RP-HPLC Method for Estimation of Ferrous Ascorbate

RP-HPLC Method for Estimation of Ferrous AscorbateDevelopment and substantiation of RP-HPLC mode for estimation of ferric ascorbate and folic erosiveulated in bulk and in pharmaceutical face regardA simple, sensitive, precise and reproducible RP-HPLC method has been developed and corroborated for estimating the contents of ferric ascorbate and folic pungent combining in bulk and pharmaceutical formulation. The analysis was performed with Gemini-NX-C18 (2504.60 mm, 5m) column utilize smooth variant inorganic phosphate buffer (pH 7) methanol (982, v/v) for ferric ascorbate whereas phosphate buffer methanol (8812, v/v) for folic vitriolic. Mobile bod was delivered at f wiped out(p) rate of 1.0 ml/min for ferrous ascorbate and folic vinegarish. The detection wavelength was 264 nm for ferrous ascorbate and 280 nm for folic cutting. The retention time of ferrous ascorbate and folic pane of glass was run aground to be 2.7 and 6.8 min, respectively. The linearity was obey ed in the concent ration telescope of 10-50 g/mL for ferrous ascorbate (r2 = 0.999) and 10-50 g/mL for folic acid (r2= 0.999). Tablet content of ferrous ascorbate and folic acid was found 100.44 % and 98.69 %, respectively.INTRODUCTIONIron deficiency and sickle cell is the most common cause of anemia worldwide, accounting for about half of in totally anemia cases. Prevalence of anemia in India is high because of low dietary intake, poor availability of iron and chronic blood loss due to hookworm pest and malaria. An exogenous source of folic acid is necessary for the maintenance of normal erythropoiesis. Study was carried out to assess efficacy, guard duty and tolerability of combination of ferrous ascorbate and folic acid (Phosfomin-XT) in patients with iron deficiency anemia, the report sho wn the increase in bioavailability of ferrous in presence of ascorbate salt. ferrous ascorbate (Fig. 1.A) is newly introduced salt of ferrous having more bioavailability than other salts o f ferrous ascorbate 1. however till date no more literary works available for estimation of ferrous ascorbate. The use of cure multivitamins are indicated in cases of deficiency in pathological counterbalancetings in which the nutritional requirements are greatly increase or in conditions in which absorption, utilization, or excretion of vitamins are abnormal 2. Folic acid (Fig. 1.B) deficiency is the end point of megaloblastic anemia 3.Ion pair chromatographic method was reported for simultaneous determination of seven pee soluble vitamins nicotinamide, thiamine, riboflavin, pyridoxine, pyridoxal, pyridoxamine, cyanocobalamine and folic acid 2. HPLC method was developed for estimation of folic acid 2-4. RP-HPLC method for estimation of folic acid was in addition reported in Indian pharmacopoeia (2010) 5. But till date, no report is available for estimation of ferrous ascorbate in combination with folic acid in bulk and pharmaceutical formulation. An attempt has do to devel op and validate a new, rapid and sensitive method for estimating the content of ferrous ascorbate and folic acid.Experimental ConditionsInstrumentationThe HPLC chromatographic schema Agilent Technologies 1200 series, assembled with a G1315D quaternary pump, a G1315D diode array detector, a rheodyne injector fitted with a 20 L draw in and EZ Chrome Elite software.Reagents and chemicalsFerrous ascorbate and folic acid were obtained as a gift samples from Curex Pharmaceutical Pvt. Ltd., Jalgaon (MS) and S. kant Healthcare Ltd, Wapi (gujrat). HPLC clique acetonitrile, and methanol was purchased from Rankem Ltd., India. Double distilled water was generated in house. IPHARED tablets (ferrous ascorbate equivalent to elemental iron and folic acid in ratio of 1001.5 mg) were purchased from a local market. precedent inception solving of ferrous ascorbate (100 g/mL)An accurately weighed touchstone of about 10 mg ferrous ascorbate was dissolved in 100.0 mL of phosphate buffer (pH 7). work ing(a) standard outcome of ferrous ascorbateWorking standard solution of ferrous ascorbate in the concentration guide of 1050 g/mL was prepared by diluting an aliquot deal of about 1 5 mL ferrous ascorbate bourgeon solution (100 g/mL) to 10 mL with nimble phase.Standard stock solution of folic acid (100 g/mL)An accurately weighed quantity of about 10 mg folic acid was mixed with 0.5 mL 2.86% solution of sodium carbonate and thin it upto 100 ml with mobile phase 5.Working standard solution of folic acidSimilarly working standard solution of folic acid in the concentration range of 1050 g/mL was prepared by diluting an aliquot portion of about 15 mL folic acid stock solution (100 g/mL) to 10 mL with mobile phase. archetype preparation for Tablet analysisAn accurately weight 20 tablets were crushed to obtained exquisite powder.Sample preparation of ferrous ascorbateA quantity