Although it is known that alkaloid compounds can be found in both Papaver somniferum L. Table 1. The dried latex product is called opium from which morphine and other alkaloid compounds can be extracted. However, if left to fully mature, the plant will form poppy seeds within the capsule which can be mechanically harvested and collected by a sieving process Kapoor, ; Schiff, Harvesting methods have been shown to greatly affect the level of alkaloids in the final opium product Mahdavi-Damghani et al.
If the opium is harvested too early in the process the product is found to be watery and if too late the opium contains significantly lower levels of alkaloids Mahdavi-Damghani et al. It was initially thought that the seeds and any products derived from them would not contain any alkaloid compounds due to the fact that the seeds develop after the latex Duke, ; Schiff, However, in the late 's, it was noted that poppy seeds contained alkaloids found in opium Grove et al.
Meadway et al. Since then, it has become increasingly apparent that the presence of alkaloids in the food chain is a problem and can potentially lead to serious repercussions Garnock-Jones and Scholes, ; Cassella et al. In Germany in particular, where poppy seeds are used and readily incorporated into food products, measures have been taken to reduce the amount of morphine present in poppy seeds intended for the food chain.
The Report highlighted the requirement to estimate the dietary morphine exposure. From evaluation of extensive scientific literature sources, the EFSA panel concluded that it was possible for an individual to suffer effects from ingestion of poppy seed products. It is known that the ingestion of poppy seeds has caused positive opiate drug test results with positive opiate results have been found in urine, blood, and oral fluid Cassella et al. In this study, the aim was to establish if thermal processing methods, the food matrix employed and the source of poppy seeds would affect the levels of opium alkaloids identified pre- and post-baking.
The main reason for this was to establish if normal food preparation techniques, employed when using poppy seeds, would ultimately affect the opium alkaloids reaching the food chain. Ultimately this will influence drug-testing results or have a potential clinical impact on an individual. Organic solvents acetonitrile, chloroform, isopropyl alcohol, diethyl ether, and methanol of HPLC grade were purchased from Sigma-Aldrich Poole, Dorset.
Poppy seeds were purchased from a number of retail outlets in the UK with the country of origin identified where available Table 2. The mass spectrometer was operated in positive mode ionization with the specific instrument parameters shown in Table 4 Carlin et al.
Table 4. Figure 2 shows the extracted ion chromatograms for each of the alkaloids being analyzed in this work, including deuterated morphine as an internal standard. An overall run time of 12 min was employed, with all analytes of interest eluting before 10 min. Figure 2. Extracted chromatograms from a mixed injection of a morphine, b morphine-d3, c codeine, d thebaine, e papaverine, and f noscapine. A minimum of six calibration points, plus a blank, were used with the concentration range and associated linear equations and R 2 values Table 5.
A calibration set was analyzed alongside every data set. Analyte specific validation data is shown in Table 5. Table 5. Validation parameters for morphine, codeine, thebaine, papaverine, and noscapine.
For each solvent, extractions were carried out at a different pH's i. To avoid contamination by plastic residues from pipette tips by the solvents, glass pipettes were employed. Poppy seeds from a portion of the 1 seeds were homogenized using a spice blender and approximately mg of the seeds were weighed into glass Durham tubes four tubes for each of the solvents.
For each solvent, the pH was altered to produce a solution of poppy seeds and solvent 1 mL at the specified pHs. To each solution, deuterated internal standard morphine-d 3 was added. The tubes were then capped and placed into an ultrasonic bath for 10 min, centrifuged at 4, rpm for 10 min. Calibration solutions were prepared in the mobile phase on the day of analysis for each of the extractions.
The ingredients were mixed into a batter and added to each of the dimples of mini-muffin trays purchased from Lakeland Ambleside, Cumbria. The final weight of poppy seeds in each mini-muffin was approximately 1. They were then left to cool to room temperature.
The poppy seed muffins were immersed in liquid nitrogen, crushed using a mortar and pestle and transferred to a spice blender for homogenisation prior to extraction and analysis by LC-MS. The liquid nitrogen method was found to be easiest to apply to the muffins: this was due to the fact that the poppy seeds were incorporated into the sponge of the muffin.
