Alternative Biological Specimens Research Articles

Development of a GC-MS Method for the Analysis of Selected Opioids in Human Hair Samples

Background: Hair samples are recognized as alternative biological specimens in forensic and clinical toxicology for detecting drug abuse and poisoning. Forensic testing for opioids in hair has become a useful diagnostic measure for assessing chronic drug usage through segmental analysis. However, accurate, sensitive and specific analytical methods are needed. The aim of this study was to introduce a simple, sensitive and specific GC-MS method for the identification and quantitation of selected and commonly abused opioids (tramadol, methadone, morphine, and codeine) in hair samples. Methods: After external decontamination, a 50 mg portion of powdered hair sample was combined with hydrochloric acid (0.1 M) and incubated on a magnetic stirrer at 56°C for 16 hours. Then, 1 mL of sodium hydroxide (0.1 M) and 2 mL of phosphate buffer (1 M, pH=8.4) were added. Chloroform-isopropanol (ratio: 80:20 V/V) was utilized as the extracting solvent and the sample was homogenized and centrifuged for 5 minutes (at 3500 rpm). After centrifugation, the organic phase was dried using dry nitrogen gas. The sample was derivatized with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA). The blank, standards, and real samples were subsequently analyzed by GC-MS.Results: The limits of detection in the linearity experiments ranged from 0.12 to 0.21 ng/mg. According to the validation results, the method exhibited linearity in the concentration range of 0.1-2.5 ng/mg for all analytes, with calibration curve slopes ranging from R2=0.98 to 0.99. Good inter and intra-day precision relative standard deviations (RSDs) were observed to be <3.5% for all compounds. Extraction efficiency varied from 91.8 to 102.4%. Conclusion: The validation and analysis of actual samples indicate that this method is straightforward, sensitive, and specific for the analysis of opioids in routine hair analysis.

SPE-UHPLC-ESI-MS/MS Analysis of Cocaine and Its Metabolites in Conventional and Alternative Biological Specimens: Application to Real Samples

SPE-UHPLC-ESI-MS/MS Analysis of Cocaine and Its Metabolites in Conventional and Alternative Biological Specimens: Application to Real Samples

Assessing the applicability of cerebrospinal fluid collected from the spinal cord for the determination of ethyl alcohol in post-mortem toxicology

This paper presents the results of a study on the applicability of cerebrospinal fluid (CSF) collected from the spinal canal in the post-mortem determination of ethyl alcohol. The present study reviewed data of autopsy cases (n = 45), in which ethyl alcohol was detected in CSF using gas chromatography with a flame ionization detector (HS-GC-FID), to investigate ethyl alcohol concentrations in CSF, compared with blood. As a result of statistical analysis of the obtained data, a high positive correlation was found between blood ethanol concentration and cerebrospinal fluid collected from the spinal canal ethanol concentration. The Pearson correlation coefficient was statistically highly significant (p < 0.001) (r = 0.9503). The data obtained allowed us to conclude that cerebrospinal fluid collected from the spinal canal can be collected during an autopsy as an alternative biological specimen to assess the ethanol content. Cerebrospinal fluid collected from the spinal canal can corroborate and lend credibility to the results obtained for blood and, in special cases, when blood is drawn from putrefied bodies and may even be a superior specimen to blood for assessing ethyl alcohol intoxication status.

Optimization of a real time PCR methodology for HCV RNA quantification in saliva samples

Saliva may be an alternative biological specimen to expand HCV detection. This study aims to evaluate an in-house quantitative RT-PCR for HCV RNA quantification in saliva. A total of 80 individuals (56 anti-HCV/HCV RNA + and 24 negative controls) donated serum and saliva, that were tested using an in-house quantitative PCR for HCV RNA. The median viral load was 4.77 log10 copies/mL (1.04–7.0 log10 copies/mL) in serum and 2.31 log10 copies/mL (1.0–3.84 log10 copies/mL) in saliva. A sensitivity of 80 % and specificity of 100 % were observed for HCV detection in saliva, which demonstrates the usefulness of in-house real-time PCR to quantify HCV RNA in saliva samples, which might increase the access of molecular diagnosis of HCV in laboratories that lack complex infrastructures for molecular testing and in individuals with poor venous access.

Hair Testing for Classic Drugs of Abuse to Monitor Cocaine Use Disorder in Patients Following Transcranial Magnetic Stimulation Protocol Treatment.

