3.5.2.1 Principle

Creatinine responds with picric corrosive in antacid medium to frame an orange hued complex. The rate of arrangement of this complex is estimated by perusing the adjustment in absorbance at 520 nm in a chose interim of time and is relative to the centralization of creatinine. The response time and the grouping of picric corrosive and sodium hydroxide have been streamlined to maintain a strategic distance from obstruction from keto acids [16].

$$\text{creatinine} + \text{picric acid} \rightarrow \text{orange coloured complex} \tag{11}$$

3.5.2.2 Reagents

Reagent 1: Picric acid. Reagent 2: Sodium hydroxide 0.75 N Reagent 3: Stock Creatinine Standard, 150 mg

## 3.5.2.3 Working solution preparation

Dilute 0.1 mL of Reagent 3 to 10 mL with purified water and mix well.

## 3.5.2.4 Procedure

Step A. Deproteinization of test sample


Mix well, keep in a boiling water bath exactly for 1 min. Cool immediately under running tap water and centrifuge or filter. Step B. Colour development.


Mix well, allow to stand at room temperature exactly for 20 min and measure immediately the optical density of blank (B), standard (S) and test (T) against purified water at 520 nm.

Calculation:

Addition sequence Blank Standard Test Urea standard — 10 μL — Specimen — — 10 μL

2-Urea reagent 1000 μL 1000 μL 1000 μL

1.Mix well and incubate for 5 min at 37°C or 15 min at room temperature.

2.Measure the absorbance of standard (Abs. S) & test sample (Abs. T) against

3.Colour is stable for 45 min when protected from light, so absorbance should be

Urea concentration in mg=dL of test specimen ¼ Abs T þ Abs S � 50 (9) Blood urea nitrogen BUN ð Þ in mg=dL ¼ urea in mg ð Þ� =dL 0:467 (10)

Creatinine responds with picric corrosive in antacid medium to frame an orange hued complex. The rate of arrangement of this complex is estimated by perusing the adjustment in absorbance at 520 nm in a chose interim of time and is relative to the centralization of creatinine. The response time and the grouping of picric corrosive and sodium hydroxide have been streamlined to maintain a strategic distance from

creatinine þ picric acid ! orange coloured complex (11)

Mix well and incubate for 5 min at 37°C or 15 min at room temperature

3.5.2 Estimation of creatinine (Span Diagnostic Ltd., Surat, India)

the blank at 578 nm.

obstruction from keto acids [16].

Reagent 1: Picric acid.

3.5.2.3 Working solution preparation

Reagent 2: Sodium hydroxide 0.75 N

Step A. Deproteinization of test sample

Reagent 3: Stock Creatinine Standard, 150 mg

Dilute 0.1 mL of Reagent 3 to 10 mL with purified water and mix well.

Serum/plasma 0.5 mL Purified water 0.5 mL Reagent 1: Picric acid 3.0 mL

3.5.1.5 Calculation

3.5.2.1 Principle

3.5.2.2 Reagents

3.5.2.4 Procedure

60

measured within that period.

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$$\text{serum creatinine in mg/100 mL} = \frac{\text{O.D.test} - \text{O.D.blank} \times \text{3}}{\text{O.D.std.} - \text{O.D.blank}} \tag{12}$$

## 3.6 Statistical analysis

The results are expressed in mean � standard deviation (SD). Statistical analysis was performed by using Students 't 'test. p values < 0.05 were considered to be statistically significant.
