Patterns of drug abuse among drug users with regular and irregular attendance for treatment as detected by comprehensive UHPLC‐HR‐TOF‐MS

The most severe consequences of drug abuse include infectious diseases, overdoses, and drug‐related deaths. As the range of toxicologically relevant compounds is continually changing due to the emergence of new psychoactive substances (NPS), laboratories are encountering analytical challenges. Current immunoassays are insufficient for determining the whole range of the drugs abused, and a broad‐spectrum screening method is therefore needed. Here, the patterns of drug abuse in two groups of drug users were studied from urine samples using a comprehensive screening method based on high‐resolution time‐of‐flight mass spectrometry. The two groups comprised drug abusers undergoing opioid maintenance treatment (OMT) or drug withdrawal therapy and routinely visiting a rehabilitation clinic, and drug abusers with irregular attendance at a harm reduction unit (HRU) and suspected of potential NPS abuse. Polydrug abuse was observed in both groups, but was more pronounced among the HRU subjects with a mean number of concurrent drugs per sample of 3.9, whereas among the regularly treated subjects the corresponding number was 2.1. NPS and pregabalin were more frequent among HRU subjects, and their abuse was always related to drug co‐use. The most common drug combination for an HRU subject included amphetamine, cannabis, buprenorphine, benzodiazepine, and alpha‐pyrrolidinovalerophenone. A typical set of drugs for treated subjects was buprenorphine, benzodiazepine, and occasionally amphetamine. Abuse of several concurrent drugs poses a higher risk of drug intoxication and a threat of premature termination of OMT. Since the subjects attending treatment used fewer concurrent drugs, this treatment could be valuable in reducing polydrug abuse. Copyright © 2015 John Wiley & Sons, Ltd.     

Profiles of urine samples taken from Ecstasy users at Rave parties: analysis by immunoassays, HPLC, and GC-MS

The abuse of the designer amphetamines such as 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) is increasing throughout the world. They have become popular drugs, especially at all-night techno dance parties (Raves), and their detection is becoming an important issue. Presently, there are no MDMA- or MDA-specific immunoassays on the market, and detection of the designer amphetamines is dependent upon the use of commercially available amphetamine assays. The success of this approach has been difficult to assess because of the general unavailability of significant numbers of samples from known drug users. The objectives of the present study are to characterize the drug content of urine samples from admitted Ecstasy users by chromatographic methods and to assess the ability of the available amphetamine/methamphetamine immunoassays to detect methylenedioxyamphetamines.We found that, when analyzed by high-performance liquid chromatography with diode-array detection (HPLC-DAD), 64% of 70 urine samples (by gas chromatography-mass spectrometry [GC-MS]: 88% of 64 urine samples) obtained from Rave attendees contained MDMA and/or 3,4-methylenedioxyamphetamine (MDA) alone or in combination with amphetamine, methamphetamine, or other designer amphetamines such as 3,4-methylenedioxyethylamphetamine (MDEA). This suggests that the majority of the Ravers are multidrug users. At the manufacturer's suggested cutoffs, the Abbott TDx Amphetamine/Methamphetamine II and the new Roche HS Amphetamine/MDMA assays demonstrated greater detection sensitivity for MDMA than the other amphetamine immunoassays tested (Abuscreen OnLine Hitachi AMPS, Abuscreen OnLine Integra AMPS, Abuscreen OnLine Integra AMPSX, CEDIA AMPS, and EMIT II AMPS). There is 100% agreement between each of the two immunoassays with the reference chromatographic methods, HPLC-DAD and GC-MS, for the detection of methylenedioxyamphetamines.     

Role of drug testing as an early warning programme: the experience of the Republic of Korea

Drug testing plays an important role in the provision of information to health authorities on trends in drug abuse. In the Republic of Korea, the testing of urine and postmortem specimens has been used as part of a programme to monitor and control the abuse of non-controlled drugs, i.e., substances that were not originally included in the lists of controlled substances in that country. Zipeprol, dextromethorphan, carisoprodol and nalbuphine are examples of such drugs, which are widely used as medicines. Increasing levels of abuse of these drugs, including abuse that resulted in fatalities, were confirmed in the Republic of Korea by the results of drug testing. Based on the accumulated data from postmortem specimens, the health authorities in the Republic of Korea subsequently introduced controls on these drugs. A significant drop in fatalities related to the abuse of these non-controlled drugs underlined the importance of timely action for improving community health. In the context of drug testing, the analysis of non-controlled and new drugs always presents a scientific challenge, because specific analytical methods for testing for those drugs are not available. In the Republic of Korea, as part of the drug abuse warning programme, it was necessary to establish methods for the detection and quantification in biological fluids of all four non-controlled drugs and their metabolites in order to monitor the trends in drug abuse. The present paper puts forward epidemiological and clinical data on abuse and fatalities associated with zipeprol, dextromethorphan, carisoprodol and nalbuphine, as well as details of the analytical methods developed.     

