P(III) vs P(V): A P(V) Reagent for Thiophosphoramidate Linkages and Application to an Asymmetric Synthesis of a Cyclic Dinucleotide STING Agonist.

A highly stereoselective synthesis of a cyclic dinucleotide (CDN) STING agonist containing two chiral thiophosphoramidate linkages is described. These rare yet key functional groups were, for the first time, installed efficiently and with high diastereoselectivity using a specially designed P(V) reagent. By utilizing this strategy, the CDN was prepared in greater than 16-fold higher yield than the prior P(III) approach, with fewer hazardous reagents and chromatographic purifications.

Total insulin and IGF-I resistance in pancreatic beta cells causes overt diabetes.

An appropriate beta cell mass is pivotal for the maintenance of glucose homeostasis. Both insulin and IGF-1 are important in regulation of beta cell growth and function (reviewed in ref. 2). To define the roles of these hormones directly, we created a mouse model lacking functional receptors for both insulin and IGF-1 only in beta cells (betaDKO), as the hormones have overlapping mechanisms of action and activate common downstream proteins. Notably, betaDKO mice were born with a normal complement of islet cells, but 3 weeks after birth, they developed diabetes, in contrast to mild phenotypes observed in single mutants. Normoglycemic 2-week-old betaDKO mice manifest reduced beta cell mass, reduced expression of phosphorylated Akt and the transcription factor MafA, increased apoptosis in islets and severely compromised beta cell function. Analyses of compound knockouts showed a dominant role for insulin signaling in regulating beta cell mass. Together, these data provide compelling genetic evidence that insulin and IGF-I-dependent pathways are not critical for development of beta cells but that a loss of action of these hormones in beta cells leads to diabetes. We propose that therapeutic improvement of insulin and IGF-I signaling in beta cells might protect against type 2 diabetes.

Peak purity assessment in a triple-active fixed-dose combination drug product related substances method using a commercial two-dimensional liquid chromatography system.

Pharmaceutical formulations containing multiple active components challenge the development of analytical methods, especially as the individual active ingredients diverge in their physicochemical properties. Establishing specificity, especially peak purity, is one of the major evaluation criteria when developing a related substances method for drug substances or products. Fixed-dose combination products may not be amenable to common strategies for assessing peak purity, such as performing orthogonal separations, due to the complexity of the separation and/or diversity of the active ingredients. An alternate approach to evaluating peak purity is demonstrated for a triple-active component fixed-dose combination product under development. A commercially available automated two-dimensional liquid chromatography system was used to perform a selective comprehensive multidimensional separation of an active ingredient peak. The first dimension performed the drug product impurity/degradant profiling method; the second dimension assayed these fractions using the drug substance profiling method, which was pseudo-orthogonal to the first dimension. A total of 14 targeted fractions were sampled across the first dimension main peak, with 11 containing detectable analytes and the remaining fractions bracketing the main peak. This degree of sampling allowed profiling of a coeluting degradant present at a 0.2% w/w level throughout the main peak.

A simulation-guided approach to the selection of RPLC columns for platform small molecule separations.

Simulations were conducted to evaluate the potential of several hundred reversed-phase columns to separate small molecules. By calculating the retention factor of compounds in randomly generated virtual mixtures via the HSM (hydrophobic subtraction model) and applying basic chromatography theory, the simulation can estimate the retention time and peak width of every virtual compound and calculate the resolution between every adjacent pair of compounds. A preferred column set based on the number of successful separations of randomly generated virtual mixtures was developed. The tandem-column liquid chromatography (TC-LC) approach can separate 53.2 % of the 16-compound samples using 20 tandem-column pairs, while a single-column approach can only separate 42.6 % of the 16-compound samples with 20 single columns. The preferred set of columns obtained from the simulation was almost the same as the empirical set of columns previously obtained. In screening applications, TC-LC can achieve a comparably successful separation factor (selectivity) with a smaller column inventory (nine 50-mm columns) compared to the larger inventory needed by single-column LC (twenty-one 100-mm columns).

Improved hydrophobic subtraction model of reversed-phase liquid chromatography selectivity based on a large dataset with a focus on isomer selectivity.

