Tutorial review on validation of liquid chromatography–mass spectrometry methods: Part I

This is the part I of a tutorial review intending to give an overview of the state of the art of method validation in liquid chromatography mass spectrometry (LC–MS) and discuss specific issues that arise with MS (and MS/MS) detection in LC (as opposed to the “conventional” detectors). The Part I briefly introduces the principles of operation of LC–MS (emphasizing the aspects important from the validation point of view, in particular the ionization process and ionization suppression/enhancement); reviews the main validation guideline documents and discusses in detail the following performance parameters: selectivity/specificity/identity, ruggedness/robustness, limit of detection, limit of quantification, decision limit and detection capability. With every method performance characteristic its essence and terminology are addressed, the current status of treating it is reviewed and recommendations are given, how to determine it, specifically in the case of LC–MS methods.     

Towards the chiral metabolomics: Liquid chromatography–mass spectrometry based dl-amino acid analysis after labeling with a new chiral reagent, (S)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidine-2-carboxylate,and the application to saliva of healthy volunteers

A novel triazine-type chiral derivatization reagent, i.e., (S)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl) pyrrolidine-2-carboxylate (DMT-(S)-Pro-OSu), was developed for the highly sensitive and selective detection of chiral amines and amino acids by UPLC–MS/MS analysis. The enantiomers of amino acids were easily labeled with the reagents at room temperature within 40 min in an alkaline medium containing triethylamine. The diastereomers derived from proteolytic amino acids, except serine, were well separated under isocratic elution conditions by reversed-phase chromatography using an ODS column (R_s = 1.2–9.0). dl-Serine was separated by use of an ADME column which has relatively higher polar surface than the conventional ODS column. The characteristic product ions, i.e., m/z 195.3 and m/z 209.3, were detected from all the diastereomers by the collision-induced dissociation of the protonated molecule. A highly sensitive detection on the amol–fmol level was obtained from the selected reaction monitoring (SRM) chromatogram. The chiral amines (e.g., adrenaline and noradrenaline) labeled with DMT-(S)-Pro-OSu were also well separated and sensitively detected by the present procedure. The method using DMT-(S)-Pro-OSu was used for the determination of dl-amino acids in the human saliva from healthy volunteers. Various l-amino acids were identified in the saliva. Furthermore, d-alanine (d-Ala) and d-proline (d-Pro) were also detected in relatively high concentrations (>5%). The ratio was higher in male saliva than in female saliva. However, the difference in the ratio of d-Ala for one day was not very high and the effect of foods and beverage seemed to be negligible. Based on the results using l-Ala-d₃, the d-Ala in saliva seemed to be produced due to the racemization with some enzymes such as racemase. The racemization reaction was reversible, i.e., d-Ala-d₃ was also racemized to l-Ala-d₃ in saliva. Thus, care should be taken during the analysis of dl-amino acids in saliva. The present method using DMT-(S)-Pro-OSu may be applicable for the determination of chiral amine metabolomics, because the resulting derivatives produce the same product ions without relation to the compounds and show highly sensitive detection in the SRM mode of MS/MS. Consequently, DMT-(S)-Pro-OSu seems to be a useful chiral derivatization reagent for the determination of amines and amino acids in biological samples.     

Analysis of peptides and protein digests by reversed phase high performance liquid chromatography–electrospray ionisation mass spectrometry using neutral pH elution conditions

In this study, the advantages of carrying out the analysis of peptides and tryptic digests of proteins under gradient elution conditions at pH 6.5 by reversed-phase liquid chromatography (RP-HPLC) and in-line electrospray ionisation mass spectrometry (ESI-MS) are documented. For these RP separations, a double endcapped, bidentate anchored n-octadecyl wide pore silica adsorbent was employed in a capillary column format. Compared to the corresponding analysis of the same peptides and protein tryptic digests using low pH elution conditions for their RP-HPLC separation, this alternative approach provides improved selectivity and more efficient separation of these analytes, thus allowing a more sensitive identification of proteins at different abundance levels, i.e. more tryptic peptides from the same protein could be confidently identified, enabling higher sequence coverage of the protein to be obtained. This approach was further evaluated with very complex tryptic digests derived from a human plasma protein sample using an online two-dimensional (2D) strong cation-exchange (SCX)-RP-HPLC-ESI-MS/MS system. Again, at pH 6.5, with mobile phases of different compositions, improved chromatographic selectivities were obtained, concomitant with more sensitive on-line electrospray ionisation tandem mass spectrometric (ESI-MS/MS) analysis. As a consequence, more plasma proteins could be confidently identified, highlighting the potential of these RP-HPLC methods with elution at pH 6.5 to extend further the scope of proteomic investigations.     

