Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers

Sphingolipids are a highly diverse category of bioactive compounds. This article describes methods that have been validated for the extraction, liquid chromatographic (LC) separation, identification and quantitation of sphingolipids by electrospray ionization, tandem mass spectrometry (ESI-MS/MS) using triple quadrupole (QQQ, API 3000) and quadrupole-linear-ion trap (API 4000 QTrap, operating in QQQ mode) mass spectrometers. Advantages of the QTrap included: greater sensitivity, similar ionization efficiencies for sphingolipids with ceramide versus dihydroceramide backbones, and the ability to identify the ceramide backbone of sphingomyelins using a pseudo-MS³ protocol. Compounds that can be readily quantified using an internal standard cocktail developed by the LIPID MAPS Consortium are: sphingoid bases and sphingoid base 1-phosphates, more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides, and these complex sphingolipids with dihydroceramide backbones. With minor modifications, glucosylceramides and galactosylceramides can be distinguished, and more complex species such as sulfatides can also be quantified, when the internal standards are available. JLR LC ESI-MS/MS can be utilized to quantify a large number of structural and signaling sphingolipids using commercially available internal standards. The application of these methods is illustrated with RAW264.7 cells, a mouse macrophage cell line. These methods should be useful for a wide range of focused (sphingo)lipidomic investigations.     

Analysis of mammalian sphingolipids by liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS)

Sphingolipids are a highly diverse category of molecules that serve not only as components of biological structures but also as regulators of numerous cell functions. Because so many of the structural features of sphingolipids give rise to their biological activity, there is a need for comprehensive or "sphingolipidomic" methods for identification and quantitation of as many individual subspecies as possible. This review defines sphingolipids as a class, briefly discusses classical methods for their analysis, and focuses primarily on liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS). Recently, a set of evolving and expanding methods have been developed and rigorously validated for the extraction, identification, separation, and quantitation of sphingolipids by LC-MS/MS. Quantitation of these biomolecules is made possible via the use of an internal standard cocktail. The compounds that can be readily analyzed are free long-chain (sphingoid) bases, sphingoid base 1-phosphates, and more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides, sulfatides, and novel compounds such as the 1-deoxy- and 1-(deoxymethyl)-sphingoid bases and their N-acyl-derivatives. These methods can be altered slightly to separate and quantitate isomeric species such as glucosyl/galactosylceramide. Because these techniques require the extraction of sphingolipids from their native environment, any information regarding their localization in histological slices is lost. Therefore, this review also describes methods for TIMS. This technique has been shown to be a powerful tool to determine the localization of individual molecular species of sphingolipids directly from tissue slices.     

Eicosanomics: targeted lipidomics of eicosanoids in biological systems

Recent advancements in mass spectrometry, especially the development of electrospray tandem mass spectrometry (ESI/LC/MS2) and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI/TOF), have greatly facilitated analysis of complex biomolecules. It has now become possible to profile, in relatively short periods of time, large multicomponent groups of compounds biosynthesized by biological systems. The efficiency and accuracy of analysis have led to the development of new concepts of mass spectrometric profiling, mapping, and imaging. Profiling of proteins in biological material (proteomics) has become a widely accepted strategy for identification of mechanisms involved in the biochemistry of disease processes, and has become a novel tool for unraveling new drug targets. Evolution of proteomics has relied on ESI/LC/MS2 and MALDI/TOF, techniques that are also useful in the novel area of quantitative proteomics.     

