Determination of protease inhibitors using liquid chromatography-tandem mass spectrometry

A method for the analysis of six protease inhibitors and one metabolite has been developed and validated. Amprenavir, ritonavir, saquinavir, lopinavir, indinavir, nelfinavir, and an active metabolite of nelfinavir (M8) are quantitated using reversed-phase liquid chromatography coupled to tandem mass spectrometry, equipped with an electrospray ionization source (ESI-LC-MS-MS). The validation data presented here shows that the method allows the rugged analysis of these species from one aliquot. The evolution of complex drug interactions assessments and the clinical use of therapeutic drug monitoring for these antiretrovirals will be a potential immediate application of this method.     

Simultaneous determination of six urinary porphyrins using liquid chromatography-tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method without sample pretreatment was developed and validated for determination of porphyrins in samples of canine urine. Acidified urine samples were directly injected into the LC-MS system and a gradient elution program was applied. The mass spectrometer was operated in the multi-reaction monitoring (MRM) mode and six porphyrins were detected with excellent sensitivity and selectivity. The lower limits of quantification were 0.014 nmol/mL for mesoporphyrin IX, coproporphyrin I, 5-carboxylporphyrin, 6-carboxylporphyrin and 7-carboxylporphyrin, and 0.029 nmol/mL for uroporphyrin I. Good ln-quadratic responses of calibration standards over the range 0.01 to 1.0 nmol/mL for mesoporphyrin IX, coproporphyrin I, 5-carboxylporphyrin, 6-carboxylporphyrin and 7-carboxylporphyrin, and 0.02 to 1.0 nmol/mL for uroporphyrin I were demonstrated. This method should be easily adapted through cross-validation for use in determining the effects of chemicals and pharmaceuticals on the urinary excretion profile of porphyrins in preclinical studies with other species, and in assisting the diagnosis of porphyria in clinical studies.     

Determination of benidipine in human plasma using liquid chromatography-tandem mass spectrometry

Fumonisins are water soluble mycotoxins produced by the fungus Fusarium verticillioides (formerly F. moniliforme). Fumonisin B(1) (FB(1)) is a diester of propane-1,2,3-tricarboxylic acid and 2-amino-12, 16-dimethyl-3,5,10,14,15-pentahydroxyeicosane, and is the most abundant of the naturally occurring fumonisins. Upon removal of the two tricarballylic acid side chains, the structure is referred to as hydrolyzed FB(1) (HFB(1)). FB(1) and HFB(1) are structurally similar to sphinganine, a sphingoid base. The fumonisins do not absorb UV light or fluoresce; therefore, derivatizing reagents are used for detection when separation is by high performance liquid chromatography (HPLC). The standard derivatizing reagent used for HPLC is ortho-phthalaldehyde (OPA) plus 2-mercaptoethanol (ME) reaction partner, however, the OPA-FB(1) derivative is not stable at room temperature. The objectives of this study were to: (1). determine the effect of temperature on the stability of the OPA-FB(1) derivative and (2). determine which structural characteristics of FB(1) contribute to the instability of the OPA-FB(1) derivative. The results indicate that OPA-FB(1), OPA-FB(3) and OPA-HFB(1) derivatives are unstable at 24 degrees C but that their stability improves significantly at 4 degrees C. The OPA-sphinganine derivative is stable for at least 24h at 24 degrees C. Thus, the instability of the OPA-FB(1) derivative may be attributed to its lack of a hydroxyl group at the carbon 1 position.     

Multi-component plasma quantitation of anti-hyperglycemic pharmaceutical compounds using liquid chromatography-tandem mass spectrometry

Type-2 diabetes is a disorder characterized by disrupted insulin production leading to high blood glucose levels. To control this disease, combination therapy is often used. Hypoglycemic agents such as metformin, glipizide, glyburide, repaglinide, rosiglitazone, nateglinide, and pioglitazone are widely prescribed to control blood sugar levels. These drugs provide the basis for the development of a quantitative multianalyte bioanalytical method. As an example, a highly sensitive and selective multi-drug method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed. This rapid, automated method consists of protein precipitation of 20 microL of plasma coupled with gradient HPLC elution of compounds using 10 mM ammonium formate buffer and 0.1% formic acid in acetonitrile as the mobile phases. MS/MS detection was performed using turbo ion spray in the positive ion multiple reaction monitoring (SRM) mode. A lower limit of quantitation (LLQ) in a range of 1.0-5.0 ng/mL was achieved for all analytes. The linearity of the method was observed over a 500-fold dynamic range. Drug recoveries ranged from 86.2 to 94.2% for all analytes of interest. Selectivity, sample dilution, intra-day and inter-day accuracy and precision, and stability assessment were evaluated for all compounds.     

