Preparation and application of a molecularly imprinted monolith for specific recognition of domoic acid.

In this work, a molecularly imprinted monolithic column was synthesized by a facile procedure and was applied for specific recognition of domoic acid, an amnesic shellfish poison. The poly(4-vinylpyridine-co-ethylene glycol dimethacrylate) molecularly imprinted monolith was synthesized in a stainless steel column by in situ polymerization. Pentane-1,3,5-tricarboxylic acid was used as a dummy imprinting template instead of the highly toxic and expensive target molecule. It is the first time that a molecularly imprinted monolith is introduced for separation and detection of domoic acid. After optimizing the preparation conditions, the prepared imprinted monolith was systematically characterized and exhibited excellent stability and permeability as a HPLC stationary phase. The results of chromatographic analysis demonstrated that the molecularly imprinted monolith exhibited specific retention and selective recognition toward domoic acid, with an imprinted factor up to 3.77. Furthermore, the molecularly imprinted monolith was successfully applied for selective enrichment of domoic acid from biological samples. Graphical abstract A molecularly imprinted monolith was prepared by using a dummy imprinted template and was successfully applied for specific recognition of domoic acid.

Spectroscopic determination of succinylcholine in dosage forms using eosin Y.

Two simple and sensitive analytical assay methods using spectrophotometry and spectrofluorimetry techniques were developed for the estimation of succinylcholine chloride (SUC) in pharmaceutical preparations. The suggested methods are based on the formation of an ion pair complex formed between the drug and eosin Y spectrophotometrically (Method I), or the suppressive effect of succinylcholine on the native fluorescence property of eosin Y (Method II). The spectrophotometric method (Method I) involves measuring the absorbance of the complex between succinylcholine and eosin Y at 550 nm in Britton Robinson buffer of pH 3. However, the spectrofluorimetric method (Method II) involves measuring the quenching effect of the studied drug on the native fluorescence property of eosin Y at the same pH at 550 nm after excitation at 480 nm. The absorbance versus concentration of the drug is rectilinear over the range of 0.5 to 15 µg/ml. The formation constant was 3.5 × 104 and the Gibb's free energy change was -2.5 × 104  J/mol. In Method II, the relative fluorescence intensity was directly proportional to SUC concentration over the range of 0.05 to 1 µg/ml. The proposed methods allowed a successful application to the estimation of succinylcholine ampoules. An explanation of the reaction pathway was postulated.

Stability of suxamethonium in pharmaceutical solution for injection by validated stability-indicating chromatographic method.

STUDY OBJECTIVE: To assess the stability of pharmaceutical suxamethonium (succinylcholine) solution for injection by validated stability-indicating chromatographic method in vials stored at room temperature. METHODS: The chromatographic assay was achieved by using a detector wavelength set at 218 nm, a C18 column, and an isocratic mobile phase (100% of water) at a flow rate of 0.6 mL/min for 5 minutes. The method was validated according to the International Conference on Harmonization guidelines with respect to the stability-indicating capacity of the method including linearity, limits of detection and quantitation, precision, accuracy, system suitability, robustness, and forced degradations. RESULTS: Linearity was achieved in the concentration range of 5 to 40 mg/mL with a correlation coefficient higher than 0.999. The limits of detection and quantification were 0.8 and 0.9 mg/mL, respectively. The percentage relative standard deviation for intraday (1.3-1.7) and interday (0.1-2.0) precision was found to be less than 2.1%. Accuracy was assessed by the recovery test of suxamethonium from solution for injection (99.5%-101.2%). CONCLUSION: Storage of suxamethonium solution for injection vials at ambient temperature (22°C-26°C) for 17 days demonstrated that at least 95% of original suxamethonium concentration remained stable.

Three homicides with darts tainted with succinylcholine: autopsy and toxicology.

