Rapid and sensitive analysis of baclofen by high-performance liquid chromatography with UV-Vis and FD detection

Abstract

Summary. Two rapid, sensitive and reproducible methods for the determination of baclofen(BAL) in urine and plasma based on high-performance liquid chromatography (HPLC) with UV-vis and fluorescent detection, respectively, were developed for the first time using a new synthesized fluorescent label, 6-oxy-(N-succinimidylacetate)-9-(2'-methoxycarbonyl) fluorescein (SAMF). The optimal derivatization yield was achieved in borate buffer (pH 8.0) for 15 min at room temperature (25 °C). With a mixture of methanol and water containing 5 mmol L −1 sodium citrate buffer (pH 5.0) as mobile phase, BAL was determined at λ = 455 nm with UV-vis detection and at λ ex /λ em = 488/520 nm with FD detection. The detection limits are 1.065 × 10 −3 mg mL −1 and 1.065 × 10 −2 mg mL −1 with HPLC-UV-vis and HPLC-FD, respectively. The proposed method has been successfully applied to the analysis of BAL in human urine and plasma samples. The established method is rapid (15 min of derivatization process and 10 min of chromatographic run), reproducible and sensitive.

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“…Then, centrifugation at 8000 rpm for 15 min and then the supernatant liquid was collected and finally filtered through a 0.45 μ millipore membrane filter. [13] The resultant plasma solution (200 μT) followed by adding 10 μ0 of internal standard and then 20 μl of the final mixture (210 μo) was injected into the HPLC column.…”

“…[13] There are several small unknown peaks of plasma and the retention time of an unknown peak was near to the baclofen peak that not affect the separation. To make them far separated, the separation was studied carefully by increasing the content of methanol in methanol-water mixture from 50% to 55% and using a column of 300 mm instead of 200 mm.…”

“…Analysis was run at a flow rate of 2.0 ml/min for 10 minutes, the column was kept at room temperature, and the detection wavelength was 220 nm. [13] …”

“…The method was fully validated with respect to adequate sensitivity, linearity, recovery, accuracy, and precision (within-day and between-day). [13] …”

Enantioresolution of (RS)‐baclofen by liquid chromatography: A review

“…To date, Bac has been determined in various biological materials using techniques based on gas (Giovanni & d'Aloja, ; Kochak & Hunc, ) or liquid (Ban, Park, & Kim, ; Fraser, MacNeil, & Isner, ; Tosunoglu & Ersoy, ) chromatography, liquid chromatography–tandem mass spectrometry (Di Rago et al, ; Dukova et al, ; Flardh & Jacobson, ; Sørensen & Hasselstrøm, ), HPLC with UV–vis and fluorescent detection (Cao & Li, ; Harrison, Tonkin, & Mclean, ), HPLC‐fluorescence detection after derivatization (Tosunoglu & Ersoy, ; Wuis, Dirks, Vree, & van der Kleyn, ) and HPLC‐electrochemical detection (Millerioux, Brault, Gualano, & Mignot, ). In addition, separation of naphthalene‐2,3‐dicarboxaldehyde‐derivatves of ( RS )‐Bac has been obtained (Chang & Yang, ; Chang, Zheng, & Chen, ; Chiang, Chang, & Whang, , ) using capillary electrophoresis (CE), and a chiral capillary column was employed to separate N ‐heptafluorobutyryl isobutyl ester derivatives of ( RS )‐Bac in human plasma and urine (Sioufi, Kaiser, Leroux, & Dubois, ).…”