ALM. Doffield, B. Zeitman and J.G. Lawless. (58) Mass
Spectrometry in Structural and Stereochemical Problems CLXXXKIII.

A Study of the Blectrorn Impact Induced Fragmentation of Aliphatic
Alilehyies. dg. Amer. Chem. Soc., 91, 6814 (1969). By R.J. Liedtke
anid oc. ~Pijerassi.

(59) Mass Spectrometry in Structural and Stereochemical Problems -
CXCVII. Flectron-Impact Induc2d Functional Group Interaction in
4-Renzyloxycyclohexyl Trimethylsilyl Ether. Org. Mass Spectrom.
4, 257 (1970). By Paul DB. Woodgate, Robin T. Gray and Carl
Di2zrassi.

(67) Mass Spectrometry in Structural and Stereochemical Problems ~
CYCVIII. A study of the Fragmentation Processes of Some
a,B-Insaturated Aliphatic Ketones. Org. Mass Spectrom., 4, 273
(1979). By Younus M. Sheikh, A.M. Duffield and Carl Djerassi.

(61) Mass Svectrometry in Structural and Stereochemical Problems
“CIT. Interaction of Pemote Punctional Groups in Acyclic Systems
agpon Flectron Impact. J. Org. Chem., 36, 1796 (1971). By M.
Sheehan, R.J. Spangler, M. Ikeda and C. Djerassi.

(62) Mass Spectrometry in Structural and Stereochemical Problems
™"VIT. Fragmentation of Unsaturated Ethers. Org. Mass Spectrom.,
5, 895 (1971). By J. P. Morizur and C. Djerassi.

(63) Mass Spectrometry in Structural and Stereochemical Problems
“CVIIT. The Effect of Double Bonds Upon the McLlafferty
Rearrangamant of Carbonyl Compounds. J. Amer. Chem. Soc., 94, 473
(1972). By J.8. Dias, Y.M. Sheikh and Cc. Djerassi.

(64) Mass Spectrometry in Structural and Stereochemical Problems
CCOX¥. Behavior of Phenyl-Substituted a,B-Unsaturated Ketones Upon
Flectron Tmpact. Promotion of Hydrogen Rearrangement Processes.
J. Orq. Chem., 37, 776 (1972). Ry R.J. Liedtke, A.F. Gerrard, J.
Diekman ani C. Djerassi.

(55) Mass Spectrometry in Structural and Stereochemical Problems
CCXXTI. Dalineation of Competing Fragmentation Pathways of
Complex Molecules from a Study of Yetastable Ion Transitions of
Deuterated Derivatives. Orq. Mass Spectron., 7, 367 (1973). By
D.H. Smith, A.M. Duffield and <. Djerassi.

(455) The Carbon-13 Maqnetic Pesonance Spectra of Acyclic Aliphatic
Amines. J. Amer. Chem. Soc., 95, 3710 (1973). By H. Eggert and
c. Djerassi.

(67) The Carbon-13 Nuclear Maqnetic Resonance Spectra of Keto
Steroids. J. Org. Chem., 38, 3788 (1973). By H. Fqgert and C.

Djerassi.

(68) Mass Spectrometry in Structural and Stereochemical Problems
CCXYXYXVIII. The Fffect of Heteroatoms Upon the Mass Spectrometric
Fragnentation of Cyclohexanones. J. Org. Them., in press. By
J.H. Block, D.H. Smith, and C. Djerassi.

(49) Mass Spectrometry in Structural and Stereochemical Problems
“CX¥LIIT. Applications of DADI, a Technique for Study of Metastable
Tons, to Mixture Analysis. J. Amer. Chem. Soc., Submitted for
publication. By D.H. Smith, C. Djerassi, K.H. Maurer, and U.

76
RAODD.

(70) Mass Spectrometry in Structural and Stereochemical Problems.
The Fragmentation of Progesterone and Alkyl-Substituted
Proaasterones, in preparation, by S. Hammerum and C. Djerassi.

(71) Applications of Molecular Orbital Theory to the
Interoretation of Mass Spectra: Prediction of Primary
Praqgnentation Sites in Organic Molecules," Org. Mass Spectrom., 7,
1241 {1973), by G. Loew, M. Chadwick, and 5.4. Smith.

77
PROGRESS REPORT

78
TEXT OF 1973 ANNUAL REPORT FOR RESEARCH

PROJECT:

RESOURCE-RELATED RESEARCH -- COMPUTERS
AND CHEMISTRY
Progress Report

Part A. APPLICATIONS OF ARTIFICIAL INTELLIGENCE TO MASS SPECTROMETRY
JBJECTIVES:

Research activities carried out under Part A of this project have been
directed toward extending the reasoning power of Heuristic DENDRAL.
Heuristic DENDRAL reresentsS a paradigm for attacking problems in one of
the major areas of importance to any scientific discipline dealing with
molecules, the area of structure elucidation. We have focused our
attention on the use of heuristic programming techniques for analysis of
mass spectra and ancillary analytical data which can be obtained
utilizing a mass spectrometer. It is convenient to discuss objectives,
progress and plans by examining three broad areas of activity in
researzth connected with Part A. We wish to note that these areas
conform to our overall strategy of PLAN-GENERATE-TEST. We have shown,
Parlier, how powerful this strategy is when applied to the task of
structure elucidation utilizing mass spectral data. The areas and their
objectives are the following:

(I) PLANNER:

(a) Extend the programs used for structure elucidation to
structural analysis of complex molecules.

(b) Assess the capabilities andi limitations of the PLANNER.

(c) Generalize the programming techniques to reduce compound
Class dependence.

(d) Explore the utility of ancillary data available from the mass
spectrometer.

(II) STRUCTURE GENERATOR:

(a) Complete the exhaustive, irredundant generator ot molecular
structures. .

{b) Develop efficient constraints 9n the generator to exploit its
potential utility.

(c) Exploit the concepts developed for the structure generator in
solving various structure-problems (related to m.s. and
others) and isomer-problems.

(III) PREDICTOR
(a) Extend the Predictor to still more complex molecular structures.
{b) Explore the design of experimental strategies, utilizing
Predictor functions, tos differantiate among candidate solutions.

We point out that the PLAN-GENERATE-TEST strategy, although applied to
structure elucidation, has potential utility as a strategy for solving
other chemical problems. Similarly, although we utilize mass spectral
data aluost exclusively, the same heuristic programming techniques
allow facile extension to analysis of data from other types of
analytical instrumentation. These were not objectives of the

original research proposal but seem logical extensions for future
work. We have illustrated the patential of these techniques for
analysis of 13C NMR data (Carhart and Djerassi, 1973). This is
discussed briefly under the PLANS section, below.

PROGRESS:

(I) PLANNER
The fuaction of the Planner is to analyze mass spectral data acquired
dn a compound. The Planner attempts to derive structural information
from these data using the rules of behavior of compounds in the mass

spectrometer.
Jbjective (a): Extend Programs.