Studies on the metabolic fate of isepamicin sulfate (HAPA-B). III. Intramuscular, intravenous and drip intravenous administration of HAPA-B in rabbits

Absorption, distribution, metabolism and excretion of isepamicin sulfate (HAPA-B) were studied following intramuscular, intravenous and drip intravenous administration at doses of 6.25, 25 and 100 mg/kg to rabbits. Plasma concentrations of HAPA-B following intramuscular, intravenous and drip intravenous administration depended on dose levels. Biological half-lives (T1/2), body clearance (Clt) and areas under plasma concentration-time curves (AUC) for different routes of administration were similar in all 3 routes. A theoretical curve for drug concentrations vs. time was obtained using pharmacokinetic parameters calculated from drug concentrations in plasma following a 45-minute drip intravenous administration. From the curve, it was estimated that 60 to 90 minutes would be required to achieve a similar maximum drug concentration in plasma by drip intravenous administration to that obtained by intramuscular administration. Thus, drug concentration patterns obtained following intramuscular administration could be duplicated in drip intravenous administration by regulating the length of time for infusion. The concentration of HAPA-B in tissues obtained following a 15-minute drip intravenous administration reached maximum after 15 minutes at a level higher than that achieved by intramuscular administration, but an hour later, concentrations in tissues including the kidney decreased to similar levels obtained following intramuscular administration and patterns of concentration decrease for drip intravenous administration and intramuscular administration were quite similar to each other thereafter. The drug was rapidly excreted into the urine following any of the 3 routes, and urinary recoveries in 24 hours were 75 approximately 92% of dose amounts for all dose levels tested. Bioautograms on thin-layer chromatographs of 0 approximately 6 hours urine samples obtained following an intramuscular administration of the drug showed a single biologically active bands with similar Rf values to HAPA-B itself. No active metabolite of the drug was detected in the urine.     

Applications of liquid chromatography-mass spectrometry in doping control

This paper reviews liquid chromatographic-mass spectrometric (LC-MS) procedures for the screening, identification and quantification of doping agents in urine and other biological samples and devoted to drug testing in sports. Reviewed methods published approximately within the last five years and cited in the PubMed database have been divided into groups using the same classification of the 2004 World Anti-Doping Agency (WADA) Prohibited List. Together with procedures specifically developed for anti-doping analysis, LC-MS applications used in other fields (e.g., therapeutic drug monitoring, clinical and forensic toxicology, and detection of drugs illicitly used in livestock production) have been included when considered as potentially extensible to doping control. Information on the reasons for potential abuse by athletes, on the requirements established by WADA for analysis, and on the WADA rules for the interpretation of analytical findings are provided for the different classes of drugs.     

Hair as a biological indicator of drug use, drug abuse or chronic exposure to environmental toxicants

In recent years hair has become a fundamental biological specimen, alternative to the usual samples blood and urine, for drug testing in the fields of forensic toxicology, clinical toxicology and clinical chemistry. Moreover, hair-testing is now extensively used in workplace testing, as well as, on legal cases, historical research etc. This article reviews methodological and practical issues related to the application of hair as a biological indicator of drug use/abuse or of chronic exposure to environmental toxicants. Hair structure and the mechanisms of drug incorporation into it are commented. The usual preparation and extraction methods as well as the analytical techniques of hair samples are presented and commented on. The outcomes of hair analysis have been reviewed for the following categories: drugs of abuse (opiates, cocaine and related, amphetamines, cannabinoids), benzodiazepines, prescribed drugs, pesticides and organic pollutants, doping agents and other drugs or substances. Finally, the specific purpose of the hair testing is discussed along with the interpretation of hair analysis results regarding the limitations of the applied procedures.     