Enantioselective determination of azelnidipine in human plasma using liquid chromatography-tandem mass spectrometry

A sensitive and simple method was developed for determination of the enantiomers of azelnidipine, (R)-(-)-azelnidipine and (S)-(+)-azelnidipine, in human plasma using chiral liquid chromatography with positive ion atmospheric pressure chemical ionization tandem mass spectrometry. Plasma samples spiked with stable isotope-labeled azelnidipine, [(2)H(6)]-azelnidipine, as an internal standard, were processed for analysis using a solid-phase extraction in a 96-well plate format. The azelnidipine enantiomers were separated on a chiral column containing alpha(1)-acid glycoprotein as a chiral selector under isocratic mobile phase conditions. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, monitoring the transitions from m/z 583-->167 for (R)-(-)-azelnidipine and (S)-(+)-azelnidipine, and from m/z 589-->167 for [(2)H(6)]-azelnidipine. The standard curve was linear over the studied range (0.05-20 ng/mL), with r(2)>0.997 using weighted (1/x(2)) quadratic regression, and the chromatographic run time was 5.0 min/injection. The intra- and inter-assay precision (coefficient of variation), calculated from the assay data of the quality control samples, was 1.2-8.2% and 2.4-5.8% for (R)-(-)-azelnidipine and (S)-(+)-azelnidipine, respectively. The accuracy was 101.2-117.0% for (R)-(-)-azelnidipine and 100.0-107.0% for (S)-(+)-azelnidipine. The overall recoveries for (R)-(-)-azelnidipine and (S)-(+)-azelnidipine were 71.4-79.7% and 71.7-84.2%, respectively. The lower limit of quantification for both enantiomers was 0.05 ng/mL using 1.0 mL of plasma. All the analytes showed acceptable short-term, long-term, auto-sampler and stock solution stability. Furthermore, the method described above was used to separately measure the concentrations of the azelnidipine enantiomers in plasma samples collected from healthy subjects who had received a single oral dose of 16 mg of azelnidipine.     

A liquid chromatography-tandem mass spectrometry method for measurement of N-modified phosphatidylethanolamines

N-Acyl phosphatidylethanolamines (NAPEs) are synthesised in response to stress in a variety of organisms from bacteria to humans. More recently, nonenzymatic modification of the ethanolamine headgroup of phosphatidylethanolamine (PE) by various aldehydes, including levuglandins/isoketals (which are γ-ketoaldehydes [γKAs] derived from arachidonic acid), has also been demonstrated. The levels of these various N-modified PEs formed during stress and their biological significance remain to be fully characterized. Such studies require an accurate, facile, and cost-effective method for quantifying N-modified PEs. Previously, NAPE and some of the nonenzymatically N-modified PE species have been quantified by mass spectrometry after hydrolysis to their constituent N-acylethanolamine by enzymatic hydrolysis, most typically with Streptomyces chromofuscus phospholipase D. However, enzymatic hydrolysis is not cost-effective for routine analysis of a large number of samples, and hydrolytic efficiency may vary for different N-modified PEs, making quantitation more difficult. Therefore, we sought a robust and inexpensive chemical hydrolysis approach. Methylamine (CH₃NH₂)-mediated deacylation has previously been used in headgroup analysis of phosphatidylinositol phosphates. Therefore, we developed an accurate assay for NAPEs and γKA-PEs using CH₃NH₂-mediated deacylation and quantitation of the resulting glycerophospho-N-modified ethanolamines by liquid chromatography-tandem mass spectrometry.     

