Development and Validation of an Analytical Method for Determining Citrinin in Red Rice and Red Yeast Rice-Based Food Supplements by UHPLC–MS/MS

Citrinin, a hepato-nephrotoxic mycotoxin produced by fungal species, plays a crucial role in the fermentation of red rice to produce red yeast rice-based food supplements, which represent the primary source of human exposure to this contaminant. A recent study conducted by the University of Valencia and the Public Health Laboratory of Valencia (Spain) developed and validated a simple and sensitive analytical method for the citrinin determination in these products. The extraction process involved a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) step and citrinin determination by ultrahigh-pressure liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS). LCGC International spoke to Olga Pardo Marin and Francesc A. Esteve-Turrillas of the University of Valencia, the corresponding authors of the resulting paper based on this research, about their team’s efforts.

Your paper (1) presents the results of a study determining levels of the mycotoxin citrinin in rice-based food supplements. Why is determining this so important? What are the health ramifications of citrinin in our diet?

The determination of citrinin levels in red-based food supplements is a matter of public health concern due to the nephrotoxic properties of this mycotoxin. Red rice-based food supplements have been identified as the primary source of human exposure to citrinin. This mycotoxin is produced through the fermentation of rice by the Monascus Purpurea fungus strain if conditions are not carefully monitored. Nowadays, these food supplements have gained popularity for their ability to help maintain normal blood cholesterol levels, as they produce monacolin K during the fermentation process. Therefore, determining the levels of citrinin in red-based food supplements allows the assessment of the human exposure to this mycotoxin and the characterization of the resulting risk by comparing it with the level of no concern for nephrotoxicity, which is set at 0.2 µg/kg bw/day (micrograms per kilogram of body weight per day).

In addition to nephrotoxicity, citrinin has associated with various harmful effects on human health, including human genotoxicity, embryotoxicity, teratogenicity and carcinogenicity.

Are mycotoxins a serious health concern in the food chain?

Certainly. Mycotoxin contamination remains a significant food safety concern. The presence of mycotoxins in food and feed can lead to adverse health effects in both humans and animals, ranging from acute poisoning to long-term consequences such as immune deficiency and cancer. According to the FAO (Food and Agriculture Organization of the United Nations), around 25% of the world’s agricultural produce is contaminated with mycotoxins. This indicates that mycotoxins are prevalent in foods if they are not stored under favorable conditions, including appropriate levels of moisture, water activity, and temperature. According to data published by the Rapid Alert System for Food and Feed in 2020, mycotoxins accounted for the highest number of reported incidents among toxic substances.

Are there multiple fungal species that produce mycotoxins in various natural food products, for example peanuts, corn, and other grains?

Several fungi can produce mycotoxins on agricultural products during harvest or in postharvest. The most prevalent mycotoxins found in food commodities are aflatoxins and ochratoxins, produced by Aspergillus species, ochratoxins and patulin produced by Penicillium, and fumonisins, deoxynivalenol, and zearalenone produced by Fusarium species. Certain mycotoxins are produced by more than one fungal species, while some fungi can produce more than one mycotoxin. Furthermore, an infected substrate can contain more than one mycotoxin.

Why was UHPLC–MS/MS your technique of choice in your analysis?

UHPLC–MS/MS technique offers a highly promising system for separation and determination of mycotoxins. UHPLC provides the advantages of chromatography applications, such as shorter columns and higher flow rates, which enhance speed, resolution, and sensitivity. On the other hand, the main advantage of MS/MS is the high sensitivity offered and the unambiguous identification of target analytes.

Briefly state your findings in this study.

In this study, the development and validation of a simple and sensitive analytical method for determining citrinin in red rice and food supplements based on red yeast rice is presented, offering a rapid and straightforward procedure. The experimental procedure involves QuEChERS extraction followed by UHPLC–MS/MS determination of citrinin, with no need for using immunoaffinity sorbents, thus reducing analysis time, cost and environmental impact associated with the analytical procedure. The proposed method was validated, fulfilling the performance criteria established by the European Commission for the analysis of citrinin in red rice based-food supplements. Therefore, it is a useful methodology for routine use in official control purposes.

Do your findings correlate with what you had hypothesized?

