IN-FIELD COLOUR TEST FOR SCREENING FENTANYL AND ANALOGUES THEREOF
20230349935 · 2023-11-02
Inventors
- Shanlin FU (Ultimo, New South Wales, AU)
- Ronald Gibrail SHIMMON (Ultimo, New South Wales, AU)
- Mélanie Zoe LAURIA (Ultimo, New South Wales, AU)
- Joshua Charles KLINGBERG (Ultimo, New South Wales, AU)
- Laura Jane CLANCY (Ultimo, New South Wales, AU)
Cpc classification
International classification
Abstract
A method of detecting the presence of fentanyl or a fentanyl analogue in a sample which comprises contacting the sample with a 1,2 naphthoquinone (such as 1,2-naphthoquinone itself) in a solvent, preferably acetonitrile, and observing a colour change, which when present, correlates with the presence of fentanyl or a fentanyl analogue. Optionally, a buffer may be provided to maintain an alkaline environment and/or a metal halide catalyst and/or light source may be added to accelerate colour development.
Claims
1. A method of detecting the presence of fentanyl or a fentanyl analogue in a sample, the method comprising the step of contacting the sample with a reagent comprising a 1,2 naphthoquinone in a solvent and observing a colour change, which when present, correlates with the presence of fentanyl or a fentanyl analogue.
2. A method according to claim 1 wherein the 1,2-naphthoquinone is unsubstituted 1,2-naphthoquinone.
3. A method according to claim 1 wherein the solvent is a nitrile.
4. A method according to claim 1 wherein the solvent is acetonitrile
5. A method according to claim 1 further comprising the step of adding a buffer to maintain an alkaline environment.
6. A method according to claim 5 wherein the buffer is added to the sample prior to contacting the sample with the reagent comprising a 1,2 naphthoquinone.
7. A method according to claim 5 wherein the buffer is sufficient to provide freebase fentanyl or a freebase fentanyl analogue, for example, is pH 8.
8. A method according to claim 5 wherein the buffer is a phosphate buffer.
9. A method according to claim 1 wherein the reagent further includes a catalyst.
10. A method according to claim 9 wherein the catalyst is a metal halide.
11. (canceled)
12. A method according to claim 1 wherein the colour change is to green and occurs within 2 minutes of adding the sample to the reagent.
13. (canceled)
14. A method according to claim 1 wherein the colour change is to blue and occurs within 10 minutes of adding the sample to the reagent.
15.-16. (canceled)
17. A method according to claim 1 wherein the further comprising the step of exposing the reagent to a light source after the sample has been added.
18. A method according to claim 17 wherein the light source has enhanced emission at 365 nm.
19. (canceled)
20. A method according to claim 1 wherein the fentanyl or fentanyl analogue is a fentanyl analogue without a free OH group, such as β-hydroxyfentanyl
21. A reaction product of fentanyl and 1,2-naphthoquinine having the following structure: ##STR00049##
22.-23. (canceled)
24. A method according to claim 1 wherein the sample is an unknown drug sample.
25. A method according to claim 1 when used as a presumptive test for a suspected illicit substance, a suspected overdose, or in sport or workplace testing.
26.-27. (canceled)
28. A test reagent for detecting the presence of fentanyl or a fentanyl analogue in a sample, the reagent comprising 1,2-naphthoquinone, a solvent, and a buffer.
29. A test reagent according to claim 28 wherein the solvent is acetonitrile and the buffer is a phosphate buffer.
30.-33. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0041]
[0042]
[0043]
[0044]
[0045]
DESCRIPTION
[0046] There is currently no presumptive colour test that will correctly and rapidly identify fentanyl and fentanyl analogues compounds selectively from other illicit compounds.
[0047] The structure common to fentanyl and all fentanyl analogues contains two major functional groups that can be targeted as a possible site for the formation of coloured adducts: a piperidine ring (tertiary amine) and a tertiary amide.
[0048] Amines are known to react with quinones. A number of quinones were therefore screened as possible agents for presumptive testing. In each screening test, the fentanyl sample (150 μg) was treated with the quinone solution (400 μl of a 25 or 50 ppm solution) in n-propanol and toluene as solvents and observing any colour change. The quinones tested are shown in Table 1:
TABLE-US-00001 TABLE 1 Quinones investigated and their structures Benzoquinones 2,3,5,6-tetrafluoro-1,4- benzoquinone
[0049] Of the quinones tested, only four, namely 2,6-dichloro-1,4-benzoquinone, 2,5-dichloro-3,6-dihydroxy-1,4-benzoquinone (chloranilic acid), 2-hydroxy-1,4-naphthoquinone (lawsone) and 1,2-naphthoquinone (1,2-naphthoquinone) gave colour changes when tested on fentanyl. The remaining quinones showed no visible reaction of any interest.
