bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2022‒12‒04
forty-five papers selected by
Sofia Costa

  1. Metabolomics. 2022 Nov 27. 18(12): 97
      INTRODUCTION: The structural identification of metabolites represents one of the current bottlenecks in non-targeted liquid chromatography-mass spectrometry (LC-MS) based metabolomics. The Metabolomics Standard Initiative has developed a multilevel system to report confidence in metabolite identification, which involves the use of MS, MS/MS and orthogonal data. Limitations due to similar or same fragmentation pattern (e.g. isomeric compounds) can be overcome by the additional orthogonal information of the retention time (RT), since it is a system property that is different for each chromatographic setup.OBJECTIVES: In contrast to MS data, sharing of RT data is not as widespread. The quality of data and its (re-)useability depend very much on the quality of the metadata. We aimed to evaluate the coverage and quality of this metadata from public metabolomics repositories.
    METHODS: We acquired an overview on the current reporting of chromatographic separation conditions. For this purpose, we defined the following information as important details that have to be provided: column name and dimension, flow rate, temperature, composition of eluents and gradient.
    RESULTS: We found that 70% of descriptions of the chromatographic setups are incomplete (according to our definition) and an additional 10% of the descriptions contained ambiguous and/or incorrect information. Accordingly, only about 20% of the descriptions allow further (re-)use of the data, e.g. for RT prediction. Therefore, we have started to develop a unified and standardized notation for chromatographic metadata with detailed and specific description of eluents, columns and gradients.
    CONCLUSION: Reporting of chromatographic metadata is currently not unified. Our recommended suggestions for metadata reporting will enable more standardization and automatization in future reporting.
    Keywords:  Data reuse; LC-MS; Metabolomics; Repositories; Retention time
  2. Anal Bioanal Chem. 2022 Nov 28.
      Liquid chromatography mass spectrometry (LC-MS) has emerged as a mainstream strategy for metabolomics analyses. One advantage of LC-MS is that it can serve both as a biomarker discovery tool and as a platform for clinical diagnostics. Consequently, it offers an exciting opportunity to potentially transition research studies into real-world clinical tools. One important distinction between research versus diagnostics-based applications of LC-MS is throughput. Clinical LC-MS must enable quantitative analyses of target molecules in hundreds or thousands of samples each day. Currently, the throughput of these clinical applications is limited by the chromatographic gradient lengths, which-when analyzing complex metabolomics samples-are difficult to conduct in under ~ 3 min per sample without introducing serious quantitative analysis problems. To address this shortcoming, we developed sequential quantification using isotope dilution (SQUID), an analytical strategy that combines serial sample injections into a continuous isocratic mobile phase to maximize throughput. SQUID uses internal isotope-labelled standards to correct for changes in LC-MS response factors over time. We show that SQUID can detect microbial polyamines in human urine specimens (lower limit of quantification; LLOQ = 106 nM) with less than 0.019 normalized root mean square error. Moreover, we show that samples can be analyzed in as little as 57 s. We propose SQUID as a new, high-throughput LC-MS tool for quantifying small sets of target biomarkers across large cohorts.
    Keywords:  Diagnostics; High-throughput screening; LC–MS; Metabolomics
  3. Anal Chem. 2022 Nov 28.
      This work presents an advanced analytical platform for untargeted enantioselective amino acid analysis (eAAA) by comprehensive achiral × chiral 2D-LC hyphenated to ESI-QTOF-MS/MS utilizing data-independent SWATH (sequential window acquisition of all theoretical fragment-ion spectra) technology. The methodology involves N-terminal pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ) as retention, selectivity, and MS tag, supporting retention and UV detection in RPLC (1D), chiral recognition, and thus enantioselectivity by the core-shell tandem column composed of a quinine carbamate weak anion exchanger (QN-AX) and a zwitterionic chiral ion-exchanger (ZWIX(+)) (2D) as well as the ionization efficiency during positive electrospray ionization due to a high proton affinity of the AQC label. Furthermore, the urea-type MS tag gives rise to the generation of AQC-tag characteristic signature fragments in MS2. The latter allows the chemoselective mass spectrometric filtering of targeted and untargeted N-derivatized amino acids or related labeled species. The chiral core-shell tandem column provides a complete enantioselective amino acid profile of all proteinogenic amino acids within 1 min, with full baseline separation of all enantiomers, but without resolution of isomeric Ile/allo-Ile (aIle)/Leu, which can be resolved by RPLC. The entire LC × LC separation occurs within a total run time of 60 min (1D), with the chiral 2D operated in gradient elution mode and a cycle time of 60 s. A strategy to mine the 2D-LC-SWATH data is presented and demonstrated for the qualitative eAAA of two peptide hydrolysate samples of therapeutic peptides containing common and uncommon as well as primary and secondary amino acids. Absolute configuration assignment of amino acids using template matching for all proteinogenic amino acids was made feasible due to method robustness and the inclusion of an isotopically labeled L-[U-13C15N]-AA standard. The quantification performance of this LC × LC-MS/MS assay was also evaluated. Accuracies were acceptable for the majority of AAs enabling AA composition determination in peptide hydrolysates simultaneously with configuration assignment, as exemplified by oxytocin. This methodology represents a step toward truly untargeted 2D enantioselective amino acid analysis and metabolomics.
  4. Front Plant Sci. 2022 ;13 1038161
      The lipidome comprises the total content of molecular species of each lipid class, and is measured using the analytical techniques of lipidomics. Many liquid chromatography-mass spectrometry (LC-MS) methods have previously been described to characterize the lipidome. However, many lipidomic approaches may not fully uncover the subtleties of lipid molecular species, such as the full fatty acid (FA) composition of certain lipid classes. Here, we describe a stepwise targeted lipidomics approach to characterize the polar and non-polar lipid classes using complementary LC-MS methods. Our "polar" method measures 260 molecular species across 12 polar lipid classes, and is performed using hydrophilic interaction chromatography (HILIC) on a NH2 column to separate lipid classes by their headgroup. Our "non-polar" method measures 254 molecular species across three non-polar lipid classes, separating molecular species on their FA characteristics by reverse phase (RP) chromatography on a C30 column. Five different extraction methods were compared, with an MTBE-based extraction chosen for the final lipidomics workflow. A state-of-the-art strategy to determine and relatively quantify the FA composition of triacylglycerols is also described. This lipidomics workflow was applied to developing, mature, and germinated pennycress seeds/seedlings and found unexpected changes among several lipid molecular species. During development, diacylglycerols predominantly contained long chain length FAs, which contrasted with the very long chain FAs of triacylglycerols in mature seeds. Potential metabolic explanations are discussed. The lack of very long chain fatty acids in diacylglycerols of germinating seeds may indicate very long chain FAs, such as erucic acid, are preferentially channeled into beta-oxidation for energy production.
    Keywords:  LC-MS/MS; Thlaspi arvense; lipidomics; mass spectrometry; oilseed; pennycress
  5. Clin Chem Lab Med. 2022 Dec 05.
      OBJECTIVES: In this study we describe the development and validation of a liquid chromatography mass spectrometry method (LC-MS/MS) to quantify five tryptophan (TRP) metabolites within the kynurenine- and serotonin pathway and apply the method to serum samples of women in the first trimester of pregnancy. A secondary aim was to investigate the correlation between body mass index (BMI) and the five analytes.METHODS: A LC-MS/MS was developed for the analysis of TRP, kynurenine (KYN), 5-hydroxytryptophan (5-HTP), hydroxytryptamine (5-HT), and 5-hydroxyindole acetic acid (5-HIAA). Serum samples (n=374) were analyzed of pregnant women (median gestational age: 8 ± 2 weeks) participating in a subcohort of the Rotterdam Periconceptional Cohort (Predict study).
    RESULTS: The LC-MS/MS method provided satisfactory separation of the five analytes (7 min run). For all analytes R2 was >0.995. Within- and between-run accuracies were 72-97% and 79-104%, and the precisions were all <15% except for the between-run precisions of the low QC-samples of 5-HTP and 5-HT (both 16%). Analyte concentrations were determined in serum samples of pregnant women (median (IQR)); TRP (µmol/L): 57.5 (13.4), KYN (µmol/L): 1.4 (0.4), 5-HTP (nmol/L): 4.1 (1.2), 5-HT (nmol/L): 615 (323.1), and 5-HIAA (nmol/L): 39.9 (17.0). BMI was negatively correlated with TRP, 5-HTP, and 5-HIAA (TRP: r=-0.18, p<0.001; 5-HTP: r=-0.13, p=0.02; natural log of 5-HIAA: r=-0.11, p=0.04), and positively with KYN (r=0.11, p=0.04).
    CONCLUSIONS: The LC-MS/MS method is able to accurately quantify kynurenine- and serotonin pathway metabolites in pregnant women, providing an opportunity to investigate the role of the TRP metabolism in the (patho)physiology of pregnancy.
    Keywords:  liquid chromatography; mass spectrometry; pregnancy; tryptophan metabolism
  6. Front Mol Biosci. 2022 ;9 1057709
      In recent years, mass spectrometry-based metabolomics has been established as a powerful and versatile technique for studying cellular metabolism by comprehensive analysis of metabolites in the cell. Although there are many scientific reports on the use of metabolomics for the elucidation of mechanism and physiological changes occurring in the cell, there are surprisingly very few reports on its use for the identification of rate-limiting steps in a synthetic biological system that can lead to the actual improvement of the host organism. In this mini review, we discuss different strategies for improving strain performance using metabolomics data and compare the application of metabolomics-driven strain improvement techniques in different host microorganisms. Finally, we highlight several success stories on the use of metabolomics-driven strain improvement strategies, which led to significant bioproductivity improvements.
