Drug Testing and Analysis

A robust and highly sensitive (picogram level) analytical method for quantification of Curcumin (cur), Dementhoxy curcumin (DMC), Bisdemethoxycurcumin (BDMC) in Wistar rat brain homogenate by UHPLC/ESI-QTOF-MS/MS was successfully developed and validated.

1,3 dimethylamylamine (DMAA) concentrations were determined in Geraniaceae, geranium oil and dietary supplements in order to establish whether DMAA contained in DS is of a synthetic origin. Findings show DMAA is not present in the plant species Geranium and Pelargonium; thus the ‘natural’ origin and use of DMAA as an ingredient in DS was not substantiated.

Although synthetic cannabinoids have been intensively investigated in recent years and oral fluid testing is continuously becoming popular in suspected driving under the influence of drugs cases, only scarce data on their stability in authentic neat oral fluid samples are yet available. In this article, the results of investigations on the recovery of 11 synthetic cannabinoids from authentic neat oral fluid samples stored over 72 h in glass and polypropylene tubes at 4 and 25 °C are presented.

Although filter paper is usually employed for Dried Blood Spots, we report here the extension of this alternative sampling support to the analysis of other body fluids and tissues collected after a fatal overdose with colchicine. The developed LC-MS/MS platform, including automated 96-well extraction of filter paper, represents a rapid and versatile approach for drug determination in multiple forensic media.

In this experiment, 16 different ELISA reagents from four commercial suppliers were evaluated to determine the cross-reactivity of 30 designer drugs, including cathinones, tryptamines, and piperizines. Cross-reactivity towards most drugs by these assays was minimal, with the exception of one supplier's mephedrone/methcathinone kit, where cathinone derivatives demonstrated cross-reactivity at concentrations as low as 150 ng/ml. Given these results, a need clearly exists for broader-range screening techniques to be applied when analyzing specimens for the majority of newer designer drugs.

A nurse administered succinylcholine (SUX) and gave first aid in the therapy of unexpected respiratory depression. SUX and succinylmonocholine (SMC) were found in plasma and urine. She wanted to present herself spectacularly as the first and decisive rescuer.

A UPLC-PDA-TOF/MS was proposed and validated for rapidly analyzing and evaluating Ginkgo biloba leaves from different origin by using multivariate statistical analysis. By comparing the mass/UV spectra and retention times with those of reference compounds and/or tentatively assigned by matching empirical molecular formulae with those of the known compounds published in the literature.

Traditional herbal medicines (THMs) are gaining popularity worldwide as an alternative approach to prescription drugs for many reasons; one general perception is that they are safe. Recently there have been number of reported studies that reveal adulteration of THMs with undeclared synthetic drugs, which may potentially cause serious toxic adverse effects. The main focus is to highlight newer analytical tools used to detect adulteration. This review gives an overview of health-related risks after consuming such spurious products and notes the challenges for future perspectives to control such type of malpractice.

Six death cases caused by diabetic coma are described with special focus on biochemical (and histological) findings. Estimations of glucose concentrations in vitreous humour or cerebrospinal fluid and of ketone bodies in blood or other matrices is obligatory while measurements of HbA1c, fructosamine or anhydroglucitol can only provide additional information on the long-term adjustment of diabetes in the deceased.

A case of serial killing by poisoning by a practical nurse is reported. In the absence of ordinary post-mortem toxicology samples, a special kind of evidence, paraffin-embedded liver tissue samples (originally taken for histology at autopsy), was successfully recovered in two cases and analyzed for drugs. In all five homicides, drugs not prescribed for the patient were detected, including digoxin, dixyrazine, citalopram, venlafaxine, and benzodiazepines (diazepam, chlordiazepoxide, temazepam and oxazepam).

This study presents the validation of immunofluorometric (IF) and immunochemiluminometric (ICL) assays for LH immunoreactivity measurements of urine samples after prolonged frozen storage. Samples from a clinical study were assayed before and after four years of storage at -20 °C without addition of any preservatives. The study describes the deviations in performance of these two immunoassays and their ultimate applicability for reliability clinical research and for anti-doping purposes.

The concentration of prednisolone in porcine urine can be used as a first indication of an animal's treatment with prednisolone. As prednisolone was also detected up to 2.8 ng/ml in the urine of untreated pigs, the ratio of prednisolone/cortisol in liver samples seems to be more suitable as an indicator for detecting illicit prednisolone administration to pigs, together with the research of prednisolone metabolites.

