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Determination of benzyl methyl ketone - A commonly used precursor in

amphetamine manufacture

Article  in  Analytical Methods · October 2012

DOI: 10.1039/C2AY25772F

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Analytical
Methods
www.rsc.org/methods Volume 4 | Number 11 | November 2012 | Pages 3499–3898
Published on 22 August 2012 on http://pubs.rsc.org | doi:10.1039/C2AY25772F
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Showcasing research into high-throughput sialic acid As featured in:

assays by Dr Shashi Prajapati and colleagues from the
Cell Culture Development – High-Throughput Analytical
Group, Biogen Idec Inc, Cambridge, MA, USA.

Improvement in accuracy and specificity of high-throughput

sialic acid assay

The efficacy of many therapeutic proteins is influenced by their sialic

acid content. Here, we used high-throughput protein purification
and denaturation to improve the accuracy and specificity of a high-
throughput sialic acid assay. The assay can accurately, precisely, rapidly
and specifically analyze 80 crude culture samples in parallel, with
See Prajapati et al., Anal. Methods,
a quantitation limit of 1 μM sialic acid, and could be used for many
2012, 4, 3565–3569.
applications in cell line and bioprocess development.

ISSN 1759-9660
www.rsc.org/methods
PAPER Registered Charity Number 207890
D’Auria et al.
Determination of benzyl methyl ketone – a commonly used precursor in
amphetamine manufacture
 Analytical Dynamic Article Links < C
Methods
Cite this: Anal. Methods, 2012, 4, 3558
www.rsc.org/methods PAPER
Determination of benzyl methyl ketone – a commonly used precursor in
amphetamine manufacture
Published on 22 August 2012 on http://pubs.rsc.org | doi:10.1039/C2AY25772F

Stefano Di Giovanni,†a Antonio Varriale,†a Vincenzo Manuel Marzullo,a Giuseppe Ruggiero,a Maria Staiano,a
Alberto Secchi,b Luigi Pierno,b Anna Maria Fiorellob and Sabato D’Auria*a
Received 18th July 2012, Accepted 21st August 2012
DOI: 10.1039/c2ay25772f

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,
Downloaded on 07 December 2012

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 an
immunological carrier was used for producing antibodies against the BMK molecules. A fluorescence
polarization-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 than
100 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 BMK is also found in cleaning agents and stain removers. In

addition, BMK is used as a precursor for the illicit manufacture of
Among the European countries, Poland, Belgium and the amphetamine and methamphetamine. It is estimated that nearly
Netherlands are the main countries in which amphetamine is 100% of amphetamine and approximately 10% of methamphet-
manufactured for the illicit market. Several clandestine labora- amine are manufactured from illicit sources of BMK.2
tories manufacturing amphetamine by means of Leuckart1 In order to mislead law enforcement authorities, criminals
synthesis are closed down every year. In order to prevent illicit often organize illicit deliveries via countries that are not usually
drug manufacture, law enforcement authorities strive to reach related to illicit drug production. Quantities of BMK smuggled
illicit producers and ‘‘retailers’’ of the main drug precursor, 1- in a single shipment range from a few kilograms to several tons.
phenyl-2-propanone (P-2-P), also known as benzyl methyl As regards the modus operandi, there have been instances in
ketone (BMK).1 which criminal organizations used names of existing companies
BMK is a colorless or slightly yellowish liquid, even if the color that were completely unaware that they were named as end-
of the illicitly manufactured BMK may vary from yellow to dark recipients in the customs documents or letters of conveyance. In
brown. It has a density similar to that of water as well as a pleasant the majority of cases, however, criminals employed the names of
scent. BMK is most frequently used as a precursor for the companies regularly importing goods from the countries
manufacture of amphetamine. The legitimate use of BMK in the producing BMK, and the goods declared in the customs docu-
chemical and pharmaceutical industries is limited to the manu- ments were consistent with the companies’ business profiles.3
facture of amphetamine and methamphetamine and their deriv- Actually, the methods used to detect BMK are mainly based on
atives. An additional legitimate use of BMK is the production of gas-chromatography methodologies, NMR and additional time-
benzyl radicals, through photolysis, which in turn are used for the consuming analyses. Recently, a new method based on profiling
production of propyl-hexedrine. In Turkey and the United States, of the impurities of BMK has been developed. This method
allowed to ascertain from which country BMK was synthesized.4
a
Laboratory for Molecular Sensing, Institute of Protein Biochemistry, It appears evident that there is an urgent necessity to develop a
CNR, Via Pietro Castellino 111, 80131 Naples, Italy. E-mail: s.dauria@ new easy methodology for the fast detection of BMK.
ibp.cnr.it; Fax: +39-0816132277; Tel: +39-0816132250
b
Selex-SI, Via Tiburtina, Rome, Italy
In this work we present a new polarization fluorescence assay
† These authors equally contributed to the work. for BMK detection. The assay is based on the use of an ad hoc

