Basic Concepts in Clinical Biochemistry-A Practical Guide.7z

For Urine Dilute urine 1:10 with distilled water and prepare protein-freefiltrate the
same way as for serum.

24.5 Procedure

1. Take a set of nine test tubes, and label them as blank, standard (S 1 – S 6 ), test for
   serum sample (TS), and test for urine sample (TU).


2. Add 10, 20, 40, 60, 80, and 100μl of 20 mg % phosphorus standard in tubes
   labelled as S 1 to S 6 , respectively, and make thefinal volume 2.5 ml with 20%
   TCA. In blank add 2.5 ml of 20% TCA.


3. Add 2.5 ml of protein-freefiltrate of serum and urine into the tubes labelled at TS
   and TU, respectively.


4. Add 1 ml of ammonium molybdate reagent and 0.2 ml ANSA reagent to all the
   tubes and vortex. Keep at room temperature for 20 min and read absorbance at
   700 nm.

Reagents Blank S 1 S 2 S 3 S 4 S 5 S 6 TS TU

Phosphorus standard,

20 mg % (μl)

• 10 20 40 60 80 100 ––

Phosphorus amount (μg) – 2 4 8 12 16 20 ––

Protein-freefiltrate of

serum/urine (ml)

–––––––2.5 2.5

20% TCA (ml) 2.5 2.49 2.48 2.46 2.44 2.42 2.40 ––

Ammonium molybdate

(ml)

1 11111111

ANSA (ml) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2

Mix well and keep for 20 min at room temperature, read absorbance at 700 nm

22.8 Calculations

Plot a graph by taking phosphorus amount at x-axis vs absorbance at y-axis, and
extrapolate its concentration from the given serum/urine sample from the graph.The
amount of phosphorus extrapolated from graph will be in 250μl of serum/urine
sample since 2.5 ml of protein-freefiltrate was used for analysis. The phosphorus
amount can also be calculated in serum and urine samples by using equation:

Phosphorus amount in serum

¼

ODof testamount of standardðÞμg 100

ODof standardvolume of sample 0ðÞ:25ml 1000

¼xmg=dl

22.8 Calculations 91