Exposure of urine containing morphine, codeine, morphine-3-glucuronide and morphine-6-glucuronide to nitrite resulted in the formation of 2-nitro-morphine and 2-nitro-morphine-6-glucuronide. These oxidation products can potentially be used for the indirect monitoring of morphine and morphine-6-glucuronide in urine specimens adulterated with nitrite.

The study was carried out to investigate to what extent exposure to sidestream marijuana smoke can lead to positive hair results for cannabinoids. Three participants were exposed to sidestream marijuana smoke and the concentrations of Δ9-tetrahydrocannabinol, Δ9-tetrahydrocannabinolic acid A and cannabinol were analyzed in hair using an LC-MS/MS method. Δ9-tetrahydrocannabinol and cannabinol concentrations similar to the ones found in hair of daily consumers were detected.

This paper focused on the method development for screening of residues of the four veterinary drugs, mebendazole, clorsulon, diaveridine, and tolfenamic acid in various food samples based on the maximum residue limit. With conditioning of liquid-liquid extraction and HPLC-UV analysis, the determinative methodology that consisted of common sample preparation step and instrumental analysis step was developed. Finally, the method was validated in accordance with the guidelines for Single-laboratory Validation of Analytical Methods for Trace-level Concentration of Organic Chemicals.

A total number of six PCP and PCPy analogues have been synthesized and analytically characterized. Some of these substances have recently been observed within the recreational context and this study aimed to provide the corresponding data to facilitate their identification which included analogues that have not yet been reported.

This paper describes the identification by UHPLC -MS/MS and -HRMS of main urinary metabolites of zolpidem and zopiclone. These compounds, often used in Drug Facilitated Sexual Assaluts (DFSA), are in fact excreted mainly as metabolites. A method was set up for their routinary determination by LC/MS/MS, that can be applied in forensic toxicology cases.

Naloxone can be detected and used as an indicator of compliance in urine samples from opioid-dependent patients on maintenance treatment with buprenorphine/naloxone coformulation (BNX). We suggest that the urine naloxone residual concentration combined with the high naloxone/buprenorphine ratio can help to differentiate therapeutic sublingual use of BNX from its parenteral abuse.

Plasma-derived microRNA can be used to differentiate between individuals receiving therapeutic replacement doses of recombinant human growth hormone (rhGH) and all others, including individuals with acromegaly. Four microRNAs, expressed differently in individuals receiving therapeutic rhGH, were identified via microarray analysis and confirmed on a larger sample set using real-time quantitative PCR analysis. Future studies on individuals receiving larger doses of rhGH, including a washout period during which the window of detection can be determined, should be performed.

New classes of recreational drugs include a synthetic cannabinoid detected in sewage for the first time. Analysis of the metabolites of synthetic cannabinoids and amphetamine-like drugs in sewage can reveal their use in discrete populations. The study demonstrates that sewage biomarker analysis can be applied to evaluate not only the use the traditional illicit drugs (cocaine, cannabis and amphetamines), but also the use of new synthetic drugs.

The development, validation and application of a method based on SPE and LC-MS/MS is presented to measure concentrations of new psychoactive substances in influent wastewater in the low ng/l range.

Ten nucleobases and nucleosides were measured simultaneously by HPLC-PDA to explore their profiles in Alpinia oxyphylla obtained from different cultivation regions. Hierarchical cluster analysis was performed to classify tested samples according to the contents of nucleobases and nucleosides.

This study aimed to evaluate the potential sublethal effects of two human pharmaceuticals (diclofenac and gemfibrozil), commonly found in the aquatic environment, on the protein profiles of marine mussels (Mytilus spp.).Twelve spots were significantly increased or decreased by gemfibrozil and/or diclofenac, seven of which were successfully identified by LC-MS/MS analysis.These proteins were involved in energy metabolism, oxidative stress response, protein folding and immune responses.

Meconium samples for drug testing were collected from 143 infants as part of routine clinical work. The drug testing findings were combined with medical records including clinical background and follow-up data. Medicinal opioids, especially the opioid substitution treatment drugs buprenorphine and methadone, comprised the majority of the findings of both admitted and unexpected drug misuse.

Glycerol was administered before an endurance exercise and plasma and urinary glycerol concentrations were assessed along with blood parameters. Plasma glycerol was highly increased after glycerol administration but was also elevated in the control group post-exercise. Urinary glycerol concentrations were significantly higher than after placebo administration until 13.6 ± 0.9 h. Glycerol-induced changes in plasma volume, haemoglobin, and haematocrit were small and clinically irrelevant.

