Proteomic LC-MS systems using nanoscale liquid chromatography with tandem mass spectrometry

Current nano-scale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) approaches in proteome research are reviewed from an analytical perspective. For comprehensive analysis of cellular proteins, analytical methods with higher resolution, sensitivity, and wider dynamic range are required. Miniaturized LC coupled with tandem mass spectrometry is currently one of the most versatile techniques. In this review, the current status of nanoLC-MS/MS systems as well as data management systems is addressed. In addition, the future prospects for complete proteomics are discussed.     

Multiresidue analysis of avermectins in cattle liver by liquid chromatography/tandem mass spectrometry

A simple multiresidue analysis method was developed for the confirmation and quantification of eprinomectin, abamectin, doramectin, and ivermectin in cattle liver. The target analytes were extracted with acetonitrile and cleaned up with C18 and C8 solid-phase extraction cartridges before analysis by liquid chromatography combined with positive ion electrospray tandem mass spectrometry. The residue mean recoveries of the 4 drugs were 64.16-99.53%. The limit of detection was 2.5 ng/g for eprinomectin, abamectin, doramectin, and ivermectin.     

Ultra high performance liquid chromatography with quadrupole time-of-flight tandem mass spectrometry and liquid chromatography with tandem mass spectrometry combined with chemometric analysis as an approach for the quality evaluation of Mume Fructus

Mume Fructus is an important traditional Chinese medicine that has been widely used in the treatment of intestinal diseases and asthma for thousands of years. In order to evaluate the quality of Mume Fructus in different processing methods, the main chemical components in Mume Fructus were investigated and a method was established for simultaneous quantification of organic acids of Mume Fructus. First, an optimized ultra-performance liquid chromatography-quadrupole-time of flight tandem-mass spectrometry method was used to identify the structures of main components in Mume Fructus. A total of 41 chemical compounds were identified, including 11 organic acids, 13 flavonoids, and three fatty acids. The contents of 11 organic acids in 18 batches of Mume Fructus from different processing methods were simultaneously determined by a liquid chromatography with tandem mass spectrometry method. The results of quantitative and hierarchical cluster analysis indicated that Mume Fructus under different processing methods were rich in the above 11 organic acids and the contents were obviously different. Taken together, the proposed quality evaluation method was fast and comprehensively reflects the content of the main chemical components in Mume Fructus under different processing methods, and provides a useful reference for the quality control and evaluation of Mume Fructus.     

Aldehyde analysis by high performance liquid chromatography/tandem mass spectrometry

This paper describes the development of a high performance liquid chromatography/tandem mass spectrometric (MS/MS) procedure for the specific qualitative and quantitative analysis of lipid aldehydes in biological matrices. A derivatisation method, which results in molecules that exhibit a common product ion on MS/MS, permits informative precursor ion scans, at high sensitivity. This has been applied to the examination of plasma in order to examine the production of aldehydes consequent on in vitro lipid oxidation. Quantitative analysis of target molecules using multiple reaction monitoring has been developed to permit quantitation in the same matrices.     

Multiresidue pesticide analysis of fruits by ultra-performance liquid chromatography tandem mass spectrometry

Ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS–MS) has been used for screening and quantification of 32 pesticides and metabolites in two fruit matrices. The compounds investigated belonged to different chemical families of insecticides, acaricides, fungicides, and herbicides; several metabolites were also included. Quantification was conducted using matrix-matched standards calibration; response was a linear function of concentration in the range tested (10–500 ng mL⁻¹). The method was validated with blank samples of lemon and raisin spiked at 0.01 and 0.1 mg kg⁻¹, and recoveries were satisfactory, between 70 and 110%, for most of the pesticides tested and relative standard deviations were below 15% (n = 5 at each spiking level). Excellent sensitivity resulted in limits of detection for all compounds well below 0.01 mg kg⁻¹, with the limit of quantification being validated at 0.01 mg kg⁻¹. The UPLC system generates narrow peaks (approx. 5 s), thus increasing peak height and improving sensitivity. This improved separation efficiency facilitates adequate resolution not only of the analytes but also of matrix interferences compared with conventional HPLC. The method developed could also resolve some geometric isomers. The main advantage of this approach is the high sample throughput achieved because of the short analysis time, which enables satisfactory separation of all the compounds in less than 5 min per sample.     

