All known naturally occurring linear cationic peptides adopt an amphipathic ␣-helical conformation upon binding to lipids as an initial step in the induction of cell leakage. We designed an 18-residue peptide, (KIGAKI) 3 -NH 2 , that has no amphipathic character as an ␣-helix but can form a highly amphipathic ␤-sheet. When bound to lipids, (KIGAKI) 3 -NH 2 did indeed form a ␤-sheet structure as evidenced by Fourier transform infrared and circular dichroism spectroscopy. The antimicrobial activity of this peptide was compared with that of (KI-AGKIA) 3 -NH 2 , and it was better than that of GMASKA-GAIAGKIAKVALKAL-NH 2 (PGLa) and (KLAGLAK) 3 -NH 2 , all of which form amphipathic ␣-helices when bound to membranes. (KIGAKI) 3 -NH 2 was much less effective at inducing leakage in lipid vesicles composed of mixtures of the acidic lipid, phosphatidylglycerol, and the neutral lipid, phosphatidylcholine, as compared with the other peptides. However, when phosphatidylethanolamine replaced phosphatidylcholine, the lytic potency of PGLa and the ␣-helical model peptides was reduced, whereas that of (KIGAKI) 3 -NH 2 was improved. Fluorescence experiments using analogs containing a single tryptophan residue showed significant differences between (KIGAKI) 3 -NH 2 and the ␣-helical peptides in their interactions with lipid vesicles. Because the data suggest enhanced selectivity between bacterial and mammalian lipids, linear amphipathic ␤-sheet peptides such as (KIGAKI) 3 -NH 2 warrant further investigation as potential antimicrobial agents.

Performance of the Roche Online KIMS (kinetic interaction of microparticles in solution) benzodiazepine (BZD) immunoassay (IA) with and without beta-glucuronidase treatment was evaluated on a Hitachi Modular automated IA analyzer calibrated using nordiazepam at 100 ng/mL. Reproducibility, linearity, accuracy, sensitivity, and interferences were evaluated. Precision of the assay (percent coefficient of variation (%CV)) with and without addition of the enzyme was less than 6% and 9%, respectively, with linearity (r(2) value of 0.9578 and 0.9746), respectively. Between-run precision of a 125 ng/mL nordiazepam control (n = 287) over 67 days, produced a %CV of 13.6% for the hydrolytic assay. Modification of the BZD assay to include automated hydrolysis of urinary BZD glucuronide conjugates was evaluated using three glucuronidated BZD standards prepared at concentrations ranging from 250 to 10,000 ng/mL. With hydrolysis, temazepam, oxazepam, and lorazepam glucuronides, produced cross-reactivities of 25%, 15%, and 20%, respectively. Without hydrolysis, the glucuronidated BZD standards produced less than 1% cross-reactivity in the assay. The ability of the assay to differentiate between positive and negative samples was evaluated by assaying 20 negative urine samples and serial dilutions of certified drug-free urine fortified with 28 different BZDs. All of the negative and positive urine samples produced the appropriate screening result. Cross-reactivities of 27 different BZDs, calculated as the normalized IA response divided by the BZD concentration that produced a response approximately equivalent to the response of a 100 ng/mL nordiazepam standard and multiplied by 100, ranged from 15% to 149%. Human urine samples (n = 28) that were previously found to contain BZDs by gas chromatography-mass spectrometry (GC-MS) also produced a positive BZD IA result. The IA was challenged with 78 potentially interfering compounds, and none produced a positive BZD response. As a part of the validation, a large number of human urine samples (29,500) were assayed using the modified Online BZD IA method to evaluate the performance of the method in production. Of the 29,500 samples tested, 80 produced a positive IA result. Analysis by GC-MS confirmed the presence of at least 1 BZD compound in 61 of the samples corresponding to a confirmation rate of 76%. The Online BZD IA modified by the automatic addition of beta-glucuronidase appears well adapted for the rapid detection of BZDs and their metabolites in human urine.

The performances of four immunoassays (DRI amphetamines, DRI ecstasy, Abuscreen ONLINE amphetamines, and a modified Abuscreen ONLINE amphetamines) were evaluated for control failure rates, sensitivity, and specificity for amphetamine (AMP), methamphetamine (MTH), 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxymethamphetamine (MDMA). The two DRI reagents and the ONLINE reagents were run according to manufacturer specifications using a Roche Hitachi Modular DDP system. The modified ONLINE reagent was calibrated with MDMA and had 16mM sodium periodate added to the R2 reagent. These assays were run on approximately 27,500 human urine samples and 7000 control urine samples prepared at 350 and 674 ng/mL over the course of 8 days. All assays were calibrated using a single point, qualitative cutoff standard with the manufacturer-recommended compound at the Department of Defense cutoff (500 ng/mL). Gas chromatography-mass spectrometry (GC-MS) confirmation was conducted on screened-positive samples. Control performance for the manufacturer recommended assays was excellent, with a maximum qualitative control failure rate of 2.03%. The modified ONLINE reagent demonstrated poor control performance with a maximum failure rate of 38.3% and showed no improved MDMA sensitivity when compared with the ONLINE reagent; the confirmation rate (20%) was improved when compared with the production ONLINE reagent (8%). The DRI ecstasy reagent provided improved sensitivity for MDMA as compared with the ONLINE reagent, with approximately 23% more samples screening and confirming positive for MDMA and a confirmation rate of approximately 90%. The DRI methamphetamine reagent had a low confirmation rate (6% or less) and produced numerous positives for samples with only ephedrine or pseudoephedrine present.

In an effort to determine a practical, efficient, and economical alternative for the use of a radioimmunoassay (RIA) for the detection of lysergic acid diethylamide (LSD) in human urine, the performance of two photometric immunoassays (Dade Behring EMIT II and Microgenics CEDIA) and the Diagnostics Products Corp. (DPC) RIA were compared. Precision, accuracy, and linearity of the 3 assays were determined by testing 60 replicates (10 for RIA) at 5 different concentrations below and above the 500-pg/mL LSD cut-off. The CEDIA and RIA exhibited better accuracy and precision than the EMIT II immunoassay. In contrast, the EMIT II and CEDIA demonstrated superior linearity r2 = 0.9809 and 0.9540, respectively, as compared with the RIA (r2 = 0.9062). The specificity of the three assays was assessed using compounds that have structural and chemical properties similar to LSD, common over-the-counter products, prescription drugs and some of their metabolites, and other drugs of abuse. Of the 144 compounds studied, the EMIT II cross-reacted with twice as many compounds as did the CEDIA and RIA. Specificity was also assessed in 221 forensic human urine specimens that previously screened positive for LSD by the EMIT II assay. Of these, only 11 tested positive by CEDIA, and 3 were positive by RIA. This indicated a comparable specificity performance between CEDIA and RIA. This also was consistent with a previously reported high false-positive rate of EMIT II (low specificity). Each of the immunoassays correctly identified LSD in 23 out of 24 human urine specimens that had previously been found to contain LSD by gas chromatography-mass spectrometry at a cut-off concentration of 200 pg/mL. The CEDIA exhibited superior precision, accuracy, and decreased cross-reactivity to compounds other than LSD as compared with the EMIT II assay and does not necessitate the handling of radioactive materials.