Document Change Log Table: Neutro Pharma Quality Control Laboratory

Neutro Pharma Quality Control Laboratory
Document No: NP/QCL/6.2/FP-266 Department: QC Department Page 1 of 8

Issue Date: 20-04-2022 Effective Date: 20-04-2022 Revision No: 01
Title: Product Test Method for OSS-PRO Tablet
Description Name/designation Signature Date
Prepared by 20-03-2022
Reviewed by 10-04-2022
Approved by 20-04-2022
Document Change Log Table
Description of
Revision No Page No Approved by Date
amendment
Generic Name: Ossein Mineral Complex + Vitamin D

Composition: Each Film Coated Tablet contains
Vitamin D……………………….400 I.U
Ossein Mineral Complex………..830mg eq. to Calcium…..177.6mg
Phosphorus ……………………..82.2mg
Residual mineral Salts…………..24.9mg
Collagen…………………………224.0mg
Other proteins……………………66.4mg
Ref. Inhouse specs

Sr. # Tests Specifications
1. An orange color, oblong, film coated tablet, blistered
Description
as 10’s x 3 packed in unit carton with leaflet.
2. Identification As per procedure for assay
3. Average Weight/Tablet 1230mg ± 5%
4. Disintegration Time NMT 30 Minutes
5.
Leakage Test To meet test requirement
90.0% - 120.0%.of the stated amount
Assay (Vitamin D Contents)
360 i.u to 480 i.u / tablet
Assay (calcium Contents)
159.8mg to 213.1mg / tablet
Assay (Phosphorus Contents)
6. 73.98mg to 98.64mg / tablet
Residual Mineral Salts
22.4mg to 29.88mg / tablet
90.0% - 120.0% of the stated amount
Collagen
201.6mg to 268.8mg
90.0% - 120.0% of the stated amount
Other Protein
59.76mg to 99.68mg
1. PURPOSE:
It is established to describe the requirement for testing method of Tablet.
2. SCOPE:
It is applicable in the Quality Control/Assurance department for the description of specification &
method of analysis for Tablet.
3. REFERENCE:
Inhouse
4. DESCRIPTION:
An orange color, oblong, film coated tablet, blistered as 10’s x 3 packed in unit carton with leaflet.
5. IDENTIFICATION:
As per procedure for assay contents.
6. AVERAGE WEIGHT/TABLET:
Take 20 tablets, weigh each tablet on calibrated balance, calculate average weight per tablet
7. DISINTEGRATION TIME:
Place 0.1N HCl in beaker so as to assure the tubes baskets should be dipped when on down position
(when up and down movement), set the temperature as 37oC ±0.5oC put a Tablet/bolus in each of six
basket tubes, put over a disk in each basket, run the DT apparatus for up and down movement and note
the time when first tablet/bolus is disintegrated completely, then again note the time when last
tablet/bolus is disintegrated completely.
8. LEAKAGE TEST:
Take 10 to 20 strips and dip in the water, having methylene blue, in vacuum up to 200mmHg for
1minuts with the help of vaccum:
9. ASSAY (CALCIUM CONTENTS)

Weigh and Grind 20 tablets / granules eq. to about 60mg of calcium, add 15ml HCl 20%, add 10ml of
water. Take 10ml of this solution + 20ml of Ammonium chloride buffer, add few drops of eriochrome
black indicator and titrate with 0.10M EDTA VS. and calculate the contents of calcium. Each ml of
0.10M EDTA is equivalent to 0.004gm of Ca++.
Calculation:
%age = volume used X Factor X 100 X Strength of 0.1M EDTA = A
Wt. taken (gm)
mg/tablet = %age x 177.6 =
10. OSSIEN MINERAL COMPLEX
mg/tablet = _830_ x mg of Ca /tablet

177.6
11. RESIDUAL MINERALS

Heat a silica crucible allow cooling in desiccator and weighing. Powder of crush tablet equivalent to
the weight of one tablet of substance in silica crucible and add 2ml of sulphuric acid and heat on hot
plate or burner, till cessation of fumes and black dried mass appears, then keep in furnace at about
600oC till off white to grayish residues appear, and allow to cool in desiccator. Allow to cool and
weigh, incinerate 15 minutes and repeat this procedure to constant weigh (if black residues persist).
Weigh the residues with crucible and calculate the %age of residues.
Weight of empty silica dish + weight of sample = gm
>
Weight of empty silica dish = gm
>
Weight of empty silica dish + weight of residues = gm
Total Residues:- weight of residues X 100 =

Weight of sample taken (gm)
Residual of Calcium = 15% (against one tablet)

Residual minerals = Total Residue – 15%
12. ASSAY (VITAMIN D CONTENTS):
12.1 By UV method: IHM

