Development And Validation Of HPLC Method To Measure Active Amines In .
Food Control 46 (2014) 136e142Contents lists available at ScienceDirectFood Controljournal homepage: www.elsevier.com/locate/foodcontDevelopment and validation of HPLC method to measure activeamines in plant food supplements containing Citrus aurantium LChiara Di Lorenzo, Ariana Dos Santos, Francesca Colombo, Enzo Moro, Mario Dell'Agli,Patrizia Restani* degli Studi di Milano, Via Balzaretti 9, 20133 Milan, ItalyDipartimento di Scienze Farmacologiche e Biomolecolari, Universitaa r t i c l e i n f oa b s t r a c tArticle history:Received 6 February 2014Received in revised form11 April 2014Accepted 12 May 2014Available online 21 May 2014Citrus aurantium L. (bitter orange) is frequently used as an ingredient of food supplements aimed toreduce body weight or improve general physical performances. The most active compounds ofC. aurantium are amines having adrenergic activity: octopamine, synephrine, tyramine, N-methyl-tyramine and hordenine. The quantification of these amines is critical since their content in food supplements is regulated by national/international rules. Some methods for the quantification of C. aurantiumamines have been published, including the official method developed by AOAC, but most of them are nottotally satisfactory for the analysis of complex matrixes, such as extracts or food supplements.A new HPLCeUVefluorescence procedure has therefore been developed; the method is quick andsimple, and allows the analysis of samples after a rapid extraction procedure without any furthercleaning step. The assay, using one or two detectors, showed good results during the validation testsperformed according to the FDA guidelines. 2014 Elsevier Ltd. All rights reserved.Keywords:SynephrineCitrus aurantiumHPLCAdrenergic aminesPlant food supplements1. IntroductionBotanicals and botanical preparations are widely consumed inWestern diets from various sources; among them plant food supplements (PFS) are the most important. Unlike drugs, these products are generally perceived positively by consumers, since“natural” is often considered synonymous with safety (Hung,Hillier, & Ernst, 2011).The popularity and the ease of access in shops or via the Internethave made for rapid diffusion of these products, and there areconcerns about their quality, composition, and safety. The problemis particularly significant, when they are obtained from unregulatedmarkets where illicit activities are not infrequent (Angell &Kassirer, 1998; Gurley, Gardner, & Hubbard, 2000).Labeling of PFS is a further problem, since the accuracy of thedeclared composition requires reliable analytical methods (Gurleyet al., 2000). Cianchino, Acosta, Ortega, Martinez, and Gomez(2008) analyzed four herbal dietary supplements aimed at weightcontrol and identified undeclared active compound such asephedrine and norephedrine. The claimed properties of these food* Corresponding author. Tel.: þ39 (0)2 50318371; fax: þ39 (0)250318284.E-mail address: patrizia.restani@unimi.it (P. .05.0170956-7135/ 2014 Elsevier Ltd. All rights reserved.supplements derived from the illegal additions, not the declaredingredients (Gurley et al., 2000). In 2010, Vaysse et al. (2010)analyzed 20 herbal medicines and dietary supplements marketedas 'natural' slimming products; 14 of them were adulterated withsibutramine and synephrine.Citrus aurantium L., also known as bitter orange, is a botanicalingredient frequently used in food supplements aimed at reducingbody weight or improving general performance. The most important active ingredients of C. aurantium are amines having adrenergic activity: octopamine, synephrine, tyramine, N-methyltyramine and hordenine (Fig. 1). The C. aurantium extract ispermitted in food supplements, but several countries have established limits for the content of active amines (among others ItalianMinistry of Health, 2012).The background is that the FDA's ban of ephedrine-containingsupplements led to increased use of C. aurantium as an alternative to Ephedra, with possible risk for consumers (Haller &Benowitz, 2000; Stohs, Preuss, Keith, Keith, Miller, & Kaats, 2011).Quality control of PFS containing C. aurantium is therefore veryimportant and a suitable analytical method is needed, to enableproducers to check the raw material and extracts.Several analytical approaches have been developed for thedetection and quantification of the amines contained inC. aurantium. Pellati, Benvenuti, Melegari, and Firenzuoli (2002)