    Keywords:  bioproductivity; enzyme engineering; metabolic engineering; metabolomics; strain improvement
  7. J Pharm Biomed Anal. 2022 Nov 21. pii: S0731-7085(22)00580-5. [Epub ahead of print]224 115159
      Currently, the use of targeted drugs such as tyrosine kinase inhibitors (TKIs) plays an important role in clinical therapy. As the number of approved TKIs continues to increase, existing analysis methods will not be able to meet the growing needs, and will hamper the development of therapeutic drug monitoring (TDM) of TKIs. Based on LC-MS/MS technology, this study tends to develop and validate a multi-component analysis method for simultaneous determination of the concentrations of 39 TKIs in plasma. Spiked plasma was blended with isotope labelled internal standards, and injected into the LC-MS/MS system after protein precipitation by acetonitrile. Chromatographic separation was achieved using an ODS-4 column with gradient elution of formic acid/water (1:1000; v/v) and acetonitrile. Analytes detection was conducted in positive ionisation mode using MRM. The total run time was 8 min. The method validation was conducted by assessing the following parameters: selectivity, linearity and the lower limit of qualification, accuracy and precision, stability, matrix effect and recovery. The concentrations of 39 TKIs showed good linearity within the range of their respective standard curves in plasma, the accuracy of all quality control samples ranged from 85.9% to 114.1%, and the precision was lower than 13.3%. The extraction recovery ranged from 92.6% to 114.7%, and the matrix effect of plasma was lower than 11.3%. This new method was successfully developed, can be used for the determination of drug concentrations in multiple patients with different kinds of TKIs, and will therefore be suitable for TDM of 39 TKIs.
    Keywords:  LC-MS/MS; Multi-component analysis; Plasma drug concentration; Tyrosine kinase inhibitor
  8. Front Mol Biosci. 2022 ;9 930204
      Untargeted metabolomics studies are unbiased but identifying the same feature across studies is complicated by environmental variation, batch effects, and instrument variability. Ideally, several studies that assay the same set of metabolic features would be used to select recurring features to pursue for identification. Here, we developed an anchored experimental design. This generalizable approach enabled us to integrate three genetic studies consisting of 14 test strains of Caenorhabditis elegans prior to the compound identification process. An anchor strain, PD1074, was included in every sample collection, resulting in a large set of biological replicates of a genetically identical strain that anchored each study. This enables us to estimate treatment effects within each batch and apply straightforward meta-analytic approaches to combine treatment effects across batches without the need for estimation of batch effects and complex normalization strategies. We collected 104 test samples for three genetic studies across six batches to produce five analytical datasets from two complementary technologies commonly used in untargeted metabolomics. Here, we use the model system C. elegans to demonstrate that an augmented design combined with experimental blocks and other metabolomic QC approaches can be used to anchor studies and enable comparisons of stable spectral features across time without the need for compound identification. This approach is generalizable to systems where the same genotype can be assayed in multiple environments and provides biologically relevant features for downstream compound identification efforts. All methods are included in the newest release of the publicly available SECIMTools based on the open-source Galaxy platform.
    Keywords:  batch effects; experimental design; mass spectrometry; meta-analysis; model organism; nuclear magnetic resonance spectroscopy; untargeted metabolomics
  9. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2022 Dec 02. 1-12
      The lethal neurotoxins, paralytic shellfish toxins (PSTs), and tetrodotoxin (TTX) have recently been found in marine shellfish from many coastal states. Herein, we applied a sensitive and reliable ultra-performance hydrophilic interaction chromatography (HILIC)-tandem mass spectrometry (MS) method to determine 13 PSTs and TTX in marine shellfish using a porous carbon solid-phase extraction (SPE). This in-house validation study required the development of a novel chromatographic separation using a HILIC-Z column, which was necessary to retain highly polar compounds. Using acetonitrile as the organic phase and ammonium formate-formic acid buffer as the aqueous phase, the quantitative analysis was carried out with an external standard method in the multiple reaction monitoring modes using positive electrospray ionization. To reduce interference, 1% aqueous acetic acid extracts of the shellfish samples were cleaned up by ion-pair SPE using a porous graphitic carbon cartridge. The calibration curves for PSTs and TTX were linear (R2 > 0.995), and the sensitivity was good, with limits of detection (LODs) of 1.7-13.7 µg/kg, and limits of quantitation (LOQs) of 5.2-41.0 µg/kg. The recoveries were 76.5-95.5% with RSDs of 3.1-12.0%. Finally, We applied the method for the determination of PSTs and TTX in three batches of Nassarius showing excellent method accuracy against expected values.
    Keywords:  LC-MS/MS; PSTs; TTX; hydrophilic interaction chromatography (HILIC)
  10. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Nov 11. pii: S1570-0232(22)00439-1. [Epub ahead of print]1213 123534
      The indole alkaloid N,N-dimethyltryptamine (DMT) induces psychedelic effects in humans. In addition to ceremonial and recreational use, DMT is subject to clinical investigations. Sensitive bioanalytical methods are required to assess the pharmacokinetics of DMT and its metabolites in human plasma. Here, a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of DMT and its major metabolites indole-3-acetic acid (IAA) and DMT-N-oxide (DMT-NO) was developed and validated. As IAA is an endogenous component of human plasma, 13C6-IAA was used to determine IAA concentrations. After simple protein precipitation with methanol, analytes were separated on a pentafluorophenyl column. A gradient consisting of 0.1% (v/v) formic acid in a methanol-water mixture was applied for analyte separation. The analytes were detected by positive electrospray ionization followed by multiple reaction monitoring. The calibration range of the assay was 0.25-250 ng/mL for DMT, 0.1-100 ng/mL for DMT-NO, and 25-25,000 ng/mL for 13C6-IAA. The intra- and inter-assay accuracy was 93-113% for all analytes at all quality control levels, with coefficient of variation ≤ 11%. All analytes were stable under storage conditions relevant for the analysis of large batches of study samples. The validated method was capable of assessing pharmacokinetic (PK) parameters of DMT and its metabolites in study participants intravenously perfused with 1 mg/min DMT for 90 min. Overall, the developed method is easy-to-use, has a short run time, and qualifies for PK and metabolism studies of DMT in clinical settings.
    Keywords:  Ayahuasca; Bioanalysis; HPLC; Metabolism; Pharmacokinetics; Psychedelic
  11. Forensic Toxicol. 2022 Jan;40(1): 102-110
      PURPOSE: Paraquat and diquat are well-known toxic herbicides, at least responsible for hundreds of fatal poisoning events worldwide. However, the determination of diquat and paraquat in plasma and urine is very challenging because of their high polarity and double charge characteristics. In this study, we aim to develop a rapid and reliable method for the determination of paraquat and diquat in human plasma and urine by ultraperformance liquid chromatography-tandem mass spectrometry.METHOD: The chromatographic separation of paraquat and diquat was tested with different chromatographic columns and different mobile phase conditions. The mass parameters were optimized by product ions, source gas flow, cone flow, desolvation temperature, and capillary voltage. The isocratic elution mode gave rapid appearance of peak of paraquat and diquat.
    RESULTS: The sharp peak shapes for paraquat and diquat were achieved with CORTECS® UPLC® HILIC (100 × 2.1 mm, 1.6 μm) column by adding formic acid and ammonium acetate in mobile phase in isocratic elution mode. The lower limit of quantification of 1.0 ng/mL for paraquat and diquat were achieved using only 50 μL of human plasma or urine. The running time for analysis of both paraquat and diquat was as short as 3.5 min per sample.
    CONCLUSIONS: A rapid and reliable method for the determination of paraquat and diquat was developed and applied to 387 clinical poisoning cases and 22 poisoning cases were found to be paraquat or diquat poisoning.
    Keywords:  HILIC column; High throughput; Isocratic elution; Paraquat and diquat; Poisoning cases at emergency departments; UPLC–MS/MS
  12. Clin Chim Acta. 2022 Nov 27. pii: S0009-8981(22)01392-4. [Epub ahead of print]
      BACKGROUND AND AIMS: Androgens play important roles in polycystic ovarian syndrome (PCOS). However, measures of androgens based on mass spectrometry (MS) remain complex due to endogenous inferences of isomers or compounds with similar structures. Lack of sensitivity can also affect the accurate quantification of androgens, especially for very low level of 11-oxygenated androgens.MATERIALS AND METHODS: We developed a fast and sensitive high-performance liquid chromatography-differential mobility spectrometry tandem mass spectrometry (HPLC-DMS/MS/MS) method for the simultaneous determination of seven androgens and 17-hydroxyprogesterone. Dispersive magnetic solid phase extraction (DMSPE) was conducted with core-shell structured nanoparticles of magnetic graphene oxide (Fe3O4@GO). In situ derivatization was performed using Girard's Reagent P.
    RESULTS: Linear ranges of the eight analytes were set in terms of clinical use. Intra- and inter-run precisions were <16.7% and 12.9% for all the analytes and relative error was -14.7%-13.3% and -9.3%-11.0%, respectively. Extraction recoveries were 54.0%-92.7% for different analytes. The method was validated and was applied to assay 432 clinical samples.
    CONCLUSION: The developed method is green, fast, sensitive and accurate for the determination of endogenous androgens. It can be readily implemented in medical laboratories to provide superior analytical performance over the traditional electrochemiluminescence immunoassay method.
    Keywords:  11-Oxygenated androgens; Classic androgens; DMSPE; HPLC-DMS/MS/MS; PCOS; in situ derivatization
  13. Forensic Toxicol. 2022 Jul;40(2): 312-321
      PURPOSE: Methorphan exists in two enantiomeric forms including dextromethorphan and levomethorphan. Dextromethorphan is an over-the-counter antitussive drug, whereas levomethorphan is strictly controlled as a narcotic drug. Chiral analysis of methorphan could, therefore, assist clinicians and forensic experts in differentiating between illicit and therapeutic use and in tracing the source of the drug.METHODS: A method for enantiomeric separation and quantification of levomethorphan and dextromethorphan in human hair was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hair was extracted in hydrochloric acid/methanol (1:20, v/v). The supernatant were separated using a Supelco Astec Chirobiotic™ V2 column (250 × 2.1 mm, i.d., 5 μm particle size) and analyzed on a triple quadrupole linear ion trap mass spectrometer in multiple reaction monitoring mode.
