Topical phenylephrine for mydriasis affects rabbit retinal pigment epithelial transport.

Rabbit retinal pigment epithelium (RPE) has an alpha-1 adrenoreceptor that substantially reduces RPE short circuit current (Isc) when stimulated. Since phenylephrine is a potent alpha adrenergic agonist frequently used to obtain pupil dilation, we examined the effect of topically applied phenylephrine for pupil dilation on RPE transport. Naive black dutch-belted rabbits received either one drop of 10% phenylephrine applied at t = 0 and t = 30 min or one drop of 1% cyclopentolate applied at t = 30 min. At t = 50 min, RPE-choroid-sclera explants from these animals were sealed in an Ussing chamber. At t = 60-70 min, 1.4 X 10(-4) M epinephrine was introduced into both sides of the Ussing chamber. The change in Isc produced by epinephrine was measured. The Isc reduction in rabbits receiving phenylephrine was 26% (+/- 5% SD, n = 6). The Isc reduction in rabbits receiving cyclopentolate was 39% (+/- 6% SD, n = 5). These values are significantly different (P less than 0.02, student two-tail t-test). These results indicate that topical phenylephrine reached the RPE in vivo and prestimulated the alpha-1 adrenoreceptors.

Initial observations of rabbit retinal pigment epithelium-choroid-sclera preparations.

Retinal pigment epithelium (RPE)-choroid-sclera preparations from black dutch-belted rabbits were sealed in an Ussing chamber maintained at 37-39 degrees C. Typical preparations produced a spontaneous voltage (Ve) of 12.5 mV (retina side positive) and possessed an electrical resistance (R) of 350 ohm-cm2. Both of these values can be attributed to the RPE. Ouabain and amiloride diminished the Ve without affecting R. Ouabain was effective when applied to the apical but not to the basolateral side of the preparation, suggesting the presence of a Na-K ATPase on rabbit RPE apical membrane similar to that found in bullfrogs, embryonic chickens, cats and dogs. Dinitrophenol also reduced Ve. Digoxin, furosemide, bumetanide, ethacrynic acid and chlorothiazide had no apparent effect upon Ve and R. The lack of response to furosemide, bumetanide and ethacrynic acid strongly suggests that, unlike RPE from other species, rabbit RPE does not possess Na-dependent Cl transport and/or does not possess furosemide receptors on its apical membrane.

Alpha-1 adrenergic receptors on rabbit retinal pigment epithelium.

Retinal pigment epithelium (RPE)-choroid-sclera preparations from black, dutch-belted rabbits were sealed in an Ussing chamber. The RPE-generated trans-RPE voltage (Ve) and electrical resistance (R) were monitored. Epinephrine (an alpha and beta adrenergic agonist) reduced Ve by as much as 39% without affecting R. A response to epinephrine was noted at concentrations as low as 1.4 X 10(-7) M. Phenylephrine (an alpha adrenergic agonist) had essentially the same effect as epinephrine at identical concentrations. Clonidine (an alpha-2 adrenergic agonist) had a very slight effect but only at 10(-4) M. Isoproterenol (a beta adrenergic agonist) had no apparent effect upon Ve or R. The RPE response to epinephrine and phenylephrine was blocked by the alpha adrenergic antagonist phentolamine and by the alpha-1 adrenergic antagonist prazosin but not by the alpha-2 adrenergic antagonist yohimbine or by the beta adrenergic antagonist propranolol. Dibutyryl cyclic AMP (in the presence and absence of IBMX) and cyclic GMP (as cGMP and in the dibutyryl form) had no apparent effect upon Ve or R. These results indicate that rabbit RPE possesses an alpha-1 adrenoreceptor which, when stimulated, substantially reduces the RPE generated trans-RPE electrical current.

Furosemide-sensitive Cl transport in bovine retinal pigment epithelium.

Bovine retinal pigment epithelium (RPE)-choroid explants were sealed in an Ussing chamber. Typical preparations produced a transepithelial voltage (Ve) of 12 mV (retina side positive) and had an electrical resistance (R) of 300 ohm-cm2. These values can be attributed to the RPE. Furosemide and ouabain reduced the Ve without affecting R when applied to the apical side of the RPE, but had no effect upon Ve and R when applied to the choroidal side. Acetazolamide had no effect upon Ve and R when applied to either side of the tissue. In Cl-free medium, ouabain reduced Ve without affecting R, while furosemide had no effect upon Ve and R. In Na-free medium, ouabain and furosemide had no effect upon Ve and R. Unidirectional isotope flux studies performed under open circuit conditions showed a net retina-to-choroid Cl flux that was abolished by furosemide. These results indicate that bovine RPE possesses a furosemide-sensitive Cl transport system.