Stable isotope liquid chromatography-tandem mass spectrometry assay for fatty acid amide hydrolase activity

Fatty acid amide hydrolase (FAAH) is the main enzyme responsible for the hydrolysis of the endocannabinoid anandamide (arachidonoyl ethanolamide, AEA) to arachidonic acid (AA) and ethanolamine (EA). Published FAAH activity assays mostly employ radiolabeled anandamide or synthetic fluorogenic substrates. We report a stable isotope liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for specific, sensitive, and high-throughput capable FAAH activity measurements. The assay uses AEA labeled with deuterium on the EA moiety (d₄-AEA) as substrate and measures the specific reaction product tetradeutero-EA (d₄-EA) and the internal standard ¹³C₂-EA. Selected reaction monitoring of m/z 66 → m/z 48 (d₄-EA) and m/z 64 → m/z 46 (¹³C₂-EA) in the positive electrospray ionization mode after liquid chromatographic separation on a HILIC (hydrophilic interaction liquid chromatography) column is performed. The assay was developed and thoroughly validated using recombinant human FAAH (rhFAAH) and then was applied to human blood and dog liver samples. rhFAAH-catalyzed d₄-AEA hydrolysis obeyed Michaelis–Menten kinetics (K_M = 12.3 μM, V_max = 27.6 nmol/min mg). Oleoyl oxazolopyridine (oloxa) was a potent, partial noncompetitive inhibitor of rhFAAH (IC₅₀ = 24.3 nM). Substrate specificity of other fatty acid ethanolamides decreased with decreasing length, number of double bonds, and lipophilicity of the fatty acid skeleton. In human whole blood, we detected FAAH activity that was inhibited by oloxa.     

Quantification of lysophosphatidylcholines and phosphatidylcholines using liquid chromatography-tandem mass spectrometry in neonatal serum

We established an improved method for quantification of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) molecular species in neonatal serum using high-performance liquid chromatography coupled tandem mass spectrometry (LC-MS/MS). A multiple reaction monitoring (MRM) mode of positive ionization for MS/MS was used. The method involved purification of phospholipids by solid phase extraction (SPE) from a 20-microl minimum specimen of serum. The assayed values of authentic 16:0-LPC and 18:0-LPC showed a linear response, and our quantitative results showed high precision for the all species of PC and LPC. Then, we quantified PC and LPC in adult and neonatal serum and compared them. Day 0-1 neonatal serum 16:0-, 18:0-, 18:1-, 18:2-LPC levels were significantly lower than adult ones. All species LPC levels in the day 0-1 neonates were significantly lower than day 4-8 neonates. Day 0-1 neonatal serum 16:0/18:2-, 18:0/18:2-PC levels were significantly lower than adult ones. Our method is advantageous for precise assessments of the relationships between PCs/LPCs levels and neonatal infectious diseases.     

Determination of cabergoline and L-dopa in human plasma using liquid chromatography-tandem mass spectrometry

We determined cabergoline and L-dopa in human plasma using liquid chromatography-mass spectrometry with tandem mass spectrometry (LC-MS-MS). The deproteinized plasma samples with organic solvent or acid were analyzed directly by reversed-phase liquid chromatography. Using multiple reaction monitoring (MRM, product ions m/z 381 of m/z 452 for cabergoline and m/z 152 of m/z 198 for L-dopa) on LC-MS-MS with electrospray ionization (ESI), cabergoline and L-dopa in human plasma were determined. Calibration curves of the method showed a good linearity in the range 5-250 pg/ml for cabergoline and 1-200 ng/ml for L-dopa, respectively. The limit of determination was estimated to be approximately 2 pg/ml for cabergoline and approximately 0.1 ng/ml for L-dopa, respectively. The method was applied to the analysis of cabergoline and L-dopa in plasma samples from patients treated with these drugs. The precision of analysis showed coefficients of variation ranging from 3.8% to 10.5% at cabergoline concentration of 13.8-26.2 pg/ml and from 2.9% to 8.9% at an L-dopa concentration of 302.5-522.1 ng/ml in patient plasma. As a result, the procedure proved to be very suitable for routine analysis.     

Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography-tandem mass spectrometry

An increasing number of proteomic strategies rely on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect and identify constituent peptides of enzymatically digested proteins obtained from various organisms and cell types. However, sample preparation methods for isolating membrane proteins typically involve the use of detergents and chaotropes that often interfere with chromatographic separation and/or electrospray ionization. To address this problem, a sample preparation method combining carbonate extraction, surfactant-free organic solvent-assisted solubilization, and proteolysis was developed and demonstrated to target the membrane subproteome of Deinococcus radiodurans. Out of 503 proteins identified, 135 were recognized as hydrophobic on the basis of their calculated hydropathy values (GRAVY index), corresponding to coverage of 15% of the predicted hydrophobic proteome. Using the PSORT algorithm, 53 of the proteins identified were classified as integral outer membrane proteins and 215 were classified as integral cytoplasmic membrane proteins. All identified integral cytoplasmic membrane proteins had from 1 to 16 mapped transmembrane domains (TMDs), and 65% of those containing four or more mapped TMDs were identified by at least one hydrophobic membrane spanning peptide. The extensive coverage of the membrane subproteome (24%) by identification of highly hydrophobic proteins containing multiple TMDs validates the efficacy of the described sample preparation technique to isolate and solubilize hydrophobic integral membrane proteins from complex protein mixtures.     

