From experimental design to uncertainty estimation for the European Pharmacopoeia HPLC analysis of human insulin

In this paper the process from experimental design (e.g. ruggedness test) to uncertainty estimation is described. The uncertainty estimate was calculated for the peak area of insulin plus A21 desamido insulin resulting from an HPLC analysis of a sample of an injectable human insulin preparation, Actrapid HM 100 IU ml(-1) (Novo Nordisk A/S). The analytical method used was the European Pharmacopoeia assay. (4) An expanded uncertainty (1) of 1.8% (of the area from the HPLC analysis) at an approximately 95% confidence level was found and confirmed by a validation study. This uncertainty refers to the peak area of the analyte from a single injection of the sample. The input parameters to the uncertainty estimate were found from a factorial experimental design (e.g. ruggedness test) consisting of 9 factors applied to the HPLC analysis. The input parameters were chosen to cover the probable contributors to the variability of the measured area of the HPLC analysis, including the sample preparation, but excluding uncertainty deriving from the reference material(s).     

Quantification of phosphorylation of insulin receptor substrate-1 by HPLC-ESI-MS/MS

Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) regulates the function and subsequent insulin signaling of this protein. Human IRS-1 has 1242 amino acid residues, including 182 serines and 60 threonines. The size, complexity, and relatively low abundance of this protein in biological samples make it difficult to map and quantify phosphorylation sites by conventional means. A mass spectrometry peak area based quantification approach has been developed and applied to assess the relative abundance of IRS-1 phosphorylation in the absence or presence of stimuli. In this method, the peak area for a phosphopeptide of interest is normalized against the average of peak areas for six selected representative IRS-1 peptides that serve as endogenous internal standards. Relative quantification of each phosphopeptide is then obtained by comparing the normalized peak area ratios for untreated and treated samples. Two non-IRS-1 peptides were added to each digest for use as HPLC retention time markers and additional standards as well as references to the relative quantity of IRS-1 in different samples. This approach does not require isotopic or chemical labeling and can be applied to various cell lines and tissues. Using this method, we assessed the relative changes in the quantities of two tryptic phosphopeptides isolated from human IRS-1 expressed in L6 cells incubated in the absence or presence of insulin or tumor necrosis factor-alpha. Substantial increases of phosphorylation were observed for Thr(446) upon stimulation. In contrast, no obvious change in the level of phosphorylation was observed for Ser(1078). This mass spectrometry based strategy provides a powerful means to quantify changes in the relative phosphorylation of peptides in response to various stimuli in a complex, low-abundance protein.     

Phenomena of insulin peak fronting in size exclusion chromatography and strategies to reduce fronting

Insulin peak fronting in size exclusion chromatography (SEC) results in more than 10% yield loss in the production of insulin. The goal of this study is to understand the mechanisms of peak fronting and to develop strategies to reduce fronting and increase insulin yield. Chromatography experiments ruled out pressure surge, viscous fingering, and adsorption as the cause for peak fronting. Theoretical analysis based on a general rate model indicated that reversible dimerization is the major cause for peak fronting and reducing the dimerization equilibrium constant is the most effective method for reducing fronting. Two strategies were developed and tested to reduce the degree of dimer formation. The first strategy was to use 0.1N acetic acid as the presaturant and eluent. The second strategy was to use 0.8 or 2.8N acetic acid in 20vol.% denatured ethanol as the mobile phase. The experimental results showed that both strategies can reduce insulin peak fronting in SEC, maintain desired product purity, and increase insulin yield to higher than 98%.     

Affinity probe capillary electrophoresis of insulin using a fluorescence‐labeled recombinant Fab as an affinity probe

Affinity probe CE (APCE) separates and detects a target molecule as a complex using a fluorescence‐labeled affinity probe (AP) by CE. The electrophoretic separation of the complex ensures accurate identification of a specific signal among nonspecific ones, which often compromises the credibility of immunoassays. APCE of insulin using a recombinant Fab (rFab) as an AP was demonstrated as a model system in this report. Anti‐insulin rFab was expressed in Escherichia coli and labeled at a cysteine residue in the hinge region with a thiol‐reactive rhodamine dye. Electrophoretically pure labeled rFab was recovered from a focused band in slab‐gel IEF and used as an AP. A mixture of standard insulin and the AP with carrier ampholyte was introduced into a neutral‐polymer coated fused silica capillary (50 μm id, 120 mm long). IEF was carried out at 500 V/cm, and the capillary was scanned for laser‐induced fluorescence under focusing conditions. The insulin‐AP complex focused at pH 6.6 within 6 min along with the free AP at pH 7.6. The complex peak decayed according to the first‐order reaction kinetics with a half life of 3.8 min. A linear calibration line was obtained for standard insulin at a concentration range of 20 pM to 5 nM using the AP at 50 nM. These results demonstrate that rFab is useful for the preparation of an AP for APCE.     

