High-throughput quantitative bioanalysis by LC/MS/MS

This review article discusses the most recent significant advances in the sample preparation and mass spectrometry aspects of high-throughput bioanalysis by LC/MS/MS for the quantitation of drugs, metabolites and endogenous biomolecules in biological matrices. The introduction and implementation of automated 96-well extraction has brought about high-throughput approaches to the biological sample preparation techniques of solid-phase extraction, liquid-liquid extraction and protein precipitation. The fast-flow on-line extraction technique is a different high-throughput approach that has also significantly speeded up analysis by LC/MS/MS. The use of pierceable caps for biological tubes further enhances the analysis speed and improves the safety in handling biological samples. The need for adequate chromatographic separation in order to eliminate interferences due to metabolites and/or matrix effects in LC/MS/MS is discussed. To highlight our limited understanding of atmospheric pressure ionization mass spectrometry, results from recent investigations that appear to be counter-intuitive are presented. Looking ahead to the future, multiplexed LC/MS/MS systems and capillary LC are presented as areas that can bring about further improvements in analysis speed and sensitivity to quantitative bioanalysis by LC/MS/MS.     

LC/MS/MS方法筛查新生儿苯丙酮尿症

苯丙酮尿症 [1～ 4 ] ( PKU )发病原因是患者基因缺陷使肝脏不能合成苯丙氨酸羟化酶而导致体内苯丙氨酸 ( Phe)不能正常代谢为酪氨酸 ( Tyr) ,前者在体内大量堆积并氧化为对人体有害的苯丙酮酸 . PKU是目前筛查范围最广的氨基酸代谢遗传疾病 ,在全世界每年 [5]约有一千万婴儿接受 PKU筛查 ;在我国 ,PKU也是卫生部要求重点筛查的病种 .Chace[5]等在 1 993年报道了 MS/ MS方法筛查新生儿PKU:直接使用 MS/ MS的中性碎片丢失扫描方式检测 Phe和 Tyr,通过氘代内标与待测氨基酸的质谱峰高比来定量 .MS/ MS方法速度快、准确性好、可以…     

LC/MS and LC/MS/MS determination of protein tryptic digests

Tryptic digests were analyzed by means of online microbore liquid chromatography combined with mass spectrometry (LC/MS) for some common proteins. Following conventional enzymatic digestion with trypsin, the freeze-dried residues were dissolved in high-performance liquid chromatography (HPLC) eluent and subjected to gradient reversed-phase microbore HPLC separation with mass spectrometric detection. The latter was done in the full-scan single or tandem (MS/MS) mass spectrometry mode. The formation of gas-phase ions from dissolved analytes was accomplished at atmospheric pressure by pneumatically assisted electrospray (ion spray) ionization. This produced field-assisted ion evaporation of dissolved ions, which could then be mass-analyzed for molecular mass or structure. In the full-scan LC/MS mode, the masses for the peptide fragments in the tryptic digests can be determined as either their singly or multiply charged ions. When the molecular weights of the peptides lie outside the mass range of the mass spectrometer, the multiply charged feature of these experimental conditions still provides reliable molecular weight determinations. In addition, collision-activated dissociation (CAD) on selected peptide precursor ions provides online LC/MS/MS sequence information for the tryptic fragments. Results are shown for the tryptic digests of horse heart cytochrome c, bovine β-lactoglobulin A, and bovine β-lactoglobulin B.     

Determination of estrogens and progestogens by mass spectrometric techniques (GC/MS, LC/MS and LC/MS/MS)

Steroid sex hormones and related synthetic compounds have been shown to provoke alarming estrogenic effects in aquatic organisms, such as feminization, at very low concentrations (ng/L or pg/L). In this work, different chromatographic techniques, namely, gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS), are discussed for the analysis of estrogens, both free and conjugated, and progestogens, and the sensitivities achieved with the various techniques are inter-compared. GC/MS analyses are usually carried out after derivatization of the analytes with bis(trimethylsilyl)trifluoroacetamide (BSTFA). For LC/MS and LC/MS/MS analyses, different instruments, ionization techniques (electrospray (ESI) and atmospheric pressure chemical ionization (APCI)), ionization modes (negative ion (NI) and positive ion (PI)) and monitoring modes (selected ion monitoring (SIM) and selected reaction monitoring (SRM)) are generally employed. Based on sensitivity and selectivity, LC/ESI-MS/MS is generally the method of choice for determination of estrogens in the NI mode and of progestogens in the PI mode (instrumental detection limits (IDLs) 0.1-10 ng/mL). IDLs achieved by LC/ESI-MS in the SIM mode and by LC/ESI-MS/MS in the SRM mode were, in general, comparable, although the selectivity of the latter is significantly higher and essential to avoid false positive determinations in the analysis of real samples. Conclusions and future perspectives are outlined.     

