We at Quinta-Analytica are getting ready to celebrate our 25th Anniversary, and what a better way to do so than in the company of Dr Miroslav Ryska, founder of Quinta and world-renowned mass spectrometry expert. We often talk about our ever-expanding service portfolio, currently spanning from early pharmaceutical innovation, formulation development, clinical trials, bioanalytical services, quality control for small and large molecules, GMP production and much more as part of the Conscio Group. But this is just the outcome of an exceptional journey that began long before Quinta’s foundation. Dr. Ryska is the true embodiment of what makes us special: Overwhelming technical expertise, unquenchable proactiveness and a lifelong dedication to tackling new challenges. We wish to share with you the article that Dr. Ryska wrote for the monograph “Origins and History of Czechoslovak Mass Spectrometry”, published by the Czech Society for Mass Spectrometry (ČSHS) and translated into English for the first time with permission from the author. Dr. Ryska’s trajectory is an inspiring story, even for the non-versed in mass spectrometry, as we come to share his passion through turbulent times: From his early student days, his strive to balance science with politics during the days of the Eastern Bloc, his groundbreaking discoveries, the changes after the 1989 Velvet Revolution and his successful efforts to establish one of the best-equipped private enterprises in Europe. Join us to discover how Dr. Ryska managed to make his dream come true and the secret behind 25 years of excellence:
Miroslav Ryska: Mass spectrometry – My Destiny
Miroslav Ryska was born on February 2, 1938, in Nový Dům, in the Rakovník district. He began his chemistry studies in 1955 at the Faculty of Natural Sciences of the Charles University with Professor Běhounek, a well-known Czech radiochemist and writer, and in the years 1956-1961 he stayed as a student at the Lomonosov University (Московский государственный университет имени М. В. Ломоносова), today in various quality rankings the best rated university of the former Eastern bloc countries. His diploma thesis on the hydrogenation of cyclohexene on Palladium was created in the group of the famous Soviet chemist and Nobel Prize winner N.N Semyonov. It was also here that he encountered mass spectrometry for the first time, which proved crucial for him. After returning to Prague, he worked as a PhD student at the Institute of Macromolecular Chemistry, where he worked with Otto Wichterle on the kinetics of heterogeneous polymerization of vinyl chloride. After defending his dissertation in 1966, he completed a postdoctoral fellowship with Professor Hummel in Cologne (Universität zu Köln) and at the same time had the opportunity to work with one of the most important European mass spectrometrist of the time, Hans-Dieter Beckey in Bonn. From 1969, he led the newly established mass spectrometry laboratory at the Institute of Macromolecular Chemistry of the Czechoslovak Academy of Sciences (ÚMCH), where he had at his disposal the MS 902 instrument of the AEI company. It was the only doubly focusing mass spectrometer in Czechoslovakia at the time. However, after academician Wichterle was fired for political reasons from the leadership, the situation at ÚMCH became difficult to bear, so in 1978 Miroslav Ryska transferred to the then Research Institute for Pharmacy and Biochemistry (VÚFB). However, VÚFB de facto disappeared as a result of privatization process after 1989, so Miroslav Ryska and his colleagues were forced to operationally establish the private company Quinta-Analytica in 1996, which is now a successfully company specializing in analytical and clinical testing of drugs.
Moscow State University my first step into the field of mass spectrometry
When in September 1955, after a short consultation with my favorite high school chemistry professor, I accepted the offer of the VŠCHT rectorate to study in the Soviet Union, I had no idea that my decision would result in the lifelong connection of my scientific and research career with the field of mass spectrometry. I was supposed to study radiochemistry in Moscow, a field which was all the rage those days. That was the time when the Faculty of Technical and Nuclear Physics of Charles University was newly founded in order to keep up with the world around us. Nuclear power, however, already was in a more advanced state in the Soviet Union back then. I started my studies at the Faculty of Chemistry of Moscow State University in the second semester in February 1956, after graduating from the 1st semester at Charles University in Prague. Very soon already in the 5th semester in 1957, my friend Karel Vacek and I decided to apply to one of the faculty’s departments. We chose the department of chemical kinetics, which was headed by the Nobel Prize winner academician N.N Semjonov. Perhaps his name and the good reputation of his department were the main motivating factors in our decision to launch our future scientific careers alongside his team. With our decision, we slowly but surely detached ourselves from the original plan to study radiochemistry. At the department of chemical kinetics, there were two specializations: Reaction kinetics in the gas and liquid phases. I did not really understand this division, but I was assigned the position of auxiliary scientific force in the laboratory of the department dealing with kinetics of gas phase reactions, while Karel was sent to the liquid phase kinetics laboratory. Our group leader was R.E. Mardalejšvili. During my first visit to his laboratory, I was struck by an instrument in the middle of the room which was huge in size, around 250x100x180cm, and which produced a great noise. It was explained to me that it was an MS4 mass spectrometer of the Nier type, which was produced in quite large series in the USSR. Later on, I would find the same device in other laboratories. It was actually a copy of the English Metropolitan Vickers MS2 instrument. Hungarian postgrad Lájoš, whose knowledge I came to greatly admire during my time there, was in charge of managing the device. From the point of view of a student in the field of mass spectrometry, my arrival to this laboratory was fateful for me.
Moscow University 1958: M. Ryska and supervisor R.E. Mardalejšvili
I started visiting the lab regularly. Already during my second visit, I was given the practical task of cleaning the ion source. It was not an easy task at the time, as first I had to carefully check the connection of all electrical wires, then thoroughly clean with sandpaper and ethyl alcohol all individual electrodes and insulators and finally assemble the source to its original state. But right when I was tightening the last electrode with a small screw and I was about to complete my task successfully, I applied a little too much force and wrenched off the head of the stainless-steel screw. Fearing that my stay in the lab was doomed, I broke out in a cold sweat. Much to my surprise, however, my boss kept his composure and calmly called the fine mechanic of the department, who corrected my mistake by drilling a new hole in the electrode and threading it for a new screw which he himself made from stainless-steel wire.