    RESULTS: The limits of detection for dextromethorphan and levomethorphan were 2 and 1 pg/mg, respectively; the lower limit of quantification was 2 pg/mg for both drugs. Good linearity (r > 0.995) was observed for both analytes over the linear range. Precision values were below 10% for both analytes; accuracy values ranged from 87.5 to 101%. The extraction recoveries were 78.3-98.4%, and matrix effects were 70.5-88.6%. This method was applied to human hair samples from 120 people suspected of methorphan use to further distinguish the drug chirality. Dextromethorphan was detected in all 120 samples at a concentration range of 2.7-19,100 pg/mg, whereas levomethorphan was not detected in any sample.
    CONCLUSIONS: A sensitive quantitative method was established for the enantiomeric separation of dextromethorphan and levomethorphan in hair. This is the first study to achieve chiral analysis of methorphan in human hair.
    Keywords:  Chiral separation; Dextromethorphan; Hair; LC–MS/MS; Levomethorphan; Methorphan
  14. Se Pu. 2022 Dec;40(12): 1087-1094
      Determining the presence of paraquat (PQ) and diquat (DQ) in urine samples through physical and chemical testing is challenging. As PQ and DQ have characteristics such as high molecular polarity and good water solubility, they are difficult to be retained by conventional reversed-phase columns. Most of the methods in the literature use hydrophilic interaction chromatography (HILIC) for the retention of PQ and DQ, but they often require high concentrations of buffer salts as the mobile phase, which increase the contamination of the mass spectrometer. In view of the above problems, a rapid and accurate analysis method was developed for the determination of PQ and DQ residuals in urine samples based on weak cation exchange (WCX) solid-phase extraction (SPE) and ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) in this study. Urine samples were first diluted with phosphate buffer (pH=6.86) and pretreated using the WCX SPE method. Chromatographic separation was performed on a Syncronis HILIC column (100 mm×2.1 mm, 1.7 μm). An electrospray ion source in the positive (ESI+) mode and full mass-data dependent MS2 (full mass-ddMS2) mode was used for quantification by matrix-matched external standard method. In this study, the concentration of ammonium formate in the mobile phase in the HILIC mode was effectively reduced to 10 mmol/L by the continuous optimization of the chromatographic conditions. MS optimization results indicated that the molecular ion (M+·) of PQ and DQ had the strongest response. In addition, sample pretreatment conditions were also optimized. The obtained results indicated that the hydrophobic polytetrafluoroethylene (PTFE) filter membrane, acetonitrile-water (1∶1, v/v) as a fixing solution, and polypropylene vials were suitable for PQ and DQ analysis. Under the optimal conditions, the linearity of PQ and DQ was good with correlation coefficients (r2) greater than 0.998. The limits of detection (LODs, S/N≥3) and limits of quantification (LOQs, S/N≥10) were 0.2 μg/L and 0.6 μg/L, respectively. Mean spiked recoveries of PQ and DQ at the four spiked levels (1.0, 20.0, 100.0, and 200.0 μg/L) were in the range of 85.8%-101% and 80.3%-86.9%, with the RSDs of 0.8%-5.1% and 0.9%-4.2%. The established method was employed for the analysis and confirmation of PQ and DQ for clinical poisoning cases. In one case, a 23-year-old male who had taken approximately 20 mL of pesticide orally was confirmed as DQ poisoning by the developed method. DQ concentration monitoring of the urine samples was conducted for this case during the clinical treatment process. The patient was successfully discharged from the hospital after five times of blood perfusion and other treatments until the DQ concentration was low in the urine samples. In conclusion, the method developed in this study based on WCX SPE-UPLC-HRMS can be used for the confirmation of poisoning cases and concentration monitoring during clinical treatment, providing strong technical support for clinical precision treatment. The method is rapid, simple, sensitive, and accurate, and it is suitable for the detection of PQ and DQ in urine samples.
    Keywords:  diquat (DQ); matrix effect; paraquat (PQ); ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS); urine samples; weak cation exchange (WCX) solid-phase extraction (SPE)
  15. Crit Rev Anal Chem. 2022 Nov 29. 1-16
      Bioanalytical methods are used to quantify drugs and their metabolites in biological samples in order to determine bioequivalence, perform pharmacokinetic and bioavailability studies, and complete therapeutic drug monitoring. The objective of this review paper is to describe bioanalytical methods based on Liquid Chromatography that are used to quantify antitubercular drugs and their metabolites in different biological samples, utilizing scientific literature from 1992 to 2021.
    Keywords:  Bioanalytical; chromatographic method; liquid chromatography; mass spectrometry; tuberculosis drug
  16. Se Pu. 2022 Dec;40(12): 1076-1086
      Special attention must be paid to children and infants because health issues stemming from a poor immunologic system and low weight make them a vulnerable risk group. Complementary foods for infants and young children, which are a class of special dietary foods, are essential transitional foods for infants from lactation to adaptation to ordinary food. Some complementary foods for infants and young children are of animal origin such as fish, meat, and the liver, which may contain veterinary drug residues. Veterinary drugs are usually small-molecular-weight chemicals that are essential for treating infections, increasing production, and improving animal husbandry. However, abuse of these substances can provoke transfer to the food chain, leading to negative consequences for humans, especially infants and young children. For a more comprehensive safety supervision of infant supplementary foods, a method based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed and used for the determination of 50 antibiotics and antivirals (grouped into six categories: fluoroquinolones, sulfonamides, macrolides, nitroimidazoles, chloramphenicols, and antivirals) in complementary foods for infants and young children. The matrix of complementary foods for infants and young children is complex and contains a large number of proteins and lipids, which poses a serious challenge for sample pretreatment. The Captiva EMR-Lipid solid-phase extraction (SPE) column is a new type of product that allows for selective and efficient lipid/matrix removal without negatively affecting the recovery of the analyte. In this study, samples were extracted with acidified acetonitrile and then purified using a Captiva EMR-Lipid SPE column. The target analytes were separated on a BEH C18 column by gradient elution using acetonitrile and water containing 0.1% (v/v) formic acid as the mobile phases. MS detection was performed with an electrospray source in the positive and negative modes in the multireaction monitoring (MRM) mode. The ion spray voltages were set at 5500 V and -4500 V in the positive and negative modes, respectively. The source temperature for both the ionization modes was set to 500 ℃. Instrumental parameters such as collision energy and declustering potential were optimized. The samples were quantified using the external standard method with matrix calibration curves to reduce the influence of the matrix effect on the quantitative results.The results showed that the 50 veterinary drug residues had good linear relationships in the range of 0.5 to 50 μg/L, with correlation coefficients higher than 0.995. The limits of detection (LODs) and quantification (LOQs) were in the range of 0.03-0.70 μg/kg and 0.09-2.33 μg/kg, respectively. The average recoveries for all the compounds under different matrices ranged from 64.37% to 119.3% at spiked levels of 5 μg/kg and 50 μg/kg, with relative standard deviations of less than 15%. Compared to QuEChERS, this method has a better purification effect. The recoveries of the 50 veterinary drugs extracted by this method were also much higher than those in the case of QuEChERS. This method was applied to the detection of 14 domestic and six imported infant supplementary foods. Sulfaquinoxaline, sulfamethazine, and tilmicosin were detected in one imported meat-based baby food. With its simple operation, high sensitivity and accuracy, and low sample quantity consumption, this method is suitable for the determination of multiveterinary drug residues in complementary foods for infants and young children. This study provides an effective analysis method for risk monitoring and troubleshooting of complementary foods for infants and young children, which is of great significance in ensuring the healthy growth of the next generation.
    Keywords:  complementary foods for infants and young children; multi-veterinary drug residues; solid-phase extraction purification; ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)
  17. J Sep Sci. 2022 Nov 22.
      Immunoassays are currently not available in commercial kits for the quantification of valproic acid, vigabatrin, pregabalin, and gabapentin, which also cannot suffer the limitations of interferences of substances with similar structures. Chromatography is a good alternative to immunoassay. In this study, a simple and robust non-derivatization gas chromatography-mass spectrometry method for simultaneous determination of the above four drugs in human plasma was developed and validated for therapeutic drug monitoring purposes. This method employed benzoic acid as the internal standard with hydrochloric acid for plasma acidification and ACN for precipitate protein. The supernatant was directly injected into gas chromatography-mass spectrometry for analysis. Good linearity was obtained with linear correlation coefficients of the four analytes of 0.9988-0.9996. Extraction recoveries of valproic acid, vigabatrin, pregabalin, and gabapentin were respectively in the ranges of 91.3%-94.5%, 90.0%-90.9%, 90.0%-92.1%, and 88.0%-92.2% with the relative standard deviation values less than 12.6%. Intra- and inter-batch precision and accuracy, and stability assays were all acceptable. Taken together, the novel method developed in this study provided easy plasma pretreatment, good extraction yield, and high chromatographic resolution, which has been successfully validated through the quantification of valproic acid in the plasma of 46 patients with epilepsy.
