Matrix effects in ultra-high performance supercritical fluid chromatography-mass spectrometry analysis of vitamin E in plasma: The effect of sample preparation and data processing.

The approaches to matrix effects determination and reduction in ultra-high performance supercritical fluid chromatography with mass spectrometry detection have been evaluated in this study using different sample preparation methods and investigation of different calibration models. Five sample preparation methods, including protein precipitation, liquid-liquid extraction, supported liquid extraction, and solid phase extraction based on both "bind and elute" and "interferent removal" modes, were optimized with an emphasis on the matrix effects and recovery of 8 forms of vitamin E, including α-, ß-, γ-, and δ-tocopherols and tocotrienols, from plasma. The matrix effect evaluation included the use and comparison of external and internal calibration using three models, i.e., least square with no transformation and no weighting (1/x0), with 1/x2 weighting, and with logarithmic transformation. The calibration model with logarithmic transformation provided the lowest %-errors and the best fits. Moreover, the type of the calibration model significantly affected not only the fit of the data but also the matrix effects when evaluating them based on the comparison of calibration curve slopes. Indeed, based on the used calibration model, the matrix effects calculated from calibration slopes ranged from +92% to - 72% for α-tocopherol and from -77% to +19% in the case of δ-tocotrienol. Thus, it was crucial to calculate the matrix effect by Matuszewski's post-extraction approach at six concentration levels. Indeed, a strong concentration dependence was observed for all optimized sample preparation methods, even if the stable isotopically labelled internal standards (SIL-IS) were used for compensation. The significant differences between individual concentration levels and compounds were observed, even when the tested calibration range covered only one order of magnitude. In methods with wider calibration ranges, the inappropriate use of calibration slope comparison instead of the post-extraction addition approach could result in false negative results of matrix effects. In the selected example of vitamin E, solid-phase extraction was the least affected by matrix effects when used in interferent removal mode, but supported liquid extraction resulted in the highest recoveries. We showed that the calibration model, the use of a SIL-IS, and the analyte concentration level played a crucial role in the matrix effects. Moreover, the matrix effects can significantly differ for compounds with similar physicochemical properties and close retention times. Thus, in all bioanalytical applications, where different analytes are typically determined in one analytical run, it is necessary to carefully select the data processing in addition to the method for the sample preparation, SIL-IS, and chromatography.

A new stability-indicating HPLC-UV method for determination of amlodipine besylate and its impurities in drug substance.

A new fast stability-indication high performance liquid chromatography method was developed and validated for the determination of amlodipine besylate and its organic impurities in drug substance. The separation of amlodipine and its seven impurities was achieved on a core shell C18 column, 100 mm × 4.6 mm; 2.6 µm, within 15 min. The mobile phase comprised of 0.4% ammonium hydroxide in water and methanol delivered in a gradient mode; the method detection wavelength is 237 nm. The selected column is stable at high pH and provided a good peak shape for basic compounds. Amlodipine besylate was subject to acid, base, oxidative, thermal, and photolytic stress conditions. The degradation products were well resolved from the amlodipine peak and its impurities. Major degradants were analyzed by liquid chromatography coupled with single-quadrupole mass detector. Amlodipine peak was shown to be free of co-elution by mass spectral analysis in all stress conditions. The method was validated in terms of specificity, linearity, accuracy, precision, and robustness. The developed method could be applied for routine quality control analysis of amlodipine besylate drug substance.

A simple and robust LC-ESI single quadrupole MS-based method to analyze polyphenols in plant extracts using deep eutectic solvents.

Currently, the interest in polyphenols is increasing due to their significant properties in health. Polyphenols exist in a range of natural products, however their extraction as well as their characterization are important issues as they are mainly present in complex matrices. Therefore, sensitive and selective analytical methods based on liquid chromatography coupled to tandem mass spectrometry are essential. Nevertheless, access to such high-resolution techniques is quite rare. Thus, in this work we present a simple, selective and robust method based on a single-quadrupole (Q) MS technique) for the analysis of a wide range of polyphenols such as flavonoids, phenolic acids and anthocyanins. Specifically, we present:•A simple liquid chromatography electro-spray ionization (LC-ESI) single-quadrupole mass selective (MS) method for the analysis of 18 different polyphenols.•Application of the method to three plant-based extracts that are derived after green extraction methods.

