AnFar 2013 Vitamin

Slide 1

Analisis SEDIAAN FarmasiGolongan Vitamin1Mei-Juni 2013broto_santoso[at]yahoo.comAnalisis Farmasi VitaminGeneric nameVitamer chemical name(s) (list not complete)SolubilitylogPVitamin B1ThiamineWater-2.96Vitamin B12Cyanocobalamin, hydroxycobalamin, methylcobalaminWater-3.52Vitamin B2RiboflavinWater-0.85Vitamin B3Niacin, niacinamideWater-0.61Vitamin B5Pantothenic acidWater-1.31Vitamin B6Pyridoxine, pyridoxamine, pyridoxalWater-1.05Vitamin B7BiotinWater0.20Vitamin B9Folic acid, folinic acidWater0.16Vitamin CAscorbic acidWater-2.34Vitamin ARetinol, retinal & 4 carotenoids including beta caroteneFat4.32Vitamin DCholecalciferolFat6.58Vitamin ETocopherols, tocotrienolsFat9.60Vitamin Kphylloquinone, menaquinonesFat8.432 Mei [email protected]

2 Mei [email protected]


Vitamin larut air (water soluble)

Vitamin C dan B2 Mei [email protected] from literatureCapillary electrophoresis: Vitamin B1, B2, B6 dan nikotinamida (S. Boonkerd, M.R. Detaevernier, dan Y. Michotte, 1994; J. Chromatography A, 670: 209-214)Cyclic voltammetry: Vitamin B6 (Teixeira et al., 2003; J. Braz. Chem. Soc., 14(2): 316-321)Fluorimetric: Vitamin B12 (Li dan Chen, 2000; Fresenius J Anal Chem, 368: 836-838)HPLC-DAD: Vitamin B (Thiamine, Nicotinic acid, Pyridoxal, Pyridoxine, Nicotinamide, Pantothenic acid, Cyanocobalamin, Riboflavin dan Thioctic acid) dan Vitamin C (Klejdus et al., 2004; Analytica Chimica Acta 520: 57-67)Catalytic voltammetric: Vitamin C (M.H. Pournaghi-Azar dan R. Ojani, 1996; Talanta 44: 297-303)Spectrophotometric: Vitamin C (Backheet et al., 1991; Analyst 116: 861-865)Non-spectrophotometric methods: Vitamin C (S.P. Arya, M. Mahajan, dan P. Jain, 2000; Analytica Chimica Acta, 417: 114)2 Mei [email protected] C (asam askorbat)

Human Glutathione Transferase O1-1 C32S Mutant in Complex with Ascorbic Acid (3VLN)created by Chimera and POV-Ray2 Mei [email protected] MetodeNoMetodeSampelSumber1.Catalytic voltammetricpharmaceutical preparations & complex matrices of freshfruit juicesTalanta 44: 297-303(1997)2.HPLC-DADpharmaceutical preparationsAnalytica Chimica Acta 2004 520: 57-67J. Agric. Food Chem. 2005, 53, 1370-13733.Spectrophotometricpharmaceutical preparations & fresh fruit juicesAnalyst 1991 116: 861-8654.Non-spectrophotometricnatural and fortified food samples & pharmaceuticalsAnalytica Chimica Acta 2000 417: 1-142 Mei [email protected] voltammetric2 Mei [email protected]

Sample tablets containing ascorbic acid as a single component (chewable tablets);tablets containing sodium carbonate combined with ascorbic acid (effervescent tablets);powdered vitamin C;ampoules containing ascorbic acid as the single component;multivitamin capsules containing vitamins A, B complex, D2 and E, nicotinamide, calcium pantothenate, and folic acid together with minerals (Fe2+, Ca2+, Mg2+, Mn2+, Cu2+, Zn2+ and Mo2+ );multivitamin tablets containing vitamins A, B complex, D2 and E, nicotinamide, calcium panthothenate, folic acid.2 Mei [email protected]&G potentiostat/galvanostat model 273 coupled with an IBM personal computer connected to an Epson model FX-850 printer was used.A conventional three-electrode cell thermostatted at 25+0.1C with calomel electrode as reference electrode, a platinum wire as auxiliary electrode and a glassy carbon disk as working electrode (A=0.126 cm2 from EG&G) was used.The working electrode was polished with alumina powder (0.05 /tm) and then washed with water and acetone in turn before each voltammetric measurement.2 Mei [email protected] tablets, capsules or powdersAn accurately weighed portion of finely powdered sample equivalent to about 100 mg of ascorbic acid was transferred to a 10 mL assay tube and ascorbic acid was extracted with two 5 mL portions of 0.5% citric acid in bidistilled water. The extracts were combined in a 50 mL flask and diluted to volume. A 1 mL portion of extract was diluted with 10 mL of glycine buffer pH 4, containing 0.1 M LiCIO4 and 0.1 mM ferrocenecarboxylic acid, in a voltammetric cell and a cyclic voltammogram was recorded using a well polished glassy carbon (GC) electrode. The amount of ascorbic acid was determined by means of a calibration graph.2 Mei [email protected] sampel (serbuk) yang ditimbang seksama setara dengan 100 mg asam askorbat dipindahkan ke dalam tabung 10 mL dan dilarutkan dengan 5 mL asam sitrat 0,5% dalam akuabides. Larutan ini dipindahkan ke dalam labu takar 50 mL dan ditambahkan pelarut hingga tanda. Diambil 1 mL dan dilarutkan dengan 10 mL dapar glisin pH 4 (larutan mengandung 0,1 M LiCIO4 dan 0,1 mM ferrocenecarboxylic acid) kemudian diukur dengan voltammeter sehingga diperoleh voltammogram. Konsentrasi asam askorbat ditentukan dari rerata dari grafik kalibrasi.electrochemical oxidationSome electrogenerated ferricinium derivatives (ferriciniumcarboxylic acid)are able to catalyse the electrochemical oxidation of ascorbic acid in buffered aqueous solutions at pH 4-5 via a homogeneous process when the ferrocene derivatives (ferrocenecarboxylic acid) are present in dissolved forms (see Scheme 1)