Health response by questionnaire in arsenic-exposed populations.

The health status of populations exposed to arsenic through drinking water was determined by a mailed questionnaire. Participants were selected from three communities located in Nevada with 1977 tap water arsenic levels of approximately 0.1 mg/l and one California community with 1977 levels around 0.39 mg/l. The questionnaire responses were obtained in 1979 from the four exposed communities and compared to those of a Wyoming community whose tap water levels of arsenic were less than 0.001 mg/l in 1979. No difference in health status for gastrointestinal, neurological, musculoskeletal, circulatory and skin disorders was found. The average number of years of consumption given by length of residence was 6-16 years. We conclude that the health status of these arsenic-exposed populations has not been adversely affected.

Electromagnetically induced focused heat in the treatment of surgically created aneurysm models.

A hand-held radiofrequency (rf) probe of a novel design based on the principle of the induced current convergence was used to treat aneurysm models using focused hyperthermia. Aneurysms were created surgically in rats by a side-to-side anastomosis between the inferior vena cava and the abdominal aorta or by grafting a donor abdominal aorta from one rat onto the abdominal aorta of another rat. Aneurysms were treated by inserting the 0.3-mm diameter probe tip into the fundus and applying the power for brief periods (0.5-1.5 sec) using a foot pedal. Collapse of the fundus was observed as the result of the heat-induced thrombosis. Thermal distribution in the immediate vicinity of the probe as well as the heating rate were measured in a uniformly dielectric phantom and in rat vessels. The aneurysms were histologically examined immediately, three days, and three weeks after the treatment. Complete obliteration of the aneurysms and patency of the parent arteries were confirmed. Partial integrity of the vessels around the lesion was also confirmed.

Chronopharmacokinetics and chronopharmacodynamics of dextromethamphetamine in man.

Stimulants, in particular the amphetamines, have been studied as countermeasures to fatigue induced by circadian desynchronosis and extended flight operations. To make recommendations concerning the use of dextromethamphetamine for operational tasks, its chronopharmacokinetic and chronopharmacodynamic profiles and influence on circadian rhythms as a countermeasure to performance deficits and fatigue were studied. Ten male volunteers, divided into two groups of five each, were given 30 mg/70 kg of oral dextromethamphetamine during two test sessions one week apart and were evaluated with cognitive (dichotic listening, pattern recognition, and compensatory tracking), subjective (fatigue scale), and physiologic (blood pressure) testing. Session order was counterbalanced with dextromethamphetamine administration at either 8:40 AM or 8:40 PM during session one and a crossover to the other time during session two. Subjective and cognitive testing was begun 1.5 hours before dextromethamphetamine administration and continued every half hour until 12.5 hours after administration. Blood pressure was measured immediately before behavioral testing. Serum and urine were collected at regular intervals for gas chromatography/mass spectrometer analysis of methamphetamine and one of its metabolites, amphetamine. No differences were found in the day-versus-night pharmacokinetic profile of dextromethamphetamine. Cognitive performance and subjective fatigue improved after daytime administration of dextromethamphetamine in comparison to performance before drug administration. This effect was suppressed during the circadian trough, which occurred approximately 8 hours into the night sessions (4:30 AM). No correlations were seen between serum concentration of methamphetamine and measured behavioral parameters.

Electromagnetic field focusing system in the treatment of brain tumors.

The electromagnetic field focusing (EFF) probe, which is based on the principle of eddy current convergence, produces intense pinpoint heat at its point of contact with tissue. This allows cutting and vaporization of tissue and coagulation of vessels. The present experiments were conducted to study heat distribution to the surrounding tissue in brain and phantom and the effect on the brain of vaporization of intracerebral tumors in 19 rabbits. The follow-up period was as long as 47 days. The heating pattern showed a rise of temperature up to 250 degrees C at the probe tip, with minimal or no temperature increase at 2 mm and beyond. Minimal or no change was noted in the surrounding brain after tumor vaporization, indicating that this system would be safe in the vaporization of brain tumors in clinical neurosurgery.

