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NOVEL RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF THIAMINE MONONITRATE, CALCIUM PANTOTHENATE, L-CYSTINE AND PARA AMINO BENZOIC ACID IN MULTI VITAMIN DOSAGE FORMS

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NOVEL RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF THIAMINE MONONITRATE, CALCIUM PANTOTHENATE, L-CYSTINE AND PARA AMINO BENZOIC ACID IN MULTI VITAMIN DOSAGE FORMS Powered By Docstoc
					                                   Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242



                                                                                                 ISSN:2249-5347
                                                                                                           IJSID
                          International Journal of Science Innovations and Discoveries                An International peer
                                                                                                 Review Journal for Science


 Research Article                                                     Available online through www.ijsidonline.info

        NOVEL RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF THIAMINE
     MONONITRATE, CALCIUM PANTOTHENATE, L-CYSTINE AND PARA AMINO BENZOIC ACID
                          IN MULTI VITAMIN DOSAGE FORMS
 Thamma Narendra kumar a*, R. Sreenivasulu b, Dr. NSV Raju a, Useni Reddy Mallub and D. Sandeepa

 a   Genovo Development Services Ltd. (R&D), Bommasandra Industrial Estate, Bengaluru-560099, Karnataka, India,b
            Department of Chemistry, Sri Krishnadevaraya University, Anantapur, Andhra Pradesh-515003,


                                                                         ABSTRACT
Received: 10.08.2011
                                     Objectives: Thiamine Mononitrate, Calcium Pantothenate, L-Cystine and Para
Modified: 16.10.2011
                                     Amino Benzoic acid are essential dietary components for animals and humans.
Published: 27.10.2011
                                     The main objective of this research is to develop a simple, accurate RP-HPLC
Keywords: Simultaneous               method for the quantification of Thiamine Mononitrate, Calcium Pantothenate, L-
estimation; Vitamins;
High-performance liquid              Cystine and Para Amino Benzoic acid in drug substances as well as drug product.
chromatography;
                                     Methods: Chromatographic separation was achieved by using Phenomenex
Pharmaceutical preparation.
                                     Synergi Max-RP 80 A° 150 × 4.6 mm; 4.0 µm column, a gradient programme of
*Corresponding Author                mobile phase A & B are used, Mobile phase-A is 100 % buffer (dissolved 3 g of 1-
                                     Hexane sulphonic acid sodium salt and 2 ml of Orthophosphoric acid in 1000 ml of
                                     Milli-Q water). Then filtered the solution through 0.45 µm nylon filter and
                                     degassed. Whereas Mobile phase-B 100 % Methanol. The flow rate of the mobile
                                     phase is 1.0 ml/min. Column temperature maintained at 25°C. Injection volume is
                                     10 µl and run time is 35 minutes. Analytes absorbance was measured at 210 nm.
                                     Results/Conclusion: The developed method was validated as per ICH guidelines

Address:                             with respect to specificity, precision, linearity, accuracy, robustness and system
                                     suitability. Satisfactory results found from method validation and the method is
Name: Tamma Narendra Kumar
                                     applicable for determination of assay of Thiamine Mononitrate, Calcium
Place: Bangalore, India
E-mail:                              Pantothenate, L-Cystine and Para Amino Benzoic acid in drug substances and
narendrakumar.thamma@gmail.com
                                     different pharmaceutical dosage forms, Nutritional supplements, Multi vitamin
                                     preparations.

