Green Analytical Chemistry Principles Research Articles

Automated H2O2 monitoring during photo-Fenton processes using an Arduino self-assembled automatic system

A cheap and easy to use Arduino self-assembled automatic system was employed to continuously monitor the hydrogen peroxide consumption during the photo-Fenton degradation of caffeine, selected as model target compound. The automatic system made it possible to measure the H2O2 concentration in the reaction cell via a colorimetric reaction and to take samples for HPLC analysis minimising the operator manual intervention and exposure to UV radiation. The obtained results were compared in terms of LOD and LOQ with H2O2 measurements manually performed using UV–Vis spectrophotometry, evidencing better analytical performance when using the automatic system; LOD and LOQ were respectively 0.032 mM and 0.106 mM for the automatic system against 0.064 mM and 0.213 mM for UV–Vis spectrophotometry. Furthermore, the photo-Fenton treatment was optimised by means of a Design of Experiments (DoE) investigating the effect of added H2O2 concentration, iron concentration and caffeine initial concentration on system performances. The use of the automatic device for such monitoring provided several advantages: automation (with consequent reduction of the workload), measurement increased precision, reduced reagents consumption and waste production in agreement with the principles of Green Analytical Chemistry.

Search for new green natural solid phases for sample preparation for PAHs determination in seafood samples followed by LC and GC–MS/MS analysis

Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic organic pollutants found in various environments, notably aquatic ecosystems and the food chain, posing significant health risks. Traditional methods for detecting PAHs in food involve complex processes and considerable reagent usage, raising environmental concerns. This study explores eco-friendly approaches suing solid phases derived from natural sources in matrix solid phase dispersion. We aimed to develop, optimize, and validate a sample preparation technique for seafood, employing natural materials for PAH analysis. Ten natural phases were compared with a commercial reference phase. The methodology involved matrix solid phase dispersion and pressurized liquid extraction, followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Three solid phases (perlite, sweet manioc starch, and barley) showed superior performance in LC-MS/MS and were further evaluated with gas chromatography-tandem mass spectrometry (GC–Ms/MS), confirming perlite as the most effective phase. Validation followed Brazilian regulatory guidelines and European Community Regulation 2021/808/EC. The resulting method offered advantages in cost-effectiveness, reduced environmental impact, cleaner extracts, and enhanced analytical performance compared to the reference solid phase and LC-MS/MS. Proficiency analysis confirmed method reliability, with over 50% alignment with green analytical chemistry principles. In conclusion, this study developed an environmentally sustainable sample preparation technique for seafood analysis using natural solid phases, particularly perlite, for PAH determination.

Natural kapok fiber as a green and efficient adsorbent for in-syringe solid-phase extraction in the determination of antidepressants in biofluids

Effective antidepressant monitoring is crucial for safe treatment, with liquid chromatography being the primary detection method. Due to the complexity of biofluids, meticulous sample preparation is vital to remove interferences and concentrate analytes. In this study, a novel solid-phase extraction method utilizing unprocessed kapok fiber (KF) as an eco-friendly and efficient sorbent, ingeniously incorporated within an in-syringe device for the extraction of antidepressants from biofluids was introduced. The extraction process displayed several advantages, including simplicity and rapidity (less than 3 min with minimal manual intervention), as well as the potential for automation, which represents significant advancements in sample preparation techniques. The method demonstrated excellent analytical performance when coupled with high-performance liquid chromatography-ultraviolet detection, achieving good linearity (R2 ≥ 0.991), excellent relative recovery (92.6–107.5 %), high precision in intraday and interday analyses (RSDs within 0.8–6.0 % and 1.2–7.9 %, respectively), and good sensitivity with limits of quantification between 5–10 ng/mL. Notably, given the use of a renewable and biodegradable material with KF, and a low-toxicity desorption solvent with ethanol, the developed method stands out for its environmental sustainability, aligning with green analytical chemistry principles. Its application to real biological samples validates its efficacy as a rapid and reliable tool for clinical and forensic monitoring of antidepressants. Moreover, the low cost of this method facilitated by the economical nature of KF and the reusability of syringes further enhances its practicability. This study presents a unique approach by employing natural and renewable KF as adsorbents, thereby eliminating the need for complex material synthesis and making a pioneering contribution to the field of analytical chemistry for drug analysis in complex matrices.

