Page 85 - GTM-2-3

Global Translational Medicine HPLC-UV lacosamide quantification

For the system suitability test, chromatographic different samples. Each sample was processed as a blank
parameters such as retention time, capacity factor, tailing to evaluate the presence of endogenous substances that
factor, resolution, and column efficiency were assessed. might elute with lacosamide or the internal standard.
These parameters were chosen to determine the method’s (ii) Analysis of forced degradation: Lacosamide solutions,
acceptability and were calculated according to the USP devoid of the internal standard, were subjected to
[14]
guidelines , utilizing typical chromatograms as the basis various reactions (oxidation, acid hydrolysis, alkaline
for evaluation. hydrolysis, aqueous hydrolysis, and photolytic
degradation). The resulting solutions were then used
2.5. Working solutions to prepare solutions with known concentration levels.
Approximately 50 mg of lacosamide reference standard (iii) Analysis of specificity against other drugs: Considering
was accurately weighed and placed into a 50 ml volumetric the possibility of polypharmacy in patients, some
flask. It was then dissolved and brought to volume with commonly used drugs, such as ibuprofen, paracetamol,
MilliQ water, creating a stock solution with a concentration diclofenac, and clonazepam, among others, were
of 1.0 mg/ml solution. Further dilutions were prepared evaluated for specificity.
from this stock solution. 2.7.2. Linearity
In a 100 ml volumetric flask, exactly 50 mg of propranolol To establish a linear-response range of the instrument within
was added and brought to volume with acetonitrile. the clinically relevant concentration range (10 – 20 µg/mL),
Subsequently, it was further diluted with MilliQ water to obtain six lacosamide levels (2.5 µg/ml, 5 µg/ml, 10 µg/ml, 15 µg/ml,
a concentration of 50 µg/ml (internal standard solution). 20 µg/ml, and 30 µg/ml) were prepared in triplicate, with

2.6. Samples and controls the addition of the internal standard for all samples. The
calibration curve for lacosamide was constructed by plotting
Freshly obtained plasma samples were stored at −20°C the area ratio (lacosamide area/propranolol area) against the
for subsequent use. A plasma pool was then generated lacosamide concentration.
to prepare control solutions with predetermined
concentrations of lacosamide, using the stock solution Subsequently, the calculation of correlation coefficient
2
at the concentrations of 2.5 µg/ml, 5 µg/ml, 10 µg/ml, (r) and coefficient of determination (R ) was performed,
15 µg/ml, 20 µg/ml, and 30 µg/ml. followed by a linear regression analysis. This analysis
included an assessment of the response factor, an evaluation
To each solution, 200 µL aliquots were taken, and 10 µL of the statistical significance of the slope variance (Sb), and
of an internal standard was added. The resulting mixture a test for proportionality (calculation of the relative zero
was vortexed for 15 s. Subsequently, 2 mL of ethyl acetate intercept, calculation of its variance, and calculation of its
was added as the extraction solvent, followed by 1 min of confidence interval [CI]).
vortexing and centrifugation at 3500 rpm for 10 min. The
resulting organic phase was transferred to a glass tube and 2.7.3. Precision
subjected to evaporation to dryness within a thermostatic This parameter was evaluated in two ways:
bath set at 37°C , all conducted under an extraction hood. (i) Repeatability: Six independent lacosamide samples
Finally, the dried extract was reconstituted in 200 µL of the were prepared at a concentration of 10 µg/ml
mobile phase, and 20 µL of this solution was injected into (considered as 100% concentration). The internal
the chromatographic system.
standard was added to each sample, and they were
2.7. Validation analyzed in triplicate. The repeatability of the
bioanalytical method was determined by calculating
The validation parameters evaluated included specificity, the coefficient of variation (CV%) of the response
linearity, repeatability, intermediate precision, factors for the six evaluated samples.
accuracy, and stability, with adherence to the stipulated (ii) Intermediate precision: Two analysts quantified
international [15,16] and national guidelines. Data analysis lacosamide in two aliquots from the same sample on
[17]
was conducted using statistical software R . different days. Each analyst used a different instrument
[18]
2.7.1. Specificity and chromatographic column, and the samples
were processed following the technique explained
Specificity was evaluated through three tests: earlier. The intermediate precision of the method was
(i) Analysis of plasma interferents: Five plasma samples with determined by calculating the CV% of the lacosamide
varying characteristics, including normal appearance, concentrations obtained in the samples analyzed by
hemolysis, lipemia, and jaundice, were sourced from the two analysts.

Volume 2 Issue 3 (2023) 3 https://doi.org/10.36922/gtm.1265