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ods for the quantitative determination of coenzyme Q10 CoQ10 standard (98%) was purchased from Sigma (USA). A
in dairy products. The results showed that MS detection stock solution of CoQ10 was prepared by accurately
is more sensitive than UV detection [17]. weighing 5.4 lg of powder and dissolving it in 10 mL of
It should be borne in mind that signal suppression or the mobile phase. The stock solutions were stored in the
enhancement of the target extracts by matrix compo- dark at – 208C and were stable for at least one year.
nents is a common phenomenon in LC-MS/MS analysis. Tobacco leaves were collected from the arboretum in
Moreover, interfering matrix components can affect the Northeast Forestry University of China and were stored
accuracy of the proposed method and may lead to some at 48C. The eight varieties of tobacco are Hong Hua Da Jin
compromising or erroneous results [18, 19]. In order to Yuan, Long Jiang 911, Yun Yan 87, Piao He 1, Yun Yan 85,
avoid problems related to matrix effects, some authors K346, K326, and NC89, respectively.
have undertaken optimization of sample preparation
and of the chromatographic system and MS/MS detection 2.3 LC-MS/MS conditions
[20 – 22]. In addition, some workers have adopted the Chromatographic analysis was carried out on a Symme-
standard addition method as a way of eliminating try Shield RP18 column (5 lm/93 /66% porosity/
matrix effects [23, 24]. 3.96150 mm). The column temperature was maintained
The aim of this study is to propose a validated LC-MS/MS at 258C. The mobile phase was a mixture of acetonitrile
method with multiple reaction monitoring (MRM) for and isopropanol (8 : 7, v/v) containing 0.5% formic acid.
separation and determination of CoQ10 in tobacco leaves. Elution was performed at a flow rate of 1 mL/min with
So far, no report has been published on the determina- the split ratio set at 7 : 3 (the real flow rate used was
tion of CoQ10 in tobacco leaves by the LC-MS/MS method. 0.3 mL/min). The injection volume was 5 mL. For opera-
Because the matrix components in tobacco leaves are tion in MS/MS mode, a mass spectrometer fitted with an
complex, in order to avoid problems arising from the electrospray ion source interface was used for analysis.
matrix effect each step mentioned above (sample pre- The source was operated at a temperature of 3508C. The
paration, chromatographic system, and MS/MS detec- infusion experiment was performed using a Harvard
,
tion) was carefully optimized in the present study. At the Model 11’ single syringe pump. The mass spectrometer
same time, the standard addition method was used as a was operated in the positive ion mode with the following
quantitation method to further minimize the matrix parameters: declustering potential (DP), 120 V; focusing
effect. Based on this work, the contents of CoQ10 in the potential (FP), 400 V; entrance potential (EP), 10 V; colli-
leaves of eight varieties of tobacco were determined and sion cell exit potential (CXP), 8 V; collision energy (CE),
compared. 38 V; ionspray voltage (IS), 4000 V; channel electron mul-
tiplier (CEM), 2000 V. Nitrogen was used for the nebuliz-
ing (setting 12), curtain (setting 10), collision (setting 5),
2 Experimental and auxiliary gas (setting 6 L/min). CoQ10 was monitored
2.1 Equipment using the MRM mode and MRM was performed with
150 ms dwell time. The mass spectrometer was pro-
LC-MS/MS analysis was performed on an API3000
grammed to monitor the protonated molecule [M+H]+ at
(Applied Biosystems, Canada) triple-stage quadrupole
m/z 864.0 via the first quadrupole filter (Q1), the product
mass spectrometer equipped with an electrospray ioniza-
ion at m/z 197.0 was monitored via the third quadrupole
tion (ESI) interface and an Agilent 1100 series HPLC from
, filter (Q3). Finally, all MS parameters were manually fine-
Agilent technologies (Agilent, CA). A Model 11’ single
tuned to obtain the highest MRM signals. The MRM tran-
syringe pump (Harvard Apparatus Inc., Holliston, USA)
sition m/z 864.0/197.0 was monitored for the detection of
was also used.
CoQ10. Under the above optimum conditions, the ion
The Agilent HPLC system consisted of a G1312A HPLC bin- source was thermally stabilized for 30 min before injec-
ary pump, a 7725i manual injector, and a G1379A degas- tion. The peak areas of CoQ10 obtained from the MRM
ser. A reverse phase HPLC column (Symmetry Shield were utilized for the quantitation. Data were processed
RP18, 5 lm, 93 /66% porosity/3.96150 mm; Waters, by Analyst Software version 1.4.
USA) was used.
