Ion-pair Solid-phase Extraction Research Articles

Simple solid-phase colorimetry for trace Cr(VI) by combination of complexation with diphenylcarbazide and ion-pair solid-phase extraction with sedimentable dispersed particulates.

A highly sensitive and simple solid-phase colorimetry for Cr(VI) was proposed. It was based on the ion-pair solid-phase extraction of Cr-diphenylcarbazide (DPC) complex with sedimentable dispersed particulates. The concentration of Cr(VI) was measured from the color tones obtained by image analysis of the photo of sediment. Various conditions, e.g., material and amounts of adsorbent particulates, chemical properties and concentration of counter ions, and pH, were optimized for the formation and quantitative extraction of the complex. In the recommended procedure, 1mL of sample was put into a 1.5mL microtube where powder form adsorbent and reagents, i.e., XAD-7HP particles, DPC, sodium dodecyl sulfate, amido sulfuric acid, and sodium chloride had been packed. The analytical operation was completed within 5min by gently shaking the microtube and allowing it to stand until enough amounts of particulates were deposited to take a picture. Chromium (VI) up to 2.0ppm was determined, and the detection limit was 0.0034ppm. The sensitivity was enough to determine Cr(VI) at lower concentrations than the water quality of standard (0.02ppm). This method was successfully applied to the analysis of simulated industrial wastewater samples. The stoichiometry of the extracted chemical species was also investigated by applying the same equilibrium model as the ion-pair solvent extraction.

Simultaneous determination of 13 paralytic shellfish toxins and tetrodotoxin in marine shellfish by porous graphitic carbon solid-phase extraction and hydrophilic interaction chromatography-tandem mass spectrometry

The lethal neurotoxins, paralytic shellfish toxins (PSTs), and tetrodotoxin (TTX) have recently been found in marine shellfish from many coastal states. Herein, we applied a sensitive and reliable ultra-performance hydrophilic interaction chromatography (HILIC)-tandem mass spectrometry (MS) method to determine 13 PSTs and TTX in marine shellfish using a porous carbon solid-phase extraction (SPE). This in-house validation study required the development of a novel chromatographic separation using a HILIC-Z column, which was necessary to retain highly polar compounds. Using acetonitrile as the organic phase and ammonium formate-formic acid buffer as the aqueous phase, the quantitative analysis was carried out with an external standard method in the multiple reaction monitoring modes using positive electrospray ionization. To reduce interference, 1% aqueous acetic acid extracts of the shellfish samples were cleaned up by ion-pair SPE using a porous graphitic carbon cartridge. The calibration curves for PSTs and TTX were linear (R 2 > 0.995), and the sensitivity was good, with limits of detection (LODs) of 1.7–13.7 µg/kg, and limits of quantitation (LOQs) of 5.2–41.0 µg/kg. The recoveries were 76.5–95.5% with RSDs of 3.1–12.0%. Finally, We applied the method for the determination of PSTs and TTX in three batches of Nassarius showing excellent method accuracy against expected values.

Combination of Sea Sand Disruption Method and Ion-Pair Solid-Phase Extraction for Effective Isolation and Purification of Chlorogenic Acid from Plants Prior to the HPLC Determination.

Chlorogenic acid (CQA) is one of phenolics commonly found in higher plants, possessing numerous health-promoting effects on humans. Unfortunately, it is easily degraded/transformed into other substances during extraction. Therefore, its reliable analysis requires a special approach that does not involve high temperatures. This paper presents a very simple method of CQA isolation using the sea sand disruption method with subsequent purification of the extract using the ion-pair solid-phase extraction process, followed by HPLC–DAD detection. It was found that control of the ion pairing reagent concentration and sample pH is crucial to improve purification, and that the best results, with recovery exceeding 98%, were obtained for 0.05 M tetrabutylammonium bisulfate at pH 7 when the ion pairs were formed directly in the extract and eluted from the C18 sorbent using an acidified methanol–water mixture. The practical potential of the developed procedure was verified by using it for CQA isolation from different plants. The approach represents one of the contemporary analytical trends and current advances in the solid phase extraction, in which several sorption extraction techniques are combined to ensure high-quality analytical results.

