bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2023‒10‒15
twenty-six papers selected by
Sofia Costa, Matterworks

  1. Anal Chim Acta. 2023 Oct 23. pii: S0003-2670(23)01012-7. [Epub ahead of print]1279 341791
      Metabolomics is the study of small molecules, primarily metabolites, that are produced during metabolic processes. Analysis of the composition of an organism's metabolome can yield useful information about an individual's health status at any given time. In recent years, the development of large-scale, targeted metabolomic methods has allowed for the analysis of biological samples using analytical techniques such as LC-MS/MS. This paper presents a large-scale metabolomics method for analysis of biological samples, with a focus on quantification of metabolites found in blood plasma. The method comprises a 10-min chromatographic separation using HILIC and RP stationary phases combined with positive and negative electrospray ionization in order to maximize metabolome coverage. Complete analysis of a single sample can be achieved in as little as 40 min using the two columns and dual modes of ionization. With 540 metabolites and the inclusion of over 200 analytical standards, this method is comprehensive and quantitatively robust when compared to current targeted metabolomics methods. This study uses a large-scale evaluation of metabolite recovery from plasma that enables absolute quantification of metabolites by correcting for analyte loss throughout processes such as extraction, handling, or storage. In addition, the method was applied to plasma collected from adjuvant breast cancer patients to confirm the suitability of the method to clinical samples.
    Keywords:  Chromatography; Mass spectrometry; Targeted metabolomics
  2. Methods Enzymol. 2023 ;pii: S0076-6879(23)00135-0. [Epub ahead of print]689 433-452
      Stable isotope dilution (SID) methodology coupled with liquid chromatography-tandem mass spectrometry (LC-MS) is rapidly becoming the gold standard for measuring estrogens in serum and plasma due to improved specificity, high accuracy, and the ability to conduct a more comprehensive analysis. A general consideration of the problems associated with measuring estrogens and two detailed derivatization methods are described in this chapter. These methods quantify estrogens and their metabolites in serum and plasma samples using this state-of-art technology, which is applicable to the routine clinical laboratory.
    Keywords:  Estradiol; Estrone; Liquid chromatography; Mass spectrometry; Multiple reaction monitoring
  3. Methods Enzymol. 2023 ;pii: S0076-6879(23)00133-7. [Epub ahead of print]689 355-376
      The quantitation of androgens is necessary to diagnose and monitor the development of diseases such as prostate cancer and polycystic ovary syndrome. Androgen measurements also support the laboratory-based study of androgen metabolism in cellular and animal models. The methods described in this chapter combine chemical derivatization of hydroxy- and keto-androgens with stable isotope dilution liquid chromatography mass spectrometry (SID-LC-MS). Chemical derivatization of hydroxy-androgens by picolinic acid and keto-androgens by Girard P enhances the ionization and detection sensitivity of androgens, while chromatographic separation and [13C]-labeled internal standards add specificity that allow for simultaneous quantitation of multiple androgens. This chapter describes the materials and protocols necessary for chemical derivatization, enzymatic synthesis of internal standards, and LC-MS detection of keto- and hydroxy-androgens.
    Keywords:  Girard P derivatization; Hydroxy-androgens; Keto-androgens; Liquid chromatography mass spectrometry; Picolinic derivatization
  4. Int J Mol Sci. 2023 Sep 28. pii: 14691. [Epub ahead of print]24(19):
      In recent years, oligonucleotides have become more important in research, drug approvals and medical therapies. Due to this growing interest in pharmaceutical applications, it is essential to develop reliable analytical methods for this substance class. In this work, we present a quantification method using liquid chromatography coupled with tandem mass spectrometry by applying an isobaric oligonucleotide standard. In addition to a proof of principle, we perform a method qualification to assess its readiness for validation according to ICH Q2 guidelines. In addition to good linearity, sensitivity, accuracy and recovery, the method showed no significant matrix effects. Furthermore, we demonstrated the application of the method by applying the quantification in a biological matrix, as well as an exemplary degradation of an oligonucleotide in bovine plasma.
