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Matrix effects are probably the Achilles heel of most quantitative liquid chromatography mass spectrometry (LC-MS) methods based on electrospray ionization. This work reports the evaluation of matrix effects in challenging matrices such as food extracts, human urine or wastewater at different dilution factors using nanoflow liquid chromatography-high resolution mass spectrometry (LC-MS). For this purpose, a suite of representative low-molecular weight compounds such as pesticides, drugs of abuse, sport drugs or environmental contaminants were selected. The approach is based on the use of reversed-phase C18 nano columns furnished with an integrated emitter tip. The nanoflow LC system was combined with full-scan high resolution mass spectrometry using a HRMS (orbitrap) instrument operated at a resolution of 70000. The sensitivity achieved with this configuration enables the implementation of high dilution factors (e.g. 1:20, 1:50 or beyond). When combining nanoflow LC-MS analysis with such high dilution factors (e.g. 1:50), signal suppression was negligible in most cases, so that matrix-matched standards may eventually be skipped, simplifying laboratory workflows by using external calibration in demanding applications such as drug analysis in urine, environmental contaminants in wastewater or pesticide testing in food, thus, eliminating the need for standard addition, matrix-matched calibration or isotopically-labelled standards.

Dynamics of the purification process of a CHO derived monoclonal antibody by ion exchange chromatography (IEC), hydrophobic interaction chromatography (HIC) and their integration has been investigated. To quantify the adsorption behavior of the target protein (IgG1) and impurities contained in the supernatant, their elution course on IEC and HIC columns has been analyzed versus pH and/or the salt concentration in the mobile phase. A short-cut method has been proposed for mathematical modeling and determining underlying kinetic and thermodynamic parameters. The accuracy of the model predictions has been verified by comparing the simulated and experimental band profiles recorded in both chromatographic processes. After verification, the model was used to optimize operating conditions for the column loading and chromatographic elution in the integrated process IEC/HIC. Two alternative loading techniques based on the upstream and downstream feed dilution were taken into account in the optimization routine. In the first one the feed stream was diluted with the loading buffer prior to the column loading, while in the latter one the feed dilution was realized inside the column using the multiple-injection technique. It was shown that the downstream dilution allowed significant reduction of the contact time between the protein and the loading buffer.

A capillary electrophoresis system for the simultaneous determination of cations in honey samples has been developed. The complete separation and quantification of K+, Ca2+, Na+, Mg2+, Mn2+, Ni2+ and Li+, which represent more than 99% of the total content of cations in honey, can be achieved in 4 min with only a dilution and filtration of the honey sample. Electrolyte solution was composed by 10 mM imidazole as the carrier buffer and background absorbance provider and acetic acid as the complexing agent (pH 3.60). The running voltage was + 25 kV at 25 degree C. Indirect UV detection was achieved at 185 nm. Under the optimum conditions the detection limits ranged from 0.02 to 48.2 mg/kg and the quantification limits have ranged from 0.41 to 48.7 mg/kg. Precision data in honey samples analysed have shown repeatability and reproducibility RSD (%) lower than 2.84 and 6.62%, respectively. Recoveries of cations in honey samples analysed have ranged from 88.5 to 101.8%. These cations were identified by their relative migration times with regard to Ba2+ migration time used as reference standard and they were quantified by using an external standard calibration. Twenty-five honey samples were analysed to test the proposed method. Mean contents of 1.22 x 10(3), 93, 85, 54, 11, 1.9 and 2.3 mg/kg were found, respectively, for K+, Ca2+, Na+, Mg2+, Mn2+, Ni2+ and Li+ cations in analysed honeys. These results were similar than the obtained by other authors.

