LC-MS/MS approach for quantification of therapeutic proteins in plasma using a protein internal standard and 2D-solid-phase extraction cleanup

The bioanalysis of plasma samples generated from in vivo studies of therapeutic proteins is of increasing interesting in the biopharmaceutical industry. The conventional ELISA approach has a long assay development time which can limit use in the early discovery and development of protein-based drugs. In this study, an LC-MS/MS bioassay was developed for the quantification of somatropin and a therapeutic human monoclonal antibody. The assay used bovine fetuin as an internal standard and a two-dimensional solid-phase extraction for the cleanup of the plasma digest. Sample extracts were resolved on an analytical size column using a 6 min LC gradient and analyzed using a triple-quadruple mass spectrometer. The linearity of the assay for somatropin was established from 1 to 1000 microg/mL with accuracy and precision within 15%. This LC-MS approach was also applied to a rat pharmacokinetic study of the therapeutic monoclonal antibody with a lower quantitation limit of 0.5 microg/mL. The LC-MS assay had improved accuracy and precision, and the results from analysis of in vivo study samples showed good agreement with the data obtained with an ELISA. The results from this study indicate that the LC-MS bioassay is a simple and feasible approach for the bioanalysis of therapeutic proteins to support in vivo studies during early drug discovery and development.     

Accelerated solvent extraction followed by on-line solid-phase extraction coupled to ion trap LC/MS/MS for analysis of benzalkonium chlorides in sediment samples

Benzalkonium chlorides (BACs) were successfully extracted from sediment samples using a new methodology based on accelerated solvent extraction (ASE) followed by an on-line cleanup step. The BACs were detected by liquid chromatography/ion trap mass spectrometry (LC/MS) or tandem mass spectrometry (MS/MS) using an electrospray interface operated in the positive ion mode. This methodology combines the high efficiency of extraction provided by a pressurized fluid and the high sensitivity offered by the ion trap MS/MS. The effects of solvent type and ASE operational variables, such as temperature and pressure, were evaluated. After optimization, a mixture of acetonitrile/water (6:4 or 7:3) was found to be most efficient for extracting BACs from the sediment samples. Extraction recoveries ranged from 95 to 105% for C12 and C14 homologues, respectively. Total method recoveries from fortified sediment samples, using a cleanup step followed byASE, were 85% for C12BAC and 79% for C14BAC. The methodology developed in this work provides detection limits in the subnanogram per gram range. Concentrations of BAC homologues ranged from 22 to 206 microg/kg in sediment samples from different river sites downstream from wastewater treatment plants. The high affinity of BACs for soil suggests that BACs preferentially concentrate in sediment rather than in water.     

Extraction and quantification of phytoestrogens in foods using automated solid-phase extraction and LC/MS/MS

Phytoestrogens are a group of polyphenolic plant metabolites that can induce biological responses. Their bioactivity is based on their similarity to 17beta-estradiol and their ability to bind to the beta-estrogen receptor. Although epidemiological data are inconclusive, phytoestrogens are considered to be beneficial for a variety of conditions, for example, hormone-related cancers like breast and prostate cancer. To investigate the biological effects of these compounds and to assess the exposure of larger cohorts or the general public, reliable data on the phytoestrogen content of food is necessary. Previously, food analysis for phytoestrogens was performed using either HPLC-UV or GC/MS. Here, we describe the development of the first generic method for the analysis of phytoestrogens in food, using automated solid-phase extraction and liquid chromatography-tandem mass spectrometry. The presented method shows a good reproducibility and can be easily adapted to other phytoestrogens if required.     