Micro sequential injection: automated insulin derivatization and separation using a lab-on-valve capillary electrophoresis system

Automated sampling and fluorogenic derivatization of islet proteins (insulin, proinsulin, c-peptide) are separated and analyzed by a novel lab-on-valve capillary electrophoresis (LOV-CE) system. This fully integrated device is based on a micro sequential injection instrument that uses a lab-on-valve manifold to integrate capillary electrophoresis. The lab-on-valve manifold is used to perform all microfluidic tasks such as sampling, fluorogenic labeling, and CE capillary rejuvenation providing a very reliable system for reproducible CE separations. Fluorescence detection was coupled to an epiluminescence fluorescence microscope using a customized capillary positioning plate. This customized plate incorporated two fused-silica fiber optic probes that allow for simultaneous absorbance and fluorescence detection, extending the utility of this device. Derivatization conditions with respect to the sequence of addition, timing, injection position, and volumes were optimized through iterative series of experiments that are executed automatically by software control. Reproducibility in fluorogenic labeling was tested with repetitive injections of 3.45 mM insulin, yielding 1.3% RSD for peak area, 0.5% RSD for electromigration time, and 2.8% RSD for peak height. Fluorescence detection demonstrated a linear dynamic range of 3.43 to 6.87 microM for insulin (r2 = 0.99999), 0.39 to 1.96 pM for proinsulin (r2 = 0.99195) and 260 to 781 nM for c-peptide (r2 = 0.99983). By including hydrodynamic flushing immediately after the detection of the last analyte, the sampling frequency for islet protein analysis was increased. Finally, an in vitro insulin assay using rat pancreatic islet excretions was tested using this lab-on-valve capillary electrophoresis system.     

Cyclic voltammetry study of glucose and insulin interactions with supported lipid membrane

The effect of D-glucose and insulin on conducting properties of supported bilayer lipid membranes (s-BLM) modified by anthraquinone-2-sulphonic acid (AQS) at the presence of potassium ferricyanide was studied by means of cyclic voltammetry (CV). Both the oxidation and the reduction current peaks were found to decrease at the presence of glucose in concentration range varying from 10 to 320 mM. The influence of insulin on membrane properties is ambiguous. While the pretreatment of membrane with 20 mU l(-1) of insulin evoked slight increase of the current with unchanged course of the dependence of peak current on glucose, the decrease of conductance was observed above 10(5) mU l(-1) of insulin.     

Pressure-induced change in permeation of insulin through a polymer alloy membrane for an implantable insulin pump

Pressure-induced change in insulin permeability through a polymer alloy membrane for an implantable insulin pump was investigated. The polymer alloy membrane was composed of a segmented polyurethane (SPU) and poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-2-ethylhexyl methacrylate) (PMEH). The polymer alloy membrane had a pathway for insulin permeation because the hydrophilic PMEH became the domain structure in the polymer alloy. The functions of the SPU/PMEH alloy membrane were characterized in terms of insulin permeability and water permeability with an applied pressure, mechanical properties, and strain and volume changes under the pressured condition. The insulin permeability synchronized with the applied pressure, that is, the insulin permeability increased 3.4 times with the applied pressure (pressure-on state) of 18 kPa in comparison with no applied pressure (pressure-off state). The permeability changed reversibly without lag time between the pressure-on and -off states. The phenomenon was caused by an increase in water permeation with the applied pressure. From the observation of volume change in the insulin reservoir in the pressure-on state, the effective pressure advancing water permeation was produced by the elasticity of the polymer alloy membrane. The polymer alloy membrane had excellent mechanical properties resisting the applied pressure, as was indicated by stress–strain measurements. It was concluded that the SPU/PMEH alloy membrane had a useful function for an implantable insulin pump.     

