Easy and accurate high-performance liquid chromatography retention prediction with different gradients, flow rates, and instruments by back-calculation of gradient and flow rate profiles

Isocratic retention data should make a suitable foundation for an accurate, cross-instrument LC retention prediction system. Our previous work suggested that in order to accurately calculate (or "project") gradient retention times on a wide range of HPLC systems using a single set of isocratic retention data, the precise shape of both the gradient and flow rate profiles produced by each instrument must be properly taken into account. However, accurate measurement of these system properties is difficult and time-consuming. In this work, we describe an approach that uses the measured gradient retention times of a set of standard solutes spiked into the sample along with their known isocratic retention vs. eluent composition relationships to determine the effective gradient and flow rate profiles by back-calculation. Retention "projections" of 20 other solutes using these back-calculated profiles, under various chromatographic conditions typical of metabolomics experiments, were remarkably accurate (as good as 0.23% of the gradient time, R2 up to 0.99996), being very near the level of retention reproducibility. Our calculations suggest that this level of accuracy will allow a quadrupole MS to identify 38-fold more compounds out of a simulated mixture of 7307; it would allow an FTICR-MS to improve its identification rate nearly two-fold with the same mixture. Moreover, very little effort is required of the user. This approach provides a simple way to correct for all instrument-related factors affecting retention, allowing dramatically streamlined and improved retention projection across gradients, flow rates, and HPLC instruments.     

High-throughput purification of combinatorial libraries I: a high-throughput purification system using an accelerated retention window approach

We have developed a high-throughput purification system to purify combinatorial libraries at a 50-100-mg scale with a throughput of 250 samples/instrument/day. We applied an accelerated retention window method to shorten the purification time and targeted one fraction per injection to simplify data tracking, lower QC workload, and simplify the postpurification processing. First, we determined the accurate retention time and peak height for all compounds using an eight-channel parallel LC/UV/MS system, and calculated the specific preparative HPLC conditions for individual compounds. The preparative HPLC conditions include the compound-specific gradient segment for individual compounds with a fixed gradient slope and the compound-specific UV or ELSD threshold for triggering a fraction collection device. A unique solvent composition or solvent strength was programmed for each compound in the preparative HPLC in order to elute all compounds at the same target time. Considering the possible deviation of the predicted retention time, a 1-min window around the target time was set to collect peaks above a threshold based on UV or ELSD detection. Dual column preparative instruments were used to maximize throughput. We have purified more than 500 000 druglike compounds using this system in the past 3 years. We report various components of this high-throughput purification system and some of our purification results.     

Standardized GC–MS (EI) procedure for monitoring the detection and identification performance applied to herbal remedies

A procedure for monitoring the identification performance of GC–MS instrumentation as applied to herbal remedies was established, using eugenol (extracted from cloves) as a control compound. The following parameters were monitored: retention time (acceptable variability 0.5%); signal-to-noise ratio (at least 40% of the initial value); ion intensity ratio (acceptable variability 20%); and identity search result (reverse match, with minimum match value of 850 for quadrupole instruments and 800 for ion trap instrument). Other candidates for control compounds (pulegone, caffeine, and methoxsalen) as well as other parameters (relative retention time, second ion intensity ratio, peak area, and direct match) did not give any additional information concerning variability, observed trends, and sensitivity.     

Improved reversed-phase gradient retention modeling

An empirical nonlinear equation is used to describe the relationship between chromatographic retention and solvent composition in both isocratic and gradient chromatography. The integration of the gradient equation can be solved with our equation, allowing a simple transition between isocratic and gradient conditions. Precise interpolation between experimental data points and reasonable extrapolation outside the directly measured data range are possible. Also, the dependence of the parameters of the equation on temperature has been explored, allowing the simultaneous exploration of temperature, gradient starting composition and gradient slopes. A unique quality criterion is used to judge the quality of the separation.     

