Original Acta Chromatographica 27(2015)2, 355–372

Research Paper DOI: 10.1556/AChrom.27.2015.2.11

Development Micellar HPLC Method for

Simultaneous Determination of Ephedrine,
Pseudoephedrine, and Methylephedrine in
Ephedra Herb and Traditional Chinese
Medicinal Preparations
Y.–M. DONG*, Q. AN, N.-W. LU, AND N. LI
School of Pharmacy, Lanzhou University, Lanzhou 730000, P. R. China
*E-mail: dongym@lzu.edu.cn

Summary. A micellar high-performance liquid chromatography (HPLC) method has

been described for simultaneous determination of ephedrine, pseudoephedrine, and
methylephedrine in Ephedra Herb and two traditional Chinese preparations. The sepa-
ration and determination of ephedrine, pseudoephedrine, and methylephedrine were
performed using a mobile phase containing 1.75 × 10−1 mol·L−1 sodium dodecyl sulphate
and 0.02 mol·L−1 potassium hydrogen phosphate with 10% (v/v) methanol at pH 3.0,
running at 1.5 mL·min−1 by a Venusil XBP C18 (250 × 4.6 mm, 5 μm) column at 40 °C.
The detected wavelength was set at 210 nm. The method was validated according to the
International Conference on Harmonization of Technical Requirements for Registration
of Pharmaceuticals for Human Use (ICH) guidelines. The main analytical parameters
were linearity (r > 0.9990), intra- and inter-day precisions (relative standard deviation
[RSD %], 0.33–1.63, and RSD %, 1.26–2.20, respectively), limit of quantifications [LOQs],
and limit of detections [LODs] (2.6 × 10−4 and 7.8 × 10−5 mg·mL−1 for ephedrine,
6.8 × 10−4 and 2.0 × 10−4 mg·mL−1 for pseudoephedrine, and 5.0 × 10−4 and 1.5 × 10−4
mg·mL−1 for methylephedrine). RSDs of recoveries were <5.5% in the three samples.
Based on the optimized chromatographic conditions and the eluted orders, a model of
separation mechanism for the analytes was established. The results indicated that the
proposed method was an accurate, “green” and cheap method.

Key Words: ephedrine, pseudoephedrine, methylephedrine, micellar high-performance

liquid chromatography

Introduction
Ephedra Herb, also called Ma Huang in China, originates from one of the
dried herbaceous stems of Ephedra sinica Stapf., Ephedra intermedia Schrenk
et. C. A. Mey., and Ephedra equisetina. Bge. [1]. Its major effective compo-
nents are alkaloids [2], including ephedrine (E), pseudoephedrine (PE), me-
thylephedrine (ME), and norephedrine. These alkaloids have similar

0231–2522 © 2014 Akadémiai Kiadó, Budapest

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356 Y.-M. Dong et al.

chemical structures (Fig. 1) and show diverse pharmacological actions, for

instance, increasing the cardiac rate, raising the heart contractility,
strengthening the peripheral vasoconstriction, and reinforcing the central
nervous system (CNS) stimulation [3]. It has been widely used as a tradi-
tional Chinese medicine (TCM) for the treatment of common cold, flu,
asthma, and cough [4], and commonly used as a raw material in many TCM
preparations. The quality of TCM is susceptible to affection of climatic con-
ditions, different cultivation areas, and different processing methods. Thus,
the level of these effective components may be varied obviously in Ephedra
Herb and its TCM preparations. The variation of these effective components
will cause the difference in the quality. In addition, the action of CNS
stimulation of the ephedrine would cause adverse events and fatalities.
Hence, it is essential to evaluate the quality and safety of raw materials and
its preparations.

