Pentafluorophenyl Research Articles

Dual-reactive single-chain polymer nanoparticles for orthogonal functionalization through active ester and click chemistry

Glucose has been extensively studied as a targeting ligand on nanoparticles for biomedical nanoparticles. A promising nanocarrier platform are single-chain polymer nanoparticles (SCNPs). SCNPs are well-defined 5–20 nm semi-flexible nano-objects, formed by intramolecularly crosslinked linear polymers. Functionality can be incorporated by introducing labile pentafluorophenyl (PFP) esters in the polymer backbone, which can be readily substituted by functional amine-ligands. However, not all ligands are compatible with PFP-chemistry, requiring different ligation strategies for increasing versatility of surface functionalization. Here, we combine active PFP-ester chemistry with copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) click chemistry to yield dual-reactive SCNPs. First, the SCNPs are functionalized with increasing amounts of 1-amino-3-butyne groups through PFP-chemistry, leading to a range of butyne-SCNPs with increasing terminal alkyne-density. Subsequently, 3-azido-propylglucose is conjugated through the glucose C1- or C6-position by CuAAC click chemistry, yielding two sets of glyco-SCNPs. Cellular uptake is evaluated in HeLa cancer cells, revealing increased uptake upon higher glucose-surface density, with no apparent positional dependance. The general conjugation strategy proposed here can be readily extended to incorporate a wide variety of functional molecules to create vast libraries of multifunctional SCNPs.

Development of a rapid HPLC-fluorescence method for monitoring warfarin metabolites formation: In vitro studies for evaluating the effect of piperine on warfarin metabolism and plasma coagulation

Warfarin, a widely prescribed anticoagulant, is highly effective for various coagulation disorders. However, its efficacy is limited by a narrow therapeutic index and frequent drug interactions, especially those involving metabolism by Cytochrome P450 (CYP450) enzymes. Piperine, found in black and long pepper, possesses blood-thinning properties and has been observed to inhibit CYP3A and CYP2C enzymes linked to warfarin metabolism. This study investigated the effect of piperine on warfarin metabolism in liver microsomes using a rapid and sensitive HPLC-Fluorescence method. The use of PFP (pentafluorophenyl) column with core shell particles provided the selectivity and resolution to resolve warfarin and its 4-, 6-, 7-, and 10-hydroxy metabolites in addition to the internal standard naproxen in less than 3 min. This is the fastest analytical assay for warfarin and its major metabolites reported to date, making it ideal for metabolic studies. The applicability of the method was demonstrated by monitoring the metabolism of S-warfarin in human and rat liver microsomes, and evaluating the inhibitory effect of piperine on metabolite formation. The results showed that piperine inhibited the formation of the major metabolite, 7-hydroxywarfarin, with half-maximal inhibitory concentration (IC50) 14.2 μM and 3.2 μM in human and rat liver microsomes, respectively. Furthermore, coagulation studies in vitro using rat plasma showed that piperine does not affect prothrombin time (PT) and activated partial thromboplastin time (aPTT). This study suggested that piperine may present a potential drug interaction with warfarin at the metabolism level, but has no direct effect on the activation of the extrinsic or intrinsic coagulation cascades. Further clinical investigation is therefore required, as piperine may increase the bioavailability of warfarin, thus increasing risk of serious adverse events in patients.

DEVELOPMENT AND VALIDATION OF AN LC-MS/MS METHOD FOR THE DETERMINATION OF TENOFOVIR AND EMTRICITABINE

Objective: Therapeutic drug monitoring of tenofovir and emtricitabine, two commonly used antiretroviral drugs, is important to maximize effectiveness while minimizing side effects. Materials: A Liquid Chromatography-Mass spectrometry (LC-MS/MS) method was developed to quantify tenofovir and emtricitabine in human plasma samples. The method involves a simple solid phase extraction procedure followed by liquid chromatography separation using a Penta Fluoro Phenyl (PFP) column with a Phenomenex C18 column and a mobile phase of ammonium formate, acetonitrile, and methanol, achieving separation in under 4 min. Results: The method showed good accuracy, low limits of quantification, adequate recovery, minimal matrix effects, and specificity. Analyte stability under multiple storage conditions was demonstrated. Conclusion: The validated LC-MS/MS method provides a reliable tool for therapeutic drug monitoring and pharmacokinetic studies of anti-Human Immuno-deficiency Virus (HIV) regimens. The assay can be applied to large populations, especially in resource-poor settings, to help individualize dosing and improve clinical outcomes while reducing toxicity.

