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With the organizational stuff out of the way, I am wondering how many of you have grown to like the new world we live in. For me, going to the home office to work irritated me at first. The colleagues are all so far away, there is no chat at the water cooler, no conversations about work at lunch and so on. Then, as time passed, video chats with the team and the colleagues from other parts of the com-pany became the new social interactions.

The fact of having a commute-free way to get to the working place (a desk in an un-used living room) made my life better. I began to wonder if the time spent in my car driving to and from work was really necessary. the effectiveness of what my colleagues and I do at the home office seems to be at least up to par with the normal operation. Sometimes I feel that doing things that require focus are more easily done here than in the office that is shared with several colleagues.

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NMR

Software for Reaction Monitoring by NMR 32Acquisition and Processing of Time-Resolved 2D SpectraM. Urbańczyk1,2, et al.

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Programmable Phages for Medicine 35How Synthetic Biology Is Fuelling a New Generation of Phage TherapyM. Dunne1 and S. Kilcher1

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Photocatalysis in the Removal of Pharmaceuticals from Waste Streams 18The Need for Robust Analytical MethodsS. Murphy1, A. Morrissey2 and K. Nolan1

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Omics

Hunting Gut Microbiota Metabolites 25Elucidating the Metabolic Interplay with Their Human HostL. Conway1, M. Correia1 and D. Globisch1

Technology and Application of Gut-On-Chip 28Gut Epithelial Mimicking in Integrated Sensor Platforms L. Giampetruzzi1, et al.

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Editorial

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Chromatoraphy and Separation

Determination of Intra- and Extra-Cellular Vitamin C Dynamics 7A Simple, Label-Free Chromatographic Technique for Studying Vitamin CT. Miyazawa1, A. Matsumoto1,2 and Y.Miyahara1

Universal Carbon Detector for High-Performance Liquid Chromatography 10Quantitative Analysis without the Analyte StandardsS.Ohira

Ion Chromatography in Water Analysis 12Application in Analysis of Anions in Water and Waste WaterK.-H. Bauer1 and T. Gluhak1

Mass Spectrometry

Back to the Roots in Disinfection 16Ion Chromatography Helps to Understand the DetailsM. Rudolph1, et al.

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G.I.T. Laboratory Journal 2/2020

Advanced Biotechnology SeriesTo reflect the substantial advances in all areas of biotechnology, Sang Yup Lee, Jens Nielsen, and Gregory Stephanopoulos have joined forces as the editors of Wiley-VCH book series, Advanced Biotechnology. This series covers all pertinent aspects of the field and each volume is prepared by eminent scientists who are experts on the topic in question.

Industrial Biotechnology:MicroorganismsChristoph Wittmann, James C. Liao

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ISBN: 978-3-527-34075-0656 pages | February 2017

Advanced Biotechnology

Edited by Toshiomi Yoshida

Applied Bioengineering

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Innovations and Future Directions


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Edited by Christina Smolke

Synthetic Biology



Parts, Devices and Applications


Edited by Gyun Min Lee and Helene F. Kildegaard

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Cell Culture Engineering: Recombinant Protein ProductionGyun Min Lee, Helene Faustrup Kildegaard

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Micro- and Nanosystems for Biotechnology



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Industrial Biotechnology



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Background

Vitamin C is one of the most fundamental nutrients to sustain life. Distributed ubiq-uitously and the most abundant of all vi-tamins (vitamin A, B, C, D, E and K), it plays a vital role in regulating redox bal-ance in the body. [2] Vitamin C involves several vitamers that are inter-convertible depending on the redox state, among

which ascorbic acid (ASC) and its oxi-dized form dehydroascorbic acid (DHA) represent two dominant species. [3] ASC is oxidized in the extracellular space by reactive oxygen species (ROS), producing ascorbate radical, which can be oxidized to DHA. Unlike ASC, DHA is transported via glucose transporters (GLUTs). Among 12 different GLUTs, GLUT1 and GLUT3 have higher affinity for DHA than for glu-

cose. [4] DHA is taken up by cells via GLUTs or degraded to 2,3-l-diketoglu-tonate (2,3-DKG), which is further de-graded into oxalic acid and threonic acid.

The spatiotemporal pattern of this vita-mer pair underlies both specificity and kinetic aspects for several important cel-lular events and, therefore, quantitative analysis of their inter-conversion is of continuous research interest. A debate on high-dose (at the millimolar scale) vita-min C cancer therapy may best illustrate the importance of accurate determination of the dynamics of the intracellular DHA–ASC pair. That is to say, the contradictory clinical data (some studies have indicated anticancer activity of vitamin C while oth-ers have shown little effect [5]) have, at least in part, been attributed to differ-

Determination of Intra- and Extra-Cellular Vitamin C Dynamics.A Simple, Label-Free Chromatographic Technique for Studying Vitamin C

▪ Taiki Miyazawa1, Akira Matsumoto1,2, Yuji Miyahara1

The redox-sensitive inter-conversion of ascorbic acid (ASC) and its oxidized form dehy-droascorbic acid (DHA) in intracellular and extracellular environments is of exceptional interest at the forefront of metabolomics and pharmaceutical research, including high-dose vitamin C cancer therapy. A chromatographic protocol to instantly determine these vitamers in both forms from cellular extracts, without any labeling or pretreatment has been reported earlier. [1]

Cellular uptake of dehydroascorbic acid (DHA, purple color) via glucose transporter 1 (GLUT1, green color) and accumulation of its reduced form, ascorbic acid (ASC, blue color) in the cell. Here, a chromatographic protocol to instantly determine these vitamins in the cell is reported.

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ences in dose, implying the existence of a threshold vitamin C value at the millimo-lar concentration range exerting effective cytotoxicity, which is only achievable via intravenous administration, not via oral administration. High-dose vitamin C kills cancer cells by increasing oxidative stress via two controversial possible mecha-nisms (Fig. 1). In the first proposed mech-anism, extracellular H2O2 directly kills cancer cells by generating •OH via the Fenton reaction (Fig. 1, Hypothesis (1)). [6] Alternatively, in the second possible mechanism, DHA was recently reported

to efficiently enter cells via GLUT1 and consume the intracellular reducing poten-tial of reduced glutathione (GSH) and NADPH, resulting in increased levels of intracellular ROS (Fig. 1, Hypothesis (2)). [7] If hypothesis (2) is feasible, high-dose vitamin C therapy could be extended to a variety of cancers presenting high GLUT1 expression and high glycolytic activity. A recent study showed that DHA treatment gave stronger anticancer effects in gastric cancer cells with high GLUT1 expression than in cells with low GLUT1 expression. [8] However, owing to the unstable nature

of DHA and the chemical and biological equilibrium between ASC and DHA, it is difficult to quantify the exact amount of DHA generated from ASC. Thus, exploring intracellular and extracellular DHA–ASC dynamics is of exceptional interest at the forefront of metabolomics, pharmaceuti-cal, and clinical research, including high-dose vitamin C therapy.

Challenges

Isotope labeling and mass-spectroscopic techniques are the gold-standard methods for monitoring the dynamics of ASC and DHA in biological samples. [7, 8, 9] How-ever, these methods need costly instru-mentation and complicated parameter op-timization. A more cost-effective and widely accessible alternative is based on high-performance liquid chromatography coupled with diode array UV detection (HPLC-DAD). However, the striking simi-larity in the size and polarity of DHA and ASC along with the fact that UV absorption of DHA is much weaker compared to ASC makes this technique poorly reliable, espe-cially for discrimination purposes. Alterna-tively, taking advantage of the poor UV sensitivity of DHA, reductant pretreatment using glutathione or dithiothreitol to con-vert DHA into ASC has been widely ap-plied; the initial amount of DHA in the sample extract can then be estimated from the increase in the total vitamin C amount (ASC + DHA). However, this indirect assay (“reduction method”) lacks true specificity and is prone to interference by other fac-tors, such as biological compounds and the conditions of the reduction reaction. [10] It is only feasible under the assumption of quantitative DHA–ASC conversion, which is not always the case; in fact, it has been found that the presence of a widely used vitamin C stabilizer (metaphosphoric acid: MPA) and variation of the pH can severely interfere with the reductant’s efficiency. [1]

Solution

A modified and remarkably simple HPLC-DAD protocol has been that enables, to our knowledge for the first time, simulta-neous absolute quantitative measurement of the DHA–ASC pair dynamics in the in-

Fig. 1: Two controversial mechanisms for the vi-tamin C cytotoxicity. Hypothesis 1: extracellular H2O2 derived from ascorbyl radical production, directly kills cancer cells by generating •OH via the Fenton reaction. Hypothesis 2: DHA efficient-ly enters cells via GLUT1 and consume the intra-cellular reducing potential of reduced glutathi-one and NADPH, resulting in inhibition of ATP production and cell death.

Fig. 2: Flow chart of (A) extraction from the cells and (B) analytical conditions for determination of cel-lular ASC and DHA by HPLC-DAD. [1]

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tracellular and extracellular environment, thus eliminating the abovementioned re-ductant pretreatment (“reduction meth-od”). [1] Our protocol (hereinafter re-ferred to as the “direct method”) commences with a cellular vitamin C ex-traction process using MPA, a commonly used stabilizer during vitamin C extrac-tion from food samples (Figure 2). [1, 10, 11] This treatment not only ensures the stability of vitamin C in the sample but also facilitates the simple removal of pro-teins from samples by centrifugation. Of note, there are no other reported proto-cols harnessing MPA as the cellular vita-min C extraction reagent be-cause of its severe interference with the “reduction method”; in-stead, existing protocols usually involve organic solvents as the extraction media. The MPA-com-patible and organic solvent-free protocol, hence, provides strik-ing advantages over the “reduc-tion method” in terms of the ro-bustness of measurement, sample storage, and ease of pu-rification. It has been demon-strated that this “direct method” can be readily applied to in vitro assay both on erythrocytes, the most extensively studied target, as well as pancreatic cancer cell line (MIA PaCa-2), to the au-thors’ knowledge the first-ever study on nucleated cell types, to trace in detail their GLUT1 (glu-cose transporter)-dependent or DHA-specific cellular uptake and, concomitantly, time- and dose-dependent intracellular conversion into ASC. [1] Yun et al. recently reported oxidative stress synchronized with intra-cellular DHA–ASC conversion, which eventually causes gluta-thione depletion, as a proposed modality for cell death, support-ing Hypothesis (2). [7, 8] Indeed, also such intracellular ASC-in-duced cytotoxicity was observed. Of note, any preceding reports only provided up to 30 minutes of kinetic information. In con-trast, owing to the simplicity of our “direct method”, the re-searchers were able to readily continue the monitoring for hours, the timescale over which the intracellular ASC conversion takes place in connection with the resultant cytotoxicity. There-fore, the presented technique should aid in providing quanti-tative bases for those therapeu-tic approaches.

Affiliations

1 Institute of Biomaterials and Bioengi-neering, Tokyo Medical and Dental Uni-versity, Tokyo, Japan2 Kanagawa Institute of Industrial Science and Technology (KISTEC-KAST), Kawasa-ki, Japan

ContactAkira MatsumotoTokyo Medical and Dental University Tokyo, [email protected]

Related Articles: http://bit.ly/GLJ-Food

References: http://bit.ly/GLJ-Matsumoto

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Background

Chemical analysis is important for envi-ronmental, biological and industrial themes. Quantitative analysis is strongly recommended for the understanding and discussion of various phenomena. For quantitative analysis, calibration is re-quired for almost all instrumental analy-ses. Ideal standards for the calibration are highly stable and pure chemicals.

However, there are many compounds that are not commercially available. Further-more, being commercially available does not mean that the compound will have sufficient stability and purity. In other word, many kinds of compounds cannot be quantified because of the lack of an analytical standard of suitable quality. Moreover, calibration is a complex pro-cess, especially in chromatography, which can analyze many analytes with a single

injection. Using a calibration-free detec-tion system for chromatography, a univer-sal detector, is strongly recommended.

Universal Carbon Detector for High-Performance Liquid Chromatography

The universal carbon detector (UCD) has been proposed to achieve calibration-free quantification of analytes. In this system, analytes eluted from the separation column are quantitatively converted to CO2 then de-tected by means of diffusion scrubber col-lection followed by conductivity detection. The obtained CO2 signals are directly relat-ed to the number of carbons in the analyt-es. The oxidation of analytes is performed using acidic persulfate coupled with a UV-lamp. This oxidation procedure is the same as that used for total organic carbon analy-sis. Under the optimized conditions, many kinds of organic compounds, including car-boxylates, amino acids, sugars, hard to oxi-dize compounds (benzoquinone), and high-carbon-density compounds (sucrose), are quantitatively converted to CO2. Nitrogen and sulfur oxides generated from amino acids do not interfere at all with the present CO2 detection method.

Firstly, the developed detector has been applied to carboxylate analysis. Carboxyl-ate ions with linear carbon chain lengths from 1 to 5 were separated with ion exclu-sion chromatography and detected with the present system, the UCD, and a tradi-tional UV detector for comparison pur-poses. The obtained chromatograms and calibration curves are shown in Fig. 1. The chromatogram for the UCD shows a linear increase in peak area with the same concentration of the mixture because the UCD response is linear to the number of carbons in the analytes. On the other hand, the UV detector responses were not related to each other and may also be af-fected by eluent pH. The calibration curves show the difference between UV and UCD clearly. The calibration curve for UV de-pends on the kinds of carboxylates. How-ever, the UCD calibration curve is on a single line if the x-axis is carbon concen-tration in mmolC/L. Additionally, the line is the same as the line obtained with car-bonates as the standard. This clearly

Universal Carbon Detector for High-Performance Liquid ChromatographyQuantitative Analysis without the Analyte Standards

▪ Shin-Ichi Ohira

A universal carbon detector, which does not require analyte standards, has been devel-oped for high performance liquid chromatography. The new detector counts the carbon in the analyte based on the chemical conversion of the analyte to carbon dioxide. The obtained carbon dioxide is collected in ultra-pure water followed by conductivity detec-tion. The system has been successfully applied to the detection of carboxylates, sugars and formaldehyde in formalin. The system performance and capability will be reported in this article.

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shows that the UCD can quantitate organ-ic compounds with a single calibration curve obtained with the primary standard of carbonates.

