. One output port of the microcomputer was wired directly to a 10-V, 60-mA relay (MHJX 6070, Allied Control Co.). The relay was wired directly to the scan switch of the mass spectrometer so that the down scan and return function of the magnetic scan circuitry could be controlled in a continuous manner by the microprocessor. A 10-bit analog-to-digital (A/D) converter (No. AD 571, 2 5 ~ s conversion time, Analog Devices, Norwood, Mass. 02062) was attached to one input port of the microcomputer and to the most sensitive galvanometer of the mass spectrometer's oscillographic recorder. The A/D converter circuit is shown in Figure 2 and was configured so as to continuously sample analog ion intensities
and present digital data to one port of the microcomputer. The digitally converted data were accessed using software machine code commands that continuously looked at the appropriate 8-bit parallel input port. Using only an 8-bit word to represent 0 to 10 mV, an accuracy of one part in 256 (h0.470) was achieved for ion intensities. This degree of accuracy was considered acceptable only for mass spectral data acquisition applications when analyzing low levels of organic compounds. Intense spectra could easily saturate the A/D converter. The last two bits of the 10-bit A/D converter were wired so that if a very intense ion were recorded, all 8 bits remained high which did not reset (or zero) the A/D device. The digital readout of a DuPont 21-093 mass marker was wired directly to two parallel input ports of the microcomputer. This allowed direct acquisition of binary-coded-decimal (BCD) representations of fragment ion mass numbers. These BCD data were then converted to a true decimal equivalent within micro-
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seconds and in real time using Z-80 machine codes, during the recycling of the instrument magnet. In this manner, a complete mass spectrum could be acquired, assembled, and stored on disks between scans. Programs to perform all aspects of mass spectral data acquisition were written using Z-80 assembler. A modified version of Dartmouth College Basic (Digital Group) was used only to initiate the run, interface with the operator, set up control parameters, and then to jump to Z-80 machine code subroutines. This approach allowed both the highest speeds and most programming versatility for microcomputer assisted mass spectral data acquisition. (Complete listings are available of the 2-80 assembler listings and BASIC programs upon written request.) In addition a calibration mixture of PFTBA (perfluorotributylamine, PCR Chemicals) and PFA (perfluoroalkane,PCR Chemicals) was used prior to each mass spectral run to ensure calibration up to 614 mass units. When allowed to settle (approximately 10 min), the mass marker system (which included a Hall probe) would remain stable for the entire run. Calibration routines were written to check the linearity of the mass marker and conditions of the mass spectrometer. These calibration programs in addition would not allow the operator to continue with the run until the correct calibration criteria had been met.
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RESULTS AND DISCUSSION Although a complete and workable computer system had been interfaced to a magnetic sector instrument, certain programming problems, inherent to high speed mass spectral data acquisition, had to be addressed. First, an approach to the correct assignment of ion intensities to a particular mass number had to be considered. Second, it was discovered that a method to correctly obtain ion intensities had to be devised, irrespective of noise spikes and daily instrument fluctuations. Previous work concerning mass identification has been reported (25) and has been implemented with some computer systems. The technique requires an external clock that is activated as the magnet begins its scan. Ion intensities are then recovered and stored along with a particular clock pulse. Later, as the mass spectra are processed, a program is executed to convert these ordered clock pulses to the correct mass number. This technique would also be amenable to microcomputer assisted data acquisition. However, a solution to this problem was more easily solved by taking advantage of a mass marker utilizing a Hall probe similar to most commercial systems which was an integral part of the mass spectrometer described here. Because the mass marker of the instrument continuously presented a digital display of the mass number, it seemed appropriate to attach the display of the mass marker directly to the microcomputer (through two 8-bit parallel input ports) and record the fragment ion mass numbers directly. This technique eliminated the time-tomass-number conversion routines previously reported (26), saved valuable program memory allocations, and could display correlated ion intensities and mass numbers in real time between scans. T h e second problem of noise discrimination appeared to be a more difficult one to solve. The very high frequency response needed to acquire fragment ion intensities rapidly allows the mass spectrometer to also record noise spikes. Noise, inherent to the instrument, is typically overlooked when analog data are recorded on an oscillographic recorder. However, for microcomputers to discriminate a fragment ion intensity from a noise spike, special parameters had to be considered. Figure 3 reveals the typical raw analog data of an intense ion a t m / e 131 for perfluoroalkane (PFK) when recorded on analog paper a t both normal speeds and a t a magnetic scan of 40 s/decade and a paper speed of 22 cm/s without noise damping. At normal paper speeds the upper trace is so compressed that noise associated with the system does not interfere with the interpretation of the data. However, the expanded picture reveals significant noise spikes