Evaluation of a multidimensional solid-phase extraction platform for highly selective on-line cleanup and high-throughput LC-MS analysis of triazines in river water samples using molecularly imprinted polymers

A novel highly selective sample cleanup procedure based on the use of molecularly imprinted polymers (MIPs) as solid-phase extraction materials has been evaluated with respect to its applicability and routine use in environmental analysis. The method comprises the combination of a restricted access material (RAM) and a MIP allowing a selective sample preparation to be achieved in the online mode. This combination is called the size-selective sample separation and solvent switch (six-SPE). The RAM column combines size exclusion and adsorption chromatography, reducing the concentration of matrix molecules by a cutoff of 15 kDa. The MIP column selectively retains the triazine analytes whereas the residual matrix is not retained and separated completely. Thus, the automated RAM-MIP is capable of excluding all matrix and nontarget compounds. The cleaned and enriched extract is subsequently eluted to an HPLC column and analyzed by LC-MS. A complete on-line analysis cycle including multidimensional solid-phase extraction, separation, and detection takes less than 15 min. Terbuthylazine, atrazine, propazine, simazine, ametryn, prometryn, irgarol, and also the metabolites deethylatrazine and deisopropylatrazine can be determined without any matrix interferences, e.g., by humic acids. The whole setup is fully automated and may be continuously operated. Nonspecific interactions with the polymer are below 1% in all cases. The accuracy of the LC-MIP-LC-MS system was controlled using a certified reference material (Aquacheck). The applicability of the method to the cleanup of real samples was demonstrated by injection of contaminated river water samples. The stability of different polymers was tested by consecutive injections, and it was shown that the performance of the materials did not vary even after more than 300 enrichment and desorption cycles.     

Ultrasensitive proteomics using high-efficiency on-line micro-SPE-nanoLC-nanoESI MS and MS/MS

Ultrasensitive nanoscale proteomics approaches for characterizing proteins from complex proteomic samples of <50 ng of total mass are described. Protein identifications from 0.5 pg of whole proteome extracts were enabled by ultrahigh sensitivity (<75 zmol for individual proteins) achieved using high-efficiency (peak capacities of approximately 10(3)) 15-microm-i.d. capillary liquid chromatography separations (i.e., using nanoLC, approximately 20 nL/min mobile-phase flow rate at the optimal linear velocity of approximately 0.2 cm/s) coupled on-line with a micro-solid-phase sample extraction and a nanoscale electrospray ionization interface to a 11.4-T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (MS). Proteome measurement coverage improved as sample size was increased from as little as 0.5 pg of sample. It was found that a 2.5-ng sample provided 14% coverage of all annotated open reading frames for the microorganism Deinococcus radiodurans, consistent with previous results for a specific culture condition. The estimated detection dynamic range for detected proteins was 10(5)-10(6). An improved accurate mass and LC elution time two-dimensional data analysis methodology, used to both speed and increase the confidence of peptide/protein identifications, enabled identification of 872 proteins/run from a single 3-h nanoLC/FTICR MS analysis. The low-zeptomole-level sensitivity provides a basis for extending proteomics studies to smaller cell populations and potentially to a single mammalian cell. Application with ion trap MS/MS instrumentation allowed protein identification from 50 pg (total mass) of proteomic samples (i.e., approximately 100 times larger than FTICR MS), corresponding to a sensitivity of approximately 7 amol for individual proteins. Compared with single-stage FTICR measurements, ion trap MS/MS provided a much lower proteome measurement coverage and dynamic range for a given analysis time and sample quantity.     

General LC-MS/MS method approach to quantify therapeutic monoclonal antibodies using a common whole antibody internal standard with application to preclinical studies

Ligand binding assays (LBAs) are widely used for therapeutic monoclonal antibody (mAb) quantification in biological samples. Major limitations are long method development times, reagent procurement, and matrix effects. LC-MS/MS methods using signature peptides are emerging as an alternative approach, which typically use a stable isotope labeled signature peptide as the internal standard (IS). However, a new IS has to be generated for every candidate, and the IS may not correct for variations at all processing steps. We have developed a general LC-MS/MS method approach employing a uniformly heavy-isotope labeled common whole mAb IS and a common immunocapture for sample processing. The method was streamlined with automation for consistency and throughput. Method qualification of four IgG(2) and four IgG(1) mAbs showed sensitivity of 0.1 μg/mL and linearity of 0.1-15 μg/mL. Quality control (QC) data of these eight mAbs were accurate and precise. The QC performance of the whole molecule labeled IS was better than those of synthetic labeled IS peptides tested. The pharmacokinetic results of two mAbs (an IgG(2) and IgG(1) candidate) dosed in rats were comparable to those of LBA. The general LC-MS/MS method approach overcomes the limitations of current methods to reduce time and resources required for preclinical studies.     

