An Analytical Strategy for Quaternary Ammonium Neuromuscular Blocking Agents in a Forensic Setting Using LC-MS/MS on a Tandem Quadrupole/Time-of-Flight Instrument

Strategies for the Analysis of Pharmaceutical Cocrystals Using HPLC with Charged Aerosol Detection

Determination of adrenaline, noradrenaline and corticosterone in rodent blood by ion pair reversed phase UHPLC-MS/MS.

An ion-pair liquid chromatography-mass spectrometry/mass spectrometry method was developed for the determination of chlormequat in fruit samples. A solid-phase extraction cleanup procedure with C18 cartridges was used. Sample preparation was simple and the achieved detection limit (0.03 mg/kg) and quantitation limit (0.08 mg/kg) were below the maximum residue levels legislated by the European Union. The chromatographic separation was performed by using a C8 column and heptafluorobutyric acid as ion pair reagent. The detection was conducted with an electrospray source and an ion trap as a mass analyzer. The reproducibility of the method gave good run-to-run (8-9%) and day-to-day (9-13%) precision values, and its applicability to the determination of chlormequat in pears and grapes, purées, and pear, apple, and grape juices was demonstrated.

Perfluorobutanoic acid (PFBA) belongs to the complex group of synthetic perfluoroalkyl substances (PFAS) which have led to ubiquitous environmental contamination. While some of the long-chain compounds accumulate in the human body, the short-chain compound PFBA was found to have a relatively short half-life in blood of a few days, in agreement with relatively low PFBA serum/plasma levels of roughly 0.01ng/ml in European studies. Surprisingly, very high median levels of PFBA of 807 and 263ng/g tissue for human lung and kidney autopsy samples, respectively, were reported in a paper of Pérez et al. (2013). This would question the concept of PFAS blood analysis reflecting the body burden of these compounds. To verify the results of high PFBA tissue accumulation in humans, we have analyzed PFBA in a set of 7 lung and 9 kidney samples from tumor patients with a different method of quantification, using high-resolution mass spectrometry with the accurate mass as analytical parameter. The only human sample with a quantifiable amount of PFBA (peak area more than twice above the analytical background signals) contained approximately 0.17ng/g lung tissue. In the light of our results and considering the analytical problems with the short-chain compound PFBA exhibiting only one mass fragmentation, it appears to be likely that PFBA is not accumulating on a high level in human lung and kidney tissue. In general, the analysis of short-chain PFAS in complex matrices like food or tissue is very challenging with respect to instrumental quantification and possible sample contamination.

Hydrophilic-interaction liquid chromatography (HILIC) of intact proteins offers high-resolution separations of glycoforms of glycoproteins differing in the number of (neutral) glycans. However, to obtain efficient separations it is essential that the positively charged sites of the proteins are shielded by acidic (negative) ion-pair reagents (IPRs), so as to enhance the contribution of the hydroxyl groups of the (neutral) sugars in the glycoprotein. Here, we studied the influence of various IPRs that differ in physico-chemical properties, such as hydrophobicity and acidity, on the capillary-scale HILIC separation of intact (glyco)proteins. We evaluated the use of fluoroacetic acid (MFA), difluoroacetic acid (DFA), trifluoroacetic acid (TFA), and heptafluorobutyric acid (HFBA) as diluents for sample preparation, as solvents for sample loading on a reversed-phase trap prior to the HILIC separation, and as mobile-phase components for HILIC and HILIC-MS. To reduce the contribution of ion-exchange interaction with the (silica-based) stationary phase, we used an acrylamide-based monolithic column. We studied the influence of the different IPRs on each step of the separation of a mixture of proteins of different size and hydrophilicity and on the separation of the five glycoforms of ribonuclease B. The content of IPR in the sample was shown not to affect the separation and the MS detection. However, a low content of TFA and DFA in the mobile phase is favourable, as it reduces adduct formation and leads to higher signal intensity. The optimized HILIC conditions successfully resolved nine major glycoforms groups of a ∼40 kDa glycoprotein horseradish peroxidase (HRP), as an example of a complex glycoprotein.

Sensitive and accurate methods are described for the determination of phosphate, arsenate and arsenite in natural water using Malachite Green as an ion-pair reagent. The ion pair of Malachite Green with molybdophosphate is floated as a membrane-like substance at the phase boundary between the aqueous phase and a diethyl ether phase. After discarding the aqueous phase, methanol is added to dissolve the floated ion pair. The determination of phosphate is performed by measuring the absorbance of the organic phase. The apparent molar absorptivity of phosphate is 3.3 × 105 l mol–1 cm–1 at 620 nm. Arsenate is also determined using the same procedure. An oxidative or a reductive reaction of arsenic with potassium dichromate or sodium thiosulphate is utilised for the determination of phosphate, arsenate and arsenite. An extraction-indirect atomic absorption spectrometric determination method was also developed in which the atomic absorption of molybdenum in the ion pair of Malachite Green with molybdophosphate is measured. The proposed methods were applied to the determination of phosphate, arsenate and arsenite in some natural water samples.

To determine the comparative toxicology of death by homicide and suicide by means other than substance toxicity. Cross-sectional (autopsy reports). Sydney, Australia. A total of 1723 cases of violent death were identified, comprising 478 homicide (HOM) cases and 1245 non-substance toxicity suicide (SUI) cases. Substances were detected in 65.5% of cases, and multiple substances in 25.8%, with no group differences. Illicit drugs were detected in 23.9% of cases, and multiple illicit in 5.3%. HOM cases were significantly more likely to have an illicit drug [odds ratio (OR) 2.09] and multiple illicits (OR 2.94), detected, HOM cases being more likely to have cannabis (OR 2.39), opioids (OR 1.53) and psychostimulants (OR 1.59) present. HOM cases were, however, significantly less likely to have benzodiazepines (OR 0.53), antidepressants (OR 0.22) and antipsychotics (OR 0.23) present. Alcohol was present in 39.6% of cases (median blood alcohol concentration = 0.12), with no group difference in prevalence. The role drugs play in premature death extends far beyond overdose and disease, with illicit drugs associated strongly with homicide.

