Indications from the case history, the site of the incident, the symptoms of a patient, or external and internal postmortem investigation as shown in Sect. “Indications from case history, clinical symptoms, or autopsy” are not sufficient for proving an intoxication in the forensic context. The poison must unambiguously be identified and quantified by chemical analysis. The systematic search for the poison is very difficult and requires a special strategy for the following reasons:

There are a large number of possible toxic substances as shown in Sect. “Diversity of toxic substances and toxicological relevance”.

These substances exhibit very different analytical properties and there is no general structural feature that discriminates highly toxic from less or nontoxic substances.

Because of the different toxicity, the substances are present in very different concentrations. For example, lethal concentrations in blood of the heart glycoside digoxin (> 10 ng/mL) are by 5 orders of magnitude lower than those of the antipyretic drug acetylsalicylic acid (> 1 mg/mL) but both occur in fatal poisonings.

The additional or even exclusive presence of metabolites of the parent substance essentially increases the number of possible analytes.

There are interferences by endogenous substances. Especially putrefaction products provide often similar analytical signals as exogenic drugs. These endogenous compounds must be recognized as such, otherwise it could be an unknown poison.

There are often combined intoxications with more than one toxic substance.

It is obvious that this analytical task cannot be solved by parallel tests for each of the many possible poisons. Instead, the vast majority of toxic substances must be detectable in a limited number (e.g., three or four) of screening procedures. For the remaining poisons which do not suit one of these procedures, specific single or group tests must be applied.

In view of the available analytical techniques and of the frequency of substance occurrence in poisonings, the systematic toxicological analysis is conducted in four branches:

(A)

Preliminary tests and specific single or group tests for frequently occurring poisons which cannot be tested in branches B, C and D

(B)

Test for toxic metals—AAS, ICP/EOS, ICP/MS

(C)

Tests for gases and volatile substances—Headspace GC and GC/MS techniques

(D)

Tests for nonvolatile organic substances—Combined chromatography/spectroscopy techniques

In practice, not all analytical procedures must be applied in every case, and the analytical effort can be limited according to the information described in Sects. “Diversity of toxic substances and toxicological relevance” and “Indications from case history, clinical symptoms, or autopsy”. From economicals point of view (specialized personnel and expensive equipment), it is also not possible that each laboratory can have all corresponding methods at hand. Therefore, close cooperation between to a certain degree differently specialized laboratories is necessary. In particular, this concerns expensive tests for seldom-occurring poisons of branch A.

Preliminary tests and specific single or group tests for frequently occurring poisonsAlcohol

Alcohol (ethanol) is omnipresent in the population, and survived or fatal intoxications with alcohol as the only harmful substance occur very often. Combination with medicines or illicit drugs is common. It was shown that when alcohol is present, relatively small overdoses of drugs may result in fatal poisoning in comparison to drugs alone [49]. Therefore, determination of alcohol in blood according to validated methods is routine in every forensic case. For better assessment of the actual drinking event, the alcohol concentration is also determined in urine. In forensic laboratories, two independent methods must be used, the enzymatic alcohol dehydrogenase (ADH) method and the headspace gas chromatographic method (HS-GC). Clinical laboratories confine themselves to the ADH method. The HS-GC method includes regularly the determination of methanol and acetone and can be extended to volatile poisons such as diethyl ether or chloroform (cf. Sect. “Toxic gases and volatile substances”). If the long-term alcohol consumption of the individual needs to be assessed, the determination of the alcohol markers ethyl glucuronide (EtG) and ethyl palmitate (EtPa) in a hair sample is the method of choice [50].

Carbon monoxide, CO-Hb

In history, town gas (coal gas) with a carbon monoxide concentration of 9–18% was generally used for cooking in towns until around 50 years ago when it was step by step replaced by natural gas with methane as the main component. As a result of this easy access and the relatively soft and painless way of dying, inhalation of carbon monoxide was the most frequent suicide method in towns at that time. But carbon monoxide poisonings are also not seldom at present. Besides its industrial use as an intermediate product, it is formed always if carbon-containing material burns under limited access to oxygen. Fire in houses and apartments, defects in chimneys of stoves and gas heating, exhaust fumes from cars in closed garages, or inside barbecues with charcoal are frequent reasons for accidental deaths by carbon monoxide. Moreover, methods to produce CO for suicide purposes from charcoal or formic acid were practiced [51, 52].

