The potential of serially coupled alkyl-bonded silica monolithic columns for high resolution separations of pharmaceutical compounds in biological fluids

Summary In this paper we investigate the potential of alkyl-bonded silica monolithic columns for the isolation and identification of drug-related components in biological fluids. Up to 6 columns have been connected in series to produce a chromatographic system with up to 40,000 plates. This high-resolution chromatography system has been coupled to both MS and NMR to enable efficient detection and characterisation of drug-related components in biological fluids. The use of six coupled columns has been shown to give enhanced resolution over a high quality silica particulate column packed with 3 μm material which exhibits the same back pressure. The effect of volume and mass load on the performance of monolithic columns for semi-preparative chromatography of biological fluids has also been investigated. In these studies it was possible to inject up to 100 mL of neat urine with no loss of chromatographic performance. Furthermore, upon re-testing, the columns showed similar chromatographic performance. Again several columns were serially connected, producing enhanced resolution in the semi-preparative mode.     

Determination of quaternary ammonium pesticides by liquid chromatography-electrospray tandem mass spectrometry

A method for the direct determination of paraquat, diquat, chlormequat and difenzoquat in water samples, using an on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry system was developed. No sample preparation was required and the detection limits were below the European Union maximum residue levels. The chromatographic separation was performed using an XTera MS C8 column. The concentration of the ion pair reagent, the pH and the gradient elution were optimized to give high recoveries and good chromatographic resolution between quats. The detection was carried out using an ion trap as mass analyzer. Parameters such as the magnitude and duration of the resonant excitation voltage and the magnitude of the trapping RF voltage for full scan tandem mass spectrometry (MS-MS) experiments were studied to establish the optimal experimental conditions. Moreover, the accurate optimization of these parameters allowed MS-MS experiments of low mass ions, below m/z 200, providing unambiguous peak identification. Finally, the reproducibility of the proposed method was shown by good run-to-run and day-to-day precision values and its applicability to the determination of quats in drinking water was evaluated using spiked samples.     

Determination of molecular mass distribution of silicone oils by supercritical fluid chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and their off-line combination

Silicone oil samples were characterized by supercritical fluid chromatography (SFC), matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI--TOF MS), and their off-line combination. SFC was used to separate samples of silicone oils on micropacked capillary columns. The fractions for the identification studies were obtained from SFC runs at defined time intervals, when the restrictor was pulled out from the chromatographic flame ionization detector (FID) and inserted into a glass vial with acetone. MALDI--TOF MS was used for the identification of individual oligomers in the fractions separated. The molecular mass distributions determined based on SFC and MALDI--TOF MS measurements were compared. From this comparison, it follows that the results are in good agreement. However, certain differences were observed: MALDI--TOF MS was capable of detecting somewhat larger oligomers than the SFC-FID, but the lower molecular mass oligomers were not present in the MALDI spectra. Differences in the region of lower molecular masses can be explained by evaporation of the more volatile low molecular mass oligomers resulting from heating of the sample during the MALDI--TOF MS measurements as a result of the absorption of the laser shot energy. The fact that no high mass discrimination effects of the MALDI--TOF MS measurements, compared with SFC, were observed is very promising for further applications of MALDI--TOF MS in characterizing synthetic polymers of moderate polydispersity.     

Recent progress in the analysis of sugar monomers from complex matrices using chromatography in conjunction with mass spectrometry or stand-alone tandem mass spectrometry

Analysis of trace levels of carbohydrate monomers in complex matrices requires excellent discrimination of the peaks of interest from background noise. Minimizing contaminating peaks introduced during sample preparation and chromatography is extremely important. However, the exquisite selectivity of the mass spectrometer is essential as a chromatographic detector in this regard. Traditionally gas chromatography-mass spectrometry (GC-MS) has been the method of choice for trace analysis of derivatized carbohydrates. Recent improvements in commercial tandem mass spectrometers (MS-MS) are encouraging the use of GC-MS-MS for improved specificity in trace analysis. There has also been an explosion in applications of electrospray ionization (ESI) for sensitive introduction of polar molecules (including sugars) into the mass spectrometer. This has encouraged ongoing developments in high-performance liquid chromatography-mass spectrometry (LC-MS) and MS-MS of underivatized carbohydrates. This has the potential to dramatically simplify sample preparation. However, as yet LC-MS and MS-MS do not match the sensitivity of GC-MS or GC-MS-MS. Developments in analysis of sugar monomers from complex matrices using chromatography (GC/LC) in conjunction with mass spectrometry (MS, MS-MS) or stand-alone MS-MS are discussed.     

