bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2019‒12‒01
eighteen papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Curr Protoc Protein Sci. 2019 Dec;98(1): e98
      Metabolomics refers to study of metabolites in biospecimens such as blood serum, tissues, and urine. Nuclear magnetic resonance (NMR) spectroscopy and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS; mass spectrometry coupled with liquid chromatography) are most frequently employed to analyze complex biological/clinical samples. NMR is a relatively insensitive tool compared to UPLC-MS/MS but offers straightforward quantification and identification and easy sample processing. One-dimensional 1 H NMR spectroscopy is inherently quantitative and can be readily used for metabolite quantification without individual metabolite standards. Two-dimensional spectroscopy is most commonly used for identification of metabolites but can also be used quantitatively. Although NMR experiments are unbiased regarding the chemical nature of the analyte, it is crucial to adhere to the proper metabolite extraction protocol for optimum results. Selection and implementation of appropriate NMR pulse programs are also important. Finally, employment of the correct metabolite quantification strategy is crucial as well. In this unit, step-by-step guidance for running an NMR metabolomics experiment from typical biospecimens is presented. The unit describes an optimized metabolite extraction protocol, followed by implementation of NMR experiments and quantification strategies using the so-called "targeted profiling" technique. This approach relies on an underlying basis set of metabolite spectra acquired under similar conditions. Some strategies for statistical analysis of the data are also presented. Overall, this set of protocols should serve as a guide for anyone who wishes to enter the world of NMR-based metabolomics analysis. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Metabolite extraction from different biospecimens Basic Protocol 2: Preparation of dried upper fraction for NMR analysis Alternate Protocol: Preparation of urine samples for NMR analysis Basic Protocol 3: NMR experiments Basic Protocol 4: Spectral processing and quantification of metabolites Basic Protocol 5: Statistical analysis of the data.
    Keywords:  NMR; metabolites; metabolomics; targeted profiling
  2. Anal Bioanal Chem. 2019 Nov 23.
      There is evidence of a positive association between per- and polyfluoroalkyl substances (PFASs) and cholesterol levels in human plasma, which may be due to common reabsorption of PFASs and bile acids (BAs) in the gut. Here we report development and validation of a method that allows simultaneous, quantitative determination of PFASs and BAs in plasma, using 150 μL or 20 μL of sample. The method involves protein precipitation using 96-well plates. The instrumental analysis was performed with ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS), using reverse-phase chromatography, with the ion source operated in negative electrospray mode. The mass spectrometry analysis was carried out using multiple reaction monitoring mode. The method proved to be sensitive, robust, and with sufficient linear range to allow reliable determination of both PFASs and BAs. The method detection limits were between 0.01 and 0.06 ng mL-1 for PFASs and between 0.002 and 0.152 ng mL-1 for BAs, with the exception of glycochenodeoxycholic acid (0.56 ng mL-1). The PFAS measured showed excellent agreement with certified plasma PFAS concentrations in NIST SRM 1957 reference serum. The method was tested on serum samples from 20 healthy individuals. In this proof-of-concept study, we identified significant associations between plasma PFAS and BA levels, which suggests that PFAS may alter the synthesis and/or uptake of BAs. Graphical Abstract.
    Keywords:  Bile acids; Human serum; LC; MS; PFAS; Perfluoroalkyl substances
  3. Metabolites. 2019 Nov 26. pii: E289. [Epub ahead of print]9(12):
      Routine diagnostic screening of inborn errors of metabolism (IEM) is currently performed by different targeted analyses of known biomarkers. This approach is time-consuming, targets a limited number of biomarkers and will not identify new biomarkers. Untargeted metabolomics generates a global metabolic phenotype and has the potential to overcome these issues. We describe a novel, single platform, untargeted metabolomics method for screening IEM, combining semi-automatic sample preparation with pentafluorophenylpropyl phase (PFPP)-based UHPLC- Orbitrap-MS. We evaluated analytical performance and diagnostic capability of the method by analysing plasma samples of 260 controls and 53 patients with 33 distinct IEM. Analytical reproducibility was excellent, with peak area variation coefficients below 20% for the majority of the metabolites. We illustrate that PFPP-based chromatography enhances identification of isomeric compounds. Ranked z-score plots of metabolites annotated in IEM samples were reviewed by two laboratory specialists experienced in biochemical genetics, resulting in the correct diagnosis in 90% of cases. Thus, our untargeted metabolomics platform is robust and differentiates metabolite patterns of different IEMs from those of controls. We envision that the current approach to diagnose IEM, using numerous tests, will eventually be replaced by untargeted metabolomics methods, which also have the potential to discover novel biomarkers and assist in interpretation of genetic data.