Absorption enhancement of polypeptide drugs by cyclodextrins. I. Enhanced rectal absorption of insulin from hollow-type suppositories containing insulin and cyclodextrins in rabbits.

The absorption of insulin (from porcine pancreas) from the rectum of rabbits after the administration of hollow-type suppositories containing insulin and five kinds of cyclodextrins (CyDs) was investigated. Three types of suppositories were employed: suppository I containing insulin (approximately 26 IU/mg) and various amounts of each CyD in citric buffer solution at pH 3.0 or powder in its cavity, suppository II containing CyD without insulin, and suppository III containing insulin without CyD. Without CyD, the insulin and glucose levels in plasma were unchanged, whereas a significant increase in the plasma insulin concentration and a marked decrease in the glucose levels were found following simultaneous administration of insulin and CyDs by suppository I. The enhancing effect of CyD on rectal insulin absorption (absorption-enhancing effect) by chemically modified CyDs (heptakis(2,6-di-O-methyl)-beta-CyD (DM-beta-CyD) and 2-hydroxypropyl-beta-CyD (HP-beta-CyD)) was higher than those by natural CyDs (alpha-, beta-, and gamma-CyD). The area under the plasma concentration-time curve (AUC) and Cmax of insulin significantly decreased with the preadministration (administration of CyD 6, 24 and 48 h before rectal insulin administration) of DM-beta-CyD. The absorption-enhancing effect disappeared 24 h after preadministration. These results suggest that CyDs enhance insulin absorption from the rectum, and that attenuation of the membrane transport barrier function in the rectum recovers at a maximum of 24 h after administration of CyDs.     

Adsorption of human insulin and AspB28 insulin on a PTFE-like surface

The interactions of human insulin, Zn-free human insulin, and AspB28 insulin with a hydrophobic surface were studied by ellipsometry. All three insulin types investigated adsorbed with high affinity onto the hydrophobic surface, as the plateau of the adsorption isotherm, represented by the irreversible bound fraction, was reached at concentrations >10(-3) mg/ml. The plateau values for human insulin and Zn-free human insulin could not be distinguished with statistical significance, whereas the plateau value for AspB28 insulin was lower than those for the two others, with an adsorbed amount corresponding to a monolayer of insulin monomers. The results observed may be explained by differences in self-association patterns of the insulin types or by enhanced charge repulsion between the AspB28 analog and the negatively charged surface.     

Characterization of insulin adsorption in the presence of albumin by time-of-flight secondary ion mass spectrometry and X-ray photoelectron spectroscopy

With a view to develop an encapsulation membrane for a bioartificial pancreas, we have studied the adsorption of insulin and human serum albumin (HSA) on it. The aim of this study was to determine the possibility of insulin detection on a polycarbonate membrane surface in the presence of HSA, an abundant blood protein. The first step of the work consisted in the identification of time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS) specific signals for insulin and albumin. For this purpose, adsorption isotherms in physiological conditions (pH = 7.2, T = 37 degrees ) were established for the two proteins by looking at the SIMS intensity variations of the characteristic protein and substrate fragments when increasing the protein concentration in the solution. The CHS+ ToF-SIMS fragment and the S2p XPS peak were identified as representative insulin signals. The second step of the work consisted in performing simultaneous adsorption of the two proteins with increasing insulin concentration. We observed an increase of the insulin signal in ToF-SIMS and XPS for insulin concentration beyond 5 microg/mL. Principal component analysis (PCA) of the ToF-SIMS results permits us to obtain information about the protein layer composition. The results show that at low relative insulin concentration in solution, the mixed adsorbed layers are enriched in insulin compared to the solution.     

Enhancing effect of cyclodextrins on nasal absorption of insulin and its duration in rabbits.

The absorption of insulin (from porcine pancreas) in rabbits after the nasal administration of aqueous preparations containing insulin and five kinds of cyclodextrins (CyDs) in phosphate buffer solution at pH 7.0 was investigated. Without CyD, the insulin and glucose levels in plasma were unchanged, whereas a marked increase in the plasma levels of insulin and a decrease in glucose concentrations were observed following the simultaneous administration of insulin and CyD such as alpha- and heptakis (2,6-di-O-methyl)-beta-CyD (DM-beta-CyD). The largest enhancing effect on the nasal absorption of insulin was obtained by DM-beta-CyD. To evaluate the duration of the absorption-enhancing effect of CyDs, preadministration (administration of CyD 0.5, 6, 12 and 24 h before insulin administration) was performed. The area under plasma concentration-time curve (AUC) and Cmax of insulin significantly decreased with the preadministration of DM-beta-CyD 6, 12 and 24 h before nasal administration. The absorption-enhancing effect disappeared 24h after the preadministration. These findings demonstrate that CyDs enhance the nasal absorption of insulin, and the recovery of the membrane transport barrier function in nasal mucosa is achieved, at the latest, 24 h after the administration of CyDs.     