[0050] The mere ability to form a coloured adduct is of course simply the first hurdle in providing a presumptive colour test. In order for a presumptive test to be useful, it must also have, among other things, a relatively low incidence of false positives against other substances, particularly those likely to be found in illicit drugs, for example, cutting agents and the like. Thus, the quinones which showed some evidence of a colour change with fentanyl were further tested against common contaminants such as lidocaine, quinine, 6-monoacetylmorphine base (6-MAM), paracetamol and caffeine in order to assess the quinone's specificity.
[0051] Lawsone, chloranilic acid and 2,6-dichloro-1,4-benzoquinone resulted in the same colour change observed with fentanyl when tested on lidocaine, quinine and 6-MAM (see Table 2), demonstrating that they were not fentanyl specific.
[0052] 1,2-naphthoquinone showed a colour change with fentanyl in both toluene and n-propanol. When tested in toluene it gave a pale orange colour and when in n-propanol it developed a pale blue colour overnight.
TABLE-US-00002 TABLE 2 reactions of quinones with fentanyl and others Quinone Blank Fentanyl 6-MAM Quinine Lidocaine 2,6-dichloro-1,4-benzoquinone p. yellow purple purple purple purple (in n-propanol) chloranilic acid pink purple purple purple purple (in n-propanol) 2-hydroxy-1,4-naphthoquinone yellow red-orange red-orange red-orange red-orange (in n-prOH and toluene) 1,2-naphthoquinone yellow p. orange NC NC p. orange (in toluene) 1,2-naphthoquinone p. yellow blue NC NC NC (in n-propanol) (overnight) NC: no colour change, p.: pale
[0053] The present inventors have shown that the nature of the substituents and the symmetry of the quinone are important factors driving the ability of such quinones to form coloured reaction products with fentanyl and its analogues with sufficient specificity and clarity to be useful as a presumptive test. Importantly, it seems that it is not possible to predict with any certainty which quinones, if any, would be useful in such a test. Surprisingly, the present inventors found that of all the quinones tested, only one, 1,2-naphthoquinone, showed promise as a presumptive test for fentanyl.
[0054] The structure of the blue reaction product was further investigated and is later detailed below.
[0055] A very important requirement for a presumptive test is reliability. It is important that the test does not have too many false negatives (in this case, testing negative for a significant number of fentanyl derivatives), or too many false positives (leading to the detention of too many suspects that will ultimately not lead to convictions).
[0056] Any solvent used is required to have good solvency for the quinone and the fentanyl or fentanyl analogue, be inert to the other agents present and ideally non-hazardous for human health. The solvent selection criteria were determined by considering both the shortest reaction time and the best contrast between the blank and the fentanyl colours.
[0057] As well as the toluene and n-propanol used as screening solvents, acetonitrile, dichloromethane, ethyl acetate, isopropanol and water were also tested to understand which one would provide the best environment for the reaction with fentanyl as well as the longest stability for 1,2-naphthoquinone. Solvents with high innate toxicity were not considered for that reason.
[0058] Toluene was not considered to be ideal given that the colour change from yellow to orange was not specific to fentanyl.
[0059] 1,2-naphthoquinone solutions in n-propanol were prepared and tested at concentrations of 50, 100, 150, 200, 300, 600 ppm in order to determine the optimal concentration for the reaction with fentanyl. A concentration of 50 ppm required 2 hours to provide a positive result, whereas a 600 ppm concentration gave a positive result after 45 minutes.
[0060] However, a 600 ppm 1,2-naphthoquinone solution led to an orange coloured blank. The blue reaction product was thus masked at high excesses of quinone and a dark green positive result with fentanyl resulted, which became dark when develop for additional time. 300 ppm gave a result after 30 minutes while 200 ppm and 150 ppm gave results around 20 minutes. The 150 ppm concentration was considered the best compromise between time and colour, given the yellow colour of the blank and the green colour of the positive result, which turns blue after approximately 2 hours. A lower concentration at 100 ppm was tested but it gave a result that was considered to be too pale, making it harder to distinguish a colour change.
[0061] 1,2-naphthoquinone in n-propanol (and also isopropanol) was found not to be suitably stable as a pre-prepared reagent. After 2-3 days storage, the reagent darkened and the reaction with fentanyl became slower, to the point where 1,2-naphthoquinone in n-propanol was not useable as a reagent after a few days.
[0062] For this reason, dichloromethane, ethyl acetate, water and acetonitrile were also tested. Of the solvents tested, only acetonitrile gave the desired colour change from clear/yellow to green/blue in a reasonable time frame. Acetonitrile was thus selected as the solvent of choice because it provided a sufficiently stable environment for 1,2-naphthoquinone while still enabling a sufficiently rapid reaction with fentanyl.
[0063] Various concentrations of acetonitrile were tested and again, as with n-propanol, 150 ppm was found to the preferred reagent concentration. The preferred final reagent was therefore a solution of 150 ppm of 1,2-naphthoquinone in acetonitrile.
