Download Routine Use of a Flexible Gas Chromatograph

CLIN. CHEM. 20/2,
255-265
(1974)
Routine Use of a Flexible Gas Chromatograph-Mass
Spectrometer-Computer System to Identify Drugs and Their
Metabolites in Body Fluids of Overdose Victims
C. E. Costello,
H. S. Hertz, T. Sakai, and K. Biemann
We have assembled a Computer-searchable
collecspectra of drugs, metabolites,
normal body-fluid
constituents,
and common
sample
contaminants,
for use in conjunction
with a gas
chromatograph-low
resolution
mass spectrometer
system. The system is designed for analysis of drugs
in body fluids of comatose
victims of accidental
or
deliberate
ingestion of a drug overdose.
The speed
and generality
of the method make it particularly
useful for such emergency
situations.
It has now
been in routine use for longer than two years, and its
usefulness
is illustrated by results obtained for several actual samples.
This goal has been achieved
in the intervening
time and the results have been presented periodically in preliminary
form.2 The present paper represents a detailed report and discussion of the results
of about two years of use of the technique
for some
600 patients.
In many cases a drug ingested in an overdose can
easily be identified
by rather simple, conventional
techniques
such as color reactions,
thin-layer
chromatography,
gas chromatography,
or ultraviolet
spectroscopy,
alone or in combination.
Very often
there is some direct or circumstantial
evidence
avail-
Additional Keyphrases: emergency
ed, which then merely needs to be confirmed in the
laboratory.
In many instances,
however, there is no
information
available;
or a number of drugs have
been ingested, which may mutually interfere in the
reliable identification;
or the specimen may contain
tion of 300 mass
able
toxicology
#{149}library
#{149} rapid
identification
of drugs and
of mass spectra
drug metabolites
An earlier
paper
(1) described
chromatograph-mass
to rapidly
the
use
of a gas
spectrometer-computer
system
the toxic agent-a
large dose of
the body fluids of a patient who had
identify
“Darvon”1-in
attempted
suicide.
Although
the
instrumentation
and techniques
(2, 3) used had been developed
for
quite different problems in chemical research, they
served well in that emergency.
In that case the resulting mass spectra had to be
interpreted
from
basic
principles,
and
it was
imme-
diately obvious, as stated then, that the technique
could be highly automated
and accelerated
if a collection of the mass spectra of commonly used drugs
and related substances
were available for immediate
automatic
comparison
gas-chromatographic
recorded
in real time
with
fractions,
the
mass
which
by a computer.
spectra
were
of the
already
concerning
the
only metabolized
a compound
nature
drug;
that
of the
or, finally,
compound
one may
is not a common
as is often the case with illicit
require
a more sophisticated
ingest-
drugs.
deal with
medicinal
Such
drug,
situations
analytical
technique;
the cases dealt with in our laboratory belong to these
categories exclusively.
It is, of course, desirable to use an approach that
not only identifies any one of a predetermined
set of
compounds,
if present, but also reveals the identity
of all the other components
that are extracted from
the original specimen. This not only assures the investigator
that no potentially
harmful
component
has been overlooked
but also leads to detection
identification
of unexpected
compounds,
be they
drugs (particularly
illicit ones), new or known
and
new
me-
The availability
of such a system would not only greatly shorten the
time required
to identify these substances
but it
would also reduce the need for involvement
of personnel intimately
acquainted
and experienced
with
the interpretation
of mass spectra of organic compounds.
Department
of Chemistry,
Massachusetts
Institute
of Technology, Cambridge,
Mass. 02139.
1 Darvon
is the trade name for 4-dimethylamino-3-methyl-1,2diphenyl-2-butanol
proprionate hydrochloride
(Eli Lilly, Inc., Indianapolis,
md.).
Received Nov. 30, 1973; accepted
Dec. 2, 1973.
2
()
Biemann,
K., Biller,
J.
E., Costello,
C. E.,
and
Sakai,
T.,
The potential
of GC-MS-computer
systems
in biomedical
and
clinical
research,
Pittsburgh
Conf. on Anal. Chem.
and Applied
Spectroscopy,
Cleveland,
Ohio, March
1972. (b) Costello,
C. E.,
Sakai,
T., and Biemann,
K., Identification
of drugs
in body
fluids,
particularly
in emergency cases of acute poisoning. 20th
Annu.
Conf. on Mass Spectrometry
and Allied Topics, Dallas,
Texas, June 1972. (c) Costello,
C. E., and Biemann,
K., On call
for poisonings:
The mass spectrometry
facility as adjunct to the
emergency room. Pittsburgh
Conf. on Analytical
Chemistry. and
Applied Spectroscopy, Cleveland, Ohio, March 1973. (d) Biemann, K., Costello,
C. E., and Andresen,
B. D., Mass spectrometnc identification
of drugs and their metabolites
in body fluids.
166th National
Meeting,
American Chemical Society, Chicago,
Ill., August 1973.
CLINICAL CHEMISTRY, Vol. 20, No. 2, 1974
255
tabolites that
help in the identification
or confirmation of the original
drug, or even artifacts
and contaminants
introduced
into
the sample
before
it
reached
the laboratory.
The identifications
of all
these
for
materials
the
represent
continuous
analysis,
an important
improvement
because
once
they
prerequisite
of the
are
recognized
their
sponsive
to
human
analysis
intervention,
that is re-
supervision,
and
improvement-is
provided
by designing
the data
processing along two parallel lines: (a) the continuous mass-spectrometric
matogram is compared,
with
the
authentic
record of the entire gas chroone spectrum
after another,
data
tion of all components
for the
automatic
of which
identifica-
authentic
spectra
are
available and can be successfully matched; (b) at the
same time, the data are also processed in the normal
manner,
well
i.e., presented
as individual
as individual
mass
mass
chromatograms
spectra
(plots
as
of in-
m/e values
over the entire
gas chromatogram) (4) and reproduced
on 16-mm microfilm
for
individual
inspection
by the investigator.3
This process is the most convenient,
practical
way of surveying and making detailed
interpretation
of such a set
dividual
of
data.