My blunder was quickly forgotten, and I learned to weld the glass parts of the device’s all-glass inlet system, which I was then able to repair by myself. A few months later I was able to improve my technical skills, when I was given the much more difficult task of trying to connect this mass spectrometer to a gas chromatograph column (only packed metal columns were known at that time). If I had been as capable as Gohlke and McLafferty I would probably be famous since my attempt was around the same time as theirs. However, my efforts ended in complete failure. I solved the transition between the high vacuum of the mass spectrometer and the atmospheric output of the metal packed column with a diameter of 4 mm in the simplest way: With an ordinary metal valve; not even a needle one. It never occurred to me that I should solve the connection using some sort of carrier gas separator, which was quite common in later years. That valve was difficult to regulate, and my second attempt ended with the device suddenly aerating and contaminating it with mercury from the diffusion pumps, which eventually penetrated into the rotary pumps and into the laboratory. Decontaminating the device, the vacuum pump and the laboratory took almost a whole week, and I no longer tried to connect the gas chromatograph with the mass spectrometer.
As I already mentioned, the two diffusion pumps of the mass spectrometer were mercury ones and were equipped with large traps filled with liquid nitrogen. They were supposed to reduce mercury vapor pressure and prevent their penetration into the source and the analyzer. One pump ensured the vacuum of the ion source and analyzer while the other that of the all-glass (unheated) inlet system. The analyzer was a magnetic 60° sector. The detector was a Faraday cage with a very high time constant amplifier whose output was connected to a line recorder. The scanning of the magnet was from lower masses to higher, the feedback of the power supply of the amplifier of the magnet source was provided by linearly increasing the voltage of the mechanically scanned potentiometer on the principle of the Kohlrausch drum. One scan in the mass range of m/z 18-100 took around 15 minutes. The resolution m/Δm of the instrument was about 100. Setting the analyzer to a specific mass was also possible manually using a potentiometer. Mercury pumps had one advantage. Mercury residuals always present in the ion source made it possible to calibrate each mass spectrum based on the presence of the mercury multiplet of the isotope ions ¹⁹⁸Hg, ¹⁹⁹Hg, ²⁰⁰Hg, ²⁰¹Hg, ²⁰²Hg and ²⁰⁴Hg. At the lower range of the spectrum, peaks of water, nitrogen, and oxygen ions, together with mercury isotopes, were the only markers that allowed the correct assignment of masses to the individual peaks of the spectrum. Even so, the unambiguous marking of masses in the spectra was not a very simple matter, because peaks of double charged ions or peaks of metastable ions appeared in the spectra, which easily led to errors in determining the correct mass. This, however, was a general approach for evaluating spectra and attributing masses to individual ions; an approach used for instance by Dr. Hanuš who always amazed me with his infallibility.
At the academic department of N.N Semjonov, I dealt with the kinetics and hydrogenation mechanisms of olefines on a palladium catalyst. Specifically, it was the hydrogenation of cyclohexene at room temperature. Later on, this was also a topic in my diploma thesis. Vapors of cyclohexene together with hydrogen at a pressure of a few torr were introduced into a reactor with a palladium catalyst. Parallel to this hydrogenation reaction, however, even in the absence of hydrogen, a disproportionation reaction took place, where 3 molecules of cyclohexene produced 1 molecule of benzene and 2 molecules of cyclohexane. By using deuterium instead of hydrogen, it was possible to distinguish the formation of cyclohexane as a product of direct hydrogenation of cyclohexene and as a product of a disproportionation reaction alongside cyclohexane deuterated to varying degrees as a product of direct hydrogenation.
Visiting Hanuš and Čermák’s workplace: My second milestone
Around April 1958, my supervisor, Dr. Mardalejšvili told me about the group of Hanuš and Čermák, responsible for constructing the first Czechoslovak mass spectrometer. He highly appreciated their work and recommended me to meet them in Prague. I first visited the Čermák and Hanuš’s laboratory in the famous building in Máchova Street during my vacation in the summer of 1958. I was surprised at the excellent results this group had achieved in their very confined apartment building. Compared to them, our facilities at the Faculty of Chemistry in Moscow State University were downright royal. But they were true masters who constructed their own instruments, while I could only boast of working with a commercial device. The scientific atmosphere in this group was simply fantastic and reminded me of other renowned European and American universities during the 1930s of the 20th century, as described by Robert Jungk in his famous book “Brighter Than a Thousand Suns”. Since then, I went to Čermák’s and Hanuš’ laboratory every time I visited Prague. I remember how whenever Dr. Čermák saw me at the door, he usually greeted me with the words “Mr. Ryska, I have a problem here, how would you solve it?”. And I, practically an apprentice next to him, blurted out something incoherent so as not to look too stupid. But he was always keenly interested in what I was doing at the university.
Wichterle Institute of Macromolecular Chemistry CSAV: Third milestone
My dream, from that moment on was to join the group of Čermák and Hanuš, whom I respected immensely. Unfortunately, this dream never came true. I asked them directly after my return from Moscow in March 1961. Dr. Hanuš told me that, unfortunately, there were no vacancies in the Institute of Physical Chemistry and Electrochemistry of the Czechoslovak Academy of Sciences (as it was then called). Instead, he recommended me to apply for a position with Prof. Wichterle, who had just founded the Institute of Macromolecular Chemistry of the Czechoslovak Academy of Sciences (ÚMCH). At that time, the ÚMCH had about 4 detached workplaces, with a secretariate located in Chotkova street, under the castle.