Doping control analysis of trenbolone and related compounds using liquid chromatography-tandem mass spectrometry

Trenbolone (17β-hydroxy-estra-4,9,11-trien-3-one) and its derivatives such as 17α-methyltrenbolone represent a class of highly potent anabolic-androgenic steroids, which are prohibited in sports according to the regulation of the World Anti-Doping Agency (WADA). Due to marginal gas chromatographic properties of these compounds but excellent proton affinities resulting from a large and conjugated π-electron system, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been the method of choice for the detection of these analytes in sports drug testing. Recent findings of trenbolone and methyltrenbolone in doping control urine samples of elite athletes demonstrated the importance of a sensitive and robust analytical method, which was based on an enzymatic hydrolysis of target compounds, liquid-liquid extraction and subsequent LC-MS/MS measurement. Diagnostic product ions obtained after collision-induced dissociation of protonated molecules were found at m/z 227, 211, 199 and 198, which enabled targeted screening using multiple reaction monitoring. Using 7 model compounds (trenbolone, epitrenbolone, methyltrenbolone, ethyltrenbolone, propyltrenbolone, 17-ketotrenbolone and altrenogest), the established method was validated for specificity, lower limits of detection (0.3-3 ng/mL), recovery (72-105%), intraday and interday precision (≤20%).     

Highly sensitive determination of saquinavir in biological samples using liquid chromatography-tandem mass spectrometry

A selective and sensitive method for the determination of the HIV protease inhibitor saquinavir in human plasma, saliva, and urine using liquid-liquid extraction and LC-MS-MS has been developed, validated, and applied to samples of a healthy individual. After extraction with ethyl acetate, sample extracts were chromatographed isocratically within 5 min on Kromasil RP-18. The drug was detected with tandem mass spectrometry in the selected reaction monitoring mode using an electrospray ion source and 2H(5)-saquinavir as internal standard. The limit of quantification was 0.05 ng/mL. The accuracy of the method varied between -1 and +10% (SD within-batch) and the precision ranged from +4 to +10% (SD batch-to-batch). The method is linear at least within 0.05 and 87.6 ng/mL. After a regular oral dose (600 mg) saquinavir concentrations were detectable for 48 h in plasma and were well correlated with saliva concentrations (r(2)=0.9348, mean saliva/plasma ratio 1:15.1). The method is well suited for low saquinavir concentrations in different matrices.     

Quantification of riboflavin in human urine using high performance liquid chromatography-tandem mass spectrometry

We developed a selective method to measure riboflavin in human urine. Sample preparation involved solid phase extraction and concentration of the target analyte in urine. The urine concentrate was analyzed using high performance liquid chromatography-tandem mass spectrometry. Riboflavin concentrations were quantified using an isotopically labeled internal standard. The limit of detection was 11 ng/mL, and the linear range was 4.4-20,000 ng/mL. The relative standard deviation at 100, 1000, and 5000 ng/mL was 17%, 17%, and 12%, respectively. The accuracy was 90%. On average, 100 samples, including calibration standards and quality control samples, were prepared per day. Using our method, we measured concentrations of riboflavin in human urine samples that were collected from participants in a study where riboflavin was used as a surrogate chemical to simulate exposure to an environmental toxicant.     

Urinary 5-HIAA measurement using automated on-line solid-phase extraction-high-performance liquid chromatography-tandem mass spectrometry

