Determination of caprolactam and 6-aminocaproic acid in human urine using hydrophilic interaction liquid chromatography-tandem mass spectrometry.

A simple and rapid assay based on hydrophilic interaction liquid chromatography with tandem mass spectrometry has been first developed and validated for simultaneous determination of caprolactam (CA) and 6-aminocaproic acid (6-ANCA) in human urine using 8-aminocaprylic acid as internal standard. A 20µL aliquot of urine was injected directly into the liquid chromatography tandem mass spectrometry (LC-MS-MS) system. The analytes were separated on a Phenomenex Luna HILIC column with gradient elution. Detection was performed on Triple Quadrupole LC-MS in positive ions multiple reaction monitoring mode using electrospray ionization. The calibration curves were linear (r(2)≥0.995) over the concentration range from 62.5 to 1250ng/mL for CA and 31.25 to 1000ng/mL for 6-ANCA. The detection limits of CA and 6-ANCA were 62.5 and 15.6ng/mL, respectively. The intra-day and inter-day precisions were within 8.7% and 9.9%, respectively. The intra-day and inter-day accuracy were between 5.3% and 3.5%, and between 6.1% and 6.6%, respectively. The method proved to be simple and time efficient, and was successfully applied to evaluate the kinetics of caprolactam in one unusual case of caprolactam poisoning.

Simultaneous measurement of the cell-differentiating agent hexamethylene bisacetamide and its metabolites by gas chromatography.

Hexamethylene bisacetamide (HMBA) is a potent in vitro differentiating agent that has clinical potential as an anticancer drug both as a single agent and as a component of combination therapy. A sensitive and efficient GC method for the isolation, derivatization, and measurement of both HMBA and its two major metabolites in plasma and urine in a single analysis is described. In situ carbamylation of the biological sample with diethylpyrocarbonate forms the urethane derivative of the basic N-acetyl diaminohexane metabolite and allows analyte isolation and concentration by solid-phase extraction. Subsequent formation of the n-butyl ester of 6-acetamidohexanoic acid, the major metabolite, provides a derivatized biological extract that can be rapidly analyzed by temperature-programmed GC. The quantitative extraction and the efficient derivatization steps provide a limit of quantitation of 0.05 mM (10 micrograms/ml) for all analytes with a precision better than 8% for the range of in vitro activity (0.1-2.0 mM). This method is amenable to automation and is well-suited for the analysis of clinical samples.

High performance liquid chromatographic assay of Amicar, epsilon-aminocaproic acid, in plasma and urine after pre-column derivatization with o-phthalaldehyde for fluorescence detection.

A simple, reliable and highly sensitive procedure was devised for measuring the levels of Amicar in blood and urine. 100 microL of serum or urine sample was added to 10 microL of a 10% w/v zinc sulfate solution and 100 microL of methanol, as previously described (Lam et al., 1980) for the removal of proteins by precipitation. 50 microL of the supernatant was then mixed with 300 microL of 1 M borate buffer containing D-valine as the internal standard before derivatization with o-phthalaldehyde. The amino acids were then separated by a stereoselective reversed-phase system using a mobile phase containing 10% of acetonitrile in 2.5 mM Cu(II) complexes of L-proline. The chromatography is highly selective, resolving Amicar from L-valine which in turn is resolved from its unnatural D-antipode, the internal standard. The procedure including sample preparation and separation required a total of 15 min. As little as 50 ng/mL of Amicar in body fluids could be detected as the o-phthalaldehyde derivative by fluorescence.

Effect of probenecid on the kinetics of epsilon-aminocaproic acid.

Healthy male subjects received, 1 wk apart, single oral doses of epsilon-aminocaproic acid (EACA) 100 mg/kg alone, EACA within probenecid (0.5 gm), or EACA 2 hr after 2.0 gm probenecid. Probenecid (2.0 gm) reduced the 8-hr urinary clearance and recovery of EACA by 50% without affecting plasma kinetics. Recovery of EACA in urine rose to 78% of the dose 48 hr after EACA. Plasma clearance of EACA did not differ from control EACA urinary clearance when 0.5 gm probenecid was given with EACA. In both cases all the EACA dose was recovered in urine within 8 hr.