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Objectives Contamination of workplace surfaces by antineoplastic drugs presents an exposure risk for healthcare workers. Traditional instrumental methods to detect contamination such as gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) are sensitive and accurate but expensive and incapable of producing results in real time. This limits their utility in preventing worker exposure. We are currently developing monitors based on lateral flow immunoassay that can detect drug contamination in near real time. In this report, we describe the laboratory performance of a 5-fluorouracil (5-FU) monitor. Methods The monitor was evaluated by spiking ceramic, vinyl, composite, stainless steel, and glass surfaces of 100 cm2 area with 5-FU masses of 0, 5, 10, 25, 50, and 100 ng. The surface was sampled with a wetted cotton swab, the swab was extracted with buffer, and the resulting solution was applied to a lateral flow monitor. Two ways of evaluating the response of these monitors were used: an electronic method where a lateral flow reader was used for measuring line intensities, and a visual method where the intensity of the test line was visually compared to the control line. Results The 5-FU monitor is capable of detecting 10 ng/100 cm2 (0.1 ng/cm2) using the electronic reader and 25 ng/100 cm2 (0.25 ng/cm2) using the visual comparison method for the surfaces studied. The response of the monitors was compared to LC-MS/MS results for the same samples for validation and there was good correlation of the two methods but some differences in absolute response, especially at higher spiking levels for the surface samples. [ABSTRACT FROM AUTHOR]

Objectives Contamination of workplace surfaces by antineoplastic drugs presents an exposure risk for healthcare workers. Traditional instrumental methods to detect contamination such as liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) are sensitive and accurate but expensive. Since immunochemical methods may be cheaper and faster than instrumental methods, we wanted to explore their use for routine drug residue detection for preventing worker exposure. Methods In this study we examined the feasibility of using fluorescence covalent microbead immunosorbent assay (FCMIA) for simultaneous detection and semi-quantitative measurement of three antineoplastic drugs (5-fluorouracil, paclitaxel, and doxorubicin). The concentration ranges for the assay were 0–1000 ng/ml for 5-fluorouracil, 0–100 ng/ml for paclitaxel, and 0–2 ng/ml for doxorubicin. The surface sampling technique involved wiping a loaded surface with a swab wetted with wash buffer, extracting the swab in storage/blocking buffer, and measuring drugs in the extract using FCMIA. Results There was no significant cross-reactivity between these drugs at the ranges studied indicated by a lack of response in the assay to cross analytes. The limit of detection (LOD) for 5-fluorouracil on the surface studied was 0.93 ng/cm2 with a limit of quantitation (LOQ) of 2.8 ng/cm2, the LOD for paclitaxel was 0.57 ng/cm2 with an LOQ of 2.06 ng/cm2, and the LOD for doxorubicin was 0.0036 ng/cm2 with an LOQ of 0.013 ng/cm2. Conclusion The use of FCMIA with a simple sampling technique has potential for low cost simultaneous detection and semi-quantitative measurement of surface contamination from multiple antineoplastic drugs. [ABSTRACT FROM AUTHOR]

Context. Healthcare worker exposure to antineoplastic drugs continues to be reported despite safe handling guidelines published by several groups. Sensitive sampling and analytical methods are needed so that occupational safety and health professionals may accurately assess environmental and biological exposure to these drugs in the workplace.Objective. To develop liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analytical methods for measuring five antineoplastic drugs in samples from the work environment, and to apply these methods in validating sampling methodology. A single method for quantifying several widely used agents would decrease the number of samples required for method development, lower cost, and time of analysis. Methods for measuring these drugs in workers’ urine would also be useful in monitoring personal exposure levels.Results. LC-MS/MS methods were developed for individual analysis of five antineoplastic drugs in wipe and air sample media projected for use in field sampling: cyclophosphamide, ifosfamide, paclitaxel, doxorubicin, and 5-fluorouracil. Cyclophosphamide, ifosfamide, and paclitaxel were also measured simultaneously in some stages of the work. Extraction methods for air and wipe samples were developed and tested using the aforementioned analytical methods. Good recoveries from the candidate air and wipe sample media for most of the compounds, and variable recoveries for test wipe samples depending on the surface under study, were observed. Alternate LC-MS/MS methods were also developed to detect cyclophosphamide and paclitaxel in urine samples.Conclusions. The sampling and analytical methods were suitable for determining worker exposure to antineoplastics via surface and breathing zone contamination in projected surveys of healthcare settings. [ABSTRACT FROM PUBLISHER]

