Methods of Analysis – Ante Mortem


Drug screening in antemortem specimens of forensic cases has many uses, all of which assist the investigating authorities in providing relevant information pertaining to the cases (Table 1). Ultimately, toxicology testing results will assist the courts in establishing the truth by either providing evidence of drug use, or by refuting the use of relevant drugs. This latter observation is important, as drug use is often suspected, but can only be confirmed by toxicology testing procedures.
Toxicology testing is particularly important in victims of sexual assault, who may have been given drugs by the assailant to reduce consciousness and memory. Drugs used in these cases are typically one of the benzodiazepines (flunitrazepam, alprazolam, etc.), barbiturates and, more recently, y-hydroxy-butyrate (GHB). Perpetrators of violent crime may
Table 1 Reasons for drug testing in forensic cases.
Establishing drug use in victims of sexual and physical assaults Establishing drug use in drivers of motor vehicles Establishing drug use in persons involved in workplace accidents Establishing workplace or environmental exposure of workers Assisting investigations for deaths occurring in hospital Assisting investigators with estimation of timing of drug use also have consumed alcohol or illicit drugs, or may even be under medication. In practice, drug users committing crimes are likely to be under the influence of two or more drugs. Drivers involved in motor vehicle accidents or traffic infringements are also frequently under the influence of two or more drugs. Toxicology testing on specimens taken soon after the incident may assist in establishing the sobriety, or otherwise, of these drivers at the time of the accident or traffic infringement. Drug testing in antemortem specimens of persons dying in hospital, and who come under a medical examination order, reduces the problems associated with postmortem redistribution. Postmortem processes can falsely elevate blood concentrations, frustrating any interpretation of postmortem toxicology.
Since the great majority of cases (>70%) involve more than one drug, it is advisable to conduct a broad drug screen to include most of the common drugs of abuse, rather than target the analysis to one or a limited range of drugs suggested by the circumstances.


Specimens collected ante mortem are most often blood, for plasma or serum, or urine; however, other specimens, such as hair, sweat, and saliva, have also been used to assess drug use.

Blood and plasma

Blood contains predominately red blood cells, white cells, and plasma. Plasma is obtained from non clotted blood by removal of the cells following centrifugation; serum is the liquid phase remaining after blood is allowed to clot. In this article, plasma and serum are considered to be one specimen, unless otherwise differentiated.
Blood, or plasma, or serum derived from blood, is the most useful specimen that can be collected, as drugs present in this fluid can best be related to a physiological effect and can be used to assess the likelihood of recent drug use or exposure to chemicals. Programs of therapeutic drug-monitoring of plasma are frequently conducted in clinical toxicological laboratories and form the basis of therapeutic drug compliance and optimization of drug doses. Typically, immunoassays are used in drug monitoring and screening, although high-performance liquid chroma-tography (HPLC), gas chromatography (GC), and mass spectrometry (MS) techniques are equally well suited.


This is, with blood or plasma, the most frequently collected specimen. Since concentrations ofdrugs and metabolites of drugs are usually much higher than in blood, urine provides a valuable specimen for assessing drug use over the previous day or two. Relatively large volumes (50 ml or more) can be collected, allowing sufficient specimen even for less sensitive techniques; however, the presence of a drug in urine does not necessarily imply recent drug use, let alone allow the prediction of possible drug effects. It is therefore advisable to include blood testing if an assessment of possible drug effects is required.


Hair has long been used to test for exposure to heavy metals, such as arsenic, mercury, and lead. Hair has also proved to be a useful specimen for the analysis of drugs. It is particularly useful for establishing drug use many weeks or months prior to collection.
Drug entry into hair is complicated and is likely to involve a number of processes. Incorporation by entrapment from the blood bathing the growing follicle is a major mechanism, although incorporation through direct contact of mature hair with sweat and/or sebaceous secretions is also a significant source of drug entry.
Because of the ability of hair to directly absorb drug, contamination of hair by direct environmental exposure should also be reasonably excluded if hair results are to be used. For example, nicotine is found in the hair of nonsmokers and cocaine is found in the hair of children of cocaine users. This is arguably the major limitation of this specimen.
The target analytes in hair are predominantly the parent drugs. Cocaine, A9-tetrahydrocannabinol (THC), heroin, and 6-acetylmorphine for heroin, and benzodiazepines are found in higher concentrations than their corresponding metabolites. In this respect, hair concentrations parallel more closely sweat concentrations than those of other specimens.
There are a number of factors that affect retention of drugs into hair: hair colour is one well-known factor. Pigmented hairs, particularly those found in black-haired Africans and Asians, have higher levels of cocaine than the often weakly pigmented Caucasian counterparts. This is likely to be true for all basic drugs which bind to forms of melanin, the major pigment in hair. Acidic drugs tend to have lower concentrations than basic drugs. Bleaching and the excessive use of shampoo and conditioners can also reduce the concentration of drugs in hair. For this reason, and because of the various routes of drug intake to hair, quantitative results in hair are rarely useful.