Quantification of 5-hydroxyindole-3-acetic acid (5-HIAA) in urine is useful for diagnosis and follow-up of patients with carcinoid tumors and for monitoring serotonin (5-hydroxytryptamine) metabolism in various disorders. We describe an automated method (XLC-MS/MS) that incorporates on-line solid-phase extraction (SPE), high-performance liquid chromatography (HPLC) and tandem mass spectrometric (MS/MS) detection to measure urinary 5-HIAA. Automated pre-purification of urine was carried out with HySphere-Resin GP SPE cartridges containing strong hydrophobic polystyrene resin. The analyte (5-HIAA) and internal standard (isotope-labelled 5-HIAA-d(2)) were, after elution from the cartridge, separated by reversed-phase HPLC and detected with tandem MS. Total cycle time was 5 min. 5-HIAA and its deuterated internal standard (5-HIAA-d(2)) were retained on and eluted from the SPE cartridges in high yields (81.5-98.0%). Absolute recovery was 96.5-99.6%. Intra-assay (n=20) and inter-assay (n=20) CVs for the measurement of 5-HIAA in urine in three concentration levels ranged from 0.8 to 1.4% and 1.7 to 4.2%, respectively. For urine samples from patients (n=78) with known or suspected metastatic carcinoid tumors, results obtained by XLC-MS/MS were highly correlated (R(2)=0.99) with the routinely used fluorometric method. This XLC-MS/MS method demonstrated lower imprecision and time per analysis (high-throughput) than manual solvent extraction methods and higher sensitivity and specificity than non-mass spectrometric detection techniques.     

Statistical and computational methods for comparative proteomic profiling using liquid chromatography-tandem mass spectrometry

The combined method of LC-MS/MS is increasingly being used to explore differences in the proteomic composition of complex biological systems. The reliability and utility of such comparative protein expression profiling studies is critically dependent on an accurate and rigorous assessment of quantitative changes in the relative abundance of the myriad of proteins typically present in a biological sample such as blood or tissue. In this review, we provide an overview of key statistical and computational issues relevant to bottom-up shotgun global proteomic analysis, with an emphasis on methods that can be applied to improve the dependability of biological inferences drawn from large proteomic datasets. Focusing on a start-to-finish approach, we address the following topics: 1) low-level data processing steps, such as formation of a data matrix, filtering, and baseline subtraction to minimize noise, 2) mid-level processing steps, such as data normalization, alignment in time, peak detection, peak quantification, peak matching, and error models, to facilitate profile comparisons; and, 3) high-level processing steps such as sample classification and biomarker discovery, and related topics such as significance testing, multiple testing, and choice of feature space. We report on approaches that have recently been developed for these steps, discussing their merits and limitations, and propose areas deserving of further research.     

Determination of 17-hydroxyprogesterone in plasma by stable isotope dilution/benchtop liquid chromatography-tandem mass spectrometry

An assay based on stable isotope dilution liquid chromatography-tandem mass spectrometry (ID/LC-MS-MS) was developed for the quantification of 17-hydroxyprogesterone, the most important indicator of 21-hydroxylase deficiency in human plasma. Plasma was extracted using ethyl acetate and Extrelut columns. LC was performed on a reversed-phase C18 column using a water/methanol gradient. A benchtop triple quadrupole mass spectrometer, operating in selected reaction monitoring mode, served as mass detector. The analytical run time was 9 min per sample. The sensitivity was high: 0.06 pmol of 17-hydroxyprogesterone yielded a signal-to-noise ratio of 13. Precision (CV) and accuracy (relative error) derived from the analyses of unspiked and spiked validation samples were 7.4-12.0% and 6.4%, respectively. When analyzing the same samples - median (range), in nanomoles per liter - from neonates and adults independently by ID/LC-MS-MS as well as by ID/gas chromatography (GC)-MS, corresponding results were obtained: neonates (n = 10), ID/LC-MS-MS 3.99 (0.48-16.05), ID/GC-MS 5.39 (1.57-13.02); adults (n = 10), ID/LC-MS-MS 2.66 (1.39-6.15), ID/GC-MS 2.54 (0.51-5.12). The technique permitted reliable detection of classical and nonclassical forms of 21- hydroxylase deficiency. The much simpler sample preparation, the faster analytical run time and the operational ease possible with ID/LC-MS-MS permit a considerable increase of sample testing per day without compromising on analytical sensitivity and specificity. We expect that benchtop tandem mass spectrometry will open new avenues in clinical steroid analysis.     

Global analysis of the membrane subproteome of Pseudomonas aeruginosa using liquid chromatography-tandem mass spectrometry

Pseudomonas aeruginosa is one of the most significant opportunistic bacterial pathogens in humans causing infections and premature death in patients with cystic fibrosis, AIDS, severe burns, organ transplants, or cancer. Liquid chromatography coupled online with tandem mass spectrometry was used for the large-scale proteomic analysis of the P. aeruginosa membrane subproteome. Concomitantly, an affinity labeling technique, using iodoacetyl-PEO biotin to tag cysteinyl-containing proteins, permitted the enrichment and detection of lower abundance membrane proteins. The application of these approaches resulted in the identification of 786 proteins. A total of 333 proteins (42%) had a minimum of one transmembrane domain (ranging from 1 to14) and 195 proteins were classified as hydrophobic based on their positive GRAVY values (ranging from 0.01 to 1.32). Key integral inner and outer membrane proteins involved in adaptation and antibiotic resistance were conclusively identified, including the detection of 53% of all predicted opr-type porins (outer integral membrane proteins) and all the components of the mexA-mexB-oprM transmembrane protein complex. This work represents one of the most comprehensive proteomic analyses of the membrane subproteome of P. aeruginosa and for prokaryotes in general.     