Reversed-phase (RP) liquid chromatography is an important tool for the characterization of materials and products in the pharmaceutical industry. Method development is still challenging in this application space, particularly when dealing with closely-related compounds. Models of chromatographic selectivity are useful for predicting which columns out of the hundreds that are available are likely to have very similar, or different, selectivity for the application at hand. The hydrophobic subtraction model (HSM1) has been widely employed for this purpose; the column database for this model currently stands at 750 columns. In previous work we explored a refinement of the original HSM1 (HSM2) and found that increasing the size of the dataset used to train the model dramatically reduced the number of gross errors in predictions of selectivity made using the model. In this paper we describe further work in this direction (HSM3), this time based on a much larger solute set (1014 solute/stationary phase combinations) containing selectivities for compounds covering a broader range of physicochemical properties compared to HSM1. The molecular weight range was doubled, and the range of the logarithm of the octanol/water partition coefficients was increased slightly. The number of active pharmaceutical ingredients and related synthetic intermediates and impurities was increased from four to 28, and ten pairs of closely related structures (e.g., geometric and cis-/trans- isomers) were included. The HSM3 model is based on retention measurements for 75 compounds using 13 RP stationary phases and a mobile phase of 40/60 acetonitrile/25 mM ammonium formate buffer at pH 3.2. This data-driven model produced predictions of ln α (chromatographic selectivity using ethylbenzene as the reference compound) with average absolute errors of approximately 0.033, which corresponds to errors in α of about 3 %. In some cases, the prediction of the trans-/cis- selectivities for positional and geometric isomers was relatively accurate, and the driving forces for the observed selectivity could be inferred by examination of the relative magnitudes of the terms in the HSM3 model. For some geometric isomer pairs the interactions mainly responsible for the observed selectivities could not be rationalized due to large uncertainties for particular terms in the model. This suggests that more work is needed in the future to explore other HSM-type models and continue expanding the training dataset in order to continue improving the predictive accuracy of these models. Additionally, we release with this paper a much larger data set (43,329 total retention measurements) at multiple mobile phase compositions, to enable other researchers to pursue their own lines of inquiry related to RP selectivity.

Trapping mode two-dimensional liquid chromatography for quantitative low-level impurity enrichment in pharmaceutical development.

Trapping mode two-dimensional liquid chromatography (2D-LC) has recently found applications in pharmaceutical analysis to clean, refocus, and enrich analytes. Given its enrichment capability, 2D-LC with multiple trappings is appealing for low-level impurity monitoring that cannot be solved by single dimensional LC (1D-LC) or unenriched 2D-LC analysis. However, the quantitative features of multi-trapping 2D-LC remain largely unknown at impurity levels from parts-per-million (ppm) to 0.15% (w/w). We present a simple heart-cutting trapping mode 2D-LC workflow using only common components and software found in typical off-the-shelf 1D-LC instruments. This robust, turn-key system's quantitative capabilities were evaluated using a variety of standard markers, demonstrating linear enrichment for up to 20 trapping cycles and achieving a recovery of over 97.0%. Next, the trapping system was applied to several real-world low-level impurity pharmaceutical case studies including (1) the identification of two unknown impurities at sub-ppm levels resulting in material discoloration, (2) the discovery of a new impurity at 0.05% (w/w) co-eluted with a known impurity, making the undesired summation above the target specification, and (3) the quantification of a potential mutagenic impurity at 10-ppm level in a poorly soluble substrate. The recovery in all studies was better than 97.0% with RSD lower than 3.0%, demonstrating accuracy and precision of the 2D-LC trapping workflow. As no specialized equipment or software is required, we envision that the system could be used to develop low-impurity monitoring methods suitable for validation and potential execution in quality-control laboratories.

Development of tandem-column liquid chromatographic methods for pharmaceutical compounds using simulations based on hydrophobic subtraction model parameters.