3558 | Anal. Methods, 2012, 4, 3558–3564 This journal is ª The Royal Society of Chemistry 2012
 synthesized fluorescence BMK derivative and specific antibodies Antibody production and IgG purification
generated against the analyte. The limit of detection (LOD) of
A rabbit was immunized following a standard protocol by intra-
the assay is 50 nM. Importantly, the whole apparatus used for
dermal injection. After the immunization period, the rabbit was
our experiments could be replaced by a small and cheap optical
sacrificed and its blood collected and centrifuged to separate
device, which is capable of revealing polarization fluorescence
blood cells from serum. A 2.0 mL sample of serum of the rabbit
changes upon interaction between the BMK derivative and anti-
was diluted 1 : 1 in 50 mM Tris–HCl at pH 7.0 (binding buffer)
BMK antibodies.5,6
and applied to 0.5 mL of resin protein A SepharoseTM 4 Fast
Flow (GE Healthcare). The IgG fraction was purified according
Materials and methods to the manufacturer’s instructions. The IgG fraction was eluted
Published on 22 August 2012 on http://pubs.rsc.org | doi:10.1039/C2AY25772F

All reagents were of the highest commercially available quality with glycine (0.1 M) at pH 2.8 and immediately buffered with
and used as received. 1-[3-(Dimethylamino)-propyl]-3-ethyl- 1.0 M Tris–HCl at pH 8.8. Elution of IgG proteins was moni-
carbodiimide (EDC), bovine serum albumin (BSA; fraction V), tored by absorbance at l ¼ 278 nm and SDS PAGE was carried
carboxymethoxylamine hemihydrochloride, benzyl methyl out to evaluate the purity of the samples (data not shown). The
ketone (BMK), EAH Sepharose 4B resin and buffers were obtained samples were collected and dialyzed against 10 mM
purchased from Sigma-Aldrich. The fluorescent probe CF488A PBS at pH 7.4.
was purchased from Biotium Inc. Nitrocellulose transfer
membrane Protran from Schleicher & Schuell and ECL detection Affinity column preparation of BMK–EAH Sepharose 4B
reagents from Amersham Biosciences were used in Western blot
The affinity column was obtained by conjugating the derivative
experiments. Goat polyclonal to rabbit IgG–HRP conjugate
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BMK-oxime to EAH Sepharose 4B as follows. A 1.2 mL sample

