Quality control, safety assessment and preparation approaches of low molecular weight heparin.

Low Molecular Weight Heparins (LMWHs) are well-established for use in the prevention and treatment of thrombotic diseases, and as a substitute for unfractionated heparin (UFH) due to their predictable pharmacokinetics and subcutaneous bioavailability. LMWHs are produced by various depolymerization methods from UFH, resulting in heterogeneous compounds with similar biochemical and pharmacological properties. However, the delicate supply chain of UFH and potential contamination from animal sources require new manufacturing approaches for LMWHs. Various LMWH preparation methods are emerging, such as chemical synthesis, enzymatic or chemical depolymerization and chemoenzymatic synthesis. To establish the sameness of active ingredients in both innovator and generic LMWH products, the Food and Drug Administration has implemented a stringent scientific method of equivalence based on physicochemical properties, heparin source material and depolymerization techniques, disaccharide composition and oligosaccharide mapping, biological and biochemical properties, and in vivo pharmacodynamic profiles. In this review, we discuss currently available LMWHs, potential manufacturing methods, and recent progress for manufacturing quality control of these LMWHs.

Qatar's National Mental Health Survey-The World Mental Health Qatar: Sampling design, instrument adaptation, quality control, and fielding procedures.

OBJECTIVES: The World Mental Health Qatar (WMHQ) study, the first national general population mental health survey in Qatar, was conducted as part of the World Health Organization (WHO) World Mental Health (WMH) Survey Initiative. It was one of the few WMH survey conducted during the COVID-19 pandemic. This paper presents the methodological advances and challenges encountered while conducting the survey by telephone during the pandemic. METHODS: Disproportionate stratified sampling using a national-level cellular telephone frame selected a representative sample of Arabic-speaking adults. Participants were initially contacted via Short Message Service text, followed by telephone interviews. WMH training materials supported a comprehensive training program, and data quality was ensured through a quality control indicator system and extensive monitoring. RESULTS: Over 234 days, 5195 interviews in Arabic were completed, averaging 77 min each. In line with Qatar's population, the majority of participants were non-Qatari residents living in Qatar (72.2%). CONCLUSIONS: A distributed remote Computer Assisted Telephone Interviewing system facilitated centralized quality monitoring and data security. However, the pandemic intensified challenges such as remote management of interviewer productivity, low response rates, and rising survey costs. The findings will inform Qatar's mental health policymakers, and the strategies used to address these challenges offer valuable insights for researchers worldwide.

Quality assessment of oral antimalarial and antiretroviral medicines used by public health systems in Sahel countries.

Malaria and Human Immunodeficiency Virus infections are among the top 10 causes of death in low income countries. Furthermore, many medicines used in these treatment areas are substandard, which contributes to the high death rate. Using a monitoring system to identify substandard and falsified medicines, the study aims to evaluate the quality of antimalarial and antiretroviral medicines in Sahel countries, assessing site conditions, compliance of medicines with pharmacopoeia tests, formulation equivalence with a reference medicine, and the influence of climate on quality attributes. Ultra Performance Liquid Chromatography methods for eight active pharmaceutical ingredients were validated following the International Conference for Harmonization guideline for its detection and quantification. Quality control consists of visual inspections to detect any misinformation or imperfections and pharmacopeial testing to determine the quality of pharmaceutical products. Medicines which complied with uniformity dosage units and dissolution tests were stored under accelerated conditions for 6 months. Artemether/Lumefantrine and Lopinavir/Ritonavir formulations failed uniformity dosage units and disintegration tests respectively, detecting a total of 28.6% substandard medicines. After 6 months stored under accelerated conditions (40 °C // 75% relative humidity) simulating climatic conditions in Sahel countries, some medicines failed pharmacopeia tests. It demonstrated the influence of these two factors in their quality attributes. This study emphasizes the need of certified quality control laboratories as well as the need for regulatory systems to maintain standards in pharmaceutical manufacturing and distribution in these countries, especially when medicines are transported to rural areas where these climatic conditions are harsher.

Phase I quality control framework for monitoring organ-at-risk dose.