The liquid chromatography (LC) analysis of small molecule pharmaceutical compounds and related impurities is crucial in the development of new drug substances, but developing these separations is usually challenging due to analyte structural similarities. Tandem-column LC (TC-LC) has emerged as a powerful approach to achieve alternative separation selectivity compared to conventional single column separations. However, one of the bottlenecks associated with use of tandem column approaches is time-consuming column pair screening and selection. Herein, we compared critical resolution (Rc) in single column vs. TC-LC separations for a given set of small molecule pharmaceutical compounds and developed a column selection workflow that uses separation simulations based on parameters from the hydrophobic subtraction model (HSM) of reversed-phase selectivity. In this study, HSM solute parameters were experimentally determined for a small molecule pharmaceutical (Linrodostat) and ten of its related impurities using multiple linear regression of their retentions on 16 selected RPLC columns against in-house determined HSM column parameters. Rc values were calculated based on HSM database column parameters for a pool of about 200 available stationary phases in both single-phase column (2.1 mm i.d. × 100 mm) or tandem column paired (two 2.1 mm i.d. × 50 mm) formats. Four column configurations (two single and two tandem) were predicted to achieve successful separations under isocratic HSM separation conditions, with a fifth tandem pair predicted to have a single co-elution. Of these five potential candidates, one tandem pair yielded compete baseline resolution of the 11-component mixture in an experimental separation. In this specific case, the tandem column pairs outperformed single-phase columns, with better predicted and experimental Rc values for the Linrodostat mixture under the HSM separation conditions. The results reported in this study demonstrated the enormous selectivity potential of TC-LC in pharmaceutical compound separations and are consistent with our previous study that examined the potential of tandem column approaches using purely computational means, though there is room for substantial improvement in the prediction accuracy. The proposed workflow can be used to prioritize a small number of column combinations by computational means before any experiments are conducted. This is highly attractive from the point of view of time and resource savings considering over 200,000 different tandem column pairings are possible using columns for which there are data in the HSM database.

Dynamic monitoring of glucagon secretion from living cells on a microfluidic chip.

A rapid microfluidic based capillary electrophoresis immunoassay (CEIA) was developed for on-line monitoring of glucagon secretion from pancreatic islets of Langerhans. In the device, a cell chamber containing living islets was perfused with buffers containing either high or low glucose concentration. Perfusate was continuously sampled by electroosmosis through a separate channel on the chip. The perfusate was mixed on-line with fluorescein isothiocyanate-labeled glucagon (FITC-glucagon) and monoclonal anti-glucagon antibody. To minimize sample dilution, the on-chip mixing ratio of sampled perfusate to reagents was maximized by allowing reagents to only be added by diffusion. Every 6 s, the reaction mixture was injected onto a 1.5-cm separation channel where free FITC-glucagon and the FITC-glucagon-antibody complex were separated under an electric field of 700 V cm(-1). The immunoassay had a detection limit of 1 nM. Groups of islets were quantitatively monitored for changes in glucagon secretion as the glucose concentration was decreased from 15 to 1 mM in the perfusate revealing a pulse of glucagon secretion during a step change. The highly automated system should be enable studies of the regulation of glucagon and its potential role in diabetes and obesity. The method also further demonstrates the potential of rapid CEIA on microfluidic systems for monitoring cellular function.

Measuring atropisomers of BMS-986142 using 2DLC as an enabling technology.

BMS-986142 has been developed as an innovative Bruton's tyrosine kinase inhibitor for treatment of several autoimmune diseases. The drug substance of BMS-986142 may contain three potential atropisomeric impurities due to its unique structural characteristics. Developing a single liquid chromatography (LC) method to separate all four highly structurally related atropisomers and other process impurities from each other turned out to be a daunting task. Two-dimensional LC (2DLC) was found to be an extremely powerful enabling technology for extracting purity information out of the complex sample impurity profile and facilitated process development before a final single dimension method was discovered. The off-the-shelf 2DLC instrument could be configured to allow injection of the targeted first dimension peak through either no-loss multiple heart-cutting fractions or as a large, single volume fraction with on-line dilution. Excellent precision (relative standard deviation of 0.3 %) and recovery (101.2 ± 0.2 %) was achieved for an atropisomer impurity at a 10 % monitoring level in the first configuration with sensitivity down to 0.2 % w/w. With the second instrument configuration, which eliminated the need for fraction recombination, similar figures of merit were maintained for the second dimension at the cost of losing the ability to collect and park multiple fractions.

High-molecular weight impurity screening by size-exclusion chromatography on a reversed-phase column.

