this is a problem with quadrupole instruments as well, and could probably be handled by an intensity calibration program. Locating peaks by rapid scanning and then spending more time on the peaks and less time between them offers the possibility of increased sensitivity and accuracy. McLafferty et al. (14) have recently presented a system for signal enhancement of high resolution mass spectra by scanning repeatedly each ion peak in turn with the accelerating voltage while a magnet scan is in progress. Finally, the possibility of accurately measuring fractional masses with a low resolution instrument can be raised. The electronics appears to be adequate in resolution, although the guaranteed accuracy of the Fluke 5205A might be preferable in such an application. However, the subject is controversial. Since the relatively broad ion peaks may hide traces of other fragments which would significantly shift the peak center without obvious peak asymmetries, ions must be chosen judiciously for such applications. That such an approach is feasible has been shown by Smith e t al. (Is),using a high resolution mass spectrometer turned down to a resolution of 2500. A marked increase in sensitivity has been obtained by signal averaging. However, a simple circuit addition can provide a still better method of noise reduction. If the output of the electronmultiplier is integrated during the sample period, then the final integrator output will be the sum of the ion current during the sample period. The integrator can be reset to zero output during the settling period set aside for the accelerating voltage to stabilize after it is switched to a new fragment ion peak center. The interface that we have designed can generate a control level for resetting an integrator, using one of the spare bits in the storage registers. If the integrator output uses different channels of the A/D converter, the standard am(14) F. W. McLafferty. J. A. Michnowicz, R. Venkataraghavan, P. Rogerson, and 8 . G. Giessner. Anal. Chern., 44, 2282 (1972) ( 1 5 ) D. H . Smtth, R. W. Olsen, F. C. Walls, and A . L. Burlingame, Ana/. Chern.. 43, 1796 (1971).
plifier output will still be available for the fragment ion peak sweep, as well as for magnetic and electric scanning. This setup would seem to offer much the same advantage as fragment ion pulse counting (16), while requiring a rather simple circuit addition. However, ion counting does allow for threshold detection to eliminate small noise pulses, and for the removal of residual noise due to random variations in pulse size and shape. On the other hand, ion counting is limited in dynamic range by pulse overlap a t high pulse rates. Although an integrator amplifier will also have a limited dynamic range, this range can in effect be extended by changing the electronmultiplier gain. This has important practical implications for stable tracer assays using gas chromatography. There is often enough sample (hundreds of nanograms) to produce strong fragment ion signals. Yet there is still a need for noise reduction to improve the precision of the measurements, so that a small per cent excess abundance can be measured.
Note added in proof. A revised version of the program has been completed that allows the user t o select the relative times spent sampling each mass channel. The data can be stored on tape, and plotted by an incremental or electrostatic plotter. ACKNOWLEDGMENT The authors wish to thank Bo Holmstedt for providing a high voltage amplifier used in the preliminary phase of this research. Received for review March 19, 1973. Accepted May 14, 1973. The authors wish to acknowledge financial support from NIH grants RR-00396, MH20717, NS05159, and 5SOlSO-RRO5389, and a USPHS Fellowship from the National Institute for Arthritis and Mental Disease. (16) D. A . Schoeller. J. M. Hayes, C. A. MacPherson, R. F. Blakely, and W. G. Meinschein, R o c . 20th Annual Conf. Mass Spectrorn. Allied Topics. Dallas, Texas, 1972, p 231
Display-Oriented Data System for Multiple Ion Detection with Gas Chromatography-Mass Spectrometry in Quantifying Biomedically Important Compounds J. T h r o c k Watson, Donald R. Pelster, Brian J. S w e e t m a n , J. C. Frolich, and John A. Oates D e p a r t m e n t of P h a r m a c o l o g y , School of Medicine, Vanderbilt University, Nashville, Tenn. 37232
A display-oriented data system for multiple ion detection ( M I D ) has been developed which combines data handling options (ion profile identification, quantitative calculations, etc.) with optimum intervention and control by the investigator in examining the data. The computer can resolve and unequivocally identify individual ion current profiles which would otherwise overlap on a conventional data record of the composite profiles. Furthermore, this data system permits amplification of individual ion profiles after they have been recorded to facilitate operator decisions in quantitative calculations and to avoid errors in manual indication of channel attenuations. The interactive nature of the software options permits efficient data
interpretation by the investigator who can assess various display parameters of the data (e.g., retention time, peak height or area, etc.) from switches and adjustable potentiometers on the computer console or via a teletype which produces a “hardcopy” of quantitative results in tabular form. Some of the operational features of this MID-data system are exemplified with analytical results of prostaglandins or catecholamine derivatives in biological samples. While this data system has been refined during the past two years of use in M I D data acquisition from a magnetic mass spectrometer, the data handling features should be adaptable to any instrumental variant of a M I D operation.