Automatic
searching
simple and reliable
are
available,
techniques
are
very
if mass spectra
of high quality
the extract
of a blood or urine
but
specimen
represents
a complex mixture.
Because
these are emergency situations,
there is no time to
separate the extract into various sub-fractions
and to
optimize
choice
pound
the
gas
chromatographic
separation
of varied
column
materials
types
or by derivatization
be “contaminated”
This
a problem
poses
for different
comof the materials
to
terpretation,
and
with other materials.
in effecting
various
means
the
automatic
to remedy
are represented
handled
to date
Sample-Handling Procedures
the
Gastric
body
contents,
fluids
clotted
usually
blood,
supplied
and (or) urine are
by the hospital,
although spinal fluid and bile have also been used.
Quantities
available
vary, but the recommended
amounts
are 10 ml of gastric contents
or urine or 5
ml of blood. Much smaller quantities
can be used
successfully
when necessary,
but less conveniently.
The extraction
procedure
is illustrated
in Scheme
1. Methylene
chloride
was chosen because of its good
solvent
property
for most drugs;
its availability
in
high purity at a reasonable
cost (“Nanograde,”
Mallinckrodt
Chemical
Works, St. Louis, Mo. 63160); its
simple, unconfusing
mass spectrum;
and its low boil-
ing point,
tract
which
without
facilitates
losses.
concentration
Solid
sodium
of the ex-
bicarbonate
sure
that
the more
basic
drugs
(amphetamines,
sodium
orator.
sulfate, and concentrated
with a rotary evapThe water bath used during
evaporation
is
kept at or slightly below ambient temperature
and
the time for this step is kept as short as possible, to
prevent loss of volatile drugs such as ethchlorvynol
or amphetamines.
The
of methylene
chloride.
subjected
computer
residue is dissolved
in 100 /41
A 2- to 5-/4l portion
is then
to gas chromatograph-mass
analysis
(Scheme
Gastric
contents
(10 ml)
2).
filter
Blood (5 ml)
or
\ centrifuge
spectrometerCo-injection
Urine
centrifuge
this
(10 ml)
ntrifuge
extract
with
5 vol CH2C12
will
data to permit the investigator
to either verify
the results of the computer interpretation
or inspect
and interpret the mass spectra of fractions that may
add solid NaHCO3
to aqueous
phra8e
not
re-extract
with
5 vol CH2C12
automatic
proce-
dures.
Materials
The
hospitals
and Methods
body
fluids
affiliated
investigated
with
the
were
Boston
obtained
Poison
combine
dry with
from
3Biller,
J. E., Hertz,
H. S., and Biemann,
K., The application
of a GC-MS-computen
system
and various
interpretive
programs
to problems
in natural
products
chemistry.
19th Annu. Conf. on
Mass Spectrometry
and Allied Topics, Atlanta,
Ga., May 1971.
CLINICAL
CHEMISTRY.
Vol. 20, No.2,
1974
CH2C12 fractions,
Na2SO4
Informa-
tion Center at the Children’s Hospital Medical Center. Only a few hospitals submit most of the samples,
256
of a
in-
all the
by the
mor-
phine) are removed from the aqueous layer. The organic extracts are combined,
dried over anhydrous
discussed.
It also makes
it even more important
to provide
the fast and efficient
means
of processing
characterized
(5 g,
ACS Certified, Fisher Scientific Co., Pittsburgh,
Pa.
15219) is added before the second extraction, to en-
be
be fully
among
by this
by
be separated
(5). Thus,
ideal
gas-chromatographic
separation
is rarely achieved,
and the system has to
be able to cope with mass spectra
that may to vari-
ous degrees
30 hospitals
600 cases
laboratory.
automatic
spectra
are added to the computer-searchable
collection. From then on they will be automatically
identified whenever
they are present
in a specimen.
One
may say the system “learns.”
This flexibility-an
automated
but more than
the approximately
Concentrate
CH2C12 solution
100
/41
extract
Scheme
1
2-5
of solution
1/41
p1 extract
of standard
Gas chromatrograph
Mass
Mass
hydrocarbons
To facilitate
ranged
455
-
4-Sec scan cycle
Total ion plot
Barplots
of spectra
Mass chromatromatograms
Library
search
and
spectrometer
is
manner
then
can be evaporated
which
is inserted
via a
the ion source
of the mass
slowly
continuously
as during
it possible
(6).
the presence
of a drug
behavior
(e.g., aspi-
2-pl portion
sample-holder,
directly
into
spectrometer
makes
vaporized
scanned
while
in
the
the gas-chromatographic
the
same
run.
Instrumentation
The
puter
gas chromatograph-mass
system
used
in this
spectrometer-com-
work
consists
of a Model
990 gas chromatograph
(Perkin-Elmer
Corp.,
walk, Conn. 06852), a Hitachi
RMU 6L mass
trometer
(Perkin-Elmer
Corp.),
and
an
Nor-
specIBM
1800
Corp., Ar-
data acquisition
and control system (IBM
monk,
N. Y. 10504). The computer
is configured
with 32 768 words of core memory,
six IBM 2315
magnetic
disk packs (capacity:
512 000 16-bit words
each), and two IBM 2401 magnetic
tape drives.
The
gas chromatograph
is coupled
to the
mass
spectrometer
by a low-volume
splitter
linked to both
a flame ionization
detector
and a porous fritted-glass
tube pressure-reduction
unit (7) by a heated
stainless-steel
capillary
and valve. The exit of the pressure-reduction
unit
is connected
leads into the ionization
trometer.
This
configuration,
ber of research
more powerful
to a glass
chamber
which
was
line
that
of the mass specdesigned
available.
lection was distributed
in 1972 and is now
indices
of hydrocarbons
of pertinent
in computer-searchable
they became
2
to determine
retention
indices
If there is reason to suspect
with poor gas-chromatographic
a second
a collec-
tained from two other laboratories
(Baylor College of
Medicine
and the National
Heart
and Lung Institute, through
the courtesy
of Dr. Marjorie
Horning
and Dr. Henry Fales, respectively)
were added when
m/e 28
-
Scheme
onto
a glass
vacuum
lock
compiled
MS
Identification
rin, LSD),
we have
of mass spectra
drugs and
their metabolites.
