Recent advances in the detection of drugs of abuse by dried blood spots.

The purpose of this review study was to sum up the main recent advances reported in the scientific literature about the detection of common drugs of abuse in biological samples upon their collection by dried blood spot devices. The most recent, innovative and fully validated methods for the qualitative and/or quantitative detection of common drugs of abuse are reported, including alprazolam, clonazepam, diazepam, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methyl-enedioxyethylamphetamine, amphetamine, methamphetamine, cocaine, tetrahydrocannabinol, 6-monoacetylmorphine, morphine, codeine, hydromorphone, hydrocodone, oxycodone, noroxycodone, tramadol, methadone, buprenorphine, fentanyl, ketamine and their respective metabolites and γ-hydroxybutyric acid. Dried blood spot proved to be extremely promising for routine analysis of forensic cases, although large-scale experiments on real samples need to be performed to confirm the emerging advantages of the technique and remove the potential limitations still affecting its widespread application.

Targeted and untargeted detection of fentanyl analogues and their metabolites in hair by means of UHPLC-QTOF-HRMS.

Detection of new psychoactive substances and synthetic opioids is generally performed by means of targeted methods in mass spectrometry, as they generally provide adequate sensitivity and specificity. Unfortunately, new and unexpected compounds are continuously introduced in the illegal market of abused drugs, preventing timely updating of the analytical procedures. Moreover, the investigation of biological matrices is influenced by metabolism and excretion, in turn affecting the chance of past intake detectability. In this scenario, new opportunities are offered by both the non-targeted approaches allowed by modern UHPLC-HRMS instrumentation and the investigation of hair as the matrix of choice to detect long-term exposure to toxicologically relevant substances. In this study, we present a comprehensive and validated workflow that combines the use of UHPLC-QTOF-HRMS instrumentation with a simple hair sample extraction procedure for the detection of a variety of fentanyl analogues and metabolites. A simultaneous targeted and untargeted analysis was applied to 100 real samples taken from opiates users. MS and MS/MS data were collected for each sample. Data acquisition included a TOF-MS high-resolution scan combined with TOF-MS/MS acquisition demonstrating considerable capability to detect expected and unexpected substances even at low concentration levels. The predominant diffusion of fentanyl was confirmed by its detection in 68 hair samples. Other prevalent analogues were furanylfentanyl (28 positive samples) and acetylfentanyl (14 positive samples). Carfentanil, methylfentanyl, and ocfentanil were not found in any of the analyzed samples. Furthermore, the retrospective data analysis based on untargeted acquisition allowed the identification of two fentanyl analogues, namely ß-hydroxyfentanyl and methoxyacetylfentanyl, which were not originally included in the panel of targeted analytes.

Optimization and validation of a GC-MS quantitative method for the determination of an extended estrogenic profile in human urine: Variability intervals in a population of healthy women.

An analytical method based on GC-MS was developed for the determination of a wide panel of urinary estrogens, together with their principal metabolites. Because of the low concentration of estrogens in urine, an efficient sample pre-treatment was optimized by a design of experiment (DoE) procedure to achieve satisfactory sensitivity. A second DoE was built for the optimization of the chromatographic run, with the purpose of reaching the most efficient separation of analytes with potentially interfering ions and similar chromatographic properties. The method was fully validated using a rigorous calibration strategy: from several replicate analyses of blank urine samples spiked with the analytes, calibration models were built with particular attention to the study of heteroscedasticity and quadraticity. Other validation parameters, including the limit of detection, intra-assay precision and accuracy, repeatability, selectivity, specificity, and carry-over, were obtained using the same set of data. Further experiments were performed to evaluate matrix effect and extraction recovery. Then the urinary estrogen profiles of 138 post-menopausal healthy women were determined. These profiles provide a representation of physiological concentration ranges, which, in forthcoming studies, will be matched on the base of multivariate statistics with the urinary estrogenic profile of women with breast or ovarian cancer.

Untargeted Metabolomics in Forensic Toxicology: A New Approach for the Detection of Fentanyl Intake in Urine Samples.