(secondary antibody) was from Abcam. The NMR spectra of the
of the resin was washed with H2O at pH 4.5 (160 mL), with 0.5 M
BMK derivative were recorded on a Varian Gemini 200
NaCl (100 mL), and again with H2O at pH 4.5 (100 mL). The
(200 MHz).
Sepharose resin was finally packed into a polystyrene column
(10 mL, BIORAD) suspended in 2.0 mL of H2O at pH 4.5 and
Synthesis of (1-methyl-2-phenyl-ethylideneaminooxy)acetic acid
the resulting suspensions were gently shaken. 15.3 mmol BMK-
Benzyl methyl ketone (99% Sigma Aldrich, 612 mL, 4.56 mmol) oxime was diluted in 2 mL of ethanol and 400 mmol EDC (H2O
was refluxed with ten equivalents of o-carboxymethoxylamine pH 4.5) to a final concentration of 0.1 M. The reaction solution
hemihydrochloride (99% Sigma Aldrich, 5.00 g, 45.7 mmol) in was mixed and incubated for 10 min at RT. The solution of BMK
6 mL of pyridine–H2O–methanol (1 : 1 : 4) for 2 h. The reaction oxime–EDC was added to the slurry resin and incubated with
was followed by thin layer chromatography (n-hexane–ethyl gentle shaking for 2 h at room temperature. The pH of the
acetate 8 : 2 + HCOH drops). Pyridine and other solvents were solution was monitored and 40 mmol EDC was added to the
removed by toluene co-distillation by a rotavapor. The product solution and the slurry resin solution (50% ethanol) was incu-
of the reaction was re-suspended in 5 mL of H2O at pH 9.00 bated with gentle shaking overnight at 4  C. The slurry resin
basified with NaOH and washed with 3
10 mL of dichloro- solution was extensively washed with H2O at pH 4.5, 0.5 M NaCl
methane. Then the aqueous phase was acidified with HCl to pH in 50% ethanol (15 mL). The slurry resin in 50% ethanol was
3.0 and the product extracted in 3
15 mL of dichloromethane. previously treated with 15 mL of 0.1 M AcOH at pH 4.0, 0.5 M
The solvent was dried over NaSO4 and vacuum distilled to leave NaCl in 50% ethanol (blocking buffer) and later with 0.1 M Tris–
an oily product. In order to remove reaction by-products and the HCl at pH 7.0, 0.5 M NaCl in 50% ethanol (wash buffer). After
excess of carboxymethoxylamine, flash chromatography was this step the resin was washed with the blocking buffer and
performed on a silica column (silica gel 60 M 0.04–0.0063 mm) incubated for 30 min at room temperature. Then the slurry resin
with eluent CH2Cl2–CH3OH 9 : 1 + HCOOH drops (isolated solution was treated with 15 mL of the wash buffer, 15 mL of the
yield 45%). 1H NMR (400 MHz, CDCl3): d 1.82 ppm (s, 3H), d blocking buffer and again with the wash buffer and finally
3.48 ppm (s, 1H), d 3.76–3.78 ppm (m, 1H), d 4.67 ppm (s, 2H), d washed with 10 mL of 50 mM Tris–HCl at pH 7.0.
7.40–7.70 ppm (m, 5H).
Antibody purification by affinity chromatography
Synthesis of BSA conjugate (BMK–BSA)
In the affinity chromatography purification on EAH–BMK
To avoid interference by the carrier protein in the polyclonal resin, 8.0 mg of IgG (2.0 mg mL1) was diluted 1 : 1 in 50 mM
antibody detection process, BMK-oxime was conjugated to the Tris–HCl at pH 7.0 (binding buffer) and incubated with the
serum albumin (BSA). The following procedure was used: 0.484 affinity column. The column, before elution, was washed with 20
mmol BMK-oxime was dissolved in ethanol and mixed with an column volumes of 50 mM Tris–HCl at pH 7.0 to eliminate
aqueous solution of 4.84 mmol EDC. The solution was diluted up unspecific antibodies. For the elution step was used 0.1 M
to 840 mL with 10 mM phosphate buffer at pH 6 and incubated glycine–HCl at pH 3.0 (5 mL) and sample fractions of 1 mL were
for 20 min at room temperature. Finally the solution was incu- collected and monitored by absorbance measurements at l ¼
bated with 4.84 nM BSA dissolved in 160 mL of 10 mM phos- 278 nm. The fractions containing the antibodies were collected
phate buffer at pH 6. The reaction mixture was incubated at and dialyzed against 10 mM buffer phosphate at pH 7.4. The
room temperature under continuous stirring and then dialyzed concentration of the antibodies was spectrophotometrically
against 0.5 L of 10 mM phosphate buffer at pH 7.4 for 4 days determined by absorbance measurements at l ¼ 278 nm, using
with daily buffer changes. 31% ¼ 14.

This journal is ª The Royal Society of Chemistry 2012 Anal. Methods, 2012, 4, 3558–3564 | 3559
 Western blot experiments dialysis tubes with a cut-off of 3500 Da (Spectrum Labs) over-
night at 4  C.
The proteins (4.0 mg of each protein) were loaded, separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%
Steady-state fluorescence measurements
SDS-PAGE), and then transferred overnight at 4  C onto a
PVDF membrane. The membrane was blocked by 50 mL of the Steady state fluorescence experiments were carried out on an FP-
blocking buffer (TBS containing 5% skimmed milk) for 1 h at 8600 Fluorescence Spectrometer (Jasco) equipped with a one-cell
room temperature. After three washings with TBS–Tween (TBS- temperature controlled sample holder. For BMK–BSA-488
Tween 0.05%, 10 minutes per washing), the filter was incubated (Abs488 0.018 OD), the excitation wavelength was fixed at 488 nm
with a purified specific antibody against BMK, obtained by and emission spectra were recorded between 500 nm and 600 nm
Published on 22 August 2012 on http://pubs.rsc.org | doi:10.1039/C2AY25772F