In addition,
the mass spectra
of
abundant
normal
constituents
of human
body fluids
and the mass spectra
of commonly
occurring
contaminants
were included.
This “library”
was ar-
for a num-
applications,
is more elaborate
and
than that needed
for the work de-
format.
Spectra
(The first version
ob-
of the col-
to the biomedical
community
also being used in commercial
are
unambiguously identified
in actual
extracts,
or are
reported
in the literature,
their
spectra
are also
added to the collection.
The 304 spectra
in the collection at this time include
240 drugs,
24 metabolites, 18 normal constituents
(e.g., cholesterol,
dimethyl
sulfone,
piperidone,
fatty acids), 19 artifacts
(e.g., phthalates,
adipates,
and other
plasticizers,
and antioxidants
such as 2,6-di-t-butyl-4-methylphenol or “lonol”)
and three other harmful
materials
(e.g., pinene, the major constituent
of turpentine).
When a series of similar
non-drug-related
materials is likely
to be encountered,
only one typical
member
of the set is added to the collection,
which
systems.)
mixture
analyses,
tion (library)
Retention
standard
Library
GC on 150 cm X 3 mm
column
3% OV-17 on
100/200 mesh GC-Q
12 or 16 #{176}C/min
from
80 to 330 #{176}C
spectrometer
Computer
Spectra
As
additional
drugs
identifies
such materials
from
consideration
as
or
metabolites
sufficiently
potential
to exclude
them
causative
agents
without
adding unnecessarily
large numbers
of nondrug spectra
to the collection.
For example,
the
entry “BHA-type
antioxidant,”
which appears
in the
search output
in Figure 7, is of this type and covers
the class of butylated
hydroxyanisole
food additives,
all of which produce
mass spectra
having
m/e 135
and 197 as the most abundant
ions. Only one hydro-
carbon
spectrum,
collection and
that of n-C24H50, is included
it is labeled
as n-alkane
in the
(Figures
2
and 3).
Computer
Comparison Algorithm
(TPLI B)
The computer-searchable
collection (“library”)
of
drug mass spectra
is stored on magnetic
tape. The
routine
TPLIB
(tape library) is designed for library
generation,
(i.e.,
library
addition
searching,
of new
The user states
to the
the mode of operation
execution
of the program.
Each compound,
with
data, occupies
one record
cluded
in the
its abbreviated
and library
spectra
record
mass
updating
existing
tape).
desired
upon
its essential
spectrometric
on the magnetic
tape. In-
is the name
of the compound,
spectrum,
the most
abundant
scribed
in this
paper;
most
of the procedures
and
programs
we describe
here can be adapted for use
with
more
limited
systems
such
as those
now also
peak, to which all other peaks are normalized
(“base
peak”),
the number
of peaks
in the abbreviated
commercially
ments.
of time
ed system
available.
or flexibility
provides
Some compromises
are necessary,
much
useful
data
but even
(8).
in terms
a limit-
spectrum,
and the five highest
The
description
are published
rectangular-array
details
of these
concepts
of the
spectrum-comparison
and
ele-
a detailed
algorithm
(9), and will only be summarized.
CLINICAL CHEMISTRY, Vol. 20, No. 2, 1974
257
The abbreviated
mass spectrum
contains
the two
most intense
peaks
in each 14 mass-unit
interval
(i.e., the intervals
6-19, 20-33, 34-47, etc.). Use of
the abbreviated
spectrum
allows one to retain
the
significant
details
of the full mass spectrum,
while
minimizing
the size of the searchable
data base. The
elements of the rectangular
array are obtained
by
summing
the intensities
of the homologous
ions at
m/el+14n,2+14n,...14+14n,wheren0,1,
2....
These
fourteen
sums
are then arranged
in decreasing
order and the homologous
series represented
by the five highest sums are stored on tape.
Comparison
of the unknown
and known
mass
spectra
is accomplished
by the computer
in two
parts:
(a) the pre-search
and (b) the detailed
comparison of abbreviated
mass spectra.
The pre-search
is performed
to eliminate
obviously
dissimilar
spectra, for which
a detailed
comparison
would
be a
waste of time. Basically
the pre-search
requires that:
the largest peak in the known spectrum
be at least
25% relative
intensity
in the unknown
spectrum;
the
mass range
covered
by the known
and unknown
spectra
differ by not more than a factor of approximately
three
(calculated
by using the number
of
peaks in the abbreviated
spectrum);
and the total
abundance
of homologous
series of ions be similar in
the known and unknown
spectra
(using the rectangular array elements).
For each known spectrum
that satisfies
the three
successivepre-search
requirements,
a detailed
comparison
with the unknown
spectrum
is performed.
The result
of this detailed
comparison
is a “similari-
ty index,”
which
ranges
between
0.000 (complete
mismatch)
and 1.000 (perfect
match).
Essentially,
the similarity
index is the weighted
ratio of the intensities
in the known abbreviated
spectrum
to those
in the unknown
abbreviated
spectrum
taken
mass
for mass.
Any or all of the pre-searches
can be bypassed
when it seems preferable
to do so. For example,
if a
spectrum
has a high background
because
of incomplete gas-chromatographic
separation,
the rectangular array pre-search
could remove from consideration
reference
spectra
that are actually
quite similar
to
the unknown.
Alternatively,
removing
the base peak
or reverse base peak requirements
permits
similarity
index calculations,
to help identify
metabolites
that
still
have
much
of the
structure
of the
parent
drug
but whose spectra
exhibit
a very significant
shift in
the base peak. For example,
demethylation
or dealkylation
of the amino side chain of tricyclic
drugs
converts
these drugs to metabolites
that retain the
ring structure
and hence give rise to mass spectra
similar
in some respects
to that of the parent drug,
but for which the base peak derived from the amine
moiety
is shifted
to a lower m/e value.
Because
removal of pre-searches
results in the computation
of
many more similarity
indices,
and a longer search
time, the pre-searches
are retained
unless preliminary results
indicate
that such manipulation
of the
search procedure
would yield additional
information.
