Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65.

At least 120 non-olfactory G-protein-coupled receptors in the human genome are 'orphans' for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs.

Development of CT-guided biopsy sampling for time-dependent postmortem redistribution investigations in blood and alternative matrices--proof of concept and application on two cases.

The postmortem redistribution (PMR) phenomenon complicates interpretation in forensic toxicology. Human data on time-dependent PMR are rare and only exist for blood so far. A new method for investigation of time-dependent PMR in blood as well as in alternative body fluids and tissues was developed and evaluated using automated biopsy sampling. At admission of the bodies, introducer needles were placed in liver, lung, kidney, muscle, spleen, adipose tissue, heart, femoral vein, and lumbar spine using a robotic arm guided by a computed tomography scanner (CT). Needle placement accuracy was analyzed and found to be acceptable for the study purpose. Tissue biopsies and small volume body fluid samples were collected in triplicate through the introducer needles. At autopsy (around 24 h after admission), samples from the same body regions were collected. After mastering of the technical challenges, two authentic cases were analyzed as a proof of concept. Drug concentrations of venlafaxine, O-desmethylvenlafaxine, bromazepam, flupentixol, paroxetine, and lorazepam were determined by LC-MS/MS, and the percentage concentration changes between the two time points were calculated. Concentration changes were observed with both increases and decreases depending on analyte and matrix. While venlafaxine, flupentixol, paroxetine, and lorazepam generally showed changes above 30% and more, O-desmethylvenlafaxine and bromazepam did not undergo extensive PMR. The presented study shows that CT-controlled biopsy collection provides a valuable tool for systematic time-dependent PMR investigation, demanding only minimal sample amount and causing minimal damage to the body.

Trace analysis of 47 psychotropic medications in environmental samples by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS).

Psychotropic medications are one of the most prescribed pharmaceuticals in the world. Given their frequent detection and ecotoxicity to the no-target organism, the emission of these medications into environments has gradually draw attention. The study developed a sensitive and reliable analytic method to simultaneously investigate 47 psychotropic medications in four matrices: wastewater, surface water, activated sludge, and sediment by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS). These 47 target analytes include 24 antidepressants, 17 antianxiety drugs, 5 anticonvulsants, and 1 relevant hormone. Solid phase extraction (SPE) was employed to extract analytes from water-phase samples. Ultrasonic Solvent Extraction method with Enhanced Matrix Removal clean-up (USE-EMR) was utilized to extract target compounds from solid-phase samples, which requires more straightforward and convenient procedures than previous methods. The extraction recoveries of all analytes ranged from 80 % to 120 % in these four sample matrices. In this study, The limit of quantitation for 47 psychotropic medications were 0.15 ng/L (estazolam) to 2.27 ng/L (lorazepam), 0.08 ng/L (desvenlafaxine) to 2 ng/L (mianserin), 0.22 ng/g (dry weight, dw) (nordiazepam) to 3.65 ng/g (dw) (lorazepam), and 0.07 ng/g (dw) (carbamazepine) to 2.85 ng/g (lorazepam), in wastewater, surface water, sludge, and sediment, respectively. In addition, the developed method was employed to analyse actual samples in two wastewater treatment plants and their receiving rivers. Carbamazepine, escitalopram, clozapine, desvenlafaxine, diazepam, lamotrigine, sertraline, temazepam, and venlafaxine were nearly ubiquitous in all matrices. Moreover, this study indicated that the inadequate removal efficiencies of psychotropic medications in wastewater treatment plants (WWTPs) had resulted in a persistent discharge of these contaminants from human sources into environments.

Occurrence of Z-drugs, benzodiazepines, and ketamine in wastewater in the United States and Mexico during the Covid-19 pandemic.

