Enhancing Quantitative Analysis of Xenobiotics in Blood Plasma through Cross-Matrix Calibration and Bayesian Hierarchical Modeling.

This study addresses the challenges of matrix effects and interspecies plasma protein binding (PPB) on measurement variability during method validation across diverse plasma types (human, rat, rabbit, and bovine). Accurate measurements of small molecules in plasma samples often require matrix-matched calibration approaches with the use of specific plasma types, which may have limited availability or affordability. To mitigate the costs associated with human plasma measurements, we explore in this work the potential of cross-matrix-matched calibration using Bayesian hierarchical modeling (BHM) to correct for matrix effects associated with PPB. We initially developed a targeted quantitative approach utilizing biocompatible solid-phase microextraction coupled with liquid chromatography-mass spectrometry for xenobiotic analysis in plasma. The method was evaluated for absolute matrix effects across human, bovine, rat, and rabbit plasma comparing pre- and postmatrix extraction standards. Absolute matrix effects from 96 to 108% for most analytes across plasma sources indicate that the biocompatibility of the extraction phase minimizes interference coextraction. However, the extent of PPB in different media can still affect the accuracy of the measurement when the extraction of small molecules is carried out via free concentration, as in the case of microextraction techniques. In fact, while matrix-matched calibration revealed high accuracy, cross-matrix calibration (e.g., using a calibration curve generated from bovine plasma) proved inadequate for precise measurements in human plasma. A BHM was used to calculate correction factors for each analyte within each plasma type, successfully mitigating the measurement bias resulting from diverse calibration curve types used to quantify human plasma samples. This work contributes to the development of cost-effective, efficient calibration strategies for biofluids. Leveraging easily accessible plasma sources, like bovine plasma, for method optimization and validation prior to analyzing costly plasma (e.g., human plasma) holds substantial advantages applicable to biomonitoring and pharmacokinetic studies.

Matrix effects demystified: Strategies for resolving challenges in analytical separations of complex samples.

Matrix effects can significantly impede the accuracy, sensitivity, and reliability of separation techniques presenting a formidable challenge to the analytical process. It is crucial to address matrix effects to achieve accurate and precise measurements in complex matrices. The multifaceted nature of matrix effects which can be influenced by factors such as target analyte, sample preparation protocol, composition, and choice of instrument necessitates a pragmatic approach when analyzing complex matrices. This review aims to highlight common challenges associated with matrix effects throughout the entire analytical process with emphasis on gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and sample preparation techniques. These techniques are susceptible to matrix effects that could lead to ion suppression/enhancement or impact the analyte signal at various stages of the analytical workflow. The assessment, quantification, and mitigation of matrix effects are necessary in developing any analytical method. Strategies can be implemented to reduce or eliminate the matrix effect by changing the type of ionization, improving extraction and clean-up methods, optimization of chromatography conditions, and corrective calibration methods. While development of an effective strategy to completely mitigate matrix effects remains elusive, an integrated approach that combines sample preparation, analytical extraction, and effective instrumental analysis remains the most promising avenue for identifying and resolving matrix effects.

Design and Preclinical Evaluation of Nicotine-Stearic Acid Conjugate-Loaded Solid Lipid Nanoparticles for Transdermal Delivery: A Technical Note.

This study aimed to develop and evaluate nicotine--stearic acid conjugate-loaded solid lipid nanoparticles (NSA-SLNs) for transdermal delivery in nicotine replacement therapy (NRT). Nicotine conjugation to stearic acid prior to SLN formulation greatly increased drug loading. SLNs loaded with a nicotine-stearic acid conjugate were characterized for size, polydispersity index (PDI), zeta potential (ZP), entrapment efficiency, and morphology. Pilot in vivo testing was carried out in New Zealand Albino rabbits. The size, PDI, and ZP of nicotine-stearic acid conjugate-loaded SLNs were 113.5 ± 0.91 nm, 0.211 ± 0.01, and -48.1 ± 5.75 mV, respectively. The entrapment efficiency of nicotine-stearic acid conjugate in SLNs was 46.45 ± 1.53%. TEM images revealed that optimized nicotine-stearic acid conjugate-loaded SLNs were uniform and roughly spherical in shape. Nicotine-stearic acid conjugate-loaded SLNs showed enhanced and sustained drug levels for up to 96 h in rabbits when compared with the control nicotine formulation in 2% HPMC gel. To conclude, the reported NSA-SLNs could be further explored as an alternative for treating smoking cessation.