In emergency departments, intoxication with the muscle relaxant succinylcholine (SUX) often leads to a potentially lethal respiratory paralysis or other deleterious side effects. However, homicide cases with SUX poisoning are very rare because the toxic or lethal concentration ranges of SUX have not yet been determined. We described three uncommon homicide cases due to acute poisoning by darts contaminated with SUX. All the victims died quickly (less than 30 min) after being shot by an especially designed dart gun. Succinylmonocholine (SMC), a metabolite of SUX, was used as a marker to detect the latter. HPLC-MS/MS analysis demonstrated the presence of SUX in the droplet residues of the darts and SMC in the blood and urine in all cases. SMC concentrations of 0.45, 14.0, and 17.9 ng/ml were detected in the victims' blood and 259.0 ng/ml in the urine from the third case. The main pathological changes consisted of hemorrhage of the injured soft tissues, visceral congestion, severe pulmonary edema, and multifocal petechial hemorrhage of the heart and lungs. Taken together, the findings supported a diagnosis of fatal SUX poisoning. Futhermore, our study provided a reference for the lethal concentrations of SUX poisoning.

Amnesic shellfish poisoning biotoxin detection in seawater using pure or amino-functionalized Ag nanoparticles and SERS.

Domoic acid (DA) biotoxin responsible for the amnesic shellfish poisoning (ASP) has been unambiguously detected in seawater in a broad range of concentration, with both pure and amino-functionalized Ag nanoparticles employed for surface enhanced Raman scattering (SERS). To achieve this, a comprehensive SERS study on DA dissolved in distilled water has been conducted. SERS of DA dissolved in seawater in concentrations ranging from 3.3 × 10(-4) to 3.3 × 10(-8) mol l(-1) exhibited specific signal, completely different to those of the corresponding DA aqueous solutions, due to the seawater interference in the overall SERS effect. In order to assess the capability of the technique as a cheaper alternative for rapid and unambiguous detection of the DA biotoxin in seawater, three detection schemes have been proposed. DA was detectable at 0.33 nmoll(-1) concentration (0.33) dissolved in distilled water and 0.033 nmol l(-1) (0.033 ppb) in seawater respectively, much lower than the admitted level by the current regulation. A solvent specific interaction of DA with the NPs was concluded, since DA aqueous solution added to Ag nanoparticles provided different SERS signal compared to that of DA directly dissolved in seawater. Employing amino-functionalized Ag nanoparticles with 4-aminothiophenol as SERS tag, SERS signal of DA on amino-AgNPs revealed significant specificity associated with the aromatic primary amine interaction of the SERS tag with DA, thus allowing DA detection in seawater at 4.16 × 10(-4) mol l(-1) concentration, much higher than in the case of pure NPs. To highlight the findings, a brief literature review to date on the DA biotoxin detection was also provided.

[Progress on suxamethonium chloride analysis].

Abstract: Suxamethonium chloride is a depolarizing muscle relaxant used in general anesthesia. In overdose, it causes adverse reactions such as bradycardia, arrhythmia, cardiac arrest, and death. The article reviews the progress on testing methods of suxamethonium chloride such as infrared spectroscopy, chemical color reaction, chemical titration, enzyme electrode, chromatography and mass spectrometry.

Progress on suxamethonium chloride analysis / 法医学杂志

Abstract: Suxamethonium chloride is a depolarizing muscle relaxant used in general anesthesia. In overdose, it causes adverse reactions such as bradycardia, arrhythmia, cardiac arrest, and death. The article reviews the progress on testing methods of suxamethonium chloride such as infrared spectroscopy, chemical color reaction, chemical titration, enzyme electrode, chromatography and mass spectrometry.

Applicability of succinylmonocholine as a marker for succinylcholine administration--comparative analysis of samples from a fatal succinylcholine-intoxication versus postmortem control specimens.

