Enhancing breast cancer screening with urinary biomarkers and Random Forest supervised classification: A comprehensive investigation.

OBJECTIVES: Urinary sex hormones are investigated as potential biomarkers for the early detection of breast cancer, aiming to evaluate their relevance and applicability, in combination with supervised machine-learning data analysis, toward the ultimate goal of extensive screening. METHODS: Sex hormones were determined on urine samples collected from 250 post-menopausal women (65 healthy - 185 with breast cancer, recruited among the clinical patients of Candiolo Cancer Institute FPO-IRCCS (Torino, Italy). Two analytical procedures based on UHPLC-MS/HRMS were developed and comprehensively validated to quantify 20 free and conjugated sex hormones from urine samples. The quantitative data were processed by seven machine learning algorithms. The efficiency of the resulting models was compared. RESULTS: Among the tested models aimed to relate urinary estrogen and androgen levels and the occurrence of breast cancer, Random Forest (RF) proved to underscore all the other supervised classification approaches, including Partial Least Squares - Discriminant Analysis (PLS-DA), in terms of effectiveness and robustness. The final optimized model built on only five biomarkers (testosterone-sulphate, alpha-estradiol, 4-methoxyestradiol, DHEA-sulphate, and epitestosterone-sulphate) achieved an approximate 98% diagnostic accuracy on replicated validation sets. To balance the less-represented population of healthy women, a Synthetic Minority Oversampling TEchnique (SMOTE) data oversampling approach was applied. CONCLUSIONS: By means of tunable hyperparameters optimization, the RF algorithm showed great potential for early breast cancer detection, as it provides clear biomarkers ranking and their relative efficiency, allowing to ground the final diagnostic model on a restricted selection five steroid biomarkers only, as desirable for noninvasive tests with wide screening purposes.

Metabolic profile of N-ethylhexedrone, N-ethylpentedrone, and 4-chloromethcathinone in urine samples by UHPLC-QTOF-HRMS.

Forensic laboratories are constantly required to identify new drugs and their metabolites. N-ethylhexedrone (NEH, HEXEN), N-Ethylpentedrone (NEP), and 4-Chloromethcathinone (4-CMC, clephedrone) are synthetic substances structurally related to natural cathinone, alkaloid present in the leaves of the Catha edulis (Khat) plant. These synthetic cathinones (SC) are members of the heterogenous family of new psychoactive substances (NPS) that raised major concerns in scientific and forensic communities over the past years due to their widespread consumption. In this context, we investigated their metabolic profile using of UHPLC-QTOF-HRMS to elucidate the distribution of the parent drug and its metabolites in urine samples over time. Initially, both male and female volunteers were divided into three groups and eight subjects of each group were administered intranasally or orally with one SC (20-40 mg of NEH or NEP intranasal, 100-150 mg of 4-CMC oral). Urine samples were collected at 0-2 and 2-4 or 2-5 h. Urine (50 µL) was diluted 1:2 with acetonitrile/methanol (95:5) and injected into the UHPLC-QTOF-HRMS. Phase-I and phase-II metabolites were identified on the basis of fragmentation patterns and exact masses. Several phase-I and glucuronide-phase-II metabolites were identified in urine samples. Keto group reduction, hydroxylation and dealkylation were the common metabolic pathways identified for all cathinones and the presence of NEH-glucuronide, NEP-glucuronide and 4-CMC-glucuronide was also relevant. Significant is the slower metabolite formation for 4-CMC, which was detected at high concentrations in its original form even 5 h after administration, due to its long half-life and low intrinsic clearance compared to the other SCs. UHPLC-QTOF-HRMS demonstrated a considerable capability to semi-quantify the three synthetic cathinones and identify the target metabolites with high reliability. The introduction of new target compounds improves the efficiency of toxicological screening analysis on real samples and extends the window of detection of the SCs in biological matrices.

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.

Five cases of unintentional exposure to BZO-4en-POXIZID among nightclub attendees in New York City.

A new class of synthetic cannabinoids called OXIZIDs has emerged in recent years. This class consists of compounds with oxindole cores and hydrazide/hydrazone linker moieties and has often been described as being designed to circumvent a Chinese class-wide ban that was effective as of 1 July 2021. However, through hair testing of nightclub attendees in New York City-a high-risk population for recreational drug use-we have evidence suggesting exposures to an OXIZID called BZO-4en-POXIZID (4en-pentyl MDA-19) prior to the effective ban. Through analysis of 6 cm segmented hair samples from attendees collected in 2021, we detected five cases of exposure. Specifically, we detected a cluster of three cases based on hair samples collected on 20 June 2021, and then two additional cases from samples collected on 16 July 2021. Four of these hair samples were long enough to analyze two 6 cm hair segments (representing approximately two 6-month timeframes) and three of four of these cases tested positive for repeated exposure (for an estimated exposure over 6 months prior to hair collection). All cases included young adult females reporting past-year cannabis use but all tested negative for tetrahydrocannabinol exposure. Three cases also reported past-year use of cocaine, ecstasy, and/or ketamine, and four cases tested positive for exposure to cocaine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), methamphetamine and/or eutylone. These subjects were exposed to BZO-4en-POXIZID-likely as an adulterant in other drugs, and these cases are among the first documented cases which occurred approximately half a year before the Chinese legislative ban.

