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Determination of Synephrine and Octopamine in Bitter Orange Peel by HPTLCwith Densitometry
Eman Shawky*
Faculty of Pharmacy, Department of Pharmacognosy, Alexandria University, Alkhartoom Square, Alexandria 21521, Egypt
*Email: shawkyeman@yahoo.com
Received 21 March 2013; revised 11 May 2013
This paper presents the development and validation of an improved
method for the simultaneous analysis of synephrine and octopamine
using high-performance thin-layer chromatography with densitomet-
ric detection. Separation was performed on silica gel 60F254 plates.
The mobile phase is comprised of methanol, ethylacetate, methylene
chloride and concentrated ammonia (2:2:1:0.05, v:v:v:v). The Rfvalues were 0.292+++++0.0083 and 0.413+++++0.0089 for synephrine and
octopamine, respectively (n5 9). Ultraviolet absorbance detection
at 277 nm was used for the alkaloids detection. Specificity, accuracy
(recovery rates were between 96 and 99%) and precision (in both
cases intra-day precision and inter-day precision were 2.0%) of themethod were determined. Their amounts were calculated using the
regression equations of the calibration curves which were linear in
the range 0.2 1.2mg/spot. The amounts of alkaloids in basic metha-nolic extracts of bitter orange peel measured by the method were
0.253 and 0.142% for synephrine and octopamine, respectively. Most
of the factors evaluated in the robustness test were found to have an
insignificant effect on the selected responses at 95% confidence
level. The method was validated giving rise to a dependable and
high-throughput procedure well suited to routine application.
Introduction
Citrus aurantium (bitter orange) is a plant belonging tothe family Rutaceae, whose fruit extracts have been usedrecently for the treatment of obesity. The most important
biologically active constituents of the C. aurantium fruits arephenethylamine alkaloids (i.e., octopamine, synephrine, tyram-
ine, N-methyltyramine and hordenine). The unripe fruits ofC. aurantium contain several different adrenergic amines whichdiffer in the number and position of hydroxyl substituents and
include synephrine, octopamine, tyramine, N-methyltyramine andhordenine) (1). Synephrine is the primary alkaloid found in theimmature fruits, whereas the other alkaloids are present at lower
levels. Of these alkaloids, synephrine and octopamine (Figure 1)exhibit the greatest activity (24).
Synephrine is present in the peel and the edible part of Citrus
fruit. The banning of ephedrine (and related alkaloids fromEphedra sinica) in dietary supplements by the US Food andDrug Administration (5) has resulted in a dramatic increase in
the use ofC. aurantium(6). In response to the rapid increase inthe use of C. aurantium extracts in weight-loss products andassociated concerns from health professionals (7, 8), numerous
new methods for the determination of synephrine and relatedadrenergic amines in citrus plant extracts and commerciallyavailable herbal products have emerged in the scientific
literature. These include high-performance liquid chromatog-raphy (HPLC) with ultraviolet (UV) absorbance (915), fluores-cence (11), electrochemical (15), electrospray ionization mass
spectrometry (ESI-MS) (8), tandem mass spectrometry (MS/MS)(16) detection and electrophoresis with UV-absorbance detec-tion (1719). Nowadays, the modern high-performance TLC(HPTLC) technique is an efficient instrumental method and
optimized quantitative HPTLC using a densitometric evaluationproduces results that are analogous to those obtained with gaschromatography as well as HPLC (20). Especially for plant
extracts which are complex mixtures, HPTLC seems to have
some advantages over other analytical methods. The HPTLCmethod allows for the simultaneous analysis of numeroussamples using small quantities of solvents, automation and
simple sample preparation, reducing the time and cost ofexperiment and allowing for fast changing of chromatographicconditions, scanning and simultaneous development of severalchromatograms. The HPTLC procedure does not require exten-
sive clean-up procedures even for quantitative analysis; more-over, the availability of many sensitive and selective reagents inpost-chromatographic derivatization, for the confirmation of
specific groups or classes of analyzed compounds, is also a great
advantage of HPTLC (2123).To the best of our knowledge, there is no report on the simul-
taneous quantitative determination of synephrine and octopa-mine in C. aurantium by HPTLC. The present work reports thedevelopment and validation of an analytical method that allows
for selective determination of synephine and octopamine inC. aurantium by means of HPTLC according to the ICH guide-lines (24, 25).