Sweating is a physiological process providing a mechanism of reducing body temperature. Sweat is produced by eccrine glands, located in the transder-mal layer of most skin surfaces and apocrine glands located in axillary and pubic regions. Approximately 50% of all sweat is produced by the trunk, 25% by the legs, and 35% by the head and upper extremities. Sweat is approximately 99% water, the remainder being sodium chloride. A rate of sweating of over 20mlh_1 is common. Sweat glands are often associated with hair follicles, and therefore, it is sometimes difficult to differentiate between the presence of drugs in hair and sweat.
Sweat is normally collected by using suitable absorbent devices such as sweat patches. Contact time may vary from a simple swipe over a portion of skin, to days for a sweat patch to absorb accumulated sweat. The device used and collection time will affect the detection of excreted drugs.
Advantages of sweat analysis are the high tolerability of patches, with correspondingly low incidence of allergic reactions, the ability to monitor drug use over several weeks, and the ability to withstand tampering. Disadvantages include ensuring cleanliness of skin and excluding other environmental contamination.
Drugs detected in sweat include alcohol (ethanol), amphetamines, cocaine, benzodiazepines, barbiturates, opioids, and phencyclidine.


Saliva is excreted primarily by three glands - the parotid, submaxillary, and sublingual – and by other small glands such as the labial, buccal, and palatal glands. Mixed saliva used for drug analysis is approximately 65% submandibular, 23% parotid, and 4% sublingual; the remaining 8% is from the other three glands.
The daily flow of saliva in an adult ranges from 500 to 1500 ml. Saliva flow is mediated by a number of physiological factors, paticularly emotional factors, age, gender, and food intake.
Saliva is not an ultrafiltrate of blood, rather it is a complex fluid formed by different mechanisms against a concentration gradient, by pinocytosis, by ultrafiltration through pores in the membrane, and by active transport. Passive diffusion is apparently the dominant mechanism.
Saliva is best collected by absorption onto an absorbent material or a device which stimulates production of saliva. A number of such devices are available to facilitate the collection process. It is also essential that collection of saliva takes place at least 30 min after a meal, or consumption of a beverage or drug, and the oral cavity is free from food material and other objects prior to collection.
The main disadvantage is that saliva volumes are usually small, hence there will be limited ability to repeat analyses. Additionally, not all subjects will be able to provide saliva on demand.
Interpretation of saliva drug concentrations is more difficult than in blood because saliva concentrations are subject to a greater number of variables, such as the degree of protein-binding, pKa of the drug, and the pH of the saliva. For some drugs (e.g. barbiturates, benzodiazepines), saliva concentrations are much lower than for blood, whereas, for others (e.g. amphetamines), concentrations are higher.


A variety of techniques is available for the detection of drugs in specimens collected ante mortem. These range from commercial kit-based immunoassays and traditional thin-layer chromatography (TLC) to sophisticated instrumental separation techniques, such as HPLC, GC, and capillary electrophoresis (CE). MS is the definitive technique to establish proof of structure of an unknown substance, although a number of other detectors can be used to identify the presence of unknown substances in biological specimens.


