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Journal of Chromatography A, 1218 (2011) 7371–7376

Contents lists available at SciVerse ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Coupling frontal elution paper chromatography with desorption corona beam
ionization mass spectrometry for rapid analysis of chlorphenamine in herbal
medicines and dietary supplements
Yun-Qing Huang a,1 , Jing-Qing You a,1 , Junsheng Zhang b , Wenjian Sun b , Li Ding b , Yu-Qi Feng a,∗
a
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, China
b
Shimadzu Research Laboratory (Shanghai) Company, Ltd., Shanghai 201201, China

a r t i c l e i n f o a b s t r a c t

Article history: We developed a convenient method by coupling frontal elution paper chromatography with desorption
Received 30 June 2011 corona beam ionization mass spectrometry (DCBI-MS) for rapid determination of chlorphenamine added
Received in revised form 22 August 2011 in herbal medicines or dietary supplements. In this method, the ethanol extract of the herbal products was
Accepted 22 August 2011
spotted directly onto an isosceles triangular filter paper sheet, and then the paper sheet was developed
Available online 25 August 2011
under strong elution condition with the sample zone migrating at the solvent front. The analyte was
finally condensed at the V-shaped tip which could then be placed under the visible plasma beam of DCBI
Keywords:
for ionization. The overall procedure took less than 5 min. The frontal elution paper chromatography on
Frontal elution paper chromatography
Desorption corona beam ionization
a triangular plate used in this work improved the signal intensity of chlorphenamine by 30-fold due to
Chlorphenamine the analyte condensing at the tip and the reduction of the background suppression. Furthermore, the
Herbal medicines paper sheet also functioned as a filter in the analysis of solid or powder samples, which can increase the
analytical throughput by omitting the step of centrifugation. The proposed method in current study was
successfully applied in the determination of chlorphenamine in herbal medicines. Chlorphenamine was
detected in four of the twelve types of herbal medicines examined in this study. The limit of detection was
200 ng/mL (2.0 ng absolute) in full-scan positive-ion mode and the linear range was from 5.0 ␮g/mL to
50 ␮g/mL with satisfactory linear coefficient (R2 (the square of the correlation coefficient) = 0.895). Good
reproducibility was achieved with relative standard deviations (RSDs) less than 15.0% and the recoveries
of chlorphenamine ranged from 84.3 to 90.6%.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction (ELDI) invented by Shiea et al. used electrospray droplets to
post-ionize neutral protein molecules generated by laser des-
Ambient ionization, pioneered with desorption electrospray orption [23]. Based on ambient plasma ionization, several novel
ionization (DESI) [1] and direct analysis in real time (DART) [2], designs of ambient plasma ionization which differentiate in elec-
has received more and more interest in recent years [3,4]. These tric current, discharge gas, plasma temperature and the voltage
techniques showed good potential in rapid analysis of samples applied have been reported [15–19]. One of the plasma based ion-
in their native environment with little or no sample preparation ization sources, named desorption corona beam ionization (DCBI)
[5–12]. So far, different energy carriers such as charged droplets for direct analysis, has been reported recently [11,20], and in this
[1,13,14], plasma [15–21], UV light [22], laser [23] and heat [12] source the plasma was produced by applying high DC voltage
have been employed in ambient ionization mass spectrometry. In at the tip of a hollow needle with helium as the discharge gas.
some cases, the analytes were desorbed and ionized by a common A visible thin corona beam extending out around 1 cm can be
energy carrier. However, it is also possible to use different types of observed in DCBI, which can facilitate localizing specific sampling
energy carriers for desorption and ionization [22,23]. For instance, areas.
the method of electrospray-assisted laser desorption/ionization Ambient ionization mass spectrometry can usually meet the
requirements for rapid and direct screening of analytes in urine,
saliva, plasma, skin and hair [24–26]. However, some practical
∗ Corresponding author. Tel.: +86 27 68755595; fax: +86 27 68755595. problems still exist in the application of this popular technique. For
E-mail address: yqfeng@whu.edu.cn (Y.-Q. Feng). instance, it is still difficult to apply these ion sources directly for
1
These authors contributed equally to this work. the determination of organic substances in complex background