    Keywords:  HPLC-MS/MS; bioanalytic; biological matrix; fragmentation reaction; internal standard; isobaric standard; method validation; multiple reaction monitoring; oligonucleotides
  5. J Lipid Res. 2023 Oct 06. pii: S0022-2275(23)00126-8. [Epub ahead of print] 100453
      Metabolic changes in adrenocortical steroids and medullary catecholamines characterize adrenal tumors, but they are measured using different analytical protocols. To increase bioanalytical validity while maintaining sample homogeneity, liquid chromatography-mass spectrometry (LC-MS)-based profiling of 29 cortical steroids and 6 medullary amines, including catecholamines and metanephrines, in a single run was developed. Alkyloxycarbonylation with isobutyl chloroformate was employed together with our comprehensive steroid assay, and all adrenal hormones were separated on a reversed-phase C18 column (50 × 2.1 mm, 1.9 μm) at a flow rate of 0.3 mL/min. The lower limits of quantification for all analytes ranged from 0.1 to 2.0 ng/mL, with extraction recoveries of 58.5-109.5%, while the imprecision and accuracy were 1.6-14.8% and 89.2-114.9%, respectively. The validated LC-MS assay was applied to serum samples obtained from 60 patients with adrenal Cushing syndrome (CS), primary aldosteronism (PA), and pheochromocytoma/paraganglioma (PPGL). In addition to the characteristic metabolic changes in glucocorticoids, mineralocorticoids, catecholamines, and metanephrine, the molecular ratios of dehydroepiandrosterone sulfate and 20α-dihydrocortisol indicated CS and PA (p < 0.01 for all compounds), respectively. Moreover, the interactive molecular ratios of 11-deoxycortisol with normetanephrine, metanephrine, norepinephrine, and epinephrine (p < 0.01 all compounds) were proposed to characterize the metabolic features of PPGL. Novel LC-MS-based quantitative profiling of steroids, catecholamines, and metanephrines in human serum was successfully established and characterized metabolic features of individual adrenal tumors that could be used for clinical purposes.
    Keywords:  adrenal cortex; adrenal medullar; adrenal tumor; chemical derivatization; mass spectrometry
  6. Anal Chim Acta. 2023 Oct 23. pii: S0003-2670(23)01051-6. [Epub ahead of print]1279 341830
      Nanospray desorption electrospray ionization (nano-DESI) is an ambient ionization technique that enables molecular imaging of biological samples with high spatial resolution. We have recently developed an integrated microfluidic probe (iMFP) for nano-DESI mass spectrometry imaging (MSI) that significantly enhances the robustness of the technique. In this study, we designed a new probe that enables imaging of biological samples with high spatial resolution. The new probe design features smaller primary and spray channels and an entirely new configuration of the sampling port that enables robust imaging of tissues with a spatial resolution of 8-10 μm. We demonstrate the spatial resolution, sensitivity, durability, and throughput of the iMFP by imaging mouse uterine and brain tissue sections. The robustness of the high-resolution iMFP allowed us to perform first imaging experiments with both high spatial resolution and high throughput, which is particularly advantageous for high-resolution imaging of large tissue sections of interest to most MSI applications. Overall, the new probe design opens opportunities for mapping of biomolecules in biological samples with high throughput and cellular resolution, which is important for understanding biological systems.
  7. J Chromatogr A. 2023 Sep 30. pii: S0021-9673(23)00638-6. [Epub ahead of print]1710 464413
      Steroid hormones have been reported to be associated with endocrine system diseases. This paper proposes a novel procedure of deep eutectic solvent (DES)-assisted liquid-liquid extraction (LLE) to extract six steroid hormones (including cortisone, cortisol, androstenedione, testosterone, 17-hydroxyprogesterone, and progesterone) from serum coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of five types of L-proline, choline chloride, and citric acid-based DESs were tailored; the DES from L-proline and ethylene glycol at a molar ratio of 1:4 with 20 % acetonitrile was selected as the best-fit assisted solvent for the six steroid hormones compared with other DESs. The parameters for extraction by selected DES were optimized using Box-Behnken design (BBD), and the optimal extraction conditions are 200 µL of acetonitrile, 100 µL of the sample, and 80 µL of DES. Under optimum conditions, the method has good linear calibration ranges (between 0.07 ng mL-1 and 600 ng mL-1), correlation coefficients of determination (r2>0.99), and low limits of quantification (between 0.02 and 0.60 ng mL-1). The extraction recoveries were in the range of 81.84-114.43 %, and the intra-day and inter-day relative standard deviations (RSDs) were less than 10 %.In general, the DES-LC-MS/MS method is a simple and environmentally-friendly method, which can be complementary to the presently available methods for determining steroid hormones in serum.
    Keywords:  Deep eutectic solvent; Liquid chromatography-tandem mass spectrometry; Liquid-liquid extraction; Steroid hormones
  8. Analyst. 2023 Oct 11.
      Secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS) is an innovative analytical technique for the rapid and non-invasive analysis of volatile organic compounds (VOCs). However, compound annotation and ion suppression in the SESI source has hindered feature detection, stability and reproducibility of SESI-HRMS in untargeted volatilomics. To address this, we have developed and optimized a novel pseudo-targeted approach, database-assisted globally optimized targeted (dGOT)-SESI-HRMS using the microbial-VOC (mVOC) database, and spectral stitching methods to enhance metabolite detection in headspace of anaerobic bacterial cultures. Headspace volatiles from representative bacteria strains were assessed using full scan with data dependent acquisition (DDA), conventional globally optimized targeted (GOT) method, and spectral stitching supported dGOT experiments based on a MS peaks list derived from mVOC. Our results indicate that spectral stitching supported dGOT-SESI-HRMS can proportionally fragment peaks with respect to different analysis windows, with a total of 109 VOCs fragmented from 306 targeted compounds. Of the collected spectra, 88 features were confirmed as culture derived volatiles with respect to media blanks. Annotation was also achieved with a total of 25 unique volatiles referenced to standard databases allowing for biological interpretation. Principal component analysis (PCA) summarizing the headspace volatile demonstrated improved separation of clusters when data was acquired using the dGOT method. Collectively, our dGOT-SESI-HRMS method afforded robust capability of capturing unique VOC profiles from different bacterial strains and culture conditions when compared to conventional GOT and DDA modes, suggesting the newly developed approach can serve as a more reliable analytical method for the sensitive monitoring of gut microbial metabolism.