    Keywords:  antiepileptic drugs; gas chromatography-mass spectrometry; human plasma; non-derivatization; therapeutic drug monitoring
  18. Chirality. 2022 Dec 02.
      Lenalidomide (LND) is an analogue of thalidomide that is second generation immunomodulatory drugs (IMiDs). LND contains asymmetric carbon atom and exist R and S enantiomer. S (-) form of enantiomer are considered to be more potent and biologically active in tumor cell. It is available in racemic form for clinical use. The study aims to develop and validate enantiomer separation of LND in human plasma. The chromatographic enantiomeric separation was achieved on a Daicel-CSP, Chiralpack IA 4.6 × 250 mm_5 μm. The mobile phase was constituted in combination of methanol:glacial acetic acid at a concentration of 499.50 ml: 50 μl. UV wavelength detection was 220 nm. The RSD% for all validation parameters was found to be within the acceptable limit. The chiral chromatographic (chiral stationary phase-high-performance liquid chromatography [CSP-HPLC]) method developed and validated for the quantitative estimation of LND enantiomers S (-) and R (+) in human plasma sample is accurate, precise, robust, stable and selective.
    Keywords:  CSP-HPLC; chirality; enantiomers; lenalidomide; validation
  19. J Chromatogr A. 2022 Dec 20. pii: S0021-9673(22)00845-7. [Epub ahead of print]1686 463654
      Metabolic reprogramming of cancer cells is a hallmark of cancer, in which the polar metabolites involving aerobic glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, and glutaminolysis play a crucial role in the occurrence and development of cancer. Therefore, targeted analysis of the polar metabolites in these pathways is of great value for understanding cancers, finding diagnostic biomarkers, and identifying therapeutic targets. However, it is still challenging to directly determine polar metabolites in these pathways without derivatization due to their diverse chemical properties, isomers, and strong polarity. Herein, a highly selective and sensitive HILIC-MS/MS method was developed for direct determination of the polar metabolites in aerobic glycolysis, PPP, TCA cycle, and glutaminolysis pathways. Without derivatization, 19 polar metabolites and their isomers with carbonyl, carboxyl, or phosphoryl groups in human plasma and cell extracts of prostate cancer (PC) were determined with strong retention and high resolution. This method has been widely verified by measuring linearity, precision, sensitivity, repeatability, matrix effect, and accuracy. The analysis of plasma samples by HILIC-MS/MS revealed distinct PC-specific metabolic signatures compared to a healthy control. In addition, this method could also be used to screen the targets of metabolic inhibitors at the cellular level. We conclude that the developed HILIC-MS/MS method provides a valuable means to study the cancer metabolic reprogramming or energy metabolism in living organisms.
    Keywords:  Cancer metabolic reprogramming; HILIC-MS/MS; Human plasma; Polar metabolites; Prostate cancer
  20. J Pharm Biomed Anal. 2022 Nov 21. pii: S0731-7085(22)00588-X. [Epub ahead of print]224 115167
      Ampicillin in combination with sulbactam is a widely used drug choice for infection prophylaxis, especially in oral and maxillofacial surgery. Clindamycin serves as an alternative in patients with known allergy to β-lactam antibiotics. To ensure effective prophylaxis, it is important to achieve sufficiently high concentrations of active antibiotic substances in the tissues affected by the surgery. To this end, a LC-MS/MS method was developed and validated that allows the quantification of ampicillin, sulbactam and clindamycin in jawbone, plasma, and so-called platelet-rich fibrin (PRF). Validation was performed in accordance with the European Medicines Agency guidelines for bioanalytical method validation. For all matrices, sample processing was carried out by protein precipitation with acetonitrile or methanol 80%, containing the isotope labelled internal standards (IS) of the three drugs. Analytes were separated on a pentaflourophenyl column at 20 °C using gradient elution. Furthermore, detection was accomplished by electrospray ionisation in positive-ion mode (ampicillin, clindamycin and corresponding IS) and negative-ion mode (sulbactam and corresponding IS) in combination with multiple reaction monitoring. Depending on the analyte and the matrix under investigation, calibration curves ranged from 0.14 to 59.8 µg/g (jawbone - ampicillin), 2.0-1000 µg/mL (plasma - ampicillin), and 1.0-495 µg/mL (PRF - ampicillin). All analytes fulfilled the requirements of the guideline regarding sensitivity, linearity, selectivity, carryover, within-run and between run accuracy and precision, matrix effect and extraction recovery in all matrices. The method was successfully applied to measure concentrations of ampicillin, sulbactam and clindamycin in real-life samples obtained in routine clinical practice.
    Keywords:  Jawbone; LC-MS/MS; Plasma; Platelet-rich fibrin; Validation; β-lactam
  21. Anal Bioanal Chem. 2022 Nov 26.
      Non-targeted analysis (NTA) using high-resolution mass spectrometry allows scientists to detect and identify a broad range of compounds in diverse matrices for monitoring exposure and toxicological evaluation without a priori chemical knowledge. NTA methods present an opportunity to describe the constituents of a sample across a multidimensional swath of chemical properties, referred to as "chemical space." Understanding and communicating which region of chemical space is extractable and detectable by an NTA workflow, however, remains challenging and non-standardized. For example, many sample processing and data analysis steps influence the types of chemicals that can be detected and identified. Accordingly, it is challenging to assess whether analyte non-detection in an NTA study indicates true absence in a sample (above a detection limit) or is a false negative driven by workflow limitations. Here, we describe the need for accessible approaches that enable chemical space mapping in NTA studies, propose a tool to address this need, and highlight the different ways in which it could be implemented in NTA workflows. We identify a suite of existing predictive and analytical tools that can be used in combination to generate scores that describe the likelihood a compound will be detected and identified by a given NTA workflow based on the predicted chemical space of that workflow. Higher scores correspond to a higher likelihood of compound detection and identification in a given workflow (based on sample extraction, data acquisition, and data analysis parameters). Lower scores indicate a lower probability of detection, even if the compound is truly present in the samples of interest. Understanding the constraints of NTA workflows can be useful for stakeholders when results from NTA studies are used in real-world applications and for NTA researchers working to improve their workflow performance. The hypothetical ChemSpaceTool suggested herein could be used in both a prospective and retrospective sense. Prospectively, the tool can be used to further curate screening libraries and set identification thresholds. Retrospectively, false detections can be filtered by the plausibility of the compound identification by the selected NTA method, increasing the confidence of unknown identifications. Lastly, this work highlights the chemometric needs to make such a tool robust and usable across a wide range of NTA disciplines and invites others who are working on various models to participate in the development of the ChemSpaceTool. Ultimately, the development of a chemical space mapping tool strives to enable further standardization of NTA by improving method transparency and communication around false detection rates, thus allowing for more direct method comparisons between studies and improved reproducibility. This, in turn, is expected to promote further widespread applications of NTA beyond research-oriented settings.
    Keywords:  Chemical space; Cheminformatics; Mass spectrometry; Non-targeted analysis; Quality assurance/control
  22. Se Pu. 2022 Dec;40(12): 1128-1135
      A method was developed for the determination of 10 organic acids in liquor, yellow rice wine, and dry red wine by ion chromatography-triple quadrupole mass spectrometry (IC-MS/MS). First, the liquor samples were diluted with deionized water, degassed with nitrogen, and analyzed by IC-MS/MS. Then, the yellow rice wine and dry red wine samples were purified with different solid-phase extraction cartridges. Finally, the GCB solid-phase extraction cartridge was selected for purification, diluted with deionized water, and analyzed by IC-MS/MS. The samples were separated using a Dionex IonPac AS11-HC anion analysis column with high capacity and strong hydrophilicity, with an KOH aqueous solution as the eluent, which was produced by an automatic generator for gradient elution. After being suppressed using a suppressor, the eluent was injected directly into the electrospray ionization tandem mass spectrometry (ESI-MS/MS), ionized in negative ion mode, detected in multiple reaction monitoring (MRM) mode, and quantified using an external standard method. Oxalic acid, fumaric acid, maleic acid, malic acid, tartaric acid, citric acid, quinic acid, and aconitic acid showed good linear relationships in the range of 0.05-2 mg/L. Succinic acid and lactic acid showed good linearities in the range of 0.05-5 mg/L and 0.05-10 mg/L, respectively. The correlation coefficients (r2) were >0.99. The limits of detection (LODs) and limits of quantification (LOQs) were 1.0-8.0 μg/L and 3.5-26.5 μg/L, respectively. The average recoveries ranged from 83.0% to 112.1%, and the relative standard deviations (RSDs) were <9.1% in spiked samples at three levels. The proposed method allowed easy pretreatment without using organic solvents or derivatization processing. Overall, the proposed method is accurate, rapid, sensitive, and it is suitable for the qualitative and quantitative analyses of the 10 organic acids in three wine samples. Moreover, it can be used for the determination of flavor and quality of alcoholic products.
    Keywords:  dry red wine; ion chromatography-tandem mass spectrometry (IC-MS/MS); liquor; organic acids; yellow rice wine
  23. J Anal Toxicol. 2022 Dec 01. pii: bkac093. [Epub ahead of print]
      Liquid chromatography coupled to mass spectrometry (LC/MS) has been increasingly used for screening purposes in forensic toxicology. High versatility and low time/resource consuming are the main advantages of this technology. Numerous multi-analytes methods have been validated in order to face the analytical challenge of the new psychoactive substances (NPS). However, forensic toxicologists must keep the attention also on "classical" psychoactive substances and medicines, such as benzodiazepines (BDZ) and prescription opioids. In this paper, a new method for simultaneous detection of 169 substances (120 NPS and 49 other drugs) in hair by LC-MS/MS was described. After decontamination of hair sample with dichloromethane, a 20-mg aliquot was added with 1 mL of methanol (0.1% of formic acid) and then sonicated at room temperature for 2 h. The mixture was then dried under nitrogen stream and reconstituted with 100 µL of methanol. The LC separation was achieved with a 100-mm long C18 column in 35 min and the mass acquisition was performed in dynamic MRM mode and in positive ionization. The analysis resulted very sensitive, with limits of quantification from 0.07 to 10.0 pg/mg. Accuracy and precisions were always within the acceptable criteria. Matrix effect and recovery rate ranges were -21.3 - +21.9% and 75.0 - 99.3%, respectively. The new method was successfully applied in a preliminary study on the prevalence of NPS, BDZ and other substances in case on driving licence issues. In 14% of cases, BDZ/antidepressants (mainly trazodone, diazepam/nordiazepam and flunitrazepam) were found. Codeine, ketamine, methylone and mephedrone were also detected.
