Drug Testing Methods Research Articles

Rapid analysis of ketamine with in-house antibody conjugated boronic acid modified silver chip on MALDI-TOF MS measurement

An antibody conjugated boronic acid modified silver chip (ABAS ship) is fabricated as a simple, rapid, accurate, sensitive and cost-effective sample preparation method for abused drug quantification in human urine. Ketamine, one common abused drug, was applied as proof of concept for ABAS chip with high resolution matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF MS) analysis. The overall testing process required 10 min at part per billion (ppb) sensitivity level, where current drug testing method necessitated several hours with similar sensitivity. The ABAS chip manufacture process started with slide glass by way of silver mirror reaction to form silver conductive glass for further chemical conjugation. Boronic acid functional group was decorated on silver conductive glass through the formation of silver-thiol (Ag–S) bond. Anti-ketamine antibody was covalently conjugated to boronic acid modified silver conductive glass through the formation of cyclic boronate ester between the boronic acid and the cis-diol groups on the glycans of antibody, which maintain the correct orientation to maximally capture its antigen. The resulting ABAS chip were designed to specifically capture ketamine in human urine samples, that could be directly analyzed by addition of MALDI α-Cyano-4-hydroxycinnamic acid (CHCA) matrix solution. The linear dynamic range of concentration in this method was 10–500 ng/mL with coefficient of determination 0.996. The limit of detection (LOD) and limit of quantification (LOQ) were 2.0 and 7.0 ng/mL, respectively. Importantly, the proposed method allows rapid and accurate quantification of ketamine from suspects’ urine samples in 10 min and small sample volume of 1 μL was required. The resulting data were consistent with traditional gas chromatography-mass spectrometry (GC-MS) analysis. Our homemade ABAS chip could thus provide a powerful tool not only for forensic science but also for most clinical diagnosis of disease as many expression antibodies for the occurrence of diverse diseases could be simply produced and purchased.

Analysis of cobalt for human sports drug testing purposes using ICP- and LC-ICP-MS.

Due to the current demands in the fight against manipulation of blood and blood components, commonly referred to as "blood doping" in sports drug testing, specific and sensitive detection methods enabling the detection of prohibited substances and methods of doping are required. Similar to illicit blood transfusions, erythropoiesis stimulating agents have been shown to be misused in sport, aiming at improving an athlete's aerobic capacity and endurance performance. Amongst other strategies, the administration of ionic cobalt (Co2+ ) can increase the number of erythrocytes by stimulating the endogenous erythropoietin (EPO) biosynthesis. Conversely, several organic Co-containing compounds such as cyanocobalamin (vitamin B12) are not prohibited in sports, and thus, an analytical differentiation of permitted and banned contributions to urinary Co-concentrations is desirable. An excretion study with daily applications of either 1 mg of CoCl2 or 1 mg of cyanocobalamin was conducted with 20 volunteers over a period of 14 consecutive days. Urine, plasma, and concentrated red blood cells were analyzed for their cobalt content. The samples were collected starting 7 days before the administration until 7 days after. Total Co concentrations were analyzed by using inductively coupled plasma mass spectrometry (ICP-MS), which yielded significantly elevated levels exclusively after inorganic cobalt intake. Furthermore, a liquid chromatography (LC)-ICP-MS approach was established and employed for the simultaneous determination of organically bound and inorganic cobalt by chromatographic separation within one single run. The analytical approach offers the option to further develop detection methods of illegal Co2+ supplementation in sport.

A novel smartphone-based device for rapid on-site methamphetamine detection

Drug abuse and drug-driving have posed serious threats to the public security. For the urgent need to rapidly on-site screen drug evidence and drug abusers, we developed a novel, sensitive and smartphone-based drug detection device. The core component of this device was a time-resolved fluorescent immunoassay (TRFIA) methamphetamine test strip. A custom APP with embedded algorithm was designed to streamline the detection process and analyze the fluorescence information. This device could reach a detection limit of 3.34 × 10– 9mol/L and a linear range from 3.91 ×10–9mol/L to 1 ×10–6mol/L. It had an excellent stability (CV =2.35%) and a favourable specificity. A single test took no more than 10 minutes. The whole device was integrated and compact. It was feasible to use this device to test methamphetamine abusers through hair. This device provided an effective method for roadside drug testing and clinical diagnosis, and could give helpful assistance to law enforcement officers.

