Catching Fakes: New Markers of Urine Sample Validity and Invalidity.

Urine drug testing is common for workplace drug testing, prescription management, emergency medicine and the criminal justice system. Unsurprisingly, with the significant consequences based upon the results of urine drug testing, a donor in need of concealing the contents of their sample is highly motivated to cheat the process. Procedures and safeguards ensuring sample validity are well known, and include measuring sample temperature at the time of collection, and laboratory measurements of creatinine, specific gravity and pH. Synthetic urine samples are available and are designed to deceive all aspects of urine drug testing, including validity testing. These samples are sophisticated enough to contain biological levels of creatinine, and are at a physiological pH and specific gravity. The goal of our research was to develop new procedures designed to distinguish authentic samples from masquerading synthetic samples. We aimed to identify substances in commercial synthetic urines not expected to be present in a biological sample distinguishing fake specimens. Additionally, we aimed to identify and employ endogenous compounds in addition to creatinine for identifying biological samples. We successfully identified two compounds present in synthetic urines that are not present in biological samples and use them as markers of invalidity. Four new endogenous markers for validity were successfully evaluated. Validity assessment was further aided by monitoring metabolites of nicotine and caffeine. When the method was applied to patient samples, 2% of samples were identified as inconsistent with natural urine samples, even though they met the current acceptance criteria for creatinine, pH and specific gravity.

Analysis of total and transferrin-bound iron in human serum for pharmacokinetic studies of iron-sucrose formulations.

BACKGROUND: Patients with iron-deficiency anemia benefit from intravenous iron therapies. Development of these pharmaceutical agents requires pharmacokinetic studies monitoring levels of both the administered agent and transferrin-bound iron (TBI). Successful pharmacokinetic methods must discriminate iron species. RESULTS: Routine colorimetric procedures were used to reliably measure total iron and TBI following iron-sucrose administration. Iron was liberated from iron-sucrose allowing the determination of all circulating iron. Solid-phase sample processing allowed the measurement of TBI. Circulating iron-sucrose could then be calculated as the difference between total iron and TBI. CONCLUSION: A reproducible and robust spectrophotometric method was developed and validated for measuring total iron and TBI in human serum following iron-sucrose therapy.

Synthesis and reactivity of 6,7-dihydrogeranylazides: reagents for primary azide incorporation into peptides and subsequent staudinger ligation.

Protein farnesyltransferase (PFTase) catalyzes the attachment of a geranylazide moiety to a peptide substrate, N-dansyl-GCVIA. Because geranylazide is actually a mixture of isomeric, interconverting primary and secondary azides, incorporation of this isoprenoid into peptides can potentially result in a corresponding mixture of prenylated peptides. Here, we first examined the reactivity of geranyl azide in a model Staudinger reaction and determined that a mixture of products is formed. We then describe the synthesis of 6,7-dihydrogeranylazide diphosphate and demonstrate that this compound allows exclusive incorporation of a primary azide into a peptide. The resulting azide-containing peptide was derivatized with a triphenylphosphine-based reagent to generate an O-alkyl imidate-linked product. Finally, we show, using a series of model reactions, that the Staudinger ligation frequently produces small amounts of O-alkyl imidate products in addition to the major amide-linked products. Thus, the alkoxyimidates we have observed as the exclusive products in the reactions of peptides containing prenylated azides also appear to be a common type of product formed using other azide-containing reactants, although at greatly reduced levels. This method for chemical modification of the C-terminus of a protein should be useful for a variety of applications in protein chemistry.