Experimental
Chemicals and standards
HPTLC analyses were performed on Merck 20 10 cm HPTLC
silica gel 60F254 (0.25 mm) plates. Synephrine and octopaminewere supplied by Sigma, Aldrich, Germany. All the reagents usedin the experiment were of analytical grade and were supplied byMerck, Dramstadt, Germany.
Preparation of standard solutions
Synephrine standard stock solution was prepared by dissolving6 mg of accurately weighed synephrine standard in 10 mL of
methanol solution. Octopamine standard stock solution was pre-pared by dissolving 10 mg of accurately weighed octopaminestandard in 10 mL of methanol. Before dilution of the solutions
#The Author [2013]. Publishedby OxfordUniversity Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
Journal of Chromatographic Science 2013;1 6doi:10.1093/chromsci/bmt113 Article
Journal of Chromatographic Science Advance Access published August 2, 2013
7/29/2019 Chromsci.bmt113.Full
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to volume, they were alkalinized with 0.05 ml of concentrated
ammonium hydroxide solution, 28% (w/v) to render themedium alkaline ( pH8).
Sample preparation and extraction method
Unripe bitter orange fruits were harvested in August 2012 andprocessed and peeled and dried in an air oven at 408C toproduce a dried, powdered material, which was used to prepare
samples of fruit peel.Development, optimization and validation of the alkaloid ex-
traction procedure from bitter orange have been completely
described in a previous publication (26). An ultrasonic bath withtemperature control was used for sonication extraction.Approximately 5 g sample material was dissolved in 200 mL
solvent. Extraction time was adjusted to 30 min. 0.37% (massfraction) hydrochloric acid in water (Sample A) and 10% (massfraction) ammonium hydroxide in methanol (Sample B) were
evaluated as extraction solvents. The flasks were capped andsonicated at 408C. The samples were extracted three times in
succession with fresh solvent. Sample A was neutralized by theaddition of concentrated ammonium hydroxide solution. The
extracts were evaporated to dryness and re-dissolved in methanolcontaining 5% ammonium hydroxide. The samples were filteredbefore analysis.
HPTLC: densitometric procedure
Instrumentation
Sample solutions for HPTLC analyses were applied by means of a
CAMAG Wilmington, NC, USA Linomat IV automated spray-onband applicator. Zones were quantified by linear scanning at227 nm with a CAMAG TLC scanner 3 with a deuterium source
in the reflection mode, slit dimension settings of length 6 andwidth 0.1, a monochromator bandwidth 20 nm and a scanningrate of 15 mm s21. The peak areas of chromatograms were
determined using the CATS TLC software (version 4.X).
Chromatographic procedure
Standard solutions were applied in the form of bands on pre-coated HPTLC silica gel plates 60F254 (20 10 cm with250 mm thickness) by means of a Linomat IV automated
spray-on band applicator operated with the following settings:band length 6 mm, application rate 15 s mL21, distance betweeneach 2 bands 4 mm, distance from the plate side edge 1 cm and
distance from the bottom of the plate 1.5 cm. Twenty milliliters
of mobile-phase methanol-ethyl acetate-methylene chloride-
ammonium hydroxide (2:2:1:0.05, v/v/v/v) was used per devel-opment. Ascending development of the plates was carried out ina 20 20 cm CAMAG HPTLC twin trough chamber saturated
with the mobile phase. The optimized chamber saturation timefor the mobile phase was 20 min at room temperature. Plateswere developed to a distance of 8 cm beyond the origin. The de-
velopment time was 13 min. After development, the plates were
air-dried for 5 min. Densitometric scanning was performed on aCAMAG TLC scanner 3 in the reflectance/absorbance modeat 227 and 277 nm. The source of radiation utilized was adeuterium lamp emitting a continuous UV spectrum between190 and 400 nm. The slit dimension was kept at 6 0.1 mm.
