Chromatographic Window Research Articles

Retention-directed and selectivity controlled chromatographic resolution: Rapid post-hoc analysis of DMPK samples to achieve high-throughput LC-MS separation

High quality chromatographic separation underpins robustness in LC-MS, frequently the analytical method of choice for pharmaceutical drug discovery work. The potential improvements in chromatographic selectivity afforded by serial column coupling (SCC), provide a useful means to enhance the resolution of complex samples. In this work, we present a revised high-throughput form of SCC, in which just two individual mixed phase columns were coupled together and combined with a gradient-optimised, retention-directed ultra-high pressure method to achieve rapid separations, with no further method optimisation necessary. The overall performance was evaluated from an open access DMPK analytical working environment perspective; where in anticipation of bioanalytical or metabolite identification chromatography challenges, or with the knowledge that stronger resolution was required for in-vitro sample analysis, the methodology could be immediately implemented by the analyst.Retention-directed selection of a shallow SCC gradient method was successful in separating peaks throughout the chromatographic window, resulting in a runtime still congruent to high-throughput analyses (3.5 min). In-vitro assay sample interferences were resolved 44–72% of the time, and the overall resolving power for isomeric separations significantly improved against single column comparisons (1.7-fold mean RS improvement). Over a sustained period of time in our laboratory, SCC methods have been used for metabolite identification and bioanalytical samples, where both convenience and effectiveness in solving analytical challenges has been consistently demonstrated. Examples that highlight SCC chromatography, and a guided discussion of the main high-throughput considerations, are included. The technique offers wide applicability, and we would recommend it as a toolbox consideration to the laboratory analyst.

Benchmarking machine learning methods for comprehensive chemical fingerprinting and pattern recognition

Machine learning (ML) has been used previously to recognize particular patterns of constituent compounds. Here, ML is used with comprehensive chemical fingerprints that capture the distribution of all constituent compounds to flexibly perform various pattern recognition tasks. Such pattern recognition requires a sequence of chemical analysis, data analysis, and pattern analysis. Chemical analysis with comprehensive multidimensional chromatography is a maturing approach for highly effective separations of complex samples and so provides a solid foundation for undertaking comprehensive chemical fingerprinting. Data analysis with smart templates employs marker peaks and chemical logic for chromatographic alignment and peak-regions to delineate chromatographic windows in which analytes are quantified and matched consistently across chromatograms to create chemical profiles that serve as complete fingerprints. Pattern analysis uses ML techniques with the resulting fingerprints to recognize sample characteristics, e.g., for classification. Our experiments evaluated the effectiveness of seventeen different ML techniques for various classification problems with chemical fingerprints from a rich data set from 126 wine samples of different varieties, geographic regions, vintages, and wineries. Results of these experiments showed an accuracy range from 58% to 88% for different ML methods on the most difficult classification problems and 96% to 100% for different ML methods on the least difficult classification problems. Averaged over 14 classification problems, accuracy for the different methods ranged from 80% to 90%, with some relatively simple ML techniques among the top-performing methods.

Chemometric strategy for untargeted lipidomics: Biomarker detection and identification in stressed human placental cells

A lipidomic study was developed in a human placental choriocarcinoma cell line (JEG-3) exposed to tributyltin (TBT) and to a mixture of perfluorinated chemicals (PFCs). The method was based on the application of multivariate curve resolution-alternating least squares (MCR-ALS) to data sets obtained by ultra-high performance liquid chromatography coupled to time-of-flight mass spectrometry (UHPLC–TOF-MS) using an untargeted approach. Lipids from exposed JEG-3 cells were solid–liquid extracted and analyzed by UHPLC–TOF-MS in full scan mode, together with control samples. Raw UHPLC–TOF-MS data of the different cell samples were subdivided into 20 distinct chromatographic windows and each window was further organized in a column-wise augmented data matrix, where data from every sample was in an individual data matrix. Then, the 20 new augmented data matrices were modeled by MCR-ALS. A total number of 86 components were resolved and a statistical comparative study of their elution profiles showed distinct responses for the lipids of exposed versus control cells, evidencing a lipidome disruption attributed to the presence of the xenobiotics. Results from one-way ANOVA followed by a multiple comparisons test and from discriminant partial least squares (PLS-DA) analysis were compared as usual strategies for the determination of potential biomarkers. Identification of 24 out of the 33 proposed biomarkers contributed to the better understanding of the effects of PFCs and TBT in the lipidome of human placental cells. Overall, this study proposes an innovative untargeted LC–MS MCR-ALS approach valid for -omic sciences such as lipidomics.