Development and evaluation of a HILIC-MS method for the determination of amino acid and non-amino acid impurities in histidine.

Developing analytical methods to assure and control the quality of amino acids has long been a challenge for food ingredient, dietary supplement, and pharmaceutical industries due to the high polarity and the absence of chromophores in many amino acids; the situation worsens further by the lack of information of impurities that could potentially be introduced during the manufacturing processes. Herein we utilize a four-step strategy including impurity identification, method development, sample analysis, and targeted impurity detection and quantitation to demystify the impurity profiles of amino acids. The effectiveness of the approach is highlighted using histidine as an example. Analysis of histidine manufacturing and degradation processes led to the identification of 12 potential impurities of histidine, including amino acids (arginine, lysine, asparagine, aspartic acid, alanine, and glycine) and non-amino acid impurities (histamine, histidinol, 4-imidazoleacrylic acid, 4-imidazoleacetic acid, ß-imidazolelactic acid, and urea). A HILIC method using Poroshell 120 HILIC-Z column (2.1 × 100 mm, 2.7 µm) and a mobile phase system consisting of ammonium formate buffer at pH 3.2 in water and 0.1% formic acid in acetonitrile coupled with a single quadrupole mass spectrometer was developed for the detection and quantitation of the proposed impurities. Evaluation of 11 commercial histidine samples using the developed method revealed distinct impurity profiles, as a fingerprint for each sample; seven of the 12 proposed impurities were detected in histidine samples tested. The developed method was evaluated in terms of specificity, linearity, range, accuracy, precision, and sensitivity (LOQ: 2.5-60.6 ng/mL) for its suitability for compendial applications. Given the high degree of overlap between the proposed and the detected impurities, the approach could be utilized to strengthen USP standards for controlling the quality of histidine. Extension of the strategy to the analysis of other amino acids is currently underway.

Implementation of orbitrap mass spectrometry for improved GC-MS target analysis in lithium ion battery electrolytes.

The implementation of orbitrap mass spectrometry for target analysis of volatile species in aged lithium-ion batteries was performed in a case study on butyl carbonates. In comparison to previously applied single quadrupole-based methods, major improvements were obtained.•Sensitivity was improved by effectively background free extracted ion chromatograms of identified marker fragment ions.•Typical isobaric interferences of typical carbonate fragment ions e.g. caused by column bleeding were identified and false positive identification avoided.•Analysis of isotope labeled electrolytes was optimized regarding mass spectrometric data reliability with mass accuracies <0.5 ppm and mass resolutions >100,000.

Quantification of ustalic acid, a chemotaxonomic marker, in Tricholoma ustale using liquid chromatography-mass spectrometry.

The development of methods for the detection and qualification of toxic substances in mushrooms is a rapidly growing research area in forensic toxicology. This study aimed to determine liquid chromatographic and mass spectrometric conditions applicable to the analysis of ustalic acid (UA) in Tricholoma ustale. We used HPLC coupled with single quadrupole mass spectrometry (Q-MS) with electrospray ionization in its negative ion mode to analyze UA. We performed HPLC separations on a C18 reversed-phase column with gradient elution using mobile phases containing water, acetonitrile, and formic acid. The MS showed that UA formed the deprotonated molecular ion [M-H]- at m/z 337, which was sufficient for the quantitative analysis of the compound. The average recovery rates of UA from four edible mushrooms (Flammulina velutipes, Pleurotus eryngii, Lentinula edodes, and Grifola frondosa) to which 10.0 µg/g of UA was added were 108%, 104%, 108%, and 107%, respectively, and the relative standard deviation values ranged from 4.1 to 6.4%. Quantitative analysis of UA in three systematically collected individual mushrooms of T. ustale revealed 41.9-155.7 ppm in each dry material. We also explored the fragmentation behaviors of UA in triple quadrupole mass spectrometry and the proposed structures for the product ions. The data suggest that conventional Q-MS with authenticated UA would be able to identify this compound in T. ustale when used for the immediate inspection of incidences of poisoning. Confirmation of the presence of UA in T. ustale would ultimately allow for the chemotaxonomic discrimination of Tricholoma species.