The system was also applied to the anal-ysis of formaldehyde (HCHO) in formalin. The chemical purity is important for han-dling and usage. However, it is impossible to analyze the chemical purity with the an-alyte itself. Thus, the purity of chemicals is analyzed by techniques such as titration and quantitative NMR. Here, the purity of HCHO in formalin was evaluated using the UCD. Formalin is well known as the aque-ous solution of HCHO gas. However, the gas dissolved solution concentration depends on the storage conditions, such as tempera-ture, and pressure, and HCHO is an unsta-

ble compound. It is difficult to obtain a suit-able quality analytical standard for HCHO. Furthermore, formalin contains methanol as a stabilizer to avoid polymerization. Thus, purity evaluation via instrumental analysis can only be achieved with UCD. For comparison purposes, titration was also performed and the results are present-ed in Fig. 2. The separation of HCHO and methanol was achieved with an ion exclu-sion chromatography column with water as the eluent. In addition, primary stan-dard carbonates were separately analyzed for the calibration curve. The quantities ob-tained with UCD and titration agreed well.

The UCD system has also been applied to sugar analysis. Sugars show none or poor UV absorptivity. Thus, most sugar analysis was achieved with a refractive index (RI) detector. The RI detector can detect almost all compounds. However, the sensitivity is poor and strongly affect-ed by eluent composition and tempera-ture. Recently developed universal detec-tors, such as the ELSD and Corona CAD, have achieved highly sensitive sugar de-tection. However, these methods also de-tect inorganic compounds and desalting of samples is strongly recommended for accurate analysis. The UCD was applied to analyze sugar in juices. For compari-son purposes, an RI detector was also connected in series. The sensitivity of the UCD and RI detectors was compared with standard compounds. The limits of detec-tion of the UCD was 1/30~1/10 better than the RI detector. In the juice analysis, the results from the UCD and RI detector showed good agreement with a regres-sion slope of 1.02 and R2 = 0.998.

Conclusions and Outlook

The universal carbon detector achieved quantitative analysis without an analyte standard. If the present universal detector is combined with qualitative analysis such as (high resolution) mass spectrometry, the combined system could even measure un-known compounds. This kind of system can dramatically advance many kinds of sci-ence. In chemistry, reaction or equilibrium intermediates, for which we cannot obtain high-quality standards, can be analyzed. This will strongly help to understand and improve the reaction efficiency. In pharma-ceutical research, the search for new func-tional compounds will be effectively ad-vanced. When new kinds of functional compounds are sought from natural mate-rials, the compounds can be simultaneously qualified and quantified. This information will strongly support effective drug discov-ery. In health sciences, the understanding of the relationship between disease and metabolomics will be dramatically ad-vanced with the present UCD system. Many of this type of study use mass spectral pat-terns. The present UCD system will help to provide quantitative data. As described above, the newly developed UCD detector will open a new door on chemical analysis.

AcknowledgementsThe original paper was published in Ana-lytical Chemistry (doi: 10.1021/acs.analchem.7b04849) [1]. This project was funded by JSPS KAKENHI Grant Number JP23750089.

ContactDr. Shin-Ichi OhiraDepartment of Chemistry, Kumamoto University Kumamoto, [email protected]

Related Articles: http://bit.ly/GLJ-HPLC

References: http://bit.ly/GLJ-Ohira

Fig. 2: HCHO in formalin analysis. The chromatogram obtained with formalin shows two peaks for HCHO and CH3OH. The cali-bration was separately perfor-med with a primally standard, NaHCO3. Three of formalin solu-tions were analyzed with HPLC-UCD, and iodide titration. Two of the formalin was purchased with the certification. These numbers are compared in the bottom. The obtained with present UCD for HCHO and methanol were well agreed with other methods.

Fig. 1: The response comparison of UCD and UV detector. The chromatograms (a) obtained with the mixture solution of linear carbon chain car-boxylates, 10 mM ea. The calibration curves for UV detector and UCD are also shown. The cali-bration curve for UCD was plotted carbon con-centration (mmolC/L) vs. peak area.

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Routine Analysis

Since ion chromatography found its way into water laboratories the routine analysis of nitrate and sulfate presented one of the major problems in water analysis. The determination of nitrate in water by photometric analysis required the use of high con-centrated sulfuric acid to nitrify an aromatic compound. The analysis of sulfate was time consuming and labor intensive. In-stead, ion chromatography was a fast and easy method to deter-mine nitrate and sulfate in a large number of samples. Thus, the determination of fluoride, chloride, nitrate and sulfate by ion chromatography was the first well established IC method in wa-ter analysis. The anions were detected by conductivity. In some cases UV-detection and inverse UV-detection were used. The suppressor systems, necessary for sensitive measurements using conductivity detection, were improving. Nowadays the measure-

ment of concentrations in ppb-range is possible. Furthermore, the separation columns for anion determination are much more efficient in comparison to the beginnings of ion chromatography; a wide offer of different columns allows to solve nearly all sepa-ration problems in routine analysis.

Thus, it is possible to analyze bromide, chloride, fluoride, ni-trate, nitrite, o-phosphate and sulfate in one run (method ac-cording to DIN EN ISO 10304-1). By following the routine of the standard DIN EN 10304-3 it is possible to analyze chromate, io-dide, sulfite, thiocyanide and thiosulfate. Chlorite, chloride and chlorate are monitored according to DIN EN 10304-4 and bro-mate to EN ISO 15061, using separation columns with high ca-pacity. With these columns it is possible to combine the stan-dards to determine as many anions as possible in one run.

In cases where the analyte absorbs UV-light, an UV-detector is useful to ensure reliable results. Interferences with chloride can be reduced by using silver cartridges, interferences with sulfate by barium cartridges. C18 columns are sometimes helpful if the matrix contains a high amount of organic substances.

Special Methods

Alongside the method for routine analysis, several special meth-ods are applied in the laboratory of this article’s authors.

Waste water: Here, the specification is not a low determination limit but a wide linear working range. Thus, an Ion Chromato-graph without a suppressor system is used, leading to linear working ranges for chloride from 0.1 to 200 mg/L, nitrate from

Ion Chromatography in Water AnalysisApplication in Analysis of Anions in Water and Waste Water

▪ Karl-Heinz Bauer1 and Tatjana Gluhak1

Since the 1980’s, Ion Chromatography was developed into a powerful and sensitive tool for Ion Analysis. Nowadays, anion- and cation-chromatography is a well-established technique and used for the determination of a large number of ions in different matrices. As the presentation of an overview of the wide field of Ion Chromatography (IC) is clearly beyond the scope of this short paper, the book of Joachim Weiss [1] is strongly recom-mended. This paper focuses on the analysis of anions in water and waste water and aims to motivate to use this technique.

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0.5 to 300 mg/L and sulfate from 1 to 600 mg/L. By using an elu-ent with a high concentration of acetone the lifetime of the col-umn can be extended. No sample preparation with C18-cartridg-es is necessary, only filtration by a 0.45 µm membrane filter.

Cyanide: An easy way to measure cyanide is to deflate the cya-nide and absorb it in an alkaline solution. This solution is inject-ed in an IC-system with an alkaline eluent. Cyanide is quantified using amperometric detection with a silver working electrode (DIN 38406 Part 7).

Bromate: During water treatment by ozonization bromide can be oxidized to more toxic bromate. Due to the drinking water limit 10 µg/L and a quantification limit of 1 µg/L a very sensitive IC method is necessary here. As direct IC determination with conductivity detection is not sensitive enough, a post column re-action technique with UV-detection and a quantification limit of 0.5 µg/L is used (DIN EN ISO 11206).

Iodide: In some cases, the analysis of iodide in water and waste water samples is necessary. Due to its long retention time at room temperature the column temperature should be in-creased to higher temperatures (e.g. 55°C), so that iodide eluates much earlier and moves in front of sulfite and sulfate. An inter-fering peak can appear in waste water samples; thus, the iodide results which are achieved by conductivity detection have to be confirmed by UV-detection (at 226 nm).

Perchlorate: To monitor perchlorate in swimming pool water, river water or tap water a very sensitive IC method is required. Very low quantification limits (lower than 1 µg/L) are reached by hyphenation of IC and mass spectrometry. When IC and con-ductivity detection is utilized, a large volume injection should

be possible and a separation column with a high capacity should be used. When perchlorate has to be analyzed in waters with high matrix concentrations of chloride, nitrate and/or sul-fate different techniques to reduce the matrix influence can be applied. The authors recommend Ag- or Ba-cartridges to re-duce the matrix concentration of chloride and sulfate respec-tively, the column cut technique, or the reinjection technique. It is also possible to use the mathematical method and to calcu-late and subtract the baseline resulting from the interfering peak.

Trifluoroacetic Acid: Like perchlorate, the trifluoroacetic acid peak eluates in the shoulder of a matrix peak. Thus, in the case of high matrix concentrations and a corresponding interfering peak the reinjection technique to eliminate the interference is used. This allows for quantification limits in the range of 1 µg/L. The results of this technique are comparable to those that have been achieved by high resolution LC-MSMS.

Chromium(VI): When the sample contains higher concentra-tions of Chromium(VI) (> 100 µg/L), it can be determined using IC with UV-detection, based on the yellow color of chromate. In very low concentrations Chromium(VI) can be identified highly sensitive by post column reaction with 1,5-diphenylcarbazide (Draft: DIN 38405-D52), where quantification limits of 20 ng/L can be achieved. The same quantification limits can be reached by hyphenating IC with ICP-MS. Theoretically, this technique en-ables a Chromium(VI) – Chromium(III) speciation using EDTA as complexing agent for Chromium(III). However in “real” samples the concentrations of calcium-, magnesium- or iron-ions are too high to allow for a stable Cr(III)-EDTA complex.

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Sum Parameters

IC is also used to monitor sum parameters:Total Nitrogen (TNb): Normally the TNb is determined by high

temperature burning of the sample and quantified by fluorescent measurement. An alternative is the alkaline microwave digestion of the sample with the addition of potassium persulfate. During microwave digestion all nitrogen (inorganic and organically bound nitrogen) is transformed to nitrate, which can be quanti-fied by IC (EN ISO 11905-1). The potassium persulfate causes a high sulfate content; thus, a column and eluent have to be se-lected which permits a good separation of Nitrate and Sulfate.

AOX, AOF, AOCl, AOBr, AOI: The increasing sensitivity of the IC system facilitates the determination of organic bound halogens. Organic bound halogens are enriched on activated carbon, burned in a wet stream of oxygen, absorbed in water (or water with hydrogen peroxide) and measured using an IC-system (Combustion IC/CIC). When a natural activated carbon contain-ing sulfur is used, ultrapure water is sufficient to absorb the ha-logenes as halogenides. When a synthetic charcoal with a very low sulfur content is used, is the risk exists that the organic bound iodide is transformed to iodine (I2) and escapes. In this case an absorption solution containing hydrogen peroxide in necessary. Routine measurements of the AOI demonstrate that inorganic iodide is also adsorbed on activated carbon and mea-sured as AOI. Thus, the content of inorganic iodide should be controlled by an additional direct IC measurement of the sample. If the concentration of inorganic iodide is > 10 µg/L, a sample pretreatment by solid phase absorption is essential. Further-more, it was found that inorganic fluoride is also enriched on activated carbon (acid absorption, acid washing as AOX) in the cases of sample concentrations higher than 0.2 mg/L. If the ad-sorption is accomplished under neutral conditions inorganic flu-oride < 10 mg/L is tolerable. The CIC determination of the AOX yielded values comparable to the standard method (DIN EN ISO 9562). By applying CIC instead of the classical AOX determina-tion, more detailed information about organic bound chlorides, bromides and iodides can be gained. Last but not least, the par-allel use of conductivity and UV detection allows for reliable re-sults and excludes interfering peaks like those of fluoride and iodate.

Conclusion

Nowadays the ion chromatographic determination of anions is a well-established method in analytical chemistry. IC can be used for separation, sample preparation and quantification. IC is a re-liable and sensitive method with a good reproducibility, is fully automated and moderate in costs. IC can be used with different detectors (from conductivity to mass spectrometry) and many different separation columns are available. IC solves many prob-lems of daily lab work.

Affiliation1Hessenwasser GmbH & Co.KG, Zentrallabor, Darmstadt, Ger-many

ContactDr. Karl-Heinz BauerHessenwasser GmbH & CO. KGDarmstadt, [email protected]

Advanced Liquid Chromatography: http://bit.ly/adlichrom

References: http://bit.ly/GLJ-Bauer

Fig. 1: The AOF, AOCl, AOBr and AOI of the river Rhine at 3 different sam-pling dates. The high value of AOF in Rhine water was observed for the sample taken near the village Biebesheim.

Fig. 2: Correlation of the measured AOF with organic micro pollutants.

Fig. 3: Correlation with inorganic analytes and indicates that AOF is trans-ported by clay particles.

14 Chromatography

and Separation

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IC is often used in addition to other meth-ods such as ICP-MS or ESI-MS to monitor anions in aqueous systems [2]. Most com-monly the EPA methods 300.0 and 300.1 are applied [3,4], which use a sodium car-bonate eluent, a column separating the seven standard anions (F-, Cl-, Br-, NO2

-, NO3

-, SO42-, PO4

3-) as well as the halooxo anions, and a suppressor. In recent years, numerous improvements have been made to further optimize these methods and to improve the detection limits for inorganic DBPs [2]. In classical method develop-ment, not only the correct column materi-al must be selected, but also parameters such as temperature, flow rate and eluent composition must be optimized in order to achieve ideal selectivity, resolution and detection limit for the respective analyti-cal problem. In addition to the continuous optimization of existing methods and the combination of several measuring tech-niques, sampling conditions are crucial to

guarantee good comparability of the indi-vidual measurements. For this purpose, the compliance with standardized sam-pling protocols is an ideal instrument to obtain reproducible results from highly representative samples and to document their alteration during the measurement period.

Sampling

When it comes to sampling and storage for water analysis, best practice can be found in ISO 5667-1 and ISO 17025 [5,6]. Disinfection-related samples are prone to change quickly upon storage because of the high chemical reactivity. Several reac-tion paths can be followed depending on temperature, pH value and the presence of other compounds. The most frequent sources of error occurring during sam-pling are contamination of the sample by dirty sample vessels (especially traces of

organic compounds or Cl-), cross-contam-ination during dilution and the loss of volatile substances due to incorrect stor-age. These volatile compounds can either be the disinfection reagents themselves (Cl2, HOCl, ClO2) or break-down products, such as O2. This typically occurs at low pH values and can lead to strong deviations in the analytical results. At higher pH val-ues, the disproportionation to Cl- and ClO3

- and ClO4- becomes fast, what not

only reduces the disinfection power but can render the solutions harmful for hu-mans and the environment. To prevent or at least reduce these changes, cooling of the samples and in particular exclusion of light are necessary. Also, the adjustment of the pH value by basic stable buffers such as phosphate-based systems can be effective.