Fig. 1. The structures of methylephedrine (ME), pseudoephedrine (PE), and

ephedrine (E)

In fact, various analytical methods have been developed to simultane-

ously separate and determine these effective components in Ephedra Herb
and its TCM preparations, such as reversed phase high-performance liquid
chromatography (RP-HPLC) [5, 6], two-dimensional HPLC (2-D HPLC) [7],
capillary electrophoresis (CE) [8–11], and two-dimensional gas chromatog-
raphy (2-D GC) [12]. Using RP-HPLC methods, the triethylamine (TEA) was
usually employed to reduce tailing of peaks. Unfortunately, the residue of
TEA on the chromatographic column might cause irreversible change or
damage to the column [13, 14] and might also cause contamination to the
pumps and capillaries of HPLC system [15]. Today, although better chro-
matographic columns have been applied for such separation without a need
of adding TEA or other amines in the mobile phase, the price of the chro-
matographic column is higher than that of common chromatographic col-
umns. Using 2-D HPLC method, the retention times of E, PE, and ME were
48.01 min, 51.12 min, and 64.12 min, respectively, which were too long. Al-
though CE had a good resolution in the separation of E, PE, and ME, the

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Development Micellar HPLC Method 357

derivative processes of E, PE, and ME in these methods were complicated

and the derivative regents, p-vinylbenzyltriethylammonium chloride and
Ru(bpy)3 C12·6H2O, were expensive. Using 2-D GC method, the quantita-
tion in 2-D GC was carried out by the accumulation of modulated peak [16],
whose quantitation was not as direct as in 1-D GC.
Therefore, a simple and economical analytical method for simultaneous
determination of E, PE, and ME in Ephedra and TCM preparations is badly
in need of establishment.
The micellar high-performance liquid chromatography (MHPLC) is a
reversed phase liquid chromatographic separation technology, whose mo-
bile phase employed the surfactant at a higher concentration than the criti-
cal micellar concentration (CMC) and little organic modifier such as ace-
tonitrile (ACN), methanol (MeOH), propanol, or pentanol, that has widely
received attention in recent years for some of its peculiar features such as
high selectivity, low toxicity, and simultaneous separation of charged and
uncharged solutes [17]. The biological samples could be directly injected
without any pretreatment into chromatographic system [18], which reduced
the time and the cost. Sodium dodecyl sulphate (SDS) is an anionic surfac-
tant and usually is used as a micellar media because it is easily dissolved in
water and could be removed from the chromatographic system. MHPLC
methods based on the use of SDS and a small amount of organic modifier
have been applied as a novel technology of separation in different disci-
plines such as food science [19, 20], pharmacy [21, 22], and environmental
science [23] since it was put forward by Armstrong [24].
Furthermore, banned drugs in sport, ephedrine and norephedrine,
were also detected by micellar liquid chromatography [25, 26]. However,
the retention time of E and norephedrine was 48.13 and 58.28, respectively,
and the theoretical plate number of E and norephedrine was less than 2000.
In addition, as far as we know, simultaneous analysis of these groups of al-
kaloids by using micellar liquid chromatography has not been reported.
In this paper, we developed an easy, accurate, green, low price, and re-
liable analytical method to simultaneously separate and determine E, PE,
and ME in Ephedra Herb and TCM preparations (Xiao’er Qingre Koufuye
(oral liquid) and Maxing Zhike Pian (tablet) by MHPLC using SDS as the
micellar media in the mobile phase. The proposed method has been vali-
dated following the International Conference on Harmonization of Techni-
cal Requirements for Registration of Pharmaceuticals for Human Use (ICH)
guidelines [27]. The separation mechanism of E, PE, and ME in the MHPLC
system was also briefly discussed. The suggested procedure provided the
main benefit of it being associated with the use of micellar media, that is,
fast and easy preparation, and minimizing the use of pollutant organic
modifier.

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358 Y.-M. Dong et al.