LC/MS分析大鼠体内氧化苦参碱及其主要代谢物LC/MS分析大鼠体内氧化苦参碱及其主要代谢物

目的研究氧化苦参碱在大鼠体内的主要代谢产物。方法以氧化苦参碱和苦参碱为对象优化液相色谱/电喷雾离子阱质谱(LC/ESI-ITMSⁿ)实验条件，分析总结其电喷雾质谱的一级电离规律和二级质谱裂解规律，作为氧化苦参碱大鼠体内代谢物结构分析的依据。健康大鼠腹腔肌注40 mg·kg^-1氧化苦参碱，收集0~24 h的尿样，尿样中的代谢物经C₁₈小柱进行富集与纯化后，在优化的LC/ESI-ITMSⁿ条件下进样分析。代谢物的结构推导主要依据代谢物的色谱保留时间及其电喷雾离子阱质谱(ESI-ITMSⁿ)电离规律。结果在大鼠尿样中有原药及其6种I相氧化及还原代谢产物，且主要代谢物为苦参碱。未发现II相代谢物。结论本法不仅操作简便、快速，而且灵敏度高、专属性强。该分析技术是研究药物代谢最有效的方法之一。     

GC-MS analysis of methamphetamine and amphetamine in hair of Thai drug addicts]

Psycho-stimulant dependence in young individuals has become a serious problem in Thailand, where consumption of the so-called YaBa methamphetamine tablets has become a fashionable trend. Due to its easy availability in the form of a tablet, young individuals abuse methamphetamine. Methamphetamine tablets are known to be potently addictive and its difficulty in cessation of drug use and to be abstinent from the drug. We herein report the results obtained from GC-MS analysis of methamphetamine and amphetamine in 33 samples of urine and hair from patients with psycho-stimulant dependence. These samples were collected from patients registered at the outpatient clinic in the Department of Psychiatry, Chiang Mai University Hospital and were sent to Nippon Medical School, Department of Legal Medicine (Tokyo, Japan) for further analysis by Dr. Werawan Ruangyttikarn, Department of Forensic Medicine, Chiang Mai University. Sample preparation: Hairs samples were cropped near the hair root. After washing, they were cut into 1.2-cm sections and extracted with methanol/5N HCl (2:1) for an hour and then, solid-phase extraction was conducted using Bond-Elut Certify. Following extraction, GC-MS analysis was performed. Urine samples were subjected to GC-MS analysis after preparation with Bond Elut Certify. Results and Discussion: In 6 samples, both urine and hair samples analyzed were negative for detection of the stimulant drugs. In those cases individuals might stop taking drug for about 5 months. In 18 samples, urine samples were negative whereas hair samples were positive. These results suggest that individuals might stop using drugs for a few days before they went to the hospital but they abuse drugs continuously. In 9 samples, both urine and hair samples were positive. These results show that individuals always abuse drugs. In order to treat drug dependence effectively it is necessary to obtain the patient history of drug use and to evaluate and determine short-term and long-term drug use with urinalysis and hair analysis, respectively. Our present data revealed that useful information concerned with the long term drug abuse can be obtained from hair analysis, and that this method of analysis is applicable not only to forensic cases but also for evaluating clinical cases.     

Disposition of drugs of abuse and their metabolites in wastewater as a method of the estimation of drug consumption

The occurrence of drugs and trace amounts of their metabolites in the aqueous environment has become a global problem. Nowadays, general information about drug use patterns results from indirect methods such as anonymous surveys and police crime statistics. Unfortunately, these sources of information determine drug use consumption indirectly and give only rough estimations about drug use trends. Therefore, in order to assess the real extent of this phenomenon, new objective tools are needed to monitor drug abuse on a large social and international scale. Several analytical methods have been developed to diagnose illicit drug consumption. GC-MS and HPLC-MS are the techniques of choice for the quantitative analysis of illicit drugs and their metabolites in clinical and forensic toxicology. These separation methods have been widely used for the determination of the occurrence of stimulatory drugs in different biological matrixes such as blood, urine, sweat, saliva and hair. Recently, a new direct and objective approach of monitoring drug use patterns has been proposed to estimate illicit drug consumption involving the measurement of urinary breakdown products in waste- and surface water. The approach proposed seems to be suitable for monitoring consumption in real time so that it is possible to identify trends in drug use patterns. The measurement of illicit drug residue in wastewater and surface water might become a standardized tool for the comprehensive assessment of drug abuse in populations.     

