13

Sampling, storage and stability

S Kerrigan

Introduction 335 Contamination 346

Specimen selection considerations 335 Stability 350 Sample handling 345 References 354

Introduction of Pathologists has published guidelines for handling medicolegal specimens and preserving the chain of Appropriate selection, sampling and proper storage of evidence, the Faculty of Forensic and Legal Medicine biological evidence are important, yet sometimes over- has published recommendations for the collection of looked, steps in forensic toxicology. These factors, forensic specimens from complainants and suspects, in combination with drug stability, can profoundly and the European Workplace Drug Testing Society impact the interpretation of results and the outcome has published guidelines on the collection of urine, of forensic casework. Criteria surrounding each of oral fluid and hair. these are presented and discussed in the material that Timing is an important factor in specimen collec- follows. Further reference to tissue sampling will also tion, particularly in antemortem cases where some be found in other chapters in this book and will be drugs have short detection times and therefore limited cross-referenced within the text where appropriate. detection windows; examples include detection of an elevated concentration of 9-tetrahydrocannabinol (THC) in blood from an impaired driver, or of Specimen selection considerations gamma-hydroxybutyric acid (GHB) following an alleged drug-facilitated sexual assault. Selection of the appropriate specimen is a critical Postmortem specimens pose additional challenges component of any toxicological investigation. Cir- owing to autolytic and putrefactive changes. Timing cumstances surrounding the case, the availability of is also important in death investigations because it specimens, the nature of the investigation and even becomes increasingly difficult to obtain good-quality legal or statutory issues may dictate which specimens specimens as the time between death and sampling are selected, and for what purpose. The role of the (postmortem interval) increases. Factors such as toxicologist is to recommend the most appropriate embalming of the body, decomposition or burial samples to be collected and tested for specific inves- can further complicate interpretation if tissues have tigations with various guidelines available on how been preserved, if specimens are putrefied, or if to collect different sample types. The Royal College exhumation is necessary. In order to be able to

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are preferred by many, disposable plastic containers Table 13.1 Specimen selection are used routinely for a wide variety of postmortem considerations tissues and antemortem samples, particularly urine. • Ease of use If glass containers are used, it is important to make use of appropriate racks for storage and trans- • Ease of specimen collection portation. One of the major disadvantages of glass is • Presence of interferences the possibility of breakage, particularly during stor- age at low temperatures. In order to minimise sample • Matrix effects loss, glass containers are preferred if volatile analytes • Parent drug and/or metabolites such as solvents or anaesthetic gases are suspected.

• Detection time Plastic containers are more susceptible to interfer- ences by plasticisers such as phthalates that might • Stability of the drug(s) in the specimen interfere with the analysis. The use of an inert plastic

• Putrefaction such as Nalgene decreases the likelihood of chemi- cal interference, but it is good laboratory practice to • Potential for automated analysis evaluate all new specimen containers prior to rou- • Sample volume tine use in the laboratory. If plastic containers are chosen, their integrity at low temperatures should be • Indication of short-term or long-term drug use evaluated. Polystyrene is more susceptible to cracking • Reference data at frozen temperatures than polypropylene vessels. Rubber septa or liners in screw-cap containers should • Interpretive value be avoided and replaced with inert liners (e.g. polyte- trafluoroethylene (PTFE), or Teflon) to reduce leakage select the appropriate specimen(s) the toxicologist and minimise drug adsorption. should have access to the case history, autopsy Antemortem blood samples are generally collected records/pathologist’s report and all other relevant into evacuated glass tubes such as Vacutainer or Veno- documents. ject for forensic toxicology purposes. Collection of Some of the important specimen selection consid- blood into similar glass collection vessels is also good erations are listed in Table 13.1. practice in postmortem blood sampling. These tubes allow the sample to be collected into a vessel that already contains necessary additives to stabilise and Specimen containers preserve the matrix. Proper mixing is necessary when It is important that the specimen container is appro- sodium fluoride or other additives are used to ensure priate for the intended use and does not compro- that dissolution is complete. mise the analytical findings. Container size should be appropriate for the volume or weight of the specimen Blood so that headspace is minimised. Typical specimen col- lection quantities are given in Table 13.2. Excessive Blood is one of the most important specimens of tox- headspace in the container can increase the chance of icological interest as it provides unique advantages oxidative loss, volatilisation of analyte (e.g. ethanol over other matrices in terms of the wide variety of and other low-boiling-point compounds) or salting analytical methodologies available, the vast amount out, which may occur if preservatives are present. of published reference data for both antemortem and Some analytes have a tendency to adhere to plastic postmortem drug concentrations, and the interpretive or glass surfaces depending on their physicochemi- value of the matrix from a pharmacological stand- cal properties. Silanisation of glassware can reduce point. However, antemortem and postmortem blood adsorptive losses for drugs that are present at trace samples are notably different, and the site of the post- levels (10 μg/L or less). Although glass containers mortem blood draw (central or peripheral) can be

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Table 13.2 Typical specimen collection quantities

Postmortem Antemortem

Specimen Quantity Specimen Quantity

Blood, heart 25 mL Blood 10–20 mL

Blood, peripheral 10–20 mL Urine 25–100 mL

Urine All Amniotic fluid 5–30mL

Bile All Breast milk 10–20 mL

Vitreous humour All Meconium All

Cerebrospinal fluid All Hair Pen-size lock

Gastric contents All Saliva 1–5mL

Liver 50 g Sweat Microlitres (insensible sweat); 1–5 mL (sensible sweat)

Kidney 50 g

Spleen 50 g

Brain 50 g

Lung 50 g

Hair 50 g Pen-size lock (150–200 hairs or 50 mg)

Sources: Dinis-Oliveira et al. (2010); Hepler and Isenschmid (2007); Kerrigan (2002); Kidwell et al. (1998); Skopp (2004); SOFT/AAFS (2006). of critical importance. Determination of parent drug plastic tubes have also been evaluated (Karinen et al. and metabolite concentrations (and their ratios) may 2010). also yield useful information pertaining to acute or chronic use. A summary of the common advantages Postmortem blood and disadvantages of various specimens is given in Postmortem blood collected at autopsy is quite Table 13.3. different from antemortem blood collected by Antemortem blood is collected by venepuncture, venepuncture from both qualitative and quantitative typically from the antecubital region of the arm, using standpoints. Postmortem blood may be more viscous, a syringe or evacuated container (e.g. Vacutainer, may contain numerous small clots or sedimented Venoject). Prior to collection, an antiseptic wipe cells, has a lower pH (as low as 5.5 owing to protein is often used to clean the collection site. Non- degradation), may contain 60–90% water, and is alcohol-containing antiseptic wipes such as Betadine subject to varying degrees of haemolysis. The site (povidone–iodine) are preferred to avoid any con- of blood collection should be clearly identified on tamination that could interfere with alcohol analysis. postmortem specimens and blood from different Although evacuated blood tubes are typically glass, sources should never be combined.

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Table 13.3 Advantages and disadvantages of antemortem (AM) and postmortem (PM) biological specimens

Specimen Advantages Disadvantages

Amniotic fluid • Determination of prenatal drug exposure • Invasive collection • Not readily adulterated • Risk of complications • Minimal sample preparation • Limited reference data • Relatively few interferences • Collection by medical personnel

Bile • Ease of detection of certain drugs (accumulation) • Complex matrix • Particularly useful for conjugated drugs • Interferences due to bile salts and fats • Requires sample preparation/pretreatment • Limited reference data

Blood (AM) • Widely accepted matrix • Invasive collection • Determines recent drug use (hours–days) • Collection by medical personnel • Related to pharmacological effect • Shorter detection time • Not readily adulterated • Extensive reference data

Blood (PM) • See above (AM) • Susceptible to postmortem redistribution (central) • Reference data widely available • Susceptible to postmortem artefacts and interfer- • Central/peripheral blood drug ratios known for some ences drugs • Susceptible to contamination (e.g. trauma) • Cardiac blood typically in plentiful supply but • Quality of specimen highly dependent on collection requires caution with interpretation protocol • Limited volume of peripheral blood

Brain • Particularly useful for lipophilic drugs, volatiles and • Non-homogeneous matrix centrally acting drugs • Drug concentrations vary by region • Complex matrix • Requires sample preparation/pretreatment • Limited reference data

Breast milk • Determination of neonatal drug exposure • Privacy, invasive collection • Not readily adulterated • Limited reference data • Many drugs present • Interferences due to high lipid content • Drug content varies with milk composition • Variable matrix

