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. 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.
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BMC Bioinformatics 13 , Download references. Anthony Gray Ph. Anderson M. This project was supported by contract No. Michael A. Marciano, Garrett D. You can also search for this author in PubMed Google Scholar. All authors reviewed the manuscript. Correspondence to Michael A. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Reprints and Permissions. Marciano, M.
DNA from Heroin. Sci Rep 8, Download citation. Received : 24 August Accepted : 29 January Published : 07 February Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Scientific Reports Phytochemistry Reviews By submitting a comment you agree to abide by our Terms and Community Guidelines.
If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Advanced search. Skip to main content Thank you for visiting nature. Download PDF. Subjects Biological techniques Biotechnology. Abstract This study is the first to report the successful development of a method to extract opium poppy Papaver somniferum L.
Introduction The heroin epidemic has become a widespread domestic issue for the American people; it has touched the lives of families who have lost loved ones to heroin overdoses and is ubiquitous in the mainstream media. Full size table. Table 3 Evaluation of extraction methods and sample mass on heroin samples of varying purities.
Figure 1. Full size image. Figure 2. Figure 3. Heroin samples following the addition of P3 buffer Qiagen Plant Minikit. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Figure EE opium sample. Discussion To the best of our knowledge, this paper is the first to demonstrate successful DNA extraction of opium poppy DNA from heroin samples.
DNA extraction from opium gum and latex DNA was extracted from opium gum by first mechanically pulverizing via mortar and pestle, either 1. DNA extraction from Black Tar Heroin Black tar heroin is particularly difficult to handle due to the gummy yet dense nature of the sample.
Table 11 Omni KlenTaq master mix components. Table 12 Omni KlenTaq master mix cycling parameters. References 1. Google Scholar Article Google Scholar PubMed Google Scholar Sweder U. Panicker Authors Michael A. Marciano View author publications.
View author publications. Ethics declarations Competing Interests The authors declare no competing interests. Additional information Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Electronic supplementary material. Supplementary information. About this article. Cite this article Marciano, M. Copy to clipboard.
Further reading OpiumPlex is a novel microsatellite system for profiling opium poppy Papaver somniferum L. Comments By submitting a comment you agree to abide by our Terms and Community Guidelines. Publish with us For authors Submit manuscript. Search Search articles by subject, keyword or author. Show results from All journals This journal. Some of these latter, after securing permits, did not even plant poppies, others had crop failures. It is said that only acres, registered under state permits, were actually grown in this year.
To these growers, however, the crop was enormously profitable. The Commissioner of Narcotics issued an order to "warn all persons concerned of the danger, inseparably connected with the cultivation of the opium poppy for any purpose, of providing a new source of supply of contraband morphine to drug peddlers and addicts". The poppy growers were not much impressed.
They were not interested in possible narcotic dangers but in the high price of poppy-seed. Their fields were unguarded and sometimes quite open to the public road. Capsules obtained for analyses showed, on the dry basis, from 0. The Federal poppy control bill was passed by both Houses of Congress and was signed by the President on Saturday, 12 December It was headed:.
The term 'opium poppy' includes the plant Papaver somniferum , any other plant which is the source of opium or opium products, and any part of any such plant. It shall be unlawful for any person who is not the holder of a license authorizing him to produce the opium poppy, duly issued to him by the Secretary of the Treasury in accordance with the provisions of this Act, to produce or attempt to produce the opium poppy, or to permit the production of the opium poppy, in or upon any place owned, occupied, used, or controlled by him.
All licenses issued under this Act shall be limited to such number, localities, and areas as the Secretary of the Treasury shall determine to be appropriate to supply the medical and scientific needs of the United States for opium or opium products, with due regard to provision for reasonable reserves: Provided, however, that nothing contained in this Act shall be construed as requiring the Secretary of the Treasury to issue or renew any license or licenses under the provisions of this Act.
Any opium poppies which have been produced or otherwise obtained heretofore, and which may be produced or otherwise obtained hereafter in violation of any of the provisions of this Act, shall be seized by and forfeited to the United States.
In most states the Opium Poppy Control Act immediately ended commercial poppy cultivation, but in California it only produced protests and demands that the cultivation be licensed. The law was misunderstood by some at first, and was interpreted in the same sense as the California state law, which had resulted only in expansion of the poppy culture.
