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.

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.

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.

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.

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.

Chemometric approach for development, optimization, and validation of different chromatographic methods for separation of opium alkaloids.

The excessive and continuously growing interest in the simultaneous determination of poppy alkaloids imposes the development and optimization of convenient high-throughput methods for the assessment of the qualitative and quantitative profile of alkaloids in poppy straw. Systematic optimization of two chromatographic methods (gas chromatography (GC)/flame ionization detector (FID)/mass spectrometry (MS) and reversed-phase (RP)-high-performance liquid chromatography (HPLC)/diode array detector (DAD)) for the separation of alkaloids from Papaver somniferum L. (Papaveraceae) was carried out. The effects of various conditions on the predefined chromatographic descriptors were investigated using chemometrics. A full factorial linear design of experiments for determining the relationship between chromatographic conditions and the retention behavior of the analytes was used. Central composite circumscribed design was utilized for the final method optimization. By conducting the optimization of the methods in very rational manner, a great deal of excessive and unproductive laboratory research work was avoided. The developed chromatographic methods were validated and compared in line with the resolving power, sensitivity, accuracy, speed, cost, ecological aspects, and compatibility with the poppy straw extraction procedure. The separation of the opium alkaloids using the GC/FID/MS method was achieved within 10 min, avoiding any derivatization step. This method has a stronger resolving power, shorter analysis time, better cost/effectiveness factor than the RP-HPLC/DAD method and is in line with the "green trend" of the analysis. The RP-HPLC/DAD method on the other hand displayed better sensitivity for all tested alkaloids. The proposed methods provide both fast screening and an accurate content assessment of the six alkaloids in the poppy samples obtained from the selection program of Papaver strains.

New Validated Method for the Determination of Six Opium Alkaloids in Poppy Seed-Containing Bakery Products by High-Performance Liquid Chromatography-Tandem Mass Spectrometry after Magnetic Solid-Phase Extraction.

Bakery products containing poppy seeds are increasingly being commercialized. These seeds may be contaminated with latex from the Papaver somniferum L. plant rich in opium alkaloids (OAs). Therefore, health authorities demand the development of analytical methods to control them. In this study, an efficient and simple method was developed and validated for the first time to analyze six OAs in bakery products by high-performance liquid chromatography-tandem mass spectrometry. For this purpose, a solid-liquid extraction was optimized, and then a magnetic material [magnetite surface-modified with Fe(III) terephthalate, denoted as Fe3O4@TPA-Fe] was used for a fast magnetic solid-phase extraction. The method has been validated with adequate recoveries (70-110%) and relative standard deviations (<20%) and without matrix effects. Nine bakery samples (five breadsticks and four sliced bread) were analyzed; breadsticks showed low amounts of OAs, but two sliced bread showed higher amounts of OAs than the new amount (1.5 mg/kg) set by the Commission Regulation (EU) 2021/2142.

Opium, Street Opium, and Cancer Risk.

Opium is defined as the air-dried latex obtained by incision from the unripe capsules of Papaver somniferum L. Opium is a complex mixture that contains approximately 10% morphine and 2% codeine. It is commonly used to prepare opium tinctures for people with chronic diarrhea. Morphine and related opioids are powerful but highly addictive analgesics; designing less addictive opioids is an active area of pharmaceutical research that may lead to significant improvements in chronic pain management. Recently, the International Agency for Research on Cancer (IARC) has classified opium consumption as carcinogenic to humans (Group 1) based on sufficient evidence of carcinogenicity in human studies. However, all human studies analyzed by the IARC Working Group included participants who consumed opium that was mixed, adulterated, and/or contaminated with known and probable human carcinogens (e.g., tarry residues of combusted opium, arsenic, lead, and chromium). The working group considered that these carcinogens were part of the complex mixture that opium is, rather than co-exposure or confounders. No evidence of carcinogenicity was available for pure opium in human, animal, or mechanistic studies. To avoid confusion and concern among health professionals and patients using medicinal opium preparations and in scientists involved in the design and development of new opium derivatives, opium should be classified in Group 3 (not classifiable as to its carcinogenicity to humans). The term 'street opium' could be used to refer to opium that probably contains human carcinogens not present in pure opium and should remain in Group 1 (carcinogenic to humans).