The probable human carcinogen 4,4′-methylene-bis(2-chloroaniline) (MOCA) was utilized to develop biomarkers of exposure to occupational carcinogens. The 32P postlabeling assay, utilizing the nuclease P1 enhancement procedure, was used to evaluate MOCA-DNA adduct formation in target tissues. Male Sprague-Dawley rats were treated with different dosing regimens of MOCA, and DNA was isolated from the liver. Additionally, a human uroepithelial cell (HUC) line was treated with N-hydroxy MOCA for 24 hr, cells were harvested, and DNA was isolated. DNA was analyzed for MOCA-DNA adduct formation by the 32P postlabeling assay. Five MOCA adducts were detected in rat liver DNA. Adduct A, which corresponded to N-(deoxyadenosin-8-yl)-4-amino-3-chlorobenzyl alcohol, was the major adduct in rat liver DNA appearing in all treatment groups. Levels of adduct A were higher when MOCA was administered by ip injection versus oral gavage. Phenobarbital pretreatment increased the amount of adduct A approximately 12-fold. The pathway leading to the formation of adduct A in DNA from HUC appeared to be saturated at the concentrations used: 2.5, 5, and 10 µM. However, an additional adduct (E) was observed at the 10 µM treatment level only. A major DNA adduct was detected in the target tissue of rats and target human cells for MOCA-induced carcinogenesis, thus making it useful as a biomarker of exposure. Other DNA adducts were also observed with the different doses and routes of exposure investigated. [ABSTRACT FROM AUTHOR]

Hemoglobin (Hb) and albumin (Alb) adducts of the suspect human carcinogen -toluidine (OT) were quantified in blood samples collected from rats after a single i.p. injection. Mild alkaline hydrolysis of Hb adducted with [C]OT followed by extraction with ethyl acetate resulted in recovery of 63% of the bound radioactivity. HPLC analysis revealed a single radiolabeled peak which was identified as OT by GC-MS. In subsequent experiments Hb and Alb adduct levels were determined by HPLC analysis of this cleavage product using fluorescence detection. 4-Ethylaniline was used as internal standard. The detection limit for OT was approximately 450 pg/injection or 5 pmol/mg Hb. Mean adduct levels for Hb increased rapidly over the first 4 hr with the highest (ng/mg Hb ± SD) 3.7 ± 0.5 detected 24 hr after OT administration at 50 mg/kg body wt. In contrast, adduct levels for pooled Alb samples increased from 0.7 ng/mg Alb at 2 hr to 2.5 ng/mg Alb at 4 hr, but were not detectable 24 hr after dosing. Hb adducts showed a linear relationship for OT doses of 10, 20, 40, 50, and 100 mg/kg body wt. The Hb adduct ½ (11 days) was determined after a single 100 mg/kg OT dose. Hb adduct levels were quantifiable (1.3 ± 0.2 ng/mg Hb) by HPLC/fluorescence 28 days after 100 mg/kg OT. Although Hb and Alb adducts differ in stability, a ratio of such OT adducts may be useful in long-term industrial biomonitoring for evaluation of OT exposure. [ABSTRACT FROM PUBLISHER]

The effect of multiple oral administration of MOCA, a suspect human carcinogen, was studied in the adult male rat. As many as 28 consecutive daily doses of [C]MOCA at 28.1μmol/kg body wt (5 μC1/day) were administered and rats were euthanized at weekly intervals for 7 weeks. MOCA adduct formation for globin and serum albumin was evaluated by determination of [C]MOCA covalent binding. The covalent binding associated with globin showed a linear increase over the 28-day exposure period with 342 fmol/mg globin 24 hr after the final dose. More extensive covalent binding was detected for albumin with 443 fmol/mg albumin after the final dose, but increases were not linear. After cessation of dosing, the albumin adduct levels decreased rapidly ( =4.6 days) in relation to globin adduct levels =16.1 days). The MOCA-globin adduct is consistent with that determined after a single 281 μmol/kg oral dose of MOCA. Significant differences related to route of administration were detected for 24-hr globin covalent binding with ip > po > dermal. Distribution of undifferentiated [C]MOCA was highest in the liver at 24 hr with tissue levels for liver > kidney > lung > spleen > testes > urinary bladder. Induction of cytochrome P450 enzymes by administration of phenobarbital (100 mg/kg/day/3 days) resulted in a significant 0.05) increase in MOCA-globin adduct formation detected with 33.5 pmol/ mg globin for induced rats versus 13.6 pmol/mg globin for control rats. Although MOCA-globin and albumin adducts show differing stability, quantification of such MOCA adducts may be useful for long-term industrial biomonitoring of MOCA. [ABSTRACT FROM PUBLISHER]

