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CHEMISTRY I
MKEB 2404
Title:
Determination of the accuracy of the assay
Objectives:
To determine the accuracy of the assay by calculation recovery and
percentage of recovery.
Develop the calibration curve and determine the value of samples that
have been made
To assess the accuracy of a method; it, tests whether a method can
measure an analyte in the presence of other substances contained
within the sample matrix
Principle:
Recovery is the ability of an analytical method to correctly measure an
analyte when known amount of it is added to samples. Recovery studies
can also test for interferences by substances contained in the sample; like
competition of protein for calcium in some dye binding methods of
calcium estimation.
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Calculation of recovery:
• Concentration added = [standard] X standard volume (ml)
Standard vol + serum vol
Methods:
1. Preparation for standard BSA with concentration 0, 10, 20, 40, 60, 80 100
in mg/dl
Concentration Calculation of volume BSA required
Set total volume is 200µl
To determine volume of Bovine Serum Albumin (BSA) need
use M1V1 = M2V2.
M1 = actual concentration of BSA
M2 = final concentration of BSA
0X
V1 = volume need
V2 = final volume
(100mg/dl)( V1) = (0)(200µl)
V1 = 0 µl
Volume water need: 200µl - 0µl = 200µl
Set total volume is 200µl
To determine volume of Bovine Serum Albumin (BSA) need
use M1V1 = M2V2.
M1 = actual concentration of BSA
M2 = final concentration of BSA
10X
V1 = volume need
V2 = final volume
(100mg/dl)( V1) = (10)(200µl)
V1 = 20 µl
Volume water need: 200µl - 20µl = 180µl
Set total volume is 200µl
To determine volume of Bovine Serum Albumin (BSA) need
use M1V1 = M2V2.
M1 = actual concentration of BSA
M2 = final concentration of BSA
20X
V1 = volume need
V2 = final volume
(100mg/dl)( V1) = (20)(200µl)
V1 = 40 µl
Volume water need: 200µl - 40µl = 160µl
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Set total volume is 200µl
To determine volume of Bovine Serum Albumin (BSA) need
use M1V1 = M2V2.
M1 = actual concentration of BSA
M2 = final concentration of BSA
40X
V1 = volume need
V2 = final volume
(100mg/dl)( V1) = (40)(200µl)
V1 = 80 µl
Volume water need: 200µl - 80µl = 120µl
Set total volume is 200µl
To determine volume of Bovine Serum Albumin (BSA) need
use M1V1 = M2V2.
M1 = actual concentration of BSA
M2 = final concentration of BSA
60X
V1 = volume need
V2 = final volume
(100mg/dl)( V1) = (60)(200µl)
V1 = 120 µl
Volume water need: 200µl - 120µl = 80µl
Set total volume is 200µl
To determine volume of Bovine Serum Albumin (BSA) need
use M1V1 = M2V2.
M1 = actual concentration of BSA
M2 = final concentration of BSA
80X
V1 = volume need
V2 = final volume
(100mg/dl)( V1) = (80)(200µl)
V1 = 160 µl
Volume water need: 200µl - 160µl = 40µl
Set total volume is 200µl
To determine volume of Bovine Serum Albumin (BSA) need
use M1V1 = M2V2.
M1 = actual concentration of BSA
100X M2 = final concentration of BSA
V1 = volume need
V2 = final volume
(100mg/dl)( V1) = (100)(200µl)
V1 = 200 µl
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20µl of each diluted
BSA
+
1000µl color reagent
2. Preparation of samples:
• This method using standard (BSA) with certain concentration that
have been prepared before
• Serum A is supplied for use as base specimen:
• To the baseline specimen, different concentration (but same volume
(25µl)) of standard was added as follows:
Standard Serum
Standard Total
Specimen volume dH2O (µl) volume
(mg/ml) volume (µl)
(µl) (µl)
A0 0 0 25 475 500
A1 100 25 0 475 500
A2 80 25 0 475 500
A3 60 25 0 475 500
A4 40 25 0 475 500
A5 20 25 0 475 500
A6 10 25 0 475 500
Specimen A0 = baseline
Specimen A1 – A5 = diluted test
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3. Make a double dilution technique using above specimen / samples
20µl of each
specimen
+
1000µl color reagent
Results:
Table below showed the results for standard:
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From the graph that has been plotted the concentration value for each
sample was listed below:
Discussion:
Below is the calculation of recovery for each sample:
Concentration Concentration
% of recovery
Samples added (refer recovered (refer
(refer formula)
formula) formula)
A0 Baseline Baseline Baseline
A1 25 84 mg/dl – 83 1
10 × = 0.5 × 100 = 200%
500 mg/dl = 1 mg/dl 0.5
A2 25 80 mg/dl – 83 3
20 × =1 × 100 = 300%
500 mg/dl = -3 mg/dl 1
A3 25 90 mg/dl – 83 7
40 × =2 × 100 = 350%
500 mg/dl = 7 mg/dl 2
A4 25 90.5 mg/dl – 83 7.5
60 × =3 × 100 = 250%
500 mg/dl = 7.5 3
mg/dl
A5 25 93 mg/dl – 83 10
80 × =4 × 100 = 250%
500 mg/dl = 10 mg/dl 4
A6 25 92 mg/dl – 83 9
100 × =5 × 100 = 180%
500 mg/dl = 9 mg/dl 5
From all calculations, the results are invalid. Usually the % of recovery should be
below than 100%. If the results are more or higher than 100% the results are
invalid.
In determine the % of recovery, the percentage that near to 100% showed that
the method that was used is more effective.
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The results from this experiment are invalid because of certain factor:
Interference that occurs in the samples
The cuvette that was used are not cleaned properly
The interference that occur in the spectrophotometer
The serum that was used are contaminate with other substances
Error in taking the absorbance value.
The graph that was plotted are not really accurate
Conclusion:
From this experiment, we can’t make any conclusion about the % of recovery
because the results exceed 100%, therefore it can’t be used. We suggest that
proper in handling and making the method of preparation are important in order
to prevent any error from occur that may cause a false results.
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