An update on the current status and prospects of nitrosation pathways and possible root causes of nitrosamine formation in various pharmaceuticals.

Over the last two years, global regulatory authorities have raised safety concerns on nitrosamine contamination in several drug classes, including angiotensin II receptor antagonists, histamine-2 receptor antagonists, antimicrobial agents, and antidiabetic drugs. To avoid carcinogenic and mutagenic effects in patients relying on these medications, authorities have established specific guidelines in risk assessment scenarios and proposed control limits for nitrosamine impurities in pharmaceuticals. In this review, nitrosation pathways and possible root causes of nitrosamine formation in pharmaceuticals are discussed. The control limits of nitrosamine impurities in pharmaceuticals proposed by national regulatory authorities are presented. Additionally, a practical and science-based strategy for implementing the well-established control limits is notably reviewed in terms of an alternative approach for drug product N-nitrosamines without published AI information from animal carcinogenicity testing. Finally, a novel risk evaluation strategy for predicting and investigating the possible nitrosation of amine precursors and amine pharmaceuticals as powerful prevention of nitrosamine contamination is addressed.

Current status and prospects of development of analytical methods for determining nitrosamine and N-nitroso impurities in pharmaceuticals.

Nitrosamine impurities in pharmaceuticals have recently been concerned for several national regulatory agencies to avoid carcinogenic and mutagenic effects in patients. The demand for highly sensitive and specific analytical methods with LOQs in the ppb and sub-ppb ranges is among the most significant challenges facing analytical scientists. In addition, artifactual nitrosamine formation during sample preparation and injection leading to overestimation of nitrosamines has received considerable attention. Numerous analytical methodologies have been reported for quantifying nitrosamine impurities in active pharmaceutical ingredients and medicinal products at the interim limit criteria as preventive measures. In this review, we meticulously discuss those reported gas and liquid chromatographic methods for nitrosamine determination in pharmaceuticals in aspects of chromatographic conditions and sensitivity of detection. We also introduce the potential of novel fluorescence-based methods recently developed to rapidly screen nitrosamine impurities. In addition, the review assesses the nitrosation assay procedure (NAP test), which is expected to be a future preventive measure for screening potential nitrosation and identifying suspected contamination with N-nitroso or other potential mutagenic impurities during the drug development process.

Development of a Sensitive Headspace Gas Chromatography-Mass Spectrometry Method for the Simultaneous Determination of Nitrosamines in Losartan Active Pharmaceutical Ingredients.

Nitrosamine impurities in angiotensin II receptor antagonists (sartans) containing a tetrazole group represent an urgent concern for active pharmaceutical ingredient (API) manufacturers and global regulators. Regarding safety, API manufacturers must develop methods to monitor the levels of each nitrosamine impurity before individual batch release. In this study, we developed and validated a sensitive, selective, and high-throughput method based on headspace gas chromatography-mass spectrometry (HS-GC-MS) for the simultaneous determination of four nitrosamines in losartan potassium API with simple sample preparation. N-Nitrosodimethylamine (NDMA, m/z 74), N-nitrosodiethylamine (NDEA, m/z 102), N-nitrosoethylisopropylamine (EIPNA, m/z 116), and N-nitrosodiisopropylamine (DIPNA, m/z 130) levels were quantified using an electron impact, single quadrupole mass spectrometer under a selected-ion-monitoring acquisition method. The method was validated according to the Q2(R1) ICH guidelines. The calibration curves of the assay ranged from 25 to 5000 ng/mL with limits of quantitation of 25 ppb for NDMA and NDEA and 50 ppb for DIPNA and EIPNA. The accuracy of the developed method ranged from -7.04% to 7.25%, and the precision %CV was ≤11.5. Other validation parameters, including specificity, stability, carryover, and robustness, met the validation criteria. In conclusion, the developed method was successfully applied for the determination of nitrosamines in losartan potassium APIs.

A new alternative assay for sensitive analysis of ethylenethiourea and propylenethiourea in fruit samples after their separation.