Z-drugs, benzodiazepines and ketamine are classes of psychotropic drugs prescribed for treating anxiety, sleep disorders and depression with known side effects including an elevated risk of addiction and substance misuse. These drugs have a strong potential for misuse, which has escalated over the years and was hypothesized here to have been exacerbated during the COVID-19 pandemic. Wastewater-based epidemiology (WBE) constitutes a fast, easy, and relatively inexpensive approach to epidemiological surveys for understanding the incidence and frequency of uses of these drugs. In this study, we analyzed wastewater (n = 376) from 50 cities across the United States and Mexico from July to October 2020 to estimate drug use rates during a pandemic event. Both time and flow proportional composite and grab samples of untreated municipal wastewater were analyzed using solid-phase extraction followed by liquid chromatography-tandem mass spectrometry to determine loadings of alprazolam, clonazepam, diazepam, ketamine, lorazepam, nordiazepam, temazepam, zolpidem, and zaleplon in raw wastewater. Simultaneously, prescription data of the aforementioned drugs were extracted from the Medicaid database from 2019 to 2021. Results showed high detection frequencies of ketamine (90 %), lorazepam (87 %), clonazepam (76 %) and temazepam (73 %) across both Mexico and United States and comparatively lower detection frequencies for zaleplon (22 %), zolpidem (9 %), nordiazepam (<1 %), diazepam (<1 %), and alprazolam (<1 %) during the pandemic. Average mass consumption rates, estimated using WBE and reported in units of mg/day/1000 persons, ranged between 62 (temazepam) and 1100 (clonazepam) in the United States. Results obtained from the Medicaid database also showed a significant change (p < 0.05) in the prescription volume between the first quarter of 2019 (before the pandemic) and the first quarter of 2021 (pandemic event) for alprazolam, clonazepam and lorazepam. Study results include the first detections of zaleplon and zolpidem in wastewater from North America.

[Changes in Test Methods for Internationalization in the Japanese Pharmacopoeia (Part 2): Establishment of a Quantitative Method for Lorazepam Using HPLC Analysis].

The Japanese Pharmacopoeia (JP) is an official normative publication that is referred to, for establishing the authenticity and properties and maintaining the quality of pharmaceutics in Japan. Partial amendments are periodically made to these guidelines to keep up with the progress of science and technology, and the international harmonization is revised every 5 years. Thus, "Internationalization of the JP" is one of the more important issues to address for the revision of the JP. For example, the incorporation of the test methods that have been used in other pharmacopeias, such as the United States Pharmacopeia (USP) and the European Pharmacopoeia (EP), into the JP is a useful approach. In light of this, we have recently reported changes in test methods in the 17th JP, "Establishment of a quantitative test method for clonidine hydrochloride from using a potentiometric titration method to using HPLC". As a part of our ongoing research to change test methods for internationalization, we selected lorazepam. Lorazepam is analyzed using a potentiometric titration method as listed in the 17th JP; however, both the USP and EP use HPLC for quantitative analysis of this drug. In this study, we synthesized the related impurities of lorazepam listed in the USP and the EP and determined their purities using quantitative NMR. The separation conditions of these compounds, including lorazepam, were examined using HPLC and simultaneous analyses were performed. In addition, lorazepam extracted from the tablets was analyzed using conditions similar to those used for the analysis of the related impurities.

Detection of trace drugs of abuse in baby formula using solid-phase microextraction direct analysis in real-time mass spectrometry (SPME-DART-MS).

The intentional or unintentional adulteration of baby formula with drugs of abuse is one of the many increasingly complex samples forensic chemists may have to analyze. This sample type presents a challenge because of a complex matrix that can mask the detection of trace drug residues. To enable screening of baby formula for trace levels of drugs, the use of solid-phase microextraction (SPME) coupled with direct analysis in real-time mass spectrometry (DART-MS) was investigated. A suite of five drugs was used as adulterants and spiked into baby formula. Samples were then extracted using SPME fibers which were analyzed by DART-MS. Development of a proof-of-concept method was completed by investigating the effects of the DART gas stream temperature and the linear speed of the sample holder. Optimal values of 350°C and 0.2 mm/s were found. Once the method was established, representative responses and sensitivities for the five drugs were measured and found to be in the range of single ng/mL to hundreds ng/mL. Additional studies found that the presence of the baby formula matrix increased analyte signal (relative to methanolic solutions) by greater than 200%. Comparison of the SPME-DART-MS method to a traditional DART-MS method for trace drug detection found at least a factor of 13 improvement in signal for the drugs investigated. This work demonstrates that SPME-DART-MS is a viable technique for the screening of complex matrices, such as baby formula, for trace drug residues and that development of a comprehensive method is warranted.