Urine thebaine determination by liquid chromatography tandem mass spectrometry after poppy seed consumption.

Laboratories are challenged to distinguish whether a positive urine morphine result is due to heroin use or possible poppy seed consumption. Thebaine is an opium alkaloid that has been shown to be present in the urine of individuals who have consumed poppy seeds, as well as those who have used opium. It is not present in heroin. We present a sensitive, specific liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for thebaine. We show that thebaine is detectable after consumption of two different poppy seed-containing products for up to 72 h in urine. We discuss limitations of the assay and suggest how the test might best be used.

Opium consumption and coronary atherosclerosis in diabetic patients: a propensity score-matched study.

There is a traditional belief among Eastern people that opium may have ameliorating effects on cardiovascular risk factors, especially diabetes; thus, it is widely used among diabetic patients. We attempted to investigate the association of opium consumption with coronary artery disease (CAD) in diabetic patients. A cross-sectional study was conducted on diabetic patients undergoing coronary angiography in our center. Out of 1925 diabetic patients included in the study, 228 were opium users, and the remaining 1697 non-opium users were used as a pool of potential comparators. Propensity scores were used to match the 228 opium consumers with 228 matched comparators for age, sex, and smoking status. The Gensini score and extent score were respectively used to assess the angiographic severity and extent of CAD. The mean Gensini score (86.9 ± 62.7 vs. 59.6 ± 43.4, p < 0.0001) and extent score (7.1 ± 2.9 vs. 5.9 ± 2.9, p < 0.0001) were significantly higher in opium user diabetic patients than in non-opium users. After adjustment for potential confounders, a dose-response relationship was observed between dose of opium and the Gensini score ( ß = 0.27, p = 0.04). There were no significant differences between the routes of opium administration (inhalation vs. oral) regarding the severity and extent of CAD. In conclusion, exposure to opium in diabetic patients may be positively associated with the risk of CAD, and with the angiographically determined severity and extent of the disease. Furthermore, dosage of opium consumption may correlate with severity of CAD.

Concentrations of the Opium Alkaloids Morphine, Codeine, and Thebaine in Poppy Seeds are Reduced after Thermal and Washing Treatments but are Not Affected when Incorporated in a Model Baked Product.

Limited information exists on the effectiveness of potential treatments to reduce levels of opium alkaloids that may be present in seeds from poppy (Papaver somniferum L.). Poppy seeds containing morphine at relatively lower (14.7 mg kg-1) and higher (210.0 mg kg-1) concentrations were subjected to dry heat and steam treatments, water washing, and baking. Sample extracts were then analyzed using liquid chromatography-tandem mass spectrometry for the opium alkaloids morphine, codeine, and thebaine. The results indicated that thermal treatment promoted opium alkaloid degradation in poppy seed samples, with a 50% loss of morphine observed after 30-40 min at 200 °C. Water washing reduced concentrations of opium alkaloids in poppy seeds by approximately 50-80%, while steam treatment resulted in reduction of morphine in only one sample type. Importantly, baking had no significant effect on concentrations of opium alkaloids. Overall, these results indicate that opium alkaloids may not be significantly affected by baking or steam application and that poppy seeds may require water washing or extended thermal treatment to promote reduction of these compounds.

Development of a Method to Extract Opium Poppy (Papaver somniferum L.) DNA from Heroin.

This study is the first to report the successful development of a method to extract opium poppy (Papaver somniferum L.) DNA from heroin samples. Determining of the source of an unknown heroin sample (forensic geosourcing) is vital to informing domestic and foreign policy related to counter-narcoterrorism. Current profiling methods focus on identifying process-related chemical impurities found in heroin samples. Changes to the geographically distinct processing methods may lead to difficulties in classifying and attributing heroin samples to a region/country. This study focuses on methods to optimize the DNA extraction and amplification of samples with low levels of degraded DNA and inhibiting compounds such as heroin. We compared modified commercial-off-the-shelf extraction methods such as the Qiagen Plant, Stool and the Promega Maxwell-16 RNA-LEV tissue kits for the ability to extract opium poppy DNA from latex, raw and cooked opium, white and brown powder heroin and black tar heroin. Opium poppy DNA was successfully detected in all poppy-derived samples, including heroin. The modified Qiagen stool method with post-extraction purification and a two-stage, dual DNA polymerase amplification procedure resulted in the highest DNA yield and minimized inhibition. This paper describes the initial phase in establishing a DNA-based signature method to characterize heroin.