Thus a "United Press" dispatch published in some papers even had the heading "Opium Poppy to be grown", and went on to say: "President Roosevelt has signed legislation designed to promote production of the opium poppy in the United States, under close supervision of the Treasury and Agriculture Departments, to furnish a domestic source of supply of the narcotic for medical purposes". The real purpose of the law was the exact opposite-not to "promote production of the opium poppy" but to end it.
The Narcotics Bureau replied to inquiries as follows:. There is no immediate or presently prospective need for the growth of the opium poppy to supply medical and scientific needs, and, therefore, it is not now anticipated that any licenses will be issued. The California growers contended that their situation had not been sufficiently taken into consideration by Congress: the law, they said, was all right for mid-western or northern growers, but their seed was already in the ground when the law was passed 11 December , to say nothing of its effective date, 9 February The officials of the Narcotics Bureau had to admit some justice to this view.
Privately, they blamed the state authorities who had issued permits for poppy growing, even when the legislation was before Congress. Moreover, it was now learned that the United State Department of Agriculture had approved the poppy cultivation! Because of the war, the farmers were required to file, with this Department, their plans for the year's crop; when these plans included the production of poppy-seed for food purposes, they had been approved by the local representatives of the Department of Agriculture, without any question having been raised.
Nine permit-holders were not present; two wrote that they had not planted, two that they could not attend that day, and five had not been heard from at that time. The rather casual attitude of the farmers is illustrated by the explanation of one grower, without a permit, who attended the conference.
He said he had not asked for the state permit as he was very busy and wished to see first whether the seed would sprout or not. It was finally decided that the growers would be permitted to harvest their crop, under state and Federal supervision, and with destruction, as before, of all plant residues left after obtaining the seeds. Of course, neither the Federal nor the state officials anticipated that poppies would again be planted without Federal licences.
The Federal officials also expected that the state permits would henceforth be refused. Both these assumptions proved to be incorrect, leading to an apparent conflict between state and Federal authority, and to the so-called "Poppy Rebellion" of California.
Under date of 6 July , the Commissioner of Narcotics received a letter from the California Chief of the Division of Narcotics Enforcement no longer the same man as the one who had held this office when the cases began , stating:. This promise was not destined to be fulfilled. The last clause may have indicated a hope that the Narcotics Bureau would relent and license poppy cultivation: there was considerable political pressure toward this, but the Bureau remained adamant.
Early in December , the Chief of the Division of Narcotics Enforcement of California informed the Federal officials that the Attorney General of the state had handed down a decision which obliged him to issue state permits for the growing of opium poppies in California. This decision, which led to a conflict between state and Federal authority, was not addressed to the Division of Narcotics Enforcement, but was contained in a letter to the State Director of Agriculture, in reply to an inquiry of the latter.
The letter cast doubt on the Federal law:. You state that for some years growers have been engaged in the State in the production of blue poppy seeds for edible use pursuant to the provisions of Chapter 1, Division 10, of the Health and Safety Code. It appears from your letter that the variety so grown is distinct from that grown for the production of opium. It is suggested that while such poppy may come within the classification of Papaver Somniferum, it is actually not suitable for producing opium The state Narcotic Enforcement Office decided that they were obliged to issue state "permits", but attached the following warning to them:.
The office of the Attorney-General of California attempted to get this "warning" clause left off the permits, because it might seem to make a. At any rate, the growers were also warned by the United States Narcotics Bureau, as will shortly be related.
As of 5 February , the state Division of Narcotic Enforcement bad issued twenty-three permits to grow a total of 1, acres of opium poppies. Heretofore the growers had generally conceded that their poppies were opium poppies, i. The statement in the Attorney-General's letter, that the seed-producing poppy "is actually not suitable for producing opium", though not originating in his office, marked the inauguration of an intensified campaign to convince the public and Congress of the complete harmlessness of the seed-producing poppy-even in defiance of the facts-and to force the Narcotics Bureau to withdraw its opposition to poppy cultivation.
Some of the propagandists were self-deceived; some of the growers were undoubtedly convinced because they were now being told what they wanted to believe. The letter to the Attorney-General in November from the state Department of Agriculture had actually stated, "Apparently, under California climatic conditions, the morphine content is practically non-existent". Already, in December , an official of the state Department of Agriculture admitted; "We might have been a little over optimistic as to the lack of morphine content in said poppies"-but the campaign kept on.