Disinfectants

IC is ideally suited for the analysis of dis-infectants and DBPs, as special sample preparation for injection into the system is usually not necessary. The chlorine-based disinfectants (Cl2, ClO2, HOCl) and their chemistry have been intensively in-vestigated in Frankfurt in recent years and more effective methods for the detec-tion of the individual chlorine-derived species (Cl-, ClO-, ClO2

-, ClO3-, ClO4

-) have been developed. By optimizing various

Back to the Roots in DisinfectionIon Chromatography Helps to Understand the Details

▪ Michael Rudolph1, Sebastian Schneider2, Mathias Rößling1, Andreas Terfort1

In times of growing water scarcity and the formation of bacterial resistance against standard antibiotics [1], classical chemical disinfectants, such as chlorine dioxide, ele-mental chlorine and hypochlorite, experience a renaissance in water treatment. A prob-lem of these disinfectants is the formation of so-called disinfection by-products (DBPs), such as chlorate (ClO3

-) or perchlorate (ClO4-), which are undesirable and need to be

monitored. Ion chromatography (IC) is best suited for this type of analysis as it can be applied to various systems, such as drinking water, pools or waste water, combined with excellent detection limits (LOD) for important contaminants.

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16 Mass Spectrometry

Articles


parameters (Fig. 1), it was possible to re-duce the total IC run time for the detec-tion of ClO2

-, ClO3- and ClO4

- to ≤ 30 min-utes, whereby the individual detection limits could be improved significantly [7].

Remaining Challenges

A major challenge remains the detection of OCl-, as the corresponding acid, which is formed in the suppressor, does not readily dissociate (pKa = 7.54) and thus is undetectable in the conductivity sensor of the IC set-up. The authors are currently exploring methods, such as other kinds of detection principles as well as pre- and post-column derivatization reactions. This would open the opportunity for a com-plete anion analysis in a single IC run, what is also important for the automation of the developed analyses. Thus, at pro-duction sites or disinfection plants, sam-ples should be taken at different stages of

the processes and measured in- or on-line for continuous quality control.

A question that has not really been ad-dressed yet is the analysis of bromine- and iodine-based disinfectants, with the latter being frequently used in the medi-cal and pharmaceutical sector. The oxo anions of bromine and iodine are much more reactive than the respective chloro-oxo anions and thus are prone to decom-position during sample transport and storage. A severe problem is bromate, which has been identified as canceroge-neous and nephrotoxic [8]. The detection of bromate is usually carried out by HPLC or photometrically [8]. Analysis of iodide and iodate can be accomplished by IC [9], but the detection of periodate species still

needs further improvement, as periodates are undesirable by-products during pro-duction and are highly dangerous for the environment and health. In order to be able to exclude contamination by such substances, the development of a new de-tection method is indispensable. The problem is much more complex than in the case of the chlorine-based systems, because in addition to metaperiodate (IO4

-

) further periodates like mesoperiodate (IO5

3-) have to be detected and character-ized individually. To solve this problem, the development of new column materials and detection methods for periodates is necessary. The ultimate goal must be to find a qualified and validated IC method for all halogen species in disinfectant-containing samples.

Affiliations1Institute of Inorganic and Analytical Chemistry, Goethe University Frankfurt, Frankfurt, Germany2Umicore, Hanau, Germany

ContactProf. Dr. Andreas TerfortInstitute of Inorganic and Analytical ChemistryGoethe University FrankfurtFrankfurt, [email protected]

Further articles on water analysis: http://bit.ly/GLJ-water

References: http://bit.ly/GLJ-Rudolph

Fig. 1: Optimization of the separation of chlorooxo anions on a Metrohm IC 883 Plus with MSM anion suppressor. The concentration of anions was 1 mmol L-1 each. 1) Cl-, 2) ClO2

-, 3) ClO3-, 4) ClO4

-. The ar-rows and the bar indicate the position of the ClO4

-. While a simple increase of temperature, a typical strategy to reduce retention times, leads to a extreme broading of the ClO4

- peak, a multi-parameter optimization led to excellent peak separation in a significantly shorter time [7].

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TiO2 is an effective photocatalyst and is cheap, safe and regularly used in formu-lations in the pharmaceutical, food and cosmetic industries. The photochemical process for TiO2 (P25 TiO2 will be dis-cussed here) is outlined in figure 1 where under UV illumination of TiO2 the promotion of an electron from the va-lence TiO2 band to the TiO2 conduction band occurs. Upon promotion of an elec-tron, a charged separated state is formed with an electron in the conduction band and a hole in the valence band. The elec-tron in the conduction band is then ca-pable of reacting with dissolved oxygen to generate the superoxide radical anion while the hole in the valence band can react with water to generate the highly reactive oxidant hydroxy radical. It is these two oxidants that will mineralise organic compounds – that is to com-pletely convert the target pharmaceuti-cal and the formed intermediate com-pounds in the reaction into CO2, NO3

-, SO4

2- and H2O. Complete mineralisation of pharmaceuticals is necessary as, while less is known about the ecotoxicity of many pharmaceuticals once released into the environment, even less is known about the intermediate products poten-tially formed during the photocatalytic process. If incomplete mineralisation oc-curs (i.e. particularly stable intermediate compounds are formed) the potential lies therein for these compounds to bio-accumulate in the environment creating a new potentially greater environmental concern [3].

Unfortunately, many reports in the area of AOP development with TiO2 (in-cluding dyes and pesticides) fail to detail intermediate formation of the target ana-lytes during the photocatalytic process, relying mainly on UV-visible spectro-scopic analysis of the reactions, leaving open the possibility that complete miner-alisation has not occurred. Elucidating the structure and fate of both the target analyte and the intermediates in the photocatalytic process is of extreme val-ue in both the development of an effi-cient photocatalytic process and for later quality control for remediating real waste streams.

Photocatalysis in the Removal of Pharmaceuticals from Waste Streams The Need for Robust Analytical Methods

▪ Sharon Murphy1, Anne Morrissey2, Kieran Nolan1

In recent years, the presence of pharmaceuticals in the environment has become a seri-ous cause for concern and the problem is continuing to grow with the on-going devel-opment of more potent and more metabolically resistant drugs. A significant body of research has been devoted to tackling this issue, including the employment of ad-vanced oxidation processes (AOPs) to remove pharmaceuticals from waste streams [1]. Of these AOP processes, those which use the photocatalyst TiO2 are some of the most studied for pharmaceutical abatement [2].

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Fig. 1: Overview of the photocatalytic process involving TiO2 with Famotidine.


Articles


LC-MS/MS Method Development and Monitoring of Oxidation Intermediates

Herein it is outlined a simple user-friendly LC-MS/MS protocol, with the ulcer drug fa-motidine as target analyte, to analyse and identify intermediate formation and eluci-date the fate of famotidine under photocat-alytic conditions. The analytical equipment required are standard LC and LC-MS/MS (low resolution MS), equipment that is common to most modern laboratories [4].

The initial photocatalytic system select-ed for the degradation of the ulcer drug famotidine with TiO2 under UV irradiation used a medium pressure mercury lamp as light source in a pyrex immersion well. Famotidine was found to be stable to light >300 nm as proven by the minimal deg-radation achieved in control studies with UV alone light (photolysis). Additional control experiments showed that light is required to photocatalytically degrade fa-motidine and that a ‘thermal degradation’ does not occur or compete with photodeg-

radation. The first stage of the protocol was to optimize the photochemistry by measuring the disappearance of famoti-dine using UV-Vis spectroscopy and it was found that optimum conditions were: 0.1 g/320 mL of Degussa P25 TiO2 for photo-catalyst concentration with a concentra-tion of 0.083 mM of famotidine (UV-Visi-ble spectra shown in fig. 2a).

To further monitor these reactions a simple HPLC method was also developed for famotidine (tab. 1), in developing this method it has been kept in mind that the majority of intermediates being formed by oxidation will be more water soluble than famotidine therefore it has been tar-geted a retention time of greater than ten minutes for famotidine. Method develop-ment and validation was performed on a Varian Prostar HPLC-PDA and the opti-mized method using a Phenomenex PFP (Luna) C18 150 mm x 4.6 mm 5 μm par-ticle size with a mobile phase of 9% methanol:water 0.1% formic acid a detec-tion wavelength at 265 nm and a run time of 15 minutes. Representative chromato-grams of a time study of the photocatalyt-ic degradation of famotidine are shown in figure 2b.

It is evident that within ten minutes of re-action time intermediates are being rapidly formed as famotidine is being consumed. It should also be noted that as predicted the observed intermediates have shorter reten-tion times than famotidine, unfortunately it would also appear that intermediate com-pounds are still present even after three hours under these conditions.

Table 1: LC Method

API Wavelength nm Mobile Phase Inj.Vol. μL Run Time (mins) tR (mins)

Famotidine 265 9% MeOH:H2O 0.1% F.A 20 15 10.5

Tab.2: LC-MS/MS Method Transfer and Re-optimi-zation. Standards for each pharmaceutical and samples from a previous photocatalytic experi-ment were employed such that the retention times of intermediates could be adjusted.

Settings Famotidine

Capillary, V - 4500

End Plate Offset, nA - 500

Nebuliser, psi 50

Dry Gas, L/min 8

Dry Temp, °C 325

Skim 1, V 15

Skim 2, V 8.1

Cap Exit, V 65

Cap Exit Offset, V 50

Octopole, V 5

Octopole Δ, V 2.05

Oct RF, Vpp 177.1

Lens 1, V - 2.2

Lens 2, V - 49.5

Trap Drive 50.1

Fig. 2: (a) UV-vis spectroscopic analysis of the photocatalytic degradation of Famotidine with 0.1 g P-25 TiO2. [FAM] = 0.083 mM, TiO2 = 0.1 g, Time = 5 h. (b) HPLC chromatogram of Famotidine with TiO2/UV at 0mins, 0mins Pads, 5, 10, 20, 30 and 40 mins. [FAM] = 0.083 mM, P-25 TiO2 0.1 g/320 mL. (c) HPLC chro-matogram of Famotidine with TiO2/UV/H2O2 (5 mM) at 0 mins, 30, 40, 60, 120 and 180 mins. [FAM] = 0.083 mM, H2O2 = 5 mM, TiO2 P-25 0.1 g/320 mL (d) HPLC chromatogram of Famotidine at 0 mins (blue) and 180 mins (red) showing solar photocatalysis with Tetracarboxyphenylporphyrin-TiO2 composite.

Fig. 3: (a) Famotidine’s confirmed intermediates (degradation products), their fragments, retention times, molecular weight (b) proposed photocatalytic degradation pathway with TiO2 of FAM.


Articles


The next key stage of the process is the elucidation of the structures of the pre-dominant intermediates being formed. To achieve this goal the developed HPLC method was transferred to a Bruker LC-MS/MS system. At this point further opti-mization was required specifically longer run times (1 hour) which simply required the reduction of MeOH content in the mo-bile phase to 1% and the column switched to a 150 mm x 2.1 mm (5 μm) Phe-nomenex PFP (Luna) C18. MS ion signals were also optimized using the Bruker sys-tem software automated optimization function. A series of standards from 0.1 μM to 100 μM were run and a standard curve was generated good linearity was obtained without the incorporation of the 100 μM standard, with an R2 value of 0.997 for famotidine (tab. 2-4)

(The optimum ESIMS settings for fa-motidine along with other method de-velopment data are in supplementary information- see online version of this article).

Once optimized the LC-MS/MS analysis of four separate famotidine photo-degra-dation experiments ,including one photo-catalytic experiment performed at a high-er concentration of 1 mM famotidine and 0.1 g/320 mL TiO2, were carried out and analyzed (The higher concentration ex-periment was performed in order to see intermediates which may be formed in low concentrations.) Table 5 (shown in the article online) presents all of the inter-mediates found in each chromatogram from these experiments including their retention times, MS/MS fragments and their molecular weights. At an early stage in data analysis some of the ions found by LC-MS/MS analysis were ruled out as in-termediates for a number of reasons: 1) the ion was present at too low an intensi-ty, 2) the ion was present at a good inten-sity but did not give a successful MS/MS

Tab. 3: LC-MS/MS method.

API Famotidine

Column PFP 150 mm x 2.1 mm 5 μm

Mobile Phase A 1:99 MeOH:H2O 0.1% F.A

Mobile Phase B N/A

Injection Volume µL

10

Pump Composi-tion (A:B)

N/A

Wavelengths scanned (nm)

265, 205, 254, 280, 300

Flow Rate (mL/min)

0.15

Run Time (mins) 63

Retention Time tR (mins)

47

Tab. 4: Table of MS parameters for Famotidine DI-MS and LC-MS Studies.

FAM DI-MS FAM LC-MS

MODE

Mass Range Mode STD/Normal STD/Normal

Ion Polarity Positive Positive

Ion Source Type ESI ESI

Current Alternating Ion Pol N/A N/A

Alternating Ion Polirity N/A N/A

DETECTOR AND BLOCK VOLTAGES

Multiplier Voltage 1750 V 1850 V

Dynode Voltage 7 kV 7 kV

Scan Delay 0 µs 0 µs

Skimmer 1 Block 100 V 100 V

Skimmer 2 Block 300 V 300 V

TUNE SOURCE

Trap Drive 32.9 50.1

Skim 1 34.9 V 15 V

Skim 2 6 V 8.1 V

Octopole RF amplitude 150 Vpp 177.1 Vpp

Octopole delta 2.4 V 2.05 V

Lens 1 -5 V -2.2 V

Lens 2 -60 V -49.5 V

OCtopole 2.49 V 5 V

Capillary Exit 108.2 V 65 V

Cap Exit Offset 73.2 V 50 V

HV endplate Offset -500 V -500 V

Current Endplate 237.671 nA 1327.5 nA

HV Capillary 4000 V 4500 V

Current Capillary 41.605 nA 91.938 nA

Dry Temp (measured) 302°C 327°C

Dry Gas (measured) 5 L/min 8.01 L/min

Nebuliser (measured) 15.14 psi 50.5 psi

TRAP

Scan Begin 50 m/z 50 m/z

Scan End 800 m/z 800 m/z

Averages 5 Spectra 5 Spectra

Charge Control ON ON

ICC Target 20000 20000

ICC Actual 6278 491

Accummulation Time 1814 µs 50000 µs

Max. Acc. Time 50000 µs 50000 µs

MS/MS MANUAL MODE

Fast Calc ON ON

ISTD OFF OFF

MS/MS AUTOMATIC

Auto MS/MS OFF ON

ROLLING AVERAGING

Rolling ON 2cts OFF

COMPRESSED SPECTRA

Compressed Spectra OFF OFF


Articles


fragmentation or 3) the ion appeared at erratic retention times.