Electrochemical sensors and biosensors based on redox polymer/carbon nanotube modified electrodes: A review

The aim of this review is to present the contributions to the development of electrochemical sensors and biosensors based on polyphenazine or polytriphenylmethane redox polymers together with carbon nanotubes (CNT) during recent years. Phenazine polymers have been widely used in analytical applications due to their inherent charge transport properties and electrocatalytic effects. At the same time, since the first report on a CNT-based sensor, their application in the electroanalytical chemistry field has demonstrated that the unique structure and properties of CNT are ideal for the design of electrochemical (bio)sensors. We describe here that the specific combination of phenazine/triphenylmethane polymers with CNT leads to an improved performance of the resulting sensing devices, because of their complementary electrical, electrochemical and mechanical properties, and also due to synergistic effects. The preparation of polymer/CNT modified electrodes will be presented together with their electrochemical and surface characterization, with emphasis on the contribution of each component on the overall properties of the modified electrodes. Their importance in analytical chemistry is demonstrated by the numerous applications based on polymer/CNT-driven electrocatalytic effects, and their analytical performance as (bio) sensors is discussed.     

Analytical capabilities of high performance liquid chromatography – Atmospheric pressure photoionization – Orbitrap mass spectrometry (HPLC-APPI-Orbitrap-MS) for the trace determination of novel and emerging flame retardants in fish

A new analytical method was established and validated for the analysis of 27 brominated flame retardants (BFRs), including so called “emerging” and “novel” BFRs (EBFRs and NBFRs) in fish samples. High performance liquid chromatography (HPLC) coupled to Orbitrap mass spectrometry (Orbitrap-MS) employing atmospheric pressure photoionization (APPI) interface operated in negative mode was used for the identification/quantitation of contaminants. HPLC-Orbitrap-MS analysis provided a fast separation of selected analytes within 14 min, thus demonstrating a high throughput processing of samples. The developed methodology was tested by intralaboratory validation in terms of recovery, repeatability, linear calibration ranges, instrumental and method limits of quantitation (i-LOQ and m-LOQ), and where possible, trueness was verified by analysis of certified reference materials (CRMs). Recoveries of analytes were between 80 and 119%, while the repeatability in terms of relative standard deviations (RSDs) was in the range from 1.2 to 15.5%. The measured values for both analyzed CRMs agreed with the provided consensus values, revealing the recovery of reference concentrations in 72–119% range. The elaborated method met the sensitivity criterion according to Commission Recommendation 2014/118/EU on monitoring of BFRs in food products for majority of the compounds. The concentrations of polybrominated diphenyl ethers (PBDEs) in real samples determined by HPLC-APPI-Orbitrap-MS method and validated gas chromatography–high-resolution mass spectrometry (GC–HRMS) method were found to be in a good agreement.     

A novel approach for LC-MS/MS-based chiral metabolomics fingerprinting and chiral metabolomics extraction using a pair of enantiomers of chiral derivatization reagents

Chiral metabolites are found in a wide variety of living organisms and some of them are understood to be physiologically active compounds and biomarkers. However, the overall analysis of chiral metabolomics is quite difficult due to the high number of metabolites, the significant diversity in their physicochemical properties, and concentration range from metabolite-to-metabolite. To solve this difficulty, we developed a novel approach for chiral metabolomics fingerprinting and chiral metabolomics extraction, which is based on the labeling of a pair of enantiomers of chiral derivatization reagents (i.e., DMT-(S,R)-Pro-OSu and DMT-3(S,R)-Apy) and precursor ion scan chromatography of the derivatives. The multivariate statistics is also required for this strategy. The proposed procedures were evaluated by the detection of a diagnostic marker (i.e., d-lactic acid) using the saliva of diabetic patients. This method was used for the determination of biomarker candidates of chiral amines and carboxyls in Alzheimer's disease (AD) brain homogenates. As the results, l-phenylalanine (L-Phe) and l-lactic acid (L-LA) were identified as the decreased and increased biomarker candidates in the AD brain, respectively. Therefore, the proposed approach seems to be helpful for the determination of non-target chiral metabolomics possessing amines and carboxyls.     

