Sample handling

Sample handling is an important consideration during the pre-analytical phase. Unlike a clinical setting, where the time between sample collection and testing is often very short, significant delays are common in a forensic setting. The pre-analytical phase may be considerable, spanning the time of death and/or discovery of a victim, autopsy and collection of specimens, sample storage, transport to the laboratory and subsequent storage prior to analytical testing. In antemortem toxicology settings, the time delay between an alleged offence and specimen collection may be short (e.g. minutes to hours in the case of most impaired driving cases) or long (e.g. hours to days in the case of some sexual assault cases).

Following collection, antemortem specimens may be subject to similar delays due to shipping or transport of specimens, requests for testing made by the submitting agency and storage of samples prior to actual testing. Although the toxicologist must consider the time delay between the event (i.e. death, or committing or being the victim of an offence) and collection of a specimen for interpretation purposes, these delays are beyond the control of the laboratory. Measures can be taken, however, to preserve and maintain the integrity of specimens after collection. Sample quality plays an important role in the validity or usefulness of subsequent analytical determinations. Inappropriate sample preservation or storage may have a deleterious effect on qualitative and quantitative determinations.

Preservation and storage

Specimens should be stored at appropriate temperatures, with adequate preservative and in an environment accessible only to authorised personnel to ensure security and integrity. Short-term storage at refrigerated temperature (4° C) is recommended for most samples, or frozen (-20 ° C or lower) during long-term storage (more than 2 weeks). Exceptions to this include hair, nails or dried blood swatches on filter paper, which can be stored at ambient temperatures.

Whereas clinical specimens are typically unpreserved, the use of a chemical preservative is often warranted in forensic specimens. Preservation of blood samples with sodium fluoride (2% w/v) is routine in most laboratories. Commercial evacuated blood collection tubes (e.g. grey-top tubes) contain sodium fluoride as the preservative and potassium oxalate as the anticoagulant. These are the preferred evacuated blood tubes for antemortem forensic toxicology casework. Inhibition of microorganisms and enzymes with sodium fluoride is important for commonly encountered analytes such as ethanol, cocaine and others. Fluoride acts as an enzyme inhibitor and helps prevent glycolysis.

Commercial blood tubes may contain a wide variety of additives (citrate, heparin, EDTA, thrombin, acid citrate dextrose mixtures, clot activator, etc.). Although these tubes are designed for a variety of clinical uses, they are not the preferred specimen containers for drug-testing purposes. Laboratories frequently encounter these blood tubes when they are submitted from a hospital setting and special care must be taken when interpreting their results (LeBeau et al. 2000; Toennes, Kauert 2001). If an anticoagulant is to be used, potassium oxalate is preferred rather than alternatives such as EDTA, heparin or citrate. Antioxidants such as ascorbic acid (0.25% w/v) or sodium metabisulfite (1% w/v) are sometimes used to prevent oxidative losses, but these agents have the potential to act as reducing agents towards some drugs, in particular N-oxide metabolites, which may be transformed into the parent drug. In a similar fashion, adjustment of specimen pH is not generally 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 used as a preservative and antimicrobial agent in urine samples. Sodium azide should not be used if samples are to be analysed by enzyme-linked immunosorbent assay because it can interfere with horseradish peroxidase-mediated colorimetric detection.

Although the addition of preservative should be routine for most antemortem and postmortem blood samples, an aliquot of unpreserved postmortem blood is sometimes collected. For example, fluoride preservatives should not be used if organophosphorus chemicals are suspected since this accelerates chemical degradation (Skopp, Potsch2004). Some drugs are known to be photolabile (e.g. ergot alkaloids such as lysergic acid diethylamide and the phenothiazines). Specimens known to contain photolabile drugs should be stored in amber vials or foilcovered containers, or otherwise protected from direct sources of light. Storage of tightly sealed appropriate containers at low temperature further inhibits sample loss. Short-term storage at refrigerated (4 ° C) and frozen (-20 ° C) temperatures is commonplace in most laboratories and repeated freeze–thaw cycles should be avoided.

