Patent Description:
The present invention relates to calibrants for spectrometry and/or chromatography that separate materials based on their sizes and shapes and methods for calibrating such spectrometry and/or chromatography.

Ion mobility spectrometry (IMS) is capable of separating molecules that may have the same mass to charge ratio (m/z) but different conformational arrangements, such as those in metabolomic, proteomics, peptidomics, and exposomic analyses. IMS has become an important analytical characterization technique because of recent advances in the field, specifically the incorporation of electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) sources to IMS and improvements in analytical resolution. The combination of ESI with high-resolution IMS systems has been shown to be an important characterization techniques for a wide range of analytes (drugs, chemical warfare agents, peptides, and proteins). IMS is also extremely fast and can provide additional information to present technologies through coupling schemes with front end separations (i.e. liquid or gas chromatography) or back end characterizations (mass spectrometry (MS)), thereby allowing multi-dimensional sample characterization with increased sensitivity and no additional time needed.

Ion mobility-mass spectrometry (IM-MS) is an analytical technique that separates gas-phase ions based on their molecular weight (more specifically, their mass to charge ratio, m/z) and size/shape (more specifically, their collisional cross section (CCS)). In this method, ions are introduced into a drift tube The application of a static uniform electric field then propels these ions in the direction of the applied field The tube is filled with a drift gas, typically helium or nitrogen. The time taken for an ion to drift through the tube is related to its rotationally averaged cross-sectional area-that is, the area covered by a particle, or more simply its collision cross-section (CCS). Compact structures travel faster than more elongated (extended) ions, due to fewer interactions with the drift gas.

<CIT> discloses a method for determining the structure of a target carbohydrate by ion mobility-mass spectrometry in negative ionization mode. CCS estimations were performed using an established protocol and dextran as calibrant (Dextran MW = <NUM> and Dextran MW = <NUM>). The calibrant and each sample were measured on a travelling wave Synapt instrument at five wave velocities in negative ion mode. Drift times where extracted from raw data by fitting a Gaussian distribution to the arrival time distribution of each ion and corrected for their m/z dependent flight time. CCS reference values of dextran were corrected for charge and mass and a logarithmic plot of corrected CCSs against corrected drift times was used as a calibration curve to estimate CCSs. One calibration curve was generated for every wave velocity and each ion polarity. The resulting five estimated CCSs for each sample ion were averaged.

There remains a need for calibrants that can provide a wider range for both the CCS and the m/z dimensions while exhibiting minimal dispersity in CCS dimension (narrow peak width) using spectrometry and/or chromatography to separate materials based on their sizes and shapes.

The solution to this technical problem is provided by the embodiments characterized in the claims.

In a first aspect the present invention relates to a composition comprising at least two calibrant compounds or salts thereof, or cationic complexes thereof, or anionic complexes thereof, wherein the at least two calibrant compounds or the salts thereof or the cationic complexes thereof, or the anionic complexes thereof comprise an alcohol functionalized core, and peripheral functionalities.

The composition of the present invention is defined in claim <NUM>.

In another aspect, the present invention relates to methods of manufacturing the composition of the present invention, including mixing two or more of the cores with <NUM>, <NUM>, <NUM> or <NUM> alcohol functionalities, and subjecting the mixture to an esterification reaction.

The method of manufacturing the composition of the present invention is defined in claim <NUM>.

In another aspect, the present disclosure relates to methods of calibrating a mass spectrometer, including providing the composition of the present disclosure comprising at least one calibrant compound or salt thereof or cationic complex thereof, or anionic complex thereof, ionizing the at least one calibrant compound to provide at least one charged ion, collecting mass spectrometry data from the at least one charged ion, and calibrating the mass spectrometer based on the mass spectrometry data.

In another aspect, the present disclosure relates to methods of calibrating an ion mobility spectrometer, including providing the composition of the present disclosure comprising at least one calibrant compound or salt thereof, or cationic complex thereof, or anionic complex thereof, ionizing at least one calibrant compound to provide at least one charged ion, collecting ion mobility data from at least one charged ion in a drift gas, and calibrating the ion mobility spectrometer based on the ion mobility data.

In another aspect, the present disclosure relates to methods of calibrating an ion mobility-mass spectrometer, including providing the composition of the present disclosure comprising at least one calibrant compound or salt thereof or cationic complex thereof, or anionic complex thereof, ionizing at least one calibrant compound to provide at least one charged ion, collecting ion mobility data in a drift gas and mass spectrometer data from the at least one charged ion, and calibrating the ion mobility-mass spectrometer based on the ion mobility data and the mass spectrometer data.

In another aspect, the present disclosure relates to methods of calibrating a light scattering spectrometer, including providing the composition of the present disclosure comprising at least one calibrant compound, dissolving the at least one calibrant compound to provide a solution of at least one calibrant compound, collecting light scattering data from at least one calibrant compound, and calibrating the light scattering spectrometer based on the light scattering data.

In another aspect, the present disclosure relates to methods of calibrating a size exclusion chromatograph, including providing the composition of the present disclosure comprising at least one calibrant compound or salt thereof or cationic complex thereof, or anionic complex thereof, dissolving at least one calibrant compound to provide a solution of at least one calibrant compound, collecting size exclusion data from at least one calibrant compound, and calibrating the size exclusion chromatograph based on the size exclusion data.