  9. Plant Methods. 2023 Oct 13. 19(1): 107
      The field of plant hormonomics focuses on the qualitative and quantitative analysis of the hormone complement in plant samples, akin to other omics sciences. Plant hormones, alongside primary and secondary metabolites, govern vital processes throughout a plant's lifecycle. While active hormones have received significant attention, studying all related compounds provides valuable insights into internal processes. Conventional single-class plant hormone analysis employs thorough sample purification, short analysis and triple quadrupole tandem mass spectrometry. Conversely, comprehensive hormonomics analysis necessitates minimal purification, robust and efficient separation and better-performing mass spectrometry instruments. This review summarizes the current status of plant hormone analysis methods, focusing on sample preparation, advances in chromatographic separation and mass spectrometric detection, including a discussion on internal standard selection and the potential of derivatization. Moreover, current approaches for assessing the spatiotemporal distribution are evaluated. The review touches on the legitimacy of the term plant hormonomics by exploring the current status of methods and outlining possible future trends.
    Keywords:  Hormonomics; Internal standard; Liquid chromatography; Mass spectrometry; Matrix effect; Metabolomics; Omics; Plant hormone; Solid phase extraction
  10. Rapid Commun Mass Spectrom. 2023 Nov 30. 37(22): e9638
      RATIONALE: Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) utilizes a 2970 nm mid-IR laser to desorb samples with depth resolutions (Z) on the order of micrometers. Conventionally, 5-20 μm thick tissue sections are used to characterize different applications of the IR-MALDESI source, but an optimal thickness has not been systematically investigated.METHODS: Mouse liver was sectioned to various thicknesses and analyzed using IR-MALDESI mass spectrometry imaging (MSI). Height profiles of tissue sections of various cryosectioned thicknesses were acquired to affirm tissue thickness. Tissue sections of each thickness were measured using a Keyence microscope. Paraffin wax was cryosectioned, mounted on microscope slides, and measured using a chromatic confocal sensor system to determine the cryostat sectioning accuracy.
    RESULTS: Analyzing sectioned tissues at higher thickness (>10 μm) leads to lower ion abundance, a decrease in signal over long analysis times, and more frequent instrument cleaning. Additionally, increasing tissue thickness above the optimum (7 μm) does not result in a significant increase in lipid annotations.
    CONCLUSIONS: This work defines an optimal sample thickness for IR-MALDESI-MSI and demonstrates the utility of optimizing tissue thickness for MSI platforms of comparable Z resolution.
  11. J Chromatogr A. 2023 Sep 30. pii: S0021-9673(23)00641-6. [Epub ahead of print]1710 464416
      Contamination of active pharmaceutical ingredients (APIs) and pharmaceutical preparations with carcinogenic N-nitrosamines has led to recalls of these products and supply shortages to patients. The present study describes the development of a highly sensitive method for simultaneous analysis of seven N-nitrosamines using on-line in-tube solid-phase microextraction (IT-SPME) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine their actual contamination in metformin tablets. Using a Carboxen 1006 PLOT capillary as the extraction device for IT-SPME, these compounds were efficiently extracted and concentrated 6‒24-fold by subjecting 40 µL of sample to 25 repeated draw/eject cycles at a rate of 0.2 mL/min. The seven N-nitrosamines were separated within 11 min by gradient elution with 0.1 % formic acid solution and acetonitrile as the mobile phase using a CAPCELL PAK C18 MGII column and detected by multiple reaction monitoring in positive ion mode. The calibration curve showed linearity in the range 0.2‒50 ng/mL and detection limits (S/N = 3) in the range 3‒112 pg/mL. The intra-day and inter-day precisions were less than 5.5 % and 7.0 % (n = 6), respectively, with accuracies ranging from 93‒117 %. Following ultrasonic extraction with water, centrifugation and filtration of the supernatant liquid through a membrane filter, the N-nitrosamine impurities in metformin tablets could be analyzed by IT-SPME/LC‒MS/MS. Their limits of quantification (S/N = 10) were 0.1‒5.1 pg/mg API and recoveries ranged from 87‒102 %. Analysis of eight metformin tablets from eight manufacturers showed that 5.8‒7.5 pg/mg N-nitrosodimethylamine were present in three tablets, with no other N-nitrosamines detected in any of the eight tablets. This method may be useful in testing for N-nitrosamine impurities in pharmaceutical preparations.