C. Di Lorenzo et al. / Food Control 46 (2014) 136e142137(model UV-875) and an injection valve (Rheodyne, Cotati, CA, USA)with a 100 mL loop. The ChromNAV software Jasco was used for dataacquisition and processing.2.3. Chromatographic conditionsThe chromatographic separations were carried out on a reversedphase LiChrospher RP-18 column (250 4 mm ID, particle size5 mm, Merck, Darmstadt, Germany) in association with a LiChrospher 100 RP-18, 5 mm guard column, both maintained at 24 C. Thefluorescence detector was set at 270/305 nm (lEx/lEm), and UVeVisdetector at 224 nm.Gradient elution at a flow rate of 1 mL/min used the followingmobile phases: A, 2.9 g/L sodium dodecyl sulfate in water adjustedto pH 4.2 with 85% ortho-phosphoric acid; B, 2.9 g/L sodiumdodecyl sulfate:acetonitrile (62:38, v/v) adjusted to pH 4.2 with85% ortho-phosphoric acid. The gradient is illustrated in Table 1.At the end of the gradient program, the column was maintainedat the initial conditions for 10 min before the next injection. Thetotal run time was 40 min.Fig. 1. Chemical structure of octopamine, synephrine, tyramine, N-methyl-tyramine,hordenine.analyzed by reversed-phase only synephrine, octopamine andtyramine. Ephedrine alkaloids and synephrine were assayed usingcolumn-switching technique coupled to a cation exchange columnwith scanning wavelength ultraviolet and fluorescence detector(Niemann & Gay, 2003). Penzak, Jann, Cold, Hon, Desai, Gurley, andSeville (2001) analyzed synephrine and octopamine using IPC (IonPair Chromatography). None of these methods, however, demonstrated the ability to analyze the compounds of interest withacceptable validation data. The AOAC official method (Roman et al.,2004) allows the separation of most Citrus amines, but there areseveral problems in applying this method to PFS: efficacy in resolution is incomplete, the analysis takes too long and is costly, andchromatographic performance quickly deteriorates. Among published methods, that by Putzbach, Rimmer, Sharpless, and Sander(2007) based on ion-pair HPLC and fluorimetric detection (FD)was considered the most promising, being more sensitive andspecific than other methods using UV detection (Arbo et al., 2008;Gurley, Wang, & Gardner, 1998) However, problems appeared whenit was applied to complex matrices and multi-ingredient samples,in particular PFS.Our aim was to develop a simple, inexpensive HPLC method forquality control of products containing C. aurantium, including PFS.2. Material and methods2.1. Reagents and chemicalsStandards of synephrine, octopamine hydrochloride, hordenine,tyramine and N-methyl-tyramine were purchased from SigmaeAldrich (St. Louis, MO, USA), with purity 95%. The standardswere stored according to the supplier's instructions. LC-grade water, methanol and acetonitrile were purchased from SigmaeAldrich(St. Louis, MO, USA). Other reagents including 85% orthophosphoric acid, 0.1 N hydrochloric acid and sodium dodecyl sulfate were from Merck KGaA (Darmstadt, Germany).2.2. InstrumentationThe HPLC equipment was from Jasco (Tokyo, Japan) and consisted of two pumps (Intelligent HPLC Pump model PU-880), afluorimetric detector (model FP-1520), a UV/Visible detector2.4. Preparation of stock and working solutionsA stock solution of each amine was prepared at a final concentration of 1 mg/mL in 0.1 N HCl and further diluted with 0.1 N HCl toobtain working solutions in the range 0.1e10.0 mg/mL; for synephrine only the range was 5.0e125.0 mg/mL. All solutions werestored at 20 C until use.2.5. Sample preparationAbout 100 mg of finely ground and homogenized samples wereprecisely weighed and added to 25 mL of 0.1 N HCl plus 75 mL of awater:methanol 75:25 solution (v/v). Extraction efficiency wasassessed