Screen printed potentiometric sensor for therapeutic monitoring of rocuronium at the point of care.

Rocuronium bromide (ROC) is currently regarded as the 'gold-standard' in emergency medicine and anesthesia. Globally, millions of human beings are daily administered ROC at emergency settings where it is favored among all the neuromuscular blockers, particularly succinylcholine, for both its fast onset of action and short duration. However, it has been reported that 45% of patients in the post-anesthesia care unit are susceptible to residual postoperative paralysis, undesired ventilator effects and incomplete recovery after ROC administration. From an analytical chemistry perspective, direct determination of ROC is a difficult approach due to the complexity in isolation from biological specimens as well as the lack of a sensitive detection techniques and detectable chromophore. This contribution describes the development of a calix[6]arene-based screen-printed electrode (SPE) that is capable of ROC detection in biological samples at the point of care. This fabricated SPE (sensor 1) exhibited superior performance characteristics (slope, LOD and life time) with respect to an ionophore-free liquid-contact electrode, LCE, (sensor 2). The proposed SPE showed a linear response over a concentration from 1 µM to 10 mM, with a Nernstian slope of 57.9 mV/decade and a detection limit of 0.39 µM. Moreover, this sensor showed a considerable selectivity towards ROC in presence of the anticipated interfering ions. To investigate the ability of the SPE to detect ROC in real biological specimens, ROC has been spiked at a concentration comparable to its anticipated level in human plasma (Cmax~ 40 µM) and the proposed SPE displayed an excellent platform for therapeutic drug monitoring (TDM) of ROC with respect to UV-spectrophotometry and LC/MS. Finally, the developed SPE was used for the determination of ROC in its commercial pharmaceutical formulation.

Physician suicide.

Pathology files at Forensic Science South Australia were examined over a 14-year period, from January 1997 to March 2011 for cases of physician suicide. Nine cases were identified (ages, 30-69 years; median = 41 years; M:F = 3:1). Three cases (33.3%) had a history of prescription drug abuse, and eight cases (88.9%) died from lethal drug self-administration. Intravenous administration of drugs was the favored method in seven cases. In only one case was an alternative method used, involving jumping from a high building. A history of depression and/or suicidal ideation was noted in six cases (66.7%). The methods of self-destruction contrasted with the general population where hanging, carbon monoxide poisoning, and gunshot wounds are more common. Availability and knowledge of lethal effects have been shown to be important determinants in the choice of methods of suicide. Access to drugs should therefore be carefully monitored in physicians with histories of depression and/or substance abuse.

Determination of muscle relaxants pancuronium and vecuronium bromide by capillary electrophoresis with capacitively coupled contactless conductivity detection.

A novel capillary electrophoretic method for the separation of pancuronium (PM) and vecuronium (VM) ions utilizing capacitively coupled contactless conductivity detection was devised and validated. The separation was carried out in bare fused-silica capillaries (50 µm id, 75/45 cm) at 25°C. Optimal BGE was 50 mM borate buffer of pH 9.5 containing 12.5 mg/mL of (2-hydoxypropyl)-γ-CD. The samples were injected hydrodynamically at 1000 mbar for 3 s. Separation was performed at +30 kV. Under such conditions the PM and VM were base-line resolved and the separation took < 4 min. For quantification phenyltrimethylammonium iodide was used as internal standard. Calibration curves were linear for both pancuronium bromide (PMB) and vecuronium bromide (VMB) in the range 25-250 µg/mL with r> 0.9968. The limits of detection were 7 and 6 µg/mL for PMB and VMB, respectively. The accuracy tested by recovery experiment at three concentration levels of added PMB and VMB was satisfactory (95.7-102.7%, n =3, with RSD < 2.61%). The method was successfully applied to the assay of PMB and VMB in commercial injection solutions.

Feasibility of microdialysis to determine interstitial rocuronium concentration in the muscle tissue of anesthetized neurosurgical patients.