Characterization and origin of the 'B' and 'C' compounds in the acid/neutral forensic signatures of heroin - products from the acylation of porphyroxine and subsequent hydrolysis.

Two significant compounds often found in the gas chromatographic analysis of the acid/neutral extracts from illicit heroin have remained uncharacterized for 30 years. The unknown compounds are referred to as the 'B' and 'C' compounds. It has been postulated that these compounds arise from acetylation of porphyroxine, a rhoeadine alkaloid found at trace levels in the opium poppy, Papaver somniferum. Porphyroxine was isolated from opium and acetylated to produce N,O8 -diacetylporphyroxine. Mild hydrolysis produced N,O8 -diacetyl-O14 -desmethyl-epi-porphyroxine (the C compound) and N-acetyl-O14 -desmethyl-epi-porphyroxine (the B compound). Both N,O8 -diacetyl-O14 -desmethyl-epi-porphyroxine and N-acetyl-O14 -desmethyl-epi-porphyroxine were determined to be C-14 epimers of porphyroxine and N,O8 -diacetylporphyroxine. The non-epimerized isomers of the B and C compounds were also detected in illicit heroin, but at much lower levels. Chromatographic and spectroscopic data are presented for the aforementioned compounds. The presence/absence and relative concentrations of these compounds is presented for the four types of heroin (Southwest Asian, South American, Southeast Asian, and Mexican). The prevalence of detection for the B and C compounds are Southwest Asian = 92-93%, South American = 64-72%, Southeast Asian = 45-49%, and Mexican ≤ 3%. When detected, the overall trend of relative concentrations of dicaetylporhyroxine, the B-compound, and C-compound is Southwest Asian > South American > Southeast Asian, each by an order of magnitude. These compounds were rarely detected in Mexican heroin. The presence/absence and relative concentrations of these compounds provide pertinent forensic signature characteristics that significantly enhance the final regional classifications. Copyright © 2015 John Wiley & Sons, Ltd.

Gene actions for yield and its attributes and their implications in the inheritance pattern over three generations in opium poppy (Papaver somniferum L.).

The gene actions for yield and its attributes and their inheritance pattern based on five parameter model have been explored in four single crosses (NBIHT-5 × NBIHT-6, NBIHT-5 × NBMHT-1, NBMHT-1 × NBIHT-6 and NBMHT-2 × NBMHT-1) obtained using thebaine rich pure lines of opium poppy (Papaver somniferum L.) for three consecutive generations. All the traits showed nonallelic mode of interaction, however, dominance effect (h) was more pronounced for all the traits except thebaine and papaverine. The dominance × dominance (l) effects were predominant over additive × additive (i) for all traits in all the four crosses except for papaverine. The seed and opium yield, and its contributing traits inherited quantitatively. The fixable gene effects (d) and (i) were lower in magnitude than nonfixable (h) and (l) gene effects. The estimates of heterosis were also higher in comparison to the respective parents which suggested preponderance of dominance gene action for controlling most of the traits. The phenotypic coefficient of variation was marginally higher than those of genotypic coefficient of variation for all the traits. The traits thebaine, narcotine, morphine and opium yield had high heritability coupled with high genetic advance. The leaf number, branches per plant and stem diameter showed positive correlation with opium and seed yields. The selection of plants having large number of leaves, branches and capsules with bigger size would be advantageous to enhance the yield potential.

Determination of morphine and related alkaloids in crude morphine, poppy straw and opium preparations by micellar electrokinetic capillary chromatography.