Strategy for Comprehensive Detection and Annotation of Gut Microbiota-Related Metabolites Based on Liquid Chromatography-High-Resolution Mass Spectrometry.

Gut microbiota, widely populating the mammalian gastrointestinal tract, plays an important role in regulating diverse pathophysiological processes by producing bioactive molecules. Extensive detection of these molecules contributes to probing microbiota function but is limited by insufficient identification of existing analytical methods. In this study, a systematic strategy was proposed to detect and annotate gut microbiota-related metabolites on a large scale. A pentafluorophenyl (PFP) column-based liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method was first developed for high-coverage analysis of gut microbiota-related metabolites, which was verified to be stable and robust with a wide linearity range, high sensitivity, satisfactory recovery, and repeatability. Then, an informative database integrating 968 knowledge-based microbiota-related metabolites and 282 sample-sourced ones defined by germ-free (GF)/antibiotic-treated (ABX) models was constructed and subsequently used for targeted extraction and annotation in biological samples. Using pooled feces, plasma, and urine of mice for demonstration application, 672 microbiota-related metabolites were annotated, including 21% neglected by routine nontargeted peak detection. This strategy serves as a useful tool for the comprehensive capture of the intestinal flora metabolome, contributing to our deeper understanding of microbe-host interactions.

Multifunctional Poly(2-ethyl-2-oxazoline) Copolymers Containing Dithiolane and Pentafluorophenyl Esters as Effective Reactive Linkers for Gold Surface Coatings.

Surface functionalization with biological macromolecules is an important task for the development of sensor materials, whereby the interaction with other biological materials should be suppressed. In this work, we developed a novel multifunctional poly(2-ethyl-2-oxazoline)-dithiolane conjugate as a versatile linker for gold surface immobilization of amine-containing biomolecules, containing poly(2-ethyl-2-oxazoline) as antifouling polymer, dithiolane for surface immobilization, and activated esters for protein conjugation. First, a well-defined carboxylic acid containing copoly(2-ethyl-2-oxazoline) was synthesized by cationic ring-opening copolymerization of 2-ethyl-2-oxazoline with a methyl ester-containing 2-oxazoline monomer, followed by postpolymerization modifications. The side-chain carboxylic groups were then converted to amine-reactive pentafluorophenyl (PFP) ester groups. Part of the PFP groups was used for the attachment of the dithiolane moiety, which can efficiently bind to gold surfaces. The final copolymer contained 1.4 mol% of dithiolane groups and 4.5 mol% of PFP groups. The copolymer structure was confirmed by several analytical techniques, including NMR spectroscopy and size-exclusion chromatography. The kinetics of the PFP ester aminolysis and hydrolysis demonstrated significantly faster amidation compared to hydrolysis, which is essential for subsequent protein conjugation. Successful coating of gold surfaces with the polymer was confirmed by spectroscopic ellipsometry, showing a polymer brush thickness of 4.77 nm. Subsequent modification of the coated surfaces was achieved using bovine serum albumin as a model protein. This study introduces a novel reactive polymer linker for gold surface functionalization and offers a versatile polymer platform for various applications including biosensing and surface functionalization.

A column screening study: Validation of a RP-LC method for determination of teriflunomide with pentafluorophenyl stationary phase