Doubts concerning the applicability of succinylmonocholine (SMC) as a succinylcholine (SUX) marker have been issued. A comparative analysis of previously discussed tissues, i.e. brain, liver and kidney, was conducted to further elucidate this question by searching for diagnostically useful differences in analyte content in samples of SUX- versus non-SUX-associated fatalities. Furthermore, possible advantages of vitreous humor as a novel and promising target matrix for SUX analytics were assessed. Sample material of SUX-negative controls as well as the fatal SUX-intoxication was derived from frozen archive material and current autopsies. Samples were analyzed according to a modified protocol of a previously published and validated method employing ion-pairing solid-phase extraction and subsequent HPLC-MS/MS analysis. Standard addition was employed for quantification as well as an estimation of the analytical limits of the method. In all tested matrices, the method was proven to be sufficiently sensitive for the intended application. No indication of native SMC was found in controls of fresh tissues, nor in fresh or frozen vitreous humor. However, most of the samples were found to be positive for a previously reported interference with SMC's main ion transition, thereby falsely suggesting an SMC content of up to 139 ng/g, 126 ng/g, 165 ng/g and 93 ng/ml in brain, liver, kidney and vitreous humor, respectively. Contrasting the results for fresh sample material, SMC was detectable in some of the initially non-putrefied liver samples after long-term storage, as well as in massively decomposed SUX-negative control bodies. In this context, a microbial origin of the analyte may be assumed. All tissues as well as the vitreous humor of the fatal SUX-intoxication were negative for SUX and SMC. Just like serum, tissue and vitreous humor samples therefore do not allow a reliable diagnosis of a SUX-intoxication: in tissues this is due to the pronounced instability of both target analytes in these esterase-containing matrices, for vitreous humor an additional reason could be their insufficient incorporation into this medium.

A protozoal-associated epizootic impacting marine wildlife: mass-mortality of southern sea otters (Enhydra lutris nereis) due to Sarcocystis neurona infection.

During April 2004, 40 sick and dead southern sea otters (Enhydra lutris nereis) were recovered over 18km of coastline near Morro Bay, California. This event represented the single largest monthly spike in mortality ever recorded during 30 years of southern sea otter stranding data collection. Because of the point-source nature of the event and clinical signs consistent with severe, acute neurological disease, exposure to a chemical or marine toxin was initially considered. However, detailed postmortem examinations revealed lesions consistent with an infectious etiology, and further investigation confirmed the protozoan parasite Sarcocystis neurona as the underlying cause. Tissues from 94% of examined otters were PCR-positive for S. neurona, based on DNA amplification and sequencing at the ITS-1 locus, and 100% of tested animals (n=14) had elevated IgM and IgG titers to S. neurona. Evidence to support the point-source character of this event include the striking spatial and temporal clustering of cases and detection of high concentrations of anti-S. neurona IgM in serum of stranded animals. Concurrent exposure to the marine biotoxin domoic acid may have enhanced susceptibility of affected otters to S. neurona and exacerbated the neurological signs exhibited by stranded animals. Other factors that may have contributed to the severity of this epizootic include a large rainstorm that preceded the event and an abundance of razor clams near local beaches, attracting numerous otters close to shore within the affected area. This is the first report of a localized epizootic in marine wildlife caused by apicomplexan protozoa.

Amnesic shellfish poisoning (ASP) toxins in plankton and molluscs from Luanda Bay, Angola.

The presence of ASP toxins in Luanda Bay, an area 2700 km apart from the closest record of this type of toxicity and with a different hydrographic regime, was studied. Two outbreaks were confirmed by LC/MS/MS with presence of domoic acid and some isomers both, in plankton and in three of the most important bivalve species from the area. Domoic acid levels in the studied bivalves were below the regulatory limits for most countries and the first estimations indicate that they depurated the toxin quickly. It is, therefore, unlikely that any intoxication would have taken place by consumption of these bivalve species. Notwithstanding, the relatively high annual frequency of the blooms together with possibility that other bivalve species could retain this compound more strongly, suggest that this kind of intoxication might pose a significant risk in Angola.

Effect of some habitual cooking processes on the domoic acid concentration in the cockle (Cerastoderma edule) and Manila clam (Ruditapes philippinarum).

The effect of cooking on the concentration and burden of domoic acid in two bivalve molluscs was studied. The Manila clam (Ruditapes philippinarum) and cockle (Cerastoderma edule) were subjected to steaming and boiling, respectively. In both cases, factorial plans were used to evaluate the effects of common cooking methods and the variations likely to take place during the cooking procedure (cooking time and salt concentration in both species, in addition to ethanol percentage in Manila clam). The domoic acid concentration and toxin content were affected by cooking in very different ways in the two species studied. The cockle lost a significant part of its domoic acid content, while the clam did not. Since the weight of the soft tissues in cooked bivalves was lower than in the raw samples in both species, the toxin concentration decreased less than the toxin burden in the cockle, while it increased in the clam, where the toxin burden did not change significantly. Among the cooking variables tested, only cooking time had a noticeable effect on the domoic acid content in the clam and cockle, with the bivalves that were cooked for a longer time having smaller amounts of toxin. It is clear that cooking affects the toxin concentration in bivalves in a way that is species specific. This characteristic must be taken into account when evaluating epidemiological information, establishing allowable toxin levels and in cases where pre-processing treatments such as cooking or similar methods are used in monitoring systems.