Yes. The use of UHPLC–MS/MS allows a rapid, selective, and sensitive determination of citrinin that allows monitoring its levels in target samples, such as red rice and red yeast rice supplements. Furthermore, the use of an abbreviated QuEChERs method for the extraction of citrinin from foods allows a simple and fast sample treatment with a reduced impact on the environment, since it reduces the number of steps of the analytical process, and with it, the consumption of reagents, disposable materials, and energy. Moreover, the proposed analytical methodology is compatible with the analysis of other mycotoxins in foods, so it can be implemented in the development of multitoxin determination.

Was there anything particularly unexpected that stands out from your perspective?

The unexpected finding was that the use of a QuEChERS extraction kit, containing 4 g MgSO₄ anhydrous, 1 g NaCl, 1 g trisodium citrate dihydrate, and 0.5 g of disodium hydrogen citrate sesquihydrate followed by a 40-fold dilution provided a clean extract for the determination of citrinin in red rice and red yeast rice supplements, effectively mitigating the matrix effects and avoiding any additional purification step.

Were there any limitations or challenges you encountered in your work?

The main challenge was to find blank red yeast rice supplement samples, crucial to perform the validation of the developed method at the low levels raised. In any case, the proposed methodology provides accurate and precise results at common citrinin levels.

What best practices can you recommend in this type of analysis for both instrument parameters and data analysis?

To maximize the MS/MS signal for citrinin, it is fully recommended to optimize the ion source parameters and to select the citrinin-methanol adduct [M + MeOH-H]⁻ (280.8 m/z) as precursor ion, because it enhances the analyte response and improves the method sensitivity. Regarding data analysis, the use of conventional software for the analysis of chromatographic data provides fast and accurate results in a simple and systematized way. Moreover, the use of internal standard calibration provides an additional level of reliability, since it ensures the quality of the result obtained for each sample analyzed.

The study specifically targeted red rice. Am I correct in presuming that is because there’s something in the red pigment that makes those beans rich in citrinin? Are there any other natural foods or minerals that you think might benefit from this sort of analysis?

The key question is not the red pigment, but the fermentation of rice by the Monascus Purpurea fungus strain if conditions are not carefully monitored. It is presumed the applicability of this analytical methodology to foodstuffs in general. However, future research will be addressed to check this.

Similarly, can this technique be used to detect other specific mycotoxins in other foods?

Preliminary studies made in our research team have check that the developed procedure is also adequate for the analysis of other food matrix. However, it would require of a specific validation process for each sample type. Regarding the analysis of other mycotoxins, the proposed analytical methodology is based on a QuEChERS extraction, which is widely employed in the literature for the analysis of other mycotoxins in foods, so it can be easily implemented in current analytical procedures for multitoxin determination.

Can you please summarize the feedback that you have received from others regarding this work?

Some authors have been interested in the applicability of this methodology for official control purposes and in monitoring levels of citrinin in a wide number of red rice and red rice-based supplements, to evaluate the prevalence and the exposure of the population. Other researchers have asked us about the applicability of this methodology for the analysis of different foodstuffs.

What are the next steps in this research and are you planning to be involved in improving this technology?

Citrinin occurs mainly in stored grains but also in other products such as olives, apples, spices, fruit and vegetable juices, beer, cheese, infant formulae, and dry meat products, such as fermented sausages. Therefore, the next step is to check the applicability of the developed methodology to the analysis of foodstuffs in general.

Are there other researchers or regulatory groups that are concerned about the issue of mycotoxins in foods?

Nowadays, it is commonly accepted that the presence of mycotoxins in food raises issues for public health, as they may be involved in the onset and maintenance of several illnesses, physiological alterations, and dysfunctions. Therefore, numerous international institutions, such as FAO or European Commission have recognized the potential health risks associated with citrinin exposure and have taken measures to address the issue by establishing regulatory limits. The European Commission set a maximum limit for citrinin in food supplements based on fermented red yeast Monascus Purpurea at 100 μg/kg. Research in the field of mycotoxins is of increasing interest due to several challenges such as the consequences of climate change for agriculture, namely elevated temperatures, increased CO₂concentrations, and drought stress or the global shortage of grain due to the Ukraine war. The interdisciplinary scientific community is challenged to develop solutions for all these issues to produce enough high-quality grain and grain products without significant mycotoxin contamination to feed the world with a population of more than 8 billion people.

