Comparison of direct analysis in real-time mass spectrometry, atmospheric solids analysis probe-mass spectrometry, and ion mobility spectrometry for ensuring food safety by rapid screening of poppy seeds.

The opium poppy (Papaver somniferum) is a global commercial crop that has been historically valued for both medicinal and culinary purposes. Naturally occurring opium alkaloids including morphine, codeine, thebaine, noscapine, and papaverine are found primarily in the latex produced by the plant. If the plant is allowed to fully mature, poppy seeds that do not contain the opium alkaloids will form within the pods and may be used in the food industry. It is possible for the seeds to become contaminated with alkaloids by the latex during harvesting, posing a potential health risk for consumers. In the USA, there have been more than 600 reported adverse events including 19 fatalities that may be linked to the consumption of a contaminated poppy-containing product such as home-brewed poppy seed tea. Unwashed poppy seeds and pods may be purchased over the Internet and shipped worldwide. The Forensic Chemistry Center, US Food and Drug Administration (FDA) has evaluated several mass spectrometers (MS) capable of rapid screening to be used for high-throughput analysis of samples such as poppy seeds. These include a direct analysis in real-time (DART) ambient ionization source coupled to a single-quadrupole MS, an atmospheric solids analysis probe (ASAP) ionization source coupled to the same MS, and ion mobility spectrometers (IMS). These instruments have been used to analyze 17 poppy seed samples for the presence of alkaloids, and the results were compared to data obtained using liquid chromatography with mass spectral detection (LC-MS/MS). Results from the 17 poppy seed samples indicate that the DART-MS, ASAP-MS, and IMS devices detect many of the same alkaloids confirmed during the LC-MS/MS analyses, although both the false-positive and false-negative rates are higher, possibly due to the non-homogeneity of the samples and the lack of chromatographic separation.

Computational insights into diverse aspects of glutathione S-transferase gene family in Papaver somniferum.

Plant glutathione S-transferases are an ancient protein superfamily having antioxidant activity. These proteins are primarily involved in diverse plant functions such as plant growth and development, secondary metabolism, signaling pathways and defense against biotic and abiotic stresses. The current study aimed to comprehensively identify and characterize the GST gene family in the medicinally important crop Papaver somniferum. A total of 93 GST proteins were identified belonging to eight GST classes and found to be majorly localized in the cytoplasm. All GST genes were found on eleven opium chromosomes. Gene duplication analysis showed segmental duplication as a key factor for opium GST gene family expansion under strong purifying selection. Phylogenetic analysis with gymnosperm, angiosperm and bryophyte revealed the evolution of GSTs earlier than their division into separate groups and also prior to the divergence of monocot and dicot. The secondary structure prediction showed the dominance of α-helices indicative of PsomGSTs as structurally stable and elastic proteins. Gene architecture showed the conservation of number of exons across the classes. MEME analysis revealed only a few class specific and many across class conserved motifs. Ser was found to be the active site residue of tau, phi, theta and zeta class and Cys was catalytic residue of DHAR, lambda and GHR class. Promoter analyses identified many cis-acting regulatory elements related to hormonal, cellular, stress and light response functions. Ser was the key phosphorylation site. Only three glycosylation sites were found across the 93 PsomGSTs. 3D structure prediction was also performed and was validated. Interactome analyses revealed the correlation of PsomGSTs with glutathione metabolizing proteins. Gene enrichment analysis and KEGG pathway analyzed the involvement of PsomGSTs in three major pathways i.e. glutathione metabolism, tyrosine metabolism and ascorbate metabolism. The outcome revealed high model quality of PsomGSTs. The results of the current study will be of potential significance to understand the functional and structural importance of the GST gene family in opium, a medicinally important crop.

Neopinone isomerase is involved in codeine and morphine biosynthesis in opium poppy.