The research focuses on column screening in order to develop a novel validation method to separate Teriflunomide significantly using comparative and equivalent column selection studies. This gives stationary phase efficiency relative to height equivalent to a theoretical plate (HETP) (>16000) and symmetry factor (<2.0%). After extensive column chemistry and technique development, the advanced Pentafluorophenyl (PFP) column separates halogenated drugs better. Teriflunomide is fluorinated drug, hence PFP is optimal for analysis. For statistical data comparison, the same chromatographic method was developed and transferred to another YMC Pack Pro C18 column that gives almost the same results as PFP column in terms of carbon load, pore size, and surface area. The refined and validated method yielded equivalent results with two stationary phases using a single analysis procedure. Column screening, technique creation, transfer, and validation with comparison studies with correlation coefficients over 0.999 are unique. The statistical data from both columns meets ICH requirements. The sensitive, reliable, robust, and reproducible approach is easy to apply in regular laboratory analysis. Data analysis showed that the validation study with PFP is better (LoD - 0.0015 ppm) for column ageing due to smaller injection volume and amount loaded in the column, shorter run time, and cheaper analysis cost. This research helps with column selection and method transfer with diverse column specifications. With less than 2.0% of RSD, the column screening analytical method validation is linear, precise, accurate, rugged, and efficient.

Highly oxidized flavones in Artemisia species – structure revisions and improved UHPLC-MSn analysis

In course of our studies of the aerial parts of Artemisia abrotanum the major methoxyflavonol could be isolated. However, by NMR structural analysis it became obvious that the substitution pattern in ring B differs from reports for casticin (2). The position of methoxyl and hydroxyl groups are interchanged, i.e., the major flavone is actually chrysosplenetin (1). Three structures in A. abrotanum and A. frigida had to be revised. Use of pyridine-d5 instead of DMSO‑d6 made the resolution of the B-ring 1H and 13C NMR signals possible and enabled correct structural assignment by 2D NMR experiments.Results from NMR structure elucidation for A. abrotanum were confirmed by LC-PDA-ESI-MSn analysis when a PFP (pentafluorophenyl) stationary phase with an optimized gradient elution was applied for separation of 1 and 2 instead of a corresponding C-18 phase. Electrospray mass spectrometry (positive and negative mode) with subsequent fragmentation (ESI-MSn) revealed distinctive mass spectral features of both compounds, especially at MS4 level. Several Artemisia extracts including A. annua were analysed on the PFP phase for the presence of 1 and 2.

B-183 The Impact of 3-EPI-25-Hydroxyvitamin D on 25-Hydroxyvitamin D Measurement by Liquid Chromatography/Mass Spectrometry

Abstract Background 25-hydroxyvitamin D is the recommended target to evaluate vitamin D status. The accurate measurement of 25-hydroxyvitamin D by liquid chromatography/mass spectrometry (LC/MS/MS) is impacted by the presence of the biologically inactive epimer of 25-hydroxyvitamin D2 and D3, 3-EPI-25-hydroxyvitamin D2 and D3. The epimers are isomers and share the same mass and fragment transitions as 25-hydroxyvitamin D2 and D3. For an accurate measurement the epimer should be chromatographically separated from 25-hydroxyvitamin D. Herein we assess the significance of 3-EPI-25-hydroxyvitamin D in our patient population. Methods A LC/MS/MS method was developed to chromatographically separate 25-hydroxyvitamin D2 and D3 from 3-EPI-25-hydroxyvitamin D2 and D3 on a Waters Xevo TQ-S Micro with an Acquity UPLC System using a 2.6 μm Pentafluorophenyl (PFP) column (Phenomenex). There were 231 residual serum samples analyzed. Results The cohort tested consisted of 16 infant samples (&amp;lt;1 years), 62 pediatric samples (1–17 years), adult samples (≥18 years), and 20 pregnant samples. 3-EPI-25-hydroxyvitamin D2 was not detected in any sample analyzed (&amp;lt;1 ng/mL). The prevalence and concentration of 3-EPI-25-hydroxyvitamin D3 was variable (Fig. 1). Conclusion The specificity of 25-hydroxyvitamin D measurement by LC/MS/MS is not affected by 3-EPI-25-hydroxyvitamin D2 as it is not present in our patient population. However, 3-EPI-25-hydroxyvitamin D3 is a significant interference. The presence of 3-EPI-25-hydroxyvitamin D3 is variable with the highest prevalence and concentration in infants. The prevalence and concentration decrease with age and is minimal in pregnancy. For infant and pediatric samples, an assay that chromatographically separates 3-EPI-25-hydroxyvitamin D3 from 25-hydroxyvitamin D3 is needed. For adult samples, it may be appropriate not to separate 25-hydroxyvitamin D3 from the epimer. For this patient population a balance between accuracy and throughput must be established.