ČSHS 1961: Čoupek and Ryska with Prof. Wichterle
For the first time in my life, I met Prof. Wichterle, one of the most important leading scientific figures of our nation. Without announcing myself in advance, I was warmly welcomed in his temporary director’s office in Chotkova street. We started talking about what I did at university. The discussion was very thorough, especially when covering the topic of chemical kinetics. But while I had a very good understanding of chemical kinetics, I was somewhat disheartened that I could not entirely follow the field of mass spectrometry, which I had already liked back at the university. However, I was promised that once the decision was made to purchase a mass spectrometer, my experience (if you could call it that) would definitely be counted on. I was not only disappointed, but also skeptical that mass spectrometry would ever be introduced at ÚMCH at all. After all, the Institute’s main focus at the time, as far as physico-chemical assays were concerned, was the determination of polymer structures, and the possibility that mass spectrometry could ever be used for direct measurement of macromolecules had not occurred to anyone yet, and even less to me. And so, for more than 4 years I devoted myself to the kinetics of heterogeneous polymerization of vinyl chloride, and I could only dream about mass spectrometry. In the spring of 1966, I defended my PhD thesis on this subject. Soon after, Professor Wichterle had me called into his office. He told me that the institute received the first 2 postdoctoral fellowship offers from West Germany: one at the University of Mainz and one at the University of Cologne. He told me that he had thought about me for a postdoc position at the Institute of Physical Chemistry in Cologne because they studied pyrolytic decomposition of polymers and copolymers by means of mass spectrometry. Were I to accept, then when returning I would be able to use that experience to build my own mass spectrometry laboratory at the ÚMCH ČSAV. Prof. Wichterle’s proposition literally put new life in me. I now firmly believed that I would return to mass spectrometry and the possibility that the device would actually be purchased was quite high, even if financially demanding. Already back then, the institute had its own US dollar account where considerable sums of foreign currency were transferred from the patents which Prof. Wichterle had registered abroad. In fact, only a fraction of the profits earned by the academy and the State.
In September 1966, I went on a one-year fellowship (which was later extended to 16 months) at the Institute of Physical Chemistry at the University of Cologne. The mass spectrometry laboratory was located in the macromolecular chemistry department, where a number of spectral instruments could be found: Mainly IR, Raman, and others. The mass spectrometry laboratory itself only had a Krupp MAT CH4 spectrometer. PhD student Horst Dieter Shüddemage worked on it. We became friends right away and have remained friends to this day. Horst performed polymer decompositions directly in the heated direct inlet of the ion source and analyzed the gaseous pyrolytic products using the field ionization method. It was fascinating to me. The MAT CH4 was a single focusing sector magnetic instrument with a relatively high resolution (5000 per 10% peak valley). On the device, in addition to the electron ionization source a field ionization was installed, I was fascinated by its unusually fast line recorder, which at the time was able to record one mass peak per second. The Head of Department, Prof. Hummel, was an expert in IR spectrometry. He published an extensive atlas of IR spectra of polymers, and my task was to prepare a whole series of copolymers of styrene with vinyl chloride, which would then be used for the determination of pyrolysis products by mass spectrometry. Moreover, their IR spectra, as well as the spectra of new unknown copolymers, were to be also included in the publication of his monograph. Bearing in mind the huge difference in copolymerization parameters, the idea of preparing copolymers of styrene and vinyl chloride, let alone their possible use, was utterly outlandish. Copolymers would necessarily have to have a block character with longer styrene and vinyl chloride sequences. Then again, looking at the characteristics of the pyrolytic products, these copolymers were rather interesting. Thermal decomposition of polystyrene sequences (or blocks) produced three products: A monomer, a dimer and a trimer of styrene. Degradation of polyvinyl chloride sequences (blocks) was more difficult, but a sequence of three contiguous vinyl chloride monomer units provided benzene in good yields after thermal dehydrochlorination. It was therefore possible, on the basis of semi-quantitative determination of benzene, monomer, dimer, and trimer of styrene, to characterize up to three adjacent monomeric units of vinyl chloride quite successfully, at least semi-quantitatively. For this purpose, field ionization, providing mostly molecular ions of all four components, was a very suitable method. However, the preparation of platinum emitters for field ionization was difficult. They were thin platinum wires with a diameter of 2 μm, which were prepared from Wollaston wires by stripping their outer silver layer by dissolving it in nitric acid. Then the thin platinum emitter was activated for several hours in an ion source in acetone vapor at a pressure of around 10⁻⁴ torr with a gradual voltage increase up to 11 kV. The thin platinum emitters were extremely sensitive both to mechanical stress and to electrical discharges. If electric discharges occurred, they were invariably destroyed. Later on, we started using platinum wires with larger diameters of up to 10 μm and thus achieved greater resistance to both mechanical and electric stress. The preparation and activation of the emitters was time demanding. If performed directly in the source of the mass spectrometer, the instrument would practically be rendered unusable. We often went to the nearby University of Bonn to see Professor Beckey, the creator of a method employing activated thin platinum wires in field ionization. Not only did he share his rich experience with us; he also helped us prepare emitters outside the source of the mass spectrometer for the device he developed. Soon after, the activation was carried out in benzonitrile instead of acetone. This resulted in much more active emitters. Our meetings with Prof. Beckey always were as interesting as they were stimulating.
Soon after returning from my stay at the University of Cologne, Professor Wichterle entrusted me with the selection of a suitable mass spectrometer for the ÚMCH ČSAV. Furthermore, I was also entrusted with building a mass spectrometry laboratory at the institute. In 1968, I travelled to England and the Federal Republic of Germany, were I met with several mass spectrometer manufacturers and negotiated possible conditions for the purchase of the instrument. This was quite unusual at the time, given that any negotiations with importers of instruments were completely under the jurisdiction of the State’s Foreign Trade Corporation for Export and Import (PZO KOVO) and employees from research institutes such as the Academy of Sciences were not allowed at all to conduct such negotiations.
