Composition 2

Label :

Mecobalamin ,Alpha Lipoic Acid, Benfothiamine ,Vitamin B6 , Calcium Pantothenate, Folic Acid
Zinic Oxide , Chromium picolinate, Inositol and leutin Capsules

Composition

Each capsule contains

Mecobalamin 1500 mcg
Alpha Lipoic Acid USP 100 mg
Benfotiamine 15 mg
Vitamin B6 IP 3 mg
Calcium Pantothenate IP 25 mg
Folic Acid IP 1.5 mg
Zinic Oxide IP 22.5 mg
Chromium picolinate USP 65 mcg
Inositol BP 10 mcg
Leutin USP 5 mg

Manufacturing Procedure
Batch Size : 1,00,000 Capsule
Batch Formula
Qty in Gm
Mecobalamin 1500 mcg 150
Alpha Lipoic Acid USP 100 mg 10,000
Benfotiamine 15 mg 1,500
Vitamin B6 IP 3 mg 300
Calcium Pantothenate IP 25 mg 2,500
Folic Acid IP 1.5 mg 150
Zinic Oxide IP 22.5 mg 2,250
Chromium picolinate USP 65 mcg 7
Inositol BP 10 mcg 1
Leutin USP 5 mg 500

1. Weigh the above materials .

2. Mix the materials in a cone blender for 15 mins.
3. The material is then filled in Capsule Shells by semi automatic capsule filling machine.
4. The filled capsules are then polished and then packed using blister.

STANDARD TESTING PROTOCOL

Test for Mecobalamin
(Reference Japanese Pharmacoepia)
Test for Alpha Lipoic Acid USP
Assay—
0.005 M Phosphate solution— Dissolve 1.36 g of monobasic potassium phosphate in 2000 mL of
water.

Phosphoric acid solution— Transfer 8.3 mL of phosphoric acid to a 100-mL volumetric flask, and
dilute with water to volume.

Mobile phase— Prepare a suitable filtered and degassed mixture of methanol, 0.005 M Phosphate
solution, and acetonitrile (1160:920:180). Adjust with Phosphoric acid solution to a pH of 3.0 to 3.1.

Solvent buffer— Prepare a suitable filtered and degassed mixture of 0.005 M Phosphate solution and
acetonitrile (1:1). Adjust with Phosphoric acid solution to a pH of 3.5 to 3.7.

Standard preparation— Dissolve an accurately weighed quantity of USP Alpha Lipoic Acid
RS in Solvent buffer to obtain a solution having a known concentration of about 1.0 mg per mL.

Assay preparation— Dissolve an accurately weighed quantity of Alpha Lipoic Acid in Solvent buffer to
obtain a solution having a concentration of about 1.0 mg per mL.

Chromatographic system (see Chromatography 621 )— The liquid chromatograph is equipped

with a 215-nm detector and a 4.6-mm × 250-mm column that contains packing L1. The flow rate is
about 1.2 mL per minute. The column temperature is maintained at 35 . Chromatograph
the Standard preparation, and record the peak responses as directed for Procedure: the retention
times for alpha lipoic acid and 6,8-epitrithiooctanoic acid are about 6.5 minutes and 13 minutes,
respectively; the column efficiency is not less than 15,000 theoretical plates; the tailing factor for the
alpha lipoic acid peak is not more than 2; and the relative standard deviation for replicate injections
is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and
the Assay preparation into the chromatograph, record the chromatograms, and measure the areas
for the major peaks. Calculate the percentage of C 8H14O2S2 in the portion of Alpha Lipoic Acid taken
by the formula:
PS (CS / CU)(rU/rS)
in which PS is the percentage of alpha lipoic acid in USP Alpha Lipoic Acid RS; CS is the concentration,
in mg per mL, of USP Alpha Lipoic Acid RS in theStandard preparation; CU is the concentration of
Alpha Lipoic Acid in the Assay preparation; and rU and rS are the peak areas for alpha lipoic acid
obtained from the Assay preparation and the Standard preparation, respectively.