Deep eutectic solvents as a green alternative to organic solvents for β-cyclodextrin pseudo-stationary phase in capillary electrophoresis

β-cyclodextrin (β-CD), as an important pseudo-stationary phase (PSP) in capillary electrophoresis (CE), frequently confronts challenges stemming from its limited water solubility, particularly when high concentrations are required for resolving complex analytes. Traditionally, researchers often resort to the use of (toxic) organic solvents to enhance the solubility of β-CD, establishing non-aqueous capillary electrophoresis (NACE) for specific separations. However, such practices are hazardous to health and run counter to the principles of green analytical chemistry. In this study, we demonstrate a deep eutectic solvent (DES), Proline:Urea (PU), as a promising alternative to conventional organic solvents for β-CD-based CE separations. The DES exhibits a solubility of up to 30% for β-CD, a significant improvement compared to the 1.8% solubility in the aqueous phase. Utilizing this DES-type separation medium, we achieved simultaneous baseline separation of a complex analyte composed of eight structurally similar naphthoic acid derivatives. Furthermore, we conducted a systematic comparison of β-CD's performance in aqueous CE buffers, organic solvents, and DESs, highlighting the superiority of this novel and environmentally friendly CE separation medium.

Quantitation of three benzimidazole compounds in milk combining effervescence-assisted dispersive liquid–liquid microextraction with electrochemical detection using carbon nanotube-electropolymerized L-DOPA modified sensor

Benzimidazoles constitute a group of compounds known for their applications in pharmacy, agriculture and food industry. In recent decades, researchers have dedicated significant efforts to improving microextraction and detection methods that align with the principles of green analytical chemistry. In the present study, we have devised an effervescence-assisted dispersive liquid–liquid microextraction method and built an electrochemical sensor for the detection of albendazole, fenbendazole, and thiabendazole using GCEs (glassy carbon electrode) modified with multiple walled carbon nanotubes (MWCNTs) and poly (L-Dopa) film, allowing benzimidazole detection in complex matrices such as milk. Optimization of the microextraction procedure and electrochemical performance of the sensor was done by response surface methodology. The electrochemical sensor enabled the detection of albendazole, fenbendazole, and thiabendazole in a range of 0.37 to 100 μg mL−1 with a detection limit ranged between 0.14 to 0.28 μg mL−1. The developed sensor was tested in commercial milk. Results show satisfactory analytical performance, achieving a sensitive and selective quantification, especially considering the complexity of the matrix in which it was applied. The newly created sensor facilitates the simultaneous quantitation of the desired analytes, delivering an exceptionally praiseworthy analytical performance with detection limits lower than most existing methods.

Two green UV-spectrophotometric techniques applying successive and progressive resolution with new concepts for simultaneous determination of completely overlapping ternary mixture

A spectrophotometric platform based on two smart techniques namely Double Divisor ratio difference spectra spectrophotometry (DDRD) and Derivative ratio amplitude difference method (DR-AD) was applied and validated. These techniques were used for determining and resolving the overlapped spectra of the 3 drugs paracetamol (PAR), pseudoephedrine (PSE) and pholcodine (PHO) in their ternary mixture. DDRD is a successive technique while DR-AD is a progressive one. In DDRD, the mixtures’ spectrum underwent division by a double divisor, and then the ratio difference method was applied. For DR-AD, consequent steps were performed to resolve each drug stepwise. DR-AD technique included division using a single divisor. The linearity ranges encompassed 5-25, 100-800 and 5-50 µg/mL for PAR, PSE and PHO, respectively. These methods were conducted according to ICH guidelines to ascertain accuracy, precision, and specificity. The analysis involved varied concentrations of drug mixtures and a pharmaceutical product. Statistical evaluation revealed no notable distinction between the suggested methodologies and those previously documented. Additionally, adherence to green analytical chemistry principles was demonstrated, establishing them as environmentally friendly analytical approaches.