Quantification of domoic acid in shellfish tissues by pressurized capillary electrochromatography.

A method was developed to quantify domoic acid (DA), the chemical responsible for amnesic shellfish poisoning (ASP), by pressurized CEC (pCEC). The effect of different experimental conditions on the separation of DA and matrix solutes, such as the content of ACN in mobile phase, pH and concentration of buffer, supplementary pressure and applied voltage, were investigated. Under the optimal conditions, the pCEC method separated DA from shellfish matrices within 6 min. By using supplementary pressure, bubble formation in the capillary column was completely suppressed. The method was repeatable, sufficient accurate and sensitive for rapid screening of DA in shell seafood.

Determination of suxamethonium in a pharmaceutical formulation by capillary electrophoresis with contactless conductivity detection (CE-C(4)D).

A simple method based on capillary electrophoresis with a capacitively coupled contactless conductivity detector (CE-C(4)D) was developed for the determination of suxamethonium (SUX) in a pharmaceutical formulation. A hydro-organic mixture, consisting of 100mM Tris-acetate buffer at pH 4.2 and acetonitrile (90:10, v/v), was selected as background electrolyte (BGE). The applied voltage was 30kV, and the sample injection was performed in the hydrodynamic mode. All analyses were carried out in a fused silica capillary with an internal diameter of 50 microm and a total length of 64.5cm. Under these conditions, a complete separation between SUX, sodium ions and the main degradation products (choline) was achieved in less than 4min. The presence of acetonitrile in the BGE allowed a reduction of SUX adsorption on the capillary wall. The CE-C(4)D method was validated, and trueness values between 98.8% and 101.1% were obtained with repeatability and intermediate precision values of 0.7-1.3% and 1.2-1.6%, respectively. Therefore, this method was found appropriate for controlling pharmaceutical formulations containing suxamethonium and degradation products.

Determination of domoic acid in mussels by HPLC with post-column derivatization using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and fluorescence detection.

A new, sensitive method was developed for the determination of the neurotoxin domoic acid (DA) using a reversed phase separation followed by post-column derivatization (PCD) with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and subsequent fluorescence detection. The PCD conditions which involves a two-step reaction was fully optimized for the lowest detection limit. The first reaction occurs between DA and NBD-Cl while the second makes possible the detection of the derivative causing the destruction of the interfering fluorescent 4-hydroxy-7-nitrobenzo-2-oxa-1,3-diazole (NBD-OH) which is the hydrolysis product of NBD-Cl. Kainic acid a similar base structure compound with DA was used as an internal standard. The developed post-column method provides the ability for a fully automated analysis, low detection limits (LOD 25 ppb in real samples of mussel extracts), it requires less sample preparation, and it gives clean simple chromatograms without chromatographic interferences from coeluting compounds such as tryptophan. The method was successfully applied to for the quantitative determination of DA in mussel tissues at quantities as low as 75 microg/kg tissue.

Identification and analysis of drugs in the solid state by 13C CPMAS NMR: suxamethonium chloride and hydrocortisonum (Corhydron).

Cross-polarization (CP) magic angle spinning (MAS) 13C NMR spectroscopy has become a routine tool in pharmacy, employed to identify and characterize drugs in the solid phase. 13C CPMAS NMR spectra were recorded for solid hydrocortisone 21-hemisuccinate and suxamethonium chloride. White crystalline substances, such as these two drugs, can be easily distinguished; and solid-state 13NMR spectra of remarkably good quality are obtained in less than half an hour. 13C CPMAS chemical shifts for solid suxamethonium chloride and hydrocortisone sodium hemisuccinate are given, as well as cross-polarization kinetic parameters for suxamethonium chloride.

