Liquid Chromatography-mass Spectrometry Conditions Research Articles

Mitigating matrix effects for LC-MS/MS quantification of nitrosamine impurities in rifampin and rifapentine

Since the discovery of potentially mutagenic nitrosamine impurities in pharmaceuticals in 2018, liquid chromatography-mass spectrometry (LC-MS) methods have been widely applied for the trace level quantification of nitrosamines. As more nitrosamines and drug samples are analyzed, method accuracy is increasingly recognized as a major analytical challenge. Matrix effects can have a significant adverse impact on method accuracy but have not attracted much attention in nitrosamine quantification literature. In this study, we observed severe matrix effects when adapting LC-MS method conditions from a published and validated method for the analysis of nitrosamines in tuberculosis drugs. Several approaches were explored to mitigate these matrix effects, including using stable isotope-labeled internal standards, modifying LC conditions and sample concentration, and quantifying with standard addition instead of an external calibration method. The results underscore the importance of monitoring matrix effects and validating nitrosamine quantification procedures for specific sample matrices. Finally, two LC-MS/MS methods are presented with validation data for the quantification of 1-methyl-4-nitrosopiperazine (MNP) in rifampin and 1-cyclopentyl-4-nitrosopiperazine (CPNP) in rifapentine and revealed high nitrosamine levels in the tested samples that far exceeded recommended acceptable intake limits.

Preparative agarose gel electrophoresis for reducing matrix interferences of organoid cell medium prior to LC-MS analysis of insulin

Organoids are 3D cell cultures with microanatomies mimicking aspects of real organs, useful for e.g. animal-free studies of development, disease, and drug discovery. The cell medium of organoid models of Langerhans islets, regulating blood glucose levels by insulin secretion, can be analyzed by liquid chromatography-mass spectrometry (LC-MS). However, organoid medium complexity is a major challenge, as matrix interferences can reduce sensitivity and selectivity, even with optimized LC-MS conditions. By applying preparative agarose gel electrophoresis-electrodialysis (PGE-ED), we were able to decrease the cell medium background signal, allowing for reduced interferences affecting LC-MS analysis of human insulin.

An Ultra-Fast and Green LC-MS Method for Quantitative Analysis of Aesculin and Aesculetin in Cortex Fraxini

This study aims to develop a fast and eco-friendly liquid chromatography–mass spectrometry (LC-MS) method for the determination of aesculin and aesculetin in Cortex Fraxini. Ultrapure water was used as the solvent during the microwave-assisted extraction process to prepare the Cortex Fraxini sample. This extraction method reduces the cost of the harmful solvent (only ultrapure water was used) and microwave extraction time (1 min). The LC separation was conducted using an Agilent InfinityLab Poroshell 120 EC-C18 column (2.1 mm × 30 mm, 2.7 µm) with a mobile phase consisting of 0.1% formic acid and acetonitrile (90:10, v/v) at a flow rate of 0.6 mL/min. Isocratic elution was employed, and the analytes were detected by MS. Through careful optimization and selection of LC-MS conditions, the analysis time was reduced to 1 min, demonstrating the method’s efficiency. The developed method was validated and exhibited excellent specificity, linearity, limit, precision, accuracy, and stability in quantifying aesculin and aesculetin in the Cortex Fraxini samples. The analysis result revealed the presence of aesculin (ranging from 3.55 to 18.8 mg/g) and aesculetin (ranging from 1.01 to 16.2 mg/g) in all ten batches of Cortex Fraxini samples. Compared to the reported LC methods, this approach substantially reduces the total analysis time and requires a minuscule volume of organic solvents. An “Analytical Eco-Scale” assessment was used to evaluate the different assay methods of Cortex Fraxini. The current LC-MS method scored an impressive 90; it was better than the other four reports’ LC methods. Thus, the developed LC-MS method is rapid and green, which is helpful for the quality evaluation of Cortex Fraxini.

Impact of Matrix Species and Mass Spectrometry on Matrix Effects in Multi-Residue Pesticide Analysis Based on QuEChERS-LC-MS

