Determination of bupivacaine tissue concentration in human biopsy samples using high-performance liquid chromatography with mass spectrometry.

In the present study, we developed a simple and rapid analytical method for the quantification of bupivacaine hydrochloride in human biopsy samples of adipose, muscle, neural, connective and cartilage tissue using liquid chromatography-mass spectrometry. Anesthetics were extracted from the tissue samples using 0.1% formic acid in acetonitrile for protein denaturation and hexane for removal of lipophilic impurities. Analytes were separated adequately on Phenomenex Luna Omega polar C18 column using a gradient mobile phase 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The lower limits of quantification were ≤ 97 ng g-1 tissue for all studied tissues. Intra-day recoveries were between 48.2% and 82.1% with relative standard deviations (RSDs) between 1.47% and 14.28%, whereas inter-day recoveries were between 52.2% and 77.6% with RSDs between 2.98% and 14.79%. The calibration curve showed a linear fit with R2 higher than 0.99 in the concentration range from 0.16 to 100 µg g-1 . Lidocaine hydrochloride was tested as internal standard because its recoveries and matrix effects were comparable to bupivacaine hydrochloride. Post-analytical corrections of measured bupivacaine tissue concentrations can accordingly be made based on recovery of lidocaine as internal standard, with recoveries between 51.2% and 86.9% and RSDs between 1.99% and 16.88%. The developed method could be used to study time-dependent spread of bupivacaine locally or to more distant locations across tissue barriers.

Integration of Solid-Phase Extraction and Reversed-Phase Chromatography in Single Protein-Coated Columns for Direct Injection of Bupivacaine in Human Serum.

A rapid, reliable and precise integrated solid-phase extraction (SPE) and reversed-phase liquid chromatography method was developed and validated to determine bupivacaine in human serum using single protein-coated analytical columns. The protein-coated columns were packed with four different sorbents: TSK-ODS, LiChrosorb RP-8, LiChrosorb RP-2 and µ-Bondapak CN-bonded silica. The method involved direct injection of serum sample onto the columns for trapping of the analyte, clean-up from weakly retained serum endogenous components, as well as the final separation. The protein-coated columns operated in two different chromatographic modes. Serum proteins were extracted and cleaned up by SPE, whereas the final separation of bupivacaine was based on reversed-phase chromatography. The protein-coated TSK-ODS column resulted in more accurate peak integration and more reproducible results. A linear relationship between the concentrations of drug and peak areas was confirmed in the range of 100-2000 ng/mL. Detection and quantification limits were 24.85 and 85.36 ng/mL, respectively. The average recovery for bupivacaine ranged from 96.48% to 98.81%. The present methodology was successfully applied, with a high degree of confidence, to analyze clinical samples obtained from patient receiving 0.5% bupivacaine therapy.

Modeling the chiral resolution ability of highly sulfated ß-cyclodextrin for basic compounds in electrokinetic chromatography.

Despite the fact that extensive research in the field of enantioseparations by capillary electrophoresis has been carried out, it is difficult to predict whether a concrete chiral selector would be useful for the separation of a racemic compound. Hence, several experimental effort is necessary to test the abilities of individual chiral selectors, usually by trial and error procedures. Thus, the enantioseparation of a new racemate becomes a time- and money-consuming task. In this work, the ability of highly sulfated ß-cyclodextrin (HS-ß-CD) as chiral selector in electrokinetic chromatography (EKC) is modeled for the first time, using exclusively directly-available structural data of forty compounds (structurally unrelated basic drugs and pesticides). A discriminant partial least squares (PLS)-based quantitative structure-property relationship (QSPR) approach is simplified, resulting in a consistent, predictive and descriptive model. It is converted into an explicit equation able to predict the enantioresolution level (Rs) of new compounds, from four structure properties available in an on-line open database: logarithm of octanol-water partition coefficient estimated at pH 7.4 (lgD), polar surface area (PSA), number of hydrogen bond donors (HBD) and acceptors (HBA). For the cases in which the model predicts good Rs only in concrete experimental conditions, a Box-Behnken experimental design is proposed for the fast PLS-based optimization of the most influential experimental variables: cyclodextrin concentration, temperature and pH.