In another aspect, the present disclosure relates to methods of determining physical properties of a sample, including providing the composition of the present disclosure comprising at least one calibrant compound or salt thereof or cationic complex thereof, or anionic complex thereof, providing the sample, collecting physical data from at least one calibrant compound, calibrating an instrument capable of measuring the physical properties based on the physical data, and determining the physical properties of the sample.

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the description of specific embodiments presented herein.

Detailed descriptions of one or more preferred embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.

Wherever any of the phrases "for example," "such as," "including" and the like are used herein, the phrase "and without limitation" is understood to follow unless explicitly stated otherwise. Similarly, "an example," "exemplary" and the like are understood to be non-limiting.

The term "substantially" allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term "substantially" even if the word "substantially" is not explicitly recited. Therefore, for example, the phrase "wherein the lever extends vertically" means "wherein the lever extends substantially vertically" so long as a precise vertical arrangement is not necessary for the lever to perform its function.

The terms "comprising" and "including" and "having" and "involving" (and similarly "comprises", "includes," "has," and "involves") and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of "comprising" and is therefore interpreted to be an open term meaning "at least the following," and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, "a process involving steps a, b, and c" means that the process includes at least steps a, b and c. Wherever the terms "a" or "an" are used, "one or more" or "at least one" is understood, unless such interpretation is nonsensical in context.

Although a range of calibration sets have been explored for ion mobility characterization, they exhibit a relatively narrow range of m/z and CCS values. The calibration systems employed for ion mobility-mass spectrometry (IM-MS) have largely been those already explored for ESI-MS, including Ultramark, polyalanine (Poly-Ala), and tetra(alkyl) ammonium salts (TAA salts).

<FIG> shows that these compounds all have a similar degree of compactness (slope of CCS relative to m/z, and shown by the shaded area), with the fluorinated Ultramark calibrants exhibiting slightly more compact nature due to higher density of fluorine. The high proportion of ionizable atoms (e.g., O and N) in these calibrant systems result in limited m/z range for <NUM>+ species, as higher masses tend to be multiply charged. For ions of these traditional calibrants in the <NUM>+ charge state, the calibration range covered is limited. As shown by the small area occupied in the two-dimensional space by these two axes, these existing calibrants exhibit limited range in the CCS dimension (for N<NUM>): <NUM>-<NUM>Å<NUM> (top CCS boundary), and limited range in the m/z dimension (typically m/z range: <NUM>-<NUM>) (right m/z boundary). Furthermore, they exhibit limited diversity in compactness (e.g. slope of CCS relative to m/z).

Therefore, there is a need for IM-MS calibrants that can overcome these limitations. The calibrants should provide a wider range for both the CCS and the m/z dimensions while exhibiting minimal dispersity in CCS dimension (narrow peak width). Additionally, the calibrants should be compatible with both positive and negative ion modes, should be technically simple to use, and should exhibit long shelf-lives.

In an aspect, the present disclosure relates to compositions containing at least one calibrant compound, or cationic complex thereof, or anionic complex thereof, in which the at least one calibrant compound or the salt thereof or cationic complex thereof, or anionic complex thereof, may include an alcohol or an amine functionalized core and peripheral functionalities.

The at least one calibrant compound or the salt thereof or cationic complex thereof, or anionic complex thereof, may include at least one core with at least one alcohol functionality, which may be selected from the group consisting of mono-functional cores, di-functional cores, tri-functional cores, tetra-functional cores, penta-functional cores, hexa-functional cores, octa-functional cores, and dendrimer-based cores comprising a plurality of alcohol functionalities.

For example, mono-functional cores with at least one alcohol functionality may be selected from the group consisting of methanol, ethanol, <NUM>-propanol, <NUM>-propanol, <NUM>-butanol, <NUM>-butanol, <NUM>-methyl-<NUM>-butanol, <NUM>-methyl-<NUM>-butanol, <NUM>-methyl-<NUM>-butanol, and <NUM>,<NUM>-dimethyl <NUM>-propanol; di-functional cores may be selected from the group consisting of ethylene glycol, <NUM>,<NUM>-propane diol, <NUM>,<NUM>-propane diol, <NUM>,<NUM>-butane diol, <NUM>,<NUM>-butane diol, <NUM>,<NUM>-butane diol, <NUM>,<NUM>-butane diol, <NUM>,<NUM>-pentane diol, <NUM>,<NUM>-pentane diol, <NUM>,<NUM>-hexane diol, and <NUM>,<NUM>-hexane diol; tri-functional cores may be selected from the group consisting of <NUM>,<NUM>,<NUM>-butane triol, <NUM>,<NUM>,<NUM>-butane triol, <NUM>,<NUM>,<NUM>-tris-(hydroxymethyl)ethane, and <NUM>,<NUM>,<NUM>-tris-(hydroxymethyl)propane; tetra-functional cores may be selected from the group consisting of pentaerythritol, erythritol, and threitol; penta-functional cores may be selected from the group consisting of xylitol, arabinitol, arabitol, adonitol, and triglycerol; hexa-functional cores may be selected from the group consisting of dipentaerythritol, allitol, dulcitol, iditol, talitol, sorbitol, galactitol, and mannitol; octafunctional core may be tripentaerythritol; and dendrimer-based cores may contain at least one layer of bis-MPA repeating units bonding to any one of mono-functional cores, di-functional cores, tri-functional cores, tetra-functional cores, penta-functional cores, hexa-functional cores, and/or octa-functional cores by esterification,.