The catalytic peak current is linearly dependent on the ascorbic acid concentration and the range of linearity depends on the amount of mediator (ferrocenecarboxylic acid) in the solution2 Mei [email protected]

Cyclic voltammograms(a) 2 mM ferrocenecarboxylic acid in 0.5 M glycine buffer + 0.1 M LiCIO4 at pH 4, (b) as for (a) but with addition of 5 mM ascorbic acid, (c) 5 mM ascorbic acid in buffer solution pH 4.Scan rate: 5 mV s-1.

anodic peak current for cyclic voltammograms with a scan rate of 10 mV s-1 was proportional to the ascorbic acid concentration within the range 5 x 10-5 s/d 1 x 10-3 M with only a small intercept.

The regression equation was: i (mA) = - 1.28 + 9757C (M); r = 0.999, n = 6.2 Mei [email protected]

ProcedureInjectionAn accurate ampoule volume equivalent to about 100 mg of ascorbic acid was transferred to a 50 mL flask and diluted to volume with 0.5% citric acid solution. A 1 mL portion of the solution was diluted in a voltammetric cell to 10 mL as described for tablets and the cyclic voltammogram was recorded using the GC electrode2 Mei [email protected] volume larutan dari ampul diambil dengan pipet volume yang setara dengan 100 mg asam askorbat, dipindahkan ke dalam labu takar 50 mL dan ditambahkan larutan asam sitrat 0,5% hingga tanda. Diambil 1 mL dan diperlakukan seperti yang telah dijelaskan untuk tablet. Voltammogram direkam menggunakan elektroda GC.Result from article2 Mei [email protected]

InterferenceSome reducing ions, such as Fe(II) exceeded 3 mM, Cu(I), Sn(II) and Sulphite

The experimental investigation showed that these ions did not interfere in buffered solution up to 50, 20, 100 and 100 mM respectively

The results of the analysis of some pharmaceutical preparations using the proposed method compared favourably with those obtained by the USP method confirming that no interference was observed from concomitant mineral ions, other vitamins, glucose, sucrose, nicotinamide and tablet excipients2 Mei [email protected] PembandingA 0.05 M iodine solution was standardized in the usual way with a primary standard of As203 or titrisol thiosulphate solution. For pharmaceutical analysis an iodimetric procedure described in the US Pharmacopeia (USP) was used.

Larutan standar Iod 0,05 M yang telah dibakukan dengan As203 atau larutan titrisol tiosulfat digunakan dalam metode pembanding seperti yang tercantum dalam US Pharmacopeia (USP).2 Mei [email protected] Mei [email protected]

Sample Vitamin drink (Penko, Krnov, Czech Republic) consisted of vitamins (thiamine, riboflavin, pyridoxine, pantothenic acid, nicotinamide, -tocopherol, cyanocobalamin, l-ascorbic acid, folic acid) and adjuvants (saccharose, glucose, E330, maltodextrine, natural identical aroma, milk proteins, lemon pectin, soy lecithin, ZnO, MnSO4 and Na2SeO3).

2 Mei [email protected] sampelThe homogenised samples of Vitamin drink (1 g of powder) were dissolved in 10 mL of solution consisted of 0.010% trifluoroacetic acid (TFA):methanol (50:50) and stirred on Vortex for 15 min.2 Mei [email protected] HP 1100 liquid chromatographic system (Hewlett-Packard, Waldbronn, Germany) was equipped with a vacuum degasser (G1322A), a binary pump (G1312A), an auto sampler (G1313A), a column thermostat (G1316A), and a UV-Vis diode array detector (model G1315A) working at 190690 nm.