Simultaneous quantitation of aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine and benzoylhypaconine in human plasma by liquid chromatography-tandem mass spectrometry and pharmacokinetics evaluation of "SHEN-FU" injectable powder

A rapid, specific and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed for simultaneous quantitation of six Aconitum alkaloids, i.e. aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylaconine (BAC), benzoylmesaconine (BMA) and benzoylhypaconine (BHA) in human plasma collected from 18 healthy volunteers after intravenous drop infusion of "SHEN-FU" injectable powder in three different dosages. Lappaconitine was selected as the internal standard (IS). LC/MS/MS system coupled with an electrospray ionization (ESI) source was performed in multiple-reaction monitoring (MRM) mode. The transitions of the Aconitum alkaloids executed as following: m/z 646.3-->586.0 for AC; m/z 632.4-->573.1 for MA; m/z 616.2-->556.1 for HA; m/z 604.2-->104.8 for BAC; m/z 590.1-->104.8 for BMA; m/z 574.1-->104.8 for BHA; m/z 585.2-->161.8 for IS. Sample preparation was performed with solid-phase extraction (SPE) on a 1 mL HLB cartridge prior to analysis. The separation was applied on a Waters C(18) column (1.7 microm, 2.1 mm x 100 mm) and a gradient elution of methanol and 0.1% formic acid-water was used as mobile phase. The retention time was less than 4.5 min. The concentrations ranged from 0.1 to 1000 ng/mL for all six Aconitum alkaloids and showed a good linearity with the correlation coefficient (r(2)) >0.995. The validated method was employed to simultaneous quantitation and successfully used for the first time for the pharmacokinetic evaluation of the six Aconitum alkaloids after intravenous drop administration of "SHEN-FU" injectable powder in phase I clinical trial.     

Rapid confirmatory method for the determination of sixteen synthetic growth promoters and bisphenol A in bovine milk using dispersive solid-phase extraction and liquid chromatography–tandem mass spectrometry

A rapid liquid chromatographic–tandem mass spectrometric (LC–MS/MS) multi-residue method for the simultaneous quantitation and identification of sixteen synthetic growth promoters and bisphenol A in bovine milk has been developed and validated. Sample preparation was straightforward, efficient and economically advantageous. Milk was extracted with acetonitrile followed by phase separation with NaCl. After centrifugation, the extract was purified by dispersive solid-phase extraction with C18 sorbent material. The compounds were analysed by reversed-phase LC–MS/MS using both positive and negative ionization and operated in multiple reaction monitoring (MRM) mode, acquiring two diagnostic product ions from each of the chosen precursor ions for unambiguous confirmation. Total chromatographic run time was less than 10 min for each sample. The method was validated at a level of 1 μg L^?1. A wide variety of deuterated internal standards were used to improve method performance. The accuracy and precision of the method were satisfactory for all analytes. The confirmative quantitative liquid chromatographic tandem mass spectrometric (LC–MS/MS) method was validated according to Commission Decision 2002/657/EC. The decision limit (CCα) and the detection capability (CCβ) were found to be below the chosen validation level of 1 μg L^?1 for all compounds.     

Advances of infrared spectroscopy in natural product research

Natural product's properties are related to certain classes of compounds such as alkaloids, flavonoids, essential oils and others. Traditionally, separation techniques including thin layer chromatography (TLC), liquid chromatography (LC), gas chromatography (GC) and capillary electrophoresis (CE) even hyphenated to mass spectrometry (MS) were used for the elucidation, qualitative and quantitative analysis of individual compounds.In food industry, spectroscopic investigations using infrared radiation have been used to monitor and evaluate the composition and quality already since the early sixties. During the last four decades near-infrared spectroscopy (NIR; 800–2500 nm; 12,500–4000 cm⁻¹) has become one of the most attractive and used methods for analysis for the following reasons: it represents a non-invasive analytical tool allowing a fast and simultaneous qualitative and quantitative characterization of natural products and their constituents. Additionally, the development of custom-made hand-held instruments enables in-field measurement for determining the optimum harvest time.Attenuated total reflection (ATR) and Fourier transform infrared (FTIR) spectroscopic imaging are suitable not only for the differentiation of different plant species, but also to distinct various ingredients within a plant. FTIR spectroscopic microscopy enables molecular imaging of complex botanical samples and therefore the detection and characterization of the molecular components of biological tissue.In the present contribution, the principle, technique and methodology of the different infrared spectroscopic methods are described followed by a discussion of quantitative and qualitative application possibilities in the field of natural product analysis.     