Trying to extract each poppy seed from the matrix proved very time consuming and was also considered that any alkaloids that may have interacted with the muffin matrix may also be included in results. Less fatty emulsion was also formed during the extraction method, when liquid nitrogen was employed. A comparative study was carried out to establish if there was a difference between alkaloid levels resulting from poppy seeds incorporated into the matrix of the muffin to those resulting from poppy seeds coated onto a bread roll.
The dough was then split into 4 equal portions and each one was pressed into poppy seeds. The rolls were left to cool to room temperature and the poppy seeds were scraped from the surface using a metal spatula and homogenized in a spice blender prior to extraction and analysis by LC-MS. It was found that at the extremes of the polarity scale diethyl ether, dichloromethane, and water , the chromatograms produced were complex and with poor peak shape for the alkaloids; these observations were independent of pH.
In contrast, the optimum result, in terms of alkaloid presence and peak shape, was obtained using the solvent mixture i. For this reason, this extraction solvent mixture was used for all subsequent extraction's. For each source, the mean weight of each of the alkaloids in poppy seeds was calculated. When the levels of morphine in poppy seeds from each of the different sources was compared Table 7 , it was found that there was much variation within batch but also between sources of poppy seeds.
There is much variation in the extracted opiate compounds, which is primarily due to the environmental differences of the seeds Katrine et al. The country of origin for both of these poppy seed sources is unknown.
When the same comparison was carried out for codeine Table 7 it was also found that there was much variation within different portions of the same batch and between sources of poppy seeds as was the case with morphine.
Source 2, which was found to have a level of morphine much higher than the other sources, was also found to have a higher level of codeine. No other similarities can be drawn from the data. It was also found that the same source with the highest levels of morphine and codeine also exhibited the highest levels of thebaine.
Noscapine was identified in only two of the eight sources of poppy seeds Table 7. It was found that the seeds from source 7 contained the highest levels of noscapine of the two sources where noscapine was identified.
Papaverine was detected in some of the analyzed seeds but peaks were so small that it was not possible to quantify them. It has been identified that sub-varieties of Papaver somniferum L. However, this taxonomic information was not available from the suppliers of the seeds.
It has been known since Annett, that factors, such as the season in which the plants are grown, weather conditions, and quality and type of fertilizer used can greatly affect the levels of alkaloids biosynthesised by Papaver somniferum L.
In turn, the levels of alkaloids found in opium latex will also be affected. No data currently exist that compares levels of alkaloids in opium latex and alkaloids from the same plant but it is assumed that the levels would correlate. On this basis, the country of origin, where the plant was grown in the field e.
This means that if a batch of poppy seeds is harvested from one field, naturally there will be variation in the levels of alkaloids from each of the plants. It has also been shown that the alkaloids present in the opium latex may contaminate the poppy seeds as part of the growing process and that a batch of poppy seeds is the combination of multiple fields in one country: all of these factors may explain why there is such variation within batch and between sources of poppy seeds.
However, the muffin matrix greatly interfered with the extraction process. During the extraction process, a fatty emulsion was formed which affected further sample preparation techniques Figure 3. These aliquots were filtered twice prior to being transferred into HPLC vials however when the chromatograms were analyzed for these muffin extractions, no alkaloids were identified. For this reason, it was not possible to include the poppy seed muffins extract results in the comparison between harvested poppy seeds, thermally processed seeds on their own and poppy seeds on the top of bread buns.
In addition, seed portions from three randomly selected sources were extracted and analyzed with the results shown in Table 8. Again, as was established with extractions of harvested poppy seeds there was much variation in the alkaloids identified and in the levels of those alkaloids present, Deuterated morphine was added prior to extraction of the alkaloids from the seeds and percentage extractions were incorporated into the calculations.
Figure 3. Sample tubes containing poppy seed muffin and extraction solvent, post agitation, and centrifugation. Table 8. Opiates have been used successfully to control pain, but they are also abused because of their mind-altering effects. The opiates that come from the poppy plant include morphine, opium , heroin, and codeine. After they enter the body, opiates stimulate specific receptors in the brain.