The isomerization of neopinone to codeinone is a critical step in the biosynthesis of opiate alkaloids in opium poppy. Previously assumed to be spontaneous, the process is in fact catalyzed enzymatically by neopinone isomerase (NISO). Without NISO the primary metabolic products in the plant, in engineered microbes and in vitro are neopine and neomorphine, which are structural isomers of codeine and morphine, respectively. Inclusion of NISO in yeast strains engineered to convert thebaine to natural or semisynthetic opiates dramatically enhances formation of the desired products at the expense of neopine and neomorphine accumulation. Along with thebaine synthase, NISO is the second member of the pathogenesis-related 10 (PR10) protein family recently implicated in the enzymatic catalysis of a presumed spontaneous conversion in morphine biosynthesis.

Improved UAV Opium Poppy Detection Using an Updated YOLOv3 Model.

Rapid detection of illicit opium poppy plants using UAV (unmanned aerial vehicle) imagery has become an important means to prevent and combat crimes related to drug cultivation. However, current methods rely on time-consuming visual image interpretation. Here, the You Only Look Once version 3 (YOLOv3) network structure was used to assess the influence that different backbone networks have on the average precision and detection speed of an UAV-derived dataset of poppy imagery, with MobileNetv2 (MN) selected as the most suitable backbone network. A Spatial Pyramid Pooling (SPP) unit was introduced and Generalized Intersection over Union (GIoU) was used to calculate the coordinate loss. The resulting SPP-GIoU-YOLOv3-MN model improved the average precision by 1.62% (from 94.75% to 96.37%) without decreasing speed and achieved an average precision of 96.37%, with a detection speed of 29 FPS using an RTX 2080Ti platform. The sliding window method was used for detection in complete UAV images, which took approximately 2.2 sec/image, approximately 10× faster than visual interpretation. The proposed technique significantly improved the efficiency of poppy detection in UAV images while also maintaining a high detection accuracy. The proposed method is thus suitable for the rapid detection of illicit opium poppy cultivation in residential areas and farmland where UAVs with ordinary visible light cameras can be operated at low altitudes (relative height < 200 m).

Detection of opium alkaloids in a Cypriot base-ring juglet.

A method has been developed for extracting poppy alkaloids from oily matrices, specifically lipid residues associated with archaeological ceramics. The protocol has been applied to fresh and artificially aged poppyseed oil and to residue from a Late Bronze Age Cypriot juglet in the collections of the British Museum. The juglet is of a type that has been linked with ancient trade in opium due to its poppy-head shape and wide distribution; it is a rare example of an intact vessel with contents sealed inside. Bulk analysis of the residue by GC-EI-MS and pyGC-EI-MS indicated a degraded plant oil and possible presence of papaverine. Analysis of the alkaloid extracts by HPLC-ESI-MS using both triple quadrupole and FTICR mass spectrometers detected the five primary opium alkaloids in fresh poppyseed oil and papaverine in most of the aged samples. Papaverine and thebaine were detected in the juglet residue, providing the first rigorous chemical evidence to support a link between this vessel type and opium, or at least poppies. The association of opium with oil raises new questions about the ancient purpose of the commodities within these vessels, and the low levels (ng g-1) of opiates detected in this unusually well-preserved residue shed doubt on the scope for their detection in more fragmentary ceramic remains (potsherds). Papaverine was found to exhibit challenging carryover behaviour in all the analytical methods used in this study. The phenomenon has not been reported before and should be considered in future analyses of this analyte in all application areas.

Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy.

The gateway to morphine biosynthesis in opium poppy (Papaver somniferum) is the stereochemical inversion of (S)-reticuline since the enzyme yielding the first committed intermediate salutaridine is specific for (R)-reticuline. A fusion between a cytochrome P450 (CYP) and an aldo-keto reductase (AKR) catalyzes the S-to-R epimerization of reticuline via 1,2-dehydroreticuline. The reticuline epimerase (REPI) fusion was detected in opium poppy and in Papaver bracteatum, which accumulates thebaine. In contrast, orthologs encoding independent CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2-dehydroreticuline were isolated from Papaver rhoeas, which does not accumulate morphinan alkaloids. An ancestral relationship between these enzymes is supported by a conservation of introns in the gene fusions and independent orthologs. Suppression of REPI transcripts using virus-induced gene silencing in opium poppy reduced levels of (R)-reticuline and morphinan alkaloids and increased the overall abundance of (S)-reticuline and its O-methylated derivatives. Discovery of REPI completes the isolation of genes responsible for known steps of morphine biosynthesis.

