Mechanistic Study of Diketopiperazine Formation during Solid-Phase Peptide Synthesis of Tirzepatide.

This study focused on investigating diketopiperazine (DKP) and the formation of associated double-amino-acid deletion impurities during linear solid-phase peptide synthesis (SPPS) of tirzepatide (TZP). We identified that the DKP formation primarily occurred during the Fmoc-deprotection reaction and post-coupling aging of the unstable Fmoc-Pro-Pro-Ser-resin active pharmaceutical ingredient (API) intermediate. Similar phenomena have also been observed for other TZP active pharmaceutical ingredient (API) intermediates that contain a penultimate proline amino acid, such as Fmoc-Ala-Pro-Pro-Pro-Ser-resin, Fmoc-Pro-Pro-Pro-Ser-resin, and Fmoc-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-resin, which are intermediates for both hybrid and linear synthesis approaches.

Multiplicative On-Column Solute Focusing Using Spatially Dependent Temperature Programming for Capillary HPLC.

The benefits of capillary liquid chromatography columns are truly realized when small, limited sample volumes require signal enhancement, but the available sample volume does not permit on-column focusing during injection onto a larger column. This dilemma is common when samples are naturally small or precious (such as in biological, forensic, art, and archeological investigations) and analyte concentrations are low. Signal enhancement by solvent-based focusing is effective with capillary columns, but it is limited to a single band-compression step and can only be achieved at the inlet.

Evaluation of three temperature- and mobile phase-dependent retention models for reversed-phase liquid chromatographic retention and apparent retention enthalpy.

Predicting retention and enthalpy allows for the simulation and optimization of advanced chromatographic techniques including gradient separations, temperature-assisted solute focusing, multidimensional liquid chromatography, and solvent focusing. In this paper we explore the fits of three expressions for retention as a function of mobile phase composition and temperature to retention data of 101 small molecules in reversed phase liquid chromatography. The three retention equations investigated are those by Neue and Kuss (NK) and two different equations by Pappa-Louisi et al.

Development of a 1.0 mm inside diameter temperature-assisted focusing precolumn for use with 2.1 mm inside diameter columns.

On-column solute focusing is a simple and powerful method to decrease the influence of precolumn band spreading and increase the allowable volume injected increasing sensitivity. It relies on creating conditions so that the retention factor, k', is transiently increased during the injection process. Both solvent composition and temperature control can be used to effect solute focusing.

Temperature-assisted solute focusing with sequential trap/release zones in isocratic and gradient capillary liquid chromatography: Simulation and experiment.

In this work we characterize the development of a method to enhance temperature-assisted on-column solute focusing (TASF) called two-stage TASF. A new instrument was built to implement two-stage TASF consisting of a linear array of three independent, electronically controlled Peltier devices (thermoelectric coolers, TECs). Samples are loaded onto the chromatographic column with the first two TECs, TEC A and TEC B, cold.

Graphical Method for Choosing Optimized Conditions Given a Pump Pressure and a Particle Diameter in Liquid Chromatography.

The general limitations on liquid chromatographic performance in isocratic and gradient elution are now well understood. Many workers have contributed to this understanding and to developing graphical methods, or plots, to illustrate the capabilities of chromatographic systems over a wide range of values of operational parameters. These have been invaluable in getting a picture, in broad strokes, about the value of changing an operational parameter or the value of one separation approach over another.

Improving the Sensitivity, Resolution, and Peak Capacity of Gradient Elution in Capillary Liquid Chromatography with Large-Volume Injections by Using Temperature-Assisted On-Column Solute Focusing.

Capillary HPLC (cLC) with gradient elution is the separation method of choice for the fields of proteomics and metabolomics. This is due to the complementary nature of cLC flow rates and electrospray or nanospray ionization mass spectrometry (ESI-MS). The small column diameters result in good mass sensitivity.

Quantitative evaluation of models for solvent-based, on-column focusing in liquid chromatography.

On-column focusing or preconcentration is a well-known approach to increase concentration sensitivity by generating transient conditions during the injection that result in high solute retention. Preconcentration results from two phenomena: (1) solutes are retained as they enter the column. Their velocities are k'-dependent and lower than the mobile phase velocity and (2) zones are compressed due to the step-gradient resulting from the higher elution strength mobile phase passing through the solute zones.

