Chapter 29. Quantitative Chemical
Analysis, Daniel C. Harris, 6th Edition,
New to this edition and a very important topic
in industry today.
GLP/GMP in the pharmaceutical industry. Other
quality standards ISO, CE and other quality
How do we know that we have done a good
job in lab? We are well trained scientists so
we should be doing good work.
This is where Quality Assurance comes in.
It will vary from lab to lab but it should be
something that is taken very seriously.
Why has this become so important?
Let us look at the next three examples
There are many testing labs that one can
have analyses done at. Let’s see how they
Pb in polyethylene film (lab self
Pb in River Water (all having a
quality management system)
Pb in River Water (National
QC / QA
Quality Control – measures taken ensure that
there is accuracy and precision in an
Quality Assurance – Assessment that the
quality standards have been met.
In Pharmaceuticals this is usually the process
of verifying the product quality. That the
products meets specification.
That the label claim is correct. ( 1 mg table)
That impurities are below certain limits
That raw materials are in manufacturer specs
That returned materials are within specs
Governed by detailed procedures.
Called SOPs – Standard Operating Procedures
There will be SOPs for 1) Equipment maintenance
and repair, 2) Method Development and
Validation, 3) Data tracking and record keeping, 4)
Computer validation and data issues, 5) Setting of
shelf life and other lab management issues, 6)
Staff training requirements, 7) chain of custody of
samples etc. etc. etc.
The real issue is record keeping and adherence to
the procedures of the lab.
An independent function within the company
to assure that the QC functions are working
They hold routine audits and internal
They will be the staff that host visits from the
FDA and coordinate the FDA site visit (every
They are inspected first
GLP / GMP
Good Laboratory and Good
Other agencies have different rules.
EPA will certify labs doing
environmental tests. Many of these
tests are specified and must be followed
When rules are changed this might
require that better methods be
Perhaps with better detection limits or
One must work to get the best
information from the resources at hand.
It is not required that the best possible
analysis be carried out. Only that it provide
the data required, meet the data quality
Cost considerations of course are important
here. This must be considered when
developing new methods such as new HPLC
The process of proving that the analytical method is
acceptable for the intended purpose. (Issues to
Limit of detection
Limit of quantization
The ability to distinguish the analyte from
everything else that might be in the sample.
Lets look at a separation for the drug
Cefotaxime by MEKC.
The drug is seen with the impurities that
might show up in an assay.
Ideally you should get baseline resolution.
This is when R > 1.5.
Failing this you might state that the unresolved
impurities at their maximum level not effect the
assay of the drug by more than 0.50%
A impurity assay might be specified with all
expected impurities > 0.1% be resolved
from the major component.
Just were do you get the impurities to test?
What is likely to be present in a drug formulation of bulk
For a compound like aspirin this is fairly simple. We know what
things to expect since it has been around for a long time.
What about an NCE?
This is not a trivial challenge.
Subject the compound to heat (solid phase), heat (in solution),
light, humidity, acidic media, basic media and oxidants to the
point of consuming about 20% of the parent.
Add Synthetic starting materials, by-products, synthesis
intermediates and known synthesis degradation products.
Add all expected excipients.
For bulk component you will want you standard curve
to have five standards spanning 0.5 to 1.5 expected
range. Include a blank
For impurity assay you will wish you standards to
span 0.05% to 2% (wt/wt). Given that impurities
might fall in the 0.5% to 1% range.
These ranges would be fixed in the SOP for the
developed analysis and based on the product
We have discussed this.
R2 is a poor measure as we have stated
Look at residuals
For zero intercept
Bulk assay - 2% from the target value of the
Impurity assay – 10% of the target value of the
Analyze – Standard Reference Materials (if available)
Results of two or more different analytical methods.
Blank sample spiked with analyte. Ranges as in
Spikes should be recovered at (100 + 2)% for major
components and 0.1% absolute or + 10% relative for
Expressed as relative standard deviations for
at least 10 experiments.
Instrument precision (< 1%)
Intra-assay precision – prep each sample from
start, same person, same day. (< 2%)
Intermediate precision (Ruggedness) – Different
people, different days and different instruments.
Interlaboratory precision is also called just
reproducibility (3% to perhaps 40% or more)
(1 0.5 log C )
CV (%) 2
Where C is the weight fraction
The range is the concentration interval where
linearity, accuracy and precision are all
Limits of Detection and Quantitation
Also called the lower limit of detection.
Smallest quantity that gives a signal
significantly different from the blank.
This term had lead to much disagreement
Finding the detection limit.
Prepare a sample that is 1 to 5 larger than
the estimated detection limit.
Measure the signal for n replicate analyses (n
Calculate s for this data set.
Measure the signal from n replicate analyses
of the blank. (n > 7) Calculate the mean
Finding the detection limit.
ydl = yblank + ts
Where t is the Student’s t value for degrees of
freedom at 98% confidence.
This gives you the signal detection limit.
Concentration Detection Limit.
Minimum detectable concentration = (ts)/slopecalib
Limit of Quantitation
We can find the analyte at this low end but it
would be difficult to quantify it accurately so
we have another term
Limit of Quantitation.
yloq = yblank + 10s
Limit of Detection (Alternate way)
ydl = yblank + 3s
From Least Squares Line.
Signal detection limit
ydl = b + 3sy
Instrument detection limit
Method detection limit
A level below which you may report not
detected. Regulatory specified.
This level might be different in different matrix
The ability for a method to be unaffected by
Shelf life of solutions used
SOP directs how you keep an assay under
Directs how samples are handled and stored.
Will dictate how and when the method should
Can be purchased from NIST.
Must be handled with care.
Care should be taken with matrix effects.
Compound used to make up solutions should
have no analyte.
Elemental purity measured on nines scale.
Four nines is 0.9999 pure. Only in terms of other
metal and not other contaminants.
You may elect to purchase NIST traced
There are many pitfalls to avoid and many
are discussed on page 732.
Analysis of blanks can be very informative.
Will detect carry over.
Method Blank – all steps except addition of
Field Blank – all steps including being taken
to the field.
Quality Control Samples
A sample that is known and analyzed along
with the rest of the samples and standards.
Blind samples are best.
Collaborative and round robin testing will help
assess analysis proficiency.
Plot the results for a process or an analytical
procedure. (with your quality control
When do you shut down.
Single observation outside action lines
2 out of 3 consecutive measurements between the
warning and action lines
7 consecutive measurement all or above the center
6 consecutive measurements all increasing or
14 consecutive measurements alternating up and
An obvious nonrandom pattern.