Development and validation of a stability indicating HPLC method for organic impurities of erythromycin stearate tablets.

A rapid, sensitive, and accurate high-performance liquid chromatography (HPLC) method was developed and validated for the separation and analysis of organic impurities in erythromycin stearate tablets. The method separates Erythromycin, Erythromycin B, Erythromycin C and nine impurities (EP Impurity A, B, C, D, E, F, H, I and M). The chromatographic separation was achieved on a Waters XBridge C18 (100 mm × 4.6 mm, 3.5 µm) column. The mobile phase comprised of 0.4 % ammonium hydroxide in water and methanol delivered in a gradient mode. The compounds of interest were monitored at 215 nm. The stability-indicating capability of this method was evaluated by performing stress studies. Erythromycin was found to degrade significantly under acid, base, and oxidative stress conditions and it was only stable under thermal and photolytic degradation conditions. The degradation products were well resolved from the erythromycin peaks. In addition, the major degradants formed under stress conditions were characterized by ultra-high-performance liquid chromatography coupled with Single-Quadrupole Mass Spectrometer (UHPLC-QDa). The method was validated to fulfill International Conference on Harmonization (ICH) requirements and this validation included specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, and robustness. The developed method could be incorporated into the USP monograph and applied for routine quality control analysis of erythromycin stearate tablets.

An evaluation of instrument types for mass spectrometry-based multi-attribute analysis of biotherapeutics.

Multi-attribute methods (MAM), based on proteolytic digestion followed by liquid chromatography-mass spectrometry analysis of proteolytic peptides, have gained substantial attention in the biopharmaceutical industry for quantifying a variety of quality attributes for therapeutic proteins. Most MAM developed so far have been based on high-resolution mass spectrometers, due to their superb resolving power to distinguish analyte signals from interferences. Lower-resolution instruments, if demonstrated suitable, may further promote the adoption of the technology due to their low cost, small footprint, and ease of use. In this work, we compared the performance of a high-resolution instrument with a few low-resolution quadrupole-type instruments in quantifying a diverse set of quality attributes in a monoclonal antibody product. Different modes of operation for the quadrupole instruments, including scan mode, selected-ion monitoring and multiple-reaction monitoring, were evaluated. The high-resolution instrument has superb performance, with a quantitation limit of 0.002%. Single-quadrupole instruments in scan mode, on the other hand, provide a quantitation limit of about 1%, which may be fit-for-purpose for many routine MAM applications.

A simple and robust LC-ESI single quadrupole MS-based method to analyze neonicotinoids in honey bee extracts.

Over the past years, neonicotinoids such as thiacloprid and flupyradifurone have gained considerable scientific and public interest. These molecules used as active compounds in pesticides are known due to cause drastic negative long-time effects on pollinators and even human health. Therefore, determining trace amounts of neonicotinoid in different environmental matrices by liquid chromatography coupled with mass selective detectors (LC-MS/MS or LC-Q-TOF/MS) has become an important methodology. However, not every scientific group has unlimited access to high-resolution mass-selective detectors (e.g., MS/MS). It becomes more apparent that the analytics of neonicotinoids are already a global issue. Research groups and organizations with a limited financial budget often depend on using cheap and robust equipment to do their analytical work. We demonstrate a single-quadrupole (Q) MS-based method with single-class residue methods (SRMs) for the analysis of neonicotinoids, applicable without the requirement of a high-end MS system. For an adequate sample clean-up strategy, QuEChERS (Quick, Easy, Cheap, Efficient, Rugged, safe) extraction and purification methods were modified and applied to eliminate residual matrix after honey bee extraction steps to analyze thiacloprid and flupyradifurone. •Simple liquid chromatography electro-spray ionization (LC-ESI) single-quadrupole mass selective (MS) method for neonicotinoid analysis.•Efficient sample pretreatment by a modified QuEChERS extraction and purification method.•Limit of detection (LOD) and limit of quantification (LOQ) for thiacloprid was 19.72 ng g-1 and 7.61 ng g-1, for flupyradifurone 65.73 ng g-1 and 25.36 ng g-1, respectively.

