Voltammetry of local anesthetics: theoretical and practical aspects.

Local anesthetics (LAs) are widely used in anesthesiology, ophthalmology, and otolaryngology as well as for treatment of chronic and oncological pain. However, anesthetics can cause adverse effects up to lethal ones. In this work, we cited reviews on chromatographic and spectroscopic methods of local anesthetics determination published earlier, and the main purpose was to review the possibilities and advantages of voltammetric methods used for the LAs determination. The electrochemical behavior, mechanism of LAs transformation on the various working electrodes and analytical parameters of voltammetric methods used for their determination were reviewed in the work. Vast majority of these methods were developed for the most widely used anesthetics in medicine like benzocaine, lidocaine and procaine. Special attention was paid to possible mechanisms of electrochemical oxidation and in some cases reduction of LAs or their derivatives. Voltammetry is used for the determination of LAs in pharmaceutical formulations and in biological fluids. The analytical characteristics in terms of sensitivity, selectivity, reproducibility also were discussed in the article.

Detection of Cutting Agents in Drug-Positive Seized Exhibits within the United States.

The following report summarizes a study performed on seized drug exhibits collected in two U.S. states to evaluate the presence and identification of cutting agents. Aliquots of seized drug materials from Kentucky (n = 200) and Vermont (n = 315) were prepared using a dilute-and-shoot procedure. Initial analysis was performed using gas chromatography-mass spectrometry (GC-MS) followed by analysis using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF). Active compounds detected overall included caffeine (31.0%), quinine/quinidine (24.7%), levamisole (11.6%), acetaminophen, (8.2%) and procaine (8.2%). These compounds were found with several drugs of abuse, such as heroin, fentanyl, methamphetamine, and cocaine. This novel information about cutting agents used to dilute or alter drugs of abuse is important to criminal investigations and in the management of acute intoxications at health centers. However, common methodologies for analysis and standard reporting practices frequently do not include cutting agents, resulting in lacking or inadequate information regarding prevalence of these substances.

Polydiacetylene Liposomal Aequorin Bioluminescent Device for Detection of Hydrophobic Compounds.

In this study, a polydiacetylene liposomal aequorin bioluminescent device (PLABD) that functioned through control of the membrane transport of Ca(2+) ions was developed for detecting hydrophobic compounds. In the PLABD, aequorin was encapsulated in an internal water phase and a calcium ionophore (CI) was contained in a hydrophobic region. Membrane transport of Ca(2+) ions across the CI was suppressed by polymerization between diacetylene molecules. On addition of an analyte, the membrane transport of Ca(2+) ions across the CI increased, and Ca(2+) ions from the external water phase could diffuse into the internal water phase via the CI, which resulted in bioluminescence of the aequorin. Lidocaine, procaine, and procainamide were used as model compounds to test the validity of the detection mechanism of the PLABD. When each analyte was added to a suspension of the PLABD, bioluminescence from the aequorin in the PLABD was observed, and the level of this bioluminescence increased with increasing analyte concentration. There was a linear relationship between the logarithm of the analyte concentration and the bioluminescence for all analytes as follows: R = 0.89 from 10 nmol L(-1) to 10 mmol L(-1) for lidocaine, R = 0.66 from 10 nmol L(-1) to 100 µmol L(-1) for procaine, and R = 0.74 from 100 nmol L(-1) to 100 µmol L(-1) for procainamide. Compared to the traditional colorimetric method using polydiacetylene liposome, the PLABD was superior for both the sensitivity and dynamic range. Thus, PLABD is a valid, simple, and sensitive signal generator for detection of hydrophobic compounds that interact with PLABD membranes.

Simple and Sensitive Paper-Based Device Coupling Electrochemical Sample Pretreatment and Colorimetric Detection.

We report the development of a simple, portable, low-cost, high-throughput visual colorimetric paper-based analytical device for the detection of procaine in seized cocaine samples. The interference of most common cutting agents found in cocaine samples was verified, and a novel electrochemical approach was used for sample pretreatment in order to increase the selectivity. Under the optimized experimental conditions, a linear analytical curve was obtained for procaine concentrations ranging from 5 to 60 µmol L(-1), with a detection limit of 0.9 µmol L(-1). The accuracy of the proposed method was evaluated using seized cocaine samples and an addition and recovery protocol.

Competitive homolytic and heterolytic decomposition pathways of gas-phase negative ions generated from aminobenzoate esters.

