Gradient Liquid Chromatography Research Articles

Development and validation of an assay for a novel ghrelin receptor inverse agonist PF-5190457 and its major hydroxy metabolite (PF-6870961) by LC-MS/MS in human plasma

PF-5190457 is a selective and potent ghrelin receptor inverse agonist presently undergoing clinical trials to treat alcohol use disorder (AUD). We describe the development and validation of a selective and sensitive liquid chromatography-tandem mass spectrometry-based method for quantification of PF-5190457 and its recently discovered hydroxy metabolite PF-6870961 in human plasma. Analytes were extracted after simple protein precipitation using methanol (2.5 ng mL−1 tacrine as an internal standard). A gradient liquid chromatography method was used to separate the analytes on an Acquity UPLC BEH C18 analytical column. The separation was achieved at a flow rate of 0.25 mL min−1 and the total chromatographic runtime was 11.30 min. Positive electrospray ionization and multiple reaction monitoring mode were used for the quantification of all the analytes. The calibration curves from six validation runs were linear with a correlation coefficient of ≥0.996 for the concentration range of 1–1000 ng mL−1 and 2–250 ng mL−1 for PF-5190457 and PF-6870961, respectively. The retention time for PF-5190457, PF-6870961 and tacrine were 4.4, 3.8, and 4.6 min, respectively. The lower limit of quantification for PF-5190457 and PF-6870961 was 1 and 2 ng mL−1, respectively. The inter-assay precision and accuracy results obtained were within the Food and Drug Administration recommended ±15% limit of nominal values. All the analytes were found to be stable under varied stability conditions. The recovery of PF-5190457 and PF-6870961 ranged from 95 to 103%. Further, the application of the method was demonstrated by measuring the concentration of PF-5190457 and its hydroxy metabolite in patient plasma samples from 100 mg dose.

Mismatch between sample diluent and eluent: Maintaining integrity of gradient peaks using in silico approaches

The mismatch of elution strength between the sample diluent and the eluent causes undesirable peak deformations for large sample volumes in gradient liquid chromatography. The solution to that problem consists in diluting the sample solution in a weak solvent. But the minimum dilution factor has to be determined by the user given some performance objectives. In silico approaches are applied in this work to find such adequate dilution factors. Two calculations methods are proposed for the prediction of peak distortions. The first comprehensive method is based on solving numerically the mass balance equations for all the analytes and the strong solvent. An excellent agreement between the experimental and the calculated gradient chromatograms is observed (sample diluent: acetonitrile/water, 50/50, v/v; injection volume: 15 μL; linear gradient: 5%–95% acetonitrile during 3 min) for five compounds (acetanilide, coumarin, benzoin, bi-naphthol, and dibutylphthalate) injected into a 2.1 × 50 mm column packed with 1.7 μm XBridge-C18 particles. This first method happens to be highly time-consuming and impractical for common users. Experimental work and calculation times are then minimized by applying a second method based on the basics of retention and dispersion of injected pulses. Despite being less accurate than the first method, the agreement between the experimental and calculated peak width remains physically meaningful allowing the experimenter to rapidly guess the required sample dilution factor for any combination of injected volume and strong solvent concentration in the sample solution.

Investigation of the inhibition of eight major human cytochrome P450 isozymes by a probe substrate cocktail in vitro with emphasis on CYP2E1

1. A protocol has been developed and validated for the high-throughput screening of eight major human cytochrome P450 (CYP) isozymes inhibition (CYP 1A2, 2C9, 2C19, 2D6, 3A4, 2B6, 2C8 and 2E1) using an in vitro probe cocktail containing eight substrates by overcoming the unfavorable effect of assay conditions on CYP2E1 inhibition data.2. The cocktail consisting of selective probe substrates like tacrine (CYP1A2), diclofenac (CYP2C9), S-mephenytoin (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A4), bupropion (CYP2B6), paclitaxel (CYP2C8) and chlorzoxazone (CYP2E1) was incubated with human liver microsomes.3. The method was investigated by incubating well-known CYP inhibitors {alphanaphthoflavone (CYP1A2), sulfaphenazole (CYP2C9), N-3-benzylnirvanol (CYP2C19), quinidine (CYP2D6), ketoconazole (CYP3A4), ticlopidine (CYP2B6), quercetin (CYP2C8) and 4-methylpyrazole (CYP2E1)} with the substrate cocktail. A fast gradient liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for this study.4. The IC50 values determined for typical CYP inhibitors were reproducible and consistent with those in the literature. DMSO has significant effect and itself inhibits CYP2E1. DMSO should not exceed 0.1% for the determination of reliable CYP2E1 inhibition profile. This cocktail assay offers an efficient and robust method to determine the CYP450 isoforms inhibition profiles of large numbers of compounds in a quick turnaround time.