Implementation of the HIF activator IOX-2 in routine doping controls - Pilot study data.

Early in 2020, racehorse doping cases revolved around the hypoxia-inducible factor (HIF) activator IOX-2. While the composition of IOX-2 has also been known and monitored in human doping controls for several years, the testing capability of routine sports drug testing methods was revisited for this newly surfaced doping agent. IOX-2 and the analytically well-established HIF activator roxadustat (FG-4592) share identical precursor/product ion pairs, enabling their co-detection in existing initial testing procedures in routine doping controls for the intact unconjugated analytes. In addition, hydroxylated IOX-2 and the corresponding glucuronic acid conjugates were identified as major metabolites in a microdose elimination study, contributing to enhanced initial testing and confirmation procedures.

Rapid Detection Device of Methamphetamine Based on Improved Light Emitting Diode Induced Fluorescence Detector

In recent years, drug abuse has become a major public health problem, drug-driving poses serious threats to the public security. For the urgent need to rapidly on-site screen drug evidence and drug abusers, we have developed a fluorescent drug detection device based on LED induction (FD-LED). The core part of the device is 365 nm high-intensity LED excitation light source, FD-LED detector' performance was tested by the fluorescent brightener OB. In this study, the FD-LED detector was evaluated, and the influence of baseline noise, baseline drift, sensitivity, detection limit and quantitative limit on the detector was analyzed, the result showed that the proposed detector had good stability and sensitivity, at the same time, the chromatographic detection conditions were set, the solution based on methamphetamine C1 sample was collocated, and combined with liquid chromatography, a detection system for methamphetamine was developed. The results showed that the minimum detection amount of the sample was 1.0 × 10–9 g/ml, and the signal-concentration relationship was obvious in the range of 1.0 ng/ml∼15.6 ng/ml. This device could provide an effective method for roadside drug testing and clinical diagnosis, and give helpful assistance to law enforcement officers.

Optimized procedures for simultaneous quantitation of low concentration levels of morphine and codeine in urine using gas chromatography–mass spectrometry

Background: Detection techniques with high specificity, precision and accuracy are required for evidence of illicit drug intake. The study aim was to develop a low-concentration drug testing method for morphine and codeine in urine samples using gas chromatography-mass spectrometry (GC-MS) to establish a precise quantitative analytical method that improves upon limit of detection (LOD), limit of quantitation (LOQ), precision, and accuracy of currently available detection methods. Methods: Using a 300 ng/mL urine sample, solid phase extraction was performed using an automated solid phase method. All analyses were performed using a Hewlett-Packard (Palo Alto, CA) HP 6890 gas chromatograph interfaced to a HP 5973 mass selective detector (MSD) equipped with a DB-5MS column to acquire full-scan and SIM mass spectrometric data. Solid phase extraction was optimal at pH 9.0 in a 2 ml sample volume. An internal standard concentration of 100 ng/mL yielded optimal results. Results: Standard solution ranges (40-450 and 40-1500 ng/mL) significantly influenced LOQ. Calibration methods were not associated with LOD and LOQ for either MOR or COD. The intra- and inter-day precision values did not exceed 2% and were not different within groups. The accuracy of the examined method ranged from 97.8% to 103.3%. All parameters were validated in 33 clinical urine specimens. Conclusions: This developed method was successfully used for the determination of morphine and codeine in human urine for forensic identification. The examined protocol can be applied to simultaneous quantification of morphine and codeine at low concentration levels in urine.

High-throughput generation of a concentration gradient on open arrays by serial and parallel dilution for drug testing and screening

In this study, we successively and parallelly slide a solvent strip on a polydimethylsiloxane microwell array to dilute the solution within the microwells. A total of 60 concentration gradients were generated one by one along the sliding direction within minutes while simultaneously forming 60 microwells with the same concentration perpendicular to the sliding direction. Using theoretical calculations, simulated predictions and experimental verification, we could regulate the concentration gradient on the microwell array by tuning the parameters of the sliding head and sliding speed. Afterwards, we cultured human non-small cell lung cancer cells and produced an anticancer drug concentration gradient on the microwell array by this sliding method. The cell viability was examined to demonstrate the potential of this method for high-throughput cell-based drug testing and screening, and this open microwell array method may enable further analysis during a drug-screening process.

