Polymers and fluorescence Balance of power 360° drinking water analysis trilogy Fluorescence spectroscopy LCMS-8060NX: performance of industrial base polymers and robustness without Automatic, simultaneous and compromising sensitivity rapid analysis of and speed CONTENT

APPLICATION »Plug und Play« disease screening solution? – The MALDI-8020 in screening for Sickle Cell Disease 4 Customized software solutions for any measurement – Macro programming for Shimadzu UV-Vis and FTIR 8 Ensuring -free food supplements – Identification of in pharmaceuticals and food supplements with LCMS-8045 11 MSn analysis of nonderivatized and Mtpp-derivatized – Two recent studies applying LCMS-IT-TOF instruments 18 Polymers and fluorescence – Part 2: How much fluorescence does a polymer show during quality control? 26

PRODUCTS The balance of power – LCMS-8060NX balances enhanced performance and robustness 7 360° drinking water analysis: Episode 2 – Automatic, simul- taneous and rapid analysis of pesticides in drinking water by online SPE and UHPLC-MS/MS 14 Versatile testing tool for the automotive industry – Enrico Davoli with the PESI-MS system (research-use only [RUO] instrument) New HMV-G3 Series 17 No more headaches! A guide to choosing the perfect C18 column 22 Validated method for monoclonal antibody drugs – Assessment of the nSMOL methodology in Global solution through the validation of bevacizumab in human serum 24 global collaboration LATEST NEWS Global solution through global collaboration – Shimadzu Cancer diagnosis: Shimadzu innovation centers as innovation centers as a partner of scientific research 2 a partner of scientific research

MARKETS eginning with one man in Kyoto, Japan, in 1875, the Chemical, Petrochemical, Shimadzu Corporation Biofuel and Energy B now has more than 10,000 em - Clinical ployees worldwide working under the company’s philosophy, “con- Environment tributing to society through sci- ence and technology.” For its Food, Beverages, Agriculture next generation of scientific excel- lence, Shimadzu has established Pharmaceutical Shimadzu Innovation Centers (SICs) across the globe, with the Plastics and Rubber European Innovation Center (EUIC) opening in 2017 in Duis - Automotive burg, Germany. Silvia Giordano and Enrico Davoli in front of the Mario Negri Institute, Milan

SHIMADZU NEWS 2/2020 LATEST NEWS

Based on the growing network 2018). In the project between England, where EUIC is support- The information including affili- Shimadzu has nurtured since its Davoli and Shimadzu, AI is ing Ph.D. grants for three years. ates and titles of the persons in arrival in Europe in 1968, the trained to analyze a specimen from this article are current as of the EUIC has worked on over a PESI-MS data without any histol- Shimadzu believes that no matter time of interviewing (December dozen on going projects that aim ogy or other laborious prepara- the excellence of its instruments, 2018). to change how societies preserve tion. AI will output a binary deci- the primary goal of its SICs is to food, practice , and pro- sion – normal or cancer – with a benefit society. SICs therefore All the instruments in this article tect the environment. Here we statistical probability. The whole view their research collaborations are research-use only (RUO) introduce a project between EUIC process takes no more than two as academic partnerships that instruments and not for use in and an academic institution that minutes from the time the surgeon never compromise academic inde- diagnostic procedures. Shimadzu will advance cancer treatment. acquires the specimen to the AI pendence, encouraging research is an analytical equipment pro - decision. publications and supporting aca- vider and in certain regions, Research to improve demic education. including the USA, Shimadzu cancer detection After Takeda and Shimadzu had shown the effectiveness of this There is no doubt who proposed approach in Japan, the question to collaborate on the cancer proj- lingering was whether it could ect between Shimadzu and Dr. extrapolate to other countries, Enrico Davoli at the Mario Negri since the AI was originally trained Institute for Pharmacological with data from Japanese speci- Research in Milan, Italy, and the mens. That was when Shimadzu first president of the Italian Mass contacted Davoli, asking if he Spectrometry Society. After its could acquire tissue samples from successful collaboration with Italian hospitals. Davoli was very Professor Sen Takeda of Yamana - excited to take part. shi University, Japan, to study a new system that combines arti - “For a researcher, having a proto- ficial intelligence (AI) with probe type and the first to try something electrospray ionization mass spec- is a desire your whole life. The trometry (PESI-MS) to enhance instrument is brand new. The research-based detection of cancer approach is brand new. It was a cells of the (Yoshimura et very good opportunity,” he said. al., 2012), Shimadzu proposed to do the same with Davoli in Eu - The early results are promising, rope. and Davoli is extending his collec- tion to samples for other solid Hepatocellular carcinomas are tumors with funding from a leading cause of cancer death. Shimadzu. “Our plan is to work Common treatment involves with other European nations to resecting the cancer cells, but test PESI-MS on other popula- there is argument in the field on tions,” he says, adding that EUIC just how much healthy tissue has been instrumental at building Prof. Sen Takeda of University of Yamanashi at Shimadzu Global Innovation Summit 2017 taken in the resection is best for the continental research network. patient survival, with some re- The success of SICs is built on products may be regulated as for searchers arguing a small buffer Working like an academic new ideas, which is why our research purposes only. If you zone that removes healthy tissue partner teams are always in consultation have questions on approved use of is acceptable and others arguing with university researchers to a Shimadzu device, please contact nothing but cancerous tissue In this way, SICs can be viewed learn about scientific problems your regional office. should be removed. During the as a hybrid of industry and acade- that Shimadzu innovation can operation, the surgeon will pass a mia. All SICs aim to create new help solve. It is also why SICs References specimen to a pathologist who commercial markets, but explains build research networks, having Kudo, M. (2018). Management of Hepato - will perform histology to deter- Dr. Ann-Christin Niehoff, EUIC introduced Davoli to Takeda. cellular Carcinoma in Japan as a World- mine whether more resection is Product Manager of Imaging, mar- Takeda was invited to speak at Leading Model. Liver Cancer 7, 134-147. needed. Each specimen takes kets do not only grow from eco- our hosting event, Global Inno - about 30 minutes to prepare and nomic need, but also training vation Summit 2017 in Kyoto, Yoshimura, K., Chen, L.C., Mandal, analyze, which not only cause expertise. where he got to meet our other M.K., Nakazawa, T., Yu, Z., Uchiyama, higher costs, but also the likeli- partners in academia as well and T., Hori, H., Tanabe, K., Kubota, T., Fujii, hood of infection. “More than 90 % of Ph.D. chem - share their latest findings in ana- H., et al. (2012). Analysis of renal cell carci- ists do not stay in academia. We lytical research. noma as a first step for developing mass New science for our help build their career,” she says, spectrometry-based diagnostics. J Am Soc partners which is why Shimadzu funds By “contributing to society Mass Spectrom 23, 1741-1749. Ph.D. students and will even be through science and technology”, Japan ranks near the top of patient co-investigators on grants. Exam - Shimadzu and its SICs want to outcomes for liver cancer, in large ples include collaborative projects translate academic research into part because of Japan’s innovative at Limoges Hospital, France, and scientific solutions for some of the diagnostic technologies (Kudo, the University College London, world’s greatest challenges.

SHIMADZU NEWS 2/2020 3 APPLICATION »Plug und Play« disease screening solution? The MALDI-8020 in screening for Sickle Cell Disease

3D illustration of sickle cell

n 2006, the World Health Assembly passed a resolution I recognizing the Sickle Cell Disease (SCD) as a public health priority and called on countries to tackle the illness. This resolution was also adopted by the United Nations in 2009. In this context, numerous screening programs have been set up in many labora- tories around the world.

SCD is a group of inherited disorders. It is caused Figure 1: Workflow of automated screening

4 SHIMADZU NEWS 2/2020 APPLICATION

ants and HbS homozygotes. The meet the criteria of automation, Resolution is an extremely second level is based on a second sensitivity, resolution, robustness, important criterion in standard reference method and is speed of analysis, resistance to the analyses of hemoglo- used to confirm the first results. high analytical rates and ease of binopathies use. Screening for hemoglobinopathies Due to abnormal hemoglobin, can be performed with newborns, Through the implementation of SCD arises from mutations in the in order to ensure early care for screening for sickle cell disease, hemoglobin beta gene (HbS) in children. This care at an early age Biomaneo company has shown which the glutamic acid is substi- enables a considerable increase in that the MALDI-8020 benchtop tuted by valine at position 6, life expectancy. The second option mass-spectrometer meets the qual- resulting in 30 Da lowering of the of screening can be carried out in ity criteria mentioned above and mass. The first NeoSickle solution young adults or adults in the case makes the use of MALDI-MS in is based on the whole protein of primary prevention, enabling laboratories easier, convenient and analysis which needs a sufficient Figure 2: Specific support for Fleximass people to know their status of fast. resolution at the mass of approxi- target homozygous AA or heterozygous mately 15 kDa. Figure 3 shows XS. People who inherit one sickle New method, high-throughput, the resolution capability of the by abnormal hemoglobin (HbS) cell gene and one normal gene easy to use MALDI-8020 in line with expecta- resulting from mutations in the have sickle cell trait (SCT) and are tions to detect the different hemo- hemoglobin beta. The condition heterozygote XS. People with Biomaneo has developed a ‘plug globin chains HbS, HbA and HbF. was first described as early as 1910. SCT do not usually have any SCD and play’ analytical solution that Today, approximately 80 % of symptoms but can pass the trait combines a sample treatment kit, With this first approach, it is not sickle cell disease cases are found on to their children. the MALDI-MS technology and a possible to separate the other in tropical regions, particularly software to interpret results auto- chains of hemoglobin variants sub-Saharan Africa, but have also Efficient analytical matically by combining them with such as HbC and HbE which have increased in Europe in recent method clinical data (figure 1). a mass difference of 1 Da com- decades due to migration. pared to the normal HbA chain. Neonatal screening is usually per- The first NeoSickle® kit was For this reason, the second ap - How to meet the needs of formed centrally in specialized developed in two versions: the proach used by Biomaneo consists public health measures laboratories. The biological sam- first is dedicated to high-through- of analyzing hemoglobin chains ple used for this screening is a dry put analysis using a robot; for this peptides, and the resolution and Disease screening programs are blood spot on blotting paper. a support to use the Fleximass tar- dynamic range of the MALDI- front-line public health measures, get of MALDI-8020 was specifi- 8020 benchtop allows for separa- and as such must be based on The use of MALDI-MS in hospi- cally created (figure 2), and the tion of the peptides of interest. robust analytical methods and tal laboratories has already proved robot script adapted. Figure 4 (page 6) illustrates the data-processing software. Cost its value, in particular through the possibility to separate the sample effectiveness is a further require- identification of micro-organisms The second version is a kit for groups homozygote AA or het- ment and has prompted the imple- (use of VITEK®MS for example). labs which analyses nearly 200 erozygotes AE. mentation of high-throughput MALDI-MS techniques meet the samples per day manually. The screening units that reduce item requirements of screening. Spe - Fleximass target is adapted for costs. Lastly, the greatest possible cifically, the techniques used must both of these options. use of automation enables the medical team to focus exclusively on abnormal samples. The Shimadzu MALDI-MS (Mass Spectrometer) together with tech- nology packages from Biomaneo Hb S 15837 Da Hb A 15867 Da Hb S 15837 DaHb A 15867 Da Hb S 15837 Da Hb A 15867 Da company in Dijon, France includ- 2.0 1.2 ing sample processing kit, data 0.12 1.0 processing and management soft- 1.5 0.10 0.8 ware were designed to address 0.08 these challenges. 1.0 0.6 0.06 Intensity 0.4 Two-tier procedure for more 0.04 0.5 0.2 accurate results 0.02 0.0 Screening programs are typically 15,840 15,860 15,880 15,900 15,840 15,860 15,880 15,900 15,840 15,860 15,880 15,900 organized in two-tier procedures: m/z m/z m/z The first tier is normally a routine method which must be able to AS SS S␤+ detect the pathological samples with a single mutation and classify unambiguously into three groups: Figure 3: Spectrum obtained from Heterozygote AS, Sbeta+ and Homozygote SS samples with the detection of hemoglobin HbA heterozygotes without HbS vari- and HbS chains ants, heterozygotes with HbS vari-

