Analysis of baclofen by capillary electrophoresis with laser-induced fluorescence detection

A new analytical method for baclofen (4-amino-3-p-chlorophenylbutyric acid) based on capillary electrophoretic separation and laser-induced fluorescence detection has been developed. Naphthalene-2,3-dicarboxaldehyde was used for precolumn derivatization of the non-fluorescent drug. Optimal separation and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH 9.5) and a He-Cd laser (excitation at 442 nm, emission at 500 nm). Linearity (r > or = 0.99) over three orders of magnitude was generally obtained and the concentration limit of detection was in the nanomolar level. Coupled with a simple cleanup procedure, the method was successfully applied to the analysis of baclofen in human plasma. Recovery of spiked baclofen in plasma was 98%. The relative standard deviation values on peak size and migration time were 7.9% and 0.4%, respectively. The limit of detection of baclofen in plasma was 10 ng/ml.     

Capillary electrophoresis of baclofen with argon-ion laser-induced fluorescence detection

A capillary electrophoretic method with laser-induced fluorescence detection for baclofen (4-amino-3-p-chlorophenylbutyric acid) has been developed. 6-Carboxyfluorescein succinimidyl ester was used for precolumn derivatization of the non-fluorescent drug. Optimal separation and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH 9.5) and an air-cooled argon-ion laser (excitation at 488 nm, emission at 520 nm). Linearity (r > or = 0.99) over three orders of magnitude was generally obtained and the lowest derivatizable concentration limit for baclofen in aqueous solution was 10 nM (2 ng baclofen/ml). Coupled with a simple clean up procedure, the method can be applied to the analysis of baclofen in human plasma at micromolar level. Recovery of spiked baclofen in plasma was 95%. The relative standard deviation values on peak size (0.5 microM level) and migration time were 8.2 and 1.0% (n=7), respectively. The limit of detection of baclofen in plasma was 0.1 microM (21 ng/ml).     

Sensitive method for the determination of baclofen in plasma by means of solid-phase extraction and liquid chromatography-tandem mass spectrometry.

A simple and sensitive method for the determination of baclofen in plasma is described. Baclofen and the internal standard, KM 08205, were isolated from plasma by solid-phase extraction using C18 material. After separation by reversed-phase liquid chromatography, the analytes were detected with tandem mass spectrometry. The extraction procedure was optimised regarding the solid-phase extraction material, the pH in the conditioning solution and the washing volume. The method was proven to be selective and sensitive with an absolute recovery of about 95%, a relative standard deviation below 5% and a limit of quantification of 10 nmol/1.     

Chiral analysis of baclofen by alpha-cyclodextrin-modified capillary electrophoresis and laser-induced fluorescence detection

An enantioselective method for baclofen (4-amino-3-p-chlorophenylbutyric acid) based on capillary electrophoresis (CE) separation and laser-induced fluorescence (LIF) detection has been developed. Naphthalene-2,3-dicarboxaldehyde (NDA) was used for precolumn derivatization of the nonfluorescent drug. alpha-Cyclodextrin (alpha-CD) was included in the buffer as a chiral selector for the separation of NDA-labeled S-(+)- and R-(-)-baclofen. Optimal resolution and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH 9.5) containing 7 mM alpha-CD and a He-Cd laser (lambda ex = 442 nm, lambda em = 500 nm). Combined with a simple cleanup procedure, this method can be applied to the analysis of baclofen enantiomers in human plasma. The relative standard deviation (RSD) values on peak areas of a plasma sample containing 1.0 microM racemic baclofen were 6.4 and 4.9% (n = 8) for the S-(+)- and R-(-)-enantiomer, respectively. The RSD value on migration times of both enantiomers was 0.5% (n = 8). Calibration graphs for S-(+)- and R-(-)-baclofen in plasma showed a good linearity (r > or = 0.999) in the concentration range of 0.1-2.0 microM. The limit of detection of baclofen in plasma was about 10 ng/mL.     

Liquid chromatography high resolution mass spectrometry for the determination of baclofen and its metabolites in plasma: Application to therapeutic drug monitoring

Baclofen is used to manage alcohol dependence. This study describes a simple method using liquid chromatography coupled to high‐resolution mass spectrometry (LC‐HR‐MS) developed in plasma samples. This method was optimized to allow quantification of baclofen and determination of metabolic ratio of its metabolites, an oxidative deaminated metabolite of baclofen (M1) and its glucuronide form (M2). The LC‐HR‐MS method on Exactive® apparatus is a newly developed method with all the advantages of high resolution in full‐scan mode for the quantification of baclofen and detection of its metabolites in plasma. The present assay provides a protein precipitation method starting with 100 μL plasma giving a wide polynomial dynamic range (R ² > 0.999) between 10 and 2000 ng/mL and a lower limit of quantitation of 3 ng/mL for baclofen. Intra‐ and inter‐day precisions were <8.1% and accuracies were between 91.2 and 103.3% for baclofen. No matrix effect was observed. The assay was successfully applied to 36 patients following baclofen administration. Plasma concentrations of baclofen were determined between 12.2 and 1399.9 ng/mL and metabolic ratios were estimated between 0.4 and 81.8% for M1 metabolite and on the order of 0.3% for M2 in two samples.     

