Development of a HPLC-FL method to determine benzaldehyde after derivatization with: N -acetylhydrazine acridone and its application for determination of semicarbazide-sensitive amine oxidase activity in human serum

Article abstract of DOI:10.1039/c8ra10004g

A novel fluorescence labeling reagent N-acetylhydrazine acridone (AHAD) was designed and synthesized. A highly sensitive high performance liquid chromatography (HPLC) method coupled with fluorescence detection to determine benzaldehyde after derivatization with AHAD was developed. Optimum derivatization was obtained at 40 °C for 30 min with trichloroacetic acid as catalyst. Benzaldehyde derivative was separated on a reversed-phase SB-C18 column in conjunction with a gradient elution and detected by fluorescence detection at excitation and emission wavelengths of 371 nm and 421 nm. The established method exhibited excellent linearity over the injected amount of benzaldehyde of 0.003 to 5 nmol mL^-1. The method was successfully applied to the determination of serum semicarbazide-sensitive amine oxidase (SSAO) activity in humans. SSAO is a significant biomarker because serum SSAO activity is elevated in patients with Alzheimer's disease, vascular disorders, heart disease and diabetes mellitus. It was demonstrated that the SSAO activity of the hyperglycemic group (60 ± 4 nmol mL^-1 h^-1) was significantly higher than that of normal blood sugar group (44 ± 4 nmol mL^-1 h^-1) with P < 0.05.

Full text of DOI:10.1039/c8ra10004g

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PAPER
Development of a HPLC-FL method to determine
benzaldehyde after derivatization with N-
acetylhydrazine acridone and its application for
determination of semicarbazide-sensitive amine
oxidase activity in human serum
Cite this: RSC Adv., 2019, 9, 6717
*
Xiuli Dong, Jiayuan Tang, Yan Ren and Xiangming Chen
A novel fluorescence labeling reagent N-acetylhydrazine acridone (AHAD) was designed and synthesized. A
highly sensitive high performance liquid chromatography (HPLC) method coupled with fluorescence
detection to determine benzaldehyde after derivatization with AHAD was developed. Optimum
derivatization was obtained at 40 ꢀC for 30 min with trichloroacetic acid as catalyst. Benzaldehyde
derivative was separated on a reversed-phase SB-C18 column in conjunction with a gradient elution and
detected by fluorescence detection at excitation and emission wavelengths of 371 nm and 421 nm. The
established method exhibited excellent linearity over the injected amount of benzaldehyde of 0.003 to 5
nmol mLꢁ1. The method was successfully applied to the determination of serum semicarbazide-sensitive
amine oxidase (SSAO) activity in humans. SSAO is a significant biomarker because serum SSAO activity is
elevated in patients with Alzheimer's disease, vascular disorders, heart disease and diabetes mellitus. It
Received 5th December 2018
Accepted 15th February 2019
DOI: 10.1039/c8ra10004g
was demonstrated that the SSAO activity of the hyperglycemic group (60 ꢂ 4 nmol mLꢁ1
h
ꢁ1) was
rsc.li/rsc-advances
significantly higher than that of normal blood sugar group (44 ꢂ 4 nmol mLꢁ1
h
ꢁ1) with P < 0.05.
Therefore, SSAO activity could be used as an important
biomarker for diabetic angiopathy and other diseases.14
1. Introduction
Numerous methods for the determination of SSAO activity
have been reported. In these methods, Radio-enzymatic assay
(REA) was used by many studies.15–20 Radiometric method can
give accurate data about SSAO activity, but radioactive
compound [14C]-benzylamine is used in the method. LC-MS can
determine the content of SSAO directly in samples,21,22 but the
instrument of the method is sophisticated and expensive. Other
methods employed to the determination of SSAO activity are
light scattering (LS)23 and HPLC.24–27 In these methods, benzyl-
amine is usually used as the preferred substrate that is oxidized
and deaminated by SSAO into benzaldehyde, and then benzal-
dehyde reacted with labeling reagent to form the corresponding
hydrazone. In the method of LS, benzaldehyde was labeled by
2,4-dinitrophenylhydrazine (DNPH) and the derivative can
enhance light scattering intensity. The method based on the
change of light scattering facilitated the assessment of SSAO
activity in vitro sensitively. But for blood plasma sample, the
complex matrix would interfere with the determination of
benzaldehyde for the poor selectivity of the method. Further-
more, the accuracy of SSAO activity measurement would be
decreased. To overcome the disadvantage, HPLC with UV
detection was utilized for the activity determination of SSAO.24
HPLC is the method with best selectivity, and it can effectively
eliminate the inuence of coexisting substances in biological
Benzaldehyde widely exists in Rosaceae plants. It has a special
odor of bitter almond and is used as an additive in food industry.
