Rapid screening of 2-benzylbenzimidazole nitazene analogs in suspect counterfeit tablets using Raman, SERS, DART-TD-MS, and FT-IR.

Developing methods to rapidly screen for novel synthetic 2-benzylbenzimidazole opioids, also known as nitazenes, has become increasingly important due to their high potency. These compounds have potency comparable or exceeding that of fentanyl by up to 10 times and have been implicated in approximately 5% of all drug overdose deaths in the United States in 2021. This paper details the authenticity determination of suspect tablets and the identification of three nitazene analogs (N-pyrrolidino etonitazene, isotonitazene, and etodesnitazene) in suspect tablets seized at a mail facility using Raman and surface-enhanced Raman scattering (SERS) with handheld devices, portable Fourier transform infrared spectrometer (FT-IR), and a direct analysis in real-time ambient ionization coupled to a thermal desorption unit and a mass spectrometer (DART-TD-MS). These methods are rapid and excellent for screening opioids in suspect tablets but could not fully determine the exact structure of some of the nitazene analogs present due to spectral similarities or similar fragmentation patterns. Liquid chromatography-mass spectrometry (LC-MS) confirmed the presence of these nitazene compounds in addition to other opioids/drugs that were in trace quantities. The quantitative high-performance liquid chromatography coupled with ultraviolet (HPLC-UV) detection experiments determined that the suspect tablets contained an average of 0.817 mg of N-pyrrolidino etonitazene per tablet. The results obtained reveal that the simultaneous deployment of these complementary and orthogonal portable analytical techniques as part of a workflow allows suspect tablets to be screened and nitazene-type drugs to be identified in suspect counterfeit tablets at remote sampling sites.

Screening suspect pharmaceuticals for illicit designer benzodiazepines using raman, SERS, and FT-IR prior to comprehensive analysis using LC-MS.

The emergence of illicit designer benzodiazepines with high dependency and no approved clinical use are of great US public health concern. Due to the increasing numbers of illicit designer benzodiazepines encountered in the US supply chain, there is a need to develop robust analytical methods that can rapidly detect these chemicals. Suspect counterfeit tablets, powders, or liquid formulations were first screened using Raman spectroscopy and surface-enhanced Raman scattering spectroscopy (SERS) for the presence of legal or illicit benzodiazepines, and then further analyzed using Fourier-transform infrared (FT-IR) spectroscopy and liquid chromatography with tandem mass spectrometric detection (LC-MS). Several microextraction procedures were developed and used to extract benzodiazepines from samples prior to SERS, FT-IR, and LC-MS analysis. Conventional Raman analyses using handheld Raman spectrometers afforded the ability to examine samples through enclosed plastic bags but were only able to detect high concentrations of various benzodiazepines in the suspect samples. The developed SERS methods were sufficient for detecting at least one benzodiazepine in the low-dose suspect samples, thereby allowing prioritization using other analytical tools that require more sample preparation and time-consuming analyses. The use of FT-IR spectroscopy coupled with extraction and spectral subtraction was found to be selective to multiple benzodiazepines and various excipients in the analyzed samples. This study demonstrated that the developed SERS and FT-IR procedures could be used in satellite laboratories to screen suspect packages at ports of entry and prioritize samples for additional laboratory-based analyses in an effort to prevent dangerous and illicit pharmaceutical products from reaching the US supply chain.

An evaluation of M2+ interference correction approaches associated with As and Se in ICP-MS using a multi-day dataset along with ICP-MS/MS/HR-ICP-MS based analysis and hierarchical modeling as a means of assessing bias in fortified drinking waters and single component matrices.