How LC-MS/MS Monitoring Aids the Process Optimization

The final stage of the protocol involves the rationalization of a degradation route for the photocatalytic degradation path-way of famotidine. Outlined in figure 3 is a proposed pathway of degradation of fa-motidine based on the predominant inter-mediates determined by LC-MS/MS.

At this point a few things become clear from both the LC and LC-MS/MS analy-sis; first oxidative degradation is pre-dominant with very little photoreduction processes being observed and perhaps most importantly complete mineralisa-tion of famotidine does not occur after three hours of reaction time with TiO2 under UV irradiation. It is important to highlight that if UV-visible spectroscopy was to be used alone as the analytical tool (fig. 2a) to monitor this reaction it maybe concluded that complete conver-sion of famotidine in the absence of in-termediates has been achieved. This fact highlights the necessity to monitor these reactions using LC techniques. On real-ization of incomplete mineralization, ad-justments were made to the reaction conditions to achieve complete mineral-ization of famotidine. To achieve this goal a supplementary oxidant, hydrogen peroxide, was added to the reaction mix-ture with TiO2. The optimum hydrogen peroxide concentration required was 5 mM concentration (it should be noted that peroxide with UV light in the ab-sence of TiO2 was found to be ineffective for the degradation of famotidine) at this concentration complete removal of fa-motidine and intermediates was achieved after 3 hours of irradiation. The chromatographic results of this experi-ment are shown in figure 2c. It is quite apparent that the addition of peroxide enhances the photooxidative process, fa-motidine is completely consumed after 5 minutes – compared to 40 minutes with-out peroxide addition - and the quantity of observed intermediates is greatly re-duced (due to the fast rate of reaction). From a process perspective the addition of a second reagent is not ideal, since it is an additional cost and the peroxide will need to be quenched post reaction complicating the process however, with-out the addition of peroxide mineralisa-tion of famotidine is incomplete thereby

leaving potential culprit compounds un-reacted that will be released into the en-vironment.

Using LC-MS/MS to Evaluate New Photocatalyst

After developing an optimized photocata-lytic process for the degradation of fa-motidine with TiO2/peroxide we explored the possibility of developing a new cata-lyst that could work effectively in the visi-ble region of the solar spectrum. Such a catalyst would be more energy efficient than TiO2 (high energy UV light only) and could be potentially used with sun light – a true green catalyst. A porphyrin/TiO2 catalyst was developed and screened for activity under visible light conditions, so-lar light and UV light conditions (same conditions as described above) [5]. Again, based on UV-Visible spectroscopy famoti-dine was consumed however, when the above described LC method (fig. 2d) was used it was found that the degradation pathway was completely different than the TiO2/UV process found with famoti-dine. A dominant intermediate correlating to the sulfoxide of famotidine was pro-duced, as a matter of fact this was the ‘product’ of the reaction under all light sources screened. Once observed by LC the predominant product was then identi-fied by the LC-MS/MS method described above and was confirmed as the sulfoxide of famotidine at a molecular weight of 353 (fig. 3 for structure). From the ana-lytical data obtained it becomes apparent that the predominant oxidant that is gen-erated by this composite catalyst is the superoxide radical anion, which is inef-fective on its own to mineralize famoti-dine, demonstrating that visible light driv-en porphyrin-TiO2 composites will not be effective for the photocatalytic mineralisa-tion of famotidine and other compounds (it was found that the porphyrin/TiO2 composite had no degradative effect on two other pharmaceuticals Tamsulosin and Solifenacin that were screened under the same conditions). However, the resul-tant product formed under these condi-tions may be of interest with respect to selective sulfoxidation in synthetic chem-istry.

Conclusions and Outlook

In the application of any AOP for the re-moval of target analytes from waste streams it is essential that a proper meth-

od of analysis be developed to determine the fate of both target analyte and inter-mediates formed under the AOP condi-tions. If only simple analysis, such as UV-visible spectroscopy are used to evaluate these reactions, which is the case with many reports in the literature, proper op-timisation of the AOP process may not be achieved leading to the production of po-tential culprit products that maybe re-leased into the environment. Further-more, with a proper method of analysis it is also possible to rapidly screen new photocatalytic systems and evaluate their performance with respect to application and mechanism of action.

The area of photocatalysis using TiO2 based materials for the photochemical re-mediation of pollutants has been well in-vestigated since the 1990s however, most studies have been carried out under labo-ratory conditions typically with single an-alyte in water or with model waste water. For the area to prove itself as a viable tool for pollutant remediation, especially in in-dustrial waste streams, more screening on real waste water must be undertaken. The need for proper analytical monitoring of these processes in real waste water will even take on more importance in the fu-ture. The presence of multiple solutes (in-cluding common salts, small organics) that will also be present with the target pollutant in real waste water has the po-tential to complicate the photocatalytic process by introducing new possible reac-tion routes between formed intermediates and other solutes present in the waste water. Furthermore, the possibility of cat-alyst fouling by solutes present in the waste water matrix may also be signifi-cantly detrimental to the application. The only way to understand and overcome these potential problems is to have robust analytical methods that can reliably mon-itor the photochemistry by identifying re-action intermediates and by using this mechanistic information can proper ac-tion be taken to overcome these problems arising in the photodegradation process.

Affiliations1School of Chemical Sciences, Dublin City University, Dublin, Ireland2School of Mechanical and Manufacturing Engineering, Dublin City University, Dub-lin, Ireland

ContactDr. Kieran NolanSchool of Chemical SciencesDublin City UniversityDublin, [email protected]

More on water analytics: http://bit.ly/GLJ-water

Additional information and references: http://bit.ly/GLJ-Nolan


Articles


The Importance of VOCs

Volatile Organic Compounds (VOCs) represent a wide range of small (molecular weight 50-200 Dalton) stable molecules, vola-tile at ambient temperature, often lipophilic with a high vapour pressure [1]. Volatile composition in all biological matrices is composed by two important and heterogeneous fractions: en-dogenous and exogenous compounds. The endogenous one re-gards molecules which are produced by cell metabolism (in vivo and/or in vitro) and could potentially be markers of physiological and pathological conditions. Instead, exogenous VOCs originate from current or previous environmental exposures [2-3] which indicate the substances to which we are daily exposed, and

which induce alterations at our normal metabolism; other exter-nal sources are related to lifestyle, diet, microbiome, drugs. What changes is the cause-effect relationship: in the first group, VOCs are the effect; in the second, the cause.

Analysis of endogenous compounds of cell culture or biofluids can help to understand physiopathological mechanisms which are at the basis of a disease, such as bacterial infection [4], in-flammation [5], cancers [6]. On the other side, exogenous are im-portant in exposome research for assessing the environmental impact on human health, gaining insight into pharmacokinetics and monitoring the health-related effects due to other external factors. In a new research paradigm for such complex scenario both endogenous and exogenous VOCs can provide important clinical information and may revolutionize our understanding of later onset diseases.

How Can We Study VOC Fingerprinting?

The principal system for VOC detection and quantification is the gas-chromatography (GC) coupled by mass spectrometer (MS) associated with a good extractive method. One of the most used is solid phase microextraction (SPME) [7]. Another approach is based on the use of gas sensors [7].

The Potential of Volatile Organic Compounds Study of VOCs in Biological Matrices by GC-MS & Gas-Sensors

▪ Valentina Longo1, Angiola Forleo1, Simonetta Capone1, Pietro Siciliano1

The analysis of VOCs emanating from biological samples ap-pears as one of the most promising approaches in metabolo-mics for the study of diseases. The presence of volatile markers has been explored in exhaled breath, urine, blood, saliva, feces, human semen and cell cultures. Detection and quantification of VOCs are carried out by gas chromatograph-mass spectrome-ter system (GC-MS) and/or gas sensors.

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For the analysis of volatile compounds, usually experiments with three methodologies are set in parallel: SPME-GC/MS, SPME-GC/sensor gas and gas sensor array.

The first two are carried out thanks to a particular rearrange-ment of traditional GC-MS system in which the gas chromato-graphic column is linked by a splitter to MS and a gas sensor. In this way, the eluted compound is detected by the two detectors at the same time [8]. The overlapping of chromatograms and cor-responding “sensorgrams” (variation of resistance in relation to

Fig. 1: Example of VOC analysis through SPME-GC/MS and GC/gas sensor (A) and 4-sensor array (B).

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01/2020

European Journal of Organic Chemistry

01/2020

Front Cover: Place your cover credit here over a maximum of 3 lines

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Chemistry—MethodsNew Approaches to Solving Problems in Chemistry

ChemElectroChem

ChemPhotoChem

ChemMedChem

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run time) permitted both to connect the sensor responses to a specific VOCs’ pat-tern both to eliminate general background contamination, such as column bleed, that is not perceived by VOC sensor (Fig. 1A).

Using exclusively sensor resistance pro-files, we have an high discrimination of our samples (for example, human semen ones) by the most well-known classifica-tion procedures in chemometrics, the Par-tial Least Squares-Discriminant Analysis (PLS-DA) [9].

The third approach is based on a 4-sen-sor array placed in a home-made cham-ber. Also, this electronic nose-based ap-proach allows a good reproducibility and classification power.

SPME-GC/MS System

Solid-phase microextraction (SPME) was invented by Pawliszyn and co-workers [10] in 1989 to redress limitations inher-ent in solid-phase extraction and liquid–liquid extraction techniques. SPME inte-grates sampling, extraction, concentration and sample introduction into a single sol-vent-free step thanks to a fused-silica fi-bre that is coated on the outside with an appropriate stationary phase. It has been routinely used in combination with GC/MS and successfully applied to extraction of volatile and semi-volatile organic com-pounds from environmental, biological and food samples.

VOC collection is allowed thanks to dis-tribution of volatile compounds between liquid fraction (biofluid or cell medium) and headspace (HS). Headspace concen-tration values are based on Henry’s law constants and depend to specific chemi-cal-physical characteristics of the sample.

By SPME, the amount of VOC removed by the fibre is proportioned to the concen-tration of the compounds in the sample. For this reason, it is possible to make a quantitative as well as a qualitative analy-sis.

To qualitative analysis is necessary the use of spectral libraries such as NIST 14 (National Institute of Standards and Tech-nology, USA) which permit to identify (based on mass spectrum) the specific compound. Usually, in our laboratory the identification is confirmed by external standards, using the same method of the samples.

To lead a quantitative study is obligato-ry the addition to the samples of specific internal standards. We ordinarily used

commercial multiple standards (EPA8260) in which are present molecules not pre-sented in our samples in order to avoid contamination and additive effect. Stan-dard curves were created based on the peak areas, which were obtained from Enhanced Data Analysis software. The data were analysed in triplicate. Due to the different density and viscosity of sam-ples which in general we study, for each type of biological fluid we draw a specific calibration curve.

GC/Gas Sensor System

Chromatographic column is connected si-multaneously to two detectors, mass spec-trometer and metal-oxide (MOX) gas sen-sors, which work in parallel by a two-way splitter, that splits the helium (He) flow in 1:1 ratio towards the two detectors through two segments of defunctionalized chromatographic column. For GC/gas sensor analysis the capillary column was inserted, by a splitter (Agilent G3180B Two-Way Splitter), into a tiny chamber hosting a VOC sensor (MiCS-5521, e2v technologies, UK) and it was positioned near sensor surface. The MOX sensor op-erating temperature was 400°C. The MOX sensor traces (resistance vs. time) (Fig. 1A) were used for data analysis. In par-ticular, to ensure that each profile is on the same scale, resistance profiles were standardized using range-scaling be-tween 0 and 1 [11].

4-Sensor Array

The third and last approach that we apply to study VOCs in biological matrices is based on a 4-sensor array with 4 MOX sensing elements operating at a tempera-ture of 250 °C which are positioned in a home-made gas-tight chamber. The sen-sor responses towards the volatile com-pounds of the different samples were ac-quired by applying a constant voltage of 1 V across the electrodes and measuring the electrical current by an electrometer Keithley mod. 6517A equipped with an internal multiplexer module (Keithley mod. 6521).

The baseline was acquired in dry air in a continuous total flow of 25 sccm, where-as, for the measurement, the sample headspace, was stripped by means of a deviation of flow into the vial, kept at room temperature for 4 min, into the sen-sor chamber. All fluxes were controlled by mass flow controllers (MFCs) and a multi-

channel mass flow programmer (MKS mod. 647B). LabView software controlled all the gas-mixing protocol and the sensor signal acquiring [12]. The gas-sensing re-sponse of the device is defined by the ra-tio Rair/Ranalyte, where Ranalyte and Rair de-noted the measured resistance in the presence of the VOCs and in dry air carri-er, respectively (Fig. 1B).

Conclusion and Outlook

Several methods to analyse volatile com-ponent of biological matrices exist, point-ing out the importance of this metabo-lome fraction.

The methods to detect VOCs are very fast, economic, reproducible and allow population screening on a large scale. The implementation of protocols to VOC anal-yse is an important step on this long and uphill way, but several research group (such as our laboratory staff) they are working hard in this direction.