The ChemStation software (Rev. A 08.01) controlled the whole liquid chromatographic system. Spectra were registered in the range of 190400 nm (SBW 100 nm). Chromatograms were registered at 280 nm.2 Mei [email protected] KromatografiReversed-phase chromatographic columns Zorbax AAA (150mm4.6 mm, 3.5 m particle size, Agilent Technologies, USA), Atlantis dC18 (150mm2.1 mm, 3 m particle size, Waters, Milford, MA, USA) and MetaChem Polaris C18-A (150mm 4.6 mm, 3m particle size, Ansys Technologies, Torrance, CA, USA) were tested for the separation of vitamins. The flow rate was 0.7 mLmin1. Auto sampler injection was 3 L. Gradient of mobile phase and its composition was tested, too. See next slide.2 Mei [email protected] profile(A, mobile phase methanol) for simultaneous vitamin separation that started at 95:5 (0.010% TFA:methanol) and was constant in the first 4 min, then decreased to 2:98 during the next 10 min and finally linearly increased up to 95:5 from 32 to 35 min is shown on (A); pH value was 3.9. Simultaneous HPLCUV chromatograms of water- and fat-soluble vitamins at three detection wavelengths: 230 nm (B), 266 nm (C) and 280 nm (D). Vitamin concentrations were 100 gmL1. Other conditions are the same as in Fig. 2.2 Mei [email protected]

Simultaneous HPLCUV determination2 Mei [email protected]

Dependences of retention (A) and symmetry (B) factorsDependences of retention (A) and symmetry (B) factors of water- and fat-soluble vitamins (100 gmL1 of each) on pH values, respectively. MetaChem Polaris C18-A column, mobile phase consisted of 0.010% TFA and methanol; gradient profile started at 92:8 (TFA:methanol; (v/v)) and was constant in the first 2 min, then decreased to 2:98 during the next 10 min and finally linearly increased up to 92:8 from 32 to 35 min. Vitamin concentrations were 100 gmL1. Autosampler injection was 3 L. HPLCUV parameters: MetaChem Polaris C18-A (150mm 4.6 mm, 3 m particle size, Ansys Technologies, Torrance, CA, USA; isocratic flow rate of 0.7 ml min1; column temperature 20C; detection wavelength of DAD is280 nm.2 Mei [email protected]

Kromatogram2 Mei [email protected]

Validation2 Mei [email protected]

a Retention times in min. b Regression coefficients. c Limits of detection (3.S/N). d Limits of quantitation (10.S/N). e Relative standard deviations. f Non-vitaminous substances.Result from article2 Mei [email protected]

Spectrophotometric2 Mei [email protected]


Sample tablets containing ascorbic acid as a single component: Vitamin-C (Alexandria) , Vitascorbol (Specia) and Vitacid-C effervescent tablets (Cid);tablets containing ascorbic acid in combination with salicylamide: Cidal-C (Cid) ; with phenylephrine, paracetamol and caffeine: Rhino-C (Cid); with rutin: Ruta-C-60 (Kahira); and with menadiol dibutyrate and rutin: Styptobion (Nile/Merck);tablets containing ascorbic acid with vitamins A, B complex, D2 and E, nicotinamide and calcium pantothenate together with minerals: Chewa-vit (Pfizer-Egypt), Totacid (Cid) , Optical Compound (Eipico/ Leo) and Thereagran Hematic (Squibb-Egypt);effervescent tablets containing calcium carbonate combined with ascorbic acid: Vitacid-calcium (Cid);multivitamin capsules containing vitamins A, B complex, D2 and E with minerals: Mineravit (Eipico) , Obron (Pfizer-Egypt) and Viterra (Pfizer-Egypt);syrups containing vitamins A, B complex, D2 and E, nicotinamide and calcium pantothenate: Beco-C (Misr), Medivit (Adco) and Fruital (Cid);ampoules containing either ascorbic acid alone: Cevarol (Memphis); or combined with novalgine: Cevagine (Memphis); with menadion sodium diphosphate and rutin: Styptobion (Nile/Merck); with calcium glubionate: Calcium-C Sandoz (Sandoz); or with other vitamins and nicotinamide: Polyvit (Misr);Drops containing ascorbic acid as a single component: Ceviline (Kahira).2 Mei [email protected] procedure is based on the reaction of ascorbic acid with the zinc chloride salt of diazotized I-aminoanthraquinone (Fast Red AL salt) in an acidic medium, followed by development of a blue colour (max 630 nm) in alkaline solution.

The mechanism of the reaction between the diazonium salt of 1-aminoanthraquinone (I) and ascorbic acid (II) to form the lactone (III), which in an alkaline medium, yields the xalohydrazide derivative (IV) , is shown in Scheme 12 Mei [email protected]