Ultra-fast tandem mass spectrometry scanning combined with monolithic column liquid chromatography increases throughput in proteomic analysis

Liquid chromatography combined with electrospray ionization mass spectrometry (LC/ESI-MS) has been used successfully for the characterization of biomolecules in proteomics in the last few years. This methodology relied largely on the use of reversed-phase chromatography, in particular C18-based resins, which are suitable for separation of peptides. Here we show that polymeric [polystyrene divinylbenzene] monolithic columns can be used to separate peptide mixtures faster and at a higher resolution. For 500 fmol bovine serum albumin, up to 68% sequence coverage and Mascot Mowse scores of >2000 were obtained using a 9 min gradient on a monolithic column coupled to an ion trap mass spectrometer with ultra-fast MS/MS scan rates. In order to achieve similar results using C18 columns, it was necessary to extend gradient times to 30 min. In addition, we demonstrate the utility of this approach for the analysis of whole Escherichia coli cell lysates by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) in combination with LC/MS/MS using 4 min gradients on monolithic columns. Our results indicate higher throughput capabilities of monolithic columns (3-fold gain in time or more) for conventional proteomics applications, such as protein identification and high sequence coverage usually required for detection of post-translational modifications (PTMs). Further optimization of sensitivity and quality of sequence information is discussed, in particular when combined with mass spectrometers that have very fast MS-MS/MS switching and scanning capabilities.     

Negative atmospheric pressure chemical ionisation low-energy collision activation mass spectrometry for the characterisation of flavonoids in extracts of fresh herbs

The flavonoid composition of commonly eaten fresh herbs such as dill, oregano and parsley was analysed by combined LC, MS and low-energy collision induced dissociation (CID) MS-MS. Negative atmospheric pressure chemical ionisation (APCI) MS and MS-MS were used to provide molecular mass information and product-ion spectra of the glycosyl compounds. The most prominent fragment was found to arise from the aglycone ion, which provides molecular mass information about the glycosyl substituent and the aglycone. Product-ion spectra of the aglycone verified the identity by comparison with product-ion spectra of authentic standards. Methoxylated flavonoids provide characteristic fragmentation, i.e., loss of *CH3, which add to the usefulness of the method for identifying unknown flavonoids. Negative-mode APCI-MS is thus demonstrated to be a good alternative to the commonly employed positive mode operation.     

Evaluation of column length and particle size effect on the untargeted profiling of a phytochemical mixture by using UHPLC coupled to high‐resolution mass spectrometry

Liquid chromatography coupled to high‐resolution mass spectrometry is the technique of choice for the untargeted profiling of food matrices. Despite the high potential of high‐resolution mass spectrometry, when dealing with complex mixtures, an efficient separation technique is also needed. The novel core‐shell chromatographic columns packed with sub‐2 μm sized particles are claimed to show very good resolution. However, the analytes retention can be significantly altered when working under ultra‐high performance chromatographic conditions. In this work, an evaluation of four chromatographic systems, with either a single or two in‐series Kinetex™ C₁₈ columns, either packed with 2.6 or 1.7 μm particles, is presented for the targeted analysis of a standard mixture and the untargeted analysis of a strawberry extract. An ultra‐high performance chromatographic system coupled via an electrospray source to a hybrid quadrupole‐Orbitrap mass spectrometer was used. From the extensive comparison, a surprising result was obtained, namely, that the system identifying the largest number of features was the one with two in‐series connected columns with the larger particle size. The inconsistency among the theoretical assumptions and the applicative findings points out the importance of an extensive chromatographic evaluation for the comprehensive untargeted profiling of complex real samples.     

Multidimensional capillary array liquid chromatography and matrix-assisted laser desorption/ionization tandem mass spectrometry for high-throughput proteomic analysis