Standard Preparation:
Take colecalceferol Vitamin D3 Working Std. 20 mg (eq. to 2000 I.U) in 50 ml volumetric flask
dissolve and make up the volume with ethanol (0.4mg/ml)
Sample Preparation:
Weigh and Grind 20 tablets / grains finely. Take powder eq. to colecalceferol (Vit. D3) 20mg (eq. to
2000 I.U) in 50 ml volumetric flask dissolve and make up the volume with ethanol, sonicate for 15
minutes, filter. (0.4mg/ml)
Measure the absorbance of solutions @ 265 nm on U.V Spectrophotometer.
Calculation:
Absorbance of Sample X Concentration of Standard X 100 =
Absorbance of Standard Concentration of Sample
12.2 By HPLC Method:

Standard preparation:
Transfer 25 mg (eq. to 2500 I.U) of Cholecalciferol working standard, accurately weighed, to a 100 ml
volumetric flask, dissolve in ethanol, mix to dissolve and make up the volume with the same solvent
filter and use.
Sample preparation:
Transfer sample equivalent to 25mg (eq. to 2500 I.U) in 100 ml volumetric flask, dissolve in ethanol,
mix to dissolve and make up the volume with the same solvent filter and use.
Mobile phase:
Acetonitrile : Methanol : water 25:25:1 mix well filter and degas.
Chromatographic system:
Column: 4.6 mm x 25 cm, 5 micron Packing L1
Detector: 254 nm
Injection volume: 20 µl
Procedure:
Set the chromatographic system as mentioned above. Inject equal volumes of sample and standard
preparations and record the response for major peaks of each ingredient and Calculate the contents of
Colecalciferol using following formula.
Calculation:
Avg. peak area of sample. X Conc. of Std. X 100 = % age of the label claim
Avg. peak area of std. X Conc. of Sample.
Determined quantity in I.U / tablet
= %age of label claim X purity of standard = I.U

100
13 ASSAY (PHOSPHORUS CONTENTS):

By Atomic absorption: IHM
Sample preparation:
Transfer sample, equivalent to about 100 mg of Phosphorus, to a 100 ml volumetric flask, add 2%
Nitric acid solution to dissolve and sonicate for about 10 minutes. Then make up volume up to mark
with same solvent (1.0 mg per ml).
Standard preparation:
Transfer an accurately weighed quantity of Potassium dihydrogen phosphate (KH2PO4) equivalent to
about 100 mg of P, to a 100 ml volumetric flask, add 2% Nitric acid solution and sonicate for about
10 minutes. Then make up volume up to mark with same solvent (1.0 mg per ml). Measure the
absorbance at 213.45nm using the respective lamp for P.
Calculation:
% age of the stated claim
= Absorbance of Sample X Conc. of Std. x 100 =
Absorbance of Standard X Conc. of Sample
Determined quantity (of Phosphorus)
X 82.2 = mg / tablet
100
or
DETERMINATION OF PHOSPHORUS:
Sulfuric acid solution:
Cautiously add sulfuric acid to water (37.5: 100), and mix.
Ammonium Molybdate solution:
50 mg/mL of Ammonium Molybdate in Sulfuric acid solution and water (2:3). [NOTE—Dissolve in
water first, and then dilute with Sulfuric acid solution to volume.]
Hydroquinone solution:
5 mg/mL of hydroquinone in water. Add one drop of sulfuric acid per 100 mL of solution.
Sodium bisulfite solution:
200 mg/mL of sodium bisulfite in water
Phosphorus standard stock solution:
Weigh 4.395 g of monobasic potassium phosphate, previously dried at 105° for 2 h and stored in a
desiccator, and transfer to a 1000-mL volumetric flask. Dissolve in water, add 6 mL of sulfuric acid
as a preservative, dilute with water to volume, and mix to obtain a solution with a concentration of
1000 µg/mL of phosphorus.
Standard solution:
20 µg/mL of phosphorus from the Phosphorus standard stock solution diluted with water
Sample solution:
Finely powder and weigh a counted number of Tablets.] Transfer a portion of the powder, equivalent
to 100 mg of phosphorus, to 25 mL of nitric acid, and digest on a hot plate for 30 min. Add 15 mL of
hydrochloric acid, and continue the digestion to the cessation of brown fumes. Cool, and transfer the
contents of the flask to a 500-mL volumetric flask with the aid of small portions of water.
Dilute with water to volume. Transfer 10.0 mL of this solution to a 100-mL volumetric flask, and
dilute with water to volume.
Instrumental conditions
Mode: Vis
Analytical wavelength: 650 nm
Cell: 1 cm
Analysis
Samples:
Standard solution and Sample solution
To 3 separate 25-mL volumetric flasks transfer 5.0 mL each of the Standard solution, the Sample
solution, and water to provide the blank. To each of the 3 flasks add 1.0 mL each of Ammonium
molybdate solution, Hydroquinone solution, and Sodium bisulfite solution, and swirl to mix. Dilute
the contents of each flask with water to volume, and allow the flasks to stand for 30 min. Determine
the absorbances of the solutions against the blank.
Calculate the percentage of the labeled amount of phosphorus (P) in the portion of Tablets taken:
Result = (AU/AS) × (CS/CU) × 100.
AU = absorbance of the Sample solution