The misuse of fentanyl, and novel synthetic opioids (NSO) in general, has become a public health emergency, especially in the United States. The detection of NSO is often challenged by the limited diagnostic time frame allowed by urine sampling and the wide range of chemically modified analogues, continuously introduced to the recreational drug market. In this study, an untargeted metabolomics approach was developed to obtain a comprehensive "fingerprint" of any anomalous and specific metabolic pattern potentially related to fentanyl exposure. In recent years, in vitro models of drug metabolism have emerged as important tools to overcome the limited access to positive urine samples and uncertainties related to the substances actually taken, the possible combined drug intake, and the ingested dose. In this study, an in vivo experiment was designed by incubating HepG2 cell lines with either fentanyl or common drugs of abuse, creating a cohort of 96 samples. These samples, together with 81 urine samples including negative controls and positive samples obtained from recent users of either fentanyl or "traditional" drugs, were subjected to untargeted analysis using both UHPLC reverse phase and HILIC chromatography combined with QTOF mass spectrometry. Data independent acquisition was performed by SWATH in order to obtain a comprehensive profile of the urinary metabolome. After extensive processing, the resulting datasets were initially subjected to unsupervised exploration by principal component analysis (PCA), yielding clear separation of the fentanyl positive samples with respect to both controls and samples positive to other drugs. The urine datasets were then systematically investigated by supervised classification models based on soft independent modeling by class analogy (SIMCA) algorithms, with the end goal of identifying fentanyl users. A final single-class SIMCA model based on an RP dataset and five PCs yielded 96% sensitivity and 74% specificity. The distinguishable metabolic patterns produced by fentanyl in comparison to other opioids opens up new perspectives in the interpretation of the biological activity of fentanyl.

Effects of truck transportation and slaughtering on the occurrence of prednisolone and its metabolites in cow urine, liver, and adrenal glands.

BACKGROUND: The recognition of illegal administration of synthetic corticosteroids in animal husbandry has been recently challenged by the case of prednisolone, whose occasional presence in the urine of bovines under strong stressful conditions was attributed to endogenous biosynthesis, not to exogenous administration. The study of the natural stress sources possibly inducing endogenous prednisolone production represents a stimulating investigation subject. The biochemical effects of transportation and slaughtering were verified in untreated cows by studying the possible occurrence of prednisolone and its metabolites in urine, liver and adrenal glands, and the cortisol/cortisone quantification. RESULTS: Cortisol, cortisone, prednisolone and its metabolites were measured in urine, collected at farm under natural micturition and then at the slaughterhouse. The study was performed on 15 untreated cows reared in different farms at the end of their productive cycle. 2-3 days after the first urine collection, the animals were transported by trucks to the abattoir, slaughtered, and subjected to a second urine sampling from the bladder. Specimens of liver and adrenal gland were also collected and analysed by means of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) validated method. The stressful conditions of transportation and slaughtering proved to increase considerably the urinary levels of cortisol and cortisone as compared to those collected at farm. Prednisolone was detected in the urine collected at the slaughterhouse of two cows only, at a concentration level (≈0.6 µg L- 1) largely below the official cut off (5.0 µg L- 1) established to avoid false non-compliances. These two animals exhibited the highest urinary cortisol levels of the series. Prednisolone and prednisone were also detected in the adrenal glands of a different cow. Prednisolone metabolites were not detected in any urine, liver, and adrenal gland sample. CONCLUSION: Within the constraints of the condition adopted, this study confirms the sporadic presence of prednisolone traces (2 samples out of 15) and the consistently increased concentration of cortisone and cortisol in the urines collected from cows subjected to truck transportation and subsequent slaughtering. No prednisolone metabolites were detected in any liver and adrenal gland samples, nor in urine specimens, unlike what was previously reported for cows artificially stressed by pharmacological treatment.

Determination of several synthetic cathinones and an amphetamine-like compound in urine by gas chromatography with mass spectrometry. Method validation and application to real cases.

Most routine practices for drugs-of-abuse testing do not include screening procedures for new psychoactive substances, despite their increasing diffusion, preventing clear knowledge of the real consumption of these drugs in the populations. To make up for this shortcoming, a gas chromatography with mass spectrometry method was developed for the simultaneous determination of 18 synthetic cathinones and one amphetamine-like compound in human urine. The sample preparation was based on liquid-liquid extraction under alkaline condition followed by derivatization with trifluoroacetic anhydride. The separation of the 19 analytes was achieved in less than 10 min. The whole methodology was validated according to national and international guidelines. Selectivity, linearity range, limit of detection and limit of quantitation, precision and accuracy were evaluated. For all the analytes, the calibration curve was linear in the 100-1000 ng/mL concentration range. The limits of detection ranged from 10 to 30 ng/mL and limits of quantitation from 30 to 100 ng/mL. Precisions were in the ranges 0.1-10.4%, and 1.0-12.1% for low (100 ng/mL) and high (1000 ng/mL) concentration, respectively. The accuracy, expressed as bias% was within ±20% for all the analytes. The present method was successfully applied to urine samples originating from autopsies, drug abuse/withdrawal controls, clinical investigations, roadside controls, driving re-licensing, and workplace testing.