Trends in reported and biologically confirmed drug use among people who use ecstasy in the nightclub/festival-attending population, 2016-2022.

Background: Nightclub/festival attendees are a population with high rates of party drug use, but research is needed to determine whether there have been shifts in unintended drug exposure in this population (e.g., via adulterants) to inform prevention and harm reduction efforts. Methods: Adults entering nightclubs and festivals in New York City were asked about past-year drug use in 2016 through 2022, with a subset providing a hair sample for testing. We focused on the 1943 who reported ecstasy use (of which 247 had a hair sample analyzed) and compared trends in self-reported drug use, drug positivity, and adjusted prevalence (adjusting for unreported use). Results: MDMA positivity decreased from 74.4 % to 42.3 %, and decreases occurred regarding detection of synthetic cathinones ("bath salts"; a 100.0 % decrease), MDA (a 76.9 % decrease), amphetamine (an 81.3 % decrease), methamphetamine (a 64.2 % decrease), and ketamine (a 33.4 % decrease) (ps < .05). Although prevalence of MDA and synthetic cathinone use was comparable between self-report and adjusted report in 2022, gaps in prevalence were wider in 2016 (ps < .01). Adjusted prevalence of synthetic cathinone use decreased more across time than prevalence based on self-report (a 79.4 % vs. 69.1 % decrease) and adjusted report for MDA use decreased more than prevalence based on self-report (a 50.6 % vs. 38.9 % decrease). Conclusions: Combining self-report and toxicology tests helped us determine that decreases in drug use/exposure were steeper regarding adjusted prevalence. Underreported drug exposure-possibly due to exposure to adulterants-appears to have had less of an effect on prevalence in 2022 than it did in 2016.

Detection of fentanyl, synthetic opioids, and ketamine in hair specimens from purposive samples of American and Italian populations.

With the current crisis related to the diffusion of fentanyl and other novel opioids in several countries and populations, new and effective approaches are needed to better elucidate the phenomenon. In this context, hair testing offers a unique perspective in the investigation of drug consumption, producing useful information in terms of exposure to psychoactive substances. In this research, we applied targeted ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) analytical methods to detect novel synthetic and prescription opioids and other common controlled psychoactive drugs in the keratin matrix. A total of 120 hair samples were analyzed from the United States (US) and Italy, segmented when longer than 6 cm, and then analyzed. In the 60 samples (83 segments in total) analyzed from a purposive sample of data collected in the US, fentanyl was detected in 14 cases (16.9%), with no detection of nitazens or brorphine. We also detected fentanyl metabolites, despropionyl-p-fluorofentanyl, and prescription opioids. In the 60 samples collected in Italy (91 segments in total), ketamine was the most prevalent compound detected (in 41 cases; 45.1%), with ketamine demonstrating a strong correlation with detection of amphetamines and MDMA, likely due to co-use of these substances in recreational contexts. Several common drugs were also detected but no exposure to fentanyl or its analogs were detected. Results of this retrospective exploration of drug use add to increasing evidence that hair testing can serve as a useful adjunct to epidemiology studies that seek to determine biologically confirmed use and exposure in high-risk populations.

Wastewater surveillance of 105 pharmaceutical drugs and metabolites by means of ultra-high-performance liquid-chromatography-tandem high resolution mass spectrometry.

Water pollution from pharmaceutical drugs is becoming an environmental issue of increasing concern, making water quality monitoring a crucial priority to safeguard public health. In particular, the presence of antidepressants, benzodiazepines, antiepileptics, and antipsychotics require specific attention as they are known to be harmful to aquatic biota. In this study, a multi-class comprehensive method for the detection of 105 pharmaceutical residues in small (30 mL) water samples was developed according to fit-for-purpose criteria and then applied to provide wide screening of samples obtained from four Wastewater Treatment Plants (WWTPs) in northern Italy. The filtered samples (0.22 µm filters) were extracted by SPE, and then eluted. 5 µL of the concentrated samples were analyzed by a UHPLC-QTOF-HRMS method validated for screening purposes. Adequate sensitivity was recorded for all target analytes, with limits of detection below 5 ng/L for 76 out of 105 analytes. A total of 23 out of the 105 targeted pharmaceutical drugs was detected in all samples. Several further compounds were detected over wide concentration intervals, ranging from ng/L to µg/L. In addition, the retrospective analysis of full-scan QTOF-HRMS data was exploited to carry out an untargeted screening of some drugs' metabolites. As a proof of concept, it was investigated the presence of the carbamazepine metabolites, which is among the most frequently detected contaminants of emerging concern in wastewater. Thanks to this approach, 10,11-dihydro-10-hydroxycarbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine and carbamazepine-10,11-epoxide were identified, the latter requiring particular attention, since it exhibits antiepileptic properties similar to carbamazepine and potential neurotoxic effects in living organism.

