Evaluate the calibration curve
Exam : use COMMAS and UNITS
STEP 1 : Evaluate the conept of the calibration curve
Concept : does not require calculations. Evaluate what you see. You make a plan
based on what is given to you.
• Threshold range :½-2
• Calibration points : at least 3 but best >5
• Calibration levels : ≥ 6 levels (low-mid-high)
• Replicates : 3 per level
• Use of internal standard
STEP 2 : Determining calibration curve
Use headers so everything is clear when a mistake is made, to understand your
logic
1) Calculate signal (drug) / ISTD
2) Select level and ratio
3) Insert
4) Scatterplot
5) Select a dot in curve
6) Right mouse
7) Add trendline (trendlijn toevoegen)
8) Click box Display equation on chart (vergelijking in grafiek geven)
9) Click box Display R-squared value on chart
𝑦+𝒐𝒓−𝑏
Equation 𝑦 = 𝑎𝑥 + 𝒐𝒓 − 𝑏 𝑥= 𝑎
y : signal/ISTD
x : concentration
a : slope
b : intercept
Watch out : y = ax + b when you set over number
1
, STEP 3 : Calculate the goodness of fit factor g(%)
1) Calculate concentration : = (signal/ISTD + b) / a
2) Calculate error(%) : = (level – concentration)/ (level *100)
3) Calculate error(%)^2
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4) Calculate sum of values : =sum(all values)
Under all the error^2 values
5) Calculate count :n-1 (n = contributors)
Under the sum
6) Calculate g : =SQRT(sum / n – 1)
Under the n-1
7) Evaluate : <10% if majority of calibrators > 10 ng/ml : OK
: <20% if majority of calibrators <10 ng/ml : OK
STEP 4 : Calculate trueness(%), bias(%) and repeatability(%)
Trueness (%)
Agreement of mean to the true value.
1) Calculate average : =AVERAGE(per level)
rd
In the 3 cell of the column
Copy first value and paste formula right click
2) Calculate trueness(%) : =AVERAGE(per level)/ level * 100
Next to average
3) Evaluate : <20% for the lowest (first) : OK
: <15% for the rest : OK
Bias (%)
1) Calculate bias(%) : = 100 – trueness(%)
2) Evaluate : <20% for the lowest (first) : OK
: <15% for the rest : OK
Repeatability (%)
1) Calculate SD : =STEDV(per level)
2) Calculate Repeatability : =(STEDV/AVERAGE(per level)) * 100
3) Calculate C : =level E-6
For evaluation we need the Horwitz equation : RSDmax = 2^(1-0.5 log C)
We want in absolute terms : no units
for example : 1 kg/l = 1; 1g/ml = 1; 1mg/ml = 1 E-3; 1µg/ml = 1 E-6 .
Important : in Excel 1 E-3 is not the same as 1 x E-3
4) Calculate log C : =LOG(C)
5) Calculate 0,5 logC : =0,5* log(C)
6) Calculate 1- : = 1- 0,5*log(C)
7) Calculate RSDmax(%) : =2^(1-0,5*log(C))
8) Calculate 2/3 : =2/3 * RSDmax
Copy > paste only values > copy paste to the correct cell next to repeatability
9) Evaluate : repeatability < 2/3 RSDmax : OK
2
Exam : use COMMAS and UNITS
STEP 1 : Evaluate the conept of the calibration curve
Concept : does not require calculations. Evaluate what you see. You make a plan
based on what is given to you.
• Threshold range :½-2
• Calibration points : at least 3 but best >5
• Calibration levels : ≥ 6 levels (low-mid-high)
• Replicates : 3 per level
• Use of internal standard
STEP 2 : Determining calibration curve
Use headers so everything is clear when a mistake is made, to understand your
logic
1) Calculate signal (drug) / ISTD
2) Select level and ratio
3) Insert
4) Scatterplot
5) Select a dot in curve
6) Right mouse
7) Add trendline (trendlijn toevoegen)
8) Click box Display equation on chart (vergelijking in grafiek geven)
9) Click box Display R-squared value on chart
𝑦+𝒐𝒓−𝑏
Equation 𝑦 = 𝑎𝑥 + 𝒐𝒓 − 𝑏 𝑥= 𝑎
y : signal/ISTD
x : concentration
a : slope
b : intercept
Watch out : y = ax + b when you set over number
1
, STEP 3 : Calculate the goodness of fit factor g(%)
1) Calculate concentration : = (signal/ISTD + b) / a
2) Calculate error(%) : = (level – concentration)/ (level *100)
3) Calculate error(%)^2
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4) Calculate sum of values : =sum(all values)
Under all the error^2 values
5) Calculate count :n-1 (n = contributors)
Under the sum
6) Calculate g : =SQRT(sum / n – 1)
Under the n-1
7) Evaluate : <10% if majority of calibrators > 10 ng/ml : OK
: <20% if majority of calibrators <10 ng/ml : OK
STEP 4 : Calculate trueness(%), bias(%) and repeatability(%)
Trueness (%)
Agreement of mean to the true value.
1) Calculate average : =AVERAGE(per level)
rd
In the 3 cell of the column
Copy first value and paste formula right click
2) Calculate trueness(%) : =AVERAGE(per level)/ level * 100
Next to average
3) Evaluate : <20% for the lowest (first) : OK
: <15% for the rest : OK
Bias (%)
1) Calculate bias(%) : = 100 – trueness(%)
2) Evaluate : <20% for the lowest (first) : OK
: <15% for the rest : OK
Repeatability (%)
1) Calculate SD : =STEDV(per level)
2) Calculate Repeatability : =(STEDV/AVERAGE(per level)) * 100
3) Calculate C : =level E-6
For evaluation we need the Horwitz equation : RSDmax = 2^(1-0.5 log C)
We want in absolute terms : no units
for example : 1 kg/l = 1; 1g/ml = 1; 1mg/ml = 1 E-3; 1µg/ml = 1 E-6 .
Important : in Excel 1 E-3 is not the same as 1 x E-3
4) Calculate log C : =LOG(C)
5) Calculate 0,5 logC : =0,5* log(C)
6) Calculate 1- : = 1- 0,5*log(C)
7) Calculate RSDmax(%) : =2^(1-0,5*log(C))
8) Calculate 2/3 : =2/3 * RSDmax
Copy > paste only values > copy paste to the correct cell next to repeatability
9) Evaluate : repeatability < 2/3 RSDmax : OK
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