Testing hair for fentanyl exposure: a method to inform harm reduction behavior among individuals who use heroin.

BACKGROUND: Deaths from fentanyl exposure continue to increase in the US. Fentanyl test strips are now available to test urine for presence of fentanyl, but additional testing methods are needed to determine past exposure and to determine exposure to specific analogs. OBJECTIVES: To investigate exposure to such analogs through hair testing. METHODS: Forty individuals in inpatient detoxification (7.5% female) reporting past-month heroin use were surveyed and provided a hair sample to be tested at a later date. While results could not be provided to patients, they were asked how they would respond if informed that their hair tested positive for fentanyl. UHPLC-MS/MS was used to test for past exposure to fentanyl, six other novel synthetic opioids, and fentanyl biomarkers/metabolites. RESULTS: 27.5% reported known fentanyl use in the past year and 67.5% reported suspected exposure. 97.5% (39 of 40) tested positive for fentanyl, 90.0% tested positive for 4-ANPP (a biomarker) and norfentanyl (a metabolite); 82.5% tested positive for acetyl-fentanyl, 47.5% tested positive for furanyl-fentanyl, and 7.5% tested positive for U-47700. Most participants (82.5%) reported they would warn others about fentanyl if they learned their hair tested positive; 75.0% reported they would try to stop using heroin, and 65.0% reported they would ensure that someone nearby has naloxone to reverse a potential overdose. CONCLUSIONS: Hair testing is useful in detecting past exposure to fentanyl, its analogs, and other novel synthetic opioids. Further research is needed to determine whether individuals who use heroin learning about exposure affects drug-taking and treatment-seeking behavior.

Untargeted Metabolomic Profile for the Detection of Prostate Carcinoma-Preliminary Results from PARAFAC2 and PLS-DA Models.

Prostate-specific antigen (PSA) is the main biomarker for the screening of prostate cancer (PCa), which has a high sensibility (higher than 80%) that is negatively offset by its poor specificity (only 30%, with the European cut-off of 4 ng/mL). This generates a large number of useless biopsies, involving both risks for the patients and costs for the national healthcare systems. Consequently, efforts were recently made to discover new biomarkers useful for PCa screening, including our proposal of interpreting a multi-parametric urinary steroidal profile with multivariate statistics. This approach has been expanded to investigate new alleged biomarkers by the application of untargeted urinary metabolomics. Urine samples from 91 patients (43 affected by PCa; 48 by benign hyperplasia) were deconjugated, extracted in both basic and acidic conditions, derivatized with different reagents, and analyzed with different gas chromatographic columns. Three-dimensional data were obtained from full-scan electron impact mass spectra. The PARADISe software, coupled with NIST libraries, was employed for the computation of PARAFAC2 models, the extraction of the significative components (alleged biomarkers), and the generation of a semiquantitative dataset. After variables selection, a partial least squares-discriminant analysis classification model was built, yielding promising performances. The selected biomarkers need further validation, possibly involving, yet again, a targeted approach.

Does the Abiotic Formation of Oligopeptides on TiO2 Nanoparticles Require Special Catalytic Sites? Apparently Not.

The oligomerization of non-activated amino acids catalyzed by nanostrucrured mineral oxide surfaces holds promises as a sustainable route for the industrial production of polypeptides. To analyze the influence of the surface type on the catalytic process, we performed, via a mild Chemical Vapor Deposition approach, the oligomerization of Glycine on two samples of TiO2 nanoparticles characterized by different relative amounts of defective surface terminations. Based on infrared spectroscopy and mass spectrometry data, we show herein that the formation of peptide bonds on titania nanoparticles does not require highly energetic surface terminations, but can occur also on the most abundant and thermodynamically most stable {101} facets of nanosized anatase.

Chromium, nickel, and cobalt in cosmetic matrices: an integrated bioanalytical characterization through total content, bioaccessibility, and Cr(III)/Cr(VI) speciation.