An alkyl-radical loss and an alkene loss are two competitive fragmentation pathways that deprotonated aminobenzoate esters undergo upon activation under mass spectrometric conditions. For the meta and para isomers, the alkyl-radical loss by a homolytic cleavage of the alkyl-oxygen bond of the ester moiety is the predominant fragmentation pathway, while the contribution from the alkene elimination by a heterolytic pathway is less significant. In contrast, owing to a pronounced charge-mediated ortho effect, the alkene loss becomes the predominant pathway for the ortho isomers of ethyl and higher esters. Results from isotope-labeled compounds confirmed that the alkene loss proceeds by a specific γ-hydrogen transfer mechanism that resembles the McLafferty rearrangement for radical cations. Even for the para compounds, if the alkoxide moiety bears structural motifs required for the elimination of a more stable alkene molecule, the heterolytic pathway becomes the predominant pathway. For example, in the spectrum of deprotonated 2-phenylethyl 4-aminobenzoate, m/z 136 peak is the base peak because the alkene eliminated is styrene. Owing to the fact that all deprotonated aminobenzoate esters, irrespective of the size of the alkoxy group, upon activation fragment to form an m/z 135 ion, aminobenzoate esters in mixtures can be quantified by precursor ion discovery mass spectrometric experiments.

Separation and determination of anesthetics by capillary electrophoresis with mixed micelles of sodium dodecyl sulfate and Tween 20 using electrochemiluminescence detection.

A simple and new method for the simultaneous determination of procaine (Pro), lidocaine (Lid), ropivacaine (Rop) and bupivacaine (Bup) was developed using capillary electrophoresis separation with mixed micelles and electrochemiluminescence detection. The use of mixed micelles of 2.0 × 10(-3) mol/L sodium dodecyl sulfate (SDS) and 8.0 × 10(-3) mol/L Tween 20 greatly improved separation selectivity. The detection sensitivities of four drugs with a Pt working electrode were increased by modification of the Pt electrode with europium(III)-doped Prussian Blue analog (Eu-PB). Under optimal conditions, the four local anesthetics were well separated and detected. The limits of detection (LOD, S/N = 3) of Pro, Lid, Rop and Bup in standard solution are 2.5 × 10(-8) , 1.3 × 10(-8) , 3.0 × 10(-8) and 4.1 × 10(-8) mol/L, respectively. The limits of quantitation (LOQ, S/N = 10) of Pro, Lid, Rop and Bup are 2.3 × 10(-7) , 1.2 × 10(-7) , 3.7 × 10(-7) and 5.6 × 10(-7) mol/L in a human urine sample, and 8.5 × 10(-7) , 6.9 × 10(-7) , 2.8 × 10(-6) and 1.1 × 10(-6) mol/L in a human serum sample, respectively. The recoveries of four drugs at different spiked concentrations in human urine and serum samples were between 86.5 and 107.6%. The proposed method has been successfully applied to determine local anesthetics in biofluids.

Rapid classification of simulated street drug mixtures using Raman spectroscopy and principal component analysis.

The ability to accurately and noninvasively analyze illicit drugs is important for criminal investigations and prosecution. Current methods involve significant sample pretreatment and most are destructive. The goal of this work is to develop a method based on Raman spectroscopy to classify simulated street drug mixtures composed of one drug component and up to three cutting agents including those routinely found in confiscated illicit street drug mixtures. Spectra were collected on both a homebuilt instrument using a HeNe laser and on a handheld commercial instrument with a 785 nm light source. Mixtures were prepared with drug concentrations ranging from 10 to 100 percent. Optimal preprocessing for the data set included truncating, Savitzky-Golay smoothing, normalization, differentiating, and mean centering. Using principal component analysis (PCA), it was possible to resolve the spectral differences between benzocaine, lidocaine, isoxsuprine, and norephedrine and correctly classify them 100 percent of the time.

Rapid analysis of tetracaine for a tape stripping pharmacokinetic study using short-end capillary electrophoresis.

A rapid and simple short-end (reverse) capillary zone electrophoresis method was developed and validated for the separation and quantification of tetracaine in skin using tape samples. The separation was performed in a 485 mm (400 mm to window) x 50 microm internal diameter fused silica capillary using a background electrolyte of phosphoric acid-Tris pH2.5 at -25 kV. The extraction of tetracaine from tape samples was achieved using methanol diluted to 50% with water before injection. Procaine was the internal standard. The migration times for procaine and tetracaine were 1.25 and 1.36 min, respectively. The limit of quantification for tetracaine was 50 microg, with a signal-to-noise ratio greater than 10. The calibration curve was linear from 50 to 1200 microg with r(2) greater than 0.99. The CV for both within- and between-assay imprecision and the percentage inaccuracy for the quality control samples including lower and upper limits of quantitation were <12.1% and <11%, respectively. The absolute mean recovery of tetracaine was >97%. The accuracy and selectivity of this method allowed the rapid measurement of tetracaine in tape samples obtained from a skin tape stripping study of local anaesthetics in healthy subjects.

