Quantitative analysis:

Basic concepts
Dr. Le Hong Luyen
Department LS, USTH
Email: [email protected]

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Content
1. Introduction
2. Criterion of QA
3. Properties of QA
4. Error resources
5. Validation method
6. Sample preparation

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 1. Introduction
• Qualitative analysis simply determines if an
analyte is present of not in the sample
• E.g.: Test for illicit substances in an athlete

• Quantitative analysis determines the quantity of a

particular molecule
• E.g.: A breath alcohol test: Before, >= 0.025 mg/100 ml
in breath air or 50 mg in 100 ml of blood is forbidden.
• How about now?

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1. Introduction
• Quantitative analysis answers the question:

• ‘How much’ expressed:

• In amount: mg, µg, ng…
• In concentration: molarity (M-mol/L or µM) or as mass/volume (mg/L,
ng/mL…)

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1. Introduction
• A quantitative analysis is generally a targeted analysis
• One or a very limited number of compounds are quantified in a
sample
• For each quantitative analysis, we normally know:
• Range of concentration of the analyte
• Specific matrix (plasma, blood, urine…)
• Developed method

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 2. Criterion for a good quantitative analysis
•An analytical method is good only for
• A specific analyte: each type of compound must follow a specific method of analysis
• A specficic matrix: A perfectly good method for one matrix may not be acceptable for another
• A specific species: same molecular formula but different stereostructures will need different
analytical methods
• A specific range of concentrations: ‘’validation range’’

•Several criterion:
• Accuracy
• Precision
• Sensitivity
• Specificity

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2.1. Accuracy

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 2.2. Precision
•

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 2.2. Precision
How to compare the precision of 2 sets of measurements?
• Sample A: 32.56, 32.55, 32.48, 32.49, 32.48.
• Sample B: 15.38, 15.37, 15.36, 15.33, 15.32.
🡪 Measure the range (the difference between the highest and lowest
scores).

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 2.2. Precision
How to compare the precision of 2 sets of measurements?
• Sample A: 32.56, 32.55, 32.48, 32.49, 32.48.
• Sample B: 15.38, 15.37, 15.36, 15.33, 15.32.
🡪 Measure the range (the difference between the highest and lowest
scores).

Subtract the lowest data point from the highest:

•Sample A: 32.56 – 32.48 = .08.
•Sample B: 15.38 – 15.32 = .06.
Sample B has the lowest range (.06) and so is the more precise.
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Difference: accuracy and precision?
• The level of accuracy and precision shows whether or not quantitative
analysis is sufficient for the measurement of a particular analyte.

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Difference: accuracy and precision?
• The level of accuracy and precision shows whether or not quantitative
analysis is sufficient for the measurement of a particular analyte.

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Difference: accuracy and precision?
The shooter is aiming for the right corner of the back of the net.
Which one of the following shows:
- high accuracy and high precision?
- low precision but high accuracy

A B

C D

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 2.3. Sensitivity
• Sensitivity: the ability to distinguish the smallest difference that we can
measure between two signals.

• It is sometime useless to have a very sensitive method.

• Sensitivity is often confused with a method’s detection limit

What is needed is the right sensitivity for the range of concentrations measured.

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2.4. Specificity
• A method is specific if it measures only the analyte targeted.
• No interference from other compounds.
• For example:
• A method that cannot differentiate between a drug and its metabolites is not
specific.
• Weighing may be accurate, reproducible and sensitive but is not specific
because the mass of a sample does not tell you if the sample is pure or not.

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False positive/ False negative result
• A method can give a false positive result if it is not specific. It means
that the signal is not due (entirely or partially) to the analyte.

• A false negative means that the analyte is not detected (entirely or

partially) because of lack of sensitivity or signal suppressed by the
matrix.

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False positive/ False negative result
• False negative: When the test says you don’t have it but you actually
have it.
• False positive: When the test says you have it but you actually don’t
have it.

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Signal vs Concentration
• A signal (S) in a quantitative analysis is an experimental measurement that is
a function of concentration (c) of the analyte
• S = f(c)
• An analytical method gives a total signal S.
• S = f(c) + N
• N: noise
• The noise has to be subtracted to have accuracy on the response due to the
analyte

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 Signal vs Concentration
The signal may be: Absorbance (Spectroscopy), peak height or area (Chromatography),
intensity (Mass spectrometry)…

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 Signal vs Concentration
Linear
Curve S = f(c)

Exponential

Logarithmic

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Limit of detection
• The limit of detection (LOD) is defined as the lowest quantity
of a substance that can be detected with confidence.
• It is usual assessed by the measurement of the signal/noise
ratio (S/N).
• There is no formal value but we may reasonably say a peak is
a peak if the S/N ratio ≥ 3.