The presence of certain metals naturally contained inside raw materials (e.g., pigments) used to produce cosmetics for make-up may represent a serious concern for the final quality and safety of the product. The knowledge of the total concentration of metals is not sufficient to predict their reactivity and their toxicological profile. For these reasons, we set up a comprehensive approach to characterize the content of Co, Cr, and Ni in two raw materials for cosmetic production, a black iron oxide and a pearly pigment, and in a finished product, pearly powder eye shadow. Namely, besides the total metal concentrations, the speciation of chromium and the bioaccessibility of the three metals were assessed. Since no standard method is so far available for hexavalent chromium extraction from cosmetic samples, three approaches were compared (EPA 3060A method, IRSA 16 method, and a Na3PO4 extraction). Results show that Na3PO4 extraction is the most selective one. Cr(VI) was undetectable in black iron oxide and present at very low concentrations (about 0.3 mg/kg) in pearly pigment and in the pearly powder eye shadow samples. The extracted Cr(VI) concentrations are not related to the total Cr content in the samples. Bioaccessibility studies were performed by in vitro extractions with synthetic lacrimal fluids and sweat. Despite the wide range of metal concentrations in the samples, the amounts of bioaccessible elements were undetectable or very low (less than 0.4 mg/kg), thus suggesting that metals in the three samples are present in inert forms. Graphical abstract The possible leaching of metals from cosmetics to biological fluids. Spectroscopic and chromatographic techniques provide complementary information for an integrated bioanalytical approach to risk characterization.

Determination of cathinones and other stimulant, psychedelic, and dissociative designer drugs in real hair samples.

The detection of new psychoactive substances (NPS) in hair proved to provide insight into their current diffusion among the population and the social characteristics of these synthetic drugs' users. Therefore, a UHPLC-MS/MS method was developed in order to determine 31 stimulant and psychedelic substituted phenethylamines, and dissociative drugs in hair samples. The method proved to be simple, fast, specific, and sensitive. The absence of matrix interferents, together with excellent repeatability of both retention times and relative abundances of diagnostic transitions, allowed the correct identification of all analytes tested. The method showed optimal linearity in the interval 10-1000 pg/mg, with correlation coefficient values varying between 0.9981 and 0.9997. Quantitation limits ranged from 1.8 pg/mg for 4-methoxyphencyclidine (4-MeO-PCP) up to 35 pg/mg for 6-(2-aminopropyl)benzofuran (6-APB). The method was applied to (i) 23 real samples taken from proven MDMA and ketamine abusers and (ii) 54 real hair samples which had been previously tested negative during regular drug screening in driver's license recovery. Six samples tested positive for at least one target analyte. Methoxetamine (MXE) was found in three cases (range of concentration 7.7-27 pg/mg); mephedrone (4-MMC) was found in two cases (50-59 pg/mg) while one sample tested positive for methylone at 28 pg/mg. Other positive findings included 4-methylethcathinone (4-MEC), alpha-pyrrolidinovalerophenone (α-PVP), 4-fluoroamphetamine (4-FA), 3,4-methylenedioxypyrovalerone (MDPV), and diphenidine. The present study confirms the increasing diffusion of new designer drugs with enhanced stimulant activity among the target population of poly-abuse consumers.

In vitro metabolism studies on the selective androgen receptor modulator (SARM) LG121071 and its implementation into human doping controls using liquid chromatography-mass spectrometry.

LG121071 is a member of the tetrahydroquinolinone-based class of selective androgen receptor modulator (SARM) drug candidates. These nonsteroidal compounds are supposed to act as full anabolic agents with reduced androgenic properties. As SARMs provide an alternative to anabolic androgenic steroids, they represent an emerging class of potential doping substances abused by athletes for illicit performance enhancement. According to the World Anti-Doping Agency's regulations, SARMs are banned substances and part of the Prohibited List since 2008. In consideration of the increasing number of adverse analytical findings in doping controls caused by SARMs abuse, potential drug candidates such as LG121071 have been proactively investigated to enable a timely integration into routine testing procedures even though clinical trials are not yet complete. In the present approach, the collision-induced dissociation (CID) of LG121071 was characterized by means of electrospray ionization-high resolution/high accuracy mass spectrometry, MS(n), and isotope labeling experiments. Interestingly, the even-electron precursor ion [M + H](+) at m/z 297 was found to produce a radical cation at m/z 268 under CID conditions, violating the even-electron rule that commonly applies. For doping control purposes, metabolites were generated in vitro and a detection method for urine samples based on liquid chromatography-tandem mass spectrometry was established. The overall metabolic conversion of LG121071 was modest, yielding primarily mono-, bis- and trishydroxylated species. Notable, however, was the identification of a glucuronic acid conjugate of the intact drug, attributed to an N-glucuronide structure. The sample preparation procedure included the enzymatic hydrolysis of glucuronides prior to liquid-liquid extraction, allowing intact LG121071 to be measured, as well as the corresponding phase-I metabolites. The method was characterized concerning inter alia lower limit of detection (0.5 ng mL(-1) in urine), recovery (40%), and intra-/interday precision (2.3% to 11.7%) to assess its fitness for purpose. Prospectively, the assay can serve as detection method for LG121071 in drug testing and/or doping controls.