by preparing PFS samples with and without the additionof 2.5 mg/g of analytes and extracting for 5, 10, 15, 20, 25 and 30 minunder stirring by a magnetic device: 20 min was sufficient foroptimal extraction. After extraction, samples were filtered througha 0.45-mm filter and injected into the chromatographic equipment.2.6. ValidationA full validation of the method with two detectors was performed according to the current FDA Guidelines on BioanalyticalMethod Validation (FDA, 2013).2.6.1. System suitability testThe following parameters were calculated using the ChromNAVsoftware: retention factor (K), separation factor between twoneighboring peaks (a), peaks' tailing factor and column efficiency(number of theoretical plates).2.6.2. LinearityFor each run, five standard stock solutions were prepared andanalyzed with three independent injections; the concentrationsranged between 5 and 125 mg/mL for synephrine and between 0.1Table 1Gradient elution used in HPLC separation.Total timePhase A (%)Phase B (%)0 min11 min25 min30 min4000406010010060
138C. Di Lorenzo et al. / Food Control 46 (2014) 136e142Fig. 2. Chromatograms of a PFS containing Citrus aurantium analyzed by the new method and Putzbach's protocol. Legend: Figure A: new method with UV detector; B: Putzbach'smethod with UV detector; C: new method with fluorimetric detector; D: Putzbach's method with fluorimetric detector.Table 2Results of system suitability tests for the five amines obtained during test analysis (n ¼ 3).Retention times (min)(mean rdenineab17.6819.0021.4023.26 0.360.541.001.2824.95 1.53Ka(mean SD)8.669.3710.6811.69 0.250.210.190.2712.61 0.37ab(mean SD)1.081.081.101.08 0.010.010.010.011.08 0.01Symmetry factor(mean SD)0.8700.9030.9321.129 0.020.010.040.0041.150 0.01K (Retention factor) ¼ (tR t0)/t0, where tR and t0 are retention times of sample components and sample solvent, respectively.a (Separation factor) ¼ (tR2 T0)/(tR1 t0), where tR2 and tR1 are retention times of two neighboring peaks.Theoretical plates (N)(mean SD)42,23243,20841,54229,020 9582888812,798453215,845 3635
33.315.04.551,388 3881500.00.9989 0.00120.9989 0.0007150.06.21.919,075 280100.00.99231 0.00480.9991 0.000430.06.01.8200.00.9998 0.00020.9999 0.0000160.042,962 301116.116.15762 17822,557 236214,466 107729,178 19746.21.9100.00.9964 0.00320.9997 0.001930.064,007 136033.34.048,482 274825.07.519,451 ine0.2e5.0TyramineSynephrine5.0e125m ¼ 1,676,305 69,517q ¼ 7790 85,015m ¼ 397,861 5707q ¼ 1,197,631 287,316m ¼ 2,722,364 50392q ¼ 60,438 27,098m ¼ 2,270,365 24,515q ¼ 805,378 133,821m ¼ 2,246,598 102,821q ¼ 135,339 176,818Octopaminem ¼ 252,843 6977q ¼ 18904 7613m ¼ 185,596 388q ¼ 1,681,704 101,893m ¼ 285,408 216q ¼ 10,113 614m ¼ 275,075 17,782q ¼ 2746 34,305m ¼ 249,605 10,318q ¼ 23,019 21,8180.9998 0.0003Area(mean SD)ng/mL0.1e50.9987 lationcoefficient(R2)FDCorrelationcoefficient (R2)UVLinearity equationFDLinearity equationUVLinear range(mg/mL)AnalyteTable 3Limit of Detection (LOD), Limit of Quantitation (LOQ), sensitivity and results of linear regression analysis of calibration curves (n ¼ 3) determined for the five amines using the UV or FD.Area(mean SD)FD/UVSensitivityC. Di Lorenzo et al. / Food Control 46 (2014) 136e142139and 5 mg/mL for the other amines. Linear regressions were obtained by plotting the areas of analyte peaks vs the nominalconcentrations. The calibration curve equations and the corresponding correlation coefficients (R2) were calculated using bothdetectors.2.6.3. Limits of Detection and QuantitationThe Limit of Detection and Quantitation (LOD and LOQ,respectively) were determined from calibration curve: the LOQ wasdefined as the lowest concentration of the calibration curves andwas estimated after 5 injections based on a signal-to-noise ratio of10. A signal-to-noise ratio of 3 was considered acceptable for estimating the LOD.2.6.4. PrecisionIntra-day precision was determined by preparing andanalyzing, on the same day, five replicates of three different concentrations in the range 0.1e3.0 mg/mL for octopamine, tyramine,N-methyl-tyramine, hordenine and 10.0, 40.0, 80.0 mg/mL forsynephrine.Inter-day precision