    Keywords:  Benzodiazepine; LC-MS/MS; New psychoactive substances; hair; prescription opioids; screening
  24. Food Chem. 2023 Mar 15. pii: S0308-8146(22)02568-7. [Epub ahead of print]404(Pt A): 134606
      Since the global demanding of natural melatonin-enriched milk has been significantly increased in the populations of children and elder, the accurate and quick melatonin detection from milk is urgently required. Thus, the regular methods no longer satisfy this requirement. In the current study, we reported a novel method to extract melatonin from milk for liquid chromatography-tandem mass spectrometry melatonin detection. This novel method was to use cold methanol (-20℃) to precipitate proteins and fat in milk with one step to extract melatonin. Compared to the regular methods, it was devoid of procedures of sample drying, solid phase extraction and sample reconstitution. It could short the extraction time from the regularly 150 min to 60 min/per 24 milk samples. We believe that this novel method provides a possibility to detect large scale of milk samples in relatively short time with more efficiency and less cost compared to the regular method.
    Keywords:  Cold temperature; Liquid chromatography-tandem mass spectrometry; Melatonin; Methanol; Milk; Serum
  25. Anal Chim Acta. 2023 Jan 02. pii: S0003-2670(22)01154-0. [Epub ahead of print]1237 340583
      Hydrogen sulfide is a toxic gas but also established as a naturally occurring gaseous signaling molecule in humans, playing key physiological roles with particular involvement in lung disease including COVID-19. Thiosulfate is the conventional biomarker of hydrogen sulfide and is excreted in human urine at low micromolar levels. Thiosulfate is amenable to detection by the element-selective inductively coupled plasma tandem mass spectrometry (ICPMS/MS), but sulfur speciation in human samples at trace levels is challenging due to the high complexity of human sulfur metabolome and the utility of this detector under such settings has not been demonstrated. We report a method for thiosulfate determination in human urine at trace physiological levels by HPLC-ICPMS/MS. The method involved one-step derivatization to improve chromatographic behavior followed by direct injection. The instrumental limit of detection was 1.4 μg S L-1 (0.02 μM or 0.1 pmol). In a group of samples from volunteers (n = 24), measured thiosulfate concentrations in the diluted urine matrix were down to 8.0 μg S L-1 with a signal-to-noise ratio >10. The method was validated for recovery (80-110%), repeatability (RSD% <5%), and linearity (r2 = 0.9999, at a tested working concentration range of 0.01-1.0 mg S L-1), and the accuracy was assessed by comparing with HPLC-ESIMS/MS which showed agreement within ±20%. This work demonstrates the applicability of HPLC-ICPMS/MS for sulfur speciation at trace levels in a matrix with complex sulfur metabolome as human urine and provides a sensitive method for the determination of the hydrogen sulfide biomarker.
    Keywords:  HPLC; Hydrogen sulfide; ICPMS/MS; Sulfur speciation
  26. Se Pu. 2022 Dec;40(12): 1119-1127
      The main methods currently used to detect illegally added chemicals in cosmetics include thin-layer chromatography, high performance liquid chromatography (HPLC), gas chromatography (GC), and liquid chromatography-mass spectrometry (LC-MS). Compared with other analytical techniques, these methods have the advantages of high sensitivity, specificity, and accuracy, all of which are required in practical detection work. However, they also present a number of limitations, such as long analysis times and requirements for skilled operators and strictly controlled laboratory environments. Supervision, a growing trend in market surveillance, requires rapid and effective methods to screen illegally added chemicals. The suspected samples are sealed for some time and then sent to the laboratory for further testing. Ion mobility spectrometry (IMS) is a new type of trace gas separation technology that was developed in recent years. The principle behind IMS is the separation and characterization of chemical species based on differences in the migration speed of their gas-phase ions under an electric field. As this technology has the advantages of miniaturization, easy operation, and quick responses, it is widely used in food and drug quality testing, as well as other related fields. However, it is rarely used in cosmetic detection, likely because the cosmetics matrix is highly complex, which can interfere with ion determination. Thus, optimizing the pretreatment process of cosmetics for IMS is important. In this work, solid-phase extraction (SPE) is combined with IMS to establish a method for the rapid screening of 14 antibacterial drugs in anti-acne cosmetics. The IMS detection parameters, sample extraction conditions, and SPE clean-up conditions (SPE column, type of leachate, type and volume of eluent) were studied and optimized in detail. The sample was extracted with 80%(v/v) acetonitrile aqueous solution (containing 0.2% (mass fraction) trichloroacetic acid), loaded onto an activated Oasis® MCX SPE column, leached with 3.0 mL of methanol, and eluted with 1.0 mL of 2% ammonia methanol solution. The eluate was then directly injected into the IMS instrument. The IMS parameters were as follows: positive ion source voltage=2200 V, transfer tube voltage=8000 V, inlet temperature=180 ℃, transfer tube temperature=180 ℃, ion gate voltage=50 V, gate voltage pulse width=85 μs, and migration gas flow rate=1.2 L/min. The migration times for the 14 antibacterial drugs ranged from 11 to 17 ms, and the detection limits for the target compounds ranged from 0.2 to 1.2 μg/g. Owing to the narrow linear range of IMS, a quantitative method employing HPLC was also established to optimize the SPE pretreatment step and verify the positive samples. Chromatographic separation was conducted on a Phenomenex Luna C18 column (250 mm×4.6 mm, 5 μm), with a column flow rate of 1.0 mL/min and gradient elution with mobile phases A (0.01 mol/L potassium dihydrogen phosphate adjusted to pH 4.0 with phosphoric acid) and B (acetonitrile). The column temperature was set to 35 ℃, and the injection volume was fixed at 5 μL. A total of 25 cosmetics samples were screened, and one positive sample was found to be consistent with the results of HPLC. The proposed method is fast, simple, and efficient, and it can be used for the rapid screening of the 14 antibacterial drugs in anti-acne cosmetics. Pretreatment can significantly reduce the influence of the cosmetic matrices on the determination results, improve instrument sensitivity, and effectively decrease the occurrence rate of false positives and negatives. The technique developed in this work can improve the efficiency of screening for illegally added chemicals and expand the applications of IMS for detecting various chemicals in complex matrices, such as cosmetics.
    Keywords:  anti-acne cosmetics; antibacterial drugs; ion mobility spectrometry (IMS); rapid screening; solid-phase extraction (SPE)
  27. Forensic Toxicol. 2022 Jul;40(2): 357-365
      PURPOSE: Prevalence measures of sociological interest concerning cocaine presence on banknotes are fraught with (i) the extreme variability of its concentration (seven orders of magnitude); (ii) the high number of banknotes needed for the statistical significance. Banknote dust from counting machines from a large and representative number of banknotes in circulation in a specific area represents the most eligible sample to ascertain cocaine circulation. No chromatographic method is available in this respect. This study aims at developing the first analytical methodology for the determination of cocaine in banknote dust samples.METHODS: This novel and straightforward approach consists of a simple methanol extraction followed by analytical determinations via ultra-high performance liquid chromatography coupled to Orbitrap high-resolution mass spectrometry.
    RESULTS: Satisfactory analytical performance was obtained with a coefficient of determination of 0.996; maximum within-run and between-run precisions were, respectively, 1.85% and 5.20%. Limits of detection and quantification were, respectively, 3 and 9 ng/mL with an overall process efficiency of 93.2%. The method developed was successfully applied to 9 banknote dust samples from local banknote counter machines. The found concentrations ranged from 2.18E + 02 to 2.31E + 03 μg of cocaine per gram of banknote dust and varied only one order of magnitude, much less than cocaine concentration on banknotes.
    CONCLUSIONS: To have an idea of cocaine circulation in a geographical area, the sampling of banknote dust, compared to banknotes, consists of tremendous advantages in terms of statistical significance, higher cocaine concentrations, and lower variability: this is crucial from the sociological point of view.
    Keywords:  Bills; Higher-energy collisional dissociation; Illicit drugs; Inclusion list; Ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry; Validation
  28. Forensic Toxicol. 2022 Jul;40(2): 322-331
      PURPOSE: We have developed and validated a high-sensitivity method to quantify lysergic acid diethylamide (LSD) and 2-oxo-3-hydroxy-LSD (OH-LSD) in oral fluid samples using liquid-liquid extraction and liquid chromatography-tandem mass spectrometry (LC‒MS/MS). The method was applied to the quantification of both substances in 42 authentic oral fluid samples.METHODS: A liquid-liquid extraction was performed using 500 µL each of samples (oral fluid samples collected using Quantisal™ device) and dichloromethane/isopropanol mixture (1:1, v/v). Enzymatic hydrolysis was evaluated to cleave glucuronide metabolites.
    RESULTS: The limit of quantification was 0.01 ng/mL for both LSD and OH-LSD. The linearity was assessed between 0.01 and 5 ng/mL. Imprecision and bias were not higher than 10.2% for both analytes. Extraction recovery was higher than 69%. The analytes were stable in the autosampler at 10 °C for 24 h, and up to 30 days at 4 and -20 °C. The method was applied to the analysis of 42 oral fluid samples. LSD was detected in all samples (concentrations between 0.02 and 175 ng/mL), and OH-LSD was detected in 20 samples (concentrations between 0.01 and 1.53 ng/mL).
