Chromsci.bmt113.Full

7/29/2019 Chromsci.bmt113.Full

1/6

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: [email protected]

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: [email protected]

Journal of Chromatographic Science 2013;1 6doi:10.1093/chromsci/bmt113 Article

Journal of Chromatographic Science Advance Access published August 2, 2013


2/6

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.

2Shawky


3/6

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


4/6

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.

4Shawky


5/6

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.

References

1. Ganzera, M., Lanser, C., Stuppner, H.; Simultaneous determination ofEphedra sinica and Citrus aurantium var. amara alkaloids byion-pair chromatography; Talanta, (2005); 66: 889894.

2. DAndrea, G., Terrazzino, S., Fortin, D., Farruggio, A., Rinaldi, L., Leon,A.; HPLC electrochemical detection of trace amines in human plasmaand platelets and expression of mRNA transcripts of trace aminereceptors in circulating leukocytes; Neuroscience Letters, (2003);346: 8992.

3. Carpene, C., Galitzky, J., Fontana, E., Atgie, C., Lafontan, M., Berlan, M.;Selective activation of beta3-adrenoceptors by octopamine: com-

parative studies in mammalian fat cells; Naunyn-SchmiedebergsArchives of Pharmacology, (1999); 359: 310 321.

4. Airriess, C.N., Rudling, J.E., Midgley, J.M., Evans, P.D.; Selectiveinhibition of adenylyl cyclase by octopamine via a human cloneda2A-adrenoceptor; British Journal of Pharmacology, (1997); 122:191198.

5. Pellati, F., Benvenuti, S.; Chromatographic and electrophoreticmethods for the analysis of phenethylamine [corrected] alkaloidsin Citrus aurantium; Journal of Chromatography A, (2007);1161(12): 7188.

6. U.S. Food and Drug Administration Website; Sales of supplementscontaining ephedrine alkaloids (ephedra) prohibited. http://

www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/2004/ucm108379.htm (accessed September 2012).

7. Avula, B., Upparapalli, S.K., Navarrete, A., Khan, I.A.; Simultaneous

quantification of adrenergic amines and flavonoids in C. aurantium,various Citrusspecies, and dietary supplements by liquid chromatog-raphy; The Journal of AOAC International, (2005); 88: 1593.

8. Fugh-Berman, A., Myers, A.; Citrus aurantium, an ingredient ofdietary supplements marketed for weight loss: current status of clin-ical and basic research; Experimental Biology and Medicine,(2004); 229: 698 704.

9. Allison, D.B., Cutter, G., Poehlman, E.T., Moore, D.R., Barnes, S.; Exactlywhich synephrine alkaloids does Citrus aurantium (bitter orange)contain?; International Journal of Obestetrics, (2005); 29: 443.

10. Hashimoto, K., Yasuda, T., Ohsawa, K.; Determination of synephrinefrom Chinese medicinal drugs originating from Citrus species

by ion-pair high-performance liquid chromatography; Journal of Chromatography, (1992); 623: 386.

11. Pellati, F., Benvenuti, S., Melegari, M., Firenzuoli, F.; Determinationof adrenergic agonists from extracts and herbal products of Citrus

aurantium L. var. amara by LC; Journal of Pharmaceutical andBiomedical Analysis, (2002); 29: 11131119.

12. Niemann, R.A., Gay, M.L.; Determination of ephedrine alkaloids andsynephrine in dietary supplements by column-switching cationexchange high-performance liquid chromatography with scanning-

wavelength ultraviolet and fluorescence detection; Journal of Agriculture and Food Chemistry, (2003); 51: 56305638.

13. Ganzera, M., Lanser, C., Stuppner, H., Ganzera, M., Lanser, C.,Stuppner, H.; Simultaneous determination of Ephedra sinica andCitrus aurantium var. amara alkaloids by ion-pair chromatography;Talanta, (2005); 66: 889894.

14. Tang, F., Tao, L., Luo, X., Ding, L., Guo, M., Nie, L.; Determination ofoctopamine, synephrine and tyramine in Citrusherbs by ionic liquidimproved green chromatography; Journal of Chromatography A,(2006); 1125: 182.

15. Kusu, F., Li, X.-D., Takamura, K.; Determination of synephrine andN-methyltyramine in Zhishi and Zhike (Immature Citrus Fruits)by high-performance liquid chromatography with electrochemicaldetection; Chemical and Pharmaceutical Bulletin, (1992); 40:32843286.