BACKGROUND: Microdialysis is used to determine the concentrations of substances in the extracellular fluid of tissues. To date, it has not been used to measure rocuronium concentrations in human muscle. We determined the ability of microdialysis to recover rocuronium from muscle interstitial tissue for the purpose of assessing the effect of chronic phenytoin therapy on muscle concentrations of rocuronium. METHODS: In a first phase, an in vitro study was performed to establish the ability of the assay to recover rocuronium. In a second phase, 17 patients undergoing brain surgery were enrolled. Eight patients were on chronic phenytoin therapy and the remaining 9 patients were not taking any antiepileptic agent (controls). Rocuronium was administered intravenously and muscle tissue samples for microdialysis were collected. RESULTS: The recovery rate of the in vitro assay was 36% at a pump rate of 1 microL/min. Rocuronium muscle tissue concentrations could be measured in 25 microdialysate samples. Rocuronium concentrations were similar in patients treated with phenytoin and in controls, although the doses required to obtain a similar effect were significantly higher in patients on chronic phenytoin treatment. CONCLUSIONS: Quantification of drug concentrations in muscle by means of microdialysis is technically feasible in the clinical setting and it might help in studying pharmacologic mechanisms of drug action. Based on our results the decrease in the degree of effect of rocuronium in the presence of chronic phenytoin therapy might seem to be due mainly to a pharmacokinetic mechanism.

Determination of atracurium, cisatracurium and mivacurium with their impurities in pharmaceutical preparations by liquid chromatography with charged aerosol detection.

The Corona CAD (charged aerosol detection) is a new type of detector introduced for LC applications that has recently become widely applied in pharmaceutical analysis. The Corona CAD measures a physical property of analyte and responds to almost all non-volatile species, independently of their nature and spectral or physicochemical properties. The LC method with charged aerosol detection was developed for the determination of three isomers of atracurium, cisatracurium and also three isomers of mivacurium with their impurities. The limit of quantitation for laudanosine was 1 microg ml(-1). The elaborate method for the analysis of those active substances and laudanosine proved to be fast, precise, accurate and sensitive. All other impurities were identified using time-of-flight mass spectrometry with electrospray ionization.

Quantitative analysis of the aminosteroidal non-depolarizing neuromuscular blocking agent vecuronium by LC-ESI-MS: A Postmortem Investigation.

The apparent recreational use of an aminosteroidal non-depolarizing neuromuscular blocking agent, vecuronium, is reported in this postmortem investigation. A quantitative method for the analysis of vecuronium and its active metabolite, 3-desacetylvecuronium, in blood and tissue samples was developed using liquid chromatography-electrospray ionization mass spectrometry operated in positive selected ion monitoring mode. Chromatographic separation was performed on a Gemini 5-microm C18 column using a mobile phase of 0.1% formic acid/acetonitrile at 0.700 mL/min. The method was linear from 0.01 to 1.00 mg/L with correlation coefficients of 0.999 and greater for both compounds. The limits of detection and quantitation were determined in blood to be 0.005 and 0.010 mg/L, respectively. The coefficients of variation were less than 10% for both intra- and interday assays. Vecuronium was quantitated in blood at 0.070 mg/L and in the kidney, liver, and spleen at 0.224, 0.045, and 0.080 mg/kg, respectively. The active metabolite 3-desacetylvecuronium was quantitated in blood at 0.100 mg/L, in the urine at 0.040 mg/L and in the kidney, liver, spleen, and lung at 0.271, 0.100, 0.082, and 0.164 mg/kg, respectively.

Simultaneous determination of atracurium and its metabolite laudanosine in post-mortem fluids by liquid chromatography/multiple-stage mass spectrometry on an ion trap.

A method was developed to accurately quantify atracurium (a non-depolarizing skeletal muscle relaxant) and its metabolite laudanosine in post-mortem specimens. Analytes were isolated from blood and tissues by liquid/liquid extraction after adding vecuronium as an internal standard. Chromatographic separation was accomplished by gradient elution in a Synergy Max RP 150 x 2.1 mm column. Positive ion electrospray ionization and mass spectrometric analyses were carried out in an ion trap mass spectrometer under collision-induced dissociation conditions. The method proved selective and sensitive, and was validated in post-mortem blood, heart, lung and liver in the range of 1-2000 ng/mL (blood) and 5-5000 ng/g (tissues); the limits of quantification obtained were 1 ng/mL in blood and 5 ng/g in tissues.

A modification of the kinetic determination of pancuronium bromide based on its inhibitory effect on cholinesterase.