血液中佐匹克隆全自动固相萃取高效液相色谱质谱检测方法研究

目的:建立血液中佐匹克隆全自动固相萃取高效液相色谱-三重四级杆串联质谱(LC/MS/MS)检测方法。方法:采用全自动固相萃取进行提取,用Oasis HLB固相萃取柱,使用二氯甲烷洗脱。洗脱物浓缩后用Eclipse Plus C18柱(3.5μm×2.1×100mm)分离,LC/MS/MS分析。结果:采用本文方法检测血液中佐匹克隆的检出限为0.1ng/ml,回收率大于90.0%。结论:本方法具有操作简单、灵敏度高和重现性好等优点,可应用于血液中佐匹克隆的检验鉴定。     

GC-MS/MS同位素内标法测定肉制品中N-二甲基亚硝胺

采用同位素内标定量,建立一种快速、准确测定各种肉制品N-二甲基亚硝胺的GC-MS/MS法。样品中加入D6-N-二甲基亚硝胺内标,经水蒸气蒸馏、二氯甲烷提取、分离、浓缩,用多反应监测模式(MRM)进样分析。色谱柱为HP-Innowax毛细管柱(30 m×0.32 mm,0.25μm),氦气为载气,氮气为碰撞气,流量为1.4 m L/min。结果表明:该方法检测范围在10~300 ng/m L内,线性关系良好y=77.16x-500.16(r2=0.999 6),精密度RSD为2.36%;回收率为82.9%~99.5%,检出限0.2μg/kg。对4种不同来源的肉制品共40批次进行检测,检出13批次,但均未超过限量(3μg/kg)。该方法灵敏、简便、准确、重复性好,可作为肉制品中N-二甲基亚硝胺检测的好方法。     

Isolation and quantification of dissolved lignin from natural waters using solid-phase extraction and GC/MS

Solid-phase extraction (SPE) was tested for the isolation of dissolved lignin from diverse natural waters (fresh, estuarine, and marine) in preparation for CuO oxidation. Capillary GC coupled to selected-ion monitoring mass spectrometry (SIM-MS) of CuO oxidation products provides the high sensitivity and precision required for the identification and quantification of trace levels of lignin in seawater. The low blanks and quick cleanup of C18 cartridges support SPE for processing such samples. Comparison of SPE with other isolation procedures (direct dry-down and ultrafiltration) has shown that this method quantitatively recovers dissolved lignin and preserves its compositional parameters. The concentration and nature of dissolved organic matter appear to be primary factors that constrain the amount of water that should be processed to obtain quantitative and reproducible recoveries of dissolved lignin using SPE. Highest recoveries of dissolved lignin were obtained at low pH (1.5-4.0) with substantial decreases at pH > 4. Extraction efficiencies were independent of flow rate within a range of five to fifteen bed volumes per minute (50-150 mL min(-1)), and both refrigeration and freezing were appropriate long-term storage methods for processed cartridges prior to elution of retained dissolved lignin.     

Gas injection membrane extraction for fast on-line analysis using GC detection

Membrane extraction has been interfaced with gas chromatography and mass spectroscopy for the analysis of volatile organics in water. The vacuum in a mass spectrometer provides fast mass transport. The time required to complete permeation in a GC interface can be fairly long, because the positive pressure of the carrier gas on the permeate side slows down the analyte permeation. The aqueous boundary layer formed on the membrane is considered to be the biggest contributor to the resistance to mass transfer. Another issue is the dispersion of analyte in the aqueous stream, which broadens the input pulse to the membrane. The overall effect of these two factors is to increase the analysis time. Gas injection of aqueous samples is presented in this paper to address these issues. Gas injection reduces the formation of boundary layer, and increases the overall diffusion coefficient seven times. Axial mixing of the sample with a gaseous eluent is minimal, and this eliminates the tailing in permeation profiles. The overall membrane extraction is found to be significantly faster when a gas is used to inject an aqueous sample. This method is also simpler in terms of instrumentation and operational procedures.     