Simple SummaryHair testing for classic drugs of abuse offers the possibility of longer detection times (for drug substances) when compared to urine analysis. Hair analysis is routinely used to detect drug abuse in forensic cases and clinical toxicology, whereas it is rarely used at addiction clinics to monitor the clinical efficacy of therapeutic interventions. Here, we explore for the first time whether hair analysis might represent a valid tool to track the clinical improvements in a population of patients with cocaine use disorder (CocUD) who underwent a repetitive transcranial magnetic stimulation (rTMS) treatment.In recent years, hair has become an alternative biological specimen for drug testing in the fields of forensic and clinical toxicology. The advantages of hair testing include larger detection windows (months/years), depending on the length of the hair shaft, compared to those of urine/blood (hours to 2–4 days for most drugs). Segmental hair analysis can disclose a month-to-month (considering 1 cm segment cuts) information of drug exposure (single or repeated) and potentially identify patterns of drug use/administration. Repetitive transcranial magnetic stimulation (rTMS) was recently proposed as a valid tool for therapeutic purposes in addictions, including cocaine use disorder (CocUD). Here, we proposed hair testing analyses of classic drugs of abuse in a clinical setting to monitor the clinical changes in treatment-seeker CocUD patients undergoing protocol treatments with rTMS stimulating the left dorsolateral prefrontal cortex (l-DLPFC). We collected hair samples from nine CocUD patients at different stages from the beginning of treatments. Hair sample analyses revealed significant changes in the patterns of cocaine use, according to the negativity of urine screening tests and the clinical reductions of craving. These data, albeit preliminary, suggest that hair testing analysis of classic drugs of abuse could be extended to clinical settings to monitor the clinical efficacy of innovative therapeutic interventions, such as rTMS.

Comment on "High Correlation between Ethanol Concentration in Postmortem Femoral Blood and in Alternative Biological Specimens, but Large Uncertainty When the Linear Regression Model Was Used for Prediction in Individual Cases".

Comment on "High Correlation between Ethanol Concentration in Postmortem Femoral Blood and in Alternative Biological Specimens, but Large Uncertainty When the Linear Regression Model Was Used for Prediction in Individual Cases".

Characterization of Cannabidiol in Alternative Biological Specimens and Urine, After Consumption of an Oral Capsule.

Among the hundred cannabinoids present in cannabis sativa indica, cannabidiol is a phytocannabinoid discovered in 1940, which can account for up to 40 % of the plant's extract. Medically, it has been proposed to treat convulsions, inflammation, anxiety and nausea. Contrary to the hallucinogenic ingredient of the plant, delta-9-tetrahydrocannabinol, cannabidiol does not seem to have a sedative effect, which can increase its popularity among users. The identification of cannabidiol in blood and urine has been widely described in the scientific literature for several years. Only few data after cannabis use has been reported regarding cannabidiol identification in alternative specimens, such as oral fluid, sweat, exhaled breath and hair. Cannabidiol capsules were purchased in the United States, from a grocery store and a green capsule containing 22 mg of cannabidiol was orally administered to a 59-year-old healthy man. Oral fluid was collected over 8 hours using the NeoSal™ device. Sweat was collected with PharmCheck™ sweat patch technology over 7 days. Exhaled breath was collected with the ExaBreath® DrugTrap device over 8 hours. Beard hair was collected 7 and 14 days after administration. Finally, urine specimens were collected over 48 hours in plastic tubes without preservative. Cannabidiol was only detected in oral fluid at 15 minutes, at 20 pg/mL. Increasing concentrations, up to 96 pg/patch of cannabidiol, were detected in the sweat patches. Cannabidiol was detected during 45 minutes in exhaled breath (Cmax 302 pg/filter at 30 minutes). Cannabidiol produced a very low but significant chromatographic signal in beard hair, with concentrations lower than 1 pg/mg. Finally, cannabidiol tested positive in urine after enzymatic hydrolysis with a Cmax at 70 ng/mL, after 6 hours.