A two-dimensional capillary array liquid chromatography system coupled with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) was developed for high-throughput comprehensive proteomic analysis, in which one strong cation-exchange (SCX) capillary chromatographic column was used as the first separation dimension and 10 parallel reversed-phase liquid chromatographic (RPLC) capillary columns were used as the second separation dimension. A novel multi-channel interface was designed and fabricated for on-line coupling of the SCX to RPLC column array system. Besides the high resolution based on the combination of SCX and RPLC separation, the developed new system provided the most rapid two-dimensional liquid chromatography (2D-LC) separation. Ten three-way micro-splitter valves used as stop-and-flow switches in transferring SCX fractions onto RPLC columns. In addition, the three-way valves also acted as mixing chambers of RPLC effluent with matrix. The system enables on-line mixing of the LC array effluents with matrix solution during the elution and directly depositing the analyte/matrix mixtures on MALDI plates from the tenplexed channels in parallel through an array of capillary tips. With the novel system, thousands of peptides were well separated and deposited on MALDI plates only in 150min for a complex proteome sample. Compared with common 2D-LC system, the parallel 2D-LC system showed about 10-times faster analytical procedure. In combination with a high throughput tandem time of flight mass spectrometry, the system was proven to be very effective for proteome analysis by analyzing a complicated sample, soluble proteins extracted from a liver cancer tissue, in which over 1202 proteins were identified.     

The identification of 3,4-MDMA from its mass equivalent isomers and isobaric substances using fast LC-ESI-MS-MS

3,4-methylenedioxymethamphetamine (3,4-MDMA, "Ecstacy") and its 17 isomers and isobaric substances are studied using liquid chromatography (LC)-positive electrospray ionization-mass spectrometry (MS). 3,4-MDMA is a controlled substance, whereas in many countries the other studied isobaric compounds are not. A method for confirmation of the presence of 3,4-MDMA in drug seizures is developed and validated. Using single MS, the compounds produce an intense protonated molecule and some characteristic fragments; but tandem MS (MS-MS) is applied to enhance specificity. The MS-MS fragmentation is studied in order to distinguish 3,4-MDMA from the other 17 related compounds. However, the MS-MS spectra of 3,4-MDMA and six related compounds are very similar. Therefore, the LC-MS-MS method is developed for the unambiguous identification of 3,4-MDMA. The use of a monolithic column allows for 5-min gradient runs. This qualitative method is tested with 49 Ecstacy samples seized by the police. All results are congruent with the ones obtained with other methods.     

HPLC-electrospray ionization-MS-MS analysis of Cephalotaxus harringtonia leaves and enhancement of the extraction efficiency of alkaloids therein by SFE

A high-performance liquid chromatographic method is developed for use in the electrospray mass spectrometric (MS)-MS analysis of alkaloids contained in Cephalotaxus harringtonia leaves. Nine alkaloids having ester groups can be separated and detected with good sensitivity. The MS and MS-MS spectra obtained provides information on their chemical structures. Supercritical fluid extraction is also applied in order to improve the extraction efficiency of Cephalotaxus alkaloids such as cephalotaxine, harringtonine, homoharringtonine, and isoharringtonine. When carbon dioxide-methanol-water (80:18:2, v/v) is used the extraction yield is found to be higher than that using the other supercritical solvents evaluated and conventional organic solvent extraction.     

A comparative study of large volume injection techniques for microbore columns in HPLC

Summary This paper focuses attention on the potentially larger signal-to-noise ratios produced by microbore columns in comparison with conventional columns. The increased chromatographic signals by the application of microbore columns are due to the lower chromatographic dilution of elution profiles which are proportional to the square of the column inner radius. Generally less than 1μl sample should be injected into microbore systems to obtain the full benefit of the column performance. However, since more sample can be loaded on conventional columns compared to microbore columns the advantage of improved signal-to-noise ratio can only be realised in situations where very little sample is available. To inject more than 1μl sample, at the same time avoiding extra band-broadening effects, suitable injection techniques must be available. In this study three injection methods for microbore systems that meet this condition, are studied and compared.     

Assessing a novel microfluidic interface for shotgun proteome analyses

Microfluidic interfaces coupled to ESI mass spectrometers hold great potential for proteomics as they have been shown to augment the overall sensitivity of measurements and require only a minimum of operator manipulations as compared to conventional nano-LC interfaces. Here, we evaluated a new type of HPLC-Chips holding larger enrichment columns (thus an increased sample loading capacity) for gel-free proteome studies. A tryptic digest of a human T-cell proteome was fractionated by strong cation exchange chromatography and selected fractions were analyzed by MS/MS on an IT mass spectrometer using both the new HPLC-Chip as well as a conventional nano-LC-MS/MS interface. Our results indicate that the HPLC-Chip is capable of handling very complex peptide mixtures and, in fact, leads to the identification of more peptides and proteins as compared to when a conventional interface was used. The HPLC-Chip preferentially produced doubly charged tryptic peptides. We further show that MS/MS spectra of doubly charged tryptic peptide ions are more readily identified by MASCOT as compared to those from triply charged precursors and thus argue that besides the improved chromatographic conditions provided by the HPLC-Chip, its peptide charging profile might be a secondary factor leading to an increased proteome coverage.     