Studies on the in vivo metabolism of methylstenbolone and detection of novel long term metabolites for doping control analysis.

The anabolic-androgenic steroid methylstenbolone (MSTEN; 2α,17α-dimethyl-17β-hydroxy-5α-androst-1-en-3-one) is available as a so-called designer steroid or nutritional supplement. It is occasionally detected in doping control samples, predominantly tested and confirmed as the glucuronic acid conjugate of methylstenbolone. The absence of other meaningful metabolites reported as target analytes for sports drug testing purposes can be explained by the advertised metabolic stability of methylstenbolone. In 2013, a first investigation into the human metabolism of methylstenbolone was published, and two hydroxylated metabolites were identified as potential targets for initial testing procedures in doping controls. These metabolites were not observed in recent doping control samples that yielded adverse analytical findings for methylstenbolone, and in the light of additional data originating from a recent publication on the in vivo metabolism of methylstenbolone in the horse, revisiting the metabolic reactions in humans appeared warranted. Therefore, deuterated methylstenbolone together with hydrogen isotope ratio mass spectrometry (IRMS) in combination with high accuracy/high resolution mass spectrometry were employed. After oral administration of a single dose of 10 mg of doubly labeled methylstenbolone, urine samples were collected for 29 days. Up to 40 different deuterated methylstenbolone metabolites were detected in post-administration samples, predominantly as glucuronic acid conjugates, and all were investigated regarding their potential to prolong the detection window for doping controls. Besides methylstenbolone excreted glucuronidated, three additional metabolites were still detectable at the end of the study on day 29. The most promising candidates for inclusion into routine sports drug testing methods (2α,17α-dimethyl-5α-androst-1-ene-3β,17β-diol and 2α,17α-dimethyl-5α-androst-1-ene-3α,17β-diol) were synthesized and characterized by NMR.

Abstract 1074: The PDX Data Commons and Coordinating Center (PDCCC) for PDXNet in support of preclinical research

Abstract Patient-Derived Xenografts (PDX) are proven models to study novel drugs or drug combinations and test hypothesis in preclinical studies. The overarching goal of the PDXNet is to coordinate the development of appropriate PDX models and methods for preclinical drug testing to advance CTEP clinical development of new cancer agents. The PDXNet is an NCI-funded consortium of six PDX Development and Trial Centers (PDTCs) and one PDCCC. Four PDTCs are responsible for developing PDXs and executing specific preclinical trials focused on cancer types including breast cancer, melanoma, and lung cancer. The other two recently awarded centers are specifically focused on minority PDX models and preclinical trials. Besides the PDTCs, the NCI Patient-Derived Models Repository (PDMR) at the Frederick National Laboratory for Cancer Research (FNLCR) is also providing models and data to the PDXNet. The PDCCC is responsible for coordination and developing standards for PDX generation as well as data analysis and metadata harmonization. The PDX Data Commons is built on top of existing NCI resources, leveraging the Cancer Genomics Cloud maintained by Seven Bridges Genomics, where PDXNet data is co-located with TCGA and other large-scale datasets. The PDCCC is co-led by experts from the Jackson Laboratory, providing scientific leadership in xenograft methods and cancer biology to ensure the promulgation of standards that are well-suited for the PDX community. A new portal has been set up at https://www.pdxnetwork.org/ to serve as the point of access to PDXNet resources. In addition, we established ongoing network-wide meetings to facilitate knowledge exchange, held PDXNet portal trainings, and set up working groups to tackle specific challenges. For instance, the Data Ontology working group has been working towards building a common data ontology model specifically for PDX datasets. We are in the process of annotating the very first dataset using this new ontology on the PDXNet portal. Also, the Workflows working group has been working on building and benchmarking various RNA-seq and whole exome sequencing analysis workflows to standardize data processing between PDXNet grantees and create a harmonized PDXNet dataset. These PDX models and the accompanying data will be opened to the community for data mining and/or preclinical research. The PDXNet is a strong step toward building a consensus around PDX models, so that the power for discovery can be expanded by making multi-institutional PDX cohorts a reality. As the coordination center, we are also working closely with the EuroPDX project to exchange standards and knowledge to support the PDX community with a set of standards going forward. The PDCCC is a central part of this process to systematically capture and analyze the variables most influential to PDX models and share protocols and tools to make PDXs an interchangeable research currency for preclinical discovery. Citation Format: Jacqueline Rosains, Anuj Srivastava, Wingyi Woo, Vishal Sarsani, ZiMing Zhao, Javad Noorbakhsh, Ogan D. Abaan, Christian Frech, Jack DiGiovanna, Ryan Jeon, Steve Neuhauser, Peter Robinson, Yvonne A. Evrard, Carol Bult, Jeffrey A. Moscow, Brandi Davis-Dusenbery, Jeffrey H. Chuang. The PDX Data Commons and Coordinating Center (PDCCC) for PDXNet in support of preclinical research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1074.

