Reversed phase liquid chromatography for the enantioseparation of local anaesthetics in polysaccharide-based stationary phases. Application to biodegradability studies.

A comprehensive study on the chiral separation of bupivacaine, mepivacaine, prilocaine and propanocaine with eight commercial polysaccharide-based chiral stationary phases (CSPs) in reversed phase conditions compatible with MS detection is performed. Methanol and acetonitrile are used as organic modifiers. Retention and resolution values obtained for each compound in the different CSPs and mobile phases are compared. The polysaccharide-based CSPs tested present different enantioselectivity towards the analytes. From the results, the experimental conditions for determining the enantiomers of bupivacaine, mepivacaine, prilocaine and propanocaine in saline aqueous samples using MS detection are used, for the first time, to perform an enantioselective biodegradability study.

Selective extraction of cocaine from biological samples with a miniaturized monolithic molecularly imprinted polymer and on-line analysis in nano-liquid chromatography.

We report the on-line coupling of a monolithic molecularly imprinted polymer to nano-liquid chromatography for the selective analysis of cocaine and its main metabolite, benzoylecgonine, in complex biological samples. After the screening of different synthesis conditions, a monolithic molecularly imprinted polymer was in situ synthesized into a 100 µm internal diameter fused-silica capillary using cocaine as template, methacrylic acid as functional monomer, and trimethylolpropane trimethacrylate as cross-linker. Scanning electron microscopy was used to assess the homogeneous morphology of the molecularly imprinted polymer and its permeability was measured. Its selectivity was evaluated by nano-liquid chromatography-ultraviolet, leading to imprinting factors of 3.2 ±â€¯0.5 and 2.2 ±â€¯0.3 for cocaine and benzoylecgonine, respectively, on polymers resulting from three independent syntheses, showing the high selectivity and the repeatability of the synthesis. After optimizing the extraction protocol to promote selectivity, the monolithic molecularly imprinted polymer was successfully on-line coupled with nano-liquid chromatography-ultraviolet for the direct extraction and analysis of cocaine present in spiked human plasma and saliva samples. The repeatability of the obtained extraction recovery, between 85.4 and 98.7% for a plasma sample spiked at 100 ng mL-1, was high with relative standard deviation values lower than 5.8% for triplicate analyses on each of the three independently synthesized molecularly imprinted polymers. A linear calibration range was achieved between 100 and 2000 ng mL-1 (R2 = 0.999). Limits of quantification of 14.5 ng mL-1 and 6.1 ng mL-1 were achieved in plasma and urine samples, respectively. The very clean-baseline of the resulting chromatogram illustrated the high selectivity brought by the monolithic molecularly imprinted polymer that allows the removal of a huge peak corresponding to the elution of interfering compounds and the easy determination of the target analyte in these complex biological samples.

A systematic investigation of forensic hair decontamination procedures and their limitations.

The effectiveness of decontamination procedures used for the removal of external drug contamination in forensic hair analysis is an ongoing debate. This investigation evaluated wash methods complying with Society of Hair Testing (SoHT) guidelines and their capacity to remove cocaine (COC) and methamphetamine (MA) from artificially contaminated hair. The most effective decontamination method was determined using a systematic approach, involving (1) an initial washing solvent screen, (2) optimization of wash duration, (3) comparison of sequential wash methods, and (4) reanalysis of clinical hair samples. For analysis, hair was subjected to micro-pulverized methanolic extraction prior to quantitation by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Methanol (MeOH) and 0.1 M phosphate buffer (pH 6) were the most effective organic and aqueous solvents, respectively, removing 28%-38% of COC and 16%-31% of MA. Wash durations longer than 30-60 minutes did not remove additional amounts, and a more efficient sequential wash method was subsequently developed. Despite this, the interpretation of reportable results relative to the SoHT cut-off levels was unchanged for most clinical hair samples reanalyzed after washing by agitation for 30 minutes with MeOH. These findings highlight the inability of decontamination solvents to completely remove external COC and MA contamination from hair, including wash methods adhering to SoHT guidelines.