Development of a monoclonal antibody against domoic acid and its application in enzyme-linked immunosorbent assay and colloidal gold immunostrip.

A monoclonal antibody (mAb) specific to domoic acid was produced from a stable hybridoma cell line, 9F1F11, generated by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells isolated from a Balb/c mouse immunized with domoic acid--keyhole limpet hemocyanin. The 9F1F11 mAb belongs to the immunoglobulin G1 (kappa-chain) isotype. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA were established for antibody characterization. In the cdELISA, the concentration causing 50% inhibition (IC50) of binding of domoic acid-horseradish peroxidase to the antibody by domoic acid was found to be 0.58 ng/mL. A sensitive and rapid mAb-based colloidal gold immunostrip was also developed. The immunostrip assay, which has a detection limit of 5 ng/mL for domoic acid, can be completed in 10 min. Analysis of domoic acid in blue mussel samples revealed that data obtained from immunostrip were in a good agreement with those obtained from cdELISA. The mAb-based cdELISA and immunostrip assay established in this study were sensitive and accurate for rapid screening of domoic acid in shellfish samples.

Simultaneous determination of rocuronium and its eight impurities in pharmaceutical preparation using high-performance liquid chromatography with amperometric detection.

A sensitive and selective HPLC method with amperometric detection (HPLC-ED) for the determination of rocuronium bromide and its eight impurities has been developed. The analysis was performed on Hypersil 100 Silica column 5 microm (250 mm x 4.6 mm; Thermo Electron). The mobile phase consisting of 4.53 g l(-1) solution of tetramethylammonium hydroxide adjusted to pH 7.4 with 85% phosphoric acid:acetonitrile (1:9), was found the best for the separation and determination of the studied compounds. The chromatograms were recorded over 10 min using the amperometric detection at a potential +0.9 V of the glassy carbon electrode versus the reference electrode Ag/AgCl. The limit of quantitation was 45 ng ml(-1) for rocuronium and from 25 to 750 ng ml(-1) for the examined impurities. The proposed HPLC-ED method was successfully applied to the analysis of rocuronium and its impurities in Esmeron solution for injection.

Qualitative identification of doxacurium and its breakdown products in postmortem fluids by liquid chromatography-tandem mass spectrometry.

During a death investigation at the Office of the Cuyahoga County Coroner in Cleveland, OH, doxacurium became a drug of interest. The Coroner's Office enlisted the aid of the Federal Bureau of Investigation Laboratory for the doxacurium analysis. Following the request, a method for the extraction and qualitative analysis of the drug in biological fluids was developed. The procedure relies on a simple solid-phase extraction procedure followed by qualitative analysis with liquid chromatography-tandem mass spectrometry. During the development of the new analytical procedure, two breakdown products of doxacurium were detected. Structures for these breakdown products are proposed. This procedure was used to analyze heart blood, cerebrospinal fluid, and bile specimens from the decedent. Doxacurium and its breakdown products were identified in all three specimens.

Tissue distribution and effects of heat treatments on the content of domoic acid in blue mussels, Mytilus edulis.

The effect of heat treatment on domoic acid (DA) content in soft tissues of mussels Mytilus edulis was investigated using high performance liquid chromatography. DA concentrations in whole flesh, hepatopancreas and tissue remainder were measured in fresh, steamed and autoclaved mussel flesh. Relative decreases in DA and tissue fluid following heat treatments of whole flesh were similar resulting in approximately equal concentrations of DA pre- and post-treatment. DA concentration decreased in the hepatopancreas and increased in tissue remainder suggesting some organ disruption of mussels during heat treatment. These findings suggest that heat treatments using either conventional steaming or autoclaving at 121 degrees C are not appropriate techniques to reduce DA in mussels during commercial processing. We also conclude that sample pre-treatment has a minimal effect on the result of a DA analysis on whole mussel tissues. The stability of DA at different temperatures within a shellfish matrix was separately tested. Reductions in DA concentration (ca. 3-7%) compensate for some of the discrepancies between what was found in the cooking fluids in the initial study and what was expected based on the whole flesh concentration of the uncooked material.