Cerebrospinal fluid • Determines recent drug use (hours–days) • Invasive collection • Minimal sample preparation • Limited reference data • Relatively few interferences

Gastric contents • Identification of acute ingestion/delayed absorption • Non-homogeneous matrix • Identification of pill fragments possible • Complex matrix • Particularly useful for orally administered • Requires sample preparation/pretreatment drugs/poisons • Requires total specimen collection for interpretation

Hair • History of drug use (months) • New technology • Readily available, easy collection • Recent drug use not detected • Low potential for donor manipulation • Environmental contamination • Useful for drug and non-drug analytes, e.g. metals • Potential for ethnic bias • Limited reference data

Kidney • Particularly useful for non-drug analytes, e.g. metals • Complex matrix • Requires sample preparation/pretreatment

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Table 13.3 (continued)

Specimen Advantages Disadvantages

Liver • Ease of detection of certain drugs (accumulation) • Complex matrix • Interpretive value for some drugs • Requires sample preparation/pretreatment • Reference data available

Lung • Particularly important for volatile analyses • Complex matrix • Requires sample preparation/pretreatment

Meconium • Long-term window of drug exposure • Non-homogeneous matrix • Non-invasive sample collection • Complex matrix (waxy) • Interferences • Requires sample preparation/pretreatment • Limited reference data

Nails • Easy collection • Limited data • History of drug use (months) • New technology • Particularly useful for metals • Not yet widely accepted • Recent drug use not detected • Environmental contamination

Saliva (oral fluid) • Readily available, easy collection • New technology • Parent drug present • Short drug detection time • Related to free drug concentration in plasma • Small sample volume (1–5 mL) • Minimal sample preparation • Potential for oral contamination • Many drugs determined • Collection method influences specimen pH and drug • Indicates recent drug use content

Spleen • Particularly useful for certain analytes if no blood is • Complex matrix available • Requires sample preparation/pretreatment • Limited data for most analytes

Sweat • History of drug use (weeks) • Newer technology • Cumulative measure of drug use • Potential for environmental contamination • Parent drug present • High inter-subject variability • Non-invasive collection • Requires special collection device • Less frequent drug testing required • Skin irritation and discomfort • Not readily adulterated • Small sample volume • No pharmacological interpretation possible • Non-homogeneous matrix (sweat/sebum)

Urine • Widely accepted matrix • Potential for donor manipulation • Easy collection • Minimal parent drug • Plentiful supply • Not useful for quantitative analysis • Amenable to automated analysis • Not related to impairment or pharmacological effect • Longer detection window than blood (days–weeks)

Vitreous humour • Determines recent drug use (hours–days) • Limited data compared with blood • Related to pharmacological effect • Small sample volume • Resistant to putrefaction • Interpretive value for ethanol-related investigations • Minimal sample preparation • Relatively few interferences • Useful for postmortem chemistry

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Central blood wherever possible. Blood collection from a ligated vein Cardiac blood samples are ideally collected following that has been ‘tied off’ is least likely to be contami- opening of the pericardial sac, removal of the nated by other sources of blood or a result of release pericardium, and removal of the blood from the of drug from tissues and organs. Typically, however, left or right chamber after the heart has been dried. a ‘femoral stick’ involves the collection of femoral Collection of central blood by insertion of a needle blood from an unligated femoral vein in the groin through the chest wall (‘blind stick’) is practised but is area. Only a small volume of blood should be collected discouraged. Although central blood collected in this to avoid ‘milking’ the vein and drawing blood from manner may be identified to the laboratory as ‘heart other sources. Typically 10–20 mL of femoral blood can be collected. Over-sampling of blood from the blood’, it may be contaminated with pericardial fluid, femoral vein will draw blood from the inferior vena fluid from the pleural cavity, or blood that has drained cava, and hence the liver, and from the larger iliac from the pulmonary vein or artery or the inferior vena vein. Although sampling from a ligated vein is gener- cava (Jones 2007). Blood collected in this manner ally preferred, a comparison of drug concentrations in is considered non-homogeneous. Central blood may clamped and unclamped femoral vessels showed good contain elevated drug concentrations as a result of correlation for eight drugs including selective sero- postmortem redistribution or contamination (diffu- tonin reuptake inhibitors, benzodiazepines, antihis- sion) from other body compartments (Prouty and tamines and one opioid (Hargrove and McCutcheon Anderson 1990; Yarema and Becker 2005), especially 2008). If femoral blood is not available, subclavian following blunt force trauma. Passive drug release or iliac blood may be an alternative. from reservoirs such as the gastrointestinal tract, liver, lungs and myocardium may occur immediately Blood clots after death; later, cell autolysis and the putrefactive Following a fall or blunt trauma to the head, a victim process participate in redistribution (Pelissier-Alicot may survive with circulation intact for several hours. et al. 2003). Drug properties such as volume of dis- Owing to the decreased circulation in the damaged tribution, lipophilicity, protein binding and pKa play region of the brain, drug or alcohol concentrations in a role in the site- and time-dependent mechanisms blood clots (e.g. subdural, subarachnoid and/or epidu- responsible for postmortem redistribution. Drugs ral) may be influenced by incomplete metabolism. It with high volumes of distribution and basic character has been suggested that intracranial blood clots may appear particularly susceptible to postmortem serve as ‘time capsules’ prior to death, because they redistribution and their cardiac blood concentrations may reflect drug concentrations several hours prior to should be interpreted accordingly. Postmortem redis- death, when an injury may have taken place. tribution can account for central/peripheral blood drug concentrations that differ by 10-fold or more. Vitreous humour Redistribution is time and concentration dependent and is very difficult to predict. Cardiac blood is typ- Direct aspiration of vitreous humour using a hypo- ically more plentiful than peripheral blood. Although dermic syringe may yield 2–3 mL of fluid per eye. cardiac blood can be a very useful specimen for The needle should be placed in the central globe screening purposes, the relationship between cardiac and aspirated with gentle suction. Preservation with blood drug concentrations and antemortem blood sodium fluoride is generally recommended. The eye drug concentrations is complex. Many toxicologists is located within the protective environment of the therefore advise against the use of cardiac blood for orbit and, being essentially outside the body, is quantitative and interpretative work. remote from other tissues. Vitreous fluid is therefore a particularly useful specimen owing to its anatomical Peripheral blood isolation, affording it notable resistance in terms of Femoral blood is the best specimen for use in post- microbial invasion and degradation, as well as being mortem testing (Chapter 7) and should be sampled remote from the central organs and subsequently less

Sample chapter from Clarke’s Analytical Forensic Toxicology, second edition Sampling, storage and stability | 341 susceptible to postmortem redistribution phenomena. plentiful than vitreous humour, the lack of plentiful Vitreous humour is particularly useful for cases reference data limits its usefulness. However, CSF may involving digoxin or hydrophilic analytes including be particularly useful in surgical death investigations. paracetamol (acetaminophen) and salicylates. The equilibrium that exists between blood and vitreous fluid is slower than with other extracellular fluids, Bile which can result in a slight delay in uptake. Further- Bile is generally aspirated from the gallbladder using more, only free drugs are able to leave the blood and a hypodermic syringe. It may be necessary to tie enter the vitreous humour. Since eye fluid is sterile off the gallbladder prior to collection if contamina- and less susceptible to microbial contamination tion appears to be an issue. Bile should be collected and hence postmortem alcohol production, it is prior to the liver specimen to avoid contamination. routinely used for ethanol determination owing to Many drugs of forensic interest accumulate in the bile, its interpretive value from the standpoint of post- particularly those that are heavily conjugated, such mortem alcohol production and the determination as opiates, benzodiazepines and cannabinoids. Bile of the pre- or post-absorptive phase of ethanol use may also be used in cases where chronic heavy-metal (Honey et al. 2005). Vitreous humour is particularly poisoning is implicated. However, owing to the pres- useful for postmortem analysis of glucose, urea ence of bile salts and fats, drug extraction from this nitrogen, uric acid, creatinine, sodium and chloride. matrix can be complicated and extensive extraction These are important analytes for the evaluation of and clean-up procedures are often required. diabetes, degree of hydration, electrolyte imbalance, postmortem interval and the state of renal function prior to death (Coe 1977, 1993). Sodium, calcium Gastric contents and chloride concentrations in vitreous humour during the early postmortem interval can be used Gastric content is a potentially valuable specimen to estimate antemortem serum concentrations. It is for analysis in postmortem and clinical cases. therefore important that sodium fluoride is not added Unabsorbed drug or tablet fragments in the gastric to specimens requiring vitreous chemistries. For that contents may provide valuable information concern- reason, vitreous humour is frequently collected into ing ingested compounds and provide an excellent two separate containers: one preserved (for drug and material for preliminary screening (Chapter 7) owing alcohol testing) and one unpreserved (for clinical to the potentially large amounts of drug that may be purposes). present. The absence of a drug in the gastric contents does not necessarily preclude oral administration. Odours emanating from the gastric content can Cerebrospinal fluid provide valuable clues about what may have been Cerebrospinal fluid (CSF) can be collected either by consumed, e.g. pesticides and cyanides. The entire lumbar puncture at the base of the spine using a hypo- contents of the stomach should be collected and dermic syringe or by withdrawal of cisternal fluid by weighed. Gastric contents are non-homogeneous and puncturing the base of the neck. Although there are should be homogenised prior to sampling. Quantita- limited published reference data for quantitative drug tive drug determinations should be interpreted within concentrations in CSF, this clear fluid comprising the context of the entire contents (total quantity, mostly water is amenable to most routine methods rather than concentration) and it is important to take of toxicological analysis. CSF may be of particular into consideration the differing absorption rates of importance in alcohol-related cases where no vitreous drugs based on their physicochemical properties as humour is available, particularly if postmortem alco- well as their formulations and coatings. hol production is suspected. Like vitreous humour, The presence of a drug in gastric contents, par- CSF is anatomically isolated and less prone to con- ticularly at low concentration, does not necessarily tamination and bacterial invasion. Although it is more indicate oral administration. Drugs may be absorbed