The Planner is presently embodied in a program which also contains a
set of functions to assemble this structural information into complete
molecules (a primitive Structure Generator) and to test these
molecular structures with other, not necessarily mass spectral, rules
(a pcinitive Predictor). This performance program was written in
this way to provide a useful tool for chemical studies while more
general versions of the Structure Generator and Predictor were being
jeveloped. This program and its performance have been described in
some detail in a publication and ia previous progress reports. A
manuscript (Smith, et.al., 1973) has now appeared describing the
application of this prograa to the analysis of mixtures of compounds
without prior separation.

Objestive (b): Assess Capabilities.

He have extended the capabilities of the Planner so that we can

analyz2 both low and high resolution mass Spectral data. A low

resolution mass spectrum is regarded by the program as a pseudo high
resolution spectrum wherein possible elemental compositions of each

peak are limited only by the inferred molecular tormula of the

compound. This results in more ambiguity with a commensurate increase

in number of candidate solutions as would be expected considering the lower
specificity of low resolution data as compared to high resolution

data.

We have extended our capabilities for molecular ion determination
utilizing a heuristic search technique through the space of plausible
noleculac ions. This techaique has had significant success even when
dealing with the low resolution mass spectra of compounds which
display no molecular ion, for example the class of derivatized amino
acids {trifluoracetyl, n-butyl esters) important to studies carried
out unier Pact B, below. ;
We have segmented the performance program to decrease the amount of
memoty required for its operation. This should increase the chances
for other groups to make use of the program.

The linitations of the present performance program are primarily the
requirement that some information about the class of conpounds be
kaown, and that, for each class, relatively detailed rules about the
nass spectral fragmentation of this class be available. The former
limitation results primarily from the nature of the program in that a
complete structure generator is not incorporated. The primitive
structure generator available to the program can only place substituents
about an assumed skeleton. This Limitation will be alleviated when

a structure generator with SOODLIST and BADLIST constraints is
available (see Structure Ganerator, below). The latter limitation

is mor2 fundamental, but is characteristic of every spectroscopic
technigue to one degree or another. It must be assumed that analysis
of a mass spectrum, alone, may not lead to sufriciently unambiguous
information about the structure of the compound yielding the spectrun.
It is For this reason that extensions of the programming techniques
to encompass data from other spectroscopic techniques are attractive.

3 S/
Qbjective (cj): Generalize Techniques.

we have carried out several successful experiments to ensure that the
perfornance program, used originally for analysis of estrogenic
steroids, retains only procedures which are compound-class independent.
By supplying fragmentation rules for other classes of compounds, we nave
successfully carried out structure elucidation of molecules in several
diverse classes including other steroiial hormones and related compounds
{progesterones, testosteroaes, androsterones), steroidal sapogenins and
derivatized amino acids.

Objective (dj): Explore Utility

Previous progress reports kave sumnarized in some detail the ways in
which data from ancillary techniques in mass spectrometry (metastable
ion ani low ionizing voltage data, labile hydrogen exchange) can be
used by the program. The utility of metastable ions for aid in
stucture elucidation continueS as an active area of interest.
Experience with the program has inspired studies on metastable ions,
first, to help delineate the course of fragmentation of molecules with
the purpose of extending and refining fragmentation rules used by the
program (Smith, Duffield and Djerassi, 1973). Experience with the
iacreased specificity of structural information with concomitant
reduction in analysis tine when metastable ion information is
availavle (Smith, et.al., 1973) has led to a study of a new technique
for detection and analysis of netastable ions (Direct Analysis of
Daughter Ions, or DADI) and has illustrated the utility of this
technique in mixture analysis (Smith, Djerassi, Maurer and Rapp,
1973).

Experience with the PLANNER has led to several research activities
relatei to, but not supportei by, this grant. Our studies of estroyen
nixtuc2s isolated from pregnancy urine have suggested new compounds
likely to be important in the human metabolism of estrogens. Some of
these compounds are hitherto unreported structures and a synthesis
progran is underway in Professor Djerassi’s laboratory to produce sone
of these compounds. The Planner will be used as one method of
comparison of the synthesized, authentic standards with those isolated

from pregnancy urine. -

Work is also being carried out to explore the fragmentation of model
systems possessing two heteroatoms in close proximity. It is clear
from the first of these studies (Block, Smitno, and Djerassi, 1973)
that the fragmentation of these difunctional systems does not reflect
that of monofunctional analogs. More groundwork is required in this
area td obtain better fragmentation rules for these systems.

II. STRUCTURE GENERATOR
Objective {a): Complete the Generator

The last progress report discussed the completion of both the basic
structure generator algorithm and program, which provide the capability
for exhaustive generation of graph isomers of a given empirical fornmala,
with prospective avoidance of duplicate structures. Since the time of
the submission of that report, manuscripts describing the structure
generator, directed specifically to an audience of chemists, have been
submitted (Masinter, Sridharan, Lederberg, and Smith, 1973; Masinter and
Sridharan, 1973). Some effort over the past year has been devoted to

/ Gor
verification of the completeness and irredundancy of the method. He
have extended existing combinatorial countiny algorithms to check that
“the numbers of isomers generated are correct. we have used an
interactive version of the generator to verify that variations (allowed
by the algorithm) of the mechanism of generation yield the same set of
isomers. In this way we are now increasingly confident that the
progran's performance accurately reflects the mathematically proven
algorithm on which it is based.

The Structure Generator has been briefly described, and placed in its
context within Heuristic DENDRAL, in an invited paper presented at a
NATO/CRS sponsored conference on Computer Representation and
Manipulation of Chemical Infornation, held in Amsterdam in June, 1973
(Smith, Masinter and Sridharan, 1973).

we have also begun to develop techniques to expand the scope of the
jenerator. One example, which has been completed, is adding extensions
to the CATALOG. The CATALOG contains the set of vertex-graphs fror
which structures are assembled. The original CATALOG was not sufficient
to generate all isomers of some potentially interesting compositions,
2.g-, those involving graphs possessing nodes of degree >3. We now have
a program which constructs complete sets of vertex-graphs containing
nodes of degree >3 from the set of trivalent graphs in the original
CATALIS. We have thus extended the capabilities of the generator.

Other such extensions are discussed in the PLANS Section, below.

Objective {b): Develop Constraints

It is absolutely essential that we provide the mechanism for
constraining the Structure Generator: without constraints it is
merely a legal move generator, as in a chess-playing program. For
structure elucidation problems, the Planner can determine many
features of the molecular structure from various types of experimental
jJata such as presence of functional groups, and the numbers of doubie
bonds and rings. Partial information of this sort can be used to
constrain the Structure Generator to the Space of plausible candidate
structures. From a graph-theoretic point of view, however,
constraining the graph generating algorithm is a difficult unsolved

problen.

We are presently formulating several types of constraints to apply to
the Structure Generator. Some types of constraint await the development
of new mathematical tools {see PLANS), while others can be immediately
implemented with relatively minor alterations to the algoritnm. The
class of constraints presently receiving attention deals with types

of unsaturation (rings or double bonds) desired in the final

structures. Related to this constraint is the constraint of number of
quaternary carbons present. The rormer information (number and

nature of multiple bonds) is readily available from several spectroscopic
techiigues, while the latter may be obtained from 13C NMR. The
implementation of this class of constraints will be used as the model
for future implementation of a GOODLISI {structural features known to

be present) and a BADLIST {structural features known to be absent).