Affiliation1Institute for microelectronics and micro-systems of National Research Council (IMM-CNR), Lecce, Italy

ContactValentina Longo Institute for microelectronics and microsystems of National Research Council (IMM-CNR), Lecce, [email protected]

Further articles on chromatography: http://bit.ly/GLJ-Chroma

References: http://bit.ly/GLJ-Longo


Articles


Metabolomics Techniques for Mi-crobiota Metabolism Analysis in Human Samples

One of the most exciting scientific devel-opments in the past decade has been the growing understanding that the microbio-ta is part of the human “ecosystem” and directly impacts physiology. This complex community of trillions of bacteria, fungi, and viruses is highly metabolically active and has co-evolved with the human host. Microbiota populate every surface of the

human body, most notably the gastroin-testinal tract and the skin. Not only do the microbes of the microbiota outnumber human cells by a factor of about two to five, they possess approximately one hun-dred times more genes than are present in the human genome. This large increase in human genetic information corre-sponds to a large number of additional enzyme classes with biochemical poten-tials which may differ radically from those of the host. These additional genes in turn give rise to a diverse range of bio-

chemical and metabolic activities, pro-ducing a vast number of metabolites that are taken up by the human host and re-sulting in complex, intertwined metabolic networks (Fig. 1).

As a result of this shared metabolism, the human microbiota has been referred to as an additional organ or even a “sec-ond brain” as it significantly influences diverse human metabolic pathways in-cluding nutrition, detoxification, hormon-al homeostasis, immune tolerance, and especially inflammation. Mounting evi-dence indicates that a dysregulated gut microflora initiates or contributes to a va-riety of diseases, including cancer, diabe-tes, obesity, cardiovascular diseases, and inflammatory bowel disease among oth-ers. Recently, several studies have begun to shed light on microbiota dysbiosis, however, our understanding of the overall metabolic interactions of microbial com-

Hunting Gut Microbiota MetabolitesElucidating the Metabolic Interplay with Their Human Host

▪ Louis Conway1, Mario Correia1, Daniel Globisch1

Recent metagenomic studies have revealed the impact of dysregulated gut microbiota on human physiology and disease development. The fact that an altered gut microbiota composition is linked to non-bowel diseases can be explained through metabolic inter-action of these microbes with their human host. Metabolomics is the method of choice to elucidate this metabolic interaction and new methods are required for selective in-vestigation of this complex co-metabolism.

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munities with their host with respect to disease development is still limited. De-tailed elucidation of the metabolic inter-play between the host and its gut micro-biota in the context of disease onset through analysis of the metabolome has a tremendous potential for discovery of di-agnostic markers of disease, improved drug efficacy and clinical and lifestyle in-terventions. Moreover, greater under-standing of the influence of the gut micro-biota on the host’s metabolism will provide access to novel disease treatment and drug development opportunities.

Global Metabolomics Analysis

Mass spectrometric metabolomics is the method of choice for the investigation of metabolism due to its sensitivity to me-tabolites over several orders of magni-tude. Global metabolomics analysis can be divided in three major parts, namely metabolite extraction, mass spectrometric detection, and data analysis. This stan-dard workflow has become routine in the analysis of human samples, however, a detailed analysis of microbial metabolism requires more advanced techniques due to the low concentrations and potentially

unusual structures of the metabolites in-volved. For this reason, analysis of me-tabolites produced or altered by microbes has generally required targeted metabolo-mics approaches, often dictated by the relative limitations and strengths of the analytical platform. Common classes of microbial metabolites are bile acids, short-chain fatty acids, trimethylamine N-oxide (TMAO), tryptophan metabolites, and anthocyanins [1,2]. Targeted analyses of bile acids, for example, has revealed

numerous previously unknown metabo-lites. The use of germ-free mice has also proven fruitful in elucidating the physio-logical effects of certain microbes in the gut microbiota, for example, the link be-tween obesity, short-chain fatty acids, and the gut microbiome. The availability of tools aimed at advancing the data analy-sis part of the metabolomics workflow has also improved dramatically, from open-source software packages for peak detection and alignment such as XCMS

249817

ISBN 978-3-527-34327-0

Hans H. Maurer, Karl P� eger, Armin A. Weber

Mass Spectral Library of

DRUGS, POISONS, PESTICIDES, POLLUTANTS, AND THEIR METABOLITES

5th Edition


CD-ROM

The Maurer/Pfleger/Weber Mass Spectral Library

This innovative reference library for clinical and forensic toxicologists has once more been extensively updated.

The 5th Edition of Mass Spectral Library of Drugs, Poisons, Pesticides, Pollutantsand their Metabolites sees the addition of 1,780 data sets, bringing the total to 10,430 mass spectra and GC retention indices. Featured compounds of 175 categories include older and new psychoactive substances, almost all relevant therapeutic drugs as well as 7,800 of their metabolites.

This reference is made up of an electronic database accompanied by two hardbound volumes.

Find out more at

www.wiley.com/go/databases

Fig. 1: Representation of the formation of sulfated metabolites in the human gut microbiota and four microbiota derived sulfated molecules [9] - published by The Royal Society of Chemistry.

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and MZmine to comprehensive and strati-fied databases of metabolites including Metlin, the Human Metabolome Database (HMDB), and Global Natural Product So-cial Molecular Networking (GNPS), which have significantly improved general me-tabolite identification [3-7].

Selective Chemical Biology Tools

Chemical biology tools have made major contributions to biological and medically relevant findings in proteomics, tran-scriptomics and genomics analysis. This is in stark contrast to metabolomics re-search, for which these tools are rare. We have recently published two new methods at the interface of chemistry and biology that permit a more informative analysis of biological samples [8,9]. Both methods have been developed for selective investi-gation of microbial metabolism in hu-mans and aim to identify previously un-

known metabolites. One approach was the development of chemoselective probes that combine immobilization to magnetic beads with a bioorthogonal cleavage site, which we have newly adapted from a pro-tecting group that is labile under mild, palladium-catalyzed conditions (Fig. 2). This architecture allows metabolites to be captured, tagged with a strongly ionizable conjugate, removed from the sample ma-trix, and released. The optimized probe was utilized on fecal samples, the sample type most directly influenced by metaboli-cally active microbial communities. Anal-ysis revealed previously unknown metab-olites and due to conjugation of the mass-spectrometric tag and separation from the sample background, the sensitiv-ity towards some metabolites increased more than 2000-fold.

A second approach focused on selective investigation of sulfated metabolites, which result from the phase II clearance

of xenobiotics by the human body and is linked to microbiota-human host co-me-tabolism [9]. The method is based on pre-treatment of the sample with a purified and highly promiscuous arylsulfatase. A very high degree of enzymatic purity is critical, in order to avoid undesired meta-bolic background conversions. Compari-son of the pretreated sample with an un-treated sample after analysis using ultra high-performance liquid chromatogra-phy-mass spectrometric techniques led to the identification of 206 sulfated metabo-lites, exceeding the number of sulfated metabolites previously catalogued by a factor of three to four. Seven previously reported metabolites derived from hu-man-microbiota co-metabolism were among these sulfated compounds, dem-onstrating the efficacy of the technique. This concept has also been successfully applied to the analysis of glucuronidated metabolites, the most common phase II modification leading to the identification of several polyphenolic metabolites that are derived from microbiota metabolism of dietary macromolecules in the human body [10].

Conclusion

Detailed investigation of microbiota-de-rived metabolites has a high potential to identify unknown bioactive compounds. These molecules can have diverse func-tions of interest for different application fields, e.g. biomarker discovery, antimi-crobial drugs, quorum sensing, and di-etary markers. A more comprehensive microbiota-derived metabolite analysis will require new chemical, bioinformatic and analytical methods to identify, eluci-date and validate metabolite structures. These new techniques will play key roles in improving our understanding of the metabolic interaction of microbiota with their human host, allowing us to utilize and manipulate microbiota metabolism to improve healthcare.

Affiliation1Department of Medicinal Chemistry,Science for Life Laboratory, UppsalaUniversity, Uppsala, Sweden

ContactAssoc. Prof. Dr. Daniel GlobischUppsala University, Department of Medicinal ChemistryUppsala, [email protected]

Further articles on mass spectrometry: http://bit.ly/GLJ-massspec

References: http://bit.ly/GLJ-Globisch

Fig. 2: a) Chemical probe design. The reactive site (green) is connected by a linker (purple) to the Noc-cleavage site (red). This probe is conjugated to magnetic beads (orange) by a spacer (black). b) The ge-neral workflow for analysis of metabolic amines using the chemical probe [8].

249817

ISBN 978-3-527-34327-0

Hans H. Maurer, Karl P� eger, Armin A. Weber


DRUGS, POISONS, PESTICIDES, POLLUTANTS, AND THEIR METABOLITES

5th Edition


CD-ROM

The Maurer/Pfleger/Weber Mass Spectral Library

This innovative reference library for clinical and forensic toxicologists has once more been extensively updated.

The 5th Edition of Mass Spectral Library of Drugs, Poisons, Pesticides, Pollutantsand their Metabolites sees the addition of 1,780 data sets, bringing the total to 10,430 mass spectra and GC retention indices. Featured compounds of 175 categories include older and new psychoactive substances, almost all relevant therapeutic drugs as well as 7,800 of their metabolites.

This reference is made up of an electronic database accompanied by two hardbound volumes.

Find out more at

www.wiley.com/go/databases

Omics 27

Articles

http://www.wiley.com/go/databases


The Genesis of GOC

Organs-on-chip (OOC) are the future of personalized medicine and pharmaceuti-cals research. The concept aims at reduc-ing time and costs associated to in vivo and in vitro analyses as in preclinical screening for drug development research. However, in vivo analyses are often limit-ed by ethical issues, and provide results that are usually non reproducible in hu-mans. [1-3] Whereas, in vitro models are static cell cultures that do not mimic many crucial features of the living tissues, thus resulting in low-predictive models. [1]

An important issue of OOC preparation is the accurate reproduction from the mi-croscopic to the macroscopic features of (a) tissue/organ district(s). The physiologi-cal key structure in organs or tissues se-

lected could thus be better investigated, helping to define disease onset and/or de-velopment still unexplored to date. [4]

Given the increasing interest on drug development, discovery and screening, a focus on the mechanisms underlying drug absorption and metabolism is mandatory for the success of future drug testing in humans. And, in drug oral absorption and metabolism, understanding the physio-logical activity of e.g. the small intestine, with all the issues regarding intestinal epithelial transport processes and factors which affect them, is central. [5]

The GOC Model and the Microenvironment Parameters

To date, GOC devices in literature are rep-resented by micro-devices, generally

adapted from e.g. lung- and heart-on-chip, to mimic the mechanical/structural prop-erties of the intestine. [6] Other studies are based on micro-engineered, biomimetic systems containing proper microfluidic channels that simulate the human intes-tine as an intestinal epithelial monolayer eventually combined with microbial sym-bionts. [7] However, these devices imply some disadvantages and open issues, such as the necessity to better reproduce and mimic the in vivo epithelium. The main advantages of these advanced models with respect to static cultures are in the more accurate reproduction of the in vivo environment, by controlling the microflu-idics parameters, and the option to oper-ate in continuum, thus reducing the delays and costs of research. [8]

However, recent microfabrication tech-niques (that tend to replicate plain func-tional units of the small intestine) still have major limitations in the inaccurate simula-tion of the peristalsis dynamics (mem-brane curvature, stretching, bending) and the lack of a sensing network in situ [9].

A multifunctional GOC device must de-tect intertwined parameters that are at the basis of various physiological process-es and responses to e.g. nutrients and to drug-based treatments. In situ automated sensing of the intestinal microenviron-mental parameters in real time has been projected, in a multifunctional device for continuous detection of e.g. cell adhesion epithelial-type processes. [10]

According to recent research that high-lights the urgent need for more physiolog-ically relevant models of human organs, GOC is presented as an integrated model with both electromechanical and sensing elements, able to reproduce/detect the key structural and functional outputs of a human gastrointestinal (GI) epithelial sys-tem. [11] Figure 1

Micromechanical stimulation sensors are used to mimic the mechanical pro-cesses and incorporated electrodes are able to control pH gradients, plasma membrane potentials and solute/ion tran-sepithelial uptake or translocation.

An Integrated Multiparameter Device for Personalized Medicine

GOC devices are integrating different types of electrodes for the detection in re-al-time of several parameters. (Fig.1) In

Technology and Application of Gut-On-Chip Gut Epithelial Mimicking in Integrated Sensor Platforms

▪ Lucia Giampetruzzi1, Amilcare Barca2, Luca Francioso1, Tiziano Verri2, Pietro Siciliano1

Gut-on-chip (GOC) platforms are microfluidic devices with micro-chambers and chan-nels to support and control media volumes and flow through a living cell culture gener-ally seeded on membranes that create 2 chambers (lower and upper). This technology mimics the physiological architecture and function as well as the pathological states of human epithelial organs. Several models reproducing human GOC have underlined their potential value as for disease and drug screening modelling.

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recent studies this has been achieved with the utilization of e.g. ITO (indium tin ox-ide) or gold electrodes (Au) patterned by wet etching technique, located at the two lower/upper interfaces of the cell culture or near the membrane seeded with cells. [9-12] ITO and Au electrodes have shown some important results in the detection of TEER (transepithelial electrical resistance) and of transepithelial transport, in moni-toring the membrane-trafficking/exchange processes and nutrient/drug fluxes. In particular, TEER is a widely used experi-mental readout and quality control assay, such as for measuring the formation and integrity of epithelial monolayers. [12] This is generally applied to cells cultured under static conditions in vitro, being an important index of intestinal barrier integ-rity; so, there is a necessity to translate this standard methodology into the micro-fluidic GOC. [13] The TEER values extrap-olated by software and electronic support platforms are worth to measure the tight junction dynamics of cultured cell mono-layers. In some works, it has been demon-strated that a stable and robust electrode set-up revealed TEER values of intestinal monolayers very closed to the traditional static experiments, providing interesting features in terms of real time monitoring. The embedded electrodes have also al-lowed to a real time optical investigation, given by their low fluorescence emission and higher transparency. So, this provides for qualitative assessing of cell cultures using optical and confocal microscopy. An-other important advantage in addition to ITO/Au electrodes is the possibility to in-duce software-controlled mechanical de-flection of the membrane suspended be-tween the two fluidic chambers, both above and below the membrane, and si-multaneous measurements by the embed-ded sensors. [9] This helps in reproducing local mechanical stretching and bending, comparable to an in vivo situation, moni-toring the effects in continuum. [14]

Finally, there is growing interest in the impact of microbial symbionts on GI health. Only recently, pathogenic bacterial chal-lenges have been developed in in vitro plat-forms to study how they influence the nor-mal microbiota and intestinal epithelial cells (thus contributing to disease develop-ment), and to design possible drug thera-pies. Considering bacteria and the microbi-ome, a GOC could help elucidating the integration of GI and immune functions. [15] Together, all these considerations sug-gest that a GOC scheme that emulates the

dynamic intestinal microenvironment, more faithfully could mimic intestinal func-tion or disorders than previously described by adopting standard in vitro models.

