Standard Testing Procedure for Borage seed oil tablet

Name of Compound: Thiamine(B1), Riboflavin(B2) Nicotinaminde(B3) and Pyrodoxine(B6)

        Assay by HPLC:


Standard solution: Transfer accurately weighed about 25.0 mg of Thiamine(B1)/ Riboflavin(B2)/ Nicotinaminde(B3)/ Pyrodoxine(B6) working standard in 25 ml volumetric flask separately. Add 10 ml of diluent, sonicate to dissolve, make up to volume with diluent. Make further dilution equivalent to sample in diluent

Note: Second dilution of standard is made separately or in combination that depends on the formulation.

Test solution: Weight the powder equivalent to average weight and transfer it in 50 ml of volumetric flask add diluent to dissolve and sonicate for 10 minutes. Filter and dilute further by taking 1 ml of sample in 25 ml of diluent.

Chromatographic condition:

Buffer: 1.4 gm Hexasulphonic Acid in 1000ml of water

Diluent                             :  Mobile phase

Mobile phase                   :  Buffer:MeOH:Glacial acetic acid(730:270:1ml)

Column                            :  C18 150 X 4.6 mm, 5 μm

Flow rate                          :  1.5 ml/ min

Wavelength                     :  280 nm

Injection volume              :  20 μl

Column temperature       :  Ambient

Run time                          :  20 minutes

Retention Time                : Ist peak of Nicotinaminde (B3)  IInd peak of Pyrodoxine (B6)  IIIrd peak of Riboflavin (B2) and IVth peak of Thiamine (B1)

Calculation: As Per Excel Sheet

Development of Gas Chromatography/ Mass Spectrometry for the Determination of Essential Fatty Acids in Food Supplemental Oil Products Borage seed Oil and Evening Primerose Oil

The GC/MS system used in this study was an Agilent GC/MSD (model No. 5973, Palo Alto, CA, USA). In the initial stage of this study, a non-polar DB-5MS colum (J & W Scientific, length 60 m, i.d. 0.25 mm, thickness 0.25 μm), a polar DB-1701 column (J & W Scientific, length 60 m, i.d. 0.25 mm, thickness 0.5 μm), and a HP-88 FAMEs column (Agilent, length 60 m, i.d. 0.25 mm, thickness 0.2 μm) were tested for their chromatographic performance for FAMEs analysis. The HP-88 column was chosen and used throughout this study [see results and discussion section]. Helium was used as carrier gas at a flow rate of 1.0 mL/min. Its injection port was kept at 250 ℃ in splitless mode. The temperature of the GC oven started at 180 ℃ and maintained for 25 minutes and was ramped to 230 ℃ at the rate of 20 ℃/min and held for 10 minutes. The interface to the mass spectrometer was maintained at 250℃.

The mass spectrometer was operated under electron impact ionization condition with a source temperature of 250℃. For roughly screening samples, scan mode was used. For quantitative analysis, the MS was operated in selected ion monitoring (SIM) mode with detecting molecular ions of methylated ester forms of linoleic acid at m/z 294, α- and γ-linolenic acids at m/z 292, stearic acid-d35 at m/z 333, 13C18-linoleic acid at m/z 312, and 13C18-α-linolenic acid at m/z 310.

among the three target analytes, we decided to determine only the dominant components in each oil samples. summarizes the measurement results of the three target analytes in Total ion chromatograms of a standard solution containing the three target analytes with three different GC columns. (a) and (b) were obtained with GC conditions optimized to obtain best separation among the three analytes. Optimized GC conditions for (c) is described in experimental section.

 Compensate y-Linolenic acid and Palmitic acid with placebo standard.

 Vitamic C Coated Ascorbic Acid


  1. 10% v/v sulphuric acid in water.
  2. 10% w/v solution of ammonium molybdate in water.

Standard: 100 mcg/ml of ascorbic acid in water.

Sample: Dilute or extract the sample with water to get 100 mcg/ml


To 2 ml of sample and standard solutions, add 2ml of sulphuric acid and 4 ml of ammonium molybdate solution. Keep at room temperature for 60 min and dilute with water to 25 ml. measure the absorbance of both at maximum at about 650 nm against reagent blank. Since colour starts fading after 1 hour, reading must be taken within this period.