[0064] Because the coloured adduct is believed to be very highly coloured, it will be detectable at small concentrations. The lower limits of the sample sizes typically expected for fentanyl and fentanyl analogues are of the order of a few tens of micrograms.
[0065] It is not unusual for fentanyl or fentanyl analogues to be present in salt form, given the presence of the amine group, so to ensure these are sufficiently soluble and reactive, a buffer can greatly facilitate the test procedure.
[0066] A variety of different buffers (0.1 M solutions) were tested: [0067] sodium phosphate buffer pH 8, [0068] potassium phosphate buffer pH 8, [0069] glycine-NaOH buffer pH 9.0, [0070] carbonate-bicarbonate buffer pH 9.2, [0071] borax-NaOH buffer pH 9.3, [0072] carbonate-bicarbonate buffer pH 10, [0073] Na.sub.2HPO.sub.4—NaOH buffer pH 10.9 and [0074] NaHCO.sub.3—NaOH buffer pH 11.
[0075] A few weak bases were also tested for the same purpose as 5% aqueous solutions (w/v): [0076] L-alanine (pH 8), [0077] ammonium hydroxide (NH.sub.4OH, pH 10), [0078] sodium carbonate (Na.sub.2CO.sub.3, pH 12).
[0079] A blank test was performed with buffer solution and 1,2-naphthoquinone reagent to determine whether the buffer reacted with 1,2-naphthoquinone. L-alanine was excluded due to the formation of a pasty yellow salt on contact with 1,2-naphthoquinone.
[0080] The buffers were tested against fentanyl. 150 μl of buffer or weak base solution was added to a vial containing 150 μg of analyte and the mixture was well shaken before adding the reagent (and catalyst as described below). The carbonate buffer pH 9.2 and 10, the glycine-NaOH buffer pH 9.0, the borax-NaOH buffer pH 9.3, the Na.sub.2HPO.sub.4—NaOH buffer pH 10.9 and the NaHCO.sub.3—NaOH buffer pH 11, and the weak bases ammonium hydroxide and sodium carbonate all failed to yield positive results with fentanyl and were thus excluded.
[0081] It was found that sodium phosphate pH 8 and potassium phosphate pH 8 buffers did not disturb the reaction between fentanyl freebase and its analogues with 1,2-naphthoquinone. The reaction of 1,2-naphthoquinone with fentanyl analogue salts in the presence of these bases provided very similar results to those observed with fentanyl. Sodium phosphate was chosen arbitrarily to be used for the further development and validation of the test, but a potassium phosphate buffer works equally well.
[0082] Ideally, presumptive or in-field tests provide not only a sensitive and specific test, but also a test which is relatively rapid in providing a colour change. A number of approaches were therefore tested with a view to reducing the time required to develop a visually detectable colour.
[0083] The effect of temperature on the reaction was tested. Conducing the reaction at temperatures of 40° C., 80° C. or 100° C. was not observed to produce any change in the speed of reaction.
[0084] Copper (II) chloride (CuCl.sub.2, aqueous) was tested as a possible catalyst for the reaction at concentrations of 0.19 mM, 1 mM, 1.9 mM, 3 mM, 4 mM, 5 mM and 10 mM.
[0085] A number of potential catalysts for the reaction were investigated, including copper (II) sulphate (CuSO.sub.4), copper (II) acetate (Cu(CH.sub.3COO).sub.2), iron (II) chloride (FeCl.sub.2) and cerium (III) chloride (CeCl.sub.3) catalysts as aqueous 2 mM solutions. When performing a test, 40 μl of a 2 mM catalyst solution was added to the vial containing the analyte and the reagent.
[0086] The investigation into a possible catalyst started with copper (II) chloride. The CuCl.sub.2 solutions were first tested with a reagent comprising 1,2-naphthoquinone 200 ppm in n-propanol which, without CuCl.sub.2, was taking 20 minutes to give a result. As a first screening 0.19 mM, 1.9 mM solutions were tested giving results after 20 minutes and after 10 minutes, respectively. A 10mM solution gave no colour change. This suggests that the ratio between fentanyl and CuCl.sub.2 is significant, and if there is a large excess of CuCl.sub.2 the reaction will not take place. Concentrations of 1.0 mM, 1.9 mM, 3.0 mM, 4.0 mM and 5.0 mM were tested using the chosen 1,2-naphthoquinone 150 ppm in acetonitrile and the 1.9 mM solution appeared to develop a colour change faster than the other concentrations.
[0087] The other catalysts were tested and yielded comparable results, FeCl.sub.2 was seen to provide a faster reaction (about 7 minutes) without buffer, but when a buffer was used, the speed of reaction was similar to that observed with CuCl.sub.2. A CuCl.sub.2 solution (1.9 mM) was finally chosen as the best way to increase the speed of reaction, bringing it from 20 minutes down to 10 minutes.