Relative sensitivities and relative specificities were calculated at 99.7 % and 98.4 % for direct ELISA and 66 % and 91.4 % for CEDIA respectively for drugs of abuse screening when applied to the German abstinence cut-offs at 10 ng/mL cannabinoids, 50 ng/mL amphetamines and designer amphetamines, 25 ng/mL opiates, 30 ng/mL cocaine metabolite, 50 ng/mL methadone metabolite and benzodiazepines at 50 ng/mL in urine.

Formestane, a steroidal aromatase inhibitor, can be used for building muscles by stopping the conversion of testosterone into estradiol, resulting in the increase of testosterone level in the body. While it was banned in human and equine sports, little is known about its metabolic fate in horses. This paper describes the metabolic studies of formestane in horses, with an aim to identify the most appropriate target metabolites to be monitored for controlling the misuse or abuse of formestane in racehorses.

Steroids reference values from a Latin-American population to control doping are presented. A comparison between sexes referred to urinary steroids is presented. A link between Caucasian and Latin-Americans is made referred to WADA limits.

The synthetic cannabinoid, UR-144, was identified in commercial 'legal high' products. A related compound was also detected and identified as product of electrophilic addition of water to cyclopropane moiety. Separate pyrolysis of this hydrated products and UR-144 resulted in the formation of hexenyl compound.

Siemens Immulite 2000 XPi hCG-assay for initial testing and PerkinElmer DELFIA Xpress hCG-assay for confirmatory testing showed adequate sensitivity and were proven fit-for-purpose in routine doping control. Population-based distribution of the assays was in good agreement with results of earlier studies and supported well the current threshold of 5 IU/L.

Gamma-hydroxybutyric acid (GHB) is a drug-of-abuse that has recently become associated with drug-facilitated sexual assault, known as date rape. For this reason the drug is commonly found ‘spiked’ into alcoholic beverages. When GHB is in solution, it may undergo conversion into the corresponding lactone, Gamma-butyrolactone (GBL); therefore both GHB and GBL have been studied in the present work to monitor the interconversion of GHB and GBL in solution with different pH conditions and temperature.

We performed GC-MS analysis, including sample preparation, of nine MC analogs. This study identified several key points required for the accurate analysis such as the prevention of thermal degradation and the loss of analytes during evaporation. In addition, this study has demonstrated analytical conditions and can successfully discriminate between three FMC positional isomers.

A robust analytical method for dopamine was successfully developed and validated. The detection limit in pictogram proves the potential of this method, which might be an effective tool for estimating the level of dopamine in human being.

Concentrations of alcohol and illicit and medicinal drugs in seriously injured drivers and drivers selected randomly at the roadside are compared. Significant differences in concentrations between the two populations were observed for some drugs. Alcohol was found in much higher concentrations in the injured drivers.

In recent years, the analysis of synthetic cannabinoids in human specimens has gained enormous importance in the broad field of drug testing. Although the analysis of blood samples is the current method of choice, the need for alternative matrices arises whenever blood sampling is not possible. In this paper, we present a fully validated LC/ESI-MS/MS method for the analysis 30 synthetic cannabinoids in oral fluid samples collected with the Dräger DCD 5000 collection device.

There are six regioisomeric dimethoxybenzylpiperazines that can be prepared directly from the corresponding substituted benzaldehydes. These six piperazine derivatives yield very similar fragment ions in their mass spectra with only the 2,3-dimethoxybenzylpiperazines showing one unique major fragment at m/z 136. Chemical derivatization as well as GC-TOF exact mass measurements did not provide any discrimination among these compounds. GC-IRD offered unique and characteristic IR spectra that allowed discrimination among these six regioisomeric compounds.

Relaxin is a peptide hormone known for reproduction and pregnancy, but it has also been demonstrated to play a vital role in other therapies. Its ability to dilate blood vessels surrounding the smooth muscles via induction of nitric oxide result in the increase of blood and oxygen supplies to the muscles. As such, it is banned in horseracing, equestrian competitions, and human sports. This paper describes the first liquid chromatography-high resolution mass spectrometry method for the identification of recombinant human relaxin-2 in equine plasma.

Endogenous matrix components that may be extracted from DBS samples and contribute to matrix effects in DBS-LC-MS/MS analysis have not been investigated before. The aim of this study is to investigate various organic solvents for their abilities to extract glycerophosphocholines (e.g. phosphatidycholine), cholesterols and triacylglycerols as the main endogenous matrix components that may result in matrix effects in DBS samples, as a general guide.

