Amino Acid Fingerprinting of Authentic Nonfat Dry Milk and Skim Milk Powder and Effects of Spiking with Selected Potential Adulterants.

A collaborative study was undertaken in which five international laboratories participated to determine amino acid fingerprints in 39 authentic nonfat dry milk (NFDM)/skim milk powder (SMP) samples. A rapid method of amino acid analysis involving microwave-assisted hydrolysis followed by ultra-high performance liquid chromatography-ultraviolet detection (UHPLC-UV) was used for quantitation of amino acids and to calculate their distribution. The performance of this rapid method of analysis was evaluated and was used to determine the amino acid fingerprint of authentic milk powders. The distribution of different amino acids and their predictable upper and lower tolerance limits in authentic NFDM/SMP samples were established as a reference. Amino acid fingerprints of NFDM/SMP were compared with selected proteins and nitrogen rich compounds (proteins from pea, soy, rice, wheat, whey, and fish gelatin) which can be potential economically motivated adulterants (EMA). The amino acid fingerprints of NFDM/SMP were found to be affected by spiking with pea, soy, rice, whey, fish gelatin and arginine among the investigated adulterants but not by wheat protein and melamine. The study results establish an amino acid fingerprint of authentic NFDM/SMP and demonstrate the utility of this method as a tool in verifying the authenticity of milk powders and detecting their adulteration.

The determination of the plate count for large molecules under reversed-phase gradient conditions.

The true performance of HPLC columns in the gradient separation of macromolecules can be assessed by measuring the true plate count, if we know the retention factor of the analyte at the point of elution. We are demonstrating in this short communication how this can accomplished in a straightforward fashion. The procedure used here is a significant simplification over previous approaches, and enables the chromatographer to do so without complex algebra or additional experiments.

Differences in preparative loadability between the charged and uncharged forms of ionizable compounds.

Ionizable compounds experience a drastic difference in preparative loadability as a function of pH. It can be shown that the preparative loadability of a compound in the ionic form is by a factor of 20 or more inferior to the loadability of the same compound in the unionized form. In this paper, we demonstrate the reason for this behavior, and show practical applications of the principle.

IRDye78 conjugates for near-infrared fluorescence imaging.

The detection of human malignancies by near-infrared (NIR) fluorescence will require the conjugation of cancer-specific ligands to NIR fluorophores that have optimal photoproperties and pharmacokinetics. IRDye78, a tetra-sulfonated heptamethine indocyanine NIR fluorophore, meets most of the criteria for an in vivo imaging agent, and is available as an N-hydroxysuccinimide ester for conjugation to low-molecular-weight ligands. However, IRDye78 has a high charge-to-mass ratio, complicating purification of conjugates. It also has a potentially labile linkage between fluorophore and ligand. We have developed an ion-pairing purification strategy for IRDye78 that can be performed with a standard C18 column under neutral conditions, thus preserving the stability of fluorophore, ligand, and conjugate. By employing parallel evaporative light scatter and absorbance detectors, all reactants and products are identified, and conjugate purity is maximized. We describe reversible and irreversible conversions of IRDye78 that can occur during sample purification, and describe methods for preserving conjugate stability. Using seven ligands, spanning several classes of small molecules and peptides (neutral, charged, and/or hydrophobic), we illustrate the robustness of these methods, and confirm that IRDye78 conjugates so purified retain bioactivity and permit NIR fluorescence imaging of specific targets.