Three 1 2 mass oriented rare earth element (REE) M2+ correction approaches (fixed factor, a dual internal standard, and an in-sample) are evaluated for use in an ICP-MS environmental method update. The multi-variant-based evaluation includes analyzing the same 19 REE-fortified matrices on eight different days over a two-month period using two instrument tunes. These REE-fortified matrices were also analyzed using HR-ICP-MS and ICP-MS/MS to estimate the reference value for use in the principal component analysis (PCA) and hierarchical modeling evaluation. A fixed factor is unable to compensate for matrix and mass dependent drift and because of this it generates the largest across matrix, tune, and day 95th percent confidence bounds for the REE corrections on both As (1.1 ppb) and Se (23 ppb) using samples fortified with 100 ppb Nd, Sm & Gd. The PCA analysis indicated that M2+ ions cluster together across matrix, tune and day better than M1+ and these tighter correlations are reflected in reduced 95th percentile confidence bounds for dual M2+ internal standards (M2+; As = 0.3 ppb; Se = 5.4 ppb; n = 704) relative to M1+ internal standards (M1+; As = 0.6 ppb; Se = 12.0 ppb; n = 1056). The use of an in-sample M2+ correction produced comparable 95th percent confidence bounds (As = 0.2 ppb; Se = 3.4 ppb; n = 352) relative to the M2+ internal standard approaches. Finally, the hierarchical modeling indicated M2+ ions as internal standards tend to minimize the across day variability induced by cone changes and the daily reoccurring matrix shifts in the M2+/M1+ ratio associated with 250 ppm matrices of Na, Ca, and Mg. This internal standard driven reduction in variability can be beneficial in compliance monitoring methods.

Analytical considerations associated with implementing M2+ correction factors to address false positives on As and Se within U.S. EPA method 200.8.

Rare earth elements (REE) can produce M2+ ions in ICP-MS and 150Nd2+, 150Sm2+, and 156Gd2+ can produce false positives on 75As and 78Se. Alternative instrumental tuning conditions, that utilize lower He flows within the collision cell, reduce these false positives by a factor of 2 (to 0.8 ppb As and 19 ppb Se in solutions containing 50 ppb Nd and Gd) with comparable 16O35Cl+ reduction (<100 ppt false 51V in 0.4% HCl) and Se sensitivity (DL < 1 ppb). Further reduction of these false positives is achieved by estimating the M2+ correction factors and utilizing them in the interference-correction software. Approaches to estimating the M2+ correction factor were evaluated with an emphasis on techniques that tolerate daily variability in end-user backgrounds and their ability to reduce the initial and ongoing purity requirements associated with the rare earth standards used to estimate the M2+ correction factor. The direct elemental and polyatomic overlaps associated with unit-mass approaches tend to overcorrect as non-rare-earth signals as small as 30 cps at the unit mass can induce bias relative to the <300 cps signals associated with the M2+ from a 50 ppb REE standard solution. Alternatively, shifting the M2+ estimate to a half mass (i.e., m/z 71.5: 143Nd2+) eliminates the direct overlap source of bias and allows the unit mass signal to approach 150000 cps before it bleeds over on the 1/2 mass because of abundance sensitivity limitations. The performance of the half-mass approach was evaluated in reagent water and regional tap waters fortified with Nd, Sm, and Gd at 2 ppb and 50 ppb. In addition, a half-mass in-sample approach was also evaluated. This approach was found to be beneficial relative to the external or fixed-factor half-mass approach as it could compensate for instrument drift and matrix-induced shifts in the M2+ factors. Finally, all results were evaluated relative to the As and Se concentrations determined using an ICP-QQQ in mass shift mode and a high-resolution ICP-MS.

Base-modified thymidines capable of terminating DNA synthesis are novel bioactive compounds with activity in cancer cells.

Current FDA-approved chemotherapeutic antimetabolites elicit severe side effects that warrant their improvement; therefore, we designed compounds with mechanisms of action focusing on inhibiting DNA replication rather than targeting multiple pathways. We previously discovered that 5-(α-substituted-2-nitrobenzyloxy)methyluridine-5'-triphosphates were exquisite DNA synthesis terminators; therefore, we synthesized a library of 35 thymidine analogs and evaluated their activity using an MTT cell viability assay of MCF7 breast cancer cells chosen for their vulnerability to these nucleoside derivatives. Compound 3a, having an α-tert-butyl-2-nitro-4-(phenyl)alkynylbenzyloxy group, showed an IC50 of 9±1µM. The compound is more selective for cancer cells than for fibroblast cells compared with 5-fluorouracil. Treatment of MCF7 cells with 3a elicits the DNA damage response as indicated by phosphorylation of γ-H2A. A primer extension assay of the 5'-triphosphate of 3a revealed that 3aTP is more likely to inhibit DNA polymerase than to lead to termination events upon incorporation into the DNA replication fork.