Molecular weight: 134.1751
IUPAC Standard InChI: InChI=1S/C9H10O/c1-8(10)7-9-5-3-2-4-6-9/h2-6H,7H2,1H3
IUPAC Standard InChIKey: QCCDLTOVEPVEJK-UHFFFAOYSA-N CAS Registry Number: 103-79-7 Chemical structure:
This structure is also available as a 2d Mol file or as a computed3d SD file
1-Phenyl-2-propanone (P-2-P), also known as benzyl methyl ketone (BMK), is a colorless or slightly yellowish liquid. It presents a density similar to that of water as well as a pleasant scent. Even if there are few legitimate uses of BMK such as in the production of the pharmaceutical drug propyl-hexedrine, most frequently BMK is used as an illicit compound for the illegal manufacture of amphetamine.Actually, BMK is identiﬁed by classical methods such as gas chromatography, NMR or HPLC. These methods are costly, time-consuming and require the presence of trained operators. It appears obvious that there is an urgent need to develop a new easy and fast method that allows us to detect the presence of traces of BMK.
In this work, a new chemically synthesized BMK derivative covalently attached to animmunological carrier was used for producing antibodies against the BMK molecules. A ﬂuorescencepolarization-based bioassay was developed by using the produced anti-BMK antibodies and the BMK derivative. The assay exhibits interesting analytical performances with a limit of detection of less than100 nM and an almost linear response up to 600 nM. Interestingly, the proposed assay could be performed using a customizable portable instrumentation and could be used by non-instructed personnel at custom borders and checkpoints or for quick spot-checks. Introduction Among the European countries, Poland, Belgium and the Netherlands are the main countries in which amphetamine is manufactured for the illicit market. Several clandestine laboratories manufacturing amphetamine by means of Leuckart1synthesis are closed down every year. In order to prevent illicit drug manufacture, law enforcement authorities strive to reach illicit producers and ‘‘retailers’’ of the main drug precursor, 1-phenyl-2-propanone (P-2-P), also known as benzyl methylketone (BMK).1BMK is a colorless or slightly yellowish liquid, even if the colorof the illicitly manufactured BMK may vary from yellow to dark brown. It has a density similar to that of water as well as a pleasant scent. BMK is most frequently used as a precursor for the manufacture of amphetamine. The legitimate use of BMK in thechemical and pharmaceutical industries is limited to the manufacture of amphetamine and methamphetamine and their derivatives. An additional legitimate use of BMK is the production of benzyl radicals, through photolysis, which in turn are used for the production of propyl-hexedrine.
In Turkey and the United States, BMK is also found in cleaning agents and stain removers. In addition, BMK is used as a precursor for the illicit manufacture of amphetamine and methamphetamine. It is estimated that nearly100% of amphetamine and approximately 10% of methamphetamine are manufactured from illicit sources of BMK.2In order to mislead law enforcement authorities, criminal soften organize illicit deliveries via countries that are not usually related to illicit drug production. Quantities of BMK smuggled in a single shipment range from a few kilograms to several tons. As regards the modus operandi, there have been instances in which criminal organizations used names of existing companies that were completely unaware that they were named as end-recipients in the customs documents or letters of conveyance. In the majority of cases, however, criminals employed the names of companies regularly importing goods from the countries producing BMK, and the goods declared in the customs documents were consistent with the companies’ business proﬁles.3Actually, the methods used to detect BMK are mainly based ongas-chromatography methodologies, NMR and additional time-consuming analyses. Recently, a new method based on proﬁlingof the impurities of BMK has been developed. This method allowed to ascertain from which country BMK was synthesized. It appears evident that there is an urgent necessity to develop anew easy methodology for the fast detection of BMK. In this work we present a new polarization ﬂuorescence assay for BMK detection. Synthesized ﬂuorescence BMK derivative and speciﬁc antibodies generated against the analyte. The limit of detection (LOD) ofthe assay is 50 nM. Importantly, the whole apparatus used forour experiments could be replaced by a small and cheap optical device, which is capable of revealing polarization ﬂuorescencechanges upon interaction between the BMK derivative and anti-BMK antibodies.
Materials and methods All reagents were of the highest commercially available qualityand used as received. 1-[3-(Dimethylamino)-propyl]-3-ethyl-carbodiimide (EDC), bovine serum albumin (BSA; fraction V),carboxymethoxylamine hemihydrochloride, benzyl methylketone (BMK), EAH Sepharose 4B resin and buffers. Goat polyclonal to rabbit IgG–HRP conjugate(secondary antibody) was from Abcam. The NMR spectra of the BMK derivative were recorded on a Varian Gemini 200(200 MHz). Synthesis of (1-methyl-2-phenyl-ethylideneaminooxy)acetic acid Benzyl methyl ketone (99% Sigma Aldrich, 612 mL, 4.56 mmol)was reﬂuxed with ten equivalents of o-carboxymethoxylaminehemihydrochloride (99% Sigma Aldrich, 5.00 g, 45.7 mmol) in6 mL of pyridine–H2O–methanol (1 : 1 : 4) for 2 h. The reaction was followed by thin layer chromatography (n-hexane–ethylacetate 8 : 2 + HCOH drops). Pyridine and other solvents were removed by toluene co-distillation by a rotavapor. The product of the reaction was re-suspended in 5 mL of H2O at pH 9.00basiﬁed with NaOH and washed with 3 10 mL of dichloro-methane. Then the aqueous phase was acidiﬁed with HCl to pH3.0 and the product extracted in 3 15 mL of dichloromethane.The solvent was dried over NaSO4and vacuum distilled to leave an oily product. In order to remove reaction by-products and the excess of carboxymethoxylamine, ﬂash chromatography was performed on a silica column (silica gel 60 M 0.04–0.0063 mm)with eluent CH2Cl2–CH3OH 9 : 1 + HCOOH drops (isolated yield 45%).1H NMR (400 MHz, CDCl3): d 1.82 ppm (s, 3H), d3.48 ppm (s, 1H), d 3.76–3.78 ppm (m, 1H), d 4.67 ppm (s, 2H), d7.40–7.70 ppm (m, 5H).