I, however, was not aware of any of that and did not know that I could cause any inconvenience to our institute with my actions. That aside, I gained much experience while being abroad in tough business discussions with instrument suppliers. I managed to negotiate very favorable terms with Manchester-based AEI for the purchase of the then top double focusing high resolution instrument of the MS-902 type, with Nier-Jonson geometry. In addition to the classic EI source, the device was equipped with a combined FI/EI source. In contrast to the MAT devices, emitters prepared from special razor Wilkinson blades were used. These were mechanically more resistant than thin wires, but their disadvantage was that they could not be heated electrically. An instrument of this type had not yet been introduced to Czechoslovakia, and its direct import from England to an Eastern Bloc country was not possible due to the embargo. At that moment, all mass spectrometers whose magnetic analyzer radius exceeded a certain prescribed limit were subject to embargo (I no longer remember its exact value). Due to the embargo, the device would have to be imported through a third neutral country, preferably through Liechtenstein. I was proud of myself for having managed to negotiate such favorable conditions plus I also received praise from Prof. Wichterle; which made me doubly happy. The feeling, however, was not to last, as it soon became clear that my independent actions caused huge problems not only to myself, but to the entire institute: I was accused by PZO KOVO of having exceeded my own competences and denied me any authority to deal with importers of devices to Czechoslovakia. KOVO took the matter into its own hands, and asked AEI in Manchester to send a new quote for a price 15% higher than originally negotiated. KOVO then “managed to reduce” it to the same level I had previously reached and ÚMCH was required to pay an extra 15% to PZO KOVO for their “successful business negotiations”. Still, all is well that ends well, and the institute received the first double focusing mass spectrometer with a resolution up to 40,000 (at 10% peak valley), delivered to Czechoslovakia from England via Liechtenstein. I built a special laboratory for the device in our second basement because its weigh made it impossible for it to be placed on the upper floors (only the magnet weighed 1.5 tons!). It was a truly cutting-edge device at the time. All electronics were based on vacuum valves, oil-diffusion pumps were, separated from the high-vacuum area by traps with automatic liquid nitrogen filling from a 120-liter Dewar container. The ion source was equipped with a total of 4 sample inlet systems: a non-heated glass inlet for the introduction of vapors, reference substances for mass scale calibration and for precise ion mass measurements using the “peak matching” method. An all-glass heated system was important for handling substances with a lower vapor pressure at normal temperatures. Another input was intended for connection with a gas chromatograph or with the output of the helium separator (the separator was Biemann type). The separator was connected to the output of the gas chromatograph via an electrically heated metal capillary. The most important part was the direct inlet, either with electric regulated heating or an unheated probe. For the heated inlet, solids were placed in a glass capillary, which was inserted into the glass pocket of the direct inlet probe filled with glass wool. The pocket was then wrapped with an electric spiral and electrically heated, either manually or continuously increasing with automatic temperature control. However, the construction of such a heated direct inlet was quite imperfect and the inlet had a relatively significant failure rate. Therefore, preference was given to an indirectly unheated input, which was equipped with a quartz microtube inserted through the insulator into a metal probe. The diameter of the microtube corresponded to the diameter of the hole in the ionization chamber, so that the sample could be inserted into the close vicinity of the electron beam by means of magnetically controlled adjustment of the probe. This made it possible to achieve high ion yields and the sensitivity of the source was exceptionally higher compared to other device types. Sample temperature corresponded to the temperature of the ion chamber, which could be changed with relative ease, or adjusted to any required value before inserting the sample into the ionization chamber of the source. Mass spectra were obtained by scanning the magnetic field strength of the magnetic analyzer from higher to lower mass when the initial high value was set by charging the capacitor to the initial value in the feedback amplifier of the magnet power supply. The speed of the scans was then given by the rate of discharge of the capacitor through the resistors and was determined by the value of these resistors. The mass scan was therefore non-linear due to the basic quadratic function of the dependence of the m/z ratio on the magnetic field strength, but also to the non-linear dependence of the amplifier feedback voltage as a time function of the voltage across the resistance of the discharged capacitor. Orientation in the spectra was therefore extremely difficult. It was facilitated by the mass marker, which sensed the intensity of the magnetic field using a coil placed between its poles. Nevertheless, the calibration of such a mass tracer was not simple. When using a set of about 18 potentiometers it was necessary to calibrate the marker using the characteristic ions of perfluorotributylamine, which served both as a calibrator for the marker and as a reference substance for determining the exact mass of the analyte ions by the “peak matching” method. The advantage of perfluorotributylamine consisted both in its favorable vapor pressure at laboratory temperature and in providing characteristic peaks of the spectrum every 100 mass units (C₂F₄ difference), starting with the ion peak m/z 1066. The stability of the mass marker setting was not general. The ion accelerating voltage amplifier feedback (540 V) was powered by a set of series-connected dry cell batteries (1.5 V). Their continuous depletion and voltage drop also caused a continuous shift in the mass scale. This shift could be compensated for with additional potentiometers, but even then, compensating for other influences was difficult. In the optimal case, the marker had to be recalibrated several times a day. However, this marker was a significant help in orientation in the spectra. Scanning of the analyzer’s magnetic field was possible up to a speed of 1 mass decade per 2 seconds. It was therefore possible to cover the mass range m/z 500 – 50 even in 2 seconds. This speed was also sufficient for a GC/MS application, although the GC/MS mode could only be considered as supplementary only and could not be compared to other instruments at the time, such as the LKB 8000 or the emerging quadrupole instruments. The ion detector was a 32-stage secondary electron dynode multiplier and the ion spectra were recorded using a 4 channel UV recorder on sensitive photographic paper. The galvanometers were able to faithfully record the rapid course of scans with the bandwidth up to 10 kHz. One channel of the amplifier was connected to the ion mass marker. Every 5th m/z value was marked with a short line, every 20th line with a medium line, and the longest line then belonged to the value of m/z 200. Despite its imperfections, judging by today’s spectral digital recording standards, the mass marker was a very important tool for quick spectral evaluation.