Carbon monoxide binds 200–300 times stronger to hemoglobin than oxygen, leading to carboxyhemoglobin (CO-Hb, Fig. 8a). In this way, blood loses its ability to transport oxygen with the consequence of internal suffocation. The degree of intoxication depends on the percentage of CO-Hb in this reversible competition between CO and O2. First symptoms (headache, racing heart) occur at 10–20%, blackout and circulatory collapse at 30–40%, and death between 40% and 70%.

Fig. 8

a Structure of carboxyhemoglobin in equilibrium with CO and O2. b Visible absorption spectra of O2-Hb, deoxy-Hb, and CO-Hb. c Visible absorption spectra at increasing CO-Hb concentration in a method based on the different stability of deoxy-Hb and CO-Hb [55]

CO-Hb should be determined in heart blood (cf. Sect. “Heart blood”). In principle, the concentration of CO dissolved in blood can be determined by infrared spectroscopy methods [53]. However, in practice, the percentage of CO-Hb is determined and much more suitable for interpretation. The methods are based on the different absorption spectra of the hemoglobin species deoxy-Hb, O2-Hb, and CO-Hb (Fig. 8b). It is usually included in the automatic determination of the hemogram in clinical laboratories whereas in legal medicine separate spectrophotometric methods are often used.

CO-Hb is much more stable against heat and alkaline pH than deoxy-Hb or O2-Hb. This is used in preliminary tests during the autopsy. On heating a postmortem blood sample in a test tube for 5 min in a boiling water bath, the cherry-red color of CO-Hb remains stable and is visible above a content of 40% whereas it turns gray-brown in the absence of CO-Hb. The higher stability of CO-Hb in alkaline medium is also used in a spectrophotometric method for its determination (Fig. 8c).

CO-Hb is regularly determined in fire victims. In these cases, the cause of death is usually complex. Besides CO, fire gases may contain HCN (Sect. “Cyanide”), SO2, NO2, HCl, and organic irritants such as formaldehyde, depending on the burning material. Inhalation of soot, heat, and decreased oxygen content in air add to these hazards to a different extent. High CO-Hb and soot in the airways prove that the victim was still alive during the fire. On the other hand, burning outside (victims of self-burning or of murder by burning) showed only low CO-Hb values.

Methemoglobin (Met-Hb)-forming substances

Met-Hb is formed from hemoglobin by oxidation of the iron ion of the hemin moiety from Fe2+ to Fe3+. As a result, similar to the formation of CO-Hb, hemoglobin loses is ability to bind and to transport oxygen. Small amounts of Met-Hb in blood are steadily formed by endogenous oxidation processes, but this is limited to a proportion of about 1.5% by the NADH-dependent enzyme methemoglobin reductase.

There are two mechanisms for the formation of toxic high Met-Hb concentrations. Inorganic oxidizing substances such as NaClO3 (previously used as total herbicide) or NaNO2 (used as pickling salt) directly oxidize hemoglobin to Met-Hb. Nitrate in well water which mainly originates from inorganic fertilizers has the same effect. NO3− > 10 mg/L is particularly dangerous for siblings whose methemoglobin reductase is not yet developed. The second mechanism is the oxidation of hemoglobin with oxygen under the catalytic action of aromatic amino or nitro compounds. The essential step in this complex mechanism is the reaction of O2-Hb with the arylhydroxylamine to form Met-Hb and the corresponding nitroso aromate from which the arylhydroxylamine is regenerated by the NADPH/NADP+ redox system. The required arylhydroxylamine can arise from both the amino and the nitro compound in the biological environment. On the other hand, injection of methylene blue as the antidote operates as a catalyst for the enzymatic reduction of Met-Hb.