A modification of the existing spectrophotometric kinetic method for the determination of pancuronium bromide (PCBr), based on pooled human serum cholinesterase (ChE, EC 3.1.1.8 acylcholine acylhydrolase) inhibition, was developed. Butyrylthiocholine iodide (concentration 1.667 mmol/L) was used as substrate and determination was performed at pH 7.6. Essential basic kinetic parameters were also determined: Michaelis-Menten's constant KM=0.33 mmol/L, maximal reaction rate Vmax=42.29 micromol/L min, inhibition constant KI=0.34 micromol/L, and IC50=0.235 micromol/L. Linear dependence between the reaction rate and the inhibitor concentration exists in PCBr concentration range 8.29-265.28 nmol/L, which corresponds to the real sample concentrations from 0.166 to 5.306 micromol/L. The method detection limit was established to be 1.86 nmol/L and the quantification limit was 6.18 nmol/L. Precision of the method was tested for three pancuronium concentrations (16.58, 99.48, and 198.96 nmol/L). The relative standard deviation (RSD) was in the range 0.78-5.13%. Accuracy was examined by the standard addition method. The influence of substances usually present in serum and urine on the reaction rate was determined. The method developed was applied for PCBr determination in spiked serum and urine samples and in the urine taken during surgery. The method was proven to have good sensitivity, accuracy, and precision and can be considered suitable for clinical practice.

The identification of mivacurium and metabolites in biological samples.

Mivacurium is a muscle relaxant that is used in hospitals and has been implicated in a number of homicides. A validated, sensitive method for the analysis of mivacurium and metabolites from biological specimens is presented here. Sample cleanup involves the precipitation of proteins with acetonitrile followed by analyte isolation using a commercially available solid-phase extraction column. Screening is performed by liquid chromatography with fluorescence detection, and confirmation is performed by liquid chromatography with tandem mass spectrometric detection. This method has been used to analyze dozens of forensic samples, including clinical specimens from living patients, as well as autopsy specimens from exhumed bodies.

Instability of pancuronium in postmortem blood and liver taken after a fatal intramuscular Pavulon injection.

The present study was designed to determine the stability of pancuronium in postmortem blood and liver during storage. Results were obtained using the method by Kerskes et al. [C.H.M. Kerskes, K.J. Lusthof, P.G.M. Zweipfenning, J.P. Franke, The detection and identification of quaternary nitrogen muscle relaxants in biological fluids and tissues by ion-trap LC-ESI-MS, J. Anal. Toxicol. 26 (2002) 29-34.], modified and validated in our laboratory. Target analytes were isolated after enzymatic hydrolysis followed by solid phase extraction (BondElut C18 column). Internal standardisation was carried out using laudanosine and the target ions were monitored by LC-ESI-MS (monitoring ions m/z 358 for IS and 286 for pancuronium). Materials were taken from a 46-year-old woman, who had been found dead. A syringe (2 ml) and an empty ampoule of Pavulon (4 mg/2 mL) were found in her hand. The residual volume of fluid in the syringe was 0.7 ml. An autopsy was performed six days after death. It revealed a needle mark on the left thigh. Postmortem materials (muscle from the injection site, blood and liver) and the syringe with fluid were stored for four months in a freezer at -20 degrees C. The initial pancuronium concentrations were 81 ng/mL in blood and 532 ng/g in liver. The analyte was stable when stored at -20 degrees C in blood even up to seven months. In liver samples its concentrations were variable. Pancuronium in blood stored at 20 degrees C underwent degradation very rapidly. After three months of storage these blood samples had concentrations not greater about 10% of the initial value. The degradation patterns of pancuronium depended on temperature and the biological matrix.

Determination of interstitial rocuronium concentrations in the muscle tissue of anesthetized dogs by microdialysis.

INTRODUCTION: The objective was to establish and validate a microdialysis technique for the quantification of interstitial concentrations of the neuromuscular blocker, rocuronium, in the muscle tissue of dogs under steady-state conditions. METHODS: The standard and combined retrodialysis approaches were used for in vivo microdialysis probe calibration. After induction of anesthesia with pentobarbital (30 mg/kg), the left femoral vein was cannulated and blood drawn for protein binding determination. Microdialysis probes were inserted in the muscle and calibrated in vivo, using vecuronium as the calibrator. Each dog received a short 2-min infusion followed by a 120-min infusion of rocuronium via the right jugular vein and three microdialysis samples were collected at steady-state during a 2-h period. Samples were stored at -70 degrees C until HPLC analysis. RESULTS: Using combined retrodialysis, rocuronium unbound interstitial (C(ISFu)) and venous plasma (C(pssuv)) concentrations are in good agreement; with a ratio C(ISFu)/C(pssuv) of 100+/-11%. Using standard retrodialysis, this ratio was 47+/-7%. CONCLUSIONS: Combined retrodialysis is a more reliable and accurate technique for quantitative assessment of rocuronium interstitial concentrations especially for lengthy anesthetic procedures. These findings have potential implications, as drug concentrations in the site of action would be more relevant for concentration-effect relation of muscle relaxants.

