Dose formulation and analysis of diapocynin

Article abstract of DOI:10.1021/jf072792n

Procedures based on high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection and liquid chromatography-mass spectrometry (LC-MS) are described for analyzing diapocynin. Diapocynin was synthesized by oxidative coupling of two apocynin monomers, through the in situ generation of sulfate radicals. It was purified by washing 3 times each with boiling water, followed by boiling methanol. HPLC was used to determine the concentration of unreacted apocynin and other impurities and the purity of the diapocynin that had been synthesized. Negative-ion, atmospheric pressure chemical ionization (APCI) LC-MS was used to determine the molecular weights of impurities. The method using HPLC with UV detection provided a calibration curve that was linear from 0.16 to 24 μg/mL. The LC-MS method was linear from 0.005 to 2 μg/mL. It was found that diapocynin has low solubility in deionized water and corn oil but is soluble in dimethylsulfoxide (DMSO) and alkaline aqueous solutions. Also, diapocynin is 13 times more lipophilic than apocynin, even though both compounds have the same pK_a of 7.4. The log of the octanol/water partition coefficient (log P) was 1.01 for apocynin and 1.82 for diapocynin. A solution of 5.5 mg/mL (16.7 mM) diapocynin in DMSO was found to be stable for at least 30 days when stored at room temperature.

Full text of DOI:10.1021/jf072792n

J. Agric. Food Chem. 2008, 56, 301–306 301
Dose Formulation and Analysis of Diapocynin
†
‡
‡
§
RON LUCHTEFELD, RENSHENG LUO, KEITH STINE, MIKAELA L. ALT,
,†,§
§
PATRICIA A. CHERNOVITZ, AND ROBERT E. SMITH*
Total Diet and Pesticide Research Center, United States Food and Drug Administration (FDA),
1
1510 West 80th Street, Lenexa, Kansas 66214, Department of Chemistry and Biochemistry,
University of Missouri—St. Louis, One University Drive, St. Louis, Missouri 63121, and Chemistry
Department, Park University, 8700 Northwest River Park Drive, Parkville, Missouri 64152
Procedures based on high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection
and liquid chromatography-mass spectrometry (LC-MS) are described for analyzing diapocynin.
Diapocynin was synthesized by oxidative coupling of two apocynin monomers, through the in situ
generation of sulfate radicals. It was purified by washing 3 times each with boiling water, followed by
boiling methanol. HPLC was used to determine the concentration of unreacted apocynin and other
impurities and the purity of the diapocynin that had been synthesized. Negative-ion, atmospheric
pressure chemical ionization (APCI) LC-MS was used to determine the molecular weights of
impurities. The method using HPLC with UV detection provided a calibration curve that was linear
from 0.16 to 24 µg/mL. The LC-MS method was linear from 0.005 to 2 µg/mL. It was found that
diapocynin has low solubility in deionized water and corn oil but is soluble in dimethylsulfoxide (DMSO)
and alkaline aqueous solutions. Also, diapocynin is 13 times more lipophilic than apocynin, even
a
though both compounds have the same pK of 7.4. The log of the octanol/water partition coefficient
(log P) was 1.01 for apocynin and 1.82 for diapocynin. A solution of 5.5 mg/mL (16.7 mM) diapocynin
in DMSO was found to be stable for at least 30 days when stored at room temperature.
KEYWORDS: Diapocynin; triapocynin; HPLC; LC-MS
INTRODUCTION
S2O82- + 2H+ + 2e f 2HSO -
-
(1)
4
Diapocynin is a metabolite of apocynin, which has antioxi-
dative and anti-inflammatory properties (1–6). Apocynin (4-
hydroxy-3-methoxyacetophenone) was identified during activity-
guided isolation of immunomodulatory constituents from
Picrorhiza kurroa, a native plant grown in the mountains of
India, Nepal, Tibet, and Pakistan. This compound specifically
blocks the activity of NADPH oxidase, a cell-membrane enzyme
known to protect against reactive oxygen species (ROS) (7). It
has also been suggested that apocynin is metabolized to
diapocynin, which may be more active than apocynin (6).
Sodium persulfate can also be converted to a sulfate radical,
SO4• , with a standard oxidation–reduction potential of 2.6 V
(10). Ferrous sulfate is a common initiator.