    Keywords:  In-tube solid-phase microextraction; Liquid chromatography–tandem mass spectrometry; Metformin; N-nitrosamines; Pharmaceutical impurities
  12. Anal Chim Acta. 2023 Oct 23. pii: S0003-2670(23)01069-3. [Epub ahead of print]1279 341848
      BACKGROUND: Recent increase in public acceptance of cannabis as a natural medical alternative for certain neurological pathologies has led to its approval in different regions of the world. However, due to its previous illegal background, little research has been conducted around its biochemical insights. Therefore, in the current framework, metabolomics may be a suitable approach for deepening the knowledge around this plant species. Nevertheless, experimental methods in metabolomics must be carefully handled, as slight modifications can lead to metabolomic coverage loss. Hence, the main objective of this work was to optimise an analytical method for appropriate untargeted metabolomic screening of cannabis.RESULTS: We present an empirically optimised experimental procedure through which the broadest metabolomic coverage was obtained, in which extraction solvents for metabolite isolation, chromatographic columns for LC-qOrbitrap analysis and plant-representative biological tissues were compared. By exploratory means, it was determined that the solvent combination composed of CHCl3:H2O:CH3OH (2:1:1, v/v) provided the highest number of features from diverse chemical classes, as it was a two-phase extractant. In addition, a reverse phase 2.6 μm C18 100 Å (150 × 3 mm) chromatographic column was determined as the appropriate choice for adequate separation and further detection of the diverse metabolite classes. Apart from that, overall chromatographic peak quality provided by each column was observed and the need for batch correction methods through quality control (QC) samples was confirmed. At last, leaf and flower tissues resulted to provide complementary metabolic information of the plant, to the detriment of stem tissue, which resulted to be negligible.
    SIGNIFICANCE: It was concluded that the optimised experimental procedure could significantly ease the path for future research works related to cannabis metabolomics by LC-HRMS means, as the work was based on previous plant metabolomics literature. Furthermore, it is crucial to highlight that an optimal analytical method can vary depending on the main objective of the research, as changes in the experimental factors can lead to different outcomes, regardless of whether the results are better or worse.
    Keywords:  Cannabis sativa L.; Data mining; Experimental factors; LC-HRMS; Metabolomic coverage; Plant metabolomics
  13. Anal Chim Acta. 2023 Oct 23. pii: S0003-2670(23)00961-3. [Epub ahead of print]1279 341740
      The chemical exposome consists of environmental exposures experienced throughout a lifetime but to date analytical approaches to investigate the plethora of low-abundance chemicals remain very limited. Liquid chromatography high-resolution mass spectrometry (HRMS) is commonly applied in untargeted exposome-wide analyses of xenobiotics in biological samples; however, human biomonitoring approaches usually utilize targeted low-resolution triple quadrupole (QQQ) mass spectrometry tailored to a small number of chemicals. HRMS can cover a broader chemical space but the detection of molecules from low-level exposure amidst a background of highly-abundant endogenous molecules has proven to be difficult. In this study, a triple quadrupole (QQQ) and a high-resolution mass spectrometer (HRMS) with identical chromatography were utilized to determine the limits of quantitation (LOQ) of >100 xenobiotics and estrogenic hormones in pure solvent and human urine. Both instrumental platforms are currently applied in exposure assessment studies and were operated in their most frequently used acquisition mode (full scan for HRMS and multiple reaction monitoring for QQQ) to mimic typical applications. For HRMS analyses, the median LOQ was 0.9 and 1.2 ng/mL in solvent and urine, respectively, while for low-resolution QQQ measurements, the median LOQ was 0.1 and 0.2 ng/mL in solvent and urine, respectively. To evaluate the calculated LOQs in complex biological samples, spot urine samples from 24 Nigerian female volunteers were investigated. The higher LOQ values for HRMS resulted in less quantified low-abundance analytes and decreased the number of compounds detected below the LOQ. Even at chronic low-dose exposure, such compounds might be relevant for human health because of high individual toxicity or potential mixture effects. Nevertheless, HRMS enabled the additional screening for exposure to unexpected/unknown analytes, including emerging compounds and biotransformation products. Therefore, a synergy between high- and low-resolution mass spectrometry may currently be the best option to elucidate and quantify xenobiotics in comprehensive exposome-wide association studies (ExWAS).
  14. J Am Soc Mass Spectrom. 2023 Oct 09.
      Several analytical challenges make it difficult to accurately measure coenzyme A (CoA) metaboforms, including insufficient stability and a lack of available metabolite standards. Consequently, our understanding of CoA biology and the modulation of human diseases may be nascent. CoA's serve as lipid precursors, energy intermediates, and mediators of post-translational modifications of proteins. Here, we present a liquid chromatography-mass spectrometry (LC-MS) approach to measure malonyl-CoA, acetyl-CoA, and succinyl-CoA in complex biological samples. Additionally, we evaluated workflows to increase sample stability. We used reference standards to optimize CoA assay sensitivity and test CoA metabolite stability as a function of the reconstitution solvent. We show that using glass instead of plastic sample vials decreases CoA signal loss and improves the sample stability. We identify additives that improve CoA stability and facilitate accurate analysis of CoA species across large sample sets. We apply our optimized workflow to biological samples of skeletal muscle cells cultured under hypoxic and normoxia conditions. Together, our workflow improves the detection and identification of CoA species through targeted analysis in complex biological samples.
