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Techniques & Tools Environmental, Mass Spectrometry

The Secret Life of Plants

I was born in Moscow, Russia, and was the only child of parents who were also only children; we were a very small family. My mother was a chemist, and my father was a physicist and lawyer – an expert in ballistics. With two scientist–parents, I passed through childhood between books and test tubes, and learned discipline, the conviction of the superiority of reason, and the importance of science for the progress of humanity. Although we didn’t have a lot of money, I never lacked for books or culture (we often visited museums, theaters, exhibitions). The example of my parents – their love for science and reading, their responsible and dedicated work – was a defining influence both in my ideals and the path that my life took in science.

In my young life, my two great passions outside of my academic classes were sport and art, and the lessons I learned from both have enriched my life and career. For over 10 years, I practiced speed skating. Daily training taught me not only to become more disciplined and persistent, but to respect time and, when necessary, to compress it. Dedication to skating eventually displaced my painting classes, although I never lost my love for art. Today, in my classes, I try to combine art and chemistry in my presentations. Graphic design has helped me a lot in my classes to transmit scientific concepts and information to students, in a manner that is both accurate and attractive.

Choosing chemistry

At school, I had excellent teachers: serious, responsible and self-sacrificing. For the rest of my life, I have carried their memory in my heart as a true example of dedicated educators. After school, I wanted to study biology or become a veterinarian, having grown up in the company of dogs – those faithful and unconditional friends. However, my mother always wanted me to be a chemist. Ultimately, I loved and respected my mother very much, so I applied to study chemistry in the Faculty of Natural Sciences at the Peoples’ Friendship University in Moscow. My father was more open when it came to my career, but he advised against his own field of criminalistics, telling me that it could be a hard and sometimes bitter profession. Nevertheless, I now teach a forensic chemistry course, which shows how instrumental analytical chemistry can be applied to solve many forensic problems and legal cases, such as drug testing, residues of explosives, arson investigation.

My university days were a time of many good memories, and of fascinating lectures with strict but respectful teachers. Organic chemistry was love at first glance – I felt like a demiurge, creating previously nonexistent molecules with unknown properties. Every time a shapeless mass in my flask precipitated as beautiful, shiny crystals, it felt like a miracle.

My love for instrumental analytical chemistry came much later, during a PhD devoted to mass spectrometry – a technique whose ‘philosophy’ is quite the opposite of organic synthesis. Molecules in the ionization chamber are destroyed and cease to exist as a whole; they dissociate and the record of the resulting fragments is the basis for establishing the original molecular structures – often unknown. It is fascinating work, similar to that of an archaeologist recombining remnants to restore an original object to its former glory. During this time (the early 1980s), the dominant themes in mass spectrometry were electron ionization, chemical ionization, fast atom bombardment (FAB) and secondary ion mass spectrometry (SIMS), along with the first experiments with glass capillary columns directly coupled to mass spectrometers. In my dissertation, I studied fragmentation patterns and stereo-specific effects of pyridine and piperidine derivatives.

A wonderful new world

The end of my PhD studies saw a new chapter in my life begin; I fell in love, got married, and gave birth to my first daughter, Juliana. My husband was Colombian and so, at the beginning of the 1980s, I found myself a newly graduated resident of Colombia. A tropical country in the northeast of South America, Colombia has beautiful cordilleras with high and imposing mountains, wide rivers, mighty forests and expansive savannas. I came to a city surrounded by mountains, with a difficult name for a foreigner to pronounce – Bucaramanga. There, I had my second daughter, Laura, and in 1983,  began my work at the Industrial University of Santander (UIS), a public university with over 20,000 students, and a strong emphasis on engineering.

In vivo sampling of flower scent with solid-phase micro extraction.
The Passiflora edulis flower.
Visiting Tumaco (SW Colombia) to learn about ethnobotanical uses of aromatic plants.
“When I arrived in Colombia, almost everything was new and different to me: people, the language, customs, tastes, and lush tropical vegetation.”

When I arrived in Colombia, almost everything was new and different to me: people, the language, customs, tastes, and lush tropical vegetation. These days, we have access to the Internet, which allows us to travel ‘digitally’ and learn about places, their histories and customs, before ever setting foot there. But back then, a lot of things were a surprise to me. There are so many plant species – so different, rare and opulent, that a lifetime would not be enough to learn them all. Huge, hairy insects with hundreds of legs frequently enter the house without permission (and used to terrify me). There are no seasons: plants sprout and flower throughout the year. The sun rises at 6 am; at 6 pm, after a short twilight, the darkness comes. People usually go to bed early and get up very early; some classes at the university start at 6 am, when in Russia I would still be asleep. Celebrating Christmas and New Year without snow, among exuberant flowers – and in 30 °C heat – was very strange to me at first.

