Dansylation isotope labeling liquid chromatography mass spectrometry for parallel profiling of human urinary and fecal submetabolomes

Human urine and feces can be non-invasively collected for metabolomics-based disease biomarker discovery research. Because urinary and fecal metabolomes are thought to be different, analysis of both biospecimens may generate a more comprehensive metabolomic profile that can be better related to the health state of an individual. Herein we describe a method of using differential chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) for parallel metabolomic profiling of urine and feces. Dansylation labeling was used to quantify the amine/phenol submetabolome changes among different samples based on ¹²C-labeling of individual samples and ¹³C-labeling of a pooled urine or pooled feces and subsequent analysis of the ¹³C-/¹²C-labeled mixture by LC-MS. The pooled urine and pooled feces are further differentially labeled, mixed and then analyzed by LC-MS in order to relate the metabolite concentrations of the common metabolites found in both biospecimens. This method offers a means of direct comparison of urinary and fecal submetabolomes. We evaluated the analytical performance and demonstrated the utility of this method in the analysis of urine and feces collected daily from three healthy individuals for 7 days. On average, 2534 ± 113 (n = 126) peak pairs or metabolites could be detected from a urine sample, while 2507 ± 77 (n = 63) peak pairs were detected from a fecal sample. In total, 5372 unique peak pairs were detected from all the samples combined; 3089 and 3012 pairs were found in urine and feces, respectively. These results reveal that the urine and fecal metabolomes are very different, thereby justifying the consideration of using both biospecimens to increase the probability of finding specific biomarkers of diseases. Furthermore, the CIL LC-MS method described can be used to perform parallel quantitative analysis of urine and feces, resulting in more complete coverage of the human metabolome.     

Development of an isotope labeling ultra-high performance liquid chromatography mass spectrometric method for quantification of acylglycines in human urine

Acylglycines play a crucial regulatory and detoxification role in the accumulation of the corresponding acyl CoA esters and are an important class of metabolites in the diagnoses of inborn errors of metabolism. Sensitive quantification of a large number of acylglycines not only improves diagnosis but also enables the discovery of potential new biomarkers of diseases. We report an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC–MS) method for quantifying acylglycines in human urine with high sensitivity. This method is based on the use of a newly developed isotope labeling reagent, p-dimethylaminophenacyl (DmPA) bromide, to label acylglycines to improve detection sensitivity. Eighteen acylglycines, namely acetylglycine, propionylglycine, isobutyrylglycine, butyrylglycine, 4-hydroxyphenylacetylglycine, 2-furoylglycine, tiglylglycine, 2-methybutyrylglycine, 3-methylcrotonylglycine, isovalerylglycine, valerylglycine, hexanoylglycine, phenylacetylglycine, phenylpropionylglycine, glutarylglycine, heptanoylglycine, octanoylglycine and suberylglycine, were measured. This method uses calibration standards prepared in surrogate matrix (un-derivatized urine) and stable-isotope labeled analytes as the internal standards. The analysis was carried out in the positive ion detection mode using multiple reaction monitoring (MRM) survey scans. The calibration curves were validated over the range of 1.0–500 nM. The method achieved a lower limit of quantitation (LLOQ) of 1–5 nM for all analytes, as measured by the standard derivations associated with calibration curves and confirmed in surrogate matrix; the signal-to-noise ratio at LLOQ ranged from 12.50 to 156.70. Both accuracy (% RE or relative error) and precision (% CV) were <15%. Matrix effects were minimized using the surrogate matrix. All eighteen analytes were stable in urine for at least 5 h at room temperature, autosampler (4 °C) for 24 h, 7 weeks at −20 °C and after three freeze/thaw cycles. This surrogate matrix approach was validated using a standard addition experiment. As an example of applications, the endogenous concentrations of all eighteen analytes in urine samples of 20 healthy individuals collected in three consecutive days (i.e., 60 samples) were determined; there was no significant correlation found between the acylglycine profile and gender or body mass indices.     