Figure 3. Analog data from mass spectrometer revealing significant noise spikes
which are easily recorded by the A/D converter operating without noise damping and a t a sampling rate of 25 ps. Also presented on the expanded picture are the unit mass trace (bottom line) from the mass marker which also reveals significant noise spikes as the mass marker changes from one mass to the next. Noise also present on the digital display of the mass marker added additional problems when the microcomputer assigned the appropriate mass number to the corresponding ion intensity. To circumvent the degree of uncertainty as to when an ion peak top maximum was recorded, as well as to ensure proper assignments of mass numbers, 2-80 machine language microcomputer programs were written. Figure 4 reveals a flowchart diagram of a microcomputer program to acquire a single m / e ion, discriminate noise spikes, assign the correct mass number, and store the corresponding maximum intensity value and mass number in memory. With the appropriate software commands, the time for mass spectral data acquisition was very short. Because single microcomputer loops could be executed within 205 ps, an entire mass spectrum (from mass 500 to 10) could be initiated, recorded, assembled, moved within memory, and stored on disk in under 3 s. An entire spectrum could also be displayed on the video monitor between scans within 5-10 s depending upon the number of fragment ions recorded. The means to achieve high speed data acquisition were obtained by allowing the A/D converter to be free-running and employing machine code subroutines
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@
(SORT)
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Figure 4. Flowchart diagram to record a mass number and ion intensity
maximum which sampled the incoming A/D-converter data and rejected information until certain criteria were met. For example, from Figure 4, if the incoming ion intensity is zero, the program loops back (to HOME) to check the mass number. If it is equal to m / e 10 (or any preset mass), the program ends by stopping the scan. If the mass number does not equal 10, then the program updates the mass number and checks the ion intensity again. If the incoming ion intensity is not zero (or any preset threshold value), a comparison to zero is made from the value previously placed in the B register of the microprocessor (initially set to zero). If the incoming ion intensity is now sampled and determined to be a larger value than the value in the B register, it is exchanged and a new intensity taken from the A/D converter. Comparisons are continually made, always placing the largest value into B. Finally when the incoming A/D value is not larger than the value in the B register, a counter of five (or any appropriate value) is set and decremented by one each time the loop is entered. If five samplings of the A/D converter fail to produce an ion intensity value greater than the maximum stored temporarily in the B register, then an ion maximum has been recorded. At this point, considerations were given to the fact that only the ion intensity maximum was recorded with this configuration and possible errors in data sampling may be present. However, the hardware design presented here utilized the signals generated by the oscillographic recorder. The signals were usually sufficiently damped by the recorder circuitry so that the ion signal was digitized a t a maximum corresponding to the value and error inherent in the oscillographic recorder (h0.570). Any noise apparent in the system was then filtered prior to microcomputer sampling and the data were of much better quality than if taken directly from the preamplifier of the mass spectrometer. Finally the program checks to see what mass is present at the mass marker, determines if the mass has changed, and finally stores both the ion intensity and its mass number in
memory when a new number is encountered. Various control functions are also depicted in Figure 4, such as magnet “turn on” and “off”, as well as a scan number counter and memory size checks. By numerous loops and comparisons, the program attempts to discriminate against mass spectrometer and mass marker noise, yet operates with very good time efficiency. In this manner, complete mass spectral scans can be acquired in the shortest possible time. The faster scan speeds are important for gas chromatographic-mass spectrometric data handling in which GC peaks need short sampling time periods for maximum GC peak resolution. Although the m / e vs. ion intensity values had been stored in the computer memory, conversion from machine code to a readable form had to be made. To accomplish this task, a short machine language program was written to convert binary-coded-decimal (a compressed value of the ion mass) to ASCII (American Standard Code for Information Interchange). This program simply converts the mass ion number, which is input to the computer initially from the mass marker as packed data, to ASCII and moves the converted data to a new memory location. The program is set up for a typical mass spectrum of 500 masses. The mass spectral data which had been acquired, sorted, and moved to a new memory location, was next stored on a floppy disk. The disk storage device could store an average of 450 full mass spectra on one blank disk and an additional 400 spectra on the program disk. These values could be greatly increased when mass spectra were recorded which contained only a few fragment ions. Once the mass spectral data had been obtained, they could easily be stored on permanent storage disks or cassette tapes, displayed in a variety of ways, or manipulated in any fashion using a combination of assembler code and BASIC. FORTRAN or