:
AS: = absorbance of the Standard solution
CS: = concentration of phosphorus in the Standard solution (µg/mL)
CU: = nominal concentration of phosphorus in the Sample solution (µg/mL)
Acceptance criteria:
90.0%–125.0% of the labeled amount of phosphorus (P)
14 COLLAGEN:
Determination of Hydroxyapatite content
Calculate for the determination of Hydroxy-apatite is based on the values of calcium and phosphorus.
Proportion of P = 165.85 = 18.496%
Proportion of Ca = 400.80 = 339.890%
Calculation:
From the calcium:
% Hydroxy-apatite (A) = a x 1004.66 / 400.8
A = %value found in the calcium determination
From the phosphorus
% Hydroxy-apatite (B) = b x 1004.66 / 185.85
B = % value found in the phosphorus determination
X=A+B/2
Determination of Total Collagen
Hydroxyproline Standard
Solution:
Dissolve 100mg Hydroxyproline in 1000 ml distilled water (stock solution) and make further dilutions
for standard curve.
1ml of stock solution + water to 100ml
Method:
Take about 100mg of MCHC, accurately weighed in a test tube, add 10ml 6N HCl and hydrolyzed in a
drying oven at 105oC for 20 hours.
After cooling transfer the test solution to a 100ml volumetric flask, add 2 drops of phenol Phthalene
and neutralize with 32% NaOH, Decolorize the pink shade with dilute HCl, and make up the volume
with water on cooling. Filter the solution if required and dilute 10ml to 100ml with water.
Pippete 1ml water (blank), 1ml of standard solution hydrolysate and 1ml if test solution hydrolysate in
a test tube. Mix and shake with 1ml chloramine T. After 20 minute add 1ml Perchloric acid 3M, shake
and allow to stand for 5 minute. A possible yellow color disappears at this moment. Shake with 1ml of
5% solution of p-dimethyl amino benzaldehyde. Heat in a bath at 60oC for 16 minutes and measure the
absorbance of test and standard solution at 555nm in a spectrophotometer with blank as reference
solution.
Calculation:
Read the contents of Hydroxyapatite in mcg from the standard curve and multiply the hydroxyapatite
value with 7.46 to obtain the percentage collage of sample.
15 AMINO ACIDS
Amino Acids Analysis
The APAF quantitative amino acid analysis procedure is used for the analysis of amino acids in
complex samples such as foodstuffs.
It involves as the first step a liquid acid (6M HCl) hydrolysis which converts protein-bound amino
acids into individual amino acids. In the next step the amino acids in the hydrolysate undergo
precolumn derivatisation with 6 Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) (ref 1, 2)
the amino acids derivatives are then separated and quantified by reversed phase (C18) HPLC.
The procedure employed is based on the waters AccQTag amino acid analysis methodology (ref 3) but
has been adapted to run on an high performance liquid chromatography (HPLC) system . it does not
analyse for tryptophan or cysteine which are acid sensitive and require special conditions for analysis.
Sample Preparation:
The samples (about 100mg) were weighed out in duplicate into hydrolysis vials and 5ml of 20% HCl
was added. These were then incubated at 110 oC for 24 hrs. after hydrolysis, the samples were
derivatised using AccQTag reagent then analyse using a high resolution RP-HPLC column on a HPLC
system with 10min run times.
Equipment – the instruments consisted of an HPLC system with UV detector for all analyses a column
(C18 4.6 x 100mm: 4.5µm) was used. Column temperature employed was 55oC, detection was at
260nm and the flow rate 1.0ml/min.
Standard Preparation:
The standard (about 100mg) were weighed out in duplicate into hydrolysis vials and 5ml of 20% HCl
was added. These were then incubated at 110 oC for 24 hrs. after hydrolysis, the samples were
derivatised using AccQTag reagent then analyse using a high resolution RP-HPLC column on a HPLC
system with 10min run times.
Equipment – the instruments consisted of an HPLC system with UV detector for all analyses a column
(C18 4.6 x 100mm: 4.5µm) was used. Column temperature employed was 55oC, detection was at
260nm and the flow rate 1.0ml/min.
Calculation:
%age = Peak area of sample X Conc. of standard X 100

Peak area of standard Conc. of sample

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Neutro Pharma Quality Control Laboratory

Document No: NP/QCL/6.2/FP-266 Department: QC Department Page 1 of 8

Description Name/designation Signature Date

Document Change Log Table

Generic Name: Ossein Mineral Complex + Vitamin D

Ref. Inhouse specs

9. ASSAY (CALCIUM CONTENTS)

10. OSSIEN MINERAL COMPLEX

mg/tablet = _830_ x mg of Ca /tablet

11. RESIDUAL MINERALS

Total Residues:- weight of residues X 100 =

Residual of Calcium = 15% (against one tablet)

12. ASSAY (VITAMIN D CONTENTS):

12.1 By UV method: IHM

12.2 By HPLC Method:

Determined quantity in I.U / tablet

= %age of label claim X purity of standard = I.U

13 ASSAY (PHOSPHORUS CONTENTS):

Determined quantity (of Phosphorus)

AU = absorbance of the Sample solution

%age = Peak area of sample X Conc. of standard X 100

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