Laboratory Testing Case Studies

Challenge: A sensitive and specific bioanalytical method suitable for quantification of an immunomodulator compound in plasma matrix for human, rat and mouse samples at a low detection level, LLOQ = 10 pg/mL, was developed by Diteba's scientists for pre-clinical and clinical studies.  This compound is a member of a class of immunomodulatory drugs that possess γ-glutamyl or β-aspartyl moieties, which was discovered by Russian scientists and is being examined for efficacy in several indications.  The synthesized molecule contains two chiral centers and therefore can exist as four configurations: two enantiomers and two diastereoisomers.

Challenge: A sponsor had developed a promising group of "first in class" potent small molecule compounds for cancer therapy.  The research scientists and medicinal chemists used structure-based drug design to derive compounds that target important proteins and therapeutic pathways in cancer.  One of these candidates has shown to be a very potent, yet highly selective growth inhibitor of many cancer types, including non-small cell lung cancer, colon cancer and leukemia.

Challenge: Diteba was contracted to develop and validate a specific quantitative analytical method capable of simultaneously detecting and quantifying all three analytes (vitamers) in different biological fluids and support a clinical study as well as for toxicological studies on small animal models.  It should be noted that previously made attempts by a reputable US-based contract research organization to develop a suitable method for quantification of PLP, PL and 4-PA by Liquid Chromatographic Tandem Mass Spectrometry (LC/MS/MS) technique had failed due to the complexity of the project.  All three analytes appear at high endogenous levels in biological fluids which in turn complicates the analytical procedure.

Challenge: Diteba was contracted to develop and validate an appropriate analytical method capable of quantifying the levels of L-tryptophan (0.06 μmol/L to 222 μmol/L) and kynurenine (0.09 μmol/L to 9.84 μmol/L) in human serum.  Several HPLC methods have been developed over time to determine serum tryptophan and kynurenine.  However, none of this research has met desirable levels of sensitivity or reproducibility.  The methods described in the literature were complicated by the interference of other amino acids with the kynurenine UV absorption in HPLC chromatography.  In addition to the analytical method development and validation exercise, it was necessary to develop a simple and accurate method of extraction for tryptophan and kynurenine from patients' blood serum.  The extraction procedures were complicated because of the nature of the samples.  The patients' samples were infected with a chronic hepatitis C which required extreme precaution in drawing and handling these samples.

1. Exhibiting linear pharmacokinetics, i.e., proportional to dose administrated from approximately 10 pg/mL to 5 000 pg/mL;
2. Assessing clinical studies conducted for a population of patients with inoperable chronic pulmonary hypertension (which are usually dosed 45-125 ng/kg/min); and
3. Proving that the synthetic prostacyclin analogue plasma concentrations remain linearly proportional to doses up to 125 ng/kg/min.

This compound is a long-acting analogue of prostacycline and it is administered in low doses (1.25-15 ng/kg/min) by continuous intravenous infusion to patients with pulmonary hypertension.  The drug improves survival and the hemodynamics in these patients.  However, the treatment is limited by a very short half-life (1-2 minutes) and chemical instability requiring continuous intravenous infusion and refrigeration during administration.  Some research has been published with regards to biotransformation and pharmacokinetics for Beraprost, a synthetic analog of prostacyclin.

Challenge: Typical oligonucleotides used as therapeutics or potential therapeutic compounds are 15 to 30 nucleotides (nt) long. Oligonucleotides <15 nt can be readily resolved by HPLC technology, however the separation of longer sequences are more challenging. Ion-pair reversed-phase (IP-RP) HPLC has been traditionally used for oligonucleotides analysis.  The traditional IP-RPLC eluent system typically uses TEA or TEAA or HFIP and the ion-pairing agent triethylammonium ion and a C18, column at 60^oC. Under such conditions, the column's hydrolytic stability becomes crucial and is important to prevent the potential contribution of the oligonucleotide secondary structure from impacting retention.

The primary technological objective of this project was to develop a more sensitive and reproducible analytical method for detection and quantification of insulin and its metabolites in animal blood serum.  The secondary technological objective was to develop a procedure to monitor drug dose uniformity as well as the in-vitro release rate.