Benzaldehyde also has been used in the determination of
semicarbazide-sensitive amine oxidase (SSAO) activity, because it
is an important product of benzylamine oxidized and deami-
nated by the enzyme. SSAO activity could be indirectly measured
by determinating the content of benzaldehyde with benzylamine
as the substrate of enzyme reaction. SSAO is a group of copper-
containing enzymes which are sensitive to semicarbazide and
other hydrazines.1 These enzymes exist in vascular smooth
muscle cells, cartilage, kidney, liver and brown fat cell of
mammalian,2,3 and they are also found in serum.4 Various amine
compounds including methylamine, allylamine, aminoacetone,
dihydroxybenzylamine and benzylamine could be oxidized and
deaminated by SSAO to generate corresponding aldehydes,
ammonia and hydrogen peroxide1,2 which are all potentially
cytotoxic and commonly associated with numerous diseases.5 It
has been reported that serum SSAO activity is elevated in patients
with Alzheimer's disease,6,7 vascular disorders and heart
disease.8–10 Serum SSAO activity of diabetics has also been found
to be obviously higher than those with normal blood sugar.11–13
School of Pharmacy, Binzhou Medical University, Yantai, 264003, P. R. China. E-mail:
xmch913@163.com; Tel: +86-535-6913406
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samples to the determination of benzaldehyde. In order to the column temperature was kept at 30 ꢀC. The injection volume
further improve method sensitivity, benzaldehyde was derivat- was 20 mL and the excitation and emission wavelengths were set
ized by uorescence labeling reagent such as dimedone25–27 and at 371 nm and 421 nm, respectively.
the derivative was determined by HPLC with uorescence
detection. However, the reaction of benzaldehyde with dime-
done was performed at a higher temperature (95 ꢀC) for
45 min.25 As a result, a fast, safe and sensitive method for the
determination of serum SSAO activity is needed.
2.3 Synthesis of AHAD
2.3.1 Synthesis of 2-(9-acridone)-acetic acid. Acridone
(9.76 g, 50 mmol) was dissolved in dimethylformamide (150
mL) and anhydrous potassium carbonate (30 g) was added, the
mixture was stirred at 90 ꢀC for 30 min. Then, ethyl chlor-
oacetate (10.6 mL, 200 mmol) dissolved in dimethylformamide
(20 mL) was added dropwise within 2 h. The mixture solution
was stirred at 90 ꢀC for 20 h. Aer cooled to room temperature,
the solution was ltered and potassium hydroxide solution (200
mL, 10%) was added to the lter liquor. Then the solution was
heated to boiling for 2 h. At the end of the reaction, the pH of
the solution was adjusted to 2–3 using HCl and the precipitate
was ltered off and washed with water-methanol (20 mL, 9 : 1)
for 3 times. The crude product was recrystallized from acetic
acid to yield brown crystal (9.12 g, 72.1% yield).
2.3.2 Synthesis of 2-(9-acridone)-ethyl acetate. Ethyl
alcohol (100 mL), 2-(9-acridone)-acetic acid (6 g) and concen-
trated sulfuric acid (5 mL) were added to a 250 mL ask
successively. The mixture was heated to reux with stirring for
5 h. The solution was poured into ice water (400 mL) with
shaking for 5 min and ltered using HPLC lter paper. The
crude product was recrystallized from methanol to yield yellow
crystal (5.8 g, 85.6% yield).
2.3.3 Synthesis of N-acetylhydrazine acridone. Ethyl
alcohol (30 mL), 2-(9-acridone)-ethyl acetate (2 g) and hydrazine
hydrate (10 mL, 85%) were added to a 100 mL ask successively.
