Simultaneous determination of succinylcholine and its metabolite in animal-derived foods by solid-phase extraction combined with ultra-performance liquid chromatography/mass spectrometry.

RATIONALE: Succinylcholine has been increasingly used in the theft of animals. Because of the presence of residual levels of succinylcholine in poisoned animals, it is harmful for people to eat foods derived from these animals. Therefore, a method should be immediately established to determine succinylcholine and its metabolite in animal-derived foods. METHODS: A fast, highly sensitive method, combining solid-phase extraction (SPE) with ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS), was developed for the determination of succinylcholine and its metabolite in animal-derived foods. The sample was initially extracted with heptafluorobutyric acid and then further cleaned up using an SPE cartridge. Succinylcholine and its metabolite were separated using acetonitrile: 0.1% formic acid in 5 mmol L-1 ammonium acetate as the mobile phase. Quantitative results were based on positive ion ESI multiple reaction monitoring mode. RESULTS: The results show good linearity over a wide range with correlation coefficients of determination of more than 0.998. Both the limits of detection of succinylcholine and succinylmonocholine are 0.2 µg kg-1 . The intra- and inter-day accuracies of the method are in the range 91.4%-104.6%, and the intra- and inter-day precisions are in the range 2.5%-6.6%. CONCLUSIONS: This method can be used for the determination of succinylcholine as an illicit drug in animal-derived foods. It was successfully applied to the identification and quantification of succinylcholine and succinylmonocholine in animal-derived foods collected from a local farmers market in Jilin Province of China.

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.

Nurse induced respiratory depression by succinylcholine--the 'hero syndrome'.

A nurse administered the neuromuscular blocking agent succinylcholine (SUX) to at least one patient and gave first aid in the therapy of unexpected respiratory depression. SUX is regarded as an undetectable and thus perfect poison due to its short half-life and degradation to the endogenous compounds choline and succinic acid. However, SUX and especially its metabolite succinylmonocholine (SMC) were found in plasma and urine a few hours after administration by means of high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Compared to clinical studies, the window of detection was sufficient to gain definite proof; in other cases no samples were collected. The nurse enjoyed high reputation with the doctors. According to the court she wanted to present herself spectacularly as the first and decisive rescuer to demonstrate her special abilities and capacities, perhaps to receive a better job in the hospital. Considering the actual case, the hero syndrome is not limited to fire-fighters.

Degradation and elimination of succinylcholine and succinylmonocholine and definition of their respective detection windows in blood and urine for forensic purposes.

The muscle relaxant succinylcholine (SUX) evokes respiratory paralysis, and numerous cases of fatal SUX intoxication have been reported. Detection of SUX and its metabolite succinylmonocholine (SMC) is difficult, both due to their (bis-) quaternary structure and the extreme hydrolytic susceptibility of SUX, and data on degradation kinetics of SUX and SMC is scarce. The present study investigates the in vivo and in vitro degradation as well as elimination of both target analytes using authentic blood and urine samples from anesthetized patients. With a special focus on the urinary data and stabilization issues, this work intends to considerably enhance the forensic knowledge concerning SUX intoxications and to present the reader with practical analytical strategies to cope with such difficult cases. Eighteen subjects undergoing surgery and requiring arterial as well as bladder catheters were included in this study. Muscle relaxation was initialized with a bolus injection of 80-100 mg SUX. Blood and urine samples were either collected using paraoxonized (n = 15) or non-modified (n = 3) tubes. Sampling was performed within 6 h after SUX application following a pre-assigned schedule. Samples were processed according to a validated isotope dilution HPLC-MS/MS method using ion-pair solid-phase extraction. In blood, SUX was usually detectable for up to 10 min post-injection, while detection of SMC was possible over the whole observation period of 6 h. Effectiveness of organophosphate stabilization was proven for both analytes and is therefore recommended. In freshly secreted urine, detection windows of a minimum of 2 h as opposed to 6 h have been determined for SUX versus SMC, respectively. Considering SMC plasma kinetics, detection of the metabolite in blood and freshly secreted urine appears to be possible over a period of at least 8-24 h. Paraoxon did not enhance the stability of either target substance in urine, stabilization of urine samples is nonetheless recommended. In summary, SMC was proven to be the most promising target analyte in SUX analysis, with urine being the proposed matrix of choice for forensic applications. Furthermore, our work defines meaningful detection windows for SUX and SMC in blood and urine as routine matrices and presents sampling recommendations as well as guideline values for forensic toxicological analysis.

Pharmacokinetic properties of succinylmonocholine in surgical patients.

