A simple and cheap design for interfacing thin layer chromatography (TLC) with electrospray ionization mass spectrometry (ESI/MS) was developed to scan and characterize compounds on TLC plate. The ...developed TLC plate was rapidly and easily modified into two sawtooth-edged pieces that were positioned on an XYZ stage so that one of the triangular tips was pointed toward the MS inlet. A drop of methanol and high DC voltage was applied at the tip to induce ESI. After the analytes in the first tip were analyzed, the TLC piece was moved so that the second triangular tip was pointed toward the MS inlet for analysis. The process was repeated until all the triangular tips on the piece were analyzed. In this manner, the analytes, no matter visible or non-visible bands, were scanned and characterized. Since a 4.8 cm long TLC track were cut to 32 triangles on two sawtooth pieces for analysis, the spatial resolution of using the sawtooth TLC-ESI/MS for analysis is 1.5 mm/band. A mixture of dye standards and Datura metel flower extract was analyzed to demonstrate the capability of sawtooth TLC-ESI/MS on scanning and characterizing chemical compounds on the TLC plates. The limits of detection of the dye standards were between 0.25 and 2.5 ng/band. TLC bands containing alkaloids such as scopolamine and norscopolamine from the Datura metel flower extract were not visualized on the developed TLC track, but were successfully detected at different triangular tips using sawtooth TLC-ESI/MS. Based on these results, the Rf values of scopolamine and norscopolamine were determined.
Display omitted
•Sawtooth TLC-ESI/MS was developed to characterize a mixture.•The developed TLC plate was modified into two sawtooth-edged pieces for inducing ESI at the triangular tips.•The entire TLC track was scanned with minimal gel particle detachment during analysis.•Visualizable and non-visualizable bands were both characterized, so that their Rf information was elucidated.
Rationale
Solid‐phase microextraction coupled with thermal desorption electrospray ionization tandem mass spectrometry (SPME‐TD‐ESI‐MS/MS) is proposed as a novel method for the rapid quantification ...of acetaminophen in plasma samples from a pharmacokinetics (PK) study.
Methods
Traces of acetaminophen were concentrated on commercial fused‐silica fibers coated with a polar polyacrylate (PA) polymer using direct immersion SPME. No agitation, heating, addition of salt, or adjustment of the pH of the sample solution was applied during the extraction. Any acetaminophen absorbed on the SPME fibers was subsequently desorbed and detected by TD‐ESI‐MS/MS.
Results
Parameters of the absorption, sensitivity, reproducibility, and linearity for the SPME‐TD‐ESI‐MS/MS method were evaluated. The time required to complete a TD‐ESI‐MS/MS analysis was less than 30 seconds. Matrix‐matching calibration was performed to calculate the concentration of acetaminophen in the sample. A linear calibration curve with a concentration range of 100–10,000 ng/mL was constructed to calculate the quantity of acetaminophen. The SPME‐TD‐ESI‐MS quantification results for acetaminophen in plasma were in good agreement with those obtained by the conventional LC/MS/MS method.
Conclusions
With the proposed method, a 10‐min SPME time was enough to achieve the lower limit of quantitation (i.e. 100 ng/mL) and for a complete PK profiling of acetaminophen. A shorter extraction time could be achieved by applying agitation, heating, adding salt, or adjusting the pH of the sample solution to enhance analyte absorption efficiency. The time required to detect acetaminophen on the SPME fiber was less than 30 s, allowing the rapid quantification of acetaminophen in plasma with good accuracy.
Flame‐induced atmospheric pressure chemical ionization (FAPCI) has been used to directly characterize chemical compounds on a glass rod and drug tablet surfaces. In this study, FAPCI was further ...applied to interface thin layer chromatography (TLC) and mass spectrometry (MS) for mixture analysis.
Methods
A micro‐sized oxyacetylene flame was generated using a small concentric tube system. Hot gas flow and primary reactive species from the micro‐flame were directed toward a developed TLC gel plate to thermally desorb and ionize analytes on the gel surface. The resulting analyte ions subsequently entered the MS inlet for detection.
Results
A 1–1.5‐mm‐wide light‐brown line was observed on the TLC plate after the desorption FAPCI/MS (DFAPCI/MS) analysis, revealing that the gel surface withstood a high temperature from the impact of the micro‐flame. Volatile and semi‐volatile chemical compounds, including amine and amide standards, drugs, and aromatherapy oils, were successfully desorbed, ionized, and detected using this TLC/DFAPCI/MS. The limit of detection of TLC‐DFAPCI/MS was determined to be 5 ng/spot for dibenzylamine and ethenzamide.
Conclusions
TLC/DFAPCI/MS is one of the simplest TLC‐MS interfaces showing the advantages such as low costs and an easy set up. The technique is useful for characterizing thermally stable volatile and semi‐volatile compounds in a mixture.