A number of different immunoassay methods are available for drugs of abuse. Numerous commercial kits now exist for this purpose. These include enzyme immunoassays (EIA) (e.g. EMIT) and enzyme-linked inmmunosorbent assays (ELISA), fluorescent immunoassays (FPIA) (e.g. Abbott TDx and ADx), agglutination or kinetic interaction of microparticles, immunoassays (e.g. TRIAGE and ONLINE), cloned enzyme donor immunoassay (CEDIA), and radio-immunoassays (RIA) (DPC assays). These kits also include devices for rapid on-site testing of blood, urine, and sweat without the need for analyzers.
These tests have the advantage of recognizing more than one member of a class of drugs, e.g. amphetamines, benzodiazepines, opioids. However, not all members are detected with equal sensitivity, which will not only be dependent on the crossreactivities of the antibodies to the benzodiazepines, but also on the profile of metabolites present in urine, and the amount of target drug. Different batches of antibody will also influence the sensitivity and selectivity to benzodiazepines and their metabolites.
The overall sensitivity can also be increased by prior hydrolysis of urine to convert glucuronide and sulfate conjugates to substances that are detectable by the kit, although reducing recommended cutoff concentrations can accommodate most of the loss of sensitivity. This technique is particularly useful for cannabis, morphine, and the benzodiazepines that are metabolized to conjugates.
Most kits are directed to urine, although many are available for plasma. Urine-based kits can be used for all types of antemortem specimens by appropriate modification. Precipitation of blood proteins by treatment with methanol, acetonitrile, dimethylformamide, or acetone, and direct analysis of the supernatant are frequently used techniques; however, the high-potency drugs are not always detected. Prior extraction of blood with a solvent (e.g. butyl chloride) provides improved detectability because a concentration step can be employed and most interferences have been removed. With all of these techniques, not all drugs are extracted. Individual validation must be conducted to ensure adequate detectability.
False-positive results with immunoassays occur, either from structurally related drugs, from metabolites of other drugs that are recognized by the antibodies, or occasionally by artifacts, such as adulterants affecting pH, detergents, and other surfactants. For this reason, any positive result must be confirmed by an alternative technique, preferably chromatography with mass spectral identification.

Thin-layer chromatography

This is the oldest of the chromatographic techniques and is still widely used in forensic laboratories as a screening technique. The movement of an organic-based solvent on a plate containing an absorbent material is based on the separation of drugs (and their metabolites). The stationary absorbent phase is typically silica, although other supports are used.
Chromatography is usually rapid (less than an hour) and a number of samples can be run simultaneously at little cost. Drugs are identified by visualization under ultraviolet light (as a darkspot), or by spraying with one of a number of reagents, which are directed to specific chemical moieties (as a colored spot), or to organic compounds generally.
The retention factor is calculated by dividing the distance moved from the origin over the distance moved by the solvent front. Characteristic colors of the spots, presence of metabolite patterns and the retention factor values provide a good means of identifying drugs in biological specimens. Unfortunately, the technique is relatively insensitive and is usually limited to urine analysis, although analysis of gastric contents and liver extracts (in postmortem analysis) is also possible. Densitometry of TLC plates can provide some quantitation of the amount of drug present in an extract. Detection limits of 500ngml_1 are possible from 5 ml of urine.
The use of high-performance TLC plates (HPTLC) has been shown to provide higher sensitivity and can detect some drugs down to 100ngml_1 from 1 ml of blood. Since specificity is not very high, it is still advisable to confirm any positive result by an alternative technique, preferably MS identification.

High-performance liquid chromatography

HPLC is a commonly used chromatographic system that involves the separation of compounds by partitioning between a pressurized moving liquid phase and a solid support containing very fine silica (410 um diameter particles) or bonded silica. The bonded lig and acts as a pseudoliquid phase. Bonded groups include C2, C8, C18, CN-alkyl, and phenyl-alkyl chains. The physiochemical properties of the bonded phase and the moving phase determine the separation process.
Moving phases are often hydroalcoholic solvent systems, such as methanol/unbuffered water to solvent/buffered phosphate solutions, the base modifier triethylamine and ion-pairing reagents, such as methane sulfonic acid, tetramethyl ammonium hydrogen sulfate, and tetrabutyl ammonium bromide. Gradient programming, in which the composition of solvent is altered with time, provides an ability to separate compounds of widely differing polarity. Normal phase chromatography on a CN-, OH-bonded column or a silica column functions in a similar way to TLC, except that resolution and sensitivity is far higher.
Detection of the sample is most often by ultraviolet spectrophotometry at or near the maximum absorption wavelength. Alternatively, other physiochemical properties of the compound(s) can be exploited. These include infrared, fluorescence, phosphorescence, electrochemical properties, and conductivity (for ionically charged substances). Compounds with functional groups can be reacted with reagents to impart greater detectability with one or more detectors, or to allow resolution of stereoisomers (Table 2).
Photodiode array detection (to supplement ultraviolet light detection) offers real advantages to analysts in identifying peaks and assisting in establishing peakpurity. Photodiode array detection can be a very useful technique if MS instrumentation is not readily available, or if absolute proof of structure is not required.
Detection limits around 10-50ngml_1 are expected for most compounds by HPLC, depending on the physiochemical properties of the drug, the volume of specimen extracted, and the method used. Lower detection limits are possible if larger amounts of sample are extracted and when a concentration step is employed.
Solid-phase extraction using small columns to selectively absorb drug from the matrix (e.g. Extrelut, Sep-Pak, Bond-Elut, etc.) provides an excellent alternative to conventional liquid-liquid extraction techniques. Solid-phase techniques have been published for most analytes and tend to be quick, often provide clean extracts, and can be readily automated.
Narrow-bore columns (~ 1-2 mm internal diameter) require less specimen and can easily be interfaced with MS. The combination of HPLC with MS (LC-MS) and tandem MS (LC-MS-MS) provide good examples of the separation power of HPLC with the sensitivity and specificity of MS. Detection limits range from 10 pg on-column, resulting in detection limits of better than 1 ngml-1 for many compounds using a thermospray or electrospray interface.

