Fast gradient elution reversed-phase liquid chromatography with diode-array detection as a high-throughput screening method for drugs of abuse. II. Data analysis

In Part I of this work, we developed a method for the detection of drugs of abuse in biological samples based on fast gradient elution liquid-chromatography coupled with diode array spectroscopic detection (LC-DAD). In this part of the work, we apply the chemometric method of target factor analysis (TFA) to the chromatograms. This algorithm identifies the target compounds present in chromatograms based on a spectral library, resolves nearly co-eluting components, and differentiates between drugs with similar spectra. The ability to resolve highly overlapped peaks using the spectral data afforded by the DAD is what distinguishes the present method from conventional library searching methods. Our library has a mean list length (MLL) of 1.255 and a discriminating power of 0.997 when both retention index and spectral factors are considered. The algorithm compares a library of 47 different compounds of toxicological relevance to unknown samples and identifies which compounds are present based on spectral and retention index matching. The application of a corrected retention index for identification rather than raw retention times compensates for long-term and column-to-column retention time shifts and allows for the use of a single library of spectral and retention data. Training data sets were used to establish the search and identification parameters of the method. A validation data set of 70 chromatograms was used to calculate the sensitivity (correct identification of positives) and specificity (correct identification of negatives) of the method, which were found to be 92% and 94%, respectively.     

An overview on the standardization of chromatographic methods for screening analysis in toxicology by means of retention indices and secondary standards

Summary A review is presented on the methods of standardization of HPLC data as used in systematic toxicological analysis. In straight-phase HPLC, the best results were obtained with a series of selected drugs as retention standards. In reversed-phase HPLC, various retention index systems were introduced. However, these systems alone cannot compensate large differences in selectivities of nominally identical, but commercially different reversed-phase column packings. Much better results were achieved with selected drugs as retention index markers. The practical applicability of such a standardized HPLC system is demonstrated.Part I see [1]Dedicated to Prof. Dr. Dr. Marika Geldmacher-von Mallinckrodt in recognition of her pioneering contributions in clinical and forensic toxicology     

A method of calculating the second dimension retention index in comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry

A method was developed to calculate the second dimension retention index of comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC/TOF-MS) data using n-alkanes as reference compounds. The retention times of the C(7)-C(31) alkanes acquired during 24 isothermal experiments cover the 0-6s retention time area in the second dimension retention time space, which makes it possible to calculate the retention indices of target compounds from the corresponding retention time values without the extension of the retention space of the reference compounds. An empirical function was proposed to show the relationship among the second dimension retention time, the temperature of the second dimension column, and the carbon number of the n-alkanes. The proposed function is able to extend the second dimension retention time beyond the reference n-alkanes by increasing the carbon number. The extension of carbon numbers in reference n-alkanes up to two more carbon atoms introduces <10 retention index units (iu) of deviation. The effectiveness of using the proposed method was demonstrated by analyzing a mixture of compound standards in temperature programmed experiments using 6 different initial column temperatures. The standard deviation of the calculated retention index values of the compound standards fluctuated from 1 to 12 iu with a mean standard deviation of 5 iu.     

The Analysis of volatile compounds by purge and trap with whole column cryotrapping (WCC) on a fused silica capillary column

The purge and trap (P&T) method of analysis has been interfaced with fused silica capillary column gas chromatography. This interfacing has been accomplished without splitting the P&T trap desorption carrier gas. Thus, 100% of the purged compounds are transferred to the column. The analytes are cryofocussed on the column using whole column cryotrapping (WCC) at ?80°C. The resulting P&T/WCC procedure is extremely well-suited to the analysis of trace purgeable aqueous organic compounds. Samples and standards containing a variety of aromatic standard compounds were analyzed. The standards included benzene, toluene, ethylbenzene, xylenes, C₃-C₄-benzenes, and naphthalene, as well as three P&T internal standard compounds. Chromatographic peak widths were uniformly less than 6 s at the base and excellent precision was obtained in the relative retention time data for all compounds. The chromatogram of a groundwater sample contaminated with aromatic gasoline compounds is also presented. Since P&T/WCC works well with fused silica capillary columns, the full sensitivity and chromatographic efficiency of capillary gas chromatography is made available to P&T analyses.     

A simple,fast, and accurate thermodynamic‐based approach for transfer and prediction of gas chromatography retention times between columns and instruments Part III: Retention time prediction on target column

This is the third part of a three‐part series of papers. In Part I, we presented a method for determining the actual effective geometry of a reference column as well as the thermodynamic‐based parameters of a set of probe compounds in an in‐house mixture. Part II introduced an approach for estimating the actual effective geometry of a target column by collecting retention data of the same mixture of probe compounds on the target column and using their thermodynamic parameters, acquired on the reference column, as a bridge between both systems. Part III, presented here, demonstrates the retention time transfer and prediction from the reference column to the target column using experimental data for a separate mixture of compounds. To predict the retention time of a new compound, we first estimate its thermodynamic‐based parameters on the reference column (using geometric parameters determined previously). The compound's retention time on a second column (of previously determined geometry) is then predicted. The models and the associated optimization algorithms were tested using simulated and experimental data. The accuracy of predicted retention times shows that the proposed approach is simple, fast, and accurate for retention time transfer and prediction between gas chromatography columns.     