A new quantitative LC tandem mass spectrometry assay for serum 25-hydroxy vitamin D

BackgroundThe accurate measurement of 25-hydoxy vitamin D (25OH-D) in serum has been a challenge for many years. We developed a liquid chromatography tandem mass spectrometry (LC Tandem MS) assay for the quantitative determination of 25OH-D₂ and 25OH-D₃ in serum. The new method was compared with two widely used commercially available immunoassays.MethodsSample preparation involved protein precipitation with acetonitrile containing deuterated forms of the target species as internal standards. An API 5000 mass spectrometer coupled with a photoionization source was used for quantitation. The performance of the new LC Tandem MS assay was compared with a radioimmunoassay (RIA, Diasorin) and a chemiluminescence immunoassay (ECLIA, Roche Diagnostics), analysing serum obtained from 152 individuals.ResultsUsing 100 μl of serum, the LC Tandem MS assay had a limit of quantitation of 1.3 nmol/L for both 25OH-D₂ and 25OH-D₃ with a linear response between 1.3 and 625 nmol/L and accuracy of between 95 and 124%. Intra- and inter-assay precision were ≤7% and ≤4%, respectively. Measurement of 25OH-D levels in 152 serum samples gave run averages of 71, 56 and 62 nmol/L for LC Tandem MS, ECLIA and RIA, respectively. Correlations between the various methods were: LC Tandem MS vs. RIA: r = 0.931; LC Tandem MS vs. ECLIA: r = 0.784; RIA vs. ECLIA: r = 0.787. The LC Tandem MS method had a positive proportional bias of 26% over the RIA, whereas the ECLIA showed variable differences.ConclusionThe new LC Tandem MS assay is accurate and precise at physiologically relevant 25OH-D concentrations, and compares favourably with the RIA. In contrast, the ECLIA shows variable bias with the other assays tested.     

Nearline acquisition and processing of liquid chromatography-tandem mass spectrometry data

Liquid chromatography–mass spectrometry (LC–MS) is a commonly used analytical platform for non-targeted metabolite profiling experiments. Although data acquisition, processing and statistical analyses are almost routine in such experiments, further annotation and subsequent identification of chemical compounds are not. For identification, tandem mass spectra provide valuable information towards the structure of chemical compounds. These are typically acquired online, in data-dependent mode, or offline, using handcrafted acquisition methods and manually extracted from raw data. Here, we present several methods to fast-track and improve both the acquisition and processing of LC–MS/MS data. Our nearly online (nearline) data-dependent tandem MS strategy creates a minimal set of LC–MS/MS acquisition methods for relevant features revealed by a preceding non-targeted profiling experiment. Using different filtering criteria, such as intensity or ion type, the acquisition of irrelevant spectra is minimized. Afterwards, LC–MS/MS raw data are processed with feature detection and grouping algorithms. The extracted tandem mass spectra can be used for both library search and de-novo identification methods. The algorithms are implemented in the R package MetShot and support the export to Bruker, Agilent or Waters QTOF instruments and the vendor-independent TraML standard. We evaluate the performance of our workflow on a Bruker micrOTOF-Q by comparison of automatically acquired and extracted tandem mass spectra obtained from a mixture of natural product standards against manually extracted reference spectra. Using Arabidopsis thaliana wild-type and biosynthetic gene knockout plants, we characterize the metabolic products of a biosynthetic pathway and demonstrate the integration of our approach into a typical non-targeted metabolite profiling workflow.     