Simultaneous determination of ten paralytic shellfish toxins and tetrodotoxin in scallop and short-necked clam by ion-pair solid-phase extraction and hydrophilic interaction chromatography with tandem mass spectrometry

Paralytic shellfish toxins and tetrodotoxin (puffer-fish toxin), the latter of which was recently found in bivalves from Europe, Japan, and New Zealand, are potent neurotoxins. A simple and effective clean-up procedure was developed for the simultaneous determination of ten paralytic shellfish toxins (gonyautoxins 1–6, decarbamoylgonyautoxins 2 and 3, and N-sulfocarbamoylgonyautoxins 2 and 3) and tetrodotoxin in the scallop, Mizuhopecten (Patinopecten) yessoensis, and the short-necked clam, Ruditapes philippinarum. To reduce matrix effects, 1% aqueous acetic acid extracts of the bivalves were cleaned up by ion-pair solid-phase extraction using a graphite carbon cartridge with tridecafluoroheptanoic acid as the volatile ion-pair reagent, followed by fourfold dilution. The ten paralytic shellfish toxins and tetrodotoxin were then separated on a hydrophilic interaction chromatography column and quantified by tandem mass spectrometry. The limits of detection and the limits of quantification for the ten PSTs ranged from 0.09 to 13.0 µg saxitoxin equivalents/kg and from 0.26 to 39.4 µg saxitoxin equivalents/kg, respectively. The limit of detection and the limit of quantification for tetrodotoxin ranged from 27.4 to 27.9 µg/kg and from 83.1 to 84.4 µg/kg, respectively. The proposed method yielded minimal matrix effects for the 11 analytes, thus allowing their quantification by simple external calibration. The proposed method also gave good mean recoveries of the 11 analytes ranging from 75.7 to 96.2% with relative standard deviations less than 16% at three fortification levels for the ten paralytic shellfish toxins (total concentrations of 277, 554, and 1107 µg saxitoxin equivalents/kg) and tetrodotoxin (100, 200, and 400 µg/kg) in the two bivalve samples. Finally, the proposed method was applied for the determination of the ten paralytic shellfish toxins and tetrodotoxin in scallop and short-necked clam samples.

Reversed-phase ion-pair solid-phase extraction and ion chromatography analysis of pyrrolidinium ionic liquid cations in environmental water samples.

A method of reversed-phase ion-pair solid-phase extraction combined with ion chromatography for determination of pyrrolidinium ionic liquid cations (N-methyl-N-ethyl pyrrolidinium, N-methyl-N-propyl pyrrolidinium, and N-methyl-N-butyl pyrrolidinium) in water samples was developed in this study. First, ion-pair reagent sodium heptanesulfonate was added to the water samples after static, centrifugation and filteration. Then, pyrrolidinium cations in the samples were enriched and purified by a reversed-phase solid-phase extraction column, and eluted from the column with methanol aqueous solution as eluent. Finally, the eluate collected was analyzed by ion chromatography. The separation and direct conductivity detection of these pyrrolidinium cations by ion-exchange column using 1.0mM methanesulfonic acid (in water)/acetonitrile (97:3, v:v) as mobile phase was achieved within 10min. By using this method, pyrrolidinium cations in Songhua River and Hulan River were successfully extracted with the recoveries ranging from 74.2 to 97.1% and the enrichment factor assessed as 60. Pyrrolidinium cations with the concentration of 0.001-0.03mg/L can be enriched and detected in the water samples. The developed method for the determination of pyrrolidinium ionic liquid cations in water samples is simple and reliable, which provides a reference for the study of the potential impact of ionic liquids on the environment.

A Rapid and Sensitive Method for the Pharmacokinetic Study of Janumet (Sitagliptin and Metformin) Tablets by LC-MS/MS Coupled with Ion-Pair Solid Phase Extraction