The mixture was heated to reux with stirring for 3 h. The
solution was poured into ice water (200 mL) with shaking for
5 min and ltered using HPLC lter paper. The crude product
was recrystallized from methanol to obtain yellow crystal (1.2 g,
65.9% yield). Found (%): C 67.41, H 4.88, N 15.77, O 11.94;
calculated (%): C 67.40, H 4.90, N 15.72, O 11.97.1H NMR (500
MHz, DMSO) d 8.37 (d, J ¼ 7.8 Hz, 1H), 7.82 (t, J ¼ 7.5 Hz, 1H),
7.65 (d, J ¼ 8.7 Hz, 1H), 7.37 (t, J ¼ 7.4 Hz, 1H), 5.18 (s, 1H), 4.40
(s, 1H). m/z [M + H]: 268.19.
In this study, we designed and synthesized a novel uores-
cence labeling reagent N-acetylhydrazine acridone (AHAD) which
can emit strong uorescence (lem ¼ 421 nm) when exposed to
excitation light (lem ¼ 371 nm). AHAD can react with carbonyl
compounds in the presence of a acid catalyst, and a highly
sensitive method for the analysis of benzaldehyde using AHAD as
labeling reagent was developed. The optimum derivatization of
benzaldehyde with AHAD was obtained at 40 ꢀC for 30 min with
trichloroacetic acid as catalyst. Benzaldehyde reacted with AHAD
to form the corresponding hydrazone quantitatively. AHAD is
stable enough for derivatization and the derivative was also stable
enough for chromatographic analysis. The developed method
was successfully applied to determine SSAO activity in human
serum with benzylamine as substrate.
2. Experimental sections
2.1 Chemicals and reagents
Benzylamine and benzaldehyde were purchased from Shanghai
Aladdin Bio-Chem Technology Co. Ltd. (Shanghai, China).
Benzaldehyde was acquired from China National Pharmaceutical
Group Corporation (Shanghai, China). Clorgiline was obtained
from Sigma-Aldrich (St. Louis, MO, USA). AHAD was synthesized
in our laboratory. MilliQ water was used throughout the experi-
ment aer ltering through a 0.22 mm nylon membrane.
Chromatography-grade acetonitrile was obtained from Yucheng
Chemical Reagent Co. (Shandong Province, China). Other
reagents such as hydrochloric acid, sodium hydroxide, dime-
thylformamide, potassium carbonate, phosphoric acid, acetic
acid, sulfuric acid, ethyl alcohol, hydrazine hydrate and tri-
chloroacetic acid of analytical grade were supplied by Sinopharm
Chemical Reagent Co. Ltd. (Shanghai, China).
2.4 Preparation of solutions
2.2 Instrumentation and conditions
The standard stock solution (1 ꢃ 10ꢁ2 mol Lꢁ1) of benzaldehyde
Experiments were performed using Agilent 1260 series high was prepared by dissolving 102 mL of benzaldehyde with
performance liquid chromatography (Agilent Technologies, acetonitrile up to 100 mL. The standard solutions for derivati-
Waldbronn, Germany). The HPLC system was equipped with zation and HPLC analysis were prepared by diluting the stock
a quaternary pump (model G1311B), an autosampler (model solution with acetonitrile. Phosphate buffer solution (PBS, 1 ꢃ
G1329B),
a
thermostated column compartment (model 10ꢁ4 mol Lꢁ1, pH 7.8) was prepared by dissolving 0.13 g of
G1316A), and a uorescence detector (FLD) (model G1321B). Na2HPO4 and 0.11 g of NaH2PO4 in 100 mL water and a Mettler-
The HPLC system was controlled by Agilent Chemstation so- Toledo FE20 pH-meter was used to check the pH of the buffer.
ware. Derivatives were separated on a SB-C18 column (150 mm Stock solutions of clorgiline (2 ꢃ 10ꢁ3 mol Lꢁ1) and benzyl-
ꢃ 4.6 mm, 5 mm i.d., Agilent, USA).
amine (5.6 ꢃ 10ꢁ3 mol Lꢁ1) were prepared in PBS. The AHAD
The chromatographic separation of the derivative was solution (1 ꢃ 10ꢁ2 mol Lꢁ1) was prepared by dissolving 26.8 mg
accomplished using gradient elution. Solvent A was water and B AHAD with acetonitrile up to 10 mL. The corresponding low
was 100% acetonitrile. The gradient condition of the mobile concentration of AHAD solution (5 ꢃ 10ꢁ4 mol Lꢁ1) was diluted
phase was as follows: 30–100% B from 0 to 15 min and then by acetonitrile. All solutions were sealed and stored at 4 ꢀC until
held for 5 min. The ow rate was constant at 1.0 mL minꢁ1 and analysis.