High Correlation between Ethanol Concentrations in Postmortem Femoral Blood and in Alternative Biological Specimens, but Large Uncertainty When the Linear Regression Model Was Used for Prediction in Individual Cases

In connection with medicolegal autopsies peripheral blood (e.g. from a femoral vein) is the specimen of choice for toxicological analysis, although alternative specimens are also sometimes submitted, such as bile, cerebrospinal fluid (CSF), vitreous humor (VH), bladder urine, pleural effusions and/or lung fluid. Ethanol concentrations were determined in duplicate in femoral blood and in various alternative biological specimens by headspace gas chromatography. The analysis was carried out on two different fused silica capillary columns furnishing different retention times for ethanol and both n-propanol and t-butanol were used as internal standards. The results were evaluated by linear regression using blood alcohol concentration (BAC) as dependent or outcome variable and the concentrations in an alternative specimen as independent or predictor variable. The Pearson correlation coefficients were all statistically highly significant (P<0.001); r=0.94 (bile), r=0.98 (CSF), r=0.97 (VH), r=0.92 (urine), r=0.94 (lung fluid) and r=0.96 (pleural cavity effusions). When the regression model was used to predict femoral BAC from the mean concentration in an alternative specimen the mean and 95% prediction intervals were 1.12±0.824g/L (bile), 1.41±0.546g/L (CSF), 1.15±0.42g/L (VH), 1.29±0.780g/L (urine), 1.25±0.772g/L (lung fluid) and 0.68±0.564g/L (pleural cavity effusions). This large uncertainty for a single new observation needs to be considered when alcohol-related deaths are evaluated and interpreted. However, the analysis of alternative specimens is recommended in medical examiner cases to provide supporting evidence with regard to the origin of ethanol, whether this reflects antemortem (AM) ingestion or postmortem (PM) synthesis.

Cell-free Circulating Tumor DNA (ctDNA) in Metastatic Renal Cell Carcinoma (mRCC): Current Knowledge and Potential Uses

Historically, tumor biopsies and clinical laboratory testing have been the gold standard for diagnosis and prognosis in metastatic renal cell carcinoma (mRCC). Genomic profiling in mRCC has traditionally been performed on tumor tissue; however, challenges and limitations in obtaining tissue biopsies led to the discovery of alternative biological specimens, namely circulating cell-free DNA (cfDNA). Rapidly evolving technologies, with increased sensitivity and specificity, have been used to query cfDNA in the clinical research setting. These investigations are rapidly establishing cfDNA and liquid biopsies as valuable complementary specimens to the gold standard, and in some instances surpassing these with unique insight into the contemporary genomic landscape and tumor heterogeneity. In this review, we will discuss recent research into the prognostic, diagnostic, and predictive utility of liquid biopsies in mRCC. We will explore their potential role in precision treatment of mRCC and conclude with what is needed in order to translate them to clinical practice.

Characterization of metizolam, a designer benzodiazepine, in alternative biological specimens

Designer benzodiazepines provide an attractive alternative to prescribed benzodiazepines for abuse purposes as they are readily available over the Internet at a low price. Although these compounds have been developed by pharmaceutical companies, at this time, they do not have a legal status. Their detection in biological fluids is challenging owing to the very few available data. For instance, there is no information about metizolam (also known as desmethyletizolam) analysis, distribution and metabolism in the scientific literature. Metizolam was ordered via Internet and a 2 mg blue tablet was orally administered to a 54-year-old healthy man (85 kg). Oral fluid (saliva) was collected over 8 hours using the NeoSal™ device. Sweat was collected with the PharmChek™ sweat patch technology over 72 hours. Exhaled breath was collected with the ExaBreath ® DrugTrap device over 15 hours. Beard hair was collected 4 and 10 days after administration. Finally, head hair was collected 3 weeks after administration. Metizolam was detectable in saliva during 8 hours, with concentrations lower than 1 ng/mL (max at 0.77 ng/mL after 2 hours). Increasing concentrations up to 186 pg/patch (after 72 hours) of metizolam were detected in the sweat patches. Metizolam produced for 4 hours a very low but significant chromatographic signal in exhaled breath, with concentrations always lower than 10 pg/filter. Finally, metizolam tested positive in beard hair (0.73 and 0.28 pg/mg after 4 and 10 days, respectively) and head hair (0.27 pg/mg). From the data obtained from one subject, metizolam showed very low concentrations in various alternative matrices, attesting that the potential forensic implications (drug-facilitated crime, addiction, DUID…) of metizolam abuse must be considered as a new challenge for toxicologists.