Objective.The aim of this work was to develop a Phase I control chart framework for the recently proposed multivariate risk-adjusted Hotelling'sT2chart. Although this control chart alone can identify most patients receiving extreme organ-at-risk (OAR) dose, it is restricted by underlying distributional assumptions, making it sensitive to extreme observations in the sample, as is typically found in radiotherapy plan quality data such as dose-volume histogram (DVH) points. This can lead to slightly poor-quality plans that should have been identified as out-of-control (OC) to be signaled in-control (IC).Approach. We develop a robust iterative control chart framework to identify all OC patients with abnormally high OAR dose and improve them via re-optimization to achieve an IC sample prior to establishing the Phase I control chart, which can be used to monitor future treatment plans.Main Results. Eighty head-and-neck patients were used in this study. After the first iteration, P14, P67, and P68 were detected as OC for high brainstem dose, warranting re-optimization aimed to reduce brainstem dose without worsening other planning criteria. The DVH and control chart were updated after re-optimization. On the second iteration, P14, P67, and P68 were IC, but P40 was identified as OC. After re-optimizing P40's plan and updating the DVH and control chart, P40 was IC, but P14* (P14's re-optimized plan) and P62 were flagged as OC. P14* could not be re-optimized without worsening target coverage, so only P62 was re-optimized. Ultimately, a fully IC sample was achieved. Multiple iterations were needed to identify and improve all OC patients, and to establish a more robust control limit to monitor future treatment plans.Significance. The iterative procedure resulted in a fully IC sample of patients. With this sample, a more robust Phase I control chart that can monitor OAR doses of new plans was established.

Research on the Development of Methods for Detection of Substandard and Falsified Medicines by Clarifying Their Pharmaceutical Characteristics Using Modern Technology.

The existence of substandard and falsified medicines threatens people's health and causes economic losses as well as a loss of trust in medicines. As the distribution of pharmaceuticals becomes more globalized and the spread of substandard and falsified medicines continues worldwide, pharmaceutical security measures must be strengthened. To eradicate substandard and falsified medicines, our group is conducting fact-finding investigations of medicines distributed in lower middle-income countries (LMICs) and on the Internet. From the perspective of pharmaceutics, such as physical assessment of medicines, we are working to clarify the actual situation and develop methods to detect substandard and falsified medicines. We have collected substandard and falsified medicines distributed in LMICs and on the Internet and performed pharmacopoeial tests, mainly using HPLC, which is a basic analytic method. In addition to quality evaluation, we have evaluated the applicability of various analytic methods, including observation of pharmaceuticals using an electron microscope, Raman scattering analysis, near-IR spectroscopic analysis, chemical imaging, and X-ray computed tomography (CT) to detect substandard and falsified medicines, and we have clarified their limitations. We also developed a small-scale quality screening method using statistical techniques. We are engaged in the development of methods to monitor the distribution of illegal medicines and evolve research in forensic and policy science. These efforts will contribute to the eradication of substandard and falsified medicines. Herein, I describe our experience in the development of detection methods and elucidation of the pharmaceutical status of substandard and falsified medicines using novel technologies.

MobiLipid: A Tool for Enhancing CCS Quality Control of Ion Mobility-Mass Spectrometry Lipidomics by Internal Standardization.

Ion mobility-mass spectrometry (IM-MS) offers benefits for lipidomics by obtaining IM-derived collision cross sections (CCS), a conditional property of an ion that can enhance lipid identification. While drift tube (DT) IM-MS retains a direct link to the primary experimental method to derive CCS values, other IM technologies rely solely on external CCS calibration, posing challenges due to dissimilar chemical properties between lipids and calibrants. To address this, we introduce MobiLipid, a novel tool facilitating the CCS quality control of IM-MS lipidomics workflows by internal standardization. MobiLipid utilizes a newly established DTCCSN2 library for uniformly (U)13C-labeled lipids, derived from a U13C-labeled yeast extract, containing 377 DTCCSN2 values. This automated open-source R Markdown tool enables internal monitoring and straightforward compensation for CCSN2 biases. It supports lipid class- and adduct-specific CCS corrections, requiring only three U13C-labeled lipids per lipid class-adduct combination across 10 lipid classes without requiring additional external measurements. The applicability of MobiLipid is demonstrated for trapped IM (TIM)-MS measurements of an unlabeled yeast extract spiked with U13C-labeled lipids. Monitoring the CCSN2 biases of TIMCCSN2 values compared to DTCCSN2 library entries utilizing MobiLipid resulted in mean absolute biases of 0.78% and 0.33% in positive and negative ionization mode, respectively. By applying the CCS correction integrated into the tool for the exemplary data set, the mean absolute CCSN2 biases of 10 lipid classes could be reduced to approximately 0%.