Students at the university, not much accustomed to foreign teachers with funny accents, were friendly, curious and very patient with my then-limited Spanish. To live in a foreign country, to understand it and to love it, you have to understand its history, geography, literature, music, and culture in general. I traveled a lot through Colombia, studying its history and customs, and fell in love with Colombian literature, especially the magical realism represented by the great Nobel Prize Laureate Gabriel García Márquez.

Science against the odds

I traveled back to my home country in 1984 to carry out doctoral studies at the University of Moscow. After graduating in the field of instrumental analysis, with emphasis on mass spectrometry, I returned to Bucaramanga, and the Industrial University of Santander, at the beginning of 1989. At this time, I could not even dream of continuing my research in mass spectrometry: in the whole country, there were only two mass spectrometry instruments and only a few chromatographs. Many students and teachers were skeptical about the possibilities of doing good science in Colombia. Equipment, infrastructure, and people with ideas and know-how are all required to develop science. The 1990s were difficult in Colombia: economically complicated, and with very little investment in education. The country was suffering at the hands of drug traffickers, who created a lot of insecurity, along with several insurgent and paramilitary groups; massacres and kidnappings were everyday news. It was a time of anxiety and distress, which led many Colombians to emigrate. The best students got their scholarships and left the country to study in the United States, Canada or Europe; most did not return to Colombia. Funding universities was not seen as a priority. Even now, there are scant resources for scientific research: in Colombia, less than 0.3 percent of GDP is devoted to the development of science and technology – much less than other Latin American countries (Brazil, Mexico, Chile, Argentina), not to mention developed countries.

Collecting plant materials.
Our research center.
Volatile substances are critical in the plant–insect dialog.

The bad state of affairs led students, and even some professors, to say that it was not possible to do science in Colombia. But I felt they were wrong. My ideal and example in life has been the Polish–French scientist Marie Skƚodowska Curie, a model of dedication, scientific rigor and vehemence. With my hero in mind, I plucked up my courage, boldness and stubbornness to defy the status quo. I would not accept that it was impossible to do good basic science and good instrumental analytical chemistry in a developing country. It became my challenge and ambition to create a cutting-edge research center, to train PhD students within Colombia, and to contribute to the development of its science. Without good basic science, it would be impossible to develop the country’s industry and technology, let alone innovate. Fortunately, there were several young professors who shared my conviction and joined me on my mission.

Biodiverse = paradise

When I left Moscow, I knew the names of almost all trees, flowers, insects, and animals there. At school, I used to study black and white photos in geography books, depicting strange plants and animals – anacondas and anteaters, capybaras and cacti, hallucinogenic fungi and poisonous jellyfish. I never imagined that in a few years I would be seeing many of these wonders for myself, living and working in one of the most biodiverse tropical countries in the world.

At first, I tirelessly interrogated people about the names of plants and their uses, but I soon came to realize that it is impossible to know the name of every plant in a country where there are more than 5,000 species per 10,000 m2. During the period of the Spanish conquest, there was much ignorance (and therefore distrust) of native flora and fauna. The conquistadors brought with them many plants from their homeland, including aromatic and medicinal plants (rue, chamomile, basil, marjoram, anise, rosemary), food crops (rice, sugar cane, various cereals, apples, plum, citrus fruits, carrots, peas, beets), ornamental and ‘stimulant’ plants (coffee, tea). The New World, in its turn, gave to the Old World tobacco, corn, potatoes, tomatoes, blackberries, beans, cassava, rubber, cinchona trees, vanilla, cocoa, and other plant species of great economic importance. Nevertheless, most industrial crops in Colombia (palm oil, sugar cane, rice, sorghum, citrus, coffee) are introduced species. Unfortunately, Colombia’s native plants have occupied a more modest place in the economy and in science, and have not been studied with due attention.

So when I asked myself, “How can I apply my analytical chemistry research here in Colombia?” the answer arose naturally: biodiversity, native plants and their metabolites. By expanding our knowledge about native plants, I believe we can find ways to harness them (wisely and sustainably), by creating products based on their oils and extracts. That was the start of a path I have followed for almost three decades.

