Toxicology A Case-Oriented Approach - part 5 pot

Figure 13.12 above showed that derivatized methamphetamine and ephedrine were virtually indistinguish-able on the basis of electron impact mass spectra.. When run under identical conditi

One can distinguish R- vs S-enantiomers by GC-MS but only by a significant

additional effort Several methods are employed including the use of a chiral column

In other words, the chromatographic column can be specially selected or specially

modified so that it is sensitive to enantiomers In one type of column, for example,

L-valine tert-butylamide is covalently bonded to the polysiloxane backbone of a

regular column The modified packing forms different diastereoisomers as hydrogen

bonds to the different enantiomers passing through the column R- and S- forms

have different retention times

The most common method for GC-MS differentiation of R- and

S-methamphet-amine is by derivatizing the methamphetS-methamphet-amine with a chiral derivatizing agent For

this purpose N-trifluoroacetyl-L-prolyl chloride is frequently used The derivatizing

agent contains a chiral carbon and the drug to be detected also contains a chiral

carbon Therefore, the derivatized methamphetamine contains two chiral carbons

Substances with more than one chiral carbon may have diastereoisomers, i.e.,

struc-tures that are not mirror images (not enantiomers), but they are stereoisomers of

each other These molecules are distinguishable from each other because some

intramolecular forces are slightly different Diastereoisomers often have different

retention times on chromatographic columns and this enables us to distinguish

between them The derivatization reaction of methamphetamine with N-TFA-L-prolyl

chloride is shown in Figure 13.16

OTHER MODES OF MASS SPECTROMETRY

The most common modality in which mass spectrometry is used is in the electron

impact mode In this form high energy electrons strike molecules as they emerge

from the gas chromatograph This occurs in a very high vacuum As described earlier,

mass spectra are produced that are usually quite specific for a particular compound

Electron impact mass spectrometry (EI-MS) is a superb technology that is

satisfac-tory for the large majority of toxicological analyses

Two other forms of mass spectrometry are available and they provide additional

capabilities that are valuable in unique circumstances The first is chemical

ionization-mass spectrometry (CI-MS) In CI, a reagent gas is present at a low pressure within

the mass spectrometer The gas, for example, methane, produces reactive ions such as

CH5+that react in a variety of ways with the molecules entering the mass spectrometer

from the gas chromatograph CI is thought of as a soft-energy type of spectrometry

in which the molecules under study are fragmented to a lesser degree than in EI-MS

CI and EI generate completely different mass spectra so that complementary

informa-tion is provided and an addiinforma-tional means of molecular identificainforma-tion is possible In the

case of compounds like amphetamines that have very simple EI mass spectra, CI can

be a great help because of added spectral information Furthermore, many types of

molecules, especially heteroatom-containing species like amines and ethers, usually

give abundant (M+1)+ ions Saturated hydrocarbons often provide large amounts of

the (M-1)+ ions In both cases these ions are usually the predominant ion species

present and informed speculation about the molecular weight can be conducted

We saw earlier that the amphetamine class of drugs usually provides very simple

spectra by electron impact-mass spectrometry Identifications are often difficult

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because of the similarities of spectra among these compounds Figure 13.12 above

showed that derivatized methamphetamine and ephedrine were virtually

indistinguish-able on the basis of electron impact mass spectra Each of these compounds gave 204,

160, 119, and 91 ions The ratios of such ions were, moreover, not greatly different

between the two compounds The CI spectra of derivatized methamphetamine and

ephedrine are shown in Figure 13.17 When run under identical conditions, derivatized

methamphetamine has a large M+1 ion at m/z of 296 whereas ephedrine gives a 294

ion, a different fragment from the 204 found in standard electron impact mass

spec-trometry Chemical ionization may, therefore, serve as a tool for clarifying

identifica-tion of compounds whose electron impact spectra are not sufficiently distinguishing

A second mode of mass spectrometry is tandem mass spectrometry, also called

mass spectrometry-mass spectrometry In this mass spectrometric modality a specific

ion present in a mass spectrum is isolated and subjected to further fragmentation