was evaluated by repeating the intra-dayprecision study on five different days. Precision was assessed bycalculating the mean, standard deviation (SD), and the coefficient ofvariation (RSD%) of these values.2.6.5. AccuracyAccuracy was determined by evaluating spiked samples withtwo concentrations of octopamine, tyramine, N-methyl-tyramine,hordenine (1.0 and 3.0 mg/mL) and synephrine (8.0 and 12.0 mg/mL)and through the calculation of the RSD% between the calculatedand the nominal values.2.6.6. Extraction recoveryA PFS containing only Hypericum perforatum L. was selected as ablank matrix, since it does not contain the investigated amines. Thecontent of ten capsules (0.5 g) was homogenized, spiked with thestandard solutions and extracted as by the described procedure.The recovery for synephrine was carried out at low, medium andhigh concentrations (10.0, 50.0, 100.0 mg/mL); for octopamine,tyramine, N-methyl-tyramine, hordenine the concentrations werein the range of 0.1e5.0 mg/mL (three for each amine). Each concentration of both sets was analyzed in three replicates. The percentages of recovery were calculated by comparing the peak arearatio of the analytes measured in the samples to the peak areas ofthe corresponding standard solutions.2.6.7. StabilityThe stability of synephrine stock solutions (40.0 and 80.0 mg/mL) and of other amines (2.0 and 4.0 mg/mL) was determined ontriplicate aliquots which were kept at room temperature for 8 h.Peak areas of these samples were compared with those of freshlyprepared stock solutions. Similarly, the stability of the samestandard solutions was assessed after storage for 20 daysat 20 C.Freeze ( 20 C)-thaw stability of these solutions was assessedby comparing their concentrations after three freezeethaw cycleswith the concentrations determined at time zero.2.6.8. SpecificityPotential interference between analytes and endogenous matrix components was investigated by analyzing six batches ofHypericum perforatum L. matrices. Peak areas of compounds coeluting with the analytes should be less than 20% of the peakarea of LOQ samples.
140C. Di Lorenzo et al. / Food Control 46 (2014) 136e142Table 4Precision, accuracy and recovery of active amines determined with UV and FD (n ¼ amineHordenineIntra-day precision (RSD %)Inter-day precision (RSD %)Recovery (%) (mean 108.494.590.5Accuracy (%) (mean SD)(mg/mL)FD 05.7101.9109.598.9 D1.03.096.0 0.1103.9 6.198.5 7.3105.4 6.28.012.095.8 6.998.9 5.2100.3 10.8108.2 4.41.03.099.7 31102.5 9.6110.3 7.7105.6 5.21.03.0100.0 4.496.9 5.5103.7 5.3103.8 5.91.03.090.2 4.5100.0 0.987.8 8.1103.2 8.1provide an adequate chromatographic resolution probably becauseof the complexity of the extracted PFS matrices. After experimenting with different gradient conditions, a new ion-pair mobilephase was developed with a binary solvent system as described inMaterials and Method. The characteristic analytes of C. aurantium(five amines) were well separated within 30 min at a flow rate of1 mL/min at room temperature.The chromatographic conditions used in the method heredescribed were very different from those used in all other published methods: gradient elution, the ion-pair technique and thepH value (4.2). In particular, the pH 4.2 allowed the best separationof interfering peaks, when compared to other elution phasesassayed. Fig. 2 shows a comparison between chromatographicseparations of a PFS sample by the new developed method (left)and Puzbach's method (right). It is evident that the new methodeliminates interferences due to complex matrix.3.2. Identification of aminesThe identity of the analytes was established by comparing thepeak retention times and Relative Retention Times (RTT) with thoseof reference standards.Fig. 3. Chromatogram of a blank extract (Hypericum perforatum L.) analyzed withfluorimetric (A) and UV (B) detector.3. Results and discussion3.1. ChromatographyThe first step of the research was the application of knownmethods (Putzbach et al., 2007; Roman et al., 2004) for the detection of amines in C. aurantium, using both raw material and derivatives (extract and PFS). However, these approaches