affinity chromatography on an EAH–BMK column (1 : 500 in with an emission slit-width of 2.5 nm. Measurements were per-
TBS-Tween 0.05% 1% skimmed milk) for 1 h at room tempera- formed in 10 mM phosphate buffer at pH 7.4, at room temper-
ture. After three washings with TBS-T (TBS-Tween 0.05%, 10 ature. BMK–BSA-488 was incubated with a range of
minutes per washing), the filter was incubated with a secondary concentration of antibodies against BMK from 0.0 to 600 nM for
antibody (goat anti-rabbit horseradish peroxidase-conjugate, 10 minutes, and fluorescence spectra were carried out. The
1 : 3000 in TBS-Tween 0.05%, 1% skimmed milk) for 1 h at room polarization fluorescence measurements were carried out, by
temperature. Finally the filter was washed three times as inserting a Glan polarizer between the excitation source and the
described above and then developed with the detection reagent sample, with a vertical (0 ) excitation polarized filter and with a
ECL. horizontal (90 ) emission polarized filter.
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Antibody titration BMK competition assay

The antibody titer was determined by an indirect ELISA assay by A competition polarization assay was carried out at a fixed
the following general procedure. The antigens (BSA–BMK, concentration of antibody against BMK (460 nM) in the pres-
GlnBP and BSA, 100 mL per well) were diluted 1 mg ml1 in a ence of an increased concentration of unlabeled BMK (from
coating buffer (50 mM bicarbonate buffer, pH ¼ 9.5) and the 0.2 mM up to 1 mM) diluted in ethanol. The incubation was done
final quantity was 0.1 mg per well. The plate was incubated for 1 h at room temperature for 30 minutes. After this pre-incubation in
at 37  C and the unreacted sites on the plate were blocked by the presence of unlabeled BMK, all samples were incubated with
incubation for 30 minutes at 37  C with 0.5% BSA in PBS 1
BMK–BSA-488 (70 nM) and the polarization fluorescence
(150 mL per well). The wells were rinsed three times with TBS measurements were carried out with an ISS K2 (ISS inc. Urbana-
(30 mM Tris–HCl, 130 mM NaCl at pH 7.4) containing 0.05% Champaign IL USA) (with a Glan polarizer between the exci-
Tween (TBS-T), pH 7.4. After three washings with PBS-T, the tation source and the sample) with an excitation polarized filter
serially diluted antibody, BMK, was added to the wells, incu- set at 90 and an emission polarized filter set at 0 .
bated for 30 minutes at 37  C and then rinsed three times with
PBS-T. Antiserum anti-BMK was diluted in phosphate-buffered
Results and discussion
saline containing Tween 20 (PBS 1X/Tween20) and 100 mL of
diluted antibody per well (1 : 500, 1 mL antibody : 500 mL buffer) In this work we describe a sensitive polarization-based method
was then added and incubated for 30 minutes at 37  C. Horse- for the detection of 1-phenyl-2-propanone (BMK). BMK is a low
radish peroxidase-conjugated anti-rabbit IgG antibodies, diluted molecular weight compound (Fig. 1), too small to elicit any
1 : 2000 in PBS-T containing BSA (1%), were added to the wells immunological response. Therefore, we chemically modified its
(100 mL) and incubated for 30 minutes at 37  C. After three structure to obtain a BMK derivative (Fig. 1) that could be
washings with PBS-T, the enzyme substrate TMB was added covalently attached to an immunological carrier, as well as to
(100 ml per well), and incubated for 5 minutes at room temper- proteins, for producing the antibodies against BMK when
ature. After addition of 100 mL of stop solution to the wells the injected into rabbits.
absorbance was measured at l ¼ 450 nm. The antibody titer was
graphically determined by plotting the reciprocal of the antibody Synthesis of (1-methyl-2-phenyl-ethylideneaminooxy)acetic acid
dilution against absorbance for each dilution of antibodies. The
titer was taken as the maximum antibody dilution at which a 1-Phenyl-2-propanone (BMK) lacks anchoring sites for conju-
reading of 0.6 absorbance units was observed. The following gation with biomolecules, so a short linker arm was introduced
values were found: 1/64 000. by condensation on the carbonyl to form an oxime. Oximes from
hydroxylamine are also classical derivatives of a carbonyl
compound, widely used for GC analysis for the high coupling
Synthesis of BMK–BSA-488
efficiency in the reaction.7,8 Here, we chose the shortest
A solution of BSA–BMK conjugate at a concentration of 0.5 mg commercially available O-alkylated hydroxylamine, bearing a
mL1 in 150 mL was dissolved in 0.1 M sodium bicarbonate buffer carboxylic moiety for coupling the ketone molecule with proteins
at pH 8.3 and mixed with CF488A from Biotium. The molar or affinity resins using common bio-conjugation procedures.
ratio of the dye and the protein was kept at 500 : 1. The reaction This derivatization has minimal impact on the shape and electron
mixture was incubated for 1 h at 37  C and the labeled molecules density of the structure, since the new bond formed is quite
were separated from the unreacted probes by gel filtration and similar to that already present on the starting molecule. The
dialysis against 10 mM phosphate buffer at pH 7.4 by using facile synthesis, in good yield, of the acid derivatives (B) starts