Slow and steady wins the race

We started with small projects, funded by government science agency COLCIENCIAS. In 1993, after more than three years of waiting, we bought our first pieces of analytical equipment from Hewlett-Packard: a HP 5890 GC and a HP 5972 GC with mass selective detector, both still working until recently. With these tools, we were admitted into the kingdom of tropical plant research, studying the secondary metabolites behind their intriguing characteristics.

Gradually, with funding from the Colombian government, COLCIENCIAS, and the Ministry of Agriculture, as well as the funds we had generated ourselves by running courses and providing analytical services, we began to stock the research center with the necessary tools: extraction equipment, analytical instruments, gas chromatographs with various detectors (TCD, FID, ECD, NPD, FPD), liquid chromatographs with different detection systems (UV-Vis, DAD, FLD, ELSD), GC-MS and LC-MS systems, low-resolution (Q) and high-resolution (rTOF, Orbitrap) mass spectrometers and tandem systems (QqQ). Initially, we focused on GC-MS for analysis of essential oils, but today we largely employ LC-MS analysis to study polar molecules, flavonoids, alkaloids, anthocyanins, and other metabolites of native plants.

By expanding our knowledge about native plants, I believe we can find ways to harness them
Our research group, with over 50 members.
Collecting plant materials in the field.
Essential oils.
Moringa oleifera flower fragrance sample in vivo.
Flowers change their volatile profile upon pollination.

Case Studies

Playing Chicken

A few years ago there was a spate of chicken fatalities in local poultry farms, and we set out to find the cause. It looked like the deaths were caused by toxic contaminants in food; however, standard analytical procedures to test for organo-chlorine and organo-phosphorous pesticides found nothing in the sorghum-based chicken feed used in the farms. Tests for aflatoxins and pathological bacteria also came up negative. We subjected the sorghum grain to various extraction procedures, before concentrating the grain extract tenfold and analyzing it with GC-MS, which showed the presence of fatty acids characteristic of sorghum, and indole traces. We had recently installed a GC-MS instrument equipped with the pulsed-splitless injection mode, which reduces the residence time of the injected sample at high (250 °C) temperatures. The analysis of the same extract, using pulsed-splitless injection, discovered a pyrrolizidine alkaloid. These alkaloids are thermolabile and had not been detected in previous analyses because they decompose at the injection port. Close examination of the sorghum grains revealed that around four percent were intruder grains - the same size, but a different color and shape. They were carefully separated and subjected to soxhlet (CH2Cl2) and SFE extractions. GC-MS analysis of these extracts showed the presence of indole and monocrotaline, a very harmful pneumotoxic and hepatotoxic compound. The intruder seeds were planted in our experimental garden and were botanically identified as Crotalaria retusa. The solution was simple: farmers were warned to eliminate Crotalaria from their sorghum crop before processing the grains for chicken food.

The Sweet Smell of Success

The chemistry of flowers and their volatile substances is a particular passion for me. Flowers showcase the power of evolution and play diverse roles in plant physiology. Their main responsibility is to perpetuate the plant and they use various strategies to attract pollinators: different forms and colors, rising temperature, and release of volatiles. The emission rate and the amount and type of substances released change with the time of day, light, and whether the plant has already been pollinated. It takes weeks or months for a synthetic organic chemist to produce a single compound, but a flower can synthesize tens of them in a few minutes. What’s more, the substances released by different flower parts (e.g., petals, sepals, feminine and masculine organs) are not the same. Determining these profiles constitutes an important part of our analytical studies, required by biochemists, entomologists, ecologists and biologists who study pollination and plant fertilization. Extraction techniques such as purge and trap, and solid-phase microextraction (SPME), combined with comprehensive chromatography (GCxGC), play a fundamental role in our studies. We are also studying the relationship between flower color and the antioxidant activity of flower extracts, and we have already found a clear correlation between red color and superior antioxidant activity.