FORTH could have been employed; however, BASIC was used as a controller to interface with the operator and has the advantage of being very flexible when writing computer programs quickly, is widely known, and requires only a minimum amount of memory allocation. Figure 5 shows an overview of the complete acquisition, storage, and display programs as they work together to generate mass spectral data. Two programs were also developed using a combination of BASIC and Z-80 machine code subroutines to vary the scan. This was done to allow single ion monitoring (SIM) (27,28) using a microcomputer. Figures 6a and 6b show block diagrams of two different approaches to this technique. In one method (Figure sa), the magnet was allowed to scan rapidly for a set period of time encompassing only a contiguous group of masses. The other approach (Figure 6b) had the program sample data over the entire mass spectral scan; however, only preselected ion intensities were recorded. By applying either program (depending upon the ions selected), a group of specific fragment ions could be monitored during an entire GC-MS run and plotted in real time. As a final capability, the microcomputer system was programmed to acquire mass spectral data and search a library for the best fit. The routines used to perform this more complex task are shown in the block diagram of Figure 7 . The program shown initially acquired an entire mass spectrum but selected and organized fragment ions of a preselected intensity (e.g., greater than 50% of the A/D converter maximum). The preprocessed data are then organized and compared to a library collection which is sorted by the 8 most intense ions. Each favorable comparison of the unknown with a library spectrum sets a counter. When the minimum acceptable comparisons have been recorded, the results of that search are printed. It was determined that for a small dedicated library collection containing 261 drugs, only 4 favorable ion
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Figure 6. Flowchart diagrams to control magnetic scan for single ion monitoring
(YES
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CALCULATE 8 AND Y / E CORPSLITION
Flgure 5. Flowchart diagram to acquire and store mass spectral data
comparisons are needed to quickly identify the unknown with confidence. The library search routine presented here was written using a combination of BASIC and 2-80 assembler. For microcomputer applications, the program has the advantage that only preselected ion intensities are initially processed before they are used in the search routines. This approach allows the library search program to function in real time. For pure compounds, the search routine would require approximately 10-15 s to search 261 spectra. For a mixture contributing a variety of fragment ions, the search would require no more than 60 s with this limited library. A much faster version of this library retrieval could be realized if the entire search procedures were performed by calling a Z-80 machine code subroutine instead of following the BASIC program outlined here. Work is currently in progress in this area. The results of a run are printed in Figure 8a and 8b. Initially, all operations concerning the instrument adjustments and the calibration of the mass marker are controlled by the microcomputer to ensure accurate mass spectrometric data. The calibration mixture (PFTBA and PFK) is initially analyzed and selected masses are checked against the authentic spectrum (stored within the program). When the mass spectrometer has stabilized (approximately 10 min), a good cali-
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Figure 7. Block diagram of library research routines using a microcomputer
bration spectrum will be recorded. The programs will then allow the user to continue with the analysis only when calibration is achieved. The operator is then asked to signify which, intermittent (user defined) or repetitive (microcomputer controlled) scan mode is desired. Once selected, data acquisition is automatically controlled. The results of a mass
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* ANALYTICAL CHEMISTRY, VOL. 52, NO. 2, FEBRUARY 1980 orderly presentation of tabular data greatly aids in the rapid assessment of the mass spectral data. More sophisticated search routines could be implemented if a larger computer were used. From the design presented here, it appears feasible that such a system could cost-effectively interface to a large time shared computer facility which could easily handle many of the more complex tasks achieved only by commercial GCMS computer systems. A microcomputer-assisted mass spectral data acquisition system does appear very feasible for most routine data gathering applications. In this paper we have examined the feasibilities and capabilities of a microcomputer to assist in the organization of large amounts of high speed data. It appears that the cost effectiveness of a microcomputer system ($2000 to $5000) warrants much more work in this area. Great savings can be realized if the microcomputer is interfaced to the mass spectrometer and programmed by the actual users of the system. With the advance of microcomputer innovations, very powerful systems will soon be assembled easily which will perform many of the routine functions of mass spectral data gathering.
A
ACKNOWLEDGMENT
Figure 8. Example results from library search of compounds from unknown overdose victim. (A) Instrument adjustment and calibration. (The calibration listing has been abbreviated for the sake of space.) (B) Results of a GC peak eluting at scan number 137
spectral-microcomputer search of an intense spectrum obtained from a basic urine extract are seen in Figure 8b. The causative intoxication agent was accurately determined as methaqualone.
