Use of truncated areas to measure extent of drug absorption in bioequivalence studies: effects of drug absorption rate and elimination rate variability on this metric.

PURPOSE: To compare the applicability and accuracy of truncated area (AUCt; where t represents truncated time) versus area to the last quantifiable time point [AUC(O-T)] for assessing bioequivalence. Drugs with either very low or very high intra-subject variability in clearance (CL) were selected for study. Clearance variability was defined by the number of subjects with a quantifiable plasma value (Cp) at each collection time from 24 hrs to last collection time (T). METHODS: Data for amiodarone and danazol, drugs with different distributions of subject CL were examined. For amiodarone, the number of subject samples observed (test + reference) at the time of the last quantifiable concentrations was 60 at 240 hrs(T), 16 at 144 hrs and 4 at 96 hrs: while danazol had 4 at 96 hr(T), 3 at 72 hrs, 16 at 60 hrs, 7 at 48 hrs, 14 at 36 hrs, 11 at 24 hrs, 13 and 2 at 16 and 12 hrs, respectively. Simulations (Scenarios A and B) were performed to obtain populations (N = 24) with CL patterns similar to those of amiodarone and danazol. For scenario A (CL pattern similar to amiodarone), log-normally distributed CL values (28.8 L/HR) with intra-subject coefficient of variation (CV) of 25%, 40% and 60% gave the desired CL pattern. Scenario B (CL pattern similar to danazol) required that a subpopulation with an increase in CL of 40% from baseline (i.e., 40.32 L/HR) in 5%, 10% and 20% of the population represent the desired distribution. Power was evaluated by the percentage of times the simulated trials were declared bioequivalent (i.e., the number of times the test vs. reference 90% CI was within 80-125%), while accuracy was determined when the true difference in fraction absorbed (i.e., 1.25) was within the CI. Each simulation was repeated 300 times. RESULTS: The simulation results for Scenario A indicated that the statistical results using truncated area (AUCt) had power and accuracy equivalent to that obtained using the AUC(O-T) metric. However, results for Scenario B indicated that AUCt had less power and accuracy than that obtained using AUC(0-T). The confidence interval (CI) for amiodarone was the same whether AUC (0-T) or AUCt was used as the metric for extent, while for danazol, the AUC(0-T) and AUCt differed in the lower limit by 7%. CONCLUSIONS: The truncated area, AUCt, has the greatest power and accuracy when the population clearance is such that most subjects have measurable plasma concentrations at last collection time(T), resulting in a proportional loss of data from each subject.

Bacillus stearothermophilus disk assay for detection of residual penicillins in milk: collaborative study.

A collaborative study was performed on a Bacillus stearothermophilus paper disk method designed to detect residual levels of 4 antibiotic drugs in whole market milk. This method is a modification of an earlier procedure developed for the International Dairy Federation. Whole milk samples spiked at low levels with ampicillin, cephapirin, cloxacillin, and penicillin G were sent frozen to 11 collaborating laboratories with instructions to assay them promptly according to the method provided. Five of the laboratories reported inconclusive results due to technical difficulties encountered with the method. The 6 remaining laboratories all detected levels of 0.005-0.008 microgram or unit/mL for penicillin G, ampicillin, and cephapirin and 0.05-0.08 microgram/mL for cloxacillin. The most commonly used official methods, the Sarcina lutea (Micrococcus luteus) cylinder plate method and the Bacillus subtilis paper disk method, can detect levels of 0.01 and 0.05 unit penicillin G/mL, respectively. The B. stearothermophilus method is rapid, simple to perform, and more sensitive than present official methods. The method has been adopted as official first action for the detection of penicillins in milk.

Detection of residual penicillins in milk by using a Bacillus stearothermophilus disk assay.

A paper disk method based on a procedure described by the International Dairy Federation is presented for the detection of 4 beta-lactam antibiotics (penicillin G, ampicillin, cephapirin, and cloxacillin) in whole milk. Bacillus stearothermophilus var. calidolactis, prepared as a stable spore suspension, was used as the test organism. Levels of 0.005 unit penicillin G/ml, 0.005 microgram ampicillin or cephapirin/ml, and 0.05 microgram cloxacillin/ml were readily detected in whole milk. Results were produced in 3-4 hr. The method offers several advantages, including greater simplicity, sensitivity, and rapidity, over methods now commonly used to detect residual levels of these drugs in milk.

Evaluation of two microbiological methods for detecting residual antibiotics in milk.

Two methods described in the AOAC Official Methods of Analysis for the detection of penicillin residues in whole milk were evaluated to determine the capability of each method to detect residues of 12 antibiotics used in the dairy industry. The first method, a cylinder-plate method that uses Sarcina lutea as the test organism, detected levels of 1 ppm of 8 of the 12 antibiotics tested. The second method, using paper disks with Bacillus subtilis as the test organism, detected approximately 1 ppm of only 4 antibiotics. This disk method was unable to detect less than 40 ppm of 5 of the antibiotics tested. The data indicate that the S. lutea cylinder-plate technique is more sensitive to more antibiotics than the B. subtilis disk method and is far superior for screening purposes.