    CONCLUSIONS: A high-sensitive method was fully validated and applied to authentic samples. To our knowledge, this is the first work to report concentrations of LSD and OH-LSD in authentic oral fluid samples.
    Keywords:  2-Oxo-3-hydroxy-LSD (OH-LSD); Authentic samples; LC‒MS/MS; Lysergic acid diethylamide (LSD); Oral fluid
  29. Se Pu. 2022 Dec;40(12): 1095-1101
      Sodium nitrophenolate (SNP) is a widely used universal growth regulator consisting of 5-nitroguaiacol sodium (5NG), 4-nitrophenol sodium (PNP), and 2-nitrophenol sodium (ONP). SNP has a positive influence on plants and animals as a feed additive that accelerates growth but is potentially hazardous to humans. SNP has been reported to be cytotoxic and mutagenic, which may increase the risk of cancer and pose a great threat to food safety. There are neither mature detection nor standard methods for the trace analysis of SNP in animal food. Therefore, the development of an accurate and precise analytical method is imperative. This innovative method has theoretical and practical significance for the control of SNP residues, offering advantages such as cost-effectiveness and time efficiency. It will be beneficial for the establishment of detection standards and management measures in foodstuffs of animal origin.In this study, a reliable method for the simultaneous determination of SNP residues in animal food (porcine muscle, chicken tissue, fish, and liver) was developed. For realizing the perfect limit of quantification, the application of back extraction coupled with high performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) was proposed to combine high sensitivity and high selectivity. The optimal method was as follows. First, 2.0 g samples were extracted with 10 mL 0.5 mol/L sodium hydroxide solution, followed by adjustment of the pH to acidity with 3 mol/L hydrochloric acid and the addition of sodium chloride (5.0 g) to saturate the inorganic phase. After back-extraction twice with 16 mL acetonitrile, the solution was merged and again saturated with 5 mL of sodium chloride solution. Second, the merged organic phase was cleaned up with 10 mL of n-hexane for defatting. The middle acetonitrile layer was then concentrated to nearly 1.5 mL at 40 ℃ in a N2 stream before dilution with the mobile phase to a volume of 3.0 mL and filtered. Finally, the analytes were separated on a C18 column (100 mm×4.6 mm, 3 μm) and subjected to gradient elution with a mixed solution of methanol and water. Mass spectrometric analysis, which was quantified using the external standard method, was carried out with an atmospheric pressure chemical ionization negative ion source and based on multiple reaction monitoring (MRM) mode. The key parameters, such as the extraction solvent, extraction steps, and purification method, were optimized.The calibration curves were linear in the ranges of 0.5-10 (5NG), 1.0-20 (PNP), and 2.5-50 μg/L (ONP) with correlation coefficients greater than 0.9999. The limit of quantification (LOQ) for 5NG was 1.0 μg/kg, double for PNP, and five times for ONP. The recoveries of the three different concentration levels in all the four matrices were in the range of 81.5%-98.4%, 81.5%-102%, and 81.4%-95.1%. The repeatability, expressed as the relative standard deviations (RSDs) of the three compounds, ranged from 1.51% to 5.98%, 1.10% to 8.85% and 0.91% to 8.61% (n=6). The developed method is characterized by an excellent purification effect, sensitivity, and accuracy. This method is suitable for the simultaneous and quantitative determination of SNP residues in foodstuffs of animal origin.
    Keywords:  atmospheric pressure chemical ionization (APCI); foodstuffs of animal origin; high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS); sodium nitrophenolate
  30. J Nutr Sci Vitaminol (Tokyo). 2022 ;68(Supplement): S116-S118
      Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is commonly used in food and pharmacological sciences to visualize localization of drugs and food compounds and their metabolites in plant, animal, and human tissues. The localization of compounds obtained by MALDI-MSI images provides useful information for elucidating their physiological and pharmacological properties. Food polyphenols, naturally occurring in tea, coffee, fruits and vegetables, have health benefits owing to their preventative effects against conditions such as cancer, diabetes, and cardiovascular diseases. In order to elucidate the pharmacological properties of polyphenols, their absorption, distribution, metabolism and excretion must be investigated. However, application of MALDI-MS imaging for polyphenols is challenging due to lack of an appropriate matrix reagent to visualize polyphenols in targeted biological tissue. The present work highlights the development of MALDI-MSI for visualization of food polyphenols. Nifedipine, which produces a nitrosophenyl pyridine derivative under laser irradiation, could be a new matrix for MS detection of polyphenols. The combination of nifedipine and phytic acid (a metal-chelating agent) successfully achieved MS visualization of polyphenols in biological tissue. The inhibitor-aided MALDI-MSI has been applied for elucidation of intestinal absorption routes and metabolic behaviors of polyphenols. The MALDI-MSI technique shows great potential for visualizing absorption, distribution and metabolism processes of food polyphenols.
    Keywords:  MALDI-MSI; polyphenols; visualization
  31. Anal Chem. 2022 Nov 27.
      The current analytical methods of stable antimony isotopes are cumbersome and not suitable for rock samples with low antimony content (<1 μg/g). In this study, we propose a new protocol for antimony isotopic analysis with a single column of AG50W-X8 resin and antimony standard doping. This method separates antimony effectively from matrices and then mixes it with the Sb standard. As Te does not affect the accuracy of antimony measurement when the Te/Sb ratio is low, we can obtain an accurate Sb isotope composition of the mixture. Then, we can calculate the antimony isotope composition of natural samples. The error propagation of the mixing and calculation processes was evaluated by the Monte Carlo method, and no significant error was found. The antimony isotope compositions were measured using a Thermo Fisher Scientific Neptune Plus multicollector-inductively coupled-mass spectrometry instrument. The instrumental mass bias of Sb isotopes was corrected with a standard-sample bracketing combined with a Sn internal normalization technique. Using the standard doping method, the measured δ123Sb values of standard solutions (Alfa, SPEX, GSB, and SCP) relative to NIST SRM 3102a were 0.02 ± 0.03‰ (2SD, N = 50), 0.29 ± 0.03‰ (2SD, N = 15), 0.24 ± 0.03‰ (2SD, N = 56), and 0.30 ± 0.03‰ (2SD, N = 15), respectively. The reproducibility for δ123Sb was better than 0.03‰ (2SD) throughout one year. This methodology has been testified by geological samples, yielding δ123Sb identical to the previously reported values. The actual Sb consumption for each sample test is as low as 5 ng. This standard doping method provides new insights into the analytical strategy of stable isotopes.
  32. Forensic Toxicol. 2022 Jul;40(2): 263-277
      PURPOSE: Zolpidem (ZOL) is a hypnotic sometimes used in drug-facilitated crimes. Understanding ZOL metabolism is important for proving ZOL intake. In this study, we synthesized standards of hydroxyzolpidems with a hydroxy group attached to the pyridine ring and analyzed them to prove their presence in postmortem urine. We also searched for novel ZOL metabolites in the urine sample using liquid chromatography-triple quadrupole mass spectrometry (LC-QqQMS) and liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QqTOFMS).METHODS: 7- and 8-Hydroxyzolpidem (7OHZ and 8OHZ, respectively) were synthesized and analyzed using LC-QqQMS. Retention times were compared between the synthetic standards and extracts of postmortem urine. To search for novel ZOL metabolites, first, the urine extract was analyzed with data-dependent acquisition, and the peaks showing the characteristic fragmentation pattern of ZOL were selected. Second, product ion spectra of these peaks at various collision energies were acquired and fragments that could be used for multiple reaction monitoring (MRM) were chosen. Finally, MRM parameters were optimized using the urine extract. These peaks were also analyzed using LC-QqTOFMS.
    RESULTS: The presence of 7OHZ and 8OHZ in urine was confirmed. The highest peak among hydroxyzolpidems was assigned to 7OHZ. The novel metabolites found were zolpidem dihydrodiol and its glucuronides, cysteine adducts of ZOL and dihydro(hydroxy)zolpidem, and glucuronides of hydroxyzolpidems.
    CONCLUSIONS: The presence of novel metabolites revealed new metabolic pathways, which involve formation of an epoxide on the pyridine ring as an intermediate.
    Keywords:  Cysteine adducts; Dihydrodiol; LC-QqQMS; LC-QqTOFMS; Structural elucidation; Zolpidem
  33. J Sep Sci. 2022 Nov 30.
      Propolis is a bee product with a complex chemical composition formed by several species from different geographical origins. The complex propolis composition requires an accurate and reproducible characterization of samples to standardize the quality of the material sold to consumers. This work developed an Ultra-high-performance liquid chromatography with a photodiode array detector method to analyze propolis phenolic compounds based on the two key propolis biomarkers, Artepillin C and p-Coumaric acid. This choice was made due to the complexity of the sample with the presence of several compounds. The optimized method was hyphenated with mass spectrometry detection allowing the detection of 23 different compounds. A step-by-step strategy was used to optimize temperature, flow rate, mobile phase composition, and re-equilibration time. Reverse-phase separation was achieved with a C18 fused-core column packed with the commercially available smallest particles (1.3 nm). Using a fused-core column with ultra-high-performance liquid chromatography allows highly efficient, sensitive, accurate, and reproducible determination of compounds extracted from propolis with an outstanding sample throughput and resolution. Optimized conditions permitted the separation of the compounds in 5.50 min with a total analysis time (sample-to-sample) of 6.50 min. This article is protected by copyright. All rights reserved.