Lipidomic and metabolomic analysis reveals changes in biochemical pathways for non-small cell lung cancer tissues

Lung cancer represents one of the leading worldwide causes of cancer death, but the pathobiochemistry of this disease is still not fully understood. Here we characterize the lipidomic and metabolomic profiles of the tumor and surrounding normal tissues for 23 patients with non-small cell lung cancer. In total, 500 molecular species were identified and quantified by a combination of the lipidomic shotgun tandem mass spectrometry (MS/MS) analysis and the targeted metabolomic approach using liquid chromatography (LC) – MS/MS. The statistical evaluation includes multivariate and univariate methods with the emphasis on paired statistical approaches. Our research revealed significant changes in several biochemical pathways related to the central carbon metabolism, acylcarnitines, dipeptides as well as the disruption in the lipid metabolism observed mainly for glycerophospholipids, sphingolipids, and cholesteryl esters.     

Development and validation of a liquid chromatography/tandem mass spectrometry procedure for the quantification of sunitinib (SU11248) and its active metabolite, N-desethyl sunitinib (SU12662), in human plasma: application to an explorative study

A sensitive, precise and accurate quantitative liquid chromatography/tandem mass spectrometry (LC–MS/MS) method for the measurement of sunitinib (SU11248) and N-desethyl sunitinib (SU12662) in human plasma was developed and validated. All sample handling was done under strict light protection. The sample preparation method employed acetonitrile protein precipitation using d₅-SU11248 as an internal standard. The processed samples were chromatographed on a polymeric reversed-phase analytical column and analyzed by triple-quadrupole MS/MS in multiple reaction monitoring (MRM) mode using positive TurboIonSpray^® (TISP). The LC–MS/MS method described in this paper presents high absolute recovery (86.2% SU11248, 84.8% SU12662), high sensitivity (lower limit of quantitation of 0.06 ng/mL for both analytes), high inter-day precision (1.6–6.1% SU11248, 1.1–5.3% SU12662) and high analytical recovery (99.8–109.1% SU11248, 99.9–106.2% SU12662), as well as excellent linearity over the concentration range 0.060–100 ng/mL (r² > 0.999) with a short runtime of only 4.0 min. Results on the stability of SU11248 and SU12662 in human plasma are presented. During validation plasma from intensive care patients receiving many drugs were tested for interference and incurred samples were analyzed. The method met all criteria of the EMA and FDA guidelines during validation and was successfully applied to a pharmacokinetic study in healthy human volunteers.     

Liquid chromatography high‐resolution mass spectrometry for fatty acid profiling

Quantification of fatty acids has been crucial to elucidate lipid biosynthesis pathways in plants. To date, fatty acid identification and quantification has relied mainly on gas chromatography (GC) coupled to flame ionization detection (FID) or mass spectrometry (MS), which requires the derivatization of samples and the use of chemical standards for annotation. Here we present an alternative method based on a simple procedure for the hydrolysis of lipids, so that fatty acids can be quantified by liquid chromatography mass spectrometry (LC‐MS) analysis. Proper peak annotation of the fatty acids in the LC‐MS‐based methods has been achieved by LC‐MS measurements of authentic standard compounds and elemental formula annotation supported by ¹³C isotope‐labeled Arabidopsis. As a proof of concept, we have compared the analysis by LC‐MS and GC‐FID of two previously characterized Arabidopsis thaliana knock‐out mutants for FAD6 and FAD7 desaturase genes. These results are discussed in light of lipidomic profiles obtained from the same samples. In addition, we performed untargeted LC‐MS analysis to determine the fatty acid content of two diatom species. Our results indicate that both LC‐MS and GC‐FID analyses are comparable, but that because of higher sensitivity and selectivity the LC‐MS‐based method allows for a broader coverage and determination of novel fatty acids.     