Toxic effects of cocaine to the cardiovascular system in conscious and anesthetized rats and rabbits: evidence for a direct effect on the myocardium.

The cardiovascular toxicity of cocaine is complex because it has local anesthetic properties, central nervous system stimulatory effects, as well as cardiac effects. Recreational use of this drug has increased recently, but the precise mechanism of sudden cardiac death induced by cocaine is not known. The primary objective of this work was to test for a direct cardiovascular toxicity of cocaine. Rats and rabbits were anesthetized, and the femoral vein and artery were cannulated for drug infusion and blood pressure monitoring. Different doses of cocaine (0.3, 1, 3, 10 mg/kg) were infused. For conscious animal experiments, the animals were allowed to recover from the anesthesia and then were subjected to cocaine. Low doses of cocaine (0.3 and 1 mg/kg) increased systolic as well as diastolic pressure in conscious rats and rabbits. In anesthetized rats and rabbits, the same dose of cocaine increased blood pressure with a decrease in heart rate. With the high doses of cocaine (3 and 10 mg/kg), all cardiac parameters were reduced in both rats and rabbits. In isolated rabbit hearts, cocaine decreased all cardiac parameters. Based on the fact that high doses of cocaine severely depressed all cardiac parameters and its effect on the isolated heart, cocaine-induced sudden cardiac death appears to be due to a primary effect on the myocardium.

Cardiovascular effects of tropacocaine in conscious and anesthetized rabbits: lack of evidence for neuro-cardiac interactions and acute neurotoxicity.

The cardiovascular effects of tropacocaine, a structural analog of cocaine, were investigated in both conscious and anesthetized New Zealand white rabbits to determine if such effects were mediated through the CNS as had been demonstrated with cocaine, i.e., did a neuro-cardiac pathway exist? To facilitate the requisite cardiovascular measurements in both urethane- and pentobarbital-anesthetized animals, the right femoral artery and vein were cannulated for the measurement of arterial blood pressure and subsequent delivery of drugs, respectively. In addition, urethane-anesthetized animals had a branch of the left renal nerve isolated and multiunit renal nerve activity was monitored to obtain measures of sympathetic nerve activity originating from the CNS. Animals utilized in conscious experiments were surgically prepared 3 days prior to drug administration by placing canulae in the femoral artery and vein that were tunneled subcutaneously to the back between the scapulae. ECG and respiratory activity were also monitored in each animal. Doses of 0.3, 1, 3, and 10 mg/kg of tropacocaine were administered in both an ascending and descending fashion at 15 min intervals to 5 animals in each group, i.e., conscious, urethane-, and pentobarbital-anesthetized. In urethane-anesthetized animals a comparison was made between sympathetic renal nerve activity, systolic and diastolic blood pressure, respiratory rate, and heart rate. No pressor effects were observed and the changes in renal nerve activity could not be assigned as the cause of the observed depressor effects at the higher doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Disposition of butadiene epoxides in Sprague-Dawley rats.

1,2-Epoxybutene (BMO) and diepoxybutane (BDE) are metabolic products of 1,3-butadiene in rodents. Both BMO and BDE are suspect in the development of tumors in rats and mice. To understand the distribution and elimination of these compounds in the absence of the rate-limiting production from butadiene, the pharmacokinetics of BMO and BDE in blood were determined in adult male Sprague-Dawley rats following intravenous administration. All animals were dually cannulated in these studies. For the BMO studies, rats were dosed with 71, 143, or 286 mumol/kg BMO (n = 3 for each dose group). For the BDE studies, rats were dosed with 523 mumol/kg BDE (n = 3). All animals tolerated the BMO and BDE doses without grossly observable adverse effects. Blood was drawn at predetermined time points and extracted in methylene chloride. BDE and BMO concentrations were quantitated by gas chromatography or gas chromatography/mass spectrometry. The BMO distribution half-lives were short and ranged from 1.4 min at the lowest dose to 1.8 min at the highest dose. Volume of distribution at steady state ranged from 0.53 +/- 0.17 to 0.59 +/- 0.31 l/kg. Systemic clearances ranged from 67 +/- 17 to 114 +/- 20 ml/min per kg. The terminal elimination half-lives were also short and ranged from 5.7 to 8.5 min among the doses. The pharmacokinetic parameters after an i.v. dose of 523 mumol/kg BDE were a distribution half-life of 2.7 min, terminal elimination T1/2 of 14 min, volume of distribution at steady state of 0.73 +/- 0.06 l/kg, and systemic clearance of 76 +/- 8 ml/min per kg. These pharmacokinetic parameters demonstrate the similarity between disposition of the two epoxides in rats, that include a rapid distribution after i.v. administration into a small extravascular body compartment as well as a rapid elimination from blood. These pharmacokinetic data provide useful blood clearance information for assessing the critical physiological and biochemical determinants underlying the disposition of butadiene epoxides.