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 12, OCTOBER 1973
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Rarely does an analytical technique provide both high selectivity and sensitivity. Multiple ion detection (MID) retains the inherent high sensitivity of a mass spectrometer while achieving a high degree of selectivity by monitoring only a few ions which are characteristic of the compound of interest. Since MID was first employed to quantify deuterium and protium species of a sugar derivative ( I ) which were not completely resolved by GLC, there have been many qualitative and quantitative applications of the technique in the analysis of drugs such as chlorpromazine ( Z ) , nortriptyline (3, 4 ) , lidocaine ( 5 ) , and hormones such as acetylcholine ( 6 ) ,prostaglandins (7, 8),bile acids ( 9 ) , catecholamines (10) and metabolites ( I I ) , and 5-hydroxyindole acetic acid (12). However, methods of data acquisition and handling of MID results have not kept pace with other refinements in MID instrumentation. In most cases. the conventional oscillograph, designed to rapidly record complete mass spectra, is also used for MID in which the output from several data channels is alternately switched to this single-input recorder. The resulting oscillographic data record consists of a series of transient sweeps because the recording medium, namely a beam of light, is not interrupted as the various MID inputs are switched alternately t o the recorder. The investigator must then construct the various ion current profiles by drawing a connecting line through the end points of the transient sweeps. Recognition of these profiles on the original data record is often difficult and ambiguous, especially when trace quantities of a given compound are being detected in the presence of more abundant components which also contribute at various times to the ion profiles. Hammar and Hessling ( 3 ) have designed a sample and hold recording system which permits individual amplification and base-line offset for each ion current channel. Klein et al (9) use a digital recording system which employs a voltage divider for optimum ion counting of ion currents which may differ in abundance up to a ratio of l:lO4. Jenden and Silverman (13) and Bonelli ( 1 4 ) have employed quadrupole mass spectrometers to monitor the profiles of up to 8 ions with a multichannel recorder having off-set pens. Chapman et al (15) have adapted a magnetic instrument with a signal-stepping and -integrating MID device which records the output of 6 ions on a multichannel pen recorder. A permanent data record on which 4 to 8 ion profiles are superimposed (1) C. C. Sweeley, W. H . Elliott, I . Fries, and R. Ryhage, Ana/. Chem., 38, 1549-53 (1966). (2) C.-G. Hammar, E. Holmstedt, and R. Ryhage. Ana/. Biochem., 25, 532-48 (1968). (3) C.-G. Hammar and R . Hessling, Ana/. Chem.. 43, 298-306 (1971). (4) T. E. Gaffney, C. G . Hammar. B. Holmstedt. and R. E. McMahon, Anal. Chem., 43,307-10 (1971). (5) J . M . Strong and A. J . Atkinson, Anal. Chem., 44, 2287-90 (1972) (6) C . - G . Hammar. I . Hanin, B. Holmstedt. R. J. Kitz, D. J. Jenden. and B. Karlen, Nature (London). 220, 915-17 (1968). (7) E. Samuelsson, M . Hamberg, and C. C. Sweeley, Anal. Blochem.. 38, 301-4 (1971). (8) U . Axen, K. Green, D. Horlin. and B. Samuelsson, Biochim. Biophys. Acta, 45, 519-25 (1971) (9) P. D. Klein, J . R. Haumann, and W. J. Eisler, Ana/. Chem., 44, 490-93 (1972). (10) S. H . Koslow, F. Cattabeni, and E . Costa, Science, 176, 177-80 (1972). (11) B. Sjoquist and E. Anggard, Ana;. Chem., 44, 2297-2301 (1972). (12) L. Bertilsson, A . J . Atkinson. J. R . Althaus. A . Harfast, J.-E. Llndgren, and E. Holmstedt, Anal. Chem.. 44, 1434-8 (1972) (13) D. J . Jenden and R. W. Silverman, Proceedings of Seminar on the Use of Stable Isotopes in Clinical Pharmacology, USAEC Document CONF-711115, Technical Information Report, August 1972, p p 273-9. (14) E. J . Bonelli, Ana/. Chem., 44, 603-6 (1972). (15) J . R. Chapman, K . R. Compson. D. Done, T. 0. Merren, and P. W. Tennant, Proceedings of 20th Annual Conference on Mass Spectrometry, Dallas, 1972, pp 166-71.
