Post-column Solvent Research Articles

Determination of highly polar compounds in atmospheric aerosol particles at ultra‐trace levels using ion chromatography Orbitrap mass spectrometry

A method using ion chromatography coupled to high-resolution Orbitrap mass spectrometry was developed to quantify highly-polar organic compounds in aqueous filter extracts of atmospheric particles. In total, 43 compounds, including short-chain carboxylic acids, terpene-derived acids, organosulfates, and inorganic anions were separated within 33min by a KOH gradient. Ionization by electrospray was maximized by adding 100µL min-1 isopropanol as post-column solvent and optimizing the ion source settings. Detection limits (S/N ≥ 3) were in the range of 0.075-25μgL-1 and better than previously reported for 22 compounds. Recoveries of extraction typically range from 85 to 117%. The developed method was applied to three ambient samples, including two arctic flight samples, and one sample from Melpitz, a continental backround research site. A total of 32 different compounds were identified for all samples. From the arctic flight samples, organic tracers could be quantified for the first time with concentrations ranging from 0.1 to 17.8ngm-3 . Due to the minimal sample preparation, the beneficial figures of merit, and the broad range of accessible compounds, including very polar ones, the new method offers advantages over existing ones and enables a detailed analysis of organic marker compounds in atmospheric aerosol particles.

Recent applications of the Charged Aerosol Detector for liquid chromatography in drug quality control

High performance liquid chromatography (HPLC) methods with UV/vis detection are the most widespread analytical procedures in modern pharmaceutical applications, but reach their limitations when it comes to non-chromophore molecules. Hence, instead of using tiresome derivatization procedures, many liquid chromatography methods make use of the so-called aerosol-based universal detectors, namely the evaporative light scattering detector (ELSD), the condensation nucleation light scattering detector (CNLSD) and the charged aerosol detector (CAD). Amongst these, the CAD, being the youngest (introduced in 2005) of these three options, is often described as the most easy-to-use detector and is stated to exhibit sufficient sensitivity and good linearity of signal in a dedicated range of concentration. Therefore, this review sets its focus on the recent applications of the CAD for active pharmaceutical ingredients, excipient analysis as well as botanical applications. Alongside the post-column solvent addition techniques, the new CAD's ability to adjust the evaporation temperature and the possibility to use an integrated power function for signal linearization are reviewed as previously unavailable, new parameters for optimization.

Enhancing Sensitivity of Microflow-Based Bottom-Up Proteomics through Postcolumn Solvent Addition.

The introduction of more sensitive mass spectrometers allows researchers to adapt front-end liquid chromatography (LC) to individual needs for the analysis of complex proteomes. Where absolute sensitivity is not paramount, it is advantageous to switch from a highly sensitive nanoflow-LC setup, the de facto standard platform in mass-spectrometry (MS)-based discovery proteomics, to a more robust, high-throughput-compatible microflow or conventional-flow setup. To enhance the microflow-LC-MS electrospray process of complex proteomic samples, we tested the effects of different solvents, including 2-propanol, methanol, and acetonitrile, pure or as mixture with dimethyl sulfoxide, which were added postcolumn to the eluting sample. Postcolumn addition of organic solvents strongly enhanced the electrospray efficiency in microflow-LC-MS experiments and improved the sensitivity across the entire gradient and for early eluting peptides by up to 10-fold. Postcolumn solvent addition did not negatively affect chromatographic performance and resulted in an overall 28-36% increase in identifications at both the protein and peptide levels. The presented microflow-LC-MS workflow, including postcolumn solvent addition, can be easily adopted on any LC-MS/MS platform.

Novel zwitterionic HILIC stationary phase for the determination of ethyl glucuronide in human hair by LC-MS/MS

Some recent studies have described a shift from traditional reversed-phase to more hydrophilic LC chemistry for EtG determination in hair (hEtG). The reason relies on the poor retention of C8– and C18-based columns for polar compounds, even in presence of great amount of aqueous phase. This work presents the development, validation and application of a new hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-LC-MS/MS) method based on a novel zwitterionic stationary phase for the analysis of hEtG. The linearity was assessed in the range of 5–100 pg/mg hair, with a correlation coefficient of >0.99. The method was selective and sensitive, with a limit of detection (LOD) and limit of quantitation (LOQ) of 1.4 pg/mg and 4.5 pg/mg in hair, respectively. Suitable diagnostic sensitivity was achieved without the introduction of a sample purification step, or a post column solvent addition. The method was successfully applied to real hair samples after full validation. This method, based on a separation at neutral conditions, confirmed the optimum retention and thus selectivity for weak acids in zwitterionic HILIC columns.