Metabolic study of new psychoactive substance methoxpropamine in mice by UHPLC-QTOF-HRMS.

Methoxpropamine (MXPr) is an arylcyclohexylamine dissociative drug structurally similar to 3-methoxyeticyclidine, ketamine, and deschloroketamine, recently appeared in the European illegal market, and was classified within the new psychoactive substances (NPS). Our study investigated the metabolism of MXPr to elucidate the distribution of the parent drug and its metabolites in body fluids and fur of 16 mice. After the intraperitoneal administration of MXPr (1, 3, and 10 mg/kg), urine samples from eight male and eight female mice were collected every hour for six consecutive hours and then at 12- to 24-h intervals. Additionally, plasma samples were collected 24 h after MXPr (1 and 3 mg/kg) administration. Urine and plasma were diluted 1:3 with acetonitrile/methanol (95:5) and directly injected into the UHPLC-QTOF-HRMS system. The phase-I and phase-II metabolites were preliminarily identified by means of the fragmentation patterns and the exact masses of both their precursor and fragment ions. Lastly, the mice fur was analyzed following an extraction procedure specific for the keratin matrix. Desmethyl-MXPr-glucoronide was identified in urine as the main metabolite, detected up to 24 h after administration. The presence of norMXPr in urine, plasma, and fur was also relevant, following a N-dealkylation process of the parent drug. Other metabolites that were identified in fur and plasma included desmethyl-MXPr and dihydro-MXPr. Knowledge of the MXPr metabolites evolution is likely to support their introduction as target compounds in NPS toxicological screening analysis on real samples, both to confirm intake and extend the detection window of the dissociative drug MXPr in the biological matrices.

Development and validation of a UHPLC-HRMS-QTOF method for the detection of 132 New Psychoactive Substances and synthetic opioids, including fentanyl, in Dried Blood Spots.

Dried Blood Spots (DBS) represents a promising micro-sampling technique in the field of forensic toxicology to carry out minimally invasive blood sample collection. In DBS, cheap, fast and easy sampling is combined with effortless store and transport. These properties aimed us to develop and validate a quick and easy procedure for the detection of a large and diverse range of emerging and alarming New Psychoactive Substances (NPS). A drop of whole blood sample was collected on a DBS card and dried for 3 h, from which a total of 132 analytes (including NPS, synthetic opioids NSO and metabolites) plus 13 deuterated internal standards could be extracted using 500 µL of a methanol/acetonitrile mixture (3:1, v/v) and subsequently separated and identified by means of ultra-high-performance liquid-chromatography (UHPLC) coupled to high resolution mass spectrometry (HRMS). The extraction efficiency proved to be reproducible with yields ranging from 30% to 100% depending on the different classes of drugs. Trueness, repeatability, and intermediate precision fulfilled acceptance criteria for almost all synthetic opioids, cathinones and hallucinogens (bias and CV% below ±20%); in particular, the aggregate inter-day trueness data showed extremely limited deviation from the expected concentrations (-10% < bias% < +10%) for 114 target analytes out of 132. The calculated limits of detection ranged from 1.3 to 6.3 ng/mL, consistently exceeding the values experimentally tested. Moderate ion suppression was observed for most analytes, partly caused by blood fortification itself. Good stability of the target analytes at -20 °C, 4 °C, and 35 °C on DBS cards after drying was observed, even for long periods of time. Optimal storage condition appeared to be at 4 °C resulting in virtually no drugs degradation for up to 40 days. The novel analytical method based on DBS sampling, verified on venous whole blood real samples previously tested positive with our routine procedure, conveys remarkable potential in analytical toxicology, clinical analysis, and doping control.

Simultaneous determination of 137 drugs of abuse, new psychoactive substances, and novel synthetic opioids in meconium by UHPLC-QTOF.

New psychoactive substances (NPS) have been introduced into the market in recent years, with new analytes reported every year. The use of these substances in women can occur at any stage of life, even in the childbearing age. Drug use during pregnancy presents significant risks for the mother and the fetus, so it is important to have tools that allow to detect prenatal exposure to these substances of abuse. Therefore, an analytical method for the determination of 137 NPS and other drugs of abuse in meconium by UHPLC-QTOF was developed and validated for semi-quantitative purpose. Linearity range, limit of detection (LOD), precision, matrix effect, selectivity, and specificity were evaluated. For all analytes, the calibration curves were studied in the ranges between 2, 10, or 50 ng/g and 750 or 1000 ng/g, (depending on the analyte) and the LOD ranged between 0.04 and 2.4 ng/g. The method was applied to 30 meconium specimens from cases in which fentanyl had been administered as epidural anesthesia at the time of delivery or cases in which the maternal hair was positive to other drug of abuse. Four meconium samples tested positive for fentanyl (range concentration = 440-750 ng/g) and two samples tested positive to acetylfentanyl (range concentration = 190-1400 ng/g).