Molecularly imprinted polymer in microextraction by packed sorbent for the simultaneous determination of local anesthetics: lidocaine, ropivacaine, mepivacaine and bupivacaine in plasma and urine samples.

This study presents the use of molecularly imprinted polymer (MIP) as packing material for microextraction by packed syringe (MEPS) to achieve higher extraction selectivity. Pentycaine was used as template for MIP. Development and validation of the determination of lidocaine, ropivacaine, mepivacaine and bupivacaine in human plasma and urine samples utilizing MIP-MEPS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were carried out. The MEPS MIP-cartridge could be used for 100 extractions before it was discarded. The extraction recovery ranged from 60 to 80%. The correlation coefficients values were >0.999 for all assays using lidocaine, ropivacaine, mepivacaine and bupivacaine in the calibration range 5-2000 nmol/L. The accuracy of the studied compounds, given as a percentage variation from the nominal concentration values, ranged from -4.9 to 8.4% using plasma and urine samples. The between-batch precision, given as the relative standard deviation, at three different concentrations (quality control samples) was ranged from -4.7 to 14.0% and from 1.8 to 12.7% in plasma and urine, respectively. The lower limit of quantification and limit of detection of the studied substances were 5.0 and 1.0 nm, respectively.

Chiral separation of bupivacaine hydrochloride by capillary electrophoresis with high frequency conductivity detection and its application to rabbit serum and pharmaceutical injection.

Conductivity detection was employed to detect the enantiomers of bupivacaine hydrochloride (Bup), which were separated by high performance capillary electrophoresis. A computer-aided technique was used to calculate the binding energies, and the interaction between Bup enantiomers and cyclodextrins (CDs) is preliminarily discussed. Factors affecting the separation efficiency such as the types and concentration of chiral selectors, running buffer, pH value, separation voltage and capillary inside diameter and length were studied. Under optimized conditions, a baseline separation of Bup enantiomers was achieved in less than 15 min in 4mM NH4Ac-NaAc-HAc (pH 4.00) -0.48mM sulfobutyl ether-beta-cyclodextrin running buffer at a separation voltage of 12 kV. The lowest detectable concentration was 0.052 microg/mL. The proposed method was applied to chiral separation of Bup enantiomers in rabbit serum and pharmaceutical injections.

Surface mass spectrometry of two component drug-polymer systems: novel chromatographic separation method using gentle-secondary ion mass spectrometry (G-SIMS).

In recent years, there has been an increase in the use of time-of-flight secondary ion mass spectrometry (TOF-SIMS) for characterizing material surfaces. A great advantage of SIMS is that the analysis is direct and has excellent spatial resolution approaching a few hundred nanometers. However, the lack of the usual separation methods in mass spectrometry such as chromatography or ion mobility combined with the complexity of the heavily fragmented ions in the spectra means that the interpretation of multicomponent spectra in SIMS is very challenging indeed. The requirements for high-definition imaging, with say 256 × 256 pixels, in around 10 min analysis time places significant constraints on the instrument design so that separation using methods such as ion mobility with flight times of milliseconds are incompatible. Clearly, traditional liquid and gas chromatographies are not at all possible. Previously, we developed a method known as Gentle-SIMS (G-SIMS) that simplifies SIMS spectra so that the dominant ions are simply related to the structure of the substances analyzed. The method uses a measurement of the fragmentation behavior under two different primary ion source conditions and a control parameter known as the g-index. Here, we show that this method may be used "chromatographically" to separate the mass spectra of a drug molecule from the matrix polymer. The method may be used in real-time and is directly compatible with the majority of TOF-SIMS instruments. The applicability to other imaging mass spectrometeries is discussed.

[Fatal poisoning with the local anesthetic bupivacaine].