Short-chain dehydrogenase/reductase catalyzing the final step of noscapine biosynthesis is localized to laticifers in opium poppy.

The final step in the biosynthesis of the phthalideisoquinoline alkaloid noscapine involves a purported dehydrogenation of the narcotinehemiacetal keto moiety. A short-chain dehydrogenase/reductase (SDR), designated noscapine synthase (NOS), that catalyzes dehydrogenation of narcotinehemiacetal to noscapine was identified in opium poppy and functionally characterized. The NOS gene was isolated using an integrated transcript and metabolite profiling strategy and subsequently expressed in Escherichia coli. Noscapine synthase is highly divergent from other characterized members of the NADPH-dependent SDR superfamily involved in benzylisoquinoline alkaloid metabolism, and it exhibits exclusive substrate specificity for narcotinehemiacetal. Kinetic analyses showed that NOS exhibits higher catalytic efficiency with NAD+ as the cofactor compared with NADP+. Suppression of NOS transcript levels in opium poppy plants subjected to virus-induced gene silencing resulted in a corresponding reduction in the accumulation of noscapine and an increase in narcotinehemiacetal levels in the latex. Noscapine and NOS transcripts were detected in all opium poppy organs, but both were most abundant in stems. Unlike other putative biosynthetic genes clustered in the opium poppy genome, and their corresponding proteins, NOS transcripts and the cognate enzyme were abundant in latex, indicating that noscapine metabolism is completed in a distinct cell type compared with the rest of the pathway.

"Vintage meds": a netnographic study of user decision-making, home preparation, and consumptive patterns of laudanum.

BACKGROUND: Reviews have commented on rising clandestine manufacture of opiate drug solutions for injecting, and to a lesser extent for oral use. Very little is known about user attempts to culture poppy seeds, widely available on the internet for manufacture of long acting medium-high potency oral solutions, both as poppy seed tea or as opium tincture (laudanum). OBJECTIVES: A netnographic research methodology aimed to provide online consumer insight into user sourcing and decision influences, experiences of home manufacture of laudanum, utilization of opium tincture recipes, and consumptive patterns. METHODS: A systematic internet search was conducted using the terms: "Laudanum," "Opium tincture," and "Tincture of Opium" in combination with "forum." Following screening of 810 forum threads with exclusion criteria and removal of duplicates, 75 fora threads on 6 online drug fora were analyzed using the empirical phenomenological psychological method. Four themes were generated. RESULTS: Findings illustrated the underpinning of user reminiscing about Victorian use of standardized laudanum, long duration shelf life, and medicinal use for opiate withdrawals with intentions to prepare. Preparation of famous recipes and use of authentic storage bottles boosted nostalgia. Participants appeared well versed in kitchen chemistry processes. Discussions centered on type and amount of alcohol used, use of additives to promote palatability and intoxication effect, homogenization of poppy seeds, and double extraction using opium tincture. Lack of detail available on intoxication experiences, with tentative dosage advised. CONCLUSIONS: Development of targeted and credible "counterpublic" harm reduction initiatives situated within online consumerism of communal drug knowledge is warranted.

Dioxygenases catalyze O-demethylation and O,O-demethylenation with widespread roles in benzylisoquinoline alkaloid metabolism in opium poppy.