of tablet powder equivalent to 10 mg of ferrous ascorbate (11.31 mg) was dissolved in 100 mL phosphate b uffer pH 7, sonicated for 30 minutes. The solution was filtered. The aliquot portion of filtrate (2.0 mL) was diluted to 10.0 mL with mobile phase.Sample preparation of folic acidA quantity of tablet powder equivalent to 5 mg of folic acid (2.72 g) was mixed with 0.5 mL of 2.86% of sodium carbonate solution and then dissolved in 100 mL mobile phase. The mixed bag was sonicated for 30 minutes. The solution was filtered. An aliquot portion of filtrate (4.0 mL) was further diluted to 10.0 mL with mobile phase.Chromatographic condition ferrous ascorbate and folic acidChromatographic condition ferrous ascorbateThe optimized chromatographic conditions for ferrous ascorbate estimation were Gemini-NX- C18 (250 4.60, 5m) as a stationary phase, mixed phosphate buffer (pH 7) methanol (982, v/v) as mobile phase, conflate rate 1 mL/min with detection wavelength 264 nm. The chromatogram for ferrous ascorbate is as shown in (Fig. 2).The optimized chromatographic conditions for folic acid estimat ion were Gemini-NX- C18 (250 4.60, 5m) as a stationary phase, phosphate buffer methanol (8812, v/v) as mobile phase, emanate rate 1 mL/min with detection wavelength 280 nm. The chromatogram for ferrous ascorbate is as shown in (Fig. 3).Results and discussion Ferrous ascorbate and folic acid combination was used in management of anemia 6. In literature various HPLC methods for estimation of folic acid was reported 7-9. But no method was reported for the estimation of ferrous ascorbate and simultaneous estimation of ferrous ascorbate and folic acid combination. A numbers of trials were carried out for simultaneous estimation of ferrous ascorbate and folic acid. Initially, therefore attempt was done for simultaneous estimation of both drugs by reversed-phase, HPLC dissolution was tried exploitation phosphate buffer pH 3 and methanol (982) as the mobile phase, in which ferrous ascorbate eluted at 2.4 min which is nearer to dead time of column and folic acid was not eluted, the resolu tion and numbers of theroetical plates was alike poor. The organic content of mobile phase was also investigated in different ratio to optimize the separation of ferrous ascorbate and folic acid simultaneously. But no mobile phase was found suitable for analysis. Therefore, attempt was made to forecast ferrous ascorbate and folic acid in bulk and in pharmaceutical formulation separately using different chromatographic condition which was optimized separately for ferrous ascorbate and folic acid (Table I). Therefore attempt was made to estimate ferrous ascorbate and folic acid by using two different mobile phases and sample preparation method. The mobile phase phosphate buffer pH 7- Methanol (982) was selected for ferrous ascorbate estimation. The buffer-methanol (8812, v/v) was selected for folic acid estimation as per Indian pharmacopoeia 5. The constant flow rate 1mL/min was maintained for both analysis. Detection wavelength 264 nm for ferrous ascorbate and 280 nm were selected for folic acid. Retention time was found to be 2.7 min for ferrous ascorbate and 6.8 min for and folic acid.VALIDATION PARAMETERS 10, 11. establishment suitabilitySystem suitability parameters, such as number of theoretical plates, HETP, and peak tailing, were determined. The takingss obtained are shown in Table I the number of theoretical plates for ferrous ascorbate and folic acid was found 3219 and 2535 respectively with separate mobile phase.Linearity and range Ferrous ascorbate and folic acid follows a linearity of response between 1050 g/mL. Linearity take apart was performed separately by analyzing five different concentrations of ferrous ascorbate and folic acid. The Solution were diluted appropriately with mobile phase to obtain concentrations in the range of 10-50 g/ml. All measurements were repeated trey times for each concentration.Y = 23397x + 28729 is the equation for ferrous ascorbate with R2 place 0.999Y = 188287x +150052 is the equation for folic acid with R2 r espect 0.999PrecisionThe repeatability was studied by multiple injections of a homogenous sample of 20 g/mL of ferrous ascorbate and folic acid separately. The results of repeatability study of ferrous ascorbate and folic acid are as follows.The result of intra-day precision study, % RSD for ferrous ascorbate was found 1.31 and for folic acid was found to be 1.61.The result of Inter-day precision study, the % RSD for ferrous ascorbate was found 1.60 and for folic acid was found to be 1.54 respectively.The results of the precision study indicate that the method is reliable and reproducible, with a relative standard disagreement less than 2.0%.AccuracyRecovery study was performed to validate the accuracy of the developed method. Recovery study was performed at 80%, 100% and 120 % level. The