Lethal concentration of bupivacaine was determined based on the examination of a case of poisoning with this local anesthetic agent taken at a dose 5 times the normal value. This study is of special interest taking into consideration the lack of data on toxic and lethal doses of bupivacaine in splanchnic organs and tissues in the literature.

Enantiomeric resolution of bupivacaine by high-performance liquid chromatography and chiroptical detection.

This work reports a new analytical procedure for the separation and determination of the enantiomers of bupivacaine and the determination of the enantiomeric purity. The isomers were separated using a Chirex 3020 (250 mm x 4.6 mm) with a mobile phase of n-hexane:dichloroethane:ethanol (82:9:9, v/v/v) at a flow-rate of 1 ml min(-1) and UV, polarimetric and circular dichroism (CD) detection. Obtained retention times were 5.93 and 7.53 min (R and S) with a resolution of Rs=2.36. Precisions (RSD) were 1.83 and 2.02% (CD detection) and 3.07 and 1.26% (UV detection) for R- and S-enantiomers, respectively (at 10 microg level). Detection limits were 0.5 and 0.5 microg (R and S) with CD detection, and 0.9 and 0.3 microg with UV detection. Polarimetric detection was inadequate to perform a quantitative method at similar concentration ranges as UV and CD because of poor sensitivity. A procedure for determination of enantiomeric purity using a conventional chromatographic column (RP18, Luna) coupled to a CD detector and anisotropy factor (CD/UV) as analytical signal was also developed. Obtained results show that RSDs of 6.7-1.6% were obtained in the range of 0-100% enantiomeric purity.

High-performance liquid chromatographic separation of rolipram, bupivacaine and omeprazole using a tartardiamide-based stationary phase influence of flow rate and temperature on the enantioseparation.

Chromatographic separation of the chiral drugs rolipram, bupivacaine and omeprazole on a tartardiamide-based stationary phase commercially named Kromasil CHI-TBB is shown in this work. The effect of temperature on the chromatographic separation of the chiral drugs using the Kromasil CHI-TBB stationary phase was determined quantitatively so as to contribute toward the design for the racemic mixtures of the named compound by using chiral columns. A decrease in the retention and selectivity factors was observed, when the column temperature increased. Van't Hoff plots provided the thermodynamic data. The variation of the thermodynamic parameters enthalpy and entropy are clearly negative meaning that the separation is enthalpy controlled.

Molecularly imprinted polymers grafted to flow through poly(trimethylolpropane trimethacrylate) monoliths for capillary-based solid-phase extraction.

Monolithic molecularly imprinted polymers (mMIPs) have been synthesized in a novel way using a trimethylolpropane trimethacrylate core material photo-polymerized in situ in a 100 microm I.D. UV-transparent capillary and further photo-grafted to create specific cavities in the grafted layer. This polymerization technique allows the imprints to be directly created on the surface of the material using a minimum amount of template. Three different anaesthetics of similar structures (bupivacaine, mepivacaine and S-ropivacaine) were used as model target molecules to synthesize sample enrichment media. Hence, various mMIPs have been prepared and evaluated on a micro-system against each analyte in order to test the retention properties and cross-selectivities of the materials. The retention factors were determined and compared with the non-imprinted reference column (mNIP), yielding high imprinting factors together with good selectivity factors between the three analytes. A study with a pure enantiomeric target was carried out to assess the degree of stereo-specific imprinting for injection of racemic mixtures. Finally, one column was imprinted with an equimolar mixture of all three anaesthetics to provide further comprehension of the retention mechanism and accredit the possibility of using the material as a sample enrichment entity. Scanning electron microscopy (SEM), nitrogen absorption/desorption (BET) and mercury intrusion porosimetry were used to characterize the monolith and the mMIPs properties. Nuclear magnetic resonance (NMR) has been used to assess the similarities between the mMIP and mNIP.

Separation and determination of bupivacaine in plasma by capillary electrophoresis with tris(2,2'-bipyridyl)ruthenium(II) electrochemiluminescence detection.