It is possible that some types of constraints may not be easily
implemanted within the algorithm. Thus, retrospective tests of
isomers may be required to search for desired or unwanted features.
We have developed some new approaches to graph matching which seem to
be Significantly more efficient than previous methods. Should
prospective implementation of a constraint prove difficuit, we will

5 53
have at our disposal some powerful graph matching tools to exercise
the constraint.

Ibjective (c): Exploit the Generator for Structure Elucidation

we have demonstrated the utility of some subsystems of the structure
generator, e.g., the LABELLER, by exploring some problems of isomerism
noted in the chemical literature. We have corrected the member and
presented the identities of isomers formed by different substitutions of
alkyl chains about a porphyrin nucleus. We are presently exploring some
probleas of isomerism of carbocyclic cing systems, specifically C10H10
and (CH) 10 and C10H2n-4 tricyclic ring systems, n = 8 - 12, related to
the mechanistics of isomeric interconversion.

We have the complete list of all topologically possible 1176 6-membered
Diels-Alder ring systems, using any combination of C,N,O and S. This
list was generated using the PARTITIONER and an extended version of
the LABELLER. These are all the 6-membered ring systems that can be
embedded in structures resulting from the well-known Diels-Alder
reaction. Of the 1176 possible ring systems, approximately 80% are
unreported in the Ring Index. Many of these are chemically unstable -
undecscoring the need for a BADLIST implementation for the Cyclic
Structure Generator. However, many of these unreported ring systems
are certainly chemically plausible. Awareness of such gaps in
relatively simple synthetic categories might lead to discovery of new
categories of compounds with important biological effects.

(III) PREDICTOR

The function of the Predictor in the PLAN-GENERATE-TEST strategy is
to perform a detailed evaluation of candidate solutions (structures)
to a structure elucidation problem. It may use a more detailed model
of spectroscopic behavior than that embodied in a Planner to attempt
to differentiate among possible solutions.

Objective {a): Extend the Progran

we have extended and generalized the Predictor used previously for
saturated, aliphatic, monofunctional compounds. Given a list of
structures and rules of fragmentation processes, it will predict a mass
spectrum for each structure. Prediction of relative ion abundances is
crude, but previous work has shown that even crude measures of ion
abundance are usually satisfactory. The predicted spectrum can be
matched then with the observed and candidates ranked according to the
quality of the match. The program works with structures and rules of
any complexity. An interesting philosophical question is how much
kaowledge should be brought to bear on interpretation of the data at the
Planning vs. Predicting stages of analysis. It is our feeling that if
more can be accomplished during Planning to constrain the Structure
Senerator, the analysis will be more efficient. On the other hand, Some
knowledge can be utilized only if a complete structure is specified, so
that its use is restricted to a predictive role. Moreover, Predictor
Functions have a greater utility, as indicated in the subsequent ©
section.

Objective (b): Differentiate Structures

The Predictor has a more obvious application in the design of

& oY
axperimental strategies to differentiate among candidate structures.
Rules of spectroscopic behavior utilized during Planning demand the
presence of some data to evaluate. The Predictor can then be used to
request additional data from any source to aid in differentiation. We
have explored this approach by analyzing the spectrum of a compound
with the performance program. The Predictor was used to evaluate the
the set of candidate structures to define the minimum number of
metastable defocussing experiments necessary to achieve a unique
Solution. Thus, no time need be spent acquiring unnecessary OF
redundant data. Clearly, this has important implications for future
work in that many different types of data (e-g-, NMR, IR) might be
requested by the Predictor to facilitate identification.

PLANS

For the remaining period of this grant we propose to carry out the
following extensions of the research outlined above.

{I) PLANNER

The major area of activity related to the present version of the Planner
will be to focus our attention on using the program in support of
chemical studies outlined under Part B (see below). The chenical
extraction and derivitization procedures used in the analysis of body
fluids restricts the types of compounds present in each separated
fraction. Such Simplifications make this a problem more amenaple to
attack. Only certain classes of compounds are present in each fraction,
and we have some knowledge of the mass spectral fragmentation of these
Classes. We wish to couple the program to the results of library
matching procedures so that we direct our efforts to structure
elucidation of those components which have not been previously
identified. This is particularly important in the context of analysis
probleas such as those discussed under Part B.

We propose increasing the utility of the program by removing two
present constraints: (a) allow unspecified "dummy" atoms in the
skeleton instead of requiring a rigidly fixed structural skeleton,
and ({b) allow fragmentation processes to be specified more flexibly ~-
in particular, allow fragmentations in substituents on the skeleton
instead of cequiring all fragmentations to cut through the skeleton.

Although we are presently unconfortable with immediate coupling of

the Structure Generator to the Planner, we propose continued
2xploration of the problems of controlling the generator automatically.
Actual implementation awaits a more comprehensive treatment of the
problem of constraints.

II. STRUCTURE GENERATOR

The inclusion of a reasonable set of constraints is obviously required
and will be the subject of most of our future development work. Wwe
plan to develop an interface to the present interactive version of

the Structure Generator that speaks a more chemical language. This
interface will be designed to avoid the present reguirement that the
usec know something about the program before he can use it. As the
optinum method for implementation of a constraint is determined, the
interface will be extended to translate the usual specification of the
constraint in chemical terms into rules acting at the level of the
program. AS we stressed in development of the PLANNER, there are
Considerable advantages to building a powerful program in an

7 8S
incremental fashion. These steps are loyically directed to our longer
term goal of developing a useful structure elucidation tool for the
chemist, based on the structure generator.

There are several other areas of interest which are peripherally related
to the problem of constraints and which will occupy our attention. The
Structure Generator knows no chemistry other than atom names and their
associated valence. There are several important areas where this is an
immediate problem. For example, the program has no explicit awareness
of the aromatic resonances, leading to a remediable redundancy in the
list of isomers. An aromaticity-predictor is also indispensable for
anticipating chemical behavior of a structure.

We wish to deal with types of isomers besides simple connectivity
isomers. We need to have the facility for assembly of molecular
sub-structures (the usual type of information inferred fron
spectroscopic data) when such an assembly yields new rings or multiple
bonds. All the above questions need a reexamination of the fundamental
mathematical considerations. The present algorithm has been proven to
yield complete and irredundant solutions. In devising new algorithms or
variants of the present one, the burden of proof can be reduced to {the
usually easier) equivalence to the previous algorithm. Professor Harold
Brown, who was the mathematician instrumental in initial development of
the labelling algorithms for structure generation, will be with us again
for several months to help attack the problems outlined above.

