# Quantitation

• Created by: LBCW0502
• Created on: 22-11-18 12:12
What is qualitative analysis?
What is in the sample (identification)
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What is quantitative analysis?
Determine concentration of a known compound
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What is quantitative data used for?
Clinical chemistry, urine/plasma drug concentration, biomarkers, drug toxicity, drug PK etc.
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What are classical methods?
Wet chemistry methods such as precipitation, extraction, distillation, b.p., m.p., gravimetric and volumetric analysis
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What are instrumental methods?
Analytical measurement (conductivity, electrode potential, light absorption or emission, mass-to-charge ratio, fluorescence etc.) made using instruments
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Outline how blood samples can be measured for particular components
Blood sample centrifuged to remove erythrocytes, left with blood plasma. Measure plasma concentrations and compare with critical lab values (e.g. albumin)
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Describe features of quantitation (1)
Use peak area to determine concentration in sample. Known compound. Establish method for analysis in matrix. Analyse standards of known amount of drug in matrix. Compare unknown to standard. Data obtained from same conditions
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Describe features of quantitation (2)
Good chromatographic resolution. Response of compound within linear range of detector
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What is peak integration?
Chromatographic peak integration defines an operation where the area under the peak is measured. Integral technique - splitting peak into large set of rectangles and summing their area
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What are the principles of quantitative analysis?
Many types of detectors. Each detector will produce a response dependent on the amount of analyte. Magnitude of response depends on analyte and on matrix
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Describe the process of external standard quantitation
Chromatogram for sample and standard (volume of sample, retention time and peak area). Analysis under same conditions. Response factor = area under peak for standard/ concentration of standard. Concentration of unknown sample = aup of sample/R
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What is the issue with the external standard quantitation method?
Need a calibration curve with known concentrations of standard to determine unknown concentration of sample (cannot determine from one reference sample) - not sure constant volume of sample was used
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Describe features of a calibration curve
Graphical representation of concentration and response for single compound. Construct by analysis of standard solutions of known concentration and measuring peak area - use of regression line
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Describe the process for internal standard quantitation (1)
ISTD should be similar to analyte (chemically/physically), unreactive/pure and elute near analyte but well resolved. Prepare set of standards, add same quantity of ISTD to test samples, add known quantity of ISTD to each sample, analyse samples
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Describe the process for internal standard quantitation (2)
Internal standard added to each sample (or in equal concentration). Peak area ratio and concentration ratio plotted for each standard, plot calibration curve. Concentration ratio of sample extrapolated from peak area ratio obtained from sample
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Describe the process for internal standard quantitation (3)
Concentration of sample calculated as concentration of internal standard is known - e.g. analysis of caffeine, paracetamol, theophyline
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Which characteristics should be considered in the validation of an analytical method?
Linearity, range, accuracy, precision, recovery, specificity, limit of quantitation and robustness
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Describe features of linearity and range (1)
Linearity - ability to produce results directly proportional to concentration of analyte. Series of samples prepared in matrix (span claimed range). Results linear/evaluate using statistical methods. Minimum of five concentration used, six replicates
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Describe features of linearity and range (2)
Range - expression of lower and higher levels of analyte that has been demonstrated to be determinable for the product. Range normally derived from linearity study
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What is accuracy? (1)
Degree of agreement of test result with true value or closeness of result obtained to true value. Accuracy measurements carried out on spiked samples of matrix at 3 concentrations (over days)
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What is accuracy? (2)
Low QC (6 replicates/3 days, Mid QC (within day). High QC (between days).
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How do you calculate % error?
Actual - experimental / actual x 100 (% error should be <15%)
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What is the difference between accuracy and precision?
Accuracy - how close measured value is to true value. Precision - measure of dispersion of values
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Describe features of precision
Degree of agreement among individual results. Determine at 3 concentrations with 6 replicated within/between day. Apply to separate/identical samples from homogeneous batch
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How do you calculate the CV?
SD/mean x 100. Low QC (6 replicates/3 separate days). Mid QC (within day). High QC (between day). Precision should be lower than 15%
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Describe features of specificity (selectivity)
Ability to measure unequivocally and desired analyte in presence of components such as excipients/impurities. Use of validation of identification tests and determination of impurities/assay
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Describe features of detection limit
Smallest quantity of analyte detected (not necessarily determined in quantitative manner). Approaches - visual evaluation, signal: noise ratio, SD of response and slope, calibration curve
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Describe features of limit of quantitation
Lowest concentration of analyte in sample that may be determined with acceptable accuracy and precision (15 %)
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Compare the differences in the presence and absence of sample clean up for a standard
For sample clean up, the concentration of the standard is 9.5 mg/mL. For no sample clean up, the concentration of the standard is 10 mg/mL. Recovery = 9.5/10 x 100 = 95%
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State different application studies of quantitation
Stability testing, leucine in rat plasma, fluticasone propionate etc.
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## Other cards in this set

### Card 2

#### Front

What is quantitative analysis?

#### Back

Determine concentration of a known compound

### Card 3

#### Front

What is quantitative data used for?

### Card 4

#### Front

What are classical methods?

### Card 5

#### Front

What are instrumental methods?