The Aerobic Oxidation and C=C Bond Cleavage of Styrenes Catalyzed by Cerium(IV) Ammonium Nitrate (CAN)

The CAN‐catalyzed aerobic oxidation severed the C=C bond selectively through the single electron transfer mechanism, giving a green method to decompose olefins. Compared with the literature reported examples, this method required simple catalyst, cheap, abundant and clean oxidant and was very safe to operate due to the mild conditions.     

Regioselective One‐pot Synthesis of New Unsymmetric Spiro Dihydrofurans in the Reaction of Mixed Two Different Cyclic β‐Dicarbonyl Compounds with BrCN and Aldehydes in the Presence of Et3N

Crossed one‐pot reaction of mixed cyclic β‐dicarbonyl with various aldehydes in the presence of cyano gen bromide and triethylamine leads to the selective and efficient formation of crossed new unsymmetri cal spiro dihydrofurans at room temperature. The products were obtained in good to excellent yields. Structure elucidation was carried out by ¹H NMR, ¹³C NMR, FT‐IR spectroscopy, Mass analyses and X‐ray crystallography technique. A proposed mechanism was discussed for the formation of products.     

High-throughput characterization of sediment organic matter by pyrolysis–gas chromatography/mass spectrometry and multivariate curve resolution: A promising analytical tool in (paleo)limnology

Molecular-level chemical information about organic matter (OM) in sediments helps to establish the sources of OM and the prevalent degradation/diagenetic processes, both essential for understanding the cycling of carbon (C) and of the elements associated with OM (toxic trace metals and nutrients) in lake ecosystems. Ideally, analytical methods for characterizing OM should allow high sample throughput, consume small amounts of sample and yield relevant chemical information, which are essential for multidisciplinary, high-temporal resolution and/or large spatial scale investigations. We have developed a high-throughput analytical method based on pyrolysis–gas chromatography/mass spectrometry and automated data processing to characterize sedimentary OM in sediments. Our method consumes 200 μg of freeze-dried and ground sediment sample. Pyrolysis was performed at 450 °C, which was found to avoid degradation of specific biomarkers (e.g., lignin compounds, fresh carbohydrates/cellulose) compared to 650 °C, which is in the range of temperatures commonly applied for environmental samples. The optimization was conducted using the top ten sediment samples of an annually resolved sediment record (containing 16–18% and 1.3–1.9% of total carbon and nitrogen, respectively). Several hundred pyrolytic compound peaks were detected of which over 200 were identified, which represent different classes of organic compounds (i.e., n-alkanes, n-alkenes, 2-ketones, carboxylic acids, carbohydrates, proteins, other N compounds, (methoxy)phenols, (poly)aromatics, chlorophyll and steroids/hopanoids). Technical reproducibility measured as relative standard deviation of the identified peaks in triplicate analyses was 5.5 ± 4.3%, with 90% of the RSD values within 10% and 98% within 15%. Finally, a multivariate calibration model was calculated between the pyrolytic degradation compounds and the sediment depth (i.e., sediment age), which is a function of degradation processes and changes in OM source type. This allowed validation of the Py–GC/MS dataset against fundamental processes involved in OM cycling in aquatic ecosystems.     

Sensor–actuator system for dynamic chloride ion determination

Chloride is a crucial anion for various analytical applications from biological to environmental applications. In order to measure the chloride ion concentration, a measurement system is needed which can detect this concentration for prolonged times reliably. Chronopotentiometry is a technique which does not need a long term stable reference electrode and is therefore very suitable for prolonged ion concentration measurements. As the used electrode might be fouled by reaction products, this work focuses on a chronopotentiometric approach with a separated sensing electrode (sensor) and actuating electrode (actuator). Both actuation and sensor electrode are made of Ag/AgCl. A constant current is applied to the actuator and will start the reaction between Ag and Clˉ, while the resulting Clˉ ion concentration change is observed through the sensor, which is placed close to the actuator. The time it takes to locally deplete the Clˉ ions is called transition time. Experiments were performed to verify the feasibility of this approach. The performed experiments show that the sensor detects the local concentration changes resulting from the current applied to the actuator. A linear relation between the Clˉ ion concentration and the square root of the transition time was observed, just as was predicted by theory. The calibration curves for different chips showed that both a larger sensor and a larger distance between sensor and actuator resulted in a larger time delay between the transition time detected at the actuator and the sensor.