Application of mass spectrometry to hair analysis for forensic toxicological investigations.

The increasing role of hair analysis in forensic toxicological investigations principally owes to recent improvements of mass spectrometric instrumentation. Research achievements during the last 6 years in this distinctive application area of analytical toxicology are reviewed. The earlier state of the art of hair analysis was comprehensively covered by a dedicated book (Kintz, 2007a. Analytical and practical aspects of drug testing in hair. Boca Raton: CRC Press and Taylor & Francis, 382 p) that represents key reference of the present overview. Whereas the traditional organization of analytical methods in forensic toxicology divided target substances into quite homogeneous groups of drugs, with similar structures and chemical properties, the current approach often takes advantage of the rapid expansion of multiclass and multiresidue analytical procedures; the latter is made possible by the fast operation and extreme sensitivity of modern mass spectrometers. This change in the strategy of toxicological analysis is reflected in the presentation of the recent literature material, which is mostly based on a fit-for-purpose logic. Thus, general screening of unknown substances is applied in diverse forensic contexts than drugs of abuse testing, and different instrumentation (triple quadrupoles, time-of-flight analyzers, linear and orbital traps) is utilized to optimally cope with the scope. Other key issues of modern toxicology, such as cost reduction and high sample throughput, are discussed with reference to procedural and instrumental alternatives.

Determination by LC-MS/MS of colistins A and B in plasma and ultrafiltrate from critically ill patients undergoing continuous venovenous hemodiafiltration.

BACKGROUND: Colistin is a 50-year-old antibiotic, the use of which was ceased in the 70s and recently resumed as a "salvage therapy" against multidrug-resistant gram-negative bacteria, such as Pseudomonas aeruginosa and Acinetobacter baumannii. The narrow therapeutic range of colistin makes the choice of its correct dosage crucial, and monitoring of blood concentration is occasionally necessary for critically ill patients, including intensive care patients subjected to continuous renal replacement therapy. METHODS: Two LC-MS/MS methods were developed and fully validated for the quantitative determination of colistins A and B in plasma and dialysis ultrafiltrate (UF) samples, ultimately arising from 4 patients undergoing continuous venovenous hemodiafiltration (CVVHDF). RESULTS: The developed methods proved to be both specific and selective. They showed good fit and linearity over the entire range of interest. Trueness and accuracy proved satisfactory. Both methods have excellent intraassay precision (percent coefficient of variations were lower than 10%) and limit of detection values in the range 20-100 ng/mL, about 1-2 orders of magnitude below the concentrations commonly detected in real samples. The mean sieving coefficient (SC) values, measured after 10 minutes of CVVHDF, were 0.42 for colistin A and 0.48 for colistin B. SC values proved to be quite stable for 24 hours, but then declined to 0.24 for colistin A and 0.32 for colistin B, respectively, after 48 hours. At the median blood flow and effluent flow rate of 120 and 28 mL/min, clearance values for colistin B were higher than 15 mL/min. During the entire duration of CVVHDF sessions, the SC and clearance values for colistin A were significantly lower than colistin B. CONCLUSIONS: Two simple methods for the simultaneous determination of colistins A and B have been developed and validated. Their application in the clinical setting demonstrates that CVVHDF treatment lasting 48 hours produces a relatively constant and efficient removal of the drug.

Sensitivity and specificity of EtG in hair as a marker of chronic excessive drinking: pooled analysis of raw data and meta-analysis of diagnostic accuracy studies.

