High-resolution Mass Spectrometric Detection Research Articles

Defining Aging Marker Molecules of 1,3‐Propane Sultone for Targeted Identification in Spent LiNi0.6Co0.2Mn0.2O2||AG Cells

This cover image visualizes the “targeted fishing” of aging marker molecules in the complex matrix of spent lithium-ion battery electrolytes. Chromatographic techniques with high-resolution accurate mass spectrometric detection enable sensitive and selective identification, applicable for reverse engineering or risk assessment approaches. More details can be found in article number 2200189, Sascha Nowak and co-workers.

Detection of bazedoxifene, a selective estrogen receptor modulator, in human urine by liquid chromatography-tandem mass spectrometry.

Bazedoxifene, a selective estrogen receptor modulator, has been explicitly included in the prohibited list issued by the World Anti-Doping Agency (WADA) since January 2020. A high-resolution liquid chromatography-tandem mass spectrometric detection method was developed to identify bazedoxifene and its metabolites in human urine and to quantify bazedoxifene (free plus glucuronide) for doping control purposes. Bazedoxifene acetate (20 mg) was orally administered to seven male volunteers, and the urine samples collected were analyzed using the developed method. The linearity ranged from 0.5 to 200 ng/ml, and the limit of detection was <0.2 ng/ml. The interday precision (2.2% to 3.6%) and the interday accuracy (-10.0% to 1.9%) were adequate. Bazedoxifene, bazedoxifene-N-oxide, and bazedoxifene glucoconjugates were identified in the urine samples. The profiles of the urinary excretion indicated the presence of small amounts of free bazedoxifene and bazedoxifene-N-oxide, whereas bazedoxifene glucuronide was the predominant metabolite. The cumulative excretion amount of bazedoxifene (free form plus glucuronide conjugate) within 78 h after the administration was 0.7% to 1.3% of the total dose. In all subjects, bazedoxifene (free plus glucuronide) could be detected in urine up to 78 h after administration.

Quantitative analysis of polycyclic aromatic hydrocarbons using high-performance liquid chromatography-photodiode array-high-resolution mass spectrometric detection platform coupled to electrospray and atmospheric pressure photoionization sources.

Polycyclic aromatic hydrocarbons (PAHs) are common pollutants present in atmospheric aerosols and other environmental mixtures. They are of particular air quality and human health concerns as many of them are carcinogenic toxins. They also affect absorption of solar radiation by aerosols, therefore contributing to the radiative forcing of climate. For environmental chemistry studies, it is advantageous to quantify PAH components using the same analytical technics that are commonly applied to characterize a broad range of polar analytes present in the same environmental mixtures. Liquid chromatography coupled with photodiode array and high-resolution mass spectrometric detection (LC-PDA-HRMS) is a method of choice for comprehensive characterization of chemical composition and quantification of light absorption properties of individual organic compounds present in the environmental samples. However, quantification of non-polar PAHs by this method is poorly established because of their imperfect ionization in electrospray ionization (ESI) technique. This tutorial article provides a comprehensive evaluation of the quantitative analysis of 16 priority pollutant PAHs in a standard reference material using the LC-MS platform coupled with the ESI source. Results are further corroborated by the quantitation experiments using an atmospheric pressure photoionization (APPI) method, which is more sensitive for the PAH detection. The basic concepts and step-by-step practical guidance for the PAHs quantitative characterization are offered based on the systematic experiments, which include (1) Evaluation effects of different acidification levels by formic acid on the (+)ESI-MS detection of PAHs. (2) Comparison of detection limits in ESI+ versus APPI+ experiments. (3) Investigation of the PAH fragmentation patterns in MS2 experiments at different collision energies. (4) Calculation of wavelength dependent mass absorption coefficient (MACλ ) of the standard mixture and its individual PAHs using LC-PDA data. (5) Assessment of the minimal injected mass required for accurate quantification of MACλ of the standard mixture and of a multi-component environmental sample.