A new, rapid, selective and sensitive method is described for determination of bupivacaine by capillary electrophoresis coupled with tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)(3)2+] electrochemiluminescence detection. The influence of parameters such as detection potential, Ru(bpy)(3)2+ concentration, buffer concentration and pH, injection time and separation voltage on separation efficiency and ECL peak intensity was systematically investigated. Under optimized conditions, the calibration curve was linear in the range 0.02-10 microg/mL. The RSD was 4.0% (n = 6). The detection limit was 3 ng/mL. The recoveries obtained were about 90%. This method was tested in the analysis of plasma samples taken from a rat after it had received bupivacaine injections.

Enantiomeric separation of basic compounds using heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-cyclodextrin in combination with potassium camphorsulfonate in nonaqueous capillary electrophoresis: optimization by means of an experimental design.

The enantiomeric separation of a series of basic pharmaceuticals (beta-blockers, local anesthetics, sympathomimetics) has been investigated in nonaqueous capillary electrophoresis (NACE) systems using heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-cyclodextrin (HDMS-beta-CD) in combination with potassium camphorsulfonate (camphorSO3-). For this purpose, a face-centered central composite design with 11 experimental points was applied. The effect of the concentrations of HDMS-beta-CD and camphorSO3- on enantioresolution was statistically evaluated and depended largely on the considered analyte. The presence of camphorSO3- was found to be particularly useful for the enantioseparation of compounds with high affinity for the anionic CD. CamphorSO3- seems to act as a competitor, reducing the affinity for the CD, probably by ion-pair formation with these analytes. For compounds with lower affinity for HDMS-beta-CD, the combination of camphorSO3- and the CD appeared to have a favorable effect on enantioresolution only if the optimal CD concentration could be reached. On the other hand, for compounds characterized by a very low affinity for the anionic CD, the association of camphorSO3- and HDMS-beta-CD is always unfavorable. Finally, experimental conditions were selected by means of the multivariate approach in order to obtain the highest resolution (Rs) value for each studied compound.

A simplified synthesis of polymeric nonparticulate stationary phases with macrocyclic antibiotic as chiral selector for capillary electrochromatography.

A simplified approach to synthesize nonparticulate (continuous or monolithic) beds with embedded vancomycin chiral selectors for capillary electrochromatography is proposed. In the present approach, N,N'-diallyltartardiamide monomer with diol functionality is used, which can be readily converted to aldehyde groups via periodate treatment. Parallel to the activation of the polymeric matrix for covalent attachment of vancomycin, the periodate treatment has shown secondary effects on the polymeric bed morphology, namely the increase of the average pore size and porosity of the skeleton. Inversed size-exclusion chromatography was applied to characterize porosimetric properties of the capillary columns before and after the periodate treatment. Electroosmotic and enantioselective properties of the nonparticulate beds synthesized are presented. The approach is of more general interest attaching different affinity groups to the polymeric matrix and/or enhancing the accessibility to the active sites, for instance, in the molecular imprinting technique.

Chiral separation of bupivacaine enantiomers by capillary electrophoresis partial-filling technique with human serum albumin as chiral selector.

Capillary electrophoresis (CE) is a powerful technique for enantiomer separations due to its intrinsic high separation efficiencies, speed of analysis, low reagent consumption and small sample requirements. However, some chiral selectors present strong background UV absorption providing high detection limits. The present paper deals with the application of the partial-filling technique to the separation of bupivacaine enantiomers by capillary electrophoresis using human serum albumin (HSA) as chiral selector. In this procedure the cationic surfactant cetyltrimethylammonium bromide (CTAB) was used as a dinamic capillary coating in order to reduce the electro-osmotic flow and detect both bupivacaine enantiomers out of the chiral selector plug. Several experimental conditions such as CTAB concentration, pH, HSA concentration and plug length, background electrolyte concentration, temperature and voltage were studied. Under the selected conditions it is possible to detect the separated enantiomers out of the HSA plug in less than 4 min using 50 mM Tris pH 8 as background electrolyte with 50 microM CTAB, at 30 degrees C and using a separation voltage of 25 kV. The proposed methodology was then validated for analytical purposes and applied to the analysis of pharmaceutical preparations commercially available. The results obtained with the proposed methodology were in good agreement with those declared by the manufacturers. The simplicity, sample throughput, accuracy, reproducibility and low cost of the proposed method make it suitable for the control of the enantiomeric composition of bupivacaine in pharmaceuticals.