同系物保留指数作为脂肪酸气相色谱定性参数的优越性考察

在气相色谱分析中,通常采用相对保留值和Kovats保留指数作为化学物质的定性参数。相对保留值在使用中受到参考标准不一致的限制而难于推广,况且柱温对相对保留值的影响很大,其重现性还受到待鉴定物与参考标准保留值差距大小的影响。只有在待鉴定物与参考标准的保留值差距不大时,相对保留值才可靠。     

Prediction of retention indices. VI: Isothermal and temperature-programmed retention indices,methylene value,functionality constant,electronic and steric effects

The Kováts retention index system with n-alkanes as reference standards has properties not fully explored when single, isolated or stand-alone analytes are analyzed by isothermal gas chromatography. When a homologous series of analytes are analyzed by either linear or non-linear temperature-programmed gas chromatography, the retention data of the entire series can be treated systematically to produce an I vs. Z plot that is linear, thereby giving insight into the relationship between chemical structure and retention index. Dead time t_M is both instrument and temperature dependent. With no dead time t_M adjustment, the retention indices of analytes calculated from experimental retention times by the method of either linear or logarithmic interpolation give statistically identical values. Linear regression analysis of the data shows the slope as methylene value (A) and intercept as functionality constant or group retention factor (GRF) of the homologous series. The A and (GRF) values vary with chemical structures, intermolecular electronic and steric interactions, and polarity of column liquid phases, and can link gas chromatographic retention index to chemical structure. Examples of the influence of molecular electronic effects and steric effects on retention index are given and discussed.     

Enantiomeric separation and discrimination of 2-hydroxy acids as O-trifluoroacetylated (S)-(+)-3-methyl-2-butyl esters by achiral dual-capillary column gas chromatography

An efficient method is described for the simultaneous enantiomeric separation of 18 different racemic 2-hydroxy acids for the determination of their absolute configurations. It involves the conversion of each enantiomer into a diastereomeric O-trifluoroacetylated (S)-(+)-3-methyl-2-butyl ester for the direct separation by achiral dual-capillary column gas chromatography with subsequent identification and determination of its chirality by retention index (I) library matching. The enantiomers of each acid were well separated with high resolution values (R > or = 1.4) on DB-5 and DB-17 columns of different polarity. When temperature-programmed I values of 2-hydroxy acid enantiomers as their diastereomeric derivatives were measured on both columns, the I values were characteristic of each enantiomer. Simple I matching with the reference values was thus useful in cross-checking each acid enantiomer for the identification and chiral discrimination. When applied to urine samples, the present method allowed positive identification of most of the spiked 2-hydroxy acids from normal urine and for endogenous (S)-lactic acid and (S)-2-hydroxybutyric acid from a clinical urine specimen.     

Comparison of Retention Indices of Some Monosubstituted Benzenes Calculated by Different Mathematical Methods in RP-LC

In reversed phase liquid chromatography, the retention indices of benzene and nine mono substituted benzenes with different functionality based on the alkan-2-ones and alkyl aryl ketones retention index standards have been determined by the application of two new mathematical adaptation methods, viz. a multiparametric least-squares regression iterative method based on the determination of the adjusted retention times and a local cubic interpolation method directly using the total retention times. The two methods were applied to two types of columns. The first group includes four octadecyl-C₁₈ columns with different packing materials obtained from different manufacturers, while the second comprises an octyl-C₈ column. The retention indices have been extensively studied using either methanol–water or acetonitrile–water mobile phase systems. The influences of the concentration of the organic modifier in the mobile phase (methanol or acetonitrile), the column temperature, and the column packing material on retention indices of the set of the ten monoaromatics studied were also investigated. The calculated multiparametric retention indices values, those obtained by the local cubic interpolation and Kováts’ methods are compared. Good agreement was observed between the retention indices calculated by the three methods.     