Phospholipid deformable vesicles for buccal delivery of insulin

To investigate the possibility of the enhancing effect of deformable vesicles on buccal delivery of insulin, two kinds of vesicles with and without the presence of sodium deoxycholate (deformable vesicles and conventional vesicles) were prepared by reverse phase evaporation methods. The liposomal entrapment efficiency was determined by column chromatography. The particle size and morphology of the vesicles were also evaluated. The hypoglycemic effects, insulin concentrations, and residual amounts of insulin deposited in the buccal membrane after buccal administration of insulin vesicles to rabbits were investigated. Compared with subcutaneous administration of insulin solution, the relative pharmacological bioavailability and the relative bioavailability of buccal administration of insulin vesicles were determined. The results showed that the entrapment efficiencies of the deformable and conventional vesicles were 18.87+/-1.78% (n=3) and 22.07+/-2.16% (n=3), respectively. The particle sizes of the deformable and conventional vesicles were 42.5+/-20.5 nm and 59.7+/-33.8 nm, respectively. There were no significant differences in appearance between the two types of vesicle. Compared with subcutaneous administration of insulin solution, the relative pharmacological bioavailability and the relative bioavailability in the insulin-deformable vesicles group were 15.59% and 19.78%, respectively, which were higher than in the conventional insulin vesicles (p<0.05), blank deformable vesicles and insulin mixture groups (p<0.05). Deformable vesicles have an enhancing effect on buccal delivery of insulin and may be a better carrier than conventional vesicles for buccal delivery of protein drugs.     

胰岛素反相制备色谱的方法开发

以胰岛素反相制备色谱方法的开发和优化为目标,通过考察色谱保留参数、峰展宽及样品流出曲线的浓度分布等色谱参数,对流动相梯度、色谱填料、载样量等色谱条件进行了优化,并建立了胰岛素制备色谱峰参数的描述方法。结果表明,所建立的方法可快速筛选出最适于胰岛素分离的色谱条件(包括流动相梯度及分离填料),即流动相中的强洗脱溶剂(有机相)需采取缓梯度窄区间的变化条件,筛选出的分离填料需具备峰向两侧展宽且展宽程度较小、样品最高浓度居中分布的特点。将方法用于实际胰岛素粗品的纯化制备,获得了杂质去除效果好、胰岛素纯度高的产品。该法为胰岛素反相色谱纯化制备方法的快速建立提供了指导,具有较强的实用价值,同时为发展大分子化合物的制备色谱方法提供了参考。     

Study of the aggregation of human insulin Langmuir monolayer

The human insulin (HI) Langmuir monolayer at the air-water interface was systematically investigated in the presence and absence of Zn(II) ions in the subphase. HI samples were dissolved in acidic (pH 2) and basic (pH 9) aqueous solutions and then spread at the air-water interface. Spectroscopic data of aqueous solutions of HI show a difference in HI conformation at different pH values. Moreover, the dynamics of the insulin protein showed a dependence on the concentration of Zn(II) ions. In the absence of Zn(II) ions in the subphase, the acidic and basic solutions showed similar behavior at the air-water interface. In the presence of Zn(II) ions in the subphase, the surface pressure-area and surface potential-area isotherms suggest that HI may aggregate at the air-water interface. It was observed that increasing the concentration of Zn(II) ions in the acidic (pH 2) aqueous solution of HI led to an increase of the area at a specific surface pressure. It was also seen that the conformation of HI in the basic (pH 9) medium had a reverse effect (decrease in the surface area) with the increase of the concentration of Zn(II) ions in solution. From the compression-decompression cycles we can conclude that the aggregated HI film at air-water interface is not stable and tends to restore a monolayer of monomers. These results were confirmed from UV-vis and fluorescence spectroscopy analysis. Infrared reflection-absorption and circular dichroism spectroscopy techniques were used to determine the secondary structure and orientation changes of HI by zinc ions. Generally, the aggregation process leads to a conformation change from α-helix to β-strand and β-turn, and at the air-water interface, the aggregation process was likewise seen to induce specific orientations for HI in the acidic and basic media. A proposed surface orientation model is presented here as an explanation to the experimental data, shedding light for further research on the behavior of insulin as a Langmuir monolayer.     