I1I. PREDICTOR

Although the Predictor has been essentially finished for our own
internal use, we propose to spend a modest amount of time in the
coming months making it more usable by others. In particular, we
wish to extend the initial work on predicting the new experiments
necessary for distinguishing among candi.late structures (e.g.,
predicting that a metastable peak at mass 70.1 would confirm one
structure and disconfirm another). In addition, we plan to work ona
cataloging some existing sets of mass spectrometry rules in such a
way that the program can be easily used for different classes of
problems.
Part A ceferences (Published or submitted during year)

R. Carhart and C. Djerassi, "Applications of Artificial Intelligence
for Chemical Inference XI....

D.He Smith, B.G. Buchanan, R.S. Engelmore, H. Adlercreutz, and

c. Djerassi, "Applications of Artificial Intelligence for Chemical
Inference, IX. Analysis of Mixtures Without Prior Separation as
Illustrated for Estrogens," Je Amer. Chem. Soc., September 5, 1973.

DeH. Smith, A.M. Duffield, and C. Djerassi, "Mass Spectrometry in
Structural and Stereochemical Problems, CCXXII. Delineation of
Competing Fragmentation Pathways of Complex Molecules froma Study

of Metastable Ion Transitions of Deuterated Derivatives," Org. Mass
Spectrom, 7, 367 (1973).

D.He Smith, C. Djerassi, K.-H. Maurer, and U. Rapp, "Mass Spectrometry
in Structural and Stereochemical Problems, CCXXXIV. Applications of
DADI, A Technique for Study of Metastable Ions, to Mixture Analysis,"
Je Amer. Chem. Soc., submitted (1973).

JeM.e Block, D.H. Smith, and C. Djerassi, "Mass Spectrometry in
Structural and Stereochemical Problems, CCXXXVIII- The Effect of

Heteroatoms upon the Mass Spectrometric Fragmentation of Cyclohexanones,"
J. Org. Chem., submitted (1973).

L.M. MaSinter, N.S. Sridharan, J. Lederberg, and D.H. Smith,
"Applications of Artificial Intelligence for Chemical Inference XIl.-
Exhaustive Generation of Cyclic and Acyclic Isomers," J. Amer. Chem.
Soc., submitted (1973).

LeMe Masinter and N.S. Sridharan, "Applications of Artificial
Intelligence for Chemical Inference, XIII. Labelling Objects Having
Symmetry, J. Amer. Chem. Soce, submitted (1973).

DeH. Smith, LL.M. MaSinter, and N.S. Sridharan, "Heuristic DENDRAL:
Analysis of Molecular Structures," to be published in the proceedings
of the NATO/CRS Advanced Study Institute on Computer Representation
and Manipulation of Chemical Intormation.

N.S. Scidharan, "Computer Generation of Vertex Graphs", Stanford
Computer Science Memo CS-73-381, Stanford University, July, 1973.

NO

&7
Part B-i Gas Chromatograph - Mass Spectrometer Data System Development

IBIECTIVES AND RATIONALE

The objectives of this part of the research project are the
improvement of GC/MS data system capabilities and the coupling of
axtracted data to the Heuristic DENDRAL programs for analysis. we
ultimately seek a substantial degree of interaction between the
instrunentation and the analysis programs including computer
specification and control of the data to be collected. In addition
to the developnent goals, this portion of the project provides for the
day-to-day operation of the GC/MS systems in support of mass spectrum
interpretation computer program development {Parts A and C) and
applications of GC and MS to biomedical and natural product sample
analysis with collaborators.

Our rationale for this approach is that the overall systen should be
designed for problem solving rather than just for data acquisition.
This implies that analytical computer programs, after review of
available experimental data, could be able to specify additional
information needed to contirm a solution or distinguish between
alternative solutions. Such requests could be passed back to ah
instrunent management program to set up proper instrument parameters
and collect the additional information. Our initial objectives to
implement an on-line, closed-loop system using the ACME computer
facility have met with a number of difficulties. These grow
principally out of ACME's limited computing capacity and commitments
as a general time-sharing service. In addition, the scanning high
resolution mass spectrometer has inherent sensitivity limitations,
which do not preclude a demonstration but rather limit the practical
sample volume which could be analyzed. Until such limitations can
be overcome, particularly in terms of computing support, we have
focussed our efforts on an open-loop demonstration of such an

approach.

PROGRESS

Progress has been made in demonstrating a GC/High Resolution Mass
Spectrometry capability, in further developing automated data analysis
algorithms, and in planning for the implementation of a data system
for the collection of metastable ion information. Progress in these
and other areas directed toward the main research goals has been
impacted by a transition in computing support which is still underway.
This transition, discussed in more detail below, was occasioned by

the phase-over of the ACME computing facility, which we had been
using, from NIH grant subsidy to a fully fee-for-service operation
under Stanford University auspices.

Summaries of the results and problems encountered in each of the areas
follow.

Gas Chromatography/High Resolution Mass Spectrometry (GC/HRNS)

we have verified the feasibility of combined gas chromatography/high
resolution mass spectrometry (SC/HRMS). Using programs described in
previous reports, we can acquire selected scans and reduce them
automatically. The procedures are slow compared to "real-time"
because of the limitations of the time-shared ACME facility. We have
recorded sufficient spectra of standard compounds to show that the

JO of
system is performing well. A typical experiment which illustrates
some of the parameters involved was the following. A mixture
(approximately 1 microgran/component) of methyl palmitate and

nethyl stearate was analyzed by GC under conditions such that the GC
peaks were well separated and of approximately 25 sec. duration. The
maSS spectrometer was scanned at a rate of 10.5 sec/decade, and a
resolving power of 5000. The resulting mass spectra displayed peaks
over a dynamic range of 100 to 1 and were automatically reduced to
masses and elemental compositions without difficulty. Mass
measurament accuracy appears to be 10 ppm over this dynamic range.

We have begun to exercise the GC/HRMS system on urine fractions
containing significant components whose structures have not been
alucidated on the basis of low resolution spectra alone. Whereas
more work is required to establish system performance capabilities,
two things have become clear: 1} GC/HRMS can be a useful analytical
adjunct to our low resolution GC/MS clinical studies (Part B-ii),

and 2) the sensitivity of the present system limits analysis to
relatively intense GC peaks. This sensitivity limitation is inherent
in scanning instruments where one gives up a factor of 20-50 in
sensitivity over photographic image plane systems in return for
on-line data read-out. This limitation may be relieved by using
television read-out systems in coniunction with extended channeltron
detector arrays as has been proposed by researchers at the Jet
Propulsion Laboratory. We can nevertheless make progress in applying
SGC/HRMS techniques to accessible effluent peaks and can adapt the
improved sensor capability when available.

These experiments have also shown that the ACME computer facility
cannot reliably provide the rapid service reguired to acquire and

file repetitive spectometer scans. This problem is to be expected

in a heavily used time-shared facility without special configuration
for high rate, real time support. Excepting possible requirements for
real time data analysis (such as in a closed-loop system), this
problen could be solved by implementing a large local buffer (e.g.,
disk) at the front-end data acquisition mini-computer. We are
2xploring this possibility in conjunction with the overall planning
for computer support discussed below.