258
CLINICAL
CHEMISTRY,
Vol. 20, No.2,
1974
Data
Acquisition
The
operating
and Interpretation
Procedure
conditions
gas
of the
chromato-
graph-mass
spectrometer
system
for the analysis
of
the body fluid extracts
are shown above in Scheme 2.
Mass-spectrometric
data for the complete
gas chromatogram
are accumulated
by the computer
and
peak centers are calculated
in real time. Perfluoroalkane is introduced
from a reservoir
before each gas
chromatogram
as calibration
for time-to-mass
conversion.
After the run is completed,
the summed
intensities
of each consecutive
spectrum
are plotted
and the spectra
are processed
(2, 3).
This corresponds essentially
to a conventional
total ionization
plot (see Figure 1) and represents
a computer-generated gas chromatogram,
with the numbers
along the
abscissa
corresponding
to the spectrum
index numbers of the individual
spectra
taken
during
the
course
of the gas chromatogram.
Mass chromatograms (4) are calculated
for each m/e value and the
oscilloscope
display of each mass spectrum
and mass
chromatogram
is microfilmed
during
processing.3
The
mass
chromatogram,
of one specified
number,
can be thought
recorded
with a detector
mass value.
tensity
The processed
data
a plot
of unnormalized
in-
mass vs. spectrum
index
of as a gas chromatogram
that is specific for a single
are automatically
written
on
magnetic
tape, and are thus available
for comparison
with the drug library a very short time after data acquisition
is completed.
When TPLIB
is executed
for
searching
purposes,
the spectrum
collection
is transferred
to a temporary
file on disk for use during
searching.
(A disk file can be manipulated
much
more rapidly
than a tape file.) At this point, two alternatives
are available
to the user: individual
spectra can be compared
to the collection,
or the whole
GC-MS
run can be compared
to the collection.
In
the latter
case, the most probable
compound
for
each unknown
spectrum
is printed,
together
with its
similarity
index, along the contours
of the total ionization plot for the GC-MS run (see Figure 2). In addition, at the end of the search,
the 10 most abundant compounds
thus identified
are printed
out in
order of decreasing
abundance
(see Figure 3). Thus,
this results in a rapid, almost fully automatic
analysis of the sample
for a wide variety
of toxic substances.
In the few cases where the coma may be
caused
by a drug in so low a concentration
in the
body fluid as to be obscured
by normal constituents,
it is still often possible to detect the presence
of such
a foreign
material
by visual
examination
of the
filmed
mass chromatograms.
These
clearly
resolve
even minor peaks, as is illustrated
below (see discussion of Figure 5).
Results
The disposition
of Case 1 is typical
of the majority
of samples
handled
by this approach.
When a deeply
comatose
24-year-old
man was brought
to Massachusetts General Hospital
after first being admitted
to a
BLUIJO
595 O-l
TOTP,L IONIZMIC4
9PE(CTR :
MOST
PLOT
P1)4
O.1887E
0.5650E
O.4161E
O.2895E
O.1217E
O.4132E
O.3090E
j9Q
DUX
Fig.
tract
1. Total
ionization
from Case 1
plot
for
08
08
07
07
07
06
06
COMPOUNDS
MEPERIDINE
N-ALKANE
CHOLESTEROL
ME TH AQUA LO NE
DIAZEPAM
OLEIC
ACID
CHOLESTA-3.5-DIENE
Fig. 3. Summary
of library search results for extract of
blood sample, Case 1, for which Figure
1 is the total ionization plot
i
iU
PROBABLE
2389
M
GC/MS
run
of
blood
ex-
Fig. 2. Library
search
output
in profile
format
for the experiment
for which
the total
ionization
plot is shown
in
Figure 1.
To avoid reduction to the point of illegibility, only the section from scan 75
to scan 130 is shown
suburban
hospital,
a blood specimen
was sent to our
laboratory
for analysis.
Little history
was available;
there was some possibility
of meperidine
ingestion,
but other information
had indicated
the absence
of
meperidine
but the possible
presence
of amitriptylene. The patient’s
condition
was not consistent
with
this result and it was suspected
that he had ingested
a combination
of drugs. In our laboratory,
5 ml blood
serum was extracted
as shown in Scheme
1 and the
GC-MS data acquired
and processed
as in Scheme 2.
The total ionization
plot for this sample
is shown in
Figure
1. A section
of the library
output
along the
contours
of the total ion plot appears
in Figure
2.
This output
format consists of an entry for the result
of the library search for each individual
spectrum
in
the run. The scan number
is printed
together
with
the similarity
index found for the most similar spectrum in the reference
collection.
The name of the
matched
compound
also appears,
displaced
by a distance indicative
of the total ionization
in that spectrum. Here the most intense
spectrum
was that recorded in spectrum
number
80, which was identified
as meperidine.
The summary
of the library
search for the entire
run is shown in Figure 3. This was printed
after the
profile output
was completed,
and it lists the most
abundant
compounds
found
in the spectrum-byspectrum
search,
together
with an exponential
expression of their total ionization
in the entire run. In
this instance,
the names of significance
were meperidine, methaqualone,
and diazepam,
since they indicated ingested drugs-cholesterol
and oleic acid are
normal
blood constituents
and cholestadiene
is an
artifact
produced
by dehydration
of cholesterol
on
the injector
port of the gas chromatograph.
The nalkane (C26H54) reference
spectrum
was found most
similar
to each of the three hydrocarbon-retentionindex standards
(C14H30, C22H54, C32H66) added to
the sample,
since only one such compound
is included in the library.
It was thus apparent
that the patient
had ingested
a very large amount
of meperidine
together
with
lesser amounts
of both methaqualone
and diazepam.
There was no indication
that any other drugs,
including
amitriptylene,
were present.
In this situation, as in most cases, no further
manipulation
of the
data was necessary
and the results were reported
immediately
to the hospital,
less than an hour after the
sample had been received.
Cases 2 and 3 are among the less-common
types of
samples,
but are included
because they illustrate
the
flexibility
of the system and its potential
for rapidly
dealing
with samples
that pose difficulties
by containing
uncommon
or unexpected
drugs,
contaminants
unrelated
to drugs,
and extremely
complex
mixtures.