Typical symptoms of a Met-Hb intoxication are cyanosis and chocolate brown blood. Concentrations of Met-Hb are given as % MetHb of total hemoglobin. The analytical determination in hemolyzed blood is based on its absorption band at 630 nm, which disappears after addition of excess KCN [54] (Fig. 9a). Total hemoglobin is measured from an aliquot of the same solution after oxidation of all hemoglobin to Met-Hb with K3Fe(CN)6. At a methemoglobin level of 20–50%, patients experience dizziness, fatigue, headache, and weakness; levels above 50% cause dysrhythmia, central nervous system depression, coma, metabolic acidosis, seizure, and tachypnea. Levels above 70% are lethal.

Fig. 9

Spectrophotometric determination of Met-Hb in a p-nitroaniline poisoning case [62]. a Typical absorbance of Met-Hb at 630 nm, which disappears after addition of cyanide. b p-Nitraniline residues adhering on the brownish colored gastric mucosa. c Identification of p-nitroaniline and its metabolites p-nitroacetanilide (M3) and 2-hydroxy-4-nitroaniline (M4) by HPLC–DAD in blood of this case

If a lethal Met-Hb level is detected, the source must be found. In most cases, this becomes clear from the circumstances of death (Sect. “Site where the unconscious person or the corpse was found”). If not, further tests are necessary. Inorganic oxidants are best detected in gastric content, and ion chromatography with conductivity detector may be useful as in the case of chlorate [56], or nitrite [57] where mainly nitrate was found after metabolic oxidation. Sodium nitrite as a mild and easily accessible means of suicide has become increasingly popular recently as a result of commentary on the internet [58, 59]. Aliphatic esters of nitrous acid lead to Met-Hb formation as well, as seen for amyl nitrite, which is abused as “poppers” for sexual stimulation [60]. By the way, generation of a controlled Met-Hb level by injection of sodium nitrite as well as inhalation of amyl nitrite has been proved as an efficient antidote in cyanide poisoning [61].

For aromatic amino and nitro compounds as the reason for high Met-Hb, the methods described in Sect. “Nonvolatile organic toxic substances” are usually successful. In the case of a 15-year-old boy who took a p-nitroaniline overdose in order to try it as a recreational drug, the substance was visible as a deposit on the gastric mucosa (Fig. 9b) and was clearly identified and quantified by HPLC–DAD (Fig. 9c) and by GC–MS [62]. Acute poisonings with aniline derivatives used as components of hair coloring [63] and with nitrobenzene [64] were reported.

Cyanide

Cyanide is a classical poison which frequently occurs in accidental, suicidal, and criminal cases. Therefore, each clinical or forensic toxicological laboratory should be able to perform its detection and quantification. KCN is the most frequently used cyanide, but other alkali or alkaline-earth cyanides or liquid HCN (boiling temperature 26 °C) are common. It is practically used in electroplating, as a chemical intermediate, or in baits for killing rats or pigeons. It has a cruel history of mass killings in Nazi concentration camps during the Second World War and, e.g., in the mass suicide of the Peoples Temple sect in Jonestown Guyana in 1978 [65]. HCN was used for execution of death penalties in the USA [66], and the “German Society of Human Dyeing” sold KCN to their clients for up to 5000 DM (Deutsche Mark)/g in the 1990s [67]. A biogenic source is amygdalin, a cyanide-containing glycoside in bitter almonds. HCN is always formed if nitrogen-containing material burns under limited air access and is, therefore, regularly present in gases of burning apartments or houses.

The toxic mechanism is the binding of CN− to Fe3+ of the cytochrome oxidase in the respiratory chain of the mitochondria, with the consequence of a changed redox potential and the blockade of this enzyme. O2-Hb which is transported by blood circulation to all cells cannot be depleted. As a result, oxygen-saturated bright-red blood is also contained in the veins and leads to the red color of the skin and of the livor mortis in death cases.

The lethal dose of HCN is 70–140 mg for adults (1–2 mg/kg). After inhalation of HCN, death occurs within seconds. However, the lethal effect may be delayed after oral intake of NaCN or KCN since CN− is not resorbed from the alkaline solution and must first be transformed into HCN by the gastric acid. Rescue is impossible at high doses. But at smaller doses, antidote therapy with thiosulfate, hydroxocobalamin, and Met-Hb-forming substances (NaNO2, amyl nitrite) can be successful [68].