Luminescence method for the determination of lorazepam in tablets.

New terbium complex of 7-chloro-5-(2-chlorophenyl)-3-hydroxy-1,2-dihydro-3H- 1,4-benzdiazepin-2-one (lorazepam, L), which is highly luminescent and do not require luminescence enhancers, is reported. The luminescence intensity of the Tb-L complex was enhanced by the addition of Tergitol 7 in water solution. The Tb-L-Tergitol 7 complex with a components ratio 1:1:2 was proposed to be used as the analytical form for the luminescence determination of drug--lorazepam. The calibration curve is linear in the 0.05-20.0 pg/mL range of lorazepam (LOD is 0.016 microg/mL). This method was applied for the determination of lorazepam in dosage form--tablets "Apo-lorazepam"--2.5 mg.

The detection and quantification of lorazepam and its 3-O-glucuronide in fingerprint deposits by LC-MS/MS.

The use of fingerprints as an alternative biological matrix to test for the presence of drugs and/or their metabolites is a novel area of research in analytical toxicology. This investigation describes quantitative analysis for the benzodiazepine lorazepam and its 3-O-glucuronide conjugate in fingerprints following the oral administration of a single 2 mg dose of lorazepam to five volunteers. Creatinine was also measured to investigate whether the amount of drug relative to that of creatinine would help to account for the variable amount of secretory material deposited. Fingerprints were deposited on glass cover slips and extracted by dissolving them in a solution of dichloromethane/methanol, containing tetradeuterated lorazepam as an internal standard. The samples were evaporated, reconstituted with mobile phase and analysed by LC-MS/MS. Chromatography was achieved using an RP (C18) column for the analysis of lorazapem and its glucuronide, and a hydrophilic interaction column (HILIC) for the analysis of creatinine. Lorazepam and its glucuronide were only detected where ten prints had been combined, up to 12 h following drug administration. In every case, the amount of lorazepam glucuronide exceeded that of lorazepam, the peak amounts being 210 and 11 pg, respectively. Adjusting for creatinine smoothed the elimination profile. To our knowledge, this represents the first time a drug glucuronide has been detected in deposited fingerprints.

Organ distribution of diclazepam, pyrazolam and 3-fluorophenmetrazine.

The organ distribution of 3-fluorophenmetrazine (3-FPM), pyrazolam, diclazepam as well as its main metabolites delorazepam, lormetazepam and lorazepam, was investigated. A solid phase extraction (SPE) and a QuEChERS (acronym for quick, easy, cheap, effective, rugged and safe) - approach were used for the extraction of the analytes from human tissues, body fluids and stomach contents. The detection was performed on a liquid chromatography-tandem mass spectrometry system (LCMS/MS). The analytes of interest were detected in all body fluids and tissues. Results showed femoral blood concentrations of 10 µg/L for 3-FPM, 28 µg/L for pyrazolam, 1 µg/L for diclazepam, 100 µg/L for delorazepam, 6 µg/L for lormetazepam, and 22 µg/L for lorazepam. Tissues (muscle, kidney and liver) and bile exhibited higher concentrations of the mentioned analytes than in blood. Additional positive findings in femoral blood were for 2-fluoroamphetamine (2-FA, approx. 89 µg/L), 2-flourometamphetamine (2-FMA, hint), methiopropamine (approx. 2.2 µg/L), amphetamine (approx. 21 µg/L) and caffeine (positive). Delorazepam showed the highest ratio of heart (C) and femoral blood (P) concentration (C/P ratio = 2.5), supported by the concentrations detected in psoas muscle (430 µg/kg) and stomach content (approx. 210 µg/L, absolute 84 µg). The C/P ratio indicates that delorazepam displays susceptibility for post-mortem redistribution (PMR), supported by the findings in muscle tissue. 3-FPM, pyrazolam, diclazepam, lorazepam and lormetazepam did apparently not exhibit any PMR. The cause of death, in conjunction with autopsy findings was concluded as a positional asphyxia promoted by poly-drug intoxication by arising from designer benzodiazepines and the presence of synthetic stimulants.