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Limit of quantification
• Lower limit of quantification (LLOQ): the lowest
concentration that can be quantitated with acceptable
accuracy and precision (≤ 20% CV).
• S/N ≥ 5-10 (but normally S/N ≥ 10)

• Upper limit of quantification (ULOQ): the highest

concentration that can be entirely quantification
• No limit in terms of S/N

• Working range: LLOQ - ULOQ

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Calibration curve
• From the curve S = f (c) obtained with standards we select a working part
between two limits: LLOQ & ULOQ.
• Usually the selected part of the calibration curve is linear.
• In order to quantitate an analyte, we have to build a calibration curve with
a standard which ideally has the same structure with the analyte.

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 Calibration curve
When we have a calibration curve we determine the concentration ci from the
signal Si obtained with the sample i. However, this determination can be valid only
if LLOQ < ci < ULOQ.

ULOQ

LLOQ

Increasing concentration

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Quality control sample
• In order to assure that a test run is valid and results are reliable,
Quality control (QC) samples should be used in the performance of
each assay.
• QC samples should be treated in the exact same manner as the test
samples
• QC samples are samples in the same matrix with known
concentrations: LLOQ < QCs < ULOQ
• QC samples are processed side-by-side with test samples

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 The standard addition method
• Matrix effect or matrix interference: components in the matrix affect the
analyte’s signal (e.g.: endogenous matrix…)
🡪 Couldn’t use the traditional calibration curve to determine the analyte’s
concentration.
signal
Analyte in matrix in equal volumes

Add increasing and known quantities

of the standard to all
Unknown conc concentration

Dilute the mixture with water

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Technical issues
• External vs internal standard
• Peak height vs peak area
• Linear relationship: Regression analysis (Phân tích hồi quy)
• Outlier

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External vs internal standard
• External standard (ES) is not present in the sample.
• It is a standard solution containing a known amount of analyte, prepared and
analyzed separately from samples containing the analytes.
• Usually used for quality controls
• Rather it is run alone, as a sample, and usually at different concentrations, so you
can generate a standard curve.
• External standards do not correct for losses that may occur during preparation of
the sample, such as extraction, centrifugation, evaporation, etc.

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External vs internal standard
• An internal standard (IS) is added in a constant amount to samples.
• A compound that must be show similar behaviour to the analyte.
• To correct analyte losses during sample preparation (extraction, centrifugation,
evaporation…)
• This substance can be used for calibration by plotting the ratio of the analyte
signal to the internal standard signal as a function of the analyte standard
concentration: S=f(ca/cIS)
• The internal standard used needs to provide a signal that is similar to the analyte
signal in most ways but sufficiently different so that the two signals are readily
distinguishable by the instrument.

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What is a good internal standard?
• Structure similar to the analyte
• Never found in the sample (to keep theirs concentration is constant)
• Well resolved
• Stable (If it is isotopically labelled)
• Pure enough not to interfere
• Same behavior with the analyte: extraction, chromatography,
response

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What is a good internal standard?
• The Internal Standard corrects for many things but it also adds to the variability
(in reality, you cannot add a same amount of IS to every samples).
• Error (As/ IS) = error Aa + error IS
• The concentration of the IS should be appropriate so that it introduces as little as
possible an additional error.

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Peak height vs peak area
• Peak height is proportional to the concentration, but it varies with the chromatographic
conditions.
• In fact, peak area is preferred if a minimum of rules are respected.
• Peak area is defined as the AUC (area under the curve).

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 Peak height vs peak area
• The baseline selection may affect the value.
• When the peaks are not well resolved, the results may
be drastically different.
• The peak area represents the amount of an analyte, so if
the retention time changes, the AUC doesn’t change.

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Peak area: unresolved peaks

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Linear relationship: Regression analysis
• We take the simple case of a regression analysis: linear relationship.
• We try to fit a straight line with the data points (this line is close to most of data
points),
• We get a function y = ax + b with the parameters a and b which minimize the sum
of the square (yi – y)2.

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Linear relationship: Regression analysis
• Practically 5-6 data points are sufficient
• The goodness of fit of the regression line can be assessed by
calculating the square of coefficient of correlation R2
• Perfectly, R2 = 1 but we never get 1.
• R2 depends on the variability, is not a good judge of linearity.
• R2 is sometimes used to compare 2 quantification methods.