In February , one of the growers wrote to the local office of the Narcotics Bureau: "We were advised by our State Attorney-General's Office, by the State Department of Agriculture and by the University of California, that, because of recent analyses, it is questionable owing to the lack of opium products in our California adapted edible blue-poppy seed [ sic ] whether we need any permit of any nature".
The issue, really a straight-out one of law observance, was being confused in many ways. Of course, the narcotics officials had not claimed that the poppy seed was narcotic, only that narcotics were inevitably produced in producing the poppy-seed. Even this was now being denied: a commercial chemical laboratory was found in San Francisco, which, with no previous experience in analysing for morphine, made a perfunctory examination of poppy capsules and stems, and was then willing to report, "we can find no trace of morphine".
Poppy growers wrote to their Congressmen, and several wrote to Henry Wallace, then Vice-President, whose interest in agricultural subjects was well known, asking them to intervene in favour of poppy cultivation.
One letter to the Vice-President declared:. Narcotics Bureau was, of course, unaware of any such "admissions". So far as the contention had any basis in fact, it may have referred to statements that the legal commercial production of opium could not profitably be made, for the legitimate market; but what was feared was illicit production for the illicit market, in which narcotics commanded prices many times higher than the values in the legal trade.
In , by direction of the Commissioner of Narcotics, a scientific study was made, chiefly, though not entirely, from the published literature, on Papaver somniferum and other poppies. It was shown that all varieties of Papaver somniferum that have been examined produce morphine, while no other plant is known to do so.
The reported isolation of morphine in the guise of "hopeine" from hops, in the last century, was a fraud and a myth.
A few erroneous analyses of some plants of the poppy family were made in the early days, but these were later corrected. It is probable that Papaver setigerum -the closest relative of Papaver somniferum -does produce morphine, but no record of a careful botano-chemical study was found. In any case, Papaver setigerum is only a rather rare wild flower, found in the Mediterranean region, occasionally grown in gardens but not grown commercially even for floral purposes.
Other poppies, such as the California poppy Eschscholzia californica , the Mexican poppy Argemone mexicana , the Oriental poppy Papaver orientale , and the common "corn poppy" or "field poppy" of Europe, the progenitor of the Shirley poppies Papaver rhoeas , have been analysed and other kinds of alkaloids found in them, but not morphine.
By original work it was shown that the Tulip poppy Papaver glaucum , though listed as a close relative of Papaver somniferum , is chemically more closely related to Papaver rhoeas, and contains no morphine.
In the beginning the Narcotics Bureau had proceeded largely by suasion, because there was not a firm legal basis for more vigorous action. Even after the Opium Poppy Control Act came into effect, the California farmers had been allowed to harvest their poppy-seed, because cultivation had been started before the law was enacted, and the violations were not wilful.
Now the violations were wilful-though supported, to a degree, by some state officials-and it was evident that effective action must be taken or the law acknowledged to be a complete failure. Beginning early in January , registered letters began to go out to the growers, warning them that they were in violation of the Federal law, regardless of state permits.
Some ploughed up their poppy-fields and planted something else; but others replied with form letters claiming that they had a right to produce poppyseed. They were banding together to fight the law. The growers now left were chiefly well-to-do men who were willing to gamble on having their poppy crop destroyed by the Government, in the hope of enormous profits in case they were able, to bluff out the Narcotics Bureau, or defeat the law in a court case.
They insisted on their "right" to produce a food crop regardless of narcotic dangers. What they refused to take into consideration was the fact that the reason the price of poppy-seed remained so high was that all other farmers were obeying the law-while they proposed to profit immensely by disobeying it. Early in March , the Commissioner of Narcotics recommended that the poppy plants in several fields be destroyed by narcotic agents, as a test case.
Largely because of the state permits, it was decided to act more slowly, through the Department of Justice. In this month the Secretary of the Treasury found it necessary to say, in reply to poppy propaganda sent to the Vice-President:.
This is incorrect. We have caused analyses to be made by both government and private chemists and have abundant proof that the blue poppies grown in California are opium poppies.
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