SHIMADZU NEWS 2/2020 5 APPLICATION

Sensitivity and dynamic generally for the high-throughput range of detection analysis of complex blood sam- ples. It very clearly satisfies the The other crucial criteria in SCD needs of screening laboratories. screening are the sensitivity and dynamic range of detection. In The power and speed of MALDI fact, the sample from very prema- instruments means that they will ture newborns has a very low per- very rarely be used full time for 100 centage, less than 3 % of the adult just a single application. For hos- hemoglobin chain compared to 80 pitals or laboratories, it is effective other compounds in the sample 60 to optimize the investments made (foetal beta hemoglobin and alpha 40 in these technologies. System ver- hemoglobin chain). Detection and Intensity % satility is an important financial 20 differentiation of the HbS beta criterion to be taken into account. hemoglobin chain in these samples 0 For laboratories with a small is very important in order to number of screening analyses, the avoid re-blood sampling and/or advantage of the MALDI-8020 is misclassify the newborns. The Figure 4: Analysis of specific peptides from hemoglobin chains HbE and HbA the management of acquisition study demonstrates the high qual- parameters. Indeed, the acquisi- ity of the MALDI-8020 instru- tion methods integrate all the ment with a percentage of classifi- less than three minutes. This time and the low maintenance parameters necessary to control cation of very premature babies throughput, including the change requirements. To maintain instru- the instrumentation, making the above 90 %. of target in the instrumentation, ment performance with an objec- MALDI-8020 applicable for mul- will allow 576 analyses per hour. tive of 500,000 tests per year, tiple applications without the risk Screening for SCD is performed Shimadzu’s MALDI-8020 pro- of interfering with individual in centralized laboratories analyz- For SCD, the main French screen- vides TrueClean automated source analyses. ing a large number of samples per ing laboratory analyses about 600 cleaning. It can be used to clean day. The technology used for samples per day, meaning over the extraction electrode in-situ To enhance the power of automat- screening must therefore be auto- 150,000 samples per year or about without breaking instrument vacu- ed high-throughput data produc- mated and/or convenient to use 20 % of births. Less than six um; it is an automated and rapid tion by the MALDI-8020, for the technician. Usability is an hours of analysis per day with the (< 10 min) UV laser-based proce- Biomaneo has developed a soft- indispensable criterion for the MALDI-8020 would be sufficient dure. Moreover, the wide-bore ware package (LIMS: Laboratory establishment and use of mass to screen all newborns in a single optics minimize the risk of source Information Management System) spectrometry in analytical labora- laboratory. contamination over time, provid- to integrate the results and assist tories. The usability of the ing a robust platform. in the interpretation of mass MALDI-8020 benchtop is clearly Fully serves the needs of spectrometry data. The LIMS an advantage for the laboratory screening laboratories These characteristics and results NeoScreening® allows production where the number and turnover demonstrate the suitability of the of the files necessary to control of technicians, not specialists of Beyond the daily throughput, the MALDI-8020 bench-top mass the MALDI-8020 and launch an MALDI-MS, is very high. last criterion concerns the robust- spectrometer for the detection of analysis run, while also retrieving ness of the instrumentation over hemoglobinopathies and more data automatically and then pro- MALDI-8020 is the system cessing and classifying each sam- of choice ple result.

The MALDI-8020 meets these cri- Conclusion teria with well-calibrated and standardized methods. Use of the The speed of acquisition and the MALDI-8020 is intuitive, allow- simplicity of changing methods ing its rapid integration into labo- makes the MALDI-8020 benchtop ratory studies and facilitating the ideal for many applications and an work of technicians. In addition, indispensable measuring instru- the system is very compact and ment for biochemistry laborato- can be easily placed on any labo- ries. ratory bench. In the context of SCD screening, Throughput in screening laborato- the MALDI-8020 meets all ries is a strong requirement to be requirements. This compact, sim- considered in the introduction of ple to use and high-performance new analytical methods. The spec- instrument allows screening in tra acquisition method imple- most cases. mented in the NeoSickle solution combined with the performance NeoSickle® is a registered trademark of the MALDI-8020 benchtop of Biomaneo. allows the analysis of a MALDI Figure 5: Screenshot of the NeoScreening LIMS after analysis of one plate NeoScreening® is a registered trademark target containing 48 samples in of Biomaneo.

6 SHIMADZU NEWS 2/2020 PRODUCTS The balance of power LCMS-8060NX balances enhanced performance and robustness without compromising sensitivity and speed

The new LCMS-8060NX coupled to the Nexera LC-40 series

ince 2010, Shimadzu is acquired MRMs in post-run With enhanced ease-of-use, Under the umbrella of Analytical shaking the market in triple analysis in case of interferences. robustness and a wide range of Intelligence, Shimadzu’s analytical S quadrupole LCMS by intro- default para meters, the new sys- systems will be successively ducing game changing innova- Subsequent systems, the LCMS- tem achieves the balance between equipped with automated func- tions. The groundbreaking pro - 8060 (2016) and LCMS-8045 high sensitivity and robustness. tions, enabling users to easily cess started with the LCMS-8030 (2017) are based on that successful review instrument status, optimize featuring fast polarity switching LCMS-8050 platform. They of - With the new IonFocus ion resource allocation and achieve of 15 ms, combined with ultra-fast fered the market a range of sensi- source, the LCMS-8060NX effi- higher throughput. electronics for short Dwell Time tivity depending on individual ciently separates from neutral (DW) and Pause Time (PT), fast needs without sacrificing user particles, allowing only the ions The concept simplifies lab man- scanning speed and fast collision flexibility. into the system driven by the agement and enables higher pro- cell. These technologies are now Focus voltage. It releases the neu- ductivity, maximum reliability and well-established on the market User-friendly, robust and tral particles through the drain. better connectivity, applying IoT, under Ultra-Fast Mass Spectro - highly sensitive Artificial Intelligence and M2M. metry (UFMS). In 2015, the A newly extended range for de - LCMS-8050 introduced 5 ms A general market demand is the fault parameters in the ion source polarity switching and scan speed robustness of high-sensitivity expands the possibilities for a at 30,000 amu/s. It improved the instruments. The LCMS-8060 is high-sensitivity analysis. The market by presenting new ways of already a highly robust system improvement of the integrated ion using a tandem LC-MS. used in clinical and food safety pathway establishes the LCMS- applications. The need for more 8060NX as the class leading triple Due to the speed of all these new robustness as well as user-friendly quadrupole LC-MS on the market. technologies, multiple MRMs/ meth od development and analysis compound methods (more than is a key driving force for Analytical Intelligence for the standard 2 MRM Multiple Shimadzu, innovating existing lab simplified lab management Reaction Monitoring technique) solutions and creating new ones started to be developed, enabling to steadily support the market. In combination with the brand- better identification with the new LC-40 series and its Analy - MRM Spectrum Mode and simpli- A new addition to the LC-MS tical Intelligence concept, it offers fied multicomponent/contami- portfolio is the LCMS-8060NX an outstanding solution for rou- nants analysis review by switching Triple Quadrupole solution. tine testing.

SHIMADZU NEWS 2/2020 7 APPLICATION Customized software solutions for any measurement Macro programming for Shimadzu UV-Vis and FTIR

Figure 1: Example of an Easy Macro for a simple measurement with pre-defined Figure 2: Execute macro window. The LabSolutions IR program used for the easy macro measurement parameters is run in the background. The macro execution can be stopped at any step.

he data output of spectro - Such requests are served by cus- easy to understand building Parameters”, it will just use the photometers has come a tomized software or macros. blocks, such as “Sample Scan”, last set parameters. Steps 4 and 5 T long way since the time of Shimadzu offers different tools “Smoothing” or “Spectrum first measure the background, and mechanical writers. Today, the depending on the complexity of Search”. Customized messages can then the sample. The sample and user gets a full-blown software the task. This article gives a brief be shown between the steps, e.g. file name can be defined by the suite with lots of different meas- overview of the possibilities of telling the user to set the sample macro, but in this example a urement options and integrated controlling Shimadzu FTIR and or clean the ATR. Figure 1 gives prompt will be shown to allow data processing. While the specifi- UV-Vis instruments by a custo - an example of an Easy Macro. the user to enter sample and file cations – such as scan speed, sig- mized software. name. Each Easy Macro must start nal-to-noise ratio or resolution – In this example, the spectrum with the command “Program are still improving gradually with FTIR – Easy Macro software is used. It is started in Start” and end with the command each new instrument, the software step 1, then the instrument is ini- “Program End”. included has become an equally The starting point for most FTIR tialized in step 2 and the scan important selling point. macros is the Easy Macro tool parameters are set in step 3. When Easy Macro provides the benefit included in LabSolutions IR. It the macro is run, the spectrum that even users without any Even though LabSolutions IR and allows users to automate usual software opens in the background; knowledge of programming can UV already offer a wide variety of analysis by ‘drag and drop’ of without the command “Scan develop such a macro. Because of tools, there are cases which demand a custom solution to a Tool Easy Macro VB Macro very specific problem. Whereas Instrument IRSpirit, IRAffinity-1s, IRTracer-100 IRSpirit, IRAffinity-1s, IRTracer-100 one customer might require a sim- Used by Customer or Shimadzu Shimadzu plified user interface and automat- Application Simple automation, starting VB macros Complex automation, IRPilot, EP validation ed analyses to support the compa- Features Simple drag & drop of pre-defined code blocks Visual Basic source code in LabSolutions IR ny’s technicians in their daily rou- Advantages Easy to understand Full integration in LabSolutions IR tine, a different client might be Prevents syntax errors Includes classes from VB, such as user forms bound to a very specialized data Can load more complex macros Very complex applications possible processing which is not common Disadvantages Only spectrum or postrun applications Not accessible for the customer enough to be implemented into the LabSolutions software. Table 1: Comparison of macro tools for Shimadzu FTIR

8 SHIMADZU NEWS 2/2020 APPLICATION

Figure 3: User interface of the IRPilot Figure 4: The initialize command from Easy Macro as visual basic source code

the pre-defined building blocks, Each building block of the Easy macros, like the IR Pilot included writes the result into “response” syntax errors and malicious code Macro generates visual basic with the Shimadzu IRSpirit, text files. are prevented. The user can see in code which is interpreted by shown in figure 3. the execute window if an Easy LabSolutions IR. The syntax of these commands is Macro tries loading a potentially The visual basic macro editor easy to understand, and the only malicious basic file, and prevent FTIR – Visual Basic included in LabSolutions IR offers prerequisite of the programming the execution. (VB) macro a full development environment environment is to be able to read for the trained specialist. Finalized and write text files. As the user Most operators meet Easy Macro The visual basic code behind such macros are either added to the rights and audit trail settings by the validation functions includ- an Easy Macro can be extracted toolbar of LabSolutions IR Post - of LabSolutions DB are not cir- ed with their Shimadzu FTIR, and used as the basis for more run or Spectrum, or they are start- cumvented by such scripts, which are started by such macros. complex Lab Solutions VB ed by the “Load Basic File” com- LabSolutions UV-Vis Automatic mand of Easy Macro. Figure 4 Control can be used even in a val- shows a screenshot from the idated environment. A bi-direct - LabSolutions IR VB macro editor ional communication between with the code for the “Initialize” LabSolutions UV-Vis and a third- building block from Easy Macro. party lab management system is possible by writing command files UV – Automatic Control for the LabSolutions UV-Vis Automatic Control Option and In the world of LabSolutions UV- then reading the text or pdf report Figure 5: Schematic of the communication between LabSolutions UV-Vis and Vis, there is no counterpart for generated after each measurement. a host system by the Automatic Control option Easy Macro, since LabSolutions UV-Vis already offers a high UV – string commands degree of automation – e.g. for text export, postrun calculations The most accessible way to read or spectrum evaluation. But it is out data from UV-Vis instruments possible to further automate by customized software is with LabSolutions UV-Vis and connect the external commands document- it to customized software by the ed in the instruction manuals of Automatic Control option. A typ- UV-1280 and UV-1900i. They are ical application for this option is also known as string commands, the communication between because the communication is LabSolutions UV-Vis and an established by simple ASCII autosampler software. strings sent over a virtual COM port. The scripts use commands similar to the building blocks of LabSolutions FTIR Easy Macro. Text files with these commands are placed in a special folder which is scanned continuously Figure 6: Autosampler control as example for a LabSolutions UV Automatic by LabSolutions UV-Vis. Control application LabSolutions reads the commands from “command” text files and

SHIMADZU NEWS 2/2020 9 APPLICATION

Tool LabSolutions UV-Vis Automatic Control String Commands UV-OCX Instrument UV i-Selection (UV-1900i, UV-2600i, UV-2700i, UV-1280, UV-1900i UV-1280, UV-1900i, UV-2600i, UV-2700i UV-3600i plus, SolidSpec-3700i) Used by Customer or Shimadzu Customer or Shimadzu Shimadzu Application Simple automation, Autosampler control, PC control of UV-1280, excel-macros, Custom software with full instrument control LIMS integration third-party software Features Scripts for LabSolutions UV-Vis by simple ASCII-commands for simple instrument Indirect instrument control by Active X text files control Advantages Code independent from instrument Commands are published Nearly full control of instrument functions Easy to implement Small, efficient code Communication errors prevented by design LabSolutions DB/CS user rights still valid Free choice of programming language Developer is supported by Microsoft IntelliSense Disadvantages Only specific functions of LabSolutions UV Solid programming skills required No LabSolutions integration accessible

Table 2: Comparison of macro tools for Shimadzu UV-Vis

VBA editor is used, the UV-OCX even implements helpful func- tions, e.g. intelligent code comple- tion, to prevent syntax errors.

