Advances in testing for sample manipulation in clinical and forensic toxicology - Part A: urine samples.

In many countries, adherence testing is used to monitor consumption behavior or to prove abstinence. Urine and hair are most commonly used, although other biological fluids are available. Positive test results are usually associated with serious legal or economic consequences. Therefore, various sample manipulation and adulteration strategies are used to circumvent such a positive result. In these critical review articles on sample adulteration of urine (part A) and hair samples (part B) in the context of clinical and forensic toxicology, recent trends and strategies to improve sample adulteration and manipulation testing published in the past 10 years are described and discussed. Typical manipulation and adulteration strategies include undercutting the limits of detection/cut-off by dilution, substitution, and adulteration. New or alternative strategies for detecting sample manipulation attempts can be generally divided into improved detection of established urine validity markers and direct and indirect techniques or approaches to screening for new adulteration markers. In this part A of the review article, we focused on urine samples, where the focus in recent years has been on new (in)direct substitution markers, particularly for synthetic (fake) urine. Despite various and promising advances in detecting manipulation, it remains a challenge in clinical and forensic toxicology, and simple, reliable, specific, and objective markers/techniques are still lacking, for example, for synthetic urine.

Advances in testing for sample manipulation in clinical and forensic toxicology-part B: hair samples.

As a continuation of part A, focusing on advances in testing for sample manipulation of urine samples in clinical and forensic toxicology, part B of the review article relates to hair, another commonly used matrix for abstinence control testing. Similar to urine manipulation, relevant strategies to manipulate a hair test are lowering drug concentrations in hair to undercut the limits of detection/cut-offs, for instance, by forced washout effects or adulteration. However, distinguishing between usual, common cosmetic hair treatment and deliberate manipulation to circumvent a positive drug test is often impossible. Nevertheless, the identification of cosmetic hair treatment is very relevant in the context of hair testing and interpretation of hair analysis results. Newly evaluated techniques or elucidation of specific biomarkers to unravel adulteration or cosmetic treatment often focused on specific structures of the hair matrix with promising strategies recently proposed for daily routine work. Identification of other approaches, e.g., forced hair-washing procedures, still remains a challenge in clinical and forensic toxicology.

Updating the use of nano-biosensors as promising devices for the diagnosis of coronavirus family members: A systematic review.

Coronavidae viruses, such as SARS-CoV, SARS-CoV-2, and MERS-CoV, cause severe lower respiratory tract infection, acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea and fever, eventually leading to death. Fast, accurate, reproductible, and cost-effective SARS-CoV-2 identification can be achieved employing nano-biosensors, reinforcing conventional methodologies to avoid the spread of COVID-19 within and across communities. Nano-biosensors built using gold, silver, graphene, In2O3 nanowire and iron oxide nanoparticles, Quantum Dots and carbon nanofibers have been successfully employed to detect specific virus antigens - nucleic acid sequences and/or proteins -or host antibodies produced in response to viral infection. Biorecognition counterpart molecules have been immobilized on the surface of these nanomaterials, leading to selective virus detection by optical or electrochemical transducer systems. This systematic review assessed studies on described and tested immunonsensors and genosensors designed from distinct nanomaterials available at the Pubmed, Scopus, and Science Direct databases. Twenty-three nano biosensors were found suitable for unequivocal coronavirus detection in clinical samples. Nano-biosensors coupled to RT-LAMP/RT-PCR assays can optimize RNA extraction, reduce analysis times and/or eliminate sophisticated instrumentation. Although promising for the diagnosis of Coronavidae family members, further trials in large populations must be adequately and rigorously conducted to address nano-biosensor applicability in the clinical practice for early coronavirus infection detection.

A needle-type micro-sampling device for collecting nanoliter sap sample from plants.

In plant research, measuring the physiological parameters of plants is vital for understanding the behavior and response of plants to changes in the external environment. Plant sap analysis provides an approach for elucidating the physiological condition of plants. However, to facilitate accurate sap analysis, a sampling device capable of collecting sap samples from plants is required. In this paper, a minimally invasive, needle-type micro-sampling device capable of collecting nanoliter (~ 91 nL) quantities of sap from plants is described. The developed micro-sampling system showed great reproducibility (3%) in experiments designed to assess sampling performance. As a proof of concept, sap samples were collected continuously from target plants with the micro-sampling system, and the dynamic changes in potassium ions, plant hormones and sugar levels inside plants were analyzed. The results demonstrated the feasibility of the micro-sampling device and its potential for developing a measurement system for plant research in the future.

Interpretable machine learning model to detect chemically adulterated urine samples analyzed by high resolution mass spectrometry.