Chromatographic alignment of LC-MS and LC-MS/MS datasets by genetic algorithm feature extraction

Liquid chromatography-mass spectrometry (LC-MS) datasets can be compared or combined following chromatographic alignment. Here we describe a simple solution to the specific problem of aligning one LC-MS dataset and one LC-MS/MS dataset, acquired on separate instruments from an enzymatic digest of a protein mixture, using feature extraction and a genetic algorithm. First, the LC-MS dataset is searched within a few ppm of the calculated theoretical masses of peptides confidently identified by LC-MS/MS. A piecewise linear function is then fitted to these matched peptides using a genetic algorithm with a fitness function that is insensitive to incorrect matches but sufficiently flexible to adapt to the discrete shifts common when comparing LC datasets. We demonstrate the utility of this method by aligning ion trap LC-MS/MS data with accurate LC-MS data from an FTICR mass spectrometer and show how hybrid datasets can improve peptide and protein identification by combining the speed of the ion trap with the mass accuracy of the FTICR, similar to using a hybrid ion trap-FTICR instrument. We also show that the high resolving power of FTICR can improve precision and linear dynamic range in quantitative proteomics. The alignment software, msalign, is freely available as open source.     

LC/MS and LC/MS/MS based protocol for identification of dyes in historic textiles

Identification of dyes in historic textiles was until recently only based on reversed phase liquid chromatography and diode-array detection (RPLC–DAD). Although in the last years mass spectrometry (MS) is increasingly used as a detection system for liquid chromatography, most applications in the field are directed to identification of the molecular ions or in studies dedicated to degradation products which may be used as markers in RPLC–DAD. In the present work, an analytical protocol for the identification of dyes using RPLC/ESI/MS is presented. Atmospheric pressure electrospray ionization (ESI) was applied, in the negative ion monitoring mode. Both single stage and tandem MS (MS/MS) approaches were considered. An ion trap was used as mass analyzer. Experiments are based on the characterization of standards (natural dyes and/or dyed fibers) with the mass spectrometer sequentially working in the following modes: single MS/full scan, followed by plotting chromatograms through ion extraction (IEC) according to mass/charge ratios corresponding to molecular ions; single MS/selected ion monitoring (SIM) mode; tandem MS/single reaction monitoring (SRM) mode; tandem MS/multiple reactions monitoring (MRM) or product ion scanning modes. A faster chromatographic separation could be applied as MS detection readily balanced the selectivity of the analytical process. In a case study, 11 dyes from 3 biological sources were detected in a 0.5 mg historic sample.     

Determination of ketamine and amphetamines in hair by LC/MS/MS

A liquid chromatography-tandem mass spectrometry method was developed for the determination of ketamine (with its metabolite norketamine) and some amphetamines (amphetamine, methamphetamine, methylenedioxyamphetamine, and 3,4-methylenedioxymethamphetamine). This method was developed to determine these compounds in hair and is able to simultaneously quantify all of them in human hair. Hair samples (20 mg) were washed and pulverized, and an extraction with formic acid (0.01%) and ultrasonication for 4 h was used. Deuterated analogs of the analytes were used as internal standards for quantification. Linearity from 0.5 to 25 ng/mg was obtained for both ketamine (and norketamine) and amphetamines with correlation coefficients exceeding 0.99. The limit of detection and the limit of quantification obtained were 0.1 and 0.5 ng/mg, respectively, for ketamine and amphetamines. A total of 25 hair samples from known drug abusers (relating to designer drug consumption or consumption of amphetamines) were examined by this validated method. The results show that the proposed method is suitable for testing these drugs in a single sample of hair. In addition, it is simpler and faster than analysis by conventional methods such as gas chromatography-mass spectrometry, which usually require a more laborious extraction procedure and, in most of cases, an additional derivatization process.     

Determination of lipophilic toxins by LC/MS/MS: single-laboratory validation

An LC/MS/MS method has been developed, assessed, and intralaboratory-validated for the analysis of the lipophilic toxins currently regulated by European Union legislation: okadaic acid (OA) and dinophysistoxins 1 and 2, including their ester forms; azaspiracids 1, 2, and 3; pectenotoxins 1 and 2; yessotoxin (YTX), and the analogs 45 OH-YTX, Homo YTX, and 45 OH-Homo YTX; as well as for the analysis of 13-desmetil-spirolide C. The method consists of duplicate sample extraction with methanol and direct analysis of the crude extract without further cleanup or concentration. Ester forms of OA and dinophysistoxins are detected as the parent ions after alkaline hydrolysis of the extract. The validation process of this method was performed using both fortified and naturally contaminated samples, and experiments were designed according to International Organization for Standardization, International Union of Pure and Applied Chemistry, and AOAC guidelines. With the exception of YTX in fortified samples, RSDr below 15% and RSDR were below 25%. Recovery values were between 77 and 95%, and LOQs were below 60 microg/kg. These data together with validation experiments for recovery, selectivity, robustness, traceability, and linearity, as well as uncertainty calculations, are presented in this paper.     

