Whiteness, redness, blueness and greenness profile assessment and design of experiments approach to microwave-aided sensitive and green spectrofluorophotometric determination of baclofen.

As a gamma amino butyric acid-ergic agonist, Baclofen is often prescribed to adults and children for the treatment of severe spasticity that originates in the brain or spinal cord. Even after reviewing the literature extensively, no one has documented a method for estimating baclofen using microwave-assisted stability-indicating spectrofluorimetric techniques, despite the abundance of options for baclofen stability, assay, and bioanalysis. Organic solvents, which are typically necessary for current procedures but may be costly and toxic, have a severe effect on aquatic life and the environment. Using green solvents and 4-chloro-7-nitrobezofuran as a fluorescent probe, this work conducted a stability-indicating spectrofluorimetric estimate of baclofen. Through the use of a design-of-experiments technique, a reliable microwave-aided spectrofluorimetric method was developed, with little solvent consumption and time for sample analysis. Prior to conducting response surface analysis and optimizing important variables and responses, a fractional factorial design was used to screen method variables and responses. A central composite design was then employed for these purposes. This flexible spectrofluorimetric technique was used to assess baclofen concentrations in forced degraded samples and marketed formulations. For baclofen determination, the suggested spectrofluorimetric approach was found to be green, quick, easy to use, economical, and user-friendly.

Dermaplaning for Transdermal Drug Permeation Enhancement: A Qualitative and Quantitative Assessment.

In this study, we sought to investigate the effect of dermaplaning, a popular cosmeceutical skin rejuvenation technique on the permeation of drugs. Baclofen and diclofenac were used as hydrophilic and hydrophobic model drugs, respectively. A specific area of skin was treated with 4 strokes of a dermaplane device. Interindividual variability was assessed by having multiple users operate the device for the study. Dermaplaned skin was histologically evaluated and characterized for resistance drop and the depletion of the stratum corneum (SC). The effect of dermaplaning on drug permeation was investigated via in vitro permeation studies. Histology studies depicted the removal of SC and some parts of viable epidermis by dermaplaning. A significant drop in electrical resistance post skin dermaplaning was observed for all treatment groups, signifying the depletion of barrier properties of SC (p < 0.05). Consequently, significant drug flux and permeation were observed over 24 h for the model drugs across dermaplaned skin. However, varied absorption profile was observed in vitro for both drugs across dermaplaned skin. Dermaplaning displayed a better suitability for significantly enhancing the permeation of the hydrophilic drug, baclofen. Evidence of variation in results post dermaplaning was observed amidst multiple users as well (p < 0.05).

Baclofen impurities: Facile synthesis and novel environmentally benign chromatographic method for their simultaneous determination in baclofen.

An efficient, economic and high yielding method was described for the synthesis of baclofen (BAC) pharmacopoeial impurities (impurity A and impurity B) which can be used for gram-scale synthesis. Furthermore, a novel ecofriendly thin-layer chromatographic TLC-densitometric method was established and validated for the determination of BAC and its synthesized impurities. The developed TLC-densitometric method is based on the chromatographic separation using TLC plates (60 F254 ) using a green mobile phase of ethyl acetate-methanol-ammonia solution, 33% (8:2:0.1, by volume) with UV scanning at 220 nm. The proposed method was validated with respect to International Conference on Harmonization guidelines. The validated method was successfully applied for determination of BAC in pure form and in its commercial dosage form. Additionally, the greenness profile of the developed method was evaluated and compared with those of the reported chromatographic methods. The developed method was found to be superior to the published methods, being environmentally benign.

Stability of sufentanil and baclofen mixtures for intrathecal analgesia at different concentrations in polypropylene syringes.

BACKGROUND: Intrathecal analgesia is a method using various molecules alone or in combination. Among these, the association sufentanil/ baclofen is widely used. Instead of moving patients to the few expert centers taking charge of these specific preparations, it could be better to transport syringes to peripheral centers managing pump refilling. That is why, it is interesting to demonstrate the stability of the mixture, and so to be able to ensure the best transport conditions of syringes. METHODS: A stability indicating UPLC-DAD method was developed and validated according to the ICH guidelines. Four mixtures of sufentanil baclofen stored in 5±3°C and 25±2°C were evaluated for seven days and compared to the initial observed concentrations. RESULTS: The stability is demonstrated only for preparations stored at 5±3°C for seven days thanks to relative concentrations (95% confidence intervals of the mean of 3 samples) systematically positioned between 90% and 110%. On the other hand, after few days, degradation products of sufentanil appeared for all mixtures stored at 25°C±2°C. CONCLUSION: This study shows the stability of a weakly and a highly concentrated mixture of sufentanil and baclofen solutions in polypropylene syringes stored at 5±3°C for seven days. This result will allow the transport of the preparation under optimal conditions. Advance preparations for intrathecal pump refills could also be feasible.