Pharmacokinetics of tertiary butyl alcohol in male and female Fischer 344 rats.

Tertiary butyl alcohol (TBA) is a small aliphatic alcohol with multiple industrial and scientific uses. A comprehensive pharmacokinetic profile for TBA has not been determined in rats. The purpose of this study was to fully characterize the pharmacokinetics of TBA in male and female F-344 rats following intravenous administration of 37.5, 75, 150 and 300 mg/kg TBA. TBA was observed to undergo a rapid distribution phase followed by a slower elimination phase. The steady-state volume of distribution for TBA was roughly 4.5 times greater than total body water, and the clearance was lower than the estimated glomerular filtration rate. The elimination of TBA appears to saturate at higher doses, as evidenced by a disproportional increase in area under the concentration-time curve and decreased rate of clearance.

INTRAV and ORAL: BASIC interactive computer programs for estimating pharmacokinetic parameters.

Two interactive computer programs, INTRAV and ORAL, were written to permit pharmacokinetic modeling of experimental data and to obtain pertinent values based on derived estimates. Both programs utilize BASIC language and were developed on a microcomputer with graphics capability. Drug concentration in blood, plasma, or serum with time following either nonabsorptive (intravenous) or absorptive (oral or intramuscular) administration is input, and a semilogarithmic display of data appears on a cathode-ray tube (CRT). The user selects limits for various linear segments using a movable cursor. On command, coefficients and exponents for the differential equation which describes those limits is computed and a nonlinear curve is fitted through the data set. Results from statistical tests are available in output formats permitting the user to determine the goodness of the selected limits. Commonly used pharmacokinetic parameters are also computed and appear on the output. Numerous graphic output options are also available to permit comparisons between data sets and/or estimates derived from other computer programs. INTRAV and ORAL were compared with the widely used programs CSTRIP, ESTRIP, and NONLIN. Both INTRAV and ORAL gave estimates which were almost identical with CSTRIP and ESTRIP, whereas those obtained with NONLIN were very similar, although not identical.

GLC determination of quinidines in human plasma.

Human plasma was made alkaline and extracted with methylene chloride. To the extract was added the internal standard, cinchonine, followed by evaporation to dryness. The resultant residue was dissolved in a methanolic solution containing trimethylanilinium hydroxide. This solution was assayed by GLC for quinidines (quinidine and hydroquinidine). Evaluation of the method over a 0.5-10-mug/ml range in human plasma gave an overall precision and accuracy of +/- 4.5% (RSD and RE). Plasma of several patients was analyzed by the present method as well as by a fluorometric method for the level of quinidines. Results from the two methods were comparable.

Comparative plasma concentrations of quinidine following administration of one intramuscular and three oral formulations to 13 human subjects.

A GLC method, based on flame-ionization detection, was developed for the assay of methotrimeprazine and its sulfoxide in plasma. For a 6-ml aliquot, the sensitivity was 2-3 ng/ml for the unchanged drug and 4-5 ng/ml for the sulfoxide. The coefficient of variation, calculated from duplicate analyses of plasma samples, was 8-15% for concentrations between 10 and 100 ng/ml. Patients treated with orally administered methotrimeprazine had higher plasma levels of the sulfoxide than of unmetabolized drug. The method also was applied to the analysis of promazine and chlorpromazine in patient plasma.

High-pressure liquid chromatographic--mass spectrometric determination of delta9-tetrahydrocannabinol in human plasma following marijuana smoking.