Determination of p-Methoxyphenacyl Bromide Derivatives of Alkylmethylphosphonic Acids in Urine Using Gas Chromatography with High-Resolution Mass Spectrometric Detection

Determination of p-Methoxyphenacyl Bromide Derivatives of Alkylmethylphosphonic Acids in Urine Using Gas Chromatography with High-Resolution Mass Spectrometric Detection

Selective extraction of gambierone and related metabolites in Gambierdiscus silvae using m-aminophenylboronic acid–agarose gel and liquid chromatography–high-resolution mass spectrometric detection

Gambierdiscus spp. are epi-benthic dinoflagellates that have been associated with ciguatera poisoning. These microalgae can have complex secondary metabolite profiles including ciguatoxins, maitotoxins, and gambierones, with varying compositions and toxicities across species and strains. Given this chemical diversity there is a need to develop selective and sensitive methods for secondary metabolite profiling. In this study, we used a cultured Caribbean strain of Gambierdiscus silvae to develop sample preparation and analysis strategies for characterizing vic-diol containing secondary metabolites. A pooled cellular extract was first screened by liquid chromatography–high-resolution mass spectrometry (LC–HRMS) for ciguatoxin-related compounds, which resulted in the confirmation of gambierone (1) and a novel isomer of 44-methylgambierone (3). Treatment of the extract with periodate confirmed that the gambierones each contained one reactive vic-diol, which was exploited for the development of a selective extraction procedure using m-aminophenylboronic acid gel and the non-aqueous binding solvent chloroform. Using this non-traditional boronate affinity procedure, LC–HRMS also revealed the presence of additional sulfated polycyclic ethers in the gambierone-containing vic-diol fraction, while pigments and other contaminants were removed. The developed tools could be applied to screen collections of Gambierdiscus and other benthic algae to provide additional chemical characterization of gambierone-related compounds. The selective extraction procedure may also prove useful as a step in the isolation of these sulfated polyethers for structural, toxicological and biotransformation studies.

High-speed countercurrent chromatography for isolation and enrichment of betacyanins from fresh and dried leaves of Atriplex hortensis L. var. "Rubra".

Betacyanins and their decarboxylated derivatives from fresh and dried edible leaves of Atriplex hortensis L. var. "Rubra" were fractionated for the first time by high-speed countercurrent chromatography. Pigments present in fresh leaf extract were separated in systems: ethanol - acetonitrile - n-propanol - ammonium sulphate - water (0.5:0.5:0.5:1.2:1.0, v/v/v/v/v) (tail-to-head mode) and tert-butyl methyl ether - n-butanol - acetonitrile - water with 0.7% heptafluorobutyric acid (2:2:1:5, v/v/v/v) (head-to-tail mode). The mobile phase flow rate was 2mL/min and the retention of the stationary phase was 79.8 and 75.2%, respectively. Pigments from dried leaves were separated in a similar ion-pair system with heptafluorobutyric acid in different volume proportions 1:3:1:5 (head-to-tail mode) and the flow rate of the mobile phase 3mL/min. The stationary phase retention was 64.0%. The application of the countercurrent chromatography for the fractionation of betacyanins from leaves of Atriplex hortensis enabled to isolate and pre-concentrate the pigments for further low- and high-resolution liquid chromatographic-tandem mass spectrometric detection. This study revealed the presence of 10 betacyanins in fresh and 16 in dried leaves of Atriplex hortensis. Two compounds were not previously identified in the whole Amaranthaceae family. Additionally, instead of (iso)amaranthin, celosianin and its epimer were dominant betacyanins in the Atriplex hortensis.

The Tire Wear Compounds 6PPD-Quinone and 1,3-Diphenylguanidine in an Urban Watershed.