Data Analysis Algorithms
A. Peak Resolution

One of the significant trade-offs to be made in GC/HRMS is tnat of
sensitivity versus resolution. In maintaining high instrument
resolution (in the range of 5,000-10,000) while scanning fast enough
to analyze a Gc effluent peak (approximately 10 sec/decade), system
sensitivity is constrained as discussed above. We have worked on a
method for reducing instrument resolution requirements through more
sophisticated computer analysis of a lower resolution output. In
effect this transters the burden of overlapping peak detection and
mass determination to the computer instead of reguiring inherently
well resolved data out of the instrument. The advantage comes in
better system sensitivity.

Unresolved peaks are separated by an analytical algorithm, the
operation of which is based on a model peak derived from known
Singlet peaks in the data. Actual tabulated peak models ate used
rather than the assumption of a particular parametric shape (€.g-«,
triangular, Gaussian, etc.). This algorithm provides an effective

// 89
increase in system resolution by approximately a factor or tnree
thereby effectively increasing systen sensitivity. By measuring

and comparing successive monents of the sample and model peaks, a
series of hypotheses are tested to establish the multiplicity of the
peak, minimizing computing requirenents for the usually encountered
simple peaks. Analytic expressions for the amplitudes and positions
of component peaks have been derived in the doublet case in terms of
the first four moments of the peak complex. This eliminates time
consuming iteration procedures for this important multiplet case.
Iteration is still required for more complex multiplets.

B. GC Analysis

The application of Gc/MS techniques to clinical problems as described
in Part B(ii) of this proposal has indicated the desirability of
automating the analysis of the results of a GC/MS experiment. The
SC/MS output involves extracting from the approximately 700 spectra
sollected during a GC run, the 50 or so representing components of
the body fluid sample. The raw spectra are in part contaminated with
background "column bleed" and in part composited with adjacent
constituent spectra unresolved by the GC.

We have begun to develop a solution to this problen with promising
results. By using a disk-oriented matrix transposition algorithm, the
array of 700 spectra by 500 mass samples per Spectrum can be rotated
to gain convenient access to the "mass fraymentogran" form of the

fata. The transposition algorithm avoids many successive passes over
the input data file as would be required in a straightforward approach.
By generating a reorganized intermediate file, time Savings by

factors of 5-10 are achieved.

The fragmentogram forn of the data displayed at a few selected mass
values, has been used at Stanford, MIT, and elsewhere for some time
to evaluate the GC effluent profile as seen from these masses. Mass
fragmeatograms have the important property of displaying higher
resolution in localizing GC effluent constituents. Thus by
transposing the raw data to the mass fragmentogram domain, we can
systematically analyze these data for baselines, peak positions, and
amplitudes, and thus derive better mass spectra for the relatively
few constituent materials. These are free from background
contamination and influences of adjacent GC peaks unresolved in the
overall gas chromatogram. These spectra can then be analyzed by
library search techniques OF first principles as necessal ye

fe have applied a preliminary yersion of this algorithm to several
urine samples. These contain several apparently simple peaks which
in fact consist of multiple components. The algorithm performs well

in separating out these constituents although further testing is
required.

Closed-loop Instrument Control

In the long term, it could be possible for the data interpretation
software to direct the acquisition of data in order to minimize
ambiguities in problem solutions and to optimize system efficiency.
The task of deciding among and collecting various types of mass
spectral information (@ege, high resolution spectra, low ionizing
voltage spectra, or selected metastable ion information) under
closed-loop control during a GC experiment is difficult. Problems
arise because of the large requirements placed on computer resources

° 90
and present limitations in instrument sensitivity or data read-out
imposed by the time constraints of GC erfluent peak widths.

Solutions to these problems may not be economically feasible within
currently existing technology but seen achievable in the future. We
are studying this problem in a manner which would entail a multi (two
or three) - pass system. This permits the collection of one type of
data (e.g., high resolucion mass spectra) during the first GC/MS
analysis. Processing of these data by DENDRAL will reveal what
additional data are necessary on specific GC peaks during a subsequent
GC/MS cun. Such additional data could help to uniquely solve a
structure or at least to reduce the number of candidate structures.
This simulated closed-loop procedure could demonstrate the utility of
DENDRAL type programs to examine data, determine solutions and
propose additional strategies, but will not have the requirement of
operating in real-time. Some parameters in the acquisition of
pacticular types of information, such as metastable data, will
require computer control, even in the open-loop mode.

We have considered plans to implement two aspects of instrument
sontrol, in addition to the magnetic scan control implemented for GC
operation and reported previously. These include system resolution
control, such as would be reguired to change from normal spectrum
scanning mode to metastable scanning mode, and high voltage control
necessary to selectively measure metastable ion fragmentation data.
In addition to these we have considered the discrete switching of
various electronic mode controls which are straightforward ana not
discussed in detail.

Implementation plans for computer control of these instrument
functions have been delayei because of the ACHE computing facility
transition which diverted the necessary hardware and software
manpower.

Resolution control involves changing the widths of the slits at the
exit of the ion source and the entrance to the ion multiplier
detector. Additional source and electrostatic analyzer voltages must
be controlled to optimize performance, as discussed later. Mechanical
slit adjustment is accomplished on the MAT-711 instrument by heating
wires which support the slit jaws. The resulting expansion or
contraction of those wires move the spring-loaded jaws. AS
implemented by the manufacturer, the time constants involved in
heating the control wires are 5-10 seconds. It is possible to speed
this up to approximately 0.5 seconds by application of a controlled
over voltage decreasing to the appropriate equilibrium value for the
desired slit width. This was demonstrated by a series of experiments
on an extra slit assembly mounted in a vacuum jar in our laboratory.
Cooling of the wires is relatively fast in the way they are mounted
so no problem exists in that direction.

It is desirable to have feedback to indicate the actual slit width
achieved rather than relying on a slit assembly calibration.
Stretching of the support wires or changes in the spring tension

under temperature cycling would change this calibration. An

aptical scheme to measure slit width in situ is possible. We do not
contemplate implementing this feedback immediately because it requires
major changes to the instrument flight tube.

Two types of metastable ion relationships are obtainable by
suitable control of the double-focussing instrument. First, for a
given daughter ion, one can trace the parent ions which give rise to

0S F/
it. Second, for a given parent ion one can trace the various
daughters to which it gives rise. fhe first measurement ("metastable
defocussing") is the more straightforward for this instrument since
parent ions can be enumerated by a simple scan of the accelerating
voltage, holding the electrostatic analyzer (ESA) voltage and
magnetic tield constant. The second type of scan requires the
coordinated scan to two of the three fields. We feel that joint
computer control of the accelerating voltage and ESA voltage is the
simpler approach since the magnetic field is more difficult to set
and monitor because of hysteresis effects. For a resolution of 1000
in the metastable ion mass measurement, the voltages must be set to
approximately .01-.02% accuracy. [his requires a 14-16 bit digital-
to-analog (D/A) converter to control the input (10 volts) to the
operational amplifier which generates the high voltage. Similar

D/A controls of ion source voltages for ion current and focus
dptimization can be implemented using optical isolators to allow
verniec control of the various high voltages around the nominal

8KV values.