Enhanced rectal absorption of insulin in rabbits from hollow-type suppositories containing insulin and glyceryl-1-monooctanoate.

The absorption of two kinds of insulin (from porcine or bovine pancreas) from the rectum of rabbits after the administration of hollow-type suppositories containing insulin and glyceryl-1-monooctanoate (GMO) as an absorption-enhancing agent was investigated. Two types of suppositories were employed: type I containing insulin in an aqueous solution (approx. 25 IU/mg/100 microliters citric buffer solution at pH 3.0) in the cavity of the suppository and GMO mixed with a base material (Witepsol H-15), and type II containing insulin in a crystalline form in the same amount as in type I. Without GMO, the insulin and glucose levels in plasma were unchanged, whereas a marked increase in the plasma levels of insulin and a decrease of glucose concentrations were found following coadministration of insulin and GMO by the type I suppository. Similar enhancement of rectal absorption of insulin was obtained from porcine and bovine sources. In the case of the crystalline insulin, despite the use of the same amount of GMO, porcine insulin was more efficiently absorbed than bovine insulin by the type II suppository. GMO enhances the absorption of insulin in an aqueous solution or a crystalline form, and the dissolution rate of insulin may be an important factor in the rectal absorption of insulin.     

Quantitative analysis of the effect of zidovudine,efavirenz, and ritonavir on insulin aggregation by multivariate curve resolution alternating least squares of infrared spectra

Quantification of the effect of antiretroviral drugs on the insulin aggregation process is an important area of research due to the serious metabolic diseases observed in AIDS patients after prolonged treatment with these drugs. In this work, multivariate curve resolution alternating least squares (MCR-ALS) was applied to infrared monitoring of the insulin aggregation process in the presence of three antiretroviral drugs to quantify their effect. To evidence concentration dependence in this process, mixtures at two different insulin:drug molar ratios were used. The interaction between insulin and each drug was analysed by ¹H NMR spectroscopy. In all cases, the aggregation process was monitored during 45 min by infrared spectroscopy. The aggregates were further characterised by scanning electron microscopy (SEM). MCR-ALS provided the spectral and concentration profiles of the different insulin–drug conformations that are involved in the process. Their feasible band boundaries were calculated using the MCR-BANDS methodology. The kinetic profiles describe the aggregation pathway and the spectral profiles characterise the conformations involved. The retrieved results show that each of the three drugs modifies insulin conformation in a different way, promoting the formation of aggregates. Ritonavir shows the strongest promotion of aggregation, followed by efavirenz and zidovudine. In the studied concentration range, concentration dependence was only observed for zidovudine, with shorter aggregation time obtained as the amount of zidovudine increased. This factor also affected the aggregation pathway.     

In-capillary non-covalent labeling of insulin and one gastrointestinal peptide for their analyses by capillary electrophoresis with laser-induced fluorescence detection

The potential of the commercially available dye sypro orange for in-capillary derivatization was evaluated for the detection of insulin and one gastrointestinal peptide (Arg-Arg-gastrin) by capillary electrophoresis with laser induced fluorescence (CE-LIF). The fluorescent emission intensity (lambda(ex) = 488 nm, lambda(em) = 610 nm) of this probe is very low in aqueous medium, and increases strongly in less polar solvent, e.g. methanol. The hydrophobic character of the two analyzed peptides is too low to induce sufficient interaction with the fluorescent probe for good sensitivity when the latter is alone in the background electrolyte. Thus, the potential of several neutral, zwitterionic, cationic and anionic surfactants to favor probe/peptide interactions has been evaluated. It was demonstrated that a borate buffer (pH 8.5) containing tetradecyltrimethylammonium bromide (TTAB) in sub-micellar conditions can be considered as the most suitable buffer for insulin CE-LIF analysis. In addition, the method showed a good linearity between insulin concentration and the peak area of the labeled insulin, allowing quantitative measurements. The sensitivity achieved so far is comparable with that achieved with UV absorption detection, but even at this level it is interesting for microchip analysis, in which fluorescence detection is much more commonly available than UV absorption detection.     