Phytochemical Analysis of Pinus cembra Heartwood-UHPLC-DAD-ESI-MSn with Focus on Flavonoids, Stilbenes, Bibenzyls and Improved HPLC Separation.

The heartwood of the Swiss Stone Pine, Pinus cembra L., has been scarcely investigated for secondary metabolites for a long period of time. Considering age and relative simplicity of heartwood investigations dating back to the 1940s to 1960s, we conducted the first investigation of P. cembra heartwood by HPLC, using UHPLC-DAD-ESI-MSn and HPLC-DAD techniques in combination with isolation and NMR spectroscopy, with focus on stilbenes, bibenzyls and flavonoids. Analytical problems in the HPLC analysis of Pinus stilbenes and flavonoids on reversed stationary phases were also challenged, by comparing HPLC on pentafluorophenyl (PFP) and C18 stationary phases. Seven flavonoids (1, 2, 3, 7, 8, 11, 12), four stilbenes (4, 6, 10, 13), two bibenzyls (5, 9), three fatty acids (14, 16, 17) and one diterpenic acid (15) were detected in an ethanolic extract of Pinus cembra heartwood. HPLC comparison of reversed stationary phases in HPLC showed that the antifungal, antibacterial and chemosensitizing dihydropinosylvin monomethyl ether (9) and pinosylvin monomethyl ether (10) can be separated on PFP, but not on C18 material, when eluting with a screening gradient of 20-100% acetonitrile. Flavonoid separation showed additional benefits of combining analyses on different stationary phases, as flavonoids 7 and 8 could only be separated on one of two C18 stationary phases. Earlier phytochemical results for heartwood investigations were shown to be mostly correct, yet expandable. Substances 5 to 12 were found in alignment with these references, proving remarkable phytochemical analyses at the time. Evidence for the described presence of pinobanksin could not be found. Substances 1 to 4 and 13 have to our knowledge not yet been described for P. cembra.

Perfluorocarbons: A perspective of theranostic applications and challenges.

Perfluorocarbon (PFC) are biocompatible compounds, chemically and biologically inert, and lacks toxicity as oxygen carriers. PFCs nanoemulsions and nanoparticles (NPs) are highly used in diagnostic imaging and enable novel imaging technology in clinical imaging modalities to notice and image pathological and physiological alterations. Therapeutics with PFCs such as the innovative approach to preventing thrombus formation, PFC nanodroplets utilized in ultrasonic medication delivery in arthritis, or PFC-based NPs such as Perfluortributylamine (PFTBA), Pentafluorophenyl (PFP), Perfluorohexan (PFH), Perfluorooctyl bromide (PFOB), and others, recently become renowned for oxygenating tumors and enhancing the effects of anticancer treatments as oxygen carriers for tumor hypoxia. In this review, we will discuss the recent advancements that have been made in PFC's applications in theranostic (therapeutics and diagnostics) as well as assess the benefits and drawbacks of these applications.

Thiol-triggered deconstruction of bifunctional silyl ether terpolymers via an SNAr-triggered cascade.

While Si-containing polymers can often be deconstructed using chemical triggers such as fluoride, acids, and bases, they are resistant to cleavage by mild reagents such as biological nucleophiles, thus limiting their end-of-life options and potential environmental degradability. Here, using ring-opening metathesis polymerization, we synthesize terpolymers of (1) a "functional" monomer (e.g., a polyethylene glycol macromonomer or dicyclopentadiene); (2) a monomer containing an electrophilic pentafluorophenyl (PFP) substituent; and (3) a cleavable monomer based on a bifunctional silyl ether . Exposing these polymers to thiols under basic conditions triggers a cascade of nucleophilic aromatic substitution (SNAr) at the PFP groups, which liberates fluoride ions, followed by cleavage of the backbone Si-O bonds, inducing polymer backbone deconstruction. This method is shown to be effective for deconstruction of polyethylene glycol (PEG) based graft terpolymers in organic or aqueous conditions as well as polydicyclopentadiene (pDCPD) thermosets, significantly expanding upon the versatility of bifunctional silyl ether based functional polymers.