Conclusion

Macro programming is a powerful tool to support customers. Some macros enable applications which are not covered by the standard software, while other macros serve to automate tedious tasks and prevent user errors. The best approach depends not only on the Figure 8: Simple instrument control Figure 7: Excel-Macro for Bilirubin in Spinal Fluid as typical customized complexity of the application, but window in a VBA form made with application using string-commands also on the experience of the pro- UV-OCX grammer.

These commands are typically X control is used to translate Table 1 compares the different used in customized Excel-macros commands written in a VBA or tools to program custom software with instrument control or to pro- Microsoft.net Framework source for Shimadzu FTIR, and table 2 gram drivers for software with code into machine code for the shows the tools for Shimadzu control of instruments from mul- spectrophotometer. It is possible UV-Vis spectrophotometers. tiple vendors. Example code for to control UV-1280, UV-1900i, the implementation in Microsoft UV-2600i and UV-2700i. Visual Basic 2015 is given in the Figure 9: UV-OCX instrument control manual along with the required Compared to string commands, embedded directly in an Excel sheet settings of the virtual com port, programming with OCX com- but a much deeper understanding mands requires less understanding of interface programming is of interfaces, as the communica- required compared to developing tion with the instrument is done software with the LabSolutions by the OCX control and commu- Automatic Control. nication errors (such as setting impossible wavelength values) are UV – OCX commands prevented by this interface. The UV-OCX is implemented as a Another possibility for direct class with pre-defined methods instrument control is given by the and parameters. This spares one option UV-OCX. Here, an Active step compared to the string com- mands, where the programmers must write their own class to translate between e.g. VBA code and ASCII-strings.

When a programming environ- Figure 10: The optional VisEase software ment such as Microsoft Visual which was developed with UV-OCX Studio or the Microsoft Excel

10 SHIMADZU NEWS 2/2020 APPLICATION Ensuring steroid-free food supplements Identification of steroids in pharmaceuticals and food supplements with LCMS-8045

nabolic steroids are often encountered in samples of A nutritional supplements and pharmaceutical preparations, either due to doping-related activ- ities [1] or cross-contamination issues during production [2]. Developing methods to identify steroids in such samples can be challenging, since the method should be able to monitor numer- ous steroids with large differences in terms of polarity. Compounds to be monitored comprise rela- tively polar steroids such as anas- trozole as well as low polarity steroid which have been occurring with increasing frequen- cy in food supplement samples [3].

The method developed includes autosampler and a CTO-20AC testing for caproate column oven with a flow selection and , a valve. The valve was used to 200,000 challenging set of isomers which divert mobile phase flow to waste were partially separated (Reso - during the first 1.5 minutes and 175,000 lution 0.6 as per Eur.Ph.) enabling avoid contamination of the mass the analytical method to identify spectrometer with early-eluting 150,000 which of the two isomers is con- matrix components. tained in the sample. With respect 125,000 to , three MRM transi- Chromatographic separation was tions are reported with no need to achieved with Zorbax SB-C18 resort to derivatization, which is Rapid Resolution HT, 50 x 100,000 usually the case with tibolone and 4.6 mm, 3.5 μm (part number its metabolites. Finally, a compre- 835975-902) maintained at 40 °C. 75,000 hensive rationale is provided for The mobile phase was a mixture choosing MRM transitions for of 0.1 % aqueous formic acid and 50,000 steroid esters, based on fragments methanol, with gradient elution of the free steroid. and a flowrate of 0.4 mL/min. 25,000 This set of parameters resulted in Instrumentation, method retention times ranging from 2.4 - 0 parameters and sample 24.0 minutes and a total runtime preparation of 31 minutes including the equili- 20.50 20.75 21.00 21.25 21.50 21.75 22.00 22.25 bration step. The experiments were conducted with Nexera X2 UHPLC system Liquid chromatography coupled to LCMS-8045 triple Figure 1: Chromatogram of standard solution, exhibiting partial separation of quadrupole mass spectrometer. Method development strategy and testosterone isocaproate The system consisted of two aimed at including a broad spec- LC-30AD pumps, a SIL-30AC trum of compounds with

SHIMADZU NEWS 2/2020 11 APPLICATION

solubility of the compounds in methanol, the homogenized sam- 350,000 ple was dissolved in methanol to 300,000 ensure high recovery for all com- 250,000 pounds (Test Solution 1). Four Test Solutions (TS) were then pre- 200,000 pared (TS 2-5) for into 150,000 the instrument. 100,000 Because matrix components con- 50,000 tained in TS1 were not always sol- 0 uble in mobile phase, 1.0 mL of 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 this solution was filtered through a 0.22 μm nylon filter and diluted Minutes to volume with mobile phase to avoid precipitation inside the col- Figure 2: Chromatogram of the standard solution umn (Test Solution 2, dilution 1 : 10). 1.0 mL each of TS2, TS3, and TS4 was transferred to 10 mL Time (min) % B comparable retention times were were exposed to 4 °C, the auto - volumetric flasks and diluted to 0.0 - 5.0 35 obtained with both columns. Since sampler was kept at room temper- volume with mobile phase (TS3, 5.0 - 20.5 35 → 100 the UHPLC column was prone to ature and 24-hour stability was dilution 1 : 102; TS4, dilution 20.5 - 25.0 100 overpressure and clogging after a established for the analytes. 1 : 103; TS5, dilution 1 : 104). 2 μL 25.01 - 31.0 35 few analyses, the 3.5 μm column of TS3 and TS4 were injected into was preferred for the application, Sample preparation the instrument. TS2 would be Table 1: Gradient elution program with an injection volume of 2 μL. injected if low intensity peaks In order to keep sample pretreat- were obtained with TS3 and TS4. respect to polarity while ensuring Autosampler temperature was ini- ment simple, effortless steps were TS5 would be injected if high a reasonable runtime, with a total tially set to 4 °C to ensure stabili- employed. The solid dosage form intensity peaks were obtained of 16 steroid esters being included ty of the samples. Because heavy samples were homogenized and with TS3 and TS4. in the analytical method. Regard - precipitation of matrix compo- 100 mg was weighed in a 10 mL ing the mobile phase flowrate, nents was observed when the vials volumetric flask. Due to the high Mass spectrometry 0.5 mL/min and 0.4 mL/min were tested, with the latter being cho- Precursor Product Product The compounds to be determined Group sen due to increased sensitivity. ion ion 1 ion 2 were divided into six groups of similar structure: testosterone and Testosterone Two mobile phase compositions testosterone esters, Testosterone 289.4 109.1 97.0 were tested with gradient elution, esters, and nandrolone 303.4 109.1 97.1 the first being a mixture of 1 mM esters, methenolone esters, bolde- 345.1 109.1 97.0 aqueous /1 mM none esters and es - Testosterone phenylpropionate 421.6 105.1 97.1 methanolic ammonium acetate and ters. The fragments of steroid Testosterone isocaproate 387.1 109.0 97.2 the second being a mixture of esters of the same group were ex - Testosterone caproate 387.1 109.0 97.2 0.1 % aqueous formic acid/ pected to be, to a certain extent, 401.6 271.3 183.0 methanol. The second mobile common, which was confirmed 443.7 109.2 97.0 phase was chosen to ensure sim- during the initial mass spectrome- 457.7 109.0 97.1 pler preparation procedure. The try experiments as can be seen in Trenbolone change in mobile phase acidity table 2. Therefore, common frag- Methyltrenbolone 285.0 227.1 198.0 using formic acid appeared to ments mentioned in table 2 may 313.0 253.1 107.5 have minor effects on retention be used to choose MRM transi- 383.1 253.1 107.5 time of the compounds since early tions for other steroid esters, Trenbolone hexahydrobenzylcarbonate 411.1 253.0 107.5 eluting compounds exhibited based on fragments of the free Nandrolone approximately 20 % higher reten- steroid. Nandrolone 275.4 257.2 239.0 tion, whereas late-eluting com- Nandrolone phenylpropionate 407.1 257.1 105.2 pounds exhibited approximately With respect to testosterone group, 429.7 257.3 239.0 10 % higher retention. m/z 109 was used for quantitation Methenolone and m/z 97 was used for confir- Methenolone (For reference only) 303.0 83.0 187.0 Two analytical columns were test- mation. Both ions are characteris- Methenolone acetate 345.0 83.1 97.0 ed, namely one Zorbax SB-C18 tic fragments of testosterone and Methenolone enanthate 415.1 82.9 187.1 Rapid Resolution HT, 50 x their use is proven in the literature 2.1 mm, 1.8 μm (part number [1-3]. Moreover, because the two Boldenone (For reference only) 287.5 134.6 120.5 822700-902) and one Zorbax ions correspond to fragments of Boldenone cypionate 411.6 135.4 125.1 SB-C18 Rapid Resolution HT, 50 the steroid core of the molecule, 453.2 135.2 67.2 x 4.6 mm, 3.5 μm (part number their applicability to all testos- Drostanolone 835975-902). Given that the first terone esters was investigated and Drostanolone (For reference only) 305.0 161.0 215.0 column contained sub-2 μm parti- confirmed. A similar rationale was 361.1 269.2 215.2 cles, considerably shorter sample followed for the group of nandro - runtime was expected, however Table 2: Rationale for the choice of MRM transitions lone and nandrolone esters, m/z

12 SHIMADZU NEWS 2/2020 APPLICATION

Retention time MRM Reference Reference C std Compound S/N ratio (min) Quantitation ion 1 ion 2 (ug mL-1) Clenbuterol 2.4 277.3 → 203.3 167.5 –– 0.05 1.9 x 103 Anastrozole 10.0 294.0 → 225.1 210.1 –– 0.05 1.2 x 104 14.9 337.0 → 281.2 317.2 299.2 1.0 204 15.0 307.0 → 289.2 93.4 –– 1.0 213 Nandrolone 15.2 275.1 → 239.1 257.2 –– 5.0 87 Methyltrenbolone 15.6 285.0 → 227.1 198.0 –– 0.05 61 Methandienone 16.1 301.0 → 283.1 283.1 149.1 0.5 694 Testosterone 16.1 289.1 → 109.2 97.0 –– 0.05 44 Methyltestosterone 16.9 303.1 → 108.8 96.8 –– 0.05 207 17.4 304.5 → 269.2 229.2 –– 5.0 103 Trenbolone acetate 17.7 313.0 → 253.1 107.5 –– 0.05 388 17.6 338.4 → 148.0 121.2 –– 0.5 891 17.4 328.6 → 81.0 121.1 –– 1.0 471 Tibolone 16.6 313.3 → 295.1 91.1 105.1 5.0 252 Methenolone acetate 19.3 344.6 → 82.8 187.1 297.2 1.0 557 18.6 333.0 → 159.1 279.1 –– 1.0 110 Testosterone propionate 19.7 345.1 → 109.1 97.0 271.0 0.05 2.2 x 103 Nandrolone phenylpropionate 20.7 407.1 → 105.2 257.1 –– 0.05 616 Drostanolone propionate 21.0 361.1 → 269.2 215.2 –– 5.0 552 Testosterone phenylpropionate 21.0 420.6 → 105.0 97.1 –– 1.0 646 Testosterone isocaproate 21.4 387.1 → 97.2 109.0 –– 0.05 148 Trenbolone enanthate 21.5 383.1 → 253.1 107.5 –– 0.05 867 Testosterone caproate 21.5 387.1 → 97.0 109.1 –– 0.05 188 Trenbolone hexahydrobenzylcarbonate 21.6 411.1 → 253.0 107.5 –– 0.05 2.1 x 103 Boldenone cypionate 21.6 411.1 → 135.1 125.1 –– 0.5 3,216 Testosterone enanthate 21.9 401.1 → 96.8 109.0 ––- 0.5 194 Methenolone enanthate 22.3 415.1 → 187.1 144.9 –– 0.5 2.0 x 103 Boldenone undecylenate 22.6 453.2 → 135.2 67.2 –– 1.0 3.0 x 103 Nadrolone decanoate 23.0 429.4 → 257.3 274.6 239.0 0.2 1.5 x 103 Testosterone decanoate 23.4 443.2 → 109.2 97.0 43.1 0.2 1.6 x 103 Testosterone undecanoate 24.0 457.2 → 109.0 169.1 –– 0.2 1.1 x 103