Quantitative analysis of phosphatidylcholine in rat liver tissue by nanoflow liquid chromatography/tandem mass spectrometry

A quantitative method was developed for the determination of phosphatidylcholine (PC) species concentration using nanoflow LC-ESI-MS/MS. In this study, a calibration method is developed to determine the effect of PC carbon chain length on MS peak intensity. Using the multiple standard addition method, a relationship between the peak intensities of different PC species from nanoflow LC-MS and carbon chain length is established first using different injection amounts of PC standards. From this relationship, a calibration curve for each carbon chain length can be obtained for the concentration calculation. It was found that the MS peak area of PC species analyzed by nanoflow LC-MS linearly decreased with increased acyl carbon numbers, and that the effect of the degree of acyl chain unsaturation on MS peak intensity was minimized when the injection amount was maintained at less than 1 pmol. The method was applied for the quantitative calculation of 34 PC species from rat liver, which were identified from data-dependent MS/MS analysis during nanoflow LC separation.     

Determination of 11 aminoglycosides in meat and liver by liquid chromatography with tandem mass spectrometry

A method using liquid chromatography with tandem mass spectrometry was developed for the determination of 11 commonly used aminoglycoside antibiotics in meat. The proposed method is sufficiently sensitive (detection limits of 15 to 40 ppb for the various antibiotics) and highly selective. It is suitable for the quantitation and confirmation of aminoglycosides in a variety of matrixes (pork muscle, fish, and veal liver). Any multiresidue method for aminoglycosides must take into account their high affinity toward sample proteins and the significantly different pK values of the various analytes. The developed method uses a low-pH extraction with trichloracetic acid to ensure complete extraction of the analytes from the matrix. An anion-exchange step is used to remove the acid from the centrifuged extract. Aminoglycosides in this solution of low ionic strength can be quantitatively retained and afterwards eluted from a weak cation-exchanger solid-phase extraction (SPE) cartridge. The highly selective SPE steps produce clean extracts, which minimize possible suppression of the mass spectrometer signal.     

Detection of sulfur-fumigated paeoniae alba radix in complex preparations by high performance liquid chromatography tandem mass spectrometry

Detection of sulfur-fumigated Paeoniae Alba Radix (PAR) in different complex preparations is challenging due to the relatively lower content of PAR and interference from more complicated components in complex preparations with different multiple constituent herbs. In this study, a high performance liquid chromatography- triple-quadrupole tandem mass spectrometry method was developed for detecting sulfur-fumigated PAR in different complex preparations. Paeoniflorin, the major component of PAR, and paeoniflorin sulfonate, the characteristic artifact transformed from paeoniflorin during sulfur-fumigation of PAR, were used as chemical markers. Multiple reaction monitoring (MRM) scan was employed to maximize sensitivity and selectivity. Through optimizing full mass scan and daughter ion scan conditions, two mass transitions were selected and employed respectively for unequivocal identification of paeoniflorin and paeoniflorin sulfonate. The detection limits for paeoniflorin and paeoniflorin sulfonate using MRM were much lower than those detected with UV 270 nm. Paeoniflorin and paeoniflorin sulfonate could be simultaneously detected in different commercial PAR-containing complex preparations without interference of other components using the established method, indicating that the newly established method was selective and sensitive enough for screening sulfur-fumigated PAR in commercial complex preparations.     

Quasi-linear gradients for capillary liquid chromatography with mass and tandem mass spectrometry

Gradient elution, capillary liquid chromatography mass spectrometry was performed with linear, static gradients constructed by laminar flowing ten, 1.5 microL volume steps of decreasing organic concentration into tubing of small internal diameter. Sample loading, gradient formation, and sample elution were accomplished entirely by means of a commercially available micro-autosampler and single-syringe drive pump. The procedure was simple, fast, stable, and reproducible. Essentially linear gradients were produced without the use of additional valves, mixers, pumps or software. It took less than 10 minutes to form a gradient and less than 30 minutes to construct the set of individual buffer vials. The gradients were shown to be stable to storage. One hour after forming, peak retention times were reproduced to +/-0.5%. Long-term retention time reproducibility was found to vary by +/-2%. Chromatographic resolution was comparable or superior to that obtained by gradient elution with conventional dynamic mixing and split flow. The procedure was adapted with a 'peak parking' method which extended the time for generating peptide fragmentation data up to 10 minutes per peptide with the triple quadruple mass spectrometer. Using this technique, collision data were collected at the 25 femtomole level on nine of ten tryptic peptides in a single run.     