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Fig. 1 Derivatization scheme of AHAD with benzaldehyde.
samples. All experiments were performed in accordance with
the ethical standards in the Helsinki declaration, and approved
by the ethics committee at Binzhou Medical university.
Informed consents were obtained from human participants of
this study.
2.6 Sample preparation procedure for SSAO enzyme activity
Serum sample was prepared according to a previous procedure28
with a minor modication. An aliquot of 50 mL human serum
was pipetted into a 1.5 mL centrifuge tube. Then, 100 mL of
clorgiline (2 ꢃ 10ꢁ3 mol Lꢁ1) was added to inhibit the mono-
amine oxidases (MAO-A and MAO-B) activity in serum. Aer
vortexed for 1 min, the mixture was incubated in a thermostat-
ically controlled water bath at 37 ꢀC for 30 min, then. 250 mL of
benzylamine (5.6 ꢃ 10ꢁ3 mol Lꢁ1) was added to the tube and
Fig.
2 Excitation and emission spectra of AHAD-benzaldehyde
derivative in different mixed solvents. (1) 100% acetonitrile, (2) 90%
acetonitrile, (3) 80% acetonitrile, (4) 70% acetonitrile, (5) 60% aceto-
nitrile, (6) 50% acetonitrile, (7) 40% acetonitrile.
the nal concentration of benzylamine was 3.5 ꢃ 10ꢁ3 mol Lꢁ1
,
2.5 Serum samples
then the tube was vortexed for 1 min and incubated at 37 ꢀC for
1 h. Then, 400 mL acetonitrile was added to the mixture, then
the tube was vortexed for 1 min and centrifuged at 5000 rpm for
10 min. The supernatant was collected into a 2 mL ampoule and
evaporated to dryness at 40 ꢀC under a gentle stream of
nitrogen. The residue was dissolved in 50 mL acetonitrile and
derivatized by AHAD.
Twelve human serum samples were supplied by Yantaishan
Hospital and stored at ꢁ80 ꢀC before analyzed. All serum
samples were divided into two groups which were matched by
blood glucose level. One was normal blood sugar group (6
samples) and another was hyperglycemia group (6 samples).
Blood glucose level was determined by the supplier of serum
Fig. 3 Effect of different reaction conditions on peak area of the reaction product of AHAD with benzaldehyde. (a) Effect of catalyst species, (b)
effect of amount of catalyst, (c) effect of reaction time, (d) effect of reaction temperature, (e) effect of amount of AHAD.
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maximum wavelengths of AHAD-benzaldehyde derivative in
purity acetonitrile were 371 nm for excitation and 409 nm for
emission, respectively. There was no obvious wavelength shi
for the excitation spectra with the decreasing of the volume
ratio of acetonitrile in the mixed solvents. But for emission
spectra, the maximum emission wavelength in 90% acetonitrile
was 417 nm, and a continuous red-shi was observed with the
decreasing of the volume ratio of acetonitrile. In the study,
when AHAD-benzaldehyde derivative owed into the ow cell,
the volume ratio of acetonitrile in the mobile phase was about
60%, so 371 nm and 421 nm was select as detection wavelengths
for further experiments. The uorescence intensity of AHAD-
benzaldehyde derivative increased with the decrease of aceto-
nitrile ratio, and a 70% increase was found when acetonitrile
ratio varied from 100% to 50%.
3.2 Optimization of derivatization conditions
In order to obtain the highest yield for the derivatization of
benzaldehyde with AHAD, effects of species and amount of
catalyst, reaction temperature, reaction time, amount of AHAD
on the reaction were investigated.
AHAD can react with benzaldehyde in the presence of acid.