The pattern that results from this second fragmentation is called a daughter ion mass

spectrum The manner in which a daughter spectrum is generated is variable In one

method, several quadrupoles are arranged sequentially so that the specific ion arising

from the first fragmentation is directed into the second quadrupole to the exclusion

of all other ions The second quadrupole then separates the fragments that come

from bombardment of the major ion of the first fragmentation In a different method,

the ion trap method, specific energy is applied to the trap that contains all of the ion

fragments from the first fragmentation This results in ejection of all ions other than

the ion of interest This is then further energized to cause its dissociation and

formation of a daughter mass spectrum

Daughter mass spectra are usually not needed because of the typically high

specificity present in the first mass spectrum In some situations, however, they may

be helpful An actual case serves to explain this point Two decomposed bodies were

discovered and some evidence suggested that the manner of death was related to an

accident caused, in part, by use of marijuana It was not possible to demonstrate the

FIGURE 13.17 CI mass spectra of derivatized methamphetamine and ephedrine.

100%

75%

25%

50%

50

119

Spect 1 6.943 min Scan: 738 Chan: 1 lon: 837 us RIC: 24929

296

100%

75%

25%

50%

0%

50

Spect 1 7.369 min Scan: 749 Chan: 1 lon: 446 us RIC: 55409 BC

294

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presence of marijuana metabolite in the bodies, however, due to the advanced state

of decomposition in which products of decomposition interfered with recognition

of the usual mass spectrum of marijuana metabolite The forensic scientists

con-fronted with this problem were able to solve it by tandem mass spectrometry A

daughter spectrum was generated from the major ion of marijuana metabolite That

daughter spectrum was clean and much less subject to interference from products

of decomposition In circumstances such as these the technique of tandem mass

spectrometry can be very useful

Questions

1 Complete these reactions while showing structures for both reactants and

products:

2 Which chromatographic packing would you use for the separation of

several compounds, each of which is a moderately polar insecticide?

3 Which chromatographic detector would you use for the detection of

sev-eral compounds, each of which is a halogenated insecticide present in

very low concentration?

4 From the table below, calculate the concentration obtained for the

unknown by using the data without the internal standard Repeat the

problem while using the internal standard data to calculate a concentration

FIGURE 13.18 Structures of derivatized methamphetamine and fragments of its mass

fragmentation.

Conc of Std Integrator Response Int Std Cond Int Std Response

CH2 CH N C

O

CF3

CH3 CH3

CH3

CF3

CH3 CH3

C

O N CH

CH CH

Benzoylecgonine+MSTFA→

Methamphetamine Pentafluoropropionic acid anhydride

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5 Study the chromatogram of Figure 13.6 and calculate resolution on the

basis of the peaks at retention times 11.1 and 14.6 minutes

6 Figure 13.18 shows the structures of derivatized methamphetamine and

several fragments formed by its mass fragmentation Calculate the mass

of each and show the structures and mass for each fragment expected

from the derivatized deuterated form of the compound (Assume

deuter-ization is in the locations shown in Figure 13.13.)

Case Study 1: The Abused Child

A 5-year-old boy had a history of six previous admissions to the hospital over

a period of 4 months On each admission he was found to be vomiting, in a state

of semi-consciousness, hypoglycemic, and complaining of abdominal pain A

definitive diagnosis could not be made and he was thought to be epileptic on the

basis of earlier convulsive episodes He was given Luminalette for his seizures

At the present admission this child was fully comatose and was admitted to

Intensive Care He was noted to be bradycardic, temperature was 34°F, and

pupils were constricted The child was cyanotic and underwent a respiratory

arrest from which he was resuscitated Naloxone was administered with a very

significant improvement in the patient’s vital signs

If the patient’s symptoms were a result of poisoning, what agent among the

following is probable?