Detection times of drugs of abuse in blood, urine, and oral fluid

Data on the detection times of drugs of abuse are based on studies of controlled administration to volunteers or on the analysis of biologic samples of subjects who are forced to stop their (often chronic) use of drugs of abuse, eg, because of imprisonment or detoxification. The detection times depend mainly on the dose and sensitivity of the method used and also on the preparation and route of administration, the duration of use (acute or chronic), the matrix that is analyzed, the molecule or metabolite that is looked for, the pH and concentration of the matrix (urine, oral fluid), and the interindividual variation in metabolic and renal clearance. In general, the detection time is longest in hair, followed by urine, sweat, oral fluid, and blood. In blood or plasma, most drugs of abuse can be detected at the low nanogram per milliliter level for 1 or 2 days. In urine the detection time of a single dose is 1.5 to 4 days. In chronic users, drugs of abuse can be detected in urine for approximately 1 week after last use, and in extreme cases even longer in cocaine and cannabis users. In oral fluid, drugs of abuse can be detected for 5-48 hours at a low nanogram per milliliter level. The duration of detection of GHB is much shorter. After a single dose of 1 or 2 ng of flunitrazepam, the most sensitive methods can detect 7-aminoflunitrazepam for up to 4 weeks in urine.     

Functional involvement of cerebral diazepam binding inhibitor (DBI) in the establishment of drug dependence

Mechanisms for formation of drug dependence and emergence of withdrawal syndrome are not yet fully understood despite of a huge accumulation of experimental and clinical data. Several clinical features of withdrawal syndrome are considered to be common (i.e., anxiety) among patients with drug dependence induced by different drugs of abuse. In this review, we have discussed the possibility of the functional involvement of diazepam binding inhibitor (DBI), an endogenous neuropeptide for benzodiazepine receptors with endogenously anxiogenic potential, in the development of drug dependence and emergence of its withdrawal symptom. The levels of DBI protein and its mRNA significantly increased in the brain derived from mice dependent on alcohol (ethanol), nicotine and morphine, and abrupt cessation of these drugs facilitated further increase in DBI expression. In the cases of nicotine- and morphine-dependent mice, concomitant administration of antagonists for nicotinic acetylcholine and opioid receptors, respectively, abolished the increase in DBI expression. Therefore, these alterations in DBI expression have a close relationship with formation of drug dependence and/or emergence of withdrawal syndrome and are considered to be a common biochemical process in drug dependence induced by different drugs of abuse.     

Detectability of new psychoactive substances, ‘legal highs’, in CEDIA,EMIT, and KIMS immunochemical screening assays for drugs of abuse

The increasing number of new psychoactive substances made available for recreational drug use has created a challenge for clinical toxicology and drug testing laboratories. As a consequence, the routine immunoassay drug testing may become less effective due to an increased occurrence of false negative and false positive screening results. This work aimed to extend the knowledge about analytical cross‐reactivity of new substances in selected CEDIA, EMIT, and KIMS immunoassays for drugs‐of‐abuse screening. Urine standards were prepared by spiking blank urine with 45 new substances. Authentic urine samples from intoxication cases identified by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) were also studied. Several new psychoactive substances were demonstrated to display cross‐reactivity in the immunoassays. CEDIA Amphetamine/Ecstasy and EMIT d.a.u. Amphetamine Class tests showed the highest reactivity towards the new drugs, which was expected since many have amphetamine‐like structure and activity. In the samples from authentic cases, five new substances displayed 100% detection rate in the CEDIA Amphetamine/Ecstasy test. In conclusion, cross‐reactivity data in routine urine drug screening immunoassays for a number of new psychoactive substances not studied before were reported. In both spiked and authentic urine samples, some new substances showed significant cross‐reactivity and are thus detectable in the routine screening methods. Copyright © 2014 John Wiley & Sons, Ltd.     