A non-target chemometric strategy applied to UPLC-MS sphingolipid analysis of a cell line exposed to chlorpyrifos pesticide: A feasibility study

A non-target chemometrics study based on the application of Multivariate Curve Resolution Alternating Least Squares (MCR-ALS) method to a data set obtained by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) has been applied to the study of human prostate cancer (DU145) cell line samples treated with the organophosphate pesticide chlorpyrifos (CPF). Full scan UPLC-MS data sets were segmented in 17 different chromatographic windows and submitted to a non-target detailed study. Every one of these chromatographic windows of the different analyzed samples (treated and non-treated with CPF) was column-wise augmented in a new data matrix with their m/z values in the common column mode to preserve the fulfillment of the assumed spectral bilinear model. MCR-ALS was used to recover the elution and mass spectral profiles of the pure components present in each of the analyzed chromatographic windows. ANOVA (p≤0.05) was then applied to compare the areas under the concentration profiles of the MCR-ALS resolved components in the CPS treated and control samples. This analysis allowed the detection of those sphingolipids having their concentration in cells modified by the presence of CPS compared to control samples where this contaminant was absent. Positively identified sphingolipids included sphingomyelins, dihydrosphingomyelin and C16 ceramide. The strategy described in this work is proposed for a general non-target UPLC-MS MCR-ALS analysis of the effect of environmental contaminants in cells in lipidomic and metabonomic studies.

Enhanced Peptide Identification by Electron Transfer Dissociation Using an Improved Mascot Percolator

Peptide identification using tandem mass spectrometry is a core technology in proteomics. Latest generations of mass spectrometry instruments enable the use of electron transfer dissociation (ETD) to complement collision induced dissociation (CID) for peptide fragmentation. However, a critical limitation to the use of ETD has been optimal database search software. Percolator is a post-search algorithm, which uses semi-supervised machine learning to improve the rate of peptide spectrum identifications (PSMs) together with providing reliable significance measures. We have previously interfaced the Mascot search engine with Percolator and demonstrated sensitivity and specificity benefits with CID data. Here, we report recent developments in the Mascot Percolator V2.0 software including an improved feature calculator and support for a wider range of ion series. The updated software is applied to the analysis of several CID and ETD fragmented peptide data sets. This version of Mascot Percolator increases the number of CID PSMs by up to 80% and ETD PSMs by up to 60% at a 0.01 q-value (1% false discovery rate) threshold over a standard Mascot search, notably recovering PSMs from high charge state precursor ions. The greatly increased number of PSMs and peptide coverage afforded by Mascot Percolator has enabled a fuller assessment of CID/ETD complementarity to be performed. Using a data set of CID and ETcaD spectral pairs, we find that at a 1% false discovery rate, the overlap in peptide identifications by CID and ETD is 83%, which is significantly higher than that obtained using either stand-alone Mascot (69%) or OMSSA (39%). We conclude that Mascot Percolator is a highly sensitive and accurate post-search algorithm for peptide identification and allows direct comparison of peptide identifications using multiple alternative fragmentation techniques.

Evaluation of compound-independent calibration using gas chromatography with atomic emission detection

The evaluation of compound-independent calibration (CIC) using gas chromatography (GC) with atomic emission detection (AED) is demonstrated by the identification of several sulfur or nitrogen compounds. Among other GC detectors, the advantage of using the AED is the selectivity of detection. Because contradictory results have been reported for the determination of numeric atomic ratios of elements, we set up a study with the objectives not only of applying these techniques but of determining under which conditions they will yield satisfactory results. The column pressure dependence of AED response is demonstrated through studies performed with constant pressure of 20, 30, and 40psi. Moreover, the data collected in this study are evidence that inter-elements response factor ratios, particularly for the C/S, is very dependent on the molecular mass and concentration of the chemicals analysed whereas molecular structure seems to have less effect on the AED signal. We therefore suggest the use of a reference set of compounds covering a large chromatographic window, which enables the selection, within this set, of the most appropriate reference compound for calibration and for determination of the raw formula of an unknown analyte.

