Monitoring Exposure to Five Chemical Warfare Agents Using the Dried Urine Spot Technique and Liquid Chromatography-Mass Spectrometry/Mass Spectrometry-In Vivo Determination of Sarin Metabolite in Mice.

Dried urine spot (DUS) is a micro-sample collection technique, known for its advantages in handling, storage and shipping. It also uses only a small volume of urine, an essential consideration in working with small animals, or in acute medical situations. Alkyl-phosphonic acids are the direct and indicative metabolites of organophosphorus chemical warfare agents (OP-CWAs) and are present in blood and urine shortly after exposure. They are therefore crucially important for monitoring casualties in war and terror scenarios. We report here a new approach for the determination of the metabolites of five CWAs in urine using DUS. The method is based on a simple and rapid sample preparation, using only 50 µL of urine, spotted and dried on DBS paper, extracted using 300 µL methanol/water and analyzed via targeted LC-MS/MS. The detection limits for the five CWAs, sarin (GB), soman (GD), cyclosarin (GF), VX and RVX in human urine were from 0.5 to 5 ng/mL. Recoveries of (40-80%) were obtained in the range of 10-300 ng/mL, with a linear response (R2 > 0.964, R > 0.982). The method is highly stable, even with DUS samples stored up to 5 months at room temperature before analysis. It was implemented in a sarin in vivo exposure experiment on mice, applied for the time course determination of isopropyl methylphosphonic acid (IMPA, sarin hydrolysis product) in mice urine. IMPA was detectable even with samples drawn 60 h after the mice's (IN) exposure to 1 LD50 sarin. This method was also evaluated in a non-targeted screening for multiple potential CWA analogs (LC-Orbitrap HRMS analysis followed by automatic peak detection and library searches). The method developed here is applicable for rapid CWA casualty monitoring.

Evaluation of Matrix Complexity in Nontargeted Analysis of Small-Molecule Toxicants by Liquid Chromatography-High-Resolution Mass Spectrometry.

Complex mixtures, characterized by high density of compounds, challenge trace detection and identification. This is further exacerbated in nontargeted analysis, where a compound of interest may be well hidden under thousands of matrix compounds. We studied the effect of matrix complexity on nontargeted detection (peak picking) by LC-MS/MS (Orbitrap) analysis. A series of ∼20 drugs, V-type chemical warfare agents and pesticides, simulating toxic unknowns, were spiked at various concentrations in several complex matrices including urine, rosemary leaves, and soil extracts. Orbitrap "TraceFinder" software was used to explore their peak intensities in relation to the matrix (peak location in an intensity-sorted list). Average practical detection limits of nontargets were determined. While detection among the first 10,000 peaks was achieved at 0.3-1 ng/mL levels in the extract, for the more realistic "top 1000" list, much higher concentrations were required, approaching 10-30 ng/mL. A negative power law functional dependence between the peak location in an intensity-sorted suspect list and the nontarget concentration is proposed. Controlled complexity was explored with a series of urine dilutions, resulting in an excellent correlation between the power law coefficient and dilution factor. The intensity distribution of matrix peaks was found to spread (unevenly) on a broad range, fitting well the Weibull distribution function with all matrices and extracts. The quantitative approach demonstrated here gives a measure of the actual capabilities and limitations of LC-MS in the analysis of nontargets in complex matrices. It may be used to estimate and compare the complexity of matrices and predict the typical detection limits of unknowns.

pyAIR-A New Software Tool for Breathomics Applications-Searching for Markers in TD-GC-HRMS Analysis.

Volatile metabolites in exhaled air have promising potential as diagnostic biomarkers. However, the combination of low mass, similar chemical composition, and low concentrations introduces the challenge of sorting the data to identify markers of value. In this paper, we report the development of pyAIR, a software tool for searching for volatile organic compounds (VOCs) markers in multi-group datasets, tailored for Thermal-Desorption Gas-Chromatography High Resolution Mass-Spectrometry (TD-GC-HRMS) output. pyAIR aligns the compounds between samples by spectral similarity coupled with retention times (RT), and statistically compares the groups for compounds that differ by intensity. This workflow was successfully tested and evaluated on gaseous samples spiked with 27 model VOCs at six concentrations, divided into three groups, down to 0.3 nL/L. All analytes were correctly detected and aligned. More than 80% were found to be significant markers with a p-value < 0.05; several were classified as possibly significant markers (p-value < 0.1), while a few were removed due to background level. In all group comparisons, low rates of false markers were found. These results showed the potential of pyAIR in the field of trace-level breathomics, with the capability to differentially examine several groups, such as stages of illness.

A multiple-method comparative study using GC-MS, AMDIS and in-house-built software for the detection and identification of "unknown" volatile organic compounds in breath.