Test for Chromium Picolinate USP

Assay—
Standard stock solution— Transfer about 0.283 g of potassium dichromate, previously dried at 120
for 4 hours and accurately weighed, to a 1000-mL volumetric
flask, dissolve in and dilute with water to volume to obtain a solution having a known concentration
of about 100 μg of chromium per mL, and mix. Store in a
polyethylene bottle.
Standard preparations— Separately transfer 1.0 mL and 2.0 mL of the Standard stock solution to
100-mL volumetric flasks, and transfer 1.5 mL and 2.0 mL of the
Standard stock solution to separate 50-mL volumetric flasks. Add 1.0 mL of nitric acid to each flask,
dilute the contents of each flask with water to volume, and mix.
These Standard preparations contain 1.0, 2.0, 3.0, and 4.0 μg of chromium per mL, respectively.
Assay preparation— Transfer about 200 mg of Chromium Picolinate, accurately weighed, to a 500-
mL volumetric flask, and add 25 mL of water. Slowly add 10 mL of
nitric acid, and boil for 10 minutes with constant swirling. Cool the solution, dilute with water to
volume, and mix. Filter a portion of the solution, and transfer 5 mL of
the filtrate to a 100-mL volumetric flask. Add 1 mL of nitric acid, dilute with water to volume, and
mix.
Procedure— Concomitantly determine the absorbances of the Standard preparations and the Assay
preparation at the chromium emission wavelength of 357.9 nm,
with a suitable atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering
851 ) equipped with a chromium hollow-cathode lamp and an
air–acetylene flame, using diluted nitric acid as the blank. Plot the absorbances of the Standard
preparations versus chromium concentration, in μg per mL, and draw
the straight line best fitting the four plotted points. From the graph so obtained, determine the
chromium concentration, in μg per mL, in the Assay preparation.
Calculate the quantity, in mg, of C18H12N3O6Cr in the portion of Chromium Picolinate taken by the
formula:
(418.31 / 51.996)(10C),
in which C is the concentration, in μg per mL, of chromium in the Assay preparation; 418.31 is the
molecular weight of chromium picolinate; and 51.996 is the atomic weight of chromium.

Test for Leutin USP

Solvent: a mixture of hexanes, acetone, toluene, and dehydrated alcohol (10:7:7:6).
Mobile phase— Prepare a filtered and degassed mixture of hexane and ethyl acetate (75:25). Make
adjustments if necessary (see System Suitability under
Chromatography 621 ).
Standard solution— Dissolve a suitable quantity of USP Lutein RS in Mobile phase to obtain a
solution containing about 150 μg per mL.
Test solution— Transfer about 1 mL of Test stock solution from the test for Content of total
carotenoids, and evaporate under a stream of nitrogen to dryness. Add 1 mL of Mobile phase, and
sonicate to dissolve.
Chromatographic system (see Chromatography 621 )— The liquid chromatograph is equipped with a
446-nm detector and a 4.6-mm × 25-cm column that contains
3-μm packing L3. The flow rate is about 1.5 mL per minute. Chromatograph the Standard solution,
and record the peak responses as directed for Procedure: the relative retention times are about 1.05
for zeaxanthin and 1.0 for lutein; the resolution, R, between lutein and zeaxanthin is not less than
1.0; the tailing factor is not more than 2; and the relative standard deviation for replicate injections
is not more than 2.0%.
Procedure— Inject a volume (about 10 μL) of the Test solution into the chromatograph, record the
chromatogram, and measure the peak responses. Calculate the percentage of Lutein taken by the
formula:
T(ri / rs),
in which T is the content, in percentage, of total carotenoids as determined in the test for Content of
total carotenoids; ri is the individual peak response of lutein in the
Test solution; and rs is the sum of the responses of all the peaks: not less than 75.0% of lutein is
found.
Content of total carotenoids—
Solvent: a mixture of hexanes, acetone, toluene, and dehydrated alcohol (10:7:7:6).
Test stock solution— Transfer about 15 mg of Lutein to a 100-mL volumetric flask, and dissolve in
and dilute with Solvent to volume.
Test solution— Quantitatively dilute the Test stock solution (1 in 100) with dehydrated alcohol to
obtain a solution having a final concentration of about 1.5 μg per mL.
Procedure— Determine the absorbance of the Test solution at the wavelength of maximum
absorbance at about 446 nm, with a suitable spectrophotometer, using dehydrated alcohol as a
blank. Calculate the percentage of total carotenoids as lutein (C40H56O2) by the formula:
1000A / 255W,
in which A is the absorbance of the Test solution; W is the weight, in g, of Lutein taken to prepare
the Test stock solution; and 255 is the absorptivity of the pure lutein.

