Journal
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume 1064, Issue -, Pages 62-67Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jchromb.2017.09.010
Keywords
Carbidopa; LC-MS/MS; Plasma; Urine; Derivatization; 2,4-pentanedione
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The reliable quantification of carbidopa in biological samples at low concentrations is,challenging because of the polar and highly unstable nature of the compound. In this paper, LC-MS/MS methods are described for the determination of carbidopa in 50 mu L of human plasma and 25 mu L of human urine in the concentration ranges 1-1,000 ng/mL and 100-50,000 ng/mL, respectively. After a simple protein precipitation (plasma) or dilution (urine) step, carbidopa is derivatized at its hydrazine moiety by reaction for one hour with 2,4-pentanedione under acidic conditions and at 40 degrees C. The product is a relatively non-polar molecule that is suitable for reversed phase liquid chromatography (3.5 min run time) with detection by tandem mass spectrometry with electrospray ionization. A stable-isotope labeled internal standard is used for response normalizatibn. Precision, accuracy and selectivity of the methods meet the criteria of international guidelines for bioanalytical method validation. Acidification of urine to pH 1.5 and the addition of two anti-oxidants (5 mg/mL sodium metabisulfite and 1 mg/mL butylated hydroxytoluene) to plasma, in combination with sampling and analysis on ice and under yellow light, ensure sufficient stability of carbidopa. The methods were successfully used to determine plasma pharmacokinetics and urinary excretion of carbidopa in healthy volunteers after a single 37.5 mg oral dose.
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