General ProcedureIn a 10 mL calibrated flask were placed 1 mL of the standard or the sample preparation, 1 mL of the reagent solution and 2 mL of 10% sodium hydroxide solution. The volume was made up to the mark with distilled water, the solution mixed and the absorbance at 630 nm measured against a reagent blank prepared similarly (absorbance of the reagent blank = 0.002).2 Mei [email protected], syrups & drops procedureAn accurately measured volume, equivalent to about 20 mg of ascorbic acid, was transferred into a 100 mL calibrated flask, 0.5 g of citric acid was added and the volume was made up to the mark with distilled water2 Mei [email protected] for tabletsAn accurately weighed amount of powder obtained from 20 tablets, equivalent to about 20 mg of ascorbic acid, was transferred into a 100 mL calibrated flask, followed by 50 mL of extracting solution. The flask was shaken for 15 min and the contents were made up to the mark with distilled water The solution was filtered, the first portion of the filtrate being rejected2 Mei [email protected] for multi-vitamin capsulesTen capsules were completely disintegrated by adding 50 mL of the extracting solution to a 200 mL beaker containing the capsules and gently heating the mixture. The suspension was transferred quantitatively into a 500 mL calibrated flask and rapidly cooled to room temperature. The volume was made up to the mark with distilled water2 Mei [email protected] SpectraFast Red AL salt (dioazotized 1-aminoanthraquinone stabilized as the zinc chloride salt) reacts with ascorbic acid in an acidic medium after which a blue colour develops in alkaline solution with a maximum absorbance at 630 nm (Fig. 1); the reagent and ascorbic acid have no absorption in this region. The blue colour formed in the alkaline medium is dispelled on acidification.2 Mei [email protected]

Stability of ColourThe colour developed immediately, attained a maximum within 1 min and remained stable for at least 2 h.

Berapakah OT reaksi dalam hal ini?Kenapa air dapat mengurangi stabilitas dari produk yang terbentuk antara asam askorbat dan pereaksi?

Stability of Ascorbic AcidFifty percent of the reagent-reducing activity of a solution of ascorbic acid (200 g.mL-1) disappeared within 2 h (Fig. 2). When ascorbic acid was prepared in 5% trichloracetic acid, 0.5% oxalic acid or 0.5% citric acid, the reducing activity was stable for 2 h but it decreased steadily after that time2 Mei [email protected]

Concentration of ReagentThe colour developed strongly as the amount of Fast Red AL salt in the reaction mixture was increased from 0.75 to 1.25 mL of a 0.25% solution (Fig. 3).

Info:Oxalic and citric acids have been reported to be efficient stabilizers of ascorbic acid solution,16,17 but the resulting final solutions were turbid when oxalic acid was used; therefore, citric acid, which produces no turbidity, was used throughout this work

16 Ponting, J. D., Ind. Eng. Chem. Anal. Ed., 1943, 15, 389.17 Wills, R. B. H., Wimalasiri, P., and Greenfield, H., J. Assoc. Off.A nal. Chem., 1983, 66, 1377.2 Mei [email protected]

Type and Concentration of AlkaliIn acidic and neutral media, no development of colour was observed, whereas in an alkaline medium the blue colour developed rapidly. Different alkalis were tested (Table 1) and all produced virtually identical absorbance readings but with small shifts in the maxima. Sodium hydroxide was preferred as the max observed with this alkali was shifted to the longest wavelength. In addition, the use of an excess of sodium hydroxide is required in order to dissolve any precipitated zinc hydroxide (resulting from the zinc chloride present in the reagent)2 Mei [email protected]

Effect of Diluting SolventsMethanol, ethanol, water, propan-2-ol, dimethyl sulphoxide and dimethylformamide were tested as diluting solvents (Table2). The results revealed that water and ethanol were the best solvents. However, water was used throughout this work; this is an additional advantage of the proposed method. Carbon tetrachloride, chloroform and 1,2-dichIoroethane were not suitable as the colour produced could not be extracted into these solvents.

Table 3 represent the recovery result of ascorbic acid with other substance

Kenapa molekul lain dalam sampel tidak mengganggu perolehan kembali dari asam askorbat?2 Mei [email protected]

Result from articleAt 630 nm, the absorbance was proportional to the amount of ascorbic acid within the range 5-25 g with only a small intercept; the regression equation was: A630 = -0.0324 + 0.024% (g mL-1); r = 0.9990, n = 8. The molar absorptivity was 4.07 x 1031 mol-1 cm-1.2 Mei [email protected]

DimethoxydiquinoneDimethoxydiquinone gives a violet-colored product with ascorbic acid in a phosphate buffer (pH 6.6). The reduced indigoid quinhydrone form is perhaps responsible for the formation of violet-colored product2 Mei [email protected]

After diluting with dioxane, absorbance of the colored solution which is stable over 24 h only under dark conditions is measured at 510 nm. Heating leads to a decrease in color intensity. Beers law holds good up to 80 g ml1 with a detection limit of 10 g ml1 .DMDQ InterferencesRiboflavin and copper interfere. The interference of iron(II) sulfate responsible for precipitation can be removed by centrifugation. Though the method is not sufficiently sensitive (=1.62x103), it can still be applied to the analysis of citrus fruits after extracting the colored product into chloroform (max=530 nm).2 Mei [email protected] as dyeThe blue dye DCIP is reduced to the colorless form on addition of ascorbic acid but it gives a pink color to the acidic solutions.2 Mei [email protected]