Fluoro-Functionalized Spherical Covalent Organic Frameworks as a Liquid Chromatographic Stationary Phase for the High-Resolution Separation of Organic Halides.

The development of novel stationary phases with specific functionality is of great importance in chromatographic separation. Herein, we fabricated fluoro-functionalized spherical covalent organic frameworks (SF-COFs) via a bottom-up strategy as stationary phases for high-performance liquid chromatography (HPLC). Benefiting from the significant monodispersity, narrow size distribution, and high fluorine content, the SF-COFs packed column showed high column efficiency and excellent resolution for the separation of the organic fluorides involving polyfluorobenzenes, polychlorobenzenes, polybromobenzenes, perfluoroalkyl methacrylates, and halogenated trifluorotoluenes, which cannot be separated on the fluorine-free spherical covalent organic frameworks packed column. Especially, the column efficiency of 20 100-38 500 plates/m was obtained for polyfluorobenzenes, and the relative standard deviations of the retention time for continuous 10 separations of polychlorobenzenes and polybromobenzenes were less than 0.98%. Furthermore, the prepared SF-COFs packed column showed overwhelming superiority in the separation of organic halides compared with commercial C18 and pentafluorophenyl (PFP) packed columns. In addition, the compounds with different hydrophobicity or aromatic ring structure were also successfully separated on the SF-COFs packed column. This work extended the application of spherical COFs and provided a new way to introduce specific functional groups into the COF-based stationary phase for HPLC.

Differentiation of industrial hemp strains by their cannabinoid and phenolic compounds using LC × LC-HRMS

Cannabis is an ancient plant that has been used for therapeutic and recreational purposes. Nowadays, industrial hemp, a variety with low concentration of the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) and high concentration of non-psychoactive cannabinoids, is getting more and more interest in the food, pharmaceutical, and cosmetic industry. However, cannabis not only contains cannabinoids as bioactive components but also other metabolites like terpenes and phenolic compounds, and the content of these interesting secondary metabolites greatly differs with the genetic variety of the plant. Due to the huge complexity of composition of the cannabis matrix, in this work, a comprehensive two-dimensional liquid chromatography (LC × LC) method has been developed as a very power separation technique coupling a pentafluorophenyl (PFP) and a C18 in the first and second dimensions. Two industrial hemp strains (cookie and gelato) were analyzed to determine the difference in their content of cannabinoids and phenolic compounds. To do this, a new demodulation process was applied for the first time to transform 2D raw data into 1D data which allowed carrying out the chemometric analysis needed to determine the statistical differences between the hemp strains. The cookie strain presented a total of 41 cannabinoid markers, while the gelato strain presented more representative phenolic compounds, in total 24 phenolic compounds were detected as potential markers of this sample. These differences in the chemical composition could determine the industrial destiny of the different hemp strains.Graphical abstract

Screening Method for 23 Alkaloids in Human Serum Using LC/MS/MS with a Pentafluorophenyl Column in Dynamic Multiple Reaction Monitoring Mode

Alkaloids are nitrogen-containing organic compounds, generally basic, and found in plants, fungi, and bacteria. Some alkaloids are used in medicine, but some compounds are highly toxic. Accidental ingestion, homicide, and suicide have occurred due to plants containing alkaloids. The identification of toxic components in biological samples is important for the diagnosis and/or treatment of poisoning cases in forensic and emergency medicine. Alkaloids have a wide variety of structures, such as isoquinoline alkaloid, indole alkaloid, tropane alkaloid, and diterpene alkaloid; therefore, there are few reports of simultaneous analysis methods. We have established a method for the simultaneous analysis of 23 alkaloids in human serum with a liquid chromatograph-tandem mass spectrometer (LC/MS/MS). A liquid-liquid extraction which was modified from the first step of the QuEChERS AOAC method was used for serum pretreatment. The separation of the compounds was performed using a pentafluorophenyl (PFP) column, CAPCELL CORE PFP (2.1 mm I.D. × 100 mm, 2.7 μm) in gradient mode. Mobile phase A consisted of 10 mM ammonium formate and 0.1% formic acid in ultrapure water, and mobile phase B was 10 mM ammonium formate and 0.1% formic acid in methanol. Simultaneous analysis was performed in dynamic multiple reaction monitoring mode. The separation of 23 alkaloids was satisfactory, as PFP columns exhibited different retention behaviors than alkyl phase columns. The PFP column effectively retained polar aromatic compounds; therefore, it was suitable for alkaloid analysis. The validated method was applied to a forensic case of aconite poisoning. The present method was useful in LC/MS/MS screening for 23 alkaloids in human serum.