4,4′-Methylene-bis(2-chloroaniline) (MOCA): Comparison of Macromolecular Adduct Formation after Oral or Dermal Administration in the Rat. CHEEVER, K. L., RICHARDS, D. E., WEIGEL, W. W., BEGLEY, K. B., DEBORD, D. G., SWEARENGIN, T. F., AND SAVAGE, R. E. JR. (1990). . , 273–283. The macromolecular binding of 4,4'-methylene-bis(2-chloroaniline) (MOCA), a suspect human carcinogen, was studied in the adult male Sprague-Dawley rat after both oral and dermal administration. Rats were euthanized 1, 3, 7, 10, 14, and 29 days after a single 281 μmol/kg body wt dose of [C]MOCA (oral, 213 μCi/kg; dermal, 904 μCi/kg). DNA from various tissues and hemoglobin were isolated for determination of the time course of MOCA macromolecular binding. After oral administration adduct formation was rapid with maximum levels appearing at 24 hr. The 24-hr covalent binding associated with the globin was 7.84 pmol/mgglobin (½ = 14.3 days). More extensive 24-hr covalent binding was detected for liver DNA with 49.11 pmol/mg DNA (½ = 11.1 days). After dermal administration of MOCA the major portion of the dose, 86.2%, remained at the application site throughout the study. For these rats the 24-hr covalent binding determined for liver DNA was 0.38 pmol/mg DNA (½ = 15.6 days). Although lower levels were detected after dermal application, similar stability of MOCA-DNA adducts indicates that quantification of such MOCA adducls may be useful for the long-term industrial biomonitoring of MOCA exposure and for the evaluation of human DNA-MOCA adduct formation, a lesion thought to be associated with the production of cancer. [ABSTRACT FROM PUBLISHER]

Ion mobility (IM) is often combined with LC-MS experiments to provide an additional dimension of separation for complex sample analysis. While highly complex samples are better characterized by the full dimensionality of LC-IM-MS experiments to uncover new information, downstream data analysis workflows are often not equipped to properly mine the additional IM dimension. For many samples the data acquisition benefits of including IM separations are all that is necessary to uncover sample information and the full dimensionality of the data is not required for data analysis. Postacquisition reduction and adaptation of the dimensions of LC-IM-MS and IM-MS experiments into an LC-MS format opens the possibility to use a plethora of existing software tools. In this work, we developed data file conversion tools to reduce the complexity of IM data analysis. Three data file transformations are introduced in the PNNL PreProcessor software: (1) mapping the IM axis to the LC axis for IM-MS data, (2) converting the drift time vs m / z space to CCS/z vs m / z space, and (3) transforming All Ions IM/MS mobility aligned fragmentation data to a standard LC-MS DDA data file format. These new data file conversions are demonstrated with corresponding lipidomics and proteomics workflows that leverage existing LC-MS data analysis software to highlight the benefits of the data transformations.

Cyclic peptides are an important class of molecules that gained significant attention in the field of drug discovery due to their unique pharmacological characteristics and enhanced proteolytic stability. Yet, gastrointestinal degradation remains a major hurdle in the discovery of orally bioavailable cyclic peptides. Soft spot identification (SSID) of the regions in the cyclic peptide sequence susceptible to amide hydrolysis by proteases is used in the discovery stage to guide medicinal chemistry design. SSID can be an arduous task, traditionally performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), often resulting in complex and time-consuming manual analysis, particularly when isomeric linear peptide metabolites chromatographically coelute. Here, we present an alternative orthogonal approach that entails a high-resolution ion mobility (HRIM) system based on Structures for Lossless Ion Manipulation (SLIM) technology interfaced with quadrupole time-of-flight (QTOF) mass spectrometry to address some of the challenges associated with SSID. Two strategies were used to resolve linear isomeric peptide metabolites: labeled and label-free, both utilizing the HRIM platform. The label-free strategy leverages negative polarity to ionize the isomers which achieves better separation of the gas phase ions in the ion mobility (IM) dimension as compared to positive polarity, which is a more conventional approach when studying proteins and peptides. The second approach uses an isotope-labeled dimethyl tag on the terminal amine group, acting as a "shift reagent" to influence the mobility of isomers in the positive mode. This method resulted in baseline separation for the isomers of interest and produced unique product ions in the fragmentation spectra for unambiguous soft spot identification. Both label-free and labeled strategies demonstrated the ability to solve the challenges associated with SSID for cyclic peptides.