Migration and elution equations in gradient liquid chromatography

What is known in the literature as the fundamental equation for gradient elution (FEGE) was previously proven only for conventional gradient LC – uniform (the same at any distance from the inlet) static (fixed in time) solvent velocity (um) in a column of uniform and static internal structure, cross-section and thermodynamic properties. A published alternative to the FEGE – the general migration equation – is valid for any column-based linear chromatography (GC, LC, SFC etc.). It allows one to theoretically or numerically predict a solute migration time to any location along the column. Starting from that general equation, several migration equations in gradient LC under different operational conditions including non-uniform non-static um, Neue-Kuss retention model and others have been developed in this report. It has been shown that the conditions of validity of the FEGE can be expanded to include non-uniform um. On the other hand, the FEGE is not valid for other unconventional operations of LC including gradient LC with dynamic (changing in time) um. This implies that FEGE cannot be applied to, e.g., gradient LC operating at constant pressure where, due to the change in solvent composition, the solvent viscosity changes causing the change in um with time. Applications of newly developed equations to other unconventional operations of gradient LC were also considered. Several new time parameters of the mobile phase flow were identified, interpreted, and evaluated.

Quadrupole Dalton-Based Controlled Proteolysis Method for Characterization of Higher Order Protein Structure.

The higher order structure (HOS) of proteins plays a critical role in the efficacy and stability of biological drugs. Perturbation of the regional structure of proteins can affect biological activity and cause instability. Characterization of HOS has become an integral part of biological drug development and is expected from regulatory agencies. The commonly used techniques for HOS characterization, such as circular dichroism, Fourier-transform infrared, differential scanning calorimetry, intrinsic fluorescence, and hydrogen-deuterium exchange mass spectrometry, have their limitations ranging from lack of sensitivity and specificity to the need of high-level expertise and poor access to instrumentation due to high cost. In this study, we demonstrated a novel controlled proteolysis-based LC-QDa method for the detection of HOS change. By digesting proteins directly without denaturation and reduction, the HOS information can be revealed through the digested peptides. After optimizing the digestion conditions and the detection procedures, we identified 13 signature peptides that can monitor various antibody domains for any HOS changes caused by external stress. By comparing the peptide peak areas between unknown samples and a native control sample, any regional structural changes in unknown samples can be detected. The method was subsequently applied to a wide range of forced degradation samples to demonstrate higher sensitivity compared to the near-UV CD method that is frequently used for monitoring tertiary structural changes. By further reducing the number of signature peptides to five and optimizing liquid chromatography gradient duration, a streamlined, high-throughput, and controlled proteolysis method was successfully established. This method can be used to support process and formulation development as well as potentially for stability testing.

Optimized chemical coverage and data quality for non-targeted screening applications using liquid chromatography/high-resolution mass spectrometry

