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A reversed-phase high-performance liquid chromatography (HPLC) separation approach has been combined with ion mobility/time-of-flight (TOF) mass spectrometry in order to characterize a combinatorial peptide library designed to contain 4000 peptides of the general form NH2-Xxx-Xxx-Xxx-CO2H, NH2-Ala-Xxx-Xxx-Xxx-CO2H, NH2- Ser-Ala-Xxx-Xxx-Xxx-CO2H and NH2-Leu-Ser-Ala-Xxx-Xxx- Xxx-CO2H (where Xxx represents a randomization over 10 different amino acids: Ala, Arg, Asp, Glu, Gly, Leu, Lys, Phe, Ser, and Val). Addition of the gas-phase mobility separation between the HPLC separation and TOF measurement dimensions makes it possible to resolve many peptide isomers that have identical retention times (and masses).

The analysis of weakly basic drugs such as salmeterol xinafoate (SX) by reverse-phase liquid chromatography remains a problem, particularly when present in combination with other drugs such as steroids and weak acids. This study describes the validation of an assay for a weakly basic drug, salmeterol (SB), its weakly acidic counter-ion, 1-hydroxy-2-naphthoic acid (XA), and the neutral glucocorticoid, fluticasone propionate (FP) using a second-generation silica stationary phase (Inertsil ODS-2). The assay utilized an Inertsil ODS-2 base-deactivated 250 mm x 4.6mm, 5 microm HPLC column, with 75:25 methanol:0.6% aqueous ammonium acetate as the mobile phase. Under these near neutral conditions, SB demonstrated a good peak shape (tailing factor=1.21+/-0.02, n=85). The method provided a short analysis time: XA, t(R)=2.96 min; SB, t(R)=5.23 min and FP, t(R)=7.01 min. The assay displayed good sensitivity for both XA (LOD for SX=0.22 microgmL(-1)) and SB (LOD for SX=0.26 microgmL(-1)). The limit of detection for FP was 0.19 microgmL(-1). Neither of the drugs was found to interfere in the determination of the other and the assay accuracy (% recovery) was high (the recoveries were: 99.58+/-1.85% for XA, 99.49+/-1.88% for SB and 100.24+/-1.28% for FP). The assay reproducibility was determined with a mean coefficient of variance for the five calibration concentrations of XA=0.71+/-0.18%; SB=1.11+/-0.64% and FP=0.92+/-0.14%. Analysis of a pressurized metered dose inhaler formulation demonstrated recovery of the analytes that are within pharmacopoeial limits. It was shown that RP-HPLC was suitable for the high throughput analysis of the combination of SX and FP.

Abstract High-performance liquid chromatography (HPLC) has been combined with high-resolution ion mobility separations and time-of-flight mass spectrometry (MS) for the analysis of complex biomolecular mixtures such as those that arise upon tryptic digestion of protein mixtures. In this approach, components in a mixture are separated using reversed phase HPLC. As mixtures of peptides exit the column, they are electrosprayed into an ion mobility/time-of-flight mass spectrometer. Mixtures of ions are separated based on differences in mobilities through a buffer gas, and subsequently dispersed by differences in mass-to-charge (m/z) ratios in a mass spectrometer. The multidimensional approach is feasible because of the large differences in timescales of the HPLC (minutes), ion mobility (milliseconds), and time-of-flight (microseconds) techniques. Peak capacities for the two-dimensional liquid chromatography-ion mobility separations (LC-IMS) and three-dimensional LC-IMS-MS separations are estimated to be not, vert, similar900 to 1 200 and not, vert, similar3.7 to 4.6 × 105, respectively. The experimental apparatus and data acquisition considerations are described; data for a mixture of peptides obtained upon tryptic digestion of five proteins (albumin, bovine and pig; cytochrome c, horse; hemoglobin, dog and pig) are presented to illustrate the approach. Author Keywords: Multidimensional separations; Ion mobility spectrometry; Proteomics; Chromatography

This paper reports the first example of electrospray ion mobility spectrometry as a detection method for HPLC separation, demonstrating its potential for the quantitative and selective detection of non-volatile and non-chromophoric organic compounds. Reduced mobility constants (K0) for 21 carbohydrates, including simple sugars, sugar alcohols and amino sugars, were determined to range from 0.68 to 1.37 cm2 V−1 s−1. Minimum detectable quantities were measured from as low as 5.8·10−14 mol for Image (+)-cellobiose to as high as 8.2·10−11 mol for Image -iditol. A plot of carbohydrate mass with respect to inverse ion mobility indicated that, to a first approximation, mobility was inversely proportional to mass for most carbohydrates. However, in several cases, isomeric separation could be achieved by ion mobility spectrometry, demonstrating that ion mobility separation is based on size and shape of the molecule rather than molecular mass. Other notable exceptions to the mobility-mass relation were ß-Image -maltose and Image (+)-cellobiose, which exhibited significantly lower mobilities than expected, and maltoheptaose, which exhibited a significantly higher mobility than expected. Dimerization and multiple charges may have caused these deviations from the simple mass-mobility correlation. Parametric optimization coupled with further basic investigations of this ionization and detection method should lead to a novel approach for the analytical determination of carbohydrates.

A rapid method for profiling bacterial and cellular proteins has been developed using a combination of capillary high-performance liquid chromatography separation followed by (MALDI-MS) matrix-assisted laser desorption/ionization mass spectrometric analysis. In this method, bacteria are sonicated, the cell walls broken, and the water-soluble proteins precipitated for analysis. The proteins are separated by capillary liquid chromatography and detected on-line by a UV absorption detector. The eluents are then collected for off-line analysis by MALDI-MS. Using this method, it is demonstrated that bacteria can be discriminated based upon their protein profiles to the species level with only pmol level detection of proteins. It has also proved to be a fast and accurate means for monitoring the expression of Hsp27 in an insect cell system.