Intoxications with succinylcholine (SUX) lead to a potentially lethal respiratory paralysis, and forensic cases involving accidental or deliberate SUX-application have been reported. Detection of SUX as well as its metabolite succinylmonocholine (SMC) is difficult: both substances are analytically challenging, and the extremely short plasma half-life of SUX additionally hampers detection of the parent compound. Pharmacokinetic data are scarce on SUX and non-existent on SMC. To enhance forensic knowledge concerning SUX intoxications, plasma pharmacokinetics of SMC were investigated in anesthetized patients. Fifteen subjects scheduled for a surgical procedure were included in this study. Muscle-relaxation was initialized with a bolus injection of 80-100 mg SUX. Blood sampling was performed within 6 h after SUX application using paraoxonized tubes. Collected plasma was processed according to a validated isotope dilution high-performance liquid chromatography-tandem mass spectrometry method using ion-pair solid-phase extraction. Pharmacokinetic parameters were derived from a user-defined as well as a three-compartment model. For SMC, peak plasma concentrations were reached after 0.03-2.0 min. In contrast to SUX, SMC was more slowly and more extensively distributed, featuring triphasic plasma concentration time profiles. Pharmacokinetic key parameters were subject to interindividual variation of potential forensic importance, with terminal half-lives of 1-3 h indicating a detection interval of 8-24 h for SMC in plasma. SMC was proven to be the only realistic SUX marker in a forensic context. The present work defines meaningful detection windows for plasmatic SMC after SUX application and offers guideline values for forensic toxicological casework.

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.

Succinylmonocholine analytics as an example for selectivity problems in high-performance liquid chromatography/tandem mass spectrometry, and resulting implications for analytical toxicology.

The determination and quantitation of drugs in biological matrices using high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) is becoming increasingly popular in analytical toxicology, while at the same time a growing awareness for the limits of this technique can be observed. Our group previously developed a rapid HPLC/ESI-MS/MS method for the detection and quantitation of succinylcholine (SUX) and succinylmonocholine (SMC) using ion-pairing extraction of samples with subsequent separation by gradient chromatography on a Synergi Hydro RP C18 column (4 microm, 150 x 2 mm). Identification of analytes was achieved in the multiple reaction monitoring (MRM) mode, using two characteristic ion transitions each, the respective analytes' retention time as well as co-elution of stable isotopic analogues. In both native serum as well as urine an interference with the main MRM transition of SMC was found to co-elute with this analyte, thus severely compromising the identification and quantitation of this target analyte. The interference was further shown to be eliminated from serum and urine by exposure to alkaline conditions and hence proven to share a key physicochemical property with SMC. The observed absence of the second and third most intense ion transitions of SMC in the unknown substance was the only useful distinction between both compounds.The detailed presentation of selectivity problems encountered during method development is intended to initiate further discussion on this yet underrepresented issue in HPLC/MS/MS. The present work emphasizes the need to monitor more than just one ion transition to confidently rule out signal interferences, ensure correct analyte identification as well as quantitation, and thus avoid false-positive results. In this context, the employment of minor MRM transitions for the quantitation and identification of a given analyte is presented as a satisfactory solution to HPLC/MS/MS selectivity problems, and proposed as a possible alternative to previously published approaches.

A fully validated isotope dilution HPLC-MS/MS method for the simultaneous determination of succinylcholine and succinylmonocholine in serum and urine samples.

A high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for the simultaneous detection of succinylcholine (SUX) and its metabolite succinylmonocholine (SMC) in serum and urine is presented. For internal standardization using isotope dilution, the deuterated compounds SUX-d(18) and SMC-d(3) were employed. Full validation was performed according to international guidelines. Solid-phase extraction (SPE) of acidified samples was accomplished using Strata-X polymeric reversed phase cartridges together with heptafluorobutyric acid (HFBA) as ion-pairing reagent. Separation was achieved within 13 min on a Phenomenex Synergi Hydro RP C18 column (4 microm, 150 x 2 mm) using a gradient of 5 mM ammonium formate buffer pH 3.5 and acetonitrile.To ensure the method's applicability in forensic as well as clinical toxicology, the specific demands of both research fields were taken into account, and the method was thus validated for a low and high concentration range. For both serum and urine as sample matrix, the validation revealed good intraday and interday precisions, consistently ranging below 15% for the lowest and below 10% for elevated concentrations. Accuracy was likewise good and never exceeded 10%. Extraction recovery was excellent, ranging between 88.1 and 103.9% for SUX and SMC in both tested matrices. Matrix effects were significant, the otherwise optimized extraction and detection methods, however, allowed for a very satisfactory sensitivity of the described method: For serum, the limits of detection and quantitation were determined to be 1.9 and 6.0 ng/ml for SUX, as well as 2.5 and 8.6 ng/ml for SMC, respectively; for urine, the corresponding values were established to be 1.4 and 4.0 ng/ml (SUX), as well as 1.5 and 4.9 ng/ml (SMC).The presented method was successfully applied to authentic samples of two forensic cases investigated in the institute of forensic medicine in Bonn, allowing the diagnosis of SUX intoxications.

