Can i make opium

Ther Drug Monit. Concentrations of morphine and codeine in paired oral fluid and urine specimens following ingestion of a poppy seed roll and raw poppy seeds.

J Anal Toxicol. J Forensic Sci. Shah M, Huecker MR. Opioid Withdrawal. StatPearls Publishing. Treasure Island, FL.

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The results indicate that the 0. The target mass may vary due to the multitude of different characteristics displayed in an illicit opium sample, e.

The differences in quality of the material dried, gummy or cooked will likely play the most significant role in determining the amount of starting material Supplementary Information: Fig. A sample that is overly desiccated and exposed to high temperatures cooked will undoubtedly require an increase in the amount of starting material.

The second study focused on identifying an efficient DNA extraction method that results in an acceptable yield and purity of DNA. The Qiagen-based method will likely result in an increasingly pure extract when compared to the Promega method, with values of 1. The average ratio for the Qiagen-extracted samples improved to 1. The higher moisture content of the opium samples had a negative impact on the quality of results.

To mitigate this problem we attempted two approaches: pretreatment with liquid nitrogen and air-drying. The preferred sample pre-processing method included air-drying in a fume hood and, as needed, treatment of the sample with liquid nitrogen followed by physical disruption with a mortar and pestle and bead-beating. This greatly increased the surface area of the opium sample exposed to the lysis buffers while simultaneously decreasing the activity of any endogenous DNA degrading DNases.

We recommend extracting poppy DNA from 0. The sample quality was more advantageous in the Qiagen extraction method; however, it is also significantly more labor intensive and, when considering the ratio standard deviations, the two methods perform similarly. The DNA isolation method developed for latex is similar to the method developed for opium.

Latex samples are commonly resuspended in water for long-term storage; therefore, the opium extraction method was modified to include the concentration of biological material. Latex samples had a lower DNA yield and low quality values Table 8. Like heroin, we expect this workflow to yield high amounts of amplifiable template DNA. One gram of samples EE uncooked and OpC-1 cooked were extracted with the Qiastool kit; InhibitEX buffer and lysis reaction volumes were doubled to ensure samples were properly lysed and washed.

Quantitative PCR was performed using the 2-stage amplification and the N primer set. The results indicate that DNA originating from both cooked and uncooked opium have been successfully extracted using the Qiastool kit and amplified using the 2-stage amplification method Fig.

In contrast, the cooked opium OpC-1 had a low yield exhibiting a Cp of Although both cooked and uncooked opium were successfully amplified, the DNA yield of the cooked opium may be improved through the extraction of higher amounts of starting material; therefore, the current optimal mass of starting material for extractions from cooked opium will be 1.

The N, N and N loci contain microsatellite sequences; therefore it is possible that size polymorphisms exist within the two genomic copies of the DNA in the diploid genome of a poppy plant. Opium sample EE-2 amplified with N and N yielded a single primary allelic peak homozygote , with an amplicon length of The amplification using primer N yielded two primary allelic peaks heterozygote with amplicon sizes of and bases.

The resulting electropherograms also exhibit minimal artifactual noise, such as non-specific allelic peaks caused by electrical spikes or —A peaks caused by the non-template dependent terminal adenylyl-transferase activity inherent to many DNA polymerases, including the Roche Fast Start High Fidelity DNA polymerase.

The stutter products correspond to products that are one or two repeat units smaller than the allelic peak. For example, the N locus contains a tetra-nucleotide repeat default n which, when amplified, resulted in the expected allelic peak at bases peak height rfu and a minor stutter peak at bases peak height: rfu.

The results demonstrate that Promega-isolated opium samples are of sufficient quality and quantity to yield amplifiable DNA and interpretable results. Select electropherograms obtained from the PCR amplification and subsequent CE-analysis of raw opium samples top-primer N, middle-primer N, bottom-primer N X-axis is fragment size in bases and Y-axis is relative fluorescent units. Note, a free-dye artifact is present at approximately bases; this is independent of the sample and can be disregarded from analyses.

PCR conditions were optimized in anticipation of the low quality and quantity of DNA encountered in processed opium and heroin samples. The EE opium sample was anticipated to exhibit elevated levels of inhibition and low DNA quantities due to characteristics such as moisture level, color and texture Fig.