-
S2O8 - + initiator f SO4• + (SO4• + SO42-)
2
-
-
(
2)
However, it has been reported that this often results in the
cleavage of side chains to form compounds with lower molecular
weight (11), not coupling to form a dimer.
The synthesis of diapocynin was described in a dissertation
that was published on the Internet (8). It was based on the
synthesis of a similar compound, dehydrodivanillin (9), except
that it started with apocynin, used the potassium salt of persulfate
instead of the sodium persulfate, and reported a lower yield
In the original paper, the sulfate radical was the reactive
species in the oxidative coupling of two vanillin molecules
through the carbon that was ortho to the phenolic hydroxyl of
each vanillin and it was converted to dehydrodivanillin (9). In
the dissertation (8), apocynin was used. It was soluble in hot
water, but diapocynin was not. When diapocynin was produced,
it precipitated out of solution. However, some iron, persulfate,
and sulfate were probably coprecipitated and were present as
contaminants. The precipitate was redissolved in aqueous
NH4OH and reprecipitated by adding 6 N HCl, to remove iron,
persulfate, and sulfate contaminants (8). The structures of
apocynin and diapocynin are in Figure 1.
(
8). Persulfate is a powerful oxidizing agent, with a standard
oxidation–reduction potential of 2.1 V, compared to 1.8 V for
hydrogen peroxide.
*
To whom correspondence should be addressed. Telephone: 913-
7
52-2127. Fax: 913-752-2122. E-mail: robert.smith@fda.hhs.gov.
†
United States Food and Drug Administration.
University of Missouri—St. Louis.
‡
Diapocynin has also been synthesized in smaller quantities
using the enzymes horseradish peroxidase and myeloperoxidase
§
Park University.
1
0.1021/jf072792n CCC: $40.75  2008 American Chemical Society
Published on Web 12/20/2007

302 J. Agric. Food Chem., Vol. 56, No. 2, 2008
Luchtefeld et al.
were prepared as described (12). Data were fit to a straight line by
linear regression analysis using Microsoft Excel.
Negative-ion, atmospheric pressure chemical ionization (APCI)
LC-MS analysis was performed using a Finnigan MAT LCQ Duo
ion trap (West Palm Beach, FL), equipped with a Spectra System P4000
pump, UV6000 LP UV diode array detector, and AP3000 auto sampler.
A C18 Ace analytical column from MacMod Analytical, Inc. (Chadds
Ford, PA), 10 cm × 2.1 mm i.d. and 100 Å pore size, was used for
separation. To determine the molecular weight of impurities, such as
triapocynin, a full-scan mass spectrum over a mass range of 100–750
amu was obtained and an isocratic mobile phase of 35:65:0.38 methanol/
water/ammonium acetate (v/v/w) at pH 7 was used. For the determi-
nation of the concentration of unreacted apocynin, a gradient elution
was used. Solvent A was 480:20:0.38 methanol/water/ammonium
acetate (v/v/w) at pH 7, and solvent B was 20:480:0.38 H O/CH OH/
Figure 1. Conversion of apocynin to diapocynin. This is an oxidation–re-
duction reaction, requiring the in situ generation of sulfate radicals, which
remove one hydrogen from each molecule of apocynin, producing
diapocynin.
(
12, 13). Diapocynin was separated from apocynin using
isocratic, reverse-phase high-performance liquid chromatography
HPLC) (14). The mobile phase was strong enough to cause
2
3
ammonium acetate (v/v/w) at pH 7. The mobile phase started with 100%
A for the first min, followed by a linear increase to 100% B from 1 to
(
1
6 min. This was followed by 100% B from 16 to 31 min and then a
diapocynin to elute before apocynin (14). Still, diapocynin is
not readily available from commercial sources, and no methods
have been reported for its analysis.
linear decrease to 100% A from 31 to 40 min. The injection volume
was 20 µL, and the eluent flow rate was 0.25 mL/min. The analysis
used selected ion monitoring (SIM) of ions with m/z 163.5–166.5 and
In preparation for possible cell culture or animal studies, the
objectives of this study were to develop methods based on
reverse-phase HPLC with ultraviolet (UV) detection and liquid
chromatography-mass spectrometry (LC-MS) to determine the
purity of diapocynin, to establish ways to optimize the synthesis
and purification procedures, and to measure the log P and pKa
of apocynin and diapocynin. These methods were used to
analyze diapocynin in different stages of its synthesis and
purification, to measure its solubility in octanol and water, and
to determine the suitability of dimethylsulfoxide (DMSO) as a
vehicle or solvent for preparing doses of diapocynin.