Improved gas-liquid chromatographic method for the determination of baclofen in plasma and urine

A simple, rapid and sensitive assay for baclofen analysis has been developed. Baclofen and the internal standard are analyzed by gas-liquid chromatography with electron-capture detection after esterification of the carboxyl group to the butyl ester and acylation of the amino group to the pentafluoropropionylamide. Recovery from biological matrixes is accomplished by ion-pair extraction. The limit of quantitation of the entire assay as stated is about 10 ng/ml baclofen in plasma.     

Fluorodensitometric determination of baclofen

A fluorodensitometric method was developed for the determination of baclofen. The method is based on the reaction of baclofen with 4-chloro-7-nitrobenzofurazan and measurement of the fluorescence intensity at 540 nm after chromatographic separation on a silica-gel plate. A linear relationship was found between relative fluorescence intensity and concentration over the range 4-100 ng of baclofen per spot. The detection and determination limits are 0.4 and 4.0 ng of baclofen per spot, respectively. The method was applied to human plasma. The recovery of baclofen from plasma was 96.67%.     

A Sensitive Method for the Determination of Baclofen in Human CSF by High Performance Liquid Chromatography

Abstract

Baclofen (4-amino-3p-chlorophenylbutyric acid) is used clinically for the treatment of multiple sclerosis and other spastic conditions. The intrathecal route of administration is now prefered to the oral route. To optimize efficacy, the levels of the drug in CSF need to be monitored after intrathecal administration. In this paper, the authors describe a sensitive reverse phase high performance liquid chromatographic method for the determination of baclofen in human CSF. This assay employed cation exchange extraction, pre-column derivatization with PITC, and ultra-violet detection (254 nm). The method was shown to be highly sensitive (5 to 10 ng/ml). This method was compared to a method using direct spectrophotometric detection of baclofen at 220 nm after direct application of CSF samples onto the chromatograph without prior extraction.     

Determination of the S(+)- and R(-)-enantiomers of baclofen in plasma and urine by gas chromatography using a chiral fused-silica capillary column and an electron-capture detector

A sensitive and enantiospecific gas chromatographic method for the determination of the S(+)- and R(-)-enantiomers of baclofen (I and II) in plasma and urine has been developed and validated. The method is based on the complete resolution of the derivatized enantiomers on a chiral fused-silica capillary column. The hydrochloride salt of a (-)-fluoro analogue of baclofen (III.HCl) was used as the internal standard in plasma, the hydrochloride salt of a (+)-fluoro analogue of baclofen (IV.HCl) as the internal standard in urine. Rapid and convenient isolation of the compounds was achieved using reversed-phase Bond-Elut C18 columns. After elution, the compounds were converted into isobutyl esters and purified by base-specific solvent extraction. The isobutyl esters were then N-acylated with heptafluorobutyric anhydride. The derivatives were quantitated after separation on the chiral column using electron-capture detection. The analysis of spiked plasma and urine samples demonstrated the good accuracy and precision of the method, with limits of quantitation of 25 nmol/l for I and II in plasma and of 2 mumol/l for I and II in urine. The method appears to be suitable for use in pharmacokinetic studies of the enantiomers in plasma and urine from animals and man after administration of the racemic baclofen.     

Capillary electrophoresis of aminoglycosides with argon-ion laser-induced fluorescence detection

A new analytical method for aminoglycosides (kanamycin, bekanamycin, paromomycin and tobramycin) based on capillary electrophoretic separation and argon-ion laser-induced fluorescence detection has been developed. 6-Carboxyfluorescein succinimidyl ester (CFSE) was used for precolumn derivatization of the non-fluorescent aminoglycosides. Optimal separation and detection were obtained with an electrophoretic buffer of 30 mM sodium borate (pH 9.0) and an air-cooled argon-ion laser (excitation at 488 nm, emission at 520 nm). The concentration limits of detection in aqueous solution were 3.9-8.2 nM. Combined with a simple cleanup procedure, this method can be applied to the determination of aminoglycosides in human plasma. A calibration curve ranging from 0.15 to 30 microM was shown to be linear. The limits of detection of aminoglycosides in human plasma were between 14.4 and 24.0 nM. Recoveries of spiked aminoglycosides in human plasma were between 92 and 105%.     