Compliance testing of patients in ADHD treatment with lisdexamphetamine (Elvanse®) using oral fluid as specimen

Pharmacological treatment of the attention-deficit/hyperactivity disorder (ADHD) includes use of the psychostimulant amphetamine. Non-adherence to medication is a well-documented problem in ADHD treatment and a cause of treatment failure. The study evaluated the possibility of using oral fluid for compliance monitoring during treatment with lisdexamphetamine (Elvanse®). UPLC-MS/MS methods for general oral fluid drug testing, lisdexamphetamine and amphetamine quantification and chiral analysis of amphetamine were used. The applied measuring ranges were 1–500 ng/mL for amphetamine and 0.01–15 ng/mL for lisdexamphetamine. Amphetamine (racemic) was detected and quantified in 98 (96%) of the 102 samples. The concentrations ranged from 2 to 8410 ng/mL. In 17 of these, the chiral analysis demonstrated intake of illicit amphetamine because L-amphetamine was present. The median D- + L-amphetamine concentration in the compliant group was 280 ng/mL, while the median concentration in the non-compliant group was statistically higher, 1677 ng/mL. In the non-compliant cases where L-amphetamine was detected, the L/D-amphetamine ratios ranged from 0.75 to 13.1 with a median of 1.0. Lisdexamphetamine was detected and quantified in 76 of the 102 cases, which represent 79% of the 98 cases with detected oral fluid amphetamine. The concentrations ranged from 0.01 to 6895 ng/mL. Drug testing had a positive rate of 23% in patients not taking illicit amphetamine and 82% among non-compliant patients with detected L-amphetamine. In conclusion, the study demonstrated the value of measuring amphetamine with a chiral method to detect intake of illicit amphetamine and to perform drug testing in oral fluid as a mean for compliance monitoring.

1039 Novel mouse model of recessive dystrophic epidermolysis bullosa-squamous cell carcinoma

The high frequency and extremely aggressive behavior of squamous cell carcinoma from recessive dystrophic epidermolysis bullosa patients (RDEB-cSCC), as well as the lack of an appropriate preclinical mouse model recapitulating the RDEB-cSCC phenotype for testing effective therapies/drugs, creates an urgent need for generating such a model. Here, we established a novel mouse model for RDEB-cSCC, in which we generated 3D cSCC skin constructs (SCs) using RDEB-cSCC and RDEB fibroblasts (FB), which were then grafted onto immunocompromised mice. 16 out of 18 animals that were grafted with RDEB patient-derived cSCC and FB developed tumors within 4 weeks post-grafting, with characteristic morphological features of human RDEB-cSCC. H&E staining of RDEB-cSCC grafts revealed invading epithelial cell columns and differentiated tumor cell islands in the upper dermis. Immunofluorescence (IF) staining showed a complete lack of C7 and increased pSTAT3 expression in the RDEB-cSCC grafted skin construct. Keratin 14, keratin 10 and E-cadherin IF staining were increased within the RDEB-cSCC grafted tumor tissue, indicating the epithelial origin of RDEB-cSCC cells in the dermis and their differentiation. Further, the RDEB-cSCC grafted tumor displayed high proliferation as demonstrated by increased Ki-67+ and p63+ expression seen in IF staining and reduced expression in vimentin staining compared with normal grafts. Moreover, serial transplantation revealed that 88% of RDEB-cSCC tumors had similar biology to the primary tumor (developed on the initial immunocompetent mouse) within 2 weeks post-transplantation onto a second immunocompetent mouse. Our data established a new preclinical model RDEB-cSCC, and an important new method for drug testing in translational cancer research.