Experimental
Chemicals and Reagents

Chromatographic-grade acetonitrile (ACN) and methanol (MeOH) were

purchased from Yuwang Reagent Factory (Yucheng, China). Sodium dode-
cyl sulphate (SDS), phosphoric acid (H3PO4), potassium dihydrogen phos-
phate (KH2PO4), ethanol, 1-propanol, and 1-pentanol were of analytical
grade and bought from Tianjin Chemical Industrial Company (Tianjin,
China). Ethanol, 1-propanol, and 1-pentanol were distilled before use. Dis-
tilled water was from the Good Laboratory Practice lab of Lanzhou Univer-
sity (Lanzhou, China) and was used to prepare kinds of solution.
Reference standards of ephedrine hydrochloride (Batch No.
171241-200506), pseudoephedrine hydrochloride (Batch No. 171237-200505),
and methylephedrine hydrochloride (Batch No. 171247-2003011) were pur-
chased from the National Institute for the Control of Pharmaceutical and
Biological Products (NICPP, Beijing, China) and used without further puri-
fication.
Materials of Ephedra Herb were purchased from local medicine mar-
kets (Lanzhou, P. R. China) and were authenticated by Jian-yin Li, lecturer,
who works in the Institute of the Pharmacognosy of Lanzhou University,
Lanzhou, China. Xiao’er Qingre Koufuye (Batch No. 110702, oral liquid)
was purchased from Yantai Rongchang Pharmaceutical Co., Ltd. (Yantai, P.
R. China). Maxing Zhike Pian (Batch No. 120502, tablet) was purchased
from Jinchang Azoth Pharmaceutical Co., Ltd. (Jinchang, P. R. China).

Apparatus

The HPLC system (Ultimate 3000 model system, Dionex, USA) consisted of
a diode array detector (DAD) and a 10-μL loop manual injector. System
control and data analysis were carried out using the chromatographic
workstation Chromeleon Client (Version Chromeleon 6.80, Dionex Corpo-
ration, Sunnyvale, USA). FE20 pH meter (Mettler Toledo Instrument Co.,
Ltd., Shanghai, China) was used to measure the pH of the mobile phase.
KH-300DB ultrasonic cleaner (Kun Shan He Chuang Ultrasonic Instruments
Co., Ltd., Kunshan, China) was employed for dissolving the reference stan-
dards and the extracting plant materials. AP-01P vacuum pump (Tianjin,
Autosci Ence Instruments Co., Ltd., Tianjin, China) was used to filter solu-
tion by 0.22 μm nylon membrane.

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Development Micellar HPLC Method 359

Optimized Chromatographic Conditions

Separation and determination of E, PE, and ME were performed on a

Venusil XBP C18 (250 × 4.6 mm, 5 μm, Agela Corporation, Beijing, China)
column using a mobile phase consisting of an aqueous solution (1.75 × 10−1
mol·L−1 SDS and 0.02 mol·L−1 KH2PO4) with 10% (v/v) of methanol at pH
3.0, running at 1.5 mL·min−1 at 40 °C. The detection wavelength was set at
210 nm. The injection volume was 10 μL.

Preparation of the Mobile Phase, Standard Solutions, and

Samples

The mobile phase consisted of a buffer solution and MeOH according to the
proportion of the buffer solution: MeOH (9:1, v/v). The buffer solution was
prepared by dissolving 1.75 × 10−1 mol of SDS and 0.02 mol of KH2PO4 in
900 mL distilled water, and the pH was adjusted to 3.0 with diluted H3PO4
prior to the addition of MeOH.
Individual stock solutions of E hydrochloride, PE hydrochloride, and
ME hydrochloride prepared at a concentration of 1.0 mg·mL−1 were ob-
tained by dissolving each one in water. All standard stock solutions were
stored until use under refrigeration at 4 °C.
Samples of Ephedra Herb were ground using pestle, and the ground
powder was sieved through 80 meshes. The sieved powder (1.0 g) was ul-
trasonically extracted by 25 mL mobile phase at 45 °C for 30 min. The ex-
tracted solution was filtered. The residue was extracted again in the same
way. The filtrates of the two extractions were combined, which were trans-
ferred to a 50-mL volumetric flask and diluted with the mobile phase to
volume and mixed.
Five ampoules of Xiao’er Qingre Koufuye (the labeled volume is 10
mL/ampoule) were mixed. Ten milliliters of the mixed sample was trans-
ferred to a 50-mL volumetric flask and diluted with the mobile phase to
volume and mixed.
Eight tablets of Maxing Zhike Pian (the average weight of the tablet is
0.26 g/tablet) were weighed accurately and ground, and the ground pow-
der (2.0 g) was weighed accurately. Then, the sample of Maxing Zhike Pian
was prepared by the preparation method of Ephedra Herb.
All the preparation solutions were filtered by 0.22 μm nylon membrane
filtration, which were directly injected into the HPLC system, and were
stored at 4 °C.