Multi-criteria decision analysis: technique for order of preference by similarity to ideal solution for selecting greener analytical method in the determination of mifepristone in environmental water samples

This work proposes the use of multi-criteria decision analysis (MCDA) to select a more environmentally friendly analytical procedure. TOPSIS, which stands for Technique for Order of Preference by Similarity to Ideal Solution, is an example of a MCDA method that may be used to rank or select best alternative based on various criteria. Thirteen analytical procedures were used in this study as TOPSIS input choices for mifepristone determination in water samples. The input data, which consisted of these choices, was described using assessment criteria based on 12 principles of green analytical chemistry (GAC). Based on the objective mean weighting (MW), the weights for each criterion were assigned equally. The most preferred analytical method according to the ranking was solid phase extraction with micellar electrokinetic chromatography (SPE-MEKC), while solid phase extraction combined with ultra-high performance liquid chromatography tandem mass spectrometry (SPE-UHPLC-MS/MS) was ranked last. TOPSIS ranking results were also compared to the green metrics NEMI, Eco-Scale, GAPI, AGREE, and AGREEprep that were used to assess the greenness of thirteen analytical methods for mifepristone determination. The results demonstrated that only the AGREE metric tool correlated with TOPSIS; however, there was no correlation with other metric tools. The analysis results suggest that TOPSIS is a very useful tool for ranking or selecting the analytical procedure in terms of its greenness and that it can be easily integrated with other green metrics tools for method greenness assessment.

An Ecologically Sound RP-HPLC Method for Estimating Citicoline using AQbD with Degradation Studies

Background: Citicoline is widely used for neurological complications. This research work was carried out using reverse-phase high-performance liquid chromatography (RP-HPLC) employing green analytical chemistry principles. Materials and Methods: The method was completely developed with ethanol:water (20:80 v/v) as the mobile phase, 0.1% orthophosphoric acid was used to adjust the pH to 3.5, and an Inertsil ODS 3 (250 x 4.6 mm, 5 μm) column as the stationary phase, the flow rate was set at 1-mL/min. The column temperature was maintained at 35ºC, and an injection volume of 10 μL was used for detection at a wavelength of 250 nm. Results and Discussion: The peak was eluted with isocratic elution with a retention time of 3.996 minutes. The linear concentration range for this method was 10 to 50 μg/mL, and the correlation coefficient R2 was 0.999. The forced degradation studies have shown that 3.5% w/w degraded in acid, 1.35% w/w in alkali, and 0.2% w/w in oxidation. The products formed during degradation were characterized using gas chromatography-mass spectrometry (GC-MS). Conclusion: The International Council for Harmonisation (ICH) guidelines were followed in the development and validation of the method optimized by the use of central composite design, and the method’s greenness was assessed through the use of AGREE, AES, GAPI, and AMGS, among other green assessment tools.

“Reagent-free determination of γ-butyrolactone in beverages”

A green partial least square (PLS) attenuated total reflectance infrared spectroscopy (ATR-FTIR) method has been developed for the prediction of the γ-butyrolactone (GBL) in all type of soft and alcoholic beverages. The method involves the use of a drop (less than 5 µL) of sample and a single calibration model based on aqueous and alcoholic solutions of GBL and beverages free from GBL. A selected wavenumber range from 1225 to 1171 cm−1 and from 999 to 972 cm−1 of the second derivative (SD) spectra of samples and standards were employed. The validation set provided root mean square error of prediction (RMSEP) of the order of 300 µg g−1 GBL with a ratio of standard error of performance to standard deviation value (RPD) of 13.4. The prediction of unknown samples not considered in the calibration nor in the validation sets offered accuracy relative errors between −1.0 and 2.9 % GBL. The proposed PLS-FTIR based method allows a fast and direct determination of GBL in all type of beverages, in less than 2 min and avoiding the use of neither reagents nor solvents, without any sample treatment required, being in agreement with the principles of Green Analytical Chemistry.

Determination of Enantiomer in Tofacitinib Citrate Using Reversed-Phase Chiral High-Performance Liquid Chromatography

Tofacitinib citrate (RR-isomer) is a janus kinase (JAK) inhibitor approved for the treatment of rheumatoid arthritis (RA) in adults who have had an inadequate response or intolerance to methotrexate. The presence of the enantiomer of tofacitinib citrate (SS-isomer) is monitored for quality control as a possible impurity in the final product. In this study, a reversed-phase high-performance liquid chromatography (RP-HPLC) method based on a chiral recognition mechanism for the separation of tofacitinib citrate and its enantiomer was established based on the principles of green analytical chemistry. A CHIRALPAK IH column was used with a mobile phase of ammonium acetate buffer (pH 8.0) and acetonitrile in a gradient elution at a detection wavelength of 285 nm. The calibration curve exhibited excellent linearity over the range of 0.1002–20.04 μg/mL (r = 0.9999). The average recovery of the enantiomer was 98.6% with a relative standard deviation (RSD) of 0.7%. The limit of detection (LOD) and the limit of quantitation (LOQ) were 0.04 and 0.1 μg/mL, respectively. This RP-HPLC method was suitable for detecting the enantiomers of tofacitinib citrate in tablets. Furthermore, the method proved to be environmentally friendly based on the evaluation by Analytical Eco-Scale, Analytical GREEnness (AGREE) and Green Analytical Procedure Index (GAPI).