  15. J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Sep 26. pii: S1570-0232(23)00301-X. [Epub ahead of print]1229 123891
      Tattoos have been gaining popularity in recent years, leading to a growing interest in researching tattoo inks and the tattooing process itself. Since the exposure to soluble tattoo ink ingredients has not yet been investigated, we here present the method validation for a short-term biokinetics study on soluble tattoo ink ingredients. The three tracers 4-aminobenzoic acid (PABA), 2-phenoxyethanol (PEtOH) and iodine will be added to commercially available tattoo inks, which will subsequently be used on healthy study participants. Following the tattooing process, blood and urine will be sampled at specific time points and analysed for these tracers. For this purpose, a method using liquid chromatography separation coupled to a quadrupole time-of-flight mass spectrometer (LC-QTOF-MS) in positive and negative ESI mode for the quantification of PABA, PEtOH and selected metabolites and an inductively-coupled plasma (ICP)-MS method for the determination of iodine were developed and validated. For LC-QTOF-MS analysis, the most applicable additives for LC eluents (0.01 % formic acid for positive and 0.005 % acetic acid for negative mode) were identified. Protein precipitation with acetonitrile was chosen for sample preparation. The methods were validated for selectivity, specificity, carryover, linearity, limit of detection (LOD) and quantification (LOQ), matrix effects, accuracy and precision, stability under different conditions and dilution integrity according to national and international guidelines with an allowed maximum variation of ±15 %. The LC-QTOF-MS method met the imposed guideline criteria for most parameters, however, some metabolites showed strong matrix effects. Validation of the ICP-MS method revealed that the KED-H2 collision mode is superior to the standard analysis mode due to enhanced method accuracy. The methods were validated for the relevant matrices plasma, urine, tattoo ink and tattoo consumables and proved to be applicable for the main target substances in the short-term biokinetics study. A proof-of-concept study showed successful quantification of iodine and PABA metabolites. The PEtOH metabolite was also quantified, but showed strong matrix effects in urine. Therefore standard addition was selected as an alternative quantification method.
    Keywords:  Biokinetics; Blood; ICP-MS; LC-QTOF-MS; Skin; Urine
  16. J Steroid Biochem Mol Biol. 2023 Oct 06. pii: S0960-0760(23)00163-2. [Epub ahead of print] 106408
      Accurate quantification of 24(S)-hydroxycholesterol and 27-hydroxycholesterol holds substantial biological significance due to their involvement in pivotal cellular processes, encompassing cholesterol homeostasis, inflammatory responses, neuronal signaling, and their potential as disease biomarkers. The plasma determination of these oxysterols is challenging considering their low concentrations and similarities in terms of empirical formulae, molecular structure, and physicochemical properties across all human endogenous plasma oxysterols. To overcome these sensitivity and specificity issues, we developed and validated a quantification method using liquid chromatography coupled to a tandem mass spectrometry instrument. Validation studies were designed inspired by Clinical and Laboratory Standards Institute (CLSI) C62-A Guidelines. The linearity ranged between 20-300nM for both oxysterols with limits of quantification at 20nM and 30nM for 24(S)-OHC and 27-OHC, respectively. Inter-day precision coefficient variations (CV) were lower than 10% for both oxysterols. An optimal separation of 25-OHC was obtained from 24(S)-OHC and 27-OHC with a resolution (Rs) > 1.25. The determination and validation of ion ratios for 24(S)-OHC and 27-OHC enabled another quality check in identifying interferents that could impact the quantification. Our developed and validated LC-MS/MS method allows consistent and reliable quantification of human plasmatic 24(S)-OHC and 27-OHC that is warranted in fundamental and clinical research projects.
    Keywords:  24(S)-hydroxycholesterol; 27-hydroxycholesterol; Liquid chromatography; Mass spectrometry; Oxysterols
  17. J Pharm Biomed Anal. 2023 Oct 04. pii: S0731-7085(23)00533-2. [Epub ahead of print]237 115764
      A rapid multi-residue LC-MS/MS method for the identification and determination of banned veterinary drugs in honey was developed. A total of 31 investigated veterinary drugs belonging to 4 classes including nitrofurans metabolites, nitroimidazoles, amphenicols, and quinolones were quantified by LC-MS/MS with ESI using one single injection. The sample preparation included treatment with 5-nitro-2-furaldehyde (5-NFA) in a thermostated ultrasonic bath (80 °C, 0.5М НСl, 20 min) to liberate matrix-bound residues of nitrofurans. Magnetic hypercrosslinked polystyrene (HCP/Fe3O4) was proposed for the solid-phase extraction and clean-up of target analytes prior to LC-MS/MS analysis. To evaluate and validate the performance of method, the criteria of the Decision (EC) no 2002/657 were applied. The LOQs of the examined analytes range from 0.3 to 1 μg kg-1, which indicates good sensitivity to quantify the target compounds in honey. The recoveries of veterinary drugs from 1 g of honey with 50 mg of the sorbent are 97-109% for nitrofuran metabolites, 84-115% for nitroimidazoles, 86-103% for amphenicols, and 97-118% for quinolones. The relative standard deviations of intra-day and inter-day precision analyses (RSD) are less than 16%. This methodology was applied to real honey samples and trace levels of some veterinary drugs were detected.