Labelling and specimen transfer All samples should be properly marked for identification with the case number, donor name, date and time of collection, signature or initials of the collector and specimen description. Tamper-proof containers and/ or tape bearing the collector’s initials and date of collection should be used. Specimens should be forwarded to the laboratory in appropriate leak-proof and tamper-proof packaging/shipping materials with all appropriate documentation (chain-of-custody forms, requisitions for testing, special requests, case information, medications list, police report, donor information/identifier such as date of birth or social security number, agency case number, pathologist/police officer name and contact information). Improperly packaged or identified materials should be returned to the submitting agency. Documentation accompanying the specimen(s) should list all of the specimens that were collected or available for testing. Once received by the laboratory, the specimens should be inspected and appropriately documented in terms of condition and quantity during the accessioning process.

Contamination

There are a variety of contamination sources for both antemortem and postmortem specimens. In addition to the potential contamination issues that may result from the use of containers and external factors, a number of important exogenous and endogenous sources of contamination should be considered.

Exogenous contaminants

Specimens collected into plastic containers are sometimes susceptible to phthalate interferences. Numerous plasticiser interferences such as dibutylphthalate may co-extract and interfere with analytical detection by gas-chromatographic or mass-spectrometric techniques, yielding characteristic phthalate ions. All plastic containers should be evaluated prior to widespread implementation. It should be noted that contamination from phthalates may occur during the analytical process through use of disposable pipette tips, solvent containers, solid-phase extraction cartridges, tubing and numerous other sources. However, environmental exposure to these substances from household items, food, beverages and other sources can produce detectable quantities of phthalate esters or their metabolites in biological specimens including blood, serum, urine and breast milk (Silva et al. 2005; Hogberg et al. 2008).

Embalming fluids, which typically contain a variety of alcohols and aldehydes, are a potential source of contamination in postmortem casework. These fluids not only dilute any remaining fluid in the body, but also alter drug distribution in remaining tissues. Another potential source of contamination comes from reusable syringes and containers for postmortem specimen collection. Some cleaning fluids that are used for syringes may contain alcohols that can compromise the analysis of volatiles. This highlights the importance of analysing specimens from multiple sites and using disposable syringes wherever possible.

The principal concern with antemortem contamination arises from the intentional manipulation of the sample to mask the presence of drugs. This typically involves the substitution, dilution or adulteration of the biological specimen with a foreign substance. Donor manipulation occurs most frequently with urine samples in workplace drugtesting situations. As a result, specimen validity testing is required in some drug-testing programmes such as for federal employees under US Department of Health and Human Services (DHHS) guidelines. Initially, adulteration of urine for drug-testing purposes involved the use of crude house hold items such as soap, bleach, vinegar, ammonia or cleaning fluids. Although these substances met with some success, a wide variety of commercial adulteration reagents and kits is now widely available (Dasgupta2007). A summary of in-vitro adulteration agents is provided in the following list :

• Ascorbic acid
• Alcohols
• Amber-13 (hydrochloric acid)
• Ammonia
• Bleach
• Clear Choice (glutaraldehyde)
• Detergent or soap (surfactant)
• Drano
• Ethylene glycol
• Gasoline
• Glutaraldehyde
• Hydrogen peroxide
• Klear (potassium nitrite)
• Lemon juice
• Liquid soap
• Lime-A-Way
• Mary Jane Super Clean 13 (surfactant)
• Salt
• Stealth (peroxide/peroxidase)
• THC-Free (hydrochloric acid)
• UrinAid (glutaraldehyde)
• Urine Luck (chromium VI, oxidant)
• Vanish
• Vinegar
• Visine
• Water
• Whizzies (sodium nitrite)