Prompted by a recent report that 6PPD-quinone (6PPD-q), a by-product of a common tire manufacturing additive that is present in road runoff, is toxic to coho salmon (Oncorhynchus kisutch), extracts of water samples collected from an urban river were re-analyzed to determine if this compound was present in stormwater-influenced flows. In addition, extracts were analyzed for 1,3-diphenylguanidine (DPG), which is also used in tire manufacturing. Samples were originally collected in the fall of 2019 and winter of 2020 in the Greater Toronto Area of Canada from the Don River, a highly urbanized watershed in close proximity to several major multi-lane highways. These target compounds were analyzed using ultra-high pressure liquid chromatography with high resolution mass spectrometric detection with parallel reaction monitoring. Both 6PPD-q and DPG were detected above limits of quantification (i.e., 0.0098 µg/L) in all extracts. Maximum concentrations for 6PPD-quinone of 2.30 ± 0.05 µg/L observed in the river during storm events exceeded the LC50 for this compound for coho salmon (i.e., > 0.8 µg/L). In composite samples collected at intervals throughout one rain event, both compounds reached peak concentrations a few hours after initiation of the event (i.e., 0.52 µg/L for DPG and 2.85 µg/L for 6PPD-q), but the concentrations of 6PPD-q remained elevated above 2 µg/L for over 10-h in the middle of the event. Estimates of cumulative loads of these compounds in composite samples indicated that kg amounts of these compounds entered the Don River during each hydrological event, and the loads were proportional to the amounts of precipitation. This study contributes to the growing literature indicating that potentially toxic tire-wear compounds are present at elevated levels and are transported via road runoff into urban surface waters during rain events.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00244-021-00878-4.

Development and validation of a liquid chromatography high-resolution mass spectrometry orbitrap method for the sensitive quantification of amoxicillin, piperacillin, tazobactam and meropenem in human faeces

Beta-lactam antibiotics are of vital importance for the treatment of infections in a broad range of patients. Although most systemically administered antibiotics will be excreted renally, a fraction will reach the gastro-intestinal tract, affecting the intestinal microbiome by eradicating a wide range of bacterial species while facilitating the growth of antimicrobial-resistant species. A better understanding of the kinetics of beta-lactam antibiotics in the gastro-intestinal tract is essential to study their role in the development of antibiotic resistance in bacteria and to help develop future therapies to prevent damage to, or restore, the intestinal microbiome.Analysis of beta-lactam antibiotics in faeces is particularly challenging due to the heterogeneous nature of the matrix, rapid degradation of some beta-lactam antibiotics in faeces and very strong ion suppression when using mass spectrometry. Sample preparation was optimized using a sequential strategy of experimental designs. It resulted in lyophilization, a MOPS buffer system and the addition of the beta-lactamase inhibitor avibactam to minimize degradation of antibiotics allowing sensitive quantification. The developed liquid chromatography method with high-resolution mass spectrometric detection was successfully validated according to bioanalytical EMA guidelines and had a linear range of 1–200 μg g−1 lyophilized faeces for amoxicillin, piperacillin and meropenem; and 0.5–100 μg g−1 lyophilized faeces for tazobactam. Despite the highly complex and heterogeneous composition of faeces, the accuracy (0.1–15%) and precision (1.7–12.1%) were in line with those obtained for quantification methods of beta-lactam antibiotics in plasma, the golden standard matrix for therapeutic drug monitoring. The applicability of the method was illustrated by successful quantification of piperacillin and tazobactam in faeces from an intensive care unit patient receiving piperacillin/tazobactam in a continuous intravenous infusion. Both piperacillin and tazobactam were still present six days after discontinuation of the therapy.

Cyclic Ion Mobility Spectrometry Coupled to High-Resolution Time-of-Flight Mass Spectrometry Equipped with Atmospheric Solid Analysis Probe for the Molecular Characterization of Combustion Particulate Matter.

