A proof-of-concept study is presented on the use of comprehensive two-dimensional liquid chromatography mass spectrometry (LC × LC-MS) for the separation of intact protein mixtures using a different mobile phase pH in each dimension. This system utilizes mass spectrometry (MS) friendly pH modifiers for the online coupling of high pH reversed phase liquid chromatography (HPH-RPLC) in the first dimension ( 1 D) followed by low pH reversed phase liquid chromatography (LPH-RPLC) in the second dimension ( 2 D).Owing to the ionic nature of proteins, the use of a different mobile phase pH was successful to provide altered selectivity between the two dimensions, even for closely related protein variants, such as bovine cytochrome c and equine cytochrome c, which differ by only three amino acids. Subminute gradient separation of proteins in the second dimension was successful to minimize analysis time, while maintaining high peak capacity. Unlike peptides, the elution order of studied proteins did not follow their isoelectric points, where acidic proteins would be expected to be more retained at low pH (and basic proteins at high pH). The steep elution isotherms (on−off retention mechanism) of proteins and the very steep gradients utilized in the second-dimension column succeeded in overcoming pH and organic solvent content mismatch. The utility of the system was demonstrated with a mixture of protein standards and an Escherichia coli protein mixture.

Accurate and specific analysis of target molecules in complex biological matrices remains a significant challenge, especially when ultra-trace detection limits are required. Liquid chromatography with mass spectrometry is often the method of choice for bioanalysis. Conventional sample preparation and clean-up methods prior to the analysis of biological fluids such as liquid-liquid extraction, solid-phase extraction, or protein precipitation are time-consuming, tedious, and can negatively affect target recovery and detection sensitivity. An alternative or complementary strategy is the use of an off-line or on-line in situ derivatization technique. In situ derivatization can be incorporated to directly derivatize target analytes in their native biological matrices, without any prior sample clean-up methods, to substitute or even enhance the extraction and preconcentration efficiency of these traditional sample preparation methods. Designed appropriately, it can reduce the number of sample preparation steps necessary prior to analysis. Moreover, in situ derivatization can be used to enhance the performance of the developed liquid chromatography with mass spectrometry-based bioanalysis methods regarding stability, chromatographic separation, selectivity, and ionization efficiency. This review presents an overview of the commonly used in situ derivatization techniques coupled to liquid chromatography with mass spectrometry-based bioanalysis to guide and to stimulate future research.

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Rationale: Different supercharging agents were tested to study their effect on the intensity and charge state distributions of high-molecular-weight intact proteins.The goal of this work was to increase chargeability and ionization efficiency for proteins ranging from 66 to 150 kDa, to enable subsequent optimization of multiple reaction monitoring (MRM) mode transitions with a triple quadrupole mass spectrometer for potential top-down quantitative analysis. Methods:Supercharging agents, such as meta-nitrobenzyl alcohol (m-NBA), dimethylsulfoxide, trifluoroethanol (TFE), and sulfolane were tested in different concentrations in 50/50 acetonitrile/water with 0.5% formic acid to examine the electrospray ionization response for three model proteins: bovine serum albumin (66 kDa), holo-transferrin (78 kDa), and immunoglobulin G (150 kDa). The settings of ionization source temperature and mobile phase flow rate were also examined.MRM transitions were developed for a wide range of precursor ions for each protein, and limits of detection were determined for the proteins in the presence of favorable additive combinations.Results: For most of the proteins, m-NBA (1%) and TFE (5%) worked most effectively, both to shift the charge state and increase intensity. This is the first report of the use of TFE as an effective agent for both increasing protein chargeability and ionization response. TFE increased ionization efficiency between 3-and 14-fold for the model proteins studied. Increases in both source temperature and flow rate reduced the magnitude of the average charge state observed.The MRM transitions of six to eight different precursor ions of the proteins were optimized and limits of detection in the nanogram quantity on column were determined. Conclusions:The feasibility for top-down quantitative analysis of high-molecularweight proteins with a triple quadrupole mass spectrometer was demonstrated.Further, additives such as TFE can be highly beneficial for increased chargeability and response of the proteins.

Three sensitive methods were developed for simultaneous determination of Ezetimibe (EZB) and Atorvastatin calcium (ATVC) in binary mixtures. First derivative (D1) spectrophotometry was employed for simultaneous determination of EZB (223.8 nm) and ATVC (233.0 nm) with a mean percentage recovery of 100.23 ± 1.62 and 99.58 ± 0.84, respectively. Linearity ranges were 10.00–30.00 μg mL−1 and 10.00–35.00 μg mL−1, respectively. Isosbestic point (IS) spectrophotometry, in conjunction with second derivative (D2) spectrophotometry was employed for analysis of the same mixture. Total concentration was determined at IS, 224.6 nm and 238.6 nm over a concentration range of 10.00–35.00 μg mL−1 and 5.00–30.00 μg mL−1, respectively. ATVC concentration was determined using D2 at 313.0 nm (10.00–35.00 μg mL−1) with a mean recovery percentage of 99.72 ± 1.36, while EZB was determined mathematically at 224.6 nm (99.75 ± 1.43) and 238.6 nm (99.80 ± 0.95). TLC-densitometry was employed for the determination of the same mixture; 0.10–0.60 μg band−1 for both drugs. Separation was carried out on silica gel plates using diethyl ether–ethyl acetate (7:3 v/v). EZB and ATVC were resolved with Rf values of 0.78 and 0.13. Determination was carried out at 254.0 nm with a mean percentage recovery of 99.77 ± 1.30 and 99.86 ± 0.97, respectively. Methods were validated according to ICH guidelines and successfully applied for analysis of bulk powder and pharmaceutical formulations. Results were statistically compared to a reported method and no significant difference was noticed regarding accuracy and precision.