Conclusion and Outlook

The GOC strategy provides a better model of the intestine than the previous cell cul-ture systems by dynamically recreating and controlling multiple features of the human organ/tissue. A layout of its active microenvironment as well as the con-trolled microfluidic environment simulat-ed with a fluid flow may be essential for e.g. transport and absorption of nutrients and/or toxicity studies and allow “more

physiological” growth of the microbiome and the possibility to study pathogenic disorders along the axis from the gut to other organs and tissue districts.

Affiliations1Institute for Microelectronics and Microsystems IMM-CNR, Lecce, Italy2Applied Physiology Laboratory’, (DiSTeBA) , University of Salento, Lecce, Italy

ContactLucia GiampetruzziInstitute for Microelectronics and Microsystems IMM-CNRLecce, [email protected]

Related Articles: http://bit.ly/GLJ-Organ

References: http://bit.ly/GLJ-Giampetruzzi

Fig. 2: A model of a multi-integrated platform for investigation on a gut-on-chip.

Fig. 1: Gut-on-chip as a sensing model. It should integrate electromechanical stimuli and sensing ele-ments, important for reproducing the key structure and function of a human gastrointestinal (GI) tract.

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The kidneys play an important role of re-moving waste from the body, while adjust-ing blood pressure and homeostasis, pro-ducing hormones and ensuring bone strength [2]. CKD is a pathology character-ized by a progressive and irreversible loss of the renal function, over months or years [3]. CKD patients have increased mortality and cardiovascular disease, and such dis-ease represents an important health bur-den. Today, the diagnosis of CKD is mainly based on the estimation of the kidney function, and the evaluation of structural tissue damage [3]. The estimated glomeru-lar filtration rate (eGFR) is calculated from serum creatinine. CKD is classified into five stages according to eGFR levels and the amount of albumin in urine [4]. De-spite the usefulness of eGFR, creatinine is influenced by age, sex and muscle mass that can interfere with the estimation of GFR. In this context, more specific and sensitive biomarkers are needed.

New biomarkers can also guide the clini-cian to adapt treatment according to dis-ease severity or metabolites accumulation in blood. Metabolic profiling potentially overtakes eGFR and urinary albumin, when assessing the response to a thera-peutic treatment or renal replacement therapy, i.e. hemodialysis and kidney

transplantation. While invasive biopsies are generally needed to diagnose the un-derlying renal disease [1], the holistic ap-proach offered by metabolomics can also improve the understanding of pathological mechanisms [5]. Discovering new bio-markers bringing more or complementary information about disease severity could allow earlier medical care and better prog-nosis. In addition to readouts related to kidney filtration, pathway enrichment could help to find metabolic pathways and enzymes related to other pathological bio-chemical mechanisms. This could bring new insights into the CKD knowledge and potential therapeutic targets.

Methodology

The metabolomics workflow involves a broad range of knowledge and interdisci-plinary competences. Typically, it implies several steps from sample collection to bio-logical interpretation including chemical analysis. Metabolomics aims to catch a maximum of information related to metab-olites in order to generate hypothesis from the experimental design. In that context, all the workflow steps are essential and must contribute to study biological facts in order to generate relevant information (Fig. 1).

Modern high-resolution mass spectrom-eters (HRMS) are, with Nuclear Magnetic Resonance (NMR), the most widely imple-mented analytical platforms for data ac-quisition in metabolomic studies. HRMS benefits from high selectivity and sensitiv-ity, allowing the simultaneous detection of a large number of metabolites, even at low concentration levels, while providing structural information with m/z measure-ment and fragmentation patterns. Gener-ally, separative techniques such as liquid or gas chromatography (LC, GC), or capil-lary electrophoresis (CE) are hyphenated to HRMS offering orthogonal separation of molecule and additional information for metabolite identification. The use of com-plementary separative techniques allows a better metabolome coverage to give the best overview of a biofluid or tissue sam-ple and ensure relevant biological evalua-tion [6]. In that context, our work takes advantage of the selection of three com-plementary LC methods hyphenated to HRMS ensuring low- to high-polarity com-pounds monitoring in plasma to evaluate metabolic profile alterations associated with CKD [7,8]. Plasma samples collected from a clinical cohort composed of healthy individuals and patients at different CKD stages were analyzed to highlight the dis-criminant metabolites related to disease.

Beyond the analytical aspects, an im-portant step in metabolomics concerns data treatment and chemometrics. In-deed, the large amount and complexity generated LC-MS metabolomics data re-quire specific attention. After preliminary data filtering and baseline correction nec-essary to reduce background noise and other artefacts, data processing aims to extract relevant information from raw data to generate data matrices. Handling data matrices characterized by a large number of variables is challenging and requires specific statistical analysis. Mul-tivariate data analysis (MVA) aims at highlighting information by reducing the dimensionality, while allowing efficient display of overall variations in the data-set.

Metabolite identification remains one of the major bottlenecks in untargeted me-tabolomics. In that context, it is crucial to measure as much physicochemical and structural properties as possible to bring information for univocal identification. These properties are accurate mass, iso-

Chronic Kidney DiseaseToward a Better Understanding of the Disease Using Metabolomics

▪ Yoric Gagnebin1, Julian Pezzatti1, Pierre Lescuyer2, Julien Boccard1, Belén Ponte3, Serge Rudaz1

Efficient patient phenotyping offered by metabolomics recently gained popularity in the clinical community. Over the past decade, the number of metabolomic studies on chronic kidney disease (CKD) increased drastically, showing the interest of an extended monitoring of metabolites to investigate biochemical alterations. This trend is related to the need for better biomarkers for diagnosis, disease progression monitoring and dis-covery of new therapeutic targets [1].

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topic pattern, MS/MS fragmentation pat-terns and retention time. For that pur-pose, a reference database was built by analyzing more than 900 standards of endogenous metabolites using the same experimental conditions to cover the ma-jor human metabolic pathways. Thanks to this strategy, 278 metabolites were univo-cally annotated in each of the 160 plasma samples from three different groups of patients, namely, healthy controls (CTRL), intermediate stage patients (CKD3b/4) and end-stage patients (CKD5).

Results

The major sources of variability of the da-taset were investigated using principal component analysis (PCA), and the three groups were well separated showing a significant trend in the metabolic profiles according to disease severity (Fig. 2). This distribution revealed an increased con-centration of numerous identified metab-olites with the evolution of the pathology. This result confirms the overall accumu-lation of compounds in blood due to de-creasing kidney function. Further super-vised multivariate analyses were performed to highlight the most signifi-cant alterations which could serve as a basis for potential biomarkers identifica-tion.

Among the metabolites with increased abundance, indoxyl sulfate, myo-inositol and kynurenic acid are examples of the most discriminant metabolites constitut-ing complementary biomarkers with a CKD diagnosis ability similar to creati-nine. Regarding the full list of discrimi-nant metabolites, biologically relevant subgroups of metabolites could be high-lighted such as N-acetyl amino acids and steroid glucuronides that increase with disease progression. Interestingly, only a few metabolites were observed to de-crease with CKD severity, such as trypto-

phan. More targeted analyses with abso-lute concentration measurements in a larger cohort are required to refine the biomarker selection, validate the results and finally provide an original effective diagnostic tool to the clinicians for routine use.

In addition to biomarker discovery, me-tabolomics could bring knowledge about biological mechanisms. Indeed, pathways mapping greatly assists in the biological interpretation and helps to generate hy-potheses. For this purpose, discriminant metabolites were placed back into the corresponding metabolic pathways to give an overview of biological networks and metabolite interactions, while assess-ing enzyme activity. Tryptophan metabo-lism was found to be significantly altered, as all the detected tryptophan metabolites showed an increased concentration with disease severity, while tryptophan itself presented a decreased abundance com-pared with the control situation.

The developed analytical workflow combined with an adapted data handling

helped to generate new and unknown bi-ological hypotheses, while confirming several known metabolic pathways and biomarkers involved in CKD disease. This study demonstrated the interest of untar-geted metabolomics and, more specifical-ly, multiplatform metabolomics to assess metabolic profile alterations related to a specific pathology.

Affiliations1School of Pharmaceutical Sciences, Uni-versity of Geneva, University of Lausanne, Geneva, Switzerland2Division of Laboratory Medicine, Geneva University Hospitals (HUG), Geneva, Swit-zerland3Service of Nephrology, Geneva Universi-ty Hospitals (HUG), Geneva, Switzerland

ContactProf. Serge Rudaz School of Pharmaceutical SciencesUniversity of GenevaGeneva, [email protected]

On-demand online-talks on mass spec in proteomics: http://bit.ly/proteomicsnow

References: http://bit.ly/GLJ-Gagnebin

Fig. 1: Generic metabolomics workflow.

Fig. 2: PCA score plot of the 160 study samples modelled from the 278 annotated features [8]. Reprinted from Journal of Chromatography B, 1116, Yoric Gagnebin et al., Toward a better understanding of chronic kidney disease with complementary chromatographic methods hyphenated with mass spectro-metry for improved polar meta-bolome coverage, 9-18, Copyright (2019), with permission from Elsevier.

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Multidimensional NMR

Modern NMR spectroscopy is based on the measurement of time-domain free in-duction decay (FID) signals of nuclei hav-ing non-zero spin (most commonly 1H and 13C). Signals are converted into spec-tra by Fourier transform. Importantly, it is possible to generate interferograms – sig-nals being a function of several time vari-ables. It makes it possible to describe the topology and geometry of studied mole-cules, i.e., connections and distances be-tween atoms. Unfortunately, sampling of even one additional (“indirect”) time di-mension is costly: acquisition of a single 2D spectrum takes at least several min-utes and one needs several different spec-tra providing complementary informa-tion. Thus, it is difficult to use 2D NMR as a “snapshot” technique in reaction moni-toring. Many techniques have been pro-

posed to alleviate this problem, usually by reconstructing part of the data using mathematical algorithms instead of mea-suring it experimentally [1]. However, un-til now it was rather cumbersome to em-ploy them in the online mode, i.e., to process the data during the experiment.

Interleaved NUS Acquisition

The freely available software package that is described herein is called Trends (time-resolved N-dimensional spectrosco-py). It is based on a special type of non-uniform sampling (NUS) – a time-resolved NUS (TR-NUS, [2]).

NUS has often been used in multidi-mensional NMR experiments to acceler-ate their acquisition. It means skipping random rows of 2D or higher-dimension-al NMR data with their subsequent recon-struction (interpolation of data). Com-

pressed sensing (CS) reconstruction has been employed, which proved successful with NMR [3]: it relies on the assumption of spectral sparsity, i.e., the presence of only a few peaks in a spectrum. As shown in many studies, the assumption is usual-ly very well fulfilled.

In standard NUS, K random rows out of the full data grid of size N are sampled. In time-resolved NUS [2], there is a long (of length >> N) shuffled list of indices of rows to be measured. Then, during the reconstruction process, the long list of data lines is grouped into “frames” of length K (Fig. 1). They can also be over-lapping, which hugely enhances the tem-poral resolution.

Moreover, TR-NUS allows the inter-leaved acquisition and the software gives an opportunity to switch from one type of NMR experiment to another between lines of data. Thus, different kinds of NMR experiments can be acquired simultane-ously, which is a powerful advantage, since snapshots of the process are syn-chronized in time.

Processing

The processing contains two main parts: 1) the reconstruction of the undersampled

Software for Reaction Monitoring by NMRAcquisition and Processing of Time-Resolved 2D Spectra

▪ Mateusz Urbańczyk1,2, Alexandra Shchukina1, Magdalena Kaźmierczak3, Dariusz Gołowicz1,4, Krzysztof Kazimierczuk1

Nuclear magnetic resonance (NMR) spectroscopy is a main workhorse in the analysis of molecular structures. It can also visualize their dynamic changes, thus serving as a great reaction-monitoring tool. Here, we present a software package allowing to set up and process data from several interleaved time-resolved two-dimensional (2D) NMR spectra, extending the amount of information provided by the conventional approach based on 1D spectra.

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data, and 2) the tracking of spectral peaks through the time series of spectra.

Importantly, the reconstruction can be carried out simultaneously to the acquisi-tion, thus allowing for an early “snooping” into the chemical processes. As full reac-tion durations may reach tens of hours/days, it is highly convenient to have some preliminary information without waiting for the reaction to finish.

Another benefit of the method is that the reconstruction is separated from the acquisition: the reconstruction can be performed multiple times with various parameters without the need to re-ac-quire any data. For details on the recon-struction, see [4].

A chemist is usually interested in the dynamics of changes of definite spectral peaks. The software allows for selecting a peak in a frame and acquiring its intensi-ty and position changes throughout the available set of frames. This is done with a peak-tracking module, which takes into account the possibility of peak appear-ance/disappearance from frame to frame. Additionally, one can fit the peak intensity profiles to approximate the chemical ki-netics rates.

Data Visualization

The described software contains a visual-ization block with the following main fea-tures: ▪ Displaying up to three sets of 2D spec-

tra and one 1D spectrum ▪ Sliders for frame number, threshold

and contour levels for 2D spectra dis-play; zoom/unzoom tools

▪ A tool to select a peak for analysis ▪ Pop-up window for peak analysis re-

sults

Besides, it provides a user-friendly inter-face for data acquisition as well as recon-struction.

System requirements

The acquisition module works with Bruker TopSpin 3.2 or higher, Magritek Spinsolve Expert and Agilent/Varian VNMRJ 4.2. The processing module works on Linux, macOS and Windows 10 systems.

Conclusion

The herein described software package for chemical reaction monitoring with 2D

NMR is an all-in-one tool for acquisition, on-the-fly processing, display and analy-sis of data. It also provides incomparably better time resolution that other known methods exploiting the concept of time-resolved non-uniform sampling. It imple-ments interleaved acquisition of NMR ex-periments allowing for the extraction of complementary types of information at the same time. The software has a user-friendly graphical interface.