Inhibitory Potentials of Phytoconstituents of Phyllanthus amarus against Severe Acute Respiratory Syndrome-Corona Virus-2 Main Protease: A Computational Aided Approach

In 2019, a new strain of Coronavirus disease was identified in Wuhan, China. Specific therapies are unavailable and investigations regarding Coronavirus disease treatment are lacking. The present study aimed to assess bioactive compounds found in medicinal plant Phyllanthus amarus as potential Coronavirus disease main protease inhibitors, using a molecular docking study. Molecular docking was performed using Autodock 4.2. Coronavirus disease main protease was docked with several compounds, and docking was analysed by Autodock 4.2, Lopinavir was used as standard for comparison. The binding energies obtained from the docking of 6LU7 with native ligand, lopinavir, Phyllanthin, Hypophyllanthin, Hexanedioic acid, bis (2-ethylhexyl) ester, Benzeneethanamine,3,4-dimethoxy-n-((pentafluoro phenyl) methylene), Phenethylamine, 2-methoxy-alpha.-methyl-4,5-(methylenedioxy), Diisooctyl adipate, P-Tert-Octylresorcinol, 4-methyl-2,5-dimethoxy phenethylamine, 3-(2,4-dimethoxy-phenyl)-2-formylaminopropionic acid, ethyl ester, Rutin, Quercetin and Niranthin were -6.08, -4.44, -4.14, -2.32, -4.23, -4.07, -2.5, -4.66, -4.14, -3.38, -7.46, -6.28 and -4.62 kcal/mol, respectively. Therefore, lopinavir may represent potential treatment option and Rutin and Quercetin appeared to have the best potential to act as Coronavirus disease main protease inhibitors. Thus all the compounds have shown significant binding energy and potent inhibitory effect against Coronavirus disease main protease. However, further research is necessary to investigate their potential medicinal use.

A ligand selection strategy to customize small molecule probes for activity-based protein profiling (LS-ABPP).

Activity-based probes (ABPs) are the key components of activity-based protein profiling (ABPP). However, designing a probe that shows target-specific as well as site-selective binding can be a challenging and time-consuming task, often requiring complex synthetic procedures to provide a selection of probes from which to choose the ideal one. In this chapter, we present a ligand selection (LS) approach that allows us to rapidly diversify probe molecules in order to meet the steric and electronic demands of the binding site of any target enzyme. The central element of this method is a trifunctional LS probe synthesized from tyrosine in five steps, consisting of a highly reactive pentafluorophenyl (PFP) ester in addition to an electrophilic chloroacetamide warhead, and a bioorthogonal alkyne reporter group. By reacting a variety of primary amine ligands with the PFP ester, a probe library is created and screened for optimal binding characteristics to the target enzyme. With the optimized probe in hand, a compound library is subsequently screened by competitive profiling to identify potential enzyme inhibitors. Conveniently, this protocol is highly adaptable to a large variety of target proteins, representing a valuable tool for enzyme characterization and the discovery of enzyme inhibitors. Here, we apply this method exemplarily to the cysteine protease 3CLpro of the coronavirus SARS-CoV-2.

Mass Spectrometry-Cleavable Protein N-Terminal Tagging Strategy for System-Level Protease Activity Profiling.

Proteolysis is one of the most important protein post-translational modifications (PTMs) that influences the functions, activities, and structures of nearly all proteins during their lifetime. To facilitate the targeted identification of low-abundant proteolytic products, we devised a strategy incorporating a novel biotinylated reagent PFP (pentafluorophenyl)-Rink-biotin to specifically target, enrich and identify proteolytic N-termini. Within the PFP-Rink-biotin reagent, a mass spectrometry (MS)-cleavable feature was designed to assist in the unambiguous confirmation of the enriched proteolytic N-termini. The proof-of-concept study was performed with multiple standard proteins whose N-termini were successfully modified, enriched and identified by a signature ion (SI) in the MS/MS fragmentation, along with the determination of N-terminal peptide sequences by multistage tandem MS of the complementary fragment generated after the cleavage of MS-cleavable bond. For large-scale application, the enrichment and identification of protein N-termini from Escherichia coli cells were demonstrated, facilitated by an in-house developed NTermFinder bioinformatics workflow. We believe this approach will be beneficial in improving the confidence of identifying proteolytic substrates in a native cellular environment.