Analysis of perchlorate in water by reversed-phase LC/ESI-MS/MS using an internal standard technique

A new method was developed for the analysis of perchlorate in water by using reversed-phase liquid chromatograhy/electrospray ionization-mass spectrometry/mass spectrometry (LC/ESI-MS/MS) in the negative ESI mode. Selective and sensitive perchlorate detection was obtained by monitoring the 35ClO4- --> 35ClO3- and 37ClO4- --> 37ClO3- mass transitions. The 35ClO4- --> 35ClO3- transition was quantitated against the internal standard oxygen-labeled sodium perchlorate (NaCl18O4). Sample pretreatment for the removal of major common anions and dissolved metal ions along with internal standard quantitation sufficiently compensated for ion suppression caused by the matrix. The 37ClO4- --> 37ClO3- transition was examined to provide additional specificity. The method sensitivity, accuracy, and precision were investigated by analyzing fortified blank samples, field samples, and performance evaluation samples. The results (1.01-13.5 microg/L) for the proficiency evaluation samples differed from the certified values (1.04-14.1 microg/L) by 3-18%. The developed reversed-phase LC/ESI-MS/MS method was rapid, accurate, and reproducible. The calculated method detection limits were 0.007 microg/L for deionized reagent water and 0.009 microg/L for synthesized reagent water, respectively. The minimum reporting limit was conservatively set to 0.05 microg/L.     

Genotoxicity screening using biocatalyst/DNA films and capillary LC-MS/MS

Detection of DNA adducts can serve as a basis for genotoxicty screening of new chemicals and drugs. We report here a simple, sensitive procedure for this purpose using films containing DNA and a biocatalyst to mimic the metabolic action of human liver cytochrome P450s. DNA adducts formed from an in-situ-generated toxic metabolite (styrene oxide) were detected at subpicomole levels after neutral thermal hydrolysis of the DNA films and analysis with capillary liquid chromatography with on-line column preconcentration and MS/MS detection. An on-line column switching system allowed for increased sample loading volume and analyte preconcentration. This approach provides an estimate of the relative rate of DNA damage.     

Sample preparation based on dynamic ion-exchange solid-phase extraction for GC/MS analysis of acidic herbicides in environmental waters

The newly established enrichment technique, dynamic ion-exchange solid-phase extraction (DIE-SPE), was studied for sample preparation for GC/MS analysis of 16 acidic herbicides in environmental waters. C18 bonded silica was the solid-phase material used. The optimal sample pH was weakly acidic to neutral. However, for common tap water and surface water, which run pH 6-9, all the acidic herbicides except for Chloramben could be effectively extracted from a sample of 1,000-mL volume without pH adjustment. The humic acid could be concurrently extracted from water, but most of it was separated from the sample by using 3 mL of 10% methanol in acetone as the eluent, which would completely elute the analytes and leave a large part of the humic acid on the cartridge. The selective elution reduced the interference of humic acid and made the DIE-SPE an effective approach for the analysis of the acidic herbicides in surface water. Comparing DIE-SPE with conventional reversed-phase SPE (RP-SPE), the former gave higher recoveries for the acidic herbicides and was less affected by sample matrixes. A tandem-cartridge system combining RP- and DIE-SPE in sequence was set up for the simultaneous isolation of the acidic herbicides and removal of the interfering substances. Despite some minimal retention on the upper RP-SPE cartridge, most of the acidic herbicides could be extracted on the lower DIE-SPE cartridge with recovery over 80% except for Chloramben (50%), fenoprop (73%), MCPB (67%), and 2,4-DB (70%) when a 500-mL aqueous sample of pH 9.5 was percolated through the tandem-cartridge system. The effectiveness of the system in removing the long carbon chain fatty acids as well as the basic and neutral organic interfering substances from the sample was also demonstrated.     