Enantioseparation of baclofen with highly sulfated beta-cyclodextrin by capillary electrophoresis with laser-induced fluorescence detection

The enantioseparation of baclofen (4-amino-3-p-chlorophenylbutyric acid) was achieved by CE-LIF with highly sulfated beta-CD (HS-beta-CD) as chiral selector. Naphthalene-2,3-dicarboxaldehyde was used for the derivatization of nonfluorescent baclofen. HS-beta-CD (2%) containing 50 mM borate buffer at pH 9.5 was chosen as the optimal running electrolyte and applied to the analysis of baclofen enantiomers in human plasma. The linearity of calibration curves (R2 > or = 0.998) for R-(-) and S-(+)-baclofen was in the 0.1-2.0 microM concentration range. After a simple ACN-protein precipitation, the LOD of baclofen in plasma sample was found as low as 50 nM.     

Indirect resolution of baclofen enantiomers from pharmaceutical dosage form by reversed-phase liquid chromatography after derivatization with Marfey's reagent and its structural variants

A high-performance liquid chromatographic (HPLC) method was developed for chiral assay of baclofen enantiomers in pharmaceutical formulations using an indirect approach. Baclofen enantiomers were derivatized with Marfey's reagent (FDNP-L-Ala-NH2) and its structural variants FDNP-L-Phe-NH2, FDNP-L-Val-NH2, FDNP-L-Leu-NH2 and FDNP-L-Pro-NH2. The resultant diastereomers were separated on RP-TLC [triethylammonium phosphate buffer (pH 4.0, 50 mm)-acetonitrile, 50:50] and on a C18 column using a linear gradient (45 min) of acetonitrile and 0.01% aqueous trifluoroacetic acid (TFA) with UV detection at 340 nm. The differences in the retention times (Delta t R) of diastereomers due to the five chiral reagents were compared. The maximum and minimum difference in retention times between separated diastereomers was for FDNP-L-Leu-NH2 and FDNP-L-Pro-NH2, respectively. The effect of flow rate, acetonitrile content and TFA concentration on resolution was studied. The method was validated for linearity, repeatability, limit of detection and limit of quantification.     

Rapid and sensitive liquid chromatography-tandem mass spectrometry method for the estimation of nicorandil in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of nicorandil in human plasma. Nicorandil was extracted from human plasma using solid-phase extraction technique. Imipramine was used as the internal standard. A Betasil C18 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The method involves a rapid solid-phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection at nanogram levels. The proposed method has been validated for a linear range of 1.0-500.0 ng/mL with a correlation coefficient of > or =0.9993. The intra-run and inter-run precision and accuracy was within 10.0%. The overall recovery for nicorandil was 63.81%. The total run time was just 3.0 min.     

Developing drug literatures. 1. Bibliometrics of baclofen and dantrolene sodium.

The literatures of two antispastic drugs, baclofen and dantrolene sodium, were studied bibliometrically for their first decade and were found to be generally similar. Baclofen had 93 papers and dantrolene sodium had 70. About a quarter of the baclofen papers were in a foreign language, whereas almost all dantrolene sodium papers were in English. Baclofen literature had a lower nonscholarly content, but the scholarly increase, 5%, was the same for both. Both drugs had an average of 12 references per paper. The literatures of both drugs had a similar degree of internal cohesiveness; almost half of their papers referred to other papers in the same drug collection. The first human paper for baclofen was the second published; for dantrolene sodium, it was the eleventh. The distribution of journals carrying papers on each of these drugs followed Bradford's law. Two authors per paper was the average for each drug. The productivity of authors approximated Lotka's law for both drugs. About two-thirds of the papers of both drugs had a drug-word in their titles. The literature of both drugs contained about 15% legendary papers, typical of clinical pharmacology. The most intense papers, 15 for baclofen and 11 for dantrolene sodium, were identified, using citation, bibliographic coupling, and co-citation frequencies. A generalization predicts what might be expected from the literature of future antispastic drugs.     

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the estimation of amlodipine in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of amlodipine in human plasma. Amlodipine was extracted from human plasma by using a solid-phase extraction technique. Imipramine was used as the internal standard. A Hypersil BDS C18 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The method involves a rapid solid-phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection at sub-nanogram levels. The proposed method has been validated for a linear range of 0.1-10.0 ng/mL with correlation coefficient >or=0.9990. The intrarun and interrun precision and accuracy were within 10.0%. The overall recovery for amlodipine was 63.67%. Total run time was 3.2 min only.     