Gas chromatography

GC is based on the principle of partitioning a substance in a gaseous phase from a stationary liquid phase. The stationary phase is typically a polymeric liquid, which is either coated on to silica or chemically coated onto the glass surface of the column itself. The nature of the functional groups and polarity of the polymer, and the temperature of the column, provide the means to vary the separation conditions.
Typically, columns are flexible capillaries made of fused silica, with internal diameters of 0.1-0.5 mm, and are coated with heat resistant polymers to promote flexibility. A large range of columns is available to provide analysts with sufficient flexibility to optimize separation conditions. The type of columns range from low polarity dimethylpolysiloxane, 14% cyanopropylphenyl, 5% diphenyl methylpolysilox-ane to the polar trifluoropropylpolysiloxane to 50% diphenyl methylpolysiloxane phases. The use of a cyanopropylphenyl or 5% phenylmethylsilicone stationary phases can give better separation of a number of moderately polar compounds than a 100% methylsilicone phase. Due to the wide polarity differences of drugs, temperature programming is necessary for assays involving detection of a number of drugs.
A range of detectors is available for GC. Flame ionization detectors are workhorse detectors for any compounds containing carbon, whereas a number of detectors are available for specific functional groups. The nitrogen-phosphorus detector selectively detects compounds with either nitrogen or phosphorus, while the electron capture detector relies on the ability of a compound to capture electrons when passing through an electric field. Electron capture detectors give the best detection limits (^ 1ngml_1) from 1.0 ml plasma, although the nitrogen-phosphorus detector provides detection limits down to 5ngml_1 for nitrogenous substances, and better than 1 ngml-1 for phosphorus-containing substances (e.g. organophosphate pesticides) (Table 3). Poisonous and other gases can be detected by use of thermal conductivity detectors which do not rely on the presence of carbon or nitrogen.

Table 2 Detection systems used in HPLC analysis of selected drugs.

Drug class Detector
Amphetamines UVand F (of derivatized drug)
Analgesics (acetaminophen, salicylate) UVand photodiode array
Anions (bromide, chloride, azide, etc.) Ion conductivity
Antidepressants UVand photodiode array
Benzodiazepines UVand photodiode array
p2-stimulants (salbutamol, fenoterol, etc.) F
Cannabinoids (THC, carboxy-THC, etc.) ECD and photodiode array
Catecholamines (epinephrine, dopamine, etc.) ECD
Cocaine and metabolites UVand photodiode array
Morphine/codeine ECD, F and UV
Quinine/quinidine F and UV

For drugs to be amenable to GC they must be thermally stable to enable volatilization into an inert gas (e.g. helium, nitrogen). In many cases compounds can be derivitized to improve their thermal stability, or to alter their retention characteristics and thus, enable a separation to occur (Table 4).
Solid-phase microextraction is a relatively recent technique, enabling rapid analysis of drugs without requiring extensive sample clean-up and concentration. Direct online injection using a dialysis technique involving a copolymer precolumn for absorption has also been reported on small sample volumes.