色谱保留时间在蛋白质组研究中的应用

液相色谱与串联质谱联用(LC-MS/MS)技术是蛋白质组学研究中的常见方法。保留时间作为独立于质谱信息的参数已经被用于蛋白质的鉴定和定量工作中。在多肽鉴定领域,多肽的色谱保留时间预测与常规的二级串联质谱数据库搜索算法结合可以提高鉴定的可信度。鉴定的灵敏度也可以通过匹配多次LC-MS实验中具有相同精确质量数和保留时间的峰而提高。另一方面,由于色谱条件的微小改变即会引起保留时间的变化,因此对多次实验结果进行保留时间比对是进行非标记定量的不可或缺的步骤。另外,联合保留时间偏移和质量数信息还可以进行蛋白质翻译后修饰(post-translational modification, PTM)的鉴定。     

Prediction of liquid chromatographic retention for differentiation of structural isomers

A liquid chromatography (LC) retention time prediction software, ACD/ChromGenius, was employed to calculate retention times for structural isomers, which cannot be differentiated by accurate mass measurement techniques alone. For 486 drug compounds included in an in-house database for urine drug screening by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOFMS), a retention time knowledge base was created with the software. ACD/ChromGenius calculated retention times for compounds based on the drawn molecular structure and given chromatographic parameters. The ability of the software for compound identification was evaluated by calculating the retention order of the 118 isomers, in 50 isomer groups of 2–5 compounds each, included in the database. ACD/ChromGenius predicted the correct elution order for 68% (34) of isomer groups. Of the 16 groups for which the isomer elution order was incorrectly calculated, two were diastereomer pairs and thus difficult to distinguish using the software. Correlation between the calculated and experimental retention times in the knowledge base tested was moderate, r² = 0.8533. The mean and median absolute errors were 1.12 min, and 0.84 min, respectively, and the standard deviation was 1.04 min. The information generated by ACD/ChromGenius, together with other in silico methods employing accurate mass data, makes the identification of substances more reliable. This study demonstrates an approach for tentatively identifying compounds in a large target database without a need for primary reference standards.     

Controlling the retention in capillary LC with solvents, temperature, and electric fields

Once a suitable stationary phase and column dimensions have been selected, the retention in liquid chromatography (LC) is traditionally adjusted by controlling the mobile phase composition. Solvent gradients enable achievement of good separation selectivity while decreasing the separation time as compared to isocratic elution. Capillary columns allow use of other programming parameters, i.e. temperature and applied electric fields, in addition to solvent gradient elution. This paper presents a review of programmed separation techniques in miniaturized LC, including retention modeling and method transfer from the conventional to micro- and capillary scales. The impact of miniaturized instrumentation on retention and the limitations of capillary LC are discussed. Special attention is focused on the gradient dwell volume effects, which are more important in micro-LC techniques than in conventional analytical LC and may cause significant increase in the time of analysis, unless special instrumentation and (or) pre-column flow-splitting is used. The influence of temperature upon retention is also discussed, and applications where the temperature has been actively used for retention control in capillary LC are included together with the instrumentation utilized. Finally the possibilities of additional selectivity control by applying an electric field over a packed capillary LC column are discussed.     

Identification of polymer additives by liquid chromatography-mass spectrometry

LC-MS at different experimental conditions was used to construct a library of MS spectra of polymer additives. Combination of retention time information derived from the chromatogram with molecular mass and fragment ion information derived from MS and MS/MS was used for the identification of 20 additives. Mixtures of different additives and extracts of LDPE films were prepared and analyzed as unknowns. All 20 additives could be identified, 15 with 100% certainty.     