Non-targeted small molecule screening methods are used to analyze samples for potential compounds of interest without focusing on specific molecular species. There is great interest in these methods for metabolomic, environmental, forensic, and food safety applications, among others, to determine compounds that are responsible for a particular disease state or the presence of a harmful compound. In order for non-targeted analyses to become standardized and routine, best practices for sample preparation, data collection, and data analysis must be determined. This work focuses on optimizing specific aspects of a non-targeted workflow that utilizes high-resolution mass spectrometry using an Orbitrap instrument coupled to liquid chromatography. Sample preparation, liquid chromatography gradient length, and mass spectrometry resolving power and ionization modes were investigated to determine optimal conditions for detecting and extracting compounds from the data that cover broad molecular and polarity ranges. Infant rice cereal, orange juice, and yogurt with spiked standards were analyzed; food is inherently challenging to analyze due in part to sample complexity and diversity. Of the conditions tested, optimal conditions included a generic sample extraction using acetonitrile, water, and formic acid, a 25 min chromatographic gradient, collecting data in both positive and negative ion modes, and using 70 k resolving power. There are of course tradeoffs associated with each of these options that will be described in detail so that the appropriate conditions can be chosen for the desired application.

Senyawa 5,3'-Dihidroksi-7,4'-Dimetoksiflavon dari Kulit Batang Tanaman Akway (Drimys beccariana Gibs) dan Aktivitas Antimalarianya

Akway (Drimys beccariana Gibs) is an endemic plant of Papua. Ethnobotany is used as a medicinal plant, one of which is used as a malaria drug. This study aims to isolate, determine the chemical structure and evaluate the antimalarial activity of D. beccariana stem bark. Powder of D. beccariana bark is macerated with methanol and fractionated using n-hexane and ethyl acetate. Ethyl acetate extract was fractionated using gradient liquid chromatography (KCV) (n-hexane: ethyl acetate: methanol). The KCV fraction was separated by repeated gravity column chromatography and preparative thin layer chromatography (TLC-P) to obtain Isolate 1. Isolate 1 was determined by spectroscopic methods including UV-Vis, IR, 1D NMR, and 2D. Isolate 1 is determined by its compound structure which is 5,3'-dihydroxy-7,4'-dimethoxyflavone. 5,3'-dihydroxy-7,4'-dimethoxyflavone compounds evaluated antimalarial activity against Plasmodium falciparum in vitro with antimalarial activity better than artemisinin.

In-situ measurement of labile Cr(III) and Cr(VI) in water using diffusive gradients in thin-films (DGT)

The toxicity and bioavailability of Cr depends on its speciation in the aquatic environment. Here, we developed a new method for simultaneously obtaining in-situ data on labile Cr(III) and Cr(VI) using diffusive gradients in thin films (DGT) and high-performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry (HPLC-ICP-MS). A Zr-oxide DGT was applied to accumulate both labile Cr(III) and Cr(VI). The elution of Cr species was carried out with 50 mM EDTA-2Na at pH 9.5 for 1.5 h. Agilent Bio WAX anion-exchange chromatography was used to separate Cr species in the slightly alkaline mobile phase containing 40 mM NH4NO3 at pH 7.4. An ICP-MS was used to quantitatively measure Cr within 4.5 min. Method detection limits were 0.05 μg/L for Cr(III) and 0.02 μg/L for Cr(VI). Labile Cr(III) and Cr(VI) was accurately quantified in synthetic solutions for pH in the range of 5–8 and ionic strength ranging from 10 to 100 mmol L−1. The method allows quantification of labile Cr(III) and Cr(VI) in natural water and was consistent with results of when a separate measurement method based on DGT was used. This study was an attempt at simultaneous in-situ quantification of labile Cr(III) and Cr(VI), and will facilitate in-situ labile Cr speciation analysis in the environment.

Biomarkers identification of Lycopodiaceae and Huperziaceae species from peninsular Malaysia with HPLC chromatographic profiling and partial least square analysis

AbstractCrude herbal extracts are complex matrix consisting large quantity of plant metabolites. The identification of plant metabolite exhibiting bioactivity using the conventional bioactivity‐guided isolation process is a tedious and time‐consuming method. Club mosses from the Lycopodiceace and Huperziaceae families from Peninsular Malaysia is used as a model for this study since the alkaloid, (‐)‐huperzine A, exhibited acetylcholinesterase inhibition and used for the treatment of Alzheimer disease. The aim of this study is to evaluate the feasibility of using high‐performance liquid chromatographic profile and applying partial least square projection to identify the biomarkers. A high‐performance liquid chromatography gradient system with increasing amount of acetonitrile in deionized water and an ODS‐3 column (250 × 4.6 mm) was developed for the acquisition of the chromatographic profiles. A colorimetric assay was used to analyze the acetylcholinesterase inhibition activity. Both the pretreated high‐performance liquid chromatography integration data and acetylcholinesterase inhibition results were subjected to chemometric data analysis. From the unsupervised principal component analysis results, no outliers were detected and the data was further subjected to partial least square projections. Two biomarkers were identified and were isolated and structurally elucidated as 6β‐hydroxyhuperzine A and (‐)‐huperzine A.