SmartAnalysis: A sustainable digital-image colorimetry method for ethanol determination in alcohol-based hand sanitizer

Introduction: The consumption of alcohol-based hand sanitizers has increased significantly after the pandemic caused by SARS-CoV-2. Despite the conclusion of the health emergency declared by the WHO in 2023, the habit of sanitizing hands with ethanol-based gel sanitizers has been globally adopted by the population. Since general methods described in official compendia for ethanol determination such as gas chromatography or distillation are laborious and not-specific to carbomer-containing gels, this work proposes an alternative method based on digital image colorimetry. Method: The digital image (provided by ethanol-phenolphthalein reaction) was captured and transformed into an analytical signal based on the Red-Green-Blue system. The image acquisitions were performed using a Samsung Galaxy J6 smartphone, and the signals were generated using the Photometrix Pro® free program. The method was validated in accordance with ICH and applied in commercial samples. Additionally, the proposed method was evaluated for its environmental impact using the Green Analytical Process Index (GAPI) tool. Pictograms were generated using the ComplexGAPI® free program. Results: Green channel exhibited a linear response in the calibration curves for ethanol concentrations ranging from 5 to 40 % (w/w) in acidic medium. The method showed linearity, precision, accuracy, and robustness. Conclusions: The proposed method presented as main advantages the use of low-cost and easy-to-handle devices and reduced reagent consumption, in accordance with green analytical chemistry principles.

Activated carbon from avocado seed as sorbent phase for microextraction technologies: activation, characterization, and analytical performance.

According to green analytical chemistry principles, the use of agricultural byproducts as sorbent phases is an interesting topic due to their lignocellulosic origin, as they are biodegradable and inexpensive. To the best of our knowledge, this is the first study in which avocado seed and avocado seed activated carbon are proposed as sustainable sorbents for solid-phase microextraction technologies, which were used to assess the proof of concept. Rotating disk sorptive extraction (RDSE) was used as a model technology and ibuprofen (Ibu) and 1-hydroxy-ibuprofen (1-OH-Ibu) as representative analytes. It was found that activated carbon (AC) prepared at 600°C with an impregnation ratio (raw material/activating agent (ZnCl2), w/w) of 1:1.2 had better extraction efficiency than other ACs obtained at different temperatures, impregnation ratios, and activating agents (K2CO3). Characterization revealed several differences between natural avocado seed, biochar prepared at 600°C, and selected AC since the typical functional groups of the natural starting material begin to disappear with pyrolysis and increasing the surface area and pore volume, suggesting that the main interactions between analytes and the sorbent material are pore filling and π-π stacking. By using this AC as the sorbent phase, the optimal extraction conditions in RDSE were as follows: the use of 50mg of sorbent in the disk, 30mL of sample volume, pH 4, 90min of extraction time at a rotation velocity of the disk of 2000rpm, and methanol as the elution solvent. The extracts were analyzed via gas chromatography coupled to mass spectrometry (GC-MS). The method provided limits of detection of 0.23 and 0.07µg L-1 and recoveries of 81% and 91% for Ibu and 1-OH-Ibu, respectively. When comparing the extraction efficiency of the selected activated carbon with those provided by Oasis® HLB and C18 in RDSE, nonsignificant differences were observed, indicating that avocado seed activated carbon is a suitable alternative to these commercial materials.