Anthropogenic air pollution has a severe impact on climate and human health. The immense molecular complexity and diversity of particulate matter (PM) is a result of primary organic aerosol (POA) as well as secondary organic aerosols (SOAs). In this study, a direct inlet probe (DIP), i.e., atmospheric solids analysis probe (ASAP), with ion mobility high-resolution mass spectrometric detection is applied. Primary particulate matter emissions from three sources were investigated. Furthermore, photochemically aged emissions were analyzed. DIP introduction allowed for a direct analysis with almost no sample preparation and resulted in a complex molecular pattern. This pattern shifted through oxidation processes toward heavier species. For diesel emissions, the fuel's chemical characteristic is partially transferred to the particulate matter by incomplete combustion and characteristic alkylated series were found. Polycyclic aromatic hydrocarbons (PAHs) were identified as major contributors. Ion mobility analysis results in drift time profiles used for structural analysis. The apex position was used to prove structural changes, whereas the full-width-at-half-maximum was used to address the isomeric diversity. With this concept, the dominance of one or a few isomers for certain PAHs could be shown. In contrast, a broad isomeric diversity was found for oxygenated species. For the in-depth specification of fresh and aged spruce emissions, the ion mobility resolving power was almost doubled by allowing for three passes in the circular traveling wave design. The results prove that ASAP coupled with ion mobility spectrometry-mass spectrometry (IMS-MS) serves as a promising analytical approach for tackling the vast molecular complexity of PM.

Polybrominated dibenzo-p-dioxins and furans (PBDD/Fs) in Italian food: Occurrence and dietary exposure

Human exposure to polychlorinated dioxins and furans (PCDD/Fs) through the dietary pathway is widely recognised and regulations in some regions of the world help to limit food contamination. Similar information on the analogous polybrominated dioxins and furans (PBDD/Fs) is scarce, partly due to the higher threshold to analytical access and unavailability of some standard materials. The analytical methodology developed here determined twelve planar PBDD/F congeners using 13Carbon labelled PBDD/F surrogates and high resolution mass spectrometric detection, and was extensively validated prior to the analysis of a range of commonly consumed Italian foods. The methodology also allowed simultaneous determination of PCDD/Fs and polychlorinated biphenyls (PCBs). The results show that PBDD/Fs occurred in different foods over a range of concentrations from <0.001 pg/g to 4.58 pg/g in fish. The dietary exposure (upper bound) of different Italian population groups, resulting from these occurrence levels was estimated using the toxic equivalency (TEQ) approach that is commonly used for dioxin-like contaminants and ranged from 0.17 to 0.42 pg TEQ/kg bodyweight/day (lower bound – 0.01 pg TEQ/kg bodyweight/day) depending on the population subgroup. Although precautionary, upper bound values may provide a more realistic estimate of toxicity as not all congeners and foods were measured. As expected, children were more highly exposed than adults due to lower body weight. These exposure levels were between a quarter and a third of that arising from the sum of PCDD/Fs and PCBs (0.61 to 1.38 pg WHO-TEQ/kg bodyweight/day), but they contribute to dioxin-like toxicity. If this data is considered in view of the revised tolerable dioxin-like dietary intake published by EFSA in 2018, it is evident that the tolerable weekly intake of 2 pg/kg bodyweight/week would be exceeded by some of the assessed population sub-groups, or all sub-groups if the cumulative intake is considered.

Column bleed in the analysis of highly polar substances: an overlooked aspect in HRMS.

To close the "analytical gap" in the liquid chromatographic (LC) analysis of highly polar substances, two techniques which have been suggested earlier were tested in terms of retention factors and detection limits: hydrophilic interaction liquid chromatography (HILIC) and mixed-mode chromatography (MMC). A substance mix of 55 analytes ranging from logD - 8.2 to 3.4 and 17 different LC columns, also comprising additional reversed-phase columns were used. Contrary to most reversed-phase columns, column bleed has been identified as an important factor, which may cause serious restrictions during high-resolution mass spectrometric detection (HRMS). We found that highly abundant background masses continuously eluting from the columns heavily influence ion transmission to the detector. As a result, the linear dynamic range as well as the sensitivity decreases and thus limits the HRMS applicability of some columns. We therefore recommend a thorough investigation of ion transmission during HRMS method development. This will help to maintain the high potential of HRMS in terms of qualitative and quantitative screening analysis.