Semi-automated determination of plasma stability of drug discovery compounds using liquid chromatography-tandem mass spectrometry

A simple procedure for the measurement of stability of drug candidates in plasma was developed to eliminate the traditional labor-intensive and time-consuming sample preparation procedures that are typically used for these studies. The procedure makes use of a thermostatic autosampler as an incubator combined with the direct plasma injection method based on high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS-MS). Untreated human, monkey, mouse and rat plasma containing the test compound was directly injected into a mixed-function column for on-line protein removal and chromatography. The test compound and its biotransformation product were separated via HPLC and monitored using the tandem mass spectrometer. The need for adequate chromatographic separation of the test compound (M) from its carboxylic acid metabolite (M+1) is demonstrated. Plasma samples from four different species at specified incubation temperatures were sequentially assayed in one analytical procedure. The injection-to-injection time was about 6 min. The peak responses of the test compound in individual plasma samples were repeatedly determined every 24 min. The retention times and peak shape of all analytes were found to be consistent throughout the experiments. The stability of the test compound in plasma was found to be a function of animal species, incubation time and temperature. The test compound was rapidly degraded in rat plasma at 37 degrees C, but it could be stabilized by adding sodium thiosulfate.     

A multiplex quantitation method for eicosanoids and platelet-activating factor using column-switching reversed-phase liquid chromatography-tandem mass spectrometry

Eicosanoids and platelet-activating factor (PAF) are phospholipid-derived lipid mediators produced by various tissues and cells through a cascade pathway. For a comprehensive analysis of these lipid mediators, a simultaneous quantitation method with sensitivity and reliability is necessary. This article details a development of column-switching reversed-phase liquid chromatography-tandem mass spectrometry for multiplex quantitation of eicosanoids and PAF. The adsorptive nature of lipids caused significant loss of signal in a conventional column-switching configuration. The use of an online-dilution method allowed use of 100% methanol as a sample solvent, which prevented sample adsorption to contacting surfaces. Addition of 0.2% formic acid to the sample solvent was required for the successful introduction of LTC4 to the trapping column and minimizing its carryover. The optimized method provided rapid analysis of 14 lipid mediators with a throughput of 96 samples/24 h, lower limits of quantitation of 5 pg on column, and linear calibration ranges up to 2000-5000 pg. The system was highly compatible with solid-phase-extracted samples, as methanol-eluted fractions were directly injected without reconstitution. The analysis of lipid mediator production of macrophage-like RAW264.7 cells demonstrated that the cell-based assay can be performed in a 96-well format, suitable for metabolomics analyses and/or screening strategies.     

A multimodal metabolomics approach using imaging mass spectrometry and liquid chromatography-tandem mass spectrometry for spatially characterizing monoterpene indole alkaloids secreted from roots

Plants release specialized (secondary) metabolites from their roots to communicate with other organisms, including soil microorganisms. The spatial behavior of such metabolites around these roots can help us understand roles for the communication; however, currently, they are unclear because soil-based studies are complex. Here, we established a multimodal metabolomics approach using imaging mass spectrometry (IMS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to spatially assign metabolites under laboratory conditions using agar. In a case study using Catharanthus roseus, we showed that 58 nitrogen (N)-containing metabolites are released from the roots into the agar. For the metabolite assignment, we used ¹⁵N-labeled and non-labeled LC-MS/MS data, previously reported. Four metabolite ions were identified using authentic standard compounds as derived from monoterpene indole alkaloids (MIAs) such as ajmalicine, catharanthine, serpentine, and yohimbine. An alkaloid network analysis using dot products and spinglass methods characterized five clusters to which the 58 ions belong. The analysis clustered ions from the indolic skeleton-type MIAs to a cluster, suggesting that other communities may represent distinct metabolite groups. For future chemical assignments of the serpentine community, key fragmentation patterns were characterized using the ¹⁵N-labeled and non-labeled MS/MS spectra.