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                                                 INTRODUCTION
       Vitamins represent a group of various compounds, both chemically and analytically, because they
comprise a wide range of bio molecules (Fig. 1). They may be present in several chemically diverse but
biologically inter convertible forms. Their common properties reside solely in fact that they are essential
dietary components for animals and/or humans            [1–4].   They are needed in relatively small amounts to
sustain life and good health. Currently many vitamin supplements such as multivitamin tablets are
available for the prevention and control of Avitaminosis but also for treating some other diseases.
       L-cystine (1-6) is a naturally occurring amino acid that is classified as a protein amino acid. One of
the main functions of L-cystine is the promotion of stomach lining health and also the correction of
situations where the absorption of essential nutrients from food sources takes place. Many people are
able to obtain as much of this protein source as they need without taking any type of supplement. L-
cystine can be found in a number of foods ranging from meats to dairy and vegetable, Chicken, turkey and
pork are all good sources of L-cysteine. L-cystine as cysteine can be obtained from eggs and milk. One of
the largest applications is the production of flavors. For example, the reaction of cysteine with sugars in a
Maillard reaction yields meat flavors. it is used for permanent wave applications predominantly in Asia.
Again the cysteine is used for breaking up the disulfide bonds in the hair's keratin.
       Thiamine (7-26) or thiamin or vitamin B1 is a water-soluble vitamin of the B complex. First named
aneurin for the detrimental neurological effects if not present in the diet. Its phosphate derivatives are
involved in many cellular processes. The best-characterized form is thiamine pyrophosphate (TPP), a
coenzyme in the catabolism of sugars and amino acids. In yeast, TPP is also required in the first step of
alcoholic fermentation. All living organisms use thiamine in their biochemistry, but it is only synthesized
in bacteria, fungi, and plants. There is still much research devoted to elucidating the exact mechanisms by
which thiamine deficiency leads to the specific symptoms observed. New thiamine phosphate derivatives
have recently been discovered, emphasizing the complexity of thiamine metabolism. Thiamine
derivatives with improved pharmacokinetics have been discovered and are to be considered more
effective in alleviating the symptoms of thiamine deficiency and other thiamine-related conditions such
as impaired glucose metabolism in diabetes. These compounds include allithiamine, prosultiamine,
fursultiamine, benfotiamine and sulbutiamine, among others.
       Pantothenic acid   (27-38),   also called pantothenate or vitamin B5, it is a water-soluble vitamin. For
many animals, pantothenic acid is an essential nutrient. Animals require pantothenic acid to synthesize
coenzyme-A (CoA), as well as to synthesize and metabolize proteins, carbohydrates and fats. It is
commonly found as its alcohol analog, the provitamin panthenol, and as calcium pantothenate.
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Pantothenic acid is an ingredient in some hair and skin care products. It plays a huge role in the
functioning of the enzymes in the human body. Some of its major functions are converting food into
energy, stimulating growth, reproduction, and many other normal bodily processes. It is essential in
human growth, reproduction and many other normal bodily processes. One of its major role in the body
is to help in the metabolism and break down of carbohydrates, fats and proteins for the production of
energy in the body. Pantothenic acid or vitamin B5 also produces enzymes and helps maintain accurate
communication between the central nervous system and the brain. It is also required for the production
of steroid hormones and hormones of the adrenal gland.
       Para-amino benzoic acid      (39-43)   (PABA) is a naturally occurring substance that is often used in
sunscreen products. PABA is sometimes called vitamin Bx, but it is not a true vitamin. PABA overdose
occurs when someone accidentally or intentionally takes more than the normal or recommended amount
of this substance. PABA is used to improve the protein used in the body, it relates to red blood cell
formation as well as assisting the manufacture of folic acid in the intestines. Para-aminobenzoic acid is
used in sunscreen preparations since it can help protect the skin against ultra-violet radiation. It has been
linked to hair growth as well as reversing the graying of hair, but these results are disappointing. Oral
supplements of PABA can make the skin less sensitive to sun damage.




        Thiamine Mononitrate                                       Calcium Pantothenate




            L-cystine                                                  Para amino benzoic acid

                                        Figure–1: Structure of analytes
       UV-Vis    spectrophotometry,       Fluorimetry,     Chemiluminiscence,       Capillary    electrophoresis   ,
Microbiology and High-performance liquid chromatography have been proposed for the determination of
vitamins. Nevertheless, there is no single analytical approach to determine vitamins within a complicated