Evaluation of the impurity profile of alcuronium by means of capillary electrophoresis.

Alcuronium, a neuromuscular blocking drug, was recently introduced to the European Pharmacopoeia. A HPLC method is described to limit the impurities of alcuronium, namely the diallylcaracurine (DAC) and the allyl-Wieland-Gumlich-aldehyde (WCA), to less than 0.5%. Since alcuronium and all impurities are quaternary salts, capillary electrophoresis (CE) is highly suitable to evaluate the impurity profile. Using 12 mM heptakis-(2,6-di-O-methyl)-beta-cyclodextrin in a 50 mM phosphate buffer at pH 5.5 or 50 mM diethanolamine buffer (pH 9.2)-acetonitrile 19:1 containing heptakis-(2,3-O-diacetyl-6-sulfo)-beta-cyclodextrin the impurities could be baseline separated and quantified. The limit of detection for DAC and WCA was found to be in the same range as found with HPLC; thus, less than 0.1% of both DAC and WCA could be detected in the solution for injection in presence of alcuronium. In injection solutions of alcuronium which were stored at higher temperatures three additional, unidentified impurities were detected. In addition, the conversion of alcuronium to DAC, occurring under acidic condition, was monitored by means of the CE method developed.

The detection and identification of quaternary nitrogen muscle relaxants in biological fluids and tissues by ion-trap LC-ESI-MS.

Quaternary nitrogen muscle relaxants pancuronium, rocuronium, vecuronium, gallamine, suxamethonium, mivacurium, and atracurium and its metabolites were extracted from whole blood and other biological fluids and tissues by using a solid-phase extraction procedure. The extracts were examined by using high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). The drugs were separated on a ODS column in a gradient of ammonium acetate buffer (pH 5.0) and acetonitrile. Full-scan mass spectra of the compounds showed molecular ions, and MS-MS spectra showed fragments typical of the particular compounds. LC-ESI-MS allowed an unequivocal differentiation of all muscle relaxants involved. The method was applied in a case of rocuronium and suxamethonium administration in a Caesarian section and in a case of intoxication by pancuronium injection. In both cases, the administered drugs could be detected and identified in the supplied samples.

Application of a high-performance liquid chromatographic assay for the neuromuscular blocker gallamine to analysis of rat plasma, muscle and microdialysate samples.

A reliable high-performance liquid chromatographic method has been validated for determination of gallamine in rat plasma, muscle tissue and microdialysate samples. A C18 reversed-phase column with mobile phase of methanol and water containing 12.5 mM tetrabutyl ammonium (TBA) hydrogen sulphate (22:78, v/v) was used. The flow-rate was 1 ml/min with UV detection at 229 nm. Sample preparation involved protein precipitation with acetonitrile for plasma and muscle tissue homogenate samples. Microdialysate samples were injected into the HPLC system without any sample preparation. Intra-day and inter-day accuracy and precision of the assay were <13%. The limit of quantification was 1 microg/ml for plasma, 1.6 microg/g for muscle tissue and 0.5 microg/ml for microdialysate samples. The assay was applied successfully to analysis of samples obtained from a pharmacokinetic study in rats using the microdialysis technique.

Cathodic adsorptive stripping voltammetric determination of muscle relaxant: gallamine triethiodide (flaxedil).

A sensitive and simple voltammetric method of analysis is developed for the determination of trace amounts of gallamine triethiode in phosphate media. This method is based on controlled adsorptive preconcentration of the relaxant onto a Hanging Mercury Drop Electrode (HMDE) whereby mercurous iodide salt(s) are formed. The technique used is Cathodic Linear Sweep Stripping Voltammetry (CLSSV). The adsorptive response was evaluated with respect to preconcentration time and potential. As little as 3 x 10(-9) mol dm(-3) i.e. 2.7 ppb flaxedil (proconcentration time 300 seconds) can be determined successfully. The application of this method was tested in the determination of flaxedil in pharmaceutical preparation (ampoules).

Assessment of in vitro and in vivo recovery of gallamine using microdialysis.