2.4 Preparation of standard solutions
2.2 Reagents and materials Working standards were prepared from the stock solu-
Acetonitrile and isopropanol were of HPLC grade (Kracke- tion (0.54 mg/mL) by dilution with mobile phase to a
ler Scientific, Albany, USA). Formic acid was of HPLC final concentration of 8.4/16.9/33.8/67.5/135.0/270.0/
grade (DIMA Technology, USA). Methanol, ethanol, and 540.0 ng/mL, respectively. All standard solutions were fil-
hexane were of analytical grade (Beijing Chemical tered through a 0.45-lm membrane filter (Millipore) and
Reagents Company, China). injected directly.
panol (8 : 7, v/v) gave the best separation of CoQ10 offset size of 0.1 amu. The DP was optimized using the quanti-
only by a slightly off-maximum sensitivity for CoQ10. tative optimization function of Analyst 1.4 to achieve the
In addition, the pH value of the mobile phase has the highest signal response. [M + H]+ (at m/z 864.0) ions were
greatest effect on the separation, peak shape, and detec- observed when the data were acquired in the Q1 scan
tion sensitivity of CoQ10. To enhance the separation and mode and the ion was chosen as precursor ion of CoQ10.
ionization efficiency, different percentages of formic Secondly, we used product ion scans to look for the most
acid were used in the mobile phase. The results showed abundant product ion. The collision energy (CE) was opti-
that CoQ10 was completely separated and the peak shape mized to achieve highest sensitivity. The product ion
of CoQ10 was better when the concentration of formic spectrum obtained, which is shown in Fig. 2, consisted of
acid was in the range of 0.1 – 1%. However, when the con- an ion at m/z 197.0 and a second ion at m/z 234.9 when CE
centration of formic acid was below 0.5%, the ionization was 38 V. The two base peaks at 197.0 and 234.9 corre-
was poor. Considering both resolution and ionization, spond to tropylium and pyrylium ions, respectively.
the optimized formic acid concentration was 0.5%. As a Among the product ions, that at m/z 197.0 was the most
result, a mixture of acetonitrile and isopropanol (8 : 7, abundant and was therefore chosen for CoQ10 quantita-
v/v) containing 0.5% formic acid was confirmed as the tion.
optimum mobile phase. Under these conditions, the Finally, the precursor/product ion pair of m/z 864.0/197.0
retention time of CoQ10 was 2.91 l 0.1 min. was chosen for the MRM scan. MRM was performed with
150 ms dwell time. Peak areas obtained from the MRM of
CoQ10 standards were utilized for quantitation.
3.3 Optimization of MS/MS detection conditions
Sample solutions of tobacco leaf extract (prepared
Electrospray ionization (ESI) and atmospheric pressure
according to the procedure described in Section 2.5)
chemical ionization (APCI) techniques were respectively
were injected directly, separated, and detected under the
tested in positive and negative ion mode. The results
optimum condition mentioned earlier (Section 2.3). The
showed that ESI in positive mode was superior to ESI in
MRM chromatogram of coenzyme Q10 in tobacco leaves is
negative mode and to APCI in positive and negative
shown in Fig. 2.
mode. CoQ10 was determined with much better sensitiv-
ity on using the ESI source in positive mode. Thus, the It can be seen from Fig. 2 that the retention time of CoQ10
ESI source in positive mode was chosen for the detection was 2.93 min. The analysis procedure can be finished in a
of CoQ10. Infusion experiments were carried out to exam- shorter analysis time. The results show the proposed
ine ionization and fragmentation patterns of the CoQ10 method to be very fast.
standards using a syringe pump.
First, a standard solution of CoQ10 was chosen to obtain a 3.4 Method of standard addition
constant signal in the Q1 scan mode. A full scan spec- Quantification was based on the standard addition
trum of the CoQ10 standard was acquired with a scan- method with the tobacco extract solution spiked with
range of 700 – 900 amu, a dwell time of 1.5 s, and a step CoQ10 standards of three different concentrations (50,
Table 1. Recoveries of CoQ10 (n = 3). method is highly sensitive. Meanwhile, a LOQ of 4.0 ng/
mL has been obtained for CoQ10 (S/N = 10).
CoQ10 added Peak area Found Recovery RSD of peak
(ng) (counts) (ng) (%) current (%)
Table 2. Determination of CoQ10 in the leaves from eight varieties of tobacco (n = 3).
Variety of tobacco Standard addition Regression coefficient Content of CoQ10 RSD (%)
curvea) (R2) (lg/g)
This work was financially supported by the Project of Scientific [11] Karpilska, J., Frankowska, R., Instrum. Sci. Technol. 2004,
Research Conditions Upgrade from the Ministry of Science and 32, 281 – 290.
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