In opium poppy, the antepenultimate and final steps in morphine biosynthesis are catalyzed by the 2-oxoglutarate/Fe(II)-dependent dioxygenases, thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM). Further investigation into the biochemical functions of CODM and T6ODM revealed extensive and unexpected roles for such enzymes in the metabolism of protopine, benzo[c]phenanthridine, and rhoeadine alkaloids. When assayed with a wide range of benzylisoquinoline alkaloids, CODM, T6ODM, and the functionally unassigned paralog DIOX2, renamed protopine O-dealkylase, showed novel and efficient dealkylation activities, including regio- and substrate-specific O-demethylation and O,O-demethylenation. Enzymes catalyzing O,O-demethylenation, which cleave a methylenedioxy bridge leaving two hydroxyl groups, have previously not been reported in plants. Similar cleavage of methylenedioxy bridges on substituted amphetamines is catalyzed by heme-dependent cytochromes P450 in mammals. Preferred substrates for O,O-demethylenation by CODM and protopine O-dealkylase were protopine alkaloids that serve as intermediates in the biosynthesis of benzo[c]phenanthridine and rhoeadine derivatives. Virus-induced gene silencing used to suppress the abundance of CODM and/or T6ODM transcripts indicated a direct physiological role for these enzymes in the metabolism of protopine alkaloids, and they revealed their indirect involvement in the formation of the antimicrobial benzo[c]phenanthridine sanguinarine and certain rhoeadine alkaloids in opium poppy.

Benzylisoquinoline alkaloid biosynthesis in opium poppy.

Opium poppy (Papaver somniferum) is one of the world's oldest medicinal plants and remains the only commercial source for the narcotic analgesics morphine, codeine and semi-synthetic derivatives such as oxycodone and naltrexone. The plant also produces several other benzylisoquinoline alkaloids with potent pharmacological properties including the vasodilator papaverine, the cough suppressant and potential anticancer drug noscapine and the antimicrobial agent sanguinarine. Opium poppy has served as a model system to investigate the biosynthesis of benzylisoquinoline alkaloids in plants. The application of biochemical and functional genomics has resulted in a recent surge in the discovery of biosynthetic genes involved in the formation of major benzylisoquinoline alkaloids in opium poppy. The availability of extensive biochemical genetic tools and information pertaining to benzylisoquinoline alkaloid metabolism is facilitating the study of a wide range of phenomena including the structural biology of novel catalysts, the genomic organization of biosynthetic genes, the cellular and sub-cellular localization of biosynthetic enzymes and a variety of biotechnological applications. In this review, we highlight recent developments and summarize the frontiers of knowledge regarding the biochemistry, cellular biology and biotechnology of benzylisoquinoline alkaloid biosynthesis in opium poppy.

Utility of opium seed extract tests in preventing hypersensitivity reactions during surgery.

BACKGROUND: Anaphylaxis during anaesthesia is fatal in 3-9% of patients and analgesics, including opioids, and is the second most common medicament-related cause, although the prevalence is underestimated. We recently found that patients may generate IgE antibodies to opium seeds. OBJECTIVES: To determine the diagnostic accuracy of specific antibodies to morphine, codeine, rocuronium and oil body and aqueous fractions of Papaver somniferum seeds in the diagnosis and prevention of allergy to opioids. METHODS: Patients with hypersensitivity reactions during surgery, and severe clinical allergy (pollen, tobacco), and illicit heroin users were selected. The sensitivity, specificity and predictive values of in vivo and in vitro diagnostic techniques including oil body and aqueous fractions of P. somniferum seeds were measured. RESULTS: We studied 203 patients, with mean age 35.1±17.1 and 200 healthy controls. Patients sensitised to heroin or with hypersensitivity reactions during surgery responded to P. somniferum seed tests. Of patients not known to be sensitised to opioids, the highest positivity was in patients sensitised to tobacco (p<0.001). Opium seed skin tests and IgE, especially the oil body fraction, were more sensitive (64.2%) and specific (98.4%) than morphine, codeine and rocuronium tests for opioid sensitivity. Pollen allergy was not a risk factor for sensitisation to morphine. CONCLUSIONS: Sensitivity to opioids and intraoperative anaphylaxis can be diagnosed by routine tests. IgE and skin tests for the oil body fraction of P. somniferum had the highest sensitivity for sensitisation to opioids.