Reduced graphene oxide as an efficient sorbent in microextraction by packed sorbent: Determination of local anesthetics in human plasma and saliva samples utilizing liquid chromatography-tandem mass spectrometry.

Herein, reduced graphene oxide (RGO) has been utilized as an efficient sorbent in microextraction by packed sorbent (MEPS). The combination of MEPS and liquid chromatography-tandem mass spectrometry has been used to develop a method for the extraction and determination of three local anesthetics (i.e. lidocaine, prilocaine, and ropivacaine) in human plasma and saliva samples. The results showed that the utilization of RGO in MEPS could minimize the matrix effect so that no interfering peaks at the retention times of the analytes or internal standard was observed. The high extraction efficiency of this method was approved by mean recoveries of 97.26-106.83% and 95.21-105.83% for the studied analytes in plasma and saliva samples, respectively. Intra- and inter-day accuracies and precisions for all analytes were in good accordance with the international regulations. The accuracy values (as percentage deviation from the nominal value) of the quality control samples were between -2.1 to 13.9 for lidocaine, -4.2 to 11.0 for prilocaine and between -4.5 to -2.4 for ropivacaine in plasma samples while the values were ranged from -4.6 to 1.6 for lidocaine, from -4.2 to 15.5 for prilocaine and from -3.3 to -2.3 for ropivacaine in human saliva samples. Lower and upper limit of quantification (LLOQ, ULOQ) were set at 5 and 2000 nmol L-1 for all of the studied drugs. The correlation coefficients values were ≥0.995. The limit of detection values were obtained 4 nmol L-1 for lidocaine and prilocaine, and 2 nmol L-1 for ropivacaine.

Local Anesthetic Activity from Extracts, Fractions and Pure Compounds from the Roots of Ottonia anisum Spreng. (Piperaceae).

Piperaceae species can be found worldwide in tropical and subtropical areas and many of them have been used for centuries in traditional folk medicine and in culinary. In Brazil, species of Piperaceae are commonly used in some communities as local anesthetic and analgesic. Countrified communities have known some species of the genus Ottonia as "anestesia" and it is a common habit of chewing leaves and roots of Ottonia species to relief toothache. The purpose of this study is to report our findings on new molecules entities obtained from the roots of Ottonia anisum Spreng, in which local anesthetic activity (sensory blockage) is demonstrated for the first time in vivo guinea pig model. Phytochemical investigation led to the isolation of three amides (pipercallosidine, piperine and valeramide) and in an enriched mixture of seven amides (valeramide, 4,5-dihydropiperlonguminine, N-isobutil-6-piperonil-2-hexenamide, piperovatine, dihydropipercallosidine, pipercallosidine and pipercallpsine). Our findings demonstrated the anesthetic potential for the methanolic extract from roots, its n-hexane partition and amides from O. anisum and it is in agreement with ethnobotanical survey.

Molecularly imprinted microspheres synthesized by a simple, fast, and universal suspension polymerization for selective extraction of the topical anesthetic benzocaine in human serum and fish tissues.

A simple, fast, and universal suspension polymerization method was used to synthesize the molecularly imprinted microspheres (MIMs) for the topical anesthetic benzocaine (BZC). The desired diameter (10-20 µm) and uniform morphology of the MIMs were obtained easily by changing one or more of the synthesis conditions, including type and amount of surfactant, stirring rate, and ratio of organic to water phase. The MIMs obtained were used as a molecular-imprinting solid-phase-extraction (MISPE) material for extraction of BZC in human serum and fish tissues. The MISPE results revealed that the BZC in these biosamples could be enriched effectively after the MISPE operation. The recoveries of BZC on MIMs cartridges were higher than 90% (n = 3). Finally, an MISPE-HPLC method with UV detection was developed for highly selective extraction and fast detection of trace BZC in human serum and fish tissues. The developed method could also be used for the enrichment and detection of BZC in other complex biosamples.

