Quantitation Flashcards
Why use quantitative data?
- clinical chemistry
- urine and plasma drug conc
- biomarkers (can predict when patient has an episode in sickle cell anaemia)
- drug toxicity (trace metals in the blood)
- drug pharmacokinetics
Two types of quantitative methods
- classical methods
- instrumental methods
What are classical methods?
- wet chemistry
precipitation, extraction, distillation, boiling point, melting point, gravimetric and volumetric analysis
What are instrumental methods?
analytical measurement
conductivity, electrode potential, light absorption or emission, mass to charge ratio. fluorescence
How to determine the concentration in a sample?
use the peak area
Does it need to be a known compound
yes
matrix?
establishment of the method of analysis of drug in the matrix
analyse standard containing a known amount of drug in the matrix/ surrogate matrix
quantiation
compare the response of an unknown sample to the response of a known standard
data must be acquired and processed under identical conditions
requires good chromatographic resolution
response of the compound is within the linear range of the detector
what does the chromatographic peak integration define
an operation where the area under the peak is measured
what is the integral technique and integral method for measuring peak area
integral technique- splits the peak into a large set of rectangles and summing their area
integral method
area= change in absorbance x change in time
what is external standard quantitation?
For an external standard quantitation, known data from a calibration standard and unknown data from the sample are combined to generate a quantitative report.
It is called external standard because the standard or known material is separate or external to the unknown material.
equation for the response factor?
response factor= area/ concentration
Problems with external standard quantiation
cannot draw a line with just one point
cannot have a 1 point/2 point calibration
need an 8 pint calibration curve
how is a calibration curve made
by analysis of standard solutions of known concentration
and measuring their peak area
best fit regression line is used to join the points
what is a better method to quantify the concentration
internal standard ISTD
requirements of an ISTD
- should be as similar as possible to the analyte
- chemically and physically similar to the analyte
- unreactive
- pure
- elute near the analse but well resolved
how is an ISTD added
prepare a set of standards with different pg/ml
add a known quantity of the ISTD to each sample
add the same quantity of the ISTD to the test samples
analyse all the samples
how does a internal standard calibration curve work
- internal standard is added to each sample or standard in equal concentrations
- peak area ratio and concentration ratio is plotted for each standard
- concentration ratio of the sample s extrapolated from the peak area ratio obtained for the sample
- concentration of the sample can be calculated as the concentration of the internal standard C(is)
characteristics that should be considered in the validation of an analytical method
- linearity
- range
- accuracy
- precision
- recovery
- specificity
- limit of quantitation
- robustness
what is linearity
indicates the ability to produce results that are directly proportional to the concentration of the analyse
samples prepared in a biological/ surrogate matrix in which the analytes concentration span the claimed range of procedure
samples are extracted and analysed
min of 5 concentrations should be used in 6 replicated
what is range
an expression of the lower and highest levels of analyse that has been demonstrated to be determinable for the product
range is derived from the linearity study
what is accuracy
the degree of agreement of the test result with the true value
the closeness of the result obtained by the procedure to the true value
how are accuracy measurements carried out
on spiked samples of the matrix or surrogate matrix
at 3 concentrations within day, between day for 3 separate days in 6 replicates
equation for % error
%error= (actual - experimental)/ actual * 100
low QC- 6 replicates,3 separate days
mid QC- within day
high QC- between day
%error must be less than 15%
what is precision
the degree of agreement among individual results
how should precision be measured
- at 3 concentrations with 6 replicated within day and between day
complete procedure should be applied repeatedly to separate, identical samples drawn from the same homogenous batch of material
should be measured by the scatter of individual results from the mean and expressed as the relative standard deviation RSD or coefficient variation CV
equation for coefficient variation
CV= (standard deviation/ mean) * 100
precision <15%
what is specificity
the ability to measure unequivocally the desired analyse in the presence of excipients and impurities and may be expected to be present
an investigation of specificity should be conducted during validation of identification tests, determination of impurities and assay
detection limit
smallest quantity of n analyse that can be detected and not necessarily determined in a quantitative manner
approaches: visual evaluation signal to noise ratio standard deviation of the response and the slope calibration curve
what is limit of quantitation
lowest concentration of analyte in sample that may be determined with acceptable accuracy and precision
% recovery
recovery = (standard/ no standard) * 100