-
3
27.5–330.5, corresponding to the [M - H] ions produced by apocynin
and diapocynin, respectively. Apocynin standards (0.05–8 µg/mL or
.3–48 µM) were prepared in 1 mM NaOH. Data were fit to a straight
0
line by linear regression analysis using Microsoft Excel.
The pK of apocynin was determined by titrating it with an aqueous
a
solution with NaOH and observing the pH and midpoint. An Orion
520A pH meter was used to measure the pH. The pK
was determined by measuring the apparent log P at different pH values
2.0, 7.4, and 7.9). The diapocynin was at least 99% pure, on the basis
a
of diapocynin
(
of the HPLC and LC-MS analyses. That is, diapocynin accounted for
at least 99% of the total UV peak area by HPLC, and there was less
than 1% unreacted apocynin, as determined by LC-MS analysis. The
log P was determined by the shake flask method (15). Diapocynin in
the aqueous phase was determined using the HPLC method with UV
detection. The concentration of diapocynin in the octanol phase was
determined by extracting the octanol with 0.1 N NaOH, followed by
dilution with deionized water and analysis by HPLC. Five different
measurements of log P were made at each pH; therefore, results could
be reported as an average ( standard deviation.
The concentrations of diapocynin in aqueous suspensions in 10 mM
phosphate buffer at pH 7.0–8.5 were determined by filtering mixtures
of excess diapocynin and buffer that had been sonicated 10 min and
diluting it into 10 mM NaOH until the diapocynin concentration was
within the range of concentrations of standards used in the calibration
curve (1.8–92 µM).
MATERIALS AND METHODS
HPLC-grade methanol was from Burdick and Jackson (Muskegon,
MI). All other chemicals were from Aldrich-Sigma (St. Louis, MO).
Apocynin (acetovallinone) was recrystallized from water and dried in
a desiccator before use. Diapocynin was synthesized by dissolving 2 g
of recrystallized apocynin in 200 mL of deionized water with stirring
and heating until the solution was boiling gently. To this was added
0
.15 g of ferrous sulfate heptahydrate and 1.6 g of sodium persulfate.
A brown precipitate formed. After 5 min, the solution was cooled and
filtered. The precipitate was dissolved in 3 N NH OH and then
4
reprecipitated by adding 6 N HCl. The precipitate was filtered and
washed 3 times with 100 mL of boiling water. The diapocynin
precipitate was further purified by washing 3 times with 100 mL of
boiling methanol. The water and methanol washings were analyzed
for impurities by HPLC and LC-MS. The purified product was dried
in a desiccator before being used in measurements of solubility, log P,
Solutions of diapocynin in DMSO were prepared by sonicating
mixtures of diapocynin and solvent for 10 min. The DMSO solutions
1
did not need filtering. DMSO solutions were diluted
NaOH, followed by another /50 dilution in water.
/10 in 4 mM
1
The diluted samples were analyzed by HPLC with UV detection.
The concentrations of diapocynin in samples that had been stored
for 30 days in closed vials at room temperature were determined
by analyzing them by HPLC, using freshly prepared diapocynin
standards.
a
pK , and stability in DMSO.
H nuclear magnetic resonance (NMR) and H decoupled C NMR
spectra of diapocynin in DMSO-d were obtained using a Bruker ARX
1
1
13
6
1
5
1
00 MHz NMR. A 30° pulse width was used for the H NMR, with a
s pulse delay. A 30° pulse width was used for the C NMR spectra,
1
3
with a 2 s pulse delay. The hydrogen and carbon chemical shifts were
referenced to the DMSO peaks, which were set to 2.50 ppm for
hydrogen and 39.50 ppm for carbon, respectively. The attached proton
test (APT) was used to distinguish between two groups of signals,
methyl/methine and methylene/quaternary.
RESULTS AND DISCUSSION
1
The H NMR spectrum of diapocynin (16) contained peaks
with the following chemical shifts, in ppm, and assignments,
The IR spectrum was obtained using a Varian 800 Fourier transform
infrared spectroscopy (FTIR, Palo Alto, CA) with an attenuated total
reflectance (ATR) accessory, from PIKE Technologies (Madison, WI).