A Microfluidic Tumor-on-a-Chip for Assessing Multifunctional Liposomes' Tumor Targeting and Anticancer Efficacy.

Two principal methods for cancer drug testing are widely used, namely, in vitro 2D cell monolayers and in vivo animal models. In vitro 2D culture systems are simple and convenient but are unable to capture the complexity of biological processes. Animal models are costly, time-consuming, and often fail to replicate human activity. Here a microfluidic tumor-on-a-chip (TOC) model designed for assessing multifunctional liposome cancer targeting and efficacy is presented. The TOC device contains three sets of hemispheric wells with different sizes for tumor spheroid formation and evaluation of liposomes under a controlled flow condition. There is good agreement between time-elapsed tumor targeting of fluorescent liposomes in the TOC model and in in vivo mouse models. Evaluation of the anticancer efficacy of four PTX-loaded liposome formulations shows that compared to 2D cell monolayers and 3D tumor spheroid models, the TOC model better predicts the in vivo anticancer efficacy of targeted liposomes. Lastly, the TOC model is used to assess the effects of flow rates and tumor size on treatment outcome. This study demonstrates that the TOC model provides a convenient and powerful platform for rapid and reliable cancer drug evaluation.

Placenta-on-a-Chip: In Vitro Study of Caffeine Transport across Placental Barrier Using Liquid Chromatography Mass Spectrometry.

Due to the particular structure and functionality of the placenta, most current human placenta drug testing methods are limited to animal models, conventional cell testing, and cohort/controlled testing. Previous studies have produced inconsistent results due to physiological differences between humans and animals and limited availability of human and/or animal models for controlled testing. To overcome these challenges, a placenta‐on‐a‐chip system is developed for studying the exchange of substances to and from the placenta. Caffeine transport across the placental barrier is studied because caffeine is a xenobiotic widely consumed on a daily basis. Since a fetus does not carry the enzymes that inactivate caffeine, when it crosses a placental barrier, high caffeine intake may harm the fetus, so it is important to quantify the rate of caffeine transport across the placenta. In this study, a caffeine concentration of 0.25 mg mL−1 is introduced into the maternal channel, and the resulting changes are observed over a span of 7.5 h. A steady caffeine concentration of 0.1513 mg mL−1 is reached on the maternal side after 6.5 h, and a 0.0033 mg mL−1 concentration on the fetal side is achieved after 5 h.

Antidoping Science: Important Lessons From the Medical Sciences.

The ineffectiveness of antidoping programs in elite sport, largely due to human and political factors, is leading to a new resolve and greater transparency of antidoping authorities and those stakeholders interested in drug-free sport. The perception by the public, athletes, and the World Anti-Doping Agency (WADA) of antidoping science and current drug testing programs in elite sport varies widely from "ineffective" to "robust and reliable." Here, we discuss why a careful and considered reevaluation of the underlying premise of antidoping science is needed to bring this unique application of predictive/diagnostic science more in line with other areas of medicine. We show how the validity of doping tests are neither "stand-alone figures" generated under ideal laboratory conditions, nor figures that can be used in isolation to support the efficacy of the current drug testing program. Given the consequences of a failed doping test for the athlete, the sport, and multiple stakeholders (e.g., the sponsors), there is a need for transparent decision making to ensure those affected are well informed. We identify in this perspective the minimal essential data on drug testing that should be reported by antidoping laboratories to draw meaningful conclusions about the effectiveness of specific drug testing methods to support antidoping. In the absence of information on the validity of a doping test, it is not possible to plan or conduct "intelligent testing." It is imperative that the prevalence of doping and the likelihood of false-positive doping tests be regularly updated and made available for the wider antidoping research community to explore new approaches that could improve the validity of antidoping tests. True confirmatory testing which requires the use of different analytical technology and ideally an independent sample taken from an athlete with a positive test to transcend the present-day analysis of the B-sample. Indirect biomarkers of doping derived from new "omics"-based approaches may significantly improve the testing strategy. Biomarker molecular signatures are flexible enough to develop "normal ranges" optimized for either test sensitivity or specificity to detect a plethora of doping substances and methods.