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is cumbersome; even though each pen may have different colored ink, it can be difficult to coordinate the synchrony of the various profiles since the recording pens are necessarily offset. The additional requirement of logging the attenuation (and changes thereof) of each of 4 to 8 ion current channels further clutters the data record and increases the probability of manual error. Furthermore, since the operator has only the dynamic range of an 8- to 10-inch width of chart paper, he must make some a priori decisions on the relative attenuations of each amplifier or run the sample twice to make optimum use of the limited dynamic range of the chart paper. None of the refinements in technique of MID provides sufficiently comprehensive data acquisition and handling to facilitate unequivocal identification and examination of any m l e profile and/or rapid calculation of any designated peak area from the recorded data. To cope with the problem of data acquisition and handling of MID results, an interface was designed and built to permit on-line reduction and display of MID data by a small laboratory computer (PDP-12A with 8K core and extended arithmetic unit). All relevant data pertaining to the sample and GLC conditions, etc. can be stored and retrieved with the corresponding analog data profiles. The operator, a t any time (on or off-line) can unequivocally identify (or display singly) any one of the ion current profiles (currently up to 3 ions because of limitations in mass spectrometer hardware). When examining MID data, the operator may also establish the retention time of various peaks on each ion current-time profile. After selecting the profile peaks of interest, the investigator may call a “Calculation” subroutine which permits him to designate limits for automatic quantitative calculations. At any time, the operator may attenuate the display (8 levels of display, attenuated by factors of 2 ) of ion profiles on the CRT to facilitate recognition of peaks. Conversely, any data which are attenuated to full scale display on the oscilloscope a t its lowest gain may, of course, be amplified by factors of 2 , up to 2 7 . In this way, the dynamic range of the CRT may be increased 27 = 128 times. The inherent range of the oscilloscope is 512 bits giving an overall dy64,000 namic range for signal display of 512 X 128 which was designed to accommodate data from the interface described below.