Sample chapter from Clarke’s Analytical Forensic Toxicology, second edition 342 | Clarke’s Analytical Forensic Toxicology into the stomach via gastric juices that are in equi- Liver librium with blood or as a result of intranasal drug Liver is a particularly important organ because use. Basic drugs are more susceptible to this because of the very large number of drugs that undergo they have a tendency to become trapped in the gastric hepatic metabolism and the fairly extensive published compartment owing to the low pH. If heavy met- reference data that exist. To reduce the possibility als are suspected, gastric contents should be collected, of drug diffusion from the small bowel, tissue from together with intestinal contents. In cases of suspected deep within the right lobe is preferred (Drummer poisoning where the patient may have survived for a 2004). The concentrations of drugs and metabolites few days in hospital prior to death and where drugs in liver are often elevated; hence this specimen may have been metabolised and eliminated from the has limited interpretive value. However, liver is body prior to death, any stomach content collected particularly useful for highly protein-bound drugs and retained by medical staff may provide valuable and the comparison of liver/blood drug ratios may information concerning drugs or poisons consumed. allow the differentiation of acute overdose from chronic drug use for some drugs. Urine Kidney In antemortem settings, a mid-stream urine sample is usually collected into a plastic container containing Most drugs pass through the kidney as a result of sodium fluoride as preservative. In some settings it urinary elimination. Kidney is an important specimen may be necessary to take precautions against spec- in cases of suspected heavy-metal poisoning owing imen adulteration. In postmortem settings, urine is to accumulation in this tissue. The presence of heavy collected by insertion using a hypodermic syringe metals or ethylene glycol during toxicological tests directly into the bladder under visualisation. Puncture may be accompanied by structural changes to the kid- of the abdominal wall should be avoided to reduce ney that can be documented using histological tests. the possibility of contamination. Urine is a valu- Spleen able specimen for both antemortem and postmortem drug testing because it is a relatively uncomplicated Spleen is an important specimen for cyanide or carbon matrix. However, the multiplicity of factors influ- monoxide analyses, particularly in fire-related deaths encing urine drug concentrations (e.g. urine volume, where blood may be compromised or unavailable. clearance, metabolism, pH and time of last void) Lung and brain are valuable specimens in cases generally means that, in isolation, these results have involving volatiles or inhalants. limited quantitative value. Exceptions to this rule may Brain include ethanol determination in a second void. Care must be exercised when considering the interpreta- Brain tissue is lipid rich and has a tendency to tion of urine GHB concentrations as GHB is present concentrate some drugs, particularly lipophilic ana- as an endogenous compound formed as a by-product lytes, narcotics and halogenated hydrocarbons (Skopp of metabolism and may also be produced as a post- 2004). If quantitative drug brain concentrations are mortem artefact as a consequence of the breakdown used, it is important to know the location of the of succinic acid semialdehyde. specimen because the brain is a non-homogeneous matrix. Drug concentrations within the brain may Tissues vary several-fold from one region to another owing to its complex structure and differing composition. Brain When tissues are sampled they should be collected is not widely used in routine toxicological analysis. quickly and placed immediately into airtight con- tainers. This is particularly important if volatiles Muscle or inhalants are suspected. Liver, kidney, brain, Muscle is not routinely encountered, despite the fact lung and spleen are the most frequently collected that it frequently contains relatively high drug concen- postmortem tissues. trations, particularly for substances with high volumes

Sample chapter from Clarke’s Analytical Forensic Toxicology, second edition Sampling, storage and stability | 343 of distribution. Perfusion rates between sites and provide valuable interpretive information pertain- drug concentrations are not consistent, and drug con- ing to the chronological sequence of toxin exposure centrations must be interpreted accordingly. Muscle (Cirimele et al. 2002). Hair has also proved to be use- is encountered more frequently for ethanol deter- ful in cases where exhumation is necessary (Tsatsakis mination in the absence of blood, or during the et al. 2001). If hair is collected post mortem, it should investigation of a suspected injection site. be sampled at the very beginning of the examination to reduce the risk of contamination. Hair Hair can provide complementary toxicological Hair has been used in a variety of antemortem toxicol- information. Issues with drug testing in hair include ogy settings to provide a history of drug exposure and external contamination, ethnicity and pigmentation, has therefore found applications in workplace drug chemical treatment and the use of appropriate cut-off testing, in monitoring of persons on probation or on concentrations. Contamination of the hair with drugs parole for drug use, in insurance testing to verify the from other sources (external deposition, environ- truthfulness of statements made by applicants relat- mental contamination, sweat or sebum) is generally ing to whether they use drugs or are smokers, in child minimised by pretreatment of the sample using a var- endangerment, in re-granting of driving licences fol- iety of aqueous and organic rinses or wash steps prior lowing suspension for drug misuse, in drug-facilitated to analysis. sexual assault and in other types of criminal case- Other keratinised specimens such as nails can also work (Nakahara 1999; Kintz et al. 2006; Curtis and be used to determine long-term exposure to drugs or Greenberg 2008). Further discussion relating to the poisons, in particular heavy metals such as thallium, scenarios in which hair testing is employed is provided arsenic or lead. However, drugs are deposited into in Chapters 6 and 7. One of the major advantages nails at a much slower rate. External decontamination is the long drug detection window compared with procedures should be performed prior to analysis (see many other specimens. Hair may allow drug expo- Chapters 6 and 7). sure over several weeks or months to be determined, depending on the length of the hair. Segmenting the Injection sites hair by length may allow an approximate timeline for exposure to be determined based on head hair growth Excision of skin and tissue (muscle) may be necessary rates of approximately 1 cm per month (Clauwaert in postmortem investigation of a suspected injection et al. 2000). Hair should be cut as close as possible to site. Typically a cube of muscle and skin is removed the scalp from the posterior vertex region of the head, for this purpose. However, it is important to compare since this region shows least variation in growth rate. the drug concentrations in the suspected injection Typically a lock of hair equivalent to the thickness site with those in a control specimen from the same of a pen or pencil is collected. The colour, length, individual where there is no evidence of injection. sampling site and any obvious cosmetic treatment of Injection sites are not always reliable indicators of the hair should be recorded. The root (proximal) and drug administration since the presence or absence of tip (distal) sections of the hair should be clearly iden- drugs in injection site tissue is dependent on the type tified. Although head hair is the preferred specimen, and depth of the injection. If the injection is made hair from other sites (e.g. pubis, axillae) may be used, directly into a blood vessel, little drug is likely to but interpretation of analytical findings may be more remain in the surrounding tissue. complex. The lock of hair is typically tied, wrapped in aluminium foil and stored under dry conditions in Entomological specimens the dark at room temperature. Hair is also a useful specimen in postmortem The potential use of insects for detecting drugs and investigations where arsenic or heavy metals are sus- other toxins in decomposing tissues has been demon- pected. Although postmortem hair analysis is not yet strated and reviewed (Introna et al. 2001). If insects widespread, there is growing interest because it may or larvae are collected from human remains they

Sample chapter from Clarke’s Analytical Forensic Toxicology, second edition 344 | Clarke’s Analytical Forensic Toxicology should be frozen as soon as possible. Larvae rapidly tains the unbound fraction of drug at concentrations eliminate drugs when removed from the food source. that are typically proportional to those measured in Drugs, metals and pesticides have been identified in plasma. However, the predictable relationship that entomological specimens including larvae and pupae. theoretically exists between saliva and plasma drug Following wash steps to remove external contamina- concentrations is influenced by many factors such as tion, entomological specimens are homogenised and saliva flow rate, which can complicate pharmacolog- analysed in a manner similar to that for tissues. ical interpretation (Crouch 2005). A more detailed discussion on saliva and oral fluid testing is provided Saliva in Chapter 6.