Ultra-high-performance liquid chromatography (UHPLC) coupled with a cobalt phthalocyanine screen-printed carbon electrode (CoPc-SPCE) was developed and validated for quantitative analysis of ethylenethiourea (ETU) and propylenethiourea (PTU). CoPC-SPCE provided high catalytic properties for ETU and PTU oxidation. This fabricated electrode is inexpensive, disposable, and easy to prepare by an in-house screen-printing technique. The chromatographic separation was performed in isocratic mode on a reversed phase C18 (100 mm × 4.6 mm, 3 µm) column, using a 90 : 10 (v/v) ratio of 0.05 M phosphate buffer solution (pH 4) and methanol as the mobile phase with a flow rate of 1.0 mL min-1 at an oxidation potential of +0.7 V vs. Ag/AgCl. The separation could be achieved within 3 min, and a wide linear range of 0.01-100 µg mL-1 (r2 > 0.99) was obtained for both analytes. The limits of detection (3 S/N) were found to be 0.006 and 0.009 µg mL-1 for ETU and PTU, respectively. Furthermore, this proposed method was utilized to determine ETU and PTU in fruit samples with satisfactory results, yielding excellent intra-day and inter-day relative standard deviations and recoveries. These results demonstrated that the proposed assay can be used as a new alternative way for inexpensive, rapid, selective and sensitive determination of ETU and PTU in fruit samples.

Development and Validation of Liquid Chromatography-Tandem Mass Spectrometry Method for Simple Analysis of Sumatriptan and its Application in Bioequivalence Study.

This work demonstrated a sensitive, selective, and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitation of sumatriptan in human plasma samples. Terazosin was used as an internal standard to minimize the variability during sample processing and detection. Sample cleanup prior to chromatographic analysis was accomplished by liquid-liquid extraction (LLE) with tert-butyl methyl ether (t-BME). The separation was performed on a reversed-phase Symmetry® C18 column (150 × 4.6 mm i.d., 5 µm) under a gradient mode, using a 0.2% formic acid aqueous solution and acetonitrile at a flow rate of 0.5 mL/min. Sumatriptan (m/z 296.26→251.05) and terazosin (m/z 388.10→290.25) were quantified using a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) under the positive ion mode. The method was fully validated following US-FDA and EMA guidelines. The LC-MS/MS assay had a calibration range of 0.5-50.0 ng/mL. The assay was precise and accurate with a between-run precision of <9.51%, and between-run accuracy between -7.27 to 8.30%. The developed method was subsequently applied in the determination of plasma concentration-time profile of a sumatriptan 50-mg tablet following oral administration in healthy volunteers.

Janus electrochemistry: Simultaneous electrochemical detection at multiple working conditions in a paper-based analytical device.

The simultaneous detection of multiple analytes from a single sample is a critical tool for the analysis of real world samples. However, this is challenging to accomplish in the field by current electroanalytical techniques, where tuning assay conditions towards a target analyte often results in poor selectivity and sensitivity for other species in the mixture. In this work, an electrochemical paper-based analytical device (ePAD) capable of performing simultaneous electrochemical experiments in different solution conditions on a single sample was developed for the first time. We refer to the system as a Janus-ePAD after the two-faced Greek god because of the ability of the device to perform electrochemistry on the same sample under differing solution conditions at the same time with a single potentiostat. In a Janus-ePAD, a sample wicks down two channels from a single inlet towards two discreet reagent zones that adjust solution conditions, such as pH, before flow termination in two electrochemical detection zones. These zones feature independent working electrodes and shared reference and counter electrodes, facilitating simultaneous detection of multiple species at each species' optimal solution condition. The device utility and applicability are demonstrated through the simultaneous detection of two biologically relevant species (norepinephrine and serotonin) and a common enzymatic assay product (p-aminophenol) at two different solution pH conditions. Janus-ePADs show great promise as an inexpensive and broadly applicable platform which can reduce the complexity and/or number of steps required in multiplexed analysis, while also operating under the optimized conditions of each species present in a mixture.

Anodic stripping voltammetric determination of total arsenic using a gold nanoparticle-modified boron-doped diamond electrode on a paper-based device.

A multistep paper-based analytical device (mPAD) was designed and applied to the voltammetric determination of total inorganic arsenic. The electrodeposition of gold nanoparticles on a boron-doped diamond (AuNP/BDD) electrode and the determination of total inorganic arsenic is accomplished with a single device. Total inorganic arsenic can be determined by first reducing As(V) to As(III) using thiosulfate in 1.0 mol L-1 HCl. As(III) is then deposited on the electrode surface, and total inorganic arsenic is quantified as As(III) by square-wave anodic stripping voltammetry the potential range between -0.25 V and 0.35 V (vs. Ag/AgCl), best at around 0.05 V. Under optimal conditions, the voltammetric response for As(III) detection is linear in the range from 0.1 to 1.5 µg mL-1 and the limit of detection (3SD/slope) is 20 ng mL-1. The relative standard deviation at 0.3, 0.7 and 1.0 µg mL-1 of As(III) are 3.6, 4.3 and 3.3, respectively (10 different electrodes). The results show that the assay has high precision, a rather low working potential, and excellent sensor-to-sensor reproducibility. The method was employed to the determination of total inorganic arsenic in rice samples. Results agreed well with those obtained by inductively coupled plasma-optical emission spectroscopy (ICP-OES). Graphical abstract A multistep paper-based analytical device (mPAD) is described that integrates a AuNP/BDD electrode preparation step and a detection step into a single device. The AuNPs are easily deposited on the BDD electrode by applying electrodeposition potential. The total inorganic arsenic concentration in rice samples was determined by using square-wave anodic stripping voltammetry.