A Practical Guide for the Quality Evaluation of Fluobodies/Chromobodies.

BACKGROUND: Fluorescent proteins (FPs) are pivotal reagents for flow cytometry analysis or fluorescent microscopy. A new generation of immunoreagents (fluobodies/chromobodies) has been developed by fusing recombinant nanobodies to FPs. METHODS: We analyzed the quality of such biomolecules by a combination of gel filtration and SDS-PAGE to identify artefacts due to aggregation or material degradation. RESULTS: In the SDS-PAGE run, unexpected bands corresponding to separate fluobodies were evidenced and characterized as either degradation products or artefacts that systematically resulted in the presence of specific FPs and some experimental conditions. The elimination of N-terminal methionine from FPs did not impair the appearance of FP fragments, whereas the stability and migration characteristics of some FP constructs were strongly affected by heating in loading buffer, which is a step samples undergo before electrophoretic separation. CONCLUSIONS: In this work, we provide explanations for some odd results observed during the quality control of fluobodies and summarize practical suggestions for the choice of the most convenient FPs to fuse to antibody fragments.

Research progress on the quality control of mRNA vaccines.

INTRODUCTION: The mRNA vaccine technologies have progressed rapidly in recent years. The COVID-19 pandemic has accelerated the application of mRNA vaccines, with research and development and clinical trials underway for many vaccines. Application of the quality by design (QbD) framework to mRNA vaccine development and establishing standardized quality control protocols for mRNA vaccines are essential for the continued development of high-quality mRNA vaccines. AREAS COVERED: mRNA vaccines include linear mRNA, self-amplifying mRNA, and circular RNA vaccines. This article summarizes the progress of research on quality control of these three types of vaccines and presents associated challenges and considerations. EXPERT OPINION: Although there has been rapid progress in research on linear mRNA vaccines, their degradation patterns remain unclear. In addition, standardized assays for key impurities, such as residual dsRNA and T7 RNA polymerase, are still lacking. For self-amplifying mRNA vaccines, a key focus should be control of stability in vivo and in vitro. For circular RNA vaccines, standardized assays, and reference standards for determining degree of circularization should be established and optimized.

Analytical strategies to identify multicomponent quality markers for commercial Hua-ju-hong using multidimensional chemical analysis.

Hua-ju-hong (HJH) is a Chinese medicinal material obtained from Citrus grandis 'Tomentosa' (CGT) and Citrus grandis (L.) Osbeck (CG) with various commercial specifications. It is known for relieving cough and dispelling phlegm. To reveal the quality marker for distinguishing the various HJH, 215 batches of commercial HJH were studied systematically using multidimensional chemical analysis. Ten major components were identified by high-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry and quantified via high-performance liquid chromatography coupled with diode array detection. In this study, a rapid, efficient, and low-cost chromatographic method was established. Total coumarin-hemiterpene and total coumarin-monoterpene were first classified and analyzed in HJH. The result indicated that the main component, naringin, was not the quality marker for differentiating CGT from CG. For reflecting the unique medicinal and food value of HJH, coumarins should be the more potential quality markers. Flavonoids were the possible quality markers for distinguishing two growth stages of fruit-exocarp and young fruit. For the first time, two chemotypes of HJH were identified in CG. This study provides a convenient yet reliant chromatographic method and novel yet systematic strategies for overall quality control of commercial HJH.

[Application of process analysis technology in traditional Chinese medicine manufacturing industry].

In the traditional Chinese medicine(TCM) manufacturing industry, quality control determines the safety, effectiveness, and quality stability of the final product. The traditional quality control method generally carries out sampling off-line testing of drugs after the end of the batch production, which is incomprehensive, and it fails to find the problems in the production process in time. Process analysis technology(PAT) uses process testing, mathematical modeling, data analysis, and other technologies to collect, analyze, feedback, control, and continuously improve the critical quality attributes(CQA) in all aspects of the production of TCM preparations in real time. The application of PAT in the TCM manufacturing industry is one of the research hotspots in recent years, which has the advantages of real-time, systematic, non-destructive, green, and rapid detection for the production quality control of TCM preparations. It can effectively ensure the stability of the quality of TCM preparations, improve production efficiency, and play a key role in the study of the quantity and quality transfer law of TCM. Commonly used PAT includes near-infrared spectroscopy, Raman spectroscopy, online microwave, etc. In addition, the establishment of an online detection model by PAT is the key basic work to realize intelligent manufacturing in TCM production. Obtaining real-time online detection data through PAT and establishing a closed-loop control model on this basis are a key common technical difficulty in the industry. This paper adopted systematic literature analysis to summarize the relevant Chinese and foreign literature, policies and regulations, and production applications, and it introduced the development trend and practical application of PAT, so as to provide references for accelerating the application of PAT in the TCM manufacturing industry, the intelligent transformation and upgrading, and high-quality development of the TCM industry.