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360 Y.-M. Dong et al.

Results and Discussion

Optimization of the Chromatographic Conditions

In order to obtain good separation, short retention times, good peak shapes,
and high efficiencies for E, PE, and ME, SDS concentration, the types and
concentration of organic modifier, pH, detected wavelength, temperature of
the column, and flow rate were optimized.

Optimization of the SDS Concentration

The concentration of SDS was an important parameter for the effective

separation of E, PE, and ME, which was investigated from 0.10 mol·L−1 to
0.25 mol·L−1. The results indicated that the retention times of E, PE, and ME
were shortened from 62.12 to 23.98 min, 61.13 to 21.26 min, and 58.12 to
19.85 min, respectively, and the theoretical plate number of E, PE, and ME,
which were calculated according to N = 16(tR / W ) , where N is the theo-
2

retical plate number, t R is the analyte retention time, and W is the peak
width measured at the baseline of the extrapolated straight sides to the
baseline. They were decreased from 5858 to 4123, 5612 to 4068, and 5383 to
3789, respectively, with increased SDS concentration. Meanwhile, the vis-
cosity of the mobile phase was amplified with increased SDS concentration,
which caused the column pressure increase.
In addition, we observed that the SDS would absorb ultraviolet (UV)
light when the SDS concentration was above the CMC from chroma-
tographic instrument. The absorption maybe lead that the sensitivity of
MHPLC was lower than that of conventional RP-HPLC. As shown in Fig. 2,
when the concentration of SDS was lower than CMC (8.20 × 10−3 mol·L−1),
the lamp intensity of UV light did not show a clear weakness. As the SDS
concentration was above the CMC, the lamp intensity of UV light was re-
duced with the increase of SDS concentration. Especially, when the concen-
tration of SDS was increased from 0.10 mol·L−1 to 2.25 × 10−1 mol·L−1, the
lamp intensity of UV light indicated an obvious reduction. When the con-
centration of SDS was higher than 0.25 mol·L−1, the analytical procedure
could not run because SDS absorbed too much ultraviolet light, which
caused little ultraviolet light to arrive at the detector. The possible cause
that UV light was absorbed by SDS was that the distribution morphology of
the micellar solution was different in different concentrations of SDS [28].
When the SDS concentration (above CMC) was increased, the numbers of
the micelles increased, which led to the enhancement of electrostatic repul-
sion among the micelles. As a result, the micellar forms were changed from

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Development Micellar HPLC Method 361

spheres to layers. The layered micelles would absorb much more UV light
than that of the sphere micelles. Therefore, the UV light that could be ob-
tained by the detector became weak.

7
1.8x10
CMC

7
1.5x10
Lamp intensity (count/s)

7
1.2x10

6
9.0x10

6
6.0x10

6
3.0x10

0.0

0.00 0.05 0.10 0.15 0.20 0.25

-1
Concentration of SDS (mol.L )
Fig. 2. The concentration of SDS versus the lamp intensity

As a result of the compromise between the sensitivity and the resolu-

tion, relatively high level of SDS, which could detect the analytes and pro-
vide enough sensitivity to assay the analytes, should be used. The sacrificed
sensitivity would be amended by adding the organic modifier and increas-
ing the column temperature. Thus, 1.75 × 10−1 mol·L−1 of SDS was selected
as the optimum concentration in the experiment.

Optimization of the Organic Modifier

Adding organic modifier, the efficiency and retention time would be

changed. The elution strength of alcohols was increased with the length of
its carbon chain [29]. 1-Propanol, 1-pentanol, ethanol, ACN, and MeOH as
the organic modifier were investigated in the experiment. When the same
amount of them was used in the mobile phase, the results of effect on the
separation were different. Using 1-propanol and 1-pentanol as the organic
modifiers, the retention times of the target analytes were shortened drasti-
cally (the retention times of the analytes were less than 15 min), but the
peaks of ME and PE were overlapped. In terms of ethanol as the organic
modifier, the peaks of ME and PE could not obtain good separation. Using

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362 Y.-M. Dong et al.