Table 3: MRM transitions for each compound

257 and m/z 239. For the group of Positive ionization: (ESI capil- Nebulizing gas: 3.0 L/min Authors trenbolone esters, m/z 253 and lary voltage set to -4 kV) Heating gas: 10.0 L/min Dr. Gerasimos Liapatas, m/z 107 were used. For the group Heat block temperature: Dr. Manos Barbounis of boldenone esters, m/z 135 was 400 °C Dwell time was set to 20 ms, Applications Department of N.Asteriadis S.A. applied, and for drostanolone pro- Desolvation line temperature: except for: 31 Dervenion Str. & Poseidonos Str., 144 pionate, m/z 215 was used. 250 °C Nandrolone: (100 ms) 51 Metamorfossi – Athens, Greece Heating gas temperature: Fluoxymesterone: (50 ms) [email protected] The optimized mass spectrometry 350 °C Methyltrenbolone: (150 ms) parameters were: Drying gas: 10.0 L/ min Methenolone acetate: (50 ms) Literature Drostanolone propionate: [1] C. Borges, N. Miller, M. Shelby, (150 ms). M. Hansen, C. White, M.H. Slawson, K. Monti, D.J. Crouch, Conclusions Analysis of a challenging subset of World Anti Doping Agency-Banned steroids and An analytical method was devel- anti- by LC-MS-MS, Journal of oped to identify 31 steroids in Analytical Toxicology, 31 (2007) 125-131. pharmaceutical preparations and [2] S. Fekete, J. Fekete, K. Ganzler, food supplements. This is the first Validated UPLC method for the fast and method to identify testosterone sensitive determination of steroid caproate in the presence of its iso- residues in support of cleaning validation mer, testosterone isocaproate, and in formulation area, Journal of Pharma - vice versa. Key parameters that ceutical and Biomedical analysis, 49 affect the analysis of all 31 ste - (2009) 833-838. roids have been identified and [3] M. Thevis, Y. Schrader, A. Thomas, taken into account. G. Sigmund, H. Geyer, W. Schanzer, Analysis of confiscated black market drugs using chromatographic and mass spectrometric approaches, Journal of Analytical Toxicology 2008;32:232-240. Figure 3: Fragmentation pathway of testosterone group and boldenone group of compounds [3]

SHIMADZU NEWS 2/2020 13 PRODUCTS 360° drinking water analysis: Episode 2 Automatic, simultaneous and rapid analysis of pesticides in drinking water by online SPE and UHPLC-MS/MS

Directive 98/83/EC1 (DWD) [2]. For more than 20 years, the DWD has regulated the quality of water intended for human consumption. Evaluation of this directive was included in the Commission Work Programme 20152 as part of the Commission’s Regulatory Fitness and Performance programme (REFIT), to assess whether this instrument remains fit for pur- pose. It is the first full evaluation of the DWD and has been pub- lished as a Commission Staff Working Document in December 2016 (SWD, 2016, 428 final).

Cooperation between the DWD and agriculture is a crucial factor for providing safe and high-quali- ty drinking water. Agricultural practices like fertilization and U citizens use up to 156 chemicals from domestic, agricul- ments for effective control of plant protection with pesticides liters of water per day. tural, commercial or natural drinking water, are user-friendly have a significant impact on E Groundwater and surface sources. Defects in quality and and provide an economical solu- drinking water quality. To prevent water such as rivers, lakes, reser- quantity generate massive prob- tion for modern laboratories. This agricultural contamination, voirs and seawater are sources for lems and cause high social and Episode 2 deals with a sensitive parameters like nitrate and pesti- drinking water. Its safety and high economic costs. Strict and steady online SPE-UHPLC-MS/MS cides have been included in the quality are essential for public and controls are therefore essential. method. DWD. These standards con- individual health as well as for the tributed to reducing the release of economy. As seen in Episode 1 in the Background fertilizers and crop protection Shimadzu NEWS 1/2020, products to preserve the environ- Hazards in source waters include Shimadzu’s ICPMS-2030 and In 1998, the European Union ment. Decreasing releases of pathogenic microorganisms and TOC-L meet analytical require- (EU) adopted the Drinking Water nitrate or pesticides into the envi-

The total abstraction of fresh water across Europe is around 182 bil- lion m3/year, drawn in roughly equal amounts from groundwater and surface water sources. Drinking water in the EU stems from around 11,000 large suppliers and 85,000 small suppliers, serving around 80 % and 20 % of the population respectively.

More than 60 % of the EU water infrastructure consists of water serv- ices provided by publicly-owned companies – the rest are regulated entities with different levels of private ownership. Regulatory options range from largely decentralized management of private companies (subject to antitrust and price regulation) to public ownership, minis- terial guidance and budget control. The European water sector is a major economic player (1 % of GDP), with an annual turnover in the EU of about 80 billion Euro. It provides around 500,000 full-time jobs Figure 1: Scheme of online SPE and UHPLC-MS/MS and a yearly investment of 7 billion Euro [1]. (Number of patent: WO 2016/098169 A1)

14 SHIMADZU NEWS 2/2020 PRODUCTS

ronment is the driving force for 4,500,000 ensuring safe and high-quality 4,250,000 drinking water. 4,000,000 3,750,000 Pesticides in drinking water 3,500,000 3,250,000 Pesticides are often applied for 3,000,000 crop protection. Their intensive 2,750,000 use and slow natural degradation 2,500,000 make them serious contaminants 2,250,000 of surface and underground water 2,000,000 – the most important sources for 1,750,000 drinking water. This exposure can 1,500,000 be dangerous for animals, humans 1,250,000 and ecosystems, with an immedi- 1,000,000 750,000 ate or long-term effect. In relation 500,000 to these compounds, some studies 250,000 have revealed health impacts such 0 as alterations of the nervous sys- 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 tem, immune system diseases, fer- Minutes tility and development problems as well as cancer. This lead to the Figure 2: Chromatogram of ground water spiked at 100 ng/L DWD setting a concentration limit of 0.1 μg/L for individual pesticides and 0.5 μg/L for the Q 229.90 > 174.05 (+) 2.30e4 Q 212.90 > 88.95 (+) 1.42e3 total sum of pesticides. 100.00 100.00 Determination of 272 pesticides in water % % Reference separation techniques require a tedious pretreatment protocol to reach the thresholds 0.00 0.00 set by environmental standards. 15.6 15.7 15.8 15.9 16.0 16.1 16.2 16.3 16.4 16.5 10.5 10.6 10.7 10.8 10.9 11.0 11.1 11.2 11.3 Shimadzu proposes a fast and sen- RT RT sitive online SPE-LC-MS/MS Q 410.90 > 182.10 (+) 1.29e3 Q 331.00 > 127.10 (+) 2.22e3 method for simultaneous highly 100.00 100.00 sensitive quantification of 272 pesticides in surface and ground- water. This method uses high % % quality solvents and reagents for mobile phases (LC-MS grade Biosolve1). Two surface waters and one groundwater sample were 0.00 0.00 11.0 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9 17.0 17.1 17.2 17.3 17.4 17.5 17.6 17.7 17.8 17.9 spiked with standard pesticides RT RT purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Six Figure 3: Example of chromatogram at the LLOQ calibration levels at 1, 10, 20, 50, 100 and 500 ng/L were prepared three times in each matrix. LC flow: 0.7 mL/min Heat block temperature: 300 °C to cover the main families of pes- SPE elution flow: 0.2 mL/min Interface temperature: 350 °C ticides such as phenylureas, tri- SPE-UHPLC conditions: Oven temperature: 40 °C azines, carbamates, sulphony- Nexera X2 Injection volume: 1,000 μl An analysis demonstrated the dif- lureas and organophosphorus ferent concentration levels pre- compounds. System: Shimadzu Nexera X2 MS conditions: pared on three water samples (fig- Column SPE: Mayi-ODS C18 LCMS-8050 ure 2). The lower limits of quantification Column LC: C18AQ CS (LLOQ’s) were established for Interchim 2,6 μm 150*3 mm System: Shimadzu LCMS-8050 One of the previous matrices, each compound by a signal to A/B (SPE): water/acetonitrile ESI ionization mode: positive spiked at different levels, was noise ratio (S/N) greater than 10. + 0.002 % formic acid + 2 mM and negative selected to establish the calibra- Table 1 classifies the compounds ammonium formate Dwell time: 4 to 199 msec to tion curve. The other doped water according to their limits of quan- A: water + 0.002 % formic acid obtain at least 15 points per peak samples worked as controls. The tification: 1, 10 and 25 ng/L. The + 2 mM ammonium formate Nebulizing gas flow: 2.8 L/min 23 different isotopic standards LLOQs obtained fit the require- B: 50/50 acetonitrile/methanol Heating gas flow: 10 L/min were added to perform an internal ments of the European directives + 0.002 % formic acid + 2 mM Drying gas flow: 10 L/min calibration. To overcome the and the guidelines of the World ammonium formate Desolvation line: 150 °C matrix effect, ISTD were selected Health Organization (WHO).