The Essentials

Essential oil production requires large volumes of vegetation. Typically, around 1 kg of essential oil is obtained from 100 kg of biomass. Many of our projects in the last 10 years have been geared to production of the required biomass in the countryside by small farmers’ associations. During the projects, the farmers learn how to cultivate aromatic plants, while adhering to clean agricultural practices, plus how to carry out post-harvest tasks and obtain the essential oil using stills designed by our group and the School of Mechanical Engineering. Lemongrass, citronella, mountain oregano (Lippia origanoides), rosemary, prontoalivio (Lippia alba), damiana, palmarosa, sage and ylang-ylang are some of the species included. These pilot production projects are particularly important because they provide an alternative to coca plantations, which have declined notably in recent years. Many aromatic plants provide three or four harvests per year and the resulting essential oil is an added-value, small-volume product that can be used in many sectors of the economy. It is an example of the application of basic scientific research to benefit populations that have had fewer development opportunities. Once the essential oils have been characterized, the knowledge of their biological and physicochemical properties permits the design of products such as mouth washes, moisturizing creams and oils, massage oils, insect repellents, and many more. Thus, through the study of the plant’s chemistry we arrive at functional products based on natural ingredients. In turn, the production of these ingredients enhances the living standards of the farmers and their families.

In 1998, we started the National School for Chromatography and Related Techniques, to provide training to anyone who wanted to learn how to use chromatography and mass spectrometry, and apply them in their own field. Since that time, more than 250 courses have been given to about 2,500 people. The Research Center also offers analytical services to Colombian industry, to projects developed by other researchers, or to governmental control entities (food, environmental, forensic). It’s a different facet of work, requiring a high degree of rigor and responsibility to ensure confidence in the analytical results obtained. We obtained the Accreditation of the Laboratory Quality System 10 years ago. The funds from analytical services are reinvested in financing theses, scholarships, the purchase of reagents, accessories, equipment and their maintenance, as well as for participation in international symposia and conferences.

In late 2004, several research groups in the country came together to develop a national program for the study of biodiversity in Colombia; in particular, tropical plants. Chemists, microbiologists, plant physiologists, botanists, agronomists, biochemists, and chemical, mechanical and industrial engineers have all participated in this program. The study of plants and their metabolites needs a multidisciplinary team, as it requires an understanding of both behavior and chemistry. Together, we must interpret the ‘language’ of the plants, learn their ‘temperament’, and assess their relationships with other plants or insects, their metabolism and adaptation strategies.

The Research Center of Excellence CENIVAM (Research Center for the Agro-Industrialization of Tropical Medicinal and Aromatic Vegetal Species) was created at the beginning of 2005, dedicated to the study of tropical plants and their agro-industrialization. This government-supported project has proved very successful, bringing together more than 150 researchers (including undergraduate and graduate students, professors, and young researchers) from 20 different research groups of 10 universities in the country, coordinated by our Research Center for Biomolecules here at the Industrial University of Santander.

A Chemist’s Best Friend

Loyal and caring, dogs have accompanied mankind throughout history. Unfortunately, many dogs live on the streets of Colombian cities in miserable conditions. They barely survive. Although government and non-government organizations exist to protect them, they are not enough. We have developed an interesting program with our students that aims to help.

We have helped to find homes for many of the stray dogs that have come to our university. In fact, a few of them have remained and live in the Institute; they are part of the staff, and students, professors and researchers contribute to provide their food, care and veterinary needs. The dogs go to classes; always punctual, they sit in the front row and participate in their own way in our seminars. We also allow graduate students to bring their own dogs into the Institute, so they are not preoccupied by leaving their pets home alone all day.

Some people would say that it is impossible to have dogs in the laboratory, in the library, or in class – especially dogs that came from the streets. But our dogs have dramatically changed the work atmosphere. They have introduced a sense of harmony, relaxation and positive energy, with their wagging tails, happy disposition, and quickness to thank, love, and forgive. They have taught our heterogenous group of students, researchers and office workers a lot. Dogs act as emotional buffers, sometimes even as lightning rods. It has been an amazing experiment, a twist of socio-biology. To me it feels natural that we, who study biodiversity, share our work environment not only with a wide range of plants, but also our canine friends, who engender unity, cooperation, and positive emotions.

Essential beauty

The study of biodiversity begins with botanical expeditions and the taxonomic identification of the plant species collected. More than 1,200 botanical samples have been collected and taxonomically identified (by the Colombian National Herbarium at Bogota), during more than 30 trips to different regions in Colombia. Back at the laboratory, we carry out extractions to obtain volatile fractions, essential oils, supercritical extracts, and hydro-alcoholic extracts, which are tested for biological activity. Around 700 essential oils and 500 extracts (obtained with ethanol–water blend or supercritical CO2) have been derived from the collected plants, many of them never before studied.