Differential isotope dansylation labeling combined with liquid chromatography mass spectrometry for quantification of intact and N-terminal truncated proteins

The N-terminal amino acids of proteins are important structure units for maintaining the biological function, localization, and interaction networks of proteins. Under different biological conditions, one or several N-terminal amino acids could be cleaved from an intact protein due to processes, such as proteolysis, resulting in the change of protein properties. Thus, the ability to quantify the N-terminal truncated forms of proteins is of great importance, particularly in the area of development and production of protein-based drugs where the relative quantity of the intact protein and its truncated form needs to be monitored. In this work, we describe a rapid method for absolute quantification of protein mixtures containing intact and N-terminal truncated proteins. This method is based on dansylation labeling of the N-terminal amino acids of proteins, followed by microwave-assisted acid hydrolysis of the proteins into amino acids. It is shown that dansyl labeled amino acids are stable in acidic conditions and can be quantified by liquid chromatography mass spectrometry (LC–MS) with the use of isotope analog standards.     

Ion-pairing reversed-phase liquid chromatography fractionation in combination with isotope labeling reversed-phase liquid chromatography-mass spectrometry for comprehensive metabolome profiling

We report a novel two-dimensional (2D) separation strategy aimed at improving the detectability of liquid chromatography mass spectrometry (LC-MS) for metabolome analysis. It is based on the use of ion-pairing (IP) reversed-phase (RP) LC as the first dimension separation to fractionate the metabolites, followed by isotope labeling of individual fractions using dansylation chemistry to alter the physiochemical properties of the metabolites. The labeled metabolites having different hydrophobicity from their unlabeled counterparts are then separated and analyzed by on-line RPLC Fourier-transform ion-cyclotron resonance mass spectrometry (FTICR-MS). This off-line 2D-LC-MS strategy offers significant improvement over the one-dimensional (1D) RPLC MS technique in terms of the number of detectable metabolites. As an example, in the analysis of a human urine sample, 3564 13C-/12C-dansylated ion pairs or metabolites were detected from seven IP RPLC fractions, compared to 1218 metabolites found in 1D-RPLC-MS. Using a library of 220 amine- and phenol-containing metabolite standards, 167 metabolites were positively identified based on retention time and accurate mass matches, which was about 2.5 times the number metabolites identified by 1D-RPLC-MS analysis of the same urine sample.     

Investigation of the species-dependent in vitro metabolism of BAL30630 by stable isotope labeling and isotope exchange experiments analyzed by capillary liquid chromatography coupled to mass spectrometry

The in vitro metabolic profile of BAL30630, an antifungal piperazine propanol derivative, which inhibits the 1,3-beta-d-glucansynthase, was investigated by incubation with microsomes of several species and with rat hepatocytes. For the spotting of the metabolites, mixtures of BAL30630 with a stable isotope (deuterium) labeled analogue were incubated. The metabolic pattern comprises several oxidized metabolites. Based on isotope exchange experiments, their structures could be assigned to epoxide- and hydroxylated metabolites. In hepatocyte incubations, several glucuronides formed from these oxidized metabolites could be observed. From the analysis of the metabolic pattern in microsomes, products of carbamate hydrolysis were characterized. This hydrolysis was highly species dependent. In activated incubations and in rat hepatocytes, those metabolites were further oxidized. In incubations without NADPH activation, the resulting hydrolytic metabolites could be enriched without the subsequent oxidation. Final structural elucidation of the metabolites was performed using accurate mass determination and isotope exchange experiments, in which incubations were analyzed by deuterium exchange and capillary HPLC–QTof-MS and MS/MS. The use of non-radioactive, stabile isotope labeled drug analogues in combination with isotope exchange studies was essential in particular for a defined assignment of the functional groups in the structures of the investigated metabolites.     