    Keywords:  HRAM-MS; IEM; LC-MS; Orbitrap; PFPP; PKU; inborn errors of metabolism; metabolomics; organic aciduria; urea cycle defects
  4. J Chromatogr A. 2019 Nov 13. pii: S0021-9673(19)31141-0. [Epub ahead of print] 460709
      Steroid hormones are a type of crucial substances that mediate numerous vital physiological functions. The comprehensive detection of steroid hormones can help understand the physiopathologic mechanism of steroid hormone-related diseases. It is very difficult to determine steroid hormones in biological samples due to their low endogenous concentrations and poor ionization efficiency. In this study, an efficient and sensitive approach was developed for profiling steroid hormones by combining liquid-liquid extraction and parallel derivatization with liquid chromatography-tandem mass spectrometry. Methoxyamine and dansyl chloride were used to derivatize steroid hormones containing carbonyl and phenolic hydroxyl groups, respectively. Our established method achieved simultaneous analysis of carbonyl and phenolic hydroxyl-containing steroid hormones and could cover estrogens, androgens, corticoids and progestogens. Twenty-nine steroid hormones were detected at pg/mL levels with the sensitivity enhanced by three orders of magnitude after derivatization. The linearity (with linear range of 2-4 orders of magnitude), precision (less than 15%) and recovery (71.1-128.7%) were satisfactory for quantitative analysis of steroid hormones. Finally, the established method was successfully employed to the determination of steroid hormones in serum samples of healthy males and females as well as ovarian cancer patients. The results showed that this approach was suitable and reliable for routine test of steroid hormones containing carbonyl and phenolic hydroxyl groups.
    Keywords:  Liquid chromatography-mass spectrometry; Parallel derivatization; Simultaneous quantification; Steroid hormones
  5. J Pharm Biomed Anal. 2019 Nov 18. pii: S0731-7085(19)32127-2. [Epub ahead of print] 112996
      BACKGROUND: Newborns, admitted to the Neonatal Intensive Care Unit (NICU), are exposed to a large number of medications, the majority of which are not labeled for use in infants, especially in preterm newborns, because clinical trials on their benefits and harms are lacking. There is a huge gap in knowledge on pharmacokinetic (PK) data in sick preterm infants, including that of drug penetration to cerebrospinal fluid (CSF). One of the issues is related to the lack of reliable analytical methods for the measurement of drugs in CSF.METHODS: In this paper we describe a specific and sensitive LC-MS/MS method for the simultaneous quantification in CSF of four commonly prescribed drugs in NICUs: caffeine, betamethasone, clonidine and furosemide.
    RESULTS: The method was validated following EMA guidelines and applied to several CSF samples of preterm infants with post-hemorrhagic ventricular dilatation in which a ventricular access device was applied. The range of the concentrations of the four drugs measured in the CSF was wide.
    CONCLUSIONS: Our method can be considered useful for further clinical studies to describe the PK aspects of these drugs in neonatal medicine.
    Keywords:  Betamethasone; Caffeine; Cerebrospinal fluid; Clonidine; Drugs; Furosemide; LC–MS/MS; Preterm
  6. J Pharm Biomed Anal. 2019 Nov 20. pii: S0731-7085(19)31900-4. [Epub ahead of print] 112999
      Sjögren's Syndrome (SjS) is a complex autoimmune disease characterized by the affection of the exocrine glands and the involvement of multiple organs. Although a greater number of biomarker studies have been carried out in recent years, the origin and pathogenesis are not yet well known and therefore there is a need to continue studying this pathology. This work aims to find metabolic changes in biological samples (plasma and urine), which could help identify the metabolic pathways affected by the SjS pathogenesis. The samples collected from SjS patients and healthy volunteers were analyzed by a fingerprinting metabolomic approach based on HPLC-ESI-QTOF-MS methodology. After feature pre-selection by univariate statistical tests, an integrated PLS-DA model using data from urine and plasma was constructed obtaining a good classification between cases and controls (AUROC = 0.839 ± 0.021). 31 and 38 metabolites in plasma and urine, respectively, showed significant differences between healthy volunteers and SjS patients and were proposed for their identification. From them, 12 plasma and 24 urinary metabolites could be annotated. In general, the main metabolic pathways altered in SjS patients were related to the metabolism of phospholipids, fatty acids, and amino acids, specially tryptophan, proline and phenylalanine.