Determination of some biochemical parameters in oral fluid and evaluation of their stability under different storage conditions

Summary Oral fluid (OF) is a complex liquid secretion, which can be collected painlessly, rapidly and non-invasively by low trained persons. The sample collection could be supervised without disturbing objects providing it. Recently OF has attracted clinical and forensic toxicologists attention due to its application as an alternative biological specimen of the blood that can provide an estimation of exposure to drugs of abuse. The present research is aimed to the characterization of OF specimen in order to determine suitable parameters as markers for its identity/dilution. Further study on the effects of storage temperature, additives presence and storage period on sample stability was also performed. The data obtained revealed that the protein profile and the α-amylase activity can be used as markers of OF samples identity, while creatinine levels could serve as an indicator for dilution/stability of oral fluid. The experimental results showed that creatinine concentration depends on storage conditions. The sample storage at 4 °C for a month leads to increasing of creatinine levels up to 50%; the process is partially inhibited by the presence of Na 2 EDTA or NaN 3 and the amount of creatinine increases up to 10% as compared to the initial value. The storage of samples at −20 °C for a month leads to significant decrease of initial creatinine concentration even in the presence of stabilizer/preservative.

Application of modified QuEChERS method to liver samples for forensic toxicological analysis

In forensic toxicological analysis, liver is commonly used as an alternative biological specimen in cases in which blood and urine cannot be obtained. Liver samples are generally purified by a solid-phase extraction (SPE) technique after homogenization. The homogenizer probe cleaning process is laborious and has a risk of cross-contamination, and the SPE technique itself is tedious and time consuming. The QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) method is widely acknowledged in some fields, such as food analysis, as a simple, fast, and reliable method. We previously developed a modified QuEChERS method for forensic toxicological analysis in human whole blood and urine. In this study, we applied this method to liver samples from forensic cases and successfully detected not only targeted drugs (i.e., benzodiazepines, zopiclone, and zolpidem) but also various types of drugs. This method has no risk of cross-contamination because homogenization of the liver and extraction of the drugs are simultaneously performed in a disposable plastic tube. In addition, the total process time is approximately 5 min. We recommend the modified QuEChERS method for extraction of drugs from both fluid and solid samples, such as liver, in forensic cases.

Analytical Techniques in Androgen Anabolic Steroids (AASs) Analysis for Antidoping and Forensic Purposes

A survey on the main analytical challenges related to the analysis of Androgen Anabolic Steroids (AASs) is reported. AASs analysis is an issue regarding antidoping analyses as well as forensic toxicology applications. This paper reports an overview of the more recent literature regarding various aspects of sample preparation, analytical techniques and interpretation of results for AASs identification in biological samples. New analytical approaches, mainly for their application to the antidoping field, are reported. The application of AASs analysis in forensic cases is also described, taking into consideration mainly the different biological samples that can be analysed for forensic purposes. Particular attention was played on the application of hair analysis as alternative biological specimen for the determination of AASs abuse.

Illicit drugs in alternative biological specimens: A case report

Postmortem tissues (e.g. liver, kidney) have been long used in forensic applications especially in those cases where blood is unavailable. The aim of this paper is to demonstrate the importance of the information provided to the forensic toxicologist at the time of carrying out the toxicological analysis, especially in cases where the samples commonly used in forensic toxicology are unavailable. This work describes the toxicological findings in a violent death resulting from a man who was hit by a train. Vitreous humor, liver and kidney were sent for toxicological analysis, once it was not possible to obtain blood and urine. The validated procedures used in the routine casework of Forensic Toxicology Laboratory of the Centre Branch of the National Institute of Legal Medicine, were applied in the analysis of liver, kidney and vitreous humor, using gas chromatography–mass spectrometry after solid-phase extraction and gas chromatography-flame ionization detector for the analysis of drugs of abuse and ethanol, respectively. Morphine, codeine, cocaine, benzoylecgonine and ecgonine methyl ester were found in the liver and in the kidney and no ethanol was found in the vitreous humor. The method validation included the study of specificity, selectivity, limits of detection, recovery and carryover. Although blood and urine are the most common and preferred matrices used for toxicological studies involving drugs of abuse, sometimes the choice of specimen is determined by the case under investigation. The forensic pathologist must be aware that relevant information must be provided so that the toxicological analysis can be conducted in accordance with case history, particularly when the only samples available for analysis are these “unconventional” specimens, since the interpretation of the obtained results is more difficult.

Analytical methods for abused drugs in hair and their applications

Hair has been focused on for its usability as an alternative biological specimen to blood and urine for determining drugs of abuse in fields such as forensic and toxicological sciences because hair can be used to elucidate the long intake history of abused drugs compared with blood and urine. Hair analysis consists of several pretreatment steps, such as washing out contaminates from hair, extraction of target compounds from hair, and cleanup for instrumental analysis. Each step includes characteristic and independent features for the class of drugs, e.g., stimulants, narcotics, cannabis, and other medicaments. In this review, recently developed methods to determine drugs of abuse are summarized, and the pretreatment steps as well as the sensitivity and applicability are critically discussed.