Monitoring polymerization events leading to the discovery of new high-molecular weight (MW) impurities is challenging during chemical syntheses of active pharmaceutical ingredients. Employing reversed-phase chromatography (RPC) stationary phases (SPs) in size-exclusion chromatography (SEC) mode could be a potential solution given their high efficiency, sensitivity, and extensive solvent compatibility. However, there is a lack of generalized means for trace polymeric impurities across a wide range of physicochemical properties. Herein, we developed a SEC-based approach with a C18 SP for screening such high-MW impurities. Seven polymer standards presenting a variety of functional groups, consisting of hydrophobic, heterocyclic, ionic, and neutral hydrophilic moieties, were utilized as model impurities to establish the screening conditions. Nine mobile phases (tetrahydrofuran-based, buffered methanol, and buffered acetonitrile) were proposed to cover all model polymers and a majority of potential high-MW impurities in small molecule chemical syntheses. The established screening system demonstrated a linearity of 0.05-1.0 % w/w (R2>0.99) for the selected model impurities with proper elution conditions. Two real high-MW impurities, BMT-041910 (polymeric degradation) and poly(phenyl thiirane) (by-product polymerization), were identified from the proposed high-MW impurity screening. The successful conditions yielded a quantitative limit better than 0.1 % w/w in both cases. We believe the developed screening platform is applicable to the analysis of a wide variety of unknown high-MW impurities of low abundance potentially generated during drug substance development.

2D separations on a 1D chip: gradient elution moving boundary electrophoresis-chiral capillary zone electrophoresis.

A new method is described for two-dimensional (2D) separations using a microfluidic chip normally employed for single dimension electrophoresis. The method employs a combination of gradient elution moving boundary electrophoresis (GEMBE) and chiral capillary zone electrophoresis (CZE). The simplicity of the first dimension GEMBE method enables its implementation in the injection channel of a conventional electrophoresis chip, simplifying the design and operation of the device. The method was used for high resolution 2D chiral separations of a mixture of amino acids considered as possible signatures of extant or extinct life for solar system exploration. The enantiomers of aspartic acid, glutamic acid, serine, alanine, and valine were all resolved as well as glycine (achiral) and several unidentified impurities, giving an estimated peak capacity of 35 for the region between valine and glycine. The results highlight the need for high peak capacity separations for chiral amino acid analysis if accurate enantiomeric ratios are to be determined.

Determination of inorganic ions in mineral water by gradient elution moving boundary electrophoresis.

A sensitive method was developed for the determination of the major inorganic ions in commercial mineral waters using gradient elution moving boundary electrophoresis with capacitively coupled contactless conductivity detection. This application was the first to demonstrate the separation of cations and anions simultaneously using gradient elution moving boundary electrophoresis. Seven ionic analytes (calcium, chloride, magnesium, nitrate, potassium, sodium, and sulfate) were separated in less than 7 min with detection values in the low µmol/L to sub-µmol/L range. Calculated values of the major ions in three commercial mineral waters were compared to reported values with good correlation. In another application, phosphate and arsenate were separated in less than 2 min with limits of detection of 300 and 140 nmol/L, respectively. For all standard analyses, the RSD for migration times and peak areas were under 3%.

Capillary and microfluidic gradient elution isotachophoresis coupled to capillary zone electrophoresis for femtomolar amino acid detection limits.

In this work, gradient elution isotachophoresis was combined with capillary zone electrophoresis (GEITP-CZE) in a single microcolumn. The multistage approach addresses the issues of analyte resolution difficulties in GEITP, as well as poor concentration sensitivity in CZE. GEITP employs rapid electrophoretic focusing at a discontinuous ionic interface within a sample well generated through combined electroosmotic and hydrodynamic flows. The interface and enriched analytes are then pulled into a capillary or microchannel as the counter-flow is reduced for on-column detection. To transform GEITP-focused samples to CZE-based separation, the sample solution is replaced with CZE buffer solution while maintaining hydrodynamic flow to ensure migration toward the detector. The single solution switch and lack of polarity inversion allows for reproducible separations (typically <6% relative standard deviation in peak heights and <0.5% in migration times). Low-pressure hydrodynamic flow during CZE allowed for flexible resolution adjustment, with a linear increase versus the square root of migration time, without altering the separation column, field strength, or electrolyte system. As a first demonstration of the applicability of GEITP-CZE, a series of amino acids to be assayed for in future Mars exploration missions as indicators of biological life were studied. Separation of six amino acids, with limits of detection as low as 200 fM, were achieved using a capillary format with a total analysis time of 11 min. A glass-based microfluidic implementation is also demonstrated that can perform GEITP-CZE in 1 cm effective lengths.

Rapid, direct, and sensitive determination of aziridine and 2-chloroethylamine by hydrophilic interaction liquid chromatography-mass spectrometry.