0021-9673/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2011.08.067
7372 Y.-Q. Huang et al. / J. Chromatogr. A 1218 (2011) 7371–7376

without sample preparation and separation steps. As it is well 2. Experiments
known, proper sample preparation can increase the analyte con-
centration and eliminate background interference [27,28]. Ambient 2.1. Materials
ionization sources have been successfully coupled with some
simple and robust separation techniques such as thin layer chro- Milli-Q water was used in preparation of solutions and elu-
matography (TLC) [29–31] and paper chromatography (PC) [14] ents. Xinhua Grade 1 qualitative filter paper was purchased
to enhance the selectivity and reliability. Typically, the TLC or PC from Hangzhou Xinhua Paper Industry (Hangzhou, China). Chlor-
method employs a rectangular sheet for developing, and the sample phenamine were purchased from Sigma (St. Louis, MO, USA). All
zone locates between the spotting point and the solvent front with the other reagents were of analytical grade and purchased from
an Rf value between 0.1 and 0.8. However, sample zone broadening Shanghai Chemical Reagent Co. (Shanghai, China). The 12 kinds of
caused by eddy and molecular diffusion will occur as the spots move patent herbal medicines (Shuanghuanglian capsule, Shuanlian oral
up the plate, which may dilute the analytes on the planar plate liquid, Qingrejiedu capsule, Jingyinghua pill, Gangmaoruan capsule,
and then lead to the failure in quantitative determination. Another Gangmaoqing pill, Gangmaotuishao pill, Lianhuaqingwen pill, Shi-
shortcoming of the TLC or PC method is that the sample zone is jigangmao pill, Yingqiao pill, Fufanggangmaoling pill and Biyankan
not visible to human naked eyes if the analytes lack chromophores, pill) were purchased from a local drugstore.
which makes it difficult to locate the sample zone directly with the
probe of ambient ionization source. Staining can help to make the 2.2. Instrument
sample zone visible, but the analysis time will be prolonged. More-
over, the structures of the analytes may be damaged and cannot be All the experiments were performed using a single-quadrupole
further determined by mass spectrometry after staining. Thus, from mass spectrometer (2010EV, Shimadzu, Japan) installed with a
the practical viewpoint, to develop a facile method with advantages DCBI source. Quantification and confirmation were performed in
of sample isolation, enrichment and visual localization is of consid- full-scan mode. The optimal instrumental parameters were as fol-
erable interest for extending the scope of application of ambient lows: interface voltage, 3.5 kV; gas temperature, 250 ◦ C; gas flow
ionization mass spectrometry. rate, 0.5 L/min; CDL temperature, 200 ◦ C; CDL voltage, 2.0 kV; cone
The use of herbal medicine to maintain human health and voltage, 1.5 kV.
cure disease can be dated back more than 5000 years [32].
Herbal products, which are now used by approximately 20% of 2.3. Paper chromatographic conditions
the population in the world, have gained increasing popularity
in the last decade. It has been estimated that as many as one The paper chromatography was developed at 25 ◦ C using the sol-
third to one half of currently used medicines were originally vent of a mixture of ethyl acetate–28% ammonia water (10:1, v/v).
derived from plants [33]. Nevertheless, some medicines marketed Xinhua Grade 1 qualitative filter papers were used for the separa-
as “herbal medicine” or “dietary supplement” were adulterated tion of standards and complex mixtures of real samples. The filter
with synthetic drugs by the manufacturers in order to amelio- paper was cut into isosceles triangle with a base of 1.0 cm and a