Alpha Tocopheryl Acetate

Standard solution:     Take 100mg of Alpha Tocophenyl Acetate WS 250ml round bottom flask.  Procedures as per Vitamin A till aqueous layer is free from alkali collect the solvent ether in a dry 100ml Volumetric flask by passing through a layer of anhydrous sodium Sulphate. Dilute 5ml to 100ml in dry volumetric flask with the solvent ether.


Test Solution:               Take Sample eq to 10mg of Alpha Tocophenyl Acetate in 250ml round bottom flask, and continue as per standard procedure and dilute to 200ml with solvent ether.


Absorption Reagent:                100gm of kaolin in boiled with 500ml of 2(N) Hcl for 15minutes with stirring the solution in filtered through No.1 paper using suction to remove acid from the powder it is washed with distilled water and dried it at 90°c temperature.


Procedure:                      Pipette 5ml of both std/test solution in dry test tube dry to a low volume on water both then suction at room temperature completely add about 15ml of 40°-60° petroleum ether add about 0.5gm of absorption reagent swirl for 5 minutes filter through what man No. 1 Paper discarding initial 5ml of the filtrate


Colour development (In dry test tubes)


Std                                         test                                        blank

Dilution (std/test/blank)                           10ml                                     10ml                                     10ml


Evaporate almost to dryness on

Water bath then completely at

Room temperature under suction

Add 10ml of alcohol shake


0.5% w/v 2-2 Bipyridyl (in alcohol)              1ml                                        1ml                                        1ml

0.6% w/v ferric ammonium Sulphate

In (alcohol)                                                               1ml                                        1ml                                        1ml


After 10minutes take the reading at 520nm against blank

 Estimation Betacarotene

 Reagent required:


Ethyl acetate


Trisodium citrate dihydrate

Ascorbic acid

Dehydrated alcohol

Mobile Phase: Prepare a filtered and degassed mixer of n-hexane, ethyl acetate and acetone in the ratio of 65 : 300 : 50.

Reagents: Dissolve 20 gm of trisodium citrate dihydrate and 2 gm of

ascorbic acid in 200 ml of water.

Sample Preparation: Transfer sample equivalent to 15 mg of betacarotene, accurately weighed into dry 100 ml amber coloured volumetric flask. Add 20 ml of reagent and sonicate for 1 hour. Add 25 ml of dehydrated alcohol and dilute to 100 ml with n-hexane. Further, dilute 5 ml of this solution to 100 ml with n-hexane.

Instrumental Conditions:

Column           : Inertsil Silica, 25 cm x 4.6 mm 5µ (G L Sciences, Japan)

Flow rate         : 1.5 ml/min.

Detector          : UV at 447 nm

Attenuation     : Set appropriately

Run time         : About 15 mins.

Injection volume: 10 µl

Procedure: Inject sample preparation into HPLC system described above. The retention time of beta carotene peak is about 4 min. Calculate content of related substance by area normalization method.


Folic Acid


Standard and Test Solution        : 20mcg/ml in 3% w/v K2Hpo4

Colour development (in stopped test tube)

Std                         std blank                             test                        test blank

Standard solution:          3ml                        3ml                                        x                              x

Test Solution:                   x                              x                                          3ml                        3ml

Potassium permanganate

(0.4% w/v in water) 2ml                              x                                              2ml                        x

Water                                  x                            2ml                                            x                           2ml

Sodium nitrate (2%

w/v in water)               1ml                        1ml                                           1ml                        1ml

6 (N) HCl                        1ml                        1ml                                        1ml                        1ml

Ammonium sulphamate

(5% w/v in water       1ml                        1ml                                        1ml                        1ml

EDTA (5% in water) 1ml                        1ml                                        1ml                        1ml

N-1 napthyl, ethylene             

Diamine, dihydrogen1ml                       1ml                                        1ml                        1ml

(0.1% w/v in water)