Separation of mixtures of solvents with wide bore capillary columns connected in series

The gas chromatographic separation of the components of a complex mixture of industrial solvents, not possible on a single stationary phase owing to coelution of some of the compounds, has been achieved on two serially connected wide bore capillary columns of different polarity. The analysis of a mixture of twenty five compounds was optimized using the serial gas chromatography theory of Purnell and Williams. The capacity factors of sample components measured with the two columns coupled in series were found to agree with those predicted by theory, regardless of the order in which they were connected.     

Evaluation of quadrupole time-of-flight tandem mass spectrometry and ion-trap multiple-stage mass spectrometry for the differentiation of C-glycosidic flavonoid isomers

LC-MS-MS is becoming a very important tool for the on-line identification of natural products in crude plant extracts. For an efficient use of this technique in the dereplication of natural products, a careful study of the parameters used to generate informative MS-MS spectra is needed. In this paper, the collision-induced dissociation (CID) MS-MS spectra of ubiquitous C-glycosidic flavonoids have been systematically studied using hybrid quadrupole time-of-flight and ion-trap (IT) mass analysers under various CID energy conditions. Efficient differentiation of flavonoid C-glycoside isomers was possible, based on the comparison of CID-MS-MS spectra of particular C-glycoside unit fragments. Striking differences between 6-C and 8-C flavonoid glycosides were especially observed in the product ion spectra of their 0.2X+ fragments ([M+H-120]+). Some guidelines for the on-line characterisation of C-glycosidic flavonoids by LC-MS-MS or LC-multiple-stage MS are given.     

Application of capillary gas chromatography to automated on-line quantitative analysis of solvent vapors

Solvent vapors in air may be measured with capillary gas chromatographic columns. By using large diameter columns and sample loops of approximately the same internal diameter, the column may be connected directly to the gas sampling valve. This approach eliminates the use of a splitter or cryogenic trapping and allows low levels to be measured. By operating the column at high velocity, column efficiency is sacrificed for increase in speed.     

Design and development of a two‐dimensional system based on hydrophilic and reversed‐phase liquid chromatography with on‐line sample treatment for the simultaneous separation of excreted xenobiotics and endogenous metabolites in urine

In the present work we describe a two‐dimensional liquid chromatographic system (2D‐LC) with detection by mass spectrometry (MS) for the simultaneous separation of endogenous metabolites of clinical interest and excreted xenobiotics deriving from exposure to toxic compounds. The 2D‐LC system involves two orthogonal chromatographic modes, hydrophilic interaction liquid chromatography (HILIC) to separate polar endogenous metabolites and reversed‐phase (RP) chromatography to separate excreted xenobiotics of low and intermediate polarity. Additionally, the present proposal has the novelty of incorporating an on‐line sample treatment based on the use of restricted access materials (RAMs), which permits the direct injection of urine samples into the system. The work is focused on the instrumental coupling, studying all possible options and attempting to circumvent the problems of solvent incompatibility between the RAM device and the two chromatographic columns, HILIC and RP. The instrumental configuration developed, RAM‐HILIC‐RPLC‐MS/MS, allows the simultaneous assessment of urinary metabolites of clinical interest and excreted compounds derived from exposure to toxic agents with minimal sample manipulation. Thus, it may be of interest in areas such as occupational and environmental toxicology in order to explore the possible relationship between the two types of compounds. Copyright © 2015 John Wiley & Sons, Ltd.     

Comparison of two-stage retention index monitoring capillary gas chromatography and selected ion monitoring capillary gas chromatography – mass spectrometry as analytical tools

Two-stage capillary GC with two-stage retention index monitoring is an efficient analytical technique which can be used for detection and determination of small amounts of volatile compounds in complex mixtures of hundreds or thousands of other compounds. The system employs two capillary columns, coated with different stationary phases, connected on-line with the aid of a micro valve; the first column acts as a pre-separating unit from which unresolved fractions of interest are cut (transferred) into another column for final, interference-free separation of the compounds to be determined. This technique has been compared with selected ion monitoring capillary GC-MS using a hydrocarbon mixture as a test sample for comparing resolution, repeatability, and the practical usefulness of the techniques. Results indicate that two-stage capillary GC is very useful for mixtures containing compounds which produce mostly non-specific ions in the MS ion source whereas compounds producing specific ions can be easily analyzed by capillary GC – single ion monitoring MS even if they are not perfectly separated by a single capillary column.     