Color extractionUsing the dye, ascorbic acid present in sample has been determined. The excess dye can be extracted with xylene or butanol. Many substances which are capable of reducing the dye resulting from the preparation and processing of samples interfere.2 Mei [email protected] as dyeAscorbic acid is determined at 336 nm (=535 cm2 mol1) via a decrease in absorbance of 7.104 M tetrachlorobenzoquinone (chloranil) in 80% acetonewater (v/v) medium.With these methods, mixtures of ascorbic acid with thiols like o-mercaptobenzoic acid, mercaptosuccinic acid, 3-mercaptopropionic acid can not be resolved.2 Mei [email protected] BlueMethylene Blue has been used to determine ascorbic acid in food products. The colorless form of the dye is extracted into chloroform after its reduction with ascorbic acid; back oxidation of the dihydro derivative to Methylene Blue has been used for the assay of ascorbic acid (max=653 nm). The method is reported to be highly sensitive.2 Mei [email protected] & ResorcinolThe reaction of ascorbic acid with ninhydrin carried out on a boiling water bath using 80% aqueous solution as a medium in 0.01 M NH4OH is used for its determination in pharmaceuticals (max=415 nm), but without added advantages.

Methanolic solution of resorcinol gives a pale yellow color (max=425 nm) with ascorbic acid in hydrochloric acid medium, obeying Beers law for 80-400 g ml1.2 Mei [email protected] dyeA purplish or blue colored species is produced by these salts with ascorbic acid in alkaline medium. Diazotized-4-methoxy-2-nitroaniline couples with ascorbic acid in oxalic acid medium in the presence of ethanol or isopropanol, giving a purplish color in alkaline solutions.

Though Fe(II), Sn(II) and dehydroascorbic acid (DHAA) do not interfere, the presence of reductones and reductic acid requires formaldehyde condensation. Low contents of vitamin C in the presence of flavanoids and pectic substances are also detected.

The reaction of ascorbic acid with 4-nitrobenzene diazonium fluoroborate in acetic acid medium was used for its determination at max 415 nm. But the mixture has to be kept for 25 min in the dark, followed by the addition of sodium hydroxide.2 Mei 2013broto_santoso@yahoo.com524-Chloro-7-nitrobenzofurazane4-Chloro-7-nitrobenzofurazane forms a bluish green colored species with ascorbic acid in presence of 0.2 M NaOH.

The absorbance is measured at 582 nm after diluting the reaction contents with 50% (v/v) aqueous acetone solution. Beers law is obeyed in the concentration range 5 20 gmL1. The colored product is stable for 30 min only when kept away from direct sunlight or artificial day light.

The method is reported free from the interference of all other vitamins and minerals present in multivitamin preparations and can be applied to the analysis of pharmaceuticals, fresh fruit juices and vegetables.2 Mei [email protected],3,5-triphenyltetrazolium chlorideHashmi et al. proposed a method based on the reaction of 2,3,5-triphenyltetrazolium chloride with ascorbic acid in alkaline medium. The pink solution is allowed to stand in the dark for 30 min at 25C; it obeys Beers law over the range 5-25 g mL1. Sugars (>15 g mL1) except sucrose interfere by forming a similar color to that of the reagent. Riboflavin, cyanocobalamin and folic acid interfere due to their own color.2 Mei [email protected] chloridePhenylhydrazinium chloride produces a yellow color (max =395 nm) when treated with ascorbic acid in 0.1 M HCl medium. The reaction contents are kept for 1 h in an incubator or water bath at 502C, thus making the method time-consuming. Beers law is obeyed in the range 25100 g of ascorbic acid. No interference is observed from other vitamins, minerals, glucose, sucrose, excipients and reducing agents. However, the presence of excessive amounts of riboflavin requires the addition of 0.5 g talc, which imparts a yellow color to the solution.2 Mei [email protected] hydrazone3-Methyl-2-benzothiazolone hydrazone reacts in the presence of sodium metaperiodate to form a blue colored solution (max =630 nm) which helps in the determination of ascorbic acid over the range 6-14 meq mL1.2 Mei [email protected] green (LMG)AA reacts with potassium iodide-iodate solution under acidic conditions to liberate iodine & this compound selectively oxidizes LMG to MG dye2 Mei [email protected] reacts with potassium iodide-iodate solution under acidic conditions to liberate iodine and the liberated iodine selectively oxidizes LMG to MG dye. The colour of the dye was measured at 620 nm. Beers law is obeyed over the concentration range of 0.8-8 g AA per 25 mL of final solution (0.032-0.32 ppm). The apparent molar absorptivity and Sandells sensitivity of the method were found to be 2.98x105 l mol-1cm-1, 0.0042 g cm-2, and respectively.2 Mei [email protected] Mei [email protected]

pharmaceuticalsAll drug samples tested were fresh and purchased from local pharmacy. An AA tablet or the content of a capsule were weighed, ground in to a fine power and stirred for 2-3 min with 50 mL of deionized water. 1 mL of 5% EDTA was added and filtered through Whatman No. 41 filter paper. The insoluble mass was washed with three successive 5 mL portions of water and the filtrate plus washings were diluted to volume in a 250 mL calibrated flask. A known volume was further diluted depending on the AA content and the colour of the sample. 1 mL aliquot was analyzed as recommended above.2 Mei [email protected] aliquot of sample solution containing 0.8-8.0 g AA was transferred in to a series of 25 mL graduated tube. To this 0.4 mL of potassium iodide-potassium iodate mixture solution and 1 mL of 0.02-mol l-1 hydrochloric acid solution were added, and the mixture was gently shaken until the appearance of yellow colour, indicating the liberation of iodine. Then 1 mL of 0.05% LMG solution was added to it followed by addition of 2 mL of acetate buffer (pH-4.5).