[Research on differential quality markers of medicine herbs for Danggui Buxue Decoction before and after processing based on fingerprint and network pharmacology].

The different quality markers of Danggui Buxue Decoction before and after processing were studied based on fingerprint and network pharmacological research, and the seven screened index components were quantitatively analyzed, so as to provide an experimental basis for the quality evaluation of Danggui Buxue Decoction before and after processing. HPLC method was used to establish fingerprints of Danggui Buxue Decoction before and after processing, and a multivariate statistical method was used to analyze the cha-racteristic maps and common peak areas of Danggui Buxue Decoction before and after processing. The different characteristic components before and after processing were screened out, and related targets and pathways of their different components were constructed based on network pharmacology. Their components were quantitatively analyzed. A total of 13 common peaks were identified in the fingerprint of the Danggui Buxue Decoction sample, and seven main chemical components were identified, with similarity > 0.911. Further cluster analysis, principal component analysis, and partial least squares discriminant analysis were used to distinguish raw and processed products. According to VIP value, the main difference components 1, 2, 6, 13, and 5 of Danggui Buxue Decoction before and after processing were screened. By combining the "five principles" of TCM Q-marker and network pharmacology, 5-hydroxymethylfurfural, ferulic acid, calycosin-7-O-ß-D-glucoside, calycosin, ligusticolide, formononetin, and ligusticolide I were selected as the signature components of quality difference before and after processing. The results of the quantitative analysis showed that the content of ligustrin I, calycosin, formononetin, and ligusticum decreased after the Danggui Buxue Decoction was processed. The content of calycosin-7-O-ß-D-glucoside and ferulic acid increased. At the same time, a new chemical compound, namely 5-hydroxymethylfurfural was produced. The established fingerprint analysis method is stable and reliable. Combined with network pharmacology and quantitative research, it screens out the differential Q-marker, which provides an experimental basis for further research on processed products of Danggui Buxue Decoction.

[Consistency evaluation of multidimensional quality attributes of Atractylodis Macrocephalae Rhizoma by fusion of multi-source information].

The quality control of Chinese medicinal decoction pieces is one of the key tasks in the traditional Chinese medicine industry. In this study, multi-source information fusion was employed to fuse the data from near-infrared spectroscopy, electronic tongues, and other tests and establish an overall quality consistency evaluation method for Atractylodis Macrocephalae Rhizoma, which provided methodological support for the overall quality evaluation of Atractylodis Macrocephalae Rhizoma. The near-infrared spectroscopy information was measured in both static and dynamic states for 23 batches of Atractylodis Macrocephalae Rhizoma samples from different sources, and the electronic tongue sensory information, moisture content, and leachate content were measured. The overall quality of Atractylodis Macrocephalae Rhizoma was evaluated by multi-source information fusion. The results showed that the near-infrared spectroscopy information of 16122103, 801000509, 801000352, 701003656, HX21L01, and 160956 was different from that of other batches of Atractylodis Macrocephalae Rhizoma powder in the static state, and 701003298, 16122103, 701003656, 701003107, 801000229, and 18090404 were the different batches in the dynamic state. The moisture content showed no significant difference between batches. The leachate content in the batch 801000509 was different from that in other batches. The electronic tongue sensory information of 150721004, 151237, 160703004, HX21M01, HX21K04, HX21K01, and 601003516 was different from that of other batches. Furthermore, data layer fusion was employed to analyze the overall quality of Atractylodis Macrocephalae Rhizoma. Four batches, 150721004, HX21M01, HX21K04, and HX21K01, showed the parameters exceeding the 95% control limits and differed from the other samples in terms of the overall quality. This study integrated the information of moisture, near-infrared spectroscopy, and other sources to evaluate the quality consistency among 23 batches of Atractylodis Macrocephalae Rhizoma samples, which provides a reference for the quality consistency evaluation of Chinese medicinal decoction pieces.