Targeted identification of phosphorylated peptides by off-line HPLC-MALDI-MS/MS using LC retention time prediction

Protein phosphorylation is a type of posttranslational modification which plays an important role in cell regulation and signal transduction. Because of its biological relevance, a considerable amount of interest has been paid to the development of efficient techniques for phosphopeptide analysis. Although advances in MS control have enabled the high-throughput discovery of proteins from limited amounts of sample, automated selection of MS/MS precursor ions based on intensity alone can significantly hamper the detection of low-abundance phosphopeptides. On the basis of the observation that the introduction of a phosphate moiety does not dramatically change peptide retention time in reverse-phase chromatography, phosphopeptide specific MS/MS fragmentation attempts based on LC retention time and m/z were evaluated using a standard protein mixture, then using in vitro phosphorylated myelin basic protein. Results indicated that the majority (98%) of phosphopeptides identified eluted within a +/- 4-min window of the predicted LC elution time. While studies presented here are primarily proof of concept in nature, data suggest that the use of LC retention time prediction could be a valuable constraint for the identification of phosphopeptides within a set of off-line LC deposited sample spots. It is expected that the development of these methods will not only permit the targeted identification of protein phosphorylation sites but also allow the in-depth analysis of the dynamic events linked to the posttranslational modification.     

Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage

The goal of this work was to evaluate the improvement in proteome coverage of complex protein mixtures gained by analyzing samples using both LC/ESI/MS/MS and LC/MALDI/MS/MS. Parallel analyses of a single sample were accomplished by interfacing a Probot fractionation system with a nanoscale LC system. The Probot was configured to perform a post-column split such that a fraction (20%) of the column effluent was sent for on-line LC/ESI/MS/MS data acquisition, and the majority of the sample (80%) was mixed with a matrix solution and deposited onto the MALDI target plate. The split-flow approach takes advantage of the concentration sensitive nature of ESI and provides sufficient quantity of sample for MALDI/MS/MS. Hybrid quadrupole time-of-flight mass spectrometers were used to acquire LC/ESI/MS/MS data and LC/MALDI/MS/MS data from a tryptic digest of a preparation of mammalian mitochondrial ribosomes. The mass spectrometers were configured to operate in a data dependent acquisition mode in which precursor ions observed in MS survey scans are automatically selected for interrogation by MS/MS. This type of acquisition scheme maximizes the number of peptide fragmentation spectra obtained and is commonly referred to as shotgun analysis. While a significant degree of overlap (63%) was observed between the proteins identified in the LC/ESI/MS/MS and LC/MALDI/MS/MS data sets, both unique peptides and unique proteins were observed by each method. These results demonstrate that improved proteome coverage can be obtained using a combination of these ionization techniques.     

MSGRAPH: A program for the display of LC/MS data

The analysis of mixtures of unknowns using techniques interfacing liquid chromatography (LC) with mass spectrometry (MS) often suffers from the high abundance of background ions. To allow a quick search for components in the mixtures, a special type of display has been developed that shows all the data obtained in an analysis run simultaneously. On the screen, the m/z information from the mass scan (in the x direction) and the time (as scan number, in the y direction) are displayed with mass peaks given as single points. The relative intensity of a signal is colour coded. Thus, it is easy to recognize analyte signals as bright islands in a dark surrounding; even weak signals in the vicinity of strong background ions are identified quite clearly. Additionally, the program incorporates some features to allow a simple operation and very fast investigation of the data. This article is an electronic publication in Spectrochimica Acta Electronica (SAE), the electronic section of Spectrochimica Acta Part B (SAB). The hardcopy text is accompanied by a disk containing the program MSGRAPH, a manual, data files and additional text files.     