Comparison of three types of mass spectrometers for HPLC/MS analysis of perfluoroalkylated substances and fluorotelomer alcohols

The performance of three different types of mass spectrometers (MS) coupled to high performance liquid chromatography (HPLC) was compared for trace analysis of perfluoroalkylated substances (PFAS) and fluorotelomer alcohols (FTOHs). Ion trap MS in the full scan and product ion MS2 mode, time-of-flight (TOF) high resolution MS and quadrupole MS in the selected ion mode as well as triple quadrupole tandem MS were tested. Electrospray ionisation in the negative ion mode [ESI-] was best suited for all instruments and compounds. PFAS could only be separated by a buffered mobile phase, but the presence of buffer suppressed the ionisation of FTOHs. Therefore, two independent chromatographic methods were developed for the two compound classes. Mass spectra and product ion spectra obtained by in-source and collision induced dissociation fragmentation are discussed including ion adduct formation. Product ion yields of PFAS were only in the range of 0.3 to 12%, independent from the applied MS instrument. Ion trap MS2 gave product ion yields of 20 to 62% for FTOHs, whereas only 4.1 to 5.8% were obtained by triple quadrupole tandem MS. Ion trap MS was best suited for qualitative analysis and structure elucidation of branched isomeric structures of PFAS. Providing typical detection limits of 5 ng injected in MS2 mode, it was not sensitive enough for selective trace amount quantification. TOF high resolution MS was the only technique combining high selectivity and excellent sensitivity for PFAS analysis (detection limits of 2 to 10 pg), but lacked the possibility of MS-MS. Triple quadrupole tandem MS was the method of choice for quantification of FTOHs with detection limits in the low pg range. It is also well suited for the determination of PFAS, though its detection limits of 10 to 100 pg in tandem MS mode are about one order of magnitude higher than for TOF MS.     

Characterization of high performance glass capillary gas chromatography

Summary High resolution gas chromatography requires the highest performance characteristics of gas chromatographic systems in terms of sampling and sample handling in strumentation, columns, and data handling. This paoper describes high precision computer measurements for characterizing capillary column efficiencies which are within 75% of the theoretical limit of capillary GC. Particular emphasis is given to detailed peak shape analysis, measurement accuracy and reproducibility, and system stability. Using known instrument performance parameters, it is then possible to characterize column performance with high accuracy in a meaningful manner. It is proposed that wall-coated tubular columns be characterized in terms of their chromatographic performance by the following parameters: Trennzahl (separation number), number of theoretical plates/meter, program temperature beseline stability, acid-base ratio, and the coefficient of skewness for 1-octanol. Statistical moments (m2) and hybrid moments are used to describe capillary column chromatographic performance because they may be related to basic physico-chemical column processes. These measurements are very sensitive parameters for characterizing GC columns. Using an online computer-based data system, the limits of capillary GC are shown to be limited by the sampling and injection steps.     

Studies on azaspiracid biotoxins. I. Ultrafast high-resolution liquid chromatography/mass spectrometry separations using monolithic columns

In this study, the performance of monolithic columns was evaluated for ultrafast liquid chromatography/mass spectrometry (LC/MS) analyses and for high-resolution separations of several azaspiracid biotoxin analogs. Because of their high permeability, monolithic columns offer a number of advantages over conventional packed columns; viz., very low backpressures and relatively flat van Deemter curves at high flow rates. That is, very high flow rates can be used for ultrafast analyses or, by using longer than normal columns, high-resolution separations are possible. In a series of experiments, we varied the mobile phase flow rates between 1 and 8 mL/min, and studied their impact on chromatographic parameters such as retention time, resolution, number of plates and pressure. The chromatographic run times could be reduced to ca. 30 s without a significant change in the separation efficiency. A signal intensity comparison revealed interesting differences between atmospheric-pressure chemical ionization (APCI) and electrospray ionization (ESI) in their flow-rate dependency. An explanation with respect to the behavior as of a mass-flow or a concentration-dependent device is given in the paper. Additionally, the column length was varied between 10 and 70 cm. As a result, the number of theoretical plates increased substantially. In the example shown in the report, an increase from 13 000 plates for a 10-cm column to 80 000 for a 70-cm column is demonstrated. In addition, the potential of the monolithic columns for high-resolution LC/MS separations is shown for a complex biotoxin mixture, which was separated on a 40-cm-long column.