 Shake well and keep for 10minutes add 1gm of Nacl and extract with 10ml Iso butyl alcohol allow to separate pipette out the aqueous layer and centrifuge and add 1gm of anhydrous sodium Sulphate to the tube determine absorbance at 550nm

Determination of Vitamin K1 Phylloquinone by HPLC

 Abstract: Phylloquinone (vitamin K1) is considered to be a major determinant of vitamin K status. For this reason, measurements of tablet vitamin K concentrations provide a useful tool of vitamin K status in man. There is a growing interest in the role, biochemical function, and metabolism of vitamin K in vivo. A modified reversed phase-HPLC method with fluorescence detection after post-column zinc reduction in tablet  samples was validated for vitamin K analysis.

High sensitivity, analytical recoveries, accuracy and calibration curves linearities could be reached. Our results showed an overall coefficient of variation of less than 10% for the intra- and interday reproducibility, while the recovery ranged from 91% to 114 %. The limit of detection and quantification were 0.015 µg mL-1 and 0.15 µg mL-1, respectively. Long-term stability was verified over a period of six months. The accuracy was proven by good results from external quality assurance.

The observed pharmacokinetic differences indicate significant intra- and inter-individual variance of vitamin K fate in the tablet . This highly accurate, robust and reliable method is appropriate for the evaluation of vitamin K status in tablet . The ability to determine vitamin K has the potential to improve pharmacokinetic studies.

The isocratic HPLC system (Agilent , Germany) consisted of separation Hitachi modules with in-line vacuum degasser, L-2130 Pump, L-2200 Autosampler, L-2300 Column Oven and L-2485 Fluorescence Detector, all controlled by Elite LaChrom software

A Nucleodur C18 Gravity column (3 mm i.d. x 100 mm length, 3 µm particle size) was used for separation.

During analysis the column temperature was maintained at 22°C, the autosampler temperature was kept at 15°C. A 10 cm long,


postcolumn peek tubing was dry-packed with zinc powder (<150 micron, Sigma Aldrich, Hamburg, Germany) and connected between the analytical column and the fluorescence detector. A vortexer and a refrigerated centrifuge were used for liquid liquid extraction. The lipid compounds were separated using a solid-phase extraction vacuum manifold.

Test Sample & Standards – vitamin K1,  were dissolved in 100% HPLC-grade 2-propanol to gain a concentration of 10 mg/mL

The mobile phase contained, per litre, 880 mL methanol, 100 mL acetonitrile, 1.1 g zinc acetate, 10 mL acetic acid and 10 mL water. The flow rate was 0.8 mL/min and the detection wavelengths were 246 nm excitation, 430 nm emission.

Biotin Estimation Using Titan Column

Vitamins are important compounds and are essential for normal metabolism. They are naturally found in many foods and are often added to processed food products. Water soluble vitamins include such compounds as thiamine (B1), riboflavin (B2), niacin (B3), pyridoxine (B6), pantothenic acid (B5), biotin (B7), folic acid (B9), and cyanocobalamin (B12). Qualitative and quantitative analysis of vitamins is a routine but challenging task since vitamins are relatively unstable and affected by a number of factors such as heat, light, air, and other food components. In this report, a Titan™ C18 UHPLC column was used to analyze B vitamins in both standard mixture and in vitamin water.

An Agilent 1290 UHPLC system was used for separations. The UV detection wavelength was set to 220 nm. The column used in this study was a Titan C18, 5 cm x 2.1 mm I.D., packed with monodisperse 1.9 µm particles. The column temperature was controlled at 35° C. The B vitamins are very hydrophilic, consequently a gradient with a low concentration of methanol and acidic mobile phase was used for elution under the reversed-phase conditions.

Water-soluble vitamins were obtained from Sigma-Aldrich. Most B vitamins were dissolved in water.


Special Reagents and Solutions:

Ether— Use ethyl ether. Use within 24 hours after opening container.