The human respiratory system is a highly complex matrix that exhales many volatile organic compounds (VOCs). Breath-exhaled VOCs are often "unknowns" and possess low concentrations, which make their analysis, peak digging and data processing challenging. We report a new methodology, applied in a proof-of-concept experiment, for the detection of VOCs in breath. For this purpose, we developed and compared four complementary analysis methods based on solid-phase microextraction and thermal desorption (TD) tubes with two GC-mass spectrometer (MS) methods. Using eight model compounds, we obtained an LOD range of 0.02-20 ng/ml. We found that in breath analysis, sampling the exhausted air from Tedlar bags is better when TD tubes are used, not only because of the preconcentration but also due to the stability of analytes in the TD tubes. Data processing (peak picking) was based on two data retrieval approaches with an in-house script written for comparison and differentiation between two populations: sick and healthy. We found it best to use "raw" AMDIS deconvolution data (.ELU) rather than its NIST (.FIN) identification data for comparison between samples. A successful demonstration of this method was conducted in a pilot study (n = 21) that took place in a closed hospital ward (Covid-19 ward) with the discovery of four potential markers. These preliminary findings, at the molecular level, demonstrate the capabilities of our method and can be applied in larger and more comprehensive experiments in the omics world.

Instantaneous monitoring of free sarin in whole blood by dry blood spot-thermal desorption-GC-FPD/MS analysis.

Dry blood spot (DBS), a micro whole-blood sampling technique, enables rapid and self-blood collection; it is stable and economical. Currently, DBS filters require various sample preparation procedures specifically tailored for the target compounds, which are followed by GC-MS or LC-MS analysis. However, the small amounts of blood make the approach analytically challenging, mostly in terms of sensitivity and quantification. Herein, we introduce a new DBS concept for GC-compatible volatile to semi-volatile compounds in which DBS is directly coupled with thermal desorption analysis, thus eliminating time consuming treatments. Furthermore, to stabilize the target compound over the sampling DBS substrate, a commercial filter based on an extremely efficient trapping adsorption phase, styrene-divinylbenzene (SDVB), is first used. The performance of the new SDVB-DBS concept was demonstrated herein for monitoring the most volatile chemical warfare agent, sarin, which might be present in blood and the detection of which is usually challenging due to its rapid metabolism. This study encompasses adequate sampling and analysis method parametrization and validation, leading to a detection sensitivity of 100 pg sarin per 30 µL whole blood in 5-day-old samples, with a linear dynamic range of two orders of magnitude, adequate precision, and acceptable accuracy. Applying the method to an in-vivo mouse intranasal exposure experiment (3LD50 GB) enabled the successful detection of 25-90 ng mL-1 free sarin in blood samples drawn 2 min after exposure. The method's performance clearly emphasizes the potential of the new concept in "freezing the clock" for reactive whole blood media in pharmacokinetics and pharmacodynamics studies, as well as in applications in which informative and reliable monitoring of unstable target compounds and biomarkers is desired.

[HOW DOES A NEW MEDICAL PROFESSION COME TO LIFE?]

AIMS: In our article we seek to describe the initiation of new medical specialty fields in Israel, including the different considerations in the decision-making process and recent trends. BACKGROUND: Physicians' specialization by postgraduate training and specialty certification satisfies the publics' need for high quality medical treatment, supports medical institutions confidence in their medical staff qualifications, guarantees physicians' social accountability and is also linked with positive medical results. Nevertheless, fragmentation of medicine enfolds a hazard to continuity of medical care and loss of holistic perspectives, as well as hazards of systematic malfunction such as brain drain in vital basic specialties and high costs. METHODS: Information was gathered from the protocols of relevant deliberations conducted at the Scientific Council of the Israeli Medical Association, dealing with the initiation of new medical specialty fields and from relevant regulations. Numerical data was extracted from the IMA database. The information gathered was qualitatively analyzed by Template Analysis. RESULTS: Over two decades ranging between 1999 and 2019, three new medical specialties were initiated, including Emergency Medicine (1999), Pain Relief Medicine (2008) and Palliative Medicine (2012). The initiation of two other specialties, Invasive Neuro-Radiology and Metabolic Diseases, is still under process. The field of Child Development has joined an existing specialty in Pediatric Neurology, becoming a new specialty in Pediatric Neurology and Child Development in 2008, a new route for Pediatric Emergency Medicine branched out of Emergency Medicine (2008) and a new route for Pediatric Rheumatology branched out of Rheumatology (2013). We describe the different considerations taken into account, the prerequisites to initiation and milestones of the process, including grandfathering and accreditation. CONCLUSIONS: The trend of specialization in more medical fields will probably continue in the foreseen future. It will therefore continue to challenge the Scientific Council of the Israeli Medical Association with complex decision-making regarding the initiation of new medical professions or new routes within exciting professions.

