Stability of Drugs of Abuse in Biological Fluids

Appropriate understanding of drug-test results relies gravely on the stability of drug information in biological specimens. This chapter reviews the literature related to the stability of drugs of abuse in blood, urine and oral fluids specimens. A frequently abused drug, ethanol is also included as its steadiness in blood and urine has been researched by many. Amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine, lysergic acid diethylamide, opiates and phencyclidine are amongst the other drug classes incorporated in the chapter. Several reasons form the basis for analysis of biological specimens for example:  indicating exposure to or using of a certain drug, correlating the presence of a drug with physiological or behavioral effects and assisting in the treatment of intoxications. To demonstrate whether drugs were responsible for, or otherwise associated with, the cause of death, the analysis of postmortem specimen drugs is performed.

The reason of drug testing determines the option of the specimen to be analyzed. Monitoring the drug use is effective with the analysis of urine; though, the correlation of its test results with the pharmacotological effects or performance impairment maybe difficult. Extended analytical procedures are required under most circumstances when in cases when the blood or oral fluid analysis can be connected with effects. It’s essential that the test results accurately reflect the drug quantity present at the time of acquisition because toxicological test are regularly performed for a period of time after specimens are received and the results of these tests are used in criminal or civil litigation. Therefore, for proper interpretation of test results, knowledge about the in vitro stability of drugs is crucial.

Data published in the literature relating to the in vitro stability of drugs of abuse in biological fluids in reviewed below. Since ethanol is currently the most widely abused drug in United States society, it is also included.


Amphetamines in Urine

The stability of amphetamine and methamphetamine in urine specimens has been the subject of several studies. Hughes et al. studied over 6 months regarding the stability of amphetamine and methamphetamine in fortified specimens. At room and refrigerated temperatures, both drugs were stable over a period of 6 months. Dugan et al, twelve months after the initial quantification, quantified amphetamine and methamphetamine in 32 urine specimens by storing each specimen at -20⁰C.  After the storage period, all specimens were positive for amphetamine and methamphetamine with the average concentrations of the 2 drugs unchanged over a year’s period. Moody et al. on the other hand examined amphetamine and methamphetamine concentrations in fortified urine specimens storing them at – 20⁰C over an 18-month period; he noticed no significant changes.

Amphetamines in Other Fluids

Amphetamine and methamphetamine in blood specimens was studied by Glorgi and Meeker by storing them at ambient temperature in fluoride/ oxalate tubes over a 4-year period; a decrease in the concentration of both drugs was observed generally. However, after the time period both drugs were detectable.

Studies were conducted, observing changes over a 10- week period, on the stability of 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine(MEDA) and N-methyl 1-(3, 4-methylenedioxyphenyl)-2- butamine (MBDB)  in oral fluids stored at room, refrigerated and frozen temperature. For all compounds in the oral fluid, degradation was observed. MDA, MDMA, MDEA and MDMB faced losses of 31%, 28%, 38% and 37% respectively. Except MDA (P< 0.05), where a greater decrease occurred at room temperature than the other temperatures, all other changes were independent of temperature.


Stability of barbiturates in blood has been investigated by several researchers. Administering pentobarbital to a dog, Coutselins and Liaris obtained blood through the femoral vein and divided the sample in 4 portions. Two parts were stored at 4 °C, 1 in a pooled container and the other in 5-mL aliquots. The remaining 2 portions were similarly prepared but were stored at 25 °C. Specimens from each group were assayed spectrophotometrically every 10 days for 2 months.

The 2 groups stored at 4 °C showed no significant changes throughout the time period, but the specimens stored at 25 °C demonstrated a progressive decrease in concentration. In addition, there were significant differences between the two groups stored at 25 °C. The rate of decline in the specimens stored in a-mL portions was greater than that of the specimen stored in a pooled container. The pentobarbital concentrations dropped from 43 mg/L to 17 ml over 60 days in the pooled group, and to 11 mg/L in the aliquoted blood.

The authors attributed the differences in the 2 groups stored at 25 °C to the fact that blood in an ordinary container evaporates over time, so subsequent testing involves an analysis of a comparatively greater quantity of blood. In generals the decreases observed at 25 °C were attributed to the oxidation of barbiturates in the putrefactive process. In 8 postmortem samples containing 1 or more barbiturates, Marriott et al. compared initial blood concentration measurements to those after a 2-week storage period at room temperature. Of the 14 analyses of 5 separate drugs, only 1 showed a decrease in blood concentration after 2 weeks. One amobarbital analysis showed a minimal decrease from 1.9 to 1.5 mg/L, while the remaining analyses, including 3 amobarbital, 1 butabarbital, 3 pentobarbital, 1 phenobarbital, and 5 secobarbital cases, showed increases in drug concentration. Most of these increases were relatively small at less than 30%. The greatest observed increase was in an amobarbital specimen in which the blood concentration increased from 4.4 to 6.5 mg/L.

An extended study of the in vitro stability of barbiturates in blood was performed by Levine et al. Six barbiturates, amobarbital, butabarbital, pentobarbital, phenobarbital, secobarbital, and thiopental were studied at 2 concentration levels 1 in the therapeutic range and 1 in the toxic range. Blood was stored at 25 °C and 4 °C and tested after 3 months. At both concentration levels, more than 78% of the original concentration of each barbiturate was found at the end of the 3-Month period. Therapeutic drug monitoring of phenobarbital triggered several studies of its stability in serum. Schafer reported no change in serum phenobarbital concentrations stored at 4 °C for about 12 weeks. Wilensky studied phenobarbital-containing serum specimens stored at room temperature for 6 months and concluded that there was no change in phenobarbital concentration.

Although earlier literature offers conflicting results concerning the stability of barbiturates, biological specimens recent studies suggest that there are minimal changes in barbiturate concentrations in blood or serum stored at room temperature or at 4 °C over a 2- to 3-month period. These small changes would not affect interpretation based on drug concentrations obtained at the time of collection or after a short period of storage.

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