Emily C. Lennert
Keywords: drug testing, blood, urine, saliva, fingerprint, metabolite, bufferin plus S, New Stac Eve Ace, Allegra FX, Alesion, dihydrocodeine phosphate, acetaminophen, fexofenadine, salicylic acid, liquid chromatography, mass spectrometry, LC-MS
Article to be reviewed:
1. Kuwayama, K.; Miyaguchi, H.; Yamamuro, T.; Tsujikawa, K.; Kanamori, T.; Iwata, Y. T.; Inoue, H. Effectiveness of saliva and fingerprints as alternative specimens to urine and blood in forensic drug testing. Drug Testing and Analysis. 2016, 8, 644-651.
Disclaimer: The opinions expressed in this review are an interpretation of the research presented in the article. These opinions are those of the summation author and do not necessarily represent the position of the University of Central Florida or of the authors of the original article.
Urine and blood are common samples taken from a suspect for drug testing in forensic cases. However, each sample type has challenges. Urine samples require the suspect to be escorted to a lavatory and supervised to ensure that samples are not falsified, and blood samples require appropriate facilities and trained personnel to draw blood. Due to the sample collection methods, these samples cannot be collected immediately upon arrest of the suspect. As time passes, drugs metabolize within the body and, if a sample is not collected immediately upon arrest, the concentration of the target drug in the sample will be lower than the concentration at the time of the arrest. To avoid this, a sample that can be collected immediately is desirable. The authors of this study investigated the utility of saliva and fingerprints, as alternatives to blood and urine, for the collection of samples for subsequent testing for drug usage.
To test this, samples were obtained from three human subjects. First, in the four week period preceding the experimental period, the subjects did not take any medications. Urine, blood, saliva, and fingerprint samples were collected prior to the administration of drugs. Urine was collected in plastic cups. Saliva was collected in a plastic dish, after rinsing the mouth with water. Blood was collected by pricking the fingertip with a lancet and drawing three 5 μL samples. Fingerprint samples were collected according to a sampling procedure defined in a previous study: after washing the hands, the index finger was exposed to air for 30 seconds, then pressed onto filter paper wetted with distilled water inside a plastic dish. After control samples were collected for each subject, each subject was administered the recommended dose of four pharmaceutical products: bufferin plus S, New Stac Eve Ace, Allegra FX, Alesion. The active ingredients of these products are given in table 1 within the study, and include dihydrocodeine phosphate, acetaminophen, fexofenadine, salicylic acid, and more. Nine main active ingredients and five metabolites were targeted in this study. Although the drugs studied by the authors were not illicit drugs, the authors sought to investigate the detection of drugs and metabolites in saliva and fingerprints, which could then be applied to illicit drugs. After administration of the drugs, blood, urine, saliva, and fingerprint samples were collected at 2 and 10 hours, 1, 2, and 3 days, and 1, 2, 3, and 4 weeks.
After collection, samples were extracted. Blood, urine, and saliva samples were extracted in water containing an unspecified internal standard. An internal standard is a known concentration of a compound, not the target compound, which can be used to improve the precision of quantitative analysis. Blood samples were shaken and centrifuged (i.e. spun at a high rate) to separate layers within the sample. Subsequently the supernatant, i.e. top layer, of each sample was removed and placed in individual, clean vials to prepare for analysis. Fingerprint samples were extracted by placing the filter paper in a vial containing the mobile phase solution (described in the following paragraph) and water with the internal standard. After shaking and centrifugation, the supernatant was removed to allow for analysis. A duplicate of each sample was prepared and examined as well.
Samples were analyzed by liquid chromatography – mass spectrometry (LC-MS). A mobile phase solution of ammonium acetate with formic acid and acetonitrile was used in this study. In LC-MS, a liquid sample is injected into the instrument’s inlet, where it is taken into the mobile phase. The mobile phase is a high pressure liquid, i.e. the mobile phase solution described above, that pushes the sample through the stationary phase, which is a tube called the column. As the sample moves through the column, compounds are separated based on molecular weight and size. Compounds then elute from, or exit, the column and enter the mass analyzer, where the molecular weight (i.e. molecular mass) can be determined from the resulting mass spectrum. Separation within the column leads to analytes (i.e. molecules) eluting at different times, called retention times. Retention times are characteristic of individual analytes, and can help to identify which compound is present. The identification is further determined by the mass spectra obtained from the mass analyzer.
Blank samples, i.e. the samples collected prior to administration of the drugs, were analyzed to confirm that no target drug were present pre-administration. As anticipated, salicylic acid was present in all of the fingerprint samples, due to the presence of salicylic acid in many personal products such as soap and shampoo. Blanks were then spiked with calibration solutions of each target, then analyzed to allow for the creation of calibration curves. Each sample was analyzed via LC-MS, the created calibration curves were used to calculate the concentration of each compound within the sample.
A limit of detection (LOD), i.e. the lowest concentration of an analyte that can be detected with certainty, was determined for each compound in each sample type, as seen in table 5 within the study. The authors determined that the LOD of all analytes were similar between the blood, urine, and saliva samples. The LOD observed in the fingerprint samples were higher than in the other sample types, indicating that the other samples were more sensitive and performed better than fingerprints in identifying analytes. The authors reported some interferences between the urine matrix and the analytes acetaminophen and dihydrocodeine phosphate, as well as interference between the blood matrix and fexofenadine. Salicylic acid was not determined in fingerprint samples due to its presence on the skin prior to drug administration. No interferences were reported for saliva samples. Detection periods were also examined; generally, the longest detection periods for most compounds were observed in urine. However, three compounds were detected in fingerprints at later sampling times, compared to the other sample types. One compound was detected for approximately the same period across all sample types.
The authors conclude that most of the 14 analytes were detectable in saliva and fingerprints. However, urine samples appear to have an advantage over the other sample types, with drugs being detectable for longer periods and the ease by which large sample quantities are collected. The authors suggest that saliva and fingerprint samples may be taken immediately, to prevent loss of evidence in cases where blood or urine samples cannot be collected swiftly. Then, blood and/or urine samples may be collected according to normal procedure. The saliva and fingerprint evidence may supplement the blood and urine evidence, and detection of drugs in these samples may serve to support analytical results of blood and urine samples.
- Drugs may metabolize before urine and blood samples can be collected from a suspect, in an appropriate manner.
- Saliva and fingerprints may be collected on scene to allow for subsequent analysis of drugs.
- The drugs studied were detected at similar limits of detection in blood, urine, and saliva, indicating comparable performance between these sample types. Slightly higher limits of detection were seen for drugs the fingerprint samples, indicating that analysis of the other sample types enabled detection of lower concentrations of drugs.
- Drugs were detected in urine and fingerprints at the longest time after ingestion by the subject.
- Interferences were reported between some drugs and the urine and blood sample matrices.
Relevance: When it is suspected that an individual has taken a drug, timely sample collection is important to ensure that sample is not metabolized and lost. A sample type that can be gathered on scene can address this concern by allowing for immediate collection, preventing loss of evidence.
- To help mitigate the chance of sample loss due to metabolism of drugs, saliva and fingerprints can be collected on scene and subsequently analyzed for the presence of drugs.
- Saliva performs comparably to urine and blood for drug analysis, when analyzed by LC-MS. Fingerprints allow for analysis of drugs as well, although at higher limits of detection than saliva, blood, and urine.
- Saliva and fingerprint evidence may supplement blood and urine evidence, which generally must be collected at a later time rather than on scene.