Assay using succinyldithiocholine as substrate: the method of choice for the measurement of cholinesterase catalytic activity in serum to diagnose succinyldicholine sensitivity.

No comparative information is available concerning the ability of various cholinesterase (ChE) methods to identify succinyldicholine-sensitive patients, purely on the basis of the enzyme activity recorded in serum. Here, we evaluated six different methods for the measurement of ChE activity; 131 subjects were subdivided according to ChE phenotype and, therefore, to succinyldicholine sensitivity. ChE phenotype was determined by measuring dibucaine and fluoride numbers. DNA analysis was also performed to confirm correlation between the phenotype classification used in the study and the ChE genotype. The tested methods were significantly different in their ability to discriminate between the subjects with and without succinyldicholine-sensitive phenotypes. The succinyldithiocholine/5,5'-dithio-bis(2-nitrobenzoate) (DTNB) method showed the highest accuracy (area under the receiver operating characteristic (ROC) curve 0.97) followed by the propionylthiocholine/DTNB method (area under the ROC curve 0.94). On the other hand, the two methods using butyrylthiocholine as substrate and that employing benzoylcholine showed limited clinical utility in discriminating subjects at risk of prolonged apnea (area under the ROC curve < or = 0.9). Using the succinyldithiocholine method, a value < or = 23 U/l was approximately five times as likely to occur in a sensitive individual as in a normal one.

Role of aspartate 70 and tryptophan 82 in binding of succinyldithiocholine to human butyrylcholinesterase.

The atypical variant of human butyrylcholinesterase has Gly in place of Asp 70. Patients with this D70G mutation respond abnormally to the muscle relaxant succinyldicholine, experiencing hours of apnea rather than the intended 3 min. Asp 70 is at the rim of the active site gorge 12 A from the active site Ser 198. An unanswered question in the literature is why the atypical variant has a 10-fold increase in Km for compounds with a single positive charge but a 100-fold increase in Km for compounds with two positive charges. We mutated residues Asp 70, Trp 82, Trp 231, Glu 197, and Tyr 332 and expressed mutant enzymes in mammalian cells. Steady-state kinetic parameters for hydrolysis of butyrylthiocholine, benzoylcholine, succinyldithiocholine, and o-nitrophenyl butyrate were determined. The wild type and the D70G mutant had identical k(cat) values for all substrates. Molecular modeling and molecular dynamics suggested that succinyldicholine could bind in two consecutive orientations in the active site gorge; formation of one complex caused a conformational change in the omega loop involving Asp 70 and Trp 82. We propose the formation of three enzyme-substrate intermediates preceding the acyl-enzyme intermediate; kinetic data support this contention. Substrates with a single positive charge interact with Asp 70 just once, whereas substrates with two positive charges, for example succinyldithiocholine, interact with Asp 70 in two complexes, thus explaining the 10- and 100-fold increases in Km in the D70G mutant.

Comparison of the mode of action of succinylcholine and succinylmonocholine on rat skeletal muscle after denervation.

The effects of equimolar concentrations (3.0 X 10(-5) M) of succinylcholine (SCh) and succinylmonocholine (SMC) were studied in-vitro at 20 degrees C in rat extensor digitorum longus muscle (EDL) 0-147 days after common peroneal nerve section. Analysis of simultaneous measurements of K+ efflux (mmolL-1 g-1) and contracture tension (mN) to SCh showed that there was a rapid increase in the mean values of both parameters up to 22-28 days after denervation (7.7 mmolL-1 g-1, 36 mN). At the end of the period studied, the contracture response declined to 4.0 mN whilst the capacity for K+ efflux remained relatively high (4.8 mmolL-1 g-1) in comparison with normal contralateral EDL muscle (n = 82) K+ efflux measurements (0.62 mmolL-1 g-1). A significant correlation (r = 0.86, P less than or equal to 0.001) was found between SCh-induced K+ efflux and contracture tension 1-56 days following nerve section which indicated that the development of the contracture response and K+ efflux were concomitant during the period specified. The ratios of maximum contracture tension/K+ efflux in response to SCh and SMC, 18-22 days after denervation were similar, 4.9 and 5.9, respectively. Results indicated that the mode of action of each agent was similar in denervated rat skeletal muscle, and that they were equally potent in their hyperkalaemic potential. Results of comparative measurements of membrane depolarization and contracture tension in response to SCh and SMC showed that both agents produced quantitatively similar responses at 7 and 14 days after denervation.(ABSTRACT TRUNCATED AT 250 WORDS)

The hydrolysis of succinyldithiocholine and related thiocholine esters by human plasma and purified cholinesterase.