[Prediction of quality markers of Angong Niuhuang Pills based on LC-MS and pull-down with SPR chips].

Elucidating the quality markers(Q-markers) of traditional Chinese medicines is essential for understanding the mechanisms of action and promoting the rational use of traditional Chinese medicines as well as for developing traditional Chinese medicine-derived drugs. Studies have shown that surface plasmon resonance(SPR) is promising in this field. This study proposed a method based on pull-down with SPR chips to predict the Q-markers of Angong Niuhuang pills(AGNHP). Firstly, 71 main chemical components of AGNHP were analyzed by UPLC-Q-TOF-MS, and then network pharmacology was employed to predict the potential targets of AGNHP against stroke. Secondly, the STAT3 protein chip was constructed, and the extract of AGNHP was recovered by pull-down of the SPR system for STAT3 ligand. The potential active ingredients were collected, enriched, and identified as coptisine, palmatine, epiberberine, berberine, worenine, demethyleneberberine, jatrorrhizine, tetrahydrocoptisine, baicalein, and baicalin methyl ester. Next, the affinity constants of the 10 active ingredients were determined as 44.7, 44, 58.1, 51.3, 39.7, 32.1, 49.2, 69.1, 19.7, and 24.9 µmol·L~(-1), respectively. The molecular docking results showed that the 10 compounds could compete for binding with STAT3. This is the first report that SPR combined with UPLC-Q-TOF-MS is reliable and feasible for determining the active ingredients of AGNHP at the molecular level from complex systems. STAT3 could be used as a potential target for the biological quality evaluation of AGNHP.

[Chemical constituents and pharmacological effects of Nauclea officinalis and predictive analysis of its quality markers].

Nauclea officinalis is a Chinese medicinal material with a high medicinal value, which contains various chemical constituents such as alkaloids, pentacyclic triterpenoids and their saponins, organic phenolic acids and their glycosides, iridoids, and flavonoids. It has antiviral, antibacterial, antipyretic, analgesic, anti-inflammatory, and immunoregulatory functions. This article systematically reviewed the reported chemical constituents and pharmacological effects of N. officinalis. According to the concept of quality markers, the quality markers of N. officinalis were predicted and analyzed from the aspects of plant kinship, specificity of chemical constituents, traditional drug efficacy, measurability of chemical constituents, plasma components, and different producing areas and harvest times, in order to provide a basis for the quality evaluation of N. officinalis.

[Research progress in raw and processed Raphani Semen and prediction of quality markers].

Raphani Semen, with both edible and medicinal values, is a typical Chinese herbal medicine with different effects before and after processing. The raw helps ascending and the cooked helps descending. This paper comprehensively summarizes the differences in chemical constituents and pharmacological effects between raw and processed Raphani Semen that are reported in recent years. Based on the principle of quality markers(Q-markers) of traditional Chinese medicines, the chemical constituent sources, chemical constituent detection techniques, and correlation between bidirectional regulatory efficacy and chemical constituents are compared between raw and processed Raphani Semen. The results suggest that sulforaphene and glucoraphanin could be used as candidate Q-markers of raw and processed Raphani Semen, respectively. This review is expected to provide a reference for further research on the processing, new drug development, and improvement of safety and effectiveness of Raphani Semen in clinical application.

Regulatory Considerations for Producing mRNA Vaccines for Clinical Trials.

The approval of clinical trials by the competent authorities requires comprehensive quality documentation on the new drug to be used on the clinical trial participant. In the EU, quality data is summarized as investigational medicinal product dossier (IMPD), in the United States, as investigational new drug (IND) application. For that, several preconditions concerning production, quality control, and assurance have to be fulfilled. Here, specific requirements related to mRNA vaccines are addressed on the basis of European standards.

Quality by Digital Design for Developing Platform RNA Vaccine and Therapeutic Manufacturing Processes.