The combined enzyme master mix with a PCR buffer pH of 9. The leaf sample displayed consistent amplification C p within each assay group. This was expected and served as a benchmark for the lower quality EE opium sample. To the best of our knowledge, this paper is the first to demonstrate successful DNA extraction of opium poppy DNA from heroin samples.

The primary challenges were the low quality and quantities of DNA expected in processed opium and heroin samples coupled with the challenges posed from sample matrix issues and cutting agents.

In that regard, the successful extraction of DNA fragments is very significant. The quantities, as measured through the qPCR crossing point values, were low; however, this was not unexpected.

The most critical finding was that we were able clearly distinguish opium poppy amplification plots from negative controls and background noise. Preliminary data indicate that we have successfully sequenced poppy DNA for some of these samples data not shown.

The opium gum or latex contains a mixture of alkaloids, terpenoids, phenols, and various proteins Opium is simply the dried latex that is collected from the opium poppy. It is typically in the raw form only dried or in the cooked form where raw opium is boiled, filtered to remove impurities, and reconstituted for heroin production.

The development of a successful DNA isolation strategy relies heavily on the ability to address the complexities present in latex or opium gum samples. Despite the increased quality metrics, the Qiagen procedure remains more labor intensive and less reliable compared to the automated Promega methods. Despite this, we recommend the use of the Qiagen stool kit as it proved to be more versatile.

In order to improve the DNA yield and overall quality of heroin samples further, we employed a 2-stage amplification reaction using two DNA polymerases. This dual polymerase system attempts to balance the amplification of damaged DNA while maintaining a high fidelity and minimize errors. This method led to more consistent responses than either polymerase alone. The combined effects of exceedingly low levels of damaged template DNA and the presence of inhibitors necessitated the modification of conventional analytical methods to increase sensitivity, for example, the cycle, 2-stage amplification mentioned above.

The combined use of these methods resulted in the successful detection of opium poppy DNA from heroin and opium samples, but it also may have contributed to the low-level activity observed in the no-template controls.

The most commonly detected non-specific signals are due to the formation of primer dimers. The primer concentration can be lowered to avoid dimerization. In addition, the use of such a high concentration is likely unnecessary when amplifying low-copy samples like those obtained from heroin extracts. The activity of the NTC is on average 4. The heroin-positive samples exhibited T m s distinct from the activity detected in the NTC with clear differences in curve morphology Supplementary Information Fig.

In addition, the signal obtained from NTC samples are reproducible across amplification reactions and instrument runs. We conclude that the NTC activity is not responsible for the primary signal obtained from the heroin positive samples. While we believe that our proposed methods perform sufficiently well with high purity heroin samples i. Our inventory of opium and heroin samples included over samples consisting of white and brown powder and black tar heroin, as well as several types of opium.

However, these samples were also limited in quantity due to the nature of the seizures in source countries. In addition, few samples had ample mass to enable replicative comparisons of the methods we evaluated in this study. This limited the depth of our sub-studies and we recommend that the data presented herein be used as a starting point for further refinement of the procedure to extract opium poppy DNA from heroin. For example, our small whole genome amplification sub-study indicated that there was no benefit to the use of these methods.

Due to the nature and type of samples, any research group would face similar limitations to successful identification of poppy DNA caused by sample availability. The ability to obtain an opium poppy DNA profile from heroin opens a host of new avenues for law enforcement. This genetic information, through the use of high throughput sequencing, will likely enable the identification of the source country or region of seized heroin and positive identification of poppy-derived illicit drugs.

This lays the foundation for the information that can then be used for interdiction of the drug trade from a local to a global scale. Source information may provide valuable intelligence leading to the interruption of this terror-funding stream or other illegal activities.

Louis, MO. The reagent blanks consist of all reagents used in the extraction and without added DNA and were processed and analyzed quantified and amplified in concert with all experimental samples. DNA was extracted from opium gum by first mechanically pulverizing via mortar and pestle, either 1. Latex samples are typically suspended in water; therefore, the extraction technique in this method was similar to that used for opium, but it required removal of the aqueous phase.