Rapid and sensitive HPLC assay for simultaneous determination of procaine and para-aminobenzoic acid from human and rat liver tissue extracts.

A sensitive and rapid high-performance liquid chromatography method has been developed for simultaneous determination of procaine and its metabolite p-aminobenzoic acid (PABA) from human and rat liver tissue extracts. The method has been validated according to ICH guidelines in terms of selectivity, linearity, lower limit of detection, lower limit of quantitation, accuracy, precision and recovery from human and rat liver tissue extracts. Chromatography was carried out on a Discovery C(18) column using 10mM ammonium acetate at pH 4.0 and acetonitrile as mobile phase. Retention times for procaine and PABA were 6.6 and 5.3 min, respectively. Linearity for each calibration curve in both tissue extracts was observed across a range from 10 microM to 750 microM for procaine and PABA. The lower limit of detection for both procaine and PABA was 5 microM and the lower limit of quantitation was 10 microM in both tissue extracts. The intra- and inter-day relative standard deviations (R.S.D.) for both procaine and PABA were <6%. Recoveries of procaine and PABA from human and rat liver tissue extracts were determined by two different methods with a single-step protein precipitation technique being employed in both methods. Recoveries for both procaine and PABA were greater than 80% from both human and rat liver tissue extracts.

Comparative drug release studies of two cationic drugs from pH-responsive nanogels.

pH-responsive nanogels consisting of methacrylic acid-ethyl acrylate (MAA-EA) cross-linked with di-allyl phthalate (DAP) were synthesized via the emulsion polymerization process. Delivery systems based on pH-responsive nanoparticles can control the release of rapidly metabolized drugs and/or have the ability to protect sensitive drugs, thereby making them ideal candidates for drug delivery applications. In this study, a drug selective electrode (DSE) was used to directly measure the concentration of procaine hydrochloride (PrHy) and imipramine hydrochloride (IMI) released from MAA-EA nanogels. With a single drug delivery system, drug release for two different drugs loaded via two distinctly different interaction forces was demonstrated. Drug release was conducted using the DSE under different pHs, MAA-EA molar ratio and DAP content. The release rate increased with pH for PrHy loaded nanogels and MAA-EA molar ratio but decreased with pH for IMI loaded nanogels and DAP content. PrHy was found to be hydrophobically bounded, while IMI was found to be electrostatically bounded onto the MAA-EA nanogels, which was further enhanced by hydrogen bonding.

Multiway partial least-squares coupled to residual trilinearization: a genuine multidimensional tool for the study of third-order data. Simultaneous analysis of procaine and its metabolite p-aminobenzoic acid in equine serum.

A new third-order multivariate calibration approach, based on the combination of multiway-partial least-squares with a separate procedure called residual trilinearization (N-PLS/RTL), is presented and applied to multicomponent analysis using third-order data. The proposed chemometric algorithm is able to predict analyte concentrations in the presence of unexpected sample components, which require strict adherence to the second-order advantage. Results for the determination of procaine and its metabolite p-aminobenzoic acid in equine serum are discussed, based on kinetic fluorescence excitation-emission four-way measurements and application of the newly developed multiway methodology. Since the analytes are also the reagent and product of the hydrolysis reaction followed by fast-scanning fluorescence spectroscopy, the classical approach based on parallel factor analysis is challenged by strong linear dependencies and multilinearity losses. In comparison, N-PLS/RTL appears an appealing genuine multiway alternative that avoids the latter complications, yielding analytical results that are statistically comparable to those rendered by related unfolded algorithms, which are also able to process four-way data. Prediction was made on validation samples with a qualitative composition similar to the calibration set and also on test samples containing unexpected equine serum components.

Characterization of procaine metabolism as probe for the butyrylcholinesterase enzyme investigation by simultaneous determination of procaine and its metabolite using capillary electrophoresis with electrochemiluminescence detection.