The greatest advantage of the MS 902 instrument was its high resolving power and easy routine measurement of exact ion masses using the “peak matching” method. Resolution of 10000/10% valley was usually achieved in less than a minute by simply mechanically controlled reduction of the width of both slits, of the output slot of the ion source and input one to the magnetic analyzer. The common mode at the so-called low resolution (1000/10%) with both slits fully open was used when recording scans of full spectra. The maximum resolution of 40000/10% valley could be obtained by optimizing the position of the magnet and narrowing the so-called z-slits, which limited the width of the peak in the z-coordinate, i.e., perpendicular to the plane of the ion path. The system for determining the exact mass of ions by “peak matching” was manual with alternating projection of the analyte and reference substance peaks. While the peak of the ion with a lower peak was projected on the oscillographic display at the full accelerating voltage of 8kV, the peak of the ion with a higher mass was projected at a lower voltage so that geometrically they overlapped each other during their alternating projection. The two-channel amplifier allowed perfect comparison of peaks differing in intensity by up to 3 orders of magnitude. The system was practical enough to allow a skilled operator to determine ion masses with an accuracy of better than 10 ppm. The speed of determining the exact mass of 1 determination per minute, possibly even higher, was excellent from a practical point of view. The use of high resolution with accurate ion mass determination meant a completely revolutionary and simpler approach in solving the question of the composition and structure of individual ions. I was often visited by Dr. Hanuš whenever he was in need of unequivocal confirmation of ion composition and structure for solving ion structure problems. I always admired his infallible suggestions and his enthusiasm about the possibilities of high resolution. In fact, our regular interactions were an important incentive for him to later acquire his own high-resolution instrument.
The MS-902 instrument also enabled the detection of spectra of metastable ions to the selected parent ion by the defocusing method. In those cases, the magnetic field was set to focus the selected parent ion at a lower accelerating voltage, usually at half, i.e., 4 kV, and by scanning the accelerating voltage up to a maximum value of 8 kV, spectra of metastable ions were obtained.
The standard equipment was an electron ionization source. In an attempt to push further the experience with field ionization that I gained at the University of Cologne, a combined source with electron ionization and EI/FI field ionization was also ordered for our device. Unlike the MAT CH4 ion emitter device, the blades here were prepared from Wilkinson razor blades, which were specially modified. For field ionization, an additional voltage of -4 kV was applied to the emitters from a separate source so that their total ionization chamber voltage at the potential of the accelerating voltage (8 kV) was 12 kV. The blades had to be activated in acetone vapor at a pressure of 10⁻⁵ torr for about 12 hours at a gradually increasing voltage from 6 kV up to a maximum of 12 kV. During activation, the signal of the acetone molecular ion m/z 58 increased by more than 2 orders of magnitude. The price of those Wilkinson blades was quite high, therefore I tried replacing them with Czechoslovak-made ASTRA razors, which were commonly available in stores. However, their appearance alone was very different from the delivered Wilkinson emitters. Protection against razor corrosion was achieved by means of a thick lubricant layer. I carefully washed the blades in acetone and other solvents to remove grease before activation. Not surprisingly, with such a simplified approach I was unable to find any molecular ions of acetone during activation, nor any other ions in the region up to m/z 100. Thus, I considered such an attempt a failure. Nothing could be done: The manufacturer probably knew very well why to carefully select the material for the emitters. Moreover, I suspected that the electrical circuit for monitoring of the total ion current had failed, probably due to an electric discharge. The hand of the milliampermeter of the total ion current monitor amplifier was stuck at the full scale in all its measurement ranges from the beginning of the activation. I tried hard for two days to find and fix the amplifier fault, but to no avail. All electrical circuits of the amplifier appeared to be fine. Suddenly, I inadvertently turned on the magnetic scan, the initial value of which was set somewhere in the region of m/z 1000, and to my great surprise, the visicorder oscillographic recorder began to record very intense spectra of hydrocarbon molecular ions from preservative anti-corrosion lubricants in the region of m/z 400 – 700. If it weren’t for Beckey’s year-old publication on field desorption ionization, it would probably be a significant discovery of ionization in the solid phase, i.e., desorption by a field. The ion current was very steady and remained constant for several days before being interrupted by a sudden electrical discharge. I was then unable to repeat the experiment.
Miroslav Ryska in the cover of the original monograph
During my time at the ÚMCH, I provided a regular service to synthetic chemists; analyzing degradation products from polymers while trying to find time for field ionization experiments. This method already interested me during my work at the University of Cologne. However, working with the blades was not as simple as with platinum emitters prepared from Wollaston wires. In cooperation with Dr. Kuraš from the Department of Fuels of the Prague Technical University, we began to investigate the behavior of simple hydrocarbons, starting with alkanes, alkenes, alkynes and ending with simple aromatic hydrocarbons. Our goal was to develop a simple method for the quantitative or at least semi-quantitative determination of hydrocarbons in lower oil fractions. We found that ionization efficiency considerably changed with increasing chain length, especially for alkanes, and that ionization efficiency of individual hydrocarbons in mixtures differed considerably from the ionization efficiency of individual hydrocarbons in their pure state. This original finding brought about great doubts as to whether field ionization would be applicable for the quantitative composition of hydrocarbon mixtures at all. The highest ionization cross-section was shown for benzene, and we feared that due to the significantly lower sensitivity of others, especially alkanes, we would not be able to reliably detect these in a mixture with benzene or other aromatic hydrocarbons, let alone determine them quantitatively. To our surprise, however, ionization efficiency of alkanes and alkenes increased several times in the presence of benzene. In addition, with enough benzene in the mixture, ionization cross-sections of the individual alkanes became equal, so that that they practically did not change at all with increasing chain length. This finding was so significant for us that we proposed a method for the quantitative determination of alkanes and alkenes with the addition of about 30% benzene to their mixtures. Under these conditions, ion yields were practically independent of the carbon chain length of all observed hydrocarbons of alkanes, alkenes, and alkynes. This finding already raised doubts in us about the existing theory of the monomolecular mechanism of field ionization in the gas phase.