Various methods are used for determination of cyanide [69]. For spectrophotometric detection, HCN is separated from the acidified human sample by isotherm diffusion into NaOH solution and subsequently transformed into a colored derivate (pyridine-barbiturate reaction) [70]. The headspace gas chromatographic determination (HS-GC) with electron capture detector is based on the derivatization to cyanogen chloride Cl-CN with chloramine T in a reaction precolumn [71, 72]. Direct measurement of HCN was performed by HS-GC with nitrogen-phosphorus detector and acetonitrile as internal standard [73]. This was improved by mass spectrometric detection (HS-GC–MS) and using K13C15N as internal standard [74]. In the laboratory of the author of this paper, cyanide was determined by isotherm distillation of HCN at room temperature into 0.1 M NaOH solution and subsequent measurement on a cyanide-sensitive electrode (Fig. 10). Instead of blood, urine and tissue homogenates can be analyzed, too. Gastric content can directly be measured after, e.g., 1:100 dilution with 0.1 M NaOH. A colorimetric test is used as a second method for confirmation. The limits of determination for all methods in blood are 0.01–0.05 µg/mL.

Fig. 10

Determination of cyanide in blood by a cyanide-sensitive electrode. a Isothermal distillation of HCN from acidified blood to 0.1 M NaOH at room temperature. b Measurement setup for the cyanide-sensitive electrode with ionometer and magnetic stirrer. c Cyanide-sensitive electrode with Ag2S membrane from below. d Test tube with electrode, magnetic stirring bar, and cyanide-containing NaOH solution

Cyanide concentrations in blood of unexposed individuals are below 0.1 µg/mL. Values above 1 µg/mL are life-threatening. Values between 0.05 and 10 µg/mL were found in blood of fire victims, depending on the burning material. For instance, for a couple who died during a fire of an artificial polyamide Christmas tree the concentrations were 8.5 and 10 µg/mL. The postmortem concentrations after oral intake of KCN are usually 10–100 µg/mL.

Ethylene glycol, diethylene glycol

Ethylene glycol HO–CH2–CH2–OH and diethylene glycol O(CH2–CH2–OH)2 are viscous, water-miscible liquids of low volatility and sweet taste which are used as antifreeze, lubricants, and solvents. They are toxic and oral doses > 1 mL/kg are believed to be fatal. Accidental mixing up with beverages is the cause of most intoxications [75], for instance, of a diethylene glycol-containing smoke fluid [76], but there are also suicides and homicides with ethylene glycol [77, 78]. Mass poisonings by diethylene glycol used as an inappropriate excipient for preparation of medications were reported [79]. The toxic effect is, like methanol, caused by acidic metabolites such as glycolic acid and oxalic acid for ethylene glycol and 2-ethoxyhydroxyacetic acid for diethylene glycol which leads to severe metabolic acidosis, renal and hepatic failure, seizures, coma, and death. Postmortem concentrations in blood between 0.3 and 2.4 mg/g were reported.

Typical clinical symptoms are acidosis, high osmolar and anion gaps, and calcium oxalate crystals in urine or kidney [77, 80]. Despite the high concentrations, determination of these compounds and their metabolites in human samples needs special methods because of their high hydrophilicity and low volatility. Direct measurement from blood was possible by thermal desorption gas chromatography [81]. The simultaneous GC–MS measurement of ethylene glycol, 1,2-propylene glycol, and glycolic acid was performed after derivatization of the carboxylic groups with isobutyl chloroformate and of the aliphatic OH groups with phenylboronic acid [82]. GC–MS [83] and positive ESI-LC–MS with measurement of the Na+ adduct of ethylene glycol [84] were also described.