The at least one calibrant compound or the salt thereof or cationic complex thereof, or anionic complex thereof, may include at least one core with at least one amine functionality, which may be selected from the group consisting of mono-functional cores, di-functional cores, tri-functional cores, tetra-functional cores, and dendrimer-based cores comprising a plurality of amine functionalities.

For example, mono-functional cores with at least one amine functionality may be selected from the group consisting of methylamine, ethylamine, <NUM>-propylamine, <NUM>-propamine, <NUM>-butylamine, <NUM>-butylamine, and tert-butylamine; di-functional cores may be selected from the group consisting of ethylene diamine, <NUM>,<NUM>-propane diamine, <NUM>,<NUM>-propane diamine, <NUM>,<NUM>-butane diamine, <NUM>,<NUM>-butane diamine, <NUM>,<NUM>-butane diamine, <NUM>,<NUM>-butane diamine, <NUM>,<NUM>-pentane diamine, <NUM>,<NUM>-hexane diamine, and <NUM>,<NUM>-heptane diamine; tri-functional cores may be selected from the group consisting of <NUM>,<NUM>'-diaminoethylamine, bis(hexamethylene) triamine, triazine, and tris(<NUM>-aminoethyl)amine; tetra-functional cores may be selected from the group consisting of <NUM>,<NUM>'diaminobenzidine, triethylenetetramine, and hexamethylenetetramine; and dendrimer-based cores may be selected from the group consisting of polyamidoamine, polypropylene imine, and polytriazine dendrimers.

In another aspect, the present disclosure relates to compositions containing at least two calibrant compounds or salts thereof or cationic complex thereof, or anionic complex thereof, in which the at least two calibrant compounds or the salts thereof or cationic complex thereof, or anionic complex thereof, may contain the alcohol or amine functionalized cores and the peripheral functionalities.

In another aspect, the present disclosure relates to methods of manufacturing the composition of the present disclosure, including mixing one, two, three, four, five, six, or more of the at least one core with at least one alcohol functionality, and subjecting the mixture to an esterification reaction.

In another aspect, the present disclosure relates to methods of manufacturing the composition of the present disclosure, including mixing one, two, three, four, five, six, or more of the at least one core with at least one amine functionality, and subjecting the mixture to an amidation reaction.

Compositions of the present disclosure may be used to calibrate any instruments that can measure mass, size, shape, and/or collisional cross section area (CCS) of molecules in a gas phase.

In another aspect, the present disclosure relates to methods of calibrating a mass spectrometer, including providing the composition of the present disclosure, ionizing the at least one calibrant compound to provide at least one charged ion, collecting mass spectrometry data from the at least one charged ion, and calibrating the mass spectrometer based on the mass spectrometry data.

The at least one charged ion may be singly-charged or multi-charged ion. Mass spectrometry data may contain a mass to charge ratio (m/z) of the at least one charged ion. The mass to charge ratio (m/z) of the at least one charged ion may be from about <NUM>/z to about <NUM> m/z, from about <NUM>/z to about <NUM> m/z, from about <NUM>/z to about <NUM> m/z, from about <NUM>/z to about <NUM> m/z, from about <NUM>/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM>/z to about <NUM> m/z, from about <NUM>/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM>/z to about <NUM> m/z, from about <NUM>/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, or from about <NUM> m/z to about <NUM> m/z, from about <NUM>/z to about <NUM> m/z, from about <NUM>/z to about <NUM> m/z, from about <NUM>/z to about <NUM> m/z, from about <NUM>/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, or from about <NUM> m/z to about <NUM> m/z.

In an embodiment, the mass to charge ratio (m/z) of the at least one charged ion may be from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM>/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM>/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM> m/z, from about <NUM> m/z to about <NUM>,<NUM>/z, from about <NUM>,<NUM> m/z to about <NUM>,<NUM> m/z, from about <NUM>,<NUM> m/z to about <NUM>,<NUM> m/z, from about <NUM>,<NUM> m/z to about <NUM>,<NUM> m/z, from about <NUM>,<NUM> m/z to about <NUM>,<NUM> m/z, from about <NUM>,<NUM> m/z to about <NUM>,<NUM> m/z.

The mass spectrometry may be selected from the group consisting of accelerator mass spectrometry, isotope ratio mass spectrometry, MALDI-TOF, SELDI-TOF, electrospray ionization (ESI)-rnass spectrometry, thermal ionization-mass spectrometry, and spark source mass spectrometry, and the mass spectrometer may be selected from the group of spectrometers useful for performing said mass spectrometry.

In another aspect, the present disclosure relates to methods of calibrating an ion mobility spectrometer, including providing the composition of the present disclosure, ionizing the at least one calibrant compound to provide at least one charged ion, introducing the at least one charged ion into the ion mobility spectrometer, collecting ion mobility data from the at least one charged ion in a drift gas, and calibrating the ion mobility spectrometer based on the ion mobility data.