These doubts were confirmed with the finding of a significant influence on the ionization of alkanes in the presence of alkynes. After this finding, we monitored the field ionization of binary mixtures of n-decane in the presence of 1-octyne, both light n-decane and n-C₁₀H₂₂, perdeuterated n-decane. During these experiments, the formation of very intense peaks of ions (C₁₀H₂₁)⁺, i.e. (M-1)⁺, was observed in the case of perdeuterated decane (M-2)⁺, which were practically not present in the absence of octyne. In the presence of 30% alkyne, the intensity of the peaks of these fragments reached the intensity of molecular ions, possibly even higher. Another significant observation was the formation of relatively intensively represented fragments of ethyl ions of n-decane. In the case of perdeuterated n-decane, however, in addition to C₂H₅⁺ fragments, equally intense C₂H₄²H⁺ and C₂H₃²H₂⁺ were formed.
Mr. Slavíček worked as a technician at the ÚMCH while simultaneously studying zoology in the Faculty of Natural Sciences at Charles University. The subject of his diploma thesis was the monitoring of DDT contamination in the organs of common voles such as livers, kidneys, muscles and fat. These studies were carried out shortly after the production of DDT was banned due to its carcinogenic properties. Under the guidance of my colleague, the highly experienced chemist Karel Bouchal, graduate student Slavíček synthesized deuterium-labeled DDT, which we then used as an internal analytical standard. After adding an internal standard to the extracts of individual voles’ organs at a resolution of 10000 using the peak matching unit, we successfully measured DDT residues not only in individual voles’ organs, but also in soils from various regions in Bohemia. Extracts of multiple organ and soil samples were directly analyzed after solvent evaporation (hexane) by rapid heating within the direct inlet probe. I believe it was the first quantitative determination using isotopic dilution mass spectrometry in any Eastern European country. Due to the high resolution and significant negative mass defect of the DDT molecular ion, it was possible to quantify DDT without preliminary extract purification or chromatographic separation. As a result of the millions of tons of DDT produced after the Second World War and applied all over the world as an insecticide, mainly in agriculture, DDT could be detected practically everywhere. According to some data it could be detected even in the glaciers of the North Pole. Due to its extraordinary resistance to degradation, to this day we still encounter traces of this substance in environmental analysis, although of course on a much smaller scale. Our results were absolutely shocking. It was impossible to find DDT-free grains or soil virtually anywhere.
As already mentioned, while at ÚMCH I also provided service for synthetic chemists, who usually resorted to us when in need of testing for the presence of desired synthesis products in their reaction mixes after the reaction had been carried out. Thanks to the high resolution and elemental determination of molecular ions based on their exact mass, we could determine either intermediate or final products after the end of synthesis. For synthetic chemists, such determination was very valuable, and they greatly appreciated these services. Of course, the determination of polymer degradation products was an inherent activity.
During the second half of the 1970s at ÚMCH, I also performed analyses on a contract basis for the Research Institute for Pharmacy and Biochemistry (VÚFB), which did not possess a mass spectrometer and had to tackle a number of problems that were practically unsolvable without such instrument: Structural determination of synthesis products or metabolites after their isolation from biological materials. It was around this time that Professor Wichterle was removed from his post as director as part of a political cleansing campaign and working conditions at the ÚMCH became unbearable for many of us. At that point I gradually prepared the grounds to shift my activities entirely to the VÚFB.
Research Institute for Pharmacy and Biochemistry: My fourth milestone
On January 1, 1978, I started working full-time at VÚFB. Although at that time the institute did not possess a mass spectrometer, 2 instruments had already been ordered: A Soviet MCH 1320 for the Physical Chemistry Department and a Varian MAT 44S GC/MS for the Pharmacokinetics Department. At first, the institute’s policy was not clear to me, and I was concerned about the need for such division. I later learned that in order to obtain funds for an MAT 44S device of Western origin, it was necessary for political reasons to also purchase a Soviet device. Securing funds for the Soviet instrument proved not a problem, much in contrast with obtaining Western currency. On top of that, the KOVO Foreign Trade Corporation had significantly increased its political influence in the government. At that time both departments belonged to the pharmaceutical-analytical section, headed by Dr. Kakáč, but director Dr. Němeček was the one responsible for preventing that any politically undesirable subject like myself would change his workplace against the will of some ÚMCH comrades. Many readers of this piece, especially the younger ones, might find it hard to believe that someone like me would be regarded that way, but such were the times. Fortunately, and thanks to the bravery and leadership of Dr. Němeček, the impossible became a reality: Compared to ÚMCH, my new workplace at the VÚFB turned out to be an oasis of political tranquility, so much needed for my scientific and professional activities. My future life path and my entire career in the field of mass spectrometry, including my later decision to start a company, would be significantly influenced by this period. It turned out that the deployment of both devices in two departments was quite formal. In VÚFB, I found a key collaborator and colleague, Ivan Koruna. I believe we influenced each other very synergistically, developing a very fruitful and creative relationship that spanned over a period of 11 year until the Velvet Revolution in 1989. I look back at our collaboration with great fondness.