Chemical derivatization and mass spectral libraries in metabolic profiling by GC/MS and LC/MS/MS

An overview is presented of gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), the two major hyphenated techniques employed in metabolic profiling that complement direct 'fingerprinting' methods such as atmospheric pressure ionization (API) quadrupole time-of-flight MS, API Fourier transform MS, and NMR. In GC/MS, the analytes are normally derivatized prior to analysis in order to reduce their polarity and facilitate chromatographic separation. The electron ionization mass spectra obtained are reproducible and suitable for library matching, mass spectral collections being readily available. In LC/MS, derivatization and library matching are at an early stage of development and mini-reviews are provided. Chemical derivatization can dramatically increase the sensitivity and specificity of LC/MS methods for less polar compounds and provides additional structural information. The potential of derivatization for metabolic profiling in LC/MS is demonstrated by the enhanced analysis of plant extracts, including the potential to measure volatile acids such as formic acid, difficult to achieve by GC/MS. The important role of mass spectral library creation and usage in these techniques is discussed and illustrated by examples.     

Affinity chromatography as a method for sample preparation in gas chromatography/mass spectrometry.

Analytical chemistry aims at developing analytical methods and techniques for unequivocal identification and accurate quantitation of natural and synthetic compounds in a given matrix. Analytical methods based on the mass spectrometry (MS) technology, e.g., GC/MS and LC/MS and their variants, GC/tandem MS and LC/tandem MS, are best suited both for qualitative and quantitative analyses. GC/MS methods not only serve as reference methods, e.g., in clinical chemistry, but they are now widely and routinely used for quantitative determination of numerous analytes. However, despite inherent accuracy, analytical methods based on GC/MS commonly consist of several analytical steps, including extraction and derivatization of the analyte. In general, unequivocal identification and accurate quantification of an analyte in very low concentrations in complex matrices require further chromatographic techniques, such as high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) for sample purification. In recent years, affinity chromatography (e.g., boronate and immunoaffinity chromatography) has been developed to a superior technique for sample preparation of numerous classes of compounds in GC/MS. In this article, the application and importance of affinity chromatography as a method for sample preparation in modern quantitative GC/MS method is described and discussed, using as examples various natural and synthetic compounds, such as arachidonic acid derivates, nitrosylated and nitrated proteins, steroids, drugs, and toxins.     

A novel cytotoxic aryltetraline lactone from Bupleurum marginatum (Apiaceae)

A detailed phytochemical study of the aerial parts of Bupleurum marginatum Wall. ex DC revealed a novel aryltetraline lactone lignan identified as 9-(benzo[d][1,3]dioxol-5-yl)-6,7,8-trimethoxy-3a,4,9,9a-tetrahydronaphtho[2,3-c]furan-1(3H)-one (marginatoxin) along with nine known compounds and characterization of five other compounds either by GLC/MS or LC/MS techniques. Chemical structures of the isolated compounds were unambiguously elucidated by both 1D, 2D NMR and mass spectrometry techniques.The in vitro cytotoxic activity of both methanol and dichloromethane extracts as well as the isolated compounds was assessed in two human cancer cell lines HepG2 and HeLa using the MTT assay. The new aryltetraline lactone lignan exhibited a potent cytotoxic activity with IC₅₀ values of 12.14 and 16.90 μM after 24 h treatment for HepG2 and HeLa cells, respectively.     

A new sulphate metabolite as a long-term marker of metandienone misuse

Metandienone is one of the most frequently detected anabolic androgenic steroids in sports drug testing. Metandienone misuse is commonly detected by monitoring different metabolites excreted free or conjugated with glucuronic acid using gas chromatography mass spectrometry (GC–MS) and liquid chromatography tandem mass spectrometry (LC–MS/MS) after hydrolysis with β-glucuronidase and liquid–liquid extraction. It is known that several metabolites are the result of the formation of sulphate conjugates in C17, which are converted to their 17-epimers in urine. Therefore, sulphation is an important phase II metabolic pathway of metandienone that has not been comprehensively studied. The aim of this work was to evaluate the sulphate fraction of metandienone metabolism by LC–MS/MS. Seven sulphate metabolites were detected after the analysis of excretion study samples by applying different neutral loss scan, precursor ion scan and SRM methods. One of the metabolites (M1) was identified and characterised by GC–MS/MS and LC–MS/MS as 18-nor-17β-hydroxymethyl-17α-methylandrost-1,4,13-triene-3-one sulphate. M1 could be detected up to 26 days after the administration of a single dose of metandienone (5 mg), thus improving the period in which the misuse can be reported with respect to the last long-term metandienone metabolite described (18-nor-17β-hydroxymethyl-17α-methylandrost-1,4,13-triene-3-one excreted in the glucuronide fraction).     