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 Linear relationship: Regression analysis
• Anscombe’s quartet is 4 datasets with 11
data points:
• The same coefficient of correlation
• The same regression line
but showing clearly different graphs.

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 Outlier ?

• What is an outlier?
• An outlier is a data point that significantly differs from the
other data points in a sample
• Causes of outlier:
• Human mistake
• Instrument malfunction

• But, an outlier is not a data point we do not like !

• It is very tempting to remove an abnormal point to fit
the data. But unless there is an evident experimental
mistake, we should keep the data point.

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 Outlier: To remove or to keep?

• Sometimes an outlier just make the wrong model

• To remove: When the outlier is coming from a documented
human mistake or instrument malfunction,
• To keep: in the absence of an obvious mistake or
malfunction the data should be analysed with that outlier as
it may indicate a larger than expected uncertainty.

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Outlier
•How to Calculate Outliers?

• Biostatistics
• Or see more details on
http://www.wikihow.com/Calculate-Outliers

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Method validation
• A method is the application of a technique to a specific analyte in a specific
matrix
• Before we run the actual samples we have to validate the method.
• We need to check the performance of the method with the quality control samples (QC
samples) and a simple statistical analysis.
• Expression of the results: The results should be given with only significant numbers.
With the uncertainty, we have to write: +/- SD, CV. Results expressed usually as a
concentration.

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Method validation
• Criteria for validation:
• Interval of concentration: LLOQ, ULOQ
• Accuracy within the accepted value (e.g. 15%)
• Precision: within the accepted value (e.g. 15%)
• Specificity
• Sensitivity
• Reproducibility
• Stability of the sample before and during the analytical phase.

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Sample preparation
Protein precipitation
Liquid-liquid extraction
Liquid-solid extraction

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Introduction
• Sample preparation is an essential part of most bioanalytical methods
• Biological samples: not introduced directly into analytical instruments
• Commonly applied before LC-MS methods
• Main issues when analyzing biological samples:
o The analytes are in low concentration
o Unwanted compounds can contribute to the noise and suppress the signal of
the analytes
o Interferences can contaminate/damage the instruments

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Goals of sample preparation
• To remove interferences for:
o Better instrumental method (chromatography)
o More confident analytical results
o Longer column lifetime
o Less instrument downtime
• To enrich sample for:
o Higher detection sensitivity
• To make your analytical lab more productive:
o Lower limits of detection
o Run more samples with less time
o Minimize costs in equipment maintance

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1/ Protein precipitation
• Two opposite goals:
• To remain proteins for future analysis (e.g. proteomics)
• To remove proteins for regular bioanalysis
• In 1st case: the precipitation should be gentle and not to alter the
protein structure
• In 2nd case: don’t care what happen to the proteins. The protein may
be denaturated

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1/ Protein precipitation
• The most used biofluid is plasma from human or a variety of animal
species containing high protein content
• Protein precipitation technique used before injecting samples
• Rapidly performed
• Simple equipment and reagents
• Good extraction recovery
• Remove 90-98% of the proteins from the samples

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1/ Protein precipitation
• 4 main approaches to precipitate plasma protein:
• Organic solvents: methanol, ethanol, acetonitrile (the most used, 1:5 v:v)
• Acids: trichloroacetic acids (TCA), perchloric acid (HClO4)
• Metal ions: Zn(OH)2, copper sulphate-sodium tungstate (Na2WO4)
• Salts (less efficient): (NH4)2SO4
• After precipitation, removing the protein by centrifugation
(10,000-15,000xg) or filtration (using special membrane) to leave a
clear supernatant used for analysis

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2/ Liquid-liquid extraction (LLE)
• The analyte (drug) usually in aqueous phase (plasma, urine) is
transferred into a water-immiscible organic solvent
• The partitioning depends on:
• The lipophilicity of the analyte
• The pH of the aqueous phase
• The nature of the organic solvent
• The relative volume of the two phases

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2/ Liquid-liquid extraction (LLE)
• Between two immiscible phases (aqueous-organic phase)

Where are the analytes?