OBJECTIVES: Urine sample manipulation including substitution, dilution, and chemical adulteration is a continuing challenge for workplace drug testing, abstinence control, and doping control laboratories. The simultaneous detection of sample manipulation and prohibited drugs within one single analytical measurement would be highly advantageous. Machine learning algorithms are able to learn from existing datasets and predict outcomes of new data, which are unknown to the model. METHODS: Authentic human urine samples were treated with pyridinium chlorochromate, potassium nitrite, hydrogen peroxide, iodine, sodium hypochlorite, and water as control. In total, 702 samples, measured with liquid chromatography coupled to quadrupole time-of-flight mass spectrometry, were used. After retention time alignment within Progenesis QI, an artificial neural network was trained with 500 samples, each featuring 33,448 values. The feature importance was analyzed with the local interpretable model-agnostic explanations approach. RESULTS: Following 10-fold cross-validation, the mean sensitivity, specificity, positive predictive value, and negative predictive value was 88.9, 92.0, 91.9, and 89.2%, respectively. A diverse test set (n=202) containing treated and untreated urine samples could be correctly classified with an accuracy of 95.4%. In addition, 14 important features and four potential biomarkers were extracted. CONCLUSIONS: With interpretable retention time aligned liquid chromatography high-resolution mass spectrometry data, a reliable machine learning model could be established that rapidly uncovers chemical urine manipulation. The incorporation of our model into routine clinical or forensic analysis allows simultaneous LC-MS analysis and sample integrity testing in one run, thus revolutionizing this field of drug testing.

Development and validation of a multipurpose and multicomponent method for the simultaneous determination of six synthetic dyes in different foodstuffs by HPLC-UV-DAD.

A simple and low-cost multipurpose analytical method using HPLC-UV-DAD was developed and validated, following international guidelines, for the determination of six synthetic food dyes: Tartrazine, Sunset Yellow, Amaranth, Allura Red, Indigotine, and Brilliant Blue. The method required a simple sample preparation step that consisted of dissolution or dilution of the samples in water, followed by pH adjustment and filtering through PVDF filters. No significant matrix effect was verified. Linear working ranges varied from 0.25 to 6.0 mg L-1. Appropriate limits of quantification (0.10 to 0.15 mg L-1), mean recoveries (90.2 to 106.6%), and repeatability and intermediate precision (<4.5%) were obtained. Sixty-one samples of different types of foodstuffs were analyzed: jelly and juice powder, jelly candy, jujube candy, hard candy, ice cream syrup, sports drinks, soft drinks, energy drinks, artificially colored ready-to-drink fruit juices and flavored alcoholic beverages. All studied samples showed dye levels in conformity with Brazilian regulations.

The Probenecid-story - A success in the fight against doping through out-of-competition testing.

The effectiveness of doping control in sport has been improved continuously during the past 50 years. One of the major steps forward was the introduction of unannounced and targeted out-of-competition testing in order to control the misuse of anabolic-androgenic steroids (AAS), mainly during the end of the 1980s. It also led to the misuse of masking agents in case a surprise control was performed. Athletes tried to be "prepared", when the doping control officer showed up. The disclosure of the masking agent probenecid in 1987 is a perfect example of a memorable finding, of a suspected and purported case of performance manipulation. Probenecid and its metabolites were identified in five urine samples collected from Norwegian athletes in an out-of-competition test, while they were staying and training in the USA. Probenecid is a drug that reduces the urinary excretion of AAS from the body. It was the first time that it had showed up in a doping control sample. The athletes were sanctioned for hampering the analysis of their urine sample, although probenecid was not yet specified on the Prohibited List. Its detection was the result of a successful collaboration of laboratories and investigative diligence and enthusiasm following up suspicious observations in the actual samples. Immediately afterwards probenecid was added to the Prohibited List for 1988 as well as including the manipulation of doping control samples.

Determining the authenticity of athlete urine in doping control by DNA analysis.

The integrity of urine samples collected from athletes for doping control is essential. The authenticity of samples may be contested, leading to the need for a robust sample identification method. DNA typing using short tandem repeats (STR) can be used for identification purposes, but its application to cellular DNA in urine has so far been limited. Here, a reliable and accurate method is reported for the successful identification of urine samples, using reduced final extraction volumes and the STR multiplex kit, Promega® PowerPlex ESI 17, with capillary electrophoretic characterisation of the alleles. Full DNA profiles were obtained for all samples (n = 20) stored for less than 2 days at 4 °C. The effect of different storage conditions on yield of cellular DNA and probability of obtaining a full profile were also investigated. Storage for 21 days at 4 °C resulted in allelic drop-out in some samples, but the random match probabilities obtained demonstrate the high power of discrimination achieved through targeting a large number of STRs. The best solution for long-term storage was centrifugation and removal of supernatant prior to freezing at -20 °C. The method is robust enough for incorporation into current anti-doping protocols, and was successfully applied to 44 athlete samples for anti-doping testing with 100% concordant typing.