Quantification of 2-hydrazinopyridine derivatized steroid hormones in fathead minnow (Pimephales promelas) blood plasma using LC-ESI+/MS/MS

Fathead minnows (Pimephales promelas) comprise a species-of-choice for the hazard assessments of various environmental contaminants, including compounds capable of disrupting endocrine function. Towards this end, the use of liquid chromatography coupled with mass spectrometry (LC–MS) and/or tandem mass spectrometry (MS/MS) is gaining common use for the quantification of steroid hormones as biomarkers of endocrine stress in small-fish toxicological studies. In this work, 2-hydrazinopyridine (2-HP) was used to derivatize and quantify the physiologically relevant steroid hormones of: 17α-hydroxypregnenolone, progesterone, 11-ketotestosterone, 11-deoxycortisol and 17α,20β-dihydroxypregnenone, in the blood plasma of male and female fathead minnows. Liquid chromatographic separation was achieved using a Waters? Sunfire C₁₈ column (2.1 mm × 50 mm with a 3.5 μm particle size) and Milli-Q water:methanol (both with 0.1% formic acid) mobile phase over a gradient of 15 min. All mass analyses were conducted using electrospray ionization in the positive mode with tandem mass spectrometry (ESI+/MS/MS). This is the first such application of 2-HP derivatization for the quantifications of the structurally and functionally diverse C19 androgen of 11-ketotestosterone; C21 progestogens of 17α-hydroxypregnenolone, progesterone and17α,20β-dihydroxypregnenone; and C21 corticosteroid of 11-deoxycortisol, in fathead minnow blood plasma. The limits of detection (LOD) were set to the lowest calibration standard that gave a signal-to-background response of ≥3, and were: 0.16 ng/ml for progesterone, 0.63 ng/ml for 17α-hydroxypregnenolone, 11-deoxycortisol and 17α,20β-dihydroxypregnenone, and 1.25 ng/ml for 11-ketotestosterone. This study demonstrates the application of 2-HP derivatization for the analysis of a variety of steroid hormones representative of endocrine function in a species of fish commonly used in toxicological studies.     

Second-tier test for quantification of underivatized amino acids in dry blood spot for metabolic diseases in newborn screening

The quantitative analysis of amino acids (AAs) in single dry blood spot (DBS) samples is an important issue for metabolic diseases as a second-tier test in newborn screening. An analytical method for quantifying underivatized AAs in DBS was developed by using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The sample preparation in this method is simple and ion-pairing agent is not used in the mobile phase that could avoid ion suppression, which happens in mass spectrometry and avoids damage to the column. Through chromatographic separation, some isomeric compounds could be identified and quantified, which cannot be solved through only appropriate multiple reactions monitoring transitions by MS/MS. The concentrations of the different AAs were determined using non-deuterated internal standard. All calibration curves showed excellent linearity within test ranges. For most of the amino acids the accuracy of extraction recovery was between 85.3 and 115 %, and the precision of relative standard deviation was <7.0 %. The 35 AAs could be identified in DBS specimens by the developed LC–MS/MS method in 17–19 min, and eventually 24 AAs in DBS were quantified. The results of the present study prove that this method as a second-tier test in newborn screening for metabolic diseases could be performed by the quantification of free AAs in DBS using the LC–MS/MS method. The assay has advantages of high sensitive, specific, and inexpensive merits because non-deuterated internal standard and acetic acid instead of ion-pairing agent in mobile phase are used in this protocol.     