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matrix in a single run. In this work, we developed and optimised a high performance liquid
chromatographic method using diode array detection for determination water and fat-soluble vitamins at
a single run. The method was successfully applied to the determination of vitamins in Nutritional
supplements, Multivitamin preparations, Pharmaceutical preparations, etc.
                                         MATERIALS AND METHODS
Chemicals and reagents
       Capsules and Standards components were supplied by Medreich India limited. All solvents were
HPLC grade and High purity water was prepared by using Millipore Milli-Q plus water purification
system (Millipore, Milford, MA, USA).
Equipment
       The Shimadzu UFLC system used consists of a pump, auto sampler and a PDA detector. The output
signal was monitored and processed by using LC solutions software.
Chromatographic conditions
       The method was developed using Phenomenex Synergi Max-RP 80 A°, 150 × 4.6 mm; 4.0 µm
column. A gradient programme of Mobile phase A & B are used , Mobile phase A is 100 % buffer, which is
prepared by dissolving 3 g of 1-Hexane sulphonic acid sodium salt in 1000 ml of Milli-Q water , then
added 2 ml of Ortho phosphoric acid and mixed well. Then filtered the solution through 0.45 µm nylon
filter and degassed. Whereas Mobile phase B was 100 % Methanol. The flow rate of the mobile phase was
1.0 ml/min. The column temperature was maintained at 25oC and the wavelength was monitored at 210
nm. The injection volume was 10 µl. The run time is fixed to 35 minutes.
Preparation of stock solutions
       A stock solution was prepared by transferring accurately weighed about 60 mg of Calcium
Pantothenate, 20 mg of L-Cystine, 30 mg of Para Amino Benzoic acid and 60 mg of Thiamine Mononitrate
working standard into a 100 ml volumetric flask, added 10 ml of 1N HCl, sonicated for 5 min, then added
5 ml of Methanol, sonicated for 5 min and then added 55 ml of purified water, sonicated to dissolve, then
made up to the volume with purified water and mixed well.
Preparation of sample solutions
       Twenty capsules were weighed, then transferred the powder sample into a petri dish and weighed
empty capsule, then calculated the average fill weight of the capsule. Weighed about 410 mg of capsule
powder (equivalent to 60 mg of Thiamine Mononitrate, 60 mg of Calcium Pantothenate, 30 mg of Para
Amino Benzoic acid and 20 mg of L-Cystine) then transferred in to a 100 ml volumetric flask, 10 ml of 1N
HCl was added, sonicated for 5 min, then added 5 ml of Methanol, sonicated for 5 minutes and then added
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55 ml of purified water, sonicated for 30 minutes to dissolve, then made up to the volume with purified
water and mixed well. This solution was centrifuged at 3500 rpm for 10 min and collected the
supernatant clear solution.
Calculation of % analyte:

   a. Calculation for % Calcium Pantothenate:
                               TRCP       WCP        100          PCP
                             -----------x-----------x-----------x-----------x A
                               SRCP       100          TW         LCP
   b. Calculation for % L - Cystine:
                               TRLC       WLC        100         PLC
                             -----------x-----------x-----------x-----------x A
                               SRLC       100          TW        LLC
   c. Calculation for % Para Amino Benzoic Acid:
                               TRPB       WPB         100         PPB
                             -----------x-----------x-----------x-----------x A
                               SRPB       100          TW         LPB
   d. Calculation for % Thiamine Mononitrate:
                              TRTM        WTM        100         PTM
                             -----------x-----------x-----------x-----------x A
                               SRTM       100         TW         LTM
        Where, TRCP = Calcium Pantothenate response from test preparation; TRLC = L - Cystine response
from test preparation; TRPB = Para amino benzoic acid response from test preparation; TRTM = Thiamine
Mononitrate response from test preparation; SRCP = Calcium Pantothenate average response from
standard preparation; SRLC = L - Cystine average response from standard preparation; SRPB = Para amino
benzoic acid average response from standard preparation; SRTM = Thiamine Mononitrate average
response from standard preparation; WCP = Calcium Pantothenate working standard weight in mg, for
standard preparation; WLC = L - Cystine average working standard weight in mg, for standard
preparation; WPB = Para amino benzoic acid working standard weight in mg, for standard preparation;
WTM = Thiamine Mononitrate working standard weight in mg, for standard preparation; PCP = Calcium
Pantothenate working standard purity in %, on as such basis; PLC = L - Cystine average working standard
purity in %, on as such basis; PPB = Para amino benzoic acid working standard purity in %, on as such
basis; PTM = Thiamine Mononitrate working standard purity in %, on as such basis; LCP = Label claim of
Calcium Pantothenate; LLC = Label claim L - Cystine average; LPB = Label claim Para amino benzoic acid;
LTM = Label claim Thiamine Mononitrate; A = Average weight of test sample and TW = Test weight taken
in mg