A rapid HILIC-MS method was developed for measuring the genotoxic impurities aziridine and 2-chloroethylamine. Sample preparation was simple and direct without requiring derivatization. Paired with a 1.5 min isocratic UHPLC separation, sample analysis could be completed in less than 5 min. Linearity was established from 0.5 µg/L to 10 µg/L for both target analytes. For main components at 100 g/L, this equates to 5 parts per billion (ppb) detectability using a benchtop, single quadrupole detector. Three model matrices were evaluated (glycine, phenylalanine, and the pharmaceutical drug asunaprevir), and the method was able to provide suitable repeatability (<10% RSD) and accuracy (±10%) at 5 µg/L concentrations. •Direct sample preparation without derivatization as is needed for GC analyses•Less than 5 min required for sample preparation and HILIC-MS analysis•Part per billion sensitivity in multiple test matrices with good recovery.

Finite sample effect in temperature gradient focusing.

Temperature gradient focusing (TGF) is a new and promising equilibrium gradient focusing method which can provide high concentration factors for improved detection limits in combination with high-resolution separation. In this technique, temperature-dependent buffer chemistry is employed to generate a gradient in the analyte electrophoretic velocity. By the application of a convective counter-flow, a zero-velocity point is created within a microchannel, at which location the ionic analytes accumulate or focus. In general, the analyte concentration is small when compared with buffer ion concentrations, such that the focusing mechanism works in the ideal, linearized regime. However, this presumption may at times be violated due to significant sample concentration growth or the use of a low-concentration buffer. Under these situations the sample concentration becomes non-negligible and can induce strong nonlinear interactions with buffer ions, which eventually lead to peak shifting and distortion, and the loss of detectability and resolution. In this work we combine theory, simulation, and experimental data to present a detailed study on nonlinear sample-buffer interactions in TGF. One of the key results is the derivation of a generalized Kohlrausch regulating function (KRF) that is valid for systems in which the electrophoretic mobilities are not constant but vary spatially. This generalized KRF greatly facilitates analysis, allowing reduction of the problem to a single equation describing sample concentration evolution, and is applicable to other problems with heterogeneous electrophoretic mobilities. Using this sample evolution equation we have derived an understanding of the nonlinear peak deformation phenomenon observed experimentally in TGF. We have used numerical simulations to validate our theory and to quantitatively predict TGF. Our simulation results demonstrate excellent agreement with experimental data, and also indicate that the proper inclusion of Taylor dispersion is important for the accurate modeling of TGF. This work is an important first step towards the understanding and prediction of the more complex, nonlinear, and multi-species interactions which often occur in on-chip electrophoretic assays such as TGF.

Gradient elution isotachophoresis with direct ultraviolet absorption detection for sensitive amino acid analysis.

This work demonstrates coupling of the newly described electrophoretic enrichment technique of gradient elution isotachophoresis (GEITP) to a low-cost, conventional ultraviolet absorbance detector to realize sensitive measurements with a universal detector, eliminating the need for fluorescent analytes or derivatization. The effects of various parameters on enrichment were studied, including current density varied by leading electrolyte concentration, current density varied by applied electric field, and counter-flow acceleration across varying capillary inner diameters. Optimized parameters were applied to the enrichment and separation of the amino acids tryptophan (Trp) and tyrosine (Tyr). Limits of detection for Trp and Tyr were 51 and 215 nM, respectively, reflecting sensitivity enhancements of 860- and 1900-fold. Analysis times were less than 6 min, and peak height RSDs were less than 4%. A demonstration of enrichment and separation of these amino acids from artificial cerebrospinal fluid is additionally shown as a first step to realizing biochemical monitoring by GEITP.

Tunable normal phase enantioselectivity of amino acid esters via mobile phase composition.

The ability to tune chiral selectivity through mobile phase modifiers is a powerful tool in chiral separations. Beyond improving efficiency and/or resolution, some mobile phase systems can even invert elution order, a highly desirable result for trace analyses or preparative scale isolations. Previous work has demonstrated that acidic modifiers, such as ethanesulfonic acid (ESA), can greatly impact separations of enantiomers. However, prior studies were primarily performed on coated chiral stationary phases (CSPs), which limited the selection of the bulk mobile phase component. In this work, the effect of ESA modifier was studied for the enantioseparation of six pairs of amino acid esters on a CHIRALPAK® IA column, an immobilized amylose-based CSP, with different combinations of standard solvents (hexane and ethanol) as well as "non-standard" solvents, such as methyl t-butyl ether, ethyl acetate, tetrahydrofuran, acetone, or 1,4-dioxane. ESA generally improved selectivity, and multiple instances of elution order reversal were observed. A Van Deemter plot study reveals that ESA exerts its effect by pulling the enantiomer deeper into the chiral cavity of the chiral polymer to increase the interactions between the analytes and the stationary phase, which is the main reason for the increased enantioselectivity.