The kinetic properties of cholinesterase (ChE) present in plasma were compared with those of purified human ChE using the substrates succinyldithiocholine (SDTCh), acetylthiocholine (AcTCh) and butyrylthiocholine (BuTCh). SDTCh was hydrolysed at two sites; a site with a low Km (Km1 11.4 +/- 3.3 microM) with a Vmax of 0.06 mumol/min/ml and a site with a high Km (Km2 132.4 +/- 14.8 microM) and a Vmax of 0.107 mumol/min/ml. The Km2 site was absent in the sample of purified ChE. The related thiocholine esters, AcTCh and BuTCh were hydrolysed at two sites by both plasma and purified ChE. This indicated that the Km2 site which hydrolysed SDTCh was not ChE. The identity of this component in plasma remains unknown but it was shown not to be albumin. The anticholinesterase agents soman and pyridostigmine were used to demonstrate the direct relationship between inhibition of plasma ChE and hydrolysis of SDTCh at the low concentrations present clinically (20 microM). Whereas high concentrations of SDTCh (200 micron) could be partly hydrolysed by an enzyme present in plasma which is insensitive to ChE inhibitors. In a limited study on the plasma from two "atypical" individuals (Dibucaine number less than 20) all three substrates were hydrolysed at a single site with a higher Km than the Km2 site present in normal plasma. The clinical implications of these results are discussed.

Chronotropic effects of succinylcholine and succinylmonocholine on the sinoatrial node.

The mechanism of bradycardia caused by the administration of succinylcholine has not been fully elucidated. Accordingly, the effects of succinylcholine and succinylmonocholine on the sinoatrial node were studied in 35 mongrel dogs. The sinus node artery was selectively perfused with autologous blood from a femoral artery at a constant pressure of 100 mmHg, and 30 to 1,000 micrograms of succinylcholine or succinylmonocholine was administered directly into the artery. Succinylcholine caused a transient (63-600 s) dose-related positive chronotropic effect. The heart rate was increased to 14.4 +/- 2.1% (mean +/- SE) above the control value after the administration of 1,000 micrograms of succinylcholine. This positive chronotropic effect was inhibited by pretreatment with pindolol or reserpine. By contrast, succinylmonocholine produced a transient (30-248 s) dose-related negative chronotropic effect. The heart rate was decreased to 17.5 +/- 1.4% below the control value after administration of 1,000 micrograms of succinylmonocholine. The negative chronotropic effect was blocked partially by atropine. It was concluded that the positive chronotropic effect of succinylcholine may be mediated through beta-adrenergic receptor stimulation by catecholamine released from the adrenergic nerve endings in the sinoatrial node, and that the negative chronotropic effect of succinylmonocholine may be the result of excitation of cholinergic receptors in the sinus node. However, a direct effect of succinylmonocholine on the sinus node could not be ruled out.

[Possibility of substance selective action on the m-cholinoreceptors of a specific site]. / O vozmozhnosti izbiratel'nogo deistviia veshchestv na m-kholinoretseptory opredelennoi lokalizatsii.

Effects of a number of agents on the muscarine-sensitive acetylcholine receptors (muscarinic receptors) of different localization were investigated. Experiments on anesthetized cats revealed the following effects of acetylcholine: hypotension, bradycardia, bronchospasm, contractions of the ileum and urinary bladder, hypersalivation. It was shown that the highest sensitivity to bisquaternary ammonium derivatives of diphenyl cyclobutanedicarboxylic (truxillic) acid is exhibited by the muscarinic receptors of the heart. The sensitivity of bronchial muscarinic receptors is slightly lower. The prevailing action on the muscarinic receptors of the heart was also shown by some N-adamantyl bisquaternary ammonium compounds. Procaine and the antihistaminics mebhydroline and diphenhydramine eliminated bradycardia induced by acetylcholine without affecting the latter's hypotensive effect. The evidence obtained indicates the heterogeneity of the muscarinic receptors of different localization.