Affiliations1University of Warsaw, Centre of New technologies, Warsaw, Poland2NMR Research Unit University of Oulu, Oulu Finland

3Faculty of Chemistry, Warsaw University of Technology, Warsaw, Poland4Faculty of Chemistry, University of War-saw, Warsaw, Poland

ContactProf. Krzysztof KazimierczukLaboratory of NMR SpectroscopyCentre of New TechnologiesUniversity of WarsawWarsaw, [email protected]

Articles on spectroscopy: http://bit.ly/GLJ-spectroscopy

Download of freely available software: http://trends.spektrino.com

Fig 1: The concept of TR-NUS interleaved acquisition of 1D, 2D HSQC and 2D TOCSY spectra.

Fig. 2: The GUI of the Trends package. Panels on the left contain TR-NUS processing parameters as well as standard NMR procedures – apodization, zero-filling etc. On the right 2D spectral snapshots ob-tained with interleaved HSQC and TOCSY spectra are presented as well as a peak intensity plot. The aza-Michael addition of benzylamine and acrylamide was studied.

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Articles

Ana C. Albéniz

Uwe T. Bornscheuer

Pier G. Cozzi

Karl Gademann

Karl Anker Jørgensen

Jean-François Nierengarten

Agneta Sjögren

Annette Beck-Sickinger

Anthony J. Burke

Bas de Bruin

Piet Herdewijn

Burkhard König

Marcin Opałło

Matthieu Sollogoub

Tomás Torres

Matthias Bickelhaupt

Benoît Champagne

Ivana Fleischer

Lene Hviid

Ronald Micura

Amélia P. Rauter

Sir J. Fraser Stoddart

Anna Trzeciak

Mário N. d. M. S.Berberan E Santos

Gilberte Chambaud

Gianluca Farinola

Nicola Hüsing

Martin Kotora

Pedro J. Pérez

Peter Somfai

Silvia Bordiga

Iris Cornet

Katharina Fromm

Ferenc Joó

Viktor Milata

Vladimír Šindelář

Nikos Tagmatarchis

Please join us in congratulatingChemistry Europe Fellows Class 2018/ 2019

Chemistry Europe – a partnership of 16 European chemical societies – established this fellowship to honor extraordinary contributions.


CE_Fellows_AD_FINAL_210x297.indd 1 24.04.20 15:39



Programmable Phages for MedicineHow Synthetic Biology Is Fuelling a New Generation of Phage Therapy

J Matthew Dunne1 and Samuel Kilcher1

Phage therapy has long lived in the shadows of conventional antibiotics. However, the escalating antibiotic resistance crisis – caused by the misuse and overuse of antibiotics – has since revived global interest in this once forgotten therapy. Until now, phages were isolated from the environment, tested for their antibacterial activity and then produced for therapeutic application. With the help of new methods in synthetic biology, scientists are now generating re-engineered designer phages with enhanced antibacterial proper-ties to combat the ever-growing number of superbugs.

Bacteriophages (phages for short) are vi-ruses that specifically target bacteria. Phages play an incredibly important role in bacterial ecology; in the world’s oceans

alone, an estimated 1023 infections occur every second, leading to 20% of marine biomass being killed every day [1]. The therapeutic potential of phages was rec-

ognized shortly after their discovery in 1915, which led to the early heyday of phage therapy before the discovery of penicillin in 1928 and the development of antibiotics. Due to the ease of use and high therapeutic efficacy of antibiotics, phages fell off the medical radar as a treatment option, particularly in Western medicine. Today, phage therapy is only practiced in a few countries, for instance in Georgia where it remains a conven-tional form of treatment [2]. However, the growing threat from multi-drug resistant

Artistic representation of the production of designer phages, which are assembled from modular, custom-made individual parts. The picture shows how a single phage scaffold can be used to create chimeric phages with different baseplates and receptor binding proteins to recognize and infect different bacte-ria. Picture: Acrylic paints on canvas by Jonas Fernbach

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bacteria means alternative approaches are desperately needed to fight superbug infections, with many researchers and cli-nicians returning to the use of phage therapy to save the day. Numerous, some-times incredible, case studies give an idea of the potential of this form of therapy [3, 4], for instance, last year in the UK a com-bination of natural and engineered phag-es were used to treat a drug-resistant My-cobacterium abscessus infection in a 15-year-old girl [3]. Nevertheless, a lack of meaningful clinical studies that can scien-tifically prove the effectiveness of this therapy is holding back phage therapy from becoming a mainstream option for fighting bacterial infections [5].

Compared to conventional antibiotics, phages offer a pivotal advantage: They have a very high host specificity and can therefore specifically eliminate a given pathogen without negatively affecting the microbiome. However, there are some critical disadvantages: Phages are so spe-cific that often many different phages must be combined to cover all clinically relevant host strains. In addition, receptor mutations, restriction systems, CRISPR-Cas systems and other mechanisms can make host bacteria resistant to phages [6]. Some so-called temperate phage can also integrate into the host genome, intro-ducing potentially dangerous genes into the host (a process known as “lysogenic conversion”). In addition, bacteria can shield themselves from phages by form-ing a physical barrier called a biofilm.

Phage Therapy 2.0 - Synthetic Phages for Medicine

Through continuous advancements in synthetic biology [7], it is possible to easi-ly re-engineer phage genomes and cir-

cumvent the above-mentioned limitations (f. 1). Phage genomes can be broken up into smaller segments and produced as synthetic fragments by PCR or gene syn-thesis. These synthetic fragments can then be quickly reassembled as a whole, infectious phage genome using the Gibson technique [8]. During reassembly, it is possible to remove toxic and unwanted genes, insert “therapeutic payload” genes, or even directly modify the genetic code to improve the antibacterial potential of the phage. Nevertheless, how does one turn a tube of synthetic phage genomes into infectious phages?

For phages of enterobacteria it is often sufficient to transform the synthetic ge-nome into electrocompetent E. coli bacte-ria, which can act as a “surrogate mother” to produce the infectious phage particles [9]. In contrast, the thick outer wall of Gram-positive bacteria makes electro-transformation of large, synthetic ge-nomes into the cells a significant, if not impossible, challenge. The solution was to exploit a little-known biological curiosity:

L-form bacteria. Remarkably, as long as they are not under osmotic stress, many bacteria have the ability to grow and di-vide without a cell wall. These so-called L-form bacteria (named after the Lister Institute in London; fig. 2) are easy to transform with large pieces of DNA, and can also function as surrogate mothers for activating synthetic phage genomes [10]. Listeria L-forms have proven highly efficient at not only activating synthetic Listeria phage genomes, but also the ge-nomes of phages targeting pathogens be-yond the genus boundary (e.g., phages against staphylococci) [10]. With the help of competent surrogate mother cells (elec-trocompetent or L-forms) it is possible to activate synthetic genomes quickly – a process also known as “genome reboot-ing”.

Previously, phage re-engineering was only possible through homologous recom-bination using suitable host cells. Howev-er, the very low recombination frequen-cies combined with the difficulty of isolating recombinant phages made this

Fig. 1: Overview of methods. Synthetic phage genomes are assembled in vitro from DNA fragments about ten kilobases long and then transformed into surrogate mother cells. This is achieved using elec-troporation (Gram-negative bacteria) or by polyethylene glycol (PEG) transformation (Gram-positive bacteria, L-forms). The surrogate mother cells produce the designer phages, which are then propa-gated and isolated.

Fig. 2: L-form bacteria. Microscopic image of Enterococcus faecalis L-form bacteria. L-forms are used to activate synthetic phage genomes. L-form cells vary in size and have intracellular vesicles. Picture: Susanne Meile

Fig. 3: Receptor binding tailspike of Listeria phage PSA. Atomic-resolution model of the distal tip of the Listeria phage PSA receptor binding tailspike (RBP). A single protein chain of the homotrimeric complex is stained blue. The globular “head” domain contains the binding sites that recognize specific types of teichoic acids found on the surfaces of Listeria cell walls. The binding specificity of PSA can be reprogrammed by swapping the distal tip components (via the stem and neck domains) with those of other phage RBPs [11].

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approach a difficult and, above all, lengthy process. New methods – such as genome rebooting – are therefore a major step forward, allowing the generation of optimized designer phages within as little as one week. However, what can we achieve with these new and improved phages? The next two paragraphs de-scribe two major therapeutic applications of designer phage.

Host Specificity

As already mentioned, the high host spec-ificity of the phage is both a curse and a blessing. Several studies have shown that the methods presented above can be used to reprogram phage specificity [9, 11-13]. This is achieved by modifying the recep-tor binding proteins (RBPs) of phages, which recognize specific receptors on the surface of the cell. As the mediators of host cell recognition and attachment, the RBPs are primarily responsible for the narrow host ranges of many phages. The specificities of Enterobacteriaceae and Listeria phages have already been altered by the generation of synthetic phages with chimeric RBPs. Such chimeric RBPs are assembled following the LEGO princi-ple by re-combining the structural do-mains, or “LEGO blocks”, from different phage RBPs. This requires an atomic-res-olution model of the RBP architecture to

provide an engineering blueprint, as well as suitable “LEGO blocks” from related phages with different specificities (fig. 3). With this method, it might be possible in the future to adapt the host spectrum of known and well-characterized phages in-stead of having to isolate and characterize new phages from the environment every time (fig. 4).

In situ Production of Therapeutic Proteins

Many phage genes are under the control of highly active promoters. This is neces-sary to form new phage particles, which requires the production of large amounts of structural proteins within a short time-frame. It is now possible to reprogram a phage genome so that during infection they express therapeutic genes as well as structural proteins (fig. 4). After the death of the host cell, these therapeutic proteins are released and exert their effects locally. Phages, for example, have been pro-grammed to produce additional depoly-merases that dissolve poly-N-acetylglu-cosamines (PNAG) or capsular polysaccharides, therefore providing the

phages with unique properties to target PNAG-containing biofilms and other exo-polysaccharides [14, 15]. In another con-cept study, Listeria phages were designed to produce a bacteriocin targeting staphy-lococci. When a co-culture of Listeria and S. aureus was infected with these phages, both bacteria die. First the phages infect and kill the Listeria cells, which releases the bacteriocins that generate “collateral damage” against the remaining staphylo-coccal cells [16]. This approach opens up possibilities for the treatment of polymi-crobial infections, which are particularly common in wounds and urinary tract in-fections.

Outlook

Phage therapy has an exciting and glob-ally important future: Many companies and public institutions are investing in the development of personalized therapies and double-blind clinical trials using com-binations of natural phages isolated from the environment [5]. In addition, synthetic phages – designed to carry various thera-peutic genes – are also being put to the test: The Swiss National Science Founda-tion recently invested in a large-scale project for the treatment of urinary tract infections in catheterised patients [17]. The results of these studies will guide the development of natural and synthetic phages for treatment and define the role of phage therapy in the fight against anti-biotic-resistant germs.

Affiliation1Institute of Food, Nutrition and Health, Laboratory of Food Microbiology, ETH Zurich, Switzerland

ContactDr. Samuel KilcherGroup Leader at the Institute of Food, Nutrition and HealthLaboratory of Food MicrobiologyETH Zurich, [email protected]

Related Articles: http://bit.ly/WAS-Biotechnology

References: http://bit.ly/GLJ-Kilcher

Fig. 4: Programmable specificity and in situ production of therapeutic proteins. The binding and infec-tion specificity of phages can be modified by targeted engineering of receptor binding proteins (upper panel). By inserting “therapeutic payloads” (e.g. biofilm depolymerases or antibacterial lysins), phages can also be used as vectors to produce therapeutics directly at the site of infection (lower panel).

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This investment for safety without com-promise is questioned when the most common added accessory called aspira-tion pump is inadequate and questions the whole safety concept: ▪ Aspiration pumps with 2-4 L waste

collection bottles are too big to be placed inside the BSC.

▪ Being placed outside, potentially bio-active liquids are pumped out of the safe containment of the BSC.

Therefore, the liquid aspiration systems BioChem-VacuuCenter BVC from Vacuubrand offer uncompromised com-fort and safety: ▪ sensitive suction selection from gentle

to fast for sample protection

▪ whisper quiet vacuum pump working on demand automatically

▪ safety aspects as 0.2µm filter, leak-proof patented hand controller as well as autoclavable and shatterproof PP waste bottle or powerful bleach proof (aggressive disinfectants) glass waste bottle

▪ ready to accept a second hand control-ler for second user, sharing one pump

▪ BVC professional with self-closing quick couplings and non-contaminated liquid level sensor

To maximize safety furthermore and to cover even unlikely events of failing 0.2 micron filters, it may also be advisable to feed the exhaust of the aspiration pump

back into the BSC – preferably direct pri-or to the internal HEPA filter. This exhaust air handling port should be offered by manufactures of biosafety cabinets and is supported by Vacuubrand BioChem- VacuuCenter types BVC control and BVC professional.

Liquid Aspiration in Biosafety CabinetsBiosafety cabinets (BSC) class I or II are indispensable for cell culture work and R&D in life science. They offer a high protection level inside and outside for prices up to more than 10.000 €.

ContactVacuubrand GmbH + Co KG Wertheim, Germany [email protected]

https://www.vacuubrand.com/bvc

BVC aspiration systems offer comfort and safety

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Especially Developed for Biopharmaceuticals

YMC’s latest hydrophobic interaction chromatography column, BioPro HIC HT, is specifically designed for biopharmaceu-ticals such as antibody-drug-conjugates (ADCs). Higher flow rates can easily be applied due to an extremely high-pres-

sure stability, which allows very short run times and high sample throughput.

Optimised for Very Fast DAR Analyses

BioPro HIC HT columns are the ideal choice for drug-to-antibody ratio (DAR) determination due to a novel and opti-

mised surface chemistry. Higher resolu-tion is offered compared to conventional HIC columns as its surface modification suppresses excessive or strong absorption of ADCs. This results in highly reliable quantitation.

The rigid 2.3 µm non-porous polymer particles with butyl modification are pres-sure tolerant up to 400 bar which allow rapid analyses through increased flow rates with consistent high resolution. Such fast runs are not possible with other HIC columns due to their much lower pressure limits, which are usually only about half that of BioPro HIC HT.

Perfect for QC

BioPro HIC HT is the perfect solution for quality control applications, not only due to the possibility of high throughput sepa-rations, but also the excellent batch-to-batch reproducibility provided by YMC. The reproducibility together with the high column stability ensures the highest reli-ability for your separations!