Test for Benfotiamine

Instrumentation Analysis was performed on chromatographic system of HPLC LC-2010C HT Shimadzu with LC
Solution equipped with Shimadzu SPD-M20A Prominence Diode array detector. HPLC instrument was
controlled by LC Solution software. Chromatographic Conditions A Waters column C18, 5 μ (250 X 4.6 mm) was
used for chromatographic separation. The mobile phase composed of water (pH 3.2 adjusted with ortho-
phosphoric acid) and acetonitrile (75:25 v/v); at a flow rate of 0.8 mL min -1 with run time of 10 min. Mobile
phase and sample solutions were filtered through a 0.45 μm membrane filter and degassed. The detection of
both drugs was carried out at 254 nm. Preparation of Diluent 1000 ml of HPLC Grade water was adjusted to
pH 3.2 with o-Phosphoric acid. Preparation of Standard Stock Solutions Standard stock solutions of 2000 μg
mL-1 of MET and 300 μg mL -1 of BEN were prepared separately using diluent. The stock solution of MET was
diluted with diluent to give working standard solutions containing 200 - 600 μg mL -1 concentrations, similarly
the BEN stock solution was diluted with diluent to give working standard solutions in the range 30 - 90 μg mL -1.
These standard solutions were injected into HPLC column and calibration curves were plotted by taking drug
peak areas vs concentrations.

Test for Vitamin B6 IP

Assay. Weigh accurately about 0.15 g, dissolve in a mixture of 5 ml of anhydrous glacial acetic acid
and 6 ml of mercuric acetate solution. Titrate with 0.1 M perchloric acid, using crystal violet solution
as indicator, until a green colour is produced. Carry out a blank titration.
1 ml of 0.1 M perchloric acid is equivalent to 0.02056 g of CsHIIN03,HCI.

Test for Calcium Pantothenate IP

Assay. Weigh accurately about 0.18 g and dissolve in 5Oml of anhydrous glacial acetic acid. Titrate
O.lM perchloric acid, determining the end point potentiometrically (2A.25). Carry out a blank
titration.
1 ml of 0.1 M perchloric acid is equivalent to 0.02383 g of ClsH32CaN201O'

Test for Folic Acid IP

Test solution. Dissolve 100 mg of the substance under examination in 5 ml of a 2.86 per cent w/v
solution of sodium carbonate and dilute to 100.0 ml with the mobile phase. Dilute 2.0 ml of this
solution to 10.0 ml with the mobile phase.
Reference solution (a). Dissolve 100 mg of folic acid RS in 5ml of a 2.86 per cent w/v solution of
sodium carbonate and dilute 100 ml with the mobile phase. Dilute 2.0 ml of this solution to 10.0 ml
with the mobile phase.
Reference solution (b).To 20 mg of pteroic acid, add 5 ml of a 2.86 per cent w/v solution of sodium
carbonate, dilute to 100 ml with the mobile phase. Mix 1.0 ml of this solution with 1.0 ml of
reference solution (a) and dilute to 100 ml with the mobile phase.