Ultra performance liquid chromatography tandem mass spectrometry performance evaluation for analysis of antibiotics in natural waters

An ultra performance liquid chromatography electrospray tandem mass spectrometry (UPLC/MS/MS) method was developed and validated for the determination of 17 antibiotics in natural waters in one single extraction and chromatographic procedure. Gradient separation conditions were optimised for 17 compounds belonging to five different antibiotic groups: quinolones (oxolinic acid, nalidixic acid, pipemidic acid, flumequine), fluoroquinolones (enoxacin, ciprofloxacin, norfloxacin, ofloxacin, enrofloxacin, sarafloxacin, danofloxacin, difloxacin, lomefloxacin), sulphonamides (sulphamethoxazole, sulphamethazine), nitro-imidazole (ornidazole) and diaminopyrimidine (trimethoprim). The separation of all compounds, obtained using a 1.7 μm particle size column (100 mm?×?2.1 mm), was achieved within 10 min time. Water samples were adjusted to pH 7 and extracted using Oasis hydrophilic–lipophilic balance (HLB) solid phase extraction cartridges. After elution with methanol and concentration, extracts were injected in a C18 column (Acquity UPLC BEH C18) and detected by tandem mass spectrometry. Average recovery from 100 ng L^?1 fortified samples was higher than 70% for most of the compounds, with relative standard deviations below 20%. Performances of the method (recoveries, detection limit, quantification limit and relative standard deviation) and matrix effects were studied, and results obtained showed that method was suitable for routine analysis of antibiotics in surface water. Samples analysis from Seine River (France) confirmed the interest of antibiotic contamination evaluation in that area.

Comparison of negative and positive ion electrospray tandem mass spectrometry for the liquid chromatography tandem mass spectrometry analysis of oxidized deoxynucleosides

Oxidized deoxynucleosides are widely used as biomarkers for DNA oxidation and oxidative stress assessment. Although gas chromatography mass spectrometry is widely used for the measurement of multiple DNA lesions, this approach requires complex sample preparation contributing to possible artifactual oxidation. To address these issues, a high performance liquid chromatography (HPLC)-tandem mass spectrometric (LC-MS/MS) method was developed to measure 8-hydroxy-2'-deoxyguanosine (8-OH-dG), 8-hydroxy-2'-deoxyadenosine (8-OH-dA), 2-hydroxy-2'-deoxyadenosine (2-OH-dA), thymidine glycol (TG), and 5-hydroxy-methyl-2'-deoxyuridine (HMDU) in DNA samples with fast sample preparation. In order to selectively monitor the product ions of these precursors with optimum sensitivity for use during quantitative LC-MS/MS analysis, unique and abundant fragment ions had to be identified during MS/MS with collision-induced dissociation (CID). Positive and negative ion electrospray tandem mass spectra with CID were compared for the analysis of these five oxidized deoxynucleosides. The most abundant fragment ions were usually formed by cleavage of the glycosidic bond in both positive and negative ion modes. However, in the negative ion electrospray tandem mass spectra of 8-OH-dG, 2-OH-dA, and 8-OH-dA, cleavage of two bonds within the sugar ring produced abundant S1 type ions with loss of a neutral molecule weighing 90 u, [M - H - 90]-. The signal-to-noise ratio was similar for negative and positive ion electrospray MS/MS except in the case of thymidine glycol where the signal-to-noise was 100 times greater in negative ionization mode. Therefore, negative ion electrospray tandem mass spectrometry with CID would be preferred to positive ion mode for the analysis of sets of oxidized deoxynucleosides that include thymidine glycol. Investigation of the fragmentation pathways indicated some new general rules for the fragmentation of negatively charged oxidized nucleosides. When purine nucleosides contain a hydroxyl group in the C8 position, an S1 type product ion will dominate the product ions due to a six-membered ring hydrogen transfer process. Finally, a new type of fragment ion formed by elimination of a neutral molecule weighing 48 (CO2H4) from the sugar moiety was observed for all three oxidized purine nucleosides.     