Sensitivity of the methylbenzenes and chlorobenzenes retention index to column temperature, stationary phase polarity, and-number and chemical nature of substituents

Retention indices of methylbenzenes and chlorobenzenes on two fused silica capillary columns, HP-5 (diphenylsiloxane 5% diphenyldimethylsiloxane) and ZB-WAX (polyethylene glycol), have been calculated at various isothermal temperatures and compared with literature data. The retention index temperature effect was studied for each solute, finding greater retention index the higher the column temperature. A comparison between the straight line fit and the fit to the recently proposed equation I = A + B/T +C ln T was carried out. The effect of the stationary phase polarity on the retention index was checked. In general, a greater retention index was found for the more polar stationary phase. The retention indices of the chlorobenzenes are greater than the retention indices of the methylbenzenes, irrespective of the stationary phase and the column temperature. In addition, the influence of the methyl/chlorine substitution on the benzene molecule was investigated at each temperature. The retention indices increased as the number of substituents (methyl/chlorine) increased. The retention index increments of methyl and chloro derivatives are also discussed, which permits to compare the effect of both, methyl or chlorine, chemical functions, for a fixed substituent number in the benzene molecule.     

Developing qualitative LC-MS methods for characterization of Vaccinium berry Standard Reference Materials

Standard Reference Materials (SRMs) offer the scientific community a stable and homogenous source of material that holds countless application possibilities. Traditionally, the National Institute of Standards and Technology (NIST) has provided SRMs with associated quantitative information (certified values) for a select group of targeted analytes as measured in a solution or complex matrix. While the current needs of the SRM community are expanding to include non-quantitative data, NIST is attempting to broaden the scope of how and what information is offered to the SRM community by providing qualitative information about biomaterials, such as chromatographic fingerprints and profiles of untargeted identifications. In this work, metabolomic and proteomic profiling efforts were employed to characterize a suite of six Vaccinium berry SRMs. In the discovery phase, liquid chromatography-tandem mass spectrometry (LC-MS/MS) data was matched to mass spectral libraries; a subsequent validation phase based on multiple-reaction monitoring LC-MS/MS relied on both retention time matching of authentic standards along with fragmentation data for a qualitative overview of the most prominent organic compounds present. Definitive and putative identifications were determined for over 70 metabolites based on reporting guidelines set forth by the Metabolomics Standards Initiative (Metabolomics 3(3):211–221, 2007), and the capability of electrospray ionization mass spectrometry (ESI-MS) to profile untargeted metabolites within a complex matrix using mass spectral matching is demonstrated. Bottom-up proteomic analyses were possible using peptide databases translated from expressed sequence tags (ESTs). Homology searches provided identification of novel Vaccinium proteins based on homology to related genera. Chromatographic fingerprints of these berry materials were acquired for supplemental qualitative information to be provided to users of these SRMs. An unbounded set of qualitative data about a biomaterial is a valuable complement to quantitative information traditionally provided in NIST Certificates of Analysis.     

Estimation of Kováts retention indices using group contributions

We have constructed a group contribution method for estimating Kováts retention indices by using observed data from a set of diverse organic compounds. Our database contains observed retention indices for over 35,000 different molecules. These were measured on capillary or packed columns with polar and nonpolar (or slightly polar) stationary phases under isothermal or nonisothermal conditions. We neglected any dependence of index values on these factors by averaging observations. Using 84 groups, we determined two sets of increment values, one for nonpolar and the other for polar column data. For nonpolar column data, the median absolute prediction error was 46 (3.2%). For data on polar columns, the median absolute error was 65 (3.9%). While accuracy is insufficient for identification based solely on retention, it is suitable for the rejection of certain classes of false identifications made by gas chromatography/mass spectrometry.     

Rapid molecular weight analysis of polymers by temperature gradient interaction chromatography

Temperature gradient interaction chromatography (TGIC) has been established as a high-resolution technique for the characterization of synthetic polymers. So far, most of the TGIC investigations focused on the high-resolution analysis and little effort has been made on the reduction of the analysis time. In this study, we examined the effect of the column heating rate, the eluent flow rate, and the column length on the TGIC analysis time. We found that the heating rate is the most important experimental parameter to control the TGIC retention time. With a C18 silica column (50 mm x 4.6mm I.D.), a set of PS standards of wide molecular weight range (5 - 648 kg/mol) could be separated within 4 min at a heating rate of 8 degrees C/min.     

Comprehensive two-dimensional gas chromatography/mass spectrometric analysis of pepper volatiles

The headspace compositions of 13 pepper and peppercorn samples of different species, colloquially also referred to as pepper, were analyzed, and more than 300 compounds were tentatively characterized by means of comprehensive two-dimensional gas chromatography in tandem with flame ionization detection, quadrupole mass spectrometric detection and time-of-flight mass spectrometric detection (GC x GC-FID, GC x GC/qMS and GC x GC/TOFMS, respectively). The analysis of volatile organic compounds (VOCs) was performed after solid-phase microextraction (SPME) using a 75-microm PDMS/DVB fibre. Fingerprint comparison between the three techniques permitted peaks to be assigned in the GC x GC-FID experiment based on the analogous MS analysis, taking into account retention shifts arising from method variations. When using GC x GC/TOFMS, about five times more peaks were identified than in GC x GC/qMS. Retention indices for all peaks were calculated in the bi-dimensional column set comprising of a 5% phenyl polysilphenylene-siloxane primary column and a polyethylene glycol second column. The spectra obtained by both mass detection techniques (qMS and TOFMS) give very similar results when spectral library searching was performed. The majority of the identified compounds eluted as pure components as a result of high-resolution GC x GC separations, which significantly reduces co-elution, and therefore increases the likelihood that pure spectra can be obtained. The differences between TOFMS and qMS (in fast scanning mode) spectra were generally small. Whilst spectral quality and relative ion ratios across a narrow peak (e.g. w(b) approximately 100-150 ms) do vary more for the fast peaks obtained in GC x GC/qMS operation, than with TOFMS, in general adequate spectral matching with the library can be achieved.     