The contents were heated (~ 40C) in a water bath for 5 min, cooled to room temp and diluted to the mark with distilled water. The mixture was kept for 10 min for completion of the reaction. The absorbance of the formed dye was measured at 620 nm against the reagent blank. The concentration of AA content was established from the calibration graph.2 Mei [email protected] offers a sensitivity, selectivity, simplicity and cost-effectiveness of the method. The method involves no extraction steps, thereby the use of organic solvents, which are generally toxic in nature are avoided. The stability of formed MG dye is an added advantage of the method. The sensitivity in terms of molar absorptivity and precision in terms of relative standard deviation of the present method indicated it to be very reliable for the determination of AA in various samples. This method is good alternative to some reported costly instrumental method.2 Mei [email protected]

Non-spectrophotometric2 Mei [email protected]

Titrimetric methods2,6-Dichlorophenolindophenol (DCIP)tetrachlorobenzoquinoneN-bromosuccinimideiodine, potassium iodate, potassium bromate & iodine monochloridechloramine To-Iodosobenzoate & o-diacetoxyiodobenzoatethallium(III) perchlorate & copper(II) sulfatecerium(IV) sulfatepotassium hexacyanoferrate(III)ferricinium trichloroacetate2 Mei [email protected]

2,6-dichlorophenolindophenol (DCIP)the reduction of DCIP (Tillimans Reagent) with ascorbic acid in acidic solution.Official method: the solution containing ca. 2 mg of ascorbic acid and 5 mL of a mixture of metaphosphoric acid and acetic acid is titrated with a standard solution of DCIP. The titrant acts as a self indicator.it is not applicable to many pharmaceutical preparations containing Fe(II), Sn(II), Cu(I), SO2, SO32 and S2O32 ions which are usually associated with mineral or liver preparations.2 Mei [email protected] Mei [email protected]

J Pharmaceutical Sciences Vol. 72, No. 2, February 1983Analytical Sciences October 1998, Vol. 14Kekurangan DCIPIt is not applicable if:there are substances naturally present in fruits or biological materials such as tannins, betannins and sulfhydryl compounds are oxidized by the dyethe concentration of dehydroascorbic acid is negligiblethe alkalinity of the sample also hinders the determinationnatural or added colors render the end point difficult to judge visibly such as edible dyes like amaranth, indigotine and tetrazine present in pharmaceutical preparations need removal either by ion exchange or by addition of charcoal

2 Mei [email protected] DCIPtitrating the citrate buffered solutions (pH 3.5) with alcoholic solution (ethanol)the alcoholic solution is claimed to be superior to official solutions mainly because of its stability, but it requires storage in a refrigeratorit can avoid or reduce interferences from colors, iron, reductones and reductic acidbut the dye is useful only for solutions containing not more than negligible amounts of interfering substances and dehydroascorbic acid2 Mei [email protected]

Anal. Chem., 1982, 54 (4), pp 793796Tetrachlorobenzoquinonethe presence of EDTA which acts as an indicator as well as a masking agent for associated metal ion impuritiesthe end point is detected by the appearance of a golden yellow colorapplicable for the interferences of citric acid, oxalic acid, tartaric acid, glucose, sucrose and maltosemixtures of ascorbic acid with thiols like cysteine, o-mercaptobenzoic acid, mercaptosuccinic acid and 3-mercaptopropionic acid cannot be resolvedthe interference of thiols is reported to be avoidable by masking with acrylamide2 Mei [email protected] TCBQ2 Mei [email protected]

Remove the thiolsResolutions of mixtures of vitamin C with thiols has been successfully carried out by first titrating the vitamin C content with tetrachlorobenzoquinone till the orange-red color appears. Upon dilution of the contents, thiols can be titrated with standard chloramine-T solution. Thiols are quantitatively oxidized to their corresponding disulfides with chloramine-T in the presence of potassium iodide

CH3C6H4SO2NCl-Na+ + 2I- + 2H+ CH3C6H4SO2NH2 + Cl-Na+ + I2 2RSH + I2 RSSR + 2HI2 Mei [email protected]