How to improve the surveillance of the Taliban ban's impact on European drug markets.

In April 2023, the Taliban banned poppy cultivation and the trade of all narcotics. This caused a 95% reduction in opium production. Usually, that would be good news. But there is a substantial worry: synthetic opioids might fill the void left by heroin. This is concerning because these drugs have led to health emergencies in areas where they are prevalent. This paper highlights the limitations of the current drug surveillance system in Europe and proposes improvements. It argues that reliance on secondary data is insufficient. Instead, we need to interview a sentinel group of people who inject drugs and adjust city-level sentinel systems, such as wastewater analysis, to specifically track the spread of synthetic opioids. Without these proactive steps, we risk only noticing a transition from heroin to synthetic opioids after it has occurred, with its harmful impacts already in place.

Pollen identification of three notorious illicit drug plants and its potential applications in forensic practice.

Opium poppy, coca and cannabis are raw materials for three notorious illicit drugs. For a long time, drug lords have been growing and smuggling these drugs in a variety of ways and channels and are continually finding new ways of trafficking their wares, which has led to the increasing difficulty of global drug enforcement. In the present paper, we propose an innovative pollen identification system for these important drug plants, which provides a tool for screening and detection of the drugs to aid in drug enforcement. By utilizing the characteristics of these fine particles, their abundant production, and high resistance to decay, we believe this tool could be applied in the following scenarios: detecting and dynamically monitoring drug cultivation activities; determining whether a suspect has been to fields of drug plants and determining whether the site has ever been planted with a drug plant and/or was involved in drug production. In the future, combined with microscope automatic image acquisition technology and intelligent image recognition technology, this pollen identification system is expected to be used to screen three notorious illicit drug plants, thus enhancing the efficiency of drug related crime investigations.

Characterization of a flavoprotein oxidase from opium poppy catalyzing the final steps in sanguinarine and papaverine biosynthesis.

Benzylisoquinoline alkaloids are a diverse class of plant specialized metabolites that includes the analgesic morphine, the antimicrobials sanguinarine and berberine, and the vasodilator papaverine. The two-electron oxidation of dihydrosanguinarine catalyzed by dihydrobenzophenanthridine oxidase (DBOX) is the final step in sanguinarine biosynthesis. The formation of the fully conjugated ring system in sanguinarine is similar to the four-electron oxidations of (S)-canadine to berberine and (S)-tetrahydropapaverine to papaverine. We report the isolation and functional characterization of an opium poppy (Papaver somniferum) cDNA encoding DBOX, a flavoprotein oxidase with homology to (S)-tetrahydroprotoberberine oxidase and the berberine bridge enzyme. A query of translated opium poppy stem transcriptome databases using berberine bridge enzyme yielded several candidate genes, including an (S)-tetrahydroprotoberberine oxidase-like sequence selected for heterologous expression in Pichia pastoris. The recombinant enzyme preferentially catalyzed the oxidation of dihydrosanguinarine to sanguinarine but also converted (RS)-tetrahydropapaverine to papaverine and several protoberberine alkaloids to oxidized forms, including (RS)-canadine to berberine. The K(m) values of 201 and 146 µm for dihydrosanguinarine and the protoberberine alkaloid (S)-scoulerine, respectively, suggested high concentrations of these substrates in the plant. Virus-induced gene silencing to reduce DBOX transcript levels resulted in a corresponding reduction in sanguinarine, dihydrosanguinarine, and papaverine accumulation in opium poppy roots in support of DBOX as a multifunctional oxidative enzyme in BIA metabolism.

Isolation and characterization of a cDNA encoding (S)-cis-N-methylstylopine 14-hydroxylase from opium poppy, a key enzyme in sanguinarine biosynthesis.