The stability of 11 dependent drugs and metabolites in wastewater under different storage conditions were evaluated, with storing samples at 4 °C found to be the most cost-effective preservative way for most drugs. Labile substances can be stabilized by pH adjustment and preservative addition. No significant analyte loss was observed in the filtration process or in the reconstituted extract.

Benzoylecgonine (BE), the main metabolite of cocaine, is a new environmental contaminant and one of the most abundant drugs found in freshwaters. We studied BE's effects on Dreissena polymorpha by the application of redox proteomics. We detected a clear increase of protein carbonylation after exposure to BE suggesting the presence of oxidative stress. BE leads to modification of some proteins involved in glucose metabolism and some which are cytoskeletal components of the cell.

Pyridostigmine pretreatment before soman intoxication followed by medical treatment with HI-6 DMS, atropine and avizafone provided a superior treatment regimen in rats compared to pyridostigmine pretreatment, followed by obidoxime, atropine and diazepam post intoxication. HI-6 DMS, physostigmine and scopolamine has superior antidotal efficacy post exposure, but immediate medical treatment is necessary, and therefore not practical for use in a field or emergency situation with exposure to the nerve agent soman.

A simple, sensitive and high throughput liquid chromatography – high resolution mass spectrometry screening method has been developed and validated for the simultaneous screening of 325 prohibited substances in horse plasma using an Orbitrap mass spectrometer.

This publication reports the MS, IR and NMR spectroscopic data of three new hallucinogenic substances identified in blotter papers seized from the drug market, namely 25D-NBOMe [2-(2,5-dimethoxy-4-methylphenyl)-N-(2-methoxybenzyl)ethanamine], 25E-NBOMe [2-(4-ethyl-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine] and 25G-NBOMe [2-(2,5-dimethoxy-3,4-dimethyl-phenyl)-N-(2-methoxybenzyl)ethanamine]. These substances are highly potent agonists of serotonin receptors (5-HT_2A), with active doses at microgram levels.

The introduction of doping substances and methods in sports are supposed to trigger noticeable effects on physical performances in metric sports. Here, we use time series analysis to investigate the recent development in male and female elite sprinting performance.

This study discusses the MS, IR and NMR spectroscopic data of 2C-G, which was seized in Poland in 2011. This paper focuses on a comparison of the analytical features of 2C-G and other representatives of the 2C-series. GC-EI/MS spectra of the parent substance and its TFAA derivative, and LC-ESI/QTOFMS spectra for 2C-G are similar to those for 2C-E (4-ethyl-2,5-dimethoxy-β-phenethylamine), but specific features are presented. The study indicated that the marketing of analogues of controlled substances poses a real analytical challenge.

A simple and highly sensitive method that involves HF-LPME–GC was used for determination of narcotic analgesic drugs in biological and wastewater samples.

The key findings of present study are pharmacokinetic interaction studies between piracetam and morin (flavonoid) (C_max, T_max, AUC_0−t, AUC_0−∞, CL, t_1/2, MRT_t), enzyme kinetic (K_m, V_max, CL_{int, invitro}, Cl_{int, in vivo}, MM & LWB plot), in vitro metabolic stability (K (min⁻¹), t_1/2, Df) & correlation between in vitro and in vivo.

Microcrystalline tests for drugs of abuse in flat capillary tubes were investigated as an alternative to microscope slides. Effects on microcrystal development as well as pre- and post experiment treatment of tubes was established. A simple method for permanent preservation of microcrystalline tests was demonstrated. Concept was successfully applied to NPS samples.

Atomoxetine (ATX) is a non-stimulant drug approved for the treatment of children and adolescents with attention deficit/hyperactivity disorder (ADHD). We studied the excretion profile of ATX and its principal metabolites 4-hydroxyatomoxetine (4-OH-ATX) and N-desmethylatomoxetine (desmethyl-ATX) in oral fluid and plasma of ADHD paediatric subjects, after administration of different dosage regimens.

The correlations between ATX and 4-OH-ATX concentrations in plasma and oral fluid indicate that oral fluid concentrations of this drug and its principal metabolite may be a predictor of plasma concentrations, even if values are too low and variable to be considered an alternative to plasma.

Mephedrone is a designer-drug that has gained increasing use in recent years. In vitro studies showed that the main enzyme responsible for metabolism of mephedrone was cytochrome P450 2D6, which formed hydroxytolyl- and nor-mephedrone, of which the former was purified and identified by NMR. In forensic cases, these metabolites and 4-carboxy-dihydro-mephedrone were identified by LC-MS/MS and LC-TOF in blood or urine, additionally, two previously unreported metabolites, dihydro-mephedrone and 4-carboxy-mephedrone were detected.