INSTRUMENTATION, MATERIALS, AND PROCEDURE The combined GLC-MS (LKB-9000) was equipped with a standard accelerating voltage alternator (AVA) which consists of 2 voltage-divider circuits to reduce the effective accelerating voltage. Each circuit includes a ten-position coarse adjustment and a fine continuous adjustment of the total resistance. This prototype-MID hardware unit permits 3 ions to be monitored over a 10% mass range. The unit also includes a filter which is designed to eliminate all frequencies above 8 Hz received and amplified by the electron multiplier during AVA operation. A magnetic mass spectrometer tends to produce a drifting magnetic field during MID operation because of the thermal effects of the maintained, high-level current (10-12 A) for the electromagnet. This problem was usually avoided by allowing the magnet to reach thermal equilibrium overnight a t the desired magnetic field; the success of this procedure was demonstrated with frequent (hourly) analyses of standards. While this hardware is not the most ideal for MID, it has been used successfully in many biological applications and was suitable for the development and evaluation of our displayed-oriented data system. The interface permits computer control of the accelerating voltage alternator so that the accelerating voltage can be cycled a t any programmed rate. Figure 1 indicates that the interface also consists of a buffer amplifier with a null adjustment and active filter, and 3 output amplifiers for data acquisition. The most sig-
A N A L Y T I C A L CHEMISTRY, VOL. 45, N O . 12, OCTOBER 1973
MID INPUT
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Figure 1. Circuit diagram
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RESULTS
nificant, unsaturated output from 1 of the 3 output amplifiers having relative gains of X1, X8, X64, is sampled by the 10-bit analog to digital converter (ADC) on the computer: by this design, the system has a dynamic range of 1 in 64,000 at each gain setting of the electron multiplier. Relays No. 2 and 3 control the coils of the relays in the AVA unit. For each ion current channel, the program goes through three timed phases. First, the proper relay is activated a n d a time delay of 200 msec is initiated to ensure that the AVA reed relays have closed‘(de1ay could be shortened with better quality relays). Next the computer ADC samples the electron multiplier of the mass spectrometer 100 times a t 2-msec intervals. Finally, the relay is opened and a 25-msec time delay initiated by the computer while the average value of 100 samples is calculated and stored. T h e cycle is then repeated for the next channel. The timing is accomplished with a real-time clock under “program interrupt” to permit on-line display on a n oscilloscope of the already-acquired data. The time base for each sample is initialized by a n amplified voltage signal from the vacuum gauge on the second stage separator which receives a pressure surge from the solvent front for each injection. One of the software options (“Acquire Data”) has a stand-by subroutine which samples, averages, and tests the a m plified vacuum meter voltage; in exceeding a preset threshold, this voltage initiates acquisition of MID data. The Digital Equipment Corporation PDP-12A computer is equipped with a n additional 4K core (total 8Kj, 2 DEC tape units, and a n extended arithmetic unit as well as a 12-channel (10 bit) analog to digital converter (ADC), a non-storage oscilloscope (CRT), 12 sense lines, 6 relays, and a real-time clock. The flexible d a t a handling system should be adaptable to any mass spectrometer and the computer software will be available, upon request, to qualified users. The interface (Figure 1) can be built “in house” with less then $100 worth of components. Derivatization of prostaglandin PGBz, after conversion of PGEz to PGBz (26), was effected with diazomethane in diethyl ethermethanol, followed by evaporation of ether, and treatment with neat bis (trimethylsilyl) trifluoroacetamide (BSTFA) to form the methyl ester ( M E ) trimethylsilyl ( T M S ) derivative: (PGBz-MET M S ) . 3.3,4.4-Tetradeuterio PGEz was made available by the Upjohn Company. PGFz,,-ME-(TMS)3 was prepared by treating PGFz,, with diazomethane, removal of excess reagent, and addition of excess BSTFA. The catecholamine analogs were derivatized by treating the hydrochloride salts with pentafluoropropionic ( P F P ) anhydride and ethyl acetate (room temperature for 30 min) to produce the P F P derivatives (the pentafluoropropionic ester and amide).
The advantages of this data system can be easily appreciated by comparing (Figure 2) computer-displayed MID results with those recorded in parallel by a conventional oscillograph from the same sample. For this comparison, 1/10 the purified extract (after derivatization) of 100 ml of urine (17) originally containing 500 ng of 3,3,4,4-tetradeuterio-PGEz (subsequently converted to d4-PGBz and then to d4-PGBz-ME-TMS) was injected on to the GLC-MS (0.5 m X 3 mm 1% Dexsil 300 on Chromosorb G, SO/lOO mesh at 240 “C). The principal ions for PGBz-ME-TMS ( m / e 321), d4-PGBz-ME-TMS ( m / e 325), and PGB1ME-TMS ( m / e 323) were monitored throughout the gas chromatographic analysis. The upper left panel of Figure 2 is a reproduction of the oscillographic record in which 3 profiles should be discernible. The same data were obtained through the computer interface as indicated in the upper right panel of Figure 2 which is an actual photograph of the computer oscilloscope (CRT) displaying the composite profiles along with numerical indicators for each profile. The individual profiles in the composite display can be readily identified by merely manipulating switches on the console which “strip-away” a given profile and its identifying m / e value from the CRT. The results of this procedure are also illustrated in Figure 2; in going from upper right to lower left and then to the lower right panel, the operator has “stripped away” the display of the m / e 323 and then the mle 321 profile. Alternatively, any one of the 3 profiles may be displayed individually. Other options in the computer program are listed in Figure 3 which is a photograph of the computer oscilloscope. This display appears upon loading the MID program or it can be “called” by the operator by depressing the “M” key (main options) on the teletype (TTY). Any of the options may be activated by depressing the indicated symbol to the left of each option, e . g . , “A” for acquire date, “R” for retrieve data, etc. The “Fill Header” option is used to indicate general information about a group of samples and analytical condi-
(16) B. J. Sweetman, J. C. Frolich. and J. T. Watson, Prostaglandins, 3, 75-87 (1973).