Background: As first-line treatments for diabetes, sitagliptin and metformin have been widely prescribed as a combination to enhance the therapeutic effect. Objective: To establish a methodology to simultaneously monitor the two drugs in vivo by a reversedphase Liquid Chromatography-Tandem Mass Spectrometric (LC-MS/MS) method. Methods: The two drugs were extracted from 50 μl human plasma by ion-pair solid phase extraction. The separation of the plasma samples was implemented on an Agilent Zorbax SB-CN column (150×4.6 mm, 5.0 µm). The mobile phase was the mixture (80:20, v/v) of methanol and 5.0 mM ammonium formate in water (pH 4.5). An ion trap spectrometer equipped with an electrospray ionization source was utilized to detect the elution in positive mode. Quantification of the analytes was achieved by Multiple Reaction Monitoring (MRM) using the transitions of m/z 408.3→235.1 for sitagliptin and m/z 130.1→ 60.2 for metformin. Results: Sitagliptin and metformin demonstrated good linearity among the range of 1.00-1000 ng/mL and 5.00-4000 ng/mL. The intra-day and inter-day investigations displayed precisions of ≤ 3.6% and an accuracy range of -7.5% to 6.0% for the two drugs. The mean recovery of the two drugs was 96.0% and 98.5%. Under mandatory storage conditions, both the drugs gave an acceptable stability. The throughput of the assay was found to be more than 100 plasma samples per day ascribed to the run time of 3.0 min for each sample. Conclusion: The developed method was successfully applied to a pharmacokinetic study for a fixeddose tablet formulation containing 50 mg sitagliptin and 500 mg metformin in 12 healthy volunteers.

Challenges in simultaneous extraction and chromatographic separation of metformin and three SGLT-2 inhibitors in human plasma using LC–MS/MS

Optimization of extraction and chromatographic conditions is essential for the simultaneous analysis of drugs having different physico-chemical properties, especially for in vivo applications. The present work describes concurrent estimation of metformin (MET) and three sodium-glucose co-transporter 2 (SGLT-2) inhibitors namely canagliflozin (CANA), dapagliflozin (DAPA) and empagliflozin (EMPA) in human plasma by liquid chromatography-tandem mass spectrometry (LC–MS/MS). Sample clean-up was optimized using ion-pair solid-phase extraction with sodium lauryl sulphate on Strata-X extraction cartridges. Consistent recoveries were obtained by critically optimizing parameters such as washing and elution solvents during extraction. The extraction recovery ranged from 79 to 88% for all the analytes. Chromatographic separation were accomplished on a Cyano (150 × 4.6 mm, 5 μm) column within 5.0 min using acetonitrile and 10 mM ammonium formate buffer (75:25, v/v) as the mobile phase. The resolution factors between MET-EMPA, MET-DAPA, MET-CANA, DAPA-EMPA and CANA-DAPA were 4.92, 5.85, 7.13, 1.01 and 1.44, respectively. Calibration curves were linear in the concentration range of 2.00–2000, 3.00–3000, 0.20–200 and 1.50–1500 ng/mL for MET, CANA, DAPA and EMPA, respectively. Mass spectrometric analysis was performed using a polarity switching approach to achieve high sensitivity in multiple reaction monitoring mode. The method was validated using current regulatory guidelines and applied to study the pharmacokinetics of fixed-dose formulations of MET and SGLT-2 inhibitors in healthy subjects.

Simultaneous determination of eight underivatized biogenic amines in salted mackerel fillet by ion-pair solid-phase extraction and volatile ion-pair reversed-phase liquid chromatography-tandem mass spectrometry

A simple and accurate method was developed for the quantitative determination of eight biogenic amines (cadaverine, histamine, 2-phenylethylamine, putrescine, spermidine, spermine, tryptamine, and tyramine) in salted mackerel fillet. The eight biogenic amines in the samples were extracted with 5% trichloroacetic acid and then purified by ion-pair solid-phase extraction using a C18 cartridge and nonafluoropentanoic acid as the volatile ion-pair reagent, without the need for both derivatization and pH adjustment. Subsequently, the eight underivatized biogenic amines were separated and quantified by volatile ion-pair reversed-phase LC–MS/MS using a C18 column with the same volatile ion-pair reagent. The developed method was validated and showed good accuracy with mean recoveries of all the eight analytes in the range of 87–118% at two fortification levels (2.5 and 5 mg/kg). Finally, the proposed method was applied to the analysis of the eight biogenic amines in salted mackerel fillet samples.