The remaining procedure is identical to the opium procedure. Black tar heroin is particularly difficult to handle due to the gummy yet dense nature of the sample. The samples were then flash frozen in liquid nitrogen, scraped within the vial. The frozen material was broken up in the vial and scraped from the sides into a powder and 2. 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. Congressional Research Service R Lurie, I. Determination of heroin and basic impurities for drug profiling by ultra-high-pressure liquid chromatography.

Forensic Sciences International , — Morello, D. J of Forensic Sci 55 , 42—49 J Forensic Sci 40 , — 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. Comparison of levels of alkaloids identified in harvested poppy seeds, seeds from the surface of bread rolls and seeds heated with no matrix.

What was identified from this data was that whether the seeds were heated on the surface of the bread roll or were heated with no bread matrix, the levels of alkaloids if detected were considerably lower than in the harvested seeds. Koleva et al. When comparing the results from the current work to levels published in the literature Table 9 it can be seen that these findings are in-keeping with those published by Sproll et al.

The work published by Grove et al. However, this could have been due to the sensitivity of the GC-MS instrument employed, the lack of information regarding the presence of other alkaloids present in poppy seeds at this time as the work reported by Grove et al. The levels of alkaloids identified in the current work are generally lower than those found by Sproll et al. This research has shown that alkaloid variation exists depends on the specific alkaloids, their source and thermal processing.

It was clear from the data obtained in this current work, and from other studies published in the literature, that there is much variation in the levels of alkaloids identified in poppy seeds. This variation can be attributed to a variety of natural parameters, such as weather and soil conditions, but also in the way that the seeds are harvested Lachenmeier et al. Processing methods prior to packaging and even the baking process has been shown to greatly affect the level of alkaloids Sproll et al.

The findings of this study correlate with the studies published in the literature. When poppy seeds are consumed on a bun or roll, it has been estimated that each roll contains 1—4 g of poppy seeds Lachenmeier et al.

Assuming that the average salad contains 3—6 g 1—2 teaspoons of poppy seeds and the average bread bun has between 1. This value cannot be used when relating to young children, the elderly or individuals of poor health Sproll et al. With respect to the values of morphine obtained in this work for harvested seeds, seeds on top of a bread roll and seeds heated with no matrix Table 10 and taking into account the weights of poppy seeds used in a variety of food products mentioned above, the morphine ingested will not exceed the ARfD determined by the EFSA.

Table Comparison of alkaloids identified on harvested and thermally processed poppy seeds. This update related to the detection of morphine, codeine, oripavine, noscapine, and papaverine in poppy seed samples whereas, the previous report related only to the levels of morphine entering the food chain.

Codeine values were given in relation to morphine equivalents, using a conversion factor of 0. Noscapine and papaverine were considered in the most recent publication however, the data that was available to the EFSA did not allow for a hazard characterization but they did conclude that the presence of these compounds would not present a health concern. In relation to the presence of thebaine and oripavine not included in the work of this paper , it was concluded that there was insufficient data to make any assessment.

Based on these updated EFSA findings, the presence of the morphine and codeine in the poppy seeds analyzed in this work, would still would still fall below the recommendations outlined. There is also little or no information on packaging of poppy seeds regarding what, if any, treatment has taken place prior to packaging.

Since the ingestion of poppy seeds has been used as reasons for failure of workplace drug testing and roadside drug testing, more should be done to ensure as much information as is possible is available on the preparation methods of the seeds. The datasets generated for this study are available on request to the corresponding author. This work was approved by the Northumbria University Ethics committee.

The university holds a UK Home Office Drug License for the storage and use of controlled drug standards and for the extraction of alkaloids from poppy seeds. The laboratory work, analysis of data and writing was carried out predominantly by MC. JD and JA helped in the design and review of data and interpretation and all parties contributed to the writing and review of the article.

All authors contributed to the article and approved the submitted version. This work was funded with the support from the Department of Applied Sciences, Northumbria University. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

This work was carried out and submitted as part of a Ph. Annett, H. Factors influencing alkaloidal content and yield of latex in the opium poppy Papaver somniferum.

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