Simultaneous determination of amino acids in different teas using supercritical fluid chromatography coupled with single quadrupole mass spectrometry.

Tea is a widely consumed beverage and has many important physiological properties and potential health benefits. In this study, a novel method based on supercritical fluid chromatography coupled with mass spectrometry (SFC-MS) was developed to simultaneously determine 11 amino acids in different types of tea (green teas, Oolong tea, black tea and Pu-erh tea). The separation conditions for the analysis of the selected amino acids including the column type, temperature and backpressure as well as the type of additive, were carefully optimized. The best separation of the 11 amino acids was obtained by adding water (5%, v/v) and trifluoroacetic acid (0.4%, v/v) to the organic modifier (methanol). Finally, the developed SFC-MS method was fully validated and successfully applied to the determination of these amino acids in six different tea samples. Good linearity (r ≥ 0.993), precision (RSDs ≤ 2.99%), accuracy (91.95%-107.09%) as well as good sample stability were observed. The limits of detection ranged from 1.42 to 14.69 ng/mL, while the limits of quantification were between 4.53 and 47.0 ng/mL. The results indicate that the contents of the 11 amino acids in the six different tea samples are greatly influenced by the degree of fermentation. The proposed SFC-MS method shows a great potential for further investigation of tea varieties.

Determination of Humectants in Tobacco Filler by High Performance Chromatography/Single Quadrupole Mass Spectrometry.

Glycerol, and 1,2-propylene glycol are the humectants most commonly used by the tobacco industry. They are found in a variety of tobacco products and are often present at high levels (~2-5 % w/w). While humectants are generally considered safe, they may serve as precursors in the formation of harmful carbonyl compounds. A selective, precise, and sensitive method for the quantification of several humectants in cigarette filler was developed. The method's sample clean-up is a two-step process consisting of a mechanical extraction, followed by solid phase extraction. Individual humectants are separated, identified, and measured using liquid chromatography coupled to a single quadrupole mass spectrometer as the detector (LC/MS). Detection limits were 0.105, 0.575, and 0.039 mg/cigarette for glycerol, 1,2-propylene glycol and triethylene glycol, respectively. The quantification range for these analytes was 0.4-75.0 mg/cigarette. Twenty-seven brands of domestic commercial cigarettes were evaluated to assess typical levels of humectants in the tobacco filler.

La glycèrine et le 1,2-propylène glycol sont les humectants les plus communément utilisés par l'industrie du tabac. Ils entrent dans la composition de divers produits de tabac oùils sont souvent présents dans des quantités élevées (~2­5% p/p). Alors que les humectants sont, en règle générale, considérés comme sûrs, ils peuvent servir de précurseurs dans la formation de composés carbonylés nocifs. Une méthode sélective, précise et sensible fut mise au point afin de quantifier divers humectants parmi les composants de remplissage des cigarettes. Cette méthode repose sur une épuration de l'échantillon en deux étapes, à savoir une extraction mécanique suivie d'une extraction en phase solide. Les humectants individuels sont séparés, identifiés et mesurés par le biais, en guise de détecteur, d'une chromatographie en phase liquide couplée à une unique spectrométrie de masse quadripolaire (LC/MS). Les seuils de détection s'élevèrent respectivement à 0,105, 0,575 et 0,039 mg/cigarette pour la glycérine, le 1,2 propylèneglycol et le triéthylèneglycol. La plage de quantification de ces analytes fut de 0,4­75,0 mg/cigarette. Vingt-sept marques de cigarettes commercialisées dans le pays furent testées afin d'évaluer les quantités types d'humectants dans le tabac de remplissage.

Use of microextraction by packed sorbent directly coupled to an electron ionization single quadrupole mass spectrometer as an alternative for non-separative determinations.