    Keywords:  Honey; LC-MS/MS; Magnetic solid-phase extraction; Multiresidue analysis; Veterinary drugs
  18. Anal Chim Acta. 2023 Oct 23. pii: S0003-2670(23)01052-8. [Epub ahead of print]1279 341831
      BACKGROUND: Developing an environmentally friendly and efficient integrated analytical approach is a cutting-edge topic in current analytical science. Due to the unique properties of supercritical carbon dioxide (sc-CO2), online supercritical fluid extraction-supercritical fluid chromatography (SFE-SFC) is developing rapidly and has been widely applied in many fields. However, it still faces several challenges such as peak broadening and matrix interference. In order to solve the problems, we developed an inline phase transition trapping-selective supercritical fluid extraction-supercritical fluid chromatography (PTT-SSFE-SFC)-tandem mass spectrometry (MS/MS) method in this study.RESULTS: This method integrated extraction, purification, separation, and detection, which was applied to determine 114 prohibited substances in cosmetics within 33 min, covering ten categories. The PTT strategy trapped the extracts on the head of the column by transforming CO2 from a supercritical state to a gaseous state, preventing peak spreading and improving sensitivity. Several adsorbents were tested when analyzing aqueous samples to reduce matrix interference and absorb water. Compared with conventional online SFE-SFC, this method improved the matrix effects of 93 and 87 target substances in the toner and mask matrix, respectively. Because the integrated method reduced sample loss, it achieved high sensitivity with LODs ranging from 0.00104 μg L-1 to 3.09 μg L-1. Furthermore, compared with other reported green methods, the inline method showed advantages in automation, efficiency, sample amount, and waste volume.
    SIGNIFICANCE AND NOVELTY: With the introduction of the PTT strategy and the adsorbent, the system obtained good peak shapes, high sensitivity, low matrix effect, and good recovery. Based on the results, inline PTT-SSFE-SFC-MS/MS as a green and efficient integrated method has great potential for analyzing low abundance and multiple categories of targets in complex samples.
    Keywords:  Cosmetic; Inline SFE-SFC; Phase transition trapping; Prohibited substances; Selective supercritical fluid extraction
  19. J Pharm Biomed Anal. 2023 Oct 02. pii: S0731-7085(23)00527-7. [Epub ahead of print]237 115758
      PARP inhibitors have demonstrated marked efficacy in ovarian cancer patients with BRCA1/2 loss-of-function mutations. In this study, we established and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) based method to simultaneously quantify the four frequently prescripted PARP inhibitors, namely niraparib, olaparib,fluzoparib, and pamiparib, in ovarian cancer. The mobile phase was 50 % methanol with 0.1 % formic acid at a flow rate of 0.3 mL/min, within 8 min run time. Four PARP inhibitors were separated on a Hypersil GOLD™ aQ C18 Polar Endcapped LC column (50 × 2.1 mm, 1.9 µm) at 35 ℃ and subjected to mass analysis using positive electro-spray ionization (ESI). The linear range of this method was 10-2000 ng/mL, 25-5000 ng/mL, and 50-10,000 ng/mL for niraparib, olaparib and fluzoparib, and pamiparib, respectively, with the correlation coefficients (r2) ≥ 0.99. Accuracies ranged from 93.12 %-110.71 and the inter- and intra-batch precisions were less than 15 % for all analytes in quality control samples. There was no significant matrix effect. Twenty-eight plasma samples were obtained from Sun Yat-sen University Cancer Center. The mean plasma concentrations (±SD) of niraparib and olaparib were 424.76 (±228.35) ng/mL and 1760.47 (±1739.69) ng/mL, respectively. The validated LC-MS/MS method allows the convient and efficient determination of four PARP inhibitors' exposure levels in ovarian cancer patients.