Analysis of proteoglycans derived sulphated disaccharides by liquid chromatography/mass spectrometry

A method has been developed for the identification and quantitative determination of sulphated disaccharides derived from chondroitin sulphate (CS) and dermatan sulphate (DS) chains attached to proteoglycans (PGs). After digestion with Chondroitinase ABC, the pool of disaccharides can be directly separated by liquid chromatography on a porous graphitized carbon (PGC) column and identified by on-line electrospray mass spectrometry under negative ionization conditions. The relative intensities of the fragment ions obtained by MS/MS allow to distinguish the sulphate position. Calibration with standard disaccharides allows the quantification of the different isomers. The method showed good repeatability in terms of relative standard deviation (RSD < 2%) and linearity between 0.5 and 50 ng (total injected amount) for both 4- and 6-sulphated disaccharides. The limit of detection achieved in full scan mode was 0.1 ng. The methodology was applied to different types of biological samples obtained from patients suffering from chronic lung inflammation such as: lung tissue, bronchoalveolar lavage fluid (BALF), induced sputum and urine.     

Ultra-high performance liquid chromatography tandem mass spectrometry for comprehensive analysis of urinary acylcarnitines

We report an enabling mass spectrometric method for the analysis of lipid metabolites in order to define better the lipid metabolome in terms of chemical diversity and generate fragment ion spectra of these metabolites as a potential resource for unknown metabolite identification. This work focuses on the analysis of one important class of lipid metabolites, the acylcarnitines. Current analytical methods have only detected and identified a limited number of these metabolites. The method described herein provides the most comprehensive acylcarnitine profile in urine of healthy individuals up to date. It involves an optimized solid phase extraction technique for selective analyte extraction using cartridges containing both lipophilic and cation-exchange properties. The captured analytes are then subjected to ultra-high performance liquid chromatography (UPLC) separation, followed by tandem mass spectrometry (MS/MS) analysis using information-dependent acquisitions and selected reaction monitoring (SRM). The urine of six healthy individuals was analyzed using this method. A total of 355 acylcarnitines were detected; only 43 of them have been previously reported in the urine of healthy individuals. Detection of this large number of acylcarnitines illustrates the great diversity of the lipid metabolome as well as the usefulness of the method for profiling acylcarnitines. Furthermore, the MS/MS spectra of the 355 acylcarnitines will be uploaded to a public human metabolome database as a mass spectrometric resource for unknown metabolite identification.     

Development of versatile isotopic labeling reagents for profiling the amine submetabolome by liquid chromatography–mass spectrometry

Metabolomic profiling involves relative quantification of metabolites in comparative samples and identification of the significant metabolites that differentiate different groups (e.g., diseased vs. controls). Chemical isotope labeling (CIL) liquid chromatography–mass spectrometry (LC–MS) is an enabling technique that can provide improved metabolome coverage and metabolite quantification. However, chemical identification of labeled metabolites can still be a challenge. In this work, a new set of isotopic labeling reagents offering versatile properties to enhance both detection and identification are described. They were prepared by a glycine molecule (or its isotopic counterpart) and an aromatic acid with varying structures through a simple three-step synthesis route. In addition to relatively low costs of synthesizing the reagents, this reaction route allows adjusting reagent property in accordance with the desired application objective. To date, two isotopic reagents, 4-dimethylaminobenzoylamido acetic acid N-hydroxylsuccinimide ester (DBAA-NHS) and 4-methoxybenzoylamido acetic acid N-hydroxylsuccinimide ester (MBAA-NHS), for labeling the amine-containing metabolites (i.e., amine submetabolome) have been synthesized. The labeling conditions and the related LC–MS method have been optimized. We demonstrate that DBAA labeling can increase the metabolite detectability because of the presence of an electrospray ionization (ESI)-active dimethylaminobenzoyl group. On the other hand, MBAA labeled metabolites can be fragmented in MS/MS and pseudo MS³ experiments to provide structural information on metabolites of interest. Thus, these reagents can be tailored to quantitative profiling of the amine submetabolome as well as metabolite identification in metabolomics applications.     

Comprehensive two-dimensional liquid chromatography coupled with mass spectrometry

In this review, instrumental aspects of comprehensive two-dimensional liquid chromatography coupled with mass spectrometry are presented. The milestones of LC×LC are briefly summarized. Instrument configuration, selection of experimental conditions, the different interfaces used in the system and the current applications of LC×LC–MS systems are described.     

