RESUMO
We present a simple hardware design which reduces run time of gradient-based LC/MS applications and improves system equilibration. Our approach does not sacrifice efficiency of chromatographic separation, and does not affect analyte retention time and therefore does not require revalidation. Our technical design is based on a six-port/two-position switching valve and flow splitter installed prior to the LC column. This design minimizes time delays caused by the high-dwell volume of some LC pumps. Implementation of short-term (40-55 s) low-ratio (1:10) flow splitting reduced delay times by over four-fold in our application. This approach allowed hardware-associated time delays to be minimized. Alternative plumbing suggestions are also discussed.
Assuntos
Cromatografia Líquida/instrumentação , Cromatografia Líquida/métodos , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Solventes , Fatores de TempoRESUMO
Solvent flow, generated by HPLC pumps is consistent and accurate. This statement, while true for single column (one dimensional) liquid chromatography applications, may not apply to column switching applications. Connection of pumps and/or columns to one flow path may cause substantial pressure changes. Immediate post valve switch pressure differences between pumps can cause backflow where the mobile phase stored at higher pressure will temporary flow into the lower pressure area. A more common side effect of column switching is flow inconsistency during pump pressurization. For the duration of pump pressurization, liquid flow through the column will be smaller than expected since the HPLC column acts like a flow restrictor.
Assuntos
Cromatografia Líquida de Alta Pressão/instrumentação , Desenho de Equipamento , PressãoRESUMO
We investigated the impact of one dimension (single reverse phase (RP) column) and two dimension (two different RP columns) chromatographic methods on SIM (MS) and multiple reaction monitoring (MRM; MS/MS) performance from human plasma. We find that MRM analysis is clearly preferable for 1-D applications; however, implementation of SIM detection in conjunction with 2-D separation technique resulted in an over 60-fold increase in analyte peak area and improved S/N compared to MRM for our analyte, human C-peptide. Implementation of a 2-D RP-RP technique with SIM detection is capable of eliminating matrix effects and greatly increases signal response and data quality. For two large peptides in complex biological samples, we found that a 2-D approach performed better than high quality sample preparation together with 1-D chromatography and MRM, even on a high-end mass spectrometer.
Assuntos
Peptídeos/análise , Peptídeos/química , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodos , Biomarcadores/análise , Biomarcadores/química , Cromatografia Líquida/instrumentação , Cromatografia Líquida/métodos , Glucagon/química , Humanos , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Peptídeo C , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Peptídeo C/química , Peptídeo C/urina , Calibragem , Diabetes Mellitus/metabolismo , Humanos , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
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.