Larry D. Bowers

The clinical significance of cyclosporine metabolites.

Cyclosporine (CsA) is extensively metabolized, with over 14 metabolites having been characterized to date. The confirmation of structure and purity is a prerequisite for studies involving CsA metabolites. Analytical techniques such as fast atom bombardment/mass spectroscopy (FAB/MS), tandem mass spectrometry (MS), 1H- and 13C-nuclear magnetic resonance (NMR) can be used for such purposes. In vitro experiments indicate that metabolites are considerably less immunosuppressive and toxic than CsA. In vivo studies have been hampered by sufficient quantities of metabolites and a suitable animal model. Preliminary results in the rat suggest that CsA metabolites are less immunosuppressive and toxic than CsA, although these results must be confirmed using a more suitable animal model. Present data indicate that the routine monitoring of metabolites is not warranted in transplant patients, although additional information is required to confirm these findings.

A radiochemical study of irreversible protein loss on high-performance liquid chromatography column frits

Much success has been achieved in the separation and purification of a wide range of proteins using various high-pressure liquid chromatography techniques. Quantitative analyses of proteins which require 100% mass recovery of the protein are still beset with problems, especially when the total injected amount of protein decreases to below 10 micrograms. Stainless-steel frits have been cited for their deleterious effects on chromatography in general. In addition, the frits have specifically been found to be a significant contributor to irreversible protein loss--particularly when protein sample sizes are on the order of 1 microgram or less. The findings presented below should therefore be of concern to those using HPLC for protein work.

Solvent strength studies on polystyrene-divinylbenzene columns

Abstract The retention behavior of a series of non-polar solutes on two polystyrene-divinylbenzene high-performance liquid chromatography supports was studied. The solvent strength of several organic solvents was measured over a range of solvent compositions. Poor peak symmetry was observed for some solvents, and this appears to be correlated with the ability of the solvent to swell the polymer. Addition of a solvent that cna swell the polymer to another solvent which cannot usually improves peak shape. The observed improvement in peak symmetry also correlates with the ability of the ternary mixture to decrease the void volume of the column.

Interlaboratory Agreement of Insulin-like Growth Factor 1 Concentrations Measured by Mass Spectrometry

BACKGROUND Insulin-like growth factor 1 (IGF-1)(7) is a key mediator of growth hormone (GH) action and a well-characterized biomarker of GH abuse. Current immunoassays for IGF-1 suffer from poor concordance between platforms, which makes comparison of results between laboratories difficult. Although previous work has demonstrated good interlaboratory imprecision of LC-MS/MS methods when plasma is supplemented with purified proteins, the interlaboratory imprecision of an endogenous protein in the nanogram-per-milliliter concentration range has not been reported. METHODS We deployed an LC-MS/MS method to quantify serum IGF-1 in 5 laboratories using 5 different instruments and analyzed 130 healthy human samples and 22 samples from patients with acromegaly. We determined measurement imprecision (CV) for differences due to instrumentation, calibration curve construction, method of calibration, and reference material. RESULTS Instrument-dependent variation, exclusive of digestion, across 5 different instrument platforms was determined to be 5.6%. Interlaboratory variation was strongly dependent on calibration. Calibration materials from a single laboratory resulted in less variation than materials made in individual laboratories (CV 5.2% vs 12.8%, respectively). The mean imprecision for 152 samples between the 5 laboratories was 16.0% when a calibration curve was made in each laboratory and 11.1% when a single-point calibration approach was used. CONCLUSIONS The interlaboratory imprecision of serum IGF-1 concentrations is acceptable for use of the assay in antidoping laboratories and in standardizing results across clinical laboratories. The primary source of variability is not derived from the sample preparation but from the method of calibration.

Cocaine reduction in unmotivated crack users using carbamazepine versus placebo in a short-term, double-blind crossover design.

On the basis of cocaine‐caused kindling in animals and the usefulness of carbamazepine in treating kindling‐type seizures, carbamazepine has been tried in clinical settings with cocaine‐dependent individuals. This report presents findings of a 20‐day, double‐blind, placebo‐controlled crossover study in 32 non–treatment‐motivated, paid, chronic crack cocaine users. Carbamazepine significantly lowered the mean number of positive urine specimens compared with placebo. Of clinical importance, serum carbamazepine levels of 4 μg/ml (17 μmol/L) or more were associated with greater improvement. A consistent, clinically important trend linked therapeutic levels with improvement for all subjective and objective outcome variables. Comparison of daily acknowledged cocaine use or professed cocaine abstinence, with cocaine use indicated by daily urinalysis in these chronic cocaine users, has suggested the possibility of cocaine saturation as an important methodologic limitation inherent in outpatient studies of cocaine use in humans.