Reference solution (c). Dilute 2.0 ml of the test solution to 20 ml with the mobile phase. Dilute 1.0 ml
of this solution to 20.0 ml with the mobile phase.
Reference solution (d). Dissolve 10 mg of N-(4aminobenzoyl)- l-gulatamic acid (folic acid impurity A)
in 1 ml of a 2.86 per cent w/v solution of sodium carbonate and dilute to 100 ml with the mobile
phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase.
Reference solution (e). To 12 mg of pteroic acid (folic acid impurity D), add 1 ml ofa 2.86 per cent w/v
solution of sodium carbonate, dilute to 100 ml with the mobile phase. Dilute 1.0 ml of this solution
to 100 ml with the mobile phase.
Chromatographic system
- a stainless steel column 25 cm x 4.0 mm, packed with octylsilane bonded to porous silica (5 /lm),
- mobile phase: a mixture of 12 volumes of methanol and 88 volumes of a solution containing 1.1 per
cent w/v solution of potassium dihydrogen phosphate and 0.6 per cent w/v solution of dipotassium
hydrogenphosphate,
- flow rate. 0.6 ml per minute,
- spectrophotometer set at 280 urn,
- injection volume. 5 Ill.
Inject reference solution (b). The test is not valid unless the resolution between the peaks due to
folic acid and folic acid impurityD is not less than 4.0. The relative retention time with reference to
folic acid for N-(4-aminobenzoyl)-L-glutamic acid(folic acid impurityA) is about 0.5; for 2,5,6-
triaminopyrimidin4(IH)-one (folic acid impurity B) is about 0.6; for isofolic acid (folic acid impurity C)
is about 0.9, for pteroic acid (folic acid impurity D) is about 1.33; for 6-pterinylfolic acid (folic acid
impurity E) is about 1.27.
Inject the test solution, reference solution (c), (d) and (e). In the chromatogram obtained with the
test solution the area of peak corresponding to folic acid impurity A is not more than the area of the
principal peak in the chromatogram obtained with reference solution (d) (0.5 per cent), the area of
the peak corresponding to folic acid impurity D is not more than the area of the principal peak in the
chromatogram obtained with reference solution (e) (0.6 per cent). The area of any other secondary
peak is not more than the area of the principal peak in the chromatogram obtained with reference
solution (c) (0.5per cent), The sum of areas of all other secondary peaks is not more than twice the
area of the principal peak in the chromatogram obtained with reference solution (c) (1.0 per
cent). Ignore any peak with an area less than 0.1 times the area of the principal peak in the
chromatogram obtained with reference solution (c) (0.05 per cent).

Test of Zinc Oxide IP

Dissolve 0.15 gin 10ml of2 Macetic acidand dilute to 50 ml with water. To the resulting
solution add about 50 mg of xylenol orange triturate and sufficient hexamine to produce
violet-pink colour. Add a further 2 g of hexamine and titrate with 0.1 M disodium edetate
until the solution becomes yellow.
1 ml of0.1 M disodium edetate is equivalent to 0.008138 g of ZnO.
 Composition 3
Label :

Mecobalamin ,Alpha Lipoic Acid, Benfothiamine ,Pyridoxine , Biotin and Folic Acid Capsules

Composition

Each capsule contains

Each capsule contains
Mecobalamin 500 mcg
Alpha Lipoic Acid 200 mg
Benfotiamine 10 mg
Pyridoxine 3 mg
Biotin 5mg
Folic Acid 1.5 mg

Manufacturing Procedure
Batch Size : 1,00,000 Capsule
Batch Formula
Qty in Gm
Mecobalamin 500 mcg 50
Alpha Lipoic Acid USP 200 mg 20,000
Benfotiamine 10 mg 1,000
Pyridoxine IP 3 mg 300
Biotin 5 mg 500
Folic Acid IP 1.5 mg 150

1. Weigh the above materials .

2. Mix the materials in a cone blender for 15 mins.
3. The material is then filled in Capsule Shells by semi automatic capsule filling machine.
4. The filled capsules are then polished and then packed using blister.

STANDARD TESTING PROTOCOL

Test for Mecobalamin
(Reference Japanese Pharmacoepia)
Test for Alpha Lipoic Acid USP
Assay—
0.005 M Phosphate solution— Dissolve 1.36 g of monobasic potassium phosphate in 2000 mL of
water.

Phosphoric acid solution— Transfer 8.3 mL of phosphoric acid to a 100-mL volumetric flask, and
dilute with water to volume.

Mobile phase— Prepare a suitable filtered and degassed mixture of methanol, 0.005 M Phosphate
solution, and acetonitrile (1160:920:180). Adjust with Phosphoric acid solution to a pH of 3.0 to 3.1.