Thermodynamic studies of pressure-induced retention of peptides in reversed-phase liquid chromatography

The pressure-induced retention of peptides on reversed-phase HPLC was studied by systematically changing organic solvent composition and temperature at both low (19 bar) and high (318 bar) pressures using a homologous series of hydrophobic poly-L-phenylalanine (n = 2-7) as the model compound. Based on van' t Hoff plots under different organic solvent compositions and pressures, the enthalpy change for the solute (deltaH) was determined. Moreover, both the enthalpy and entropy change for each phenylalanine residue (deltadeltaH and deltadeltaS), which corresponds to solute retention on a microenvironment along the depth of C18 chain, were also calculated by direct subtractions. Results indicate that under acetonitrile (ACN) compositions above 35%, the pressure caused deltadeltaS value to change from a negative to a positive value and both deltaH and deltadeltaH to change from a negative to a less negative value, all leading to a thermodynamic state closer to those under 35% acetonitrile composition. This implies that the pressure-induced retention observed in this study was an entropy-favored but enthalpy-unfavored process and was explained by pressure-induced desorption of solvent molecules that were associated with the stationary phase or with the peptide solute. Under 35% acetonitrile composition, however, it was found that neither deltadeltaH nor deltadeltaS value was significantly changed by the pressure. Whereas, both deltaH value and the intercept of van't Hoff plots under 35% acetonitrile composition were increased by pressure. This indicates that under low organic solvent composition, 35%, most of the acetonitrile molecules adsorbed on the surface of the stationary phase and only little solvent molecules were dissolved in the bulk stationary phase where the phenylalanine residues were partitioned. This study has provided new thermodynamic insights to the pressure-induced retention for peptides and proteins.     

Using new structurally related additive schemes in the precalculation of gas chromatographic retention indices of polychlorinated hydroxybiphenyls on HP-5 stationary phase

A new additive scheme is proposed for the precalculation of gas chromatographic retention indices of complex organic compounds. The principal feature of this approach is the absence of previously calculated I increments for any structural fragments or functional groups in the molecule. Instead, arithmetical operations involving I values of simpler structural analogues of target compounds are used directly. I precalculation for polychlorinated hydroxybiphenyls (839 congeners) on the HP-5 stationary phase was chosen as one of the most important applications of the method under discussion. Such a large number of congeners cannot be obtained as reference samples and their gas chromatographic (GC)-mass spectrometric (MS) identification should therefore be based currently on precalculated I values.     

Relationship between thermodynamic characteristics and isothermal retention indices

Summary Thermodynamic characteristics determined from retention data measured under isothermal conditions were used for the calculation of retention indices of a number of model compound. The minimum retention index difference required for the identification of two adijacent compounds as well the temperature dependency of retention index are discussed. The possibility of alculating thermodynamic characteristics from retention indices published in the literature was also investigated.Dedicated to Professro J. F. K. Huber on the occasion of his 60th birthday.     

Extraction,separation and quantitative structure–retention relationship modeling of essential oils in three herbs

The essential oils extracted from three kinds of herbs were separated by a 5% phenylmethyl silicone (DB‐5MS) bonded phase fused‐silica capillary column and identified by MS. Seventy‐four of the compounds identified were selected as origin data, and their chemical structure and gas chromatographic retention times (RT) were performed to build a quantitative structure–retention relationship model by genetic algorithm and multiple linear regressions analysis. The predictive ability of the model was verified by internal validation (leave‐one‐out, fivefold, cross‐validation and Y‐scrambling). As for external validation, the model was also applied to predict the gas chromatographic RT of the 14 volatile compounds not used for model development from essential oil of Radix angelicae sinensis. The applicability domain was checked by the leverage approach to verify prediction reliability. The results obtained using several validations indicated that the best quantitative structure–retention relationship model was robust and satisfactory, could provide a feasible and effective tool for predicting the gas chromatographic RT of volatile compounds and could be also applied to help in identifying the compound with the same gas chromatographic RT.     

Comprehensive two-dimensional gas chromatography, retention indices and time-of-flight mass spectra of flavonoids and chalcones