Rapid analysis of MMI270B, an inhibitor of matrix metalloproteases in human plasma by liquid chromatography-tandem mass spectrometry: matrix interference in patient samples

A high-throughput method was developed and validated for the quantitative determination of MMI270B, an inhibitor of matrix metalloprotease (MMP) enzymes, in human plasma. The method was based on reverse-phase chromatographic separation of the analyte from plasma extract followed by atmospheric pressure chemical ionization (APCI) and tandem mass spectrometry in the selected reaction monitoring mode (SRM). Extraction was performed using simple protein fi ltration in the 96-well plate format to increase the throughput of the method. Optimised chromatographic separation in a short column (30 x 4.6 mm i.d.) coupled with positive APCI mode of ionization followed by selective SRM mode of detection yielded clean chromatograms with minimal signal suppression. The chromatographic conditions resolved isobaric interference peaks observed in samples from patients dosed with MMI270B. The standard curve was linear (r = 0.997) within the concentration range of 1.04 (lower limit of quanti fi cation) to 1040 ng/mL using 0.1 mL of human plasma. The accuracy of the method varied from 93.6 to 103% with a precision of 2.17-6.71% over the concentration range. The method was simple, rapid, and robust with an analyte recovery of >98%.     

Simultaneous determination of nitrite and nitrate in human plasma by on-capillary preconcentration with field-amplified sample stacking

A simple method for the determination of nitrite and nitrate in human plasma has been developed using CZE with minimal sample preparation. Field‐amplified sample stacking (FASS) was used to achieve submicromolar detection by dilution of the plasma sample with deionized water. In CZE, the separation of nitrite and nitrate was achieved within 10 min without adding EOF modifier. The optimal condition was achieved with 50 mM phosphate buffer at pH 9.3. The ninefold diluted plasma samples were injected hydrodynamically for 40 s into a 60 cm×75 μm id uncoated fused‐silica capillary. The separation voltage was 20 kV (negative potential) and UV detection was performed at 214 nm. The linearity curves for nitrite and nitrate were obtained by the standard addition method. The estimated LODs for nitrite and nitrate in ninefold diluted plasma sample were 0.05 and 0.07 μM, respectively. The LODs for nitrite and nitrate in original plasma samples were 0.45 and 0.63 μM. The intra‐ and inter‐day precisions for both analytes were <2.6% and the recovery ranged between 92.3 and 113.3%. It was found that nitrite was more stable than nitrate in the plasma after the sample preparation. This proposed method was applied to a number of human plasma samples and the measured nitrite and nitrate concentrations in human plasma were consistent with the literature ranges.     

Enantioselective high-performance liquid chromatographic method for the determination of baclofen in human plasma

A new analytical method for the separation and determination of R-(−)- and S-(+)- baclofen enantiomers in human plasma by high-performance liquid chromatography (HPLC) with UV detection was developed. Enantioselective resolution of the baclofen enantiomers was achieved by using teicoplanin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic T with a polar ionic mobile phase (PIM) consisting of methanol: glacial acetic acid: triethylamine, 100:0.1:0.1, (v/v/v) at a flow rate of 0.5 ml min⁻¹ and UV detection set at 220 nm. The analytes of interest with S-(+)-sulpiride as the internal standard were extracted from human plasma using liquid-liquid extraction procedure with ethyl ether under alkaline condition prior to HPLC analysis. Recoveries for R-(−)- and S-(+)-baclofen enantiomers were in the ranges of 96-103% at 60-2500 ng ml⁻¹ level. Intra-day and inter-day precision calculated as %RSD was in the ranges of 1.2-5.2 and 1.3-4.3% for both enantiomers, respectively. Intra-day and inter-day accuracy calculated as percentage error were in the ranges of 1.2-3.9 and 1.1-3.9% for both enantiomers, respectively. Linear calibration curves in the concentration ranges of 20-3000 ng ml⁻¹ for each enantiomer showed correlation coefficient (r) of 0.9997. The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 20 and 10 ng ml⁻¹ (S/N=3) respectively.     

高效液相色谱法测定人血浆中总高半胱氨酸含量

建立了快速检测人血浆中总高半胱氨酸含量的高效液相色谱（HPLC）方法。采用反相HPLC分析前,以巯基特异性荧光试剂ABDF对血浆中巯基进行衍生．方法简便、灵敏、准确,无干扰峰影响。平均回收率为97．75％,日内和日间精密度分别为4．49％和9．79％。     

Quantitative liquid chromatography-mass spectrometry determination of catechins in human plasma by automated on-line extraction using turbulent flow chromatography

A simple, fast and sensitive liquid chromatography-mass spectrometry (LC-MS) method with automated on-line extraction using turbulent flow chromatography (TFC) for the determination of five catechins in human plasma was developed. In this method, after on-line extraction by its injection onto an extractor column at turbulent flow, five catechins were backwashed onto a reversed phase column via on-line column switching and separated chromatographically at a laminar flow of 1 ml min(-1). Using this tandem LC-LC-MS system, the extraction, the separation and the quantitation of five catechins in human plasma could be achieved with satisfactory selectivity and sensitivity. The limit of detection (S/N = 3) ranged from 0.6 to 2 ng ml(-1). The described procedure was very simple and rapid since no off-line sample preparation was required, total analysis time being 18.5 min.