Congener-specific analysis of hexabromocyclododecane by high-performance liquid chromatography/electrospray tandem mass spectrometry

A congener-specific method based on high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ES-MS/MS) in the negative ion mode was developed for the analysis of hexabromocyclododecane (HBCDD). On a C(18) analytical column, with a methanol/water mobile phase, the alpha-isomer was completely resolved from the beta- and gamma-isomers while the beta- and gamma-isomers were sufficiently resolved at half their peak heights. The ES spray voltage strongly influenced the intensity of the ion signal. For MS, a source temperature of 500 degrees C and a collision energy of 50 eV were found to be optimum for the [M-H](-) to Br(-) transition. Run-to-run and day-to-day (n = 3) variability was minimal, with relative standard deviations of 2.6-4.1 and 2.4-4.4%, respectively. The limit of detection was 4-6 pg on-column. When applied to tissue samples from Lake Winnipeg fish both alpha- and gamma-isomers of HBCDD were found in low-ng/g (lipid corrected) concentrations.     

Enantioselective analysis of zopiclone in rat brain by liquid chromatography tandem mass spectrometry

A new high-performance liquid chromatographic method with triple quadrupole mass spectrometry detection was developed and validated for the quantification of zopiclone enantiomers in rat brain samples. Zopiclone enantiomers were resolved on a CHIRALPAK AD column with a mobile phase consisting of acetonitrile/ethanol/methanol (60:20:20, v/v/v) at a flow rate of 1.3 mL?min^-1. Moclobemide was used as internal standard. The sample treatment procedure was carried out employing solid-phase extraction, yielding mean absolute recoveries of 89.6 and 91.7 % for each zopiclone enantiomer. The validated method showed linearity in the range of 0.29–344.8 ng?g^?1, with quantification limits of 0.29 ng?g^?1 for both enantiomers. Precision and accuracy were within acceptable levels of confidence (<15 %). The method was applied in a pilot study of zopiclone kinetic disposition in rats. It could be observed that the levels of (+)-(S)-zopiclone were always higher than those of (-)-(R)-zopiclone, confirming the stereoselective disposition of zopiclone.     

Broad-spectrum drug screening of meconium by liquid chromatography with tandem mass spectrometry and time-of-flight mass spectrometry

Analysis of the major drugs of abuse in meconium has been established in clinical practice for detecting fetal exposure to illicit drugs, particularly for the ready availability of the sample and ease of collection from diapers, compared with neonatal hair and urine. Very little is known about the occurrence and detection possibilities of therapeutic and licit drugs in meconium. Meconium specimens (n = 209) were collected in delivery hospitals, from infants of mothers who were suspected to be drug abusers. A targeted analysis method by liquid chromatography–triple quadrupole mass spectrometry (LC-MS/MS) was developed for abused drugs: amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, morphine, codeine, 6-monoacetylmorphine, oxycodone, methadone, tramadol, buprenorphine, and norbuprenorphine. A separate LC-MS/MS method was developed for 11-nor-∆⁹-tetrahydrocannabinol-9-carboxylic acid. A screening method based on LC coupled to time-of-flight MS was applied to a broad spectrum of drugs. As a result, a total of 77 different compounds were found. The main drug findings in meconium were as follows: local anesthetics 82.5% (n = 172), nicotine or its metabolites 61.5% (n = 129), opioids 48.5% (n = 101), stimulants 21.0% (n = 44), hypnotics and sedatives 19.0% (n = 40), antidepressants 18.0% (n = 38), antipsychotics 5.5% (n = 11), and cannabis 3.0% (n = 5). By revealing drugs and metabolites beyond the ordinary scope, the present procedure helps the pediatrician in cases where maternal denial is strong but the infant seems to suffer from typical drug-withdrawal symptoms. Intrapartum drug administration cannot be differentiated from gestational drug use by meconium analysis, which affects the interpretation of oxycodone, tramadol, fentanyl, pethidine, and ephedrine findings.     