Abstract 1029: The PDX Data Commons and Coordinating Center (PDCCC) for PDXNet

Abstract Patient-Derived Xenografts (PDX) are powerful models to study tumors' drug-response in the context of personalized medicine. In the PDX model settings, by virtue of expanding the patient's tumor sample, testing multiple drug or drug-combinations can be executed rapidly and has no ethical limitations. However, there are major issues around standards that need to be addressed to make these models widely accessible and usable. The overarching goal of the PDXNet is to coordinate the development of appropriate PDX models and methods for preclinical drug testing to advance CTEP clinical development of new cancer agents. In an effort to standardize protocols for PDX generation as well as data analysis and metadata harmonization, we are building a data storage, sharing, and analysis platform that harmonizes PDXNet data with other large datasets and analysis workflows. The PDX Data Commons is built on top of existing NCI resources, leveraging the Cancer Genomics Cloud maintained by Seven Bridges Genomics, where PDXNet data is co-located with TCGA and other large-scale datasets. The PDCCC is co-led by experts from The Jackson Laboratory, providing scientific leadership in xenograft methods and cancer biology to ensure the promulgation of standards that are well-suited for the PDX community. In addition, the PDCCC is responsible for establishing studies to identify best-practices for PDX data analysis and metadata schemas. The data collected as part of the PDXNet is currently stored on the PDXNet portal that has a query interface for identifying models for pre-clinical trials. Simultaneously, we administer training activities and research pilots to build synergies within the PDXNet, enhancing the ability of the PDXNet to develop clinical trials from PDX studies. In PDXNet, besides the PDCCC, there are 4 PDX Development and Trial Centers (PDTCs) responsible for executing specific pre-clinical trials focused around cancer types including breast cancer, melanoma, and lung cancer. Data generated by the PDTCs will be hosted by the PDCCC, and metadata will be collected based on schemas developed by the network for systematic ontological analysis. These PDX models, in coordination with the NCI Patient-Derived Models Repository (PDMR) at the Frederick National Laboratory for Cancer Research (FNLCR) will be shared with the broader community. In addition, PDTC's will collaborate with non-PDXNet investigators for PDX studies through an administrative supplement program supported by the NCI. The PDXNet is a strong step toward building a consensus around PDX models, so that the power for discovery can be expanded by making multi-institutional PDX cohorts a reality. The PDCCC is a central part of this process to systematically capture and analyze the variables most influential to PDX models and share protocols and tools to make PDXs an interchangeable research currency for pre-clinical discovery. Citation Format: Anurag Sethi, Anuj Srivastava, Xingyi Woo, Vishal Sarsani, Ziming Zhao, Javad Noorbakhsh, Christian French, Jack DiGiovanna, Ogan D. Abaan, Steve Neuhauser, Peter Robinson, Yvonne A. Evrard, Carol J. Bult, Jeffrey A. Moscow, Brandi Davis-Dusenbery, Jeffrey H. Chuang. The PDX Data Commons and Coordinating Center (PDCCC) for PDXNet [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1029.

A Simple Pipetting-based Method for Encapsulating Live Cells into Multi-layered Hydrogel Droplets

Here, we present a simple pipetting-based approach for generating multi-layered core-shell hydrogel droplets composed of alginate for outer shell and collagen for culturing cells inside. By using a multi-hole plastic substrate and by pipetting, multi-layered hydrogel droplets were generated in a simple and rapid manner. HEK293 cells, which are human embryonic kidney cells, were cultured for 14 days in double-layered hydrogel droplet with high viability. Cancer cells were co-cultured with epithelial cells in multi-layered hydrogel droplets and applied for drug tests with curcumin. As epithelial cells protect cancer cells from anti-cancer drugs, co-cultured cells showed lower sensitivity to curcumin. We developed a simple and easy method for creating complex hydrogel particles for 3D multicellular co-culture and developed an alternative method for drug testing in vivo.