High Performance Liquid Chromatography of Dithiolthiones

Abstract

Sixteen dithiolthiones including oltipraz (5-(2-Pyrazinyl)-4-methyl-1-1, 2 dithiol-3-thione) and anethol dithiolthione (P-methoxyphenyl-1,2-dithiol-3-thione) have been studied using an isocratic high performance liquid chromatographic system. The retention characteristics of these compounds were determined using a conventional C 18 reverse phase column and a μ Bondapack phenyl column. The mobile phases used consisted of several concentrations of methanol and water. All compounds studied were adequately detected in the nanogram range (30–100 ng on column) using ultra violet detection (UV) set at 300 nm. Several analogs and isomers were separated. The retention characteristics of the 16 compounds studied are reported for both columns using 4 mobile phases and chromatograms of reference standards are presented.     

Identification of a new C28H14 polycyclic aromatic hydrocarbon as a product of supercritical fuel pyrolysis: Tribenzo[cd,ghi,lm]perylene

Tribenzo[cd,ghi,lm]perylene has been identified as a product of the supercritical pyrolysis of both toluene and Fischer-Tropsch synthetic jet fuel. This identification is based on HPLC/UV/MS data, which show that compound I, eluting immediately after five other C28H14 isomers, is also a C28H14 PAH. The UV spectrum of compound I has features of a benzenoid PAH, of which there are only eight C28H14 isomers. Four of these isomers--benzo[a]coronene, phenanthro[5,4,3,2-efghi]perylene, benzo[cd]naphtho[3,2,1,8-pqra]perylene, and benzo[pqr]naphtho[8,1,2-bcd]perylene--have already been identified as supercritical pyrolysis products by matching their UV spectra with those of respective reference standards. A fifth C28H14 PAH--benzo[ghi]naphtho[8,1,2-bcd]perylene, which does not have a reference standard--has also been recently identified through MS and UV data, use of annellation theory to predict UV spectral characteristics, and length-to-breadth ratio/retention time data. Of the remaining three isomers, bisanthene (IUPAC name phenanthro[1,10,9,8-opqra]perylene) has been determined not to be present in our product mixture, as its UV spectrum does not match that of any of our product PAH. Using annellation theory, we predict the UV spectral characteristics of the two remaining C28H14 benzenoid isomers, for which there are no reference standards (tribenzo[cd,ghi,lm]perylene and naphthaceno[3,4,5,6,7-defghij]naphthacene). Results from this analysis show that the predicted UV spectral features of tribenzo[cd,ghi,lm]perylene match those of compound I--and that those of naphthaceno[3,4,5,6,7-defghij]naphthacene are inconsistent with those of compound I. The length-to-breadth ratio of tribenzo[cd,ghi,lm]perylene also agrees with compound I's HPLC elution behavior. This is the first time that tribenzo[cd,ghi,lm]perylene (IUPAC name phenanthro[2,1,10,9,8,7-pqrstuv]pentaphene) has been identified as a product of fuel pyrolysis or combustion.     

Characterization of stationary phases based on monosubstituted benzene retention indices using correspondence factor analysis and linear solvation energy relationships in RPLC

In reversed-phase liquid chromatography (RPLC), the comparison of experimental results obtained from different columns is a complex problem. A correspondence factor analysis (CFA) and a linear solvation energy relationship (LSER) were applied on retention data to characterize second-order intermolecular interactions responsible for retention on a set of RPLC columns. Seven octadecyl-C₁₈ columns with different packing materials are obtained from different manufacturers and one octyl-C₈ column. The retention data were determined under isocratic conditions using a methanol–water (65:35, v/v) mobile phase. The chromatographic retention indices based on alkan-2-ones and alkyl aryl ketones retention index scales are calculated using a multiparametric least-squares regressions iterative method. The CFA and LSER results permitted to highlight that the retention indices were appropriate for studying the second-order retention mechanisms on the eight chromatographic systems investigated and exhibited the best reproducibility. Although many earlier studies have reported the use of chemometric methods to characterize chemical factors affecting retention in RPLC using retention factors as retention parameters, this is the first study based on retention indices.