Pluronic microemulsions as nanoreservoirs for extraction of bupivacaine from normal saline.

We hypothesized that custom-designed microemulsions would effectively scavenge compounds from bulk media. Pluronic-based oil-in-water microemulsions were synthesized that efficiently reduced the free concentration of the local anesthetic bupivacaine in 0.9% NaCl. Both the molecular nature and concentration of the constituents in the microemulsions significantly affected extraction efficiencies. Pluronic F127-based microemulsions extracted bupivacaine more efficiently than microemulsions synthesized using other Pluronic surfactants (L44, L62, L64, F77, F87, F88, P104). Extraction was markedly increased by addition of fatty acid sodium salts due to greater oil/water interface area, increased columbic interaction between bupivacaine and fatty acids sodium salt, and greater surface activity. These data suggest that oil-in-water microemulsions may be an effective agent to treat cardiotoxicity caused by bupivacaine or other lipophilic drugs.

Direct enantiomeric purity determination of the chiral anesthetic drug bupivacaine by 1H NMR spectroscopy.

The direct determination of the enantiomeric purity of the chiral anaesthetic drug bupivacaine has been developed using 1H NMR (400 MHz) spectroscopy with a chiral solvating agent. Optimization of experimental conditions in terms of temperature, substrate concentration and solvating agent to substrate molar ratio provided two significant signal splittings for chiral recognition resulting from diastereomeric solvation. Based on the relative intensities of the aliphatic methyl resonances assigned to (S)-(-)- and ($)-(+)-bupivacaine, the analysis of synthetic mixtures of the enantiomers by the proposed NMR method resulted in assay values which agreed closely with the known quantities of each enantiomer in the mixtures tested. The mean +/- SD recovery values for the (R)-(+)-enantiomer was 100.0 +/- 0.6% of added antipode (n = 7). The optically pure enantiomers were used to establish the minimum sensitivity of the NMR spectroscopic method of chiral analysis.

Dual-opposite injection electrokinetic chromatography for the unbiased, simultaneous separation of cationic and anionic compounds.

The concept of dual opposite injection in capillary electrophoresis (DOI-CE) for the simultaneous separation, under conditions of suppressed electroosmotic flow, of anionic and cationic compounds with no bias in resolution and analysis time, is extended to a higher pH range in a zone electrophoresis mode (DOI-CZE). A new DOI-CE separation mode based on electrokinetic chromatography is also introduced (DOI-EKC). Whereas conventional CZE and DOI-CZE are limited to the separation of charged compounds with different electrophoretic mobilities, DOI-EKC is shown to be capable of separating compounds with the same or similar electrophoretic mobilities. In contrast to conventional EKC with charged pseudostationary phases that often interact too strongly with analytes of opposite charge, the neutral pseudostationary phases appropriate for DOI-EKC are simultaneously compatible with anionic and cationic compounds. This work describes two buffer additives that dynamically suppress electroosmotic flow (EOF) at a higher pH (6.5) than in a previous study (4.4), thus allowing DOI-CZE of several pharmaceutical bases and weakly acidic positional isomers. Several DOI-EKC systems based on nonionic (10 lauryl ether, Brij 35) or zwitterionic (SB-12, CAS U) micelles, or nonionic vesicles (Brij 30) are examined using a six-component test mixture that is difficult to separate by CZE or DOI-CZE. The effect of electromigration dispersion on peak shape and efficiency, and the effect of surfactant concentration on retention, selectivity, and efficiency are described.

Separation of enantiomers on HPLC chiral stationary phases based on human plasma alpha1-acid glycoprotein: effect of sugar moiety on chiral recognition ability.

