M. Tech. (Biomedical technology, Faculty of Applied and Computer Science), Vaal University of Technology / Approximately 1% of the world’s population has epilepsy, the second most common
neurological disorder after stroke. In South Africa almost 1 in every 100 people has
epilepsy, affecting all ages. Levetiracetam (LEV), marketed as Keppra® is an
anticonvulsant drug used in the treatment of epilepsy. The daily dosage is 500 mg
twice daily with a maximum of 3000 mg. The therapeutic range of LEV is between
12-46 μg/ml. Therapeutic drug monitoring (TDM) should be considered for LEV in
patients with poor seizure control or long term treatment. TDM depends on accurate
drug concentration measurements. In order to provide an accurate measurement,
the High performance liquid chromatography (HPLC) method was developed,
compared with a commercially available kit, and the stability of the samples was
investigated.
Ethical approval was obtained from the Human Research Ethics Committee
(Medical), VUT (Ethics reference number: 2015024.4). The study was conducted
from January to October 2015. This study involved three groups of volunteers who
gave written consent. The first group were fifteen healthy MTech students in the
Biomedical Technology Department at the Vaal University of Technology (VUT).
Their blood samples were used for the analytical validation of the method and for the
stability studies over a 4 weeks period. The second group were six patients from
Pathcare Laboratories in Potchefstroom, Klerksdorp and Vereeniging who used
Levetiracetam. Their blood samples were used to investigate the influence of
different collection tubes as well as the handling and storage of samples on the LEV
concentration. The third group were forty four patients from Pathcare Laboratories,
Cape Town. Their blood samples were transported to Clinical Pharmacokinetic
Laboratory (CPL) for routine therapeutic drug monitoring analysis of LEV and used to
compare the newly developed HPLC method and the Commercial kit.
The HPLC method was successfully developed and validated to determine LEV in human plasma/serum samples. The calibration curves showed good linearity (r2 =
0,999) over the concentration range of 1 – 60 μg/ml. Accuracy, mean extraction
recovery, lower limit of detection (LLOD) and lower limit of quantification (LLOQ)
were 98-112%, 97,15% (±1,57), 0,5 and 1,0 μg/ml respectively, in plasma standards. The method was shown to be simple and fast, reproducible and effective for routine
laboratory analyses in the future.
The agreement between the newly developed method and the ClinRep® HPLC
complete commercial kit was the same and there was a statistical significant
correlation between the two methods (average r=0.999; p-value < 0.0001, F-test with
a true value =0). The method was much cheaper than the commercial kit, used less
sample (100 μl) and had a longer running time (15 minutes) to ensure no
endogenous interference. The costs of the developed method was 71-82% lower
than the three commercial kits available in South Africa.
Stability experiments were performed to evaluate the stability of LEV in human
plasma/serum, simulating the same conditions which occurred during study samples’
analyses. The % RSD was lower than 5% under all the conditions: freeze, fridge,
room temperature and auto sampler over the 4 week period. The results showed
that both LEV and the I.S (internal standard) were stable in human serum/plasma
under all these conditions.
The influence of five different collection tubes, Gold (SST Gel), Red, Purple (EDTA)Green (Heparin) and Blue (Sodium Citrate) was investigated. In two patients, decreased levels were observed in tubes containing blue (sodium citrate) and Green (Heparin). The decrease was not statistically significant. This is an important observation and is an indication that anticoagulants may cause some problems due to drug-protein binding and interference in the matrix effect.
A cost effective and reliable HPLC-method with minimal sample preparation time for
the routine determination of LEV in plasma/serum samples was developed. It was
also shown that the plasma/serum samples were stable at different temperatures
over a time period. The only collection tubes that may interfere with the
concentrations were the Green (Heparin) and Blue (Sodium Citrate) tubes.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:vut/oai:digiresearch.vut.ac.za:10352/314 |
| Date | 05 1900 |
| Creators | Engelbrecht, Lynette |
| Contributors | Grobler, C. J., Rheeders, M. |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | xx, 155, [45] : tables, diagrams, illustrations |
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