Saliva or oral fluid can be collected non-invasively by Sweat expectoration, by aspiration, by vacuum or by sat- uration of an absorbent swab (Kidwell et al. 1998). Moisture loss via the skin and elimination of insen- Detection times are comparable to those in blood. As sible (non-visible) sweat take place during normal much as 1.5 L of saliva per day is produced by the breathing at a rate of 0.3–0.7 L/day. Sensible sweat submandibular, parotid and sublingual glands inside refers to perspiration that is actively excreted during the mouth. Secretions from a specific gland may be stress, exercise or extreme temperature, at rates of collected using a special device or by cannulation, but 2–4 L/h. About half the total volume of sweat is elim- this is uncommon. Although specific gland secretions inated from the trunk of the body. The remaining fluid are advantageous from a standpoint of saliva:plasma is lost from the legs or upper extremities and head in ratio and reduced oral contamination, mixed saliva is approximately equal amounts (Kidwell et al. 1998). typically collected for routine drug-testing purposes. Sweat is usually collected using an adhesive Oral fluid can be collected non-invasively, conve- absorbent patch that is placed on the surface of niently and without invasion of privacy and is the clean skin or by wiping the skin with a swab or most commonly collected fluid from the oral cavity gauze. Careful preparation of the skin is necessary for the determination of drugs. Chewing an inert sub- prior to placement of a sweat patch to minimise stance, such as Teflon tape or a rubber band, may external drug contamination or bacterial degradation increase salivation for the purpose of specimen col- of the drug once it has been retained. Use of a lection. It should be verified that no adsorption takes semipermeable membrane to cover the absorbent pad place between the drug and the chewed substance. prevents non-volatile components in the environment Acidic sweets or citric acid have also been used to from penetrating the pad externally, but allows stimulate glandular secretions. Care must be taken oxygen, water and carbon dioxide to diffuse through. that residual food, drink or interfering substances Salts, solids and drugs that pass through the skin inside the mouth do not interfere with the analy- are trapped in the absorbent pad, where they are sis. This is particularly important for drugs that are temporarily stored in situ, until the patch is removed. ingested orally or smoked. Following eating or drink- Owing to the relatively small volume (μL) of ing, a 10-minute wait period is recommended before insensible sweat secreted from a small absorbent sample collection. area (typically 3 × 5 cm), patches are typically worn Owing to the ease and non-invasive nature of for several days on the outer portion of the upper specimen collection, oral fluid is of particular interest arm or back. In practice most skin wipes or sweat in workplace drug testing, for insurance testing and, patches contain a mixture of sweat and sebum, the more recently, for roadside impairment testing. The oily secretion from the sebaceous glands. As with placement of an absorbent pad into the oral cavity will saliva, increased flow rates can influence the quantity collect oral fluid, a mixture of predominantly saliva of drug eliminated into sweat. This specimen is par- and small amounts of gingival crevicular fluid, cellular ticularly useful for compliance testing or monitoring debris and blood. Saliva contains serous fluid derived long-term exposure (weeks), which might be desirable from plasma. This ultrafiltrate of interstitial fluid con- in probation or parole settings.

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Amniotic fluid during pregnancy, in particular the last 20 weeks of gestation. It provides more complete and long-term Amniotic fluid has been used to investigate prenatal information on drug exposure than neonatal urine drug exposure. Its collection (amniocentesis) typically or cord blood. The specimen is complex and takes place between weeks 16 and 20 of pregnancy. non-homogeneous. All available samples should A needle is inserted through the abdomen into the be collected and homogenised prior to analysis. uterus where there is the least chance of touching the Meconium and other important matrices involved in placenta or the fetus. The collection of amniotic fluid maternal–fetal medicine have been reviewed (Gray is typically performed in conjunction with ultrasound and Huestis 2007; Lozano et al. 2007). visualisation in order to reduce the risk of damag- ing the developing fetus. Although complications are rare, miscarriage occurs in a very small percentage Sample handling of women. Typically 5–30 mL of amniotic fluid is removed during the procedure. Sample handling is an important consideration dur- ing the pre-analytical phase. Unlike a clinical setting, Breast milk where the time between sample collection and testing is often very short, significant delays are common During pregnancy, oestrogen and progesterone, in a forensic setting. The pre-analytical phase may secreted in the ovary and placenta, cause milk- be considerable, spanning the time of death and/or producing glands in the fatty tissue of the breasts discovery of a victim, autopsy and collection of speci- to develop and become active. The pituitary hor- mens, sample storage, transport to the laboratory and mone prolactin stimulates the production of fluid subsequent storage prior to analytical testing. In ante- (600–1000 mL/day) by the milk-secreting cells. Con- mortem toxicology settings, the time delay between an traction of the myoepithelial cells surrounding the alleged offence and specimen collection may be short alveoli allows the milk to be expressed. For specimen (e.g. minutes to hours in the case of most impaired collection purposes, a breast pump can be used. The driving cases) or long (e.g. hours to days in the case of matrix is somewhat non-homogeneous. Colostrum, some sexual assault cases). Following collection, ante- a creamy white to yellow pre-milk fluid, may be mortem specimens may be subject to similar delays expressed from the nipples during the last trimester due to shipping or transport of specimens, requests of pregnancy and shortly after delivery. Many drugs for testing made by the submitting agency and stor- are excreted into breast milk and the scientific and age of samples prior to actual testing. Although the medical literature contains numerous citations of toxicologist must consider the time delay between the the presence of drugs in this matrix. Drugs that are event (i.e. death, or committing or being the victim of extensively protein bound may not readily pass into an offence) and collection of a specimen for interpre- the milk, but emulsified fats contained in the milk tation purposes, these delays are beyond the control may concentrate highly lipid-soluble drugs. The high of the laboratory. Measures can be taken, however, to lipid content and natural emulsifying agents present preserve and maintain the integrity of specimens after in breast milk mean that some sample pretreatment collection. Sample quality plays an important role is often required. in the validity or usefulness of subsequent analytical determinations. Inappropriate sample preservation or Meconium storage may have a deleterious effect on qualitative and quantitative determinations. Meconium formation begins between weeks 12 and 16 of gestation. As the first faecal matter passed by Preservation and storage the neonate, it is typically collected within 1–5 days of birth. Analysis of drugs in meconium may provide Specimens should be stored at appropriate tem- a relatively long-term history of drug exposure peratures, with adequate preservative and in an