[POSTGRADUATE MEDICAL EDUCATION ACCREDITATION IN ISRAEL].

INTRODUCTION: Accreditation of Post-Graduate Medical Education permits medical institutions to train residents, allowing them to achieve specialist certification. An accreditation system usually employs several tools such as site-visits, information gathering and occasionally self-evaluation, to determine adherence to pre-defined standards. The Scientific Council of the Israeli Medical Association is entrusted by law on this accreditation system in Israel. In our article, we briefly review the Post-Graduate Medical Education accreditation system in Israel and a number of pivotal challenges faced by the Scientific Council in this field in the 21st century. These challenges include the adaptation to different medical settings such as community based clinics and medical arrays, the adaptation of tools used for accreditation, new methods for up to date information gathering and updated structure of site-visit teams. A significant future challenge will be adapting the accreditation system to the new Competency Based Medical Education model of residency promoted in Israel by the Scientific Council.

Dry Blood Spot sample collection for post-exposure monitoring of chemical warfare agents - In vivo determination of phosphonic acids using LC-MS/MS.

Phosphonic acids are the direct and immediate metabolites of organophosphorus chemical warfare agents (OP-CWAs). Accordingly, their detection serves for evaluating exposure to OP-CWAs in a terror or war scenario. After exposure, phosphonic acids are present in the blood; however, blood drawing must be carried out by medical personnel, hence the number of samples that can be drawn in a mass-casualty event is limited. Herein, we describe a new approach developed for the determination of phosphonic acids in blood using Dry Blood Spots (DBSs) on a filter paper. The method is based on a simple sample preparation protocol, followed by LC-MS-MS targeted (MRM) analysis. The detection limits of Soman (GD), Cyclosarin (GF) and VX metabolites in whole blood were as low as 1 ng/ml, while the detection limits were 0.3 ng/ml for the GF metabolite and 0.5 ng/ml for the Sarin (GB) metabolite. Good recoveries were obtained in the range of 1-100 ng/ml for GB and GD metabolites, and 3-100 ng/ml for GF, VX and RVX metabolites, with a linear response (R2 = 0.99). The method has proven to be reliable even with DBS samples stored up to 35 days at room temperature before analysis. This method was implemented in a 24 h time-course determination of the Sarin metabolite in an in - vivo experiment, after rat exposure to 1 LD50 of Sarin. This technique is simple, rapid, sensitive, robust, long lasting and compatible with field collection and storage; hence, it can serve for large-scale sampling and reliable monitoring of potential OP-CWAs casualties. Since DBS sampling is amenable to nonprofessionals, including self-sampling, this technique is highly suitable for mass-casualty incidents.

[Mentoring newly appointed department heads--a new project review and initial findings].

BACKGROUND: In Israel, the training of a department head is based mostly on clinical and professional aspects and often does not include any training in other facets of management he or she will encounter. The newly appointed department head is expected from the start to deal with many diverse tasks, and is exposed to great physical and emotional stress. The Israeli Medical Association, taking note of this situation, initiated a mentoring program for newly appointed heads of medical departments, clinics and units. This article seeks to present a preliminary description of our experience with this mentoring project, in which senior managers mentor novices in the position. METHOD: An announcement of the new project was sent to both senior and beginning managers, detailing the project's goal. The project's content and structure were determined together with the participants, mentors and mentees. The values attending the project were delineated as full and genuine partnership, attention to needs, and personal choice of the mentee. Basic guidelines, adaptive to modification according to personal preferences, were developed based on these values. Though not readily assumed, our decision to allow mentees to choose their mentors was found to be suitable for this project. All participants, mentors and mentees, were asked to complete feedback forms in preparation for the final gathering of the group. RESULTS: The first session of the project included 8 mentor and mentee couples. Feedback indicated a high suitability rate between mentor and mentee, which resulted in high levels of satisfaction among the mentees. Responses to feedback questionnaires depicted that the relationships between the mentors and mentees included trust, openness and a non-judgmental approach, which allowed mentees to share personal difficulties and develop plans to overcome them. Most mentees described the mentorship as providing leverage to personal growth. Mentors expressed satisfaction for the opportunity to contribute of their experience. CONCLUSIONS: The success of the first session and the satisfaction expressed by its participants serve as an indication that the project met an existing need of beginning managers. The successful cooperation between mentors and mentees, closely accompanied by the professional staff, proved that beginning managers are more than willing to work alongside senior managers and learn from their vast experience. The writers believe there is a true need for mentorship for managers in our health system. The method depicted in this project was found to be efficient at this point. The next sessions of the project will allow us to identify more ways to match and oversee the mentor-mentee couples.