HPLC chiral stationary phases based on human plasma alpha1-acid glycoprotein (AGP) and partially deglycosylated AGP (pd-AGP) were prepared to investigate the effects of sugar moiety of AGP on chiral discrimination of various solutes. Removal of a sugar moiety of AGP by treatment with N-glycosidase was confirmed by high-performance capillary electrophoresis, reversed-phase HPLC and matrix-assisted laser desorption-time of flight (MALDI-TOF) mass spectrometry. The average molecular weights of AGP and pd-AGP were estimated to be ca. 33,000 and 30,600, respectively, by MALDI-TOF mass spectrometry. Next, AGP and pd-AGP were bound to aminopropyl-silica gels activated with N,N '-disuccinimidylcarbonate. The retentivity+ and enantioselectivity of the neutral, acidic and basic solutes tested on the pd-AGP column were significantly or not significantly larger in most solutes than those on the AGP column. This is ascribable to that by cleavage of a sugar chain(s) by N-glycosidase, pd-AGP could become more hydrophobic than AGP, and/ or that a solute could be easily accessible to the specific and/or non-specific binding sites of pd-AGP. It is interesting that warfarin enantiomers are not resolved on the pd-AGP column, but resolved on the AGP column. A sugar chain(s) of AGP cleaved by N-glycosidase might be involved in the enantioselective binding of warfarin enantiomers.

Enantioseparation of anaesthetic drugs by capillary zone electrophoresis using cyclodextrin-containing background electrolytes.

The enantiomers of five racemic anaesthetic drugs were resolved with cyclodextrins using capillary zone electrophoresis. Parameters which affected the chiral resolution, such as type and concentration of cyclodextrin, temperature, and addition of organic modifier were investigated. The results show that the enantiomeric discrimination of the solutes is influenced by the structural shape of the solute molecules, separation temperature, and type of cyclodextrin. It was found that alpha-cyclodextrin was the best enantioselector for resolution of prilocaine and ketamine, while the enantiomers of mepivacaine, ropivacaine, and bupivacaine were resolved with beta-cyclodextrin and/or modified beta-cyclodextrins, i.e., methyl- and 2-hydroxypropyl-beta-cyclodextrin, as chiral selectors. The length of the alkyl chain on the amino group of the drug molecule had a strong effect on the enantioresolution of mepivacaine, ropivacaine, and bupivacaine. Baseline separation of racemic ketamine was achieved with alpha- and methyl-beta-cyclodextrin at 15 degrees C. Addition of 5 M urea to the running buffer containing beta-cyclodextrin at high concentrations resulted in the enantioseparation of prilocaine, mepivacaine, and ketamine. Enantioresolution was improved upon the addition of 10% methanol to the buffer containing urea and beta-cyclodextrin. Generally, the complex formed between the S-enantiomers and modified beta-cyclodextrins was stronger than the corresponding R-forms. An exception was prilocaine where the R-form gave a more stable complex both with alpha- and beta-cyclodextrin.

Enantioseparation of local anaesthetic drugs by capillary zone electrophoresis with cyclodextrins as chiral selectors using a partial filling technique.

The enantiomers of prilocaine were successfully resolved with alpha-cyclodextrin, and those of mepivacaine and bupivacaine, with methyl-beta-cyclodextrin as chiral selectors, by means of capillary zone electrophoresis (CZE) employing a partial filling technique. By this separation mode, a discontinous separation zone is formed in the capillary. Prior to application of the actual drug substance, the capillary is partially filled with the separation solution. During the enantioseparation both ends of the capillary are dipped into the running buffer solution, i.e., without chiral selector. The consumption of chiral selector is thus very low, less than a microliter per run. The repeatibility of the electrophoretic mobility of the enantiomers was better than 1.2% relative standard deviation (RSD). The effect of the length of the separation zone on the resolution of the enantiomers was studied. The application time of the chiral selector, instead of the selector concentration, was varied in order to improve and regulate the enantioresolution and reduce consumption of the chiral selector as much as possible. It was found that the enantioseparations were directly affected by the length of the separation zone, and there was a minimal plug length where complete enantioresolution was achieved.