Peptide enrichment by ion–pair solid-phase extraction

The technique of Solid-Phase Extraction (SPE) is widely used in various fields to concentrate samples and the search for tools to improve recoveries remains of outmost importance. The use of polymer based cartridges has become prevailing in a broad range of fields to enrich peptides from biological matrices. However, the existing SPE protocols are characterized by disparity. Ion-pairing (IP) reagents are commonly used in chromatographic applications, but their combination with SPE is less known. The aim of this study was to evaluate various SPE loading conditions, including the use of IP reagents, to improve the recoveries of nine selected peptide molecules. Control of pH and the use of IP reagents were found to be crucial to improve the enrichment of the peptides, especially cationic peptides, for which an up to ten-fold increase was observed. The practical potential of the presented theoretical findings were verified by employing IP-SPE for the development of an efficient extraction method for the doping relevant peptide Synacthen. The general proof of principle was obtained by analysis of excretion study urine samples and validation was performed with focus on the limit of detection (20 pg/ml) and recovery (37%).

Analysis of UDP-Sugars from Cultured Cells and Small Tissue Samples.

UDP-sugars are important substrates for the synthesis of various cellular glycans and glycoconjugates, many of which play essential roles in the pathobiology of diseases associated with deranged glucose metabolism, such as cancer and type 2 diabetes. Hence, their analysis from cultured cells and especially from tissue samples can give valuable information. This chapter describes a method for UDP-sugar isolation from various sources utilizing ion-pair solid-phase extraction with graphitized carbon cartridges, and their analysis using anion-exchange high-performance liquid chromatography.

Simultaneous quantitation of metformin and dapagliflozin in human plasma by LC-MS/MS: Application to a pharmacokinetic study.

A single LC-MS/MS assay has been developed and validated for the simultaneous determination of metformin and dapagliflozin in human plasma using ion-pair solid-phase extraction. Chromatographic separation of the analytes and their internal standards was carried out on a reversed-phase ACE 5CN (150 × 4.6 mm, 5 μm) column using acetonitrile-15 mm ammonium acetate, pH 4.5 (70:30, v/v) as the mobile phase. To achieve higher sensitivity and selectivity for the analytes, mass spectrometric analysis was performed using a polarity switching approach. Ion transitions studied using multiple reaction monitoring mode were m/z 130.1 [M + H]+ /60.1 for metformin and m/z 467.1 [M + CH3 COO]- /329.1 for dapagliflozin in the positive and negative modes, respectively. The linear calibration range of the assay was established from 1.00 to 2000 ng/mL for metformin and from 0.10 to 200 ng/mL for dapagliflozin to achieve a better assessment of the pharmacokinetics of the drugs. The limit of detection and limit of quantitation for the analytes were 0.39 and 1.0 ng/mL for metformin and 0.03 and 0.1 ng/mL for dapagliflozin, respectively. There was no interference of plasma matrix obtained from different sources, including hemolyzed and lipemic plasma. The method was successfully applied to study the effect of food on the pharmacokinetics of metformin and dapagliflozin in healthy subjects.

Ion-pair solid phase extraction for the simultaneous separation and quantitation of metformin and canagliflozin in human plasma by LC-MS/MS

A high-throughput and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has been developed for the simultaneous determination of metformin and canagliflozin in human plasma. The optimized ion-pair solid phase extraction of the analytes was carried out using 100 μL plasma, followed by reversed-phase chromatography on COSMOSIL 5CN-MS (150 × 4.6 mm, 5 μm) column. An isocratic mobile phase consisting of methanol-20 mM ammonium formate in water, pH 5 (75:25, v/v) was used for separation of the analytes with a resolution factor of 2.18. Mass spectrometer was operated in the multiple reaction monitoring mode at unit mass resolution. Electro spray ionization was used for mass transition of m/z 130.1/60.1 and m/z 462.1/267.1 for metformin and canagliflozin, respectively. The method was successfully validated for the concentration range of 2.50–2500 ng/mL for both the analytes. The intra- and inter-batch precision determinations for metformin and canagliflozin, expressed as coefficient of variation were lower than 5.5% at all the test concentrations. Recovery of both the drugs was in the range of 78–90% and was reproducible in the quality control samples. No significant matrix effects were observed for the analytes as evident from the IS-normalized matrix factors. Stability of the drugs in whole blood and plasma samples was ascertained under different conditions. The assay showed acceptable performance when applied to clinical samples generated from healthy Indian subjects.

The determination of capreomycin in human plasma by LC-MS/MS using ion-pairing chromatography and solid-phase extraction.