Concentrations of the standards chromatographed were deter-mined from the intensity of diffusely reflected light. Evaluation
was by peak area measurement with linear regression.
Method validation
Method validation was performed on the parameters such as lin-earity, limit of sensitivities, specificity, precision, accuracy, recov-
ery and robustness as per ICH (The International Conference onHarmonisation) (24) guidelines. All the data were evaluatedusing standard statistical packages for Windows. Statistical signifi-
cance was considered at 95% probability level (P, 0.05).
Calibration and quantification
The stock standard solutions of synephrine and octopaminewere serially diluted to six standard solutions. A volume of2.0 mL of each solution was applied on the HPTLC plate to
deliver 0.21.2 mg of synephrine per spot and 1.02.0 mg ofoctopamine per spot. This was done in triplicate and repeatedfor 3 days. For each concentration, the applied spot bands were
evenly distributed across the plate to minimize possible variationalong the silica layer.
Four microliter aliquots of the prepared sample solutions Aand B were subjected to HPTLC analysis as described inChromatographic procedure section.
Precision
The precision of the method was determined by the measurementof instrumental, inter- and intra-day precision. Instrumental preci-sion was measured by scanning the same spot of a single concen-
tration seven times. The repeatability or intra-day precision was
studied by analyzing the standard solutions repeatedly, in thesame laboratory and on the same day, at three concentrations.Intermediate precision included the analysis of the standards
three times a day over 3 days by a different analyst. The results of
repeatability and intermediate precision are expressed as relativestandard deviation (RSD) (%).
Accuracy
The accuracy of the methods was determined by standard add-ition techniques. Known amounts of standard synephrine and
octopamine in a range of low, medium and high concentrationswere added to pre-analyzed samples and analyzed under opti-mized conditions. Addition experiments for each concentration
were performed in triplicate and the accuracy was calculated asthe % of analyte recovered. Three analyses per concentrationwere performed and mean+SD was determined.
Figure 1. Chemical structures of synephrine and octopamine.
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Results
Calibration curves, limits of detection/quantification
Adverse effects of overloading are to be carefully avoided inplanar chromatography. Accordingly, the calibration rangeshould be selected as low as possible, preferably starting nearthe limits of quantification (LOQs). Working stock solutions of
synephrine and octopamine were prepared by dilution withmethanol to give the concentrations 0.1, 0.2, 0.3, 0.4, 0.5 and0.6 mg/mL for synephrine and 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 mg/mLfor octopamine. Standard solutions were spotted on HPTLC platesto give absolute amounts of 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mg/bandfor standard synephrine and 1.0, 1.2, 1.4, 1.6, 1.8 and 2.0 mg/bandfor standard octopamine. This was repeated on 3 different days.Each concentration was spotted thrice. Calibration curves wereprepared by the least-squares method using absolute amount
(mg/band) as the independent variable (X) and the peak area ofsynephrine standard as the dependent variable (Y). The curves(Table I) confirmed linear relationship (r2 ! 0.98) between the
working concentration and the peak areas. Linearity was checkedfor 3 consecutive days for the same concentration range (six datapoints in triplicate) from different stock solutions.
Limits of detection (LOD) and LOQ were determined using thelinear regression equations: LOD 3.3Sy, x/b and LOQ 10Sy,x/b, where Sy, x is the standard deviation (SD) of the Y-valuedistribution around the regression line and b is the slope of thecalibration curve. LODs and LOQs for synephrine and octopamineare given in Table I.
Determination of synephrine and octopamine in bitter
orange peel extracts
Quantification of synephrine and octopamine was performedaccording to the procedure described in the Chromatographicprocedure section. The HPTLC profile of the acidic and basic
extracts (samples A and B respectively) at 277 nm showed that
acceptable separation was achieved without any interference ofthe nearby components under the specified conditions
(Figure 2). The amounts of synephrine and octopamine in bitterorange peel extracts were determined from the calibrationgraphs and the results are given in Table II. The content of
synephrine (0.253%) obtained here by the HPTLC method is inaccordance with the previous reports. Chen and Hou (27)reported 0.121.98% of synephrine, by TLC/UV absorbance inpowdered peel and fruit. 0.352% of synephrine was reported inpulverized dried fruits by the RP-HPLC/UV method (11).