The at least one charged ion may be singly-charged or multi-charged. The ion mobility data may contain a collision cross section (CCS) value of the at least one charged ion. The collision cross section (CCS) value of the at least one charged ion may be from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM><NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, or from about <NUM>Å<NUM> to about <NUM>Å<NUM>.

In an embodiment, the collision cross section (CCS) value of the at least one charged ion may be from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, from about <NUM>Å<NUM> to about <NUM>Å<NUM>, or from about <NUM>Å<NUM> to about <NUM>Å<NUM>.

The drift gas may be selected from the group consisting of helium, nitrogen, argon, and carbon dioxide.

In another aspect, the present disclosure relates to methods of calibrating an ion mobility-mass spectrometer, including providing the composition of the present disclosure, ionizing the at least one calibrant compound to provide at least one charged ion, introducing the at least one charged ion into the ion mobility-mass spectrometer, collecting ion mobility data in a drift gas and mass spectrometer data from the at least one charged ion, and calibrating the ion mobility-mass spectrometer based on the ion mobility data and the mass spectrometer data.

In another aspect, the present disclosure relates to methods of calibrating a light scattering spectrometer, including providing the composition of the present disclosure, dissolving the at least one calibrant compound to provide a solution of the at least one calibrant compound, introducing the solution of the at least one calibrant compound into the light scattering spectrometer, collecting light scattering data from the at least one calibrant compound, and calibrating the light scattering spectrometer based on the light scattering data. The light scattering data may contain the size of the at least one calibrant compound in the solution.

In another aspect, the present disclosure relates to methods of calibrating a size exclusion chromatograph, including providing the composition of the present disclosure, dissolving the at least one calibrant compound to provide a solution of the at least one calibrant compound, introducing the solution of the at least one calibrant compound into the size exclusion chromatograph, collecting size exclusion data from the at least one calibrant compound, and calibrating the size exclusion chromatograph based on the size exclusion data. The size exclusion data may contain the size of the at least one calibrant compound in the solution.

In another aspect, the present disclosure relates to methods of determining physical properties of a sample, including providing the composition of the present disclosure, providing the sample, collecting physical data from the at least one calibrant compound, calibrating an instrument capable of measuring the physical properties based on the physical data, and determining the physical properties of the sample. The physical properties of the sample may include mass, size, shape, and/or collisional cross section area of the sample in a drift gas. The instrument may be selected from the group consisting of mass spectrometer, ion mobility spectrometer, ion mobility-mass spectrometer, light scattering spectrometer, size exclusion chromatograph, and a combination thereof.

Embodiments of the present disclosure include novel calibrants and methods of making these calibrants that contain multifunctional cores and/or dimethylolpropionic acid (bis-MPA), i.e.,
<CHM>
based polyester dendrimers. These core molecules or dendrimers may be functionalized on their periphery to prepare a series of singular discrete compounds with exact molecular weights and well-defined sizes. These functionalized cores and dendrimers may then be used as IMS calibrants. Each compound within a sample set may be composed of a core molecule with at least one alcohol functionality, or at least one amine functionality. Each of these alcohol or amine functionalities can then be coupled with an activated carboxylic acid to yield multiple ester or amide bonds.

Functionalized cores or dendrimers (the latter based on the bis-MPA monomer) are prepared as singular discrete compounds that may exhibit a wider range of CCS and m/z values. In some embodiments, CCS values of greater than <NUM>Å<NUM>, and m/z values of in excess of <NUM> (for singly charges species) can be measured, more than doubling the range of the existing common calibrants.

To achieve higher CCS values, embodiments of the present disclosure may include the addition of alkyl chains onto the periphery of the cores to make "star-shaped" calibrants (with multiple alkyl arms). Fatty acids varying from short chains to long chains are coupled with the multifunctional cores or dendrimers. Though the addition of long alkyl chains may not significantly increase the propensity for charging (ease of complexation with cations), it does increase the size substantially, and therefore yields extended conformations with high CCS values but modest m/z values.

In one embodiment, the present disclosure provides functionalized polyester dendrimers based on bis-MPA monomer that may be modified by esterification of their peripheral functionalities in order to prepare a set of singular, discrete compounds that may exhibit a wider range of CCS and m/z values. In particular, CCS values in excess of <NUM>Å<NUM>, and m/z values of in excess of <NUM> (for singly charges species) can be measured, more than doubling the range of existing calibrants.

In another embodiment, the present disclosure provides functionalized core molecules by reacting activated carboxylic acid derivatives with the alcohol and/or amine functionalities of the multifunctional cores. A set of discrete compounds may be generated that expand the available range of m/z calibration points as well as CCS calibration points for IM-MS characterization.

For example, multifunctional core may have a formula I: X-[OH]n, in which n is an integer from <NUM> to <NUM>, and X is a core comprised of alkane, ether, ester, amine, and/or amide functionalities or generations of alkane, ether, ester, amine, and/or amide dendrimers. Nonlimiting examples of X-[OH]n cores are shown in Table <NUM>. In the composition of the present invention cores with n=<NUM>, <NUM>, <NUM> and <NUM> are used.

According to the present invention X-[OH]n are reacted with carboxylic acid derivatives according to Formula II:
<CHM>
where m is an integer from <NUM> to <NUM>.