Quantitative determination of pesticides in human plasma using bio-SPME-LC-MS/MS: a robust tool to assess occupational exposure to pesticides.

Analysis of biofluids, such as plasma, can be used to investigate occupational pesticide exposure in the agricultural industry. Considering the chemical complexity and variability of plasma samples, any protocol for pesticide analysis should achieve efficient sample cleanup to minimize matrix effects and enhance method sensitivity through analyte pre-concentration. In this work, a high-throughput method was developed for analysis of 79 pesticides, commonly used in agricultural practices, in human plasma, using biocompatible solid-phase microextraction (SPME) coupled to liquid chromatography-tandem mass spectrometry. An SPME method was developed using a biocompatible hydrophilic-lipophilic balance/polyacrylonitrile (HLB/PAN) extraction phase and demonstrated negligible matrix effects. The performance of the developed SPME method was compared to a QuEChERS -Quick, Easy, Cheap, Effective, Rugged, and Safe- method, the most common sample preparation and cleanup approach for pesticide analysis in complex matrices. Comparable accuracy and precision were achieved for both methods, with accuracy values within 70-120% and relative standard deviation < 15%. Overall, the developed SPME and QuEChERS methods extracted 79 out of 82 monitored pesticides in human plasma. The SPME protocol demonstrated higher sensitivity than the QuEChERS method and a drastic reduction of matrix effects.

Biocompatible SPME coupled to GC/MS for analysis of xenobiotics in blood plasma.

This work proposes a new method for biomonitoring studies focused on the screening and quantification of xenobiotics in blood-derived samples. The performance of a polydimethylsiloxane/divinylbenzene/polydimethylsiloxane (PDMS/DVB/PDMS) biocompatible extraction phase was investigated for extraction of pesticides and pharmaceuticals from plasma samples via direct immersion solid-phase microextraction (SPME) prior to gas chromatography-mass spectrometry. Under the optimum extraction settings, which included an attentive optimization of the fiber rinsing conditions, the microextraction device was able to endure 100 consecutive extractions from undiluted and diluted plasma with an overall reproducibility up to 28% for all the analytes tested, except chlorpyrifos-methyl. Optimized conditions were used to validate a quantitative method using matrix-matched calibration with isotopically labeled internal standard correction. Accuracy and precision values obtained for analysis of bovine plasma were within 96-132% and 0.05-5.82% respectively. LLOQs for all the analytes were at 1 µg L-1 and LDR ranged within 1-100 µg L-1. The applicability of this method to plasma from different species (human, rat, rabbit) was also investigated. This work represents the first step toward broader use of the biocompatible PDMS/DVB/PDMS extraction phases for analysis of multiclass xenobiotics in plasma and other complex biofluids.

Ion exchange solid phase microextraction coupled to liquid chromatography/laminar flow tandem mass spectrometry for the determination of perfluoroalkyl substances in water samples.

Per- and polyfluoroalkyl substances (PFAS) are toxic and bioaccumulative compounds that are persistent in the environment due to their water and heat resistant properties. These compounds have been demonstrated to be ubiquitous in the environment, being found in water, soil, air and various biological matrices. The determination of PFAS at ultra-trace levels is thus critical to assess the extent of contamination in a particular matrix. In this work, solid phase microextraction (SPME) was evaluated as a pre-concentration technique to aid the quantitation of this class of pollutants below the EPA established advisory limits in drinking water at parts-per-trillion levels. Four model PFAS with varying physicochemical properties, namely hexafluoropropylene oxide dimer acid (GenX), perfluoro-1- butanesulfonate (PFBS), perfluoro-n-octanoic acid (PFOA) and perfluoro-1-octanesulfonate (PFOS) were studied as a proof of concept. Analysis was performed with the use of ultra-high pressure liquid chromatography-laminar flow tandem mass spectrometry (UHPLC-MS/MS). This study proposes the use of hydrophilic-lipophilic balance-weak anion-exchange/polyacrylonitrile (HLB-WAX/PAN) as a SPME coating, ideal for all model analytes. A sample volume of 1.5 mL was used for analysis, the optimized protocol including 20 min extraction, 20 min desorption and 6 min LC/MS analysis. This method achieved LOQs of 2.5 ng L- 1 (PFOS) and 1 ng L - 1 (GenX, PFBS and PFOA) with satisfactory precision and accuracy values evaluated over a period of 5 days.