failed to3.3. System Suitability Test (SST)Table 2 shows data on suitability of the chromatographic system. Data obtained with UV and fluorimetric detectors weresimilar. The considered parameters show that the chromatographic system used in this assay is very efficient. Indeed, it yieldsretention factors (K) ranging between 8.7 and 12.6 (K acceptablevalues 2) (FDA, 2013) as well as symmetrical, sharp peaks and istherefore suitable for the quantification of the five analytes incomplex matrices, such as PFS. Similar data on efficiency andselectivity were also obtained after about 200 injections of samples, showing the long life of the column and the consequent lowcost of the assay.Table 5Extraction recovery (%) of active amines (n ¼ 3).Time of extraction (min)Octopamine2015105100.4899.9799.8392.49 2.691.743.222.05Synephrine101.8897.899.8594.33 2.273.012.851.10Tyramine100.399.8199.7094.75 .8 3.092.862.792.67Hordenine99.9298.998.995.02 3.142.993.293.34
C. Di Lorenzo et al. / Food Control 46 (2014) 136e1421413.4. Validation3.4.1. Linearity, LOD and LOQThe method was linear between 5 and 125 mg/mL for synephrineand between 0.1 and 5 mg/mL for other amines, corresponding tothe tested concentrations, as shown by the correlation coefficients(R2) being always greater than 0.99. Table 3 shows the linearityresults (no significant variation of the slope between calibrationcurves, RSD being between 0.08 and 6.5%, as well as the Limit ofDetection (LOD) and Limit of Quantitation (LOQ) values: LOD obtained by fluorescence detector was between 1.8 and 7.5 ng/mL andLOQ was between 6.0 and 25.0 ng/mL for tyramine and octopaminerespectively. The fluorimetric detection was 4e30 times as sensitive as UV detection. These ranges of sensitivity were in every casesuitable for accurate determination of the amines in C. aurantiumsamples.Fig. 4. Chromatogram of a blank extract (Hypericum perforatum L.) spiked with astandard mixture (10 mg/mL) analyzed with fluorimetric (A) and UV (B) detector.3.4.2. Precision, accuracy, specificity and recoveryTable 4 shows the results of precision, accuracy and recoveryexperiments. Intra- and inter-day precision data showed RSD%values always 15% so that the method is precise, and as theFig. 5. Chromatograms of a plant food supplement (tablets, titrated at 5% synephrine) containing Citrus aurantium analyzed with fluorimetric (A) and UV (B) detector and of aC. aurantium extract revealed with fluorimetric (C) and UV (D) detector.
142C. Di Lorenzo et al. / Food Control 46 (2014) 136e142calculated accuracy was always within 15% of the nominal concentration, the method can be considered accurate. A blank samplecontaining only H. perforatum L. was tested to confirm the absenceof peaks having retention times similar to those of C. aurantiumamines (Fig. 3). No peak at the retention times of the amines had anarea exceeding 20% of LOQ values. The recovery with both detectionsystems ranged between 80 and 110% for all amines.3.4.3. StabilityStock solutions of synephrine, octopamine, tyramine, N-methyltyramine and hordenine were stable for at least 24 h at roomtemperature: the peak areas in the stock standard solutioncompared with those for freshly prepared solution ranged between99.50% 3.6 and 91.0% 3.2 for UV detector and between90.5% 3.8 and 104.2% 0.5 for the fluorimetric detector. The samestock solutions were stable for at least 1 month at 20 C: the longterm stability ranged between 91.4% 5.9 and 102.2% 3.7 for theUV detector and between 90.8% 5.9 and 108.9% 5.6 for thefluorimetric detector.Freeze/thaw stability experiments showed that the analyteswere stable in 0.1 N HCl for at least three freezeethaw cyclesat 20 C: the stability ranged between 91.9% 1.3 and 101.9% 2.5for the UV detector and between 91.9% 1.1 and 107.8% 2.2 for thefluorimetric detector.Stability of the 5 amines in the final extract was at least 48 h at4 C.3.5. Application of the method3.5.1. Optimization of time extractionSpiked blank samples analyzed after different extraction timesshowed that the recovery of the analytes reached a percentageclose to 100% after 20 min of magnetic