Biuret method.

Evaluation of Analytical Performance of a Method

Practical 3: Determination of Analytical Range, Sensitivity, Detection Limit and Precision of the Assay

Aims and objectives:

§ To evaluate analytical performance of the Biuret method.

§ To determine the linear range, sensitivity, detection limit and precision of the Biuret method.

Introduction:

A. Analytical goals

Laboratory tests are performed to assist clinicians in diagnosis and management of patients. Certain analytical characteristics of a test are important in determining the clinical usefulness of that test. These characteristics include specificity and sensitivity of the test. Other important performance characteristics use the analytical accuracy and precision of the test procedure.

The goals set for a test will depend on the intended use of that test. For example the analytical goals for a test intended for diagnosis, both accuracy and precision are required to allow comparison of results from other patients. If a test is intended mainly for monitoring of treatment, or the course of a disease, reproducibility of results over time is the main concern.

B. Evaluation of analytical methods

Careful selection followed by a thorough evaluation of analytical methods is important steps in ensuring good quality performance. A good starting point for this exercise is the study of published data and information on the method under consideration. However, individual laboratories must proceed to carry out evaluation studies to establish performance of the method. These performance parameters include precision, accuracy, analytical sensitivity, analytical specificity and linear range.

§ Precision

Precision describes the agreement between two or more measurements that have been made in exactly the same way. Precision indicates how close the single values are to one another. Precision can be expressed by variance, standard deviation and coefficient of variation that estimate the amount of random variation in a population of values.

Variance is a statistic used to describe the spread or distribution of a population of data/values.

_

V = ∑ N (x1 - x) 2

i = 1

N - 1

Standard deviation (s) is the positive square root of the variance.

_

s = ∑ N (x1 - x) 2

i = 1

N - 1

Coefficient of variation (CV)

CV = Standard Deviation X 100%

Mean

_

CV = (SD / x) X 100%

In clinical chemistry, precision is expressed by standard deviation (SD). The smaller the SD for a specified group of values, the more precise the values are. CV of lower than 5% is to be considered precise and acceptable for the patient specimen analysis. In analytical work, the dispersion of values is generally larger, if the mean of the population of values is larger. Therefore, the dispersion is often described in relation to the mean by the coefficient of variation (CV).

Detection limit

Detection limit refers to the smallest concentration that can be accurately measured. Detection limit depends on the amplitude of the blank readings and also is related to the precision of these measurements. The detection limit, XL, defines that point at which any smaller result should not be reported, except as “less than XL”.

§ Linear range

Linear range: the range of concentration or other quantity in the sample over which the method is applicable without modification. Ideally the calibration curve should be linear and pass through the origin.

§ Analytical sensitivity

Defined as the slope of the calibration curve and the ability of an analytical procedure to produce a change in the signal for a defined change of the quantity.

§ Analytical specificity

Refers to a method's ability to measure only the analyte of interest.

Materials:

BSA (140mg/ml), Biuret reagent, control sera, distilled water, spectrophotometer, cuvettes, test tubes, test tube rack, micropipettes, disposable tips

Procedures:

1. BSA standard that is 140mg/ml is provided. A series of dilution and assay of protein are set up using the Biuret method. (The range of linearity is determined using the stock reference solution provided ~ concentration given).

2. The respective volume of diluent and stock are worked out.

3. Duplicate analysis of the solution is carried out.

4. The observed results are plotted against the expected concentration.

5. Comments are given.

§ Determine the linear range of this method

Determine the equation of line y = mx + b

§ Determine the sensitivity of this method

Sensitivity = change in OD

[ ] change

Slope of the linear regression (m) gives the sensitivity.

§ Determine the detection limit

~ Assay reagent blank readings are measured for 10X - 20X.

~ The mean and SD of the OD are determined.

~ Mean + 3SD = lower limit of detection (99% confidence).

§ Determination of intra-assay precision

Procedure:

~ Using the control sera provided, replicate analysis (10X) is carried out using the Biuret method.

~ Tabulate the data and determine:

Ø The mean of the results

Ø The SD and CV

Results are commented.

Biuret method:

Assay protocol:

Wavelength: 540nm

Measurement: against reagent blank

Only one reagent blank per series is required

Pipetting Scheme:

Reagent blank

Sample/Standard/Control

Distilled water

Sample or standard

Color reagent

20µl

-

1000µl

-

20µl

1000µl

Mixed, incubated for 5 minutes at 20 - 25ºC. The absorbance of the sample

and the standard are measured against reagent blank within 60 minutes.