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                                          RESULTS AND DISCUSSION
Method development and optimization
       Different brand of HPLC columns were tried for getting the symmetrical peak for all the actives. In
these trails found that Phenomenex Synergi Max-RP 80 A°, 150 × 4.6 mm; 4.0 µm column is the best
suitable for the peak shapes as well as for responses of the actives. By this column obtained superior peak
shape and separation because of the unique property of fully porous silica support with bonded phase of
C12 with TMS end capping. This column is the best suite for the separation of basic compounds. Diluent
was optimized based on the physical properties of the actives to get the better stability in the solution
form with superior extraction of the actives from excipients in to the solution form.
                                     Table – 1: System Suitability Results
                                                                Active Ingredient
           Standard inj N°            Calcium                              Para Amino       Thiamine
                                                        L – Cystine
                                   Pantothenate                            Benzoic Acid   Mononitrate
                  1                   3591923            1592561            15353893        16694727
                  2                   3564911            1594309            15365712        16171727
                  3                   3574612            1601222            15352225        16353795
                  4                   3571210            1599206            15398240        16340249
                  5                   3568934            1601830            15415350        16281353
                 Mean                3574318             1597826            15377084       16368370
                %RSD                     0.3                 0.3                0.2            1.2
       Theoretical Plate count         4316                5453               4779           170077
            Tailing Factor               1.4                 1.1                1.5            1.2
                                      Precision Results (% RSD of % assay)
                                                                Active Ingredient
             Sample N°                Calcium                              Para Amino       Thiamine
                                                        L – Cystine
                                   Pantothenate                            Benzoic Acid   Mononitrate
                  1                     99.5                99.5              100.3           99.6
                  2                     99.5                99.9              100.3           99.7
                  3                     99.4                99.6               99.6           99.5
                  4                     99.5                99.5              100.1           99.5
                  5                     99.5                99.9              100.1           99.6
                  6                     99.1                99.7              100.2           99.5
                 Mean                   99.4                99.7              100.1           99.6
                %RSD                     0.2                 0.2                0.3            0.1
                    Comparison b/n Precision Intermediate Precision Results (Response % RSD)
                                      Calcium                              Para Amino       Thiamine
                %RSD                                    L – Cystine
                                   Pantothenate                            Benzoic Acid   Mononitrate
              Precision                  0.2                 0.2                0.3            0.1
       Intermediate Precision            0.5                 0.2                0.2            0.3


Results of forced degradation studies
       A study was conducted to demonstrate the effective separation of degradants from the active
ingredients. Mixture of drug substance with placebo was subjected to the following stress conditions to
induce degradation.

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                                  Table – 2: Stress study conditions and results
                                                                     % Degradation from Drug Product
     Stress Study            Stress Condition
                                                            Calcium                  Para Amino      Thiamine
                                                                         L-Cystine
                                                          Pantothenate               Benzoic acid   Mononitrate
                      0.1N HCl solution for about 2 hrs
    Acid Hydrolysis                                           12.2          1.3           3.0            3.5
                                    at 60°C
                      0.1N NaOH solution for about 2
   Base Hydrolysis                                            12.5          3.0           3.1            2.6
                      hrs 30 min at 60°C
       Oxidation      1% H2O2 for 2 hrs at 25°C               4.1           3.6           6.0            4.8
       Aqueous        Purified water for about 6 hrs at
                                                              4.0           3.6           2.7            5.7
      Hydrolysis      60°C
      Fluorescent     Sun-light of 1.2 Million Lux
                                                              2.7           2.1           1.5            3.6
    Light Exposure    Hours
        UV Light
                      UV-light of 200 Watts h/m2              2.4           1.7           1.5            2.7
       Exposure
        Thermal       Dry heat at 105°C for 24hrs             4.6           3.3           2.8            5.7
       Humidity       90% RH at 25°C for 7 days               3.2           2.5           1.7            3.4
For all active peaks peak purity was evaluated by using the Photodiode array detector and purity of peaks
was passed.
VALIDATION OF THE METHOD
Specificity: Specificity is the ability of the method to measure the analyte response in the presence of its
degradants. The specificity of the developed method was carried in the presence of its degradants. Stress
studies were performed on dosage form to provide an indication of the stability-indicating property and
specificity of the proposed method. Intentional degradation was attempted with stress condition of UV
light (200 watts h/m2), Sun light (1.2 Million Lux Hours), Humidity (90% RH at 25°C), Dry heat (105oC),
Acid (0.1N HCl), Base (0.1N NaOH) and Oxidation (1% H2O2) to evaluate the ability of the proposed
method to separate Calcium Pantothenate, L-Cystine, Para Amino Benzoic acid and Thiamine
Mononitrate from its degradation products. For heat, study period was 24 hrs whereas for hydrolytic,
acid, base and oxidation study period was about 2 hrs. Peak purity test was carried out by using PDA
detector in stress samples.
       In the stressed samples % degradant products were calculated and reported.
       In the standard and sample chromatograms before 2 minutes one peak will appear and this peak
no need of quantification and it originates from the salt form of the materials have been used.




