The biggest problem we have ever had with the PicoTag unit is the Mininert
valve caps used to seal the hydrolysis tubes. They have a tendency to crack
and leak, with replacement caps at over $100 EACH to replace. Even if they
test ok under vacuum at the beginning of the hydrolysis, they can leak
slightly during heating. Waters has been giving us credit and replacing
broken caps if they fail within the first month or so of use, but it is still
frustrating to lose a set of samples due to leaking caps. One characteristic
sign of leaked caps is when you do not hear the "psst" of pressure change as
you open the cap's valve after the tube has cooled. We have not investigated
microwave hydrolysis, which is a method that is an option.
In 1994, Augustine Smith gave a workshop presentation on Amino Acid
Validation at the ABRF meeting. One of our newsletters from that time should
have a summary in it. Here are some of the issues she covered:
For validation of amino acid analysis, you can run trials of the sample at
various hydrolysis times (like you would for a time-zero study). You could
do different hydrolysis temps, too, if you felt that temperature would be a
critical variable, ie hard to control. For microwave, other variables might
be critical. A plot of the individual AA pmol recoveries vs time (or other
variable) would show you where the stable range is for each AA. Comparing
the plots of all AAs would allow you to choose one timepoint that is most
favorable to the majority of them. You will of course have lower recoveries
of some of them (Ser, Thr, Met) but you should expect that.
You might also want to validate the appropriate sample load by doing a
linearity study. Test 5-7 levels of the sample, hydrolyzing at the optimal
condition you determined above. Plot the recoveries of each AA and do
linear regression on each to obtain the coefficient of determination. The
range where r squared is greater than 0.98 or so is the linear range for each
AA. Again, determine the "consensus range" for the majority of AAs to yield
the working linear range for that sample in that buffer.
If you have a troublesome buffer (ie one that contains detergents or other
agents), the effect of the buffer components should also be investigated.
You can do this by spiking in different concentrations of these elements then
comparing the effects on AA recoveries and HPLC chromatographic
performance. You can then determine how sensitive the analysis will be to
variations in the buffer constituents. Ideally, of course, it should be
pretty rugged to minor fluctuations in composition.
Once you have established the appropriate hydrolysis conditions, sample load,
and buffer effects, you should have the finalized method. You can then do
experiments to determine precision and accuracy of the method. From this
information, you will get data to support the expected performance of the
method for the specific sample you are validating. You should then write
this method as a protocol, then test the final protocol by running several
trials (preferably with separate lots or batches) of the sample, and compare
the results obtained to those you expected. If they meet your expectations,
the method is validated. If they do not, you need to investigate why they
are different. You may need to do some re-development in order to get the
method where you need it. You should not consider the method validated until
and unless it can perform as expected when following the final written
protocol. Once it is validated, you should not make any further changes to
the protocol unless you evaluate the effects the change(s) might have on the
method's performance.
There are many excellent references on method validation, most significantly
the International Conference on Harmonisation Guidelines for Method
Validation. There are two parts, both available on the Internet. One way to
find them is to go to www.fda.gov and search the Federal Register for "ICH
Guidelines". There are several guidelines on various topics, but two on
method validation. These are very general, but are the benchmark against
which regulatory agencies are evaluating method validations.
A very easy way to dive into the validation literature is to use an Internet
search engine and query "analytical + test + method + validation". I have
gotten direct links to ICH and FDA documents, as well as websites with
validation info (like Waters) and original publications.
Also, there have been some terrific articles published on process and test
method issues in the journal BioPharm, a free publication from Advanstar
Press. Call 888-527-7088 for info on back-issues and subscriptions.
I hope this helps. Please feel free to contact me with any other questions.
Nadine Ritter
Abbott Diagnostics Division
Nadine.Ritter@add.ssw.abbott.com