The applicability of comprehensive two-dimensional gas chromatography (GC×GC) for flavonoids analysis was investigated by separation and identification of flavonoids in standards, and a complex matrix natural sample. The modulation temperature was optimized to achieve the best separation and signal enhancement. The separation pattern of trimethylsilyl (TMS) derivatives of flavonoids was compared on two complementary column sets. Whilst the BPX5/BPX50 (NP/P) column set offers better overall separation, BPX50/BPX5 (P/NP) provides better peak shape and sensitivity. Comparison of the identification power of GC×GC-TOFMS against both the NIST05 MS library and a laboratory (created in-house) TOFMS library was carried out on a flavonoid mixture. The basic retention index information on high-performance capillary columns with a non-polar stationary phase was established and database of mass spectra of trimethylsilyl derivatives of flavonoids was compiled. TOFMS coupled to GC×GC enabled satisfactory identification of flavonoids in complex matrix samples at their LOD over a range of 0.5-10 μg/mL. Detection of all compounds was based on full-scan mass spectra and for each compound a characteristic ion was chosen for further quantification. This study shows that GC×GC-TOFMS yields high specificity for flavonoids derived from real natural samples, dark chocolate, propolis, and chrysanthemum.     

Tandem mass spectrometric accurate mass performance of time-of-flight and Fourier transform ion cyclotron resonance mass spectrometry: a case study with pyridine derivatives

The interpretation of mass spectra is a key process during compound identification, and the combination of tandem mass spectrometry (MS/MS) with high-accuracy mass measurements may deliver crucial information on the identity of a compound. Obtaining accurate mass data of fragment ions in MS/MS reveals the particular problem of mass calibration when a lockmass, which is frequently used to obtain accurate masses in MS, is absent. An alternative technique is to recalibrate the MS/MS spectrum using a reference MS/MS spectrum acquired under the same conditions. We have tested and validated this approach using a hybrid quadrupole/orthogonal acceleration reflectron-type time-of-flight (TOF) mass spectrometer. The results were compared with those obtained under similar conditions on a Fourier transform ion cyclotron resonance (FT-ICR) instrument. We found that the mass accuracy observed with such an "external" recalibration on the TOF instrument in MS/MS is identical to what can be obtained on a similar instrument operating in one-dimensional MS mode using the lockmass technique. However, mass accuracy in both cases is one order of magnitude inferior to that obtained using FTMS, and also inferior to that observed using sector field MS when operated at comparable resolution. Nevertheless, for small (<200 Da) molecules, this mass accuracy was still sufficient to have the "true" elemental composition identified as the first hit in about 70% of all cases. It was possible to elucidate the fragmentation mechanism of eight azaheterocycles containing a pyridine moiety, where the accurate mass data from the TOF instrument allowed distinction between two alternative fragmentation pathways.     

Effect of mobile phase composition, pH and buffer type on the retention of ionizable compounds in reversed-phase liquid chromatography: application to method development

Optimizing separation of ionizable compounds in order to find robust conditions has become an important part of method development in liquid chromatography. This work is an attempt to explain the observed variations of retention of acid and basic compounds with the organic modifier content in the mobile phase, according to various factors: the type of modifier, the type of buffer, the temperature and of course the type of solute. This is done by considering the variation of the so-called chromatographic pKa which refers to the pH measured in the aqueous medium and is determined from retention data. A procedure is described that accurately relates, from nine experiments, retention to solvent composition and pH. The limits of such a procedure are evaluated and two examples of optimized separations of basic compounds are given.     

Metabolite profiling and beyond: approaches for the rapid processing and annotation of human blood serum mass spectrometry data

In this paper, we describe data processing and metabolite identification approaches which lead to a rapid and semi-automated interpretation of metabolomics experiments. Data from metabolite fingerprinting using LC-ESI-Q-TOF/MS were processed with several open-source software packages, including XCMS and CAMERA to detect features and group features into compound spectra. Next, we describe the automatic scheduling of tandem mass spectrometry (MS) acquisitions to acquire a large number of MS/MS spectra, and the subsequent processing and computer-assisted annotation towards identification using the R packages MetShot, Rdisop, and the MetFusion application. We also implement a simple retention time prediction model using predicted lipophilicity logD, which predicts retention times within 42 s (6 min gradient) for most compounds in our setup. We putatively identified 44 common metabolites including several amino acids and phospholipids at metabolomics standards initiative (MSI) levels two and three and confirmed the majority of them by comparison with authentic standards at MSI level one. To aid both data integration within and data sharing between laboratories, we integrated data from two labs and mapped retention times between the chromatographic systems. Despite the different MS instrumentation and different chromatographic gradient programs, the mapped retention times agree within 26 s (20 min gradient) for 90 % of the mapped features.