A rapid method for the determination of morphine and related alkaloids in crude morphine, poppy straw and opium preparations by micellar electrokinetic capillary chromatography (MEKC) has been developed. Morphine, codeine, thebaine, oripavine, papaverine, narcotine, narceine, cryptopine and salutaridine were separated in less than 10 min using a 70 cm x 50 microm I.D. uncoated fused-silica capillary column with a buffer consisting of 10% dimethylformamide, 90% 0.05 M cetyltrimethylammonium bromide, 0.01 M potassium dihydrogen orthophosphate, 0.01 M sodium tetraborate, pH 8.6. An applied voltage of -25 kV and a temperature of 28 degrees C gave the best separation of the alkaloids. Pholcodine was used as the internal standard. The compounds were detected by UV at 254 nm. The levels of morphine and related alkaloids determined by MEKC were in good agreement with those determined by high-performance liquid-chromatography (HPLC). The coefficients of variation for area calculation (%C.V.) for multiple sample and standard injections by MEKC were slightly greater than for HPLC but were still acceptable (morphine content of poppy straw: %C.V. MEKC 1.7%, %C.V. HPLC 0.3%).

[Characteristics of Papaver somniferum L. cv. ikkanshu cultivated in Izu].

The seeds of Papaver somniferum L. cv. Ikkanshu were sown in November (Autumn sowing: AS) and March (Spring sowing: SS) in a field at Izu Experimental Station for Medicinal Plants of National Institute of Health Sciencs, and both AS and SS plants were cultivated to investigate their growth, opium yield and alkaloid content in the opium. Growing periods from the sowing to the opium harvest were approximately six months for AS plants and three months for SS plants. Sizes of plants and capsules in AS were bigger than those in SS, reflecting their growth period. Opium yields per an are in AS and SS were 212.09 g and 142.03 g, respectively. The opium was able to be collected four times in the AS plants though the SS plants ceased to exude opium after the second incision. Therefore higher yield of opium in AS plants seems to be attributed to an amount of opium in the third and fourth incision. Average morphine content in the total opium was 15.61% in AS plants and 15.04% in SS plants, and the estimated amounts of morphine per an are in AS and SS plants were 33.16 g and 21.38 g, respectively.

[Influence of fertilizers on yield and yield components of opium from Papaver somniferum Linn].

The effect of fertilizer on the yield and yield components of opium from Papaver somniferum L.cv.Ikkanshu were investigated from 1993 to 1995. The weight of opium per one capsule was tendency to increase in the order of peat moss > pulverized charcoal > compound fertilizer, but the differences among the results were not significant. Furthermore, no difference in the capsule weight per 100 m2 and the yields of opium and alkaloids per 100 m2 were detected. The significant difference was only observed when the data were compared among the weights and yields of different years. The difference among the yield of opium was mainly due to the weight of opium per one capsule. The weight of opium per one capsule showed a high correlation with capsule husk weight. The alkaloids contents in opium obtained with a different fertilizer application showed no difference. Morphine content at the first lancing was about 11%, and the value decreased with the order of lancing. On the contrary, the codeine and the thebaine content did not change during lancing and the value were 6-7% and 3%, respectively. The papaverine and the noscapine content decreased in the order of lancing.

Degradation of morphine in opium poppy processing waste composting.

To investigate morphine degradation and optimize turning frequency in opium poppy processing waste composting, a pilot scale windrow composting trial was run for 55 days. Four treatments were designed as without turning (A1), every 5 days turning (A2), every 10 days turning (A3) and every 15 days turning (A4). During composting, a range of physicochemical parameters including the residual morphine degradation, temperature, pH, and the contents of total C, total N, total P and total K were investigated. For all treatments, the residual morphine content decreased below the detection limit and reached the safety standards after day 30 of composting, the longest duration of high temperature (⩾50 °C) was observed in A3, pH increased 16.9-17.54%, total carbon content decreased 15.5-22.5%, C/N ratio reduced from 46 to 26, and the content of total phosphorus and total potassium increased slightly. The final compost obtained by a mixture of all four piles was up to 55.3% of organic matter, 3.3% of total nutrient (N, P2O5 and K2O) and 7.6 of pH. A turning frequency of every ten days for a windrow composting of opium poppy processing waste is recommended to produce homogenous compost.