The use of chemical warfare agents is prohibited but they have been used in recent Middle Eastern conflicts. Their accidental exposure (e.g. arsenical lewisite) is also known and causes extensive ...painful cutaneous injury. However, their molecular pathogenesis is not understood. Here, we demonstrate that a nexus of stress granules (SGs), integrated stress, and RNA binding proteins (RBPs) Roquin and Reganse-1 play a key role. Lewisite and its prototype phenylarsine oxide (PAO) induce SG assembly in skin keratinocytes soon after exposure, which associate with various RBPs and translation-related proteins. SG disassembly was detected several hours after exposure. The dynamics of SG assembly-disassembly associates with the chemical insult and cell damage. Enhanced Roquin and Regnase-1 expression occurs when Roquin was recruited to SGs and Regnase-1 to the ribosome while in the disassembling SGs their expression is decreased with consequent induction of inflammatory mediators. SG-targeted protein translational control is regulated by the phosphorylation-dependent activation of eukaryotic initiation factors 2α (eIF2α). Treatment with integrated stress response inhibitor (ISRIB), which blocks eIF2α phosphorylation, impacted SG assembly dynamics. Topical application of ISRIB attenuated the inflammation and tissue disruption in PAO-challenged mice. Thus, the dynamic regulation of these pathways provides underpinning to cutaneous injury and identify translational therapeutic approach for these and similar debilitating chemicals.
Flame atmospheric pressure chemical ionization (FAPCI) combined with negative electrospray ionization (ESI) mass spectrometry was developed to detect the ion/molecule reactions (IMRs) products ...between nitric acid (HNO
3
) and negatively charged amino acid, angiotensin I (AI) and angiotensin II (AII), and insulin ions. Nitrate and HNO
3
-nitrate ions were detected in the oxyacetylene flame, suggesting that a large quantity of nitric acid (HNO
3
) was produced in the flame. The HNO
3
and negatively charged analyte ions produced by a negative ESI source were delivered into each arm of a Y-shaped stainless steel tube where they merged and reacted. The products were subsequently characterized with an ion trap mass analyzer attached to the exit of the Y-tube. HNO
3
showed the strongest affinity to histidine and formed (M
histidine
-H+HNO
3
)
–
complex ions, whereas some amino acids did not react with HNO
3
at all. Reactions between HNO
3
and histidine residues in AI and AII resulted in the formation of dominant M
AI
-H+(HNO
3
)
-
and M
AII
-H+(HNO
3
)
–
ions. Results from analyses of AAs and insulin indicated that HNO
3
could not only react with basic amino acid residues, but also with disulfide bonds to form M-3H+(HNO
3
)
n
3-
complex ions. This approach is useful for obtaining information about the number of basic amino acid residues and disulfide bonds in peptides and proteins.
Graphical Abstract
ᅟ
Worldwide, more than 200 million people are estimated to be exposed to unsafe levels of arsenic. Chronic exposure to unsafe levels of groundwater arsenic is responsible for multiple human disorders, ...including dermal, cardiovascular, neurological, pulmonary, renal, and metabolic conditions. Consumption of rice and seafood (where high levels of arsenic are accumulated) is also responsible for human exposure to arsenic. The toxicity of arsenic compounds varies greatly and may depend on their chemical form, solubility, and concentration. Surprisingly, synthetic organoarsenicals are extremely toxic molecules which created interest in their development as chemical warfare agents (CWAs) during World War I (WWI). Among these CWAs, adamsite, Clark I, Clark II, and lewisite are of critical importance, as stockpiles of these agents still exist worldwide. In addition, unused WWII weaponized arsenicals discarded in water bodies or buried in many parts of the world continue to pose a serious threat to the environment and human health. Metabolic inhibition, oxidative stress, genotoxicity, and epigenetic alterations including micro-RNA-dependent regulation are some of the underlying mechanisms of arsenic toxicity. Mechanistic understanding of the toxicity of organoarsenicals is also critical for the development of effective therapeutic interventions. This review provides comprehensive details and a critical assessment of recently published data on various chemical forms of arsenic, their exposure, and implications on human and environmental health.