A bioanalytical method was developed and validated for the quantification of capreomycin (Cm) analogs, Cm IA and Cm IB, in human plasma. This implemented ion-pairing solid phase extraction, followed by ion-pairing high-performance liquid chromatography, with tandem mass spectrometry detection. Chromatographic separation was achieved using a Discovery C18 , 5 μm, 4.6 × 50 mm analytical column. An isocratic mobile phase consisting of water and acetonitrile with 0.1% formic acid and 4mm heptafluorobutyric acid (80:20; v/v) was used at a flow-rate of 500 μL/min. An AB Sciex API 3000 mass spectrometer at unit resolution, in multiple reaction monitoring mode, was used for detection. Electrospray ionization was used for ion production. The method was successfully validated for the range 469-30,000 ng/mL for Cm IA and for Cm IB, with cefotaxime as the internal standard. The within- and between-day precision determinations for Cm IA and IB, expressed as the percentage coefficient of variation, were < 20.0% at the lower limit of quantification (LLOQ) and < 8.2% at all other test concentrations. Recovery of both analogs was > 72.3% and reproducible at the low, medium and high end of the calibration range. No significant matrix effects were observed for the analyte. The assay performed well when applied to clinical samples generated from children in a clinical multidrug resistant tuberculosis research study in South Africa.

Quantitative determination of acidic hydrolysis products of Chemical Weapons Convention related chemicals from aqueous and soil samples using ion-pair solid-phase extraction and in situ butylation.

Chemical warfare agents such as organophosphorus nerve agents, mustard agents, and psychotomimetic agent like 3-quinuclidinylbenzilate degrade in the environment and form acidic degradation products, the analysis of which is difficult under normal analytical conditions. In the present work, a simultaneous extraction and derivatization method in which the analytes are butylated followed by gas chromatography and mass spectrometric identification of the analytes from aqueous and soil samples was carried out. The extraction was carried out using ion-pair solid-phase extraction with tetrabutylammonium hydroxide followed by gas chromatography with mass spectrometry in the electron ionization mode. Various parameters such as optimum concentration of the ion-pair reagent, pH of the sample, extraction solvent, and type of ion-pair reagent were optimized. The method was validated for various parameters such as linearity, accuracy, precision, and limit of detection and quantification. The method was observed to be linear from 1 to 1000ng/mL range in selected ion monitoring mode. The extraction recoveries were in the range of 85-110% from the matrixes with the limit of quantification for alkyl phosphonic acids at 1ng/mL, thiodiglycolic acid at 20ng/mL, and benzilic acid at 50ng/mL with intra- and interday precisions below 15%. The developed method was applied for the samples prepared in the scenario of challenging inspection.

LC–tandem mass spectrometry method for the simultaneous determination of metformin and sitagliptin in human plasma after ion-pair solid phase extraction

A reversed-phase liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) method was established for simultaneous determination of two oral hypoglycemic drugs metformin (MET) and sitagliptin (STG) in human plasma. The analytes were extracted from 50μL human plasma by ion-pair solid phase extraction using sodium lauryl sulphate on Phenomenex Strata-X (30mg/1mL) cartridges. The chromatographic separation was accomplished on XSelect HSS CN (150×4.6mm, 5μm) column using mobile phase consisting of methanol-8.0mM ammonium formate in water, pH 4.5 (80:20, v/v) under isocratic condition. Tandem MS detection was performed on a triple quadrupole spectrometer equipped with an electrospray ionization source, operated in the positive mode. Multiple reaction monitoring (MRM) was used to quantify the analytes following transitions, m/z 130.1→60.1 and m/z 408.3→235.1 for MET and STG respectively. The method displayed acceptable linearity in the concentration range of 4.00–3200ng/mL for MET and 1.00–800ng/mL for STG. The intra-batch and inter-batch precisions were ≤5.1% and accuracy ranged from 96.5 to 103.3% for both the drugs. The mean recovery of MET and STG obtained from spiked plasma samples was 82.5% and 90.4% respectively with minimal matrix interference. Both the drugs were found to be stable under all mandatory storage conditions. The validated method was successfully applied to a clinical pharmacokinetic study for a fixed-dose tablet formulation containing 500mg MET and 50mg STG in 16 healthy volunteers.