The coevolution of liquid chromatography (LC) with mass spectrometry (MS) has shaped contemporary proteomics. LC hyphenated to MS now enables quantification of more than 10,000 proteins in a single injection, a number that likely represents most proteins in specific human cells or tissues. Separations by ion mobility spectrometry (IMS) have recently emerged to complement LC and further improve the depth of proteomics. Given the theoretical advantages in speed and robustness of IMS in comparison to LC, we envision that ongoing improvements to IMS paired with MS may eventually make LC obsolete, especially when combined with targeted or simplified analyses, such as rapid clinical proteomics analysis of defined biomarker panels. In this perspective, we describe the need for faster analysis that might drive this transition, the current state of direct infusion proteomics, and discuss some technical challenges that must be overcome to fully complete the transition to entirely gas phase proteomics.

Lipids are structurally diverse molecules that play a pivotal role in a plethora of biological processes. However, deciphering the biological roles of the specific lipids is challenging due to the existence of numerous isomers. This high chemical complexity of the lipidome is one of the major challenges in lipidomics research, as the traditional liquid chromatography-mass spectrometry (LC-MS) based approaches are often not powerful enough to resolve these isomeric and isobaric nuances within complex samples. Thus, lipids are uniquely suited to the benefits provided by multidimensional liquid chromatography-ion mobility-mass spectrometry (LC-IM-MS) analysis. However, many forms of lipid isomerism, including double-bond positional isomers and regioisomers, are structurally similar such that their collision cross section (CCS) differences are unresolvable via conventional IM approaches. Here we evaluate the performance of a high resolution ion mobility (HRIM) system based on structures for lossless ion manipulation (SLIM) technology interfaced to a high resolution quadrupole time-of-flight (QTOF) analyzer to address the noted lipidomic isomerism challenge. SLIM implements the traveling wave ion mobility technique along an ∼13 m ion path, providing longer path lengths to enable improved separation of isomeric features. We demonstrate the power of HRIM-MS to dissect isomeric PC standards differing only in double bond (DB) and stereospecific number (SN) positions. The partial separation of protonated DB isomers is significantly enhanced when they are analyzed as metal adducts. For sodium adducts, we achieve close to baseline separation of three different PC 18:1/18:1 isomers with different cis -double bond locations. Similarly, PC 18:1/18:1 ( cis -9) can be resolved from the corresponding PC 18:1/18:1 ( trans -9) form. The separation capacity is further enhanced when using silver ion doping, enabling the baseline separation of regioisomers that cannot be resolved when measured as sodium adducts. The sensitivity and reproducibility of the approach were assessed, and the performance for more complex mixtures was benchmarked by identifying PC isomers in total brain and liver lipid extracts.

Characterization and monitoring of post-translational modifications (PTMs) by peptide mapping is a ubiquitous assay in biopharmaceutical characterization. Often, this assay is coupled to reversed-phase liquid chromatographic (LC) separations that require long gradients to identify all components of the protein digest and resolve critical modifications for relative quantitation. Incorporating ion mobility (IM) as an orthogonal separation that relies on peptide structure can supplement the LC separation by providing an additional differentiation filter to resolve isobaric peptides, potentially reducing ambiguity in identification through mobility-aligned fragmentation and helping to reduce the run time of peptide mapping assays. A next-generation high-resolution ion mobility (HRIM) technique, based on structures for lossless ion manipulations (SLIM) technology with a 13 m ion path, provides peak capacities and higher resolving power that rivals traditional chromatographic separations and, owing to its ability to resolve isobaric peptides that coelute in faster chromatographic methods, allows for up to 3× shorter run times than conventional peptide mapping methods. In this study, the NIST monoclonal antibody IgG1κ (NIST RM 8671, NISTmAb) was characterized by LC-HRIM-MS and LC-HRIM-MS with collision-induced dissociation (HRIM-CID-MS) using a 20 min analytical method. This approach delivered a sequence coverage of 96.5%. LC-HRIM-CID-MS experiments provided additional confidence in sequence determination. HRIM-MS resolved critical oxidations, deamidations, and isomerizations that coelute with their native counterparts in the chromatographic dimension. Finally, quantitative measurements of % modification were made using only the m / z -extracted HRIM arrival time distributions, showing good agreement with the reference liquid-phase separation. This study shows, for the first time, the analytical capability of HRIM using SLIM technology for enhancing peptide mapping workflows relevant to biopharmaceutical characterization.