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.

Influence of the capillary diameter on the separation efficiency and sensitivity: a systematic approach.

In this study, the influence of the capillary inner diameter (id) on the efficiency and sensitivity of a capillary zone electrophoresis separation was investigated. Four local anaesthetic drugs (lidocaine, prilocaine hydrochloride, procaine and tetracaine) were separated with a validated method using capillaries of different id. The separation parameter N and the resolution of the critical peak pair were monitored. It was found that N increases in tighter capillaries, while sensitivity decreases indicated by the increased detection limit for lidocaine. This loss in sensitivity can partially be compensated by loading more sample into the capillary by means of an increased injection time and pressure. However, when it comes to the evaluation of the drug quality according to a monograph in the European Pharmacopoeia, we cannot recommend to vary the capillary id in order to meet the system suitability criteria.

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.

Automated analysis of lidocaine and its metabolite in plasma by in-tube solid-phase microextraction coupled with LC-UV for pharmacokinetic study.

A sensitive, selective, and reproducible in-tube solid-phase microextraction and liquid chromatographic (in-tube SPME/LC-UV) method for determination of lidocaine and its metabolite monoethylglycinexylidide (MEGX) in human plasma has been developed, validated, and further applied to pharmacokinetic study in pregnant women with gestational diabetes mellitus (GDM) subjected to epidural anesthesia. Important factors in the optimization of in-tube SPME performance are discussed, including the draw/eject sample volume, draw/eject cycle number, draw/eject flow rate, sample pH, and influence of plasma proteins. The limits of quantification of the in-tube SPME/LC method were 50 ng/mL for both metabolite and lidocaine. The interday and intraday precision had coefficients of variation lower than 8%, and accuracy ranged from 95 to 117%. The response of the in-tube SPME/LC method for analytes was linear over a dynamic range from 50 to 5000 ng/mL, with correlation coefficients higher than 0.9976. The developed in-tube SPME/LC method was successfully used to analyze lidocaine and its metabolite in plasma samples from pregnant women with GDM subjected to epidural anesthesia for pharmacokinetic study.

An effective way to biosynthesize α-glucosyl eugenol with a high yield by Xanthomonas maltophilia.

CONTEXT: Eugenol is known for its analgesic, local anesthetic, anti-inflammatory, antibacterial, and hair growing effects, the application of which, however, is limited by its low solubility, liability of sublimating, and its pungent smell. Compared to eugenol, its glycosylated derivate [eugenol α-glucoside (α-EG)] has more advantages in application. OBJECTIVE: The biosynthesis of α-EG by Xanthomonas maltophilia Hugh (Xanthomonadaceae) BT-112 and the optimum conditions for α-EG production are investigated here. MATERIALS AND METHODS: The α-EG was obtained by fermentation using Xanthomonas maltophilia BT-112 and purified by macroporous absorption resin. The identity of α-EG is confirmed by high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). RESULTS: The maximum yield of α-EG reached 10.62 g/L broth when the suspension of Xanthomonas maltophilia strain was incubated at 30°C with 70 mM eugenol and 1.0 M maltose. DISCUSSION AND CONCLUSION: Bio-fermentation was applied in this work to get α-EG with a high mole conversion, which is a potentially efficient and highly promising approach to modify phenolic compounds into glucosides.

Liposomes for entrapping local anesthetics: a liposome electrokinetic chromatographic study.