    Keywords:  Biomarkers; HPLC-ESI-QTOF-MS; Metabolomics; Phosphatidylinositols; Sjögren’s syndrome; Unsaturated fatty acids
  7. J Proteome Res. 2019 Nov 27.
      Whilst many laboratories take appropriate care, there are still cases where the performances of untargeted profiling methods suffer from a lack of design, control and articulation of the various steps involved. This is particularly harmful to modern comprehensive analytical instrumentations that otherwise provide an unprecedented coverage of complex matrices. In this work, we present a global analytical workflow based on comprehensive two-dimensional gas chromatography (GC×GC) coupled to high resolution time-of-flight mass spectrometry (HR-TOF-MS). It was optimized for sample preparation and chromatographic separation, and validated on in-house QC samples and NIST SRM 1950 samples. It also includes a QC procedure, a multi-approaches data (pre)processing workflow and an original bias control procedure. Compounds of interest were identified using mass, retention and biological informations. As a proof of concept, 35 serum samples representing 3 subgroups of Crohn's disease (with high, low and quiescent endoscopic activity) were analyzed along with 33 healthy controls. This led to the selection of 31 unique candidate biomarkers able to classify Crohn's disease and healthy samples with OPLS-DA Q2 0.48 and ROC AUC 0.85 (100% sensitivity and 82% specificity in cross validation). 15 of these 33 candidates were reliably annotated (MSI level 2).
  8. Anal Chim Acta. 2020 Jan 15. pii: S0003-2670(19)31201-2. [Epub ahead of print]1094 57-69
      In this study, a combined targeted/untargeted UHPLC-ESI-QTOF-MS/MS method for the targeted quantitative analysis of the primary platelet lipid mediators thromboxane B2 (TXB2), 12S-hydroxy-5Z,8E,10E-heptadecatrienoic acid (HHT) and its oxidation product 12-keto-5Z,8E,10E-heptadecatrienoic acid (KHT) was developed, which allowed simultaneous untargeted profiling for the detection of other lipid biomarkers such as other oxylipins and fatty acids (FAs) in platelet releasates. A general procedure for the synthesis of keto-analogs from hydroxylated polyunsaturated FAs (PUFAs) using Dess-Martin periodinane oxidation reagent was proposed for the preparation of KHT standard. MS detection was performed in data independent acquisition (DIA) mode with sequential window acquisition of all theoretical fragment ion mass spectra (SWATH) in the range of 50-500 Da with variable window sizes. The LC-MS/MS assay was validated for the targeted analytes and applied for analysis of supernatants derived from resting platelets and from platelets treated with thrombin. The targeted analytes KHT, HHT and TXB2 were found at highly elevated levels in the activated platelet releasates. On average, 13 ± 7, 15 ± 9, and 0.6 ± 0.2 attomols per platelet were released upon thrombin-activation. Furthermore, the simultaneous untargeted profiling (n = 8 in each group) revealed that these oxylipins are released with a pool of other (significantly upregulated) oxidized (12-HETE, 12-HEPE) and non-oxidized PUFAs. All these compounds can be considered additional biomarkers of platelet activation complementing the primary platelet activation marker thromboxane B2. The other lipids may support platelet activation or trigger other biological actions with some potential implications in thromboinflammation.
    Keywords:  Intercellular platelet signaling; Lipid mediators; Lipidomics; Oxylipins; Platelet activation; SWATH
  9. Saudi J Biol Sci. 2019 Nov;26(7): 1843-1847
      A highly sensitive and ultra-fast high performance liquid chromatography- tandem mass spectrometry (LC-MS/MS) assay is developed and validated for the quantification of Lenalidomide in human plasma. Lenalidomide is extracted from human plasma by Liquid- Liquid Extraction by Ethyl Acetate and analyzed using a reversed phase isocratic elution on a XTerra RP18, (4.6 × 50 mM, 5 µm) column. A 0.1% Formic acid: Methanol (10:90% v/v), is used as mobile phase and detection was performed by Triple quadrupole mass spectrometry LC-MS/MS using electrospray ionization in positive mode. Fluconazole is used as the internal standard. The lower limit of quantification is 9.999 ng/mL for Lenalidomide. The calibration curves are consistently accurate and precise over the concentration range of 9.999 to 1010.011 ng/mL in plasma for Lenalidomide. This novel LC-MS/MS method competes with all the regulatory requirements and shows satisfactory accuracy and precision and is sufficiently sensitive for the performance of pharmacokinetic and bioequivalence studies in humans.