Screening for basic drugs in hair of drug addicts by liquid chromatography/time-of-flight mass spectrometry

Hair analysis in forensic and clinical toxicology has been strongly focused on drugs of abuse, and comprehensive, drug class-independent screening methods based on mass spectrometric detection have not been applied to date. In this study, a qualitative drug screening method by liquid chromatography coupled to time-of-flight mass spectrometry, earlier developed and evaluated for forensic toxicological urine analysis, was adapted for screening of basic drugs in hair. The method included alkaline hydrolysis, purification with mixed-mode solid phase extraction, and analysis by liquid chromatography coupled to time-of-flight mass spectrometry with automated data analysis and reporting. Identification was based on accurate mass, isotopic pattern fit, and retention time, if available. Analysis of 32 hair samples from deceased drug addicts revealed 35 different drugs. The drug classes identified included antidepressants, antipsychotics, antiepileptics, amphetamines, opioids, beta-blockers, a benzodiazepine, a hypnotic, a local anesthetic, an antiemetic, and an antipyretic analgesic. The findings were in good agreement with the findings in blood and urine by other methods. Moreover, information about previous drug use not evident in the analysis of other matrices was obtained in the majority (72%) of the cases. Tramadol was an especially predominant finding, suggesting tramadol abuse as an opioid substitute. One apparent false-positive finding was identified. The mean and median mass accuracies of positive findings were 2.3 and 1.8 ppm, corresponding to 0.5 and 0.4 mDa, respectively. Cutoff values for tramadol and methamphetamine in hair were 100 and 200 pg/mg, respectively. The method proved to be a simple and straightforward tool for comprehensive screening of basic drugs in hair.     

Analytical validation for a series of marker compounds used to assess renal drug elimination processes

Renal drug elimination is determined by glomerular filtration, tubular secretion, and tubular reabsorption. Changes in the integrity of these processes influence renal drug clearance, and these changes may not be detected by conventional measures of renal function such as creatinine clearance. The aim of the current study was to examine the analytic issues needed to develop a cocktail of marker drugs (fluconazole, rac-pindolol, para-aminohippuric acid, sinistrin) to measure simultaneously the mechanisms contributing to renal clearance. High-performance liquid chromatographic methods of analysis for fluconazole, pindolol, para-aminohippuric acid, and creatinine and an enzymatic assay for sinistrin were developed or modified and then validated to allow determination of each of the compounds in both plasma and urine in the presence of all other marker drugs. A pilot clinical study in one volunteer was conducted to ensure that the assays were suitable for quantitating all the marker drugs to the sensitivity and specificity needed to allow accurate determination of individual renal clearances. The performance of all assays (plasma and urine) complied with published validation criteria. All standard curves displayed linearity over the concentration ranges required, with coefficients of correlation greater than 0.99. The precision of the interday and intraday variabilities of quality controls for each marker in plasma and urine were all less than 11.9% for each marker. Recoveries of markers (and internal standards) in plasma and urine were all at least 90%. All markers investigated were shown to be stable when plasma or urine was frozen and thawed. For all the assays developed, there were no interferences from other markers or endogenous substances. In a pilot clinical study, concentrations of all markers could be accurately and reproducibly determined for a sufficient duration of time after administration to calculate accurate renal clearance for each marker. This article presents details of the analytic techniques developed for measuring concentrations of marker drugs for different renal elimination processes administered as a single dose to define the processes contributing to renal drug elimination.     

Determination of thiamine and its phosphorylated forms in human plasma, erythrocytes and urine by HPLC and fluorescence detection: a preliminary study on cancer patients

In man, neurotoxicity associated to ifosfamide treatment can be reversed by intravenous thiamine administration. Trying to explain this clinical finding, we decided to study possible changes in thiamine availability and activation in patients exposed to ifosfamide. Free thiamine and its phosphate esters levels were measured in plasma, erythrocytes and urine by an ion-pair HPLC method with pre-column derivatization, which allowed separation of the fluorescent compounds in less than 10 min. The method was validated by linearity, sensitivity and reproducibility studies, whose values met the demands for bioanalytical assays. This method was applied to assess thiamine status in cancer patients exposed to ifosfamide therapy for advanced disease.     

