Chromatographic separations. The experimental conditions were chosen on the basis of literature reviews [7–13] and initial research for the separation of paclitaxel in
different conditions. The chosen stationary phase RP-18 had a low surface coverage of C-18 ligands [13]. Acetonitrile was used instead of methanol to improve the low-wavelength UV detection. Mobile phase strength was selected for better retention and resolution of paclitaxel to avoid noise and eluting of drifts in the free volume of the column. The overall chromatographic runtime was achieved within 10 min. Fi-gure 1 shows a typical chromatogram of paclitaxel (A) and its UV absorption spectra in deionized water (B).
Figure 1. The chromatographic separation of paclitaxel
Paklitaksela hromatogrāfiskais profils (A) un UV absorbcijas spektrs (B)
A
B
Ilva Nakurte, Ilze Plinte, Pēteris Mekšs, Žanete Zvirbule, Agnese Sudraba, Ivars Tolmanis, 87 Aija Line, Uldis Vikmanis, Viesturs Boka, Mārcis Leja. Development and Validation..
Figure 2. The specificity of the method for determination of paclitaxel in human serum Metodes specifiskums paklitaksela noteikšanai cilvēka asins serumā
Method validation. The first validation step was to check the specificity of the developed method. Figure 2 shows overlay chromatograms of deionized water (A), pure human serum (B), and paclitaxel standard solution (C). These chromatograms show that the developed method is specific because it does not have any troubleso-me factors.
Figure 3. The limit of detection and quantitation of paclitaxel in human serum Paklitaksela detektēšanas (A) un kvantitēšanas (B) robeža cilvēka asins serumā
Ilva Nakurte, Ilze Plinte, Pēteris Mekšs, Žanete Zvirbule, Agnese Sudraba, Ivars Tolmanis, 89 Aija Line, Uldis Vikmanis, Viesturs Boka, Mārcis Leja. Development and Validation..
Calibration curve was prepared with paclitaxel solutions with a specific concen-tration range (Table 1). The linear relationship for serum was obtained with good linearity. Figure 3 shows chromatogram samples for the limit of detection (LOD) (A) and the limit of quantitation (LOQ) (B) of paclitaxel in human serum. Relative standard deviation (RSD), %, between 3 parallel injections was 1.9% and 0.3%, res-pectively.
Table 1 Calibration curve and the limits of detection and quantitation of the promoted method
Concentration
Accuracy and precision results are summarized in Table 2. We got satisfactory accuracy results. The degree of accuracy for six results was ranging from 95.7 to 104.1%. Precision does not exceed RSD 0.2% for both retention time TR and pacli-taxel area. This part of validation gave us clear conviction that our proposed method can give accurate and precise results, with good recovery.
Table 2 Accuracy and precision of human serum
Run #1 #2 #3 #4 #5 #6 Mean RSD, %
Added concentration to human serum, 10.00 × 10-3 mg/ml Found
concen-tration 10.41 10.32 10.09 9.57 9.64 9.61 9.94 3.8
Accuracy, % 104.1 103.2 100.9 95.7 96.4 96.1 99.4 3.8
Precision, TR 7.20 7.21 7.20 7.20 7.18 7.17 7.19 0.2
Precision, area 1.77 × 106 1.78 ×
In Figure 4, the examined stability of the paclitaxel standard solution (A) and spiked human serum sample solution (B) at 25° C during 24 hours is shown. It was detected that both standard and sample solutions are not stable at room temperature.
RSD, %, between the first and last results for the standard solution is more or less acceptable − 2.9%, but degradation of sample solution occurs more often, RSD – 10.2%. The developed method needs freshly prepared solutions for analyses, which complicates these analyses. Further investigations with sample cooling would be ad-visable.
Conclusions
According to the results of the current study, we conclude that it is possible to separate and perform quantitative determination of paclitaxel both in standard
Figure 4. Stability of paclitaxel in solutions during 24 hours at 25 °C
Paklitaksela stabilitāte standartšķīdumā (A) un analizējamā parauga šķīdumā (B) 24 stundu ilgumā istabas temperatūrā
B A
solution and human serum using HPLC without any liquid-liquid extraction, in spite of paclitaxel being highly involved with blood proteins. The validation results dem-onstrate that paclitaxel concentrations can be accurately and precisely quantified in human serum, with a very low limit of detection 7.0 × 10-4 mg/ml. The proposed method is not completely adapted and the pilot validation needs improvement in sta-bility studies. Using the HPLC method with sample cooling, it is possible to avoid
Ilva Nakurte, Ilze Plinte, Pēteris Mekšs, Žanete Zvirbule, Agnese Sudraba, Ivars Tolmanis, 91 Aija Line, Uldis Vikmanis, Viesturs Boka, Mārcis Leja. Development and Validation..
degradation of sample and standard solutions. The proposed method could be ap-plied to determine paclitaxel in the blood samples of ovary cancer patents using Paxene as treatment medicament, and it might be useful in the further study of drug therapeutic interaction and monitoring of paclitaxel.
Acknowledgements
The work was supported by the research grant of the University of Latvia (2007/
ZP-83), State Research Programme in Health No. 8 (project No. 3). The authors would like to thank European Social Foundation for financial support and research fellowship (I. N.) to perform this research.
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Kopsavilkums
Paklitaksels ir viena no plašāk pazīstamajām bioaktīvām vielām, kas iegūta no īves koka un ko izmanto medicīnā kā pretvēža līdzekli apmēram 22% vēža gadījumu. Paklitakselam ir izstrādātas analītiskās noteikšanas metodes, bet katrā zāļu formā bez aktīvās vielas ir vēl citas sastāvdaļas, kas var ietekmēt paklitaksela farmakokinētiskos rādītājus. Tāpēc ikviena zāļu forma ir jāpārbauda klīniskos pētījumos, un katrai jaunai zāļu formai ir jāizstrādā aktīvās vielas un tās galveno metabolītu noteikšanas metodes cilvēka asins serumā. Šī darba mērķis
bija validēt ātru, ērtu un drošu analītisku metodi kvalitatīvai un kvantitatīvai paklitaksela noteikšanai ar apgrieztās fāzes augstefektīvās šķidruma hromatogrāfijas metodi to cilvēku asins serumā, kuru ārstēšanai izmanto zāļu formu Paxene bez īpašas parauga iepriekšējas sagatavošanas. Šī metode tika veiksmīgi validēta, un ir ieteikts to izmantot klīniskajos pētījumos iesaistīto pacientu terapeitiskam monitoringam.
LATVIjAS UNIVERSITāTES RAKSTI. 2008, 735. sēj.:
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