3560 | Anal. Methods, 2012, 4, 3558–3564 This journal is ª The Royal Society of Chemistry 2012
Published on 22 August 2012 on http://pubs.rsc.org | doi:10.1039/C2AY25772F

Fig. 1 Schematic derivatization reaction of 1-phenyl-2-propanone (A) to oxime (B).

from the ketone (A) (1-phenyl-2-propanone, 99% Sigma Conjugation of the BMK derivative to GlnBP and BSA
Aldrich), and the linker (O-(carboxymethyl)hydroxylamine
GlnBP9 was chosen as a carrier protein and the 1-methyl-2-
hemihydrochloride, 98% Sigma Aldrich) was used as received
phenyl-ethylideneaminooxy (BMK derivative) hapten was
without further purification. A refluxing ternary mixture
conjugated to it via the carboxyl reactive group by the water-
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(methanol, water, pyridine) with a slight excess of the carboxy-

soluble carbodiimide method.8–11 The BSA conjugate was
methyl-hydroxylamine allows the formation of the product
prepared by the same procedure used for the GlnBP conjugate
within two hours, as observed by TLC. An easy purification
and the BSA–BMK conjugate was tested by Western blot
work-up was sufficient to get rid of most of the linker used in
experiments performed with specific antibodies against BMK.
excess, allowing the immediate use of the product for our
purposes. However, for a better structure characterization by
NMR, flash chromatography on silica gel with a polar eluent was
Production of antibodies against the BMK derivative and ELISA
needed to completely eliminate the carboxyhydroxylamine still
test
present in the oily product after the first work-up. The structure
was confirmed by 1H NMR (shown in Fig. 2). As expected, the The GlnBP–1-methyl-2-phenyl-ethylideneaminooxy conjugate
major difference with respect to NMR spectra of the ketone was used to immunize rabbits for the production of polyclonal
precursor (not shown) was the appearance of a singlet at 4.7 ppm antibodies against BMK, following a standard protocol.10 At the
due to methylene protons on the linker arm introduced on the end of the immunization period the IgG fractions of the rabbit
original scaffold. serum were isolated by the protein A column kit by standard

1
Fig. 2 H-NMR spectra of BMK-oxime in CDCl3, 400 MHz.

This journal is ª The Royal Society of Chemistry 2012 Anal. Methods, 2012, 4, 3558–3564 | 3561
Published on 22 August 2012 on http://pubs.rsc.org | doi:10.1039/C2AY25772F
Downloaded on 07 December 2012

Fig. 3 ELISA assay of anti-BMK IgG. The assay was performed in the Tris–borate buffer in the presence of 0.005% Tween and 1% milk. Temperature
was set at 25  C.

procedures. The IgG fractions collected were pooled, concen-

trated, and dialyzed against 10 mM PBS at pH 7.4 and tested by
ELISA assay.
To exclude false reactions due to anti-GlnBP antibodies
present in the serum of the rabbit, a protein different from GlnBP
was conjugated to the BMK derivative. For this purpose, BSA
was chosen. Fig. 3 shows the results of the ELISA tests. The titre
of the anti-BMK antibodies seems to be excellent, it was still
possible to perform the ELISA test with IgG dilutions up to
64 000.

Preparation of affinity columns and purification of specific

antibodies
Antibodies specific for the BMK derivative were purified from
Fig. 4 Western blot analysis of the specific antibody anti-derivative
the IgG fraction (after protein A purification) by affinity chro-
produced in rabbit. Lane 1: 10 mg of BSA-BMK and lane 2: 10 mg of BSA.
matography on resin conjugated with the BMK derivative. After
Temperature was set at 25  C.
loading, the column was washed with binding buffers to elimi-
nate unspecific antibodies and eluted using a buffer at pH 2.8.
The fractions containing the antibodies were chosen by moni-
toring the absorbance at l ¼ 278 nm and were pooled and tested
against the BMK derivative by Western blot.