Workplace drug testing in Italy – critical considerations

Workplace drug testing (WDT) was established in Italy on 30 October 2007. Two tiers of survey are required: the first tier concerns drug testing on urine samples, the second involves both urine and hair analysis. Between July 2008 and December 2011, 10 598 workers’ urine samples and 72 hair samples for opiates, cocaine, cannabinoids, amphetamines, methylenedioxyamphetamines, methadone, and buprenorphine were tested in our laboratory. Urine analyses were performed by immunological screening (EMIT); hair analysis and confirmation tests in urine were performed by gas chromatography–mass spectrometry (GC‐MS). Employees tested positive in urine for drugs of abuse numbered 2.8% in 2008, 2.03% in 2009, 1.62% in 2010, and 1.43% in 2011. As regards the second level of analysis, we observed that only one‐third of the workers who had been tested positive for drugs of abuse were referred to an Addiction Treatment Unit in order to verify drug addiction. Our experience shows that, four years after approval of the law on WDT, the percentage of workers positive for drugs of abuse in urine has reduced in comparison to the first year. Moreover, our data show that most of the times employees who tested positive are tardily referred or not referred at all to a Public Addiction Treatment Unit to verify drug addiction. This makes us believe that the legal provisions are widely disregarded not paying the right tribute to the fact that Italy is one of few European countries with legislation on WDT. Copyright © 2013 John Wiley & Sons, Ltd.     

Testing for drug use, Part 1: Analytical methods

Issues surrounding the screening and testing of individuals for drug use, including analytical and legal aspects of the procedures and social, political, and ethical problems and concerns, are reviewed. Historically, professional and societal debate regarding drug taking, drug-use problems, and the utility of drug testing programs occurs in cycles. Analytical methods commonly used to test for drug use include breath analysis for alcohol and urine drug assays. Blood alcohol concentrations are determined by laboratory assay methods or by portable devices used in the field. While poor laboratory procedures can invalidate test results for both breath and urine tests, urine screening test results can be further invalidated by improper handling of specimens or by tampering on the part of the subject. Also, test results are meaningful only if they are correlated with a clinical state. Legal issues have been raised concerning the validity of testing procedures used and the reliability of evidence obtained, especially in relation to pre-employment drug screening. From an ethical standpoint, drug testing tends to focus efforts to combat drug abuse on the drugs themselves instead of on the social context of the problem. With a recycled interest in drug-use testing and screening, primarily attributable to technological advances, little attention is being given to other approaches to controlling drug use. Additional research is needed to better describe the nature and extent of our drug-use problems and their impact on society.     

Urine fingerprinting: detection of sample tampering in an opiate dependency program

Methadone treatment programs commonly monitor patient compliance by screening urine samples for drugs of abuse. Our experience suggests that re-submission of urine samples (for example, providing a urine sample that is either not that of the patient or was previously submitted) is often used as a method of sample tampering. We have developed an algorithm that combines urine sodium, chloride, creatinine and pH values with urine drug screening results to effectively detect resubmitted samples. Given the widespread use of urine drug screening in drug and alcohol rehabilitation programs, we believe this technique has significant practical benefits. This technique may also have an application in forensic identification of duplicate samples.     

Serum concentrations,pharmacokinetic/pharmacodynamic modeling,and effects of dexamethasone on inflammatory mediators following intravenous and oral administration to exercised horses

Corticosteroids are potent anti‐inflammatory drugs and as such are commonly administered to performance and racehorses. The objectives of the current study were to describe blood and urine concentrations and the pharmacokinetics and effects on cortisol and inflammatory mediator concentrations, following intravenous and oral administration to 12 exercised horses. Horses received an intravenous administration of 40 mg of dexamethasone sodium phosphate and 20 mg of dexamethasone tablets with a 4 week washout in between administrations. Blood and urine samples were collected prior to and for up to 96 hours post drug administration. Whole blood samples were collected at various time points and challenged with lipopolysaccharide or calcium ionophore to induce ex vivo synthesis of eicosanoids. The concentrations of dexamethasone and eicosanoids were measured using LC–MS/MS and the concentrations from both routes of administration fit simultaneously using a three‐compartment pharmacokinetic model. A turnover model with inhibition of K_in gave an adequate fit to the dexamethasone‐cortisol PKPD data. Serum and urine dexamethasone concentrations were at the limit of quantitation at 96 and 48 hours post administration, respectively. The volume of distribution, systemic clearance, and terminal half‐life was 0.907 L/kg, 7.89 mL/h/kg, and 1.34 h, respectively. The IC₅₀ for cortisol suppression was 0.007 ng/mL. Stimulation of dexamethasone treated blood with lipopolysaccharide and calcium ionophore resulted in an inhibition of inflammatory biomarker production for a prolonged period of time post drug administration. The results of this study suggest that dexamethasone has a prolonged anti‐inflammatory effect following intravenous or oral administration to horses.