Fundamentals of Analytical Toxicology. Robin Whelpton
Different urine specimens, e.g. random, early morning, end-of-shift, 24-hour (the total urine voided over a day), may be collected in the course of metabolic or other studies. In metabolic studies, it is important to note the time of the beginning and end of the collection period so that the rate of urine production can be calculated. A random urine sample is usually a midstream specimen. A preservative, for example 2 mol L–1 hydrochloric acid to prevent microbial growth and to stabilize phenols, may be added. Fresh urine is yellow/yellow-green in colour, but on storage in acidic solution the colour changes to yellow/brown and even to dark brown because of the oxidation of urobilinogen to urobilin. Crystals, particularly of uric acid and calcium oxalate, may form causing turbidity.
When random, early morning, or end-of-shift specimens are collected it is common practice to relate certain analytical results to a ‘fixed’ urinary constituent such as creatinine, which is considered to be excreted at a relatively constant rate in normal subjects. However, because creatinine is derived from creatine, there are situations such as muscle wasting, excessive exercise, or in bodybuilders dosing with creatine, when this is not strictly true. Note that many clinical chemistry laboratories report creatinine in mmol L–1 (1 mmol L–1 = 113 mg L–1).
The concentrations of many drugs and metabolites, and of some endogenous constituents, will remain the same in acidified urine for over a week at room temperature, and for up to a month at 2–8 °C. Unacidified urine undergoes microbiological attack and many changes occur, including the complete loss of amino acids. For long term storage acidified urine can be stored at –20 °C, but it may be necessary to centrifuge the sample to remove any precipitate formed during storage prior to any analysis. Dried urine spots may also be produced in an analogous manner to dried blood spots.
2.2.5 Stomach contents
This specimen encompasses vomit, gastric aspirate, and gastric lavage fluid as well as the contents of the stomach at post-mortem. The nature of this sample can be very variable and additional procedures such as homogenization followed by filtration and/or centrifugation may be required to produce a sample amenable to analysis. Sometimes tablet residues may be apparent, study of which which might help identify the drug ingested.
2.2.6 Faeces
The analysis of faeces is rarely performed in clinical chemistry, but sometimes drug and possibly metabolite analysis may be required in pharmacokinetic and metabolism studies. Analyses may also be requested if, for example, drug leakage from ingested packets of drug is suspected. Unlike plasma, urine, and other fluid samples, faeces are not homogeneous, and thus it is often necessary to analyze the whole sample or homogenize the whole sample and prove that the fraction taken for analysis is representative of the whole. It may take more than a day after dosage before a drug or a drug metabolite appears in faeces. The stability of some drugs in meconium has been assessed (Wu et al., 2017).
2.2.7 Tissues
Histology specimens are usually collected into a preservative such as formalin (37 % w/v) aqueous formaldehyde solution). Such pre-treatment must be borne in mind if toxicological analyses are requested subsequently. Samples of tissue obtained post-mortem are normally kept at 2–8 °C prior to analysis.
2.3 Guidelines for sample collection for analytical toxicology
If poisoning is suspected, a 10 mL blood sample (lithium heparin or EDTA tube) should be taken from an adult (proportionally less from a young child) as soon as possible. In addition, 2 mL of blood should be collected in a fluoride/oxalate tube if ethanol is suspected. Note that tubes of this type for clinical use contain only ca. 0.1 % w/v fluoride (Table 2.3), whereas ca. 2 % w/v fluoride (40 mg sodium fluoride per 2 mL blood) is needed to inhibit fully microbial action in such specimens. Addition of fluoride may also help protect other labile drugs such as clonazepam, cocaine, and nitrazepam from degradation. If possible, the retention of an unpreserved blood sample is also advisable.
Table 2.4 Sample requirements: general analytical toxicology
| Sample | Notesa |
| Whole blood | 10 mL (lithium heparin or EDTA tube – use fluoride/oxalate if ethanol suspected; plastic tube if paraquat suspected; glass or plastic tube with minimal headspace if carbon monoxide or other volatiles suspected) |
| or Plasma/serum | 5 mL (send whole blood if volatiles, metals, and some other compounds suspected – see above) |
| Urineb | 20–50 mL (plain bottle, no preservativec) |
| Gastric contentsd | 25–50 mL (plain bottle, no preservative) |
| Scene residuese | As appropriate |
aSmaller volumes may often be acceptable, for example in the case of young children
bNormally the only sample that is required for substance misuse screening
cSodium fluoride (2 % w/v) should be added if ethanol is suspected and blood is not available
dIncludes vomit, gastric lavage (SWO, first sample), etc.
eTablet bottles, drinks containers, aerosol canisters, etc. – pack entirely separately from biological samples, especially if poisoning with volatiles is a possibility
Collection of urine, stomach contents, and ‘scene residues’, i.e. material such as tablet bottles or drinks containers found at the scene of an incident may be helpful (Table 2.4). Samples of other appropriate fluids and tissues should also be collected as detailed below, especially when investigating deaths (Table 2.5), but may not be required for analysis unless either special investigations are required or decomposition is advanced (Dinis-Oliveira et al., 2010; Dinis-Oliveira et al., 2017; Belsey & Flanagan, 2016). However, such samples should be retained (2–8 or –20 °C) in case they are needed. The advantages and disadvantages of various specimens are detailed in Table 2.6. There are special considerations in sample collection and storage for metals/trace elements analysis (Section 21.2).
Information recorded on the sample container at the time the sample is collected should include the name of the patient (first and family or last name) and date of birth, patient/subject/animal number, the date and time of collection, and the sample type. Many laboratories will have a bespoke analysis request form to accompany the sample(s) on which this and any other appropriate information such as a note of any preservative added to the sample, site of collection of blood, etc. should be recorded (Box 2.2). The date and time of receipt of all specimens by the laboratory should be recorded and a unique identifying number assigned in each case.
Table 2.5 Sample requirements: post-mortem biochemistry and toxicology