LC tandem MS – overview of technology

Many different types of mass spectrometers are available for the analysis of biological molecules featuring different ionisation methods and technologies for mass analysis. The combination of electrospray ionisation with triple quadrupole or hybrid quad-rupole/ion trap mass spectrometry is one of the most versatile and widely-used for quantitative LC-MS analysis in clinical chemistry. Useful data can be obtained using direct injection methods without chromatography, as exemplified by high-throughput screening tests such as neonatal dried blood spot or urine screening. Ion suppression and interferences limit both the sensitivity and imprecision of direct injection methods and chromatography is required for applications requiring improved performance. Short columns are typically used to maintain throughput and columns with particle sizes <2μm are increasingly used to achieve high chromatographic resolution. Internal standards, preferably ¹³C analogues, are critical to obtaining good quantitative data and compensating for ion suppression and significant within-batch variations in absolute ion responses. Multiple reaction monitoring is used to monitor analytes within timed chromatographic windows and dozens of analytes can be multiplexed in a single run. With appropriate method design, there appear to be few molecules of clinical interest that cannot be analysed by LC-MS.

Improved Chromatographic Assessment of Chinese Medicinal Products by Multi-Chemical Classes Analysis

An improved strategy for the quality evaluation of proprietary Chinese medicines (PCM) in complex matrices is presented. It involves the simultaneous separating and displaying of multi-chemical classes and analytes within a single chromatographic elution. The developed fingerprints have the properties of better coverage of multi-chemical classes among the composing herbs. This comprehensive and more facile approach was illustrated for Lemai Keli, a commonly used PCM listed in the Chinese Pharmacopoeia with highly complex analytical matrices. Ten marker compounds belonging to monoterpene glycosides, chalcone glycosides, phenolic acids and phenolic carboxylic aldehyde groups of the analytes together with three common chromatographic peaks were successively separated and displayed within the chromatographic window as a unique HPLC-PAD fingerprint. Rigorous method validation data demonstrated the suitability of the current method for quality assessment purposes in a highly complex PCM matrix. Additional chemometric treatment further demonstrated the uniqueness of the developed fingerprint, with a distinctive chemical profile compared to a highly similar PCM formulation (Kangen Karyu) from which it was considered to be impossible to differentiate when using the conventional single marker approach.

Peak capacity estimation in isocratic elution

Peak capacity (i.e. maximal number of resolved peaks that fit in a chromatographic window) is a theoretical concept with growing interest, but based on a situation rarely met in practice. Real chromatograms tend to have uneven distributions, with overlapped peaks and large gaps. The number of resolved compounds should, therefore, be known from estimations. Several equations have been reported for this purpose based on three perspectives, namely, the intuitive approach (peak capacity as the size of the retention time window measured in peak width units), which assumes peaks with the same width, and the outlines of Giddings and Grushka, which consider changes in peak width with retention time. In this work, the peak capacity concept is discussed and three new approaches are derived based on realistic descriptions of peak shape. The first one is based on the Grushka's approach and considers the contributions of column and extra-column peak variances. The second one relies on Giddings’ and assumes asymmetrical peaks where left and right peak half-widths depend linearly on retention time. The third equation, based on the intuitive approach, uses a mean peak width obtained by integration, instead of a mean value from several representative peaks. The accuracy of the classical Giddings’ approach for ideal peaks, a modification of the Grushka's approach that considers variation of peak width at half-height, and the three new approaches were checked on combined chromatograms built by adding real peaks. The results demonstrate that the change in efficiency (and not in skewness) is the relevant factor, at least in the studied examples. Also, peak width should be measured at low peak height ratios (i.e. 10%) to better account peak deformation.

Venom landscapes: Mining the complexity of spider venoms via a combined cDNA and mass spectrometric approach

The complexity of Australian funnel-web spider venoms has been explored via the combined use of MALDI–TOF mass spectrometry coupled with chromatographic separation and the analysis of venom-gland cDNA libraries. The results show that these venoms are far more complex than previously realized. We show that the venoms of Australian funnel-web spiders contain many hundreds of peptides that follow a bimodal distribution, with about 75% of the peptides having a mass of 3000–5000 Da. The mass spectral data were validated by matching the experimentally observed masses with those predicted from peptide sequences derived from analysis of venom-gland cDNA libraries. We show that multiple isoforms of these peptides are found in small chromatographic windows, which suggests that the wide distribution of close molecular weights among the chromatographic fractions probably reflects a diversity of structures and physicochemical properties. The combination of all predicted and measured parameters permits the interpretation of three-dimensional ‘venom landscapes’ derived from LC-MALDI analysis. We propose that these venom landscapes might have predictive value for the discovery of various groups of pharmacologically distinct toxins in complex venoms.