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                                Figure – 2: Blank chromatogram




                               Figure – 3: Standard chromatogram




                                Figure – 4: Placebo chromatogram



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                  Figure – 5: Sample chromatogram with peak purity evaluation




                          Figure – 6: Purity plot of Calcium Pantothenate




                               Figure – 7: Purity plot of L – Cystine




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                                    Figure – 8: Purity plot of Para amino benzoic acid




                                    Figure – 9: Purity plot of Thiamine Mononitrate
Precision: The precision of the method verified by Repeatability and Intermediate precision.
Repeatability was checked by injecting six individual weights of placebo mixed with API (Calcium
Pantothenate, L-Cystine, Para Amino Benzoic acid and Thiamine Mononitrate). % RSD of assay results for
each ingredient was calculated. The intermediate precision of the assay method was evaluated by
different analysts and performing the analysis on different days and with different HPLC instruments and
columns.
       The % RSD of assay of Calcium Pantothenate, L-Cystine, Para Amino Benzoic acid and Thiamine
Mononitrate during the assay method precision study was 0.2 %, 0.2 %, 0.3 %, and 0.1 % respectively.
The % RSD of the assay results obtained in the intermediate precision study was within the limit (0.5, 0.2,
0.2, and 0.3) conforming good precision of the method. The % RSD values were represented in table-1.
Accuracy: Accuracy of the assay method was evaluated with dosage form equivalent to about 50% to
150% of the target assay of the Calcium Pantothenate, L-Cystine, Para Amino Benzoic acid and Thiamine
Mononitrate. The percentages of recovery at each level were calculated. The percentage recovery of

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Calcium Pantothenate, L-Cystine, Para Amino Benzoic acid and Thiamine Mononitrate was ranged from
98.2 to 101.5%. The % recovery values for Calcium Pantothenate, L-Cystine, Para Amino Benzoic acid and
Thiamine Mononitrate are represented in table-3.
                          Table – 3: Recovery of active ingredients - summary table
                       Calcium                                  Para Amino Benzoic            Thiamine
                                              L – Cystine
                    Pantothenate                                        Acid                 Mononitrate
        Level
                     %          %            %           %                    %                           %
                                                                % Recovery              % Recovery
                  Recovery     RSD        Recovery      RSD                  RSD                         RSD
         50%        99.5       0.3          99.9        1.1         99.0      0.7           98.2         0.4
         25%        98.8       0.4          100.4       0.6         99.3      0.2           98.2         0.4
         50%        99.0       0.2          100.6       0.9        101.5      0.5          100.8         0.3
        100%        98.7       0.2          100.9       0.1         98.3      0.0          100.8         0.2
        150%        98.8       0.3          101.0       0.2         99.9      0.1          100.6         0.2
Linearity: Linearity of test solutions for the assay method was verified in the range of Calcium
Pantothenate 300, 600, 900 µg/ml, L-Cystine 100, 200, 300 µg/ml, Para Amino Benzoic acid 150, 250,
450 µg/ml, Thiamine Mononitrate 300, 600, 900 µg/ml respectively. The peak area versus concentration
data was treated by least-squares linear regression analysis.
       Linearity calibration plot for the assay method was obtained over the calibration ranges tested, i.e.
Calcium Pantothenate 300-900 µg/ml, L-Cystine 100-300 µg/ml, Para Amino Benzoic acid 150-450
µg/ml, Thiamine Mononitrate 300-900 µg/ml respectively and correlation coefficient obtained was
greater than 0.999. The result shows that an excellent correlation existed between the peak area and
concentration of the analyte.
       Linearity chromatograms, graphs and results were represented in figure-10, figure-11 and table-4
respectively.




