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L-carnitine is a high polarity compound, and lack
of retention by reversed-phase HPLC. Ion pair
methods can increase the retention time of L-carnitine,
but still not enough. In complex nutriceutical
formulation the L-carnitine peak is easily to
be interfered by some other ingredients.Currently
we developed an Ion-Exchange HPLC method which
xhibits a much longer retention for L-carnitine.
The k' is about 6~10, dependant on pH an ionic
strength of the mobile phase.
L- carnitine, as its structure shows, has low
UV absorption. Its natural degradant Crotonoylbetain
on the other hand has two conjugated double-bonds,
which gives it about 200 times stronger UV absorption
than the parent compound. Chemical structures
of L-carnitine and crotonoylbetaine are shown
below:
 
Theoretically, a 0.5% of crotonoylbetaine as
impurity existing in the L-carnitine (99.0% purity)
could double the measured amount of L-carnitine,
if these two peaks are not fully separated from
each other. An example of such method would be
one that was published in the USP23.
We urge raw material suppliers and finished product
manufacturers look into the current methodology
used in the quality control testing of your L-carnitine.
Complete separation of the two compounds is imperative
for two reasons:
- Trace amount of degradant, even at 0.01%
content, would falsely increase the result on
L-carnitine by 2% when they are not separated.
- L-carnitine stored at room temperature for
one year has shown substantial increase in crotonoylbetaine.
Therefore, proper testing of L-carnitine could
assure that already degraded material would
not give a falsely "better" result
than the fresh and good products.
- We do not know the biological activity of
the degradant, therefore it would be better
to minimize its presence by testing the raw
material properly.
Our Ion-exchange HPLC methods not only gives
excellent separation within a reasonably long
retention time, it also can be easily adjusted
for the elution time by varying the pH and ionic
strength of the mobile phase, when a complex formulation
requires to do so.
The method LOD (Limit of Detection)
= 0.00228mg/ml
LOQ (Limit of Quantitation) = 0.00762 mg/ml
Linearity spans a range of 104
Spike recovery ranged from 98.1% to 101.0%
Typical chromatogram: a raw material with degradant.
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