Single-Run Mass Spectrometry Analysis Provides Deep Insight into E. coli Proteome.

Single-run mass spectrometry has enabled the detection and quantifications of E. coli proteins. A total of 2068 proteins quantified by intensity-based absolute quantification (iBAQ) Schwanhäusser et al.: (Nature. 473, 337-342, 2011) procedure were obtained with single enzyme-trypsin, without pre-fractionation, by quadruplicate long liquid chromatography runs coupled with high-resolution linear trap quadrupole (LTQ)-Orbitrap Velos mass spectrometry. The single-run of 12h has ability to cover almost 98% of the quadruplicate LC-MS/MS runs of E. coli proteome and is therefore almost equivalent to quadruplicate LC-MS/MS runs. These quantified proteins are about 52% of the total proteins present in E. coli genome according to Uniprot database. The quantified proteins covered almost all of the proteins in folate biosynthesis. Remarkably greater part of Gene Ontology (GO) Barrell et al.: (Nucleic Acids Res. 37, D396-D403, 2009), Ashburner et al.: (Nat. Genet. 25, 25-29, 2000) annotations, signaling pathways along with protein-protein interactions were covered. Some of the important biological processes-cell cycle, DNA repair, ion transport, ubiquinone biosynthetic process, pseudouridine synthesis, peptidoglycan biosynthetic process, RNA processing, and translation-revealed protein-protein interaction network generated by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) Jensen, et al.:(Nucleic Acids Res 37, D412-D126, 2009) database. Therefore, to achieve the saturation point of detection of maximum number of proteins in single LC-MS/MS run, 12-h liquid chromatography gradient is appropriate. Graphical Abstract ᅟ.

Micro-Data-Independent Acquisition for High-Throughput Proteomics and Sensitive Peptide Mass Spectrum Identification.

State-of-the-art strategies for proteomics are not able to rapidly interrogate complex peptide mixtures in an untargeted manner with sensitive peptide and protein identification rates. We describe a data-independent acquisition (DIA) approach, microDIA (μDIA), that applies a novel tandem mass spectrometry (MS/MS) mass spectral deconvolution method to increase the specificity of tandem mass spectra acquired during proteomics experiments. Using the μDIA approach with a 10 min liquid chromatography gradient allowed detection of 3.1-fold more HeLa proteins than the results obtained from data-dependent acquisition (DDA) of the same samples. Additionally, we found the μDIA MS/MS deconvolution procedure is critical for resolving modified peptides with relatively small precursor mass shifts that cause the same peptide sequence in modified and unmodified forms to theoretically cofragment in the same raw MS/MS spectra. The μDIA workflow is implemented in the PROTALIZER software tool which fully automates tandem mass spectral deconvolution, queries every peptide with a library-free search algorithm against a user-defined protein database, and confidently identifies multiple peptides in a single tandem mass spectrum. We also benchmarked μDIA against DDA using a 90 min gradient analysis of HeLa and Escherichia coli peptides that were mixed in predefined quantitative ratios, and our results showed μDIA provided 24% more true positives at the same false positive rate.