    Keywords:  Artepillin C; Fast analysis; Fused-core; Propolis; p-Coumaric acid
  34. Proc Natl Acad Sci U S A. 2022 Dec 06. 119(49): e2208458119
      Determining mechanism of action (MOA) is one of the biggest challenges in natural products discovery. Here, we report a comprehensive platform that uses Similarity Network Fusion (SNF) to improve MOA predictions by integrating data from the cytological profiling high-content imaging platform and the gene expression platform Functional Signature Ontology, and pairs these data with untargeted metabolomics analysis for de novo bioactive compound discovery. The predictive value of the integrative approach was assessed using a library of target-annotated small molecules as benchmarks. Using Kolmogorov-Smirnov (KS) tests to compare in-class to out-of-class similarity, we found that SNF retains the ability to identify significant in-class similarity across a diverse set of target classes, and could find target classes not detectable in either platform alone. This confirmed that integration of expression-based and image-based phenotypes can accurately report on MOA. Furthermore, we integrated untargeted metabolomics of complex natural product fractions with the SNF network to map biological signatures to specific metabolites. Three examples are presented where SNF coupled with metabolomics was used to directly functionally characterize natural products and accelerate identification of bioactive metabolites, including the discovery of the azoxy-containing biaryl compounds parkamycins A and B. Our results support SNF integration of multiple phenotypic screening approaches along with untargeted metabolomics as a powerful approach for advancing natural products drug discovery.
    Keywords:  metabolomics; natural products; pharmacology
  35. Talanta. 2022 Nov 14. pii: S0039-9140(22)00885-2. [Epub ahead of print]254 124089
      The use of vacuum jacketed LC columns (VJC) to minimize on- and post-column band broadening to maximize chromatographic performance has been evaluated as a potential route to improved high throughput (HT) analysis. Here the use of the "VJC" approach has been applied to the HT bioanalysis of the antidiabetic GPR40 agonist drug fasiglifam in rat plasma samples obtained following a 5 mg/kg IV dose. The data obtained from a 1 minute VJC/MS-based analysis showed significant improvements compared to that from a conventional 2 minute UHPLC method for the drug. Notably, using VJC/MS with the rapid 1 min analysis provided a ca. 50% reduction in peak width coupled with a 2-5 fold higher peak response whilst doubling analytical throughput when compared to a conventional UHPLC/MS method. In addition, the increased resolution provided by the VJC system also improved the separation of fasiglifam from common matrix interferences such as co-extracted phospholipids thereby reducing the potential for matrix effects. The concatenation of these improvements suggests that the VJC approach may indeed provide a pathway to more sensitive, robust and high throughput drug bioanalysis, with particular advantages for drug discovery applications.
    Keywords:  Bioanalysis; Fasiglifam; Sensitivity; UHPLC-MS/MS; Vacuum jacketed columns
  36. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Nov 20. pii: S1570-0232(22)00440-8. [Epub ahead of print]1213 123535
      The esters of endogenous anabolic steroids are the most frequently used doping agents for prolonging the half-life of exogenously ingested endogenous anabolic steroids. As a cost- and time-saving matrix, dried blood spots (DBSs) are valuable for directly detecting endogenous anabolic steroid esters in blood and for providing conclusive evidence of their abuse. In this study, a method for simultaneous detection of 20 endogenous anabolic steroid esters in DBSs based on ultra-high performance liquid chromatography-quadrupole-Orbitrap mass spectrometry (UPLC-Q-Orbitrap-MS) with parallel reaction monitoring (PRM) was developed and validated, and 10 of these esters were analyzed in DBSs for the first time. This method analyzes the greatest number and types of endogenous anabolic steroid esters of any current method using DBSs. Girard's Reagent P (GRP) was used for the derivatization of endogenous anabolic steroid esters in a DBS matrix for the first time, and the conditions of the derivatization reaction were optimized to achieve a higher sensitivity compared to previous methods. Selectivity, limit of detection (LOD), extraction recovery, precision (intra- and inter-), matrix effects, and carry-over were analyzed to validate the method. The LODs were lower and the recoveries were higher than those of previous studies. The relative standard deviation of the intraday precision was below 20% and the interday precision was below 35%. A product ion analysis of GRP nandrolone ester, GRP boldenone ester, GRP dehydroepiandrosterone acetate, and GRP androstanolone ester derivatives was performed, and the structures of the fragment ions were proposed for the first time.
    Keywords:  Derivatization; Dried blood spots; Endogenous anabolic steroids esters; Girard's Reagent P; Method validation
  37. J AOAC Int. 2022 Dec 01. pii: qsac154. [Epub ahead of print]
      BACKGROUND: Cyanogenic glycosides are secondary metabolites in plants. In almonds and apricot kernels, amygdalin is an abundant cyanogenic glycoside. Upon consumption, amygdalin is enzymatically metabolized into hydrogen cyanide. Depending on the number of kernels consumed and the amygdalin concentration, ingestion of amygdalin-containing kernels may result in adverse effects. To better understand the U.S. marketplace, the development and validation of analytical methods to reliably measure amygdalin in apricot kernels and almonds is needed to support the collection of occurrence and consumption data in retail products.OBJECTIVE: The aim of this study was to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitation of amygdalin in apricot kernels and almonds following the FDA Foods Program Guidelines for the Validation of Chemical Methods, 3rd Edition.
    METHODS: Apricot kernels and almonds were cryogenically homogenized and extracted using methanol containing an internal standard (IS), geniposide, followed by filtration, dilution, and LC-MS/MS analysis. Matrix effects were minimized using dilution. Quantitation was achieved using an external, solvent-based calibration.
    RESULTS: The amygdalin response was linear (r2> 0.99) over a range of 0.05-50 µg/mL. The recovery of amygdalin spiked at 10 - 10,000 µg/g in sweet apricot kernels, raw almond, and dry roasted almond ranged from 90-107% with RSDs ≤ 6%. The method limit of detection (LOD) and limit of quantitation (LOQ) was 0.8 and 2.5 ng/g, respectively. Amygdalin concentrations in 18 market samples ranged from 2- 24,000 µg/g. Corresponding estimates of cyanide concentration ranged from 0.2-1420 µg/g.
    CONCLUSIONS: Method performance meets the acceptance criteria defined by FDA guidelines and is fit for purpose for the analysis of amygdalin in apricot kernels and almonds.
    HIGHLIGHTS: An LC-MS/MS method is developed for the quantification of amygdalin in apricot kernels and almonds.
  38. Bioanalysis. 2022 Dec 01.
      Aim: IDH mutations have been identified as frequent molecular lesions in several tumor types, particularly in gliomas. As a putative marker of IDH mutations, elevated D-2-HG has been reported in glioma, acute myeloid leukemia and intrahepatic cholangiocarcinoma. Excessive production of L-2-HG has also been described in renal cell carcinoma and 2-hydroxyaciduria. Materials & methods: The authors present a fully optimized stable isotope dilution multiple reaction monitoring method for quantification of D-/L-2-HG using LC-MS/MS. This is the first method validation study performed on cerebrospinal fluid, plasma and urine demonstrating clinical applicability with samples from glioma patients. Results & conclusion: This method validation study showed high accuracy and precision with low limit of detection and limit of quantification values. The authors believe that the presented approach is highly applicable for basic and clinical research on related pathologies.
    Keywords:  D-2-hydroxyglutaric acid; HPLC; L-2-hydroxyglutaric acid; biomarker; glioma; isocitrate dehydrogenase mutation; stable isotope dilution multiple reaction monitoring; tandem mass spectrometry
  39. Oxid Med Cell Longev. 2022 ;2022 6876327
      Background: Benign essential blepharospasm (BEB) is a form of focal dystonia that causes excessive involuntary spasms of the eyelids. Currently, the pathogenesis of BEB remains unclear. This study is aimed at investigating the serum metabolites profiles in patients with BEB and healthy control and to identify the mechanism and biomarkers of this disease.Methods: 30 patients with BEB and 33 healthy controls were recruited for this study. We conducted the quantitative and nontargeted metabolomics analysis of the serum samples from 63 subjects by using liquid chromatography and Orbitrap mass spectrometry (LC-Orbitrap MS). Multivariate statistical analysis was performed to detect and identify different metabolites between the two groups. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and receiver operating characteristic (ROC) curve analysis of the altered metabolites were performed.
    Results: A total of 134 metabolites were found and identified. The metabolites belonged to several metabolic pathways including phenylalanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, arginine biosynthesis, linoleic acid metabolism, tryptophan metabolism, aminoacyl-tRNA biosynthesis, sphingolipid metabolism, glycosphingolipid biosynthesis, leucine and isoleucine biosynthesis, and vitamin B6 metabolism. Eight metabolites were identified as the potential biomarkers.
    Conclusions: These results demonstrated that serum metabolic profiling of BEB patients was significantly different from healthy controls based on LC-Orbitrap MS. Besides, metabolomics might provide useful information for a better understanding of BEB.
  40. J Chromatogr A. 2022 Nov 04. pii: S0021-9673(22)00822-6. [Epub ahead of print]1687 463631
      A fast and reliable method for the direct determination of the herbicide glyphosate, its major degradation product aminomethylphosphonic acid (AMPA) and glufosinate is presented for a variety of food matrices. The Quick Polar Pesticides in food of Plant Origin method (QuPPe-PO-Method) was used for extraction without further preconcentration or clean-up steps involving e.g. solid phase extraction (SPE). The method makes use of a commercially available high performance liquid chromatograph coupled to a tandem mass spectrometer with electrospray ionization (LC-ESI-MS/MS) - as present in many laboratories - equipped with an ion chromatography (IC)-column using an MS-compatible eluent made of 0.8% formic acid in water. Due to the absence of time-consuming clean-up procedures, strong matrix effects (ME) of up to 91% for AMPA in grapefruit can be observed, when comparing its sensitivity to that obtained for solvent-based standards. The limits of detection (LODs) were determined for the sample matrices apple, mushrooms, grapefruit, linseed, red lentils and wheat and they were found to be in the range of 0.09 to 0.8, 0.04 to 1 and 0.2 to 2 µg/kg for glyphosate, AMPA and glufosinate, respectively. For the same matrices the validation was carried out according to SANTE guidelines for different commodity groups by spiking them up prior to extraction to concentrations ranging from 10 to 400 µg/kg for matrices with high water content and from 10 to 800 µg/kg for matrices with low water content. When using solvent-based calibration under the use of isotopically labelled internal standards (ILIS) the recoveries were found to range from 84% to 120% and the relative standard deviations (RSD) range between 1% and 19% for glyphosate, AMPA and glufosinate at all fortification levels for all matrices investigated. Accordingly, the method was successfully introduced in our laboratory with limits of quantification (LOQs) of 10 µg/kg for glyphosate, AMPA and glufosinate in samples from SANTE commodity groups 1, 2, 4a and 5. The reliability and robustness of the method are demonstrated by showing a recovery control chart obtained for glyphosate in randomly selected samples from different commodity groups. Therefore, the samples were spiked up with 10 µg/kg of glyphosate during routine analysis, whereby all recoveries were found to be in the range between 70 and 120%.