Some of the most popular commercial products contain glutaraldehyde (fixative), pyridinium chlorochromate (PCC) orchromium(VI)-containing species (oxidant), nitrite(oxidant)orperoxide/peroxidase.In general, in-vitro adulterants can interfere with presumptive immunoassaytests, with the intention of producing false-negative results. However, some agents have the potential to interfere with confirmatory tests such as gas chromatography/ mass spectrometry (GC-MS) as well. Although this is less likely, studies have shown that some reagents may produce lower than expected or negative results for some analytes. Adulteration detection products are available commercially. On-site or dipstick tests are available for nitrite, glutaraldehyde, pH, specific gravity, creatinine, bleach, PCC and oxidants. Specimen dilution or in-vivo adulteration by ingestion of a substance to mask the presence of drugs is also encountered. This is commonly achieved by the ingestion of large quantities of fluid prior to the test or by administration of a diuretic. Examples of in-vivo adulteration agents are given in the following list:

Prescription
– Thiazides and thiazide-like drugs (e.g. hydrochlorothiazide, metolazone)
– Carbonic anhydrase inhibitors (e.g. acetazolamide)
– Loop diuretics (e.g. bumetanide, furosemide, torsemide)
– Osmotic diuretics (e.g. mannitol)

Over the counter (OTC)
– Aqua-Ban
– Diurex
– Fem-1
– Midol
– Pamprin
– Premsy- PMS

Other
– Alcoholic beverages
– Xanthines (e.g. caffeine, theophylline, 8-bromotheophylline)
– Herbals and aquaretics (e.g. golden seal root, juniper)

Urine specimen substitution or dilution can be detected if specimen validity tests are performed. A specimen may be considered invalid if the pH is between 3 and 4.5 or between 9 and 11. It may be adulterate less than 3 or greater than 11.The normal temperature range is 32–38 ° C. A specimen is considered dilute if the creatinine concentration is less than 200mg/L and the specific gravity is less than 1.003. Other sources of contaminants or unexpected analytes include pyrolytic breakdown products due to thermal degradation of drugs. These may be present due to pyrolysis during administration of the drug (e.g. anhydroecgonine methyl esterfollowing crack cocaine use) or occasionally in situ during analysis if conditions are not properly controlled or evaluated. Other sources of contamination may arise from pharmaceutical impurities or adulterants and cutting agents that are incorporated into illicit drugs prior to sale.

Medical artefacts

Clinical therapy can sometimes produce medical artefacts that complicate toxicological findings. Medical artefacts are most common in postmortem cases where infusion pumps may continue to run after death, introducing high concentrations of drug in local body compartments. Access to hospital records and case information, and collection of peripheral blood, vitreous fluid and liver are particularly important in these types of cases. Other sources of medical artefacts may include organ harvest drugs such as the calcium-channel blocker verapamil, or papaverine, which is used to inhibit vasoconstriction during transplant surgery.

If living patients are administered fluids (e.g. saline) during clinical care, blood is only contaminated (diluted) with the infusion solution if it is collected downstream from the intravenous line. Blood circulation and equilibrium with tissues is rapid, so the administration of fluids does not usually influence drug or alcohol concentrations in blood if normal precautions are taken. If downstream collection is suspected, careful review of the medical records and/or measurement of the haematocrit to determine specimen dilution may be necessary.

Endogenous contaminants, artefacts and interferences

By their very nature, all biological specimens are subject to endogenous interferences, regardless of whether or not they are derived from living or deceased persons. More complex biological specimens such as blood, tissue or meconium will require more extensive sample preparation to remove these interferences than less complex matrices such as vitreous humour, or cerebrospinalororalfluid.Ingeneral,however,antemortem specimens are somewhat less susceptible to endogenous artefacts or contaminants. Ethanol, GHB, carbon monoxide, cyanide and other short-chain alcohols can be metabolically produced post mortem (Skopp 2004). The formation of toxicologically significant concentrations of cyanide in postmortem tissue (Lokan et al. 1987) has been attributed to the conversion of thiocyanate to cyanide and the breakdown of protein (Curry et al. 1967). Although in some circumstances ethanol can be produced in situ in unpreserved antemortem fluids, the same is true to a far greater extent in postmortem specimens, particularly blood. Likewise, GHB is present in antemortem fluids at very low concentrations in the absence of a serious genetic disorder such as GHB uria (Knerr et al. 2007). Differentiation of exogenous and endogenous GHB is complicated by specimen type, storage conditions, preservative and other factors. Many laboratories use a cut-off concentration to help differentiate the two, for example 10mg/L in urine (Kerrigan 2002; LeBeau et al. 2007). Concentrations of GHB may increase in urine during storage, upon collection and storage of unpreserved blood, or in citrate-buffered antemortem blood (LeBeau et al. 2000). Although preserved antemortem blood GHB concentrations are typically lower than those in urine, numerous studies have shown forensically significant concentrations of GHB in postmortem blood. Postmortem urine and vitreous fluid appear to be less susceptible to this increase.