Simultaneous quantification of related substances of ezetimibe and simvastatin in combined dosage form using a novel stability-indicating liquid chromatographic method

A novel, simple, robust, and rapid reversed-phased high-performance liquid chromatographic method has been developed for the separation and quantitative determination of the related substances of ezetimibe and simvastatin in combined dosage forms. Successful separation of the drug from the process-related impurities and degradation products formed under stress conditions was achieved on Inertsil ODS-3V (150 × 4.6 mm, 5.0 μm) column. The gradient liquid chromatography (LC) method employs solution A and solution B as mobile phase. The solution A contains 0.1% orthophosphoric acid solution in water, and solution B contains 0.1% orthophosphoric acid solution in acetonitrile. Flow rate was monitored at 2.0 mL/min, and the ultraviolet (UV) detection, at 238 nm. In forced degradation studies, the effect of acid, base, oxidation, UV light, and temperature was investigated, showing that good resolution between the peaks corresponds to process-related impurities and degradation products from both analyte. The performance of the method was validated according to the present International Conference on Harmonization (ICH) guidelines for specificity, limit of detection, limit of quantification, linearity, accuracy, precision, ruggedness, and robustness. To the best of our knowledge, a rapid LC method, which separates all the impurities of ezetimibe and simvastatin in combined dosage forms, disclosed in this investigation was not published elsewhere.

Protocol for the visualisation of axial temperature gradients in ultra high performance liquid chromatography using infrared cameras

A protocol was developed for the visualisation of axial temperature gradients on a Kinetex column (1.3 μm C18 100 Å 50 × 2.1 mm) operated at near maximum pressure of the system (Pmax) using an infrared camera. Real time viscous frictional heating effects across the entire column length was observed, and showed that with increasing flow rate there was an increase in the maximum temperature of the column, and the difference between the inlet and outlet temperatures. Temperature profile data over the entire length of the column revealed the dynamics of heat exchange processes along different parts of the column, and raises the question on potential heating effects on eluents. The axial temperature gradients of eluents such as pure methanol, isopropyl alcohol and acetonitrile near Pmax were compared; finding that acetonitrile which had the highest flow velocity at Pmax gave the highest overall temperature increase for these eluents.

PH gradient-liquid chromatography tandem mass spectrometric assay for determination of underivatized polyamines in cancer cells

Altered levels of polyamines in biological specimens have been suggested as potential biomarkers for cancer. Difluoromethylornithine (DFMO, an irreversible inhibitor of ornithine decarboxylase) is reported to modulate polyamines to potentially attenuate proliferation of neuroblastoma cells. A clinical trial is being conducted to evaluate DFMO in various cancers. To determine the pharmacodynamics effect of DFMO, an analytical assay is needed to accurately measure the changes in polyamines in cancer cells. In this study, a novel pH gradient LC-ESI-MS/MS method was developed and validated for the quantitation of polyamines (putrescine, spermidine and spermine) in cancer cells. To separate polar and basic polyamines, a multi-mode column composed of ODS and weak ionic ligands was used. The pH gradient was generated from pH 5.3 to pH 2.7 with 2 mM ammonium acetate and 0.4% acetic acid in 10% acetonitrile as mobile phase. The detection of polyamines was performed utilizing multiple reaction monitoring on electrospray ionization mass spectrometry operated in positive ion mode. A pH gradient method increased resolution and decreased peak width of conventional analytical assays, resulting in 10–250-fold higher detection limits. Mobile phases without ion-pairing reagents were LC-MS compatible and eliminated possible signal suppression and MS contamination. The developed method was successfully applied to the analysis of polyamines in neuroblastoma and leukemia cells treated with DFMO. Putrescine levels were significantly (p < 0.001) decreased in CCRF-CEM (3.68 vs 1.23 ng/mg protein), SK-N-BE(2) (1.98 vs 1.31 ng/mg protein) and CHLA-20 (2.06 vs 0.90 ng/mg protein) cells treated with DFMO relative to vehicle control. The assay will provide a useful tool in determining the pharmacodynamic effect of DFMO in cancer clinical trials.