The secondary metabolites of the plants are highly complex mixtures. Their constituents have different volatilities, polarities, and concentrations; their extraction requires the use of various techniques, headspace, solid-phase microextraction (SPME), purge & trap (P&T), steam distillation or hydrodistillation, solvent or supercritical fluid extraction, or matrix solid-phase dispersion. Analysis of the fractions, oils or extracts obtained requires the use of gas chromatography, liquid chromatography and detection systems with high-resolution mass-spectrometric analyzers. After their characterization, essential oils and extracts are sent to different laboratories to study their biological properties. After performing some 5,500 assays, around 45 percent of all oils or extracts tested positive for one of the biological properties examined (antibacterial, antiviral, antifungal, anti-inflammatory, antigenotoxic, photoprotective, among others).

Research in my group follows several avenues. One is the study of essential oils, their production, their physicochemical analysis, and the determination of their biological properties. Despite the rich botanical diversity in Colombia, most essential oils are currently imported. Rural Colombia remains wedded to traditional agriculture, with little technological advancement or innovation. By strengthening the alliance between the university (know-how), business (technological capacity) and countryside (biodiversity) we hope to develop the natural ingredients industry in Colombia. Eventually, essential oils and extracts may not only supply the internal demand, but could also be exported. We are also making final products based on essential oils and extracts, including air fresheners, repellents, creams, mouthwashes, soaps, and antiseptic gels.

Of particular note is our natural insect repellent, which contains essential oils that we are already producing with a group of farmers. It is a very important product, especially now, with the rise of dengue, Zika, and chikungunya, all transmitted by the Aedes aegypti mosquito. We have patented a number of our discoveries, including a mobile essential oil still for field use, and the biotransformation of citronellol to hydroxycitronellol by means of a fungus. Another eight patent requests have been filed recently.

Other lines of research include the isolation and identification of toxic alkaloids in tropical plants, plus the study of polyphenolic compounds (flavonoids, anthocyanins) in tropical flowers and their antioxidant activity. I find the study of tropical flowers particularly fascinating. Flowers use many strategies to attract pollinators – extremes of shape and color, sweet nectar, and intense and constantly-changing fragrances. They can even vary their temperature and color over the course of the day. This wonderful complexity means that the study of flower metabolomics requires an ingenuous combination of extraction methods and highly sensitive analytical techniques. High-resolution technologies (GCxGC, GC-TOF-MS, LC-TOF-MS, and Orbitrap-related techniques), as well as tandem configurations (QqQ, Q-TOF) are needed for the complete and reliable description of secondary metabolites, which can serve as a biologically relevant signal for a pollinator at ppb or even ppt concentrations.

Insect predators or pollinators of plants are worthy of study in their own right, too. It is interesting to discover how some flower secondary metabolites are transferred to insects and can even become defensive tools; for example, some caterpillars devour plant leaves that contain pyrrolizidine alkaloids and so become toxic to their natural enemies. The study of these complex chemical relationships would not be possible without a good base of high-resolution chromatographs and mass spectrometers.

After nearly three decades, it is still exciting to study the biodiversity of tropical plant species through the prism of their chemical constituents.
‘Good science in a developing country’ is not oxymoronic

I am passionate about doing science in a developing country that is building its STEM sector – a country that suffers many socio-economic and political problems, but where young people are eager for knowledge and progress. I believe that by creating laboratories with cutting-edge technology and diverse and modern extraction systems, we are contributing to an exciting and growing science economy in Colombia.

Curiosity and motivation are both needed to push research forward in such settings – something that university teachers must inculcate in students. More than 300 undergraduate students have completed their program at CENIVAM in recent years, along with 50 Master’s degrees and 20 PhDs. In my classes, I try to convey my message in an entertaining way and pass on my love of the field. Having more ‘fans’ of instrumental analytical chemistry is very important, because this discipline permeates so many fields of science – medicinal chemistry, forensic, environmental, food chemistry, natural products, petroleum chemistry, geochemistry, and many more.

After nearly three decades, it is still exciting to study the biodiversity of tropical plant species through the prism of their chemical constituents – the products of their secondary metabolism. And discovering – through chemical analysis – a plant’s structures, origins and functions remains a great challenge. This fantastical journey begins with reliable instrumental chemical analysis, and that makes our work as rewarding as it is important.

Elena Stashenko is Director of the Research Center for Biomolecules at the Industrial University of Santander in Bucaramanga, Colombia.

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About the Author
Elena Stashenko

Elena Stashenko is Director, Research Center for Biomolecules - CIBIMOL Research Center of Excellence, CENIVAM Universidad Industrial de Santander Bucaramanga, Colombia.

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