Major changes that occur after death produce autolytic changes and putrefaction by microorganisms. Invasion of microorganisms, particularly from the gastrointestinal tract into tissues and body fluids, occurs within hours at ambient temperature. Lipids, carbohydrates and proteins are hydrolysed by microbial enzymes, the pH of blood steadily increases, and the putrefactive amines, tyramine, tryptamines, phenethylamines and other endogenous substances are liberated.

Trauma is a non-preventable source of contamination in postmortem forensic toxicology. Rupture of organs or compartments within the body can compromise quantitative drug analyses owing to the mixing of fluids (e.g. of gastric contents with blood) or from the microbial action that occurs as a result. Postmortem alcohol production canalsoresult in detectable quantities of ethanol as an artefact. Glycolysis and the presence of yeasts and microorganisms can convert a variety of postmortem substrates to ethanol. Although concentrations are typically low (<0.7g/L), concentrations of 2g/L and higher have been reported (Zumwalt et al. 1982; O’Neal, Poklis 1996).

Postmortem alcohol productionisinfluencedbymany factors,includingthetimebetweendeath and sampling, environmental conditions (temperature, humidity, location), external factors (traumaticinjury, incineration), the availability of an ethanol substrate and the extent to which microorganisms are available. Vitreous humour and urine provide complementary information that may assist with the interpretation of results. This highlights the importance of collecting a variety of specimens post mortem. Other short-chain alcohols can be produced by microorganisms in situ. Isopropanol has been documented as a postmortem artefact, particularly in drowning victims. Putrefaction can also complicate carbon monoxide determination in postmortem blood. Increases in the apparent concentration of carboxyhaemoglobin have been documented owing to the formation of methaemoglobin, a decomposition product that can interfere with spectrophotometric determination. Preservation of postmortem blood (and storage in the dark at 4 ° C or lower)has been recommended (Skopp 2004).

Stability

Drug stability can be influenced by many factors including the physicochemical properties of the drug, characteristics of the specimen or matrix, tendency to conjugate/deconjugate, specimen collection procedure (e.g. contaminationwith microorganisms), container selection (e.g. oxidation, adsorption), and the use of preservatives or other additives. The majority of published drug stability studies focus on antemortem or non-biological matrices.

Scientific findings for biological specimens are complex because drug instability is often matrix dependent andinfluencedbyfactorssuchasspecimenpHandthepresenceofother substances in addition to external factors. In general, drug instability in any toxicological specimen is due to metabolic degradation, chemical transformation, or a combination of both (e.g. cocaine). Drug stability in postmortem matrices poses an added level of complexity because conditions are less controlled and degradation of analytes may be accelerated owing to putrefactive decomposition, microbial invasion and the increased presence of bacteria, forexample, the bacterial enzymatic conversion of morphine glucuronides to free morphine in blood (Carroll et al. 2000), and the bioconversion of nitrobenzenes by enteric bacteria (Robertson, Drummer 1998). After a specimen has been collected, enzymes may remain active and continue to degrade or transform the drug in vitro. This process may take place post mortem inside the body, or after postmortem or antemortem specimens have been collected, during transportation to the laboratory and during storage. This is particularly important with esterases, which may further hydrolyse drugs post-collection unless they are inhibited by a preservative. In general, drug instability arises as a result of moieties or functional groups that are susceptible to transformation, such as esters (e.g. 6 acetylmorphine, cocaine, acetylsalicylic acid), sulfur-containing drugs, photolabile drugs(e.g.phenothiazines, midazolam, lysergicacid diethylamide) or those with functional groups that are readily oxidized or reduced.