The urine specimen
submitted
for Case 2, a young
child afflicted
with severe hallucinations,
arrived in a
plastic
tube. The gas chromatogram
obtained
with
an extract
of this sample
appeared
to be devoid of
drug-related
peaks: the peaks observed
correlated
in
both retention
time and mass spectra
with the common plasticizers
dioctyl adipate
and several isomers
of dioctyl phthalate..
Upon closer examination
it was
noted,
however,
that spectrum
348, Figure 4a, contained
ions in addition
to those caused
by dioctyl
phthalate.
Subtraction
of spectrum
357, which also
contained
the ions characteristic
of dioctyl phthalate
but lacked the additional
ions observed
in spectrum
348, gave a spectrum
of a component
that was centered at spectrum
348, as indicated
by the maximum
observed
in the mass chromatogram
of m/e 287, Figure 5. Computer
comparison
of this “cleaned”
spectrum (Figure 4b) with the drug library gave a close
fit for the anti-allergy
drug cyproheptadine
(I), and
CLINICAL
CHEMISTRY,
Vol. 20, No.2,1974
259
LPThS
94,
DSC
2
972
976--348
0
#{149}
La I
r#{149}’
.1LL
i’r
pe
e
.ri#{149}’1
visual comparison
of this spectrum
with the reference spectrum
(Figure 4c) confirmed
the identity
of
the unknown
with this drug. The presence
of cyproheptadine
had not been suspected
and it is doubtful
whether
the conventional
methods
of analysis
would
have detected
it at all, because
most methods
are
limited
to recognition
of a compound
or group of
compounds
whose presence
is anticipated
and they
are consequently
unable to detect or to identify compounds outside their limited range of application.
An
excessive
dose of cyproheptadine
would result in a
state such as that observed
for the child and its
identification
in this urine extract
satisfactorily
explained the child’s condition.
,-
.Ld.U.
-
.
aoo
N
CYPPU-[PTAOII[
Fig. 4. (a) Scan
CH3
348 from GC/MS
run of urine extract
from
Case
2. Spectrum
is predominantly
that of dioctyl
phthalate.
(b) Scan 348, above, after subtraction
of scan
357, effectively
removing
dioctyl phthalate
ions. (c) Reference spectrum
of cyproheptadine
267
TOTAL
IONZATION-
Case 3 involved
a young man, a participant
in a
methadone
maintenance
program,
who was brought
to the hospital
in a state of deepening
coma. It was
not known whether
he had consumed
an overdose
of
methadone
or had fallen back into the use of other
drugs. Blood and urine samples
were provided
to our
laboratory.
The total ionization-retention
index plot
obtained
for the urine extract
is shown in Figure 6,
labeled to show the results of the library search.
[The
hydrocarbon
standards
C14H30, C22H46 and C32H66
were coinjected
in order to provide reference
data for
the retention
index calculations
(6).] These results
indicated
the ingestion
of the drugs methadone
and
methaqualone.
The identification
of the metabolites
as well as the unchanged
drugs enabled
the analyst
to characterize
additional
major
chromatographic
fractions and thereby confirm the identifications
of
the drugs. The metabolite
spectra
had already
been
placed in the reference
collection,
and so these compounds
were completely
characterized
by the automatic procedure,
but some indication
of their structures
could
have been obtained
from the library
search even if the exact compound
spectra
had not
yet been added.
Figure 7 shows the results
of the
comparison
of the spectrum
assigned
to HI with the
library.
This output
format
presents
the 10 compounds
in the reference
collection
found to be most
like the spectrum
550
Fig. 5. Overplot
O
of m/e
ion of cyproheptadine,
550
550
287
on
plot (light trace) for the extract
260
(heavy
trace),
the
of the total
of urine, Case 2
a section
CLINICAL CHEMISTRY, Vol. 20, No.2,
1974
molecular
ionization
MW 287
under
consideration,
together
with
an index of their similarity.
For this spectrum
the
highest
similarity
was found for methaqualone
metabolite
1 (III) and indeed the retention
index also
agrees well with that previously
determined
for this
compound.
The search program
also retrieved
two
closely
related
compounds:
another
methaqualone
metabolite
(IV) and the drug itself (II). The inclusion of these two compounds
on the list of most similar compounds
would have provided
valuable
clues
LPET#{128}
.
43
DEN
_
TNN
5 23
73
DT#{128}.
METHADONE
M E TA AOL lIE
5
DEN
73
73
2O21-lP
METHADONE
7
(21)
UEIHAOUALOME
(2
METNAOUALONE
iil
(21)110)
CIMEIHOL
SULEONE
NICOTINE
LAIE
(
cnoLEsrERoL
THEOBROMINE
c.Hji
(‘a,
‘nap
Fig. 6. Total
ionization-retention
index
plot (6) for urine
extract
described
in text as Case 3
Labeled peaks indicate library search results obtained for this GC/MS
run. A mixture of hydrocarbons was coinjected and these are labeled
C14(n-C14H35),
C22(n-C22H46),
and C32(n-C32H66)
URINE
480,
161
NON
RUN
NO.
H
cTrAm[
2096
RESULTS
II).
METHAOUALONE
METAPOLITE
METHAQUALONE
METABOLITE
THIORIDAZINE
METAPOLITE
2,6-01
-T-BUTVL-4-METI,YLPHENOI..
BHA-TYPE
ANTIOXIDANT
THIORIE,AZINE
METABOLITE
NARCE
1 NE
METHAOUALONE
MEPHOBARBI
PHRNVL8UTAZ
I
2
2
I
TAL
ONE
SIR.
0.173
0.104
0.085
0.079
0.069
0.068
0.065
0.064
0.059
0.059
247
248
250
252
280
249
229
225
203
236
Fig. 7. Library search output, identifying
methaqualone
metabolite
(III),
for scan taken at maximum of GC peak,
listing
10 compounds
in collection
most
Similar
to the
spectrum
of
the compound
found in this extract,
to the identity
of the unknown,
had the
spectrum
of III not yet been in the collection.