A production prototype structures for lossless ion manipulation ion mobility (SLIM IM) platform interfaced to a commercial high-resolution mass spectrometer (MS) is described. The SLIM IM implements the traveling wave ion mobility technique across a ∼13m path length for high-resolution IM (HRIM) separations. The resolving power (CCS/ΔCCS) of the SLIM IM stage was benchmarked across various parameters (traveling wave speeds, amplitudes, and waveforms), and results indicated that resolving powers in excess of 200 can be accessed for a broad range of masses. For several cases, resolving powers greater than 300 were achieved, notably under wave conditions where ions transition from a nonselective "surfing" motion to a mobility-selective ion drift, that corresponded to ion speeds approximately 30-70% of the traveling wave speed. The separation capabilities were evaluated on a series of isomeric and isobaric compounds that cannot be resolved by MS alone, including reversed-sequence peptides (SDGRG and GRGDS), triglyceride double-bond positional isomers (TG 3, 6, 9 and TG 6, 9, 12), trisaccharides (melezitose, raffinose, isomaltotriose, and maltotriose), and ganglioside lipids (GD1b and GD1a). The SLIM IM platform resolved the corresponding isomeric mixtures, which were unresolvable using the standard resolution of a drift-tube instrument (∼50). In general, the SLIM IM-MS platform is capable of resolving peaks separated by as little as ∼0.6% without the need to target a specific separation window or drift time. Low CCS measurement biases <0.5% were obtained under high resolving power conditions. Importantly, all the analytes surveyed are able to access high-resolution conditions (>200), demonstrating that this instrument is well-suited for broadband HRIM separations important in global untargeted applications.

Defects in sphingolipid metabolism have emerged as a common link across neurodegenerative disorders, and a deeper understanding of the lipid content in preclinical models and patient specimens offers opportunities for development of new therapeutic targets and biomarkers. Sphingolipid metabolic pathways include the formation of glycosphingolipid species that branch into staggeringly complex structural heterogeneity within the globoside and ganglioside sub-lipidomes. Characterization of these sub-lipidomes has typically relied on liquid chromatography-mass spectrometry-based (LC-MS) approaches, but such assays are challenging and resource intensive due to the close structural heterogeneity, the presence of isobaric and isomeric species, and broad dynamic range of endogenous glycosphingolipids. Here, we apply Structures for Lossless Ion Manipulations (SLIM)-based High Resolution Ion Mobility (HRIM)-MS to enable rapid, repeatable, quantitative assays with deep structural information sufficient to resolve endogenous brain gangliosides at the level of individual molecular species. Analyses were performed using a prototype SLIM-MS instrument equipped with a 13-m serpentine path which enabled resolution of closely related isomeric analytes such as GD1a d36:1 and GD1b d36:1 based on recorded mass-to-charge (m/z) and arrival times. To demonstrate the power of our methodology, brain extracts derived from wild-type mice hemi-brains were analyzed by HRIM-MS using flow injection analyses (FIA) without the need for additional separation by liquid chromatography. Endogenous ganglioside species were readily resolved, identified, and quantified by FIA-SLIM-MS analyses within 2 min per sample. Thus, the FIA-SLIM-MS platform enables robust quantification across a broad range of lipid species in biological specimens in a standardized assay format that is readily scalable to support studies with large sample numbers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The pharmaceutical and clinical industries are imperative for the maintenance of global health and welfare and require accurate, reproducible, and high throughput analyses. Technological advancements, such as the development and implementation of liquid chromatography-tandem mass spectrometry (LC-MS), have allowed for improvements in these areas, however there is still room for development. One way in which current analyses may be improved is by the implementation of ion mobility technology. Ion mobility has the capability to produce much more comprehensive data sets, by providing separation of isomers, as well as improving throughput, with separations being performed as fast as 60 ms. Here we will discuss the potential for ion mobility to assist in the two specific areas of glycosylation monitoring of biological drugs, and vitamin D analysis, as representatives of ion mobility's potential in both the pharmaceutical and clinical industries, respectively, as well as the current hurdles of ion mobility adoption in both fields.

The industrial hygiene community has witnessed exponential growth in the use of sensors, especially by individuals. Remote wireless sensors are now monitoring worker health, the environment, agriculture, work sites, disaster relief, and "smart" buildings and facilities.