Dehydrated Hexane— Prepare a chromatographic column by packing a chromatographic tube, 60 cm × 8 cm in diameter, with 500 g of 50- to 250-µm chromatographic siliceous earth, activated by drying at 150 for 4 hours (see Column Adsorption Chromatography under Chromatography 621). Pass 500 mL of hexanes through the column, and collect the eluate in a glass-stoppered flask.

Butylated Hydroxytoluene Solution— Dissolve a quantity of butylated hydroxytoluene in chromatographic hexane to obtain a solution containing 10 mg per mL.

Aqueous Potassium Hydroxide Solution— Dissolve 500 g of potassium hydroxide in 500 mL of freshly boiled water, mix, and cool. Prepare this solution fresh daily.

Alcoholic Potassium Hydroxide Solution— Dissolve 3 g of potassium hydroxide in 50 mL of freshly boiled water, add 10 mL of alcohol, dilute with freshly boiled water to 100 mL, and mix. Prepare this solution fresh daily.

Sodium Ascorbate Solution— Dissolve 3.5 g of ascorbic acid in 20 mL of 1 N sodium hydroxide. Prepare this solution fresh daily.

Sodium Sulfide Solution— Dissolve 12 g of sodium sulfide in 20 mL of water, dilute with glycerin to 100 mL, and mix.

Mobile Phase A— Prepare a mixture of acetonitrile, methanol, and water (25:25:1). The amount of water and the flow rate may be varied to meet system suitability requirements.

Mobile Phase B— Prepare a 3 in 1000 mixture of n-amyl alcohol in Dehydrated Hexane. The ratio of components and the flow rate may be varied to meet system suitability requirements.

Internal Standard Solution— Transfer 15 mg of USP D4,6-Cholestadienol RS, accurately weighed, to a 200-mL volumetric flask, add a 1 in 10 mixture of toluene and Mobile Phase B to volume, and mix.

Standard Preparation— Transfer about 25 mg of USP Ergocalciferol RS or Cholecalciferol RS, accurately weighed, to a 50-mL volumetric flask, dissolve without heat in toluene, add toluene to volume, and mix. Pipet 10 mL of this stock solution into a 100-mL volumetric flask, dilute with toluene to volume, and mix. Prepare stock solution fresh daily.

Assay Preparation

For oily solutions— Accurately weigh a portion of the specimen to be assayed, preferably more than 0.5 g and equivalent to about 125 µg of cholecalciferol or ergocalciferol (5000 USP Units). Add 1 mL of Sodium Ascorbate Solution, 25 mL of alcohol, and 2 mL of Aqueous Potassium Hydroxide Solution, and mix.

For capsules or tablets— Reflux not less than 10 capsules or tablets with a mixture of 10 mL of Sodium Ascorbate Solution and 2 drops of Sodium Sulfide Solution on a steam bath for 10 minutes, crush any remaining solids with a blunt glass rod, and continue heating for 5 minutes. Cool, add 25 mL of alcohol and 3 mL of Aqueous Potassium Hydroxide Solution, and mix.

For dry preparations and aqueous dispersions— Accurately weigh a portion of the specimen to be assayed, preferably more than 0.5 g and equivalent to about 125 µg of cholecalciferol or ergocalciferol (5000 USP Units). Add, in small quantities and with gentle swirling, 25 mL of alcohol, 5 mL of Sodium Ascorbate Solution, and 3 mL of Aqueous Potassium Hydroxide Solution.

SAPONIFICATION AND EXTRACTION— Reflux the mixture prepared from the specimen to be assayed on a steam bath for 30 minutes. Cool rapidly under running water, and transfer the saponified mixture to a conical separator, rinsing the saponification flask with two 15-mL portions of water, 10 mL of alcohol, and two 50-mL portions of ether. Shake the combined saponified mixture and rinsings