400 MHz ¹H NMR spectroscopy is used to detect pharmacologically active substances in food supplements and medicines, which are widely marketed in an illegal or counterfeited way over the Internet. An example is sibutramine in anorectic formulations such as so-called slimming tea. The NMR approach has the advantage that identification and quantification is often possible, even when pure compounds as reference standards are unavailable.

The results of a study performed on samples of ‘legal highs’ seized in ‘head shops’ in Poland between mid-2008 and mid-2011 were presented. The most common ingredients were MPDV, caffeine, butylone, TFMPP, lidocaine, 4-MEC, mephedrone, pFPP, BZP and MDPBP. Cathinones and piperazines were mixed mainly within the chemical classes, caffeine was mixed both with piperazines and cathinones, whereas lidocaine only with the latter class. A great inconsistency in the qualitative and quantitative composition of products with identical labelling was shown.

Paromomycin-loaded albumin microspheres were formulated and tested for efficacy and stability in present work. Efficacy studies provided significant increase in efficacy of formulation in comparison to PM solution at equivalent concentration. Stability studies proved stability of prepared microsphere formulation at all tested conditions including accelerated conditions.

Molecularly imprinted polymers (MIPs) exhibiting high selectivity and affinity to the predetermined molecule are now seeing a fast growing research

The alkaloids of Radix Aconiti Lateralis were studied. 48 compounds were structurally identified, among them, a new kind of alkaloids was found with protonated molecules at m/z 452, 468 and 482.

Paromomycin loaded albumin microspheres were prepared to target Paromomycin to macrophages. A new bioanalytical method was developed and found to be very sensitive. Pharmacokinetic studies performed in rat showed reduction in Cmax of free Paromomycin when administered as developed microsphere formulation. Toxicity studies indicated better toxicity profile for prepared formulation as compared to marketed Paromomycin IM injection for the treatment of visceral leishmaniasis.

A rapid and sensitive liquid chromatography tandem mass spectrometry method has been developed and validated for the determination of febuxostat in human plasma. The liquid–liquid extraction technique was used for the extraction of febuxostat from human plasma using trandolapril as the internal standard (IS). Chromatography was performed on a UPLC BEH C18, 50 mm X 2.1 mm, 1.7 µm particle size column, with the mobile phase consisting of 0.1% formic acid and acetonitrile (in a 25:75 ratio), followed by detection using mass spectrometry. The method involves a simple reversed isocratic chromatography condition and mass spectrometry detection, which enables detection at sub-microgram levels. The method was validated and the lower limit of quantification for febuxostat was found to be 0.075 µg/mL. The mean recovery for febuxostat ranged from 100.92 to 106.5%. This method increased the sensitivity and selectivity; resulting in high-throughput analysis of febuxostat using commercially available IS for pharmacokinetic, bioavailability and bioequivalence studies, with a chromatographic run time of 1.5 min only.

A simple, feasible, cost effective, rapid accurate, specific and sensitive high-performance liquid chromatographic method with UV detection has been developed and validated according to the FDA guidelines for the determination of zidovudine, lamivudine and nevirapine in human plasma. It was successfully applied to analyze the plasma samples of the pharmacokinetic study of the above drugs formulations in 12 healthy human volunteers.

A simple, sensitive and selective RP-HPLC method was described for the simultaneous determination of naftidrofuryl oxalate (NF) and its hydrolytic degradation product (metabolite), naftidrofuryl acid (NFA). The method was applied as a stability-indicating assay of the parent drug NF in its tablets. Also, the degradation product NFA was determined down to a level of 0.005% in presence of large excess of the parent drug. Finally, drug monitoring of NF and its metabolite in human plasma/urine samples was carried out.

The performance of a commercial immunoassay for detection of ethyl glucuronide in urine is evaluated against a liquid chromatography tandem mass spectrometry method. The use of the immunoassay for qualitative and quantitative determinations is appraised in the context of both post-mortem and clinical samples (n=1000). The immunoassay gives a very good approximation to the EtG concentration in both sample types and its use as a more economic alternative can be justified with no detrimental effect on the determined EtG concentration.

The detection of 3,5-dimethoxyphenol (3,5-DMP), myrtenol and 1-octen-3-ol compounds is easily performed by GC-MS (SIM) after an enzymatic hydrolysis (β-glucosidase) allowing the release of volatile compounds from yew glycosides. Their presence and relative amount consitute key indicators to confirm a yew ingestion.