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 methamphetamine 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 CH 5 + 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 additional means of molecular identification 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 0371 ch13 frame Page 214 Monday, August 27, 2001 1:46 PM © 2002 by CRC Press LLC 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% 0% 65 89 137 159 178 209 223 251 315 355 100 150 200 250 300 350 m/z 50 119 Spect 1 6.943 min. Scan: 738 Chan: 1 lon: 837 us RIC: 24929 296 100% 75% 25% 50% 0% 100 150 200 250 300 350 m/z 50 Spect 1 7.369 min. Scan: 749 Chan: 1 lon: 446 us RIC: 55409 BC 294 56 94 117 154 204 244 313 0371 ch13 frame Page 215 Monday, August 27, 2001 1:46 PM © 2002 by CRC Press LLC 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 10 10,500 20 20,000 50 41,000 20 16,000 100 99,500 20 20,200 Unknown 60,500 20 23,000 CH 2 CH N C O CF 3 CH 3 CH 3 CH 3 CF 3 CH 3 CH 3 C O NCH CHCH Benzoylecgonine MSTFA+→ +→Methamphetamine Pentafluoropropionic acid anhydride 0371 ch13 frame Page 216 Monday, August 27, 2001 1:46 PM © 2002 by CRC Press LLC 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. 0371 ch13 frame Page 217 Monday, August 27, 2001 1:46 PM © 2002 by CRC Press LLC 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 Hair segment Morphine 6-AcetylMorphine Phenobarbital 1 (at scalp) 0 ng/mg 0 ng/mg 23 ng/mg 2 0.1 0.2 32 3 0.2 0.3 38 4 (furthest from scalp) 0.3 0.6 31 0371 ch13 frame Page 218 Monday, August 27, 2001 1:46 PM © 2002 by CRC Press LLC 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. 0371 ch13 frame Page 219 Monday, August 27, 2001 1:46 PM © 2002 by CRC Press LLC References 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. 0371 ch13 frame Page 220 Monday, August 27, 2001 1:46 PM © 2002 by CRC Press LLC © 2002 by CRC Press LLC 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 approximately 1 µg/mL. Thus, calcium, magnesium, and other predominantly light elements are not included in the trace designation. Our discussion here is confined 1 4 © 2002 by CRC Press LLC 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 approximately 1 µg/mL. Thus, calcium, magnesium, and other predominantly light elements are not included in the trace designation. Our discussion here is confined 1 4 Alcohols 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 0371 ch15 frame Page 231 Monday, August 27, 2001 1:48 PM © 2002 by CRC Press LLC [...]... LLC Inebriation, nausea, paralysis, acidosis, possible coma Elevated heart and breathing rates, pulmonary edema, renal failure Pain in sides, renal tubular necrosis 0371 ch 15 frame Page 251 Monday, August 27, 2001 1:48 PM 6 A child is brought to the emergency department with a blue coating around his mouth His parents bring along a can of antifreeze, which is also blue in color They suspect that the child... patient Misidentification of methanol was not possible because the laboratory ran multiple test methods including gas chromatography, an enzymatic method, calculation based on osmolal gap, and gas chromatography-mass spectrometry All methods were in good quantitative agreement The laboratory was acutely aware of the skepticism which would greet their findings and they prepared for that response in a. .. serum bicarbonate of 18 meq/L Ultrasound of the head and EEG were normal No infection was diagnosed nor were any other causes apparent for his acidosis He was discharged after 3 days The child was re-admitted 2 days later with respiratory distress and weakness Laboratory data for this admission are as noted: pH PCO2 pO2 HCO3– Organic acids 7. 35 29 mm 48 mm 16 mmol/L Normal Sodium Glucose Blood urea N 140... LLC 0371 ch 15 frame Page 252 Monday, August 27, 2001 1:48 PM A blood sample from the child was tested for volatiles and gave a result of 1148 mg/dL of methanol, an extremely high concentration At this point the child was started on folic acid and IV ethanol He was also evaluated for optic damage because methanol is known to cause severe visual problems including blindness No optic atrophy was found The... 19 95 © 2002 by CRC Press LLC 0371 ch 15 frame Page 254 Monday, August 27, 2001 1:48 PM Case Study 2: Patient with Convulsions A 42-year-old man was lying in bed with vomitus in the area where he lay His brother, who discovered him, recalled seeing his brother staggering several hours earlier and slumping to the ground several times Upon arrival at an emergency department, the patient was foaming at... treated with ethanol and folate? Q1 What is the most common method for methanol analysis? a) Enzymatic analysis b) Gas chromatography c) Liquid chromatography d) Calculation based on osmolal gap Q2 How could laboratory error be ruled out? a) Repeat the test b) Have other laboratories verify the result c) Test the specimen by different methods d) Review all aspects of specimen handling and repeat the entire... This patient did not accumulate the toxic metabolite, formic acid c) There was a defect in this patient’s conversion of methanol to formaldehyde d) The clinical problem was recognized early enough for normal treatment to prevent severe sequelae Answers and Discussion Q1 (Answer = b) The most common method for methanol analysis is gas chromatography, usually by head space Enzymatic methods are available... offending agent Lethal blood levels are reported variously as 100 to 200 mg/dL for ethylene glycol and 100 to 300 mg/dL for methanol Congenital disease appears unlikely (although not impossible) because the child has an entirely healthy 2-year-old sister and he has been tested for organic acidurias, possible causes of the observed metabolic acidosis Finally, ethanol rarely causes significant acidosis... child drank some antifreeze A laboratory result for ethylene glycol was reported as 90 mg/dL of blood Discuss Case Study 1: An Infant with Multiple Admissions At 5 weeks of age a male child who had earlier done well on infant formula, became limp, lethargic, and mildly comatose Although afebrile, he was admitted to the hospital to rule out sepsis Noteworthy findings included moderate acidosis and a serum... available Because reagents for enzymatic methods are not very stable they should © 2002 by CRC Press LLC 0371 ch 15 frame Page 253 Monday, August 27, 2001 1:48 PM be used soon after preparation, a factor which is not compatible with the low volume of testing usually done for methanol Calculation by osmolal gap is usually very approximate and quite nonspecific It should not be regarded as an adequate method . 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. was diagnosed nor were any other causes apparent for his acidosis. He was discharged after 3 days. The child was re-admitted 2 days later with respiratory distress and weakness. Laboratory data. meth- ods including gas chromatography, an enzymatic method, calculation based on osmolal gap, and gas chromatography-mass spectrometry. All methods were in good quantitative agreement. The laboratory