rate symptoms quickly, which may be particularly problematic height of 4.0 cm. The triangle paper sheet was pre-marked with
in China [34]. Those added synthetic drugs may interact with two parallel lines for origin (1.0 cm from the base line) and finish
some other ingredients in the herbal products and cause unpre- line for the end of developing (0.3 cm from the V-shaped tip). Both
dictable effects on users. Thus, it is essential to identify whether the pre-marked lines were parallel to the base line (Fig. 1). 10 ␮L
or not the synthetic drugs are present in herbal products. TLC of sample solution was spotted onto the paper sheet along the pre-
[35], high-performance liquid chromatography (HPLC) [36–38], marked origin line using a 10 ␮L-auto dispenser. Then the paper
capillary electrophoresis (CE) [39–41], gas chromatography–mass sheet was dried under airflow, and placed in a bath saturated with
spectrometry (GC–MS) [42], liquid chromatography–mass spec- the mobile phase. The developing solvent was allowed to contact
trometry (LC–MS) [43–47] and capillary electrophoresis–mass 0.3 cm lower of the origin line. Once the mobile phase moved to
spectrometry (CE–MS) [48] have been employed for the analysis the finish line near the V-shaped tip (this took less than 3 min), the
of adulterants in herbal products. However, these techniques are paper sheet was removed from the bath. The solvent front would
limited by the lack of specificity, or need time-consuming sam- bypass the marked line and arrive just at the V-shaped tip. After
ple preparation, separation (GC, LC, CE) and derivatization steps being dried under airflow, the V-shaped tip was placed under the
(GC), which makes these analyzing methods less capable of reli- visible plasma beam of DCBI for ionization.
able and high-throughput screening of large amounts of sample on
the market. 2.4. Sample preparation
In this study, a commonly adulterated synthetic drug, chlor-
phenamine, was determined in herbal medicines by coupling For solid tablets, 10 mg of the sample was weighed and placed in
frontal elution paper chromatography with desorption corona a prepared 1.5 mL FastPrep tube containing 990 ␮L ethanol. 10 ␮L of
beam ionization mass spectrometry (FEPC–DCBI-MS). The spotted ketamine (1.0 mg/mL) was added as internal standard. After mash-
sample was developed on an isosceles triangular filter paper sheet ing by a vibration mill for 30 s, 10 ␮L of the suspension was spotted
under strong elution condition with the analyte migrating at the on the filter paper sheet for development. For liquid samples, 10 ␮L
solvent front. The analyte was condensed at the tip of the triangular of the liquid was directly spotted on the filter paper sheet for devel-
filter paper sheet which was then submitted to the DCBI source for opment.
ionization. The use of PC as the primary separation technique also
reduced the interferences from chemicals originated from herbage. 3. Results and discussion
In general, the procedure of the analytical method developed in cur-
rent study included three parts: extracting analytes with ethanol, 3.1. Paper chromatography
separating analytes on a filter paper and ionizing analytes with
DCBI (Fig. 1). The overall procedure can be completed within 5 min. PC needs no expensive equipment, and is easy to handle. In
The performance of the FEPC–DCBI-MS method was evaluated by this respect, the simple and robust separation method, PC, was
the analysis of chlorphenamine in 12 kinds of patent medicines employed to combine with DCBI mass spectrometry for the deter-
purchased from the local market. mination of chlorphenamine in this study.
Y.-Q. Huang et al. / J. Chromatogr. A 1218 (2011) 7371–7376 7373