Net-like mesoporous carbon nanocomposites for magnetic solid-phase extraction of sulfonamides prior to their quantitation by UPLC-HRMS.

A net-like mesoporous carbon nanocomposite (MCN) was hydrothermally prepared by using filter paper as the raw material. The MCN contains magnetic nanoparticles of type Fe3O4 which result from the addition of Fe(NO3)3·9H2O during synthesis. The MCN was characterized by X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, Raman spectra, Brunauer-Emmett-Teller methods and vibrating sample magnetometry. The MCN is shown to be a viable material for magnetic solid-phase extraction of trace sulfonamides (SAs) including sulfadiazine, sulfapyridine, sulfamerazine, sulfamethazine, sulfamethizole, sulfamethoxypridazine, sulfachloropyridazine and sulfadimethoxine. Following desorption with acetone containing 0.5% ammonia, the SAs were quantified by UPLCwith high-resolution mass spectrometric detection. With sulfamethazine as an example, the adsorption equilibrium configurations and the major interaction mechanism between SAs and the MCN were calculated by using density functional theory. Under the optimal conditions, the calibration plots are linear in the 0.05-10ng·mL-1 SA concentration ranges. The limits of detection are between 7.2 and 13.6ng·L-1. The recoveries from spiked samples ranged from 79 to 107%, with relative standard deviations of <9.9%. Graphical abstractSchematic representation of nanocomposite preparation, adsorption mechanism of sulfonamide and magnetic solid-phase extraction (MSPE) for sulfadiazine (SDZ), sulfapyridine (SPD), sulfamerazine (SMR), sulfamethazine (SMZ), sulfamethizole (SMT), sulfamethoxypridazine (SMP), sulfachloropyridazine (SCP) and sulfadimethoxine (SDMX).Quantificationwas accomplished by UPLC with high-resolution mass spectrometric detection.

UHPLC-HRMS determination of adrenaline and dopamine dansyl derivatives in human saliva

In the last decade, the quantification of catecholamines in human biological fluids has been of great interest. Changes in catecholamine levels, such as adrenaline and dopamine, in the body lead to neurological disorders as well as several diseases, namely Alzheimer’s and Parkinson’s diseases, neuroendocrine catecholamine - producing tumors - pheochromocytoma, paraganglioma, carcinoid tumors and neuroblastoma. Moreover, different drug combinations can lead to the distortions in the human biological passport, containing the information not only about the erythropoiesis and steroid profile, but also about catecholamine levels, which are difficult to quantify in terms of sample preparation and analysis. A method has been proposed for the determination of adrenaline and dopamine dansyl derivatives in human saliva, including the derivatization and determination of analytes by ultra-high-performance liquid chromatography (UHPLC) with high resolution mass spectrometric detection. The derivatization procedure allowed obtaining less polar catecholamine derivatives, which is especially important for their quantification by reversed-phase ultra-high-performance liquid chromatography since it ensures their better retention on the sorbent. The sensitivity of these substances quantification by the proposed method was estimated; the highest sensitivity was achieved using the mobile phase consisting of the 0.1% formic acid aqueous solution and acetonitrile. The lower limit of quantification was 5 ng/mL for dansyldrenaline and 10 ng/mL for dansyldopamine respectively. The proposed technique was tested on real saliva samples obtained from volunteers to quantify catecholamine dansyl derivatives by reversed-phase high-performance liquid chromatography with high-resolution mass spectrometric detection. High sensitivity of the technique allows using it for adrenaline and dopamine determination in clinical diagnosis.

Structural Characterization of Triterpene Saponins from Manchurian Aralia by High Resolution Liquid Chromatography–Mass Spectrometry

The mass-spectrometric characteristics of triterpene saponins from Manchurian aralia were studied using high-performance liquid chromatography in combination with high-resolution mass-spectrometric detection. Conditions for the preparation of extracts for the determination of triterpene saponin concentrations in plant raw materials were optimized. The experimental mass-spectrometric characteristics of triterpene saponins were compared with those described in the literature, and the structural formulas of test compounds were proposed.