Quantification of lorazepam and lormetazepam in human breast milk using GC-MS in the negative chemical ionization mode.

Lormetazepam (Loramet is a benzodiazepine mainly used as an hypnotic to treat insomnia. Lorazepam (Temesta) is used as an anxiolytic, tranquilizer, sedative, and anticonvulsant, and it is the major metabolite of lormetazepam. In this study, we designed a method to simultaneously detect and quantify these substances in human breast milk. Solid-phase extraction of 2 mL of milk was followed by derivatization with a trimethylsilyl reagent. Separation and detection was performed using gas chromatography coupled to mass spectrometry in the negative chemical ionization mode. Calibration curves were linear in the ranges of 10-200 and 1-20 ng/mL for lorazepam and lormetazepam, respectively. Limits of detection were estimated at 0.016 ng/mL for lormetazepam and 0.100 ng/mL for lorazepam. Our method was applied to real case samples from a woman receiving both benzodiazepines. Lorazepam concentrations varied from 55.3 to 123.1 ng/mL, and lormetazepam concentrations varied from 1.7 to 7.3 ng/mL.

Investigation of the effect of mobile phase composition on selectivity using a solvent-triangle based approach in achiral SFC.

Defining a method development methodology for achiral drug impurity profiling in SFC requires a number of steps. Initially, diverse stationary phases are characterized and a small number of orthogonal or dissimilar phases are selected for further method development. In this paper, we focus on a next step which is the investigation of the modifier composition on chromatographic selectivity. A solvent-triangle based approach is used in which blends of organic solvents, mainly ethanol (EtOH), propanol (PrOH), acetonitrile (ACN) and tetrahydrofuran (THF) mixed with methanol (MeOH) are tested as modifiers on six dissimilar stationary phases. The tested modifier blends were composed to have equal eluotropic strengths as calculated on bare silica. The modifier leads to minor changes in terms of elution order, retention and mixture resolution. However, varying only the modifier composition on a given stationary phase does not lead to the creation of dissimilar systems. Therefore the modifier composition is an optimization parameter, with the stationary phase being the factor determining most the selectivity of a given mixture in achiral SFC.

Chemometric approach to open validation protocols: Prediction of validation parameters in multi-residue ultra-high performance liquid chromatography-tandem mass spectrometry methods.

The recent technological advancements of liquid chromatography-tandem mass spectrometry allow the simultaneous determination of tens, or even hundreds, of target analytes. In such cases, the traditional approach to quantitative method validation presents three major drawbacks: (i) it is extremely laborious, repetitive and rigid; (ii) it does not allow to introduce new target analytes without starting the validation from its very beginning and (iii) it is performed on spiked blank matrices, whose very nature is significantly modified by the addition of a large number of spiking substances, especially at high concentration. In the present study, several predictive chemometric models were developed from closed sets of analytes in order to estimate validation parameters on molecules of the same class, but not included in the original training set. Retention time, matrix effect, recovery, detection and quantification limits were predicted with partial least squares regression method. In particular, iterative stepwise elimination, iterative predictors weighting and genetic algorithms approaches were utilized and compared to achieve effective variables selection. These procedures were applied to data reported in our previously validated ultra-high performance liquid chromatography-tandem mass spectrometry multi-residue method for the determination of pharmaceutical and illicit drugs in oral fluid samples in accordance with national and international guidelines. Then, the partial least squares model was successfully tested on naloxone and lormetazepam, in order to introduce these new compounds in the oral fluid validated method, which adopts reverse-phase chromatography. Retention time, matrix effect, recovery, limit of detection and limit of quantification parameters for naloxone and lormetazepam were predicted by the model and then positively compared with their corresponding experimental values. The whole study represents a proof-of-concept of chemometrics potential to reduce the routine workload during multi-residue methods validation and suggests a rational alternative to ever-expanding procedures progressively drifting apart from real sample analysis.