Graphical abstract
Vesicants such as arsenicals and mustards produce highly painful cutaneous inflammatory and blistering responses, hence developed as chemical weapons during World War I/II. Here, using lewisite and ...sulfur mustard surrogates, namely phenylarsine oxide (PAO) and 2-chloroethyl ethyl sulfide (CEES), respectively, we defined a common underlying mechanism of toxic action by these two distinct classes of vesicants. Murine skin exposure to these chemicals causes tissue destruction characterized by increase in skin bifold thickness, Draize score, infiltration of inflammatory cells, and apoptosis of epidermal and dermal cells. RNA sequencing analysis identified ∼346 inflammatory genes that were commonly altered by both PAO and CEES, along with the identification of cytokine signaling activation as the top canonical pathway. Activation of several proinflammatory genes and pathways is associated with phosphorylation-dependent activation of heat shock protein 90
(p-HSP90
). Topical treatment with known HSP90 inhibitors SNX-5422 and IPI-504 post PAO or CEES skin challenge significantly attenuated skin damage including reduction in overall skin injury and clinical scores. In addition, highly upregulated inflammatory genes
, etc. by both PAO and CEES were significantly diminished by treatment with HSP90 inhibitors. These drugs not only reduced PAO- or CEES-induced p-HSP90
expression but also its client proteins NLRP3 and pP38 and the expression of their target inflammatory genes. Our data confirm a critical role of HSP90 as a shared underlying molecular target of toxicity by these two distinct vesicants and provide an effective and novel medical countermeasure to suppress vesicant-induced skin injury. SIGNIFICANCE STATEMENT: Development of effective and novel mechanism-based antidotes that can simultaneously block cutaneous toxic manifestations of distinct vesicants is important and urgently needed. Due to difficulties in determining the exact nature of onsite chemical exposure, a potent drug that can suppress widespread cutaneous damage may find great utility. Thus, this study identified HSP90 as a common molecular regulator of cutaneous inflammation and injury by two distinct warfare vesicants, arsenicals and mustards, and HSP90 inhibitors afford significant protection against skin damage.
Adenocarcinoma in Barrett's esophagus (BE) occurs more frequently between 12 and 3 o'clock at the gastroesophageal junction (GEJ).
BE patients were prospectively recruited from December 2013 to July ...2016. Expression of p53, Ki-67, cyclin-D1, COX-2 and p21 was assessed in quadrantic biopsies from the proximal and distal margins of the BE segments. Cell cycle marker association with current or subsequent dysplasia or adenocarcinoma was examined.
110 patients: median age 64 (IQR, 56-71) years; median BE segment length C4M6; and a median follow-up of 4.7 (IQR, 3.6-5.7) years. In total 13 (11.8%) had evidence of dysplasia or neoplasia (2.7% indefinite for dysplasia, 5.5% low grade, 1.8% high grade and 1.8% adenocarcinoma) at index endoscopy. Six (7%) developed dysplasia or neoplasia (1 low grade, 2 high grade and 3 adenocarcinoma) during follow-up. Ki-67 expression was highest at 3 o'clock, and overall was 49.6% higher in the 12-6 o'clock position compared to 6-12 o'clock odds ratio (OR), 1.42 (95% confidence interval (CI), 1.00-2.12). A similar pattern was found with p21 1.82 (1.00-3.47). There was increased expression of several markers in distal BE biopsies; cyclin-D1 1.74 (1.29-2.34); Cyclo-oxygenase 2 2.03 (1.48-2.78) and p21 2.06 (1.16-3.68). Expression of Ki-67 was lower in distal compared to proximal biopsies 0.58 (0.43-0.78). P53 expression had high specificity (93.8%) for subsequent low-grade dysplasia, high-grade dysplasia or adenocarcinoma.
Increased cellular proliferation was seen at 12-6 o'clock at the GEJ. Cell-cycle marker expression was increased at the GEJ compared to the proximal BE segment. These findings mirror reflux esophagitis and suggest ongoing reflux contributes to the progression of dysplasia and malignancy in BE.
Display omitted
Lewisite is a chemical warfare agent intended for use in World War and a potential threat to the civilian population due to presence in stockpiles or accidental exposure. ...Lewisite-mediated skin injury is characterized by acute erythema, pain, and blister formation. N-acetyl cysteine (NAC) is an FDA-approved drug for acetaminophen toxicity, identified as a potential antidote against lewisite. In the present study, we have explored the feasibility of rapid NAC delivery through transdermal route for potentially treating chemical warfare toxicity. NAC is a small, hydrophilic molecule with limited passive delivery through the skin. Using skin microporation with dissolving microneedles significantly enhanced the delivery of NAC into and across dermatomed human skin in our studies. Microporation followed by application of solution (poke-and-solution) resulted in the highest in vitro delivery (509.84 ± 155.04 µg/sq.cm) as compared to poke-and-gel approach (474.91 ± 70.09 µg/sq.cm) and drug-loaded microneedles (226.89 ± 33.41 µg/sq.cm). The lag time for NAC delivery through poke-and-solution approach (0.23 ± 0.04 h) was close to gel application (0.25 ± 0.02 h), with the highest for drug-loaded microneedles (1.27 ± 1.16 h). Thus, we successfully demonstrated the feasibility of rapid NAC delivery using various skin microporation approaches for potential treatment against lewisite-mediated skin toxicity.
PDF