Case 3
reference
235
H3C
MW 250
2511
‘C H3
MW 266
251
LI
iL
Scan
137. from
GC/MS run of
3, taken
at maximum
of peak
that
similar
to methaqualone;
(b) reference
methaqualone
(II);
(C) reference
spectrum
(V)
Fig. 8. (a)
Case
most
urine extract,
library
found
spectrum
of
of
cocaine
Comparison
of spectrum
number
137 in the urine
extract
chromatogram,
Figure 8a, or the corresponding spectrum
in the blood extract
chromatogram,
with the reference
spectrum
of methaqualone
(II)
(Figure 8b), reveals the presence
of extraneous
peaks
and thus points to the presence
of yet another
substance that is unresolved
from methaqualone
under
these conditions.
Inspection
of mass chromatograms
of the unmatched
masses
to determine
those that
maximize
at scan 137, as illustrated
in Figure 9 by
the mass chromatograms
of m/e 182 and m/e 303,
permits
one to assemble
a list of the more abundant
ions originating
from the unknown.
Comparison
of
this list with an eight-peak
index of our drug spectra
collection4
suggests
that the unknown
compound
is
cocaine
(V) and a visual comparison
of its spectrum
(Figure 8c) with scan 137 shows that, indeed,
all the
peaks of the reference
spectrum
of cocaine
are present in this scan, in addition
to those peaks appropriate for methaqualone.
Retention
index data
from
other samples
also indicated
the similar
behavior
of
the drugs cocaine and methaqualone
under the gas-
NkH3
I(YOH
MW 266
4 This
index was compiled
(and has been distributed upon request) by the authors’
laboratory.
It is patterned
after the Eight
Peak Index of Mass Spectra, Table 3, published by the Mass
Spectrometry
Data Centre, AWRE, Aldermaston,
Reading
RG7
4PR, U.K.
CLINICAL
CHEMISTRY,
Vol. 20, No. 2. 1974
261
LIIFE 420, 104
RiM .
TOTAL I55IZAT!55 PLOT
IF 9’#{128}CT#A200
s’cciaa
M/E t02
I1X
F o
I
I
P
WE
I.
j)Jv2I
F
z
H
LIr
55%
Fig.
(b)
I
I
l
I
II
4i
IP/’’lT”)’E
j.77
Fig.
I
chromatographic
conditions
used here. All these considerations
led to the conclusion
that cocaine
was
present,
even though it was masked
in the gas chromatogram
by the co-eluting
methaqualone,
the
major component.
The unambiguous
identification
of cocaine was important,
not only for the immediate
treatment
of the patient’s
critical condition
but also
provided
a piece of useful information
for those responsible
for the patient’s
participation
in the methadone program.
C
#{231}COCH3
-..
JJi4h
LI
20-
II
II.JIIj.
-188
MW303
1821
In the blood-serum
extract,
in addition
to methadone and methaqualone
and its metabolites,
diazepam and its N-demethyl
metabolite
were identified.
The presence of diazepam in the blood specimen
and
its absence
in the urine sample
illustrate
the importance of examining
more than one body fluid whenever possible.
Discussion
The GC-MS-Computer
system is particularly
well
suited for samples
where the poisoning
agent is unknown and could include
any or a number
of various
classes of compounds.
The extraction
procedure
and
gas-chromatographic
separation
are rapid
and will
accommodate
a wide variety of toxic agents. Because
no class separations
or derivatizations
are involved,
they are not so limited
in scope as are more specific
techniques.
The method
also tolerates
the presence
of other materials
(fatty acids, phthalates)
that comCLINICAL CHEMISTRY, Vol. 20, No.2, 1974
10. Mass spectrum
168 of urine extract,
Iiili
ii
‘‘‘‘
200
1.00
3.
Case 3
262
73
0.70
9. (a) Total ionization
plot for urine extract,
Case 3.
Mass chromatogram
for m/e
182, urine extract,
Case
(c) Mass
chromatogram
for m/e
303, urine
extract,
0
5 23
I&AR
scan
I
480, 16-I
of methaqualone
Case
300
metabolite
(IV)
3
monly occur in large concentrations
and can pose
(recognized
or unrecognized)
interferences
in other
techniques.
The result is the identification
of a specific drug, rather than simple indication
of its being
a member
of a class of drugs. This capability
can be
of significance
in discriminating
between
long- and
short-acting
barbiturates,
for example,
for which different clinical treatments
will be required.
Since the
reference
spectra
are already
in the tape library,
it
also obviates
the need to have a standard
available
for simultaneous
analysis
or coinjection.
In the case
of the less-common
drugs, this can be a critical factor, because
valuable
time could be lost locating
an
authentic
sample of a drug whose presence
was suggested by thin-layer
chromatographic,
GC, or ultraviolet data alone.
On the other hand, if the spectra
obtained
should
include
substances
not contained
in the collection,
the basic interpretability
of mass spectra
makes it
possible
for the chemist
to deduce the nature of the
substance
even in the absence
of an authentic
spectrum.
This capability
is particularly
useful
in the
analysis
of new or unusual
“street”
drugs, for which
no reference
material
may be available.
It is also essential in recognizing
that some of the components
of
the mixture
are metabolites
of drugs that may or
may not also be present
in the extract.
For example,
it is a fairly simple
matter
to recognize
that spectrum 168, Figure 10, belongs to a hydroxylated
metabolite
of methaqualone
(IV). The shift of the major
peaks in the spectrum
to m/e 251 and 266 and the
difference
in retention
index indicate
aromatic
hydroxylation
(6) .
In contrast,
the spectra
of some drug metabolites
are quite different
from those of the parent
compound.
The mass spectrum
of methadone
(VI) is
dominated
by the ion at m/e
72, resulting
from
cleavage
fi
to the
nitrogen:
CH3CH=N(CH3)2.
There are no ions at m/e values greater than 72 that
have an abundance
of more than 10%. Spectrum
116,
Figure
11, is obviously
quite different
from that of
Nau, H., and Biemann,
K., Utilization
of automatically
signed retention
indices for computer
interpretation
of mass
tra, Anal. Lett., in press.
asspec-
Lrnr’E
#{149}.