A Fatality Related to Two Novel Hallucinogenic Compounds: 4-Methoxyphencyclidine and 4-Hydroxy-N-methyl-N-ethyltryptamine.

In this case report, we present an evaluation of postmortem concentration distribution of the hallucinogenic compound 4-methoxyphencyclidine (4-MeO-PCP) in a fatality principally attributed to this drug. Another hallucinogen, 4-hydroxy-N-methyl-N-ethyltryptamine was also detected, but was not quantitated. A man--who had a history of recent 'strange' behavior--was found deceased, on his bed, in his locked room. Toxicology testing, which initially screened positive for phencyclidine (PCP) by ELISA, subsequently detected and confirmed the two hallucinogens by gas chromatography-mass spectrometry. 4-MeO-PCP concentrations were then quantified by a specific secondary testing technique. The peripheral blood concentration was 8.2 mg/L compared with the central blood concentration of 14 mg/L. The liver concentration was 120 mg/kg, the vitreous was 5.1 mg/L, the urine was 140 mg/L and the gastric contents contained 280 mg. PCP was not detected, but therapeutic concentrations of venlafaxine, olanzapine, lorazepam and hydroxyzine were confirmed. The cause of death was certified due to acute mixed drug intoxication, and the manner of death was certified as accident.

Comparative metabolism of lorazepam in man and four animal species.

The metabolic disposition of lorazepam (Wy-4036) in man, dog, cat, rat and miniature swine is compared. Except in the cat, absorption of lorazepam is rapid in these species. Absorption in humans is nearly complete. Lorazepam glucuronide is the major metabolite in all species except the rat in which a dihydrodiol derivative is the main product of lorazepam biotransformation. Lorazepam glucuronide, which has no demonstrable CNS activity, is also present in the plasma of all species investigated. The concentrations of lorazepam in rat brain correlate well with those in plasma but are about three times higher. The urinary route of excretion predominates in man, dog and miniature swine while in the rat the bulk of the drug-related material is eliminated with the feces as a consequence of biliary excretion.

Determination of bromazepam in human plasma by high-performance liquid chromatography with electrospray ionization tandem mass spectrometric detection: application to a bioequivalence study.

A simple method using a one-step liquid-liquid extraction (LLE) followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of bromazepam in human plasma, using lorazepam as internal standard. The acquisition was performed in the multiple reaction monitoring mode, monitoring the transitions: m/z 316 > 182 for bromazepam and m/z 321 > 275 for lorazepam. The method was linear over the studied range (1-100 ng ml(-1)), with r(2) > 0.98, and the run time was 2.5 min. The intra- and inter-assay precisions were 2.7-14.6 and 4.1-17.3%, respectively and the intra- and inter-assay accuracies were 87-111 and 75.8-109.5%, respectively. The mean recovery was 73.7%, ranging from 64.5 to 79.7%. The limit of quantification was 1 ng ml(-1). At this concentration the mean intra- and inter-assay precisions were 14.6 and 7.1%, respectively, and the mean intra- and inter-assay accuracies were 102.5 and 104%, respectively. Bromazepam stability was evaluated and the results showed that the drug is stable in standard solution and in plasma samples under typical storage and processing conditions. The method was applied to a bioequivalence study in which 27 healthy adult volunteers (14 men) received single oral doses (6 mg) of reference and test bromazepam formulations, in an open, two-period, randomized, crossover protocol. The 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak plasma concentration), AUC(0-96) and AUC(0-inf) (area under the plasma concentration versus time curve from time zero to 96 h and to infinity, respectively) were within the range 80-125%, which supports the conclusion that the test formulation is bioequivalent to the reference formulation regarding the rate and extent of bromazepam absorption.

Suppression of chiral recognition of 3-hydroxy-1,4-benzodiazepines during micellar electrokinetic capillary chromatography with bile salts.