Reverse-phase HPLC was performed using a Shimadzu LC-10 HPLC
in parentheses: 2.494 (-CH ), 3.895 (-OCH ), 7.451 and 7.463
3
3
1
3
(
aromatic CH), and 9.465 (-OH). The C NMR spectrum
contained peaks with the following chemical shifts, in ppm, and
assignments by comparing with the APT spectrum, in paren-
theses: 26.25 (-CH3), 55.98 (-OCH3), 109.66 (aromatic C-2,
CH), 124.46 (C-5, C), 125.25 (C-6, CH), 127.86 (C-1, C),
(
Kyoto, Japan), equipped with an autosampler and a LC-10 AV UV–Vis
detector, set at 276 nm. An Alltech Platinum EPS C18 column, with a
µm particle diameter and 100 Å pore size, was used with an isocratic
5
1
47.44 (C-3, C), 149.11 (C-4, C), and 196.15 (CdO). The FTIR
mobile phase consisting of 48:52:0.31 methanol/water/ammonium
acetate (v/v), flowing at 1.0 mL/min. The injection volume was 20
µL. For quantitative analysis, diapocynin standards (0.16–25 µg/mL
or 1.8–76 µM) were prepared in 1 mM NaOH. Diapocynin standards
-1
spectrum contained the following peaks, λmax cm : 3318 (OH),
1666 (CdO), 1588 (aryl CdC), 1286, 1204, 1127, 1083, and
910.

Dose Formulation
J. Agric. Food Chem., Vol. 56, No. 2, 2008 303
Figure 2. LC-MS analysis of diapocynin, showing the removal of apocynin by washing 3 times with boiling water. (A) Mixture of 0.5 µg/mL apocynin
and 0.5 µg/mL diapocynin standards, (B) sample before washing with water, (C) sample after washing, and (D) water blank.
When the Platinum EPS C18 column was used with 48%
methanol in the HPLC analysis, diapocynin eluted before
apocynin and the analysis time was 4 min. This might be
unexpected, because diapocynin is more hydrophobic than
apocynin. This was apparent in the synthesis of diapocynin.
Apocynin was soluble in boiling water, but diapocynin was not.
To measure hydrophobicity or lipophilicity, the octanol–water
partition coefficients of apocynin and diapocynin were measured.
Apocynin is more soluble than diapocynin in both octanol and
water at pH 2, and its octanol–water partition coefficient was
0.009, n ) 5). The log P is used to predict the bioavailability
of a compound, its ability to pass through a cell membrane,
permeate the skin, partition between soil and water, and persist
in the environment (17). The standard method for measuring
log P specifies that the pH of the aqueous phase should be
adjusted so that the compound being tested is nonionic (15).
For this reason, log P was measured first at pH 2. However,
physiological pH is close to 7.4; therefore, the apparent log P
at pH 7.4 might be a better indication of the bioavailability of
any dose of diapocynin that might reach the blood or other
organs, except the stomach, which is below pH 2. Thus,
estimates of the bioavailability of diapocynin should depend
upon the route of administration. Also, even though diapocynin
has a higher log P than apocynin, it is not as soluble in either
n-octanol or water. The ratio of its solubility in these solvents
is higher than the ratio for apocynin, but its absolute solubility
is less.
1
0.3 ( 1.5 (log P ) 1.01 ( 0.06). However, the solubility of
diapocynin in octanol-saturated water at pH 2 was much lower
0.24µg/mLor3.4µM)thantheitssolubilityinoctanol-saturated
water, and its octanol–water partition coefficient was 66 ( 13
log P ) 1.82 ( 0.08, n ) 5). The apparent log P of diapocynin
(
(
increased as the pH of the octanol-saturated water was
increased. The pKa of diapocynin was found to be 7.4, which
is also the pKa of apocynin, which was obtained by titrating an
aqueous solution of apocynin. The octanol–water partition
coefficient at pH 7.4 was 0.924 ( 0.019 (log P ) -0.034 (
2
The HPLC method produced a linear calibration curve (r )
0.9999) for diapocynin at concentrations from 0.16–25 µg/mL
or 1.8–76 µM. It was used to analyze diapocynin produced after

304 J. Agric. Food Chem., Vol. 56, No. 2, 2008
Luchtefeld et al.