[0088] Further, it was surprisingly discovered that the reaction between 1,2-naphthoquinone and the fentanyl or fentanyl analogue does not occur if the mixture is kept in the dark, e.g. if placed in a box in a cupboard immediately after addition of the reagent to the sample.
[0089] The reaction was further investigated to determine whether a specific light source could be used to accelerate the development of a detectable colour. In these tests, a mixture of fentanyl and 1,2-naphthoquinone was exposed to a light source and checked for a colour change each minute for 20 minutes. Three different light sources were tested: [0090] Polilight® PL500 ROFIN (500 W) used on the white light setting, which ranges from 400 to 680 nm, and on UV setting 350 nm, [0091] UV handheld lamp used for the reading of thin layer chromatography plaques, model Spectroline® ENF-260C7FE (6 W) which has two settings, 365 and 254 nm and [0092] UV torch from Sirchie® (1 W), which has a wavelength of 365 nm.
[0093] The UV TLC lamp was found to be the most effective way to speed up the reaction with light. In fact, even though the polilight® (UV setting) and the UV torch also gave results, the UV TLC lamp was the only one to accelerate the results without changing the colours observed so far for this test. When using this lamp, after two minutes a green colour could be observed, compared to ten minutes without a light source. After 10 minutes the colour became blue, compared to two hours without this final step. The results of using a light source as the last step of the test are summarised in Table 3.
TABLE-US-00003 TABLE 3 Use of different light sources Light Source Blank Fentanyl Polilight ®, white light pink-orange orange-brown Polilight ®, UV 350 nm pink-orange green-grey UV TLC lamp, 360 nm yellow (NC) green (2-3 min) UV TLC lamp, 254 nm yellow (NC) NC UV torch, 365 nm pink-orange grey NC: no change compared to when no light source is used
[0094] In order to further enhance the usefulness of the present method, the optimal volumes were also investigated. A small amount of fentanyl or fentanyl analogue and a large volume of reagent, when there is significantly more 1,2-naphthoquinone than fentanyl, will result in the blue colour being more dilute and also masked by the colour of 1,2-naphthoquinone than with a smaller quantity of reagent.
[0095] For this reason, the volumes of buffer, 1,2-naphthoquinone solution and catalyst were optimised to have the smallest volume, which theoretically would also give a lower limit of detection.
[0096] The following buffer volumes were tested: 150, 100, 50, 40, 30, 20 μl. After the optimal buffer volume was chosen, 400, 300 and 200 μl of 1,2-naphthoquinone solution and 40, 30 and 20 μl of catalyst solution were tested in all their possible combinations.
[0097] Ideally, an in-field colour test would require the use of a single reagent only. Minimising the number of reagents was therefore attempted by testing a pre-combined buffer, 1,2-naphthoquinone and catalyst solution as well as a 1,2-naphthoquinone and catalyst solution. The optimal volumes chosen for the different reagents of the test were 40 μl of phosphate buffer (0.1 M, pH 8), 200 μl of 1,2-naphthoquinone (150 ppm in acetonitrile) and 40 μl of CuCl.sub.2 (1.9 mM (aq.)).
[0098] The naphthoquinone solution and the catalyst can be mixed together without a change in speed of reaction or in the colour change. However, when the buffer was premixed with the other two reagents, it took 30 minutes to observe a positive result. This is not ideal and it is better to add the buffer to the fentanyl or fentanyl analogue as a separate first step, followed by a second step of adding a reagent comprising a mixture of 1,2-naphthoquinone and catalyst in acetonitrile.
Summary of Optimal Method
[0099] The following general procedure is thus recommended (see
[0106] In addition to being able to reliably identify the class of substances targeted, a presumptive test should minimise the number of false positives, i.e. should not give positive results on other substances. This is particularly the case in drug tests, where target substances are often adulterated with a variety of household chemicals.
[0107] Sensitivity of a test is the true positive rate (that is, in this case, the percentage of fentanyls that react positively to the test) and specificity is the true negative result (that is, in this case, the percentage of other substances tested that did not produce a false positive test).
[0108] To test the sensitivity of the reagent for the fentanyl class, 21 fentanyl analogues were tested: acetylfentanyl HCl, acrylfentanyl HCl, alfentanil HCl, benzodioxolefentanyl, β-hydroxyfentanyl HCl, butyrylfentanyl HCl, 4-chloroisobutyrylfentanyl HCl, cyclopentylfentanyl HCl cyclopropylfentanyl, 4-fluoroisobutirylfentanyl HCl, furanylfentanyl HCl, phenylpropionylfentanyl, remifentanil, sufentanil citrate, ocfentanil, ortho-fluoro fentanyl, benzyl fentanyl, despropionyl para-fluoro fentanyl, 4-fluorobutyryl fentanyl, N-methyl norcarfentanil and norfentanyl. Their structures can be found in Table 4.