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                 Figure-10: Linearity chromatograms of four ingredients




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                                 Table – 4: Linearity Regression Summary Table
                                             Calcium                              Para Amino         Thiamine
        Active ingredient name                                  L – Cystine
                                           Pantothenate                           Benzoic Acid      Mononitrate
                  Level – 1 (50%)              1659535            784171              7737695          7672519
   Response




                  Level – 2 (75%)              2702541            1252070            12412698         12536931
   Solutions
   Linearity




                 Level – 3 (100%)              3364230            1572471            15987709         16798448
                 Level – 4 (120%)              4038990            1931182            18609074         21433362
                 Level – 5 (140%)              4746170            2218895            22052848         25615005
                 Level – 6 (150%)              5028976            2395662            23441163         27734450
                       Slope                 5543.037981        7987.176329        51640.87933       33639.47941
   Regress

   Results




                     Intercept               61611.26695        7678.721728        286708.7734       2751597.782
     ion




               Correlation Coefficient          0.999              0.999               0.999            0.999
                  Standard Error             68892.55769        26164.82623         291318.456       377888.9883




                                 Figure-11: Linearity graphs
Robustness: To determine the robustness of the developed method, experimental conditions were
deliberately altered. To study the effect of flow rate on resolution; flow was changed by + 0.1 units from
0.9 to1.l ml/min instead of 1.0ml/min. The effect of the column temperature on resolution was studied at
+ 5°C units from 20°C and 30oC instead of 25oC. The effect of filters was conducted using two different
filters namely, 0.45µm PVDF filter and 0.45µm Nylon 66 filter.

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       In all the deliberate varied chromatographic conditions (flow rate, column temperature and
change of filters), the resolution between all pairs of compounds was greater than 2.0, tailing factor for
the components was less than 2.0 and the theoretical plate count was more than 5000. 0.45 µm PVDF
filter and 0.45 µm Nylon 66 filters are found to be suitable for filtration.
                  Table –5: Robustness Parameter Summary table – Flow rate variation
                                                 Flow Rate Variation (± 0.1 mL/min)
              Peak Name              Low (0.9 mL/min)   Actual (1.0 mL/min) High (1.1 mL/min)
                                     TF       %RSD        TF        %RSD         TF    %RSD
         Calcium Pantothenate        1.2          0.5           1.2         0.9           1.2         0.2
               L – Cystine           0.9          0.1           0.9         0.3           0.9         0.1
        Para Amino Benzoic Acid      1.3          0.1           1.3         0.2           1.3         0.2
         Thiamine Mononitrate        1.4          0.1           1.4         0.4           1.5         0.2

            Table –6: Robustness Parameter Summary table – Column oven temperature variation
                                             Column oven temperature variation (± 5°C)
              Peak Name                Low (20°C)       Actual (25°C)           High (30°C)
                                     TF     %RSD       TF        %RSD         TF        %RSD
         Calcium Pantothenate        1.2         0.4           1.2         0.9           1.2          0.2
               L – Cystine           1.0         0.1           0.9         0.3           0.8          0.1
        Para Amino Benzoic Acid      1.3         0.1           1.3         0.2           1.3          0.1
         Thiamine Mononitrate        1.4         0.1           1.4         0.4           1.5          0.1


       TF signifies the tailing factor of respective peak and %RSD signifies the % relative standard deviation of
       respective peak response from five replicate injections.
                              Table –7: Filter validation parameter Summary table
                                                                Filter validation
              Active Ingredient Name                            Similarity factor
                                                0.45 µm PVDF Filter        0.45 µm Nylon 66 Filter
               Calcium Pantothenate                     0.98                            0.98
                     L – Cystine                        0.99                            0.99
              Para Amino Benzoic Acid                   1.00                            0.99
               Thiamine Mononitrate                     1.00                            0.99


Solution stability: Solution stability in the assay method was carried out by leaving both the test
solution of sample and reference standard in tightly capped volumetric flasks at room temperature for 1
day and after 2 days.
       The % RSD of the assay of actives during solution stability experiments were within 1%. No
significant changes were observed in the content during solution stability experiments. The solution
stability experiment data confirms that the sample solutions used during assay were stable for 2 days.



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                                  Tamma Narendra Kumar et al., IJSID 2011, 1 (2), 226-242

                                                          CONCLUSION
        The simple reverse phase LC method developed for quantitative analysis of actives in
pharmaceutical dosage forms is precise, accurate, linear, robust and specific. Satisfactory results were
obtained from validation of the method. The method is stability indicating and can be used for routine
analysis of production samples and stability samples of actives in pharmaceutical dosage forms.
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Description: NOVEL RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF THIAMINE MONONITRATE, CALCIUM PANTOTHENATE, L-CYSTINE AND PARA AMINO BENZOIC ACID IN MULTI VITAMIN DOSAGE FORMS