Capillary electrophoresis

A powerful emerging technique showing widespread application in forensic science is that of CE. It is actually a number of related techniques, including capillary zone electrophoresis, micellar electrokinetic capillary chromatography, capillary electrochroma-tography, capillary isotachophoresis, capillary gel electrophoresis, and capillary isoelectric focusing, and is complementary to HPLC with high separation power.
In its most simple form, CE employs a separation capillary of 20-100 um internal diameter and up to 100 cm long, a high voltage source, electrodes, an injection system, and a detector. The capillary is often fused silica, coated with plastic polyimide to confer elasticity. The capillary ends are dipped in buffer and are held at a potential of up to 30 kV. The separation is based on migration of charged drug molecules against an electric field, and electroosmosis caused by the osmotic migration of cations and water to the cathode as a result of ionization of the silyl hydroxyl groups on the fused silica. The electroosmosis factor can be altered by changing the pH of the buffer, ionic strength of buffer, modifiers added to buffer, and type of capillary internal wall coating.
The amount of sample or biological extract applied to CE is in the nanogram scale, allowing for trace analysis with adequate sensitivity for most applications.
Electrokinetic micellar chromatography has been shown to be capable of the analysis of illicit drugs in urine and in plasma. This is a powerful technique, as it can separate a large range of compounds with high sensitivity and has the ability to separate compounds of widely differing polarity in one run.
Multiwavelength ultraviolet light detection can be used to provide an added degree of confirmation. The sensitivity is adequate for routine confirmatory analyses of presumptive positive urine specimens for drugs of abuse. CE can also be linked to other detectors, including the mass spectrometer.

Mass spectrometry

MS is the definitive technique if unequivocal identification of unknown compounds is required for forensic purposes. It is usually linked directly to a chromatographic separation process such as CE, HPLC, or GC, or even to another mass spectrometer (MS-MS).
Compounds do not always show characteristic spectral detail (e.g. amphetamines). Consequently, it is recommended that derivatives should be prepared for such compounds, or for substances which show poor chromatographic properties (Table 3). One of the derivatives most frequently described is the tri-methylsilyl ether for amines, hydroxy-, and carboxyl-containing substances. Alternatively, other silyl ethers such as £-butyl are used, and fluorinated acyl anhydrides (e.g. pentafluoropropionic anhydride) are widely used for amines and hydroxy compounds, and a combination of a perfluorinated alcohol with a perfluorinated acyl anhydride for carboxy-, hydroxy-, and amine-containing substances. Other derivatives are also known.

Table 3 Detection systems used in GC analysis of selected drugs.

Drug class Detector
Amphetamines NPD, EI-MS, NCI (as derivative)
Antidepressants NPD, EI-MS
Antipsychotics NPD, EI-MS
Benzodiazepines NPD, ECD, NCI
Cannabinoids (THC, carboxy-THC etc.) EI-MS, NCI (as derivative)
Carbon monoxide, and other gases TCD
Cocaine and metabolites NPD, EI-MS (as derivative of BE)
Heroin, morphine and other opioids NPD, EI-MS (as derivative for morphine)
Organophosphate pesticides NPD, EI-MS

Table 4 Derivatives used in GC-MS analysis of selected drugs.

Drug class Detector
Amphetamines AA, HFBA, methyl chloroformate
Barbiturates None, or iodomethane in TMAH
Benzodiazepines t-butyl-DMS, TMS, PC/PI
Cannabinoids (THC, carboxy-THC etc.) TFAA, TMS, PFPA/PFP, t-butyl-DMS
Cocaine and metabolites t-butyl-DMS, PFPA/PFP, TMS
Morphine HFBA, TMS

Positive-ion chemical ionization produces a much higher intensity molecular ion, and is often used to reduce fragmentation and to provide evidence of the molecular weight of the compound. In this mode, reagent gases, such as methane and ammonia, are used to produce different ion-molecule collisions in the ion chamber (source).
The use of negative-ion chemical ionization affords a greatly enhanced detection limit for certain compounds, compared with electron impact mass spec-trometry. In this NCI mode a single ion cluster is often observed and can provide, for some drugs (e.g. benzodiazepines and derivitized THC), a detection limit of 0.1 ngml-1.
The use of deuterated internal standards provides an ideal way of monitoring changes in chromato-graphic performance, and, most importantly, essentially eliminating matrix effects caused by poor recoveries of drug. While recoveries of drug may vary from one matrix to another, and even from calibrators, the deuterated internal standard will correct for this. For this reason, assays involving MS should use deuterated internal standards wherever possible.

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