Determination of brevetoxins in shellfish by LC/MS/MS: single-laboratory validation

A single-laboratory validation is reported for an LC/MS/MS quantification of six brevetoxins in four matrixes (Greenshell mussel, eastern oyster, hard clam, and Pacific oyster). Recovery and precision data were collected from seven analytical batches using shellfish flesh at 0.05 mg/kg. Method recoveries and within-laboratory reproducibility ranged from 73 to 112%, with an RSD between 14 and 18% for brevetoxin-3, brevetoxin B5, brevetoxin B2, and S-desoxy brevetoxin B2. The recovery and within-laboratory reproducibility for brevetoxin-2 was 61%, with an RSD of 27%. Brevetoxin B1 gave an RSD of 12%, but no reference material was available and this toxin was only recorded in a hard clam sample naturally contaminated with brevetoxins. One naturally contaminated sample of each shellfish matrix, with brevetoxin levels ranging from 0.012 to 9.9 mg/kg, was tested in multiple batches, and the RSDs were similar to those for fortified samples at 0.05 mg/kg. Comparisons with limited data for the neurotoxic shellfish poisoning mouse bioassay for four naturally contaminated shellfish samples showed that the regulatory action limit of 0.8 mg/kg is conservative with respect to the bioassay regulatory limit of 20 mouse units/100 g.     

化妆品中碘丙炔醇丁基氨甲酸酯的高效液相色谱检测及质谱确证

建立了高效液相色谱(HPLC)-二极管阵列检测器(DAD)测定化妆品中碘丙炔醇丁基氨甲酸酯的方法.化妆品样品经超声提取后,高效液相色谱-二极管阵列扫描检测,并在235 nm波长进行分析.用保留时间结合紫外光谱定性,外标法定量,并采用液相色谱-质谱法确证.碘丙炔醇丁基氨甲酸酯的回收率为92.7%～99.8%,相对标准偏差在0.8%～2.1%之间,定量限为20 mg/kg.     

Quantification of taraxasterol in rat plasma by LC/MS/MS: application to a pharmacokinetic study

Taraxasterol, a pentacyclic triterpene from Taraxacum officinale, is one of the main active constituents of the herb. This study developed and validated a highly selective and sensitive liquid chromatography/tandem mass spectrometry for the determination of taraxasterol in rat plasma over the range of 9.0–5000 ng/mL. Chromatographic separation was achieved on a C₁₈ (4.6 × 50 mm, 5.0 µm) column with methanol–isopropanol–water–formic acid (80:10:10:0.1, v/v/v/v) as mobile phase with an isocratic elution. The flow rate was 0.7 mL/min. After adding cucurbitacin IIa as an internal standard (IS), liquid–liquid extraction was used for sample preparation using ethyl acetate. The atmospheric pressure chemical ionization source was applied and operated in positive ion mode. Selected reaction monitoring mode was used for the quantification of transition ions m/z 409.4 → 137.1 for taraxasterol and m/z 503.4 → 113.1 for IS. The mean recoveries of taraxasterol in rat plasma ranged from 85.3 to 87.2%. The matrix effects for taraxasterol were between 98.5 and 104.0%. Intra‐ and inter‐day precision were both <11.8%, and the accuracy of the method ranged from ?7.0 to 12.9%. The method was successfully applied to a pharmacokinetic study of taraxasterol after oral administration of 7.75, 15.5 and 31.0 mg/kg in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Coupling techniques in LC/MS and SFC/MS

Summary Techniques and applications of analytical instruments combining a chromatographic technique, including liquid chromatography and supercritical fluid chromatography, with mass spectrometry (LC/MS and SFC/MS), that have appeared over the past five years, are reviewed and discussed. It is shown that still many different methods co-exist and have both specific advantages and limitations. SFC/MS appears easier to run for many compounds so far analysed by conventional LC/MS methods. On the other hand, new LC/MS methods that use fast atom bombardment or electrospray ionization have the greater potential for the investigation of polar biopolymers.     

液相色谱-串联质谱法测定地表水中的丙烯酰胺

建立了地表水中丙烯酰胺残留的液相色谱-串联质谱联用测定方法。结果表明,该法的检出限0．1μg,线性范围0．1～100．0μg/L,加标回收率81．7％～86．4％。     

