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Global Translational Medicine HPLC-UV lacosamide quantification

For both precision analyses, a CV% of < 15% was used 2.8. Lacosamide quantification in patients
as the acceptance criterion. Lacosamide quantification, utilizing the previously
2.7.4. Accuracy validated HPLC technique as described, was carried out on
samples collected from five patients prescribed lacosamide
Accuracy was evaluated by calculating the percentage for the treatment of epilepsy. Blood samples were collected
of recovery. Triplicate samples of lacosamide in plasma during steady-state conditions while undergoing treatment.
were prepared at three different concentrations: 5 µg/ml, The first sample was collected before drug intake (trough
10 µg/ml, and 15 µg/ml. These samples were processed level), and the second sample was collected 2 h after dosing
according to the described technique and injected in (peak level). Informed consent was obtained from all
triplicate into the chromatograph. patients as part of routine clinical care.
The percentage of recovery was determined as the ratio
between the measured concentration value and the theoretical 3. Results
concentration value. To analyze the results, a Student’s t-test 3.1. Optimization
with a significance level of P = 0.05 was employed. Data The following stationary phases were evaluated: a
variability was investigated using the CV% and Cochran’s 150 × 4.6 mm, 2.6 µm Kinetex-Phenomenex C8 column,
G test. The latter was used to determine whether the a 250 × 4.6 mm, 5 µm Thermo C8 column, and a
concentration level had any influence on the results.
150 × 4.6 mm, 2.6 µm Kinetex-Phenomenex C18 column.
2.7.5. Sensitivity Among these, only the Kinetex-Phenomenex C18 column
achieved adequate resolution of both peaks.
Sensitivity can be assessed by determining the Limit of
Detection (LOD) and the Limit of Quantitation (LOQ). The chromatographic conditions employed included
LOD and LOQ were calculated using Equation 1: a 70:30 ratio of 10 mM phosphate buffer (pH = 3.5) and
KS× 0/b (I) acetonitrile. This composition facilitated lacosamide
elution within 10 min without interferers. The column
Where k = 3 for LOD and 10 for LOQ, S0 represents the temperature was set at 30°C; deviations from this
standard deviation of the y-intercept, and b is the slope of temperature resulted in system pressure variations
the calibration curve. (e.g., increased pressure at 25°C and elution front issues at
40°C). Flow rates of 0.6 mL/min and 0.8 mL/min, except for
2.7.6. Stability the working pressure (2440 psi vs. 2880 psi, respectively).
The stability of the analyte of interest was evaluated in Nevertheless, employing a flow rate of 0.6 mL/min proves
three sample stability assays: advantageous in preventing premature aging of a column.
In addition, adjusting the pH of the phosphate buffer to
(i) Short-term and long-term sample stability: 3.0 contributes to stability and minimizes peak broadening
Unprocessed samples were stored under various compared to higher pH values .
[19]
conditions, including room temperature (for 24 h,
48 h, and 72 h), in the refrigerator at 4°C (for 24 h, Several internal standards, such as aminopyrine,
48 h, and 72 h), in a freezer at −20°C (for 24 h, 48 h, benserazide, acetazolamide, sulfanilamide, caffeine, and
72 h, 7 days, and 30 days), and in a freezer at −80°C propranolol, were examined. While most of them eluted very
(1 week, 1 month, 6 months, 1 year, and 2 years). close to the front peak, propranolol was ultimately selected.
(ii) Post-processed sample stability: Samples were The wavelengths under evaluation were 210 nm,
processed according to the analytical method and 260 nm, and 290 nm, corresponding to local maxima in
then stored under different conditions, including the UV absorption spectra. Optimal sensitivity for both
room temperature, in a refrigerator at 4°C, and in the molecules was observed at 210 nm.
freezer at −20°C for 24 h, 48 h, 72 h, and 1 week.
In the context of sample treatment, various methods
(iii) Freeze-thaw stability: Samples were subjected to three were employed. Protein precipitation was tested using
freeze-thaw cycles (freezing in a −20°C and -80°C perchloric acid at different dilutions (one-third, one-fourth,
freezer for 24 h each, followed by thawing).
and one-fifth), in addition to acetonitrile and methanol.
In each of these assays, the obtained lacosamide However, these preparations led to an underestimation
concentration was compared with the initial (time zero) of the true concentration and failed to achieve adequate
concentration. Samples were considered stable if their resolution. Higher concentrations of precipitant
final time concentration remained above 90% of the initial resulted in tailing, while lower proportions introduced
concentration. more plasma interferers. Subsequently, alternative liquid-

Volume 2 Issue 3 (2023) 4 https://doi.org/10.36922/gtm.1265