LC‐MS: a powerful tool in workplace drug testing

Workplace drug testing is a well-established application of forensic toxicology and it aims to reduce workplace accidents caused by affected workers. Several classes of abused substances may be involved, such as alcohol, amphetamines, cannabis, cocaine, opiates and also prescription drugs, such as benzodiazepines. The use of alternative biological specimens such as hair, oral fluid or sweat in workplace drug testing presents several advantages over urinalysis-mainly the fact that sample collection can be performed easily without infringing on the examinee's privacy, so the subject is more likely to perform the test. However, drugs are usually present in these alternative specimens at low concentrations and the amount of sample available for analysis is small. The use of highly sensitive techniques is therefore necessary. In fact, the successful interface of liquid chromatography with mass spectrometry (LC-MS) has brought a new light into bioanalytical and forensic sciences as it allows the detection of drugs and metabolites at concentrations that are difficult to analyse using the more commonly adopted GC-MS based techniques. This paper will discuss the importance of LC-MS in supporting workplace drug-testing programmes. The combination of LC-MS with innovative instrumentation such as triple quadrupoles, ion traps and time-of-flight mass spectrometers will also be focused.

Analytical pitfalls in hair testing

This review focuses on possible pitfalls in hair testing procedures. Knowledge of such pitfalls is useful when developing and validating methods, since it can be used to avoid wrong results as well as wrong interpretations of correct results. In recent years, remarkable advances in sensitive and specific analytical techniques have enabled the analysis of drugs in alternative biological specimens such as hair. Modern analytical procedures for the determination of drugs in hair specimens - mainly by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) - are reviewed and critically discussed. Many tables containing information related to this topic are provided.

Human Milk Research for Answering Questions about Human Health

Concerns regarding human milk in our society are diverse, ranging from the presence of environmental chemicals to the health of breastfed infants and the economic value of breastfeeding to society. The panel convened for the Technical Workshop on Human Milk Surveillance and Biomonitoring for Environmental Chemicals in the United States, held at the Hershey Medical Center, Pennsylvania State College of Medicine, on 24–26 September 2004, considered how human milk research may contribute to environmental health initiatives to benefit society. The panel concluded that infant, maternal, and community health can benefit from studies using human milk biomonitoring. Unlike other biological specimens, human milk provides information regarding exposure of the mother and breastfed infant to environmental chemicals. Some of the health topics relevant to this field of research include disorders of growth and development in infants, cancer origins in women, and characterization of the trend of exposure to environmental chemicals in the community. The research focus will determine the design of the study and the need for the collection of alternative biological specimens and the long-term storage of these specimens. In order to strengthen the ability to interpret study results, it is important to identify reference ranges for the chemicals measured and to control for populations with high environmental chemical exposure, because the amount of data on environmental chemical levels in human milk that is available for comparison is extremely limited. In addition, it will be necessary to validate models used to assess infant exposure from breastfeeding because of the variable nature of current models. Information on differences between individual and population risk estimates for toxicity needs to be effectively communicated to the participant. Human milk research designed to answer questions regarding health will require additional resources to meet these objectives. We thank the following organizations for generously providing support for the workshop: the American Chemistry Council; the Centers for Disease Control and Prevention (C13/CCU323635-01); the Department of Health and Human Services, Health Resources and Services Administration (HHSH240200415021P); Health Canada (H405-03-ExSD079/4500078209); 3M; Pennsylvania State University College of Medicine; the Research Foundation for Health and Environmental Effects; and the U.S. Environmental Protection Agency, Office of Children’s Health (CH-83213101-0). The opinions expressed in this article are those of the authors and do not necessarily reflect the views and/or policies of their affiliations.

Alternative specimens for workplace drug testing.

Recent advances in analytical techniques have enabled the detection of drugs and drug metabolites in alternative biological specimens for the purposes of workplace testing. A wide variety of specimens are available, each providing valuable information concerning prior or current drug use. The present focus is on oral fluid (saliva), hair, and sweat. An extensive evaluation by the Division of Workplace Programs of the Department of Health and Human Services is underway to determine the utility of these specimens in federally regulated programs. In future years, the testing of alternative specimens will expand our ability to understand the patterns of drug use and will become routine in all areas of forensic toxicology.

Utilization of aminoplastics as an alternative biological SEM specimen preparation technique

Utilization of aminoplastics as an alternative biological SEM specimen preparation technique