Solvent buffer— Prepare a suitable filtered and degassed mixture of 0.005 M Phosphate solution and
acetonitrile (1:1). Adjust with Phosphoric acid solution to a pH of 3.5 to 3.7.

Standard preparation— Dissolve an accurately weighed quantity of USP Alpha Lipoic Acid
RS in Solvent buffer to obtain a solution having a known concentration of about 1.0 mg per mL.

Assay preparation— Dissolve an accurately weighed quantity of Alpha Lipoic Acid in Solvent buffer to
obtain a solution having a concentration of about 1.0 mg per mL.

Chromatographic system (see Chromatography 621 )— The liquid chromatograph is equipped

with a 215-nm detector and a 4.6-mm × 250-mm column that contains packing L1. The flow rate is
about 1.2 mL per minute. The column temperature is maintained at 35 . Chromatograph
the Standard preparation, and record the peak responses as directed for Procedure: the retention
times for alpha lipoic acid and 6,8-epitrithiooctanoic acid are about 6.5 minutes and 13 minutes,
respectively; the column efficiency is not less than 15,000 theoretical plates; the tailing factor for the
alpha lipoic acid peak is not more than 2; and the relative standard deviation for replicate injections
is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and
the Assay preparation into the chromatograph, record the chromatograms, and measure the areas
for the major peaks. Calculate the percentage of C 8H14O2S2 in the portion of Alpha Lipoic Acid taken
by the formula:
PS (CS / CU)(rU/rS)
in which PS is the percentage of alpha lipoic acid in USP Alpha Lipoic Acid RS; CS is the concentration,
in mg per mL, of USP Alpha Lipoic Acid RS in theStandard preparation; CU is the concentration of
Alpha Lipoic Acid in the Assay preparation; and rU and rS are the peak areas for alpha lipoic acid
obtained from the Assay preparation and the Standard preparation, respectively.
Test for Chromium Picolinate USP
Assay—
Standard stock solution— Transfer about 0.283 g of potassium dichromate, previously dried at 120
for 4 hours and accurately weighed, to a 1000-mL volumetric
flask, dissolve in and dilute with water to volume to obtain a solution having a known concentration
of about 100 μg of chromium per mL, and mix. Store in a
polyethylene bottle.
Standard preparations— Separately transfer 1.0 mL and 2.0 mL of the Standard stock solution to
100-mL volumetric flasks, and transfer 1.5 mL and 2.0 mL of the
Standard stock solution to separate 50-mL volumetric flasks. Add 1.0 mL of nitric acid to each flask,
dilute the contents of each flask with water to volume, and mix.
These Standard preparations contain 1.0, 2.0, 3.0, and 4.0 μg of chromium per mL, respectively.
Assay preparation— Transfer about 200 mg of Chromium Picolinate, accurately weighed, to a 500-
mL volumetric flask, and add 25 mL of water. Slowly add 10 mL of
nitric acid, and boil for 10 minutes with constant swirling. Cool the solution, dilute with water to
volume, and mix. Filter a portion of the solution, and transfer 5 mL of
the filtrate to a 100-mL volumetric flask. Add 1 mL of nitric acid, dilute with water to volume, and
mix.
Procedure— Concomitantly determine the absorbances of the Standard preparations and the Assay
preparation at the chromium emission wavelength of 357.9 nm,
with a suitable atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering
851 ) equipped with a chromium hollow-cathode lamp and an
air–acetylene flame, using diluted nitric acid as the blank. Plot the absorbances of the Standard
preparations versus chromium concentration, in μg per mL, and draw
the straight line best fitting the four plotted points. From the graph so obtained, determine the
chromium concentration, in μg per mL, in the Assay preparation.
Calculate the quantity, in mg, of C18H12N3O6Cr in the portion of Chromium Picolinate taken by the
formula:
(418.31 / 51.996)(10C),
in which C is the concentration, in μg per mL, of chromium in the Assay preparation; 418.31 is the
molecular weight of chromium picolinate; and 51.996 is the atomic weight of chromium.