Although instability typically leads to decreases in drug concentration, this is not always the case. Conjugated drugs, such as the glucuronides may deconjugate under some conditions, increasing the concentration of free drug. Depending on the collection, storage conditions, use of preservative, container type,matrix and other factors, the concentration of a drug at the time of assay may not be identical to the concentration at the time of collection. All toxicological results should be interpreted within this context. Some drugs also exhibit a degree of thermal instability. This is a consideration for drugs that are subjected to elevated temperatures during administration (e.g. by smoking) and during analysis (e.g. by GC-MS). Pyrolysis products may be indicative of smoking if it can be shown that they are not produced during analysis, for example: anhydroecgonine methyl ester (AEME) following the use of crack cocaine; 1phenyl-cyclohexene following phencyclidine (PCP) use; and transphenyl propene following metamfetamine use. Ideally, drug stability should be evaluated in a number of ways:longterm stability in the specimen or matrix of interest; the effect of freeze– thaw cycles; short-term stability (typically refrigerated); and bench-top (roomtemperature) stability. The kinetic variables governing instability are often matrix and temperature dependent and an understanding of any one of these (e.g. long-term stability of a particular drug in blood) does not necessarily imply predictable results under different conditions (i.e. short-term storage of the drug in urine).

It should be noted that, although there are many published studies and reviews of drug stability, these tend to focus on longor short-term storage in common matrices such as blood, urine, serum or plasma. As the number of drugs of interest continues to grow and the variety of specimens becomes more diverse, the scientific literature becomes somewhat limited in terms of drug stability.

The stability of drugs in non-traditional and non-biological matrices is beyond the scope of this discussion, but investigations in this area are ongoing. These include studies of the stability drugs in dry stains of biological origin (DuBey, Caplan 1996), in formaldehyde solutions following embalming (Tracy et al. 2001), and in hair fibres after exposure to cosmetic treatment such as bleaching, perming or straightening (Potsch, Skopp 1996). Drug stability data are often confounded by the fact that a considerable number of studies report instability as an incidental or anecdotal finding, rather than as part of a formalised and well-designed investigation of stability. Guidelines for conducting stability experiments and the statistical evaluation of the results have been reviewed (Peters 2007).

Of all of the most commonly encountered drugs, cocaine is certainly the most notorious in terms of instability. Nevertheless, the vast majority of drugs are relatively stable or moderately so to the extent that one can make reasonable assumptions when interpreting the results. Stability for some of the most frequently encountered drugs is summarised below.

Amfetamines

Metamfetamine and amfetamine in urine samples preserved with sodium fluoride (1% w/v) were stable during long-term storage at -20 ° C for at least a year (Moody et al. 1999). A long-term study of whole blood stored over 5 years at room temperature  showed erratic but significant decreases in metamfetamine between 3 months and 5 years of storage, ranging from 9% to 38% (Giorgi, Meeker 1995). Results suggested that amfetamine was perhaps less stable than metamfetamine. Samples were collected into 10-mL grey-top Vacutainer tubes with sodium fluoride (100mg) and potassium oxalate (20mg). Although both drugs are considered to be moderately stable, storage of blood samples at room temperature is not advised owing to the production of interfering substances. The overall stability of the amfetamine class extends to many of the designeramfetamines,forexample, 3,4-methylene dioxymetamfetamine (MDMA), 3,4-methylenedioxyamfetamine (MDA) and 3,4-methylenedioxyethylamfetamine (MDEA). Stability of these drugs was investigated in urine, blood and water for 21 weeks at -20 ° C, 4 °C and 20 ° C (Clauwaert et al. 2001). Although all drugs were stable at -20 ° C, results were compromised in blood samples at 5 and 13 weeks when stored at 20 ° C and 4 ° C, respectively, owing to matrix degradation and interfering substances.