SHIMADZU NEWS 2/2020 15 PRODUCTS

Compounds with LOQ at 1 ng/L 4MBC Chlorfluazon Ethidimuron Ioxynil Oxamyl Acetamiprid Chloridazon Ethiofencarb IPPMU Oxasulfuron Simazine hydroxy Chlorsulfuron Ethiprole IPPU Oxazepam Simetryn Acide niflumique Enbuconazole Iprovalicarb Oxydemeton methyl Spirotetramat Alachlore Clethodim Fenobucarb Isoprothiolane Paclobutrazol Ametryn Coumafene Fenothiocarbe Isoproturon Penconazole Sulfosulfuron Amidosulfuron Coumatetralyl Fenpyroximate Isoxaben Penoxsulam Tebuconazole Aminocarb Fensulfothion Lufenuron Phosphamidon Tebufenoside Atenolol Cybutrine Fenuron Malaoxon Pinoxaden Tebutam Cycloxydim Fipronil sulfone Malathion Pirimicarb Atrazine desethyl Cyflufenamid Fluazinam Mandipropamid Pirimicarb desmethyl Teflubenzuron Atrazine-OH Cyproconazole Flufenacet Mecarbam Pirimicarb II Tepraloxydim Azaconazole DCPMU Fluometuron Mefluidide Terbumeton Azamethiphos Desmetryn Fluopicolide Mercaptodimethur Terbumeton desethyl Azimsulfuron Dichlorophen Fluoxastrobin Metabenzthiazuron Promecarb Azinphos ethyl Dicrotophos Flupyrsulfuron methyl Metalaxyl Prometryn Terbuthylazine desethyl Azinphos methyl Diethofencarb Fluridone Metazachlor Propachlore Terbuthylazine hydroxy Azoxystrobin Difenacoum Flurtamone Metconazole Propazine Tetraconazole Bensulfuron methyl Difenoconazole Flusilazole Methomyl Propiconazole Thiabendazole Benthiavalicarb isopropyl Difethialone Fluxapyroxad Metobromuron Propoxur Thiacloprid Diisobutylphtalate Foramsulfuron Metolachlore Propoxycarbazone Thiazafluron Buturon Dimetachlore Formetanate Metosulam Propyl Thiobencarb Cafeine Fosthiazate Metoxuron Proquinazid Thiophanate methyl Carbamazepime Dimethomorph Fuberidazole Prosulfuron Triadimefon Carbamazepine epoxyde Dimetilan Halosulfuron methyl Metropolol Pyrazophos Triazamate Carbaryl Dimoxystrobin Hexaconazole Metsulfuron methyl Pyrifenox Tribenuron methyl Carbendazim Diniconazole Hexaflumuron Pyrimethanil Trietazine Carbetamide Monuron Pyroxsulam Trietazine-2-OH Carbofuran Dinoterb Hexythiazox Myclobutanil Quizalofop ethyl Triflumizole Carbofuran 3 hydroxy Diuron Hydroxypropazine Neburon Rimsulfuron Trimetoprime Carboxin Econazole Nicosulfuron Trinexapac ethyl Chlorantraniliprole Epoxiconazole Imazaquin Oruface Sebuthylazine Triticonazole Chlorbromuron Erythromicine Imidacloprid Oryzalin Siduron Vamidothion Compounds with LOQ at 10 ng/L 245T Cyprosulfamide Imazamox Metazachlor oa Quizalofop Aldicarbe Cyromazine Fenoxycarb Imazofulfuron Methidathion Roxythromycine Atrazine desisopropyl Desethyl terbutylazine 2-0H Iodosulfuron methyl Oxadixyl Tembotrione Bentazone Desmedipham Florasulam Isoprocarb Paraoxon Testosterone Benzafibrate Dia2hydroxy Flufenacet oa Isoxadifen ethyl Phenmedipham Thiencarbazone methyl Brodifacoum -P Lorazepam Phoxim Thiodicarb Bromadiolone Diclofenac MCPA Propamocarb Tolytriazole Butyl paraben Dimetachlore oa Forchlorfenuron MCPP Propanolol Triasulfuron Haloxyfop Propyzamide Triazoxide Clofentezine Ethoxysulfuron Haloxvfop methyl Mesosulfuron methyl Prothioconazole Clorsulon Ethyl paraben Imazalil Metamitron Pymetrozine Trietazine desethyl Closantel Tylosine Compounds with LOQ at 25 ng/L 2,4-D Dea 2 hydroxy Fluquinconazole Titrosulfuron Triflumuron

Table 1: Limit of quantification in ng/L for each compound

Performance evaluation Conclusion and a perfect example of a chro- will give an overview on how to matographic method in combina- analyze anions such as nitrate and Repeatability was determined at Shimadzu offers a unique, auto- tion with mass spectrometry as a more using ion chromatography the low level of concentration for mated and sensitive method to powerful, user-friendly and time- in the next issue of Shimadzu each water with three injections. quantify a large number of pesti- saving method meeting the users’ NEWS. Regardless of the water matrix, cides in water. Limits of quantifi- needs. the repeatability was less than cation range from 1 to 25 ng/L Biosolve is a trademark of BioSolve® 15 % for all 272 targeted com- with 1 mL sample injection. In addition to contami- Company (TBC) pounds. Recovery rates ranging from 85 - nation, drinking water suppliers 115 %, while repeatability is less and water treatment plants are Referenzes The results obtained showed than 15 % for the full list. Finally, suffering from elevated concentra- [1] REFIT EVALUATION of the Drinking Water recovery rates from 85 to 115 % this method is appropriate for tion levels of nitrates released by Directive 98/83/EC, 2016. for the calibration standards and thresholds of the actual normative extensive agricultural treatment. [2] European Drinking Water Directive the control samples. environment such as the DWD, Episode 3 of our water Trilogy 98/83/EC1.

16 SHIMADZU NEWS 2/2020 PRODUCTS Versatile testing tool for the automotive industry New HMV-G3 Series: “Redesigned” with a color camera and data protection functions HMV-G3 series main unit

he future of mobility, the other drive-related parts undergo future use of cars as well as treatments which are critical T their propulsion, is multi- parameters. If the treatment is not faceted as never before. Currently, carried out properly, this can ideas and projects meeting up - cause additional costs and in a coming needs are at the R&D worst case situation, risk passen- stage in the automotive industry, ger safety while traveling by car. which is driven by ever-shorter development times. Simultane - Shimadzu’s new HMV-G3 hard- ously, it is one of the most com- ness testers target research and petitive markets with very high production processes. New func- quality standards. tions improve data reliability, accommodate ISO standards relat- As a global supplier to the auto- ed to Vickers hardness tests as motive supply chain, Shimadzu standard and offer high expand- provides the full range of analyti- ability. cal instrumentation and testing solutions in chromatography, For mechanical parts, spectroscopy, mass spectrometry, metals, layers, ceramics TOC and material testing. This and plastics Improvement of the automatic reading function covers engines, motors and power sources, bodies and interiors, The micro-hardness tester is indis- quadrangular pyramidal diamond, compliant with measurements at environmental conservation, sus- pensable in metallographic struc- referred to as an indenter, applies low test forces in accordance with pension and power transmission ture research, quality control of a force to a sample. The hardness ISO standards related to Vickers systems, electronics, batteries and manufactured products and for of the sample can be calculated by hardness tests. Protective functions fuel cells. This diversity of testing preparing of product certification measuring the indentation formed to heighten data reliability, as well methods meets the valid stan- documents. The ability to measure on the surface. as report creation functions are dards, norms, regulations and hardness in limited microscopic also included as standard. compliance requirements. areas and to do this with extreme- Accordingly, in recent years, there ly high reliability is required for has been a need for strengthened Addi tionally, a motorized microm- Treatments of drive-related small precision mechanical parts, data management functions, im - eter for adjusting the position of parts are critical parameters metal structures, processed surface provements to operability and the the stage can be added optionally, layers and plating layers. In addi- enhancement of various functions and a model equipped with a 5 Particularly hardness testing of tion to metals, it is also useful in to suit the application. megapixel camera, the best in the ferrous and non-ferrous compo- the evaluation of fine ceramics and industry, has been integrated into nents to international standards is engineering plastics. Compliant with ISO the series. The system can be con- a well-known process in the auto- standards figured according to application- motive market. The wear and The HMV-G3 series instruments and user-specific needs. strength characteristics of rotating measure Vickers hardness at very As a standard, all Shimadzu and cast parts in the engine or low test force levels. A regular HMV-G3 series models are now

By comparison: Color vs. Monochrome image

SHIMADZU NEWS 2/2020 17 APPLICATION MSn analysis of nonderivatized and Mtpp-derivatized peptides Two recent studies applying LCMS-IT-TOF instruments

due to poor ionization of some

Inten. (x100,000) peptides present at low concentra- A) ESI-MS tion in the biological sample. To 1.00 339.139(1) 379.207(2) overcome this problem, introduc- 0.75 tion of a functional group con- 0.50 taining stable positive charge into 0.25 molecule, which increases 758.417 0.00 the ionization efficiency, allows 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z sensitive detection by electrospray Inten. (x1,000,000) 2 mass spectrometry (ESI-MS). 212.138(1) B) ESI-MS 1.0 Precursor ion: 379.205 [M+H]2+ 322.166(2) 546.275(1) 0.5 273.340(2) Collision energy: 70 % Recently, researchers have devel- 0.0 oped several efficient methods for 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z peptide derivatization by quater-

Inten. (x1,000,000) C) ESI-MS2 nary ammonium salt (QAS) syn- 420.259(1) Precursor ion: 758.417 [M+H]2+ thesis [2]. The highest intensity 309.190(1) 5.0 Collision energy: 70 % 338.154(1) increase (as much as 1,000 x) was 0.0 observed for pyrylium ionization 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z reagents, which react readily with Inten. (x1,000,000) the primary amino groups inside 1.0 D) ESI-MS3 209.127 379.205 –> 546.276 –> fragments chain of lysine residue to form 0.5 Collision energy: 70 % 338.152 pyridinium salts bearing a stable 0.0 positive charge [3]. The peptides 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z derivatized with pyrylium reagent can be detected even at attomolar level (10-18). A new kind of iso- Figure 1: ESI-MS spectrum of the crude synthetic peptide Lys(Mdpp)-Pro-Pro-Pro (panel A), ESI-MS2 fragmentation spectra baric tag for peptides which is 2+ 1+ 3 2+ → + → of [M+H] (panel B) and the fragmentation spectrum of [M] (panel C) and ESI-MS analysis of [M+H] b2 (panel D). based on combination of 16O/18O (Mdpp – 4-(4-methoxyphenyl)-2,6-diphenylpyridinium salt). exchange and derivatization by isotopologues of pyridinium salts was also proposed [4]. he LCMS-IT-TOF combines make LCMS-IT-TOF the instru- biological samples, the increase QIT (Ion Trap) and TOF ment of choice for advanced sci- in sensitivity of MS analysis is one However, it is necessary to search T (Time-Of-Flight) technolo- entific studies in impurity analy- of the important research goals of for new ionization markers which gies. The QIT ion trap gives an sis, metabolic profiling and bio- the group. will show a similar degree of ion- efficient MSn capability. After marker research [1]. ization enhancement and have selecting the appropriate precur- Two examples of recent studies improved properties (higher solu- sor ions, several MSn experiments The research projects of the are presented here which due to bility, sensitivity and precision of could be performed during one Group of Chemistry and Stereo - the required MSn analysis were analysis). Researchers recently introduction of the sample, with chemistry of Peptides and Prote - possible only by application of the developed a new ionization re - product ions obtained serving as ins from the Faculty of Chemis - LCMS-IT-TOF instrument. agent (4-(4-methoxyphenyl)-2,6- parent ions for the next stage of try, University of Wrocław, diphenylpyrylium salt, Mdppl) to fragmentation. MSn spectra with Poland focus on the study of pep- 1. MSn analysis by LCMS-IT-TOF improve the applicability in pro- high mass accuracy offer im - tides and proteins by mass spec- of derivatized peptides with teomic research (unpublished proved reliability of signal assign- trometry. Professor Zbigniew 4-(4-methoxyphenyl)-2,6- data). Specific MSn transitions are ment and structural analysis by Szewczuk and coworkers investi- diphenylpyrylium salt used for unambiguous analysis of fragmentation pathways. The IT- gate the structure of natural and particular peptides derivatized TOF instrument provides high synthetic peptides, peptide conju- Tandem mass spectrometry is a with Mdppl. resolution and accuracy for all MS gates and adducts, searching for powerful tool in proteomic re - and MSn modes as well as mass posttranslational modifications search. Despite the rapid develop- The 4-(4-methoxyphenyl)-2,6- stability and excellent resolution and biomarkers. Due to low con- ment of this technique, insuffi- diphenylpyrylium salt was linked for LC-MS analysis. Such features centration of some peptides in cient sensitivity is still a problem to the side chain amino group of a