    Keywords:  Human plasma; LC-MS/MS; Lenalidomide; Liquid-liquid extraction; Validation
  10. Curr Drug Metab. 2019 Nov 24.
      BACKGROUND: Allergic diseases are considered among the major burdons of public health with increased prevalence globally. Histamine H1-receptor antagonists are the foremost commonly used drugs in the treatment of allergic disorders. Our target drug is one of this class, loratadine and its biometabolite desloratadine which is also a non sedating H1 receptor antagonist with anti-histaminic action of 2.5 to 4 times greater than loratadine. To develop and validate a novel isocratic reversed-phase high performance liquid chromatography (RP-HPLC) method for rapid and simultaneous separation and determination of loratadine and its metabolite, desloratadine in human plasma.METHODS: The drug extraction method from plasma was based on protein precipitation technique. The separation was carried out on a Thermo Scientific BDS Hypersil C18 column (5µm, 250 x 4.60 mm) using a mobile phase of MeOH : 0.025M KH2PO4 adjusted to pH 3.50 using orthophosphoric acid (85 : 15, v/v) at ambient temperature. The flow rate was maintained at 1 mL/min and maximum absorption was measured using PDA detector at 248 nm.
    RESULTS: The retention times of loratadine and desloratadine in plasma samples were recorded to be 4.10 and 5.08 minutes respectively, indicating a short analysis time. Limits of detection were found to be 1.80 and 1.97 ng/mL for loratadine and desloratadine, respectively, showing a high degree of method sensitivity. The method was then validated according to FDA guidelines for the determination of the two analytes in human plasma.
    CONCLUSION: The results obtained indicate that the proposed method is rapid, sensitive in the nanogram range, accurate, selective, robust and reproducible compared to other reported methods.
    Keywords:  Desloratadine; FDA guidelines; Human plasma; Loratadine; Protein precipitation; RP-HPLC
  11. Biomolecules. 2019 Nov 26. pii: E779. [Epub ahead of print]9(12):
      Polyamines are involved in the regulation of many cellular functions and are promising biomarkers of numerous physiological conditions. Since the concentrations of these compounds in biological fluids are low, sample extraction is one of the most critical steps of their analysis. Here, we developed a comprehensive, sensitive, robust, and high-throughput LC-MS/MS stable-isotope dilution method for the simultaneous determination of 19 metabolites related to polyamine metabolism, including polyamines, acetylated and diacetylated polyamines, precursors, and catabolites from liquid biopsies. The sample extraction was optimized to remove interfering compounds and to reduce matrix effects, thus being useful for large clinical studies. The method consists of two-step liquid-liquid extraction with a Folch extraction and ethyl acetate partitioning combined with dansyl chloride derivatization. The developed method was applied to a small gender-related trial concerning human serum and urine samples from 40 obese subjects. Sex differences were found for cadaverine, putrescine, 1,3-diaminopropane, γ-aminobutyric acid, N8-acetylspermidine, and N-acetylcadaverine in urine; N1-acetylspermine in serum; and spermine in both serum and urine. The results demonstrate that the developed method can be used to analyze biological samples for the study of polyamine metabolism and its association with human diseases.
    Keywords:  LC-MS/MS; acetylpolyamines; liquid biopsies; obesity; polyamine metabolism; polyamines
  12. Anal Methods. 2019 Jan 07. 11(1): 49-57
      Methylation aberrations play an important role in many metabolic disorders including cancer. Methylated metabolites are direct indicators of metabolic aberrations, and currently, there is no Liquid chromatography - Mass spectrometry (LC-MS) based method available to cover all classes of methylated metabolites at low detection limits. In this study, we have developed a method for the detection of methylated metabolites, and it's biological application. In this approach, we used a HILIC based HPLC with MS to measure methylated organic acids, amino acids, and nucleotides. These metabolites were separated from each other by their hydrophobic interactions and analyzed by targeted metabolomics of single reaction monitoring by positive and negative mode of electrospray ionization. These metabolites were quantified, and the interday reproducibility was <10% relative standard deviation. Furthermore, by applying this method, we identified high levels of methylated metabolites in bladder cancer cell lines compared to benign cells. In vitro treatment of cancer cells with methylation inhibitor, 5- aza-2'-deoxycytidine showed a decrease in these methylated metabolites. This data indicates that HPLC analysis using this HILIC based method could be a powerful tool for measuring methylated metabolites in biological specimens. This method is rapid, sensitive, selective, and precise to measure methylated metabolites.
