TY - JOUR
T1 - High performance liquid chromatographic determination of the stabilized cyclophosphamide metabolite 4-hydroxycyclophosphamide in plasma and red blood cells
AU - Huitema, A. D.R.
AU - Tibben, M. M.
AU - Kerbusch, T.
AU - Kettenes-Van Den Bosch, J. J.
AU - Rodenhuis, S.
AU - Beijnen, J. H.
N1 - Funding Information:
This research was supported with a grant from the Dutch Cancer Society (project NKI 97-1439). The authors would like to thank Dr. R. van Gijn and Dr. H. Rosing for their technical assistance and suggestions and Ms. J.M.A. Damen for running the mass spectra.
PY - 2000
Y1 - 2000
N2 - A reversed-phase high performance liquid chromatographic (HPLC) method for the determination of the activated cyclophosphamide (CP) metabolite 4- hydroxycyclophosphamide (4-OHCP) in human plasma and red blood cells is described. 4-OHCP is very unstable in biological matrices. Therefore, it was treated, immediately after sampling, with semicarbazide to form a stable semicarbazone derivative, which was identified with electron spray mass spectrometry. The derivative was analysed with HPLC with ultraviolet (UV) detection at 230 nm. Sample pretreatment consisted of a liquid-liquid extraction with ethyl acetate, the chromatography system was a 25 cm C8 column (particle size 5 μm) with a mobile phase of acetonitrile-0.025 M potassium phosphate buffer (15:85 v/v) pH 6.0. After assay optimisation, the method was validated and stability studies were carried out. The method proved linear in clinically relevant concentrations (50-5000 ng/mL). The lower limit of quantitation was 50 ng/mL. Accuracy and precision were below 7% over the full concentration range. A calibration curve in plasma can also be used for the quantification of 4-OHCP in red blood cells. The derivative was found to be stable for at least 11 months when stored at -70 °C. A pharmacokinetic pilot study in a patient treated with 1000 mg/m2 CP in combination with thio TEPA and carboplatin demonstrated the applicability of the assay for the determination of 4-OHCP in plasma and red blood cells.
AB - A reversed-phase high performance liquid chromatographic (HPLC) method for the determination of the activated cyclophosphamide (CP) metabolite 4- hydroxycyclophosphamide (4-OHCP) in human plasma and red blood cells is described. 4-OHCP is very unstable in biological matrices. Therefore, it was treated, immediately after sampling, with semicarbazide to form a stable semicarbazone derivative, which was identified with electron spray mass spectrometry. The derivative was analysed with HPLC with ultraviolet (UV) detection at 230 nm. Sample pretreatment consisted of a liquid-liquid extraction with ethyl acetate, the chromatography system was a 25 cm C8 column (particle size 5 μm) with a mobile phase of acetonitrile-0.025 M potassium phosphate buffer (15:85 v/v) pH 6.0. After assay optimisation, the method was validated and stability studies were carried out. The method proved linear in clinically relevant concentrations (50-5000 ng/mL). The lower limit of quantitation was 50 ng/mL. Accuracy and precision were below 7% over the full concentration range. A calibration curve in plasma can also be used for the quantification of 4-OHCP in red blood cells. The derivative was found to be stable for at least 11 months when stored at -70 °C. A pharmacokinetic pilot study in a patient treated with 1000 mg/m2 CP in combination with thio TEPA and carboplatin demonstrated the applicability of the assay for the determination of 4-OHCP in plasma and red blood cells.
UR - http://www.scopus.com/inward/record.url?scp=0034125970&partnerID=8YFLogxK
U2 - 10.1081/JLC-100100448
DO - 10.1081/JLC-100100448
M3 - Article
AN - SCOPUS:0034125970
SN - 1082-6076
VL - 23
SP - 1725
EP - 1744
JO - Journal of Liquid Chromatography and Related Technologies
JF - Journal of Liquid Chromatography and Related Technologies
IS - 11
ER -