When these opiate receptors are activated, it causes a rush of pleasure and then hours of feeling content, relaxed, or high. Some of these receptors are in the reward center of the brain. The brain starts to want to repeat the positive stimulation and this can lead to addiction. Aside from pain relief and a sense of euphoria, opiates can cause:. The seeds, stems, and pods sold to make poppy seed tea are unwashed. Poppy seeds are not created equal, and everyone processes them differently.
Poppy seeds can vary in their concentrations of codeine and morphine, meaning that the strength of the tea made by one crop of seed can be quite different from another. Additionally, people metabolize the poppy seed differently. One study gave poppy seeds to volunteers, and there was a big variation in how much codeine and morphine were excreted in their urine. Eating poppy seeds has, in fact, caused individuals to fail urine drug screenings.
In the past, even modest amounts of poppy seeds have caused people to test positive for morphine , one of the opioids contained in the poppy. DHHS increased the threshold for detecting opiate metabolites in urine to help prevent these false positives, so you or your loved one can safely enjoy a poppy seed bagel. The dried pods or straw are ground into a powder and steeped in water. This is a popular way to extract opioids from the poppy.
Research has shown it is possible to create a lethal dose of morphine in poppy seed tea brewed at home. Because it is almost impossible to tell what the concentration of the active drugs might be in any one crop of poppy pods or poppy seeds, it is impossible to control the amount of the drug enough to avoid overdose.
Opioids suppress the respiratory system, which can lead to death by respiratory arrest after an overdose of the drug. If someone you care about experiences the following symptoms of opioid overdose, call immediately and stay with the person. Paramedics carry a medication called Narcan naloxone that can counter the effects of an opiate overdose. Poppy seed tea can be addictive and deadly. There have been a number of documented cases of death from the use of poppy tea.
Opioids are extremely habit-forming and can quickly lead to tolerance, physical dependence, and addiction. Opioid withdrawal can occur when stopping cold turkey, resulting in unpleasant symptoms often described as "having a bad flu," with fever and sweating, nausea and vomiting, muscle aches and pain, and insomnia.
If a dry pellet formed, vortexing was needed. In order to maximize amplification of low copy number samples, DNA extracts were concentrated prior to amplification. DNA extracts 2. Amplification of the samples was carried out with a two-step cycling method using two different polymerases.
The first round of amplification used the Omni KlenTaq polymerase Tables 11 and This was used to combat the low quality of the DNA expected in opium and heroin samples. The Omni KlenTaq master mix was spiked with a dilution of the Roche Fast Start High Fidelity polymerase to take advantage of the proofreading activity. The second round of amplification was carried out using Roche Fast Start High Fidelity polymerase without purification of the sample Tables 13 and The fluorescence crossing point C p generated in the LightCycler software 1.
Controls were included in every quantitation and amplification. DNA extraction negative controls reagent blanks or RB consisting of all reagents used in the extraction and without added DNA were processed and analyzed quantified and amplified with samples with added material.
PCR negative controls No-template controls, NTCs consisting of the amplification reagents and specific primer sets used within each reaction were included with every qPCR run. This indicates that the extractions were contaminate free and the primers did have aberrant specificity for any DNAs that may be present in the samples that were not poppy DNA.
Alternatively, gel purification could be done in place of the AMPure procedure. The markers contain microsatellite sequences for use in potential downstream genetic identification assays Supplementary Information: Table These results indicate that these sequences are unique.
In addition, the primerBLAST query results did not return any target sequences within the nr database, therefore specificity for the Papaver somniferum L. Note, the poppy-specific primer sequences used in this study are available from the corresponding author on reasonable request.
Capillary electrophoresis was used to further characterize the DNA markers. Capillary electrophoresis was performed using an injection voltage of 1. The data relating to poppy-specific primer sequences used in this study are available from the corresponding author upon reasonable request. Finklea, K. Heroin Trafficking in the United States.
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