Am,
so-i
5
A
LJ.l.Li
Mass
spectrum
#{149}
L
I
100
Fig.
11.
e3
9i15
73
J
2121
of methadone
metabolite
(VII)
scan 116 of urine extract, Case 3
methadone.
metabolized
Methadone,
however,
is known
to be
to a cyclic compound
(VII) (10), and spectrum 116 does correlate quite well with the mass spectral behavior
expected
of such a structure.
It exhibits
an abundant
molecular
ion, reflecting
the stability
of
the phenyl-substituted
heterocyclic
ring and the
only facile loss is that of a methyl group.
\\
CH
Cli
N’
112
CH
7
CH3
CH
“CH3
-
N3
-
Cli
-N;
<CH2
262
CI
I 72
MW
While
309
during
assignments
277
such as these can be
it is more efficient
and
requires
less expertise
on the part of the individual
analyst
if these metabolite
spectra
are included
in
the reference
collection.
Accordingly,
after each of
these metabolites
was first identified
by deduction
on the basis of spectra
obtained
during
analysis
of
emergency
samples,
their authenticated
spectra were
added to the library,
so that their identification
in
the case illustrated
here was automatic.
It is essential
that metabolites
be recognized
as
such for two reasons:
(a) The parent
drug may have
been completely
metabolized
and can thus be recognized only by identifying
the resulting
metabolites.
Chloral
hydrate,
for instance,
is converted
to 1,1,1trichloroethanol
and none of the drug itself appears
in the blood or urine of the patient.
(b) An analysis
of the type described
here cannot be considered
complete until all of the significant
gas-chromatographic
peaks
are satisfactorily
accounted
for. The total
number
of drugs
ingested
is usually
unknown,
so
that unmatched
peaks in the chromatogram
must be
considered
as potential
evidence
of further
drugs unless a relationship
to a drug already
identified
in the
extract can be established.
Contaminants
may also be present
and give rise to
gas-chromatographic
peaks as large as or even larger
deduced
structure
MW
an analysis,
than the drugs
cation of these
themselves.
contaminants
Fast and reliable identifieliminates
the possibil-
ity of confusing
them
with the causative
agent(s)
and makes
it possible
to suggest
modifications
of
sample acquisition
and transfer
procedures
that minimize or eliminate
this interference
in the future. For
example, it was noted quite early in this work that
some blood samples
produced
a major peak with a
retention
index 2665 (OV-17).
Interpretation
of its
low and high resolution
mass spectra
led to its identification
as tri-(2-butoxyethyl)
phosphate
and its
source
was traced
to the rubber
stoppers
used on
blood-collection
tubes
(“B-D
Vacutainers”).
(To
eliminate
this interference,
we recommend
that all
blood samples
submitted
to our laboratory
be drawn
with glass syringes.)
Information
such as the identity
and source of contaminants
is useful not only for
GC-MS procedures
but can be of assistance
to those
using more limited
methods
in which unknown
contaminants
interfere
even more. Simple
procedural
modifications
can then be devised to eliminate
contaminants,
once their nature
and source are established by GC-MS.
The additional
dimension
of specific data provided
by mass spectra
means that drugs whose gas-chromatographic
behavior
is identical
on OV-17 (e.g.,
glutethimide
and meprobamate;
diazepam
and diphenylhydantoin)
may be easily
distinguished
by
their mass spectra.
The GC-MS
approach
to this problem
is preferable to direct admission
of the sample
into the ion
source of the mass spectrometer,
as has been discussed elsewhere
(11). Samples
of biological
origin,
extracted
by a deliberately
nonselective
procedure
to
assure that a very wide range of drug types will be
retrieved,
contain a large number
of compounds
such
as fatty acids and cholesterol,
which may be present
in concentrations
much greater than are the drugs of
interest
in these analyses.
Multiple
drug ingestion
may also result
in a very complex
extract.
If this
mixture
is admitted
directly into the mass spectrometer, ions derived from an individual
drug will be accompanied
by (and possibly
obscured
by) ions derived from all the other materials
present.
It is then
a task, which can be difficult
and may at times be
impossible,
to determine
successfully
which observed
masses correspond
to the individual
components
and
thereby
identify
the causative
agent(s).
While slow
controlled
heating
of the sample and the use of highresolution
mass spectrometry
do help to alleviate
these difficulties
somewhat,
we have found that use
of the gas chromatograph
to achieve
separation
is
the most routinely
useful
approach
for extracts
of
body fluids. The spectra obtained
from GC peaks are
those of relatively pure compounds
and are therefore
amenable
to immediate
automated
comparison,
since the data-acquisition
system provides
digital information
that is quickly
converted
to a searchable
format. The separation
of components
ensures
that
even relatively
minor components
produce
definitive
spectra.
CLINICAL
CHEMISTRY,
Vol.20,
No.2,
1974
263
In order to make maximum
use of the data provided by the gas-chromatographic
separation,
we routinely
calculate
retention
indices
for all runs (6).
This additional
dimension
of information
is useful in
several ways. It provides
a means of distinguishing
clearly between
compounds
whose common
structural feature
makes
their mass spectra
very similar,
e.g., the base peak at m/e 58 (and only major ion) of
many
compounds
containing
the -CH2N(CH3)2
grouping,
a very common
feature of many local anesthetics
and tranquilizers,
among others.
A comparison of the retention
index of the compound
retrieved
by the library search with that of the spectrum
being
searched
helps the analyst
to decide
whether
the
search has found the identical
compound
or, instead,
the most similar
compound
in the collection.
In
the event of incomplete
gas-chromatographic
resolution, the presence
of extraneous
peaks in the experimental spectrum
may cause the similarity
index between known and unknown
spectra
to be quite low,
even when the exact compound
is being matched.
Coincidence
of the retention
indices
of the known
and unknown
gives a further
degree of confidence
in
the analysis
when relatively
impure spectra
must be
used for the comparison,
as is illustrated
in Figure 8,
where the spectrum
of a small gas-chromatographic
peak has been successfully
matched
despite the presence of a relatively
high background.