TABLE-US-00004 TABLE 4 Fentanyl and Analogues tested 10 Initial Minute Colour Colour Reaction Compound Structure Change Change (Yes/no) Blank N/A Yellow Yellow Fentanyl
[0109] The amount of analyte used for testing was 150 μg for all except acetylfentanyl, acrylfentanyl, butyrylfentanyl and 4-chloroisobutyrylfentanyl, where only about 25 μg was used due to limited availability.
[0110] Four analogues tested, alfentanil, remifentanil, β-hydroxyfentanyl and N-methyl norcarfentanil did not show a shift in colour.
[0111] Alfentanil, remifentanil and sufentanil (which reacted but gave a brown colour and took longer than others) are all therapeutical fentanyls used in medicine as anaesthetics and analgesics. To date, no misuse of Alfentanil, remifentanil and sufentanil has been reported (Patil Armenian, K. T. V. J. B.-W. K. L. L., 2018. Fentanyl, fentanyl analogs and novel synthetic opioids: A comprehensive review. Neuropharmacology, Issue 134, pp. 121-132). Without wishing to be bound by theory, these analogues all have a substituent on the carbon of the piperidine ring connected to the nitrogen of the amide group. If the reaction between 1,2-naphthoquinone and fentanyl is occurring on the tertiary amine, then it is theorised that a substitution on the piperidine ring creates too much of a hindrance for the reaction to occur at all. From the results of N-methyl norcarfentanil, a similar result is expected for the similarly substituted compound carfentanil as well.
[0112] In the case of β-hydroxyfentanyl, again without wishing to be bound by theory, it is possible that an intramolecular hydrogen bond between the hydroxyl group and the nitrogen of the piperidine group is formed and makes the nitrogen unavailable for other reactions.
[0113] It is possible that the change from a tertiary to secondary amine on the piperidine ring of norfentanyl accounts for the change in coloured product. In this case, a bright orange colour was formed, as opposed to the green present for the other derivatives.
[0114] The EMCDDA reports valerylfentanyl, 4-fluoroisobutyrylfentanyl, acryloylfentanyl (acryl fentanyl), cyclopentyl fentanyl and carfentanil as the fentanyl derivatives most seized in Europe in 2017 (EMCDDA, 2018. European Drug Report 2018: trends and development. Luxembourg: Publications Office of the European Union). All except carfentanil have substitutions close to the carbonyl of the amide group, like the other analogues tested that gave positive results. Therefore, it seems that many current designer fentanyls would be detected by the test.
[0115] To test the specificity of the test for the fentanyl class, a total of 102 other substances were tested. Among these were the most common illicit drugs 3,6-diacetylmorphine (heroin HCl), cocaine HCl, dexamphetamine HCl, methylenedioxymethamphetamine HCl (MDMA HCl), 6-monoacetylmorphine (6-MAM), methamphetamine HCl and morphine HCl. The rest were new psychoactive drugs (NPS), steroids, benzodiazepines, medicinal drugs, other powdered substances and common cutting agents including sugars, paracetamol and caffeine. The results are shown in Table 5.
TABLE-US-00005 TABLE 5 Results of specificity studies Initial Colour 5 min colour Compound Class Compound Change change Reference Blank Yellow Yellow Fentanyl NC Green Common Illicit Cocaine HCl NC NC Drugs Dexamphetamine HCl NC NC Diacetylmorphine (Heroin) HCl NC NC MDMA HCl NC NC Methamphetamine HCl NC NC 6-monoacetylmorphine base NC NC Morphine HCl NC NC Phenethylamines 4-ethyl-2,5-dimethoxyphenethylamine NC NC (2C—E) 4-iodo-2,5-dimethoxyphenethylamine NC NC (2C—I) 4-bromo-2,5-dimethoxy- NC NC phenethylamine (2C—Br) 25-H-NBOMe NC NC 25-E-NBOMe NC NC 25-I-NBOMe NC NC Synthetic Methcathinone HCl NC NC Cathinones Methylone HCl NC NC Ethylone HCl NC NC 4-methylmethcathinone HCl NC NC 4-fluoromethcathinone HCl NC NC 4-methylpyrrolidinopropiophenone HCl NC Bright Green* 4-methylpyrrolidinobutiophenone HCl NC Bright Green* Pyrovalerone HCl NC Green* (.fwdarw.bright green after 10 min) Synthetic UR-144 NC NC Cannabinoids JWH-073 NC NC Synthetic U49900 NC NC Opioids AH 7563 NC NC AH 7959 NC NC AH 8507 NC NC AH 8532 NC NC UF-17 NC NC Benzodiazepines Alprazolam NC NC Flunitrazepam NC NC Oxazepam NC NC Triazolam NC NC