Fabric phase sorptive extraction: A sustainable approach in analysis of pharmaceutical product

As the pharmaceutical industry continues to evolve, there is an increasing demand for sustainable and efficient analytical techniques in the analysis of pharmaceutical products. This manuscript explores the application of Fabric Phase Sorptive Extraction (FPSE) as a novel and sustainable approach for the extraction and analysis of pharmaceutical compounds. FPSE, a recent advancement in sample preparation, offers a greener alternative by utilizing a fabric-like sorbent material. The environmentally friendly nature of FPSE, with reduced solvent consumption and waste generation, aligns with the principles of green analytical chemistry. Case studies involving the analysis of various pharmaceutical products showcase the versatility and applicability of FPSE in different matrices.

Greenness of dispersive microextraction using molecularly imprinted polymers

Abstract Molecularly imprinted polymers (MIPs) as materials with determined levels of selectivity and specificity for designated analytes have recently gained much attention in various application fields. However, with the growing adoption of green analytical chemistry (GAC) principles, it is essential to investigate the greenness of MIP synthesis and its subsequent application in sample preparation, as well as to evaluate the “green” nature of the developed analytical methodologies, such as dispersive solid-phase microextraction (DSPME). Accordingly, the main objective of this research was to evaluate the greenness of MIP-based glycidyl methacrylate synthesis and MIP use as a DSPME sorbent prior to high-performance liquid chromatography with mass spectrometry (HPLC-MS). The green perspective of MIP-DSPME prior to HPLC-MS was investigated using various analytical metric tools such as the Analytical Eco-Scale, the Green Analytical Procedure Index, and Analytical GREEnness (AGREE). Since these analytical tools are not fully implementable for the assessment of the greenness of the MIP synthesis, some alternative approaches were used to optimize the synthesis parameters to make the MIP DSPME sorbent as close as possible to the GAC principles. The calculated AGREE score (0.62) and 91 points in the Analytical Eco-Scale for the proposed DSPME technique using MIP indicated a high level of greenness.

Direct Immersion-Solid Phase Microextraction for Therapeutic Drug Monitoring of Patients with Mood Disorders.

This article discusses a new method for monitoring drug concentrations in blood samples from patients with mood disorders. The method uses solid-phase microextraction to extract analytes directly from blood samples. It has been adapted to identify the most commonly used drugs in mood disorders, including amitriptyline, citalopram, fluoxetine, paroxetine, sertraline, trazodone, duloxetine, venlafaxine, lamotrigine, quetiapine, olanzapine, and mirtazapine. The analysis is carried out using high-performance liquid chromatography coupled with mass spectroscopy. The proposed DI-SPME/LC-MS method allows for a simple and quick screening analysis while minimizing the volume of the tested sample and solvent, in line with the principles of green analytical chemistry. The method was used to analyze 38 blood samples taken from patients with mood disorders, and drug concentrations were determined and compared with therapeutic and toxic dose ranges. This allowed for better control of the course of treatment.

Development of a green analytical methodology based on a deep eutectic solvent for the determination of pesticide residues in industrial hemp

In this study, in accordance with the green analytical chemistry principles, the use of eco-friendly solvents has been proposed for the first time for the analysis of pesticide residues in industrial hemp (Cannabis sativa L.). With this aim, betaine-based natural deep eutectic solvents with lactic acid and propylene glycol were explored as extraction solvents in the solid–liquid extraction of fifteen pesticides from hemp samples. The mixture composed of betaine:propylene glycol at molar ratio 1:4 (n/n) was selected due to its better analytical performance. The separation, identification and quantification of pesticides were carried out by gas chromatography coupled to tandem mass spectrometry. The extraction protocol was optimised through the step-by-step strategy, evaluating NADES composition, water addition to the solvent, solid–liquid ratio, and re-extraction solvent. The analytical methodology was validated in terms of calibration (linear regression with R2 ≥0.9966), trueness and precision (average relative recovery of 63–77% with relative standard deviation ≤20%) and sensitivity (limits of quantification of 0.663–3.625 µg/kg). These results successfully demonstrated the convenience of the developed approach for the determination of pesticides in industrial hemp, which has industrial applications such as the production of food and supplements, cosmetics, and textiles, which should not compromise the health of consumers. In addition, the comparison with previously reported research in this field showed that not only the present method is more sensitive, but also more sustainable and economical.