ACN and MeOH, the three analytes could simultaneously obtain good
separation and symmetries of the chromatographic peaks. However, the re-
tention behaviors of them were different.
E, PE, and ME obtained simultaneous separation in the range from 3%
(v/v) to 7% (v/v) for ACN, and the retention time of the three analytes had
no obvious change within this range and the peaks of E, PE, and ME be-
came thin. When the amount of ACN was increased from 7% (v/v) to 11%
(v/v), the retention times of ME and E were slightly reduced, and the reten-
tion time of PE was reduced much more than that of the others, which led
the resolution between ME and PE to less than 1.5. Using MeOH (3% (v/v)
to 10% (v/v)), the results of the tests were similar to the chromatographic
behavior of ACN (3% (v/v) to 7% (v/v)). As the amount of MeOH was in
the range of 15% (v/v) to 20% (v/v), the retention times of the three analytes
were delayed and the sensitivity was declined. Moreover, when the amount
of organic solution was less than 5% (v/v) in the mobile phase, it would
cause the working life of the chromatographic column shorten.
Good resolutions for the analytes could be obtained when the concen-
trations of ACN and MeOH were in the range of 3%–10% (v/v), but the
price of ACN was about three times that of MeOH. Considering the factors
of the requirement of the resolution, working life of the chromatographic
column and cost, 10% MeOH was selected as the organic modifier.

Optimization of the pH

Five values of pH (2.5, 3.0, 3.5, 4.5, and 5.5) were tested. The results of the
effect on the separation were shown in Fig. 3. When the value of pH was 3.0,
the target analytes gained good separation. Their resolutions were more
than 1.5, and the tailing factors of their peaks were less than 1.2. As the
value of pH was 2.5, the retention times of E and ME were delayed, while
the retention time of PE was shortened which caused the peaks of ME and
PE overlapped. When pH was increased from 3.0 to 5.5, the retention times
of the three analytes were delayed, while the retention time of PE was de-
layed too much so that the peaks of PE and E were overlapped from part to
all. Meanwhile, the peaks of E, PE, and ME became wider with increasing
the values of pH. Therefore, the analyses were performed at pH 3.
The acid-base constants of E, PE, and ME (pKa = 9.64, 9.74, and 9.40,
respectively) were outside of the working pH range of chromatographic
column (2.5–7.5). Hence, there was equilibrium between the monoproto-
nated and the nonprotonated. For analytes, the retention on chroma-
tographic column was the same using mobile phase of SDS at pH 3.0 and
7.0 [30]. However, there was obvious variation for the retention time of

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Development Micellar HPLC Method 363

analytes. The change of pH maybe impacted the intensity of interaction

between analytes and SDS micellar or free SDS or water-organic because of
difference of chemical structures.

27

26 E
PE
Retention time (min)

25
ME

24

23

22

21
2.5 3.0 3.5 4.0 4.5 5.0 5.5
pH

Fig. 3. Effect of different pH on separation

Selection of the Detected Wavelength

The detection wavelength was set to scan from 200 to 400 nm under the
same chromatographic conditions. The results indicated that the maximum
absorption was observed near at 205 nm, but the noise was so strong that it
interfered with detection to the target analytes. When the values of wave-
length were increased, the peak heights of E, PE, and ME were reduced.
Thus, 210 nm was selected as the best detected wavelength, which could
exclude interference of noise and obtain good absorption to determine the
analytes.
Selection of the Column Temperature and Flow Rate

Low column efficiency was a shortcoming of MHPLC. Adding organic

modifier and rising column temperature would improve column efficiency.
The column temperature was investigated from 30 °C to 50 °C. Peaks of the
three analytes became thin and high (tailing factor of E, PE, and ME was
decreased from 1.01–1.20 to 0.99–1.03, 1.13–1.16 to 0.98–1.05, and 1.12–1.18
to 1.02–1.05, respectively) when the column temperature was increased.
However, when the column temperature was too high, it gave rise to the

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364 Y.-M. Dong et al.

working life of the column shortened. Hence, the column temperature was
set at 40 °C.
The retention time of the three analytes was shortened distinctly with
the increase of flow rate. Meanwhile, column pressure also increased when
the flow rate was increased. Thus, the flow rate was set at 1.5 mL·min−1.