Computing Transition

As mentioned earlier, the transition of the ACME computing facility
from NIH subsidy to Stanford-sponsored fee-for-service operation has
impacted our development efforts this past year. Both the low
resolution instrument used for routine body fluid analysis research
and the high resolution instrument are affected. All computing
support was previously obtained from the ACME facility, much of it
aS core research without explicit transfer of funds. The transition
has creyuired consideration of both technical and economic factors.
The new facility represents a combination of the previous ACME
interactive and real time computing load with various administrative
and batch computing loads on a new IBM 370/158 computer. This new
environment will have even more difficulty in supporting real tine
computing needs than ACME did. No real time support has been
available since the 360/50 service was discontinued on July 31,
although terminal service was reestablished in mid-August. Data
acquisition service via the IBM 1800 is expected to be operational
by early November.

For the high resolution instrument, this transition, as a mininun,
necessitates an interface modification (we previously sent data
through the IBM 2701 interface no longer to be supported). It also
amplifies the problems we encountered in sending and filing high
cate mass spectrometer data {particularly during GC/MS runs). These
problens would be present to some extent in any general time-Sharing
service machine without specific hardware and software configuration
provision for these needs (such provisions for real time support had
been proposed in our SUMEX computer application).

After examining a variety of alternatives, we conclude that a
dedicated mini-computer solution (built around a machine with the
arithmetic capability of a PDP-11/45) would be highly attractive
technically and relatively inexpensive. A stand-alone mini-computer
systen would cost in the range of $50,000-$60,000, augmenting
existing equipment, plus approximately $9,000 per year maintenance
and $2,000 per year for supplies. Estimates for 370/158 support,
based on current charging alyorithmss and previous utilization
experience, run from $35,000-$50,000 per year. This spread is caused
by uncertainties in the effects of planned measures to increase
operating efficiency and possible changes to the rate structure. In

~ FA
any case, the mini-computer approach pays for itself in 1 to 2 years
of operation and provides the responsiveness Of a dedicated machine
for real time support. Unfortunately our existing budget does not
provide for this solution. The budget is very marginal tor purchase
of computing support from the 370/158 as well. This later approach
is the only currently available one, however, Since it can he
implemented with relatively low start-up Cost. The effect of budget
limitations appears in terms of a reduced number of samples which
can be run. We have attempted to minimize the other budget costs
{manpower principally) to increase the computing funds available.
This will necessarily impact our development goals. We hope, in the
renewal application for DENDRAL support, to be able to implement the
more effective mini-computer approach for the high resolution
spectrometer as a longer term solution.

we have undertaken an interim mini-computer solution for the low
resolution spectrometer (Finnigan 1015 quadrupole) which is primarily
used for our body fluid analysis studies. For the Same reasons outlined
above, a Mini-computer solution is attractive. In the case of the low
resolution quadrupole instrument, a lesser capacity machine will suffice
for immediate data acquisition and display functions. We have
implemented such an interim system on a PDP~11/20 machine available fron
other funding sources. This system, which is now operational, allows
the acquisition of GC/MS data, limited by the capacity of tne DEC tape
storage medium to approximately 600 spectra, per experiment. For
sertain types of GC analyses, up to 1090 spectra per experiment are
required so this limits, to some extent, the utility of this interin
system. A calcomp plotter is supported for display purposes. A fixed
head disk ‘provides for library search procedures which are still being
converted from the ACME system. We have applied to the NIH-GMS for
funds to augment this system in order to relieve current limitations as
part of a Genetics Center research proposal.

FUTURE PLANS

Our future plans are basically to continue development along the lines
outlined above. We will complete the computing support transition steps
described. These include primarily establishing a connection to the new
370/158 facility to provide interim support for the high resolution
system. We will pursue additional software and hardware development
goals as far as possible within the limited budget available. ‘These
afforts will concentrate for the most part on bringing up a metastable
jon analysis data system. It should be reemphasized that the manpower
levels proposed in the follow-on budget have been minimized to allow for
purchasing computing time on the 370/158. The allocated manpower is
ceguired primarily for instrument operation and maintenance with mininal
provision for development efforts.
Part B(ii)- Analysis of Body Fluids by Gas Chromatography/Mass
Spectrometry.

The chemical separation of urine into the following fractions prior
to GC/MS analysis has been described in previous DENDRAL Reports:

free acids (analyzed by gc/ms as their methyl esters)

amino acids (analyzed by gc/ms as their N-trifluoroacetate
n-butyl ester)

carbohydrates (analyzed by gc/ms as their trimethyl silyl ether
derivatives)

hydrolyzed acids (analyzed by gc/ms as their methyl esters)

hydrolyzed amino acids (analyzed by gc/ms as their
N-trifluoroacetate n-butyl esters)

During the past year we have extended these methods of fractionation
to the following body fluids: blood {after an initial precipitation
of proteins by the addition of ethanol) and amniotic and
cerebrospinal fluids. The following summarizes the results obtained
from an analysis of these fluids during the past year by gas
chromatography-mass spectrometry.

URINE ANALYSIS:

A. The Development of a "Metabolic" Profile Characteristic of
Neonatal Tyrosenemia Using Combined Gas Chromatography~Mass
Spectrometry.

This work was carried out in collaboration with clinical colleagues
from the Department of Pediatrics at Stanford University anda joint
publication describing this research is in preparation.

The study was based on a total of one hundred and four 24-hour urine
samples from sixteen premature or small birthweight infants receiving
treatment in the Stanford nursery. After exclusion of infants who
becane ill, died, or left the nursery, we were able to follow nine
infants closely for periods of between 4 and 6 weeks from day 3 of
life. All nine infants had birthweights of below 1500g and three

of these were below 1000g. -

of the nine infants studied, five showed transient tyrosinemia as
shown by a marked elevation in the urinary excretion of the tyrosine
metabolites, p-hydroxyphenyllactic acid, p-hydroxyphenylpyruvic acid
and p-hydroxyphenylacetic acid. There was also a less marked but
distinct elevation in the urinary tyrosine output. Figures 1 and 2
show the metabolic profiles of the same infant {(J.L.) in the
normal({a) and tyrosinemic(b) states. Figure 1 shows the free acid
sutputs, chromatographed as the methyl ester-methyl ether derivatives
and Figure 2 is an expression of the free amino acids of the sane
urines, chromatographed as the N-trifluoroacetyl n-butyl ester
derivatives. In each case the concentration of each metabolite is a
function of the peak height as compared to the height of the internal
standard. Table 1is a summary of the ranges of urinary output of
tyrosine and metabolites observed for all the infants in the study.