Carnitine is an amino acid derivative that plays a key role in energy metabolism. Endogenous carnitine is found in its free form or esterified with acyl groups of several chain lengths. Quantification of carnitine and acylcarnitines is of particular interest for screening for research and metabolic disorders. We developed a method with online solid-phase extraction coupled to high-performance liquid chromatography and tandem mass spectrometry to quantify carnitine and three acylcarnitines with different polarity (acetylcarnitine, octanoylcarnitine, and palmitoylcarnitine). Plasma samples were deproteinized with methanol, loaded on a cation exchange trapping column and separated on a reversed-phase C8 column using heptafluorobutyric acid as an ion-pairing reagent. Considering the endogenous nature of the analytes, we quantified with the standard addition method and with external deuterated standards. Solid-phase extraction and separation were achieved within 8 min. Recoveries of carnitine and acylcarnitines were between 98 and 105 %. Both quantification methods were equally accurate (all values within 84 to 116 % of target concentrations) and precise (day-to-day variation of less than 18 %) for all carnitine species and concentrations analyzed. The method was used successfully for determination of carnitine and acylcarnitines in different human samples. In conclusion, we present a method for simultaneous quantification of carnitine and acylcarnitines with a rapid sample work-up. This approach requires small sample volumes and a short analysis time, and it can be applied for the determination of other acylcarnitines than the acylcarnitines tested. The method is useful for applications in research and clinical routine.

Optimization and application of parallel solid-phase extraction coupled with ultra-high performance liquid chromatography–tandem mass spectrometry for the determination of 11 aminoglycoside residues in honey and royal jelly

The nebramycin aminoglycoside profiles of Streptomyces tenebrarius and their characterization using an integrated liquid chromatography-electrospray ionization-tandem mass spectrometric analysis

Pralidoxime (2-PAM) is a monopyridinium oxime used as an antidote for the treatment of poisoning with organophosphorus (OP) compounds, for example, pesticides and nerve agents, reactivating OP-inhibited acetylcholinesterase. However, appropriate dosing and efficacy remains a matter of discussion requiring experimental data. Therefore, we developed and validated an ion pair chromatography-diode array detection (IPC-DAD) method suitable for quantitative analysis of 2-PAM in human and porcine urine. Before injection of 20 µl, urine was acidified with trichloroacetic acid, mixed with internal standard (pyridine-4-aldoxime, 4-PAO), and diluted with IPC solvent yielding a total dilution of 1:49.5 and a 100% recovery. Isocratic separation was carried out at 25 °C on a LiChrospher 60 RP-select B column (125 x 4.0 mm I.D.) using phosphate buffer (7.5 mM Na(2) HPO(4) , 7.5 mM KH(2) PO(4) , pH 2.6) mixed with octanesulfonate (2.5 mM) as ion pair reagent and acetonitrile (6% v/v) as organic modifier (1 ml/min). 2-PAM was detected at 293 nm and 4-PAO at 275 nm. The method is rugged, selective, and characterized by good intra-day and inter-day precision (RSD, 1.3-6.0%) and accuracy (88-100%) with a limit of detection at 4.9 µg/ml, a limit of quantification at 9.8 µg/ml, and a broad calibration range from 4.9-2500 µg/ml. The procedure was applied to urine samples obtained from dimethoate poisoned minipigs receiving 2-PAM therapy (intravenous bolus injection and infusion). Results indicate that 60-80% of infused 2-PAM is rapidly (within 1-2 h) excreted in the urine.

Rapid detection and identification of bacterial pathogens by using an ATP bioluminescence immunoassay.

A novel micro matrix solid phase dispersion method was successfully used for the extraction of quaternary alkaloids in Phellodendri chinensis cortex. The elution of target compounds was accomplished with sodium hexanesulfonate as the eluent solvent. A neutral ion pair was formed between ion-pairing reagent and positively charged alkaloids in this process, which was beneficial for selectively extraction of polar alkaloids. Several parameters were optimized and the optimal conditions were listed as follows: silica gel as the sorbent, silica to sample mass ratio of 1:1, the grinding time of 1min. The exhaustive elution of targets was achieved by 200µL methanol/water (9:1) containing 150mM sodium hexane sulfonate at pH 4.5. The method validation covered linearity, recovery, precision of intraday and interday, limits of detection, limits of quantitation, and repeatability. This established method was rapid, simple, environmentally friendly, and highly sensitive.

A novel amperometric detector for heavy metal ions has been developed and successfully applied for ion pair chromatography. The detector is based on the electrochemical transfer of the metal ions across an array of water/nitrobenzene micro interfaces. The ion transfer is facilitated by the neutral ionophores methylenebis(diphenylphosphineoxide) and methylenebis(di- phenylphosphinesulfide). More than eight metals are separated in less than 15 min on an RP18 column using octyl sulfonate as ion pair reagent. For the heavy metals, the limits of decision are 19(Pb(2+)), 9(Zn(2+)), 9l (Co(2+)), 8(Cd(2+)) and 1.6(Mn(2+)) microg/L. The applicability of the new method for water samples is demonstrated.

Targeted profiling of polar intracellular metabolites using ion-pair-high performance liquid chromatography and -ultra high performance liquid chromatography coupled to tandem mass spectrometry: Applications to serum, urine and tissue extracts