The Soviet MCH 1320 was a newly developed double-focusing, sector field mass spectrometer with Nier-Johnson geometry. At the time, however, it was a clear example of how much behind Soviet mass spectrometer technology was compared to the West. It was far from reaching the level of the MS 902 from ÚMCH, which was more than one generation older. The shortcomings of the MCH 1320 were not limited to resolution only, but above all operability and practical measurements. Perhaps its electronics were more modern (transistor), but in every other respect the instrument was incomparable to new generation devices of Western origin. We called the MCH 1320 Alyosha and in turn, German MAT 44S was named Berta. The MAT 44S was a lower-midrange quadrupole GC/MS system with a VARIAN gas chromatograph and its own small computer (64 kB RAM) as a control unit. The packed chromatographic columns were connected via an open split to the inlet of the ion source using a thin heated platinum capillary. During the analysis, spectra were recorded at a speed of 1 spectrum/sec onto a 10″ floppy disk, with a maximum capacity of 52 spectra with records of the analysis parameters. From the floppy disk, spectra were transcribed onto A3 x/y format paper using a x/y plotter. Another very valuable inlet for us was the heated direct inlet. Samples were placed in miniature aluminum crucibles into the electric heating coil. The ion source was a combined EI/CI one. Although in theory we could switch between EI and CI modes during a single analysis via a mechanically controlled system, in reality it wasn’t very convenient and therefore seldomly used. Chemical ionization was possible in both positive and negative modes. It is safe to say that we were the first group from Eastern Europe who routinely used chemical ionization in our analyses and in fact we achieved good results. We often measured chemical ionization spectra for Dr. Hanuš, and although he was a conservative supporter of purely monomolecular electron ionization mechanisms and regarded our non-monomolecular ionization method as questionable and unconventional, we eventually convinced him of its usefulness.
VÚFB 1978: M. Ryska and Dr Němeček at the Alyosha mass spectrometer
We usually serviced chemists from the organic synthesis department, and in addition we also determined the structure of drug metabolites. The identification of metabolites in complex extracts such as blood and urine was often challenging, especially if we take into account the low capacity of our floppy disks, which made it impossible to record spectra of all peaks during GC/MS analysis. We developed a method to artificially create isotopic clusters by experimentally administering a 1:1 mixture of a drug and its analogue (i.e., 3 deuterium atoms) to an animal in case of animal metabolite studies. The analysis was visually monitored on the screen, and if a peak appeared containing a cluster of ions in a 1:1 ratio with a mass difference of 3 mu, then only these spectra were recorded on a diskette.
High resolution (actually medium high) with accurate ion mass determination (no better than 10 ppm) was performed with the help of Alyosha, but only in case of utmost need, given that as I already mentioned, handling this instrument wasn’t nearly as straightforward as with the MS902.
During my time at VÚFB I tried to convince my superiors of the need to purchase a better instrument than our MAT 44S, let alone good old Alyosha. However, my efforts were not met with success: It was necessary to have significant political influence to achieve such goal; from my position it was impossible to obtain the support of others; and in this aspect VÚFB was not as agile compared to other institutes. In spite of everything I never gave up on this idea and ultimately managed to make it come true after founding my own private company (see below).
The Mass Spectrometry group of the Ioannes Marcus Marci Spectroscopic Society and other international activities
VÚFB also allowed me to participate in international scientific conferences and other events abroad, understanding, of course, that I could not count on any financial support for such trips. Once again, readers of this text may not fully appreciate that back at the ÚMCH it would have been unimaginable for someone like me, classified as an undesirable subject for political reasons, to be allowed to travel abroad, particularly to the West. At VÚFB, I was not prevented from any of these scientific activities. While still an employee at the ÚMCH after Dr. Herman had stopped his chairmanship of the mass spectrometry expert group at the Ioannes Marcus Marci Society (SSJMM) I followed him as a chairmen of the group, This group was one of the smallest within the Society. Bear in mind that in those times, the total number of mass spectrometers and therefore mass spectrometry workplaces in the entire Czechoslovakia did not even reach 10. Consequently, the expert group did not organize any of its own courses, seminars, or schools, as they do nowadays. In our pioneering lab at the J. Heyrovský Institute of Physical Chemistry, practically only Dr. Hanuš and his student Dr. Tureček were active in the field of classical organic mass spectrometry. Dr. Čermák and his PhD student Dr. Herman devoted themselves to the processes of bimolecular ion-molecule reactions, where they both achieved outstanding results and became worldwide renowned scientific leaders: Dr. Čermák in the field of Penning ionization by gas molecules in the excited state (Helium) and Dr. Herman in the topology of ion-molecule reactions. As the head of the mass spectrometry expert group, I was appointed to the scientific committee of the International Mass Spectrometry Conferences. The statutes, structure and activities of this committee were the work of Prof. Beynon, a leading voice in the European mass spectrometric community. In 1985, I participated as a representative of the Czechoslovak mass spectrometry group in the 10th International Mass Spectrometry Conference in Swansea. As already mentioned, we did not receive any financial support from VÚFB or other organizations at home, but thanks to the help of Prof. Beynon me and my colleague Ivan Koruna would be able to participate in this event. Converting our own savings into Western currency, which we would have welcomed, was legally impossible at that time. I made a huge effort to get other members of the SSJMM mass spectrometry group to participate in this conference. I even asked in writing the chairman of the SK VTIR (State Commission for Scientific, Technical and Investment Development) Obzina, who at the time was also the Minister of the Internal affairs and thus a very influential man. Unfortunately, all my efforts were in vain. Three years later I repeated my request, this time to participate in the 11th International Mass Spectrometry Conference in Bordeaux, but the outcome was the same. Apparently, my past activity at the Wichterle Institute had not gotten me any political favor. Once more we attended the conference with my colleague Ivan Koruna. We were helped financially by local organizers, and it was enough for us to have modest accommodation and even more modest meals during the event. I dreamed that one day, one of the next conferences would be hosted in Prague: A complete utopia back then. With our joint efforts, we, the representatives of the countries of the Eastern Bloc, at least managed to push through a secret vote on the decision to hold the 13th International Conference in Budapest and thus support our Hungarian colleague, friend of Dr. Hanuše Dr. Cornides. My long-time dream of hosting a conference in Prague would not be realized until 2006 by my successor, my first PhD student, Vladimír Havlíček. Nevertheless, our international contacts and foreign colleagues enabled me to organize other smaller-scale events in our country.
