Transient isotachophoresis-Capillary zone electrophoresis-Mass spectrometry method with off-line microscale solid phase extraction pretreatment for quantitation of intact low molecular mass proteins in various biological fluids.

Quantification of proteins is still predominantly done by the traditional bottom-up approach. Targeting of intact proteins in complex biological matrices is connected with multiple challenges during the sample pretreatment, separation, and detection step of the analytical workflow. In this work, we focused on the development of an on-line hyphenated capillary zone electrophoresis-mass spectrometry method employing off-line microscale solid-phase extraction based on hydrophilic lipophilic balance (HLB) sorbent as a sample pretreatment step for the analysis of low molecular mass intact proteins (<20 kDa) spiked in various biological fluids (human serum, plasma, urine, and saliva). A detailed optimization process involved the selection of a suitable capillary surface, background electrolyte (BGE), and comparison of two in-capillary preconcentration methods, namely transient isotachophoresis (tITP) and dynamic pH junction (DPJ), to enhance the sensitivity of the method. Optimum separation of the analytes was achieved using uncoated bare fused silica capillary employing 500 mM formic acid (pH 1.96) + 5 % (v/v) acetonitrile as BGE. tITP was utilized as an optimum preconcentration technique, achieving a 19- to 127-fold increase in the signal intensity when using 200 mM ammonium formate (adjusted to pH 4.00) as the leading electrolyte and BGE as the terminating electrolyte. Off-line microscale solid-phase extraction with various eluate treatment procedures was evaluated to ensure the compatibility of the sample pretreatment method with the selected in-capillary preconcentration, separation, and detection process. Achieved extraction recoveries of spiked proteins were in the range of 76-100 % for urine, 12-54 % for serum, 21-106 % for plasma, and 25-98 % for saliva when the eluate was evaporated and reconstituted in the solution of the leading electrolyte to achieve the tITP process. The optimum method was validated across different biological matrices, offering good linearity, accuracy, and precision, and making it suitable for proteomic studies (e.g., therapeutic drug monitoring, biomarker research) in different biological samples.

Capillary electrophoresis on-line hyphenated with mass spectrometry for analysis of insulin-like growth factor-1 in pharmaceutical preparations.

Insulin-like growth factor-1 (IGF-1) is a 70-amino acid single-chain polypeptide, which has found application in diagnostics as a biomarker of growth hormone disorders and as a therapy for growth failure in children and adolescents. Due to its strong anabolic effects, it is often abused by athletes for doping purposes. Here, we developed an on-line hyphenated method based on capillary zone electrophoresis (CZE) and triple quadrupole mass spectrometry (MS) detection with electrospray ionization (CZE-electrospray ionization source-MS [CZE-ESI-MS]) for the determination of IGF-1 in pharmaceutical matrices. We achieved a highly efficient, accurate, repeatable, sensitive, and selective analysis of IGF-1 with favorable migration times (<15 min). Optimized and validated CZE-ESI-MS method was successfully applied for the determination of IGF-1 in injectable solutions (Increlex®), and its presence was also confirmed in nutritional preparations (tablets and liquid colostrum). This is the first validated CZE-ESI-MS method for the determination of IGF-1 in pharmaceutical matrices revealing the potential of capillary electrophoresis for its use in drug quality control laboratories with benefits, such as high separation efficiency, high-speed analysis, low sample consumption, as well as environmental and cost aspects.

Capillary electrophoresis-mass spectrometry for intact protein analysis: Pharmaceutical and biomedical applications (2018-March 2023).

Capillary electrophoresis is recognized as a valued separation technique for its high separation efficiency, low sample consumption, good economic and ecological aspects, reproducibility, and complementarity to traditional liquid chromatography techniques. Capillary electrophoresis experiments are generally performed utilizing optical detection, such as ultraviolet or fluorescence detectors. However, in order to provide structural information, capillary electrophoresis hyphenated to highly sensitive and selective mass spectrometry has been developed to overcome the limitations of optical detections. Capillary electrophoresis-mass spectrometry is increasingly popular in protein analysis, including biopharmaceutical and biomedical research. It is frequently applied for the determination of physicochemical and biochemical parameters of proteins, offers excellent performance for in-depth characterizations of biopharmaceuticals at various levels of analysis, and has been also already proven as a promising tool in biomarker discovery. In this review, we focus on the possibilities and limitations of capillary electrophoresis-mass spectrometry for protein analysis at their intact level. Various capillary electrophoresis modes and capillary electrophoresis-mass spectrometry interfaces, as well as approaches to prevent protein adsorption and to enhance sample loading capacity, are discussed and the recent (2018-March 2023) developments and applications in the field of biopharmaceutical and biomedical analysis are summarized.

Solid phase extraction as sample pretreatment method for top-down quantitative analysis of low molecular weight proteins from biological samples using liquid chromatography - triple quadrupole mass spectrometry.

Targeting and quantifying intact proteins from biological samples is still a very challenging research area. Several crucial steps exist in the analytical workflow, including development of a reliable sample preparation method. Here, we developed and applied for the first time a non-immunoaffinity sample preparation method based on a generally widely available micro-elution solid phase extraction (µSPE) strategy for the extraction of multiple lower molecular weight intact proteins (<30 kDa) from various biological matrices. Omission of a time-consuming drying and reconstitution step after extraction resulted in a more simple and rapid sample preparation procedure. A model set of eleven intact proteins (molecular weights: 5.5-29 kDa; isoelectric points: 4.5-11.3) were analyzed in multiple biological fluids using reversed-phase liquid chromatography with a triple quadrupole mass spectrometer operated in multiple reaction monitoring mode. Various sample pre-treatment reagents, sorbent types, and washing and elution solvents were experimentally tested and optimized to obtain the µSPE clean-up condition for a broad mixture of intact proteins having variable physicochemical properties. 1% trifluoroacetic acid and 0.2% Triton 100-X were selected as suitable sample pre-treatment reagents for releasing protein-protein interactions in human serum/plasma and human urine, respectively. Hydrophilic lipophilic balanced µSPE sorbent was selected as a high performing stationary phase. Addition of 1% trifluoroacetic acid to all washing and elution solutions showed the most beneficial effect for the extraction recovery of the proteins. Under the optimized conditions, reproducible extraction recoveries >65% for all targeted proteins (up to 30 kDa) in human urine and >50% for most of the proteins in serum/plasma were achieved. The selected conditions were applied also for the analysis of clinical serum and urine samples to demonstrate the feasibility of the developed method to target intact proteins directly by more affordable µSPE sample preparation and triple quadrupole mass spectrometry, which could be beneficial in many application fields.