stirring (Table 5). Therefore,all analyzes were performed using that extraction-time.3.5.2. ApplicabilityThe applicability of the method was finally verified by usingboth a blank matrix of H. perforatum L. and two real samples (aC. aurantium extract and a commercial PFS). Standard stock solutions (10 mg/mL) of the amines were added to the blank matrix inorder to evaluate peak separation and method suitability. Figs. 4and 5 show the resulting chromatograms, where separation washighly satisfactory in all the samples.4. ConclusionThis paper describes the development, validation and application of a LC/UV/Fluorescence assay for quantitative analysis of fiveactive amines in C. aurantium raw material and derivatives. Themethod has been fully validated according to FDA Guidelines forBioanalytical Method Validation (FDA, 2013) showing accurate,precise, selective, fast and relatively inexpensive analyzes. Thequantification of these amines is particularly important since theirconcentrations in PFS are regulated by national and internationallaws and for post-market control of undeclared illegal addition ofsynephrine and octopamine. The improved chromatographic system and the use of a fluorescence detector provide a validated toolto analyze the five active amines in complex matrices, includingplant food supplements.AcknowledgmentsThe research leading to these results has received funding fromthe European Community's Seventh Framework Program (FP7/2007-2013) under grant agreement n 245199. It has been carriedout within the PlantLIBRA project (website: www.plantlibra.eu).This report does not necessarily reflect the Commission's views orits future policy on this area.ReferencesAngell, M., & Kassirer, J. P. (1998). Alternative medicine - the risks of untestedand unregulated remedies. The New England Journal of Medicine, 339,839e841.Arbo, M. D., Larentis, E. R., Linck, V. M., Aboy, A. L., Pimentel, A. L., Henriques, A. T.,et al. (2008). Concentrations of p-synephrine in fruits and leaves of Citrusspecies (Rutaceae) and the acute toxicity testing of Citrus aurantium extract andp-synephrine. Food and Chemical Toxicology, 46, 2770e2775.Cianchino, V., Acosta, G., Ortega, C., Martínez, L. D., & Gomez, M. R. (2008).Analysis of potential adulteration in herbal medicines and dietary supplements for weight control by capillary electrophoresis. Food Chemistry, 108,1075e1081.FDA e Food and Drug Administration. (2013). Guidance for industry e Bioanalyticalmethod validation. Food and Drug Administration Accessed on October urley, B. J., Gardner, S. F., & Hubbard, M. A. (2000). Content versus label claims inephedra-containing dietary supplements. American Journal of Health-SystemPharmacy, 57, 963e969.Gurley, B. J., Wang, P., & Gardner, S. F. (1998). Ephedrine-type alkaloid content ofnutritional supplements containing Ephedra sinica (Ma-huang) as determinedby high performance liquid chromatography. Journal of Pharmaceutical Sciences,87, 1547e1553.Haller, C. A., & Benowitz, N. L. (2000). Adverse cardiovascular and central nervoussystem events associated with dietary supplements containing ephedra alkaloids. The New England Journal of Medicine, 343, 1833e1838.Hung, S. K., Hillier, S., & Ernst, E. (2011). Case reports of adverse effects of herbalmedicinal products (HMPs): a quality assessment. Phytomedicine, 18, 335e343.Italian Ministry of Health. (09 July 2012). Disciplina dell'impiego negli integratorialimentari di sostanze e preparati vegetali (Regulation on the use of botanicalsand botanical preparations in food supplements). In Gazzetta Ufficiale n. erie generale/originario(Accessed on October 2013).Niemann, R. A., & Gay, M. L. (2003). Determination of ephedrine alkaloids andsynephrine in dietary supplements by column-switching cation exchange highperformance liquid chromatography with scanning-wavelenght ultraviolet andfluorescence detection. Journal of Agricultural and Food Chemistry, 51,5630e5638.Pellati, F., Benvenuti, S., Melegari, M., & Firenzuoli, F. (2002). Determination ofadrenergic agonists from extracts and herbal products of Citrus aurantium L.var. amara by LC. Journal of Pharmaceutical and Biomedical Analysis, 29,1113e1119.Penzak, S. R., Jann, F. C. P., Cold, J. A., Hon, Y. Y., Desai, H. D., & Gurley, B. J.