(17) J. C. Frolich, B. J. Sweetman. K. Carr, J. Splawinski. J. T. Watson, E. Anggard. and J. A . Oates. Advan. Biosci.. 9 , p 321-30 (1973).
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 1 2 , OCTOBER 1 9 7 3
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. ... ... . . .. .. .i graphs01 t h e computer OsciIIo!scope as t h e profiles 01 m / e 323 and then that 01 m / e 321 are “stripped” from the comnmcile rlicnl-v
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Figure 3. Photograph of computer osciiioSCOpe showing software options available to operator as described in text tions. The operator responds to a qiuestionnaire which appears on the CRT display inquiring the m l e values for up L. -”LIIc. v p ~ ’ L a L v BcJl~, to 3 different ions, the date, initials” -c *Lera1 description of the type of compounds and GLC conditions. Once the header is filled, this important general information will be stored with each set of analog data profiles until the header information is updated. In this wav. “. valuable operator and instrument time are not consumed by repetitious submission of general d;sta for each GLC analytical run. The “Acquire Data” option permits t1.- _II ”_ a pre-injection statement to the general header-data block, thereby identifying a specific sample by name, number, quantity injected, and change in GLC temperature or detector sensitivity. The operator then depresses the “return” key on the TTY which calls a standby subroutine t o initiate the actual data acquisition a t the moment of pressure surge due to solvent through the second ^_^_^
___
2074
stage molecular separator. Use of the pressure gauge output provides a consistent indication of injection time so that retention times for various runs may be compared reliably. As described earlier, each datum point on the CRT is the average of 100 samples of the electron multiplier output sampled uniformly over a 200-msec period. Several GLC runs may be recorded (up to a total of 30 minutes) with the retention-time base re-initiated by each successive injection. The acquisition mode can be terminated by depressing the “return” key on the TTY. The operator then has a n opportunity to review the data profiles before depressing the “return” key a second time which permits him to enter a post-run statement which will also be added to the general header information and stored with the ion current profiles. Depressing the “return” key a t this point displays the list of main options a t which time the operator depresses the “S” key (see Figure 3) if he wishes to save the data. Approximately 40 runs of 30-minute duration can be stored on magnetic Linc (Digital Corp.) tape. The limitation of 30 minutes of continuous data acquisition and display for 3 ion profiles is imposed by 8K of active core; if data from only 2 ion profiles were acquired, the time of continuous acquisition could be extended to 45 minutes. The screen width accomodates 256 data points (each a n average of 100 samples of the electron multiplier output) which allows a six-minute time interval of data of any (or all three) ion profile(s) to be displayed a t any time. The time axis for display is continuously variable so that the operator may select any 6-minute interval for viewing. A typical header block with pre-injection (first arrow) and post-injection comments (last 3 arrows) is shown in Figure 4. These data are stored on Linc tape (magnetic)
ANALYTICAL CHEMISTRY, VOL. 45. NO. 12. OCTOBER 1973
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Figure 6. Sequence illustrating amplification of MID profiles for display on oscilloscope These data resulted from an aliquot of urine extract containing about 100 pg normetanephrine PFP (m/e 458 and 445) under arrow in photo A and 20 ng of a-methyl dopamine-PFP ( m / e 4 4 2 ) . These same data are amplified 4 X and 8 X in photo B and C. Photo D shows the m / e 458 profile only, amplified 16X
ANALYTICAL CHEMISTRY, VOL. 45, NO. 12, OCTOBER 1973
2075
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Right panel: Same data after software filtering option