Steroids DHEA NC NC Drostanolone propionate NC NC Methandienone NC NC Stanozolol NC NC Sugars Artificial Sweetener NC NC Brown Sugar NC NC Cellulose NC NC d-Fructose NC NC d-Sorbitol NC NC Glucose NC NC Icing Sugar NC NC Inositol NC NC Lactose NC NC Maltose NC NC Mannitol NC NC Sucrose NC NC Other Drugs Allopurinol NC NC Aminophenazone Orange Red Amitriptyline HCl NC NC Aspirin NC NC Benzocaine NC Orange Caffeine NC NC Carbamazepine NC NC Codeine Phosphate NC NC Codral (paracetamol, NC NC pseudoephedrine, codeine) Doxepin HCl NC NC Ephedrine HCl NC NC Fluoxetine HCl (prozac) NC Orange Ibuprofen NC NC Lidocaine NC NC Metronidazole NC NC Panamax tablet, crushed (paracetamol NC NC and excipients) Paracetamol NC NC Phenacetine NC NC Phenobarbital NC NC Propanolol HCl NC NC Quinine NC NC Salycilamide NC NC Spronolactone NC NC Sulindac NC NC Tetramisole NC NC Tramal (tramadol HCl and excipients) NC NC Verapamil HCl NC Orange Other substances Ascorbic acid NC Clear/ Transparent Benzoic acid NC NC Boric acid NC NC Calcium chloride NC NC Citric acid NC NC Cornflour NC NC Creatine NC NC Dimethyl sulfonate NC NC Flour NC NC Indole Orange Orange Magnesium stearate NC NC Methylimidazole Bright Orange Bright Orange Methylpropionanilide NC NC Phenolphthalein NC NC Piperidine Orange Orange Potassium carbonate NC NC Pyrazole NC NC Pyridine NC NC Salt NC NC Sodium bicarbonate NC NC Sodium carbonate NC NC Sodium phosphate dibasic NC NC Starch NC NC Stearic acid NC NC Tartaric acid NC NC Thiamine NC NC
[0116] None of the illegal substances tested gave a reaction, and neither did the sugars or common cutting agent like paracetamol and caffeine.
[0117] Three cathinones produced a colour similar to the one given by fentanyl. It is noted that these three molecules contain a pyrrolidine group which is a cyclic tertiary amine, similar to the piperidine group present in fentanyl and its analogues. Nonetheless, these compounds can be distinguished because the reaction is much faster for these compounds, giving a bright green after 5 minutes for 4-methylpyrrolidinopropriophenone HCl (4-MPPP) and 4-methylpyrrolidinobutiophenone HCl (4-MPBP) and a green for pyrovalerone HCl.
[0118] To assess the selectivity of the reagent for fentanyl in the presence of other common contaminants, tests were done on mixtures of fentanyl and heroin HCl, fentanyl and cocaine HCl, fentanyl and methamphetamine HCl, fentanyl and MDMA HCl, fentanyl and paracetamol, fentanyl and caffeine, and fentanyl and maltose, where the amount of fentanyl was either 150 μg, 50 μg or 20 μg and its percentage in the mixtures varied between 66% and 5%. No difference in the positive result for fentanyl in the presence of contaminants was observed apart from paracetamol and MDMA. The results are shown in Table 6
TABLE-US-00006 TABLE 6 Purity study results Mixed Fentanyl product Fentanyl Percentage (μg) (μg) (%) Result.sup.a Heroin 300 150 66 green (10 m.) 150 150 50 green (10 m.) 75 150 33 green (10 m.) 20 20 50 green (15 m.) 60 20 25 green (15 m.) 180 20 10 p. green (15 m.), green (20 m.) 380 20 5 p. green (15 m.), green (20 m.) Cocaine 150 150 50 green (10 m.) 20 20 50 green (15 m.) 380 20 5 p. green (15 m.), green (20 m.) MDMA 150 150 50 green (10 m.) 450 150 25 p. green (20 m.), green (25 m.) 950 50 5 NC 20 20 50 p. green (20 m.), green (30 m.) 380 20 5 NC Methamphetamine 150 150 50 green (10 m.) 20 20 50 green (20 m.) 380 20 5 p. green (15 m.), green (20 m.) Paracetamol 75 150 66 p. green (10 m.), green (15 m.) 150 150 50 green (20 m.) 300 150 33 green (20 m.) 450 150 25 p. green (25 m.), green (50 m.) 950 50 5 NC 20 20 50 p. green (20 m.), green (30 m.) 380 20 5 NC Caffeine 150 150 50 green (10 m.) 20 20 50 green (15 m.) 380 20 5 green (20 m.) Maltose 150 150 50 green (10 m.) 20 20 50 green (15 m.) 380 20 5 green (20 m.) .sup.aNC = no colour change, p. = pale, m. = minutes
[0119] For fentanyl 50% in paracetamol, approximately 20 minutes was necessary to determine that a positive result was obtained. This is double the time of a test on pure fentanyl. A large amount of paracetamol can slow down or stop the reaction of 1,2-naphthoquinone and fentanyl, as it is seen when fentanyl is 5% or less of a mixture with paracetamol and no green colour is observed.