Strategy for quantifying trace levels of BMAA in cyanobacteria by LC/MS/MS

The cyanobacterial neurotoxin β-N-methylamino-l-alanine (BMAA) is an amino acid that is putatively associated with the pathology of amyotrophic lateral sclerosis/Parkinsonism–dementia complex (ALS-PDC) disease. It raises serious health risk concerns since cyanobacteria are ubiquitous thus making human exposure almost inevitable. The identification and quantification of BMAA in cyanobacteria is challenging because it is present only in trace amounts and occurs alongside structurally similar compounds such as BMAA isomers. This work describes an enhanced liquid chromatography/tandem mass spectrometry platform that can distinguish BMAA from its isomers β-amino-N-methyl-alanine, N-(2-aminoethyl) glycine (AEG), and 2,4-diaminobutyric acid, thus ensuring confident identification of BMAA. The method's sensitivity was improved fourfold by a post-column addition of acetonitrile. The instrument and method limits of detection were shown to be 4.2 fmol/injection (or 0.5 pg/one column) and 0.1 μg/g dry weight of cyanobacteria, respectively. The quantification method uses synthesized deuterated BMAA as an internal standard and exhibits good linearity, accuracy, and precision. Matrix effects were also investigated, revealing an ion enhancement of around 18 %. A lab-cultured cyanobacterial sample (Leptolyngbya PCC73110) was analyzed and shown to contain about 0.73 μg/g dry weight BMAA. The isomer AEG, whose chromatographic properties closely resemble those of BMAA, was also detected. These results highlight the importance of distinguishing BMAA from its isomers for reliable identification as well as providing a sensitive and accurate quantification method for measuring trace levels of BMAA in cyanobacterial samples.     

Nanoflow LC/IMS-MS and LC/IMS-CID/MS of protein mixtures

A simple ion trap/ion mobility/time-of-flight (TOF) mass spectrometer has been coupled with nanoflow liquid chromatography to examine the feasibility of analyzing mixtures of intact proteins. In this approach proteins are separated using reversed-phase chromatography. As components elute from the column, they are electrosprayed into the gas phase and separated again in a drift tube prior to being dispersed and analyzed in a TOF mass spectrometer. The mobilities of ions through a buffer gas depend upon their collision cross sections and charge states; separation based on these gas-phase parameters provides a new means of simplifying mass spectra and characterizing mixtures. Additionally it is possible to induce dissociation at the exit of the drift tube and examine the fragmentation patterns of specific protein ion charge states and conformations. The approach is demonstrated by examining a simple three-component mixture containing ubiquitin, cytochrome c, and myoglobin and several larger prepared protein mixtures. The potential of this approach for use in proteomic applications is considered.     

An algorithm for thorough background subtraction from high-resolution LC/MS data: application for detection of glutathione-trapped reactive metabolites

A control sample background-subtraction algorithm was developed for thorough subtraction of background and matrix-related signals in high-resolution, accurate mass liquid chromatography/mass spectrometry (LC/MS) data to reveal ions of interest in an analyte sample. This algorithm checked all ions in the control scans within a specified time window around the analyte scan for potential subtraction of ions found in that analyte scan. Applying this method, chromatographic fluctuations between runs were dealt with and background and matrix-related signals in the sample could be thoroughly subtracted. The effectiveness of this algorithm was demonstrated using four test compounds, clozapine, diclofenac, imipramine, and tacrine, to reveal glutathione (GSH)-trapped reactive metabolites after incubation with human liver microsomes supplemented with GSH (30 microM compound, 45-min incubation). Using this algorithm with a +/- 1.0 min control scan time window, a +/- 5 ppm mass error tolerance, and appropriate control samples, the GSH-trapped metabolites were revealed as the major peaks in the processed LC/MS profiles. Such profiles allowed for comprehensive and reliable identification of these metabolites without the need for any presumptions regarding their behavior or properties with respect to mass spectrometric detection. The algorithm was shown to provide superior results when compared to several commercially available background-subtraction algorithms. Many of the metabolites detected were doubly charged species which would be difficult to detect with traditional GSH adduct screening techniques, and thus, some of the adducts have not previously been reported in the literature. Copyright (c) 2008 John Wiley & Sons, Ltd.     

LC/MS quantitative study of glucose by iodine attachment

We explored the potential of iodine attachment to improve the sensitivity of glucose measurement by LC/MS. After sample preparation, glucose was separated by normal phase chromatography, followed by anionization by I⁻-attachment prior to MS by post-column addition of a methanolic solution of iodoform. Iodine is capable of forming an anionic adduct with neutral monosaccharides in negative ion mode electrospray mass spectrometry. Quasi-molecular ions [M + I]⁻ of glucose, and [6,6-²H₂]glucose (abbreviated d₂-glucose) internal standard were quantitated in selected ion monitoring (SIM) mode. Iodine attachment LC/MS analysis provided high sensitivity, superior to GC/MS. It greatly simplified sample preparation and increased throughput. The advantages of iodine attachment can be realized even on old mass spectrometers. A LOD of 50 pg glucose on column was achieved. Due to iodine's predisposition to sublimate, the iodoform concentration must be minimized, which adds complexity to method development. To optimize reagent concentration we developed an efficient and flexible gradient-based delivery platform. Strategy for method development with iodoform is given.