Accurate prediction of retention in hydrophilic interaction chromatography by back calculation of high pressure liquid chromatography gradient profiles

Gradient retention times are difficult to project from the underlying retention factor (k) vs. solvent composition (φ) relationships. A major reason for this difficulty is that gradients produced by HPLC pumps are imperfect – gradient delay, gradient dispersion, and solvent mis-proportioning are all difficult to account for in calculations. However, we recently showed that a gradient “back-calculation” methodology can measure these imperfections and take them into account. In RPLC, when the back-calculation methodology was used, error in projected gradient retention times is as low as could be expected based on repeatability in the k vs. φ relationships. HILIC, however, presents a new challenge: the selectivity of HILIC columns drift strongly over time. Retention is repeatable in short time, but selectivity frequently drifts over the course of weeks. In this study, we set out to understand if the issue of selectivity drift can be avoid by doing our experiments quickly, and if there any other factors that make it difficult to predict gradient retention times from isocratic k vs. φ relationships when gradient imperfections are taken into account with the back-calculation methodology. While in past reports, the accuracy of retention projections was >5%, the back-calculation methodology brought our error down to ∼1%. This result was 6–43 times more accurate than projections made using ideal gradients and 3–5 times more accurate than the same retention projections made using offset gradients (i.e., gradients that only took gradient delay into account). Still, the error remained higher in our HILIC projections than in RPLC. Based on the shape of the back-calculated gradients, we suspect the higher error is a result of prominent gradient distortion caused by strong, preferential water uptake from the mobile phase into the stationary phase during the gradient – a factor our model did not properly take into account. It appears that, at least with the stationary phase we used, column distortion is an important factor to take into account in retention projection in HILIC that is not usually important in RPLC.

Development and validation of reversed-phase HPLC gradient method for the estimation of efavirenz in plasma.

Efavirenz is an anti-viral agent of non-nucleoside reverse transcriptase inhibitor category used as a part of highly active retroviral therapy for the treatment of infections of human immune deficiency virus type-1. A simple, sensitive and rapid reversed—phase high performance liquid chromatographic gradient method was developed and validated for the determination of efavirenz in plasma. The method was developed with high performance liquid chromatography using Waters X-Terra Shield, RP18 50 x 4.6 mm, 3.5 μm column and a mobile phase consisting of phosphate buffer pH 3.5 and Acetonitrile. The elute was monitored with the UV-Visible detector at 260 nm with a flow rate of 1.5 mL/min. Tenofovir disoproxil fumarate was used as internal standard. The method was validated for linearity, precision, accuracy, specificity, robustness and data obtained were statistically analyzed. Calibration curve was found to be linear over the concentration range of 1–300 μg/mL. The retention times of efavirenz and tenofovir disoproxil fumarate (internal standard) were 5.941 min and 4.356 min respectively. The regression coefficient value was found to be 0.999. The limit of detection and the limit of quantification obtained were 0.03 and 0.1 μg/mL respectively. The developed HPLC method can be useful for quantitative pharmacokinetic parameters determination of efavirenz in plasma.

BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF TICAGRELOR BY RP-HPLC

Objective: The main purpose of this study was to develop a simple, precise, rapid and accurate RP-HPLC method for the quantitative determination of ticagrelor in human plasma. Methods: The separation was accomplished by the isocratic method by utilizing phenomenex C18 column on a Shimadzu binary gradient liquid chromatography system furnished with LC-20AD solvent delivery system, SPD-20-A photo-diode array detector and 20 µl loop volume in a rheodyne injector. The analyte was extracted by protein precipitation in the involvement of diethyl ether as a protein precipitator. The mobile phase was developed for the estimation of the drug in human plasma consists of acetonitrile and methanol in the ratio of 60:40% v/v. Separation was done with a flow rate of 1 ml/min at a detection wavelength of 254 nm.Results: Retention time was found to be 4.503 min with a run time 10 min. Linearity shows in a range of 20-100 µg/ml, with a correlation coefficient of 0.9992 respectively. Stability studies of ticagrelor in plasma were carried out by, short term stability, long term stability and bench top stability studies. Short term stability, long term stability and bench top stability of ticagrelor was carried out from 20 and 100 µg/ml concentration and %RSD was ascertained 0.12% and 0.08%, 0.18% and 0.15%, 1.19% and 1.30% respectively.Conclusion: The outcomes were observed to be inside the knowledge of ICH guidelines. The prepared solution was injected in triplicate, and % RSD was measured. Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for routine examination of ticagrelor in human plasma

Effect of water on the retention on diol and amide columns in hydrophilic interaction liquid chromatography.