Pseudo internal standard approach for label-free quantitative proteomics

Quantitative proteome analysis has become a versatile tool to understand biological functions. Although stable isotope labeling is the most reliable method for quantitative mass spectrometry, preparation of isotope-labeled compounds is time-consuming and expensive. Simple label-free approaches have been introduced, but intensity-based quantitation without standards is not generally accepted as reliable, especially for small molecules. We have developed a novel label-free quantitative proteome analysis using pseudo internal standards (PISs). This idea was derived from northern blotting analysis, in which housekeeping genes are used as standards to normalize and compare target gene expression levels in different samples. In many proteomics studies, most proteins do not change their expression levels under different conditions, and therefore, these proteins can be employed as pseudo internal standards. This new approach is simple and does not require additional standards or labeling reagents. The PIS method represents a novel approach for mass spectrometry-based comprehensive quantitatitation and may also be applicable to quantitative metabolome analysis.     

Broad spectrum analysis of 109 priority compounds listed in the 76/464/CEE Council Directive using solid-phase extraction and GC/EI/MS

A single multiresidue method was developed to determine 109 priority organic compounds included in the 76/464/EEC Council Directive on Pollution of the European Union. Such Directive includes 132 priority pollutants with a broad spectrum of polarities to be analyzed in drinking and surface waters, with the aim to protect water quality. From this list, the compounds analyzed included benzidines, chloroanilines, chloronitrobenzenes, chloronitrotoluenes, chlorophenols, chloronitrotoluidines, PAHs, PCBs, pesticides, phenylurea, and triazine herbicides. The method was developed in four steps. First, automated off-line solid-phase extraction using polymeric sorbent Oasis 60 mg cartridges was optimized to trap 109 compounds. Second, gas chromatography coupled to mass spectrometry with electron impact ionization (GC/EI/MS) was used in selected ion monitoring (SIM) mode for tentative identification of target analytes. Third, GC/EI/MS under full scan conditions was used for spectrum identification and analyte confirmation. Last, quantification was performed from SIM chromatogram using surrogates and internal standard. This method offered excellent sensitivity and selectivity, and the preconcentration of 200 mL permitted the achievement of limits of detection at the low nanogram/liter level and recoveries between 70 and 120%. Such methodology was applied to determine 109 organic compounds in French surface waters, and several pollutants were detected at levels from ppt to ppb. This multiresidue method developed was highly reproducible and robust and permitted a high sample throughput.     

Dimethyl isotope-coded affinity selection for the analysis of free and blocked N-termini of proteins using LC-MS/MS

Because of enhanced a(1) ion signals, dimethyl labeling is useful for identifying the N-termini of proteins or peptides. Herein, we describe a novel strategy that uses dimethyl isotope-coded affinity selection (DICAS) to isolate peptides that contain either the dimethylated or in vivo blocked N-termini of proteins for comprehensive sequence analyses by LC-MS/MS. In this method, dimethyl labeling at the protein level was first performed using formaldehyde-d(2) to label all unblocked protein N-termini and lysine residues, followed by trypsin digestion. The free N-terminal amines of internal peptides generated by digestion were captured by solid supports with aldehyde functionalities through reductive amination. The flow-through fractions, which contained either in vivo or in vitro blocked N-terminal peptides, were subjected to sequence analyses by LC-MS/MS. Owing to the unique feature of a1 signal enhancement associated with dimethylated peptides and the use of the deuterium reagent, the in vitro blocked (or in vivo free) N-termini of proteins could be easily differentiated from the in vivo blocked N-termini. Thus, their sequences and N-terminal modifications could be assigned unambiguously from MS/MS spectra. In this study, the completeness of the labeling and the efficiency of the isolation method were first confirmed by the use of a mixture of model proteins composed of hemoglobin, myoglobin, and alpha-lactalbumin. The N-termini of all three proteins, including both alpha and beta chains of hemoglobin as well as a signal sequence located in the N-termini of alpha-lactalbumin, were successfully identified. The protocol was then applied to the analysis of an unfractionated lysate of MCF-7 cells. Results indicate that more than 80% of the isolated peptides contained the N-termini of unique proteins, and many of them were consistent with known or inferred N-terminal processing such as methionine removal and acetylation. In addition, using the DICAS approach, we identified a novel N-terminal formylation for the Ig kappa chain V-III region SIE protein and a novel N-terminal signal sequence (1th-32th amino acid) for profilin.     