The application of microdialysis technique for the investigation of pharmacokinetics and pharmacodynamics of drugs requires careful assessment of probe performance to ensure validity of the data obtained using this technique. The aim of this study was to establish and validate the microdialysis technique for investigation of the pharmacokinetics and pharmacodynamics of the neuromuscular blocker, gallamine. In vitro recovery of gallamine from the microdialysis probe when different perfusion flow rates were employed was evaluated leading to selection of a flow rate of 2 microl/min with 15-min sampling intervals for the subsequent studies. In vitro recovery of gallamine from the microdialysis probe was independent of concentration, stable over an 8-h period and reproducible. Comparable in vitro recoveries were obtained by different established approaches including recovery estimation by gain, loss and the zero-net flux (ZNF) method. Recovery by loss was used to study the in vivo recovery of gallamine from rat muscle tissue. The in vivo recovery was stable over a 5.5-h sampling period. In vitro performance of the probe subsequent to the in vivo study remained stable supporting reusage of the probe. These data highlight the importance of a systematic examination of microdialysis probe validation.

Atracurio en anestesia general / Atracurio in general anesthesia

Este trabajo busca evidenciar las caracteristicas de accion relajante, interaccion relajante con los anestesicos mas empleados en el medio. Se estudiaron 122 pacientes, tomando en cuenta la medicacion preanestesica empleada, la inclusion del halotano cruzando datos con el peso de los pacientes y tiempo de duracion de accion. En general existe en este estudio mayor tiempo de duracion de accion (41-61) que el teorico 17-40'. El empleo de benzodiazepinas no prolonga significativamente esta duracion, siendo el halotano el que mas prolonga el tiempo de relajacion. En todos los casos el atracurio resulto ser un buen relajante para la intubacion, su reversion casi no es necesaria luego de 30' de accion. Los efectos adversos en este estudio practicamente fueron insignificantes

Determination of rocuronium and its putative metabolites in body fluids and tissue homogenates.

A sensitive and selective HPLC method was developed for the quantification of the neuromuscular blocking agent rocuronium and its putative metabolites (the 17-desacetyl derivative and the N-desallyl derivative of rocuronium) in plasma, urine, bile, tissue homogenates and stoma fluid. Samples were prepared by extraction of the biological matrix with dichloromethane, after mixing with a KI-glycine buffer. After evaporation of the organic solvent the samples were chromatographed on a reversed-phase HPLC column, using an aqueous buffer-dioxane (84:16, v/v) as the mobile phase. The aqueous buffer consisting of 0.1 M sodium dihydrogen phosphate, 0.11 mM 9,10-dimethoxyanthracene-2-sulphonate (DAS), 0.11 mM 1-heptane-sulfonic acid, was adjusted to pH 3 with orthophosphoric acid. After separation, the eluent was extracted with dichloroethane, and the organic phase was led to a fluorimetric detector, operating at 385 nm (excitation) and 452 nm (emission). The method was validated for the assay in plasma, urine, bile, tissue homogenates and stoma fluid, by determination of the repeatability, reproducibility, accuracy, lower limit of quantification, lower limit of detection, extraction recovery, effect of sample volume, and stability in the biological matrix. The method was found to be sensitive (lower limit of quantification for rocuronium in plasma is 10 ng/ml) and accurate. The interference of concomitant drugs with the assay of rocuronium and its putative metabolites has been studied extensively. In order to confirm the identity of rocuronium and its putative metabolites, a TLC method was developed. The method has been applied successfully in several pharmacokinetic studies with rocuronium.

[Studies on the medicinal plants of Magnoliaceae tu-hou-po of Manglietia].

Data from a survey of the drug market and investigation of the original plant of "Tu-hou-po", after careful botanical examinations, showed that the drugs were derived from 5 species of the genus Manglietia of Magnoliaceae, viz. Manglietia chingii Dandy, M. insignis (Wall.) Bl., M. duclouxii Finet et Gagnep., M. yuyuanensis Law and M. szechuanica Hu. Comparisons of the main characteristics of the plants, Tu-hou-po and Hou-po crude drugs and chemical components showed that Manglietia is taxonomically the closest to Magnolia and contained similar components (tab 1-2 and fig 1). The results of HPLC analysis demonstrated that they contained magnolol, honokiol, magnocurine and salicifoline, in different quantities. However, no magnosprengerine was detected. Besides, it was also found that the percentage of magnolol and honokiol contents were higher, while that of magnocurine was lower in Hou-po. On the contrary, the content of magnocurine was higher, while that of magnolol and honokiol were lower in Tu-hou-po. Manglietia chingii (Tu-hou-po) is being used as the Chinese traditional drug "Hou-po" in the clinic in Guangxi. Therefore, M. chingii is noteworthily exploited as a new resource of Hou-po for further research.