For example, carboxylic acid derivatives may be selected from the group consisting of methanoic (formic) acid, ethanoic (acetic) acid, propanoic (proprionic) acid, butanoic (butyric) acid, pentanoic (valeric) acid, hexanoic (caprylic) acid, heptanoic (enanthic) acid, octanoic (caprylic) acid, nonanoic (pelargonic) acid, decanoic (capric) acid, undecanoic acid, dodecanoic (lauric) acid, tridecanoic acid, tetradecanoic (myristic) acid, pentadecanoic acid, hexadecanoic (palmitic acetic) acid, heptadecanoic (margaric acetic) acid, octadecanoic (stearic acetic) acid, nonadecanoic acid, eicosanoic (arachidic) acid, heneicosanoic acid, docosanoic (behenic) acid, tetracosanoic (lignoceric) acid, hexacosanoic (cerotic) acid, octacosanoic (montanic) acid, and triacosanoic (melissic) acid.

In addition, X-[OH]n may be reacted with carboxylic acid derivatives that include a hydrocarbon chain that may contain one or more branching points, one or more double bond, and/or one or more triple bond, such as arachidonic acid, cis-<NUM>-docosenoic acid, cis-<NUM>-eicosenoic acid, elaidic acid, linoleic acid, linolenic acid, linolelaidic acid, myristoleic, oleic acid, palmitoleic acid, petroselenic acid, and cis-<NUM>-tetracosenoic acid.

A synthetic scheme for esterification of multifunctional cores or multifunctional dendrimer cores is shown below:
<CHM>
wherein m = <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>, n = <NUM> to <NUM>, and X is a core comprised of alkane, ether, ester, amine, and/or amide functionalities or generations of alkane, ether, ester, amine, and/or amide dendrimers (e.g., as shown in Table <NUM>), These reactions may be achieved using dicyclohexylcarbodiimide (DCC) and <NUM>-dimethylaminopyridine (DMAP) as a catalyst.

<FIG> shows a calibrant based on a hexafunctional alcohol core C1 (n = <NUM>) (a bis-MPA dendrimer) that has been esterified with dodecyl (lauryl) esters to yield a compound with an exact molecular weight and a well-defined size.

To evaluate the performance of the calibrants of the present disclosure in IM-MS, a mixture of the calibrants of the present disclosure shown in Table <NUM> and the existing standards, e.g., TAA salts, LCMS QC Ref, Poly-Ala, Ultramark, and SphericalCal Mix, are compared.

<FIG> shows that the addition of alkyl chains, e.g., from <NUM>-carbon chain (IMS <NUM>) to <NUM>-carbon chain (IMS <NUM>), onto cores and dendrimers provides a more extended conformation, and hence higher CCS values relative to m/z, (with values as high as <NUM>Å<NUM>, e.g., <NUM>-carbon chain IMS <NUM> C1-F1 as indicated by open arrows), than the existing standards, e.g., TAA salts, LCMS OC Ref, Poly-Ala, and Ultramark, as shown in <FIG>. The SpheriCal standards exhibit a trend in compactness similar to the existing standards, but may extend the CCS range just beyond <NUM>Å<NUM>. All data correspond to Na+ adducts.

To achieve higher m/z, an embodiment of the present disclosure may include the incorporation of multiple halogen atoms into the calibrant compositions. Achieving high m/z can be a challenge in mass spectrometry because increases in the mass (m) tends to also increase the ability to carry charge (z). However, certain elements (e.g. iodine "I'') have a high atomic mass (A. = <NUM>) but may not have an increased propensity to carry charge (e.g. relative to elements like O, atomic mass = <NUM>, and N, atomic mass = <NUM>). To a lesser extent, the same may be true for fluorine (A. Therefore, an embodiment of the present disclosure may include compounds with high I (or F) content that can exhibit very high masses in low charge states (hence high m/z) as well as very compact conformation with modest CCS values, despite high m/z values.

For example, X-[OH]n may be reacted with carboxylic acid derivatives, such as those of Formula IV:
<CHM>
in which y is an integer from <NUM> to <NUM>.

For example, carboxylic acid derivatives may be selected from the group consisting of benzoic acid, <NUM>-iodo benzoic acid, <NUM>-iodo benzoic acid, <NUM>-iodo benzoic acid, <NUM>,<NUM>-diiodo benzoic acid, <NUM>,<NUM>-diiodo benzoic acid, <NUM>,<NUM>-diiodo benzoic acid, <NUM>,<NUM>-diiodo benzoic acid, <NUM>-diiodo benzoic acid, <NUM>,<NUM>-diiodo benzoic acid, <NUM>,<NUM>,<NUM>-triiodo benzoic acid, <NUM>,<NUM>,<NUM>-triiodo benzoic acid, <NUM>,<NUM>,<NUM>-triiodo benzoic acid, <NUM>,<NUM>,<NUM>-triiodo benzoic acid, <NUM>,<NUM>,<NUM>,<NUM>-tetraiodobenzoic acid, <NUM>,<NUM>,<NUM>,<NUM>-tetraiodobenzoic acid, <NUM>,<NUM>,<NUM>,<NUM>-tetraiodobenzoic acid, and <NUM>,<NUM>,<NUM>,<NUM>,<NUM>-pentaiodobenzoic acid.