Calculation:

Concentration

(mg/ml)

Volume of BSA stock solution (µl)

Volume of distilled

water (µl)

0

0.0

100.0

20

14.3

85.7

40

28.6

71.4

60

42.9

57.1

80

57.1

42.9

100

71.4

28.6

140

100.0

0.0

The volume of stock solution and diluent required to prepare a series of dilution are calculated by using the following formula:

M1V1 = M2V2

M1 = concentration of BSA stock solution

M2 = concentration of diluted BSA solution

V1 = unknown volume of BSA stock solution

V2 = volume of diluted BSA solution

Total volume of each dilution prepared = 100µl.

BSA stock solution = 140mg/ml

Volume of distilled water (diluent) = 100µl - V1

§ 0 mg/ml dilution

M1V1 = M2V2

(140)V1 = (0) (100)

V1 = 0µl

§ 20 mg/ml dilution

M1V1 = M2V2

(140)V1 = (20) (100)

V1 = 14.3µl

§ 40 mg/ml dilution

M1V1 = M2V2

(140)V1 = (40) (100)

V1 = 28.6µl

§ 60 mg/ml dilution

M1V1 = M2V2

(140)V1 = (60) (100)

V1 = 42.9µl

§ 80 mg/ml dilution

M1V1 = M2V2

(140)V1 = (80) (100)

V1 = 57.1µl

§ 100 mg/ml dilution

M1V1 = M2V2

(140)V1 = (100) (100)

V1 = 71.4µl

§ 140 mg/ml dilution

M1V1 = M2V2

(140)V1 = (140) (100)

V1 = 100µl

Result:

Concentration

(mg/ml)

Absorbance

Mean

Set 1

Set 2

20

0.054

0.055

0.055

40

0.146

0.171

0.159

60

0.227

0.232

0.230

80

0.320

0.327

0.324

100

0.377

0.432

0.405

140

0.489

0.496

0.493

§ The linear range of Biuret method

From the graph:

Biuret method linear from 0mg/ml - 102mg/ml

y = mx + b

Gradient, m = y2 - y1

x2 - x1

= 0.31 - 0.1

80 - 26

= 0.21

54

= 0.004

Since the line passes through origin, b = 0

y = 0.004x

Absorbance = 0.004 [concentration]

§ The sensitivity of Biuret method

Sensitivity (gradient) = change in OD

[ ] change

= y2 - y1

x2 - x1

= 0.31 - 0.1

80 - 26

= 0.21

54

= 0.004 (mg/ml) - 1

§ The detection limit of Biuret method

Tube

Absorbance

Mean

Concentration

(mg/ml) (x)

­­­­­­­­­­­­­­­­ _

(x - x)2

Set 1

Set 2

1

0.008

0.012

0.010

2.50

0.4277

2

0.010

0.014

0.012

3.00

1.3317

3

-0.005

-0.001

-0.003

-0.75

3.4077

4

-0.024

-0.014

-0.019

-4.75

3.4077

5

-0.020

-0.016

-0.018

-4.50

3.4077

6

-0.026

-0.025

-0.026

-6.50

3.4077

7

0.030

0.027

0.029

7.25

29.2032

8

-0.013

-0.019

-0.016

-4.00

3.4077

9

-0.011

-0.016

-0.014

-3.50

3.4077

10

-0.012

-0.018

-0.015

-3.75

3.4077

11

0.009

0.003

0.006

1.50

0.1197

12

-0.013

-0.019

-0.016

-4.00

3.4077

13

0.037

0.040

0.039

9.75

62.4732

TOTAL

24

120.8171

Negative concentration or absorbance values will not be involved in the calculation of mean concentration of BSA and are given an arbitrary value of 0.