A comprehensive study on the performance of different retention mechanisms in sport drug testing by liquid chromatography tandem mass spectrometry

Anti-doping substances listed by the World Anti-Doping Agency (WADA) include hundreds of compounds of very different physico-chemical properties. Anti-doping control laboratories need to screen all these substances in the so-called Initial Testing Procedures (ITPs) what is very challenging from an analytical point of view. ITPs are mostly based on reversed-phase (RP) liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using C18 columns, which feature poor retention and peak tailing for polar and basic compounds, respectively. While studies on this field dealing with the comparison of stationary phases are focused on certain chemical classes, this research provides a wide multi-target approach. For this purpose, a representative group of 93 anti-doping agents (log P from −2.4 to 9.2) included in ten different classes of prohibited substances was selected. A comprehensive study on the performance of six columns and four eluents on different separation parameters (retention factors, asymmetry factors, co-elutions, total run times) and matrix effects (signal enhancement or suppression) was performed for LC-MS/MS-based ITPs. Columns working in both RP [C18, C8, phenyl hexyl (PH), pentafluorophenyl (PFP) and mixed-mode hydrophilic/RP (HILIC-RP)) and hydrophilic (HILIC)] modes were investigated. Eluents contained methanol or acetonitrile as organic modifiers, with or without the addition of ammonium acetate. The best column-mobile phase binomial for ITPs was PFP using water-methanol (0.1% formic acid) as eluent, while HILIC was the best option for highly polar non-aromatic anti-doping agents, which were poorly addressed by PFP. Excellent good peak shapes and relative acceptable matrix interferences were obtained for HILIC-RP, which was tested for the first time for the analysis of anti-doping agents, although the number of compounds eluting too fast was too high. On the whole, the alkyl phase C18 showed the worst performance and although C8 and PH were better, their performance did not surpass that of PFP. Possible retention mechanisms underlying separation in the different stationary phases were discussed. This research provides valuable information to anti-doping control labs for improving LC-MS/MS-based ITPs and it proposes PFP as a suitable alternative to the already established C18.

Preferential light-chain labeling of native monoclonal antibodies improves the properties of fluorophore conjugates

Site specific labeling methods have significant potential to enhance the properties of antibody conjugates. While studied extensively in the context of antibody-drug conjugates (ADCs), few studies have examined the impact of homogenous labeling on the properties of antibody-fluorophore conjugates (AFCs). We report the application of pentafluorophenyl (PFP) esters, which had previously been shown to be reasonably selective for K188 of the kappa light chain of human IgG antibodies, toward producing AFCs. We show that simple replacement of N-hydroxy succinimide (NHS) with PFP dramatically increases the light-chain specificity of near-infrared (NIR) AFCs. Comparing the properties of AFCs labeled using NHS and PFP-activated esters reveals that the latter exhibits reduced aggregation and improved brightness, both in vitro and in vivo. Overall, the use of PFP esters provides a remarkably simple approach to provide selectively labeled antibodies with improved properties.

One-Pot Synthesis of Pentafluorophenyl Sulfonic Esters via Copper-Catalyzed Reaction of Aryl Diazonium Salts, DABSO, and Pentafluorophenol.

Pentafluorophenyl (PFP) sulfonic esters were synthesized via a copper-catalyzed one-pot multicomponent reaction of aryl diazonium tetrafluoroborate, DABSO (DABCO·(SO2)2), and pentafluorophenol. The reaction system provided the desired pentafluorophenyl sulfonic esters in good yields and exhibited excellent functional group tolerance. In addition, the generated PFP sulfonic esters were successfully applied in Sonogashira, Suzuki, Chan-Evans-Lam, and decarboxylative coupling reactions.