Precision
Instrumental, intra and inter-day precision are given in Table III
in terms of RSD (%). The results depict that the method is
precise for the analysis of synephrine and octopamine in bitterorange peel extract.
Accuracy
The accuracy of the proposed method was expressed as the re-covery of standards added to the pre-analyzed samples. The
average percentages of recovery of synephrine at three differentlevels were 97.09, 98.25 and 98.78%, while the average percen-tages of recovery of octopamine at three different levels were
96.3, 97.95 and 98.42% (Table IV). It can be seen that the pro-posed method has an adequate degree of accuracy for the simul-taneous determination of synephrine and octopamine.
Robustness
To test the robustness of the method, small changes in the chro-matographic parameters were deliberately made, which may
Table I
Statistical Evaluation of Calibration: Calibration Equation, Linearity and Regression Diagnostics (n 9)
Analytical parameter Synephrine Octopamine
Rf value 0.292+ 0.0083 0.413+ 0.0089Intercept 3671 29611.0Slope 24 387 12 163r2 0.997 0.994
Linear range (mg/band) 0.2 1.2 1.0 2.0LOD (mg/band) 0.033 612 0.092 701LOQ (mg/band) 0.101 854 0.280 913
Figure 2. HPTLC scan densitogram at 277 nm showing selective base line separationof synephrine (3) and octopamine (4) from other sample components in bitter orange
peel extract (Sample B).
Table II
Assay of Synephrine in Bitter Orange Peel Samples (Calculated on Plant Dry Weight Basis) (n 6)
Sample Synephrine amountquantified % (w/w)
RSD (%) Octopamine amountquantified % (w/w)
RSD (%)
Acidic sample (Sample A) 0.0023+ 0.0001 4.76 0.00162+ 0.00006 3.74Basic sample (Sample B) 0.253+ 0.017 3.15 0.142+ 0.0055 3 .87
Table III
Precision (RSD (%))
Precision Synephrine (RSD (%)) Octopamine (RSD (%))
Instrumental 0.975 0.877Repeatability 1.637 1.723Intermediate precision 1.922 1.835
Determination of Synephrine and Octopamine in Bitter Orange Peel3
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affect the performance of the method such as mobile-phasecomposition, chamber saturation time, spot band size, delay
between spotting and plate development and developing dis-tance, but only negligible changes in the peak areas were found.Quantitation was not significantly affected by changing scanning
wavelength by+5 nm. Results are given in Table V.
Discussion
Method optimization
One of the major advantages of TLC is the minimal sample prep-
aration normally required. However, the existence of a possiblematrix effect should be constantly taken into account. Since C.aurantiumalkaloids can interact strongly with the free silanols
present on the surface of the stationary phase, peak tailing of theanalytes is often observed. Ammonium hydroxide was added tothe samples and standards to suppress the ionization of the alka-
loids and enhance peak resolution.Several eluent systems and chromatographic conditions, in-
cluding those reported in the literature (2830), were tried in
order to separate synephrine and octopamine from the co-occurring compounds in C. aurantium extract. After thoroughtesting, mobile phase composed of methanol, ethylacetate and
methylene chloride (2:2:1, v:v:v) was used as a starting point forthe development of HPLTC plates. The results showed good sep-aration. However, all peaks still showed some tailing, especially
the synephrine peak. Concentrated ammonia solution was intro-duced into this mobile phase in volumes of 0.025, 0.05 and0.1 mL while other components were constant. The results
showed reduced tailing of all the peaks. The combination ofmethanol, ethylacetate, methylene chloride and concentratedammonia at 10, 10, 5 and 0.25 mL, respectively, i.e., 2:2:1:0.05, v/v/v/v, resulted in well-separated, compact spots which showedsymmetrical peaks on the chromatogram (Figure 2). The
Rf-values with their SDs were 0.292+0.0083 and 0.413+0.0089 for synephrine and octopamine, respectively (n 9).