[0120] Regarding MDMA HCl, for a fentanyl mixture of 25% in MDMA, 20 minutes was necessary to see a positive result and no result is observed when fentanyl is 5% or less of the mixture.
[0121] Without any attempt at optimizing the volumes of the different reagents, it was found that a positive result was observed with as little as 50 μg of fentanyl. Therefore, to determine the lower limit of detection (LOD), 25, 20, 15, 10 and 5 μg of fentanyl were tested with the standard method with and without light source.
[0122] The lower the amount of fentanyl, the paler the green result was, and even if reacting, took a longer time to be declared as a positive result. For 25 μg and below, a positive result took around 15 minutes to be seen. When using the optimized volumes of the reagents, it was possible to lower the LOD to 15 μg. When using a light source, the LOD seems to be higher. There is no result when testing below 40 μg.
[0123] A solution of 1,2-naphthoquinone 150 ppm was prepared in acetonitrile to test its stability and was divided in 3 different scintillation vials that were closed with parafilm® and placed on a laboratory shelf, cupboard and refrigerator (5° C.) respectively. Similar samples of a 10:1 solution of 1,2-naphthoquinone 150 ppm and CuCl.sub.2 1.9 mM (aq.) were also prepared in order to understand if the 1,2-naphthoquinone and CuCl.sub.2 steps can be merged into one without compromising the stability of the reagent.
[0124] The solutions were used on fentanyl at 2 weeks' intervals and compared to the result of a fresh solution and between each other for up to ten weeks.
[0125] The results of the stability testing saw no significant change in speed of reaction for all reagents for up to 10 weeks for the 1,2-naphthoquinone solutions and for up to 8 weeks for the 1,2-naphthoquinone and CuCl.sub.2 solutions. The latter, after 10 weeks, showed a difference in the colour observed after reaction with fentanyl. In fact, the solution initially turned brown-green instead of the usual green. This change delayed the determination of a positive result by a couple of minutes.
[0126] This is mitigated after additional time and in the end all tests developed the blue colour characteristic of the reaction with fentanyl with no differences between them or a fresh reagent. The solutions can still be used to give a positive result after 10 weeks and the effect on the speed of reaction could be mitigated if a light source is used to accelerate it.
[0127] The method of the present invention are useful visual tests because of the colour change. However, the test lends itself to being conducted in a more automated or standardised manner if desired.
[0128] A device suitable for detection of the colour change is shown in
[0129] In
[0130] In
[0131] A particular embodiment of the test kit is shown in
[0132] The lower tip shown in
[0133] A potential drawback of any test that relies upon inspection by the naked eye is that there can be numerous factors impacting upon what would be considered to constitute a determinative colour change. This can be particularly challenging in the case of field tests for drugs where the lighting conditions and stresses on the analyst would generally be much less favourable than those in the laboratory. Instrumental analysis can help overcome this potential problem. For this reason, it was considered worthwhile to identify the blue adduct formed between fentanyl and 1,2-naphthoquinone.
[0134] In order to investigate the nature of the reaction occurring between fentanyl and 1,2-naphthoquinone, and propose a reaction mechanism, the blue colour compound was investigated with several techniques. The blue product was first isolated using solvent extraction by adding ethyl acetate and water, the organic (blue) layer was kept and dried under nitrogen stream, the solid was tested using FTIR, along with comparison samples of 1,2-naphthoquinone and fentanyl.
[0135] FTIR indicated that no major changes to the tertiary amine and amide of the fentanyl or ketones of the 1,2-naphthoquinone functional groups occurred. A tertiary amine nitrogen atom is not connected to a hydrogen atom and therefore does not have characteristic peaks in FTIR, unlike secondary and primary amines. Fentanyl contains a tertiary amine and it was possible to see that in the final product, no peaks that would indicate the formation of a secondary or primary amine were seen, neither were peaks indicating the presence of alcohol or aldehyde. It seems therefore that all the functional groups of fentanyl and 1,2-naphthoquinone remain unaltered in the final blue product that is formed by their reaction. For QTOF analysis, the blue isolated product was reconstituted in acetonitrile (1.5 ml total), filtered and directly injected without any previous method of separation; this was done for a reaction that occurred in the presence of CuCl.sub.2 and for one that occurred without CuCl.sub.2.