High-performance thin-layer chromatographic assay of metformin in urine using ion-pair solid-phase extraction: Application for bioavailability and bioequivalence study of new microbeads controlled release formulation

A sensitive high-performance thin-layer chromatographic (HPTLC) method was developed for the estimation of metformin in human urine. The drug was extracted from urine using ion-pair solid-phase extraction (IPSPE). Separation was carried out on Merck HPTLC aluminum sheets of silica gel 60 F254 using a mixture of chloroform-methanol-ammonia 27% w/v (5:5:0.2, v/v) as a mobile phase. This system was found to give dense and compact spots for metformin (retardation factor, RF, 0.25) following densitometric measurements of the spots at 237 nm. The proposed method was applied to generate urinary excretion data for metformin after administration of two metformin formulations (500 mg extended release, XR, Formulation R, and microbeads controlled release, Formulation T) to six healthy human volunteers in a two-treatment, crossover design. Various pharmacokinetic parameters like peak excretion rate ((dAU/dt)max), cumulative amount (Ae0–24) of metformin excreted, elimination half-life (t1/2), and terminal elimination ...

Preconcentration of ultra-trace amounts of iron and antimony using ion pair solid phase extraction with modified multi-walled carbon nanotubes

Ion pair solid phase extraction was applied to the simultaneous preconcentration of iron and antimony. The ion pairs consisting of FeCl4− or SbCl4− anions and the benzyldimethyltetradecyl ammonium cation were formed on the surface of multi-walled carbon nanotubes, then eluted with nitric acid, and the elements finally quantified by ETAAS. The adsorption capacities of the impregnated MWCNTs are 9.2 mg g−1 for iron and 27.5 mg g−1 for antimony. The following analytical figures of merit were determined for iron and antimony, respectively: Enrichment factors of 210 and 230, assay precisions of ±5.3 % and ±4.8 %, linear calibration plots from 0.7 to 9.4 and 13.0 to 190 ng L−1, and detection limits of 0.17 and 3.5 ng L−1. The method was applied to the determination of iron and antimony in human hair, synthetic sample, and to the certified reference materials gold ore (MA-1b) and trace elements in water (SRM 1643d).

Tartrazine determination from mustard sample by TLC-Photodensitometry and TLC-Digital processing of images

A facile and rapid thin-layer chromatography (TLC) method for tartrazine analysis from mustard is proposed. Silica gel precoated plates and isopropyl alcohol-ammonia mixture as mobile phase were used for tartrazine determination. The synthetic dye was extracted from solid matrix by ultrasound assisted extraction with a methanol-ammonia mixture and further purified by ion-pair solid phase extraction, using hexadecyltrimethylammonium-bromide as ion-pair reagent. Recovery determination was carried out on mustard samples spiked with tartrazine at different concentration levels. Good recoveries, around 98%, and a relative standard deviation of 17.4% were obtained. An extensive study for the quantitative evaluation of dyes was developed by comparing the photodensitometric results with those obtained by using digital processing of scanned plates with ImageDecipher-TLC software. The method was applied for the tartrazine determination in various mustard samples purchased from a local market.

Development of a liquid chromatography–tandem mass spectrometry method for plasma-free metanephrines with ion-pairing turbulent flow online extraction

Liquid chromatography-tandem mass spectrometry has become the preferred technology to measure unconjugated metanephrine and normetanephrine in plasma because of its high sensitivity and specificity over immunoassay and gas chromatography-mass spectrometry. In our earlier study, plasma metanephrines were extracted with offline ion-pairing solid-phase extraction and quantified by liquid chromatography-tandem mass spectrometry with porous graphitic carbon column based chromatography. In this study, we aim to automate the sample preparation with turbulent flow online extraction technology and maintain or improve the analytical performance previously achieved from the offline approach. The online extraction was done with a mixed-mode cation exchange turbulent flow chromatography column assisted with ion-pairing reagent and porous graphitic column was used for chromatographic separation. The total online extraction and analytical LC runtime was 12 min. This method was linear from 6.3 to 455.4 pg/mL for metanephrine; 12.6 to 954.5 pg/mL for normetanephrine with an accuracy of 80.6% to 93.5% and 80.9% to 101.7%, respectively. The lower limit of quantitation was 6.3 pg/mL for metanephrine and 12.6 pg/mL for normetanephrine. Inter-assay and intra-assay precision for metanephrine and normetanephrine at low and high concentration levels ranged from 2.0% to 10.5%. In conclusion, we have developed a fast and sensitive automated online turbulent flow extraction method for the quantitative analysis of plasma metanephrines. Ion-pairing reagent was necessary for the success of this method.

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.