Fig. 1. Analysis of chlorphenamine added in herbal medicines and dietary supplements using frontal elution paper chromatography coupled with desorption corona beam
ionization mass spectrometry (FEPC–DCBI-MS). 10 ␮L of the extract of the herbal medicine was spotted on the filter paper sheet along the pre-marked origin line. Then the
paper sheet was dried under airflow, and placed in a bath saturated with the mobile phase. After developing and drying under airflow, the V-shaped tip was placed under
the visible plasma beam of DCBI for ionization.

In order to evaluate the enrichment efficiency of solvent front-
migrating, 10 ␮L chlorphenamine (5 ␮g/mL) was spotted onto a
rectangular paper sheet (2.0 cm × 5.0 cm) along the origin line fol-
lowed by developing at 25 ◦ C using the solvent of the mixture
of ethyl acetate–28% ammonia water (10:1, v/v). DCBI-MS detec-
tion was performed before and after the development. The results
showed that the analyte was condensed at the solvent front, and
the mass spectrometry signal intensity of chlorphenamine was
improved by 2.5-fold after development (Fig. 3B). The same amount
of chlorphenamine was then spotted and developed on a triangle
filter paper sheet as described in the experimental part. The sig-
nal intensity of chlorphenamine was further improved by 10-fold
compared to that of chlorphenamine developed on the rectangular
paper sheet (Fig. 3C). In addition, the analyte and internal stan-
dard migrated at the solvent front and stopped just at V-shaped
tip, which largely facilitated localizing the sample band to the
plasma beam (Fig. 2). The results demonstrated that solvent front-
migrating on a triangle paper sheet efficiently improved the spot’s
clarity, compactness and reproducibility. Although the analyte was
not entirely separated from the potential interference, detection
of chlorphenamine in real samples was achievable using DCBI-MS
because of the great selectivity of mass spectrometry as the detec-
tor. It should be pointed out that if the paper sheet is developed
Fig. 2. The filter paper sheet stained in iodine vapor after development. The unre-
tained ingredient was condensed at the top band. too long, the analyte can escape from the paper tip and move to the
glass wall, which will prevent the successful determination of the
analyte using the DCBI-MS.
At first, we hoped to separate the sample zone from any decom-
position products, impurities or excipients in the herbal medicine. 3.2. Optimization of DCBI conditions
To this end, we tried to localize the sample zone between the spot-
ting point and the solvent front with an Rf value between 0.1 and 0.8 3.2.1. Effect of discharge gas flow rate
by adjusting the polarity of the developing solvent. Initial results Helium gas flow rate may affect the signal intensity of the ana-
showed that the sample zone broadening caused by eddy and lyte by DCBI-MS. On the one hand, the signal intensity will increase
molecular diffusion always occurred as the spot moved up the plate. with the increase of the gas flow rate. On the other hand, flush
Furthermore, the sample zone was not visible to human naked eye, away effect of the gas flow at high rate may reduce the chance to
which made it difficult to localize the sample zone directly under get into the MS inlet for the generation of ions. Helium gas with
the plasma beam. Staining can help to make the sample zone visi- different flow rates ranging from 0.1 to 1.5 L/min was tested under
ble, but it may interfere with the mass spectrometry analysis of the positive-ion mode. The results indicated that the signal intensity of
analytes and prolong the analyzing time. To circumvent the prob- the analyte increased sharply as the gas flow rate increased from
lems, the plate was developed under strong elution condition with 0.1 L/min to 0.5 L/min, and then decreased when the gas flow rate
the sample zone migrating at the solvent front. The solvents of the was higher than 0.5 L/min. Because the paper sheet will vibrate
mixture of ethyl acetate–28% ammonia water with different vol- up and down seriously when the gas flow rate was higher than
ume ratios (1:0, 50:1, 30:1, 20:1, 10:1, 5:1) were used to develop 1.0 L/min, which may largely decrease the reproducibility of the
the paper sheet. The results showed that the analytes migrated at ion signal, the helium gas flow rate of 0.5 L/min was used for the
the solvent front when the volume ratio of ethyl acetate over 28% further experiments.
ammonia water was higher than 20:1. Thus, the solvent of the mix-
ture of ethyl acetate–28% ammonia water (10:1, v/v) was selected 3.2.2. Effect of desorption temperature
for further experiments. And the unretained ingredient was appar- The process of the DCBI analysis was influenced by two fac-
ently condensed at the top band by the front elution tactic (Fig. 2). tors, desorption and ionization. Because the desorption is mainly
7374 Y.-Q. Huang et al. / J. Chromatogr. A 1218 (2011) 7371–7376

Table 1
Calibration parameters for quantification of chlorphenamine.

Analyte Calibration range (␮g/mL) Regression equation R2 LOD (␮g/mL)

Chlorphenamine 5.00–50.0 y = 0.0190x + 0.0120 0.895 0.200

Fig. 3. Comparison of ion signals intensity of chlorphenamine without development (A), developed in rectangular (B) or triangle (C) paper sheets. 10 ␮L of chlorphenamine
(5 ␮g/mL) was spotted to the paper sheet for analysis. The developing solvent was a mixture of ethyl acetate–28% ammonia water (10:1, v/v).

Fig. 4. Analysis of chlorphenamine in real sample (Ganmaoling pill) using full-scan positive-ion mode. (A) Direct desorption and ionization on the pill; (B) ionization of the
ethanol extract of the pill spotted to the filter paper without development; (C) ionization of the analyte on the filter paper sheet after development.
Y.-Q. Huang et al. / J. Chromatogr. A 1218 (2011) 7371–7376 7375

Table 2
Recoveries of chlorphenamine in extract of solid herbal medicine and liquid herbal medicine.

Samples Analyte Added (␮g/mL) Found (␮g/mL) Recovery RSD (%)

0 10.5 – 15.0
Extract of solid herbal medicine 10.0 18.9 0.843 12.8
30.0 37.7 0.906 10.7
Chlorphenamine
0 0 – –
Liquid herbal medicine 10.0 8.45 0.845 15.5
30.0 26.9 0.895 12.4

Table 3
Comparison of different methods for analysis of adulterants in herbal products from literature and this study.