N-bromosuccinimideN-bromosuccinimide was introduced as a reagent for the determination of ascorbic acid using starch as an indicatorReductones, reductic acid and iron salts do not interfere with this titration, but it was found to give unsatisfactory results with the samples of preserved juices and squashes containing metabisulfite as a preservativeQuinoline yellow solution has been used for detecting the end point in the titrations with N-bromophthalimide and N-bromosaccharinThese reagents were also used in the potentiometric titration of Vitamin C. Cysteine and glutamic acid interfereN-bromosaccharin was used for the microdetermination of ascorbic acid in pure solutions and pharmaceutical preparations2 Mei [email protected]

iodine, potassium iodate, potassium bromate & iodine monochloridestarch cannot be used in such titrations because it decreases the reaction rate between ascorbic acid and iodinevariamine blue, carbon tetrachloride or chloroform in the presence of mercuric chloride and p-ethoxychrysoidine as indicators have been recommended. naphthol blue black, amaranth or Brilliant Ponceau 5R as alternative indicatorsCu(II), As(III), Hg(II), cysteine, thiourea, thioglycollic acid, sulfide and sulfite interfere seriouslySeetharampa and Shubha proposed a titrimetric method based on the oxidation of ascorbic acid with pyridinium chlorochromate and estimating the unconsumed oxidant iodometricallyperphenazine, 1-amino-4-hydroxyanthraquinone, azine and oxazine dyes, phenosafranine, safranine T, wool fast blue and aposafranine have also been used in the bromatometric estimation of ascorbic acid2 Mei [email protected](IV) sulfate2 Mei [email protected]

Lebih detail lihat diAnalytica Chimica Acta 2000 417: 1-14

Non-spectrophotometric methods for the determination of Vitamin C2 Mei [email protected]

Human Liver Glycogen Phosphorylase a Complexed with Riboflavin, N-2 Acetyl-beta-D-Glucopyranosylamine and CP-403,700 (1L5R)created by Chimera and POV-Ray2 Mei [email protected] Analytica Chimica Acta 2004 520: 57-67 B-Komplex tablets (Lciva, Prague, Czech Republic) consisted of vitamins (thiamine, riboflavin, pyridoxine, pantothenic acid, nicotinamide) and adjuvants (lactose mohydrine, may amylose, gelatine, calcium stearate, talcum, saccharose, titanium dioxide, sodium caramel, paraffin, silica gel anhydride, natural identical aromas, fibres and additives).Vitamin drink (Penko, Krnov, Czech Republic) consisted of vitamins (thiamine, riboflavin, pyridoxine, pantothenic acid, nicotinamide, -tocopherol, cyanocobalamin, l-ascorbic acid, folic acid) and adjuvants (saccharose, glucose, E330, maltodextrine, natural identical aroma, milk proteins, lemon pectin, soy lecithin, ZnO, MnSO4 and Na2SeO3).

2 Mei [email protected] Mei [email protected]

Validation2 Mei [email protected]

a Retention times in min. b Regression coefficients. c Limits of detection (3.S/N). d Limits of quantitation (10.S/N). e Relative standard deviations. f Non-vitaminous substances.Result from article2 Mei [email protected]

FIA2 Mei [email protected]

ReactionRiboflavine forms a 1:1 complex with Ag(I) within pH 6.5 and 7.1Pyridoxine reacts with N,N-diethyl-p-phenylenediamine and potassium hexacyanoferrate(III) in phosphate buffer yielding an indophenol dye2 Mei [email protected]

Konsumsi pereaksiThe reagent consumption was in media 25-fold lower than those required in the determination of the vitamins by flow-based procedures with continuous reagent addition (Table 4)2 Mei [email protected]

Hasil2 Mei [email protected]

Application Note 251Determination of Water- and Fat-Soluble Vitamins in Nutritional Supplements by HPLC with UV Detection

usingDionex UltiMate 3000 HPLC system2 Mei [email protected] vitamin supplement samples (Brands 1 to 5) were analyzed. The ingredients are listed in Table 1. Brands 1 and 2 were from two different pharmaceutical companies, and Brands 3, 4, and 5 were from a third company that produces special vitamin tablets for women, children, and the elderly2 Mei [email protected]

Sistem KromatografiSistem gradien eluenPanjang gelombang2 Mei [email protected]

Kondisi KCKTVit Water-solubleVit Fat-soluble2 Mei [email protected]

KromatogramVC & VB group vitaminsfat-soluble vitamins & carotene2 Mei [email protected]

Hasil-method detection limit (MDL)2 Mei [email protected]


Sample preparationOne gram of the pulverized dried thiamine hydrochloride powder was dissolved in the least necessary amount of water, transferred to a 100-mL standard flask and made up to the mark with water. Ten tablets were ground and the powder was mixed, dried, and dissolved in the minimum of water, then the solution was filtered into a 100-mL standard flask and made up to the mark with water. The contents of 10 vials were transferred to a 100-mL standard flask and made up to the mark with water.2 Mei [email protected] titrationAliquots of thiamine hydrochloride sample solution (0.50-5.0 mL) were transferred to a 100-mL beaker and diluted to 10 mL with water. Ten ml of 1M sodium hydroxide were added and the solution was stirred for 2 min. The silver-silver sulphide ion-selective electrode and the double-junction silver-silver chloride reference electrode were introduced into the solution and standard 0.01M silver nitrate was slowly added from a dark-glass burette with its tip immersed in the solution. The electrode potential was monitored as a function of the titrant volume added. Titration end-points were calculated from first or second derivative titration curves (1 mL of O.OlM silver nitrate 1.535 mg of thiamine hydrochloride).2 Mei [email protected] & perolehan kembali2 Mei [email protected]