Quality by digital design (QbDD) utilizes data-driven, mechanistic, or hybrid models to define and optimize a manufacturing design space. It improves upon the QbD approach used extensively in the pharmaceutical industry. The computational models developed in this approach identify and quantify the relationship between the product's critical quality attributes (CQAs) and the critical process parameters (CPPs) of unit operations within the manufacturing process. This chapter discusses the QbDD approach in developing and optimizing unit operations such as in vitro transcription, tangential flow filtration, affinity chromatography, and lipid nanoparticle (LNP) formulation in mRNA vaccine manufacturing. QbDD can be an efficient framework for developing a production process for a disease-agnostic product that requires extensive experimental and model-based process-product interaction characterization during the early process development phase.

Systematic review on fingerprinting development to determine adulteration of Chinese herbal medicines.

BACKGROUND: It has been a current research hospots using fingerprinting technology for quality control of Chinese herbal medicines (CHMs), which provides a scientific basis for establishment of overall quality control in accordance with the characteristics of CHMs. The fingerprinting technology for CHMs is diverse, and the research field covers many disciplines, such as analytical chemistry, pharmacology, pharmaceutics, biochemistry, and molecular biology. PURPOSE: To effectively understand the key areas and future directions of research regarding the fingerprint and adulteration of CHMs. METHODS/RESULTS: this paper analyzed 879 articles in this field in the Web of Science Core Collection from 2000 to 2023 with CiteSpace and VOSviewer, and systematically assessed the research process, hotspots, topic distribution among disciplines, etc. The most prominent contributors of fingerprint and adulteration of CHMs research are mainly from China, India, the United States, England, and Brazil. The knowledge domains of fingerprint and adulteration of CHMs research focus mainly on the topics of molecular authentication, DNA barcoding, HPLC, near-infrared spectroscopy, manage data, chemometrics, and electrochemical fingerprinting. Most countries have recognized the pharmaceutical potential of natural products, and have paid more attention to the fingerprint and adulteration of CHMs in the past decade. Future the research tends to focus more on molecular identification and authentication, and electrochemical and chromatographic fingerprinting in controlling the adulteration of CHMs. CONCLUSION: This research provides a valuable reference for scholars in related fields to analyze existing research results, understand the development trend, and explore new research directions.

A robust cusum control chart for median absolute deviation based on trimming and winsorization.

Statistical quality control is concerned with the analysis of production and manufacturing processes. Control charts are process control techniques, commonly applied to observe and control deviations. Shewhart control charts are very sensitive and used for large shifts based on the basic assumption of normality. Cumulative Sum (CUSUM) control charts are effective for identifying that may have special causes, such as outliers or excessive variability in subgroup means. This study uses a CUSUM control chart problems structure to evaluate the performance of robust dispersion parameters. We investigated the design structure features of various control charts, based on currently defined estimators and some new robust scale estimators using trimming and winsorization in different scenarios. The Median Absolute Deviation based on trimming and winsorization is introduced. The effectiveness of CUSUM control charts based on these estimators is evaluated in terms of average run length (ARL) and Standard Deviation of the Run Length (SDRL) using a simulation study. The results show the robustness of the CUSUM chart in observing small changes in magnitude for both normal and contaminated data. In general, robust estimators MADTM and MADWM based on CUSUM charts outperform in all environments.

HS-SPME-GC-MS and Chemometrics for the Quality Control and Clustering of Monovarietal Extra Virgin Olive Oil: A 3-Year Study on Terpenes and Pentene Dimers of Italian Cultivars.

Terpenes and pentene dimers are less studied volatile organic compounds (VOCs) but are associated with specific features of extra virgin olive oils (EVOOs). This study aimed to analyze mono- and sesquiterpenes and pentene dimers of Italian monovarietal EVOOs over 3 years (14 cultivars, 225 samples). A head space-solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) method recently validated was used for terpene and pentene dimer quantitation. The quantitative data collected were used for both the characterization and clustering of the cultivars. Sesquiterpenes were the molecules that most characterized the different cultivars, ranging from 3.908 to 38.215 mg/kg; different groups of cultivars were characterized by different groups of sesquiterpenes. Pentene dimers (1.336 and 3.860 mg/kg) and monoterpenes (0.430 and 1.794 mg/kg) showed much lower contents and variability among cultivars. The application of Kruskal-Wallis test-PCA-LDA-HCA to the experimental data allowed defining 4 clusters of cultivars and building a predictive model to classify the samples (94.3% correct classification). The model was further tested on 33 EVOOs, correctly classifying 91% of them.