With the popularity of multi-residue pesticide analysis based on quick, easy, cheap, effective, rugged, and safe (QuEChERS) cleanup and liquid chromatography-mass spectrometry (LC-MS), matching optimal matrix-matched calibration protocols and LC-MS conditions to reduce matrix effects (MEs) has become a crucial task for analysts in their routines. However, dozens to hundreds of pesticide analytes in a single run generate increasingly multi-dimensional ME data, requiring appropriate tools to handle these data sets. Therefore, we established an ME analysis strategy by drawing on analytical thinking and tools from metabolomics analysis. Using this, matrix species-induced and mass spectrometry-induced systematic ME variations were distinguished, and pesticides contributed to the variations were scanned out. A simultaneous weakening of MEs on 24 pesticides in 32 different matrices was achieved using the time-of-flight-mass spectrometry (TOF-MS) scan under the information-dependent acquisition (IDA) mode of high-resolution mass spectrometry (HR-MS), compared to multiple reaction monitoring (MRM) scanning by tandem mass spectrometry (MS/MS). Bay leaf, ginger, rosemary, Amomum tsao-ko, Sichuan pepper, cilantro, Houttuynia cordata, and garlic sprout showed enhanced signal suppression in the MRM scan for 105 differential MRM transitions for 42 pesticides and in IDA mode for 33 pesticides, respectively. This study revealed the interference of matrix species and mass spectrometry on MEs and provided a novel strategy for ME analysis.

Qualitative Characterization of the Rat Liver Mitochondrial Lipidome Using All Ion Fragmentation on an Exactive Benchtop Orbitrap MS.

Untargeted lipidomics profiling by liquid chromatography -mass spectrometry (LC-MS) allows researchers to observe the occurrences of lipids in a biological sample without showing intentional bias to any specific class of lipids and allows retrospective reanalysis of data collected. Typically, and in the specific method described, a general extraction method followed by LC separation is used to achieve nonspecific class coverage of the lipidome prior to high resolution accurate mass (HRAM) MS detection . Here we describe a workflow including the isolation of mitochondria from liver tissue, followed by mitochondrial lipid extraction and the LC-MS conditions used for data acquisition. We also highlight how, in this method, all ion fragmentation can be used to identify species of lower abundances, often missed by data dependent fragmentation techniques. Here we describe the isolation of mitochondria from liver tissue, followed by mitochondrial lipid extraction and the LC-MS conditions used for data acquisition.

DEVELOPMENT AND APPLICATION OF A TEST MIXTURE FOR UNTARGETED LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY ANALYSIS OF URINE SAMPLES

Metabolic profiling of complex biological matrices based on liquid chromatography-mass spectrometry (LC-MS) allows detecting a wide range of metabolites with distinct concentrations and physicochemical properties. Given the complexity of samples and the necessity of a comprehensive approach in untargeted metabolomics, quality control strategies are mandatory to obtain high-quality data. The LC-MS performance must be monitored and evaluated to guarantee data reliability. In this study, a test mixture (TM) was developed, systematically evaluated, and applied to untargeted metabolomics of urine samples from individuals suspected of inborn errors of metabolism. The TM was composed of fifteen analytes that eluted across the entire gradient in reversed-phase columns and ionized in positive/negative electrospray modes. It helped set the LC-MS conditions for urine analysis, from sample reconstitution solvent to selecting the MS ion source parameters. The TM quickly indicated column stationary phase degradation during the batch analysis when employed to monitor and evaluate the LC-MS system in an untargeted metabolomic analysis. Thus, in addition to pooled QC samples, a TM can be employed in untargeted metabolomics to rapidly assess the system performance avoiding unnecessary efforts for further data treatment and multivariate analysis of poor-quality data

Quadruplex stable isotope derivatization strategy for the determination of panaxadiol and panaxatriol in foodstuffs and medicinal materials using ultra high performance liquid chromatography tandem mass spectrometry

This work proposed a novel strategy based on quadruplex stable isotope derivatization for sensitive, accurate, speedy and green determination of panaxadiol (PD) and panaxatriol (PT). This strategy integrated with ultrasound-assisted dispersive liquid-liquid microextraction, microwave-assisted derivatization, and magnetic graphene oxide dispersive solid phase extraction as efficient sample pretreatment techniques, coupling with ultra-high performance liquid chromatography tandem mass spectrometry detection in multiple reaction monitoring mode for the analytical purpose of high-throughput, sensitivity, selectivity, green and accuracy. Quadruplex mass spectrometry sensitizing derivatization reagents, 3-N-(D0-/D3-methyl-, and D0-/D5-ethyl-)-2′-carboxyl chloride rhodamine 6 G, were designed, synthesized and applied for the high-throughput derivatization of hydroxyl‑containing PD and PT for the first time. The analogue derivatives of PD and PT standards using 3-N-propyl-2′-carboxyl chloride rhodamine 6 G as derivatization reagent were employed as the internal standards for the mass spectrometry quantitation to minimize matrix effect. All rhodamine 6 G derivatives of PD and PT were easily and selectively adsorbed by magnetic graphene oxide for efficient magnetic dispersive solid phase extraction before injection. Parameters of (micro-) extractions, derivatization and liquid chromatography-mass spectrometry conditions were all optimized in details. Method validation results consisting of linearity, accuracy, precision, repeatability, matrix effect, stability and limits of detection (LODs, 0.02 ng/mL for PD and 0.03 ng/mL for PT) were determined. The analytical performance of this method benefited from efficient integration of suitable sample pretreatment techniques and characteristic structure of rhodamine 6 G with four mass tags in derivatization reagents. The developed and validated method has been applied to the quantitation of PD and PT for the quality assessment of related foodstuffs and traditional Chinese medicinal materials.