Analysis of ent-kaurenoic acid by ultra-performance liquid chromatography-tandem mass spectrometry

ent-Kaurenoic acid (KA) is a key intermediate connected to a phytohormone gibberellin. To date, the general procedure for quantifying KA is by using traditional gas chromatography–mass spectrometry (GC–MS). In contrast, gibberellins, which are more hydrophilic than KA, can be easily quantified by liquid chromatography-tandem mass spectrometry (LC–MS/MS). In this study, we have established a new method to quantify KA by LC–MS/MS by taking advantage of a key feature of KA, namely the lack of fragmentation that occurs in MS/MS when electrospray ionization (ESI) is in the negative mode. Q1 and Q3 were adopted as identical channels for the multiple reaction monitoring of KA. The method was validated by comparing with the results obtained by selected ion monitoring in GC–MS. This new method could be applicable for the quantification of other hydrophobic compounds.     

Identification of tryptic peptides from large databases using multiplexed tandem mass spectrometry: simulations and experimental results

Multiplexed tandem mass spectrometry (MS/MS) has recently been demonstrated as a means to increase the throughput of peptide identification in liquid chromatography (LC) MS/MS experiments. In this approach, a set of parent species is dissociated simultaneously and measured in a single spectrum (in the same manner that a single parent ion is conventionally studied), providing a gain in sensitivity and throughput proportional to the number of species that can be simultaneously addressed. In the present work, simulations performed using the Caenorhabditis elegans predicted proteins database show that multiplexed MS/MS data allow the identification of tryptic peptides from mixtures of up to ten peptides from a single dataset with only three "y" or "b" fragments per peptide and a mass accuracy of 2.5 to 5 ppm. At this level of database and data complexity, 98% of the 500 peptides considered in the simulation were correctly identified. This compares favorably with the rates obtained for classical MS/MS at more modest mass measurement accuracy. LC multiplexed Fourier transform-ion cyclotron resonance MS/MS data obtained from a 66 kDa protein (bovine serum albumin) tryptic digest sample are presented to illustrate the approach, and confirm that peptides can be effectively identified from the C. elegans database to which the protein sequence had been appended.     

Development and validation of a rapid method for the determination and confirmation of 10 nitroimidazoles in animal plasma using liquid chromatography tandem mass spectrometry

A rapid LC–MS/MS method has been developed and validated for the simultaneous identification, confirmation and quantitation of 10 nitroimidazoles in plasma. The method validated in accordance with Commission Decision (CD) 2002/657/EC is capable of analysing for metronidazole (MNZ), dimetridazole (DMZ), ronidazole (RNZ), ipronidazole (IPZ) and their hydroxy metabolites MNZ-OH, HMMNI (hydroxymethyl, methyl nitroimidazole), IPZ-OH. The method is also capable of analysing carnidazole (CRZ), ornidazole (ORZ) and ternidazole (TRZ) which are rarely analysed by modern methods. MNZ, DMZ and RNZ have a recommended level (RL) of 3 ng mL^?1 which this method is easily able to detect for all the nitroimidazole compounds. Plasma samples are extracted with acetonitrile, and NaCl is added to help remove matrix contaminants. The acetonitrile extract undergoes a liquid–liquid wash step with hexane; it is then evaporated and reconstituted in mobile phase. The reconstituted samples are analysed by liquid chromatography tandem mass spectrometry (LC–MS/MS). The decision limits (CCα) range from 0.5 to 1.6 ng mL^?1 and the detection capabilities (CCβ), range from 0.8 to 2.6 ng mL^?1. The results of the inter-assay study, which was performed by fortifying bovine plasma samples (n = 18) on three separate days, show the accuracy calculated for the various analytes range between 101% and 108%. The precision of the method, expressed as CV% values for the inter-assay variation of each analyte at the three levels of fortification (3, 4.5 and 6.0 ng mL^?1), ranged between 4.9% and 15.2%. A day 4 analysis was carried out to examine species variances in animals such as avian, ovine, porcine and equine.