Sample chapter from Clarke’s Analytical Forensic Toxicology, second edition 346 | Clarke’s Analytical Forensic Toxicology environment accessible only to authorised personnel Although the addition of preservative should be to ensure security and integrity. Short-term storage routine for most antemortem and postmortem blood ◦ at refrigerated temperature (4 C) is recommended samples, an aliquot of unpreserved postmortem blood ◦ for most samples, or frozen (−20 C or lower) during is sometimes collected. For example, fluoride preser- long-term storage (more than 2 weeks). Exceptions vatives should not be used if organophosphorus chem- to this include hair, nails or dried blood swatches icals are suspected since this accelerates chemical on filter paper, which can be stored at ambient degradation (Skopp and Potsch 2004). Some drugs temperatures. are known to be photolabile (e.g. ergot alkaloids such Whereas clinical specimens are typically unpre- as lysergic acid diethylamide and the phenothiazines). served, the use of a chemical preservative is often Specimens known to contain photolabile drugs should warranted in forensic specimens. Preservation of be stored in amber vials or foil-covered containers, blood samples with sodium fluoride (2% w/v) is or otherwise protected from direct sources of light. routine in most laboratories. Commercial evacuated Storage of tightly sealed appropriate containers at low blood collection tubes (e.g. grey-top tubes) contain temperature further inhibits sample loss. Short-term ◦ ◦ sodium fluoride as the preservative and potassium storage at refrigerated (4 C) and frozen (−20 C) tem- oxalate as the anticoagulant. These are the preferred peratures is commonplace in most laboratories and evacuated blood tubes for antemortem forensic repeated freeze–thaw cycles should be avoided. toxicology casework. Inhibition of microorganisms and enzymes with sodium fluoride is important for Labelling and specimen transfer commonly encountered analytes such as ethanol, All samples should be properly marked for identi- cocaine and others. Fluoride acts as an enzyme fication with the case number, donor name, date inhibitor and helps prevent glycolysis. and time of collection, signature or initials of the Commercial blood tubes may contain a wide vari- collector and specimen description. Tamper-proof ety of additives (citrate, heparin, EDTA, thrombin, containers and/or tape bearing the collector’s ini- acid citrate dextrose mixtures, clot activator, etc.). tials and date of collection should be used. Spec- Although these tubes are designed for a variety of imens should be forwarded to the laboratory in clinical uses, they are not the preferred specimen appropriate leak-proof and tamper-proof packaging/ containers for drug-testing purposes. Laboratories shipping materials with all appropriate documenta- frequently encounter these blood tubes when they tion (chain-of-custody forms, requisitions for testing, are submitted from a hospital setting and special care special requests, case information, medications list, must be taken when interpreting their results (LeBeau police report, donor information/identifier such as et al. 2000; Toennes and Kauert 2001). If an antico- date of birth or social security number, agency case agulant is to be used, potassium oxalate is preferred number, pathologist/police officer name and con- rather than alternatives such as EDTA, heparin or tact information). Improperly packaged or identified citrate. Antioxidants such as ascorbic acid (0.25% materials should be returned to the submitting agency. w/v) or sodium metabisulfite (1% w/v) are sometimes Documentation accompanying the specimen(s) should used to prevent oxidative losses, but these agents have list all of the specimens that were collected or avail- the potential to act as reducing agents towards some able for testing. Once received by the laboratory, the drugs, in particular N-oxide metabolites, which may specimens should be inspected and appropriately doc- be transformed into the parent drug. In a similar umented in terms of condition and quantity during fashion, adjustment of specimen pH is not generally the accessioning process. favoured routinely, because, just as some drugs are alkali labile (e.g. cocaine, 6-acetylmorphine), others are acid labile. Sodium azide (0.1% w/v) is sometimes Contamination used as a preservative and antimicrobial agent in urine samples. Sodium azide should not be used if samples There are a variety of contamination sources for are to be analysed by enzyme-linked immunosor- both antemortem and postmortem specimens. In addi- bent assay because it can interfere with horseradish tion to the potential contamination issues that may peroxidase-mediated colorimetric detection. result from the use of containers and external factors,

Sample chapter from Clarke’s Analytical Forensic Toxicology, second edition Sampling, storage and stability | 347 a number of important exogenous and endogenous substances met with some success, a wide variety of sources of contamination should be considered. commercial adulteration reagents and kits is now widely available (Dasgupta 2007). A summary of in Exogenous contaminants vitro adulteration agents is provided in Table 13.4. Some of the most popular commercial products Specimens collected into plastic containers are some- times susceptible to phthalate interferences. Numer- ous plasticiser interferences such as dibutylphthalate Table 13.4 In vitro adulteration agents may co-extract and interfere with analytical detection • by gas-chromatographic or mass-spectrometric tech- Ascorbic acid niques, yielding characteristic phthalate ions. All plas- • Alcohols tic containers should be evaluated prior to widespread • implementation. It should be noted that contamina- Amber-13 (hydrochloric acid) tion from phthalates may occur during the analytical • Ammonia process through use of disposable pipette tips, solvent • Bleach containers, solid-phase extraction cartridges, tubing and numerous other sources. However, environmen- • Clear Choice (glutaraldehyde) tal exposure to these substances from household • Detergent or soap (surfactant) items, food, beverages and other sources can pro- duce detectable quantities of phthalate esters or their • Drano metabolites in biological specimens including blood, • Ethylene glycol serum, urine and breast milk (Silva et al. 2005; Hogberg¨ et al. 2008). • Gasoline Embalming fluids, which typically contain a vari- • Glutaraldehyde ety of alcohols and aldehydes, are a potential source of contamination in postmortem casework. These • Hydrogen peroxide

fluids not only dilute any remaining fluid in the • Klear (potassium nitrite) body, but also alter drug distribution in remaining • tissues. Another potential source of contamination Lemon juice comes from reusable syringes and containers for post- • Liquid soap mortem specimen collection. Some cleaning fluids that • are used for syringes may contain alcohols that can Lime-A-Way compromise the analysis of volatiles. This highlights • Mary Jane Super Clean 13 (surfactant) the importance of analysing specimens from multiple • Salt sites and using disposable syringes wherever possible. The principal concern with antemortem contami- • Stealth (peroxide/peroxidase) nation arises from the intentional manipulation of the • THC-Free (hydrochloric acid) sample to mask the presence of drugs. This typically involves the substitution, dilution or adulteration of • UrinAid (glutaraldehyde) the biological specimen with a foreign substance. • Urine Luck (chromium VI, oxidant) Donor manipulation occurs most frequently with urine samples in workplace drug-testing situations • Vanish (see Chapter 5). As a result, specimen validity testing • Vinegar is required in some drug-testing programmes such as for federal employees under US Department of Health • Visine and Human Services (DHHS) guidelines (or EWDTS • Water or UKWDT guidelines). Initially, adulteration of urine for drug-testing purposes involved the use • Whizzies (sodium nitrite) of crude household items such as soap, bleach, Source: Kerrigan and Goldberger (2005). vinegar, ammonia or cleaning fluids. Although these Sample chapter from Clarke’s Analytical Forensic Toxicology, second edition 348 | Clarke’s Analytical Forensic Toxicology contain glutaraldehyde (fixative), pyridinium Table 13.5 In vivo adulteration agents chlorochromate (PCC) or chromium(VI)-containing species (oxidant), nitrite (oxidant) or peroxide/ Diuretics peroxidase. In general, in vitro adulteration can inter- fere with presumptive immunoassay tests, with the Prescription intention of producing false-negative results. How- • Thiazides and thiazide-like drugs (e.g. hydrochlorothiazide, ever, some agents have the potential to interfere with metolazone) confirmatory tests such as gas chromatography–mass spectrometry (GC-MS) as well. Although this is less • Carbonic anhydrase inhibitors (e.g. acetazolamide) likely, studies have shown that some reagents may produce lower than expected or negative results for • Loop diuretics (e.g. bumetanide, furosemide, torsemide) some analytes. Adulteration-detection products are • Osmotic diuretics (e.g. mannitol) available commercially. On-site or dipstick tests are available for nitrite, glutaraldehyde, pH, specific Over the counter (OTC) gravity, creatinine, bleach, PCC and oxidants. Specimen dilution or in vivo adulteration by inges- • Aqua-Ban tion of a substance to mask the presence of drugs is • Diurex also encountered. This is commonly achieved by the ingestion of large quantities of fluid prior to the test or • Fem-1 by administration of a diuretic. Examples of in vivo adulteration agents are given in Table 13.5. Urine • Midol specimen substitution or dilution can be detected if • specimen validity tests are performed. A specimen may Pamprin be considered invalid if the pH is between 3 and 4.5 or • Premsyn PMS between 9 and 11. It may be adulterated if the pH is less than 3 or greater than 11. The normal temperature Other ◦ range is 32–38 C. A specimen is considered dilute if • the creatinine concentration is less than 200 mg/L and Alcoholic beverages the specific gravity is less than 1.003. (Numbers differ • Xanthines (e.g. caffeine, theophylline, 8-bromotheophylline) slightly under EWDTS or UKWDT guidelines.) Other sources of contaminants or unexpected • Herbals and aquaretics (e.g. golden seal root, juniper) analytes include pyrolytic breakdown products due to thermal degradation of drugs. These may be Source: Kerrigan and Goldberger (2005). present due to pyrolysis during administration of the drug (e.g. anhydroecgonine methyl ester following death, introducing high concentrations of drug in crack cocaine use) or occasionally in situ during local body compartments. Access to hospital records analysis if conditions are not properly controlled or and case information, and collection of peripheral evaluated. Other sources of contamination may arise blood, vitreous fluid and liver are particularly impor- from pharmaceutical impurities or adulterants and tant in these types of cases. Other sources of medical cutting agents that are incorporated into illicit drugs artefacts may include organ harvesting drugs such as prior to sale. the calcium-channel blocker verapamil, or papaver- ine, which is used to inhibit vasoconstriction during transplantation surgery. Medical artefacts If living patients are administered fluids Clinical therapy can sometimes produce medical arte- (e.g. saline) during clinical care, blood is only facts that complicate toxicological findings. Medical contaminated (diluted) with the infusion solution artefacts are most common in postmortem cases if it is collected downstream from the intravenous where infusion pumps may continue to run after line. Blood circulation and equilibrium with tissues