18 SHIMADZU NEWS 2/2020 APPLICATION

completely new possibilities for cant amount of material and is of (x10,000,000) use of MSn analysis in quantitative limited use in case of highly 3.0 and qualitative proteomics. hydrophobic compounds. Various chromatographic methods includ- To conclude, the MSn method can ing RP-TLC and RP-HPLC sup- 2.0 be used successfully for sensitive plement or replace the SFM, offer- peptide sequencing at high colli- ing several practical advantages sion energies. Such fast confirma- including reproducibility, insensi- 1.0 tion of the structure of derivatized tivity to impurities and degrada- peptides based on the MS3 method tion products, broader dynamic 3 0.0 is extremely reliable. MS analysis range and reduced sample han- 2.0 3.0 4.0 5.0 6.0 7.0 8.0 allows unambiguous identification dling and sample size [6]. One of of peptide fragments resulting the main advantages of HPLC in (x10,000,000) 2.0 from MS2 experiments. The accu- lipophilicity determination is the racy of the IT-TOF instrument ability to compare directly the enables determination of the mo - properties of a series of com- 1.5 lecular formula of the fragments, pounds by analyzing mixtures. making it possible to propose 1.0 structural formulae of the ions Lipophilicity is studied thorough- obtained. The IT-TOF instru- ly in design and development of 0.5 ments not only allow the determi- neuropharmaceuticals due to nation of fragmentation pathways, blood-brain-barrier issues. Several 0.0 but also offer new and fast meth- research groups investigate anti - 3.0 4.0 5.0 6.0 7.0 8.0 ods for identifying trace amounts nociceptive activity of peptides [7, of potential biomarkers in the 8], and there is a significant inter- n Figure 2: LC-MS chromatograms of peptide library subsets. Extracted ion samples tested. MS experiments est in application of RP-HPLC to chromatograms (XIC) for +2 ions represent library components. combine two fragmentation mech- lipophilicity comparison of the anisms: charge remote and charge analogs proposed. directed. model peptide (Lys-Pro-Pro-Pro). formed using instruments equip - Figure 1 shows ESI-MS and ESI- ped with ion trap (IT). 2. Internal fragments formed IMPRINT MSn analysis of Lys(Mtpp)-Pro- during MSn for analysis of Pro-Pro. Precise analysis of the resulting isomeric peptides Shimadzu NEWS, Customer Magazine of Shimadzu Europa GmbH, Duisburg fragments requires high-resolution In MS2 experiments, a series of b spectra which can only be guaran- The importance of mass spec- Publisher and y ions were obtained. Due to teed with IT-TOF instruments. trometry analysis in peptide sci- Shimadzu Europa GmbH the increase in collision energy, The researchers noted that frag- ence is obvious at several levels – Albert-Hahn-Str. 6 -10 · D-47269 Duisburg Phone: +49 - 203 - 76 87-0 the intensity of m/z 338 signal mentation of single-charged ions from characterization of natural Fax: +49 - 203 - 76 66 25 grew although its intensity was gave information about ionization or synthetic products, sequence [email protected] still negligible. Peak at m/z 209 tag whereas the same experiment analysis to stability studies and www.shimadzu.eu after MS3 experiment correspond- for double-charged ion provided quantification. There is also a Editorial Team ed to the internal fragment of pep- more information about peptide growing interest in application of Uta Steeger tide sequence. The signal is char- sequence. When combined, these mass spectrometry to physico- Phone: +49 (0)203 76 87-410 acteristic for pyridinium salts, but experiments provide more reliable chemical studies including charge Ralf Weber, Maximilian Schulze it appears mainly in the charge and comprehensive structural variants, molecular interactions Design and Production remote fragmentation mechanism information. The m/z 338 frag- and adduct stability studies. m/e brand communication GmbH GWA [5]. ment ion can be used in MRM or Duesseldorf parent ion scan to study all pep- The main advantages of mass Circulation To confirm this statement clearly, tides containing 4-(4-methoxy - spectrometry include high sensi- German: 5,020 · English: 3,270 an MS3 experiment was performed phenyl)-2,6-diphenylpyridinium tivity and unambiguous identifica- in which for the first fragmenta- groups. tion of product, with excellent Copyright tion reaction the [M+H]2+ was detection levels and the possibility Shimadzu Europa GmbH, Duisburg, Germany – June 2020. selected, containing one mobile As a result of MS3, [M+H]2+ → of application to isotopically + proton responsible for charge b2 → fragments appear at m/z modified reference materials. Windows is a trademark of Microsoft 2+ Corporation. ©2020 directed fragmentation mecha- 209 and 338 as either [M+H] nism. The single-charged frag- or [M]+ (figure 1B, 1C). The MS3 The lipophilicity of chemical com- Apple Inc. All rights reserved. Apple, ments resulting do not contain a technique therefore leads to pounds, represented as logarithm the Apple logo, Mac OS and iOS are mobile proton and may undergo unique fragments, meaning that it of the octanol-water partition trademarks of Apple Inc. fragmentation according to charge can be used for the sensitive and coefficient (logP), is a very impor- remote fragmentation mechanism. unambiguous analysis of reporter tant parameter in medical chem- Combination of these two consec- ions in proteomic studies of com- istry. Typically, logP is measured utive fragmentation reactions pounds derivatized by Mtppl, for in the traditional shake-flask occurring according to different example: 379 → 546 → 338 and method (SFM) which may be mechanisms may only be per- 379 → 546 → 209. This gives time-consuming, requires signifi-

SHIMADZU NEWS 2/2020 19 APPLICATION

Inten. (x100,000) Inten. (x100,000) 3.0 624.297 3.0 624.300 201.122 201.122 2.0 2.0 310.173 1.0 285.151 369.229 1.0 461.237 397.627 515.254 0.0 0.0 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z Inten. (x100,000) Inten. (x100,000)

2.0 201.121 3.0 201.121 562.280 1.5 280.145 558.281 2.0 1.0 372.691 559.291 244.163 558.279 218.148 1.0 372.696 0.5 362.143 458.653 801.967 440.139 0.0 0.0 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z

Inten. (x100,000) Inten. (x100,000) 558.289 5.0 279.643 3.0 201.121 201.121 558.287 2.0 244.164 2.5 515.246 1.0 315.125 370.157 370.150 455.723 628.215 458.186 669.565 0.0 0.0 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z

Inten. (x100,000) Inten. (x100,000)

201.121 575.277 2.0 575.276 201.122 2.5 1.0 297.150 377.648 377.644 471.702 261.158 515.253 766.998 0.0 0.0 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z

Inten. (x100,000) Inten. (x100,000) 532.272 4.0 532.273 201.122 3.0 201.121 2.5 2.0 307.273 439.666 439.673 1.0 262.213 357.689 435.692 307.283 436.181 569.276 887.873 0.0 0.0 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z

Inten. (x100,000) Inten. (x10,000)

518.255 7.5 315.125 518.257 2.5 201.122 5.0 296.329 2.5 429.205 218.148 342.186 426.144 248.148 708.310 0.0 0.0 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z

Figure 3: MS2 analysis of peptide library subsets, [M+2H]2+ ions were selected as precursors

The LC-MS procedure seems ideal threonine, proline, glycine and could be studied only after a Pro peptides) or were rather weak for this application, offering the asparagine to provide a broad method to distinguish isomeric (Gly analogue). additional benefits of indication of range of changes. peptides could be established for charged state of studied peptides. analysis of sets. Analysis was performed using a More complex tandem MS2 ap - Subset Library 1: Shimadzu IT-TOF instrument, proach is needed in case of iso- Ser-Ile-Xxx-Gln-Asp-Ala-Lys The researchers subjected the sub- and it was decided to use the MSn meric peptides, to assign the peak Subset Library 2: set libraries to MS2 analysis using feature and subject the y5 ions to order to proper sample compo- Ser-Ile-Ala-Gln-Asp-Xxx-Lys [M+2H]2+ peptide ions as precur- further fragmentation (MS3). nents. In the recent project, the where Xxx represents Gly, Tyr, sors (figure 3). High mass stability The Tyr analogues were selected researchers encountered a prob- Thr, Pro, Asn or Ala of TOF allowed conducting of a for preliminary studies, and the lem which required a more advan - long series of LC-MS experiments results shown in figure 4 reveal ced approach to differentiating of The two resulting subset libraries without recalibration. The parent interesting differences in MS3 isomeric peptides. (modified in position 3 or 6, re - ions in overlapping chromato- fragment patterns. The character- spectively) were analyzed using graphic peaks were successfully istic MS3 transitions will be used To investigate the effect of se - LC-MS. The peptides in each sub- selected and fragmented due to in further lipophilicity studies of quence modification on chroma - set differ in molecular weight and high resolution and accuracy of podocin fragment peptide tographic behavior of peptides MS analysis was sufficient to the IT-TOF instrument. In practi- libraries. and, subsequently, their lipophi - assign the signals. The elution cally all cases the fragmentation licity, a peptide library was profiles represented by extracted produced b2 and y5 ions, which Based on individual properties of designed and synthesized based chromatograms (XIC) are pre- are identical in pairs of isomeric the peptide sequence studied, reg- on podocin 290-296 fragment sented in figure 2. The retention peptides and cannot be used to ular MS2 experiments could not with a sequence Ser-Ile-Ala-Gln- time differences between the iso- assign the peaks in chromatogram confirm which Ala residue was Asp-Ala-Lys. The alanine residues meric peptides were minimal and of a mixture of libraries. Some replaced in a given peptide. The were selected as diversity posi- the effect of position of the modi- observed differences were identi- isomeric peptides could be identi- tions and replaced with tyrosine, fying residue on retention time fied as +2 fragment ions (Thr and fied only after obtaining deeper

20 SHIMADZU NEWS 2/2020 APPLICATION

insight into their fragmentation 3 using MS function of the IT-TOF Inten. (x100,000) 624.298 instrument. 2.0 201.122

1.0 Conclusion 285.151 369.229 323.087 402.682 461.228 507.243 607.267 0.0 Scientific challenges could be 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z solved quickly, successfully and Inten. (x10,000) efficiently due to the MSn capabil- 2.5 333.166 2.0 ity and high mass accuracy of the 292.124 Shimadzu LCMS-IT-TOF instru- 1.5 ment. 1.0 275.100 407.154 218.142 478.195 589.256 0.5 425.203 Methods: 0.0 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z Pyrylium salt synthesis: Syn - Inten. (x10,000) 310.173 thesis of pyrylium salts involves 7.5 two steps. The first step involves 5.0 synthesis of a specific chalcone based on condensation reaction 2.5 with different commercially avail- 450.197 226.077 able aldehydes and ketones. The 346.224 407.184 478.205 589.256 0.0 reaction is carried out in alkaline 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z environment, in the presence of 10 % NaOH in ethanol. In the second step, cyclization reaction is Figure 4: MSn analysis of Tyr-containing library elements. MS2 results were not sufficient to produce characteristic ions, performed with obtained chalcone whereas MS3 experiments could be used to unambiguously identify the isomers. and proper ketone. The reaction requires the presence of trifluo- romethanesulfonic acid (TFMSA) Fmoc chemistry, and equimolar Peptide XB-C18 column (50 x Literature as a counterion. mixtures of peptide derivatives 2.1 mm, 3.6 μm) was used apply- [[1] www.shimadzu.com/an/lcms/lcmsittof/ were used at diversity steps. ing a gradient separation from 5 to ittof.html, accessed 14.01.2020 Derivatization procedure: Crude products were applied in 60 % B in 15 min, A: 0.1 % [2] M. Cydzik, M. Rudowska, P. Stefanowicz, Sample of model peptide was dis- the LC-MS experiments. HCOOH in water, B: 0.1 % Z. Szewczuk, J. Pept. Sci. 17 (2011), solved in dimethylformamide HCOOH in acetonitrile, flow rate 445-453 (DMF) and excess proper pyryli- MS and MSn experiments: MS 0.2 mL/min. The IT-TOF instru- [3] M. Waliczek, M. Kijewska, M. Rudowska, um salt and trimethylamine (TEA) and MSn was performed on a ment was operated in positive ion B. Setner, P. Stefanowicz, Z. Szewczuk, was added. The mixture was incu- Shimadzu IT-TOF mass spec- mode, and the 150 - 1,000 m/z Sci. Rep., 6 (2016), 1-12 bated at 60 °C for three hours. trometer. For the experiments range was analyzed. [4] M. Waliczek, R. Bachor, M. Kijewska, After this time, solvent was evap- using precursor ion mode, a frag- D. Gaszczyk, K. Panek-Laszczynska, orated under nitrogen and the ment ion corresponding to pyri- Acknowledgements A. Konieczny, K. Dabrowska, residue was lyophilized. dinium was chosen. The research was supported in part by W. Witkiewicz, K. Marek-Bukowiec, grant No. UMO-2016/23/B/ST4/01036 from J. Tracz, M. Luczak, Z. Szewczuk, Peptide library synthesis was LC-MS conditions: UHPLC the National Science Centre, Poland. P. Stefanowicz, Anal. Chimica Acta, 1048 performed on solid support using Nexera equipped with an Aeris The authors would like to thank O. Uysal (2019), 96-104 and P.U. Godigamuwa Acharige for library [5] M. Rudowska, D. Wojewska, A. Kluczyk, synthesis. The authors would also like to R. Bachor, P. Stefanowicz, Z. Szewczuk, thank Andrzej Reszka (Shim-Pol, Poland) J. Am. Soc. Mass Spectrom., 23 (2012), for providing access to Shimadzu’s IT-TOF 1024-1028. instrument. [6] C. Liang, J. Qiao, H. Lian, J. Chrom. A, 1528 (2017) 25-34 Authors [7] H. Liu, B. Zhang, X. Liu, Ch. Wang, J. Nib, Dr. Dorota Gaszczyk, Prof. Zbigniew R. Wanga, Bioorg. Med. Chem. 15 (2007) Szewczuk, Dr. Alicja Kluczyk 1694-1702 Faculty of Chemistry [8] K. Wtorek, R. Artali, J. Piekielna- University of Wroclaw Ciesielska, J. Koszuk, A. Kluczyk, 50-383 Wroclaw – Poland L. Gentilucci, A. Janecka, Eur. J. Med. Chem. 179 (2019) 527-536

Authors of the article (from the left): Dr. Dorota Gaszczyk, Prof. Zbigniew Szewczuk and Dr. Alicja Kluczyk

SHIMADZU NEWS 2/2020 21 PRODUCTS

peak shape deterioration of acidic compounds or long equilibration No more headaches! times required for low ionic strength acidic mobile phase.