Ultra-high capacity liquid chromatography chip/quadrupole time-of-flight mass spectrometry for pharmaceutical analysis

Nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) has attracted increasing interest in virtue of high sensitivity, low sample consumption, and minimal matrix effect. In this work a HPLC-Chip/quadrupole time-of-flight (Q-TOF) MS device with a new ultra-high capacity small molecule chip (UHC-Chip) which features a 500 nL enrichment column and a 150 mm × 75 μm analytical column, was evaluated with a drug mixture covering a wide range of polarities. Excellent chromatographic precision with 0.1-0.5% RSD for retention time and 1.7-9.0% RSD for peak area, low limit of detection, good chip-to-chip reproducibility and linearity were obtained by using this UHC-Chip. Compared with the standard HPLC-Chip with 40 nL trapping column, the UHC-Chip showed higher enrichment capability and hence gave a higher response in signal detection. Additionally, 4-30 times increase in sensitivity was obtained compared with conventional LC/MS, which indicated that UHC-Chip/MS was a valuable tool for the quantitative analysis of low level impurities and degradation products in pharmaceuticals. Moreover, satisfactory results obtained from trace drug analysis of serum samples further proved its practicality and potential for use in drug testing and development.     

Contents of selected B vitamins in NIST SRM 3280 multivitamin/multielement tablets by liquid chromatography isotope dilution mass spectrometry

There is increased interest in accurately assessing the total dietary intake of vitamins from all sources, including foods and dietary supplements. Consequently, a Dietary Supplement Ingredient Database (DSID), based upon analytical values, is being established by USDA with support of the Office of Dietary Supplements (ODS), NIH. The DSID necessitated the development of a new SRM, 3280 — Multivitamin/Multimineral Tablets, by the National Institute of Standards and Technology (NIST), with support from the ODS. As a continuation of a long-term project to develop and validate new methods of determining water-soluble B vitamins in foods and dietary supplements, and as part of a collaborative effort with NIST to characterize SRM 3280, values for the vitamin contents of SRM 3280 have been generated by a liquid chromatographic isotope dilution mass spectrometric (LC/IDMS) method. Isotope-labeled (¹³C and/or ²H) B vitamins (B1-thiamine, B6-pyridoxine, B3-nicotinamide, and B5-pantothenic acid) were obtained from commercial sources, with the support of the ODS/NIH. Our LC/IDMS method uses a C18 reversed phase column, an Agilent 1100 HPLC system, and a Quattro Micro triple-quad mass spectrometer (MS). B vitamin determination was achieved using a gradient LC profile combined with MS/MS detection in multiple reaction monitoring mode. Stock solutions of the isotope-labeled vitamins were calibrated against USP standard solutions. The SRM tablets, with added amounts of the four isotope-labeled B vitamins, were extracted and the vitamins simultaneously determined in a single LC run, in contrast with the single-component determinations performed via IDMS. Unknown vitamin concentrations were calculated by comparing the ratios of the integrated LC peaks at the different masses of the unlabeled and labeled vitamins.     

Critical review of the application of liquid chromatography/mass spectrometry to the determination of pesticide residues in biological samples