Bupivacaine is a lipophilic, long-acting, amide class local anesthetic commonly used in clinical practice to provide local anesthesia during surgical procedures. Several cases of accidental overdose with cardiac arrest and death have been reported since bupivacaine was introduced to human use. Recent case reports have suggested that Intralipid (Fresenius Kabi) is an effective therapy for cardiac toxicity from high systemic concentrations of, e.g. bupivacaine, even though the mechanism behind the interaction is not fully clear yet. Our long-term aim is to develop a sensitive, efficient, and non-harmful lipid-based formulation to specifically trap harmful substances in vivo. In this study, the in vitro interaction of local anesthetics (bupivacaine, prilocaine, and lidocaine) with Intralipid or lipid vesicles containing phosphatidylglycerol, phosphatidylcholine, cardiolipin, cholesterol, and N-palmitoyl-D-erythro-sphingosine (ceramide) was determined by liposome electrokinetic chromatography. The interactions were evaluated by calculating the retention factors and distribution constants. Atomic force microscopy measurements were carried out to confirm that the interaction mechanism was solely due to interactions between the analytes and the moving pseudostationary phase and not by interactions with a stationary lipid phase adsorbed to the fused-silica wall. The heterogeneity of the liposomes was also studied by atomic force microscopy. The liposome electrokinetic chromatography results demonstrate that there is higher interaction between the drugs and negatively charged liposome dispersion than with the commercial Intralipid dispersion.

[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.

Surface modified polypropylene pipette tips packed with a monolithic plug of adsorbent for high-throughput sample preparation.

UV-initiated poly(butyl methacrylate-ethylene glycol dimethacrylate) porous polymer monoliths were prepared in situ in polypropylene-based pipette tips for high-throughput sample preparation. Prior to the in situ polymerization, the surface of the PP tips was modified. In this work, two different surface modification approaches were tested for this purpose. First the photoinitiator benzophenone was used to generate radicals at the surface of PP by hydrogen abstraction. In the second modification approach, a thin layer of a polymer was directly grafted to the surface. The effect of surface modification was measured by contact angle measurements of a drop of water at the surface. As a result of the surface modification, scan electron microscopy images indicate a covalent attachment of the monolith to the wall of the pipette tip. Pipette tips modified with 5% BP in methanol and packed with a plug of monolith were further evaluated for high-throughput sample preparation. Using a liquid handling system, the extraction performance of packed pipette tips was tested for the analysis of ropivacaine in plasma samples. The recovery and reproducibility results were in accordance with internationally accepted criteria for qualitative and quantitative analysis of the test substance, ropivacaine.

A convenient TLC method for the identification of local anesthetics.

A TLC method for the separation of seven local anesthetics and the related antiarrhythmic drug procainamide was developed. Spraying the plates with cobalt(II) thiocyanate solution, followed by spraying with Ehrlich's reagent allowed a clear distinction between the drugs, except for the couple articaine/prilocaine. Articaine could be distinguishedfrom prilocaine and other local anesthetics by a colour reaction with copper(II) sulphate solution.

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.

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.

[Separation of oxetacaine and its metabolites] . / Zur Trennung von Oxetacain und seinen Metaboliten.

Oxetacaine (1), known as Tepilta, may be biotransformed to 2-5. After having tried to separate 1-5 by GC we succeeded in finding the HPLC method on Silicagel Si 60 with the eluent MeOH/TBME/HClO4.

1H-NMR studies of drugs with chiral solvating agent: the direct enantiomeric purity determination of the chiral anesthetic, prilocaine.

Chiral recognition of prilocaine was obtained on a 400 MHz 1H-NMR spectrometer by fast diastereomeric interactions with the chiral solvating agent (S)-(+)-2,2,2-trifluoro-1-(9-anthryl) ethanol (TFAE). Assignment of absolute configuration was based on relative field position of the resolved enantiomeric signals. Influence of temperature, substrate concentration and solvating agent to substrate molar ratio on enantiomeric resolution were studied and evaluated. Optimization of experimental conditions provided two significant resolved signals for quantitative use. Utilizing the relative intensities of the resolved enantiomeric signals of the methine proton attached to the stereogenic center assigned to (S)-(+)- and (R)-(-)-prilocaine, 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 tested mixtures. The mean +/- SD recovery values for the (S)-(+)-enantiomer was 99.9 +/- 0.2% of added antipode (n = 7). The optically pure enantiomers were used to establish the minimum amount detected by the proposed NMR spectroscopic method.