Synthesis of BSA conjugate (BMK–BSA)To avoid interference by the carrier protein in the polyclonal antibody detection process, BMK-oxime was conjugated to the serum albumin (BSA). The following procedure was used: 0.484mmol BMK-oxime was dissolved in ethanol and mixed with anaqueous solution of 4.84 mmol EDC. The solution was diluted upto 840 mL with 10 mM phosphate buffer at pH 6 and incubatedfor 20 min at room temperature. Finally the solution was incu-bated with 4.84 nM BSA dissolved in 160 mL of 10 mM phos-phate buffer at pH 6. The reaction mixture was incubated at room temperature under continuous stirring and then dialyzedagainst 0.5 L of 10 mM phosphate buffer at pH 7.4 for 4 days with daily buffer changes. Antibody production and IgG puriﬁcation A rabbit was immunized following a standard protocol by intra-dermal injection. After the immunization period, the rabbit was sacriﬁced and its blood collected and centrifuged to separate blood cells from serum. A 2.0 mL sample of serum of the rabbit was diluted 1 : 1 in 50 mM Tris–HCl at pH 7.0 (binding buffer)and applied to 0.5 mL of resin protein A SepharoseTM 4 Fast Flow (GE Healthcare). The IgG fraction was puriﬁed according to the manufacturer’s instructions. The IgG fraction was eluted with glycine (0.1 M) at pH 2.8 and immediately buffered with1.0 M Tris–HCl at pH 8.8. Elution of IgG proteins was monitored by absorbance at l¼278 nm and SDS PAGE was carried out to evaluate the purity of the samples (data not shown). The obtained samples were collected and dialyzed against 10 mMPBS at pH 7.4. Afﬁnity column preparation of BMK–EAH Sepharose 4B The afﬁnity column was obtained by conjugating the derivative BMK-oxime to EAH Sepharose 4B as follows. A 1.2 mL sample of the resin was washed with H2O at pH 4.5 (160 mL), with 0.5 MNaCl (100 mL), and again with H2O at pH 4.5 (100 mL). The Sepharose resin was ﬁnally packed into a polystyrene column(10 mL, BIORAD) suspended in 2.0 mL of H2O at pH 4.5 andthe resulting suspensions were gently shaken. 15.3 mmol BMK-oxime was diluted in 2 mL of ethanol and 400 mmol EDC (H2OpH 4.5) to a ﬁnal concentration of 0.1 M. The reaction solution was mixed and incubated for 10 min at RT. The solution of BMK oxime–EDC was added to the slurry resin and incubated with gentle shaking for 2 h at room temperature. The pH of thesolution was monitored and 40 mmol EDC was added to the solution and the slurry resin solution (50% ethanol) was incubated with gentle shaking overnight at 4 C. The slurry resin solution was extensively washed with H2O at pH 4.5, 0.5 M NaClin 50% ethanol (15 mL). The slurry resin in 50% ethanol was previously treated with 15 mL of 0.1 M AcOH at pH 4.0, 0.5 MNaCl in 50% ethanol (blocking buffer) and later with 0.1 M Tris–HCl at pH 7.0, 0.5 M NaCl in 50% ethanol (wash buffer). After this step the resin was washed with the blocking buffer and incubated for 30 min at room temperature. Then the slurry resin solution was treated with 15 mL of the wash buffer, 15 mL of the blocking buffer and again with the wash buffer and ﬁnallywashed with 10 mL of 50 mM Tris–HCl at pH 7.0.Antibody puriﬁcation by afﬁnity chromatography In the afﬁnity chromatography puriﬁcation on EAH–BMK resin, 8.0 mg of IgG (2.0 mg mL1) was diluted 1 : 1 in 50 mMTris–HCl at pH 7.0 (binding buffer) and incubated with the afﬁnity column. The column, before elution, was washed with 20column volumes of 50 mM Tris–HCl at pH 7.0 to eliminate unspeciﬁc antibodies. For the elution step was used 0.1 Mglycine–HCl at pH 3.0 (5 mL) and sample fractions of 1 mL were collected and monitored by absorbance measurements at l¼278 nm. The fractions containing the antibodies were collected and dialyzed against 10 mM buffer phosphate at pH 7.4.