HIC Columns for High Throughput: BioPro HIC HTThe drug-to-antibody ratio (DAR) of antibody-drug-conjugates (ADCs) is critical for the therapeutic efficiency as well as for the pharmacokinetic activity. The control of the DAR in the production process is therefore very important in quality control of ADCs. Hydro-phobic interaction chromatography (HIC) is typically the method of choice for the deter-mination of these ratios.

ContactDr. Daniel EßerProduct Manager Analytical ChromatographyYMC Europe GmbHDinslaken, [email protected] throughput analysis of brentuximab vedotin (Adcetris) by shortening the analysis time using YMC’s

BioPro HIC HT.

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Liquid Handling Machine Provided for UK Government’s Covid-19 Testing EffortsSygnature Group is supporting the UK Government’s Covid-19 testing strategy with the loan of a key piece of equipment. The Tecan Freedom EVO 100 will be used to help automate and ac-celerate the analysis of samples at the first of the UK Govern-ment’s new Lighthouse Labs in Milton Keynes. The liquid handling machine is designed to automate the pipet-ting of multiple samples in laboratories undertaking biological testing. Using the machine is considerably faster than manual pipetting, increasing the speed at which assays can be run and enhancing sample through-put. The equipment also facilitates better assay reproducibility and allows resources and testing equipment to be used more efficiently. The machine is normally used by the high-throughput chemistry group supporting the synthesis of the company’s corporate compound collection.

Sygnature Discoverywww.sygnaturediscovery.com

Thermoelectric Process Thermostats

Thermoelectric process thermostats, which are used in the temperature control of etching processes in the semiconductor in-dustry, have been a permanent feature of Lauda’s comprehensive portfolio since its acquisition by Noah Precision in 2014. Now the company has fully revised and ex-panded the former POU product line with the new Semistat series. The new product line comprises three models: Semistat S 1200 is an affordable entry-level device that offers an even smaller footprint than its predecessor, with the same cooling out-put of 1.2 kW at 20°C. The newly-devel-oped Semistat S 2400 retains the mechani-cal and hydraulic structure of the product line. The process thermostat also incorporates the latest generation of high-performance Peltier elements. The Semistat 4400 brings 4.4 kW cooling output to the market. This process thermo-stat is specially designed for applications using 300 mm wafers with a high cooling output requirement at low temperatures.

Laudawww.lauda.de

Instrument-Free Molecular Diagnostic Test for Covid-19Sense Biodetection has announced an accelerated program to launch the world’s first instrument-free, point-of-care molecular diagnostic test for SARS-CoV-2, the coronavirus responsible for the Covid-19 pan-demic. The company is partnering closely with Phillips-Medisize, a global medical device innovator, de-veloper and manufacturer, to scale-up production of its test to meet the grow-ing demand for rapid diagnostics. The simple disposable test uses a nasal swab sample to give an ultra-rapid result without the need for any instrumentation. As a molecular test, its performance is equivalent to Gold Standard laboratory tests but it is easy to use in any setting and results are available in under 10 minutes. The test is fully self-contained and can be widely distributed to wher-ever it is needed, overcoming the logistical and contamination problems associ-ated with machine-based testing.

Sense Biodetectionwww.sense-bio.com

Water Activity Isotherm Generation

Decagon has developed the AquaSorp to utilize the patented high-resolution DDI (Dynamic Dewpoint Isotherm) method. The re-sultant high-resolution moisture sorption iso-therms make it feasible to model and engineer food products and packaging in ways not previously feasi-ble. Manufacturers can therefore develop products that optimize safety, quality and profitability. Throughout the measurement range of 0.10 to 0.95 aw, the AquaSorp achieves an accuracy of +/-0.005 aw and a repeatability of +/-0.003 aw. A further advan-tage of the automatic measuring system is that it needs no user intervention once it has been started, so there is no risk of human error influencing the re-sults.

Labcellwww.labcell.com

Liquid Chromatograph

Scion Instruments has unveiled the LC6000 liquid chromatograph, adding to their existing range of chromatography products. The HPLC delivers confidence in re-sults through outstanding lifetime performance, a robust design maximizes up-time and productivity levels whilst minimizing cost of operation. The SCION 6000 Series offers an array of automation options for workflow optimization. It has ex-cellent flow rate and injection volume precision and an ultra-low carry-over. There is a low-volume degassing option and the device has a large solvent cabinet.

Scion Instrumentswww.scioninstruments.com

Ion Polishing Tool

The PIPS II is an ion polishing tool that allows for low energy milling with easy repeatability and high precision. The Whisperlok X, Y stage has the ability to center the region of interest for re-polishing. The device features low-energy focusing penning ion guns, for focused ion beam (FIB) prepared samples, a vari-able energy from 0.1 – 8.0 kV for improved low energy milling and a reduction in amorphous layer for corrected TEMs. The LN2 specimen cooling eliminates artifacts while a 10” color touchscreen provides simple but full control via the graphical user interface. The color image storage can store and use optical im-ages with the TEM and EELS data in the same format.

Gatanwww.gatan.com

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Vacuum Pumps for Mass Spectrometer Systems

Hena 50 and 70 are single-stage, oil-sealed rotary vane pumps from Pfeiffer Vacuum, which were specially developed for the de-manding requirements of mass spectrometer systems. They achieve pumping speeds of be-tween 32 and 59 m³/h, depending on their size and speed of rotation. Their integrated oil mist separator ensures clean exhaust air. They are equipped with a frequency convert-er that enables them to be used worldwide with single-phase input and identical pump capacity for 50 and 60 Hz. A con-stantly high flow rate in the target pressure range, adjustable pumping speeds and low final vacuum contribute to the instrument’s reliable high performance. Long maintenance intervals and operating hours are ensured by the high oil volume in the pumps and their low oil temperature during operation. Both models are certified according to UL and IEC 61010.

Pfeiffer Vacuumwww.pfeiffer-vacuum.com

High Precision Event Timer

Picoquant has released new versions of the Multiharp 150 event timer that offer im-proved timing precision with 10 ps minimum bin width, along with jitter better than 45 ps. The new models are ideal for fast and high-resolution fluorescence lifetime imaging (rapidFLIM) and multichannel photon correlation. All sixteen of the de-tector channels and the common SYNC input of a Multiharp 150 are indepen-dent and synchronized. The SYNC input can also be used as either an additional detection channel or for a periodic reference signal with repetition rates up to 1.2 GHz. Depending on the configuration of the computer, the Multiharp 150 can sustainably capture and transfer 80 million time tags per second with single channel peak rates of up to 1.5 Gcps for short times. Thanks to its hardware-inte-grated 65536 bin histogrammer, count rates up to 166 million counts per second (Mcps) in total for 4 or 8 channel versions and up to 332 Mcps for the 16 channel version can be achieved in lifetime measurements.

Picoquantwww.picoquant.com

Quadrupole Time-Of-Flight Mass Spectrometer

Shimadzu‘s LCMS-9030 Q-TOF is a quadrupole time-of-flight mass spectrometer and combines sensitive quadrupole technologies with a unique TOF architecture. The system has won an iF Design Award, one of the leading design prices worldwide for excellent aesthetics as well as for user-friendly, ergonomic and efficient features. The research grade mass spectrometer delivers high-resolution, accurate mass detection with incredibly fast data acquisition rates. It has a powerful TOF architecture to transform high mass accuracy workflows by achieving high-sensitivity, high-speed and high-res-olution detection. The LCMS-9030 provides effort-less performance with less need for calibration and easy switching between ionization units.

Shimadzuwww.shimadzu.eu

All-In-One Listeria, L. Monocytogenes Detection Method

Hygiena has introduced the Insite L. mono Glo environmental surface screening test. This is an easy-to-use, self-contained, environmental sampling and screen-ing test for Listeria species and Listeria monocytogenes (L. mono). Each device contains liquid media containing antibiotics, growth enhancers, and color-changing compounds specific to Listeria plus fluorescent compounds specific to Listeria monocytogenes. Within 48 hours, the two-step test changes color in the presence of Listeria species, while illumination with UV light reveals if L. mono-cytogenes are present. The test is used for environmental monitoring in food processing facilities after cleaning. Screening out negative samples quickly and easily provides a cost-effective way to increase surveillance, monitor hazards and control risks.

Hygienawww.hygiena.com

Trace Element Analysis Solutions

The Thermo Scientific iCAP Pro Series ICP-OES platform is designed to provide a fast, sensitive range of trace element analysis solutions capable of capturing the com-plete spectrum of high matrix samples in a single run, improving workflow productivi-ty and reducing analysis costs. From stand-by to start-up in just five minutes, the new instruments reduce gas consumption with-in a vertical dual-purged optical pathway interface that has lower installation re-quirements due to its standard wall socket and low extraction flow rate. The launch of the platform helps laboratories by-pass traditional sample preparation requirements and the need to undertake multiple measurements, providing a range of trace element analysis solutions that enhance workflow productivity and reduce cost-per-sample.

Thermo Fisher Scientificwww.thermofisher.com

Optimized Production of AAV

Lonza has launched the TheraPEAK SfAAV medium, a chemically defined, non-animal origin medium designed specifically for the production of Adeno Associ-ated Virus (AAV) in Spodoptera Fuigiperda (Sf9) insect cells for gene therapy. Due to its chemically defined nature, the new hydrolysate free medium pro-duces AAV that requires less purification, further decreasing the overall process-ing time and minimizing labor requirements. Furthermore, the medium supports consistent cell growth throughout all phases of the culturing process, consider-ably reducing process variability. As a chemically defined, non-animal origin product, the medium is safer to use than media containing animal or human components, thereby facilitating regulatory compliance. Additionally, the medi-um is supplied with a U.S. Food and Drug Administration drug master file, allevi-ating the relevant preparation and submission burden.

Lonzawww.lonza.com

Products 41

Marketplace

http://www.pfeiffer-vacuum.com

http://www.picoquant.com

http://www.shimadzu.eu

http://www.hygiena.com

http://www.thermofisher.com

http://www.lonza.com


Catalysis Cell Reactor

Linkam has recently seen an increased interest in the CCR1000 stage that was specifically designed for the analysis of catalytic reactions. The CCR1000 has a custom-designed chamber for experimental analysis, and can be used with a microscope, FT-IR or Raman spectrometer. Samples are mounted on virtually nonreactive disposable ceramic fabric filters placed inside the ceramic heating element, which is capable of heating samples from ambient to 1000°C at rates of up to 200°C/min and pressures up to 5 Bar. It has been designed to ensure the majority of components are in di-rect contact with the sample and carrier gases are as nonreactive as possible. A variety of different lid window materials are available so that bright field and Operando microscopy techniques can be used.

Linkam Scientific Instrumentslinkam.co.uk

Products for Cell Culture Workflows

Proculture, the new workflow-minded product line for cell culture from Bel-Art and Wilmad-Labglass brands, covers multiple steps of the cell culture process from isolation to harvesting. Products include an array of shaker flasks, spinner flasks with a unique impeller that increases aeration and eliminates dead spots, and an orbital shaker platform that converts an existing mag-netic stir plate into an orbital shaker at a fraction of the cost of an orbital shaker. The Proculture line includes products that can sim-plify researchers’ cell culture experiments.

Scientific Productswww.scientificproducts.com

Protein Labeling Kit

Fluidic Analytics has launched a time-saving and affordable protein labeling kit – fluidiphore rapid amine 503. Absorbing at 503 nm, the new kit can be used with a multitude of techniques including the company’s Fluidity One-W instru-ment for protein interaction analysis. Fluorescent labeling of proteins such as antibodies, DNA and lipids is widely used to investigate and better understand protein structure and function. There are various fluorescent dyes available on the market, most of which require a long labeling process over several hours and a purification step to remove excess dye. Fluidic Analytics’ fluidiphore label-ing kit is extremely rapid, taking just 30 minutes to bind proteins rather than four hours to overnight. In addition, the kit does not require a purification step. This is due to a clever ‘triple-lock’ against background effects.

Fluficwww.fluidic.com

Ultra-Cool Circulation Chillers

Lauda has introduced a new generation of circulation chillers to its Ultracool series, which have been developed with a focus on energy efficiency and in compliance with the European Ecodesign Directive. An integrated web server enables control via mobile devic-es, as well as connection to the Lauda Cloud. Industrial circulation chillers are often used in continuous operation. They cool applica-tions used in the printing industry, machine and plant construction and metal working. Companies are increasingly focusing on ener-gy-saving and sustainable measures as part of the energy revolution; the need for energy-efficient options to control the temperature of industrial processes is also con-stantly increasing.

Laudawww.lauda.de

Black Microplates that Minimize Sample Degradation

Porvair Sciences has expanded its range of high quality, black microplates that minimize sample degradation by exposure to light, even over long storage periods. Available in a choice of 48, 96 and 384-well formats, the black plates are precisely manufactured to applicable ANSI / SLAS dimensions ensuring complete compatibility with almost all read-ers and automated equipment. Manufactured from polypropylene, they offer excellent heat and solvent resistant qualities. Using only ultra-pure grade poly-mer means that each black plate has near zero leachates, ensuring long-term sample integrity. The black plates are supplied RNase / DNase-free, meaning they can be used to store even the most sensitive biological samples.

Porvair Scienceswww.porvair-sciences.com

Free corona safety signs

Safety and identification specialist Brady Corporation offers signs for download to help stop the spread of the COVID-19 virus. Anyone can freely download the print-ready files from Brady websites. All signs offered are compliant or in line with the ISO 7010 international standard to maximise recognition anywhere in the world. Each sign includes a quickly recognisable icon for almost any COVID-19 safety measure, ranging from wash hands and keep distance to wearing the appropriate personal protection equipment. Brady’s safety and iden-tification signs are usually available only on sturdy, industrial-grade materials that require a specialised thermal transfer label printer to create. However, to make the safety signs available quickly in any facility, they have now been organ-ised in a full-colour format that is easy to print on most office printers. Once printed on paper, sets of signs can be applied with tape on doors or smooth walls where needed to communicate the temporary safety measures.

Bradywww.bradyeurope.com

42 Products

Marketplace

http://www.linkam.co.uk

http://www.scientificproducts.com

http://www.fluidic.com

http://www.lauda.de

http://www.porvair-sciences.com

http://www.bradyeurope.com

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Department of Chemistry, Kumamoto University 10

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Uppsala University 25

Hessenwasser 12

Institute for Microelectronics and Microsystems of

National Research Council (IMM-CNR) 22, 28

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