BACKGROUND: To assess the debated diagnostic performance of ethyl glucuronide in the 3-cm proximal scalp hair fraction (HEtG) as a marker of chronic excessive drinking. METHODS: In July 2012/May 2013, after a systematic search through the MEDLINE, OVID/EMBASE, WEB OF SCIENCE, and SCOPUS databases, 8 studies were included in the pooled analysis that report raw single data on HEtG concentration and self-reported daily alcohol intake (SDAI). A receiver operating characteristic curve analysis and a Spearman rank-order correlation test were used. A meta-analysis was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and Cochrane recommendations, comprising quality and bias assessments. RESULTS: The pooled analysis showed that 30 pg/mg could be a useful cutoff value for HEtG to detect an SDAI >60 g/d and demonstrated a parabolic direct correlation between HEtG and SDAI data [rho 0.79; 95% confidence interval (CI), 0.69-0.87; P < 0.001]. The meta-analysis found an overall HEtG sensitivity of 0.96 (95% CI, 0.72-1.00) and a specificity of 0.99 (95% CI, 0.92-1.00); a nomogram to predict the posttest probability of exhibiting the targeted condition in the general population was built. Significant variability among the included studies was detected, which was mainly explained by true heterogeneity in the presence of publication bias. CONCLUSIONS: With the available data, we conclude that HEtG is a promising marker for identifying chronic excessive drinking. Nonetheless, larger and well-designed population studies are required to draw any definitive conclusions on the significance and appropriateness of its application in the forensic setting.

Hair analysis for long-term monitoring of buprenorphine intake in opiate withdrawal.

BACKGROUND: Buprenorphine (BUP) is a psychoactive pharmaceutical drug largely used to treat opiate addiction. Short-term therapeutic monitoring is supported by toxicological analysis of blood and urine samples, whereas long-term monitoring by means of hair analysis is rarely used. Aim of this work was to develop and validate a highly sensitive ultrahigh-performance liquid chromatography tandem mass spectrometry method to detect BUP and norbuprenorphine (NBUP) in head hair. METHODS: Interindividual correlation between oral dosage of BUP and head hair concentration was investigated. Furthermore, an intra-individual study by means of segmental analysis was performed on subjects with variable maintenance dosage. Hair samples from a population of 79 patients in treatment for opiate addiction were analyzed. RESULTS: The validated ultrahigh-performance liquid chromatography tandem mass spectrometry protocol allowed to obtain limits of detection and quantification at 0.6 and 2.2 pg/mg for BUP and 5.0 and 17 pg/mg for NBUP, respectively. Validation criteria were satisfied, assuring selective analyte identification, high detection capability, and precise and accurate quantification. Significant positive correlation was found between constant oral BUP dosage (1-32 mg/d) and the summed up head hair concentrations of BUP and NBUP. Nevertheless, substantial interindividual variability limits the chance to predict the oral dosage taken by each subject from the measured concentrations in head hair. In contrast, strong correlation was observed in the results of intra-individual segmental analysis, which proved reliable to detect oral dosage variations during therapy. CONCLUSIONS: Remarkably, all hair samples yielded BUP concentrations higher than 10 pg/mg, even when the lowest dosage was administered. Thus, these results support the selection of 10 pg/mg as a cutoff value.

Profile of the urinary excretion of prednisolone and its metabolites in finishing bulls and cows treated with a therapeutic schedule.

BACKGROUND: Prednisolone was one of the first glucocorticoids to be synthesised, but it is still widely applied to cattle. Illegal uses of prednisolone include its uses for masking a number of diseases before animal sale and, at lower dosages for extended periods of time, for the improvement of feed efficiency and carcass characteristics. Since occasional presence of prednisolone has been detected at trace level in urine samples from untreated cattle, the Italian Ministry of Health introduced a provisional limit of 5 ng/mL to avoid false non-compliances. However, this limit proved ineffective in disclosing prednisolone misuse as a growth-promoter. In the present study, prednisolone acetate was administered to finishing bulls and cows according to a therapeutic protocol (2 × 0.4-0.5 mg/kg bw i.m. at 48 h interval) to further verify the practical impact of this cut-off limit and develop sound strategies to distinguish between exogenous administration and endogenous production. Urinary prednisolone, prednisone, 20ß-dihydroprednisolone, 20α-dihydroprednisolone, 20ß-dihydroprednisone, 6ß-hydroxyprednisolone, cortisol, and cortisone were determined using a validated LC/MS-MS method. RESULTS: The urinary excretion profile showed the simultaneous presence of prednisolone, 20ß-dihydroprednisolone, and prednisone, the latter at lower concentrations, up to 33 days after the first dosing. Higher analyte levels were detected in bulls even after correction for dilution in the urine. Prednisolone concentrations below 5 ng/ml were determined in half of the samples collected at 19 days, and in all the samples obtained 26 and 33 days after the first administration. No measurable concentrations of prednisolone or its metabolites were found in the samples collected before the treatment, while cortisol and cortisone levels lower than the respective LOQs were observed upon treatment. CONCLUSIONS: The present study confirms the criticism of the coarse quantitative approach currently adopted to ascertain illegal prednisolone administration in cattle. As previously shown for growth-promoting treatments of meat cattle, the simultaneous determination of urinary prednisolone, prednisone, 20ß-dihydroprednisolone, along with cortisol and cortisone, may represent a more reliable approach to confirm the exogenous origin of prednisolone. Such a strategy would facilitate unequivocal detection of animals treated with prednisolone acetate using a therapeutical protocol, even 3 to 4 weeks after the treatment.