Metode lainnyaDerivatisasi KCKT: Analytical Biochemistry, 333 (2004) 336344 VitB6 & B12Spektofotometri derivatisasi: J.Pharm. Biomed. Analysis, 22 (2000) 915923 VitB1 & B6Voltammetri: J. Braz. Chem. Soc., 14_2 (2003) 316-321 & Talanta, 61 (2003) 743/753 VitB6 & B12Fluorometri: Fresenius J Anal Chem, 368 (2000) 836838 VitB12


Vitamin Tidak larut air (fat soluble)

Vitamin A, D, E dan K2 Mei [email protected]

Crystal Structure of Human Holo Cellular Retinol-binding Protein II (CRBP-II) from 2RCTcreated by Chimera and POV-Ray2 Mei [email protected] Mei [email protected]

Metode Analisis2 Mei [email protected]

Flow Injection- chemiluminescence2 Mei [email protected]

Tris(2,2-bipyridyl)ruthenium(II) [Ru(bpy)32+]


Interference studiesThe influence of commonly used excipients and additives in pharmaceutical formulations was investigated by analyzing solutions containing 1.0 106 mol/L vitamin A. The tolerable foreign species were taken as a relative error not greater than 8%. No interference could be found when 750-fold sorbitol and mannitol, 500-fold glycerol and cellulose, 200-fold sucrose, glucose, starch, gum acacia, magnesium stearate and glycerol monostearate, 100-fold stearic acid and tocopherol, 10-fold triglyceride and 1-fold vitamin D3 and cholesterol were added to 1.0 106 mol/L vitamin A.2 Mei [email protected] Mei [email protected]

Prosedur dan hasilFrom each standard solution of ethanol/retinol prepared in water and methanol respectively, 0.1 mL was mixed with 1.3 mL of pyrophosphate (50mM, pH 10.5) and 1.5 mL of NAD+ (10 mM). The reaction mixture was incubated for selected time periods (e.g. 5 min) in the thermostated water bath at 37C. At zero time, 0.1mL of alcohol dehydrogenase enzyme was added and the reactants were mixed gently. The liberated NADH wasmeasured at 340 nm for approximately 10 min (at 37C) using UVvis spectrophotometer equipped with 10mm silica cuvettes. A thermostated water bath was used to maintain the temperature during incubation.2 Mei [email protected]

Retinol

Crystal Structure of Human Holo Cellular Retinol-binding Protein II (CRBP-II) from 2RCTcreated by Chimera and POV-Ray2 Mei [email protected] 1975 100: 238-242A colorimetric method for the determination of vitamin D, based upon the use of anisaldehyde - sulphuric acid as a colour reagent, has been developed. The method enables 0.1-0.5 mg of calciferol to be determined with a mean percentage recovery of 100.2 1.44 percent, and it avoids the difficulties met with in the antimony(II) chloride colour reaction.Both methods, as applied to oily injections of vitamin D, are compared, and a statistical analysis of the results reveals that the proposed method is the more precise, and has an accuracy equal to that of the antimony(II) chloride method.2 Mei [email protected] kerja2 Mei [email protected]


Hasil pada minyak ikan2 Mei [email protected]

Tocopherol

Crystal Structure of Human Alpha-tocopherol Transfer Protein Bound to Its Ligand (1R5L)created by Chimera and POV-Ray2 Mei [email protected] Mei [email protected]

PreparasiTransesterifikasiSampel2 Mei [email protected]

standard additions methodFor the standard additions method three separatory funnels were used. To the first was added a 5.0-mL aliquot of the EDTA-vitamin solution. A mixture of a 5.0-mL aliquot of the EDTA-vitamin solution and 0.10 mL of standard solution (measured with a micropipette) was placed in the second. The third contained 100 mL of the standard solution alone. The tocopheryl acetate in each solution was extracted with two 10-mL portions of petroleum ether and the extracts were treated as just described for the samples. The standard additions method was repeated with 10-mL aliquots. The tocopheryl acetate content in mg/g of dry multi-vitamin (I.U.) is calculated from the standard additions method results as follows:2 Mei [email protected]

ReactionThe rate of reaction is indicated by the rate of colour development, which depends on the rate of reduction of tetrazolium blue by tocopherol. To optimize conditions, we have investigated a number of parameters such as alkalinity, temperature, time, reagent concentration, solvent, transesterification and order of additions2 Mei [email protected]

Validasi2 Mei [email protected]




Hasil2The method proposed for determining vitamin K1 combines the advantages of photochemical reactions (e.g. selectivity, sensitivity, cleanliness and easy manipulation) with those associated with the use of flow-injection systems (e.g. simplicity, rapidity and low cost)2 Mei [email protected]

Vitamin K2 Mei [email protected]

Terima Kasih2 Mei [email protected]

Documents

AnFar 2013 Vitamin