Sometimes it is not necessary to separate the individual compounds of a sample to resolve an analytical problem, it is enough to obtain a signal profile of the sample formed by all the components integrating it. Within this strategy, electronic noses based on the direct coupling of a headspace sampler with a mass spectrometer (HS-MS) have been proposed. Nevertheless, this coupling is not suitable for the analysis of non-volatile compounds. In order to propose an alternative to HS-MS determinations for non-volatile compounds, here we present the first 'proof of concept' use of the direct coupling of microextraction by packed sorbents (MEPS) to a mass spectrometer device using an electron ionization (EI) and a single quadrupole as ionization source and analyzer, respectively. As target compounds, a set of analytes with different physic-chemical properties were evaluated (2-ethyl-1-hexanol, styrene, 2-heptanone, among others). The use of MEPS extraction present many advantages, such as it is fast, simple, easy to automate and requires small volumes of sample and organic solvents. Moreover, MEPS cartridges are re-usable as samples can be extracted more than 100 times using the same syringe. In order to introduce into the system all the elution volume from the MEPS extraction, a programmable temperature vaporizer (PTV) is proposed as the injector device. Results obtained with the proposed methodology (MEPS-PTV/MS) were compared with the ones obtained based on the separative scheme, i.e. using gas chromatography separation (MEPS-PTV-GC/MS), and both methods provided similar results. Limits of detection were found to be between 3.26 and 146.6µgL-1 in the non-separative scheme and between 0.02 and 1.72µgL-1 when the separative methodology was used. Repeatability and reproducibility were evaluated with values below 17% in all cases.

Analysis of glyphosate and aminomethylphosphonic acid in leaves from Coffea arabica using high performance liquid chromatography with quadrupole mass spectrometry detection.

Glyphosate is a commonly applied herbicide in coffee plantations. Because of its non-selective mode of action it can damage the crop exposed through spray drift. Therefore, it is of interest to study glyphosate fate in coffee plants. The aim of this study was to develop an analytical method for accurate and precise quantification of glyphosate and its main metabolite aminomethylphosphonic acid (AMPA) at trace levels in coffee leaves using liquid chromatography with single-quadrupole mass spectrometry detection. The method is based on a two-step solid phase extraction (SPE) with an intermediate derivatization reaction using 9-fluorenylmethylchloroformate (FMOC). An isotope dilution method was used to account for matrix effects and to enhance the confidence in analyte identification. The limit of quantification (LOQ) for glyphosate and AMPA in coffee leaves was 41 and 111 µg kg(-1) dry weight, respectively. For the method optimization a design of experiments (DOE) approach was used. The sample clean-up procedure can be simplified for the analysis of less challenging matrices, for laboratories having a tandem mass spectrometry detector and for cases in which quantification limits above 0.1 mg kg(-1) are acceptable, which is often the case for glyphosate. The method is robust, possesses high identification confidence, while being suitable for most commercial and academic laboratories. All leaf samples from five coffee fields analyzed (n=21) contained glyphosate, while AMPA was absent. The simplified clean-up procedure was successfully validated for coffee leaves, rice, black beans and river water.

Comparison of liquid chromatography and supercritical fluid chromatography coupled to compact single quadrupole mass spectrometer for targeted in vitro metabolism assay.

