ESTIMATION OF CREATININE
Creatinine is a nitrogenous product that is produced when metabolism of creatine in the skeletal and muscle. They are filter by the kidney and excreted in the urine.
REAGENT
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SATURATED PICRIC ACID SOLUTION
COMPOSITION
Picric acid
Distilled water
PREPARATION
Place an excess of picric acid in an amber colour bottle containing distilled water, leave for a few days then discard the suppernant in another container. The suppernant is the saturated solution, store in the bottle.
NB: picric acid is explosive so it must be store always in damp.
- 10% W/V SODIUM HYDROXIDE
COMPOSITION
Sodium hydroxide
Distilled water
PREPARATION
Dissolve sodium hydroxide in a distilled water and store
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CREATININE WORKING REAGENT
COMPOSITION
Saturated picric solution 10ml
10% Sodium hydroxide 2.0ml
PREPARATION
Mix 10ml of saturated picric acid solution with 2.0ml of 10% sodium hydroxide.
It should be prepared freshly for use.
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STOCK CREATININE STANDARD 10MMOL/L REAGENT
COMPOSITION
Pure creatinine 0.113g
(0.1N) hydrochloric acid 100mmol/l
PREPARATION
Dissolved 0.113g of pure creatinine in a quantity of (0.1N) hydrochloric acid and make up the solution to 100ml with (0.1N) hydrochloric acid, the reagent is stable for two month when store at 2-8ºC
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WORKING CREATININE STANDARD
COMPOSITION
Stock creatinine standard 1%
Distilled water 200µlmol/l
PREPARATION
Dilute 1:50 of stock creatinine standard and make up to 200µlmol/l
Stable for one month when store at 2-8ºC
METHOD
STEP 1
Set up three tubes as follow;
REAGENT | TEST TUBE | STD TUBE | BLK TUBE |
Working reagent | 2.0µl | 2.0µl | 2.0µl |
Standard reagent | 0.2µl | ||
Test sample | 0.2µl | ||
Mix the tube very well leaves it at room temperature to stand for 1 hours 30 minute
STEP 2
Set the instrument to zero with the blank.
First reading, use 490nm wavelength or use blue green filter.
Record your reading down
STEP 3
Add 50µl (0.05ml) acetic acid 60% v/v to all tubes.
Set the instrument to zero with the blank and read the absorbance using 490nm, or blue green filter
Record the reading separately.
STEP 4
Subtract second reading from the first reading to give final reading.
CALCULATIION
Second reading – first reading = final reading
Then calculate
Ab. Test
Creatinine µmol/l = — x 200
Ab. Std
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