Investigation of compound-independent calibration and partial molecular formula determination by gas chromatography–atomic-emission detection for characterisation of organophosphorus and organosulfur agents related to the chemical weapons convention

Atomic-emission detection (AED) is a technique particularly-well suited to screening complex samples for multiple compounds containing heteroatoms such as phosphorus, sulfur, or nitrogen, which are especially relevant in verification of chemical disarmament. Among other GC detectors, AED has unique characteristics such as compound-independent calibration and possible raw-formula determination. Because contradictory results have been reported on these points, we set up a study with the objectives not only of applying these techniques to chemical weapons convention-related chemicals but of determining under which conditions they would yield satisfactory results. The extensive data collected in this study are evidence that the response of the detector, particularly for the phosphorus line, is very dependent on the molecular mass and concentration of the chemicals analysed whereas molecular structure seems to have less effect on the AED signal. Most interestingly, compound-independent calibration and subsequent partial molecular formula determination usually seem satisfactory when the reference compounds used to calibrate the system have GC retention times and molecular masses close to those of the unknown analytes (whose molecular mass may be determined by GC-CI-MS). We therefore suggest the use of a reference set of compounds covering a large chromatographic window, which enables the selection, within this set, of the most appropriate reference compound for calibration and for determination of the raw formula of an unknown analyte. For optimal performance, the use of a new discharge tube is also recommended.

Description and Validation of a Four-Channel Staggered LC−MS/MS System for High-Throughput in Vitro Screens

A parallel four-channel LC-MS/MS system for quantitative high-throughput in vitro screens is described. The system comprises four pumps, a four-valve autosampler equipped with a nine-port stream selector valve, and a triple quadrupole mass spectrometer. Staggered injections across the sample list are performed by four injector valves onto separate columns in such a manner that the chromatographic window of interest is always eluting from one of the columns into the mass spectrometer. To demonstrate the applicability of the system, three different compounds (verapamil, propanolol, dextromethorphan) were analyzed. The results showed that the sample throughput could be increased more than three times in comparison with a conventional single-column LC-MS/MS system. Validation results for the three compounds showed an accuracy of 85.0-108%, a precision (given as relative standard deviation) of 2.60-10.7%, and a carryover of less than 0.1%.

Gas chromatography/ion trap mass spectrometry applied for the determination of polybrominated diphenyl ethers in soil

A gas chromatography/ion trap mass spectrometry (GC/ITMS) method was developed for the determination of polybrominated diphenyl ethers (PBDEs). ITMS parameters were optimized in order to achieve the best sensitivity for the PBDE analysis. Tandem mass spectrometry, along with an isotope dilution internal standard method, was used for the quantitation. Chromatographic windows were developed for mono- to hepta-BDEs, depending on the retention times when a 30-m GC column was used. A different 15-m column was used to analyze deca-BDE. Environmental soil samples collected from an electronic waste recycling site were prepared by using Soxhlet extraction and column chromatographic cleanup. Average recoveries of 61-118% were obtained for the 13C-labeled PBDE internal standards spiked in the samples prior to sample preparation. The accuracy represented by relative analytical errors was -24% to 18%, and the precision (relative standard deviation) was 11-26% (n=8). The method detection limits ranged from 0.013-0.25 ng/g for the PBDEs in soil.

A Four-Column Parallel Chromatography System for Isocratic or Gradient LC/MS Analyses

A novel approach to parallel liquid chromatography/ tandem mass spectrometry (LC/MS/MS) analyses for pharmacokinetic assays and for similar quantitative applications is presented. Modest modifications render a conventional LC/MS system capable of analyzing samples in parallel. These modifications involve the simple incorporation of three valves and four LC columns into a conventional system composed of one binary LC pumping system, one autosampler, and one mass spectrometer. An increase in sample throughput is achieved by staggering injections onto the four columns, allowing the mass spectrometer to continuously analyze the chromatographic window of interest Using this approach, the optimized run time is slightly greater than the sum of the widths of the desired peaks. This parallel chromatography unit can operate under both gradient and isocratic LC conditions. To demonstrate the utility of the system, atorvastatin, five of its metabolites, and their deuterated internal standards (IS) were analyzed using gradient elution chromatography conditions. The results from a prestudy assay evaluation (PSAE) tray of standards and quality control (QC) samples from extracted spiked human plasma are presented. The relative standard deviation and the accuracy of the QC samples did not exceed 8.1% and 9.6%, respectively, which is well within the acceptance criteria of the pharmaceutical industry. For this particular analysis, the parallel chromatography system decreased the overall run time from 4.5 to 1.65 min and, therefore, increased the overall throughput by a factor of 2.7 in comparison to a conventional LC/MS/MS analytical method.