Sample chapter from Clarke’s Analytical Forensic Toxicology, second edition Sampling, storage and stability | 349 is rapid, so the administration of fluids does not Major changes that occur after death produce usually influence drug or alcohol concentrations in autolytic changes and putrefaction by microorgan- blood if normal precautions are taken. If downstream isms. Invasion of microorganisms, particularly from collection is suspected, careful review of the medical the gastrointestinal tract into tissues and body fluids, records and/or measurement of the haematocrit to occurs within hours at ambient temperature. Lipids, determine specimen dilution may be necessary. carbohydrates and proteins are hydrolysed by micro- bial enzymes, the pH of blood steadily increases, Endogenous contaminants, artefacts and the putrefactive amines, tyramine, tryptamines, and interferences phenethylamines and other endogenous substances are liberated. By their very nature, all biological specimens are Trauma is a non-preventable source of contami- subject to endogenous interferences, regardless of whether or not they are derived from living or nation in postmortem forensic toxicology. Rupture deceased persons. More complex biological speci- of organs or compartments within the body can mens such as blood, tissue or meconium will require compromise quantitative drug analyses owing to the more extensive sample preparation to remove these mixing of fluids (e.g. of gastric contents with blood) interferences than less complex matrices such as vit- or from the microbial action that occurs as a result. reous humour, cerebrospinal or oral fluid. In general, Postmortem alcohol production can also result in however, antemortem specimens are somewhat less detectable quantities of ethanol as an artefact. Glycol- susceptible to endogenous artefacts or contaminants. ysis and the presence of yeasts and microorganisms Ethanol, GHB, carbon monoxide, cyanide and other can convert a variety of postmortem substrates to short-chain alcohols can be metabolically produced ethanol. Although concentrations are typically low post mortem (Skopp 2004). The formation of tox- (<0.7 g/L), concentrations of 2 g/L and higher have icologically significant concentrations of cyanide in been reported (Zumwalt et al. 1982; O’Neal and postmortem tissue (Lokan et al. 1987) has been Poklis 1996). Postmortem alcohol production is influ- attributed to the conversion of thiocyanate to cyanide enced by many factors, including the time between and the breakdown of protein (Curry et al. 1967). Although in some circumstances ethanol can be pro- death and sampling, environmental conditions (tem- duced in situ in unpreserved antemortem fluids, the perature, humidity, location), external factors (trau- same is true to a far greater extent in postmortem spec- matic injury, incineration), the availability of an imens, particularly blood. Likewise, GHB is present ethanol substrate and the extent to which microor- in antemortem fluids at very low concentrations in the ganisms are available. Vitreous humour and urine absence of a serious genetic disorder such as GHB- provide complementary information that may assist uria (Knerr et al. 2007). Differentiation of exogenous with the interpretation of results. This highlights the and endogenous GHB is complicated by specimen importance of collecting a variety of specimens post type, storage conditions, preservative and other fac- mortem. Other short-chain alcohols can be produced tors. Many laboratories use a cut-off concentration by microorganisms in situ. Isopropanol has been to help differentiate the two, for example 10 mg/L in documented as a postmortem artefact, particularly urine (Kerrigan 2002; LeBeau et al. 2007). Concen- in drowning victims. Putrefaction can also compli- trations of GHB may increase in urine during storage, cate carbon monoxide determination in postmortem upon collection and storage of unpreserved blood, or blood. Increases in the apparent concentration of car- in citrate-buffered antemortem blood (LeBeau et al. 2000). Although preserved antemortem blood GHB boxyhaemoglobin have been documented owing to concentrations are typically lower than those in urine, the formation of methaemoglobin, a decomposition numerous studies have shown forensically signifi- product that can interfere with spectrophotomet- cant concentrations of GHB in postmortem blood. ric determination. Preservation of postmortem blood ◦ Postmortem urine and vitreous fluid appear to be less (and storage in the dark at 4 C or lower) has been susceptible to this increase. recommended (Skopp 2004).

Sample chapter from Clarke’s Analytical Forensic Toxicology, second edition 350 | Clarke’s Analytical Forensic Toxicology

Stability Depending on the collection, storage conditions, use of preservative, container type, matrix and other fac- Drug stability can be influenced by many factors tors, the concentration of a drug at the time of assay including the physicochemical properties of the drug, may not be identical to the concentration at the time characteristics of the specimen or matrix, tendency to of collection. All toxicological results should be inter- conjugate/deconjugate, specimen collection procedure preted within this context. (e.g. contamination with microorganisms), container Some drugs also exhibit a degree of thermal insta- selection (e.g. oxidation, adsorption), and the use of bility. This is a consideration for drugs that are preservatives or other additives. subjected to elevated temperatures during adminis- The majority of published drug stability studies tration (e.g. by smoking) and during analysis (e.g. focus on antemortem or non-biological matrices. by GC-MS). Pyrolysis products may be indicative Scientific findings for biological specimens are of smoking if it can be shown that they are not complex because drug instability is often matrix produced during analysis, for example: anhydroecgo- dependent and influenced by factors such as specimen nine methyl ester (AEME) following the use of crack pH and the presence of other substances in addition cocaine; 1-phenyl-cyclohexene following phencycli- to external factors. In general, drug instability in any dine (PCP) use; and transphenyl propene following toxicological specimen is due to metabolic degrada- methamfetamine use. tion, chemical transformation, or a combination of Ideally, drug stability should be evaluated in a both (e.g. cocaine). number of ways: long-term stability in the speci- Drug stability in postmortem matrices poses an men or matrix of interest; the effect of freeze–thaw added level of complexity because conditions are less cycles; short-term stability (typically refrigerated); and controlled and degradation of analytes may be acceler- bench-top (room temperature) stability. The kinetic ated owing to putrefactive decomposition, microbial variables governing instability are often matrix and invasion and the increased presence of bacteria, for temperature dependent and an understanding of any example, the bacterial enzymatic conversion of mor- one of these (e.g. long-term stability of a particular phine glucuronides to free morphine in blood (Carroll drug in blood) does not necessarily imply predictable et al. 2000), and the bioconversion of nitrobenzenes results under different conditions (i.e. short-term stor- by enteric bacteria (Robertson and Drummer 1998). age of the drug in urine). After a specimen has been collected, enzymes may It should be noted that, although there are many remain active and continue to degrade or transform published studies and reviews of drug stability, these the drug in vitro. This process may take place post tend to focus on long- or short-term storage in com- mortem inside the body, or after postmortem or mon matrices such as blood, urine, serum or plasma. antemortem specimens have been collected, during As the number of drugs of interest continues to grow transportation to the laboratory and during storage. and the variety of specimens becomes more diverse, This is particularly important with esterases, which the scientific literature becomes somewhat limited in may further hydrolyse drugs post-collection unless terms of drug stability. The stability of drugs in non- they are inhibited by a preservative. traditional and non-biological matrices is beyond the In general, drug instability arises as a result of scope of this discussion, but investigations in this area moieties or functional groups that are susceptible to are ongoing. These include studies of the stability transformation, such as esters (e.g. 6-acetylmorphine, drugs in dry stains of biological origin (DuBey and cocaine, acetylsalicylic acid), sulfur-containing drugs, Caplan 1996), in formaldehyde solutions following photolabile drugs (e.g. phenothiazines, midazolam, embalming (Tracy et al. 2001), and in hair fibres after lysergic acid diethylamide) or those with functional exposure to cosmetic treatment such as bleaching, groups that are readily oxidized or reduced. Although perming or straightening (Potsch¨ and Skopp 1996). instability typically leads to decreases in drug concen- Drug stability data are often confounded by the fact tration, this is not always the case. Conjugated drugs, that a considerable number of studies report instabil- such as the glucuronides may deconjugate under some ity as an incidental or anecdotal finding, rather than as conditions, increasing the concentration of free drug. part of a formalised and well-designed investigation