A critical review is made on the use of hyphenated liquid chromatography/mass spectrometry (LC–MS) for the identification and quantification of pesticides and their metabolites in human biosamples (whole blood, plasma, serum and urine). The first applications of LC–MS in this field began in the early 1990s. Since then, increasing interest has been shown in applying this powerful technique, with most applications dealing with the determination of a variety of chemically diverse metabolites in urine. The use of different LC–MS interfaces and mass spectral detection modes are discussed. Special attention is given to tandem mass spectrometry (MS/MS) due to its inherent advantages of increased sensitivity and selectivity, as well as its advantages for identification and confirmation of analytes in samples. Quantification can be severely affected by matrix effects, the most common being inhibition of the ionisation of analytes in the mass spectrometer, which leads to unacceptable errors if no correction is made. Different approaches can be employed to minimise this undesirable matrix effect, the preferred being the use of labelled internal standards (when available) in isotope dilution methods or the application of an efficient clean-up, performed off-line or automated on-line. Adequate criteria for confirming the identities of residues detected are required in order to avoid false positives. The criterion most commonly used with a triple quadrupole instrument is the monitoring of two MS/MS transitions together with the ion abundance ratio. TOF mass analysers are seldom used in pesticide residue analysis despite their improved resolution and mass accuracy characteristics, which makes them very suitable for confirmation purposes. The main reasons for the relative unpopularity of TOF MS in residue analysis are its limited sensitivity and its high acquisition cost. In this paper, we present a critical assessment on current techniques, trends and future developments, and give illustrative examples to point out the main characteristics of LC–MS for pesticide residue analysis in biological fluids.     

Thiol metabolomics of endothelial cells using capillary liquid chromatography mass spectrometry with isotope coded affinity tags

Thiol and disulfide levels are critical to maintaining the redox potential of a cell. Perturbations of these levels are important in disease pathogenesis. To improve endogenous mammalian metabolome quantitation, thiol specific tagging, extraction and relative quantitation were undertaken. Reduced and oxidized thiol (disulfide) metabolites from endothelial cells were tagged and extracted using cleavable isotope coded affinity tags (cICAT). Extracted cICAT labeled thiols were analyzed using capillary reverse phase liquid chromatography coupled to mass spectrometry (capLC-MS) with positive mode electrospray ionization. Reactions between thiol metabolite standards and the reactive group of cICAT indicate completion by 8h at pH 9 with no apparent disulfide formation. cICAT labeled reduced thiols from endothelial cells showed 1-5% RSD using ratiometric quantitation of isotopes and 6-17% RSD based on signal intensity alone. Sample injection was optimized to 16 pmol. Using high mass accuracy MS, 75 putative thiol metabolites were detected in all experimental samples. Treatment of endothelial cells with 2,3-dimethoxy-5-methyl-1,4-benzoquinone (BQ) shows decreased levels in 28 putative reduced thiols and increased levels of 27 putative disulfides. Treatment of endothelial cells with 30 mM glucose resulted in 22 putative reduced thiols with decreased levels and 7 putative disulfides with increased concentration. Thiols were identified based on accurate mass within 3 ppm and analysis of fragmentation patterns. Using higher collision induced dissociation (HCD), shared product ions between different thiols led to the analysis of thiols from the cysteine-glutathione (Cys-GSH) pathway. Specific reduced thiols and disulfides in this pathway revealed changes different from the overall trends of thiols/disulfides. This suggests varying regulation of the Cys-GSH pathway distinct from other thiol-containing pathways and dependence on the type of environmental stimulus. These results indicate the utility of analyzing reduced thiols and disulfides in eukaryotic samples.     

Alterations of the exo- and endometabolite profiles in breast cancer cell lines: A mass spectrometry-based metabolomics approach

In recent years, knowledge about metabolite changes which are characteristic for the physiologic state of cancer cells has been acquired by liquid chromatography coupled to mass spectrometry. Distinct molecularly characterized breast cancer cell lines provide an unbiased and standardized in vitro tumor model reflecting the heterogeneity of the disease. Tandem mass spectrometry is a widely applied analytical platform and highly sensitive technique for analysis of complex biological samples. Endo- and exometabolite analysis of the breast cancer cell lines MDA-MB-231, -453 and BT-474 as well as the breast epithelial cell line MCF-10A has been performed using two different analytical platforms: UPLC-ESI-Q-TOF based on a scheduled precursor list has been applied for highlighting of significant differences between cell lines and HPLC-ESI-QqQ using multiple reaction monitoring has been utilized for a targeted approach focusing on RNA metabolism and interconnected pathways, respectively. Statistical analysis enabled a clear discrimination of the breast epithelial from the breast cancer cell lines. As an effect of oxidative stress, a decreased GSH/GSSG ratio has been detected in breast cancer cell lines. The triple negative breast cancer cell line MDA-MB-231 showed an elevation in nicotinamide, 1-ribosyl-nicotinamide and NAD+ reflecting the increased energy demand in triple negative breast cancer, which has a more aggressive clinical course than other forms of breast cancer. Obtained distinct metabolite pattern could be correlated with distinct molecular characteristics of breast cancer cells. Results and methodology of this preliminary in vitro study could be transferred to in vivo studies with breast cancer patients.     