Western blot with specific antibodies

Western blot experiments of the BMK-conjugate (BSA was the
negative control) were performed by incubating the samples with
purified specific antibodies anti-BMK purified on an EAH–
BMK affinity column (1 : 500 dilution). After incubation of the
HRP conjugate secondary antibody and the development with
the ECL-specific reagent, a clear response was observed for
BSA–BMK and no response was detected for BSA (Fig. 4).

Fluorescence steady-state measurements of BMK–BSA-CF488

conjugate

Fig. 5 Polarization emission spectra of BMK–BSA–488. Temperature In order to perform polarization immunoassay for BMK, we
was set at 25  C. generate a labelled BMK–BSA with CF488 (BMK–BSA–488).

3562 | Anal. Methods, 2012, 4, 3558–3564 This journal is ª The Royal Society of Chemistry 2012
Published on 22 August 2012 on http://pubs.rsc.org | doi:10.1039/C2AY25772F
Downloaded on 07 December 2012

Fig. 6 Polarization emission spectra of BMK-BSA-488 in the presence of increasing concentration of BMK-specific antibodies. The inset shows the
variations of the polarized emission at 512 nm as a function of the antibody concentration.

To ensure that the sample does not have any free dye in the These results show that only the addition of increasing anti-
solution, that could interfere in the measurements, gel filtration body concentration of 200 nM causes an increase in BMK–BSA-
and dialysis were performed to remove the unreacted probe 488 polarized fluorescence intensity. The spectra show that the
before the polarization measurements were made. Fig. 5 shows addition of antibodies (600 nM) resulted in an approximate 36%
the polarized emission spectra of BMK–BSA-488 at 25  C. The increase in the BMK–BSA-488 intensity.
excitation was fixed at 488 nm and the spectra were acquired Fig. 6 inset shows the variation of maximum of polarized
from 500 nm to 600 nm. The maximum of emission of BMK– fluorescence emission at 512 nm as a function of the antibody
BSA-488 was centred at 512 nm. concentration. These results show that the increase of intensity
Fig. 6 shows the polarized emission spectra of BMK–BSA-488 was linear with the addition of antibodies in the concentration
in the presence of increasing concentrations of BMK-specific range from 0.0 nM to 600 nM.
antibodies. The measurements were done at 25  C and a solution
of antibodies was added in a range of concentration from 30 nM Polarization competitive immunoassay
to 600 nM.
The FP immunoassay was used to measure the competition
between the tracer of unlabeled BMK in solution and BMK–
BSA-488 for binding with specific antibodies anti-BMK.
Different samples with a fixed concentration of antibody
(460 nM) were incubated with increasing concentration of BMK
in the range of 0.2–1.0 mM. Each sample was mixed off-line and
allowed to incubate for 30 minutes before the fluorescence
polarization measurements.
Fig. 7 shows the decrease of polarized fluorescence emission as
a consequence of increase of unlabeled BMK in solution. By
analysing the data shown in the figure, it appears that with this
method it is possible to detect amounts of BMK less than 50 nM.

Conclusions
In conclusion, a new polarization fluorescence-based assay has
been developed for the detection of BMK. The materials used for
the assay have been synthesized starting from commercial sour-
ces. The molecular structure of BMK has been changed for easy
Fig. 7 Titration of FP immune-assay with increasing concentration of binding to the GlnBP protein to produce an immune-reactive
unlabeled BMK. conjugate and, subsequently, to generate specific antibodies

This journal is ª The Royal Society of Chemistry 2012 Anal. Methods, 2012, 4, 3558–3564 | 3563
 anti-BMK in rabbits. The developed assay shows interesting Acknowledgements
analytical performances with a LOD of less than 100 nM and an
almost linear response up to 600 nM. In addition, the assay This project is in the framework of the FP7 EU Project
presents a high reproducibility in the entire range of BMK ‘‘CUSTOM’’ grant agreement number: 242387. We would like to
concentrations, as shown in Fig. 7. thank Prof. Alfonso Iadonisi, University of Naples ‘‘Federico II’’
The main advantage of this assay is the possibility of con- for the NMR experiments.
structing a simple optical apparatus which is capable of detecting
changes in polarization fluorescence intensity5,6 that could be References
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3564 | Anal. Methods, 2012, 4, 3558–3564 This journal is ª The Royal Society of Chemistry 2012

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