Pharmacokinetics of FK506 After Intravenous and Oral Administration in Patients Awaiting Renal Transplantation

The authors examined the safety and pharmacokinetics of FK506, a new hepatically metabolized immunosuppressant, after single‐dose intravenous (IV) infusion (20 μg · kg−1 · 4 hours−1) and oral (80 μg/kg) administration in six nondialysis patients, aged 27 to 53 years, with chronic renal failure awaiting transplantation. A two‐period, randomized, crossover study protocol was used with blood samples drawn for 72 hours after each dose and a washout period of 4 days. Whole‐blood FK506 levels were determined using a standard, two‐step, nonspecific enzyme immunoassay. There were no significant changes in vital signs, EKG, or complete laboratory test battery for any patient during the entire study period. No side effects were noted after IV or oral FK506 dosing. Mean ± SD distribution half life was 0.9 ± 0.2 hours, elimination half life (t1/2β) 33 ± 8 hours, total body clearance (CL) 2.4 ± 1.1 L/hour, and bioavailability 14 ± 12%. There was no significant correlation between serum creatinine (Cr) and CL (r = 0.36) or between Cr and t1/2β (r = −0.30). It was found that FK506 is incompletely and erratically absorbed after oral administration and is rapidly distributed outside the blood compartment after IV dosing. An extended sampling period seems necessary to accurately characterize the slow elimination phase of FK506.

Abuse of performance-enhancing drugs in sport.

The use of stimulants such as amphetamine, cocaine, strychnine, and ephedrine in modern sport to improve focus and delay fatigue was reported in the early 1900s. In response to the death of a Danish cyclist at the 1960 Olympics, the death of a UK cyclist at the Tour de France, and other evidenc

Characterization of immobilized β-Glucuronidase in aqueous and mixed solvent systems

Abstract β-Glucuronidase (β- d -glucuronide glucuronosohydrolase, EC 3.2.1.31) was covalently attached to alkylamine-controlled pore glass via a glutaraldehyde immobilization scheme. The activity of this immobilized β-glucuronidase was studied with respect to several kinetic parameters in comparison with the behavior of the soluble enzyme. K m values for p -nitrophenyl glucuronide, estriol-3-glucuronide, and estriol-16β-glucuronide were determined. For each substrate the K m was essentially the same, 0.2 mM, and this value did not change when the enzyme was immobilized. The soluble and immobilized enzyme both displayed a relatively broad pH maximum centered at pH 6.8 for all substrates. Several organic-aqueous mixtures including methanol, ethanol, acetonitrile and ethylene glycol were tested, and their effects on the activity of immobilized β-glucuronidase were similar to those found for the soluble enzyme. Long-term (1 year) storage stability tests of the immobilized enzyme were carried out. The immobilized enzyme retained 40% of its initial activity after 1 year and was very robust towards most of the organic solvents tested.

Quantitative fractionation of serum bilirubin species by reversed-phase high-performance liquid chromatography

A rapid high-performance liquid chromatographic method is described for separating and quantifying four bilirubin species present in serum: bilirubin, bilirubin monoglucuronide, bilirubin diglucuronide, and bilialbumin. Sample preparation consists of dilution with ascorbic acid and dimethylsulfoxide and filtration to remove solid materials. The diluted serum was injected directly onto a wide-pore (300 A) reversed-phase column and the bilirubin species eluted with a water-isopropanol gradient. Excellent agreement was found between the total bilirubin concentration measured by the high-performance liquid chromatographic method and a diazotized sulfanilic acid procedure.

A radiochemical study of irreversible adsorption of proteins on reversed-phase chromatographic packing materials

A radiochemical study of the irreversible adsorption of proteins on commercial reversed-phase HPLC packing materials is reported. The conditions of study are similar to those used in HPLC separation of protein. The effects of the amount and contact time of two proteins, ovalbumin and cytochrome c, are reported. Additional results include the effect of column pretreatment with protein, silanophilic mobile-phase blocking agent, and type of packing material on the extent of irreversible adsorption. The loss process is shown to be at least biphasic and the mechanisms of loss distinct for different proteins.