Test for Leutin USP

Solvent: a mixture of hexanes, acetone, toluene, and dehydrated alcohol (10:7:7:6).
Mobile phase— Prepare a filtered and degassed mixture of hexane and ethyl acetate (75:25). Make
adjustments if necessary (see System Suitability under
Chromatography 621 ).
Standard solution— Dissolve a suitable quantity of USP Lutein RS in Mobile phase to obtain a
solution containing about 150 μg per mL.
Test solution— Transfer about 1 mL of Test stock solution from the test for Content of total
carotenoids, and evaporate under a stream of nitrogen to dryness. Add 1 mL of Mobile phase, and
sonicate to dissolve.
Chromatographic system (see Chromatography 621 )— The liquid chromatograph is equipped with a
446-nm detector and a 4.6-mm × 25-cm column that contains
3-μm packing L3. The flow rate is about 1.5 mL per minute. Chromatograph the Standard solution,
and record the peak responses as directed for Procedure: the relative retention times are about 1.05
for zeaxanthin and 1.0 for lutein; the resolution, R, between lutein and zeaxanthin is not less than
1.0; the tailing factor is not more than 2; and the relative standard deviation for replicate injections
is not more than 2.0%.
Procedure— Inject a volume (about 10 μL) of the Test solution into the chromatograph, record the
chromatogram, and measure the peak responses. Calculate the percentage of Lutein taken by the
formula:
T(ri / rs),
in which T is the content, in percentage, of total carotenoids as determined in the test for Content of
total carotenoids; ri is the individual peak response of lutein in the
Test solution; and rs is the sum of the responses of all the peaks: not less than 75.0% of lutein is
found.
Content of total carotenoids—
Solvent: a mixture of hexanes, acetone, toluene, and dehydrated alcohol (10:7:7:6).
Test stock solution— Transfer about 15 mg of Lutein to a 100-mL volumetric flask, and dissolve in
and dilute with Solvent to volume.
Test solution— Quantitatively dilute the Test stock solution (1 in 100) with dehydrated alcohol to
obtain a solution having a final concentration of about 1.5 μg per mL.
Procedure— Determine the absorbance of the Test solution at the wavelength of maximum
absorbance at about 446 nm, with a suitable spectrophotometer, using dehydrated alcohol as a
blank. Calculate the percentage of total carotenoids as lutein (C40H56O2) by the formula:
1000A / 255W,
in which A is the absorbance of the Test solution; W is the weight, in g, of Lutein taken to prepare
the Test stock solution; and 255 is the absorptivity of the pure lutein.

Test for Benfotiamine

Instrumentation Analysis was performed on chromatographic system of HPLC LC-2010C HT Shimadzu with LC
Solution equipped with Shimadzu SPD-M20A Prominence Diode array detector. HPLC instrument was
controlled by LC Solution software. Chromatographic Conditions A Waters column C18, 5 μ (250 X 4.6 mm) was
used for chromatographic separation. The mobile phase composed of water (pH 3.2 adjusted with ortho-
phosphoric acid) and acetonitrile (75:25 v/v); at a flow rate of 0.8 mL min -1 with run time of 10 min. Mobile
phase and sample solutions were filtered through a 0.45 μm membrane filter and degassed. The detection of
both drugs was carried out at 254 nm. Preparation of Diluent 1000 ml of HPLC Grade water was adjusted to
pH 3.2 with o-Phosphoric acid. Preparation of Standard Stock Solutions Standard stock solutions of 2000 μg
mL-1 of MET and 300 μg mL -1 of BEN were prepared separately using diluent. The stock solution of MET was
diluted with diluent to give working standard solutions containing 200 - 600 μg mL -1 concentrations, similarly
the BEN stock solution was diluted with diluent to give working standard solutions in the range 30 - 90 μg mL -1.
These standard solutions were injected into HPLC column and calibration curves were plotted by taking drug
peak areas vs concentrations.