known amount of standard ferrous ascorbate was added to preanalyzed sample and subjected for HPLC analysis.Procedure for ferrous ascorbateSample solutionA quantity of tablet powder equivalent to 1 0 mg ferrous ascorbate (11.35 mg) was dissolved in 100 mL phosphate buffer, sonicated for 30 minutes. The solution was filtered.Standard stock solution of ferrous ascorbate (100 g/mL)An accurately weighed quantity of about 10 mg ferrous ascorbate was dissolved in 100.0 mL of phosphate buffer pH 7.Working solution of ferrous ascorbateThree sticks containing 10.0 ml volumetricalal flask were taken. To each set, 2 ml of sample solution was added. To the first set (80%) of three volumetric flasks, a quantity of about 1.6 mL of standard stock solution of ferrous ascorbate was added. To the second set (100%) of three volumetric flasks, a quantity of about 2.0 mL of standard stock solution ferrous ascorbate was added. To the third set (120%) of three volumetric flasks, a quantity of about 2.4 mL of standard stock solution of ferrous ascorbate was added. The volume was made up to the mark with mobile phase in all flasks.The sample and standard solution were analyzed as per optimized chro matographic conditions for ferrous ascorbate.Procedure for folic acidSample solution of folic acidA quantity of tablet powder equivalent to 10 mg folic acid (5360 mg) was dissolved in 1mL of 2.86% of solution of ferrous sodium carbonate and volume was made with 100 mL buffer, sonicated for 30 minutes. The solution was filtered through whatman filter paper No.41.Standard stock solution of folic acid (100 g/mL)Weighed accurately about 10 mg folic acid mixed with 0.5 ml 2.86% solution of sodium carbonate solution and diluted with 100 ml of mobile phase.Working solutionThree sets containing 10.0 ml volumetric flask were taken. To each set, 2 ml of sample solution was added. To the first set (80%) of three volumetric flasks, a quantity of about 1.6 mL of standard stock solution of folic acid was added. To the second set (100 %) of three volumetric flasks, a quantity of about 2.0 mL of standard stock solution folic acid was added. To the third set (120 %) of three volumetric flasks, a qua ntity of about 2.4 mL of standard stock solution of folic acid was added. Then, the volume was made up to the mark with mobile phase in all flasks.The sample and standard solution were analyzed as per optimized chromatographic conditions given for folic acid. These results of accuracy are as summarized in Table no IIDetermination of the particularize of detection and quantitation (LOD and LOQ)The limit of detection and limit of quantitation is determined by using following formulae and result is given below.,WhereSD = Standard deviation of response, S = position of linearity curve equationThe result of LOD (g/ml) of ferrous ascorbate and folic acid was found to be 0.259 and 0.213 respectively.The result of LOQ (g/ml) of ferrous ascorbate and folic acid was found to be 0.784 and 0.645 respectively.RobustnessAs per ICH norms, small but heedful variations, by altering the flow rate of the mobile phase, were made to check the methods message to remain unaffected (method stability). The change was made in flow rate by altering flow rate 0.9 mL /min and 1.1 mL/min Results of analysis are summarized in Table no IIITablet analysisContents of ferrous ascorbate and folic acid found in the tablets by the proposed method are shown in Table VI the low RSD values indicate that the method is precise and accurate.Specificity studySpecificity study was conducted for ferrous ascorbate and folic acid in presence of excipients present in tablet.Preparation of physical mixture for specificity studyThe average weight of 20 tablets was found to be 804 mg. Therefore, as per labelled claim, tablet formulation excipients in tablet were 75 mg.Mixture of drug and excipients 728 g/mL ferrous ascorbate, 1.5 g/mL folic acid and 75 g/mL excipients in equal parts (like lactose monohydrate, microcrystalline cellulose, milligram stearate, starch pregelatinised etc.) was prepared. The solution was analyzed by optimized chromatographic conditions given for ferrous ascorbate and folic acid.Th e peak purity value of ferrous ascorbate and folic acid was found to be more than the input threshold value. It shows that the peak of ferrous ascorbate and folic acid was free from any impurity or co-elution in presence excipients present in tablet.The VU-spectrum of ferrous ascorbate and folic acid is as shown in Fig. 4 and Fig. 5Also the peak purity spectrum of ferrous ascorbate and folic acid was shown in Fig. 6 and Fig. 7ConclusionRP-HPLC method for estimation ferrous ascorbate and folic acid in bulk and in pharmaceutical formulation was successfully developed as per ICH guidelines. The method was found accurate, precise and sensetive hence it fuck be used for routine analysis by any analyst.