Biocompatible SPME fibers for direct monitoring of nicotine and its metabolites at ultra trace concentration in rabbit plasma following the application of smoking cessation formulations.

The ultra-trace determination of nicotine and its 4 major metabolites (cotinine, nornicotine, norcotinine and anabasine) from rabbit plasma was achieved by a newly developed solid phase microextraction-liquid chromatography-tandem mass spectrometry method. Extraction of the target analytes was performed with hydrophilic/lipophilic balance-polyacrylonitrile SPME fibers. Dual fiber extraction was necessary to guarantee improved recovery at parts-per-trillion levels. Liquid chromatographic analysis was achieved in a 6-min run using a C18 (1.9 µm C18, 50 mm x 2.1 mm) column with a mobile phase flow rate of 0.4 mL/min. Tandem mass spectrometry was used for detection and quantification in positive electrospray ionization (ESI+) mode for all the targeted analytes. Two stable isotope-labeled internal standards were used for signal correction and accurate quantification. The mass spectrometer with laminar flow ion flux transport, guaranteed improved signal stability, minimal contamination of the ion guide and reproducibility into the first quadrupole analyzer. The method was validated in line with the Food and Drug Administration (FDA) guidelines for bioanalytical method validation. The results met the acceptance criteria as proposed by the FDA: accuracy was tested at 0.35, 10 and 75 µg L - 1 and ranged between 98.3-112.2% for nicotine, 94.1-101.9% for cotinine, 94.7-107.0% for nornicotine, 81.1-107.2% for norcotinine and 94.3-115.2% for anabasine, with precision up to 14.2%. Stability tests indicated that all the targeted analytes were stable in the desorption solution for at least 1 week. LOQs ranged from 0.05 to 1 µg L-1. The method was successfully applied to analyze plasma samples obtained from rabbits following transdermal application of a smoking cessation formulation loaded with solid lipid nanoparticles containing a nicotine-stearic acid conjugate.

Use of natural sorbents as alternative and green extractive materials: A critical review.

The increasing concern about environmental degradation and resource depletion has inspired the analytical chemistry community to develop analytical methods that comply as much as possible with the principles of Green Analytical Chemistry. Significant progress has been made in greening sample preparation strategies by miniaturizing sampling devices and decreasing the amount of sorptive phase needed for efficient extraction of targeted molecules. In this context, the use of natural sorbents represents an additional and convenient option for green sample preparation. The advantages of using natural sorbents for extraction include their availability from renewable sources, low toxicity and biodegradability. In this review, we describe the use of various natural sorbents for metals and organic molecules extraction, focusing on the most innovative applications within the decade 2009-2019. Particular emphasis is given to the description of commonly used biopolymers - e.g. cellulose, chitin, and lignin - and their use in a variety of sample preparation strategies. We also refer to different functionalization approaches that enhance the extraction efficiency of natural sorbents.

Blue light-triggered photochemistry and cytotoxicity of retinal.

The chemical- and photo- toxicity of chromophore retinal on cells have long been debated. Although we recently showed that retinal and blue light exposure interrupt cellular signaling, a comprehensive study examining molecular underpinnings of this perturbation and its consequences to cellular fate is lacking. Here, we report molecular evidence for blue light excited-retinal induced oxidative damage of polyunsaturated lipid anchors in membrane-interacting signaling molecules and DNA damage in cells using live-cell imaging and in vitro experimentation. The incurred molecular damage irreversibly disrupted subcellular localization of these molecules, a crucial criterion for their signaling. We further show retinal accumulation in lipid-bilayers of cell membranes could enhance the lifetime of retinal in cells. Comparative response-signatures suggest that retinal triggers reactions upon photoexcitation similar to photodynamic therapy agents and generate reactive oxygen species in cells. Additionally, data also shows that exposing retinal-containing cells to sunlight induces substantial cytotoxicity. Collectively, our results explain a likely in vivo mechanism and reaction conditions under which bio-available retinal in physiological light conditions damages cells.