A guide to choosing the perfect C18 column The Shim-pack Arata Peptide C18 columns offer an excellent separa- tion performance of peptides, even under weak ion pairing acidic mo - bile phases, and are therefore suit- able for MS detectors. Since the adsorption of peptides to particles is minimized, the Shim-pack Arata Peptide C18 column ensures high recovery of peptides and provides excellent peptide analysis.

Shim-pack G series: for general applications

The Shim-pack G series consists of GIST, GISS and GIS, designed not only for conventional analysis but also for ultra-high speed and preparative methods due to their various particle size ranges. The Shim-pack GIST series includes standard columns for general ap - plications, while the Shim-pack GIS columns are especially suited for preparative chromatography and the Shim-pack GISS for MS analysis.

Shim-pack GIST series: he all-rounder in liquid of a complex separation, Shim- ent particle sizes (1.9 μm, 3 μm, ultra-high inertness and high chromatography: C18- pack Velox columns satisfy any 5 μm) and different column durability T columns are broadly used in users’ needs. dimensions, Shim-pack Scepter pharmaceutical, food, clinical and LC columns are fully scalable Shim-pack GIST series columns environmental industries for Due to their robustness, excellent between UHPLC, HPLC and are packed with newly developed reversed-phased chromatography. column lifetimes are obtained, preparative LC, making method high-purity porous spherical sili- With thousands of options in the even for the most challenging transfer seamless between differ- ca. Superior inertness not only market, it can be overwhelming to sample matrices. ent laboratory instrumentation. improves analytical precision but choose the right C18 column. also increases the column lifetime. Shimadzu therefore offers a guide The Shim-pack Velox SP-C18 The Shim-pack Scepter HD-C18 It provides symmetric peaks even to its portfolio of C18 columns to (Sterically Protected) series was with its high loading and for strong ionic compounds, and simplify the selection process designed specifically for applica- therefore high hydrophobicity, is undesired adsorption and peak (figure 1). No more headaches tions under low pH condition, especially suited for the analysis tailing are reduced to a minimum. through information overflow. offering a well-balanced retention of isomers. This is particularly It is capable of covering a wide profile with long lifetimes even useful for pharmaceutical applica- pH range (1 - 10) and is therefore Shim-pack Velox: Maximize under the harsh acidic conditions tions where medical substances suitable for many applications. separation performance with needed for LC-MS(/MS) analysis. need to be separated from struc- core shell technology turally similar impurities. Furthermore, the Shim-pack GIST Shim-pack Scepter: excellent series is designed with various par- Designed to maximize perform- stability and performance under Shim-pack Arata LC columns: ticle size ranges, making it easy to ance of LC systems, Shimadzu’s a wide range of conditions unprecedented resolution and transfer any analysis from ultra- Shim-pack Velox columns with peak shape of basic compounds high-speed to conventional meth- core shell technology enable users These next generation organic sili- ods. to achieve increased separations ca hybrid-based columns achieve LC columns claiming to be desig - and faster analysis on any LC excellent stability and perform- ned for basic compounds often fail Compared to general C18 co - platform. Whether developing a ance over a broad pH range. With to achieve adequate resolution of lumns, Shim-pack GIST C18-AQ high-efficiency LC separation different chemistries, Shim-pack certain compounds. This problem achieves strong retention of hy - method, transferring an existing Scepter columns are effective for is overcome successfully with the drophilic highly polar compounds method for increased throughput method development/scouting and Shim-pack Arata columns, which while maintaining high inertness while maintaining resolution or are suitable for a wide variety of solve issues such as fronting of and durability in highly or 100 % trying to improve the resolution applications. Due to their differ- highly polar basic compounds, aqueous mobile phases. Further -

22 SHIMADZU NEWS 2/2020 PRODUCTS

more, it reduces the absorption of GISS series also includes the Shim-pack GIS C18-P is designed for separations that cannot be basic and acidic compounds and 1.9 μm and 3 μm HP series for with a polymerically bonded octa - achieved by other modes, such achieves superior peak shapes in UHPLC-analyses. decyl group, providing high steric as hydrophobic interactions or the analysis of metal complexes. selectivity for separation of planar π-π interactions. Shim-pack GIS Series: high and non-planar compounds, such Shim-pack GISS Series: retentivity, lower column back as vitamin D2/D3 or PAHs. Shim-pack MC: the perfect high-speed analysis with pressure, high inertness column for Micro LC ultra-high inertness and high Shim-pack GIS RP-Shield con- durability These columns are packed with tains a polar functional group The Shim-pack MC columns are high purity silica gel and are ideal embedded between silica surface compatible with a range of mobile The Shim-pack GISS series main- as generic HPLC columns. Highly and an octadecyl group, making it phase flow rates, from one micro- tains the same ultra-high inertness uniform particles ensure stable stable in 100 % aqueous mobile liter per minute to dozens of and wider pH range as the Shim- mobile phase delivery and out- phases without phase collapse. microliter per minute, making pack GIST series while providing standing low pressure. The Shim- The embedded polar functional them the ideal columns for micro rapid separations with symmetric pack GIS columns not only help group is also extremely base-deac- LC. peaks. The optimization of surface reduce solvent costs, but also tivated, enabling the column to area, pore size and chemical bond- relieve system load. In addition, provide superior peak shape for ing delivers superior peak shapes, the uniform silica surface and sta- acids. It also provides unique More information on Shimadzu’s C18 column making these columns ideal for ble chemical modifications ensure selectivity due to hydrogen bond- range can be found at LC/MS/MS analysis. Shim-pack high analysis reproducibility. ing interactions, making it suitable www.shimadzu.eu/hplc-columns

START: Several instru- Ī Micro LC Which Scepter C18 MC C18 ments Ī (general purpose) (e.g Nexera Mikros) instrument do you use? Scale up/down Scepter HD-C18 (isomeric compounds) Instrument

Application HPLC UHPLC-like UHPLC (e.g. Prominence) (e.g. Nexera XR) (e.g. Nexera X2/X3) Column recommendation < 440 bar < 700 bar < 1,300 bar Ī Ī Ī

Scepter C18 Yes Yes NoAre your analytes Yes Ī Ī Do you want to do Would you like ≥ 5 μm Ī peptides or Ī Arata Peptide C18 GIS C18-P preparative LC? columns? larger molecules?

No Ī Ī No

Yes Do you have Yes Ī Are your analytes GIS C18-P both acidic and basic Ī Arata C18 PAHs? analytes?

No Ī Ī No

GIS RP Shield Yes Are your analytes polar No No Ī Ī Is your detector GIST C18-AQ compounds Is speed a priority? Ī an MS? Velox SP-C18 (e.g. organic acids)?

No Ī Yes Ī Ī Yes

Velox C18 (general purpose)

Scepter C18 NoAre you analytes Yes Velox SP-C18 (higher pH Ī GIST C18-AQ GIST C18 polar compounds Ī stability) Velox SP-C18 Velox C18 (e.g. organic acids)? GISS C18*

Figure 1: Selection guide to choosing the right column. *The Velox series, with core shell technology, is the first choice for high speed analysis. The columns recommended for MS are shown in bold.

SHIMADZU NEWS 2/2020 23 PRODUCTS Validated method for monoclonal antibody drugs Assessment of the nSMOL methodology in the validation of bevacizumab in human serum

assay was devel- an unknown drop in sensitivity, oped for the but 10 μL was deemed to be a analysis of beva- more than acceptable assay vol- cizumab in ume. human serum with a standard Assay performance during the curve range of validation was acceptable and well 100 ng/mL to below the ±20 % acceptance 10,000 ng/mL. criteria (table 1) with inter-assay Initially, the stan- %Dev values ranging from dard Shimadzu -5.89 % to -1.66 % (2.17 % at the sample processing LLOQ) and inter-assay precision protocol using values at ≤ 7.05 % (13.8 % at the the kit analogue LLOQ). Analyte peak response in internal standard blanks of all individual human was followed and serum lots evaluated was <20 % yielded results of the LLOQ peak response, with acceptable showing acceptable selectivity. accuracy and pre- cision. However, No substantial matrix for the chroma - effects tography devel- oped internally, A lack of substantial matrix the analogue effects is highly important for any internal standard LC-MS assay. In this validation, 3D illustration of antibodies gave atypical low and high QCs were prepared results at high in six individual lots of human evelopment and validation Evaluation of the method analyte concentrations in frozen serum as well as in a lot of hyper- of bioanalytical methods with a cancer-treating QC samples. lipemic serum and a lot of D for proteins can be hin- hemolyzed serum. Choice of eval- dered by the availability of assay Applicable for the rapid uation of QCs over matrix factor reagents. LC-MS/MS hybrid Shimadzu scientists have tested development of methods with was to test a more stringent assays enable an approach capable this technology and successfully minimal assay optimization? approach in evaluating nSMOL as of using more generalized rea - developed and validated methods these QC lots could have differing gents. For these assays, the cap- for several monoclonal antibody The method was subsequently characteristics in assay capture ture and subsequent sample pro- drugs [1, 2]. As part of an inde- optimized to a 96-well plate and trypsin digestion, as well as cessing steps are highly critical for pendent evaluation of the method- method with stable labeled pep- the typical lot to lot variation in optimal sensitivity and selectivity. ology, Intertek Pharmaceutical tide internal standard for impro - ion suppression/enhancement Services (a contract research orga - ved throughput, selectivity and caused by endogenous compounds Nano-surface and molecular- nization based in San Diego, CA) assay performance. No substantial present with chromatographic elu- orientation limited proteolysis has evaluated the technique and changes were made to the critical tion of the analyte. (nSMOL) provides a unique sam- validated an LC-MS/MS method Shimadzu protocol steps to test ple processing approach to quan- for bevacizumab using a valida- whether or not the methodology For the matrix effects experi- tification of monoclonal antibody tion protocol consistent with can be used to rapidly develop ments, at least two-thirds of the (IgGs) drugs in biological sam- industry and regulatory standards methods with minimal assay opti- individual lots were within 20 % ples. The nSMOL technique pro- for pharmaceutical drug candi- mization. Using 10 μL of serum, a of nominal concentration and the vides selective proteolysis of the dates [3, 4]. Bevacizumab is a LLOQ (lower limit of quantifica- overall mean concentration was Fab region of the antibody using medication applied to treat a num- tion) with strong peak response within 20 %, indicating that there a specialized protein A capture ber of types of cancer. (>20 : 1 S/N) was observed. Con - were no substantial matrix effects resin and trypsin-immobilized version of the assay to a 96-well (table 2). In addition, both hemo - nanoparticles for protein diges- To assess the utility of the format required a change in the lyzed and hyperlipemic lots were tion. nSMOL technique, an LC-MS aliquot size of the method due to within 20 % deviation from nomi-

24 SHIMADZU NEWS 2/2020 PRODUCTS

Quality Control Concentrations (ng/mL) Quality Control Concentrations (ng/mL)