In the laboratory of the author of this paper, a simple method for determination of ethylene glycol was applied which could easily be integrated into the HS-GC measurement of alcohol. Ethylene glycol is cleaved with periodate into two molecules of formaldehyde which are reduced to methanol with sodium borohydride.

$$}_} }_} } \mathop\limits^}_} }}}_} } = } \mathop\limits^}_} }}}_} }$$

(1)

In this procedure, 1 mL H2O and 3 mL 4% HClO4 are added to 1 mL blood or serum. After homogenization and centrifugation, 0.2 mL of the supernatant it transferred into a headspace vial, and 0.2 mL of 10% NaIO4 solution are added. After 5 min, about 2 mg solid NaBH4 is mixed with this solution, the vial is closed, and the contents are analyzed for methanol according to the routine HS-GC alcohol method with tert-butanol as internal standard. Propylene glycol yields an equimolar mixture of methanol and ethanol.

Immunoassays for drugs and poisons

Immunoassays have gained increasing importance in various bioanalytical fields such as clinical diagnostics, environmental monitoring, and food testing. The principles, methodologies, and applications were comprehensively described in books [85, 86] and reviews [87, 88]. Immunologic analysis as the essential principle of a large variety of advanced spot testings was also presented in a previous lecture of ChemTexts [89]. The principle is based on the highly sensitive and selective, quasi-irreversible reaction of the analyte (antigen) with specifically tailored antibodies.

$$} + }\left( }} \right) \to }$$

(2)

Antibodies are proteins with a Y-shaped structure and consist of four chains, two light (M ≈ 20,000) and two heavy (M ≈ 50,000–70,000) chains which are linked together by disulfide bonds (Fig. 11a). Besides a constant region, each chain has a variable region which enables the specific binding of the antigen. Under natural conditions, antibodies are formed only against antigens (peptides, proteins etc.) with a molecular mass > 3000. But the antigens carry usually a smaller determinant group (epitope) by which the antibodies recognize and distinguish them from body’s own tolerated molecules. This enables the preparation of antibodies with high affinity for smaller molecules: In an animal experiment, the target analyte is chemically bound to albumin (Fig. 11b). After reinjection, the immune system of the animal recognizes the modified albumin as foreign with the analyte moiety as the determinant group and forms specific antibodies. These have a high affinity also to the non-bound, free analyte and can be isolated and multiplied by biochemical techniques to form the analyte-specific reagent.

Fig. 11

Reagents used in immunoassays. a Structure of drug-specific antibodies. b Morphine differently bound to albumin to form opiate-specific antibodies. The cross-reactivity to individual opiates other than morphine depends on the site of binding. Binding at the 3-OH group leads to a high cross-sensitivity against codeine and morphine-3-glucuronide, where the N-CH3 group can be “seen” by the antibodies, whereas binding to the nitrogen atom leads to higher cross-sensitivity against normorphine or nalorphine with an N–H or N-allyl group and the 3-OH group “visible” to the antibody. c Immobilized or labeled antibodies and or target drugs used in immunoassays

Such antibodies often have a cross-affinity for metabolites of the analyte and to other substances with the same or similar structural features and, therefore, exhibit a group specificity for, e.g., opiates, amphetamines, or benzodiazepines. The degree of cross-sensitivity of the antibodies to other compounds depends on the kind of binding to albumin during their preparation, i.e., which part of the bound molecule the antibody “sees” during its formation (Fig. 11b).

The direct assessment of the analyte–antibody reaction is not possible. For this purpose, the following auxiliary reagents are necessary depending on the respective test assay (Fig. 11c): the immobilized analyte or the immobilized antibody, bound to the wall of a test vessel or to a test strip, and the labelled analyte or the labelled antibody for visual or instrumental determination. Practically used labels which are chemically bound to the analyte or to the antibody are 125I (radioimmunoassay, not used anymore), colloidal gold (visual test plates and strips), an enzyme in combination with a suitable substrate (enzyme immunoassay) or fluorescein (fluorescence polarization immunoassay). There are a large variety of implementations of immunoassays [86]. In forensic and clinical toxicology, enzymes are the most common labels employed in immunoassay methods. The enzyme-catalyzed reaction enables amplification of the signal and in this way increases the sensitivity of the method.