Optimization of a liquid chromatography ion mobility-mass spectrometry method for untargeted metabolomics using experimental design and multivariate data analysis

High-resolution mass spectrometry coupled with pattern recognition techniques is an established tool to perform comprehensive metabolite profiling of biological datasets. This paves the way for new, powerful and innovative diagnostic approaches in the post-genomic era and molecular medicine. However, interpreting untargeted metabolomic data requires robust, reproducible and reliable analytical methods to translate results into biologically relevant and actionable knowledge. The analyses of biological samples were developed based on ultra-high performance liquid chromatography (UHPLC) coupled to ion mobility - mass spectrometry (IM-MS). A strategy for optimizing the analytical conditions for untargeted UHPLC-IM-MS methods is proposed using an experimental design approach. Optimization experiments were conducted through a screening process designed to identify the factors that have significant effects on the selected responses (total number of peaks and number of reliable peaks). For this purpose, full and fractional factorial designs were used while partial least squares regression was used for experimental design modeling and optimization of parameter values. The total number of peaks yielded the best predictive model and is used for optimization of parameters setting.     

Accurate analysis of trace pentachlorophenol in textiles by isotope dilution liquid chromatography-mass spectrometry

A highly accurate method for measuring pentachlorophenol (PCP) concentrations in textile samples was developed. This highly accurate method for the analysis of textile samples is valuable, given the inherent challenges associated with the complexity of the sample matrix. This method can be applied to certify the concentration of pentachlorophenol in textile CRMs. A measurement procedure based on isotope dilution liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) was developed. Samples were pretreated with acid and then with n-hexane. Excellent precision was obtained. The validated concentration ranges for the method were 1.0-50 ng/g, the LOD was 1.0 ng/g, and the LOQ was 5.0 ng/g. The precision of this method is in the range of 0.80-1.40%. The method can trace to mass.     

Rapid and sensitive liquid chromatography with tandem mass spectrometry method for the simultaneous quantification of yonkenafil and its major metabolites in rat plasma

Yonkenafil is a promising drug for treatment of male erectile dysfunction. Previous studies showed that the piperazine‐N,N’‐deethylation metabolite, piperazine‐N‐deethylation metabolite, and piperazine‐N‐deethylation‐N,N’‐deethylation metabolite were the major metabolites of yonkenafil after extensive metabolism. We developed a sensitive and selective method for the simultaneous quantification of yonkenafil and its major metabolites using high‐throughput liquid chromatography with tandem mass spectrometry. Analytes and internal standard were extracted from a small quantity of plasma (50 μL) using liquid–liquid extraction with diethyl ether/dichloromethane (60:40, v/v), and the baseline separation was achieved on Zorbax SB‐C₁₈ column using ammonia/water/methanol (0.2:20:80, v/v/v) as the mobile phase. The assay was performed with an electrospray positive ionization mass spectrometry through the multiple‐reaction monitoring mode within 2 min. Calibration curve of the method was linear within the range of 1.00–1000 ng/mL for all the analytes with the intra‐ and interday precisions of 4.0–5.2 and 4.0–5.3% for yonkenafil, 3.1–4.9 and 3.1–5.2% for the piperazine‐N,N’‐deethylation metabolite, 4.8–6.8 and 4.8–7.3% for the piperazine‐N‐deethylation metabolite, and 2.9–6.1 and 5.4–6.3% for the piperazine‐N‐deethylation‐N,N’‐deethylation metabolite, respectively. The recoveries were above 90% with low matrix effects. The validated assay was successfully applied to support a preclinical pharmacokinetic study in six rats using a single oral dose of yonkenafil (8 mg/kg).     