Effect of different acids on the reaction was investigated. These
acids include formic acid, oxalic acid, acetic acid, citric acid,
and trichloroacetic acid. The concentration of all acids was
0.10 mol Lꢁ1. The result (Fig. 3a) showed that the highest
detection response was achieved when trichloroacetic acid was
used as the catalyst of the derivatization. For oxalic acid and
citric acid, the peak area was slightly smaller than trichloro-
acetic acid, so trichloroacetic acid was selected as the catalyst
for subsequent tests. Then, the effect of trichloroacetic acid
volume on derivatization was studied over the range of 20–100
mL. The maximum peak area was obtained when 60 mL of tri-
chloroacetic acid was used in the derivatization (Fig. 3b).
The effect of temperature on derivatization of benzaldehyde
with AHAD was examined over the range of 30–50 ꢀC. When
raising reaction temperature, the detection response of deriva-
tive increased, and the maximum peak area of benzaldehyde
derivative was obtained at 40 ꢀC (Fig. 3c). There was an obvious
change for detection response over 40 ꢀC which could be caused
by the decomposition of derivative. The effect of time (over the
range of 10–50 min) on the derivatization reaction was also
Fig. 4 HPLC chromatograms after applying the procedure described
in Section 2.7 (a) acetonitrile blank; (b) standard benzaldehyde (40.0
nmol mLꢁ1); (c) blank serum sample; (d) serum sample.
2.7 Derivatization procedure of AHAD with benzaldehyde
50 mL of standard benzaldehyde solution or supernatant, 350 mL
of 5 ꢃ 10ꢁ4 mol Lꢁ1 AHAD solution and 60 mL of trichloroacetic
acid (0.1 mol Lꢁ1) were added into a 2 mL ampoule, succes-
sively. The ampoule was sealꢀed and bathed in a thermostatically
controlled water bath at 40 C for 30 min. Aer derivatization,
40 mL of 50% acetonitrile was added to dilute the derivatization
solution to 500 mL. The nal solution was injected directly for
HPLC analysis without any other operations. The derivatization
scheme of AHAD with benzaldehyde is shown in Fig. 1.
3. Results and discussion
3.1 Fluorescence spectra AHAD-benzaldehyde derivative
The uorescence spectra of AHAD-benzaldehyde derivative was investigated. The maximum detection response was obtained at
determined at the wavelength range of 250 to 550 nm to nd 40 min, and the rise of reaction time lead to a slight loss of peak
uorometric detector wavelength. As was shown in Fig. 2, the intensity (Fig. 3d). So reaction time was controlled at 40 min.
Table 1 Instrument precision, accuracy, method precision and recovery for the determination of benzaldehyde
Instrument precision (RSD, %)
Retention
Method precision (RSD, %)
Benzaldehyde
(nmol mLꢁ1
)
time
Peak area
Accuracy (%)
Intra-day
Inter-day
Recovery (%)
0.1
2.0
40.0
0.036
0.052
0.059
0.97
0.82
0.69
95.8
96.9
97.2
4.9
2.5
3.6
6.9
5.0
4.2
93.5
95.2
96.1
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The amount of AHAD was tested to ensure the sufficient
reaction of benzaldehyde with AHAD. The result (Fig. 3e) indi-
cated that maximum detection response was obtained when 350
mL AHAD was used in the derivatization reaction, and further
increasing AHAD beyond this volume had no signicant change
on detection response. Therefore, 350 mL AHAD was chosen for
subsequent experiments and the derivatization action was
completed in 40 min.
3.3 Stability of AHAD and AHAD-benzaldehyde
Solution of AHAD in acetonitrile was stable enough when it was
ꢀ
stored at 4 C, and there was no obvious change for the deriv-
atization yield aer seven days. The stability of the AHAD-
benzaldehyde derivative was tested by re-analysis of the same
sample stored at 4 ꢀC for 24 h and at room temperature for 12 h.
The percent change of the peak area was less than 3.1% at 4 ꢀC
and less than 3.9% at room temperature. This results indicated
that the deꢀrivative was stable enough for at least 24 h when
stored at 4 C and for 12 h when stored at room temperature.