LC-MS/MS method for quantification of baclofen in hair: A useful tool to assess compliance in alcohol dependent patients?

To evaluate adherence to treatment, we developed and validated a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for baclofen quantification in hair.Twenty mg was washed twice with dichloromethane, incubated in phosphate buffer (pH 5) for 10 minutes at 95°C, then extracted by liquid-liquid extraction in alkaline condition. Baclofen-d4 was used as the internal standard. This method was applied to assess compliance in4 treated alcohol-dependent patients (3 dead and one living). Blood quantification of baclofen and ethanol were performed in the 4 cases. Hair ethylglucuronide (ethanol metabolite, EtG) measurement (2x3 cm) was associated in 1 patient. Baclofen quantification in hair was validated over the range 10-5000 pg/mg. The accuracy was within 96.0%-110.9% and the precision was less than 9.3%. Baclofen segmental (3x2cm) hair concentrations found in the living patient were 4420, 4260, and 4380 pg/mg, reflecting a regular exposure over the last 6 months and suggesting patient compliance. However, the high EtG level found in this patient in the analyzed segments (225 pg/mg and 215 pg/mg) showed excessive alcohol consumption during the same period, suggesting therapeutic failure. In the 3 deceased patients, the non-segmental analysis of hair showed baclofen concentrations of 15, 545, and 2475 pg/mg. The low concentrations in the 2 first cases are compatible either with a poor compliance or to a beginning of a treatment. This is the first measurement of baclofen in hair of alcohol dependent patients. It could be used as a monitoring biomarker to assess patient's compliance.

Selectivity Enhancement in Molecularly Imprinted Polymers for Binding of Bisphenol A.

Bisphenol A (BPA) is an estrogen-mimicking chemical that can be selectively detected in water using a chemical sensor based on molecularly imprinted polymers (MIPs). However, the utility of BPA-MIPs in sensor applications is limited by the presence of non-specific binding sites. This study explored a dual approach to eliminating these sites: optimizing the molar ratio of the template (bisphenol A) to functional monomer (methacrylic acid) to cross-linker (ethylene glycol dimethacrylate), and esterifying the carboxylic acid residues outside of specific binding sites by treatment with diazomethane. The binding selectivity of treated MIPs and non-treated MIPs for BPA and several potential interferents was compared by capillary electrophoresis with ultraviolet detection. Baclofen, diclofenac and metformin were demonstrated to be good model interferents to test all MIPs for selective binding of BPA. Treated MIPs demonstrated a significant decrease in binding of the interferents while offering high selectivity toward BPA. These results demonstrate that conventional optimization of the molar ratio, together with advanced esterification of non-specific binding sites, effectively minimizes the residual binding of interferents with MIPs to facilitate BPA sensing.

Identification of Drugs in Parenteral Pharmaceutical Preparations from a Quality Assurance and a Diversion Program by Direct Analysis in Real-Time AccuTOFTM-Mass Spectrometry (DART-MS).

In healthcare settings drug diversion and impairment of physicians are major concerns requiring a rapid and efficient method for surveillance and detection. A Direct Analysis in Real Time ion source coupled to a JEOL AccuTOFTM time-of-flight mass spectrometer (DART-MS) method was developed to screen parenteral pharmaceutical formulations for potential drug diversion. Parenteral pharmaceutical formulations are also known as injectable formulations and are used with intravenous, subcutaneous, intramuscular and intra-articular administration. A library was created using the mass spectra data collected by a DART-MS operated in switching mode at 20, 60 and 90 V settings. This library contained 17 commonly encountered drugs in parenteral pharmaceutical formulations that included the surgical analgesic: fentanyl, hydromorphone and morphine; anesthetic: baclofen, bupivacaine, ketamine, midazolam, ropivacaine and succinylcholine; and a mixture of other drug classes: caffeine, clonidine, dexamethasone, ephedrine, heparin, methadone, oxytocin and phenylephrine. Randomly selected 200 de-identified parenteral pharmaceutical formulations containing one or more drugs were submitted for analysis to the FIRM Toxicology Laboratory at Virginia Commonwealth University Health and were screened using the DART-MS. The drug contents of the de-identified formulations were previously confirmed by a published high performance liquid chromatography (HPLC) method. The drugs in the formulations were rapidly and successfully identified using the generated library. The DART-MS and HPLC results were in complete agreement for all 200 parenteral pharmaceutical formulations.