Sample chapter from Clarke’s Analytical Forensic Toxicology, second edition Sampling, storage and stability | 351 of stability. Guidelines for conducting stability experi- to cannabidiol, or be oxidised to cannabinol as ments and the statistical evaluation of the results have a result of exposure to air or acidic conditions. been reviewed (Peters 2007). THC has been reported to be stable in refrigerated Of all of the most commonly encountered drugs, blood for 6 months and at room temperature for 2 cocaine is certainly the most notorious in terms of months (Johnson et al. 1984). Binding of THC to instability. Nevertheless, the vast majority of drugs are hydrophilic surfaces, such as storage containers or relatively stable or moderately so to the extent that rubber stoppers should be considered. For example, one can make reasonable assumptions when inter- THC stored in blood collected in unsilanised glass preting the results. Stability for some of the most tubes was stable for 4 days at room temperature frequently encountered drugs is summarised below. ◦ and4weeksat−20 C. By comparison, similar sam- ples stored in polystyrene tubes showed 60–100% Amfetamines decreases in concentration (Christophersen 1986). Methamfetamine and amfetamine in urine samples The principal metabolite shows greater stability than preserved with sodium fluoride (1% w/v) were sta- the parent drug. In one study, 11-nor-9-carboxy-9- ◦ ble during long-term storage at −20 C for at least tetrahydrocannabinol (THCA) was stable in frozen a year (Moody et al. 1999). A long-term study of urine preserved with sodium fluoride (1% w/v) for a whole blood stored over 5 years at room temperature year (Moody et al. 1999). However, this study used showed erratic but significant decreases in metham- silanised glassware for urine sample storage, which fetamine between 3 months and 5 years of storage, may not be typical of storage containers for actual ranging from 9% to 38% (Giorgi and Meeker 1995). casework. Loss of THCA in urine is largely attributed Results suggested that amfetamine was perhaps less to adsorption, or from foaming of the sample, which stable than methamfetamine. Samples were collected can account for losses as high as 89% (Dextraze et al. into 10-mL grey-top Vacutainer tubes with sodium 1989). The use of a de-foaming agent (e.g. 2-octanol) fluoride (100 mg) and potassium oxalate (20 mg). Although both drugs are considered to be moderately can reverse these losses, but such use is not routine. stable, storage of blood samples at room temperature It has been suggested that, although the adsorptive is not advised owing to the production of interfering losses of THCA from urine stored in plastic contain- substances. ers take place quickly (within an hour of collection), The overall stability of the amfetamine class they are not significant enough to compromise the extends to many of the designer amfetamines, analysis (Stout et al. 2000). Furthermore, adsorptive for example, 3,4-methylenedioxymethamfetamine losses of THCA in urine may be pH dependent, with (MDMA), 3,4-methylenedioxyamfetamine (MDA) greater losses occurring at acidic pH (Jamerson et al. and 3,4-methylenedioxyethylamfetamine (MDEA). 2005). Stability of these drugs was investigated in urine, Deconjugation of cannabinoids, such as THCA- − ◦ ◦ blood and water for 21 weeks at 20 C, 4 Cand glucuronide, to THCA should also be considered ◦ 20 C (Clauwaert et al. 2001). Although all drugs ◦ (Skopp and Potsch 2004). Hydrolysis of the acyl- were stable at −20 C, results were compromised in ◦ glucuronide results in increased concentration of blood samples at 5 and 13 weeks when stored at 20 C ◦ the unconjugated metabolite. THCA-glucuronide and 4 C, respectively, owing to matrix degradation ◦ was unstable in urine at 4 C and above, and at and interfering substances. increasing urinary pH (Skopp 2004). Studies suggest that THCA-glucuronide is less stable in plasma than Cannabinoids in urine (Skopp and Potsch 2002). Although no Decreases in cannabinoid concentrations in refrig- significant losses in THCA-glucuronide were seen ◦ erated and frozen samples are largely attributed to in plasma or urine stored at −20 C, instability was oxidative losses, temperature effects or lipophilic documented following storage of plasma and urine binding to containers. THC may decompose when at refrigerated and room temperature, in some cases exposed to air, heat or light. It can undergo hydrolysis within 2 days (Skopp and Potsch 2002).

Sample chapter from Clarke’s Analytical Forensic Toxicology, second edition 352 | Clarke’s Analytical Forensic Toxicology

Opioids Cocaine

6-Monoacetylmorphine (6-MAM) is a labile metabo- Of all of the common drugs of abuse, cocaine is lite of diamorphine due to hydrolysis of the ester certainly notorious in terms of stability. Both chem- bonds. It may undergo deacetylation to morphine ical and enzymatic transformations occur to pro- during storage. Buprenorphine, codeine, fentanyl, duce hydrolytic products. Spontaneous conversion of hydromorphone, methadone, morphine, oxycodone, cocaine to benzoylecgonine (BE) via the ester link- oxymorphone and tramadol were all stable in frozen age occurs at physiological and alkaline pH. At pH plasma stored for almost 3 months and subjected to 5, there were no measurable decreases in cocaine ◦ two freeze–thaw cycles (Musshoff et al. 2006). Free concentration at 40 C after 21 days, compared with concentrations of morphine, codeine and methadone adecreaseof40to70%inurineatpH8(Baselt were moderately stable in frozen urine preserved with 1983). Ester linkages have a tendency to be alkali sodium fluoride over a year (Moody et al. 1999). labile, and as a result the chemical transformation However, total morphine concentrations under of cocaine to BE is increasingly favourable as the similar conditions may be less stable (Moriya and pH of the matrix increases. Although the addition Hashimoto 1997). Long-term storage in preserved of preservative does not prevent chemical hydrolysis, whole blood stored at room temperature showed the kinetics can be inhibited by storage of samples significant increases and decreases (Giorgi and at low temperature, or by pH adjustment. The lat- Meeker 1995) over 1 to 5 years. These results suggest ter is not favoured in routine casework because of important differences in stability between free and the possibility that acid-labile drugs might also be conjugated species. present. Liver methylesterases are largely responsible The stability of glucuronidated morphine is of for the enzymatic transformation of cocaine to BE importance because ratios of free and total morphine and plasma pseudocholinesterase for the conversion are sometimes used for interpretive purposes. Marked of cocaine to ecgonine methyl ester (EME). Addition differences in glucuronide stability exist between of a cholinesterase inhibitor such as sodium fluoride antemortem and postmortem blood. Morphine-3- and reduced temperature are important precautions. glucuronide was stable in refrigerated antemortem Both BE and EME undergo further transformation to blood preserved with sodium fluoride, but unstable in ecgonine, a polar metabolite. In unpreserved blood, postmortem blood under the same conditions (Carroll hydrolysis of the phenyl ester predominates, yield- et al. 2000). Hydrolysis of the glucuronidated species ing EME. Addition of sodium fluoride inhibits the to free morphine increases with temperature, storage production of EME but does not prevent chemical time and degree of putrefaction. Other studies have hydrolysis of cocaine to BE. Cocaine in blood stored ◦ confirmed the stability of morphine-3-glucuronide in at 4 C was undetectable within 3 days in the absence refrigerated antemortem blood and plasma for up to of preservative, and only 40 to 60% of the cocaine 6 months (Skopp et al. 2001). Storage of postmortem was detected in preserved blood and plasma after 21 ◦ specimens at −20 Cpreventedin vitro hydrolysis of days (Baselt 1983). There have been numerous stud- the glucuronide. ies on the stability of cocaine and its metabolites in various media, and comprehensive stability studies Phencyclidine are available (Isenschmid et al. 1989). In unpreserved blood, cocaine is hydrolysed to EME, whereas trans- Studies suggest that PCP is a relatively stable drug, formation to BE predominates in preserved blood. even when stored in blood at room temperature for Although BE exhibits greater stability than cocaine, up to 18 months (Levine et al. 1983). However, decreases in concentration are largely due to further significant decreases in concentration were measured hydrolysis to ecgonine. BE was shown to be stable in in preserved blood at room temperature over 5 years preserved urine for a period of at least 1 year when ◦ (Giorgi and Meeker 1995). stored at −20 C (Moody et al. 1999).