Conceivable Mechanism of the Separation

According to the process of optimized chromatographic conditions and the

elution orders of E, PE, and ME in the chromatogram (Fig. 4), we simulated
a conceivable separation model (Fig. 5) for the separation of E, PE, and ME.
At pH 3.0, the predominant form of E, PE, and ME in solution would
be positively charged ions according to pKa values which were 9.64, 9.74,
and 9.40, respectively. The stationary phase was modified by the mobile
phase containing SDS and the surface of it was coated with a layer of nega-
tive charges. The combination of protonated analytes to modified stationary
phase was too strong, so the retention time of analytes were long in pure
micellar mobile phase. The mobile phase contained SDS micelles, free SDS,
MeOH, and water.
100

75
Peak height (mAu)

50
ME
PE E
25

-25

-50

0 5 10 15 20 25
Retention time (min)
Fig. 4. The MHPLC chromatogram of reference standards. Chromatographic conditions:
A venusil XBP C18 column (250 × 4.6 mm, 5μm). The mobile phase: aqueous solution
(1.75 × 10−1 mol· L−1 SDS and 0.02 mol· L−1 potassium dihydrogen phosphate) – methanol
(MeOH) (90:10, v/v) at pH 3.0. Isocratic elution was used. The flow rate
was 1.5 mL·min−1. The injection volume was 10 μL

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Development Micellar HPLC Method 365

Modified stationary phase of C18

Fig. 5. Simulative model of separation mechanism

Firstly, these ions were absorbed on the modified stationary phase.

Then, E, PE, and ME were desorbed in the mobile phase and interacted with
the SDS micelles or free SDS. The outer layers of SDS micelles were negative
charge, and the interior was hydrophobic part. ME had strong hydropho-
bicity. Hence, the interaction between ME and SDS micelles was stronger
than that of ME and free SDS. This combination reduced knock number
between ME and the modified stationary phase in SDS micelles and free
SDS move. ME accompanied SDS micelles were directly flowed out the
chromatographic column and were firstly detected. Although E and PE
were isomer, PE was easy to form intermolecular hydrogen. Thus, its steric
hindrance was smaller than that of E. Hence, its ability of combination to
SDS micelles or free SDS were stronger than that of E and the knock number
between PE and the modified stationary than phase was less than that of E
with SDS micelles and free SDS move. The results were that PE was de-
tected secondly and E was detected thirdly.
Adding MeOH to the mobile phase, association of MeOH and no bond
silicon hydroxyl became less because the stationary phase was modified by

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366 Y.-M. Dong et al.

containing SDS mobile phase. MeOH interacted with the hydrophilic layer
formed by the sulfate head groups of SDS, as indicated that the SDS mi-
celles were surrounded by MeOH, which caused that the interaction of
electrostatic repulsion among the micelles was reduced and the interaction
of electrostatic attraction between the analytes and micelles was increased.
As a result, the retention times of analytes were shortened and the chroma-
tographic system efficiencies were improved.

Optimization of the Extraction Conditions

To extract the three analytes fully in the samples as possible as we can, ex-
traction conditions were optimized. According to the literatures reported,
the ultrasonic extraction method was employed [31]. Hence, we mainly in-
vestigated the influence of different solvents on the extraction of the three
analytes. The following were used as the extraction solvents, respectively, to
extract the analytes by ultrasonication bath for two times at 45 °C for 30 min:
0.10 mol·L−1 of hydrochloric acid–MeOH (0.8:99.2, v/v), 0.25 mol·L−1 of
phosphoric acid–MeOH (0.8:99.2, v/v), 0.25 mol·L−1 of phosphoric acid and
the mobile phase. Using 0.10 mol·L−1 of hydrochloric acid as the extraction
solvent, ME could not be detected and the peaks of the solvent had strong
interference. When 0.25 mol·L−1 of phosphoric acid–MeOH (0.8:99.2, v/v)
and 0.25 mol·L−1 of phosphoric acid were used as the extraction solvents,
the analytes could be detected, but the sensitivities were poor. As the mo-
bile phase was used as the extraction solvent, the sensitivities of the ana-
lytes were good and no other interference. Hence, the mobile phase was
employed as the extraction solvent.