HPLC-ESI- MS/MS analysis of beet (Beta vulgaris) leaves and its beneficial properties in type 1 diabetic rats

The phenolic profile of the leaves of Beta vulgaris subspecies vulgaris variety rubra was investigated by high-performance liquid chromatography (HPLC) coupled to electrospray ionization high resolution mass spectrometric (ESI-HRMS-MS) detection. Mass spectrometry-based molecular networking was employed to dereplicate the known compounds. Twelve known compounds, seven of which are previously undescribed as constituents in the B. vulgaris leaves were dereplicated and assigned with various levels of identification confidence. The ameliorative effects of the aqueous methanolic extract of the leaves were assessed against alloxan induced diabetic rats. It was found that the extract significantly decreased (p < 0.001) serum glucose, lipid profile, ALT, AST, TNF-α, IL-1β, IL-6, and hepatic MDA levels; and significantly increased (p < 0.001) hepatic TAO and GSH; and down-regulated the expression of hepatic NF-κB versus the untreated diabetic groups, in a dose-dependent manner. In molecular docking, all identified compounds exhibited good glide score against the PPAR-ɣ target, confirming the in vivo observed activities. In conclusion, B. vulgaris has immunomodulatory / antioxidant effects that could be helpful in slowing the progression of diabetic complications.

Development of a CE-MS method for the study of riociguat and metabolite M1 in pharmaceutical analysis.

Riociguat is a novel antihypertensive drug for the treatment of pulmonary hypertension. We present electrophoretic characterization, i.e. migration behavior of riociguat and metabolite M1 as support for optimized CZE/MS assay. Fundamental separation parameters, such as peak width, symmetry, and resolution are studied in a series of ammonium formate buffers within pH range 2.60-5.61. The narrow region of peak symmetry lies close to pH 4.0 for both analytes. Accordingly, the value of resolution maximizes in a background electrolyte adjusted to pH 4.10. Basic calibration parameters estimated from CZE experiments with absorption photometric and mass spectrometric detection of riociguat and metabolite M1 were evaluated. More than three orders lower LOD was achieved with high resolution mass spectrometric detection. The observed difference in the sensitivity of both detection techniques gives priority to the utilization of CZE/MS in practice. The values of dissociation constants of riociguat and metabolite M1, pKBH , were determined from CZE measurements in lithium formate and lithium acetate background electrolytes with constant ionic strength. The value of pKBH = 4.30±0.02 for riociguat corresponds well to the value already presented in the literature. According to our observation, metabolite M1 behaves like a slightly stronger base with estimated pKBH = 4.40±0.02.

Ovothiol A is the Main Antioxidant in Fish Lens.

Tissue protection from oxidative stress by antioxidants is of vital importance for cellular metabolism. The lens mostly consists of fiber cells lacking nuclei and organelles, having minimal metabolic activity; therefore, the defense of the lens tissue from the oxidative stress strongly relies on metabolites. Protein-free extracts from lenses and gills of freshwater fish, Sander lucioperca and Rutilus rutilus lacustris, were subjected to analysis using high-field 1H NMR spectroscopy and HPLC with optical and high-resolution mass spectrometric detection. It was found that the eye lenses of freshwater fish contain high concentrations of ovothiol A (OSH), i.e., one of the most powerful antioxidants exciting in nature. OSH was identified and quantified in millimolar concentrations. The concentration of OSH in the lens and gills depends on the fish genus and on the season. A possible mechanism of the reactive oxygen species deactivation in fish lenses is discussed. This work is the first to report on the presence of OSH in vertebrates. The presence of ovothiol in the fish tissue implies that it may be a significantly more common antioxidant in freshwater and marine animals than was previously thought.

Quantitative metabolomic analysis of changes in the lens and aqueous humor under development of age-related nuclear cataract.