W A A V P guideline for evaluating the efficacy of anticoccidials in mammals (pigs, dogs, cattle, sheep)

This guideline is intended as an aid in the design, implementation and interpretation of studies for the assessment of drug efficacy against Eimeria in cattle and sheep, Cystoisospora in pigs and dogs, and Cryptosporidium in cattle. It deals with the most important aspects of how to conduct both experimental and field studies for dose determination, dose confirmation and assessment of field effectiveness. Also, guidance on the selection of animals, diagnostic techniques, statistical evaluation and methods for the preparation, maintenance and use of parasites is provided. The specific management conditions that may influence the course of natural infections and consequently determine treatment schemes are mentioned and suggestions for best practice in sampling and evaluation of data prior to conducting of efficacy studies are given. The guideline is also intended to assist investigators in carrying out specific studies, provide relevant information for registration authorities involved in the decision-making process, assist in the approval of anticoccidial drugs in the target species, and facilitate the world-wide adoption of standard procedures. Although currently not implemented, issues of drug resistance testing and alternative methods for drug testing are also discussed as future issues in drug testing against mammalian coccidia.

Surveillance of drug abuse in Hong Kong by hair analysis using LC-MS/MS.

The aim of this study is to reveal the habits of drug abusers in hair samples from drug rehabilitation units in Hong Kong. With the application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology, a total of 1771 hair samples were analyzed during the period of hair testing service (January 2012 to March 2016) provided to 14 drug rehabilitation units including non-governmental organizations (NGOs), rehabilitation centers, and medical clinics. Hair samples were analyzed for abused drugs and their metabolites simultaneously, including ketamine, norketamine, cocaine, benzoylecgonine, cocaethylene, norcocaine, codeine, MDMA, MDA, MDEA, amphetamine, methamphetamine, morphine, 6-acetylmorphine, phencyclidine, and methadone. The results showed that ketamine (77.2%), cocaine (21.3%), and methamphetamine (16.5%) were the frequently detected drugs among those drug abusers, which is consistent with the reported data. In addition, the usage of multiple drugs was also observed in the hair samples. About 29% of drug-positive samples were detected with multiple drug use. Our studies prove that our locally developed hair drug-testing method and service can be a valid tool to monitor the use of abused drugs, and which could facilitate rehabilitation program management.

Implementation of Contingency Management at a Large VA Addiction Treatment Center.

Contingency management (CM) is an evidence-based intervention that reinforces target behaviors of patients, such as abstinence from substance use. This column discusses the experiences and lessons learned at a large U.S. Department of Veterans Affairs Addiction Treatment Center during implementation of CM as part of the VA's national rollout. Challenges are discussed related to staff reception and limited initial referrals, identifying a drug testing method and staff confidence in that method, training requirements, and financial resources needed. Local innovations and CM expansion since 2012 are also reviewed.

Potential of GHB phase-II-metabolites to complement current approaches in GHB post administration detection

Recently, phase-II-metabolites of γ-hydroxybutyric acid (GHB), namely GHB-β-O-glucuronide and GHB-4-sulfate, were implemented in the scope of drug testing methods The clearance of GHB from the circulation is extremely fast due to its incorporation into the metabolic pathway of the citrate cycle. The elimination half-life of GHB from blood was reported to be dose dependent between 30 and 50min resulting in narrow detection windows of less than 12h after illicit administration or cases of drug facilitated sexual assault regardless of the biological matrix used. As sulfated metabolites tend to show prolonged half-lives and slower elimination kinetics compared to unmodified or glucuronidated drugs, the potential of GHB-4-sulfate in prolonging the detection of GHB administration was assessed. Its urinary concentrations were determined in n=100 samples from athletes and n=50 samples from sport students, and the resulting data were used to calculate a preliminary reference population-based threshold for urinary GHB-sulfate concentration. The threshold was then compared to concentrations found in post-administration urine samples collected from 3 volunteers who administered GHB within the setting of a clinical trial. Due to the large inter-individual variability of concentrations found in the reference population, GHB-4-sulfate itself was not suitable to prolong the detection times for GHB applications, even when specific gravity-corrected values were used.Therefore, a metabolomics-based approach was applied to the reference population samples and evaluated regarding other urinary metabolites that potentially correlate with the urinary excretion of GHB-4-sulfate and GHB-β-O-glucuronide in order to find a suitable marker to normalize urinary concentrations. The most promising candidate was found at a molecular mass of 321.0696 and was preliminarily identified as β-citryl-glutamic acid.