[0136] QTOF analysis showed the molecular ion of the blue product at 491 m/z ([M+H].sup.+), therefore the mass of the blue compound is 490 Da, which is the sum of fentanyl and 1,2-naphthoquinone masses minus 4 mass units. Without wishing to be bound by theory, this suggests a reaction causing a loss of 4 hydrogen atoms which could occur by the formation of two double bonds. It is thought that the site of reaction is the nitrogen of the piperidine ring of fentanyl. A possible mechanism of reaction is that firstly a quaternary amine is formed in this site, leading to the opening of the piperidine ring and the formation of a double bond. The amine could then react in para position with 1,2-naphthoquinone, forming the blue product. However, this theory cannot explain how another double bond is formed, and two additional hydrogen atoms are lost. The tentative structure for the blue product is shown below.
##STR00048##
[0137] Even though additional investigation may be necessary to fully understand the mechanism of reaction, the QTOF analyses allow the conclusion with certainty that the same product is formed with or without CuCl.sub.2 being present, which is indeed acting as a catalyst, speeding up the reaction but not being part of the final product.
[0138] A similar reaction was modelled using pyrovalerone as a substitute for fentanyl. The results found for the pyrovalerone was analogous to those for fentanyl. The molecular mass found also differs by 4 units from the sum of 1,2-naphthoquinone and pyrovalerone mass. This synthetic cathinone contains a tertiary amine in the form of a pyrrolidine ring, which is very similar to the piperidine structure of fentanyl. These two factors suggest that the same mechanism of reaction occurs and gives more weight to the theory that the reaction occurs on the tertiary cyclic amine.
[0139] A small amount of the blue isolated solid was re-diluted in acetonitrile and tested to obtain its UV-Vis spectrum, 1,2-naphthoquinone in acetonitrile and the solution of 1,2-naphthoquinone and CuCl.sub.2 were also tested in UV-Vis to understand if the peaks of absorption of the reagent and those of the final product are well separated and could potentially be monitored by a portable spectrophotometer to determine a positive result of the test.
[0140] The reaction was also monitored to understand if the peak associated with the blue product would be visible before the blue colour becomes visible with the naked eye, which could potentially shorten the time necessary to declare a positive result.
[0141] 1,2-naphthoquinone has two peaks of maximum absorbance at 397 nm and 340 nm while the blue product has a broad characteristic peak at 584 nm. The peaks are very well separated. When the mixture of 1,2-naphthoquinone and CuCl.sub.2 is analysed there is an additional peak around 251 attributable to CuCl.sub.2.
[0142] The monitoring of the absorbance at 584 nm for the reaction of 150 μg of fentanyl with the standard recommended amount of reagent can be seen in
[0143] Other tests were performed to understand if a peak would be visible for an amount of fentanyl below the 15 μg LOD. Theoretically, when a low amount of fentanyl is present, it does react with 1,2-naphthoquinone but the blue colour of the product is not visible because it is masked by the yellow colour of large amounts of 1,2-naphthoquinone. A blank was tested alongside reagents mixed with 10, 15, 25 and 40 μg of fentanyl left to react for 15 minutes. The wavelength associated to the absorbance peak for the blue product was monitored. The blank was then compared to the responses of the mixtures to understand if a significant difference could be detected.
[0144] The reaction of a mixture of fentanyl (5%) and paracetamol was monitored with UV-Vis, paracetamol has been found to quench the reaction and no blue colour is visible to the naked eye.
[0145] When monitoring the reaction of 10 μg of fentanyl, a quantity found to be below the LOD of the test, no difference in absorbance at 584 nm was observed. The same was seen when monitoring the reaction of a mixture of fentanyl (5%) and paracetamol with 1,2-naphthoquinone. Paracetamol has been found to quench the reaction and no blue colour is visible to the naked eye, no significant increase in absorbance was observed either, only normal fluctuations of the baseline. A more sensitive instrument adapted to allocate a small cuvette would be necessary to carry out satisfactory experiments to understand if monitoring the absorbance at 584 nm could potentially detect the reaction below the 15 μg of fentanyl limit of detection or when other substances are present.
[0146] Non-limiting examples of some colorimetric analysis devices envisaged to be suitable for carrying out the tests of the present invention are shown in
[0147] The specific device in 4A and 4A′ has two opposed windows to allow a light from a light source 51 to enter from a first window 52 and exit to a detector 53 at an opposed window 54. The detector could be any sort of visible detector, or even a simple RGB detector. In
[0148] Alternatively, the device as shown in 4B and 4B′ could have a single window 60 with an opposed reflective portion 62, where the light enters and exits via the same window 60, after passing twice through the sample, before entering detector 53. 3B′ also has a region of reduced cross section 55.
[0149] Alternatively, the device as shown in 4C and 4C′ could be configured for handheld use. The device has a through passage 70 to enable visual inspection of the tube 22 (or reduced portion 55) without interference. Light passing through the sample is simply observed with the naked eye. This embodiment could advantageously contain a reference sample for side-by side comparison.
[0150] Colorimetric devices according to the present invention can be made in portable or mobile form, and the accompanying software can be configured to analyse the output of the RGB or other detector and provide simply a positive or negative result for the presence of fentanyl or a fentanyl analogue.