(2001). Seville (sour) orange juice: synephrine content and cardiovasculareffects in normotensive adults. The Journal of Clinical Pharmacology, 41,1059e1063.Putzbach, K., Rimmer, C. A., Sharpless, K. E., & Sander, L. C. (2007). Determination ofbitter orange alkaloids in dietary supplements standard reference materials byliquid chromatography with ultraviolet absorbance and fluorescence detection.Journal of Chromatography A, 1156, 304e311.Roman, M. C., Gray, D., Luo, G., McClanahan, R., Perez, R., Roper, C., et al. (2004).Determination of ephedrine alkaloids in botanicals and dietary supplements byHPLCeUV. The Journal of AOAC International, 87, 1e14.Stohs, S. J., Preuss, H. G., Keith, S. C., Keith, P. L., Miller, H., & Kaats, G. R. (2011).Effects of p-synephrine alone and in combination with selected bioflavonoidson resting metabolism, blood pressure, heart rate and self-reported moodchanges. International Journal of Medical Sciences, 8, 295e301.Vaysse, J., Balayssac, S., Gilard, V., Desoubdzanne, D., Malet-Martino, M., &Martino, R. (2010). Analysis of adulterated herbal medicines and dietary supplements marketed for weight loss by DOSY 1H-NMR. Food Additives & Contaminants. 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2.6. Validation A full validation of the method with two detectors was per-formed according to the current FDA Guidelines on Bioanalytical Method Validation (FDA, 2013). 2.6.1. System suitability test The following parameters were calculated using the ChromNAV software: retention factor (K), separation factor between two
Jun 01, 2016 · THIAMINE HYDROCHLORIDE HPLC H ilic HPLC HPLC to Hilic HPLC Add HPLC ID test Remove pH test THIAMINE MONONITRATE HPLC Hilic HPLC HPLC to Hilic HPLC Add HPLC ID test Remove pH test NIACINAMIDE n/a n/a Remove Melting Range ADENINE HPLC to UPLC HPLC to UPLC n/a CALCIFEDIOL n/a add HPL
Analytical Method Development and Validation of Bendamustine in Bulk Using RP-HPLC J Pharm Res Analytical Method Development and Validation of Bendamustine in Bulk Using RP-HPLC . Table 3: Variables in HPLC.-Hplc Method Validation is a key process for effective quality assurance. "Validation" is established documented .
BUILD BETTER HPLC METHODS WITH CRAWFORD SCIENTIFIC A collection of articles designed to help improve your HPLC Method Development knowledge and skills. VOLUME III . HPLC Method Development Volume III Page 2 TABLE OF CONTENTS P.03 P.08 P.17 P.25 UNDERSTANDING STATIONARY PHASES FOR HILIC SEPARATIONS HAVE WE FORGOTTEN THE ADVANTAGES OF CORE-SHELL PARTICLES? PRACTICAL HPLC METHOD DEVELOPMENT .
HPTLC [18] HPLC [19-20], UPLC [21] and capillary electrophoresis [22]. Two methods were only reported for estimation of this combination, the first is HPTLC [23] and the other is HPLC method [24]. The latter does not fulfill all requirements of validation which will be discussed later. Development of HPLC method for
Please cite this article as: Bharathi TG et al., RP-HPLC Method Development and Validation for the Quantitative Determination of Potential Impurities of Mirabegron. American Journal of Pharmacy & Health Research 2021. Research Article www.ajphr.com 2021, Volume 9, Issue 01 ISSN: 2321-3647(online) RP-HPLC Method Development and Validation for the
HPLC method development by QbD approach HPLC method development by Analytical QbD was as follows. Selection of quality target product profile The QTPP plays an important role for identifying the variables that affect the QTPP parameters. The retention time, theoretical plates, and peak asymmetry were iden-tified as QTPP for proposed HPLC method .
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validation Linearity The method was found to be linear in the concentration range of 80 to 12 μg mL-1 (Table1). The calibration curve of standard revealed that they had similar pattern are shown in Figure 1 and 2. Table 1: Validation parameters of the developed HPLC method for gives linear regression of the data points with the equation