In 1985, with the significant help of my colleague Koruna, I organized the 1st Seminar of Mass Spectrometry in the recreation center of the firm Elektro-Praga Hlinsko. As lecturers for the school, we recruited practically everyone who was active in mass spectrometry at the time and had a mass spectrometer in their workplace, including Dr. Hanuš, Dr. Tureček and others. The total number of school participants was about 23, with almost half of them being lecturers. The seminar´s program was truly thorough, with lectures and discussions from early morning to late evening. Lectures were published in a special edition of the Bulletin of the Czechoslovak Spectroscopic Society.
Another seminar of mass spectrometry was later held in Klučenice, near the Orlík Dam, with multiple foreign mass spectrometrists as lecturers: Prof. Gelpi, Prof. Przybylski, Prof. Schmid, Dr. Bruins, Prof. Dube. We invited increasingly important personalities to our lectures and seminars, and little by little, the Czechoslovak mass spectrometry group became known abroad. It can be said that our mass spectrometry group was the most active among all Eastern European groups.
One of the most rewarding outcomes from this period is that I eventually became the European editor of two of the world’s most important journals: The International Journal of Mass Spectrometry and Rapid Communications in Mass Spectrometry.
Mass spectrometry in the private company Quinta-Analytica s.r.o.: Conclusion
After the Velvet Revolution of 1989, the situation of VÚFB was not easy. The Institute, which in the past served as a research hub for all SPOFA companies (SPOFA was a group which comprised the entire pharmaceutical industry and R&D of Czechoslovakia), found itself in a challenging situation where many pharmaceutical companies disappeared and those that remained had not the slightest interest in research. The very structure of the VÚFB was unique in the world and unique in its own way due to the fact that it dealt with drug research at all stages of development. This unique structure was a great advantage for the future survival of the institute, provided that it could endure a crisis period of several years after 1989 or find key strategic partners. However, the huge assets of the institute, especially real estate inside and outside Prague, turned out to be a disadvantage during the ensuing period of intensive privatization given that privatization drivers were more interested in the properties than the actual research performed by the Institute. During this uncertain phase in the Institute’s history, I already tried as the head of the analytical department to raise its prestige by introducing a more extensive array of analytics in the field of clinical drug testing and bioequivalence studies. The breakthrough was our first study which, unfortunately, could not be carried out without a new GC/MS system. I managed to convince the director of the Institute, Mr. Dohnal, and in 1994 we purchased a Finnigan ITD GC/MS instrument with an ion trap analyzer. If I’m not mistaken, that was the first ion trap in Czechoslovakia, and despite its limitations, relative to today’s ion traps, the new instrument proved invaluable for the successful completion of our first project: A crucial requirement for obtaining new projects. The analytical department thus quickly became the most profitable department in the Institute, with really good financial results.
Still, by the end of 1996 it was more than clear that VÚFB’s days were numbered. I was sorry for the very successful work that we had started, and guided by the desire to preserve the continuity of our operations and save the work of at least 20 analysts, I approached three colleagues, analytical chemists from my department, and together we founded our own private company: Quinta-Analytica s.r.o. In our new rented premises at the Litochleby Policlinic we were able to continue our analytical and clinical drug testing activities, thanks to the recognition built upon our previous successes and above all, thanks to the dedication and commitment of everyone. The first instrument that we leased was the GC/MS system Finnigan MAT GCQ, back in in 1997. A year later, it was the turn of our first LC/MS Finnigan MAT LCQ; and many other instruments followed afterwards. Our company became exceedingly proficient in the development and commercial use of analytical methods for the pharmaceutical sector. Thanks to our accomplishments, in 2006 we were able to build and put into operation the Quinta-Analytica campus, comprised of two buildings and a total laboratory area of more than 8000m², for a total cost of 250 million CZK.
2004_Miroslav Ryska setting the first stone of Quinta’s new site
Nowadays, the mass spectrometry laboratory is only a small part, albeit most significant, of this complex: Our company currently possesses 4 GC/MS systems, 12 LC/MS/MS triple quads, 1 linear trap, 1 QTOF, 3 high resolution Orbitraps and 1 ICP/MS systems Quinta’s mass spectrometry laboratory ranks among the best equipped laboratories not only in Czech Republic, but also in Europe. Using our triple quads, we are able to perform over 100,000 quantitative determinations annually, being able to handle all kinds of drugs and their metabolites in biological samples, especially in blood plasma. Never before could I have imagined the continuous 24-hour fully automatic operation of so many instruments at the same time. Back when I worked at ÚMCH or VÚFB, I would have considered such mass spectrometry capacities to be pure utopia. My dream to own modern appliances and to be self-sufficient, without the need of support from superiors or even politicians, had finally come true for me. Unfortunately, the many duties and tasks associated with managing Quinta and ensuring marketing as the most important prerequisite for the successful projection of our company progressively separated me from my favorite field, to which I devoted my entire life. With that said, I now observe our younger colleagues and I am filled with immense satisfaction at their accomplishments, knowing we have overcome the wrongs of the past, and with a certain pride not only for the company’s success and equipment, but above all for the extraordinary group of analysts and colleagues, whose contribution made my lifelong dream come true.
QUINTA-ANALYTICA is an EMA and FDA-inspected European CRO, part of the Conscio group. For over 25 years Quinta has offered GCP/GLP/GMP-certified clinical, bioanalytical and CMC services for the pharma and biotech sectors. Visit us at www.quinta.cz or contact us at email@example.com for more information about our services.