The amount of water adsorbed on polar columns plays important role in hydrophilic interaction liquid chromatography. It may strongly differ for the individual types of polar columns used in this separation mode. We measured adsorption isotherms of water on an amide and three diol-bonded stationary phases that differ in the chemistry of the bonded ligands and properties of the silica gel support. We studied the effects of the adsorbed water on the retention of aromatic carboxylic acids, flavonoids, benzoic acid derivatives, nucleic bases, and nucleosides in aqueous-acetonitrile mobile phases over the full composition range. The graphs of the retention factors versus the volume fraction of water in mobile phase show "U-profile" characteristic of a dual hydrophilic interaction-reversed phase retention mechanism. The minimum on the graph that marks the changing retention mechanism depends on the amount of adsorbed water. The linear solvation energy relationship model suggests that the retention in the hydrophilic interaction liquid chromatography mode is controlled mainly by proton-donor interactions in the stationary phase, depending on the column type. Finally, the accuracy of hydrophilic interaction liquid chromatography gradient prediction improves for columns that show a high water adsorption.

Multiple Reactions Monitoring (MRM) Mass Spectrometry for Absolute Quantification of Allergens in German Cockroach (GCr) Allergen Extracts

Mass spectrometry (MS) offers a unique approach for rapid compositional characterization and absolute quantification of individual allergens and allergen isoforms in complex allergen extracts, and thus has the potential to transform the ongoing efforts of allergen standardization. We developed an assay to evaluate GCr extracts, based on liquid chromatography (LC) and MRM MS, by systematically optimizing the digestion conditions (buffer, protein denaturation, digestion time, and trypsin quantity), LC gradient, and MS parameters (collision energy, dwell time, precursor-fragment pairs). In silico simulation, followed by recombinant-allergen and extract digestion and high resolution MS analysis allowed us to identify 47 proteotypic and quantotypic reference peptides for use as calibrators and internal standards for quantification of endogenous peptides. The peptide list represents 9 allergens and 8 iso-allergens and variants. Since the initial phase of the project aimed to generate a template-method using selected GCr allergens, twenty-six peptides (13 native and 13 isotopically-labelled) representing Bla g 1, 2, 3, 4 and 5 were chemically synthesized and used in LC-MRM/MS method development. Robustness and suitability of the overall protocol was extensively assessed following the ICH (Q2(R1) and FDA guidelines. The protocol offers excellent precision (CV < 10%), linear response (r > 0.99), dynamic range (>3), detection, and recovery. The applicability of the method was further demonstrated using eight commercial GCr extracts from four different vendors. The LC-MRM/MS method proved to be a useful tool for absolute quantification of allergens in GCr extracts and has promise for the standardization of complex allergen extracts.

Discovery and Longitudinal Evaluation of Candidate Biomarkers for Ischaemic Stroke by Mass Spectrometry-Based Proteomics.

Application of acute therapies such as thrombolysis for ischaemic stroke (IS) is constrained because of diagnostic uncertainty and the dynamic nature of stroke biology. To investigate changes in blood proteins after stroke and as a result of thrombolysis treatment we performed label-free quantitative proteomics on serum samples using high-resolution mass spectrometry and long high-performance liquid chromatography gradient (5 hours) combined with a 50-cm column to optimise the peptide separation. We identified (false discovery rate [FDR]: 1%) and quantified a total of 574 protein groups from a total of 92 samples from 30 patients. Ten patients were treated by thrombolysis as part of a randomised placebo-controlled trial and up to 5 samples were collected from each individual at different time points after stroke. We identified 26 proteins differently expressed by treatment group (FDR: 5%) and significant changes of expression over time for 23 proteins (FDR: 10%). Molecules such as fibrinogen and C-reactive protein showed expression profiles with a high-potential clinical utility in the acute stroke setting. Protein expression profiles vary acutely in the blood after stroke and have the potential to allow the construction of a stroke clock and to have an impact on IS treatment decision making.