No abstract

This paper describes the hyphenation of cIEF and MALDI-TOF MS via a fractionation or spotting device. After focusing in cIEF the compounds are hydrodynamically mobilized and deposited on a MALDI target plate using a sheath liquid interface, which provides the catholyte solution and the electrical ground. From previous experiments, sample conditions that resulted in a high resolution in cIEF and acceptable protein signal intensity in MS were selected [Silvertand et al., Electrophoresis, 2008, 29, 1985-1996]. Besides the mixture of test proteins, the sample solution contains 1% Pharmalyte, 0.3% hydroxyethyl cellulose and 0.1% Tween 20 and is used for both optimization as well as characterization of the cIEF-MALDI-TOF MS system. Hyphenation problems encountered are mainly due to transfer of the liquid from the needle to the MALDI target plate and are solved by choosing the proper sheath catholyte (200 mM NH4OH in 50% methanol with 0.1% Tween20). MS electropherograms were reconstructed by plotting the intensities of the m/z values corresponding to the proteins versus migration time (related to spot number). Reproducibility, peak width and signal intensity for different focusing and spotting (fractionation) times were calculated using these reconstructed MS electropherograms as well as the UV electropherograms. The best results were obtained with focusing time of 75 min (no under- or overfocusing) and a spotting time of 5 s (highest protein signal intensity in MS). The applicability of the system is demonstrated by the analysis of a biopharmaceutical (glucagon) and its deamidation product.

Fludarabine and cyclophosphamide are anticancer agents mainly used in the treatment of hematologic malignancies. We have developed and validated an assay using high-performance liquid chromatography (HPLC) coupled with electrospray ionization tandem mass spectrometry for the quantification of fludarabine in combination with cyclophosphamide in human heparin and human EDTA plasma. Sample pre-treatment consisted of a protein precipitation with cold acetonitrile (-20 degrees C) using 250 microL of plasma. Separation was performed on an Extend C18 column (150 x 2.1 mm i.d.; 5 microm) with a stepwise gradient using 1 mM ammonia solution and acetonitrile at a flow rate of 400 microL/min. The analytical run time was 12 min. The triple quadrupole mass spectrometer was operated in the positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over a concentration range of 1 to 100 ng/mL for fludarabine and cyclophosphamide in human heparin and human EDTA plasma. The coefficients of variation were <13.9% for inter- and intra-day precisions. Mean accuracies were also within the designated limits (+/-15%). The analytes were stable in plasma, processed extracts and in stock solution under all relevant conditions.

Low repeatability of migration time, peak area, and linearity (pI vs. mobilization time) is a problem often encountered in capillary IEF (cIEF) and is mainly caused by protein precipitation and protein-wall interactions. In order to study the influence of these phenomena, the effect of different classes of additives on repeatability of migration time, peak area and linearity of a mixture of seven model proteins has been investigated. Moreover, the influence of these additives on protein signal suppression in MALDI-TOF MS has been studied. The optimal ampholyte blend (stabilizes pH gradient) to be used depends on the selected UV detection wavelength. All tested ampholyte blends show a significant and comparable signal suppression in MS. The best detergent (to prevent precipitation and wall interaction) should be determined for each sample individually, but generally polyethylene oxide and zwitterionic detergents show good repeatability for migration time (RSD <4.5%) and peak area (majority <10%). The RSD of R(2) is <1.3% for the hydrophilic protein mixture. However, these components cause severe signal suppression in MS. Therefore glucoside detergents should preferably be used for MS coupling. Viscosity-increasing agents (for hydrodynamic wall coating and to minimize diffusion) in particular cellulose derivatives, give good repeatability for migration times (RSD <4.5% at lower concentrations), peak area (except for high concentration methylcellulose and hydroxyethylcellulose all within 7.5%), and correlation (pI vs. migration time), but severe signal suppression is observed in MALDI-TOF MS. Overall, cIEF repeatability and linearity can significantly be improved by adding the appropriate components. However, when the system is coupled to a MALDI-TOF MS, compromises have to be made between high repeatability and linearity on one hand and MS signal intensity on the other.