CONCLUSIONS The intent of the work presented here was only to show the feasibility of interfacing a microcomputer to a magnetic sectior mass spectrometer in order to digitize mass spectral data. Various designs indeed could have been possible. However, a working system was achieved which showed the simplicity of a technique to obtain usable data. The microprocessorbased system presented here, in its most limited application, easily and accurately circumvented the tedious methods of data acquisition and the manual counting of each mass spectrum obtained with a conventional oscillographicrecorder. The use of a microprocessor to perform this routine task greatly aided in the rapid acquisition of mass spectral data. However, problems with this system are clearly evident. The dynamic range of the 8-bit A/D signal described here could be extended if a more sophisticated 16-bit A/D converter were employed using an autoranging circuit. Currently, such A/D converters are prohibitively expensive for microcomputer applications. However, the dynamic range of the A/D circuit presented here could be extended if an autoranging circuit were implemented using the last two bits of the 10-bit A/D converter and an additional 8-bit parallel input port. Work is currently in progress in this area. Also limitations were apparent concerning the need for sophisticated programs to subtract background spectra, to perform unique mass chromatogram plots when gas chromatographic peaks overlap, and to search large library files with a small microcomputer system. Although the library research routine presented here does nothing more than retrieve library data of compounds which appear to possess fragment ions similar to the unknown, the
The authors greatly appreciate the mass spectrometry support provided by Clyde Williams and the technical assistance offered by Donald Chichester.
LITERATURE CITED Hites, R. A.; Biemann, K. Anal. Chem. 1988, 4 0 , 1217-1221. Reynolds, W. E.; Bacon, V. A.; Bridges, J. C.; Coburn, T. C.; Halpern, 8.; Lederberg, J.; Levinthal, E. C.; Steed, E.; Tucker, R. B. Anal. Chem. 1970, 42, 1122-1 129. Sweeley, C. C.; Ray, B. D.; Wood, W. I.; Holland, J. F.; Krichevsky, M. I. Anal. Chem. 1970, 42, 1505-1515. Biller. J. E.; Herlihy, W. C.; Biemann, K. "Computer-Assisted Structure Elucidation", Smith, D. H., Ed.; ACS Symp. Ser. 1977, 5 4 , 18-25. Hites, R. A.; Biemann, K. Anal. Chem. 1970, 42, 855-860. Costello, C. E.; Hertz, H. S.; Sakai, T.; Biemann, K . Clin. Chem. 1974, 2 0 , 255-265. Hertz, H. S.;Hites, R. A.; Biemann, K. Anal. Chem. 1971, 43, 681-691. Finkle, B. S.; Taylor, D. M.; Bonelli, E. J. J . Chromatcgr. Sci. 1973, IO, 312-333. Dromey. R. G. Anal. Chem. 1979, 5 1 , 229-232. Dromey, R. G. Anal. Chem. 1978. 48, 1464-1469. Smith, D. H. Anal. Chem. 1972, 4 4 , 536-547. Crawford, L. R.; Morrison, J. D. Anal. Chem. 1988, 40, 1469-1474. Heller, S.R. Anal. Chem. 1972, 4 4 , 1951-1961. Grotch. S. Anal. Chem. 1978, 43, 1362-1370. Kelly, J. A.; Nau. H., Forester, H. J., Biemann, K. Biochem. Mass. Spectrosc. 1975, 2. 313-325. Nau, H.; Forester, H. J.; Kelly, J. A,; Biemann, K. Biochem. Mass. Spectrosc. 1975, 2 , 326-339. Legett, D. J. Anal. Chem. 1978, 5 0 , 718-722. Wu, A. H. B.; Malmstadt, H. V. Anal. Chem. 1978, 5 0 , 2090-2096. Bush, N.; Freyer, P.; Szameit, H. Anal. Chem. 1978, 5 0 , 2166-2167. O'Haver, T. C. Anal. Chem. 1978, 5 0 , 676-679. Defreese. J. D.; Walczak, K. M.; Malmstadt, H. V. Anal. Chern. 1978, 50, 2042-2046. Eaton, H. E.; Stuart, J. D. Anal. Chem. 1978, 5 0 , 587-591. Holkefoer, D. H.; Schlereth, F. H.; Parfitt, W. E. 26th Annual Conference on Mass Spectrometry and Allied Topics, St. Louis, Mo., May 1978. Godse, D. D.;Warsh, A. U.; Warsh, J. J. Presented at the 26th Annual Conference on Mass Spectrometry and Allied Topics, St. Louis, Mo.,May 1978. Waller, G. R. "Biochemical Applications of Mass Spectrometry", Waller, G. R., Ed.; Wiley-Interscience: New York, 1972; Chapter 3. Hites, R. A.; Biemann, K. Anal. Chem. 1987, 39, 965-970. Manews. D. E.; Denson, K. D.; Hayes, J. M. Anal. Chem. 1978, 50, 681-683. Sweeley, C. C.; Elliott, W.; Fries, I.; Ryhage, R. Anal. Chem. 1988. 38, 1549.
RECEIVED for review April 4,1979. Accepted November 26, 1979. This work was partially supported by a Cottrell Research Corporation Starter Grant and is in partial fulfillment of the requirements for the degree of Ph.D. for F.T.D.