Resolution of Enantiomers of (RS)-Baclofen by Ligand-Exchange Thin-Layer Chromatography.

A new chromatographic method has been developed for direct enantioresolution of (RS)-baclofen by ligand-exchange thin-layer chromatography (TLC) adopting two different approaches; (A) TLC plates were prepared by mixing the ligand exchange reagents (LER) with silica gel slurry and the chromatograms were developed with different achiral solvents or solvents having no chiral additive, and (B) the LER consisting of Cu(II)-L-amino acid complex was used as chiral mobile phase additive and the plain plates of silica gel having no chiral selector were used. Cu(II) acetate and four L-amino acids (namely, L-tryptophan, L-histidine, L-proline and L-phenylalanine) were used for the preparation of LERs. Spots were located by the use of iodine vapor. Effect of temperature and the mole ratio of Cu(II)-to-amino acid on enantioresolution were also studied. The results for the two methods have been compared, and the issue of involvement of the Cu(II) cation for the best performance of the two methods has been discussed with respect to the same mobile phase. L-Trp proved to be a good ligand using a common mobile phase in each case.

Validated Method for the Quantification of Baclofen in Human Plasma Using Solid-Phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry.

A highly sensitive and fully validated method was developed for the quantification of baclofen in human plasma. After adjusting the pH of the plasma samples using a phosphate buffer solution (pH 4), baclofen was purified using mixed mode (C8/cation exchange) solid-phase extraction (SPE) cartridges. Endogenous water-soluble compounds and lipids were removed from the cartridges before the samples were eluted and concentrated. The samples were analyzed using triple-quadrupole liquid chromatography-tandem mass spectrometry (LC-MS-MS) with triggered dynamic multiple reaction monitoring mode for simultaneous quantification and confirmation. The assay was linear from 25 to 1,000 ng/mL (r(2) > 0.999; n = 6). Intraday (n = 6) and interday (n = 15) imprecisions (% relative standard deviation) were <5%, and the average recovery was 30%. The limit of detection of the method was 5 ng/mL, and the limit of quantification was 25 ng/mL. Plasma samples from healthy male volunteers (n = 9, median age: 22) given two single oral doses of baclofen (10 and 60 mg) on nonconsecutive days were analyzed to demonstrate method applicability.

[Lethal intoxication with baclofen].

The objective of the present study was to select and develop simpler methods for the quantitative determination of baclofen in blood with the use of HPLC and tandem MS (MS-MS) techniques and its qualitative determination in cadaveric organs by the GC/MS technique. These mathods were shown to be suitable for the purpose of forensic medical analysis, clinical, toxicological, and therapeutic monitoring. The special emphasis is laid on the methods used to investigate the biological materials obtained from the subjects who died from baclofen intoxication.

Evaluation of two modified quick, easy, cheap, effective, rugged and safe (QuEChERS) sample preparation methods for the analysis of baclofen and gabapentin in feeds by liquid chromatography tandem mass spectrometry.

This paper presents a new analytical method for the simultaneous determination of baclofen and gabapentin in feeds based on two modified quick, easy, cheap, effective, rugged and safe (QuEChERS) sample preparation methods and liquid chromatography tandem mass spectrometry (LC-MS/MS). For the two modified QuEChERS methods, samples were first extracted with acidified acetonitrile (5.0% acetic acid, v/v) without using acetonitrile salting-out extraction. Then, the first modified QuEChERS method was established according to the original QuEChERS cleanup procedure. For the second modified QuEChERS method, the extract was evaporated to dryness and reconstituted in acetonitrile. Subsequently, the analytes in the reconstituted solution were retained by primary secondary amine (PSA) and released from PSA with 1.0% formic acid in methanol. Finally, the eluate was evaporated and dissolved in 0.1% formic acid solution/methanol (v/v, 80:20). All of the samples were analyzed by LC-MS/MS on a Waters Acquity BEH C18 column with 0.1% formic acid in water/methanol as the mobile phase with gradient elution. The matrix effect, recovery, and repeatability, within laboratory reproducibility, and the LODs and LOQs of the two modified QuEChERS sample preparation methods were investigated and compared. Comparative results showed that the second method was obviously superior to the first method.

Liquid chromatography-tandem mass spectrometry determination of baclofen in various biological samples and application to a pharmacokinetic study.