Isotope Labeling-Assisted Evaluation of Hydrophilic and Hydrophobic Liquid Chromatograph-Mass Spectrometry for Metabolomics Profiling.

High throughput untargeted metabolomics usually relies on complementary liquid chromatography-mass spectrometry (LC-MS) methods to expand the coverage of diverse metabolites, but the integration of those methods is not fully characterized. We systematically investigated the performance of hydrophilic interaction liquid chromatography (HILIC)-MS and nanoflow reverse-phase liquid chromatography (nRPLC)-MS under 8 LC-MS settings, varying stationary phases (HILIC and C18), mobile phases (acidic and basic pH), and MS ionization modes (positive and negative). Whereas nRPLC-MS optimization was previously reported, we found in HILIC-MS (2.1 mm × 150 mm) that the optimal performance was achieved in a90 min gradient with 100 μL/min flow rate by loading metabolite extracts from 2 mg of cell/tissue samples. Since peak features were highly compromised by contaminants, we used stable isotope labeled yeast to enhance formula identification for comparing different LC-MS conditions. The 8 LC-MS settings enabled the detection of a total of 1050 formulas, among which 78%, 73%, and 62% formulas were recovered by the best combination of 4, 3, and 2 LC-MS settings, respectively. Moreover, these yeast samples were harvested in the presence or absence of nitrogen starvation, enabling quantitative comparisons of altered formulas and metabolite structures, followed by validation with selected synthetic metabolites. The results revealed that nitrogen starvation downregulated amino acid components but upregulated uridine-related metabolism. In summary, this study introduces a thorough evaluation of hydrophilicity and hydrophobicity based LC-MS and provides information for selecting complementary settings to balance throughput and efficiency during metabolomics experiments.

An improved method for fast and selective separation of carotenoids by LC-MS.

Carotenoids are a large class of compounds that are biosynthesized by condensation of isoprene units in plants, fungi, bacteria, and some animals. They are characteristically highly conjugated through double bonds, which lead to many isomers as well susceptibility to oxidation and other chemical modifications. Carotenoids are important because of their potent antioxidant activity and are the pigments responsible for color in a wide variety of foods. Human consumption is correlated to many health benefits including prevention of cancer, cardiovascular disease, and age-related disease. Extreme hydrophobicity, poor stability, and low concentration in biological samples make these compounds difficult to analyze and difficult to develop analytical methods for aimed towards identification and quantification. Examples in the literature frequently report the use of exotic stationary phases, solvents, and additives, such as ethyl acetate, dichloromethane, and methyl tert-butyl ether that are incompatible with liquid chromatography mass spectrometry (LC-MS). In order to address these issues, we implemented the use of LC-MS friendly conditions using a low-hydrophobicity cyano-propyl column (Agilent Zorbax SB-CN). We successfully differentiated between isomeric carotenoids by optimizing two gradient methods and using a mixture of 11 standards and LC-MS in positive ionization mode. Three complex biological samples from strawberry leaf, chicken feed supplement, and the photosynthetic bacterium Chloroflexus aurantiacus were analyzed and several carotenoids were resolved in these diverse backgrounds. Our results show this methodology is a significant improvement over other alternatives for analyzing carotenoids because of its ease of use, rapid analysis time, high selectivity, and, most importantly, its compatibility with typical LC-MS conditions.

Qualitative characterization of the rat liver mitochondrial lipidome using all ion fragmentation on an Exactive benchtop Orbitrap MS.

Untargeted lipidomics profiling by liquid chromatography-mass spectrometry (LC-MS) allows researchers to observe the occurrences of lipids in a biological sample without showing intentional bias to any specific class of lipids and allows retrospective reanalysis of data collected. Typically, and in the specific method described, a general extraction method followed by LC separation is used to achieve nonspecific class coverage of the lipidome prior to high-resolution accurate mass (HRAM) MS detection. Here we describe a workflow including the isolation of mitochondria from liver tissue, followed by mitochondrial lipid extraction and the LC-MS conditions used for data acquisition. We also highlight how, in this method, all-ion fragmentation can be used to identify species of lower abundances, often missed by data-dependent fragmentation techniques. Here we describe the isolation of mitochondria from liver tissue, followed by mitochondrial lipid extraction and the LC-MS conditions used for data acquisition.