100 300 3,000 7,500 300 7,500 Amount Amount Dev. Amount Dev. Amount Dev. Amount Dev. Amount Dev. (%) Amount Dev. (%) Runfound # found (%) found (%) found (%) found (%) Lot # found found (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) 97.1 -2.90 276 -8.00 2,540 -15.3 7,110 -5.20 Lot 1 340 13.3 8,160 8.80 91.2 -8.80 264 -12.0 2,910 -3.00 6,960 -7.20 Lot 2 393 31.0 8,680 15.7 88.1 -11.9 306 2.00 3,030 1.00 8,370 11.6 Lot 3 351 17.0 8,620 14.9 1 105 5.00 276 -8.00 2,910 -3.00 7,660 2.13 Lot 4 225 -25.0 5,540 -26.1 90.2 -9.80 238 -20.7 2,860 -4.67 7,850 4.67 Lot 5 326 8.67 7,350 -2.00 79.1 -20.9 292 -2.67 3,110 3.67 8,030 7.07 Lot 6 342 14.0 8,110 8.13 a a 302 0.667 2,800 -6.67 6,970 -7.07 Mean 330 7,740 107 7.00 311 3.67 3,170 5.67 7,370 -1.73 sd 56.0 1,180 2 132 32.0 271 -9.67 2,720 -9.33 6,990 -6.80 % CV 17.0 15.2 117 17.0 286 -4.67 2,720 -9.33 7,020 -6.40 % Dev 9.83 3.24 111 11.0 297 -1.00 2,910 -3.00 6,580 -12.3 A. R. 4/6 5/6 108 8.00 281 -6.33 2,740 -8.67 6,740 -10.1 118 18.0 289 -3.67 2,750 -8.33 7,610 1.47 Table 2: Matrix effects for bevacizumab in individual lots of human serum. 3 104 4.00 292 -2.67 2,500 -16.7 7,780 3.73 A.R. = Acceptance Ratio. 86.4 -13.6 a a 3,070 2.33 4,560 b -39.2 b Quality Control Concentrations (ng/mL) 98.5 -1.50 254 -15.3 2,840 -5.33 7,590 1.20 Mean 102 282 2,850 7,380 Sample Type 300 7,500 sd 14.1 19.9 190 515 Conc. (ng/mL) Dev. (%) Conc. (ng/mL) Dev. (%) % CV 13.8 7.05 6.65 6.98 373 24.3 8,670 15.6 Hyperlipemic % Dev 2.17 -5.89 -5.04 -1.66 342 14.0 8,550 14.0 Serum 346 15.3 8,450 12.7 Table 1: Quality control results for Bevacizumab in human serum. Mean 354 8,560 a = No analyte response detected. Sample is rejected. sd 16.9 110 b = Outlier value per the Dixon Test, value is not included in statistical calculations. % CV 4.77 1.29 % Dev 17.9 14.1 nal concentration (table 3). Since labeled internal standard is highly 301 0.333 7,810 4.13 some individual lots were outside recommended. Hemolyzed 252 -16.0 7,610 1.47 20 % and since there was a bias in Serum 276 -8.00 7,420 -1.07 the hyperlipemic lot measured nSMOL is a trademark of Shimadzu Corp. Mean 276 7,610 concentrations, some chromato- sd 24.5 195 graphic adjustment or sample Authors % CV 8.87 2.56 cleanup modification might be Michael H. Buonarati, Ph.D., Senior Director % Dev -7.89 1.51 useful to improve assay perfor - Intertek Pharmaceutical Services mance in this area. Dale Schoener, Scientific Director Table 3: Matrix effects for bevacizumab in hemolyzed and hyperlipemic lots Intertek Pharmaceutical Services of human serum Conclusion References molecular-orientation limited proteoly- Recommendations for validation of Overall, the nSMOL methodology [1.] Iwamoto N., Umino Y., Aoki C., sis to LC-MS bioanalysis of Cetuximab. LC-MS/MS bioanalytical methods for resulted in acceptable assay per- Yamane N., Hamada A., Shimada T. Bioanalysis. 8 (2016), 1009-20. protein biotherapeutics. AAPS J. 17 formance in the validation of Fully validated LC-MS bioanalysis of [3.] Guidance for Industry: Bioanalytical (2015), 1-16. bevacizumab in human serum Bevacizumab in human plasma using Method Validation, U.S. Department of with minimal adjustment of stan- nano-surface and molecular-orientation Health and Human Services, Food and dard assay conditions. As individ- limited (nSMOL) proteolysis. Drug Metab Drug Administration, May 2018. ual laboratories use nSMOL, some Pharmacokinet. 31 (2016), 46-50. [4.] Jenkins R., Duggan JX., Aubry AF., adjustment of assay conditions [2.] Iwamoto N., Takanashi M., Umino Y., Zeng J., Lee JW., Cojocaru L., Dufield D., might be warranted and as with Aoki C., Hamada A., Shimada T. Garofolo F., Kaur S., Schultz GA., Xu K., any LC-MS assay, use of a stable Application of nano-surface and Yang Z., Yu J., Zhang YJ., Vazvaei F.

SHIMADZU NEWS 2/2020 25 APPLICATION How much fluorescence does a polymer show during quality control? Polymers and fluorescence – Part 2: Fluorescence spectroscopy of industrial base polymers

less, opaque and soft granules in lenticular form.

In this application, “industrial” polymers and recycled polymers are all colorless, transparent or turbid. It is expected that all poly- mers would show their natural fluorescence.

Fluorescence can occur when the molecule absorbs much energy. Considering the molecules of PC, GPPS, SAN and PET, all struc- tures contain high-energy ring systems.

Fluorescence and phosphorescence

When an electron in the ground state S0 is exposed to light, the energy of the electron in the mol- Microplastics and synthetic materials in the environment are some of the greatest challenges worldwide for mankind, ecule is changed. This elevates it animals and the environment. The example of PET bottles demonstrates that their fluorescence intensity provides information on to the excited state S1 with a high- the material composition. This Part 2 of a series of articles expands the focus on different polymers. In Part 3, which will be released er energy level (see figure 2). This in the next issue of Shimadzu NEWS, particle size and its effect on fluorescence will be examined. excited state is not stable in the long term and changes rapidly as s described in Part 1 of the rene (GPPS), styrene acrylonitrile it approaches the ground state. last issue, polyethylene (SAN) and a recyclate of polycar- The excited electron decays by A terephthalate (PET) can be bonate (PCrec). radiating the energy through heat examined easily with fluorescence or light. The radiative transition spectroscopy, simply by applying The industrial polymer variants from state S1 to S0 that emits a a piece of the PET bottle wall in listed here are used for example as photon is called fluorescence. The order to analyze the fluorescence. “glass replacements” in industry alternative excitation from T1 to This revealed fluorophores that due to their hardness (impact S0 is called phosphorescence. In might not have been suspected in resistance) and durability. Some of the T1 state, three electrons are at the transparent, colorless or slight- the granules have a cylindrical or a higher energy level (excited ly bluish-clear polymer pieces. slightly cloudy lenticular shape. triplet state). S1 is the excited sin- The biopolymer granules exam- glet state (1 excited electron in In the next step, polymers are ined, such as polylactides (PLA) higher energy levels). examined that originate not from and polybutylene succinate (PBS) the consumer sector, but rather as were transparent and colorless, Polycarbonates contain several “new” or “recycled” directly from with slight turbidity. The non- Figure 1: A typical polymer granulate in phenyl-based molecular groups, industrial production. For this aromatic polymers such as poly - crystal clear and colorless cylindrical form which are very energy-rich due to purpose, crystal clear and color- propylene (PPhomo), polyethyl- on graph paper. The orange grid in the the π electrons of the double bond less polymers were selected, such ene (PE) and polyvinyl chloride background of the granulate corresponds in the ring. In fluorescence, elec- as polycarbonate (PC), polysty - (PVCsoft), also appeared as color- to 1 mm per square. trons can be lifted into higher

26 SHIMADZU NEWS 2/2020 APPLICATION

energetic orbitals (S1) from a purposes, as these traces may con- high-energy ring system using tain fluorescent additives. hard radiation. This energy level is unstable, and the electron leaves Chemically speaking, SAN con- this orbital emitting photons sists of styrene and acrylonitrile. (luminosity = fluorescence). The dominant molecular structure is again the phenyl ring from the The importance of the styrene component. phenyl group

The general structure of the PC is

SAN where R is a placeholder for hydroxyphenole. The best known is the PC type, which is produced with using phosgene, whereby phenol is a waste prod- uct. mono-PS Figure 2: The imaginary axes would be seen as follows: The X axis corresponds to time Since the phenyl group plays an and wavelengths; the Y axis represents energy. The excitation by light (arrow upwards) important role in the molecular at the time 00:00 at a given wavelength leads after a short time (dwell time in S1 and structure of all polymers under release of first energy) to a wavelength offset to longer wavelengths. Therefore: Fluores- consideration, it can be assumed cence needs an excitation/light source and appears shortly after excitation with higher The molecular structure shows that the analytical fluorescence wavelengths. Another conclusion is that if hard radiation is reduced by energy, it becomes 2-ring systems (phenolic) and a wavelength pairs are found in the softer or changes from short wavelength (hard) to long wavelength (soft) radiation. carbonyl group with the usual same regions. -C=O double bond, which is soft- ened electronically by the position Polylactides and polybutylene compounds or aromatic elements in the group R-CO2-. There succinate are chain molecules con- are available for fluorescence. are enough π electrons available taining an ester group, which are for fluorescence. equipped with high-energy elec- trons as shown in the structures. In the polycarbonate used in this PBS PE application, the PCmonomer is based on BPA (bisphenol A). PE, PP, and PVC on the other Recycled PC samples should be hand are chain molecules in which identifiable for quality control PLA no surplus energy-rich π electron PP

PVC

Sample preparation

The sample size and position with - in the barium sulphate (BaSO4) bed in the holder for solid samples must be taken into account in the measurements. The experiment with the granules showed that the slit geometry of the light spot plays an essential role. The projection of the slit (rectangle) aims exactly at the center of the round holder. Figure 3: Fluorescence spectra of six different polymers: top left to right – GPPS, PC and PBS; bottom left to right – SAN, PCrec and The inside diameter of the BaSO4 PLA (for positions of the spots in the EEM see Table 1 [page 28]). bed is 2.5 cm.

SHIMADZU NEWS 2/2020 27 APPLICATION

Polymer Range [nm] Shape [μm] Conclusion CH2 chains requires less hard Industrial Hotspot (EM/EX) Pellet radiation. The non-aromatic PC 350/310 Cylindric Each polymer used here has its polymers such as LDPE, PPho - PC rec 305/290 Lens shape own fluorescence spectrum. In mo, and PVCsoft show fluores- 350/310 the industrial plastics PC, GPPS cence, although none is expected 435/375 and SAN, fluorescence is deter- in these π electron-poor materi- 435/400 mined by the fluorescence activi- als. SAN 315/290 Cylindric ty of the phenyl ring. In contrast, 370/315 the chain molecules of the plas- Fluorescence spectroscopy can 400/350 tics PLA and PBS fluoresce due therefore be used easily for quali- GPPS 310/290 Cylindric to the presence of the ester. Ac - ty control in order to make the Biopolymer cording to the molecular struc- “invisible” visible. PLA 370/275 Lens shape ture, steric constraints and differ- 440/360 ent excitation positions in the PBS 430/360 Lens shape compact PLA molecule, the PLA Non-aromatic molecule has hard radiation cor- LDPE 285/260 Opaque lens responding to the energy de - 330/290 mand, whereas the PBS with long PP homo 300/280 Opaque lens 330/300 PVC soft 405/325 Opaque lens 445/360

Table 1: Active fluorescence zones (hotspots) of the polymer granules in different forms – cylindrical and lenticular granules

Depending on the selected slit, also show different fluorescence. this round surface is illuminated Figures 3 (page 27) and 4 show in a more or less rectangular fash- the EEM (excitation emission ion. The granulate was embedded matrix) matrices obtained. in the center of the holder and sealed with a quartz plate. This is Remarkably, fluorescence activi- important to note because the ties could be found in all poly- granules are not flat and cannot mers. The hypothesis “no fluo- be homogenized with BaSO4 due rescence with LDPE and PP” was to their size and strength. BaSO4 not met. It appears that additives is used here as a fixing powder. are present that promote fluores- cence. Comparison be tween PC Evaluation of the and PCrec for example, demon- Figure 4: Spectra for long-chain non-aromatic polymers with the same scaling of measurements strates that by using an additive intensity. Also at top left is the control EEM from the quartz plate above the BaSO4 (for bleaching), the recyclate is bed. Top right PPhomo, bottom left PVCsoft and bottom right LDPE (low density Depending on the properties supposed to appear colorless and polyethylene). described, the different polymers transparent (table 1).

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