Method Validation

The method was validated in terms of linearity, limit of quantifications

(LOQ), limit of detection (LOD), precision, stability, and robustness.

Linearity and Sensitivity

The standard stock solutions prepared were diluted to the appropriate con-
centration. The calibration curves for the three analytes were plotted using
the areas of the chromatographic peaks (triplicate injections) obtained at
five different concentrations versus the concentrations of each analyte. Sat-

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 Development Micellar HPLC Method 367

isfactory calibration curves of E, PE, and ME were obtained. The LODs and
LOQs for the three analytes were calculated with the concentration where
the signal-to-noise ratio is 3 and 10. The results are shown in Table I.

Table I. Results of method validation linearity, LOD, LOQ, intra-day precision, and
inter-day precision

Parameters Ephedrine Pseudoephedrine Methylephedrine

A= A= A=
Linear equation
0.24497C + 0.00292 0.30703C + 0.00438 0.33697C + 0.00458
Correlation coefficient (r) 0.9999 0.9993 0.9995

Linearity range (μg·mL−1) 20.03–125.12 16.6–106.4 0.2–120.2

LOD (mg·mL−1) 7.8 × 10−5 2.0 × 10−4 1.5 × 10−4

LOQ (mg·mL−1) 2.6 × 10−4 6.8 × 10−4 5.0 × 10−4

Intra-day precision RSD %
0.33 0.52 1.63
(retention time)
Intra-day precision RSD %
1.49 1.39 1.08
(peak area)
Inter-day precision RSD %
1.26 1.34 1.83
(retention time)
Inter-day precision RSD %
2.20 1.71 1.55
(peak area)

Precision

The method precision was determined by injection of six standard samples

of E, PE, and ME at 100% level of the expected concentrations of E, PE, and
ME in Ephedra Herb and the two TCM preparations for the intra-day preci-
sion and across three different days for the inter-day precision. Relative
standard deviations (RSDs) of peak areas and retention times for E, PE, and
ME were below 2.50%, 1.75%, and 1.85%, respectively. As shown in Table I,
it suggested that the method had good precision.

Stability

Sample stability was determined with sample of Ephedra Herb during 0 h, 2

h, 4 h, 6 h, and 12 h under the optimized chromatographic conditions. RSDs

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368 Y.-M. Dong et al.

of peak areas for E, PE, and ME were 1.59%, 1.36%, and 1.01%, respectively,
which indicated that the sample solution was at least stable in 12 h.

Robustness

Robustness of the method was the measure of its capacity to remain unaf-
fected by slightly changed method parameters, which was determined by
replicate injections (n = 3) of standards mixed solution of E, PE, and ME at
concentration of 20 μg mL−1 under slightly changed chromatographic para-

Table II. Robustness evaluation of MHPLC

Changed chromatographic Retention time Peak area

Analytes Level
parameters (min) (RSD %) (RSD %)

Ephedrine Column temperature (°C) 39–41 1.02 1.67

Flow rate (mL·min )

−1 1.45–1.55 2.21 1.78

pH 2.9–3.1 1.67 2.11

MeOH (%) 9.9–10.1 0.04 1.39

Concentration of SDS
0.170–0.180 4.12 2.64
(mol·L−1)
Pseudoephedrine Column temperature (°C) 39–41 0.91 1.78

Flow rate (mL·min )

−1 1.45–1.55 2.23 2.14
pH 2.9–3.1 1.98 2.33

MeOH (%) 9.9–10.1 0.07 1.89

Concentration of SDS
0.170–0.180 5.96 2.78
(mol·L−1)
Methylephedrine Column temperature (°C) 39–41 1.68 1.52
Flow rate (mL·min−1) 1.45–1.55 2.38 1.53

pH 2.9–3.1 2.28 1.84

MeOH (%) 9.9–10.1 0.81 1.49
Concentration of SDS
0.170–0.180 4.97 3.08
(mol·L−1)

meters (column temperature, flow rate, pH, percentage of MeOH, and SDS
concentration). The changes of the retention time and the peaks area were
investigated (Table II). RSDs of peak areas and retention times for E, PE, and
ME were below 4.15%, 6.00%, and 5.00%, respectively, which indicated that

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Development Micellar HPLC Method 369

determination analytes were unaffected by slightly changed chroma-

tographic conditions.