Capillary electrophoresis with electrochemiluminescene detection was used to characterize procaine hydrolysis as a probe for butyrylcholinesterase by in vitro procaine metabolism in plasma with butyrylcholinesterase acting as bioscavenger. Procaine and its metabolite N,N-diethylethanolamine were separated at 16 kV and then detected at 1.25 V in the presence of 5.0 mM Ru(bpy)(3)2+, with the detection limits of 2.4x10(-7) and 2.0x10(-8) mol/L (S/N=3), respectively. The Michaelis constant Km value was 1.73x10(-4) mol/L and the maximum velocity Vmax was 1.62x10(-6) mol/L/min. Acetylcholine bromide and choline chloride presented inhibition effects on the enzymatic cleavage of procaine, with the 50% inhibition concentration (IC50) of 6.24x10(-3) and 2.94x10(-4) mol/L.

Flow injection amperometric determination of procaine in pharmaceutical formulation using a screen-printed carbon electrode.

A rapid and simple method for procaine determination was developed by flow injection analysis (FIA) using a screen-printed carbon electrode (SPCE) as amperometric detector. The present method is based on the amine/hydroxylamine oxidation from procaine monitored at 0.80 V on SPCE in sodium acetate solution pH 6.0. Using the best experimental conditions assigned as: pH 6.0, flow rate of 3.8 mL min(-1), sample volume of 100 microL and analytical path of 30 cm it is possible to construct a linear calibration curve from 9.0x10(-6) to 1.0x10(-4) mol L(-1). The relative standard deviation for 5.0x10(-5) mol L(-1) procaine (15 repetitions using the same electrode) is 3.2% and detection limit calculated is 6.0x10(-6) mol L(-1). Recoveries obtained for procaine gave a mean values from 94.8 to 102.3% and an analytical frequency of 36 injections per hour was achieved. The method was successfully applied for the determination of procaine in pharmaceutical formulation without any pre-treatment, which are in good accordance with the declared values of manufacturer and an official method based on spectrophotometric analysis.

Simultaneous determination of procaine and para-aminobenzoic acid by LC-MS/MS method.

A sensitive high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for simultaneous determination of procaine and its metabolite p-aminobenzoic acid (PABA). N-Acetylprocainamide (NAPA) was used as an internal standard for procaine and PABA analysis. This assay method has also been validated in terms of linearity, lower limit of detection, lower limit of quantitation, accuracy and precision as per ICH guidelines. Chromatography was carried out on an XTerra MS C(18) column and mass spectrometric analysis was performed using a Quattro Micro mass spectrometer working with electro-spray ionization (ESI) source in the positive ion mode. Enhanced selectivity was achieved using multiple reaction monitoring (MRM) functions, m/z 237-->100, m/z 138-->120, and m/z 278-->205 for procaine, PABA and NAPA, respectively. Retention times for PABA, procaine and NAPA were 4.0, 4.7 and 5.8min, respectively. Linearity for each calibration curve was observed across a range from 100nM to 5000nM for PABA, and from 10nM to 5000nM for procaine. The intra- and inter-day relative standard deviations (RSD) were <5%.

A sequential injection fluorometric procedure for the determination of procaine in human blood and pharmaceuticals.

An automated procedure for the assay of procaine hydrochloride in human blood and pharmaceuticals was developed using a sequential injection (SI) technique with fluorometric detection and fluorescamine as the fluorescence probe. A few microliters of fluorescamine and procaine hydrochloride solutions were used in the SI system leading to the formation of a derivative, which was then excited by a 400-nm LED and whose emitted fluorescence was monitored at a wavelength of 494 nm. A linear calibration graph was obtained with 10-200 ng mL(-1) (procaine) by loading 10.0 microL of sample solution and 5.0 microL of fluorescamine solution (both 0.125 % m/v). A detection limit of 2.6 ng mL(-1), defined as 3 times the blank standard deviation (3sigma), was achieved along with a sampling frequency of 25 h(-1) and a precision of 2.1 % RSD at the 50.0 ng mL(-1) level. Procaine contents in injection solutions from various pharmaceutical manufactures were analyzed and reasonable agreement was achieved between the values obtained by using the present procedure and the documented spectrophotometry, and both were coincident with the nominal concentrations. In addition, the degradation of procaine in human blood was investigated. A fast degradation of procaine in human blood was observed for the first 30 min, while afterwards the degradation was retarded.

Electrochemistry and voltammetry of procaine using a carbon nanotube film coated electrode.