Sample extraction
The effectiveness of different extraction approaches and condi-
tions was previously studied (26) to achieve maximum recovery
of bitter orange alkaloids from different sample matrixes.
Extractions were carried out under basic (pH8), neutral( pH5) and acidic (pH2) conditions. Basic extraction condi-tion was problematic in that study because of poor peak shape
which results from the injection of a basic sample extract inHPLC. In the present study, the extracts prepared under acidicand basic conditions were compared. Application of the acidic
sample extract resulted in poor peak shape. An attempt to neu-
tralize acidic extracts prior to application was done but this ledto precipitation of unidentified constituents after the addition of
ammonium hydroxide. The results of analysis of both samplesare presented in Table II. The basic samples gave by far betterresults.
Method validation
Following method development, an experimental procedure forsample treatment and analysis (fully described in the materials
and methods section) was obtained and submitted to validation.Linearity, LOD and LOQ, repeatability, accuracy and robustnesswere evaluated. According to the definition of validation, the ac-
ceptance criteria should be considered and customized to theintended use of the analytical method. No acceptance criteriaare provided for the determination of active compounds in
natural products. However, at the present time, when a methodconcerned with assaying an active ingredient in herbal matricesis designed, repeatability, intermediate precision and accuracy
set at +5% or better are considered satisfactory.The specificity of the method was assessed before starting the
validation step. A peak purity test of synephrine and octopamine
in each sample was performed comparing the UV-overlaidspectra measured (Figures 3 and 4) within the synephrine andoctopamine peaks in both the peak flanks and at peak maximum.
No interference was observed regarding the densitograms of thesample, confirming the selectivity of the method (Figure 2). The
analyte was tested for stability during development performing atwo-dimensional separation (31). Furthermore, it was verified thatthe samples were stable in solution for more than a week and onthe plate for not,12 h before and after development.
Table IV
Accuracy Results
Alkaloid Matrixamount(mg/mL)
Addedamount(mg/mL)
Totalamount(mg/mL)
Meanresult(mg/mL)
Recovery(%)
RSD(%)
Synephrine 0.0254 0.0125 0.0379 0.0368 97.09 3.630.025 0.0504 0.0495 98.25 4.780.5 0.5254 0.519 98.78 2.76
Octopamine 0.0142 0.0075 0.0217 0.0209 96.3 4.110.015 0.0292 0.0286 97.95 2.680.03 0.0442 0.0435 98.42 2.53
Table V
Robustness of the Method (n 6)
Op timisat ion condition S ynep hrine (RS D (%)) Octopa mine (RSD (%))
Mobile-phase composition 1.258 1.89Mobile-phase volume 0.696 0.78Duration of saturation 0.874 0.67
Figure 3. Superimposed UV spectra of synephrine from standard and sample zonesobtained by HPTLC spot scanning from 200 to 400 nm.
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Conclusion
A quick, precise and accurate method based on normal-phase
HPTLC has been developed for simultaneous routine analysis ofsynephrine and octopamine in bitter orange peel extracts. Toour knowledge, this is the first time HPTLC is successfully
applied to the simultaneous quantitative analysis of synephrineand octopamine. The method offers performance standardscomparable with those of HPLC.
The method was successfully validated for linearity, precision,
accuracy, specificity and robustness. It has several advantagesover HPLC methods. It consumes ,35 mL of mobile phase perrun (18 samples per plate), whereas HPLC methods would
consume not ,100 mL per run of similar number of samples. Ifwe consider the time from sample preparation to densitometricevaluation for one plate, the new method takes an average of 1 h,
whereas HPLC methods would generally take .2 h for the samenumber of samples. It is of low cost, quick and does not usechloroform, and therefore, suitable for routine analysis. In per-
spective, the method could benefit from the application ofstate-of-the-art HPTLC equipment (horizontal migration cham-
bers and automated devices for sample application and densito-metric analysis). This would likely further increase performanceand sample throughput.
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