  13. Anal Bioanal Chem. 2019 Nov 23.
      Polar lipids, especially glycerophospholipids, constitute the main components of cell membranes and are precursors of signaling molecules in many cellular and physiological processes. For this reason, the development of methods with high capability for detection of polar lipids in biological samples is required. In this research, the objective was to develop a method for comprehensive qualitative/quantitative determination of polar lipids in plasma by a combination of acquisition methods with a triple quadrupole mass analyzer. The strategy was optimized in two steps: (a) a first step for detection of lipids by monitoring selective fragmentation patterns representative of each lipid family and (b) a second step for confirmation of lipid species by detection and identification of product ions associated with the conjugated fatty acids. The acquisition list was divided into two multiple reaction monitoring (MRM) methods to ensure the detection of all transitions with suited instrumental sensitivity according to chromatographic retention time and relative abundance in plasma. The combination of the two MRM methods allowed the detection of 398 polar lipids in plasma in 64 min. Precision, estimated as within-day variability, was below 6.8% for all determined lipid families, while between-day variability was below 24.0%. This strategy has been applied to a cohort formed by 384 individuals in order to obtain a qualitative and quantitative distribution of polar lipids in human plasma. The most concentrated lipid families in relative terms were lysophospholipids, plasmalogens, and phosphatydilcholines, with mean relative concentration of 58.0, 17.1, and 8.3%, respectively. Then, sphingomyelins and phosphatidylethanolamines reported a relative concentration of 2.0%, followed by phosphatidylserines, with 1.1%. Graphical abstract.
    Keywords:  LC; Lipidomics; MS/MS; Multiple reaction monitoring; Plasma; Polar lipids
  14. Metabolites. 2019 Nov 27. pii: E292. [Epub ahead of print]9(12):
      Hydrophilic Interaction Liquid Chromatography (HILIC) chromatography is widely applied in metabolomics as a complementary strategy to reverse phase chromatography. Nevertheless, it still faces several issues in terms of peak shape and compounds ionization, limiting the automatic de-convolution and data semi-quantification performed through dedicated software. A way to improve the chromatographic and ionization performance of a HILIC method is to modify the electrostatic interactions of the analytes with both mobile and stationary phases. In this study, using a ZIC-HILIC chromatographic phase, we evaluated the performance of ammonium fluoride (AF) as additive salt, comparing its performance to ammonium acetate (AA). Three comparative criteria were selected: (1) identification and peak quality of 34 standards following a metabolomics-specific evaluation approach, (2) an intraday repeatability test with real samples and (3) performing two real metabolomics fingerprints with the AF method to evaluate its inter-day repeatability. The AF method showed not only higher ionization efficiency and signal-to-noise ratio but also better repeatability and robustness than the AA approach. A tips and tricks section is then added, aiming at improving method replicability for further users. In conclusion, ammonium fluoride as additive salt presents several advantages and might be considered as a step forward in the application of robust HILIC methods in metabolomics.
    Keywords:  HILIC; ammonium fluoride; metabolomics; mobile phase modifier
  15. Methods Mol Biol. 2020 ;2091 31-37
      The coupling of anion exchange high-pressure liquid chromatography (HPLC) with electrospray ionization mass spectrometry (ESI-MS) allows for the simultaneous detection of the six forms of inositol phosphate (InsP). Here we describe a rapid quantitative analysis of InsPs by HPLC-ESI-MS, which can be applied to a wide array of sample types. With this method, InsPs could be separated and detected within 20 min of sample injection. The detection limit was as low as 25 pmol (i.e., ca. 2 nmol/g sample) for each type of InsP, which is particularly important for analytes that are often present at low abundance in nature.