A method was developed for analyzing delta9-tetrahydrocannabinol (I), a psychotomimetic constituent found in marijuana smoke. The developed method utilizes a high-pressure liquid chromatographic (HPLC) gradient elution program to separate I from the other major cannabinoids in marijuana smoke. To achieve the sensitivity required to detect I in human plasma following marijuana smoking, a mass spectrometric quantification method was developed to analyze the HPLC eluant. To 1 ml of human plasma was added a known amount of internal standard, trideuterated I. This stable isotope provided a check on extraction efficiency, a marker for UV monitoring of the HPLC effluent and subsequent collection, and a convenient mass for mass spectrometric quantification. An ion-counting technique was used in conjunction with the peak matching accessory of the mass spectrometer to provide for a rapid comparison between molecular ions of I and the internal standard. The method was linear, accurate, and reproducible over the concentration range expected for I in plasma following marijuana smoking; 2.5 ng/ml was the lower practical limit of detection. Plasma from 11 male subjects was analyzed by the method at appropriate intervals up to 24 hr after the smoking of a marijuana cigarette containing 10.8 mg of I. Results demonstrated that levels of I could be determined accurately in the plasma of marijuana smokers in the 1-hr period following smoking.

Clinical use and time relationship of changes in affinity measurement of glycosylated albumin and glycosylated hemoglobin.

Simple techniques for measurement of glycosylated hemoglobin and glycosylated albumin by affinity chromatography on m-aminophenylboronic acid agarose columns have recently been developed. This study explored the time course of changes in glycoalbumin versus those of glycohemoglobin in response to rapid changes in ambient glucose concentration. One would predict that glycoalbumin levels would change more rapidly than glycohemoglobin levels due to the shorter half-life of albumin than hemoglobin. This was found to be the case in a group of rabbits rendered diabetic with alloxan. Glycoalbumin levels plateaued 4 weeks after alloxan administration, while glycohemoglobin levels were still rising. In a group of diabetic patients in whom glucose levels were initially poorly controlled, strict diet or intensive insulin management were used to rapidly bring glucose levels under control. In this group of patients, the glycoalbumin values entered the normal range and plateaued, while glycosylated hemoglobin levels were still falling. Glycoalbumin determination by affinity chromatography is a valuable adjunct to glycosylated hemoglobin determination in evaluating near term control of blood sugar values.

Precocious retinal adhesion is affected by furosemide and ouabain.

A "precocious" adhesive force develops between the neurosensory retina (NSR) and the retinal pigment epithelium (RPE) 15-30 minutes after eyes are removed from day 15 embryonic chickens and incubated at 37 degrees C. Precocious adhesion has been reported to be blocked by exposure to cold but to be unaffected by exposure to furosemide and ouabain. Since RPE transport is thought to play a major role in the adhesion between the RPE and NSR of adult mammals, and since ouabain and furosemide block RPE transport in embryonic chickens, it has been thought that precocious adhesion in embryonic chickens is not a good model for studying adhesion between the RPE and the NSR of adult mammals. We have found, however, that when steps are taken to ensure that ouabain and furosemide reach the transport sites on RPE apical membrane before precocious adhesion develops, that ouabain and furosemide do affect precocious adhesion.

GC-MS identification of sympathomimetic amine drugs in urine: rapid methodology applicable for emergency clinical toxicology.