High-throughput chiral liquid chromatography/tandem mass spectrometry

Chiral liquid chromatography is a well-established area of bioanalytical chemistry and is often used during the processes of drug discovery and development. The development and use of a chiral drug require the understanding of the pharmacokinetic characteristics of each of the enantiomers, including potential differences in their absorption, distribution, metabolism, and excretion. Chromatographic techniques coupled to atmospheric pressure ionization-tandem mass spectrometry have shown potential as sensitive and robust tools in the quantitative and qualitative determination of enantiomers in biologic fluids and tissue extracts. However, development of a chiral liquid chromatography method requires time-consuming procedures that are devised empirically. Clearly, there is an incentive to design chromatographic approaches that are easy to use, compatible with mass spectrometry ionization interface conditions, exhibit relatively short run times without compromising sensitivity, and offer a broad analyte specificity. For these reasons, the present paper explores the feasibility of the bonded macrocyclic glycopeptide phases (teicoplanin and vancomycin) for analysis by chiral liquid chromatography/tandem mass spectrometry. Ritalinic acid, pindolol, fluoxetine, oxazepam, propranolol, terbutaline, metoprolol, and nicardipine were tested in this study. Furthermore, an example of a simultaneous chiral LC/MS/MS detection (chromatographic run time approximately 10 min) of four pharmaceutical products resulting in baseline resolutions of all four pairs of enantiomers is presented. Methanol, an MS-compatible mobile phase, was utilized in all the experiments.     

Plasma lipid analysis by hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A novel method for the analysis of endogenous lipids and related compounds was developed employing hydrophilic interaction liquid chromatography with electrospray ionization tandem mass spectrometry. A hydrophilic interaction liquid chromatography with carbamoyl stationary phase achieved clear separation of phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, ceramide, and mono‐hexsosyl ceramide groups with good peak area repeatability (RSD% < 10) and linearity (R² > 0.99). The established method was applied to human plasma assays and a total of 117 endogenous lipids were successfully detected and reproducibly identified. In addition, we investigated the simultaneous detection of small polar metabolites such as amino and organic acids co‐existing in the same biological samples processed in a single analytical run with lipids. Our results show that hydrophilic interaction liquid chromatography is a useful tool for human plasma lipidome analysis and offers more comprehensive metabolome coverage.     

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.     

Proteome analysis in the bovine adrenal medulla using liquid chromatography with tandem mass spectrometry

Liquid chromatography/mass spectrometry (LC/MS)-based proteomics has been used to identify soluble proteins in the bovine adrenal medulla. This gland is a major source of hormones, opioids, neurotransmitters, and several vital proteins. The adrenal medulla proteins were first purified using ammonium sulfate precipitation. The resulting proteins were then pre-fractionated with a C-4 high-performance liquid chromatography (HPLC) column. Each 2-min HPLC fraction was digested with trypsin, and separated further and analyzed using capillary liquid chromatography/tandem mass spectrometry (capLC/nanospray-MS/MS) to map the proteome of the adrenal medulla. The parent mass and sequence ion information thus obtained for tryptic peptides was used to search the NCBInr database using the SEQUEST search engine. A total of 195 proteins were identified, of which 71 had good scores (delta correlation value greater than 0.1, preliminary score above 200, and cross-correlation value above 2.5). The prominent proteins thus identified are secretogranin I precursor, chromogranin A, proenkephalin A precursor, myosin X, hemoglobin beta chain, hemoglobin alpha chain, heat shock protein 10 kDa, and replicase.     

Elucidation of triacylglycerols in cod liver oil by liquid chromatography electrospray tandem ion-trap mass spectrometry

Though liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS²) has been widely used in the structural elucidation of triacylglycerols (TAG) in vegetable oils, its potentiality for the identification of TAG molecules in omega-3 rich oils remains unexplored till date. Hence, this article investigates the applicability of LC-ESI-MS² for the structural characterization of naturally occurring TAG in cod liver oil without the TAG fractionation during the sample preparation. A computational algorithm was developed to automatically interpret the mass spectra and elucidate the TAG structures respectively. The results were compared against the lipase benchmark method. A principal component analysis study revealed that it is possible to discriminate genuine from adulterated cod liver oil.