A synthetic scheme for esterification of multifunctional cores or multifunctional dendrimer cores is shown below:
<CHM>
e.g., y = <NUM> or <NUM>, n = <NUM> to <NUM>, and X as defined above. These reactions may be carried out in the presence of dicyclohexylcarbodiimide (DCC) and <NUM>-dimethylaminopyridine (DMAP).

Table <NUM> shows the chemical structures of some calibrants with triiodobenzoate functionalized cores in accordance with some embodiments of the present disclosure.

<FIG> shows that halogenated aromatics, e.g., triiodobenzoate functionalized core (IMS <NUM> C-F, IMS <NUM> C1-F1, and IMS <NUM>), exhibit higher mass to size ratio, hence higher m/z, lower CCS values, and an overall more compact trend line than the existing standards, e.g., TAA salts, LCMS QC Ref, Poly-Ala, and Ultramark, as shown in <FIG>. This may increase the m/z values in the <NUM>+ charge state from below <NUM> to as high as <NUM>, with a CCS of only <NUM>Å<NUM>, e.g., IMS <NUM> C1-F1 (as indicated by arrows). The slope of IMS <NUM> C1-F1 together with IMS <NUM> C-F (as indicated by arrow heads) and IMS <NUM> (as indicated by open arrow head) is smaller than that of IMS <NUM>-<NUM>, indicating that IMS <NUM> calibrants are more compact than IMS <NUM>-<NUM> calibrants. IMS <NUM> calibrants achieve very high masses in low charge states (hence high m/z) as well as very compact conformation with modest CCS values, despite high m/z values.

Size dispersity refers to the range of sizes that a single compound may exhibit. If the compounds are flexible, they may exhibit both very compact and very extended conformations. Embodiments of the present disclosure may include branched cores that lead to structures, which are more architecturally compact and therefore lack the ability to exhibit a wide range of shapes. This is in contrast, for example, to a long linear compound, which may exhibit either compact (wadded up) or extended (more elongated linear) conformations.

<FIG> shows that the observed peak width in the CCS dimension is a function of both instrument resolution and the dispersities of conformation, which are sampled by the molecule in the gas phase. The series of dendrimers, e.g., IMS <NUM> C-F (see Example <NUM>), IMS <NUM> C-F (with <NUM>-carbon chain), and IMS <NUM> C-F (with <NUM>-carbon chain), exhibit CCS dispersities as narrow as those observed by other calibrants, such as the trialkyl ammonium salts (TAA salts). The conformational dispersity of the dendrimer calibrants of the present disclosure may be as narrow as those measured for other calibrants.

Embodiments of the present disclosure may include calibrants that exhibit the ability to complex with a range of cations and/or anions to achieve mass spectra and ion mobility spectra in both positive and negative ion modes.

<FIG> shows an analysis in the positive mode of ionization, in which data for calibrations can be achieved, as long as the appropriate salt is used. In positive ion mode, with <NUM>% sodium formate used as a cation source in <NUM>% formic acid, IMS103 (IMS103-C, IMS103-D, IMS103-E, and IMS103-F) yields sodiated adducts.

<FIG> shows an analysis in the negative mode of ionization, in which data for calibrations can be achieved, as long as the appropriate salt is used. In negative ion mode, with <NUM>% ammonium acetate used as an anion source in <NUM>% ammonia IMS103 (IMS103-C, IMS103-D, and IMS103-E) yields acetate adducts.

In another embodiment of the invention, compounds exhibiting a range of compactness, from high CCS and low m/z (more extended) to low CCS and high m/z (more compact) are disclosed. By tuning the compactness of the calibrants (e.g., varying peripheral groups from long, extended linear fatty acids, to short, mass-dense iodinated aromatic rings) a larger area of the ion mobility mass spectrometry graph may be covered by calibration points, rather than a single linear trend as observed by most of the existing calibrations systems.

To a round bottom flask was added one or more of the following "core" compounds: ethylene glycol ("B"), tris(hydroxymethyl)ethane ("C"), pentaerythritol ("D"), xylitol ("E"), dipentaerythritol ("F"), tripentaerythritol "H"), or bis-MPA dendrimers made from the above cores. These were dissolved in tetrahydrofuran. <NUM> molar equivalents (per -OH of the hydroxyl terminated cores or dendrimers) of butanoic acid were added to the solution of cores. To these reagents were added <NUM> molar equivalents (per -OH of the hydroxyl terminated cores or dendrimers) of dicyclohexylcarbodiimide and <NUM> molar equivalents (per -OH of hydroxyl-terminated core or of dendrimer) of <NUM>-dimethylaminopyridine (DMAP).

The reaction mixture was stirred vigorously for approximately <NUM> hours at standard temperature and pressure. The reaction was monitored by MALDI-TOF MS to confirm completion of the reaction for each of the cores present in the reaction. After complete esterification is observed by MALDI-TOF MS, the flask contents were transferred to a separatory funnel, diluted with dichloromethane, extracted twice with <NUM> aqueous NaHSO<NUM> (sodium bisulfate) and extracted twice with <NUM> aqueous NaHCO<NUM> (sodium bicarbonate). The organic layer was reduced in vacuo to concentrate the sample. A MALDI-TOF MS spectra of the purified product confirmed the purity of the mixture of esterified products and is shown in <FIG>.

