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Eduard Rogatsky

Publications and source records attributed to Eduard Rogatsky.

10 recordsLinked to original sources
PubMed2006

High dead volume pumps: delay time reduction by short-term low-ratio split flow in LC/MS applications.

The aim of our work was to develop a low-cost, simple and reliable solution to reduce LC/MS analysis time by compensating for limitations inherent to high dead volume standard HPLC pumps. In our approach, we utilized a temporary (sub-1 min) low ratio flow split (approximately 1:10) at 5 mL/min pump flow before the column. During this short period, 90% of the entire pump flow is delivered to waste and used for fast pump/system equilibration. Although full-time flow splitting is widely used in capillary/nano applications (usually with high split ratios in the hundreds or thousands), to our knowledge, this is the first time that short-term low-ratio flow splitting has been used in conventional LC/MS applications.

Centrifugation, Density Gradient↗
PubMed2006

Sensitive quantitative analysis of C-peptide in human plasma by 2-dimensional liquid chromatography-mass spectrometry isotope-dilution assay.

BACKGROUND: Isotope-dilution assays (IDAs) are well established for quantification of metabolites or small drug molecules in biological fluids. Because of their increased specificity, IDAs are an alternative to immunoassays for measuring C-peptide. METHODS: We evaluated a 2-dimensional liquid chromatography-mass spectrometry (2D LC/MS) IDA method. Sample preparation was by off-line solid-phase extraction, and C-peptide separation was performed on an Agilent 1100 2D LC system with a purification method based on high-pressure switching between 2 high-resolution reversed-phase columns. Because of the low fragmentation efficiency of C-peptide, multiple-reaction monitoring analysis was omitted and selective-ion monitoring mode was chosen for quantification. Native and isotope-labeled ([M+18] and [M+30]) C-peptides were monitored in the +3 state at m/z 1007.7, 1013.7, and 1017.7. RESULTS: The assay was linear (r(2) = 0.9995), with a detection limit of 300 amole (1 pg) on column. Inter- and intraday CVs for C-peptide were < or =2%. Comparison with an established polyclonal-based RIA showed high correlation (r = 0.964). Plasma concentrations of total C-peptide measured by RIA were consistently higher than by IDA LC/MS, consistent with the higher specificity of IDAs compared with immunoassays. CONCLUSIONS: The 2D LC/MS IDA approach eliminates matrix effects, enhancing assay performance and reliability, and has a detection limit 100-fold lower than any previously reported LC/MS method. Isotope-labeled C-peptide(s) can be clearly differentiated from endogenous C-peptide by the difference in m/z ratio, so that both peptides can be quantified simultaneously. The method is highly precise, robust, and applicable to pharmacokinetic detection of plasma peptides.

C-Peptide↗
PubMed2006

Direct sensitive quantitative lC/MS analysis of C-peptide from human urine by two dimensional reverse phase/reverse phase high-performance liquid chromatography.

We describe for the first time the direct quantitative analysis of human C-peptide from urine by isotope dilution assay. Implementation of 2-D reverse phase-reverse phase chromatography (2-D RP-RP) with SIM detection resulted in high assay sensitivity (LOQ= 5 pg on column), accuracy, and method ruggedness. Our experiments demonstrate the strong resolving capability of our proposed 2-D RP-RP platform which significantly reduces strong matrix effects and their resulting quantitation error over a wide range of urine concentrations. In contrast, single column methods (both SIM and multiple reaction monitoring) were found acceptable only for strongly diluted urine samples.

C-Peptide↗
PubMed2006

Two-dimensional reverse phase-reverse phase chromatography: A simple and robust platform for sensitive quantitative analysis of peptides by LC/MS. Hardware design.

We have revised current two-dimensional RP-RP approaches and developed a new robust 2-D RP-RP platform. This platform was implemented on an Agilent 1100 2-D liquid chromatography system and is based on high pressure switching between two high-resolution RP columns. An independent binary gradient was implemented for each dimension. The powerful combination of dual analytical columns with independent gradient elution achieves high analyte purity, effectively eliminates matrix effects, and maximizes MS sensitivity in Q1 SIM comparable to the sensitivity enhancements of MS/MS-based methods. Implementation of dual simultaneous gradient profiles (overlapped gradients) reduces 2-D method run-time to the scale of 1-D method run-times. This robust and sensitive approach is particularly suitable for hydrophobic peptides and small proteins and can be used as a routine standard technique for enhanced on-line peptide purification coupled with mass spectrometric detection.

C-Peptide↗
PubMed2005

Evaluation of matrix effect and chromatography efficiency: new parameters for validation of method development.

From the viewpoint of regulatory guidelines, validation of LC-UV and LC-MS based methods have the same requirements. Matrix effects are not considered for most method validations if they do not influence reproducibility or assay linearity. Since matrix effects can strongly suppress ionizaton efficiency and therefore reduce sensitivity, they must be evaluated (and discussed in the context of method development)--prior to method qualification. The severity of matrix effects is directly dependent upon chromatographic performance. We suggest that evaluation of matrix effects and LC efficiency is essential information for method assessment, optimization and transfer to other mass spectrometers, and should be a mandatory part of routine LC/MS method validation.

Chromatography, Liquid↗
PubMed2005

Sensitive LC MS quantitative analysis of carbohydrates by Cs+ attachment.