• Rule #1: The ionized species in the water phase

• Rule #2: The unionized species distributed between two phases
according to the physico-chemical properties of the analyte, the
organic solvent and the composition of the water phase
• Rule #3: It is possible to extract ionized species with an appropriate
counterion (the ion that accompanies an ionic species)
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How to choose the organic solvent?
• Rule ‘like with like’: a highly lipophilic analyte will extract well into a
relative nonpolar solvent, a more polar analyte requires a more polar
solvent
• Criteria of solvent choice:
• Water immiscible, low boiling point (to allow for evaporation), less dense than
water (to form an upper layer for ease of transfer)
• Acceptable for use in terms of safety and environmental policies
• Solvents to avoid: toluene (high boiling point), benzene (toxic),
chloroform (environmental reason)

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Properties of organic solvents

Properties of organic solvents

to be considered:
- Polarity
- Solubility in water
- Density
- Boiling point
- Safety

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Polarity of solvents

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Acid-base extraction
• Useful for isolating and purifying amines, carboxylic acids and
phenols.
• All these functional groups can be interconverted from non-ionic
organic-soluble forms to water-soluble ionic forms by changing the pH

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Acid-base extraction
• Ladder diagram is useful
• Ex: Phenol (pKa = 10). When extracted at pH = 7.4 (plasma)

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Back extraction
• We may use the acid-base form to selectively back-extract a mixture
from an organic phase into an aqueous phase
• The charged specie is more soluble in water phase
• Example:

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Back extraction

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Distribution ratio
• At a certain temperature, the ratio of concentration of the solute between
two phases is constant
• For a given molecule, this ratio is dependent on the nature of solvent 1
(biological fluid or water phase) and solvent 2 (organic phase)
• Higher D value, higher solute’s extraction from organic solvent

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Extraction yield

(= D)

(= D)

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Effect of volume of the solvent 2

• The concentration is the same,

Case 1
D • The distribution ratio is constant
• But MORE solute is extracted with a
larger volume of solvent

Amount = Concentration x Volume

Case 2
D

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 Effect of repetitive extraction
Question: Which give the better extraction (D=2)?
- Case 1: extraction 1 time with 1 volume of solvent 2

Case 2
- Case 2: extraction 1 time with 2 volume of solvent 2
- Case 3: extraction 2 time with 1 volume of solvent 2

Case 3
Extraction yield in each case?

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 Effect of repetitive extraction
Question: Which give the better extraction (D=2)?
- Case 1: extraction 1 time with 1 volume of solvent 2

Case 2
- Case 2: extraction 1 time with 2 volume of solvent 2
- Case 3: extraction 2 time with 1 volume of solvent 2

Calculate the extraction yield:

Case 3
- Case 1: % extracted = 66.6 %
- Case 2: % extracted = 80 %
- Case 3: % extracted = 89 %

More solute is extracted with repetitive extraction

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 Extraction of partially ionized molecules
Organic phase (2) Organic phase (2)

AH B

AH A - + H+ BH+ B + H+
Acid Base
Water phase (1) Water phase (1)

• The extraction depends on the pH value of the aqueous phase

• 2 equilibria:
o In the water phase between ionized and unionized species
o Between organic and water phase: only with unionized specie

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 How to calculate extraction yield
Weak acid Weak base
Organic phase

AH

AH A - + H+
Acid
Water phase

Organic phase pH = pKa

B
pH = pKa + 1

BH+ B + H+
Base pH = pKa - 1
Water phase
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Partition coefficient P and Distribution ratio D
• In medicinal chemistry, we usually use logP
• P is the partition coefficient between n-octanol and water of an
uncharged molecule
• P is obtained at equilibrium and room temperature
• Relationship between logD (for ionized species) and logP (for
unionized species)

Weak acid Weak base

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3/ Liquid-Solid extraction
• The solutes in aqueous phase are retained on a solid support
o Usually a column, or cartridge, flat disk
o Then eluted with an appropriate solvent
• Usually we want to remove from
o Salts
o Proteins
o Polar endogenous compounds

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Principle
4 main steps:
• Conditioning the column using an
appropriate solvent
• Adding sample: percolation of the
samples through the solid sorbent
• Washing impurities
• Eluting the analyte

To achieve an optimal SPE extraction:

• Choice of sorbent
• Using suitable solvents

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 On-line SPE
• SPE is easily coupled with an HPLC or HPGC
system
• Beneficial when the amount of solutes is limited
• Advantages:
o Sensitivity,
o Reliability
o Speed

Setup of an online SPE-HPLC system 68

What is a good extraction?
• Selectivity: remove almost unwanted species
• Constant and good recovery
• As easy as possible
• Amenable to automatization
• Safety concerns
o Use of non/less toxic solvents

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 Advantages of SPE
• Its main advantages:
o Amenable to automatization
o Small volume of solvents used
o Many choices of sorbents for specific needs
o Efficient and rapid
o Preventing emulsion and disposal of large quantity of organic solvents
• Inconveniences:
o Applied for relatively small volumes of solvent

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