A new rapid, convenient and sensitive electrochemical method is described for the determination of procaine in pharmaceutical preparations, based on the unique properties of a multi-wall carbon nanotube (MWNT) thin film. The electrochemical behavior of procaine at the MWNT film-coated glassy carbon electrode (GCE) was investigated in detail, showing that the MWNT-coated GCE exhibits electrocatalytic activity to the oxidation of procaine because of the significant peak current enhancement and the lowering of oxidation overpotential. Furthermore, the mechanism for the oxidation of procaine at the MWNT-coated GCE was also studied. Finally, various experimental parameters such as solution pH value, the amount of MWNT, accumulation conditions and scan rate were optimized for the determination of procaine, and a new method with detection limit of 2 x 10(-7) mol/L was developed for procaine determination. This newly proposed method was successfully demonstrated with procaine hydrochloride injection.

[Spectral study and determination of metoclopramide and procaine hydrochloride by sequential injection analysis].

A sequential injection spectrophotometric method is proposed for the determination of metoclopramide or procaine hydrochloride, based on the reactions of metoclopramide and procaine hydrochloride with cerium(IV) in sulfuric acid medium. Red intermediate products were observed and were found unstable so that their analytical use is only possible by sequential injection technology. For metoclopramide, the detection limit is 6.5 microg x mL(-1), and the linear range of determination is 9.7-116.6 microg x mL(-1) with a sampling frequency of 45 h(-1). For procaine hydrochloride, the detection limit is 7.4 microg x mL(-1), and the linear range of the determination is 10.0-130.0 microg x mL(-1) with a sampling frequency of 45 h(-1). The method has been applied to the determination of metoclopramide and procaine hydrochloride in tablets and injections with satisfactory results as compared with standard method.

Hair analysis as a novel investigative tool for the detection of historical drug use/misuse in the horse: a pilot study.

REASONS FOR PERFORMING STUDY: Analysis of human hair for drug residues is being used increasingly as a diagnostic tool in the investigation of drug use and abuse. Hair analysis is complementary to urine/blood testing in that it can provide an extensive historical record of drug use, is noninvasive, impersonal and can facilitate retesting. However, the technique has not been studied in horses. HYPOTHESIS: That the systemic administration of drugs in horses could be identified by the detection of drug residues in hair. OBJECTIVE: To evaluate hair analysis as a potential retrospective diagnostic test for drug administration in horses by studying the deposition of systemically administered drugs in tail hair. METHODS: Tail hairs (n = 40-50) from 4 horses with known drug histories were washed, chopped into 3-5 mm fragments and extracted overnight, in 0.1 mol/l hydrochloric acid, prior to solid-phase extraction and analysis by high-performance liquid chromatography. Horse 1, a 3-year-old Thoroughbred colt (gastric ulcer), was treated for 14 days with omeprazole; Horse 2, a 3-year-old Thoroughbred colt (anaerobic infection), was treated for 5 days with metronidazole; Horse 3, an 8-year-old Thoroughbred gelding (sinusitis), was treated for 10 days with trimethoprim/sulphadiazine; and Horse 4, a 3-year-old Thoroughbred colt (respiratory infection), was treated for 5 days with procaine benzylpenicillin. RESULTS: Omeprazole was not detected in tail hair. Metronidazole was detected in tail hair at a concentration of 0.57 ng/mg, trimethoprim and sulphadiazine at concentrations of 9.14 and 2.26 ng/mg, respectively, and procaine at a concentration of 1.66 ng/mg. CONCLUSIONS: The data presented suggest that hair analysis may become a useable technique for the retrospective detection of drug administration in horses. POTENTIAL RELEVANCE: This technique could ultimately be used as part of a prepurchase veterinary examination to identify misuse of anti-inflammatory and sedative drugs, in an in-training testing programme to identify use of anabolic agents, or to provide evidence to support post race blood or urine test results. Clearly, more extensive research will be required to evaluate the effectiveness of the technique over a much broader range of drugs.

Highly accurate nephelometric titrimetry.

A method that accurately indicates the end-point of precipitation reactions by the measurement of the relative intensity of the scattered light in the titrate is presented. A new nephelometric titrator with an internal nephelometric sensor has been devised. The work of the titrator including the sensor and change in the turbidity of the titrate and intensity of the scattered light are described. The accuracy of the nephelometric titrimetry is discussed theoretically. The titration of NaCl with AgNO(3) serves as a model. A relative error as well as deviation is within 0.2% under the experimental conditions. The applicability of the titrimetry in pharmaceutical analyses, for example, phenytoin sodium and procaine hydrochloride, is generally illustrated.