    Keywords:  Fluzoparib; Niraparib; Olaparib; Pamiparib; Therapeutic drug monitoring; UPLC–MS/MS
  20. Transl Clin Pharmacol. 2023 Sep;31(3): 139-147
      Coproporphyrin (CP)-I and CP-III are the markers of organic anion-transporting polypeptides' (OATPs) activities, and they are porphyrin metabolites that originate from heme synthesis. Furthermore, CP-I and CP-III, which are OATP1B endogenous metabolites, have gradually attracted the attention of scientists and researchers in recent years. Previous studies have also observed CP-I and CP-III levels as clinical biomarkers for predicting OATP1B inhibition in drug-drug interaction studies. To establish an accurate ultra-high performance liquid chromatography-mass spectrometry method for the quantitation of CP-I and CP-III, we reviewed previous methodological publications and applied them to a clinical pharmacology study using a human urine matrix. We used 13.25 M formic acid as a working solution for internal standards (CP-I 15N4 and CP-III d8) to avoid isobaric interference. The calibration curve showed good linearity in the range of 1-100 ng/mL, with a correlation coefficient (R2) higher than 0.996 in each validation batch. Both the between-run and within-run assays achieved good precision and accuracy, and we found that both CP-I and CP-III were stable in the pre-study validation. The method exhibited suitable dilution integrity, allowing for the re-analysis of samples with concentrations exceeding the upper limit of quantification through dilution. Overall, the application of the described method in a clinical study revealed that it can be utilized effectively to monitor drug-drug interactions mediated by OATP1B.
    Keywords:  Coproporphyrin I; Coproporphyrin III; Liquid Chromatography; Organic Anion-Transporting Polypeptides
  21. J Pharm Biomed Anal. 2023 Sep 26. pii: S0731-7085(23)00519-8. [Epub ahead of print]237 115750
      In the last decade, the kynurenine pathway, which is the primary metabolic route for tryptophan (TRP) catabolism, has sparked great interest in the pharmaceutical sciences due to its role in immune regulation and cancer immunoediting. In this context, the development of cell-based assays might represent a tool to: i) characterize the cell secretome according to cell types; ii) gain more insight into the role of kynurenines in different disease scenarios; iii) screen hIDO1 (human indoleamine 2,3-dioxygenase) inhibitors and evaluate their effect on downstream TRP-catabolizing enzymes. This paper reports a validated Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) method to simultaneously quantify TRP, L-kynurenine (KYN), xanthurenic acid (XA), 3-hydroxykynurenine (3OHKYN), kynurenic acid (KA), 3-hydroxyanthranilic acid (3OHAA), anthranilic acid (AA), 5-hydroxytryptamine (serotonin, 5HT) and tryptamine (TRYP) in Dulbecco's Modified Eagle and Eagle's Minimum Essential Media (DMEM and EMEM, respectively). The quantitative method was validated according to FDA, ICH and EMA guidelines, later applied: i) to assess the impact of selective inhibition of hIDO1 or hTDO (human tryptophan 2,3-dioxygenase) on the kynurenine pathway in A375 (melanoma), MDA-MB-231 (breast cancer), and U87 (glioblastoma) cell lines using multivariate analysis (MVA); ii) to determine the IC50 values of both well-known (i.e., epacadostat, linrodostat) and the novel hIDO1 inhibitor (i.e., BL5) in the aforementioned cell lines. The proposed LC-MS/MS method is reliable and robust. Furthermore, it is highly versatile and suitable for applications in the preclinical drug research and in vitro assays.
    Keywords:  HIDO1; Kynurenine pathway; LC-MS/MS; Method validation; TDO; Targeted-metabolomics
  22. Foods. 2023 Sep 25. pii: 3558. [Epub ahead of print]12(19):
      Zearalenone and its metabolites are mycotoxins generated by Fusarium species while crops are growing and can typically be found in various foods, posing a risk to human health. Governments have implemented stricter regulations concerning the permissible levels of zearalenone in food products to safeguard public health. Stricter regulations on zearalenone levels in food have been implemented. However, detecting zearalenone and its metabolites remains challenging due to sample complexity and interference. Surprisingly few reviews of sample preparation methods for zearalenone in food have appeared in the past decade. In this overview, we outline the most recent developments in the sample pre-treatment technology of zearalenone and its metabolites in food samples based on chromatography-mass spectrometry methods since 2012. This review covers some prominent technologies, such as liquid-liquid extraction-based methods, solid-phase extraction-based methods, and QuEChERS (quick, easy, cheap, effective, rugged, and safe) extraction, providing valuable insights into their advantages and limitations for potential applications. The assessment of the methods discussed, along with an overview of current challenges and prospects, will guide researchers in advancing the field and ensuring safer food quality for consumers worldwide.