A HPLC method with automated column switching was developed and validated for the determination of Ro 63-1908 in rat and cynomolgus monkey plasma. Human plasma was used for calibration and was also included in the validation process. Ro 63-1908 belongs to a class of neuroprotective N-methyl- d -aspartate (NMDA) receptor blockers which were in development for the treatment of stroke and traumatic brain injury. The method involves deproteinisation of plasma samples with ethanol and direct injection of the supernatant (1.4 ml) into the HPLC column-switching system. To prevent a breakthrough of the analyte and the internal standard on the precolumn (Purospher RP-18, 75×4 mm) due to the high ethanol content, the injection solution was diluted, on-line, using an additional pump and a T-piece. 1% ammonium acetate–ethanol (100:2, v/v) was used as mobile phase for injection, as well as for on-line dilution, resulting in pre-concentration of the analyte and the internal standard on the precolumn. As Purospher RP-18 is a non-endcapped stationary phase with a special selectivity for amines, the analyte and the internal standard could then be selectively eluted with 30% acetonitrile (without any buffer in the mobile phase) and transferred to the analytical column [consisting of two coupled columns (125+250×4 mm) packed with Superspher 60 RP-select B], where they were separated by gradient elution and detected by fluorescence detection. Compared to the use of a 125 mm long precolumn and dilution of the supernatant with ammonium acetate prior to injection, the 75 mm precolumn and the on-line dilution procedure allowed about one third shorter run times (21 min) and, therefore, a higher sample throughput. The limit of quantification was 1 ng/ml using 0.4 ml plasma. The method was applied to more than 670 plasma samples from pharmacokinetic and toxicokinetic studies and is also suitable for other matrices and NMDA receptor blockers.

The potential of ion chromatography to serve as a new reference method principle for the determination of total sodium and potassium in human serum was investigated. Sample pretreatment consisted of acidic dilution and filtration and detection was based on conductivity. Methods for the separate and simultaneous determination of both analytes were investigated. Further, the influence of calibration (using either a single-point calibration or a standard curve) on method imprecision, inaccuracy and analysis time was examined. The best method performance was achieved by separate analysis using single-point calibration with bracketing analysis scheme. For this variant, the mean total coefficient of variation for sodium and potassium was 1.0% and the mean method bias was −0.2% for sodium and +0.2% for potassium, as determined with three control materials from the National Institute of Standards and Technology. Our results are comparable to those of reference methods based on flame atomic emission spectrometry. Therefore, we consider ion chromatography as a valuable reference methodology for the determination of total sodium and potassium in human serum.

This paper describes a direct determination of benomyl and carbendazim in a single analytical run and their confirmation by absorption spectra using diode array detection. A simple technique has been developed for preparation of benomyl calibration standard. Benomyl was dissolved in chloroform prior to a high dilution in water for HPLC. Detection limits, precision, and accuracy of data are presented. The method is simple, rapid, accurate, and precise.

Results are presented from a study in which a continuously changed gaseous sample was monitored by multiplex gas chromatography (MGC), using the exponential dilution (ED) technique of Ritter and Adams (1976) to change the composition and concentration of a gaseous mixture in such a way as to imitate changes in the atmospheric gases sampled by a descending aircraft. A calibration of the MGC system was performed with four different rates of sample dilution, and the errors resulting from various degrees of change in the sample concentration were determined.

The amount of sample entering a capillary column during split injection increases considerably if the major component (e.g., the solvent) recondenses in the column inlet. Owing to the reduction in volume during the recondensation, additional sample vapour is sucked into the column. The resulting true splitting ratiomay deviate from the pre-set ratio by a factor exceeding 30. Recondensation occurs at column temperatures below the boiling point of the major component (usually the solvent). It is favoured by a short distance between the tip of the syringe needle and the column entrance, large sample volumes, narrow glass inserts in the injector and other factor that limit the dilution of the sample vapour with carrier gas prior to the splitting process. The deviation of the true from the pre-set splitting ratio is an important source of error in quantitaion based on the external standard method because this deviation may depend sensitively on critical parameters. It may cause high standard deviations, and also the true splitting ratio may be different for, e.g., the calibration mixture and the sample, creating systematic errors that are difficult to detect.

A method for the preparation of gaseous calibration mixtures with defined contents of standard substances is described. An accurately controlled stream of gas (e.g., nitrogen) is drawn at a low flow-rate through a thermostated container filled with a chromatographic support impregnated with the standard substance, thus generating a continuous stream of saturated vapour of the substance. This stream is diluted in a mixing chamber with a large stream of pure gas, and a small fraction of the dilute mixture, separated by means of a splitter, is again diluted with a large stream of pure gas in another mixing chamber. In this two-stage dilution procedure it is possible to prepare mixture containing standard substances at parts per billion concentrations with an error of about 4%. Mixtures containing several standards are obtained through use of a separate saturator for each standard; the concentration ratio of two standard substances can be set to within the range 1:1 to 1:106.