Test for Folic Acid IP

Test solution. Dissolve 100 mg of the substance under examination in 5 ml of a 2.86 per cent w/v
solution of sodium carbonate and dilute to 100.0 ml with the mobile phase. Dilute 2.0 ml of this
solution to 10.0 ml with the mobile phase.
Reference solution (a). Dissolve 100 mg of folic acid RS in 5ml of a 2.86 per cent w/v solution of
sodium carbonate and dilute 100 ml with the mobile phase. Dilute 2.0 ml of this solution to 10.0 ml
with the mobile phase.
Reference solution (b).To 20 mg of pteroic acid, add 5 ml of a 2.86 per cent w/v solution of sodium
carbonate, dilute to 100 ml with the mobile phase. Mix 1.0 ml of this solution with 1.0 ml of
reference solution (a) and dilute to 100 ml with the mobile phase.

Reference solution (c). Dilute 2.0 ml of the test solution to 20 ml with the mobile phase. Dilute 1.0 ml
of this solution to 20.0 ml with the mobile phase.
Reference solution (d). Dissolve 10 mg of N-(4aminobenzoyl)- l-gulatamic acid (folic acid impurity A)
in 1 ml of a 2.86 per cent w/v solution of sodium carbonate and dilute to 100 ml with the mobile
phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase.
Reference solution (e). To 12 mg of pteroic acid (folic acid impurity D), add 1 ml ofa 2.86 per cent w/v
solution of sodium carbonate, dilute to 100 ml with the mobile phase. Dilute 1.0 ml of this solution
to 100 ml with the mobile phase.
Chromatographic system
- a stainless steel column 25 cm x 4.0 mm, packed with octylsilane bonded to porous silica (5 /lm),
- mobile phase: a mixture of 12 volumes of methanol and 88 volumes of a solution containing 1.1 per
cent w/v solution of potassium dihydrogen phosphate and 0.6 per cent w/v solution of dipotassium
hydrogenphosphate,
- flow rate. 0.6 ml per minute,
- spectrophotometer set at 280 urn,
- injection volume. 5 Ill.
Inject reference solution (b). The test is not valid unless the resolution between the peaks due to
folic acid and folic acid impurityD is not less than 4.0. The relative retention time with reference to
folic acid for N-(4-aminobenzoyl)-L-glutamic acid(folic acid impurityA) is about 0.5; for 2,5,6-
triaminopyrimidin4(IH)-one (folic acid impurity B) is about 0.6; for isofolic acid (folic acid impurity C)
is about 0.9, for pteroic acid (folic acid impurity D) is about 1.33; for 6-pterinylfolic acid (folic acid
impurity E) is about 1.27.
Inject the test solution, reference solution (c), (d) and (e). In the chromatogram obtained with the
test solution the area of peak corresponding to folic acid impurity A is not more than the area of the
principal peak in the chromatogram obtained with reference solution (d) (0.5 per cent), the area of
the peak corresponding to folic acid impurity D is not more than the area of the principal peak in the
chromatogram obtained with reference solution (e) (0.6 per cent). The area of any other secondary
peak is not more than the area of the principal peak in the chromatogram obtained with reference
solution (c) (0.5per cent), The sum of areas of all other secondary peaks is not more than twice the
area of the principal peak in the chromatogram obtained with reference solution (c) (1.0 per
cent). Ignore any peak with an area less than 0.1 times the area of the principal peak in the
chromatogram obtained with reference solution (c) (0.05 per cent).
TEST OF PYRIDOXINE
Assay. Weigh accurately about 0.15 g, dissolve in a mixture of 5 ml of anhydrous glacial acetic acid
and 6 ml of mercuric acetate solution. Titrate with 0.1 M perchloric acid, using crystal violet solution
as indicator, until a green colour is produced. Carry out a blank titration.
1 ml of 0.1 M perchloric acid is equivalent to 0.02056 g of CsHIIN03,HCI.

TEST OF BIOTIN USP

Assay— Mix about 500 mg, accurately weighed, of Biotin with 100 mL of water, add phenolphthalein
TS, and titrate the suspension slowly with 0.1 N sodium hydroxide VS, while heating and stirring
continuously. Each mL of 0.1 N sodium hydroxide is equivalent to 24.43 mg of C10H16N2O3S.