Semi-automatic liquid chromatographic analysis of olpadronate in urine and serum using derivatization with (9-fluorenylmethyl)chloroformate.
The semi-automatic bioanalytical assays for olpadronate [(3-dimethylamino-1-hydroxypropylidene)bisphosphonate] involves a protein precipitation with trichloroacetic acid and a double co-precipitation with calcium phosphate for serum samples and a triple calcium co-precipitation for urine samples. These manual procedures are followed by an automated solid-phase extraction on a cation-exchange phase. The procedure is continued either directly, at high olpadronate levels in urine, or after off-line evaporation under nitrogen and reconstitution in water on the same robotic workstation. The continued automatic procedure comprehends derivatization with (9-fluorenylmethyl)chloroformate, ion-pair liquid-liquid extraction and ion-pair HPLC with fluorescence detection at 274/307 nm. The intra- and inter-day precisions for urine and serum samples are typically in the 5-8% range for different olpadronate concentrations [levels near the lower limit of quantification (LLQ) excluded]. The LLQ is 5 ng/ml olpadronate for a 2.5-ml urine sample and 10 ng/ml for a 1-ml serum sample, respectively.
Sparidans
R W
RW
Leiden University Medical Center, Department of Clinical Pharmacy and Toxicology, Leiden, The Netherlands. r.w.sparidans@pharm.uu.nl
den Hartig
J
J
Cremers
S
S
Beijnen
J H
JH
Vermeij
P
P
D001331:Automation; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods; D004164:Diphosphonates / Q000032:analysis* / Q000097:blood / Q000652:urine; D005449:Fluorenes / Q000737:chemistry*; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry*; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence
D004164:Diphosphonates; D005449:Fluorenes; D007202:Indicators and Reagents; C055774:olpadronic acid; C054007:1-(9-fluorenyl)methyl chloroformate