Negative mode sheathless capillary electrophoresis electrospray ionization-mass spectrometry for metabolite analysis of prokaryotes.

Capillary electrophoresis (CE) was coupled to negative mode electrospray ionisation-mass spectrometry (MS) for separation and detection of phosphorylated and acidic metabolites in extracts of prokaryotes. Unlike previous CE-MS systems for metabolite analysis, a sheathless interface was used to improve sensitivity. To accomplish this, the separation capillary was modified by creating a porous junction near the outlet where the electrospray voltage and cathodic voltage for CE were applied. The outlet of the capillary was pulled to a 5 microm inner diameter to form an electrospray emitter and had a frit fabricated near the exit to prevent clogging. During analysis pressure was applied at the inlet of the separation column to create sufficient flow towards the detector. Limits of detection for 19 metabolites in full scan mode ranged from 20 nM for ADP ribose to 2.5 microM for alpha-ketoglutarate for 40 nL injections. Extracts of Escherichia coli, strain DH5-alpha, were analyzed using this system. In full scan mode, 118 different metabolites were detected. Tandem mass spectrometry was also employed to attempt identification. Reproducible fragmentation of 19 parent peaks was found and 10 of these produced spectra that were consistent with identification obtained from matching to compounds in the MetaCyc database. These results demonstrate the utility of a sensitive CE-MS system for large scale metabolite detection in biological samples.

Perfusion and chemical monitoring of living cells on a microfluidic chip.

A microfluidic device that incorporates continuous perfusion and an on-line electrophoresis immunoassay was developed, characterized, and applied to monitoring insulin secretion from single islets of Langerhans. In the device, a cell chamber was perfused with cell culture media or a balanced salt solution at 0.6 to 1.5 microL min(-1). The flow was driven by gas pressure applied off-chip. Perfusate was continuously sampled at 2 nL min(-1) by electroosmosis through a separate channel on the chip. The perfusate was mixed on-line with fluorescein isothiocyanate-labeled insulin (FITC-insulin) and monoclonal anti-insulin antibody and allowed to react for 60 s as the mixture traveled down a 4 cm long reaction channel. The cell chamber and reaction channel were maintained at 37 degrees C. The reaction mixture was injected onto a 1.5 cm separation channel as rapidly as every 6 s, and the free FITC-insulin and the FITC-insulin-antibody complex were separated under an electric field of 500 to 600 V cm(-1). The immunoassay had a detection limit of 0.8 nM and a relative standard deviation of 6% during 2 h of continuous operation with standard solutions. Individual islets were monitored for up to 1 h while perfusing with different concentrations of glucose. The immunoassay allowed quantitative monitoring of classical biphasic and oscillatory insulin secretion with 6 s sampling frequency following step changes in glucose from 3 to 11 mM. The 2.5 cm x 7.6 cm microfluidic system allowed for monitoring islets in a highly automated fashion. The technique should be amenable to studies involving other tissues or cells that release chemicals.

Capillary liquid chromatography with MS3 for the determination of enkephalins in microdialysis samples from the striatum of anesthetized and freely-moving rats.

In vivo microdialysis sampling was coupled to capillary liquid chromatography (LC)/electrospray ionization quadrupole ion trap mass spectrometry (MS) to monitor [Met]enkephalin and [Leu]enkephalin in the striatum of anesthetized and freely-moving rats. The LC system utilized a high-pressure pump to load 2.5 microl samples and desalt the 25 microm i.d. by 2 cm long column in 12 min. Samples were eluted with a separate pump at approximately 100 nl min(-1). A rapid gradient effectively separated the endogenous neuropeptides in 4 min. A comparison was made for operating the mass spectrometer in the MS2 and MS3 modes for detection of the peptides. In standard solutions, the detection limits were similar at 1-2 pM (2-4 amol injected); however, the reproducibility was improved with MS3 as the relative standard deviation was <5% compared with 20% for MS2 for 60 pM samples. For dialysate solutions, reconstructed ion chromatograms and tandem mass spectra had much higher signal-to-noise ratios in the MS3 mode, resulting in more confident detection at in vivo concentrations. The method was successfully used to monitor the peptides under basal conditions and with stimulation of peptide secretion by infusion of elevated K+ concentration.