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with the corresponding ion current profiles. Upon retrieval of these data, the investigator may alternately display such a header block or the accompanying ion profiles by merely depressing the “return” key on the TTY. The retention time of any peak may be ascertained by depressing one of the sense switches on the computer console which displays a vertical cursor (Figure 5) on the CRT display along with the ion current profiles. From the console, the operator can adjust the lateral position of the time cursor to coincide with the center of any given peak. The retention time corresponding to the position of the time cursor is continuously displayed in the upper left quadrant (arrow) of the CRT as indicated in Figure 5. The abundance of the data profiles may be amplified on the CRT in 7 discrete steps (factor of 2 per step or maximum of X 128). This adjustment facilitates inspection of the data profiles for discernible peaks. Figure 6 shows a sequence of photographs of the CRT display of data resulting from analysis of a human urine extract for normetanephrine as the PFP derivative which fragments upon electron ionization to produce ions a t mle 458 and 445 in the ratio of 2 : l . These ion current profiles show a barely discernible peak (arrow) in the photograph (A) a t the far left of Figure 6. The readily discernible peak in the ion 2076
current profile of mle 442 is produced by the PFP derivative of a-methyl dopamine used as an internal standard. These same data have been amplified 4~ in the next photograph (B) and 8 x in photograph C in which the peaks on the m / e 445 and 458 profiles become more discernible as the peak on the mle 442 profile goes off scale. Photograph D is the computer display in which the m/e 442 and 445 profiles were stripped away to confirm the identity of the peak on the mle 458 profile after it was amplified to 16X. These changes in amplification for display merely facilitate recognition of peak areas by the investigator and do not affect the quantitative relationship of the data as stored by the computer. When trace quantities of a substance are detected by the electron multiplier a t high gain, the data profile on the CRT may be considerably scattered. This is especially true when the profile is also magnified on the CRT as is the mle 423 profile in the left panel of Figure 7 which resulted from the injection of 200 pg of PGF*,-ME-trisTh4S into the GLC-MS. In these cases, it is helpful to use the smoothing routine (18) which is initiated by depressing the “N” key on the TI’Y. The effect of the software data-filtering operation is seen in the right panel of (18) A . Savitzsky and M J E Golay Anal Chem, 36, 1627 (1964)
A N A L Y T I C A L CHEMISTRY, VOL. 45, N O . 12, OCTOBER 1973
Figure 7. The smoothing routine facilitates recognition of the base line for purposes of quantitative measurement. Depressing the “C” key on the TTY initiates the calculation mode which displays one of the ion current profiles on the CRT along with 3 adjustable cursors which resemble 3 sides of a rectangle as seen in Figure 8. The operator adjusts the level of the horizontal cursor to designate the base line and then brackets the peak by adjusting the other 2 cursors to the leading and trailing edges of the peak as indicated in the right panel of Figure 8. The operator then depresses the “C” key again and TTY prints the value of the peak height, total peak area, and the “central” area for the selected ion. The area evaluated by the computer is the sum of data points minus the base line between the leading and trailing end points of the peak as defined by the cursors for “total” peak area, and between the coordinates of peak width a t half-height for “central” peak area. For the latter measurement, within the limits set by the operator, the computer finds the peak height, calculates the half-height, and locates the points a t which the peak profile intersects the coordinates of half-height. The computer then calculates the area limited by the base line, 2 imaginary vertical lines, and the curvature of the peak profile above half-height as depicted schematically in Figure 9. The “central” peak area should be proportional from peak to peak because it is based on the width at half-height; in general, the value of the “central” area is approximately 70% of the value for “total” area. The “central” peak-area concept is useful in coping with practical problems encountered in routine analyses such as that depected in Figure 10 involving a shoulder on the trailing edge of the peak for which the operator ordinarily e
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Sequence shows that decisions regarding the trailing edge of an unresolved peak greatly affect value of total area (2639, 2963, 3607 in going left to right) whiie the value of peak height and central area remain at 123 and 2089, respectively, in all cases (see accompanying TTY output at bottom of figure) A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 12, OCTOBER 1973