<FIG> shows MALDI-TOF MS data for IMS <NUM> C-F, the product of octanoic acid functionalization of cores C, D, E, and F.

To a round bottom flask was added one or more of the following "core" compounds: tris(hydroxymethyl)ethane ("C"), pentaerythritol ("D"), xylitol ("E"), dipentaerythritol ("F") made from the above cores. These were dissolved in tetrahydrofuran. <NUM> molar equivalents (per -OH of the hydroxyl terminated cores or dendrimers) of Octanoic Acid were added to the solution of cores. To these reagents were added <NUM> molar equivalents (per -OH of the hydroxyl terminated cores or dendrimers) of dicyclohexylcarbodiimide and <NUM> molar equivalents (per -OH of hydroxyl-terminated core or of dendrimer) of <NUM>-dimethylaminopyridine (DMAP).

The reaction mixture was stirred vigorously for approximately <NUM> hours at standard temperature and pressure. The reaction was monitored by MALDI-TOF MS to determine completion of the reaction for each of the cores present in the reaction. After complete esterification is observed by MALDI-TOF MS, the flask contents were transferred to a separatory funnel, diluted with dichloromethane, extracted twice with <NUM> aqueous NaHSO<NUM> (sodium bisulfate) and extracted twice with <NUM> aqueous NaHCO<NUM> (sodium bicarbonate). The organic layer was reduced in vacuo to concentrate the sample. A MALDI-TOF MS spectra of the purified product confirmed the purity of the mixture of esterified products and is shown in <FIG>.

<FIG> shows a scheme for the synthesis of IMS <NUM> C-F,.

To a round bottom flask was added one or more of the following "core" compounds: tris(hydroxymethyl)ethane ("C"), pentaerythritol ("D"), xylitol ("E"), dipentaerythritol ("F") made from the above cores. These were dissolved in tetrahydrofuran. <NUM> molar equivalents (per -OH of the hydroxyl terminated cores or dendrimers) of Dodecanoic Acid were added to the solution of cores. To these reagents were added <NUM> molar equivalents (per -OH of the hydroxyl terminated cores or dendrimers) of dicyclohexylcarbodiimide and <NUM> molar equivalents (per -OH of hydroxyl-terminated core or of dendrimer) of <NUM>-dimethylaminopyridine (DMAP).

<FIG> shows MALDI-TOF MS data for IMS <NUM> C-F, the product of dodecanoic acid functionalization of cores C, D, E, and F.

To a round bottom flask was added one or more of the following "core" compounds: tripentaerythritol ("H") made from the above cores. These were dissolved in tetrahydrofuran. <NUM> molar equivalents (per -OH of the hydroxyl terminated cores or dendrimers) of Octadecanoic Acid were added to the solution of cores. To these reagents were added <NUM> molar equivalents (per -OH of the hydroxyl terminated cores or dendrimers) of dicyclohexylcarbodiimide and <NUM> molar equivalents (per -OH of hydroxyl-terminated core or of dendrimer) of <NUM>-dimethylaminopyridine (DMAP).

<FIG> shows MALDI-TOF MS data for IMS <NUM>, the product of octadecanoic acid functionalization of core H (IMS018H).

<FIG> shows a scheme for the synthesis of IMS <NUM> C1-F1.

To a round bottom flask was added one or more of the following "core" compounds: the first generation of bis-MPA dendrimers from the following <NUM> cores: tris(hydroxymethyl)ethane ("C1"), pentaerythritol ("<NUM>"), xylitol ("E1"), dipentaerythritol ("F1") made from the above cores. These were dissolved in tetrahydrofuran. <NUM> molar equivalents (per -OH of the hydroxyl terminated cores or dendrimers) of Octadecanoic Acid were added to the solution of cores. To these reagents were added <NUM> molar equivalents (per -OH of the hydroxyl terminated cores or dendrimers) of dicyclohexylcarbodiimide and <NUM> molar equivalents (per -OH of hydroxyl-terminated core or of dendrimer) of <NUM>-dimethylaminopyridine (DMAP).

<FIG> shows MALDI-TOF MS data for IMS <NUM> C1-F1, the product of octadecanoic acid functionalization of cores C1, D1, E1, and F1.

To a round bottom flask was added one or more of the following "core" compounds: tris(hydroxymethyl)ethane ("C"), pentaerythritol ("D"), xylitol ("E"), dipentaerythritol ("F") made from the above cores. These were dissolved in tetrahydrofuran. <NUM> molar equivalents (per -OH of the hydroxyl terminated cores or dendrimers) of Docosanoic Acid were added to the solution of cores. To these reagents were added <NUM> molar equivalents (per -OH of the hydroxyl terminated cores or dendrimers) of dicyclohexylcarbodiimide and <NUM> molar equivalents (per -OH of hydroxyl-terminated core or of dendrimer) of <NUM>-dimethylaminopyridine (DMAP).

<FIG> shows MALDI-TOF MS data for IMS <NUM> C-F, the product of docosanoic acid functionalization of cores C, D, E, and F.