Sanguinarine is a benzo[c]phenenthridine alkaloid with potent antimicrobial properties found commonly in plants of the Papaveraceae, including the roots of opium poppy (Papaver somniferum). Sanguinarine is formed from the central 1-benzylisoquinoline intermediate (S)-reticuline via the protoberberine alkaloid (S)-scoulerine, which undergoes five enzymatic oxidations and an N-methylation. The first four oxidations from (S)-scoulerine are catalyzed by cytochromes P450, whereas the final conversion involves a flavoprotein oxidase. All but one gene in the biosynthetic pathway from (S)-reticuline to sanguinarine has been identified. In this communication, we report the isolation and characterization of (S)-cis-N-methylstylopine 14-hydroxylase (MSH) from opium poppy based on the transcriptional induction in elicitor-treated cell suspension cultures and root-specific expression of the corresponding gene. Along with protopine 6-hydroxylase, which catalyzes the subsequent and penultimate step in sanguinarine biosynthesis, MSH is a member of the CYP82N subfamily of cytochromes P450. The full-length MSH cDNA was expressed in Saccharomyces cerevisiae and the recombinant microsomal protein was tested for enzymatic activity using 25 benzylisoquinoline alkaloids representing a wide range of structural subgroups. The only enzymatic substrates were the N-methylated protoberberine alkaloids N-methylstylopine and N-methylcanadine, which were converted to protopine and allocryptopine, respectively.

Poppy APETALA1/FRUITFULL orthologs control flowering time, branching, perianth identity, and fruit development.

Several MADS box gene lineages involved in flower development have undergone duplications that correlate with the diversification of large groups of flowering plants. In the APETALA1 gene lineage, a major duplication coincides with the origin of the core eudicots, resulting in the euFUL and the euAP1 clades. Arabidopsis FRUITFULL (FUL) and APETALA1 (AP1) function redundantly in specifying floral meristem identity but function independently in sepal and petal identity (AP1) and in proper fruit development and determinacy (FUL). Many of these functions are largely conserved in other core eudicot euAP1 and euFUL genes, but notably, the role of APETALA1 as an "A-function" (sepal and petal identity) gene is thought to be Brassicaceae specific. Understanding how functional divergence of the core eudicot duplicates occurred requires a careful examination of the function of preduplication (FUL-like) genes. Using virus-induced gene silencing, we show that FUL-like genes in opium poppy (Papaver somniferum) and California poppy (Eschscholzia californica) function in axillary meristem growth and in floral meristem and sepal identity and that they also play a key role in fruit development. Interestingly, in opium poppy, these genes also control flowering time and petal identity, suggesting that AP1/FUL homologs might have been independently recruited in petal identity. Because the FUL-like gene functional repertoire encompasses all roles previously described for the core eudicot euAP1 and euFUL genes, we postulate subfunctionalization as the functional outcome after the major AP1/FUL gene lineage duplication event.

Clinical value of morphine, pholcodine and poppy seed IgE assays in drug-abusers and allergic people.

BACKGROUND: The diagnosis of anaphylactic reactions due to opiates during anaesthesia can be difficult, since in most cases various drugs may have been administered. Detection of specific IgE to poppy seed might be a marker for sensitisation to opiates in allergic people and heroin-abusers. This study assessed the clinical value of morphine, pholcodine and poppy seed skin-prick and IgE determination in people suffering hypersensitivity reactions during anaesthesia or analgesia and drug-abusers with allergic symptoms. METHODS: We selected heroin abusers and patients who suffered severe reactions during anaesthesia and analgesia from a database of 23,873 patients. The diagnostic yield (sensitivity, specificity and predictive value) of prick and IgE tests in determining opiate allergy was analysed. RESULTS: Overall, 149 patients and 200 controls, mean age 32.9 ± 14.7 years, were included. All patients with positive prick to opiates showed positive prick and IgE to poppy seeds, but not to morphine or pholcodine IgE. Among drug-abusers, 13/42 patients (31%) presented opium hypersensitivity confirmed by challenge tests. Among non-drug abusers, sensitisation to opiates was higher in people allergic to tobacco (25%), P<.001. Prick tests and IgE against poppy seed had a good sensitivity (95.6% and 82.6%, respectively) and specificity (98.5% and 100%, respectively) in the diagnosis of opiate allergy. CONCLUSIONS: Opiates may be significant allergens. Drug-abusers and people sensitised to tobacco are at risk. Both the prick and specific IgE tests efficiently detected sensitisation to opiates. The highest levels were related to more-severe clinical profiles.