An example of a homogenous competitive enzyme immunoassay is the enzyme multiplied immunoassay technique (EMIT) with the analyte labelled with the enzyme glucose-6-phosphate dehydrogenase (Fig. 12). The substrates are glucose-6-phosphate and NAD+ which is transformed to NADH with its characteristic UV absorption band at 340 nm. The increase of the absorbance at this wavelength within a definite time is used for determination of the enzymatic reaction rate. After addition of antibodies and labelled analyte to the sample, analyte and the enzyme-labelled analyte compete for the antibodies. It is crucial for this method that the enzyme is sterically blocked in the labelled analyte—antibody complex. The remaining rate of NADH formation increases with the analyte concentration as shown by the calibration curve in Fig. 12. This method can be calibrated and can be performed in all automated clinical laboratory systems.

Fig. 12

Principle of the enzyme multiplied immunoassay technique (EMIT). D = drug to be tested; AB = drug-specific antibody; E-D = enzyme-labeled drug, glucose-6-phosphate dehydrogenase chemically linked to the drug; S = substrate of the enzyme, glucose-6-phosphate + NAD+; P = product of the enzymatic reaction, NADH with specific absorption at 340 nm. This is a kinetic test in which the increase of the absorbance of NADH at 340 nm under constant conditions is used for drug determination

A prerequisite of EMIT is the optical transparency of the sample at 340 nm. For this reason, its direct application is mainly limited to urine. A suitable sample preparation also allows the use of other materials [90]. Because of the group specificity, the predominant presence of metabolites in urine, and the general problems in quantitative interpretation of urine results (Sect. “Urine”), EMIT is mainly used as a pretest in toxicological screening for methadone, opiates (target substance morphine), cocaine (benzoylecgonine), amphetamines (methamphetamine), ecstasy (MDMA), cannabinoids (THC-COOH), and benzodiazepines (oxazepam). Test kits for medical drugs and drug classes are available, e.g., tricyclic antidepressants. For interpretation, cutoffs were defined which should as far as possible exclude false negative results. Positive samples must be confirmed, specified, and quantified by mass spectrometric techniques in order to gain judicial validity.

Besides application to drug-driving cases or workplace drug testing, urine testing with EMIT or another immunoassay (fluorescence polarization immunoassay [FPIA] [91], cloned enzyme donor immunoassay [CEDIA] [92]) is regularly performed as part of the systematic toxicological analysis in emergency and death cases.

Another broadly applied method is the enzyme-linked immunosorbent assay (ELISA) [93]. Owing to extremely high sensitivity, specificity, precision, and throughput it is used for a plethora of analytes. It serves as a pretest for drug testing in large sample series of blood [94], oral fluid [95], and hair [96]. Optical transparence of the sample is not necessary. Among the different kinds of ELISA available [93], the competitive technique is usually preferred for molecules with the lower molecular size of most drugs and poisons. The principle is shown in Fig. 13.

Fig. 13

Reagents and working principle of enzyme-linked immunosorbent assay (ELISA). For explanations, see text

The test is performed in “micro wells” (small tubes) of a 96-well microplate coated with analyte-specific antibodies. The sample with the analyte and a defined amount of the enzyme-modified analyte (enzyme: horseradish peroxidase) are pipetted into the well. The enzyme-modified analyte competes with the analyte of the sample material for the available binding sites of the antibodies. In the next step, the unbound material is washed out and 3,3′,5,5′-tetramethylbenzidine (TMB) + H2O2 is added as the substrate of the enzyme. In case of a negative sample, high enzyme activity remains in the well and TMB is oxidized to a blue colored product. The reaction is stopped after a certain time by addition of sulfuric acid and the color turns yellow with an absorption maximum at 450 nm. For a positive sample, the lower enzyme concentration in the well leads to a less intense color. The absorbance at 450 nm vs. analyte concentration calibration curve is inverse.

The providers of ELISA test kits supply automated procedures by employing robotic workstations equipped with washing and dispensing modules, automated mechanical handling and analysis, and microplate readers. The conventional 96-well formats were transformed into higher-throughput 384- and 1536-well plates.

Besides the application for large series pretests, immunoassays were also developed for special poisons, which are difficult or impossible to detect by other methods and partly could also be misused as warfare agents [97, 98]. Examples are poisons with peptide or protein structure:

The toxins of the mushroom death cap (Amanita phalloides), α-amanitin and β-amanitin, are cyclic octa