To a round bottom flask was added one or more of the following "core" compounds: tris(hydroxymethyl)ethane ("C"), pentaerythritol ("D"), xylitol ("E"), dipentaerythritoi ("F") made from the above cores. These were dissolved in tetrahydrofuran. <NUM> molar equivalents (per -OH of the hydroxyl terminated cores or dendrimers) of Benzoic Acid were added to the solution of cores. To these reagents were added <NUM> molar equivalents (per -OH of the hydroxyl terminated cores or dendrimers) of dicyclohexylcarbodiimide and <NUM> molar equivalents (per -OH of hydroxyl-terminated core or of dendrimer) of <NUM>-dimethylaminopyridine (DMAP).

<FIG> shows MALDI-TOF MS data for IMS <NUM> C-F, the product of benzoic acid functionalization of cores C, D, E, and F.

<FIG> shows a scheme for the synthesis of IMS <NUM> C-F.

To a round bottom flask was added one or more of the following "core" compounds: tris(hydroxymethyl)ethane ("C"), pentaerythritol ("D"), xylitol ("E"), dipentaerythritol ("F") made from the above cores. These were dissolved in tetrahydrofuran. <NUM> molar equivalents (per -OH of the hydroxyl terminated cores or dendrimers) of Triiodobenzoic Acid were added to the solution of cores. To these reagents were added <NUM> molar equivalents (per -OH of the hydroxyl terminated cores or dendrimers) of dicyclohexylcarbodiimide and <NUM> molar equivalents (per -OH of hydroxyl-terminated core or of dendrimer) of <NUM>-dimethylaminopyridine (DMAP).

The reaction mixture was stirred vigorously for approximately <NUM> hours at standard temperature and pressure. The reaction was monitored by MALDI-TOF MS to determine completion of the reaction for each of the cores present in the reaction. After complete esterification is observed by MALDI-TOF MS, the flask contents were transferred to a separatory funnel diluted with dichloromethane, extracted twice with <NUM> aqueous NaHSO<NUM> (sodium bisulfate) and extracted twice with <NUM> aqueous NaHCO<NUM> (sodium bicarbonate). The organic layer was reduced in vacuo to concentrate the sample. A MALDI-TOF MS spectra of the purified product confirmed the purity of the mixture of esterified products and is shown in <FIG>.

<FIG> shows MALDI-TOF MS data for IMS <NUM> C-F, the product of triiodobenzoic acid functionalization of cores C, D, E, and F.

To a round bottom flask was added one or more of the following "core" compounds: tripentaerythritol ("H") made from the above cores. These were dissolved in tetrahydrofuran. <NUM> molar equivalents (per -OH of the hydroxyl terminated cores or dendrimers) of Triiodobenzoic Acid were added to the solution of cores. To these reagents were added <NUM> molar equivalents (per -OH of the hydroxyl terminated cores or dendrimers) of dicyclohexylcarbodiimide and <NUM> molar equivalents (per -OH of hydroxyl-terminated core or of dendrimer) of <NUM>-dimethylaminopyridine (DMAP).

<FIG> shows MALDI-TOF MS data for IMS <NUM>, the product of triiodobenzoic acid functionalization of core H.

To a round bottom flask was added one or more of the following "core" compounds: the first generation of bis-MPA dendrimers from the following <NUM> cores: tris(hydroxymethyl)ethane ("C1"), pentaerythritol ("D1"), xylitol ("E1"), dipentaerythritol ("F1") made from the above cores. These were dissolved in tetrahydrofuran. <NUM> molar equivalents (per -OH of the hydroxyl terminated cores or dendrimers) of Triiodobenzoic Acid are added to the solution of cores. To these reagents were added <NUM> molar equivalents (per -OH of the hydroxyl terminated cores or dendrimers) of dicyclohexylcarbodiimide and <NUM> molar equivalents (per -OH of hydroxyl-terminated core or of dendrimer) of <NUM>-dimethylaminopyridine (DMAP).

<FIG> shows MALDI-TOF MS data for IMS <NUM> C1-F1, the product of triiodobenzoate acid functionalization of cores C1, D1, E1 and F1.

<FIG> shows the MALDI-TOF mass spectrum for the product, i.e., N,N,N-Tris-(<NUM>-aminoethyl)amine-tris(triodobenzamide) or N,N',N"-(nitrilotris(ethane-<NUM>,<NUM>-diyl)tris(<NUM>,<NUM>,<NUM>-triiodobenzamide) of the amidation reaction between N,N,N-tris(<NUM>-aminoethyl)amine and <NUM>,<NUM>,<NUM>-triiodobenzoic acid.

<FIG> shows representative example of stability of IMS calibrants under ambient conditions - IMS <NUM> at <NUM> months (bottom panel) and after <NUM> months (top panel) of storage. This data shows after <NUM> months at ambient conditions (e.g., exposed to room temperature, light, and air), no sign of degradation of these calibrants was observed.

Claim 1:
A composition comprising at least two calibrant compounds or salts thereof, or cationic complexes thereof, or anionic complexes thereof, wherein the at least two calibrant compounds or the salts thereof or the cationic complexes thereof, or the anionic complexes thereof comprise an alcohol functionalized core, and peripheral functionalities,
wherein the alcohol functionalized core is selected from the group consisting of:
<CHM>
and
wherein the peripheral functionalities are esters prepared by coupling the alcohol functionalities of the cores with a carboxylic acid, or an activated ester, or an activated carboxylic acid derivative having a chemical structure of Formula II:
<CHM>
wherein m = <NUM>-<NUM>.