Temperature-based on-column solute focusing in capillary liquid chromatography reduces peak broadening from pre-column dispersion and volume overload when used alone or with solvent-based focusing.

On-column focusing is essential for satisfactory performance using capillary scale columns. On-column focusing results from generating transient conditions at the head of the column that lead to high solute retention. Solvent-based on-column focusing is a well-known approach to achieve this.

In Vivo Monitoring of Dopamine by Microdialysis with 1 min Temporal Resolution Using Online Capillary Liquid Chromatography with Electrochemical Detection.

Microdialysis is often applied to understanding brain function. Because neurotransmission involves rapid events, increasing the temporal resolution of in vivo measurements is desirable. Here, we demonstrate microdialysis with online capillary liquid chromatography for the analysis of 1 min rat brain dialysate samples at 1 min intervals.

Temperature-assisted on-column solute focusing: a general method to reduce pre-column dispersion in capillary high performance liquid chromatography.

Solvent-based on-column focusing is a powerful and well known approach for reducing the impact of pre-column dispersion in liquid chromatography. Here we describe an orthogonal temperature-based approach to focusing called temperature-assisted on-column solute focusing (TASF). TASF is founded on the same principles as the more commonly used solvent-based method wherein transient conditions are created that lead to high solute retention at the column inlet.

Development of selective comprehensive two-dimensional liquid chromatography with parallel first-dimension sampling and second-dimension separation--application to the quantitative analysis of furanocoumarins in apiaceous vegetables.

Various implementations of two-dimensional high-performance liquid chromatography are increasingly being developed and applied to the analysis of complex materials, including those encountered in the analysis of foods, beverages, and nutraceuticals. Previously, we introduced the concept of selective comprehensive two-dimensional liquid chromatography (sLC × LC) as a hybrid between the more conventional, but extreme opposite sampling modes of heartcutting (LC-LC) and fully comprehensive (LC × LC) 2D separation. The sLC × LC approach breaks the link between first dimension ((1)D) sampling time and second dimension ((2)D) analysis time that is faced in LC × LC and allows very rapid (as low as 1 s) sampling of highly efficient (1)D separations, while at the same time allowing efficient (2)D separations on the timescale of tens of seconds.

Selective comprehensive multidimensional separation for resolution enhancement in high performance liquid chromatography. Part II: applications.

In this second paper of a two-part series, we demonstrate the utility of an approach to enhancing the resolution of select portions of conventional 1D-LC separations, which we refer to as selective comprehensive two-dimensional HPLC (sLC × LC), in three quite different example applications. In the first paper of the series we described the principles of this approach, which breaks the long-standing link in online multi-dimensional chromatography between the timescales of sampling the first dimension (¹D) separation and the separation of fractions of ¹D effluent in the second dimension. In the first example, the power of the sLC × LC approach to significantly reduce the analysis time and method development effort is demonstrated by selectively enhancing the resolution of critical pairs of peaks that are unresolved by a one-dimensional separation (1D-LC) alone.

Selective comprehensive multi-dimensional separation for resolution enhancement in high performance liquid chromatography. Part I: principles and instrumentation.

An approach to enhancing the resolution of select portions of conventional one-dimensional high performance liquid chromatography (HPLC) separations was developed, which we refer to as selective comprehensive two-dimensional HPLC (sLC×LC). In this first of a series of two papers we describe the principles of this approach, which breaks the long-standing link in on-line multi-dimensional chromatography between the timescales of sampling the first dimension (¹D) separation and the separation of fractions of ¹D effluent in the second dimension. This allows rapid, high-efficiency separations to be used in the first dimension, while still adequately sampling ¹D peaks.

Targeted three-dimensional liquid chromatography: a versatile tool for quantitative trace analysis in complex matrices.

Targeted multidimensional liquid chromatography (MDLC), commonly referred to as 'coupled-column' or 'heartcutting', has been used extensively since the 1970s for analysis of low concentration constituents in complex biological and environmental samples. A primary benefit of adding additional dimensions of separation to conventional HPLC separations is that the additional resolving power provided by the added dimensions can greatly simplify method development for complex samples. Despite the long history of targeted MDLC, nearly all published reports involve two-dimensional methods, and very few have explored the benefits of adding a third dimension of separation.