a) Methamphetamine

b) Strychnine

c) Heroin

d) Cocaine

(Answer = c) All of the choices given would cause hyperstimulation, which is

not consistent with most of this patient’s findings He is manifesting primarily

symptoms of physiological depression These symptoms are consistent with

poisoning by heroin This conclusion is strongly reinforced by the fact that the

patient was improved by naloxone, a narcotic antagonist The earlier report of

convulsions by this child may have been due to a different toxin although

convulsions may also result from heroin overdose under certain circumstances

Laboratory screening of the child’s urine was positive for barbiturates and

opiates, findings that were consistent with the child’s symptoms and with his

medical history When the patient regained consciousness, he was questioned

by police The child stated that one of his relatives had forced him, on many

occasions, to consume bitter brown and white powders As a result, the child

was placed in protective custody and an investigation was launched regarding

the alleged poisoning A judge ordered that the child’s hair be tested for

barbi-turates and heroin in an attempt to corroborate the child’s testimony that he had

been subjected to this abusive treatment for a long period

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An 8-cm tuft of hair was cut as closely as possible to the scalp The hair

was cut into 2-cm segments, washed, and enzymatically digested Any drugs

present were then extracted into chloroform, derivatized with

pentafluoropropi-onic acid, and eventually injected into a gas chromatograph-mass spectrometer

The mass spectrometer was operated in the selective-ion monitoring mode in

which only ions specific to the substances being sought are measured

As seen in the table of results the child’s hair revealed the presence of

phe-nobarbital in every segment This was consistent with his continuous use of

phenobarbital The amounts of phenobarbital also correlated with his therapeutic

history Opiates were also identified in three of the segments that were tested

Based on these forensic laboratory findings, the accused relative of the child

was found guilty and sentenced to a prison term

Questions

Q1 At the trial of the alleged assailant, defense attorneys argued that the finding

of opiates in the child’s hair was due to the patient’s use of antitussive drugs

a) This is a solid argument that cannot be deflected by laboratory studies

b) Opiates are not found in antitussives

c) 6-acetyl morphine is evidence of heroin administration and heroin is

absent from pharmaceuticals

d) Antitussive drugs would not enter the hair

Q2 Why is a sensitive analytical method needed when testing hair?

a) Almost no drug enters hair

b) The forms of drugs found in hair are very unusual metabolites

c) The amount of specimen is very small compared to the amounts

avail-able in biofluids

d) Only a very small percent of the drug present in hair can be recovered

for testing

Q3 In the case discussed here, the child’s hair specimen was taken 6 weeks

after he recovered from the coma How much of his hair should be drug

free?

a) The 2-cm segment nearest to the scalp only

b) The first two segments

c) The segment most distal from the scalp

d) All of the 8-cm specimen

Testing Results

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Q4 How can we rule out external contamination of the hair?

a) It cannot be excluded and constitutes an inherent limitation of hair testing b) The hair sample is treated in the laboratory so that all drug from outside the body is removed, but no drug from inside the body is removed

c) The ions tested in mass spectrometry are indicative of the in vivo

pres-ence of drugs

d) The finding of metabolites in hair is a strong indication that contami-nation did not cause the positive result

Answers and Discussion

Q1 (Answer = c) Opiates are present in many antitussive (cough suppressant) medications They are effective for this purpose because they diminish the coughing reflex 6-Acetylmorphine is, however, a metabolite of heroin that arises only from heroin and not from other opiates It is not found in any natural source other than as a heroin metabolite Its presence is proof

of the heroin use

Q2 (Answer = c) Significant quantities of drug usually enter hair, although for some drugs the quantity is small enough that it does contribute to the analytical challenge Forms of metabolites present in hair are sometimes different from those found in urine This fact would not, however, mean that a more sensitive method is needed Recovery percents are satisfactory for hair testing The problem with hair testing is that hair is very light in the quantities usually taken for testing A 100-mg quantity is typically taken If the concentration of drug or metabolite was the same in hair as

in urine, then 100 mg is equivalent to only 0.1 mL of urine, about 2% of the mass of a urine specimen that is usually tested With hair, we are essentially testing very small quantities and, therefore, need methods with low detection limits

Q3 (Answer = a) Hair grows at an approximate rate of 1.3 cm (close to 0.5 in.) per month Although there is some interpersonal variation, one can use this figure to determine the time of drug use based on segmental analysis, i.e., cutting the hair and testing the separated pieces In the present case, the hair segment nearest to the scalp was, indeed, drug-free That segment was growing while the child was in protective custody The other segments all contained drugs and corroborated the charges against the child’s assailant Q4 (Answer = d) Contamination of hair by drugs present in the environment

is a problem with hair testing A great deal of research has been directed

at sample preparation to selectively remove from the sample drugs that are present on the hair by incidental contact Many methods have been developed that appear to be successful in eliminating external drug Most

of them, however, involve a risk of false negatives by elimination of some internal drug as well If the laboratory demonstrates the presence of metabolites of drugs, however, this is a strong indication that the person has ingested or injected the drug

Martz, R et al., The use of hair analysis to document a cocaine overdose following a

sustained survival period before death, J Analyt Toxicol., 15, 279, 1991.