Morphometrics of waterlogged archaeological seeds give new insights into the domestication and spread of Papaver somniferum L. in Western Europe.

Domesticated opium poppy Papaver somniferum L. subsp. somniferum probably originated in the Western Mediterranean from its possible wild progenitor, Papaver somniferum L. subsp. setigerum and spread to other European regions. Seeds of opium poppy have been identified in different European regions since the Early Neolithic (from the 6th millennium cal. BC onwards) period. However, until recently, the absence of morphological identification criteria has prevented the discrimination between wild and domestic morphotypes. New morphometric approaches to distinguish modern subspecies have been proven to be applicable to waterlogged archaeological remains, opening the possibility of understanding the process of domestication of the plant in both time and space. This paper applies seed outline analyses, namely elliptic Fourier transforms, combined with size and number of cells to archaeological waterlogged Papaver seeds throughout the Neolithic period in the NW Mediterranean and the surroundings of the Alps. Furthermore, one example from the Late Bronze Age (LBA) was added to see what kind of differences appeared during the >1000 years between the end of the Neolithic and the LBA. The aim of the study is to classify the archaeological seeds as domestic or wild morphotypes and observe morphometric changes in connection to geographical and chronological patterns that can explain the spread and domestication process(es) of this important crop. A total of 295 archaeological seeds coming from 10 waterlogged sites dating between 5300-2300 cal. BC (Neolithic), and one LBA site dating to 1070 cal. BC were analysed. The results indicate the presence of seeds, similar to the wild morphotype, in the Mediterranean sites and larger seeds, similar to the domestic morphotype, in the regions surrounding the Alps. The number of cells mainly increased during the Late Neolithic (3300 to 2300 cal. BC) and, finally, in the Late Bronze Age (ca. 1050-800 cal. BC), larger, morphologically domesticated seeds are clearly predominant. A change in the shape of the seeds is only clearly visible in the LBA material. Altogether our results suggest that opium poppy seeds show no sign of domestication in the early periods of the Neolithic, despite the fact that the plant was very probably already cultivated at that time in the western Mediterranean region.

Design and Optimisation of Sustainable Sample Treatments Based on Ultrasound-Assisted Extraction and Strong Cation-Exchange Purification with Functionalised SBA-15 for Opium Alkaloids in Ground Poppy Seeds.

An analysis methodology was optimised and validated for the quantification of opium alkaloids (OAs) in ground poppy seeds. This involved ultrasound-assisted extraction (UAE) and solid-phase extraction (SPE) purification before analysis using a high-performance liquid chromatography mass spectrometry detector (HPLC-MS/MS). UAE was optimised through the design of experiments with three factors and a three-level full factorial design. For SPE optimisation, a commercial material was compared with a previously synthesised material of SBA-15 silica functionalised with sulfonic groups (SBA-15-SO3-). The synthesised material demonstrated superior efficiency with only 25 mg and proved to be reusable for up to four cycles. The methodology was properly validated in terms of linearity, limits of detection and quantification, and selectivity. Matrix effects were negligible; adequate recovery values (85-100%) and inter-day and intra-day precision (≤15%) were obtained. The greenness of the method was evaluated with the AGREEprep metric scale, being more environmentally friendly compared to OA analysis methods. Finally, the method was applied to different samples of ground poppy seeds and revealed a concentration of 140 mg/kg of morphine equivalents in one of the samples, surpassing the legislatively established limits by sevenfold. This highlights the need to analyse these types of samples to mitigate potential public health issues.