Toward an Optimized Dynamic Headspace Method for the Study of Volatiles in Low-Density Polyethylene

A dynamic headspace technique for detecting C6−C14 volatile organic compounds (VOCs) in low-density polyethylene (LDPE) is discussed in which VOCs are purged with N2, trapped at ambient temperatures on Tenax-GC, and identified by gas chromatography/mass spectrometry. The experimental variables are quantitatively modeled to enhance sensitivity of the technique. The rate of extraction of VOCs from LDPE and the rate of isothermal desorption of VOCs from Tenax-GC are first-order processes with rate constants of 2.79 × 10-5 and 1.53 × 10-3 s-1 respectively. Approximately 4.2% of extractable VOCs are present in the vapor phase at ambient temperature, and 21% of VOCs are liberated instantaneously from Tenax-GC during thermal desorption at 200 °C. The chromatographic peak area associated with each VOC, and the total peak area in the chromatographic window, vary with desorption temperature in accordance with the van't Hoff isochore with an “average” desorption enthalpy of 45.5 kJ mol-1. Keywords: Low-density polye...

High sample throughput phosphoamino acid analysis of proteins separated by one- and two-dimensional gel electrophoresis

Studies on protein phosphorylation usually involve radiolabelling techniques and visualisation on gels; this limits studies to those on tissues which can be 32P labelled. With the advent of reproducible micropreparative two dimensional gel electrophoresis, the proteome (protein complement) of a subcellular fraction, cell or tissue can be displayed. We have investigated the sensitivity of limited protein hydrolysis to detect in vivo phosphorylation in proteins blotted from one or two-dimensional polyacrylamide gels onto polyvinylidene difluoride (PVDF) membranes. The method uses 9-fluorenylmethyl chloroformate (FMOC) derivatisation chemistry and a modified HPLC AMINOMATE system. Conditions were established for hydrolysis of the PVDF-blotted protein (5.7 M HCl at 110°C for 4 h) which resulted in the recovery of phosphoserine (Ser(P)), phosphothreonine (Thr(P)) and phosphotyrosine (Tyr(P)). The chromatography was carried out on the same system routinely used for amino acid compositional analysis using a gradient elution modified from that used for separation of 16 amino acids. A chromatographic window was designed where all 3 phosphoamino acids are separated with baseline resolution in the order of Ser(P), Thr(P) and Tyr(P), and eluted before the normal protein amino acids. The total separation time is 13 min and includes the elution of excess FMOC, its derivatives and incompletely hydrolysed peptides. The FMOC fluorescence of these three standard phosphoamino acids is linear in the range 10–100 pmol. The technique is shown to be successful in finding phosphoproteins separated by two-dimensional gel electrophoresis. The method is sensitive (30 pmol of a single site phosphorylated protein, 3.8 pmol of Ser(P) detected) and allows automated sample throughput. This presents an option for rapid screening of protein phosphorylation of large numbers of proteins separated by two-dimensional polyacrylamide gel electrophoresis.

Experience with a sulfonamide diuretic in a large urine drug testing program.

Gas chromatography/mass spectrometry (GC/MS) with selected ion monitoring is a common confirmation method for tetrahydrocannabinol (THC) metabolites in urine. Department of Defense laboratories use this method for analysis of 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THCA) following ion exchange column purification and methylation. Furosemide, a common diuretic which is excreted into urine, will be isolated with THCA in the extraction procedure, will appear in the chromatographic window [retention time relative to 11-nor-delta-9-tetrahydrocannabinolmethyl ether-9-carboxylic acid methyl ester (THCA-Me) of 1.13], and will exhibit the six ions monitored for THCA-Me and its trideuterated analog. The results indicate that methylated furosemide and other common diuretics can easily be distinguished from THCA-Me. They also allow one to explain in court the extraneous peaks in a chromatogram and to correct for the overutilization of derivatizing reagent.