Cannabinoids

Decreases in cannabinoid concentrations in refrigerated and frozen samples are largely attributed to oxidative losses, temperature effects or lipophilic binding to containers. THC may decompose when exposed to air, heat or light. It can undergo hydrolysis to cannabidiol, or be oxidised to cannabinol as a result of exposure to air or acidic conditions.

THC has been reported to be stable in refrigerated blood for 6 months and at room temperature for 2 months (Johnson et al. 1984). Binding of THC to hydrophilic surfaces, such as storage containers or rubber stoppers should be considered. For example, THC stored in blood collected in unsilanised glass tubes was stable for 4 days at room temperature and 4 weeks at -20 ° C. By comparison, similar samples stored in polystyrene tubes showed 60–100% decreases in concentration (Christophersen 1986). The principal metabolite shows greater stability than the parent drug. In one study, 11-nor-9-carboxy-D 9 -tetrahydrocannabinol (THCA) was stable in frozen urine preserved with sodium fluoride(1% w/v) for a year (Moody et al. 1999). However, this study used silanised glassware for urine sample storage, which may not be typical of storage containers for actual casework. Loss of THCA in urine is largely attributed to adsorption, or from foaming of the sample, which can account for losses as high as 89% (Dextraze et al. 1989). The use of a de-foaming agent (e.g. 2-octanol) can reverse these losses, but use is not routine. It has been suggested that, although the adsorptive losses of THCA from urine stored in plastic containers take place quickly (within an hour of collection), they are not significant enough to compromise the analysis (Stout et al. 2000). Furthermore, adsorptive losses of THCA in urine may be pH dependent, with greater losses occurring at acidic pH (Jamerson et al. 2005).

Deconjugation of cannabinoids, such as THCA-glucuronide, to THCA should also be considered (Skopp, Potsch 2004). Hydrolysis of the acylglucuronide results in increased concentration of the unconjugated metabolite. THCA-glucuronide was unstable in urine at 4 ° C and above, and at increasing urinary pH (Skopp 2004). Studies suggest that THCA-glucuronide is less stable in plasma than in urine (Skopp, Potsch 2002). Although no significant losses in THCA-glucuronide were seen in plasma or urine stored at -20 ° C, instability was documented following storage of plasma and urine at refrigerated and room temperature, in some cases within 2 days (Skopp, Potsch 2002). Opioids 6-Monoacetylmorphine(6-MAM) is a labile metabolite of diamorphine due to hydrolysis of the ester bonds. It may undergo deacetylation to morphine during storage. Buprenorphine, codeine, fentanyl, hydromorphone, methadone, morphine, oxycododone, oxymorphoneandtramadol were all stable in frozen plasma stored for almost 3 months and subjected to two freeze–thaw cycles (Musshoff et al. 2006). Free concentrations of morphine, codeine and methadone were moderately stable in frozen urine preserved with sodium fluoride over a year (Moody et al. 1999). However, total morphine concentrations under similar conditions may be less stable (Moriya, Hashimoto 1997). Longterm storage in preserved whole blood stored at room temperature showed significant increases and decreases (Giorgi, Meeker 1995) over 1–5 years. These results suggest important differences in stability between free and conjugated species. The stability of glucuronidated morphine is of importance because ratios of free and total morphine are sometimes used for interpretive purposes. Marked differences in glucuronide stability exist between antemortem and postmortem blood. Morphine-3-glucuronide was stable in refrigerated antemortem blood preserved with sodium fluoride, but unstable in postmortem blood under the same conditions (Carroll et al. 2000). Hydrolysis of the glucuronidated species to free morphine increases with temperature, storage time and degree of putrefaction. Other studies have confirmed the stability of morphine-3-glucuronide in refrigerated antemortem blood and plasma for up to 6 months (Skopp et al. 2001). Storage of postmortem specimens at -20 ° C prevented in vitro hydrolysis of the glucuronide. Phencyclidine Studies suggest that PCP is a relatively stable drug, even when stored in blood at room temperature for up to 18 months (Levine et al. 1983). However, significant decreases in concentration were measured in preserved blood at room temperature over 5 years (Giorgi, Meeker 1995). Cocaine Of all of the common drugs of abuse, cocaine is certainly notorious in terms of stability. Both chemical and enzymatic transformations occur to produce hydrolytic products. Spontaneous conversion of cocaine to benzoylecgonine (BE) via the ester linkage occurs at physiological and alkaline pH. At pH 5, there were no measurable decreases in cocaine concentration at 40 ° C after 21 days, compared with a decrease of 40–70% in urine at pH 8 (Baselt 1983). Ester linkages have a tendency to be alkali labile, and as a result the chemical transformation of cocaine to BE is increasingly favourable as the pH of the matrix increases.