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Table I. Precision of M I D Results. Comparison of Method of Calculation of Low-Level Prostaglandin (400 pg PGFz,-ME-fMS3) and Internal Standard (20 ng d4-PGFza-ME-TMS3) Injected into GLC-MS Actual Values of CENTRAL AREA from Samples Described Above Sample Central area Central area Ratio No. of m / e 427 of m / e 423 4271423
1 2 3 4 5 6
1750 1691 1867 2301 1964 1976
37.8 34.2 35.9 43.6 39.7 44.5
46.3 49.4 52.0 52.8 49.5 44.4
Mean = 49.1 f 1.3 Summary of Results for Different Methods of Measuring Some Data No. of Ratio of peak Ratio of total Ratio of replicates heights peak areas CENTRAL AREA
N=6
50.3 f 1.98 (4.0%)a
58.9 k 2.4 (4.1%)
49.1 f 1.3 (2.7%)
a Standard deviation of mean expressed as percentage of mean
would have to make a judgment concerning the limits of peak area integration. Using the central-peak calculation option in this case eliminates the requirement for critical placement of the area-limiting cursors a t the leading and trailing edges of the peak so long as the shoulder is resolved below the half-height of the major peak. For example, for the 3 different positions of the trailing peak-area cursor for the peak in Figure 10 which also includes the TTY output for each case, note that the value for peak height and central area was the same, while the total peak area was substantially different in each of the 3 cases. Values of central peak area are linear over the same range, 100 pg to 20 ng (16), as values for peak height and total peak area. As expected, the precision of central area measurements is better than that for total area (Table I), probably because operator decisions concerning the recognition of the leading and trailing edges of the peak are not so critical for central area; also contributions from unresolved peaks are minimized by the central area concept. Table I summarizes calculations made on data resulting from replicate injections of a low-level prostaglandin sample (20 ng cl4-PFG2, and approximately 400 pg of PGFzn as the methyl ester, T M S ether derivatives per injection.
2078
More attractive statistics are available for sample levels over 100 ng (precision less than fl%) but these are not relevant since such high levels of the compound-of-interest are rarely found in realistic, biological samples. For data derived from quantitative detection in the picrogram range, a precision of f5% is generally acceptable with current methodology. Both precision and accuracy deteriorate a t these low levels because of poor ion statistics and, in part, because of the greater relative importance of variable background contributions to the ion currents being monitored. Use of the area cursors do not, of course, completely overcome errors due to drifting base line, noise, etc., but they do permit optimum judgment on the part of the operator in evaluating the data. In this work, the computer has been integrated into the control and operation of the MID unit to circumvent ambiguities otherwise imposed by conventional means of recording. The computer acquires, reduces, and stores MID data together with all relevant analytical conditions and sample information. The data are readily retrieved and displayed for examination and computation by the investigator. This display-oriented data system has been refined several times in 2 years of laboratory operation to provide optimum interaction by the investigator in interpreting MID data for the quantification of prostaglandins (17, 19) and other biological compounds.
ACKNOWLEDGMENT Richard Manning contributed to the original interface design and Betty Fox routinely operated the GC-MScomputer system. The deuterium labelled prostaglandins dpPGE2 and d4..PGFza were kindly supplied by the Upjohn Company. Received for review January 2, 1973. Accepted May 2, 1973. Reported initially at 5th Great Lakes Regional ACS Meeting, Peoria, Ill., June 11, 1971. JTW is Recipient of Public Health Service Research Career Development Award for 1973-78 (National Institute of General Medical Sciences). This work was supported by The Research Center for Clinical Pharmacology and Drug Toxicology GM-15431. (19) 8.J. Sweetman, J. T. Watson, K. Carr, J. A. Oates, and J. C. Frolich, submitted for publication.
A N A L Y T I C A L C H E M I S T R Y , V O L . 4 5 , NO. 12, OCTOBER 1973