Nova HPLC metoda za istovremeno određivanje empagliflozina i dapagliflozina - razvoj, validacija, testiranje robusnosti i procena ekološke prihvatljivosti

Empagliflozin (EMPA) and Dapagliflozin (DAPA) are mainly recommended for the treatment of type 2 diabetes mellitus and heart failure. Based on the principles of green analytical chemistry, a simple, rapid and robust HPLC method was developed for the determination of both analytes in bulk. An isocratic protocol was developed using a C18 column, with a mobile phase consisting of just 30% organic modifier (ACN), while the remaining 70% was HPLC water. The run was completed within 4 min, with a flow rate of 1.20 mL/min, while UV detection took place at 230 nm. The method was then validated according to the ICH Q2 (R1) guidelines, and all the parameters examined were within the specified limits. Furthermore, the robustness of the method was evaluated by employing a factorial experimental design. Finally, the environmental friendliness of the proposed method was assessed by using the Analytical GREEness (AGREE) metric tool. The proposed method can be used for analysis of EMPA and DAPA in bulk, with potential application to the relevant pharmaceuticals.

"Zelena" RP-HPLC metoda za određivanje profila nečistoća amlodipina u tabletama

Increased awareness of nature preservation has encouraged the introduction of the green analytical chemistry (GAC) practice concepts concerning several important aspects, including sustainable development, environmental impact, and minimum waste. The aim of this research was to contribute to the implementation of this approach for the pharmaceutical industry while retaining the crucial aspects and strict requirements of quality control of medicines. Therefore, an ethanolbased, green and robust high-performance liquid chromatography (HPLC) method for the determination of related substances of amlodipine (AML) in film-coated tablets was developed and optimized using the Design of Experiments (DoE). The chromatographic separation was performed on an RP-select B column (250 x 4.0 mm, 5 mm), using a mixture of 0.04 M sodium dihydrogen phosphate monohydrate (pH 4.0) and ethanol (60:40 % v/v) as a mobile phase. The optimized conditions provided the separation of two specified impurities (impurity D and impurity F). The selectivity of the method was confirmed using forced degradation studies. The Analytical Eco-scale approach and AGREE metrics confirmed that the method conforms to the GAC principles. The validated method was successfully applied for the determination of related substances in three samples from the market, demonstrating the applicability of the method in routine analysis.

Critical overview on the use of hydrophobic (deep) eutectic solvents for the extraction of organic pollutants in complex matrices

During the last decades, many efforts have been devoted to the adaptation of sample preparation techniques and methods to the principles of Green Analytical Chemistry. Among them, this article review focusses on those aimed to green the solvents involved in sample treatment. Research in this field started in the late 1990s with the synthesis of room temperature ionic liquids, which were later replaced by the deep eutectic solvents (DESs). During the last years, a subclass of DESs, the so-called hydrophobic deep eutectic solvents (HDESs) have attracted attention. HDESs have contributed to circumventing some of the limitations of early-synthesised hydrophilic DESs regarding the cost of raw materials, the simplicity of synthesis, and the biocompatibility and, apparently, the biodegradability of the mixtures. In addition, these mixtures allowed the treatment of aqueous samples and the extraction of non-polar analytes.This article discusses fundamental aspects regarding the nomenclature used concerning HDESs, summarises the main physicochemical properties of these mixtures, and through discussion of key application studies, describes current progress in the use of these green solvents for the extraction of trace organic contaminants from a variety of matrices. Remaining gaps and possible lines of future development in this emerging, active and attractive research area are also identified and critically discussed.

Miniaturized Analytical Strategy Based on μ-SPEed for Monitoring the Occurrence of Pyrrolizidine and Tropane Alkaloids in Honey.

Currently, the analysis of trace-level contaminants in food must be addressed following green analytical chemistry principles and with a commitment to the sustainable development goals. Accordingly, a sustainable and ecofriendly microextraction procedure based on μ-SPEed followed by ultrahigh liquid chromatography coupled to ion-trap tandem mass spectrometry analysis was developed to determine the occurrence of pyrrolizidine and tropane alkaloids in honey samples. The μ-SPEed procedure took approximately 3 min per sample, using only 100 μL of organic solvent and 300 μL of diluted sample. The method was properly validated (overall recoveries 72-100% and precision RSD values ≤15%), and its greenness was scored at 0.61 out of 1. The method was applied to different honey samples, showing overall contamination levels from 32 to 177 μg/kg of these alkaloids. Atropine was found in all the samples, whereas retrorsine N-oxide, lasiocarpine, echimidine, and echimidine N-oxide were the main pyrrolizidine alkaloids in the samples analyzed.