Chemical profiling of constituents of Smilacis glabrae using ultra-high pressure liquid chromatography coupled with LTQ Orbitrap mass spectrometry

A reliable LC-MS method has been applied for the separation and identification of major constituents of the rhizome of Smilacis glabrae. Identification of the constituents was carried out by interpretation of their retention time, and MS and MS/MS data, especially by comparing these with Sarcandra glabra under the same LC-MS conditions, as well as the data provided by the literature. Thirty-three compounds, including catechin derivatives, flavanonols, phenolic acid derivatives and phenylpropanoid glycosides were either identified or tentatively characterized. Among them, compound 12 was deduced to be a new phenylpropanoid-substituted catechin. Fragmentation behaviors of the three major categories of compounds were also investigated. This UPLC-PDA/ESI-MS(n) method was effective for the separation and identification of the constituents and could be the basis for the comprehensive quality control of Smilacis glabrae.     

Is nontarget screening of emerging contaminants by LC-HRMS successful? A plea for compound libraries and computer tools

This review focuses on the possibilities and limits of nontarget screening of emerging contaminants, with emphasis on recent applications and developments in data evaluation and compound identification by liquid chromatography-high-resolution mass spectrometry (HRMS). The general workflow includes determination of the elemental composition from accurate mass, a further search for the molecular formula in compound libraries or general chemical databases, and a ranking of the proposed structures using further information, e.g., from mass spectrometry (MS) fragmentation and retention times. The success of nontarget screening is in some way limited to the preselection of relevant compounds from a large data set. Recently developed approaches show that statistical analysis in combination with suspect and nontarget screening are useful methods to preselect relevant compounds. Currently, the unequivocal identification of unknowns still requires information from an authentic standard which has to be measured or is already available in user-defined MS/MS reference databases or libraries containing HRMS spectral information and retention times. In this context, we discuss the advantages and future needs of publicly available MS and MS/MS reference databases and libraries which have mostly been created for the metabolomic field. A big step forward has been achieved with computer-based tools when no MS library or MS database entry is found for a compound. The numerous search results from a large chemical database can be condensed to only a few by in silico fragmentation. This has been demonstrated for selected compounds and metabolites in recent publications. Still, only very few compounds have been identified or tentatively identified in environmental samples by nontarget screening. The availability of comprehensive MS libraries with a focus on environmental contaminants would tremendously improve the situation.     

On the inter‐instrument and inter‐laboratory transferability of a tandem mass spectral reference library: 1. Results of an Austrian multicenter study

The inter‐instrument and inter‐laboratory transferability of a tandem mass spectral reference library originally built on a quadrupole‐quadrupole‐time‐of‐flight instrument was examined. The library consisted of 3759 MS/MS spectra collected from 402 reference compounds applying several different collision‐energy values for fragmentation. In the course of the multicenter study, 22 test compounds were sent to three different laboratories, where 418 tandem mass spectra were acquired using four different instruments from two manufacturers. The study covered the following types of tandem mass spectrometers: quadrupole‐quadrupole‐time‐of‐flight, quadrupole‐quadrupole‐linear ion trap, quadrupole‐quadrupole‐quadrupole, and linear ion trap‐Fourier transform ion cyclotron resonance mass spectrometer. In each participating laboratory, optimized instrumental parameters were gathered solely from routinely applied workflows. No standardization procedure was applied to increase the inter‐instrument comparability of MS/MS spectra. The acquired tandem mass spectra were matched against the established reference library using a sophisticated matching algorithm, which is presented in detail in a companion paper. Correct answers, meaning that the correct compound was retrieved as top hit, were obtained in 98.1% of cases. For the remaining 1.9% of spectra, the correct compound was matched at second rank. The observed high percentage of correct assignments clearly suggests that the developed mass spectral library search approach is to a large extent platform independent. Copyright © 2009 John Wiley & Sons, Ltd.