Online post-column solvent assisted and direct solvent-assisted electrospray ionization for chiral analysis of propranolol enantiomers in plasma samples

An Online post-column solvent-assisted ionization (OPSAI) method was developed for enhancing the ionization of the beta-blocker propranolol utilizing normal phase LC-MS/MS. Solvent-assisted electrospray ionization (SAESI) was studied by the introduction of the assistant solvents A: 0.5% Formic acid in Isopropanolol, B: 0.5% Formic acid in Isopropanolol−Water (1:1), and C: 0.5% Formic acid in water into the electrospray ionization chamber using a spray needle. Analyte molecules can be directly ionized by the aid of the assistant solvent spray. Both methods were applied to the chiral separation of propranolol enantiomers using normal phase analysis on cellulose-based chiral column. Interestingly, both methods are easy to handle and offer a wide range of assistant solvents that can be used in order to gain the optimum ionization of the analyte molecules. The both methods considerably improved the analyte signal and the peak area greatly increased. The propranolol average signal-to-noise (S/N) ratio was enhanced from 26±1 and 42±1 to 2341±61 and 1725±29 for R-propranolol and S-propranolol, respectively, when the post-column solvent method (OPSAI) was used with isopropanol-assistant solvent (A). While in case of solvent-assisted electrospray ionization method (SAESI) signal was enhanced from 26±1 and 42±1 to 2223±72 and 2155±58 for R-propranolol and S-propranolol, respectively, with water as an assistant solvent. The limit of detection was 10ng/mL and the method was linear in the range 50−2000ng/mL. The NPLC-MS method was applied for the determination of propranolol enantiomers in human plasma after microextraction by packed C18 sorbent.

Design and evaluation of a multi-detection system composed of ultraviolet, evaporative light scattering and inductively coupled plasma mass spectrometry detection for the analysis of pharmaceuticals by liquid chromatography

Reversed-phase liquid chromatography was coupled to a multi-detection system composed of ultraviolet (UV) detection, evaporative laser scattering detection (ELSD) and inductively coupled plasma mass spectrometry (ICP-MS). By applying the principle of post-column solvent compensation, the organic modifier content was kept constant in ELSD and ICP-MS under gradient elution. Chlorine ( 35Cl), bromine ( 79Br and 81Br) and sulfur ( 34S) were monitored in several pharmaceutical compounds. The limit of quantitation (LOQ) was 80 ng/mL for chlorine (chlorpropamide) and 2 ng/mL for bromine (bromazepam). Calibration graphs were linear from 1.0 μg/mL to 100 μg/mL for chlorpropamide ( r 2 0.990) and from 10 ng/mL to 500 ng/mL for bromazepam ( r 2 0.996). The low LOQ value for bromine allows to quantify bromine in pharmaceutical samples below the 0.05% level of the active pharmaceutical ingredient.

Sensitivity enhancement of liquid chromatographic–direct chemiluminescence detection by on-line post-column solvent mediated pre-oxidative chemiluminescence

The compatibility of liquid chromatography solvents with oxidizing reagents frequently employed in direct chemiluminescence reactions is examined in this study. Various oxidizing reagents were examined for their response in hydro-organic and micellar mobile phases in both isocratic and gradient elution modes. Mild oxidants like hydrogen peroxide, periodate, cerium and hypochlorite were found to be completely compatible with common reversed phase HPLC solvents posing as no threat to the detection procedure. On the other hand, stronger oxidants like acidic permanganate were found to oxidize organic solvents towards the production of an intense light signal. Although several analytical applications can emerge from this finding, the conjunction of this system with reversed phase HPLC is impractical owing to a significant baseline increase which deteriorates the sensitivity of the analysis. A convenient solution to this problem is proposed based on the regulated on-line post-column pre-oxidation of the organic solvent (SPOC) with mild oxidants that have no influence on the final signal. The analytical utility of this new approach in the determination of organic compounds after chromatographic separation is demonstrated.