Bimetallic Pt-Au nanocatalysts electrochemically deposited on boron-doped diamond electrodes for nonenzymatic glucose detection.

The enormous demand for medical diagnostics has encouraged the fabrication of high- performance sensing platforms for the detection of glucose. Nonenzymatic glucose sensors are coming ever closer to being used in practical applications. Bimetallic catalysts have been shown to be superior to single metal catalysts in that they have greater activity and selectivity. Here, we demonstrate the preparation, characterization, and electrocatalytic characteristics of a new bimetallic Pt/Au nanocatalyst. This nanocatalyst can easily be synthesized by electrodeposition by sequentially depositing Au and Pt on the surface of a boron-doped diamond (BDD) electrode. We characterized the nanocatalyst by scanning electron microscopy (SEM), X-ray diffraction (XRD), and voltammetry. The morphology and composition of the nanocatalyst can be easily controlled by adjusting the electrodeposition process and the molar ratio between the Pt and Au precursors. The electrocatalytic characteristics of a Pt/Au/BDD electrode for the nonenzymatic oxidation of glucose were systematically investigated by cyclic voltammetry. The electrode exhibits higher catalytic activity for glucose oxidation than Pt/BDD and Au/BDD electrodes. The best catalytic activity and stability was obtained with a Pt:Au molar ratio of 50:50. Moreover, the presence of Au can significantly enhance the long-term stability and poisoning tolerance during the electro-oxidation of glucose. Measurements of glucose using the Pt/Au/BDD electrode were linear in the range from 0.01 to 7.5mM, with a detection limit of 0.0077mM glucose. The proposed electrode performs selective electrochemical analysis of glucose in the presence of common interfering species (e.g., acetaminophen, uric and ascorbic acids), avoiding the generation of overlapping signals from such species.

Boron Doped Diamond Paste Electrodes for Microfluidic Paper-Based Analytical Devices.

Boron doped diamond (BDD) electrodes have exemplary electrochemical properties; however, widespread use of high-quality BDD has previously been limited by material cost and availability. In the present article, we report the use of a BDD paste electrode (BDDPE) coupled with microfluidic paper-based analytical devices (µPADs) to create a low-cost, high-performance electrochemical sensor. The BDDPEs are easy to prepare from a mixture of BDD powder and mineral oil and can be easily stencil-printed into a variety of electrode geometries. We demonstrate the utility and applicability of BDDPEs through measurements of biological species (norepinephrine and serotonin) and heavy metals (Pb and Cd) using µPADs. Compared to traditional carbon paste electrodes (CPE), BDDPEs exhibit a wider potential window, lower capacitive current, and are able to circumvent the fouling of serotonin. These results demonstrate the capability of BDDPEs as point-of-care sensors when coupled with µPADs.

A novel paper-based device coupled with a silver nanoparticle-modified boron-doped diamond electrode for cholesterol detection.

A novel paper-based analytical device (PAD) coupled with a silver nanoparticle-modified boron-doped diamond (AgNP/BDD) electrode was first developed as a cholesterol sensor. The AgNP/BDD electrode was used as working electrode after modification by AgNPs using an electrodeposition method. Wax printing was used to define the hydrophilic and hydrophobic areas on filter paper, and then counter and reference electrodes were fabricated on the hydrophilic area by screen-printing in house. For the amperometric detection, cholesterol and cholesterol oxidase (ChOx) were directly drop-cast onto the hydrophilic area, and H2O2 produced from the enzymatic reaction was monitored. The fabricated device demonstrated a good linearity (0.39 mg dL(-1) to 270.69 mg dL(-1)), low detection limit (0.25 mg dL(-1)), and high sensitivity (49.61 µA mM(-1) cm(-2)). The precision value for ten replicates was 3.76% RSD for 1 mM H2O2. In addition, this biosensor exhibited very high selectivity for cholesterol detection and excellent recoveries for bovine serum analysis (in the range of 99.6-100.8%). The results showed that this new sensing platform will be an alternative tool for cholesterol detection in routine diagnosis and offers the advantages of low sample/reagent consumption, low cost, portability, and short analysis time.