16. Gay, M.L., Niemann, R.A., Musser, S.M.; An isotopically labeled internalstandard liquid chromatography-tandem mass spectrometry methodfor determination of ephedrine alkaloids and synephrine in dietarysupplements; Journal of Agriculture and Food Chemistry, (2006);54: 285291.

17. Avula, B., Khan, I.A.; Separation and determination of ephedrine enan-tiomers and synephrine by high performance capillary electrophor-esis in dietary supplements; Chromatographia, (2004); 59: 71 77.

18. Slezak, T., Francis, P.S., Anastos, N., Barnett, N.W.; Determination ofsynephrine in weight-loss products using high performance liquidchromatography with acidic potassium permanganate chemilumin-

escence detection; Analytica Chimica Acta, (2007); 593: 98 102.19. Avula, B., Upparapalli, S.K., Khan, I.A.; Enantiomeric separation of

adrenergic amines in Citrusspecies, related general and dietary sup-plements by capillary electrophoresis; Chromatographia, (2005);62: 151157.

20. Ciesla, L=====., Waksmundzka-Hajnos, M.; Two-dimensional thin-layerchromatography in the analysis of secondary plant metabolites;

Journal of Chromatography A, (2009); 1216: 10351052.21. Medic-Saric, M., Jasprica, I., Mornar, A., Males, Z.; Application of TLC

in the isolation and analysis of flavonoids. In Waksmundzka-Hajnos,M., Sherma, J., Kowalska, T. (eds). Thin layer chromatography in

phytochemistry. CRC Press Taylor & Francis Group, Boca Raton/London/New York, (2008), pp. 405423.

Figure 4. Superimposed UV spectra of octopamine from standard and sample zonesobtained by HPTLC spot scanning from 200 to 400 nm.

Determination of Synephrine and Octopamine in Bitter Orange Peel5


6/6

22. Pereira, C.A.M., Yariwake, J.H., Lanc, F.M., Wauters, J.-N., Tits, M.,Angenot, L.; A HPTLC densitometric determination of flavonoids fromPassiflora alata, P. edulis, P. incarnate and P. caerulea and compari-son with HPLC method; Phytochemical Analysis, (2004); 15: 241 248.

23. Gunther, M., Schmidt, P.C.; Comparison between HPLC andHPTLC-densitometry for the determination of harpagoside fromHarpagophytum procumbens CO2-extracts; Journal of Pharmaceutical and Biomedical Analysis, (2005); 37: 817821.

24. ICH.; Q2B Validation of analytical procedures: methodology. In The

6th International Conference on Harmonization (ICH), Geneva,November, 2003.

25. Ermer, J.; Validation in pharmaceutical analysis. Part I: an integratedapproach; Journal of Pharmaceutical and Biomedical Analysis,(2001); 24: 755 767.

26. Putzbach, K., Rimmer, C.A., Sharpless, K.E.; Determination of bitterorange alkaloids in dietary supplements standard reference materials by

liquid chromatography with ultraviolet absorbance and fluorescencedetection; Journal of Chromatography A, (2007); 1156: 304311.

27. Chen, F., Hou, L.; Yaowu Fensi Zazhi, (1984); 4(3): 169171.Abstract from HCAPLUS 1984:497731.

28. Hardwick, B.C., Axelrod, B.; Isolation of octopamine from annual ryegrass; Plant Physiology, (1969); 44: 17451751.

29. Stewart, I., Newhall, W.F., Edwards, G.J.; The isolation and identifica-tion of synephrine in the leaves and fruit of citrus; The Journal ofBiological Chemistry, (1964); 239: 930 932.

30. Arbo, M.D., Braun, P., Leal, M.B., Larentis, E.R., Aboy, A.L., Bulcao, R.P.,et al.; Presence of p-synephrine in teas commercialized in Porto

Alegre (RS/Brazil); Brazilian Journal of Pharmaceutical Sciences,(2009); 45(2): 273278.

31. Reich, E., Schibli, A.; High-performance thin-layer chromatographyfor the analysis of medicinal plants; Thieme Medical PublishersInc., New York, NY, (2006).

6Shawky

Documents

Chromsci.bmt113.Full