Sample chapter from Clarke’s Analytical Forensic Toxicology, second edition Sampling, storage and stability | 353

Chemical hydrolysis of cocaine during analysis Benzodiazepines should also be considered. Liquid–liquid and solid- Benzodiazepines including alprazolam, chlor- phase extractions of cocaine and metabolites from diazepoxide, clonazepam, diazepam, flunitrazepam, biological specimens routinely employ alkaline con- flurazepam, lorazepam, midazolam, nitrazepam, nor- ditions during extraction or elution steps. These con- diazepam, oxazepam, temazepam and triazolam were ditions may result in chemical hydrolysis of cocaine ◦ stable in plasma for 1 month at −20 C (Kratzsch to BE as an artefact of analysis. Minimising the dura- et al. 2004). Diazepam was found to be stable in tion of exposure and appropriate use of deuterated blood stored at room temperature or refrigerated internal standards is recommended. over a period of 5 months (Levine et al. 1983). In contrast, diazepam and temazepam were unstable Antidepressants in postmortem blood under putrefying conditions. In general, benzodiazepines with a nitro group (e.g. clon- Studies in serum have shown the tricyclic antide- azepam, nitrazepam, flunitrazepam) are among the pressants to be moderately stable. Amitriptyline, most unstable owing to reduction of the nitro group. imipramine, clomipramine and doxepin are relatively ◦ Additives that inhibit reduction (e.g. 2% w/v sodium stable in serum samples stored at −25 Cfor3to6 ◦ metabisulfite) slow the degradation. Postmortem months or at 4 C for 7 days (Rao et al. 1994). Newer conversion of nitrobenzodiazepines to their respective antidepressants, including reboxetine, sertraline and 7-amino breakdown products may also occur as a venlafaxine, were also stable in frozen plasma. result of anaerobic bacterial action (Robertson and Significant decreases in concentration were seen Drummer 1995, 1998). Chlordiazepoxide, which for sertraline, desmethylsertraline and reboxetine contains an N-oxide functionality, is also unstable in when stored at room temperature for more than 7 whole blood. At room temperature, chlordiazepoxide days (Heller et al. 2004). Atomoxetine, citalopram, rapidly decreased and was undetectable by day 8 fluoxetine, mirtazepine and paroxetine have also (Levine et al. 1983). Sodium fluoride inhibits the shown moderate long-term stability when frozen degradation of chlordiazepoxide to nordiazepam (Peters 2007). and demoxepam, but does not completely prevent it. Storage at low temperatures, preferably frozen, is Neuroleptics recommended (Drummer and Gerostamoulos 2002; Peters 2007). Among the newer atypical neuroleptics, quetiapine and olanzapine have been shown to exhibit signif- Lysergide icant instability at room temperature (Heller et al. 2004). Although both were stable for a few days, Lysergide (LSD) is photolabile, and specimens sus- quetiapine concentrations decreased by as much as pected of containing LSD should be protected from 50% after 14 days and olanzapine was undetected the light. Decreases in drug concentration have been in some samples. However, the majority of neurolep- documented in blood, serum and urine, with and with- tic drugs, including olanzapine and quetiapine, were out sodium fluoride. In one study, however, LSD con- ◦ stable in frozen plasma at −20 C for 1 month after centrations in urine were stable for 4 weeks at room three freeze–thaw cycles (Kratzsch et al. 2003). Phe- and refrigerated temperatures (Francom et al. 1988). nothiazines exhibit a pH-dependent photosensitivity. Furthermore, increases in chlorpromazine and thior- Ethanol idazine concentrations in patient samples have been documented and may be attributed to a conversion of During storage, ethanol concentrations may increase the metabolite back to the parent drug during storage or decrease. Ethanol losses are largely attributed (Davis et al. 1977; Holmgren et al. 2004). to evaporation, chemical oxidation and microbial

Sample chapter from Clarke’s Analytical Forensic Toxicology, second edition 354 | Clarke’s Analytical Forensic Toxicology consumption, whereas increases are largely due to 2002; LeBeau et al. 2007). Nevertheless, storage microbial conversion of substrates to ethanol. at refrigerated temperature, use of sodium fluoride Although measured increases in blood ethanol as preservative and analysis at the earliest possible concentrations have been documented under some interval are recommended wherever possible. conditions, this is inhibited by the addition of sodium fluoride as preservative and storage at refrigerated temperatures. Ethanol was stable in fluoridated blood References for 2 months at room temperature (Glendening and Waugh 1965). Even after storage for 1 to 3 years Ball W, Lichtenwalner M (1979). Ethanol production in at room temperature, average decreases at room infected urine. N Engl J Med 301: 614. Baselt RC (1983). Stability of cocaine in biological fluids. temperature were 0.4 g/L. Average losses following J Chromatogr 268: 502–505. storage of blood at room temperature for 3 and 6.75 Berankova K et al. (2006). Gamma-hydroxybutyric acid years were 0.19 and 0.33 g/L, respectively (Chang stability and formation in blood and urine. Forensic Sci et al. 1984). In one study, loss of ethanol was evident Int 161: 158–162. Blackmore DJ (1968). The bacterial production of ethyl in blood contaminated by Pseudomonas. Although alcohol. J Forensic Sci Soc 8: 73–78. this was not prevented by 1% sodium fluoride, Carroll FT et al. (2000). Morphine-3-d-glucuronide increasing the quantity of preservative to 2% did stability in postmortem specimens exposed to bacterial prevent ethanol loss (Dick and Stone 1987). enzymatic hydrolysis. Am J Forensic Med Pathol 21: 323–329. Urine is less susceptible than blood to in vitro Chang RB et al. (1984). The stability of ethyl alcohol in ethanol production except in rare instances. Urine forensic blood specimens. J Anal Toxicol 8: 66–67. samples treated with microorganisms known to pro- Christophersen AS (1986). Tetrahydrocannabinol stability duce ethanol did not produce increases in ethanol in whole blood: plastic versus glass containers. J Anal Toxicol 10: 129–131. concentration greater than 0.2 g/L, even following ◦ Cirimele V et al. (2002). Determination of chronic abuse of incubation at 37 C (Blackmore 1968). The use of the anaesthetic agents midazolam and propofol as preservative and refrigeration of urine samples is effec- demonstrated by hair analysis. Int J Legal Med 116: tive in terms of maintaining ethanol stability. Excep- 54–57. tions have been noted, but are rare. For example, a Clauwaert KM et al. (2000). Segmental analysis for cocaine and metabolites by HPLC in hair of suspected drug dramatic increase in ethanol concentration was doc- overdose cases. Forensic Sci Int 110: 157–166. umented in the urine from a diabetic patient found Clauwaert KM et al. (2001). Stability study of the designer to contain Candida albicans. The increase in ethanol drugs ‘MDA, MDMA and MDEA’ in water, serum, concentration was also accompanied by a significant whole blood, and urine under various storage temperatures. Forensic Sci Int 124: 36–42. decrease in glucose concentration (Ball and Lichten- Coe JI (1977). Postmortem chemistry of blood, walner 1979). cerebrospinal fluid, and vitreous humor. Leg Med Annu 1976: 55–92. Gamma-Hydroxybutyric acid Coe JI (1993). Postmortem chemistry update. Emphasis on forensic application. Am J Forensic Med Pathol 14: In addition to GHB being present in a variety of 91–117. biological specimens as an endogenous substance, Crouch DJ (2005). Oral fluid collection: the neglected variable in oral fluid testing. Forensic Sci Int 150: in situ production of GHB during storage has been 165–173. documented and widely studied. In general, increases Curry AS et al. (1967). The production of cyanide in post in GHB concentration are more pronounced in post- mortem material. Acta Pharmacol Toxicol (Copenh) mortem specimens. The concentration of GHB in an 25: 339–344. Curtis J, Greenberg M (2008). Screening for drugs of abuse: unpreserved postmortem blood sample stored under hair as an alternative matrix: a review for the medical refrigerated conditions for 4 months approached toxicologist. Clin Toxicol (Phila) 46: 22–34. 100 mg/L. GHB increases in postmortem urine are Dasgupta A (2007). The effects of adulterants and selected less pronounced and typically an order of magnitude ingested compounds on drugs-of-abuse testing in urine. Am J Clin Pathol 128: 491–503. lower, even in the absence of preservative (Berankova Davis CM et al. (1977). Improved gas chromatographic et al. 2006). Antemortem samples are much less analysis of chlorpromazine in blood serum. Clin Chim susceptible to in situ production over time (Kerrigan Acta 78: 71–77.

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Sample chapter from Clarke’s Analytical Forensic Toxicology, second edition