Although the addition of preservative does not prevent chemical hydrolysis, the kinetics can be inhibited by storage of samples at low temperature, or by pH adjustment. The latter is not favoured in routine casework because of the possibility that acid-labile drugs might also be present. Liver methylesterases are largely responsible for the enzymatic transformation of cocaine to BE and plasma pseudocholinesterase for the conversion of cocaine to ecgonine methyl ester (EME). Addition of a cholinesterase inhibitor such as sodium fluoride and reduced temperature are important precautions. Both BE and EME undergo further transformation to ecgonine, a polar metabolite. In unpreserved blood, hydrolysis of the phenyl ester predominates, yielding EME. Addition of sodium fluoride inhibits the production of EME but does not prevent chemical hydrolysis of cocaine to BE. Cocaine in blood stored at 4 ° C was undetectable within 3 days in the absence of preservative, and only 40–60% of the cocaine was detected in preserved blood and plasma after 21 days (Baselt1983). There have been numerous studies on the stability of cocaine and its metabolites in various media and comprehensive stability studies are available (Isenschmid et al. 1989). In unpreserved blood, cocaine is hydrolysed to EME, whereas transformation to BE predominates in preserved blood. Although BE exhibits greater stability than cocaine, decreases in concentration are largely due to further hydrolysis to ecgonine. BE was shown to be stable in preserved urine for a period of at least 1year when stored at -20 ° C (Moody et al. 1999). Chemical hydrolysis of cocaine during analysis should also be considered. Liquid–liquid and solid-phase extraction of cocaine and metabolites from biological specimens routinely employ alkaline conditions during extraction or elution steps. These conditions may result in chemical hydrolysis of cocaine to BE as an artefact of analysis. Minimising the duration of exposure and appropriate use of deuterated internal standards is recommended.

Antidepressants

Studies in serum have shown the tricyclic antidepressants to be moderately stable. Amitriptyline, imipramine, clomipramine and doxepin are relatively stable in serum samples stored at -25 ° C for 3–6 months or at 4 ° C for 7 days (Rao et al. 1994). Newer antidepressants, including reboxetine, sertraline and venlafaxine, were also stable in frozen plasma. Significant decreases in concentration were seen for sertraline, desmethylsertraline and reboxetine when stored at room temperature for more than 7 days (Heller et al. 2004). Atomoxetine, citalopram, fluoxetine, mirtazepine and paroxetine have also shown moderate long-term stability when frozen (Peters 2007). Neuroleptics Among theneweratypicalneuroleptics, quetiapine andolanzapine have been shown to exhibit significant instability at room temperature (Helleret al.2004). Although bothwerestable fora fewdays,quetiapine concentrations decreased by as much as 50% after 14 days and olanzapine was undetected in some samples. However, the majority of neuroleptic drugs, including olanzapine and quetiapine, were stable in frozen plasma at -20 ° C for 1month after three freeze–thaw cycles (Kratzsch et al. 2003). Phenothiazines exhibit a pH-dependent photosensitivity. Furthermore, increases in chlorpromazine and thioridazine concentrations in patient samples have been documented and may be attributed to a conversion of the metabolite back to the parent drug during storage (Davis et al. 1977; Holmgren et al. 2004).