3.4 Selectivity of the method
In order to evaluate the selectivity of the developed method and
apply the method to determine serum SSAO activity, some
substances which maybe disturb the separation and quantiza-
tion of benzaldehyde were investigated. These substances
included byproducts in derivatization, endogenous compo-
nents in serum, and regents used in sample preparation. The
derivatization reaction of benzaldehyde with AHAD was
occurred in the presence of trichloroacetic acid, and some by-
products were found in derivatization procedure besides
derivatization product. In addition, excess labeling reagent
used in derivatization may also be a possible interference to
separation. A derivatization procedure was carried out with 50
mL of acetonitrile instead of standard benzaldehyde solution,
and the chromatographic isolation result (Fig. 4a) was
compared with the chromatogram of standard benzaldehyde
solution (Fig. 4b). The result showed that the retention time of
benzaldehyde derivative was 7.82 min, and no peak was found
at this time in Fig. 4a. Other peaks including excess AHAD in the
two samples' chromatograms were almost the same, and they
did not disturb the isolation and determination of benzalde-
hyde derivative. A blank sample was prepared according to the
procedure described above with 250 mL PBS instead of benzyl-
amine and analyzed on the same chromatographic conditions.
The result (Fig. 4c) was compared with serum sample (Fig. 4d),
and no interference was found. As a result, the byproducts in
derivatization, endogenous components in serum, and regents
used in sample preparation did not disturb the isolation and
determination of AHAD-benzaldehyde derivative, and the
proposed method was of highly selectivity.
3.5 Linearity and correlation coefficient
The calibration curve was established by the analysis of blank
serum samples spiked with standard benzaldehyde. The spiked
samples were prepared and derivatized according to the
procedure described above. The injected concentration of
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benzaldehyde varied from 0.003 to 5 nmol mLꢁ1. AHAD- Section 2.6 with benzylamine replaced by 250 mL of PBS. The
benzaldehyde derivative showed excellent linear relation with recovery was calculated according to the equation: recovery (%)
correlation coefficient of 0.9997 and the linear regression ¼ Aa/Ab ꢃ 100%, where Aa and Ab are the peak area values of
equation was Y ¼ 238.8X ꢁ 0.28 (X: injected concentration per each spiked concentration before and aer sample preparation.
nmol per mL, Y: peak area per mV per min).
The analyses were carried out in triplicate for each concentra-
tion, and the experimental recoveries obtained were ranged
from 93.5 to 96.1% (Table 1).
3.6 Sensitivity
The calculated LOD with uorescence detection (at a signal-to-
noise ratio of 3 : 1) was 8.9 ꢃ 10ꢁ3 nmol mLꢁ1 hꢁ1, and LOQ (at
a signal-to-noise ratio of 10 : 1) was 0.03 nmol mLꢁ1 hꢁ1. AHAD
provides lower LOD values than other derivatizing agents used
in UV or uorescence detection, such as 2,4-dinitrophenylhy-
drazine24 and dimedone,25 which permit us to determine lower
content than methods involving other reagents.
3.9 Advantage of the proposed method
Among these methods mentioned above, REA was mostly used
by some researchers, however, radioactive compound [14C]-
benzylamine was used as substrate for the enzymatic reaction
which made researchers exposed to radiological hazards.
HPLC24,25 was more sensitive and with higher selectivity. To
determine SSAO activity, benzylamine was used as the preferred
substrate. Benzylamine was deaminated and oxidized by SSAO
into benzaldehyde, and benzaldehyde was labeled by UV or
uorescent labeling reagents. AHAD was used to label benzal-
dehyde in this work, and the comparison of AHAD with other
labeling reagents was listed in Table 2. The main advantage of
the method developed using AHAD as labeling reagent was the
higher sensitivity compared with other methods. The LOD for
SSAO in this study was 8.9 ꢃ 10ꢁ3 nmol mLꢁ1 hꢁ1 which is
lower than DNPH24 and dimedone.25 The proposed method was
also more sensitivity than LS23 and FIA-FL28 with 1,2-
diaminoanthraqui-none (DAAQ) as labeling reagent. Besides
higher sensitivity, there were several other advantages including
mild reaction conditions of low temperature and weak acid,
broad linear range and high recovery. LC-MS can determine the
SSAO activity in samples with higher sensitivity,21,29 but the
instrument of this method is sophisticated and expensive.