Baclofen is a structural analogue of γ-aminobutyric acid (GABA) that has been used for the treatment of spasticity since 1977. This study describes a simple and sensitive LC/MS/MS assay for the quantification of baclofen in rat plasma, urine, as well as various tissue samples. The assay utilized a simple protein precipitation and achieved lower limit of quantification (LLOQ) of 0.25ng/mL for rat plasma and brain samples and 2ng/mL for rat urine, liver and kidney samples. The assay was validated to demonstrate the specificity, linearity, recovery, LLOQ, accuracy, precision, and stability by using matrix matched quality control samples. There is no endogenous or exogenous peaks interfering with the analytes and matrix effects were minimized by optimized separation condition. The assay was linear over a concentration range of 0.25-500ng/mL for rat plasma and brain tissue, and 2-5000ng/mL for rat urine, kidney and liver with correlation coefficients >0.999. The mean intra- and inter-day assay accuracies were 94.6-104.6 and 96.0-103.6%, respectively. The mean intra- and inter-day precisions were 5.71 and 5.70%, respectively. The developed assay was successfully applied to a pharmacokinetic study and examined urinary excretion and tissue distribution of baclofen in rats following intravenous and oral administration.

HPLC enantioresolution of (R,S)-baclofen using three newly synthesized dichloro-s-triazine reagents having amines and five others having amino acids as chiral auxiliaries.

Enantioresolution of (R,S)-baclofen was accomplished using a newly synthesized set of three chiral derivatizing reagents (CDRs) having amines [(S)-(-)-α,4-dimethylbenzylamine, (-)-cis-myrtanylamine and (R)-(-)-1-cyclohexylethylamine] as chiral auxiliaries in cyanuric chloride and another set of five CDRs having amino acids (L-Leu, D-Phg, L-Val, L-Met and L-Ala) as chiral auxiliaries. These eight CDRs were used for synthesis of diastereomers of (R,S)-baclofen under microwave irradiation. The diastereomers were separated on a reversed-phase C(18) column using mixtures of methanol with aqueous trifluoroacetic acid with UV detection at 230 nm. Chromatographic data obtained for the two sets of diastereomers were compared among themselves and among the two groups. The method was validated for limit of detection, linearity, accuracy and precision.

[Determination of baclofen using micellar electrokinetic chromatography with laser induced fluorescence detection].

A novel micellar electrokinetic chromatographic method with laser induced fluorescence detection after derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) was developed for the determination of muscle relaxant drug baclofen (BAL). After optimization, baseline separation of the derivatives of BAL and gabapentin (internal standard) was obtained within 7 min in a running buffer (pH 9.75) composed of 15 mmol/L sodium borate, 20 mmol/L sodium dodecyl sulfate and 10% (v/v) acetonitrile. The separation voltage was 17.5 kV. The column temperature was 25 degrees C. The samples were injected by a pressure of 3.45 kPa (0.5 psi) for 3 s. The method has a linear range of 0.025 - 25 mg/L for BAL with the correlation coefficient of 0.999 9. The limit of detection (LOD, S/N = 3) and the limit of quantification (LOQ, S/N = 10) were 0.90 microg/L and 6.25 microg/L, respectively. The developed method was used for the analysis of BAL pharmaceutical preparation and urine samples spiked with BAL standard. The ranges of recovery were 101.6% - 107.9% for BAL preparation and 107.0% - 109.6% for urine samples. This method can be applied to the quality assessment of baclofen drug products, and provide supplementary means for the drug metabolism research of baclofen.

Simultaneous determination of R-(-)-, S-(+)-baclofen and impurity A by electrokinetic chromatography.

A rapid method for the simultaneous analysis of R-(-)-, S-(+)-baclofen and impurity A, (4RS)-4-(4-chlorophenyl) pyrrolidin-2-one, by electrokinetic chromatography was established. The optimized condition was in 100mM sodium borate buffer (pH 9.9) containing 18mM alpha-cyclodextrin (CD) and 1% (v/v) ACN using a fused-silica capillary dynamically coated with polyethylene oxide (PEO), with an effective length of 56cm and an inner diameter of 50microm, hydrodynamic injection at 50mbar for 6s, temperature of 45 degrees C, applied voltage of 27kV and UV detection at 220nm. Baseline separation of all analytes was achieved within 9min (R(s)>2.7) with the migration order of impurity A, S-(+)- and R-(-)-baclofen. The method showed good linearity (r(2)>0.999 in a range of 5-50microg/mL for impurity A and 50-500microg/mL for baclofen enantiomers), precision (%RSDs<3.37%) and recoveries (100.3% for R-(-)-baclofen, 101.6% for S-(+)-baclofen and 96.1% for impurity A). Detection and quantitation limits were 10 and 30microg/mL for both enantiomers, and 2 and 5microg/mL for the impurity, respectively. The method was efficient for the determination of baclofen enantiomers and impurity A in pharmaceutical raw material and formulations due to its reliability, speed and simplicity.