TABLE 1 Daily Excretion in mg/kg

Tyrosine p-HPLactic p-HPPyruvic p-HPAcetic
Normal 0.2 - 3 0 - 5 0 - 0.5 0.2 - 2
ryrosinemic 3 - 15 5 - 50 0.5 - 5 0.5 - 5

AS shown by Table 1 and Figure 1 neonatal tyrosinemia is characterized
by a very large increase in the output of p-~hydroxyphenyllactic acid
and by a 10-50 fold excess of the latter over p-hydroxyphenylpyruvic
acid. Studies of the hereditary defects in tyrosine metabolism
initially indicated that p-hydroxyphenylpyruvic acid was the major
metabolite although more recently cases have been reported where
p-hydroxyphenyllactic is in a 2-5 excess over p-hydroxyphenylpyruvic.
These latter determinations were made using GC and GC/MS

methods and therefore probably retlect the improved specificity of
the analytical procedure (previously colormetric methods were used)
rather than a difference of actual metabolic profile. Apart from the
very large excess of p-hydroxyphenyllactic acid over its keto analog
we could detect no significant differences between the profiles shown
in neonatal tyrosinemia and those published for hereditary disease.
Other metabolites such as p-hydroxymandelic acid, DOPA
N-acetyltyrosine, which have previously been reported in tyrosinemic
urine were not seen to be elevated.

Be GC/MS Analysis of Urine from Children Suffering from Leukemia.

This research was carried out with twenty 24-hour urine samples
supplied by Drs. Jordan Wilbur and Tom Long of the Stanford Children's

Hospital.

The acidic fraction of all urines studied in this project showed no
abnormal metabolites nor were gross amounts of known acids detected.
The amino acid fraction, however, of six of the urine samples showed
the presence of an non-protein amino acid, beta-aminoisobutyric acid
{BAIB). In several of these instances the patients were excreting
in excess of 1 gram of BAIB per day. The literature contains many
references to increased BAIB excretion (genetic excretors, jead
poisoning, pulmonary tuberculosis, march hemoglobinuria, thalassaemia
and Down's Syndrome). The reported excretion of BAIB by leukemic
patients was not substantiated by another investigator. There are
several criticisms in the literature of the methods used for the
quantitation of BAIB in biological fluids and in order to fill this
void a sensitive, specific and rapid method for the quantitation of
BAIB has been developed. (SEE: The Quantitation of bAIB in Urine
by Mass Fragmentography; W.E. Pereira, Re Summons, WE. Reynolds,
T.C. Rindfleisch and A.M. Duffield, in press).

C. GC/MS Analysis of Urine from Patients Suffering fron Hodgkin's
Disease.

During this study 20 urine samples from patients with diagnosed
Hodgkin's Disease (Department of Oncology, Stanford University
Medical Center) were analyzed and in general, no abnormal metabolic
profile could be found in any urine. There was one exception in
which an individual was noted to excrete massive quantities of
adipic acid (of the order of 1 gram per day).

D. Detection of Metabolic Errors by GC/MS Analysis of Body Fluids.
This project results from a collaborative effort between the Departments

of Genetics and Pediatrics of the Stanford University Medical Center.
To date over 50 samples have been analyzed; the majority {35) being

7 9S
urine, while amniotic fluid (10), hlood (6), and cerebrospinal fluid (6)
were also analyzed. It has been and will continue to be our practice to
analyze aliquots of fluid samples in collaboration with clinical
investigators obtained for valid diagnostic purposes completely divorced
from this research on GC/"S analysis techniques. This investigation is
not intended to serve as a screening program for a large population but
rather to focus on those individuals who exhibit suggestive clinical
manifestations such as psychomotor retardation and progressive
neurologic disease as well as suggestive pedigrees.

In the case of amniotic fluid the hope is to be able to monitor the
condition of the fetus in those pregnancies which might be considered
at rist. To date we have investigated specimens from normal
pregnancies in order to establish the catalog of compounds to be
observed in amniotic fluid. From this base it could prove possible
to identify materials which might identify the health of the fetus.

We have been able to confirm the presence of orotic acid in a urine
from a person found to have orotic aciduria while another urine
sample was used to demonstrate our ability to identify the
characteristic metabolites present in isovaleric acidemia. The
following description refers to a urine froma child with
hypophosphatasia.

A child died 33 hours after birth in Fresno, California, with the
classical signs of hypophosphatasia. This genetic defect is marked
by high phosphoethanolamine (PEA) concentrations in urine of

affected honozygotes and unaffected heterozygotes. Atter
derivatization (in this instance the TMS ethers of the water

soluble carbohydrate fraction were prepared) we were able to detect
by GC/MS large concentrations of ethanolamine and phosphoric acid

but not PEA itself. The derivatization procedure we used most likely
hydrolyzed PEA. We were able to quantitate for this compound in the
infant's urine using an amino acid analyzer, and PEA excretion was
extremely high {over 200 times normal values for infants) confirming
the diagnosis. Next we examined urine samples from the child's
parents, presumed heterozygotes, by GC/MS and by the amino acid
analyzer. Again, no PEA was detected by the former method although
the presence of ethanolamine and phosphoric acid was demonstrated.
we determined the following excretion levels of PzA by amino acid

analyzers

Newborn infant: 94 micromoles per 100 ml. (Normal 0.21-0. 33)
Father: 269 micromoles per 24 hours (normal 17-99)
Mother: 32 micromoles per 24 hours (normal 17-99)

It is of interest that in this family the affected infant and his
unaffected father both show subnormal serum alkaline phosphatase
activity. The mother, who did not excrete increased amounts of PEA,
was found to have normal activity of this enzyme in her serum. The
following table summarizes the serum phosphatase activity measurements:

Newborn infant: 0.2 units*® (normal 2.8-6.7)

Father: 0.7 units {normal 0.8-2.3)
Nother: 3.4 units (normal 0.8-2. 3)
(* - 1 unit is that phosphatase activity which will liberate 1

millimole of p-nitrophenol per hour per liter of serum)

Sot

76
E. Drug Analysis Service Using GC/HS

We were recently contacted by physicians to rapidly identify a drug
self-administered by a patient in the Stanford University Hospital.
From the mass spectrum the drug was identified as pentazocaine
Within the hour. Although not part of the formal DENDRAL proposal
we expect that Similar cases may arise in the future and we intend
to respond positively to such requests.

Development of Library Search Routines for Mass Spectrum Identification

The analysis of a single body fluid fraction produces between 600 and
750 mass spectra. In order to cope with the interpretation of the
daily production of mass specta (about 8 body fluid fractions for a
total 2f between 4,800 and 6,000 mass spectra) we have begun the
implementation of library search routines. Concurrent with the
analysis of body fluids for metabolic content we have been recording
the mass spectra of many reference compounds. This collection
represents the beginning of the construction of a library of
reference spectra. Late in 1973 we expect to receive from Dr. 5S. Markey,
University of Colorado Medical Center, a more comprehensive library
which he has collated from contribrtors (including our own laboratory)
in the field of biological applications of gas chromatography/mass
spectrometry.

Prior to the demise of the ACME computer faciliity at Stanford
University, we ran library search routines on data collected fron
urine Fractions. Because of the ACME system being heavily loaded,
our programs took about one minute per compound identification.
However, the experience gained will be used to implement library
Search routines on our current PDP-11 GC/MS data system. In addition
we have sent mass spectra fron several urine analyses to Dre. 5- Grotch,
Jet Propulsion Laboratory, Pasadena, California, in order that he
‘could use his library search routines on real data. In this instance
the limiting factor for efficient compound identification was the
library content which was limited to a few compounds of biological
significance. In addition those compounds of interest that were
present in the library were often in a derivatized forn ditferent
from that used in our analytical methodology.