Fig. 6. The MHPLC chromatograms of Ephedra Herb (a), Xiao’er Qingre Koufuye (b),
and Maxing Zhike Pian (c). The chromatographic condition was described in Fig 4.

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 370 Y.-M. Dong et al.

Analysis of Real Samples

The newly established MHPLC method was applied to simultaneously de-

termine the three active compounds in Ephedra Herb (Fig. 6a), Xiao’er Qin-
gre Koufuye, and Maxing Zhike Pian including Ephedra Herb (Fig. 6b and

Table III. The standard addition recovery and determined contents of ephedrine (E),
pseudoephedrine (PE), and methylephedrine (ME) in the Ephedra Herb, Xiao’er Qingre
Koufuye, and Maxing Zhike Pian, respectively

Added Found
Recovery%
Samples Analytes concentration concentration RSD % Contents
(n = 6)
(μg·mL−1) (μg·mL−1)

Ephedra herb E 47.08 45.90 97.5 2.16

0.57
58.81 58.13 98.8 1.32
mg·g−1
70.56 70.84 100.4 2.57

PE 13.44 12.99 96.7 3.09

0.16
16.80 16.28 96.9 2.07
mg·g−1
20.16 19.69 97.7 2.51

ME 2.45 2.30 94.1 5.01

0.03
3.06 2.99 97.8 2.74
mg·g−1
3.67 3.73 101.5 2.45
Maxing Zhike
E 27.45 26.94 98.1 1.68
Pian
41.59
34.31 33.12 96.5 1.35
μg/tablet
41.17 40.62 98.7 2.48

PE 11.60 11.12 95.9 2.75

17.61
14.50 14.54 100.3 3.94
μg/tablet
17.40 17.13 98.4 2.84
Xiao’er
Qingre E 13.22 13.49 102.1 3.36
Koufuye
16.53
16.53 16.24 98.3 2.16
μg·mL−1
19.84 18.77 94.6 2.49

PE 8.86 8.24 93.0 2.75

11.09
11.08 10.94 98.8 1.87
μg·mL−1
13.30 13.20 99.2 1.58

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Development Micellar HPLC Method 371

6c). The recovery tests were done by the standard addition method. Low,
medium, and high amounts of the standards were added to the samples.
Then, the samples were prepared by the method described in the Sec-
tion 2.4. The results (Table III) indicated good recovery percentages for all
the concentrations obtained, and the developed MHPLC method was pre-
cise for simultaneous quantitative evaluation of the E, PE, and ME.

Conclusion

In this paper, we developed an MHPLC method for simultaneous separa-

tion and determination E, PE, and ME in Ephedra Herb, Xiao’er Qingre
Koufuye, and Maxing Zhike Pian for the first time. SDS was used as the
micellar media in the MHPLC system. The micellar mobile phase not only
improved the selectivity of the separation but also had a little amount of
organic solvent, which was friendly to the environment. In addition, the
price of SDS was cheap. For similar chemical structures of E, PE, and ME,
they could obtain good resolution using the proposed MHPLC method. The
developed method was validated according to the guidance of ICH, and it
was proved to be a practicable, precise, and accurate method. The separa-
tion mechanism of E, PE, and ME was briefly discussed, and a model of the
separation mechanism was also simulated. We found that SDS would ab-
sorb UV light in the experimental process and elaborated the possible rea-
sons for this phenomenon. The developed method could be used to evalu-
ate the quality of Ephedra Herb and TCM preparations whose raw materi-
als include Ephedra Herb.

Acknowledgments
This work was supported by the Fundamental Research Funds for the Cen-
tral Universities of China (No. lzujbky-2011-93).

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Accepted by MWH

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