vigorously for 30 seconds, and allow to stand until both layers are clear. Transfer the aqueous phase to a second conical separator, add a mixture of 10 mL of alcohol and 50 mL of solvent hexane, and shake vigorously. Allow to separate, transfer the aqueous phase to a third conical separator, and transfer the hexane phase to the first separator, rinsing the second separator with two 10-mL portions of solvent hexane, adding the rinsings to the first separator. Shake the aqueous phase in the third separator with 50 mL of solvent hexane, and add the hexane phase to the first separator. Wash the combined ether-hexane extracts by shaking vigorously with three 50-mL portions of Alcoholic Potassium Hydroxide Solution, and wash with 50-mL portions of water vigorously until the last washing is neutral to phenolphthalein. Drain any remaining drops of water from the combined ether-hexane extracts, add 2 sheets of 9-cm filter paper, in strips, to the separator, and shake. Transfer the washed ether-hexane extracts to a round-bottom flask, rinsing the separator and paper with solvent hexane. Combine the hexane rinsings with the ether-hexane extracts, add 5.0 mL of Internal Standard Solution and 100 µL of Butylated Hydroxytoluene Solution, and mix. Evaporate to dryness in vacuum by swirling in a water bath maintained at a temperature not higher than 40. Cool under running water, and introduce nitrogen sufficient to restore atmospheric pressure. Without delay, dissolve the residue in 5.0 mL of a mixture of equal volumes of acetonitrile and methanol, or in a

measured portion of the acetonitrile-methanol mixture until the concentration of vitamin D is about 25 µg per mL, to obtain the Assay Preparation.

Chromatographic System— Use a chromatograph, operated at room temperature, fitted with an UV detector that monitors absorption at 254 nm, a 30-cm × 4.6-mm stainless steel cleanup column packed with column packing L7 and using Mobile Phase A, and a 25-cm × 4.6-mm stainless steel analytical column packed with column packing L3 and using Mobile Phase B.

Cleanup Column System Suitability Test— Pipet 5 mL of the Standard Preparation into a round-bottom flask fitted with a reflux condenser, and add 2 or 3 crystals of butylated hydroxytoluene. Displace the air with nitrogen, and heat in a water bath maintained at a temperature of 90 in subdued light under an atmosphere of nitrogen for 45 minutes, to obtain a solution containing vitamin D and pre-vitamin D. Cool, add 10.0 mL of Internal Standard Solution, mix, and

evaporate in vacuum to dryness by swirling in a water bath maintained at a temperature not higher than 40. Cool under running water, and introduce nitrogen sufficient to restore atmospheric pressure. Without delay, dissolve the residue in 10.0 mL of a mixture of equal volumes of acetonitrile and methanol, and mix. Inject 500 µL of this solution into the cleanup column, and record the chromatogram as directed under Procedure. The chromatogram exhibits a peak exhibiting a retention time between 5 and 9 minutes, corresponding to the separation under a single peak of the mixture of vitamin D, pre-vitamin D, and D4,6-cholestadienol from other substances. Adjust the water content or other operating parameters, if necessary (see Mobile Phase A).

Cyanocobalamin (B12) Microbiological Assay

 Media Used

B12 assay agar Himedia (M110)


E.coli (Mutant)

 Diluted used

Distilled water

Standard’s concentration

  • 025mcg/ml
  • 035mcg/ml
  • 050mcg/ml
  • 075mcg/ml
  • 100mcg/ml


Test concentration


One level assay with standard curve Plot a graph training zone mean diameter in X-axis and Log of concentration on y axis From the graph find log concentration of the test zone diameter.

Take antilog of concentration of test zone diameter and do the calculation Antilog × test dilution × claim × potency

 ESTIMATION OF Metal Like Mangenesium , Iron , Zinc , Manganese & Copper

By Atomic Absorption Spectrophotomter

To 2 ml of the solution obtained during the estimation of copper sulphate add 2.5 ml of dilute sulphuric acid, evaporate to dryness. To the residue, add 3 ml of phosphoric acid, 6 ml of water and 0.5 g of sodium periodate and dissolve by aid of heat. Heat in a water bath for 30 min , cool , dilute to 10 ml with water. Measure the absorption of the resulting solution at the maximum at about 526 nm. The result can be calculated from a standard curve prepared by diluting 1, 2, 3, 4, 5 ml of 0.005% w/v solution of potassium permanganate to 10 ml with water. Each mg of potassium permanganate is equivalent to 1.4114 mg of Manganese sulphate. And other suitable Reference material