    Keywords:  HPLC; HPLC-ESI-MS; Inositol phosphate; Ion-exchange HPLC; Phytic acid
  16. J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Nov 12. pii: S1570-0232(19)31373-X. [Epub ahead of print]1134-1135 121876
      In this work, the physical and chemical properties of a novel zwitterionic LC stationary phase are applied to the development, validation and application of a new fast and reliable method devoted to the analysis of GHB (gamma-hydroxybutyric acid) and its relatively new discovered glucuronide metabolite in hair. The obtained sensitivity, expressed as limit of detection (LOD) and quantification (LOQ), were 0.033 and 0.10 ng/mg for GHB and 0.11 and 0.37 ng/mg, for GHB-glucuronide respectively. Linearity was assessed between LOQ and 50 ng/mg for both compounds. GHB and GHB-glucuronide extraction from hair matrix was maintained simple and consisted in an acidified-solvent incubation. No samples purification was required before LC-MS/MS analysis. The method was finally applied to 65 real hair sample, 60 adults and 5 children below 2 years old. The obtained results highlighted that GHB concentrations were in the range 0.11-0.96 ng/mg (average 0.38 ± 0.25 ng/mg) in 44 cases (68%) while in 21 samples GHB concentrations were in the range between LOD and LOQ (0.033-0.1 ng/mg). GHB-glucuronide was detected in few samples (n. 3) at levels below LOQ. The interest on these molecules relies on the fact that GHB is both a naturally occurring inhibitory neurotransmitter in the central nervous system and an illicit drug often experienced by victims of drug-facilitated sexual assault. GHB-glucuronide was firstly identified in urine by the group of Petersen in 2013 and, as per analogy to ethyl glucuronide, it was proposed as a longer biomarker for GHB intoxication.
    Keywords:  Drug-facilitate sexual assault; Forensic toxicology; GHB; GHB-glucuronide; Liquid chromatography–mass spectrometry
  17. Anal Bioanal Chem. 2019 Nov 25.
      Vitamin A deficiency (VAD) is a major micronutrient deficiency in children. Although plasma and serum retinol levels are proposed as the key indicators of VAD, collecting and transporting plasma and serum are difficult and inconvenient in field studies. Dried blood spot (DBS) retinol has been used as an alternative to plasma retinol in several epidemiological and clinical studies. A limitation of methods that use DBS retinol is the instability and apparent loss of retinol in DBSs. Therefore, an accurate, reliable method for stabilizing retinol in DBSs and quantifying and comparing DBS retinol concentrations with equivalent plasma retinol levels is required. In this study, antioxidants on paper combined with vacuum treatment were found to greatly increase the stability of DBS retinol during 120 min of air drying and 30 days of room-temperature storage. A surrogate matrix of whole blood prepared using a mixture of human erythrocytes and 2% BSA in PBS was firstly used in DBS retinol determination based on the fact that retinol is excluded from erythrocytes. The method was linear in the concentration range of 0.04-300 μg/mL. Both the between-run (n = 5) and within-run (n = 6) precision (relative standard deviations, RSD%) were below 8.42%. The spiked recoveries at 3 concentrations ranged from 86.48 to 98.13%. The internal standard (IS)-normalized matrix factor (MF) was 99.72% with a RSD% of 10.50% (n = 3). The accuracy was calibrated using two National Institute of Standards and Technology (NIST) serum-generated calibrants at concentrations of 0.1962 and 0.3948 g/mL, and relative errors (RE% values) of 0.07% and 4.95% were found, respectively. A simple calibration model was first developed to convert DBS retinol concentration to the equivalent plasma retinol concentration, thereby enabling comparisons with clinical reference ranges and with studies using serum or plasma samples. Graphical abstract.
    Keywords:  Dried blood spots; LC-MS/MS; Retinol
  18. Int J Mol Sci. 2019 Nov 27. pii: E5957. [Epub ahead of print]20(23):
      A comparative study of the impact of n-butylamine and traditionally used additives (ammonium hydroxide and formic acid) on the efficiency of the electrospray ionization (ESI) process for the enhancement of metabolite coverage was performed by direct injection mass spectrometry (MS) analysis in negative mode. Evaluation of obtained MS data showed that n-butylamine is one of the most effective additives for the analysis of metabolite composition in ESI in negative ion mode (ESI(-)) The limitations of the use of n-butylamine and other alkylamines in the analysis of metabolic composition and a decontamination procedure that can reduce MS device contamination after their application are discussed. The proposed procedure allows the performance of high-sensitivity analysis of low-molecular-weight compounds on the same MS device in both polarities.
    Keywords:  alkylamines; decontamination procedure; metabolome profiling