3.7 Precision and accuracy
Instrumental precision and accuracy were investigated by
analyzing standard benzaldehyde solution at three concentra-
tions (0.1, 2.0, and 40.0 nmol mLꢁ1), and ve determinations
were performed for each concentraion. The instrumental show
good precision in the range of 0.036–0.059% for retention time
and 0.69–0.97% for peak area of benzaldehyde, respectively (see
Table 1). The accuracy was calculated according to the equation:
accuracy (%) ¼ Ca/Cb ꢃ 100%, where Ca is the concentration of
benzaldehyde calculated by linear regression equation, and Cb
is the concentration of standard benzaldehyde solution. The
accuracy was found in the range of 95.8–97.2%. Precision (intra-
day and inter-day) of the method were investigated by spiking
benzaldehyde at three different concentrations (0.1, 2.0, and
40.0 nmol mLꢁ1) into serum sample. The serum sample was
prepared according to the procedure described above using 250
mL of PBS instead of the SSAO's substrate benzylamine before
benzaldehyde was spiked. The intra-day precision ranged from 3.10 Application
2.5% to 4.9%, while the inter-day precision ranged from 4.2% to
6.9%. The results showed good accuracy and adequate precision
for the determination of benzaldehyde in the serum sample.
The developed method was utilized to determine the serum
SSAO activity of 12 human subjects. Serum SSAO activity is
dened as benzaldehyde (nmol) formed per milliliter serum per
hour.28 The results of human serum SSAO activity were listed in
Table 3. The average SSAO activity (mean ꢂ SE) of normal blood
3.8 Recovery
Recovery experiments were performed by analyzing serum sugar group was 44 ꢂ 4 nmol mLꢁ1 hꢁ1 and hyperglycemia
samples spiked with three concentrations of benzaldehyde (0.1, group was 60 ꢂ 4 nmol mLꢁ1 hꢁ1. SE (standard error) is dened
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2.0, and 40.0 nmol mLꢁ1) before sample preparation and aer as standard deviation= sample size.30 Statistical analysis
sample preparation according to the procedure described in between the two groups was performed using “SPSS 19.0” with
Table 3 The results for determination of the SSAO activity
Normal blood sugar group
Hyperglycemia group
Blood sugar level
Blood sugar level
SSAO activity
Mean activity
SSAO activity
Mean activity
(mmol Lꢁ1
)
(nmol mLꢁ1 hꢁ1
)
(nmol mLꢁ1 hꢁ1
)
(mmol Lꢁ1
)
(nmol mLꢁ1 hꢁ1
)
(nmol mLꢁ1 hꢁ1
)
5.2
4.9
4.1
5.7
5.1
4.6
49.6
35.7
29.4
56.3
38.9
51.8
44 ꢂ 4a
7.8
8.1
6.9
9.2
8.3
9.0
68.4
72.1
57.2
50.5
61.3
48.7
60 ꢂ 4a
pffiffiffi
a
SE ¼ SD= n (n ¼ sample size).
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the independent samples t-test. F-test was performed before
t-test, and the result showed that variances of two groups have
no signicant difference (P ¼ 0.574). Statistical result of t-test
revealed that SSAO activity of hyperglycemia group was signi-
cantly higher than normal blood sugar group (P < 0.05). These
results proved that the SSAO activity may be used as a valuable
biomarker for hyperglycemia. SSAO activity value could help
doctors to diagnose whether hyperglycemia is present.
7 Z. J. Jiang, J. S. Richardson and P. H. Yu, Neuropathol. Appl.
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¨
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13 T. Obata, Life Sciences, 2006, 79, 417–422.
´
A novel uorescence labeling reagent N-acetylhydrazine acri-
done (AHAD) was designed and synthesized. AHAD which can
emit strong uorescence when exposed to excitation light is an
excellent labeling reagent for carbonyl compounds A highly
sensitive HPLC-FL method to determine benzaldehyde aer
derivatization with AHAD was developed. The optimum deriv-
atization conditions of benzaldehyde with AHAD was obtained
at 40 ꢀC for 30 min with trichloroacetic acid as catalyst. The
developed method showed good correlation, precision and
accuracy. The proposed method was utilized successfully to
determinate SSAO activity in human serum with a satisfactory
result. It is also a accurate and sensitive method to be used for
routine analysis of the SSAO activity in serum of diabetic human
subjects. AHAD also has the potential to be used for the deriv-
atization and analysis of other carbonyl compounds such as
aliphatic aldehydes, aromatic aldehydes and ketones. Further
application of AHAD will be carried out in the next work of our
laboratory.
´
´
¨
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