During the development of a micellar electrokinetic chromatographic screening method for 1,4-benzodiazepines, peak splitting and broadening were observed for some 3-hydroxy-1,4-benzodiazepines (oxazepam, lorazepam, temazepam and lormetazepam). This phenomenon occurred when the micellar phase consisted of bile salts and can be ascribed to the chiral nature of these surfactants. As the bile salts were applied in order to reduce the capacity factors to an appropriate level, enantiomer separation was not an objective and even disturbing. By increasing the analysis temperature, the chiral recognition of these compounds could be suppressed.

Separation of oxazepam, lorazepam, and temazepam enantiomers by HPLC on a derivatized cyclodextrin-bonded phase: application to the determination of oxazepam in plasma.

The enantioselective high-performance liquid chromatography (HPLC) of three racemic 3-hydroxybenzodiazepines, oxazepam (Oxa), lorazepam (Lor), and temazepam (Tem), is a difficult operation because of the spontaneous chiral inversion in polar solvent. To solve this problem, we have developed an HPLC method based on a chiral Cyclobond I-2000 RSP column, maintained at 12 degrees C, and a reversed mobile phase (acetonitrile in 1% triethylamine acetate buffer, TEAA) at a flow rate of 0.4 ml/min. Peaks were detected by a photodiode-array detector at 230 nm for quantification and by an optical rotation detector for identification of (+) and (-) enantiomers. The results showed that peak resolutions of Oxa, Lor, and Tem enantiomers, analyzed under the same conditions, were 3.2, 2.0, and 1.8, respectively. For the determination of Oxa enantiomers in plasma of rabbits, extraction with diethyl ether at pH 1.5, a polar organic mobile phase, and a Cyclobond I-2000 SP column were used. Other analytical conditions were the same as previously described. Blood samples were immediately cooled at 4 degrees C and centrifuged at 0 degrees C for the collection of plasma. The results showed a difference in plasma S(+)- and R(-)-oxazepam concentrations in rabbits. No racemization of S(+)- or R(-)-Oxa enantiomers, added alone to blank plasma, was observed after extraction and enantioselective HPLC analysis.

Study on protolytic equilibria of lorazepam and oxazepam by UV and NMR spectroscopy.

Protolytic equilibria in homogeneous and heterogeneous systems of lorazepam and oxazepam, which are sparingly soluble ampholytes from the class of 1,4-benzodiazepines, were studied at 25 degrees C and ionic strength of 0.1 M. Acidity constants and equilibrium constants in a heterogeneous system were determined. On the basis of the analysis of the corresponding 13C- and 1H-NMR spectra, deprotonation site in the molecules of the investigated compounds was predicted. Finally, the correlation between chemical shifts in the 1H-NMR spectra and the acidity of the amide proton of 1,4-benzodiazepines was established.

A selective fluorimetric method for the determination of some 1,4-benzodiazepine drugs containing a hydroxyl group at C-3.

A highly selective and sensitive fluorimetric method was developed for the determination of four 1,4-benzodiazepine drugs containing a hydroxyl group at carbon 3, namely oxazepam, lorazepam, cinolazepam and temazepam. The method is highly specific because other benzodiazepinee lacking the hydroxyl group at C-3 do not react similarly and hence do not interfere. The proposed method involves reduction of the target compound using Zno/HCl at room temperature with the formation of a highly fluorescent derivative within 15 min. The different experimental parameters were carefully studied and incorporated into the procedure. Under the described conditions, the proposed method is applicable over the concentration range of 0.1-1.2 micrograms ml-1 for both temazepam and cinolazepam, and 0.2-2.5 and 1-8 micrograms ml-1 for oxazepam and lorazepam respectively. The recoveries of the title compounds from spiked urine ranged from 90.0 to 92.0% and for serum from 94.1 to 95.4% with a limit of detection (S/N = 2) of 4 ng ml-1 for all drugs. The mechanism of the fluorimetric reaction is discussed.

Biopharmacological data and high-performance liquid chromatographic analysis of 1,4-benzodiazepines in biological fluids: a review.

A review with 123 references on the analysis of 1,4-benzodiazepines in biological samples using HPLC is presented. Some important physico-chemical and biopharmacological data for the development of analytical methods are collected. Different methods of sample pretreatment, chromatographic conditions and detection systems are discussed.