Liquid chromatography/chemical reaction interface mass spectrometry as an alternative to radioisotopes for quantitative drug metabolism studies.

Chemical reaction interface mass spectrometry (CRIMS) was coupled on-line with HPLC using a Vestec particle beam interface. A helium-assisted nebulizer provided added stability with no loss in accuracy or precision as compared to the thermospray nebulizer at flow rates of up to 1.0 mL/min using isocratic conditions. However, mass spectral response was found to be solvent-dependent for both the helium-assisted and thermospray nebulizers. Postcolumn solvent addition of methanol eliminated solvent-dependent decreases in mass spectral response. This allowed gradient HPLC elutions to be performed. Under these conditions, the flow of solvent into the particle beam interface was 2.5 mL/min, so a conventional thermospray nebulizer had to be used instead of the helium-assisted nebulizer. Experiments were conducted with the antianxiety agent buspirone in order to validate the methodology. Metabolites from in vitro incubations of [15N]/[14C]buspirone with rat liver slices were analyzed by gradient LC/CRIMS and by gradient LC/[14C] radioactivity counting. The response from LC/CRIMS analysis for individual metabolites was then compared with that obtained by LC/[14C] radioactivity counting. An excellent correlation was observed between the two methods for metabolites with quite different HPLC characteristics. Thus, gradient LC/CRIMS in combination with stable isotopes provides an alternative to using radioisotopes for carrying out drug metabolism studies.

The application of ‘Z’-shaped UV cells in open-tubular capillary column liquid chromatography

‘Z’-shaped UV cells for packed capillary columns cannot be used in open-tubular capillary column liquid chromatography (OT-LC) because of their relatively large volume. With post-column solvent make-up, a commercial ‘Z’-shaped cell (volume 100 nl) was used in our OT-LC system, resulting in 9-fold sensitivity increase against on-column UV detection and with little efficiency loss. A small volume (5 nl) ‘Z’-shaped cell allows the direct coupling of the cell to high efficiency columns. For dilute samples, 9-19-fold sensitivity enhancement could be obtained. However, column efficiency decreases with increasing signal level as a result of peak deformation.

Direct and reverse flow-through heterogeneous scintillation counting of radiolabelled amino acids using post-column solvent segmentation

A method for flow-through heterogeneous scintillation counting of 14C-labelled amino acid is described. It is based on post-column solvent segmentation of the aqueous column eluate with hexane before the eluate enters the flow cell of the radioactivity monitor. Using yttrium silicate scintillator granules of 60–80 μm diameter, counting efficiencies of 0.71( 14C) and 0.01( 3H) are obtained. Segmentation of the column eluate allows the temporary storage of the column eluate in a capillary storage loop without additional band broadening and, after the separation is completed, re-introduction of the segmented stream through the radioactivity monitor at reduced flow-rate so as to increase the sensitivity of flow-through radioactivitydetection.

Solvent-sample interactions in thermospray mass spectrometry of antineoplastic nitrogen mustards.

Conditions are reported for the optimization of thermospray mass spectrometric analysis of antineoplastic nitrogen mustard alkylating agents. In aqueous ammonium acetate mobile phase, multiple sequential solvolytic reactions occur with these highly labile compounds, and protonated molecular ions of the reaction products are observed. However, when high proportions of acetonitrile or other organic modifier are added to the mobile phase, solvolytic reactions are much reduced and abundant protonated molecular ions are detected. One exception to these observations is phosphoramide mustard, which solvolyses under all conditions attempted. A lower limit for detection of melphalan using direct injection and summing the ion current between m/z 120 and 870 is about 150 ng. Successful thermospray liquid chromatography/mass spectrometry of these compounds should be possible using high percentages of methanol in the mobile phase or increasing the organic content by post-column solvent modification.

A post column solvent trapping technique for the analysis of very volatile halocarbons

Utilisation d'un piege garni d'alumine activee entre le chromatographe et le detecteur pour l'elimination du solvant (methanol)