Low-molecular weight protein profiling of genetically modified maize using fast liquid chromatography electrospray ionization and time-of-flight mass spectrometry

In this work, the use of liquid chromatography coupled to electrospray time-of-flight mass spectrometry (LC-TOFMS) has been evaluated for the profiling of relatively low-molecular weight protein species in both genetically modified (GM) and non-GM maize. The proposed approach consisted of a straightforward sample fractionation with different water and ethanol-based buffer solutions followed by separation and detection of the protein species using liquid chromatography with a small particle size (1.8 μm) C(18) column and electrospray-time-of-flight mass spectrometry detection in the positive ionization mode. The fractionation of maize reference material containing different content of transgenic material (from 0 to 5% GM) led to five different fractions (albumins, globulins, zeins, zein-like glutelins, and glutelins), all of them containing different protein species (from 2 to 52 different species in each fraction). Some relevant differences in the quantity and types of protein species were observed in the different fractions of the reference material (with different GM contents) tested, thus revealing the potential use of the proposed approach for fast protein profiling and to detect tentative GMO markers in maize.     

Determination of biogenic amines in wines by pre-column derivatization and high-performance liquid chromatography coupled to mass spectrometry

A new HPLC method for determining biogenic amines in wines is developed. This method is based on pre-column amine derivatization, further separation of derivatives and on-line hyphenation of HPLC to atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Biogenic amines have been derivatized with 1,2-naphthoquinone-4-sulfonate at 65 °C and pH 9.2 for 5 min. The separation of derivatives has been accomplished in a C₁₈ analytical column using an elution gradient based on increasing the percentage of methanol. Derivatives have been ionized in positive mode and detected by selected ion monitoring. The operating conditions of the APCI-MS system (voltages, temperatures and gases) have been thoroughly optimized to obtain the maximum sensitivity for all analytes. In the selected conditions, APCI-MS spectra display little fragmentation and good signal-to-noise ratio. Depending on the amine characteristics, the main spectral peaks are due to mono- and di-derivative products. Figures of merit of the method have been established under the selected conditions using red wine samples. Recoveries ranging from 94% to 106% have been obtained which prove excellent accuracy of the method in the determination of histamine, putrescine, cadaverine, tryptamine, phenylethylamine, tyramine and serotonin in red wines. The proposed method has been applied to the analysis of commercial wines from different Spanish regions.     

Identification of impurities in polymyxin B and colistin bulk sample using liquid chromatography coupled to mass spectrometry

The European Pharmacopoeia (Ph. Eur.) describes liquid chromatography-ultraviolet (LC-UV) methods using C₁₈ stationary phases for the analysis of polymyxin B and colistin.Several unknown impurities were detected in commercial samples of those polypeptide complexes. However, the Ph. Eur. does not specify any related substances for polymyxin B and colistin. Since both methods use non-volatile buffers, the mobile phases were incompatible with mass spectrometry (MS). For the identification of related substances in bulk samples by LC/MS, volatile mobile phase systems were developed. However, the LC/MS methods (with volatile additives) showed inferior chromatographic separation compared to the LC-UV method (with non-volatile additives). Moreover, previously identified impurities by LC/MS could not be assigned in LC-UV methods as the separation in both systems was different.In this study, known impurities were traced in the LC-UV methods and new impurities present in polymyxin B and colistin bulk samples were characterized. To achieve this, each peak from the non-volatile system was collected separately and reinjected into an LC system with a volatile mobile phase coupled to MS. This way, collected impurity peaks were rechromatographed on a reversed phase column in order to separate the analyte from the buffer salts. Using this method, out of 39 peaks, five novel related substances were characterized in a polymyxin B bulk sample. Fourteen impurities, which were already reported in the literature were traced as good as possible in the LC-UV method. In the case of colistin, a total of 36 peaks were investigated, among which four new compounds. Additionally, 30 known impurities were traced in the LC-UV method.