    Keywords:  Direct determination; Glyphosate; Ion chromatography; LC-MS/MS; QuPPe-PO-Method
  41. Se Pu. 2022 Dec;40(12): 1064-1075
      In order to monitor the risk of pesticide pollutants in drinking water, an analytical method based on online-solid phase extraction coupled with ultra performance liquid chromatography-triple quadrupole mass spectrometry (online-SPE-UPLC-MS/MS) was established for the simultaneous rapid screening and determination of 107 pesticides and metabolites (organophosphorus, organic nitrogen, organic heterocycle, carbamate, amide, benzoyl urea, neonicotinoid, etc.) in raw water and drinking water. Different injection volumes (5, 10, and 15 mL) were compared. The detection response increased with an increase in the injection volume, but the matrix effect also became more pronounced. Under the premise of ensuring the sensitivity of the method and meeting the detection requirements, the injection volume was selected as 5 mL. Accordingly, the samples were filtered through a 0.22-μm hydrophilic polytetrafluoroethylene filter, and then, 5 mL samples were injected into the online-SPE system by the automatic sampler. After adsorption on an X Bridge C18 online-SPE column, the samples were washed with pure water and eluted by gradient elution using acetonitrile and 0.1% formic acid aqueous solution as the mobile phases, with separation on an ACQUITY HSS T3 column. The samples were detected by multiple reaction monitoring with electrospray ionization in positive and negative ion modes, and quantified by an external standard method. Using raw water and drinking water as the sample matrices, the accuracy and precision of the method were verified. The 107 pesticides and metabolites showed good linear relationships in different ranges with correlation coefficients (r2)>0.995. The limits of detection (LODs, S/N=3) of the method were 0.03-1.5 ng/L, and the limits of quantification (LOQs, S/N=10) were 0.1-5.0 ng/L. The target pesticides were spiked at concentration levels of 1, 20, and 50 ng/L. The spiked recoveries of the 107 targets in raw water and drinking water samples were 60.6%-119.8% and 61.2%-119.0%, respectively. The corresponding relative standard deviations (RSDs, n=6) were 0.3%-18.6% and 0.4%-17.1%. The pesticide residues in raw water and drinking water were determined by this method. Amide herbicides, triazine herbicides, triazole insecticides, carbamate insecticides, and neonicotinoid insecticides had high detection rates. The detected concentrations ranged from 0.1 to 97.1 ng/L in raw water and from 0.1 to 93.6 ng/L in drinking water. The sample consumption of online-SPE method was lower than that in the traditional off-line SPE methods, which greatly improved the convenience of sample collection, storage, and transportation. The samples only need to be filtered before injection and analysis. The method is simple to operate and shows good reproducibility. With this online-SPE method, only 23 min were required from online enrichment to detection completion. The developed method has the advantages of high analytical speed and high sensitivity. The method is suitable for the trace analysis and determination of 107 typical pesticides in raw water and drinking water, which effectively improves the detection efficiency of pesticides in water and has high potential for practical application. It can extend technical support for the pollution-level analysis of typical pesticides and metabolites in drinking water and provide an objective basis for human health risk assessment.
    Keywords:  drinking water; online-solid phase extraction (online-SPE); pesticides; raw water; ultra performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS)
  42. Anal Chem. 2022 Dec 01.
      Metabolic profiling is widely used for large-scale association studies, based on biobank material. The main obstacle to the translation of metabolomic findings into clinical application is the lack of standardization, making validation in independent cohorts challenging. One reason for this is sensitivity of metabolites to preanalytical conditions. We present a systematic investigation of the effect of delayed centrifugation on the levels of NMR-measured metabolites and lipoproteins in serum and plasma samples. Blood was collected from 20 anonymous donors, of which 10 were recruited from an obesity clinic. Samples were stored at room temperature until centrifugation after 30 min, 1, 2, 4, or 8 h, which is within a realistic time scenario in clinical practice. The effect of delaying centrifugation on plasma and serum metabolic concentrations, and on concentrations of lipoprotein subfractions, was investigated. Our results show that lipoproteins are only minimally affected by a delay in centrifugation while metabolite levels are more sensitive to a delay. Metabolites significantly increased or decreased in concentration depending on delay duration. Further, we describe differences in the stability of serum and plasma, showing that plasma is more stable for metabolites, while lipoprotein subfractions are equally stable for both types of matrices.
  43. Anal Chem. 2022 Nov 28.
      In comprehensive two-dimensional liquid chromatography (LC × LC), solvents of high eluotropic strength are frequently used in the first dimension (1D), which lead to peak broadening in the second dimension (2D). In the majority of the current LC × LC column combinations, analytes are less than optimally refocused upon transfer to the second column, which negatively affects sensitivity. Furthermore, the typical combination of 1 or 2.1 mm columns in the 1D paired with a 3 mm (or broader) column in the 2D leads to at least a 9- or 4-fold dilution and a corresponding loss of sensitivity when using concentration-sensitive detectors. This occurs due to the enhanced radial dilution of the analytes in a broader column, while the sensitivity problem is further exacerbated in LC × LC due to the high flow operated 2D. In this paper, we introduce a solution to neutralize and inverse this dilution problem through a reconcentrating solution using temperature-responsive liquid chromatography (TRLC) in the 1D, which is a purely aqueous separation mode. Full solute refocusing at the 2D column head is thereby obtained when TRLC is combined with reversed-phase liquid chromatography (RPLC). This is shown for the combination of a 2.1 mm I.D. TRLC column with decreasing RPLC column diameters (3-2.1-1 mm) operated at the same linear velocities, hence a resulting decrease in dilution, respectively. Ultraviolet (UV) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) detection were used to determine the experimental detection limits. Sensitivity improvements with UV detection were somewhat lower than expected, but represent ∼1.5- and 3-fold sensitivity enhancement when using a 1 mm I.D. column compared to 2.1 or 3 mm I.D. columns in the 2D, respectively. This is attributed to extra-column dispersion and the poorer performance of 1 mm I.D. columns. A major benefit of the use of 1 mm I.D. columns in the 2D is that it allows split-free coupling of 2D effluent with ESI-MS (at 450 μL/min), making the coupling robust and simple. When using ESI-MS even better, albeit more variable, sensitivity enhancements were obtained on the narrower columns. The benefits of the methodology are demonstrated for paraben test solutes and for phenolic compounds in a blueberry extract by TRLC × RPLC-UV-ESI-TOF-MS.
  44. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Nov 15. pii: S1570-0232(22)00442-1. [Epub ahead of print]1213 123537
      IBP (2,6-diisobornyl-4-methylphenol) is a small drug molecule with antioxidant properties considered to be a promising neuro-, cardio-, and retinoprotective agent. In this study, a bioanalytical LC-MS/MS method for its determination in rat plasma was developed using 11H-indeno[1,2-b]quinoxalin-11-one oxime as an internal standard (IS). The analytes were extracted from plasma by liquid-liquid extraction technique using isopropyl alcohol:chloroform mixture (1:5, v/v) followed by evaporation and reconstitution of the residues in acetonitrile. The chromatographic separation was carried out on the EC Nucleodur C8 ec column (150 × 4.6 mm, 5 μm) under an isocratic elution mode using acetonitrile and water containing 0.1% (v/v) formic acid (97:3, v/v) as a mobile phase at a flow rate of 0.55 mL/min (40 °C). The IS and IBP were eluted at 3.79 ± 0.02 and 6.30 ± 0.02 min, respectively. The total analysis time was 7.00 min. Multiple reaction monitoring was used to conduct the MS/MS detection in the negative ion mode with transitions at m/z 245.9 → 214.9 (IS) and 379.2 → 256.0 (IBP). Validation studies of the developed method revealed good linearity over the range of 10-5,000 ng/mL. Within- and between-run accuracy was in the range of 92-110%, while within- and between-run precision was below 8%. Additionally, low matrix effects and high recovery (above 98%) were observed. IBP remained stable in rat plasma at room temperature for 4 h, at -80 °C for 21 days, over three freeze-thaw cycles, under vacuum concentrator (45 °C, dried residues) and auto-sampler (15 °C, processed samples) temperatures for 1 h and 24 h, respectively. Subsequently, the validated LC-MS/MS method has been successfully applied to quantitate IBP in actual plasma samples after a single oral, intramuscular, and subcutaneous dose of IBP (10 mg/kg in the peach oil) to rats. Pharmacokinetic studies show that more rapid and complete IBP absorption with a satisfactory excretion rate were observed after oral administration route compared to the intramuscular and subcutaneous ones.
    Keywords:  2,6-Diisobornyl-4-methylphenol; LC-MS/MS; Lipophilic drugs; Method validation; Pharmacokinetics; Phenolic antioxidants; Rat plasma