Calculation:

Ø Absorbance = 0.004 [concentration]

Concentration (x) = absorbance ­

0.004

E.g. Tube 1, Concentration = 0.01

0.004

= 2.50 mg/ml

_

Ø Mean concentration (x) = ∑x

N

= 24

13

= 1.846 mg/ml

_

Ø Standard deviation, s = ∑(x - x)2

N - 1

= 120. 8171

12

= 3.173 mg/ml

Ø Lower limit of detection of Biuret method = Mean + 3SD

= 1.846 + 3 (3.173)

= 1.846 + 9.519

= 11.365 mg/ml

§ The intra-assay precision of Biuret method

Tube

Absorbance

Mean

Concentration

(mg/ml) (x)

­­­­­­­­­­­­­­­­ _

(x - x)2

Set 1

Set 2

1

0.380

0.381

0.381

95.25

42.9025

2

0.396

0.398

0.397

99.25

6.5025

3

0.424

0.428

0.426

106.50

22.0900

4

0.409

0.405

0.407

101.75

0.0025

5

0.419

0.427

0.423

105.75

15.6025

6

0.407

0.407

0.407

101.75

0.0025

7

0.414

0.418

0.416

104.00

4.8400

8

0.404

0.403

0.404

101.00

0.6400

9

0.413

0.413

0.413

103.25

2.1025

10

0.392

0.403

0.398

99.50

5.2900

TOTAL

1018

99.9750

Calculation:

Ø Absorbance = 0.004 [concentration]

Concentration (x) = absorbance ­

0.004

E.g. Tube 1, Concentration = 0.381

0.004

= 95.25 mg/ml

Ø Mean concentration (x) = ∑x

N

= 1018

10

= 101.8 mg/ml

_

Ø Standard deviation, s = ∑(x - x)2

N - 1

= 99.9750

9

= 3.333 mg/ml

_

Ø Coefficient of variation = (SD / x) X 100%

= (3.333/101.8) X 100%

= 3.274%

Discussion:

A. Determine the linear range of Biuret method

1. In this experiment, the linear range of Biuret method can be obtained from the graph, which is 0mg/ml - 102mg/ml.

2. The equation of linear line: y = mx + b can also be determined from graph:

§ y = absorbance

§ m = slope of the linear line (= sensitivity)

§ x = concentration

§ b = intercept at y-axis

Thus, concentration can be calculated by the formula when the absorbance value, slope and intercept are known. Otherwise it is also can be determined directly from the graph.

3. The International Federation of Clinical Chemistry has defined the analytical range in a qualitative sense, stating that it is “the range of concentration or other quantity in the specimen over which the method is applicable without modification”.

4. The absolute minimum number of different concentrations that must be measured for linearity verification is three. Replicate measurements, at least in duplicate, should be made on each concentration sample.

5. An initial linearity study could use aqueous standards to identify the capabilities of the method in an ideal specimen matrix. This should be followed by the analysis of the analyte in a dilution series of samples containing the biological matrix, such as urine or serum, which will be used for patient tests. The aqueous and matrix samples will provide important information about the influence of the biological matrix on the method.

B. Determine the sensitivity of Biuret method

1. Sensitivity is the ability of an analytical procedure to produce a change in the signal for a defined change of the quantity. In this experiment, the sensitivity obtained is 0.004 (mg/ml) - 1 which can be determined from the slope of the calibration curve.

2. The higher the value or the steeper the curve, the more sensitive is the experiment towards the changes in analyte quantity. Thus, by plotting absorbance values against concentration, we can compare the sensitivity of two methods being evaluated. For example:

Method A is more sensitive than method B as there is larger change in the absorbance value for a defined change of the concentration.

C. Determine the detection limit of Biuret method

1. In this experiment, the lower detection limit obtained is 11.365 mg/ml, which means that any values lower than 11.365 mg/ml should not be reported because it is the smallest value that can be accurately measured. The limit of detection is the minimum concentration of analyte whose presence can be qualitatively detected under defined conditions.

2. Most of the absorbance values obtained in this experiment are negative. There are only five tubes show positive absorbance values. Negative values are not taken into account in the calculation of mean concentration and are given an arbitrary value of zero.

D. Determination of intra-assay precision

1. Precision describes the agreement between two or more measurements that have been made in exactly the same way. Precision can be expressed by variance, standard deviation and coefficient of variation that estimate the amount of random variation in a population of values.

2. The coefficient of variation obtained from the experiment is 3.274%, which is lower than 5% and is considered to be precise and acceptable for the patient specimen analysis.

Conclusion:

The method is evaluated in terms of its analytical performance capabilities. A method-evaluation study is to determine the method with acceptably small analytical errors. In this experiment, the analytical performance of Biuret method is being evaluated by its analytical range, sensitivity, detection limit and precision of the assay. The method with good analytical performance characteristics is important for medical application of the test.

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