Measurement of 18 perfluorinated organic acids and amides in human serum using on-line solid-phase extraction

We have developed an on-line solid-phase extraction (SPE) method coupled to high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) for measuring trace levels of 18 perfluorinated chemicals (3 perfluorosulfonates, 8 perfluorocarboxylates, 7 perfluorosulfonamides) in serum. Without protein precipitation, only dilution with 0.1 M formic acid, one aliquot of 100 microL of serum was injected into a commercial column switching system that allowed for concurrent SPE and HPLC-MS/MS acquisition. First, the analytes were concentrated on a C18 SPE column. Then, this column was placed automatically in front of a C8 analytic HPLC column for chromatographic separation of the analytes. Detection and quantification were done using negative-ion TurboIonSpray ionization, a variant of electrospray ionization, MS/MS. Excellent recovery was achieved for all analytes including the volatile sulfonamide derivatives that could not be determined before using traditional off-line SPE methods. The high throughput and low limits of detection (0.05-0.8 ng/mL) using a small sample volume (100 microL of serum) and isotope dilution quantification make this method suitable for large-scale epidemiologic studies.     

High-throughput metabolic toxicity screening using magnetic biocolloid reactors and LC-MS/MS

An inexpensive, high-throughput genotoxicity screening method was developed by using magnetic particles coated with cytosol/microsome/DNA films as biocolloid reactors in a 96-well plate format coupled with liquid chromatography-mass spectrometry. Incorporation of both microsomal and cytosolic enzymes in the films provides a broad spectrum of metabolic enzymes representing a range of metabolic pathways for bioactivation of chemicals. Reactive metabolites generated via this process are trapped by covalently binding to DNA in the film. The DNA is then hydrolyzed and nucleobase adducts are collected using filters in the bottom for the 96-well plate of analysis by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). The magnetic particles facilitate simple and rapid sample preparation and workup. Major DNA adducts from ethylene dibromide, N-acetyl-2-aminofluorene and styrene were identified in proof-of-concept studies. Relative formation rates of DNA adducts correlated well with rodent genotoxicity metric TD(50) for the three compounds. This method has the potential for high-throughput genotoxicity screening, providing chemical structure information that is complementary to toxicity bioassays.     

Validated comprehensive analytical method for quantification of coenzyme A activated compounds in biological tissues by online solid-phase extraction LC/MS/MS

We report a robust, reliable, and comprehensive analytical method for the identification and quantification of the entire class of coenzyme A (CoA) activated substances, particularly short-, medium-, and long-chain acyl-CoAs derived from various biological tissues. This online SPE-LC/MS/MS-based method is characterized by a simple three-step sample preparation: (1) addition of buffer, organic solvents, and internal standards; (2) homogenization; and (3) centrifugation. The supernatant is injected directly into the SPE-LC/MS/MS system. Identification of CoA activated compounds is performed by accurate mass determination within the HPLC run. Method validation for short-, medium-, and long-chain acyl-CoA fatty acids revealed excellent quality. Accuracy was found to be between 87 and 107% and precision was between 0.1 and 12.8% in mouse skeletal muscle. The lower limit of quantification for all investigated compounds was well below 3.1% of estimated physiological levels in 200 mg of mouse tissue. Comparable results were obtained for mouse liver, mouse brown white adipose tissue and rat liver. For all investigated tissues, no matrix effect was observed.