Benzodiazepines

Benzodiazepines including alprazolam, chlordiazepoxide, clonazepam, diazepam, flunitrazepam, flurazepam, lorazepam, midazolam, nitrazepam, nordiazepam, oxazepam, temazepam and triazolam were stable in plasma for 1 month at -20 ° C (Kratzsch et al. 2004). Diazepam was found to be stable in blood stored at room temperature or refrigerated overaperiod of 5months (Levineetal.1983). Incontrast, diazepam and temazepam were unstable in postmortem blood under putrefying conditions. In general, benzodiazepines with a nitro group (e.g. clonazepam, nitrazepam, flunitrazepam) are among the most unstable owing to reduction of the nitro group. Additives that inhibit reduction (e.g. 2% w/v sodium metabisulfite) slow the degradation. Postmortem conversion of nitrobenzodiazepines to their respective 7-amino breakdown products may also occur as a result of anaerobic bacterial action (Robertson, Drummer 1995, 1998). Chlordiazepoxide, which contains an N-oxide functionality, is also unstable in whole blood. At room temperature, chlordiazepoxide rapidly decreased and was undetectable by day 8 (Levineetal.1983). Sodium fluoride inhibits the degradation of chlordiazepoxide to nordiazepam and demoxepam, but does not completely prevent it. Storage at low temperatures, preferably frozen, is recommended (Drummer, Gerostamoulos 2002; Peters 2007).

Lysergide

Lysergide (LSD) is photolabile and specimens suspected of containing LSD should be protected from the light. Decreases in drug concentration have been documented in blood, serum and urine, with and without sodium fluoride. In one study, however, LSD concentrations in urine were stable for 4 weeks at room and refrigerated temperatures (Francom et al. 1988).

Ethanol

During storage, ethanol concentrations may increase or decrease.

Ethanol losses are largely attributed to evaporation, chemical oxidation and microbial consumption, whereas increases are largely due to microbial conversion of substrates to ethanol. Although measured increases in blood ethanol concentrations have been documented under some conditions, this is inhibited by the addition of sodium fluoride as preservative and storage at refrigerated temperatures. Ethanol was stable in fluoridated blood for 2 months at room temperature (Glendening, Waugh 1965). Even after storage for 1–3 years at room temperature, average decreases at room temperature were 0.4g/L. Average losses following storage of blood at room temperature for 3 and 6.75 years were 0.19 and 0.33g/L, respectively (Chang et al. 1984). In one study, loss of ethanol was evident in blood contaminated by Pseudomonas. Although this was not prevented by 1% sodium fluoride, increasing the quantity of preservative to 2% did prevent ethanol loss (Dick, Stone 1987).

Urine is less susceptible than blood to in vitro ethanol production except in rare instances. Urine samples treated with microorganisms known to produce ethanol did not produce increases in ethanol concentration greater than 0.2g/L, even following incubation at 37 ° C (Blackmore 1968). The use of preservative and refrigeration of urine samples is effective in terms of maintaining ethanol stability. Exceptions have been noted, but are rare. For example, a dramatic increase in ethanol concentration was documented in the urine from a diabetic patient found to contain Candida albicans. The increase in ethanol concentrationwas also accompanied by a significant decrease in glucose concentration (Ball, Lichtenwalner 1979).

gamma-Hydroxybutyric acid

In addition to GHB being present in a variety of biological specimens as an endogenous substance, in situ production of GHB during storage has been documented and widely studied. In general, increases in GHB concentration are more pronounced in postmortem specimens. The concentration of GHB in an unpreserved postmortem blood sample stored under refrigerated conditions for 4 months approached 100 mg/L. GHB increases in postmortem urine are less pronounced and typically an order of magnitude lower, even in the absence of preservative (Berankova et al. 2006). Antemortem samples are much less susceptible to in situ production over time (Kerrigan 2002; LeBeau et al. 2007). Nevertheless, storage at refrigerated temperature, use of sodium fluoride as preservative and analysis at the earliest possible interval are recommended wherever possible.