Enantiomeric analysis of baclofen analogs by capillary zone electrophoresis, using highly sulfated cyclodextrins: Inclusion ionization constant pKa determination.

Using cyclodextrin-capillary zone electrophoresis (CD-CZE), baseline separation of baclofen phaclofen, saclofen, and hydroxy-saclofen, potent gamma-aminobutyric acid(B) (GABA(B)) agonist or antagonists was achieved. A method for the enantioresolution of those analogs of GABA was developed using anionic cyclodextrins (highly sulfated CD or highly S-CD) as chiral selectors and capillaries dynamically coated with polyethylene oxide (PEO). With charged CDs we observed good resolutions due to the large electrophoretic mobility of these chiral selectors opposite to the mobility of the solutes. The highly S-alpha-CD and S-beta-CD were found to be complementary and the most effective complexing agent, allowing good enantiomeric resolution in short runtimes. The complete resolution was obtained using 25 mM phosphate buffer at pH 2.5 containing 3% w/v of highly S-alpha-CD or S-beta-CD at 25 degrees C with an applied field of 0.30 kV/cm. The apparent binding constants of the inclusion complexes were evaluated and the migration order was determined. A comparison was possible to investigate the importance of the anionic group of the molecules in the separations. The pK(a) values were determined for all four compounds in order to explain relative electrophoretic migration of the solutes.

Determination of baclofen enantiomers in pharmaceutical formulations using maltodextrin-based enantioselective, potentiometric membrane electrodes.

Two enantioselective, potentiometric membrane electrodes (EPMEs) based on maltodextrins with different value of dextrose equivalent (DE) (maltodextrin I: DE 4.0-7.0; maltodextrin II: DE 16.5-19.5) were proposed for the assay of baclofen enantiomers in baclofen raw materials and from its pharmaceutical formulation, Norton-Baclofen tablets. The slopes of the electrode function of the proposed electrodes were 55.0 mV/pS-baclofen for maltodextrin I-based electrode and 59.0 mV/pR-baclofen for maltodextrin II-based electrode and the detection limits were 1.34 x 10(-6) mol l(-1) (S-baclofen) and 2.52 x 10(-10) mol l(-1) (R-baclofen), respectively. The surfaces of the electrodes are stable and easily renewable by simply polishing on alumina paper.

The bioequivalence study of baclofen and lioresal tablets using capillary electrophoresis.

A simple and robust analytical method for rapid separation and sensitive quantification of baclofen in human plasma by capillary electrophoresis technique was developed. Electrophoretic separation was optimized and successfully performed using simple sodium tetraborate aqueous solution. Observed detection limit in biological material was 10 ng. Using UV detection at 200 nm excellent linearity (r = 0.999) was observed over the concentration range from 0.025 to 1.0 microg mL(-1). The described method has been validated and applied to the quantitative determination of baclofen in human plasma. The bioavailability of Baclofen (Polpharma) and Lioresal (Novartis) in 18 healthy volunteers was investigated. The results indicate bioequivalence of the reference and Baclofen preparations.

Optimization of the chiral resolution of baclofen by capillary electrophoresis using beta-cyclodextrin as the chiral selector.

The chiral resolution of baclofen was achieved by capillary electrophoresis using a fused-silica capillary (60 cm x 75 microm ID). The background electrolyte (BGE) was phosphate buffer (pH 7.0, 50 mM)-acetonitrile (95:5 v/v) containing 10 mM beta-cyclodextrin. The applied voltage was 15 kV. The values of alpha and R(s) were 1.06 and 1.00, respectively. The electrophoretic conditions were optimized varying the pH and the ionic strength of the BGE, concentrations of beta-cyclodextrin and acetonitrile and the applied voltage.

Electrochemical analysis and analgesic behavior of Zn(II)-baclofen complex.

The electroanalytical behavior of Zn(II)-baclofen complex has been described by direct current polarographic (DCP) method. The complex of transition metal Zn(II) with baclofen, [4-amino-3(4-chlorophenyl) butanoic acid, has been prepared and characterized on the basis of elemental analysis, IR-spectral, polarographic and amperometric studies. The metal-ligand interaction in aqueous medium has been studied polarographically at 25 degrees C and at an ionic strength of mu = 1.0 M KCl. The reduction of the complex was found to involve two electrons and of diffusion controlled nature. The analytical results indicated a 1:1 (M:L) stoichiometry for Zn(II)-baclofen complex. The results on the drug and drug-metal complex revealed that the complex is more potent as compared with the parent drug.