Method Specificity Throughput Sensitivity Reproducibility Reference

TLC +a + + ++ [35]
HPLC ++ + ++ +++ [36–38]
CE ++ + ++ ++ [39–41]
GC–MS ++++ + ++++ ++ [42]
HPLC–MS +++ + ++++ +++ [43–47]
CE–MS +++ + ++++ ++ [48]
FEPC–DCBI-MS +++ ++++ ++ ++ This study
a
+, satisfactory; ++, good; +++, very good; ++++, excellent.

thermo-dependent, gas heating is usually required for desorbing took only 10–20 s. Therefore, the rapid analysis procedure facili-
the less volatile species from the sample surface for DCBI-MS anal- tated the practical application of the method for the analysis of
ysis. The optimum desorption temperature vary with the volatility large amounts of sample.
of the analytes and the strength of the interaction between analytes
and substrate. In this study, the effect of the DCBI probe tempera- 3.5. Roles of the filter paper
ture ranging from 150 ◦ C to 400 ◦ C on desorption was examined.
The results showed that the signal intensity of chlorphenamine Before developing on a paper sheet, the solid or powder sam-
first increased rapidly from 150 ◦ C to 250 ◦ C, then increased slightly ples were ground into fine powder and then suspended in ethanol
between 250 ◦ C and 300 ◦ C, and decreased when the temperature for extraction. The suspension was directly spotted onto the paper
was higher than 300 ◦ C. Thermal expansion and thermal decompo- sheet. The undissolved particles were left at the origin after devel-
sition might be the reasons for the signal intensity dropping at high opment. Besides as the PC substrate, the use of paper sheet can
temperature. Therefore, the DCBI probe temperature of 250 ◦ C was improve analysis speed by omitting the centrifugation step. In addi-
selected for further experiments. tion, because DCBI is an open air ionization source, the paper can
be directly presented for desorption and ionization without post-
3.3. Matrix effect treatment. Thus, the third role of the paper sheet served was as the
substrate of the ambient ionization.
Dozens of chemicals, including fatty acids, sterols, alkaloids,
flavonoids, glycosides and saponins, were contained in the herbal 3.6. Validation of the method
medicines or dietary supplements. The complex matrix of herbal
medicines may significantly suppress the ionization of the analytes Through the examination of the operating conditions described
or make the spectra much more complicated, which may cause above, we came to the optimized parameters (discharge gas flow
false negative or positive results in direct analysis using ambient rate, 0.5 L/min; desorption temperature, 250 ◦ C) to achieve the best
ionization mass spectrometry. For instance, in the case of direct performance of the method. Under the optimized experimental
desorption and ionization of real sample (Ganmaoling pill) using conditions, the calibration curve was constructed by plotting the
DCBI-MS, the ion signal of chlorphenamine was not observed in the ratios of peak intensities of chlorphenamine over those of the
mass spectrum (Fig. 4A). After ethanol extraction, chlorphenamine internal standard against the corresponding chlorphenamine con-
can be detected when the ethanol extract of the pill was spotted centrations. The results showed that chlorphenamine exhibited
on the filter paper without development, but the matrix inter- satisfactory linear MS response (Table 1). We also investigated the
ference was obvious since the ion signal intensity of the analyte detection limit of chlorphenamine using FEPC–DCBI-MS in full-scan
was low (Fig. 4B). Only several high ion signals such as m/z 195, positive-ion mode. A standard solution of chlorphenamine was
218, 302, 285 and 326 can be observed in the mass spectrum diluted from 10.0 ␮g/mL to certain concentration and then sep-
under positive-ion mode, whereas, after a simple separation using arated and concentrated with the triangle paper sheet prepared
frontal elution paper chromatography, the matrix interference was according to the procedure described in the experimental section.
reduced remarkably and the signal intensity of chlorphenamine The detection limit was determined as the concentration of chlor-
increased by 30-fold (Fig. 4C). phenamine at a signal-to-noise ratio (S/N) of 3. The result showed
that the detection limit was 200 ng/mL (2 ng, absolute).
3.4. Analysis speed Recoveries of chlorphenamine from solid or liquid herbal
medicines spiked with low and high concentrations of analyte
The typical analysis procedure took less than 5 min, which were studied to further validate the quantitative applicability of
included the following steps: sample mashing and extracting, the proposed method. As shown in Table 2, the recoveries of
1.0–1.5 min; application of an aliquot of sample onto filter paper, chlorphenamine ranged from 84.3 to 90.6% with RSD in the range
0.2 min; paper chromatography, 3.0 min; drying, 0.3 min; DCBI-MS of 10.7–15.5%. The results demonstrated that the FEPC–DCBI-MS
detection, 0.2 min. Once the analysis started, the speed was limited method is satisfactory for the quantification of chlorphenamine in
just by loading the paper sheet to the DCBI source, which usually herbal medicines.
7376 Y.-Q. Huang et al. / J. Chromatogr. A 1218 (2011) 7371–7376

3.7. Determination of chlorphenamine in real samples to thank Dr. Bi-Feng Yuan (Wuhan University) for advice on the
manuscript.
The FEPC–DCBI-MS method was applied to the determina-
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