Update on analytical methods for toxic pyrrolizidine alkaloids

Methods for the determination of toxic pyrrolizidine alkaloids in plants and foods are described with emphasis on the important aspects of sample extraction and clean-up and the now preferred determination by liquid chromatography-mass spectrometry. The efficiencies of different extraction solvents and methods are described, as are the methods of reduction of N-oxides. Appropriate liquid chromatography-mass spectrometry conditions are tabulated. This concise review is intended to guide analysts towards adopting a more unified and reliable approach to the analysis of these important toxins.

Development of generic liquid chromatography-mass spectrometry methods using experimental design

Standard approaches to development of liquid chromatography-mass spectrometry (LC-MS) methods, either ion-pairing or reversed-phase liquid chromatography, have been through trial and error or intentional variation of experimental factors. These approaches to method optimization fail to take into account interactions between experimental factors and therefore the results may not be optimal for the combination of experimental factors. Another approach to optimization is through the use of chemometrics. Chemometric approaches can be more efficient than trial and error or intentional variation because chemometrics make use of multivariate designs; experimental factors are varied simultaneously at the various levels. Therefore chemometrics can take into account interactions between factors. The goal of this study was to develop a generic ion-pair LC-MS method for the analysis of acidic compounds using a chemometric approach called design of experiments (DOE). Four acidic compounds which cover three classes of acidic functional groups: 1-naphthyl phosphate ( 1), 1-naphthalenesulfonic acid ( 2), 2-naphthalenesulfonic acid ( 3), and (1-naphthoxy)acetic acid ( 4) were used as model compounds to develop the generic method. This study illustrates that LC-MS conditions can be optimized efficiently with minimal amount of experimentation using a chemometric approach to experimental design.

What are we determining using gas chromatographic multiresidue methods: tralomethrin or deltamethrin?

The analytical behaviour of the relatively new pyrethroid insecticide tralomethrin has been evaluated by using gas chromatography (GC) with electron-capture and mass spectrometry (MS) detectors, and liquid chromatography (LC)–atmospheric pressure ionization mass spectrometry with electrospray interfacing. Under the GC conditions commonly used in pesticide residue analysis, it was found that tralomethrin is transformed into deltamethrin (in a reproducible way) in the injector port of the GC system. Results obtained in this work indicate that the GC multiresidue methodologies routinely applied in the analysis of pyrethroid pesticides in foods cannot distinguish between these two pesticides, and the chromatographic signal obtained at the retention time of deltamethrin/tralomethrin can be really quantified as either deltamethrin or tralomethrin, including when it is confirmed as deltamethrin by MS. Under the LC–MS conditions assessed in this work, deltamethrin and the two diasteroisomers of tralomethrin were well separated and identified.

Preliminary application of liquid chromatography–electrospray-ionization mass spectrometry to the detection of 5-methyltetrahydrofolic acid monoglutamate in human plasma

Liquid chromatography (LC) in direct combination with mass spectrometry (MS) has been shown to be a good analytical technique for the selective separation and detection of labile folate monoglutamates. Reversed-phase LC and electrospray-ionization MS conditions were developed and optimized for the separation and detection of 5-methyltetrahydrofolic acid, 5-formyl tetrahydrofolic acid, tetrahydrofolic acid, dihydrofolic acid and folic acid in aqueous samples. Representative and reproducible positive ion mass spectra were generated for each folate under mild MS conditions. The selective MS detection and identification of endogenous 5-methyltetrahydrofolic acid in human plasma was accomplished through the development of a straightforward C 18-based solid-phase extraction procedure. This procedure allows for the qualitative assessment of 5-methyltetrahydrofolic acid in plasma. Based upon an isotope-dilution internal standard calibration study with standards, the LC–MS limit of quantitation for 5M-THF was estimated to be 0.39 ng/ml.

Identification of tylosin in bovine muscle at the maximum residue limit level by liquid chromatography-mass spectrometry, using a particle beam interface.

A particle beam liquid chromatography-mass spectrometric method is presented as a confirmatory technique for analysis of tylosin residues in bovine muscle. After chloroform extraction and a diol solid-phase extraction clean-up, on-line liquid chromatography-mass spectrometry (LC-MS) of extracts is carried out on an RP-18 bonded silica column. The analyte is introduced into the ion source by a particle beam interface and identified by negative chemical ionization with selective ion monitoring. The tylosin molecular ion is obtained with this ionization mode. The response of the ion chromatogram peak areas is linear for the three levels of spiked muscle analysed (0.5, 1 and 2 maximum residue limit). Under these LC-MS conditions, other macrolide antibiotics such as spiramycin and erythromycin do not interfere with tylosin.