Role of thiol-disulfide exchange in insulin binding to its receptor.

The effects of reduction by DTT, oxidation by DTNB and treatment with NEM on the thiol contents and insulin binding to its receptor in mice liver membranes were studied. Reduction with DTT leads to a parallel increase in the thiol content and the specific binding of insulin to the membrane. Scatchard analysis of the results shows little change in the number of binding sites but a twofold increase of the binding constant. Washing the membrane with bound insulin by a DTT containing buffer results in a more marked increase in the release of bound insulin than washing with buffer alone, suggesting that part of the insulin is bound to its receptor by covalent disulfide linkages through a thiol-disulfide exchange reaction and reduction with DTT leads to a marked increase in this "disulfide-linked" insulin. Treatment with DTNB or NEM of the DTT-reduced membrane seems to reverse the effect of DTT reduction, although the reaction of the untreated membrane with DTNB or NEM had little or no effect on the specific binding of insulin. It is suggested that initially, part of the thiols responsible for the exchange reaction may not be available for reaction with DTNB and reduction with DTT generates further thiols leading to increased specific binding in general and increased insulin binding to the receptor through covalent disulfide linkages in particular.     

Microsphere based continuous-flow immunoassay in a microfluidic device for determination of clinically relevant insulin levels

This paper describes a miniaturized microsphere-based immunoassay integrated into a microfluidic device for rapid quantitation of insulin. Analysis of bionic pancreas studies have revealed that the rates of absorption of insulin analogs vary from patient to patient, and even within patients on different occasions. Thus development of an approach to monitor insulin continuously allows the pharmacokinetic characteristics of insulin analogs to be determined in real-time. The authors have developed a microsphere-based continuous flow assay in a microfluidic chip that allows for the detection of insulin within seconds with high sensitivity and specificity. The method was applied to near real-time monitoring of clinical samples. Calibration plot were established for different insulin analogs such as insulin aspart (Novolog), insulin lispro (Humalog), and regular human insulin (RHI) and the insulin detection limit was 0.26 ng.mL^?1 (44 pM). This sensitivity allows to detect the fasting insulin levels of T1D patients, which are reported in the range of 50–180 pM (0.3–1 ng.mL^?1), after treatment with subcutaneous insulin administration. This fast approach was also applied to sera collected in intervals from T1D patients after a bolus of insulin aspart delivery. The insulin profile obtained by this method is similar to the basal and peak insulin levels as determined using the standard non-continuous ELISA reference method. In our perception, this assay will improve healthcare by personalizing diagnostics for better clinical outcome and provide real-time feedback on sensing and actuation.

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Graphical abstract Schematic illustration of the microfluidic microsphere based Microfluidic Lab-On-Chip device for near real-time insulin monitoring.

     

Comparison of mass spectra of peptides in different matrices using matrix-assisted laser desorption/ionization and a multi-turn time-of-flight mass spectrometer, MULTUM-IMG.

The mass spectra of peptides obtained with different matrices were compared using a matrix-assisted laser desorption/ionization (MALDI) ion source and a multi-turn time-of-flight (TOF) mass spectrometer, MULTUM-IMG, which has been developed at Osaka University. Two types of solid matrices, alpha-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), and a liquid matrix made from a mixture of 3-aminoquinoline and CHCA were used. When measuring the peak signal intensity of human angiotensin II [M+H]+ from a fixed sample position, the liquid matrix produced a stable signal over 1000 laser shots, while the signal obtained with CHCA and DHB decayed after about 300 and 100 shots, respectively. Significant differences in the mass resolving power were not observed between the spectra obtained with the three matrices. Signal peak areas were measured as a function of the cycle number in a multi-turn ion trajectory, i.e., the total flight time over a millisecond time scale. For both [M+H]+ of human angiotensin II and bovine insulin, the decay of the signal peak area was the most significant with CHCA, while that measured with DHB was the smallest. The results of the mean initial ion velocity measurements suggested that the extent of metastable decomposition of the analyte ions increased in order of DHB, the liquid matrix, and CHCA, which is consistent with the difference in the decay of the signal peak area as the total flight time increased.