References

1. Ponz-Perelló, P.; Esteve-Turrillas, F. A.; Cortés, M. Á.; Herranz, J.; Pardo, O. Development and Validation of an Analytical Method for Determination of Citrinin in Red Rice and Red Yeast Rice-Based Food Supplements by Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry. Food Chem. 2024, 455, 139941. DOI: 10.1016/j.foodchem.2024.139941

Olga Pardo Marín, doctor in Chemistry (University of Valencia), professor of Analytical Chemistry at the University of Valencia. Her research focuses on four main areas: i) Analytical chemistry; ii) Control of food and environmental contaminants; iii) Risk assessment; and iv) Human biomonitoring. She has participated extensively in food contaminant monitoring programs (total diet studies) to evaluate population exposure to food contaminants. Throughout her research career, she has participated in 15 regional, 5 national and 5 international projects funded in competitive calls, acting as principal investigator (PI) or co-PI in6 of them. These projects have resulted in the authorship of 75 research articles in journals indexed in JCR (57 as lead/last/corresponding author). She has collaborated with institutions in Spain, Colombia, Chile, and Indonesia, as well as with the EFSA. Her scientific production accumulates more than 1800 citations, with an h index of 27. She has directed two doctoral theses on the evaluation of exposure to contaminants through diet in the population of the Valencian Community and on exposure and risk to organic contaminants persistent through the evaluation of internal exposure (biomonitoring) in the population of the Valencian Community. She has also coordinated more than 20 Final Master's Projects and has completed an international stay in the Netherlands. The main achievements of her career have been the creation of the Analytical Platform for the study of Exposome in FISABIO, the implementation of different biomonitoring programs to evaluate internal exposure to environmental chemicals in the population of the Valencian Community, and the development of strategies to evaluate the risk derived from internal or external exposure to contaminants, an essential tool to prioritize public health measures. Photo courtesy of Marin.

Francesc A. Esteve Turrillas has earned a Degree in Chemistry (2000), a Degree in Food Science and Technology (2004), and a Ph.D. in Chemistry with European mention (2006) at the University of Valencia. During his graduate and postgraduate training, he was awarded with several fellowships (2 collaborative and 1 pre-doctoral) and 4 post-doctoral contracts (including “Juan de la Cierva” program of the Spanish Ministry of Education and Science and “JAE-doc” program from the Spanish National Research Council) to support his work. His research has been performed mainly at the Department of Analytical Chemistry of the University of Valencia and at the Institute of Agricultural and Food Technology of the Spanish National Research Council (IATA-CSIC). Additionally, he has performed research stays in relevant research groups in Dr. John R. Dean’s group at Northumbria University (Newcastle-upon-Tyne, United Kingdom), Dr. Wolfgang Lindner’s group at University of Vienna (Vienna, Austria), Dr. Graham Mills’ group at University of Portsmouth (Portsmouth, United Kingdom), Dr. Alfredo Sanz-Medel’s group at University of Oviedo (Oviedo, Spain), Dr. Hedi Mattoussi’s group at Florida State University (Tallahassee, FL, USA), and Dr. Justyna Płotka-Wasylka’s group at the Gdańsk University of Technology (Gdasnk, Poland). He has joined as researcher and participated in 19 competitive research projects, including one international, six of the National Program of Research of the Spanish Ministry, and several autonomic projects, as well as transference projects with private companies. The main results from his studies have been published in 116 papers (100 in the Q1) in refereed journals included in the Science Citation Index in Analytical Chemistry, Food Science and Technology, and Environmental Sciences areas; being the first author of 39 articles and the corresponding author of 31 of them. His work has generated a total of 2766 citations and he has an h−index of 28 (Scopus). In addition, he has edited one handbook in John Wiley & Sons and published 11 book chapters, he has contributed to more than 90 communications in national and international scientific meetings, and he has two inventions protected by international PCT patents. He is currently associate professor at the University of Valencia, teaching in Degree in Chemistry and Master Degree in Experimental Techniques in Chemistry, he has codirected more than 65 undergraduate students of Chemistry, Pharmacy and Biotechnology degrees and Master Degree in Experimental Techniques in Chemistry. He has been the codirector of two PhD thesis and he is currently the codirector of three more. Throughout his scientific and teaching career he has obtained three research six-year periods and three teaching five-years periods. Since 2021, he is the head of the Master Degree in Experimental Techniques in Chemistry of the University of Valencia. Photo courtesy of Turrillas.