Metabolites are essential for the proper functioning of the eye lens, they either enter the lens from the aqueous humor (AH), or are synthesized in the lens epithelium. Antioxidants, osmolytes and UV filters are especially important for the lens protection, and their lack may cause the development of ophthalmic diseases. Comparison of the metabolomic compositions of lenses and AH taken from cataract patients with that taken from human cadavers without cataract can shed light onto molecular mechanisms underlying onset of age-related nuclear cataract. Combined use of 1H nuclear magnetic resonance and high performance liquid chromatography with optical and high-resolution mass spectrometric detection for the identification and quantification of metabolites in the lens and AH extracts. The concentrations of 86 metabolites were determined for four groups of samples, including lenses and AH from cataract patients and from human cadavers. In cataractous lens the most abundant metabolites are (in descending order): myo-inositol, lactate, acetate, glutamate, glutathione; in AH-lactate, glucose, glutamine, alanine, valine. The concentrations of the majority of metabolites in normal post-mortem samples of both lens and AH are higher than that in samples from the cataract patients. Comparison of metabolite concentrations in lens and corresponding AH reveal that the most important for the lens protection metabolites are synthesized in the lens epithelial cells. The reduced levels of antioxidants, UV filters, and osmolytes were found in the cataractous lenses what cannot be explained by post-mortem changes in normal lens; that indicates that the age-related nuclear cataract development may originate from the dysfunction of the lens epithelial cells.

Recent improvements in sports drug testing concerning the initial testing for peptidic drugs (< 2kDa) - sample preparation, mass spectrometric detection, and data review.

In this work, a novel initial testing assay based on liquid chromatography-mass spectrometry is presented, enabling the detection of peptidic drugs and drug candidates (< 2kDa) prohibited in sports. The assay covers representatives and metabolites of gonadotropin releasing hormone and its analogs (GnRHs), growth hormone secretagogues (GHS), growth hormone releasing peptides (GHRPs), and the Vasopressin-analog Desmopressin. The general objective of this work was to reduce sample preparation efforts to a minimum while preserving highest possible sensitivity and specificity of the assay, demonstrating limits of detection between 50 and 200pg/mL. Here, a "dilute-and-inject" strategy provides the simplest conceivable sample preparation procedure. Furthermore, the combination of well-established strategies for the determination of peptides, such as two-dimensional liquid chromatography, dimethyl sulfoxide (DMSO)-assisted electrospray ionization, high resolution mass spectrometric detection and a tailored reporter template, which facilitates data review enormously, provides a high-throughput initial testing assay for lower molecular mass peptidic and peptide-related analytes.

Analysis of insulin and insulin analogs from dried blood spots by means of liquid chromatography-high resolution mass spectrometry.

While dried blood spot (DBS) analysis concerning low molecular mass molecules has become more and more established in various fields of analytical chemistry, the utility of DBS in determining peptides and proteins from DBS is yet comparably limited. In consideration of the fact that the apparent benefits of DBS sampling are similar for analytes of lower and higher molecular mass, dedicated (non-generic) sample preparation procedures are required that meet the needs for detecting peptidic drugs and hormones in DBS. The analysis of insulin and its synthetic analogs by mass spectrometry has received increased attention in several fields such as doping controls, forensics, and drug metabolism and pharmacokinetics studies. Hence, a strategy facilitating the analysis of insulin and its synthetic or animal analogs (human, Lispro, Aspart, Glulisine, Glargine, Detemir, Tresiba, and porcine and bovine insulin) from DBS was developed. The successful analysis of these substances at physiologically relevant concentrations was realized after ultrasonication-assisted extraction, immunoaffinity purification, and liquid chromatographic separation followed by high resolution mass spectrometric detection (with or without ion mobility). Assay validation demonstrated adequate sensitivity (LOD 0.5ng/mL for most insulins), as well as precise (< 25%) and reproducible results for all included target insulins. Additionally, proof-of-principle data were obtained by the analysis of DBS samples obtained from healthy volunteers in non-fasting state as well as a sample from a diabetic volunteer treated with the fast acting analog insulin Aspart.