    Keywords:  chromatography; extraction; mass spectrometry; sample preparation; zearalenone
  23. Food Sci Nutr. 2023 Oct;11(10): 6288-6302
      An ionic liquid-based dispersive liquid-liquid microextraction (IL-DLLME) of 20 anthelmintic drugs followed and detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed, optimized, and validated. The parameters affecting the anthelmintic extraction efficiencies such as selection of extraction solvent (ionic liquids), selection of disperser solvent, volume of extraction solvent, volume of disperser solvent, pH of the aqueous phase, extraction time, salt addition, and centrifugation time were optimized. Validation was conducted according to ISO/IEC 17025:2017 and Commission Implementing Regulation (EU) 2021/808 of 22 March 2021. Validation parameters such as calibration function, matrix effect, limit of detection (LOD), limit of quantification (LOQ), decision limit (CCα), accuracy, and precision were established. Coefficient of determination (R 2) values ranging from .99938 to .99995 were obtained using the matrix calibration curve spiked at 0, 0.25, 1.0, 1.5, and 2.0 times MRL. The LODs and LOQs were calculated using the standard deviation of the response and the slopes of the calibration curves ranged from 0.35 to 26.1 μg/kg and from 1.2 to 87.0 μg/kg, respectively, and were dependent on calibration range. The CCα values ranged from 23 to 1022.0 μg/kg and are also dependent on the MRL concentration levels. The coefficient of variation (CV) values calculated are within the reproducibility range of 16%-30% adapted from the Horwitz Equation CV = 2(1-0.5 log C) and ranged from 1.7% to 16.9%. The developed and validated and the standard QuEChERS method were compared. The IL-DLLME LC-MS/MS method was applied to 32 small stock (18 caprine [goat] and 14 ovine [sheep]) liver samples received from municipal abattoirs at Botswana National Veterinary Laboratory for the analysis of anthelmintic drug residues. The results obtained indicated that the anthelmintic drug residues were all below the detection capability, and therefore, the samples were passed as fit for human consumption.
    Keywords:  anthelmintic drugs; caprine; green sample preparation; ionic liquid‐dispersive liquid–liquid microextraction; ovine
  24. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2023 Sep 20. 41(9): 680-684
      Objective: To develop a method for the analysis of phenylglyoxylic acid (PGA) and mandelic acid (MA) in urine by ultra-high performance liquid chromatography tandem mass spectrometry. Methods: The study was conducted in April 2022. Urine samples were directly diluted with the initial mobile phase, separated by Waters HSS T3 column after passing through the membrane, and analyzed under negative ionization mode (ESI(-)) and multiple reaction monitoring (MRM) conditions, the contents of PGA and MA in human urine were quantitatively determined by external standard method. Results: The determination of PGA and MA showed a good linear relationship within the range of 10-1000 ng/ml, with a correlation coefficient of 0.9999. The linear regression equation of PGA was y=1141.4x+2157.3, the detection limit and lower limit of quantification of the method were 0.081 ng/ml and 0.269 ng/ml, and the recovery rate was 90.47%-99.83%. The linear regression equation of MA was y=62.8x+140.3, the detection limit and lower limit of quantification of the method were 0.551 ng/ml and 1.836 ng/ml, and the recovery rate was 92.75%-101.09%. The intra and inter batch precision of PGA and MA were both<5%. Conclusion: An ultra-high performance liquid chromatography tandem mass spectrometry method for the analysis of PGA and MA in urine was established.The sample pretreatment operation is simple, and the accuracy and precision of the method meet the standard requirements. It can be used for monitoring and evaluating PGA and MA in urine of the general population and occupational contact population.
    Keywords:  Mandelic acid (MA); Phenylglycolic acid (PGA); Ultra-high performance liquid chromatography tandem mass spectrometry; Urine
  25. J Am Soc Mass Spectrom. 2023 Oct 11.
      A typical mass spectrometry imaging experiment yields a very high number of detected peaks, many of which are noise and thus unwanted. To select only peaks of interest, data preprocessing tasks are applied to raw data. A statistical study to characterize three types of noise in MSI QToF data (random, chemical, and background noise) is presented through NECTAR, a new NoisE CorrecTion AlgoRithm. Random noise is confirmed to be dominant at lower m/z values (∼50-400 Da) while systematic chemical noise dominates at higher m/z values (>400 Da). A statistical approach is presented to demonstrate that chemical noise can be corrected to reduce its presence by a factor of ∼3. Reducing this effect helps to determine a more reliable baseline in the spectrum and therefore a more reliable noise level. Peaks are classified according to their spatial S/N on the single ion images, and background noise is thus removed from the list of peaks of interest. This new algorithm was applied to MALDI and DESI QToF data generated from the analysis of a mouse pancreatic tissue section to demonstrate its applicability and ability to filter out these types of noise in a relevant data set. PCA and t-SNE multivariate analysis reviews of the top 4000 peaks and the final 744 and 299 denoised peak list for MALDI and DESI, respectively, suggests an effective removal of uninformative peaks and proper selection of relevant peaks.
  26. J Am Soc Mass Spectrom. 2023 Oct 13.
      Single-cell metabolomics has the potential to reveal unique insights into intracellular mechanisms and biological processes. However, the detection of metabolites from individual cells is challenging due to their versatile chemical properties and concentrations. Here, we demonstrate a tapered probe for pneumatically assisted nanospray desorption electrospray ionization (PA nano-DESI) mass spectrometry that enables both chemical imaging of larger cells and global metabolomics of smaller 15 μm cells. Additionally, by depositing cells in predefined arrays, we show successful metabolomics from three individual INS-1 cells per minute, which enabled the acquisition of data from 479 individual cells. Several cells were used to optimize analytical conditions, and 93 or 97 cells were used to monitor metabolome alterations in INS-1 cells after exposure to a low or high glucose concentration, respectively. Our analytical approach offers insights into cellular heterogeneity and provides valuable information about cellular processes and responses in individual cells.