The development of a sensitive assay for the quantitative analysis of carbohydrates from human plasma using LC/MS/MS is described in this paper. After sample preparation, carbohydrates were cationized by Cs(+) after their separation by normal phase liquid chromatography on an amino based column. Cesium is capable of forming a quasi-molecular ion [M + Cs](+) with neutral carbohydrate molecules in the positive ion mode of electrospray ionization mass spectrometry. The mass spectrometer was operated in multiple reaction monitoring mode, and transitions [M + 133] --> 133 were monitored (M, carbohydrate molecular weight). The new method is robust, highly sensitive, rapid, and does not require postcolumn addition or derivatization. It is useful in clinical research for measurement of carbohydrate molecules by isotope dilution assay.

Blood Chemical Analysis↗
PubMed2005

Novel, highly robust method of carbohydrate pre-purification by two-dimensional liquid chromatography prior to liquid chromatography/mass spectrometry or gas chromatography/mass spectrometry.

We describe a novel two-dimensional liquid chromatography (2D-LC) method for fast and robust isolation and concentration of low abundant carbohydrates (sorbitol, glycerol) from biological matrices (plasma and urine). Off-line pre-purified fractions, enriched by analyte of interest, were analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS-MS). Initial 2D-LC automated sample pre-purification improved MS detection, eliminated matrix effects, and achieved high sensitivity (picogram detection limit) with a 6 min runtime and increased column lifetime. Using this method we have analyzed more than 1300 samples from biological matrices without column replacement.

Carbohydrates↗
PubMed2003

Primary folding dynamics of sperm whale apomyoglobin: core formation.

The structure, thermodynamics, and kinetics of heat-induced unfolding of sperm whale apomyoglobin core formation have been studied. The most rudimentary core is formed at pH(*) 3.0 and up to 60 mM NaCl. Steady state for ultraviolet circular dichroism and fluorescence melting studies indicate that the core in this acid-destabilized state consists of a heterogeneous composition of structures of approximately 26 residues, two-thirds of the number involved for horse heart apomyoglobin under these conditions. Fluorescence temperature-jump relaxation studies show that there is only one process involved in Trp burial. This occurs in 20 micro s for a 7 degrees jump to 52 degrees C, which is close to the limits placed by diffusion on folding reactions. However, infrared temperature jump studies monitoring native helix burial are biexponential with times of 5 micro s and 56 micro s for a similar temperature jump. Both fluorescence and infrared fast phases are energetically favorable but the slow infrared absorbance phase is highly temperature-dependent, indicating a substantial enthalpic barrier for this process. The kinetics are best understood by a multiple-pathway kinetics model. The rapid phases likely represent direct burial of one or both of the Trp residues and parts of the G- and H-helices. We attribute the slow phase to burial and subsequent rearrangement of a misformed core or to a collapse having a high energy barrier wherein both Trps are solvent-exposed.

Animals↗
PubMed2002

Acute elevation of NEFA causes hyperinsulinemia without effect on insulin secretion rate in healthy human subjects.

Increased circulating levels of nonesterified free fatty acids (NEFA) have been observed in such hyperinsulinemic states as obesity, impaired glucose tolerance, diabetes, and dyslipidemia where they have been causally linked to the development of insulin resistance and hyperinsulinemia. The concentration of NEFA in plasma is believed to have direct modifying effects on insulin secretion and clearance. It remains controversial whether acute increases in NEFA potentiate insulin secretion in human subjects. We studied the effect of an acute elevation of NEFA during lipid-heparin infusion compared to a glycerol-only control on glucose-stimulated insulin secretion and clearance during a 120-min hyperglycemic (10 mM) clamp in 7 healthy normoglucose-tolerant volunteers. The metabolic clearance rate of C-peptide (MCR(CP)) was measured in each subject during the study by simultaneous infusion of C-peptide. Insulin secretion rate (ISR) was calculated from deconvolution of C-peptide data after correction for the rate of C-peptide infusion. Clearance rate of insulin (MCR(INS)) was calculated based upon endogenous ISR. Plasma glucose (mg/dL): basal (90-115 min) 90.2 +/- 2.8 vs. 90.2 +/- 2.3; clamp (150-240 min) 180.5 +/- 2.8 vs. 180.9 +/- 1.3. Plasma insulin (pmol/L): prebasal (fasting) 29.6 +/- 10.0 vs. 29.8 +/- 10.6; basal (90-115 min) 30.1 +/- 9.2 vs. 34.5 +/- 12.1; second phase clamp (210-240 min) 127.6 +/- 18.2 vs. 182.5 +/- 17.3*. Plasma NEFA (mM): prebasal 0.47 +/- 0.08 vs. 0.52 +/- 0.09; basal 0.35 +/- 0.05 vs. 0.98 +/- 0.02*; clamp (122-240 min) 0.06 +/- 0.02 vs. 0.77 +/- 0.06*. ISR (pmol/min): prebasal 72.7 +/- 7.5 vs. 72.0 +/- 7.9; second phase clamp (210-240 min) 268.5 +/- 27.2 vs. 200.2 +/- 23.7. MCR(INS) (mL/min): prebasal 3393 +/- 488 vs. 3370 +/- 511; clamp 2284 +/- 505 vs. 1214 +/- 153* (*p < 0.05 glycerol vs. intralipid/heparin). This study demonstrates that acute NEFA elevation causes hyperinsulinemia due to a significant decrease in systemic insulin clearance without increasing rates of insulin secretion.

Adult↗