Rossi, S.S et al., Application of hair analysis to document coercive heroin administration

to a child, J Analyt Toxicol., 22, 75, 1998.

Metal Analysis

(Assay of Toxic Metals)

CONTENTS

Early Colorimetric Methods

Instrumental Methods

Flame Atomic Absorption Spectroscopy (FAAS)

Theory

Possible Problems

Graphite Furnace Atomic Absorption Spectroscopy (GFAAS)

Neutron Activation Analysis (NAA)

Inductively Coupled Plasma-Mass Spectrometry (ICP-MS)

Performance

Specimen Preparation

Interferences

References

Questions

Metal testing is a common feature of toxicology testing programs It is done for several reasons One of the more common is the monitoring of employees in haz-ardous occupations where they are exposed to certain metals on a chronic basis at the workplace Such employees are protected by OSHA and other government bodies which mandate that employees be tested periodically to assure that dangerous levels

of toxic metals are not accumulating in their bodies This type of testing often is performed on urine but is occasionally conducted on whole blood The workspace

is also monitored If the toxic metal is likely to enter the air space, then air sampling

is conducted

A second kind of metal testing is in the context of clinical toxicology This is less routine and consists of physicians ordering various blood and urine testing for metals when a patient complains of symptoms that are suspicious for metal poison-ing This latter type of clinical toxicology testing is much less frequent than the routine monitoring referred to above

The term “trace element analysis” is sometimes used synonymously with metal testing Trace metals are understood to be those present in quantities less than

elements are not included in the trace designation Our discussion here is confined

14

Metal Analysis

(Assay of Toxic Metals)

CONTENTS

Early Colorimetric Methods

Instrumental Methods

Flame Atomic Absorption Spectroscopy (FAAS)

Theory

Possible Problems

Graphite Furnace Atomic Absorption Spectroscopy (GFAAS)

Neutron Activation Analysis (NAA)

Inductively Coupled Plasma-Mass Spectrometry (ICP-MS)

Performance

Specimen Preparation

Interferences

References

Questions

Metal testing is a common feature of toxicology testing programs It is done for several reasons One of the more common is the monitoring of employees in haz-ardous occupations where they are exposed to certain metals on a chronic basis at the workplace Such employees are protected by OSHA and other government bodies which mandate that employees be tested periodically to assure that dangerous levels

of toxic metals are not accumulating in their bodies This type of testing often is performed on urine but is occasionally conducted on whole blood The workspace

is also monitored If the toxic metal is likely to enter the air space, then air sampling

is conducted

A second kind of metal testing is in the context of clinical toxicology This is less routine and consists of physicians ordering various blood and urine testing for metals when a patient complains of symptoms that are suspicious for metal poison-ing This latter type of clinical toxicology testing is much less frequent than the routine monitoring referred to above

The term “trace element analysis” is sometimes used synonymously with metal testing Trace metals are understood to be those present in quantities less than

elements are not included in the trace designation Our discussion here is confined

14

CONTENTS

Ethanol Mechanism of Action Alcohol Pharmacokinetics Absorption

Distribution Metabolism Elimination Calculations on Blood Alcohol and Elimination Alcohol Toxicity

Acute Chronic Toxicity Alcohol Testing

Testing Methods Gas Chromatography Photometric Analysis Breath Testing Methanol

Metabolism Toxicity Treatment Testing Isopropanol

Pharmacokinetics Toxicity

Therapy Testing Ethylene Glycol Metabolism Toxicity Therapy Testing Crystalluria Gas Chromatography of Ethylene Glycol Enzymatic Assay

Problems and Questions For Further Reading

15

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