The formation and self-assembly of long prebiotic oligomers produced by the condensation of unactivated amino acids on oxide surfaces.

In situ IR and mass spectrometry evidence for the catalytic formation on SiO2 and TiO2 surfaces of glycine oligomers (poly-Gly) up to 16 units long by successive feeding with monomers from the vapor phase is presented. Parallel experiments carried out on hydroxyapatite resulted in the unreactive adsorption of Gly, thus indicating that the oligomerization was specifically catalyzed by the surfaces of SiO2 and TiO2 . Furthermore, the poly-Gly moved on the surface when contacted with H2 O vapor and formed self-assembled aggregates containing both helical and ß-sheet-like structural motifs. These results indicate that polypeptides formed by the condensation of amino acids adsorbed on a mineral surface can evolve into structured supramolecular assemblies.

Detecting novel psychoactive substances around the world.

PURPOSE OF REVIEW: The worldwide spread of novel psychoactive substances (NPS) in the illicit drug market and their continuous increase in number and type, for the purpose of bypassing controlled substance legislation, represents a continuing challenge for forensic scientists, clinicians and enforcement authorities. We aim to provide information regarding the most urgent harms related to NPS consumption in different world regions and the current state of the art for NPS analysis. RECENT FINDINGS: Unfortunately, the identification of NPS in biological samples is controversial, especially when samples are limited, or the drug is promptly and extensively metabolized. This causes a lack of information on their real diffusion in different parts of the world and in different populations. New technologies and instrumental detection of NPS in alternative samples are offering comprehensive information about NPS use. SUMMARY: The lack of detection and underreporting of NPS in biological samples makes it difficult to obtain complete qualitative and quantitative information about NPS prevalence. The most innovative strategies that have been proposed in the last 2 years to assist NPS analysis and possibly facilitate the understanding of the NPS diffusion around the world are presented.

Method development for the quantification of nine nitazene analogs and brorphine in Dried Blood Spots utilizing liquid chromatography - tandem mass spectrometry.

The detection of nitazenes in biological fluids is increasingly needed as they are repeatedly reported in intoxication and overdose cases. A simple method for the quantification of low levels of nine nitazene analogs and brorphine in Dried Blood Spots (DBS) was developed and validated. 10 µL of spiked whole blood is deposited on a Capitainer®B card and allowed to dry. The spot is punched out, and extracted with 500 µL methanol:acetonitrile (3:1 v/v) added with 1.5 µL of fentanyl-D5 as the internal standard. After stirring, sonication, and centrifugation of the vial, the solvent is dried under nitrogen, the extract is reconstituted in 30 µL methanol, and 1 µL is injected into a UHPLC-MS/MS instrument. The method validation showed linear calibration in the 1-50 ng/mL range, LOD values ranging between 0.3 ng/mL (isotonitazene) and 0.5 ng/mL (brorphine), average CV% and bias% within 15 % and 10 % for all compounds, respectively. The matrix effect due to blood and filter paper components was within 85-115 % while recovery was between 15-20 %. Stability tests against time and temperature showed no significant variations for storage periods up to 28 days. Room temperature proved to represent the best samples storage conditions. UHPLC-MS/MS proved capable to reliably identify all target analytes at low concentration even in small specimen volumes, as those obtained from DBS cards, which in turn confirmed to be effective and sustainable micro-sampling devices. This procedure improves the efficiency of toxicological testing and provides an innovative approach for the identification of the nitazene class of illicit compounds.