The goal of this study was to evaluate the combination of powerful chromatographic methods and compact single quadrupole MS device for simple in vitro cytochrome P450 (CYP) inhibition assay, instead of more expensive triple quadrupole MS/MS detectors. For this purpose, two modern chromatographic approaches (ultra-high pressure liquid chromatography (UHPLC) and ultra-high performance supercritical fluid chromatography (UHPSFC)) were tested in combination with simple MS detector. To show the applicability for an in vitro CYP-mediated metabolism assay using the cocktail approach, a method was first developed in UHPLC-MS to separate a mixture of 8 probe substrates and 8 CYP-specific metabolites. A screening procedure was initially applied to determine the best combination of a column, an organic modifier and a mobile-phase pH, followed by fine tuning of the conditions (i.e., gradient profile, temperature and pH) using HPLC modelling software. A similar sequential method development procedure was also evaluated for UHPSFC-MS. For method development, where peak tracking is necessary, the use of single quadrupole MS was found to be extremely valuable for following the investigated analytes. Ultimately, a baseline separation of the 16 compounds was achieved in both UHPLC-MS and UHPSFC-MS with an analysis time of less than 7 min. In a second series of experiments, sensitivity was evaluated, and LOQ values were between 2 and 100 ng/mL in UHPLC-MS, while they ranged from 2 to 200 ng/mL in UHPSFC-MS. Based on the concentrations employed for the current in vitro phase I metabolism assay, these LOQ values were appropriate for this type of application. Finally, the two analytical methods were applied to in vitro CYP-dependent metabolism testing. Two well-known phytochemical inhibitors, yohimbine and resveratrol, were investigated, and reliable conclusions were drawn from these experiments with both UHPLC-MS and UHPSFC-MS. At the end, the proposed strategy of optimized chromatography combined with simple MS device has been shown to potentially compete with the widely used combination of generic chromatography and highly selective MS/MS device for simple in vitro CYP inhibition assays. In addition, our analytical method may be easier to use in a routine environment; the instrument cost is significantly reduced and the two developed methods fit for purpose.

Evaluation of volatile metabolites as markers in Lycopersicon esculentum L. cultivars discrimination by multivariate analysis of headspace solid phase microextraction and mass spectrometry data.

To gain insights on the effects of cultivar on the volatile metabolomic expression of different tomato (Lycopersicon esculentum L.) cultivars--Plum, Campari, Grape, Cherry and Regional, cultivated under similar edafoclimatic conditions, and to identify the most discriminate volatile marker metabolites related to the cultivar, the chromatographic profiles resulting from headspace solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-qMS) analysis, combined with multivariate analysis were investigated. The data set composed by the 77 volatile metabolites identified in the target tomato cultivars, 5 of which (2,2,6-trimethylcyclohexanone, 2-methyl-6-methyleneoctan-2-ol, 4-octadecyl-morpholine, (Z)-methyl-3-hexenoate and 3-octanone) are reported for the first time in tomato volatile metabolomic composition, was evaluated by chemometrics. Firstly, principal component analysis was carried out in order to visualise data trends and clusters, and then, linear discriminant analysis in order to detect the set of volatile metabolites able to differentiate groups according to tomato cultivars. The results obtained revealed a perfect discrimination between the different Lycopersicon esculentum L. cultivars considered. The assignment success rate was 100% in classification and 80% in prediction ability by using "leave-one-out" cross-validation procedure. The volatile profile was able to differentiate all five cultivars and revealed complex interactions between them including the participation in the same biosynthetic pathway. The volatile metabolomic platform for tomato samples obtained by HS-SPME/GC-qMS here described, and the interrelationship detected among the volatile metabolites can be used as a roadmap for biotechnological applications, namely to improve tomato aroma and their acceptance in the final consumer, and for traceability studies.

Analysis of free amino acids in flue-cured tobacco leaves using ultra-high performance liquid chromatography with single quadrupole mass spectrometry.

Amino acids are one of the most important metabolites of organisms. They play an important role in plant growth, development, and product quality. A method based on RP ultra-performance LC with single quadrupole MS and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate precolumn derivatization was developed for the analysis of free amino acids in flue-cured tobacco leaves. Unlike the corresponding UV detection method, this method avoids matrix interference of complicated tobacco components, and the quantitative accuracy and resolution were improved. Twenty free amino acids were detected in flue-cured tobacco leaves. The method showed a good linearity with correlation coefficients of 0.9966-0.9998. The LODs for derivatized amino acids were 0.2-9.7 fmol/µL. Good repeatability with an RSD of 2.5-8.6% and satisfactory intra- and interday precisions were obtained. The developed method was used to investigate free amino acids in flue-cured tobacco leaves in China. The effects of aroma type, variety, and growing regions on free amino acids were investigated. The results showed that free amino acids in tobacco were affected by growing regions and varieties.