Application of GC/HRMS to Body Pluid Analysis

We reported in the Last annual report of the DENDRAL project that
the Varian MAT 711 mass spectrometer was interfaced with a gas
chromatograph for the recording of low resolution mass spectra. We
have now used this system tor the recording of HRMS of gas
chromatographic rractions from urine analyses. We were able to
record HRMS scans over several gas chromatographic peaks of interest
in a number ot urine fractions. The high resolution results were
found to be of a high quality in mass measurement accuracy. When
using the MAT 711 instrument for GC/HRMS the sensitivity of the ion
source was a limiting factor in that less intense gas chromatographic
peaks often lacked sufficient material to generate acceptable high
resolution mass spectra. Notwithstanding this limitation the HRMS
data recorded on different urine fractions was used to confirm the
identification of several netabolites. If hy chance the metabolite
of concern was available only in quantities insufficient for direct
GC/HRMS, preparative GC would be used to concentrate the component
of interest for subsequent HRMS.

C7

77
RESOURTE OPERATION

Over the term of this grant our mass Spectrometry laboratories have
provided support to numerous research projects in addition to the
DENDRAL computer program development project funded under this
grant. These cover a variety of applications at Stanford, in the
United States, and abroad. Included are problems in the study of
human netabolites, biochemistry, and natural product chemistry.
Samples have been run in collaboration with outside people both on
the MAT-711 GC/High Resolution Mass Spectrometer system and the
Finnigan 1015 GC/Low Resolution Quadrupole Mass Spectrometer system.
The low resolution system has also been supported by a NASA research
grant.

The following tables summarize the support rendered in terms of
humbers of Samples run through various types of analysis:

I. MAT-711 High Resolution System (Period covered 11/71 - 6/73).

 

Batch Batch GC/High GC/Low
High Resol. Low Resol. Resol. kesol.
KS MS MS MS
DENDRAL program devel. 317 3
Stanford Genetics (Body
fluid analysis) 39 7 13
Stanford Chemistry {non-
DENDRAL - Dr. Djerassi's
group) 91 112 50
Stanford Chemistry (non-
DENDRAL - Drs. Vantamelen,
Johnson, Mosher, Collman,
Altman, Goldstein) 29 23 y
Stanford Surgery {Dr. Fair) 8
Dr. Adlerkreutz (Finland) 10
De. Venien {France} 26
Dr. Gilbert, Mors, Baker {Brazil} 40 Qy
Dr. Orazi (Argentina) 19 1
Dr. Subramanian (India) 10 5
Dre Khastgirc (India) 5
Dr. O'Sullivan (Ireland) 5
De. Badr (Libya) 30
Dr. Mital (India) 5

oO q &
624
samples

IT)

Note the samples run are specified by fluid type.
extracted and derivatized as described in Part B (ii)
Specific discussions of the

may cepresent several GC/LRS analyses.

215

Samples

13 54
samples sanuples

FINNIGAN 1015 Low Resolution System (period covered 8/72-8/73)

Each fluid is
and therefore

results of various of the analyses run are discussed earlier in

Part B(ii).

Stanford Pediatrics (Drs. Cann, Sunshine

and Johnson)

Stanford Oncology (Dr. Rosenberg)

Stanford Psychiatry - Genetics
{Drs. Brodie and Cavalli-Sforza)

Stanford Respiratory Medicine (Dr. Robin)

Stanford Pharmacology (Dr. Kalman)

Stanford Biochemistry (Dr. Stark)
Stanford Children's Hospital
(Drs. Wilbur and Long)

uc San Francisco Medical School -
Dermatology (Dr. Banda)

Menlo Park V.A. Hospital (Dr. Forrest)

Palo Alto V.A. Hollister

and Green)

Hospital {Drs.

University of Puerto Rico School of
Medicine (Dr. Garcia-Castro)

GC/Low
Resolution MS

24

13

urines

Amniotic Fluids
bloods

cerebrospinal fluids

urines

cerebrospinal fluids
urines

bloods

extracts

extracts
urines

urines

extracts
extracts

urines

 

243

samples

77
PART B PUBLICATIONS

fhe following summarizes the publications resulting fron research in
the low resolution mass spectrometry laboratory over the past year,
including body fluid analysis. This laboratory has been jointly
supported by NIH (DENDKAL) and NASA. ‘The listed publications include
research relevant to both Sponsors.

The Determination of Phenylalanine in Serum by Mass Fragmentography.
Clinical Biochem., 6 (1973)
By WE. Pereira, V.A. Bacon, Y. Hoyano, R. Summons and A.M. Duffield.

The Simultaneous Quantitation of Ten Amino Acids in Soil Extracts by
Mass Fragmentography

Anal. Biochem., 55, 236 (1973)

By. W.eE. Pereira, Y. Hoyano, W.E. Reynolds, R-~E. Summons and

A.M. Duffield.

An Analysis of Twelve Amino Acids in Biological Fluids by Mass
Fragmentography.
Anal. Chem.,
By RE. Summons, W.E- Pereira, WeE. Reynolds, T.C. Rindfleisch ahd
A.M. Duffield.

The Quantitation of B-Amino isobutyric Acid in Urine by Mass
Fragmentography.
Clin. Chim. Acta, in press
By #.E. Pereira, K.E. Summons, aaE. Reynolds, T.-C. Rindfleisch and
AM. Duffield.

The Determination of Ethanol in Blood and Urine by Mass Fragmentography.

Clin. Chim. Acta
By W.E. Pereira, R.E. Summons, T.C. Rindfleisch and A.M. Duffield.

A Study of the Electron Impact Pragmentation of Promazine Sulphoxide
and Promazine using Specifically Deuterated Analogues.

Austral. J- Cheme, 26, 325 (1573)
By M.D. Solomon, R.~ Summons, l. Pereira and A.M. Duffield.

Mass Spectrometry in Structural and Stereochemical Problems. CCXXXVII.
Electron Impact Induced Hydrogen Losses and Migrations in Some
Acomatic Amides

Org. Mass Spectry., in press.

By A.M. Duffield, G. DeMNartino and C. Djerassi.

Spectrometrie de Masse. IX. Fragmentations Induites par Impact
Electronique de Glycols- -En Serie Tetraline
Bull Soc. Chim. France, 2105 (1973).

Spectrometric de Masse VIII. Elimination d'can Induite par Impact
Electronique dans Le Tetrahydro-1,2,3,4-Napthtal-ene-diol-1,2.
Org. Mass Spectre., 7, 357 (1973).
By P. Perros, J.P. Morizur, J. Kossanyi and A.M. Duffield.

Shlorination Studies I. The Reaction of Aqueous Hy pochlorous Acid
With Cytosine.
Biochem. Biophys. Res. Commun., 48, 880 (1972)
By W. Patton, V. Bacon, A.M. Duffield, B. Halpern, Y. Hoyano,
We Pereira and J. Lederberg.

“= /00