M.Pharm Quality Assurance-2-T (2009-2010) Title:- :- Estimation of serum paracetamol by Glenn & Kendall Method Exp.No.:Roll No:Date:Page No.

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AIM: To estimate serum paracetamol by Glenn and Kendall method. REFERENCE: Principles of Precision Colorimetry by G.N.Crawford. REQUIREMENTS:
A) Chemicals: (1) Trichloroacetic acid 10% (2) Hydrochloric acid 22% (3) Sodium Nitrite solution 10% (4) Sulphamic acid solution 15% (5) Sodium hydroxide solution 10% (6) Serum (7) Paracetamol (8) Distilled water B) Apparatus: (1) Test Tubes (2) Vortex mixer (3) 1 ml pipettes (4) 5 ml pipettes (5) 10ml, 50 ml and 10 ml volumetric flasks (6) Centrifuge (7) Centrifuge tubes (8) UV spectrophotometer

THEORY:
Various methods have been used for estimation of drugs in serum. For paracetamol, methods with high accuracy such as HPLC, mass spectrometry, UV spectrophotometry, etc have been used to estimate serum levels. But for routine laboratory practices, a highly validated colorimetric method of serum estimation has been designed. This is based on Glenn & Kendall method. Glenn and Kendall method is mostly used for extraction of DNA from biological samples. But the method can also be suitably modified for serum drug estimation. Colorimetric reactions are a type of visible spectroscopic methods where coloured analytes are produced by reaction with a suitable reagent and the intensities of coloured products are measured. In the reaction mixture, paracetamol reacts with sodium nitrite to yield 2- nitro 4 acetamidophenol. The resulting nitrophenol reacts with sodium hydroxide to form the yellow phenolate anionic chromophore which shows maximum absorbance at 430 nm. The above reaction is diazotization.

________________________________________________________________________ School of Pharmacy and Technology Management.

M.Pharm Quality Assurance-2-T (2009-2010) Title:- :- Estimation of serum paracetamol by Glenn & Kendall Method Exp.No.:Roll No:Date:Page No.:-

REAGENT:
1. Preparation of Trichloroacetic Acid Solution: Dissolve 10gm of Trichloroacetic acid in 100ml of water. 2. Preparation of Hydrochloric Acid: Mix 22ml of Concentrated Hydrochloric acid in 100 ml of water. 3. Preparation of Sodium Nitrite Solution: Dissolve 10gm of sodium nitrite in 100 ml of water. 4. Preparation of Sulphamic Acid Solution: Dissolve 15gm of sulphamic acid in 100 ml of water. 5. Preparation of Sodium Hydroxide Solution: Dissolve 10gm of sodium hydroxide in 100 ml of water. 6. Preparation of Standard Solution: Weigh accurately 30 mg of paracetamol in 100 mL volumetric flask. Add 70 ml of water and sonicate to dissolve. Dilute to volume with diluent and mix. 7. Preparation of Sample Solution: Weigh accurately 30 mg of paracetamol in 50 mL volumetric flask. Add 30 ml of water and sonicate to dissolve. Dilute to volume with water and mix. In a 10 ml flask add 3 ml of serum and 3 ml of above paracetamol solution and mix (on a vortex mixer). This solution is used as sample serum containing paracetamol. 8. Preparation of Serum (Blank) Solution: In a 10 ml flask add 3 ml of serum and 3 ml of water and mix.

PROCEDURE:
1) Take 1 ml of blank (water), 1 ml of serum solution, 1 ml of standard solution, and 1 ml of sample solution in separate 10 ml volumetric flask. 2) To each flask add 2 ml of trichloro-acetic acid solution and mix thoroughly. 3) Centrifuge sample solution and standard solution at 1500 rpm for 5 minutes. 4) Transfer 1 mL of each of supernatant fluid into a test tube and add 0.5 ml hydrochloric acid solution followed by 1 mL of sodium nitrite solution. 5) Mix and allow stand for 2 minutes. 6) Then carefully add 1 ml of sulphamic acid solution to each tube. 7) The solution will froth. After frothing stops mix and add 1 ml of sodium hydroxide. 8) Read the absorbance at 430 nm against the reagent blank.

________________________________________________________________________ School of Pharmacy and Technology Management.

M.Pharm Quality Assurance-2-T (2009-2010) Title:- :- Estimation of serum paracetamol by Glenn & Kendall Method Exp.No.:Roll No:Date:Page No.:-

OBSERVATION:
Sr. No. 1 2 Sample Standard Solution Sample Solution Concentration 300 ppm 300 ppm Absorbance 0.4871 0.2037

CALCULATION: % content in sample = absorbance of sample solution x 100 absorbance of std solution = 0.2037 x 100 0.4871 = 41.81 %

________________________________________________________________________ School of Pharmacy and Technology Management.

M.Pharm Quality Assurance-2-T (2009-2010) Title:- :- Estimation of serum paracetamol by Glenn & Kendall Method Exp.No.:Roll No:Date:Page No.:-

RESULT: The percentage content of paracetamol found in the serum sample is 41.81%.

CONCLUSION: The drug content in serum sample was successfully estimated by Glenn and Kendall method.

DATE:

GRADE:

SIGN:

________________________________________________________________________ School of Pharmacy and Technology Management.

M.Pharm Quality Assurance-2-T (2009-2010) Title:- :- Estimation of serum paracetamol by Glenn & Kendall Method Exp.No.:Roll No:Date:Page No.:-

________________________________________________________________________ School of Pharmacy and Technology Management.