A method was developed that permitted rapid identification in urine of the following sympathomimetic amines: amphetamine, benzphetamine, cathinone, desmethylsegiline, diethylpropion, ephedrine, fenfluramine, mazindol, methylenedioxyamphetamine, methylenedioxyethylamphetamine, methylenedioxymethamphetamine, mescaline, methamphetamine, methcathinone, methylaminorex, methylphenidate, pemoline, phendimetrazine, phenylepherine, phentermine, phenylpropanolamine, pseudoephedrine, and selegiline. In addition, two alpha-phenylethylamine-like monoamine oxidase inhibitors, phenelizine and tranylcypromine, were studied. Those sympathomimetic amines containing a primary or secondary amine, a hydrazine, and/or hydroxyl (except mazindol) functional groups were derivatized effectively using an on-column derivatization technique that used a reagent consisting of 10% fluoroanhydride in hexane, whereas the other sympathomimetic amines, including mazindol, were analyzed underivatized. Three different fluoroanhydrides, trifluoroacetic (TFAA), pentafluoropropionic (PFPA), and heptafluorobutyric (HFBA), and three different injection-port temperatures (160, 200, and 260 degrees C) were investigated. Both TFAA and PFPA gave sympathomimetic amine derivatives with essentially identical retention times, whereas HFBA gave longer retention times and better separation of individual compounds. The base fragmentation ion was noted to increase 50 amu (CF2) for each derivatized sympathomimetic amine as the length of the carbon-fluorine chain increased. Fragmentation ion abundance was maximized at an injection-port temperature of 260 degrees C, and this enhanced sensitivity coupled with the better chromatographic resolution of the individual sympathomimetic amines prompted the selection of HFBA as the derivatizing agent of choice. Assignments were made for the fragmentation ions produced by each derivatized drug. The developed method was adapted to analyze urine specimens that might be encountered in emergency toxicology testing. For identification of sympathomimetic amines requiring derivatization, 0.1 mL of the patient specimen had amphetamine-d5 and methamphetamine-d5 added as internal standard followed by adjustment of pH to 9.3 with borate buffer, extraction with 9:1 chloroform/isopropanol, centrifugation and separation of the organic phase, addition of 10% methanolic HCI and evaporation under nitrogen, reconstitution with HFBA reagent, and on-column derivatization during gas chromatographic-mass spectrometric (GC-MS) analysis. For those sympathomimetic amines not requiring derivatization, 1.0 mL of urine specimen had diazepam-d5 added as internal standard followed by the same extraction procedure and reconstitution accomplished with ethyl acetate. Because precolumn derivatization was eliminated and only 8 min was required for GC-MS analysis, complete analysis time was approximately 30 min, making the method suitable for clinical emergency toxicology purposes.

Identification of urinary benzodiazepines and their metabolites: comparison of automated HPLC and GC-MS after immunoassay screening of clinical specimens.

An automated high-performance liquid chromatographic method, benzodiazepines by REMEDi HS, was used to analyze benzodiazepines and their metabolites after beta-glucuronidase hydrolysis of 1-mL urine specimens from the following: 924 clinic and hospital patients whose specimens had previously been found to be presumptively positive using either EMIT or Triage immunoassay methodologies and 128 individuals whose specimens had screened negative by EMIT d.a.u.TM. REMEDi analyses did not correlate with the immunoassay results in 136 of the positive and three of the negative urine specimens. Gas chromatographic-mass spectrometric (GC-MS) confirmatory analyses were performed on these discordant specimens using 3 mL beta-glucuronidase-hydrolyzed urine followed by extraction with chloroform-isopropanol (9:1) and derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide. Two benzodiazepines, flunitrazepam and clonazepam, and their 7-amino metabolites were analyzed without prior derivatization. The analyses established 87% concordance between REMEDi and GC-MS versus 13% concordance with immunoassay for the subset. GC-MS analysis of these 142 specimens demonstrated two reasons for the nonconcurrence between REMEDi and EMIT: EMIT had given either false-negative or false-positive results and EMIT had given a positive result even though the determined metabolites were below the 200-ng/mL cutoff for the immunoassay and the 80-ng/mL cutoff for REMEDi. A total of 23 specimens were found to contain only lorazepam by REMEDi and GC-MS, 15 of which had been screened by Triage. A reevaluation of these 23 specimens by EMIT d.a.u. demonstrated that 11 were positive. This finding was in contrast to previous reports that EMIT will not detect lorazepam glucuronide in urine. An unexpected finding was the REMEDi identification and subsequent GC-MS confirmation of 7-aminoflunitrazepam, a urinary metabolite of flunitrazepam that is not available in the United States and that represented illicit use by four patients. A distinct advantage of REMEDi proved to be its capability in identifying demoxepam, a major metabolite of chlordiazepoxide; GC-MS analysis could not detect this metabolite because of its thermal decomposition to nordiazepam. To further evaluate the specificity of REMEDi, we conducted GC-MS analyses in a random fashion on 55 additional nondiscordant urine specimens that were identified as either positive or negative, as well as 22 specimens identified as containing 7-aminoclonazepam by REMEDi. Concurrence was observed between the two methods for all specimens, with the exception of one apparent false positive for alpha-hydroxyalprazolam by REMEDi. The reproducibility of the REMEDi method was found to be excellent; it was assessed by comparing results of 266 specimens that were reprocessed in different batches and for known calibrators and controls also processed with each batch. Study results demonstrated that the automated REMEDi assay for urinary benzodiazepines and their metabolites was comparable with GC-MS but had distinct advantages over GC-MS because of the following reasons: simplicity of the assay, less time required for analyses, and provision of additional information concerning the parent benzodiazepine.