Reverse-phase high performance liquid chromatography for the determination of tiopronin in human plasma after derivatization with p-bromophenacyl bromide
In the study, we developed a simple, rapid and sensitive method for the determination of tiopronin (TP) in human plasma, which was based on derivatization with p-bromophenacyl bromide ( p-BPB) followed by liquid–liquid extraction and reverse-phase HPLC-UV detection. For the first time, the p-BPB was...
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Published in: | Analytica chimica acta Vol. 565; no. 2; pp. 178 - 182 |
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Main Authors: | , , , , |
Format: | Journal Article |
Language: | English |
Published: |
Amsterdam
Elsevier B.V
21-04-2006
Elsevier |
Subjects: | |
Online Access: | Get full text |
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Summary: | In the study, we developed a simple, rapid and sensitive method for the determination of tiopronin (TP) in human plasma, which was based on derivatization with
p-bromophenacyl bromide (
p-BPB) followed by liquid–liquid extraction and reverse-phase HPLC-UV detection. For the first time, the
p-BPB was introduced into the derivatization of TP. The thiol group of TP was trapped with
p-BPB to form a TP-
p-BPB adduct, which can be very suitable for UV detection. From acidified plasma samples, the derivatized TP was extracted with 5
mL dichloromethane. Effective chromatographic separation was achieved using a C18 column (DIAMONSIL 150
mm
×
4
mm i.d., 5
μm) based on an acetonitrile–water–trifluoroacetic acid (40:59.88:0.12, v/v/v) elution at a flow-rate of 1
mL/min. The IS and the derivatized TP were detected at 263
nm. No endogenous substances were found to interfere. The limit of quantification for derivatized TP (TP-
p-BPB) in plasma was 40
ng/mL. The calibration curve for the derivatized TP showed linearity in the range 0.04–4
μg/mL with a regression coefficient corresponding to 0.9991 and the coefficient of the variation of the points of the calibration curve being lower than 10%. Extraction recoveries of the derivatized TP in plasma were greater than 72%. The method was suitably validated and successfully applied to determination of TP in human plasma samples. |
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ISSN: | 0003-2670 1873-4324 |
DOI: | 10.1016/j.aca.2006.02.049 |