Figure 3. LC-MS analysis of methanol washing of diapocynin. (A) chromatogram, using full-scan mass spectral detection. (B) m/z 164.95 negative ion
produced by apocynin, eluting at 2.48 min. (C) m/z 329.05 negative ion produced by diapocynin, eluting at 3.11 min. (D) m/z 329.02 negative ion
produced by an isomer of diapocynin, eluting at 6.31 min. (E) m/z 492.91 negative ion produced by an isomer of triapocynin, eluting at 8.03 min. (F) m/z
492.95 negative ion produced by another isomer of triapocynin, eluting at 13.7 min.
different reaction times. It was found that only 5 min at 100 °C
was needed for the reaction. More triapocynin impurity was
produced when the reaction proceeded for 30 min, which was
the reaction time used by previous workers (8, 9). Most of the
small amount of unreacted apocynin was removed by thoroughly
washing the diapocynin with boiling water 3 times, as shown
in Figure 2. However, there were two small peaks that eluted
after the apocynin in the chromatogram of the water-washed
diapocynin that had reacted for 30 min.
To determine the molecular weight of these two small peaks,
LC-MS was used with isocratic elution. The first methanol
washing of a sample that reacted for 30 min was diluted 1:1
with water and analyzed by LC-MS, and the results are in
Figure 3. With the Ace column and 35% methanol, apocynin
eluted before diapocynin and three other peaks eluted at 6.31,
8.03, and 13.72 min. The m/z values of these peaks were 329.0,
-
492.9, and 492.9, respectively. These were due to [M-1] ions;
therefore, the molecular weights of these impurities were 330
and 494, corresponding to the expected molecular weights of
an isomer of diapocynin and two isomers of triapocynin. The
coupling of two apocynin molecules with a molecular weight
of 166 occurred through the loss of two hydrogens; therefore,
the molecular weight of diapocynin was 330. Similarly, the
coupling of an apocynin with a diapocynin to produce triapo-
cynin also happened with the loss of two hydrogens; therefore,
the expected molecular weight was 494.
Almost all of the rest of the apocynin and most of the
triapocynin was removed by washing with boiling methanol.

Dose Formulation
J. Agric. Food Chem., Vol. 56, No. 2, 2008 305
Figure 4. Triapocynin was present in samples reacted for 30 min (A) but not in samples reacted for 5 min and washed with boiling methanol (B). Only
-
ions with m/z 492.5–493.5, because of the triapocynin [M-1] ion, are shown.
LC-MS chromatograms showing the removal of triapocynin
by washing with methanol are in Figure 4. A gradient elution
and SIM were used in this analysis. For quantitative analysis
of apocynin impurity in diapocynin, the calibration curve was
linear between 0.05 and 8 µg/mL for apocynin.
The reaction was conducted in boiling water for 5 min, instead
of the original 30 min in hot water (8, 9). The sodium salt of
persulfate was used, instead of the potassium salt, because the
potassium salt produced a lower yield (8). Also, the diapocynin
was redissolved in 3 M NH OH, instead of NaOH, because
4
Diapocynin was not very soluble in water, but suspensions
that passed through 1 µm filters could be made but with
relatively poor reproducibility. The concentrations of super-
saturated aqueous suspensions of the purified diapocynin in 10
mM phosphate depended upon pH. The amount of diapocynin
suspended at pH 7.0 was 14 ( 5 µg/mL. At pH 7.5, it was 42
NaOH is often contaminated with chloride and carbonate (18).
Finally, this newer method of synthesis included washing the
diapocynin 3 times each with boiling water and boiling methanol
and analysis of the purified diapocynin by HPLC and LC-MS.
ACKNOWLEDGMENT
(
15 µg/mL; at pH 8.0, it was 206 ( 29 µg/mL; and at pH 8.5,
it was 240 ( 17 µg/mL (n ) 5).
We acknowledge Ann Adams for her careful review of this
paper and her helpful suggestions.
Diapocynin was quite soluble and stable in DMSO. A solution
of 5.52 mg/mL (16.7 mM) diapocynin in DMSO was analyzed
after 1, 10, and 30 days. The concentrations of diapocynin each
day were 5.45 ( 0.09, 5.54 ( 0.13, and 5.44 ( 0.08 mg/mL.
Thus, 5.52 mg/mL diapocynin in DMSO was stable for 30 days
at room temperature.
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