Examination
of the body fluids of persons
who
have taken
abnormally
high dosages
of drugs also
provides
an opportunity
for studying
metabolism
under circumstances
that could not be induced
deliberately.
Such studies
can give valuable
information concerning
the metabolism
of therapeutic
doses
of such drugs by providing
larger amounts
of metabolites or may indicate
alternative
metabolic
pathways that become
important
at much higher doses.
The retention-index
data generated
in the GC-MS
experiment
can be used for further
studies
with gas
chromatography
alone and also have an independent
information
content,
because differences
in retention
indices
among
a related
group of compounds
give
clues to their structural
differences-a
valuable
tool
in interpreting
the mass spectra of new metabolites
The procedure
described
here has been designed
as
a qualitative
technique,
but does permit rough estimation
of quantities
involved
by comparison
of the
area of the gas-chromatographic
peak of the unknown with that of co-injected
standards.
An allowance must be made for nonquantitative
extraction
and variability
of flame detector
response to different
types
of compounds.
Usually
it suffices to report to
the attending
medical
personnel
the approximate
concentration
at the time the identification
of the
agent is reported.
If a greater
accuracy
is indeed required,
the conventional
clinical methods
of quantitative analysis
can be more reliably
used, once the
identity
of the substance
to be quantitated
and the
nature
of the contaminants
have been established.
GC-MS
methods
with stable
isotopes
have unique
advantages
for quantitative
analysis
of drugs
and
.
264
CLINICAL
CHEMISTRY,
Vol. 20, No.2,
1974
drug metabolites
in body fluids (12 ); but the simpler methods
are usually
adequate
for these emergency situations,
once the qualitative
results
have
been obtained
as described.
The selection
of drugs for inclusion
in the searchable library
was originally
based
on the likelihood
that they would be ingested
in suicide attempts.
Experience
has shown,
however,
and Poison
Bureau
data (13) also indicate
that many of the poisoning
victims
who arrive at the emergency
room of local
hospitals
are suffering
the consequences
of accidental
poisoning
either
because
of deliberate
ingestion
of
drugs purchased
from illegal sources,
or, in the case
of young children,
random
ingestion
of drugs or other
toxic materials
available
in the home or elsewhere.
We have therefore
expanded
the library to include a
number
of illicit drugs (LSD, psilocybin,
morphine,
phencyclidine,
etc.)
and
potential
contaminants
(strychnine),
as well as drugs and other substances
not usually
considered
as possibilities
in adult poisonings,
but which the unsupervised
child may ingest in toxic quantities
(doxylamine,
camphor,
etc.).
In order to make it possible
to rapidly
identify
all
the major peaks in the gas chromatogram,
the spectra of commonly-occurring
blood and urine constituents and contaminants
have also been added to the
library.
This ensures
that the chemist’s
attention
will be directed
toward those components
of toxicological significance.
Although
efforts have been made
to instruct
the attending
physicians
as to the proper
manner
of sample
handling
to minimize
contamination, the very number
of physicians
and hospitals
who may potentially
send samples
for examination
increases
the likelihood
that impurities
such as plasticizers will still be observed
occasionally.
It is a simple matter
to add such spectra
to the library
and
thereby
avoid the chance that this can cause a false
lead as to the poisoning
agent.
We have received more than 600 samples during
the past two years; we have identified
at least one
toxic substance
in about
75% of the cases. The remaining
25% includes
those patients
whose coma
was caused by factors other than ingested
substances
and those overdose
victims
whose body fluid did not
contain a detectable
amount
of the toxic agent at the
time of sampling.
Multiple
ingestions
have been
most common
with barbiturate
poisonings.
We have,
so far, found a total of 72 different
drugs, as well as
26 other toxic materials,
results
that constitute
a
strong
argument
in favor of this broad-based
approach.
The age distribution
of the patients
from whom we
have received
samples
over the same period is indicated in Table 1. In general,
we have observed
that
accidental
ingestions
account
for most of the poisonings of small children,
that young teenagers
are frequently
the victims
of illicit drugs that have been
misrepresented
to them,
while the comas of older
teenagers
usually
involve excessive
or unwise use of
known drugs and those of adults are most often the
result of deliberate
suicide attempts.
Table 1. Age Distribution of Patients
Age,
yrs
No. of patients
tional Institutes
of Health,
RR 00317 from the Biotechnology
Resources
Branch,
Division
of Research
Resources,
and Training
Grant GM 01523.
Dr. James E. Biller’s and Mr. Edward M. Ruiz’s contributions
in the continuous
improvements
of the data handling procedures
0
29
0-2
3-6
95
47
are gratefully acknowledged.
38
References
7-10
11-15
111
16-20
child, ?yrs.
adult
91
13
177
Total
601
It has to be kept in mind that both gas chromatography
and mass spectrometry
require
the substance
to have a measurable
vapor pressure.
Fortunately, the vast majority
of drugs, or at least the corresponding
free acids or bases (if the drug is administered
as a salt) or some of the major metabolites,
fall into this category.
Inorganic
poisons like arsenic
and cyanide,
however, are, of course, not detectable.
We conclude that the GC-MS-Computer
system
described
is well suited
to rapid
identification
of
toxic materials
in body fluids of poisoning
victims.
The system
is general
enough
to quickly
identify
a
wide variety
of agents responsible
for the coma or
other emergency
condition
of the patient.
The system has advantages
in both speed and specificity
over other methods
and it therefore
is expected
that
the method
will be widely applicable
and extremely
useful for clinical
chemists,
particularly
in view of
the ever-increasing
number
of drugs appearing
on
the legal and illegal market.
We are grateful
to Professor
John Hedley-Whyte
and Mr. Hans
Laasburg
of Beth Israel Hospital,
Boston, for their advice and for
providing
us with samples
of many of the drugs needed
to accumulate
the collection
of authentic
mass spectra,
and to Dr. Frederick Lovejoy, Jr. of the Boston Poison Information
Center for arranging communication
with the appropriate
personnel
in area
hospitals.
This work is supported
by research
grants of the Na-
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K., Biller, J., et al., Identification
of
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265