Ritalinic Acid Research Articles

UHPLC-QTOFMS Urine Drug Screening With Dilute-and-Shoot Sample Preparation and Vacuum-Insulated Probe-Heated Electrospray Ionization.

We developed a method for comprehensive urine drug screening by applying dilute-and-shoot extraction and vacuum-insulated probe-heated electrospray ionization with ultra-high performance liquid chromatography high-resolution quadrupole time-of-flight mass spectrometry (DS-UHPLC-VIP-HESI-QTOFMS). The method involved five-fold post-hydrolysis dilution of urine samples and chromatography on a C18 UHPLC column prior to QTOFMS analysis. The recently introduced VIP-HESI ion source was chosen due to its enhanced ionization efficiency and compatibility with UHPLC-QTOFMS. Extensive data was acquired in positive ion mode with a low collision energy (7 eV) and an elevated collision energy (30 eV), using the broadband collision-induced dissociation data acquisition scan mode that continuously generated high-resolution and accurate mass for parent and fragment qualifier ions, and parent ion isotopic patterns. Compound identification was performed against an in-house database with 1263 compound entries, using an automated post-run reverse target database search with preset identification criteria. Method validation with 56 different drugs showed acceptable results for the limit of identification (median 5 ng/mL), matrix effects (70-130%), repeatability of retention times (< 1%), mass accuracy (< 1 mDa), as well as for specificity and stability. As compared with an established UHPLC-QTOFMS method relying on solid-phase extraction and conventional electrospray ionization, DS-UHPLC-VIP-HESI-QTOFMS produced comparable results from authentic clinical urine samples for most drugs, but showed clearly improved detectability for pregabalin, gabapentin, and ritalinic acid. We anticipate that the new method will be a step forward for laboratories performing routine urine drug screening due to its fast turnaround time, reduced manual workload, cost efficiency, and broad substance coverage.

Effect of bariatric surgery on the pharmacokinetics of drugs used for attention-deficit hyperactivity disorder-A case series.

Changes in gastrointestinal physiology following bariatric surgery may affect the pharmacokinetics of drugs. Data on the impact of bariatric surgery on drugs used for attention-deficit/hyperactivity disorder (ADHD) are limited. In patients treated with ADHD medication and undergoing bariatric surgery, serial drug concentrations were measured for 24 h preoperatively and one, six and 12months postoperatively. Primary outcome was change in area under the concentration-time curve from 0 to 24 h (AUC0-24), with other pharmacokinetic variables as secondary outcomes. Eight patients treated with lisdexamphetamine (n= 4), dexamphetamine (n= 1), methylphenidate (n= 1) and atomoxetine (n= 2) were included. In total, 409 samples were analysed. Patients underwent sleeve gastrectomy (n= 5) and Roux-en-Y gastric bypass (n= 3). AUC0-24 and Cmax of dexamphetamine increased after surgery in those using the prodrug lisdexamphetamine. There was no clear-cut reduction in tmax postoperatively. For ritalinic acid and atomoxetine, no changes in AUC0-24 were observed, but for atomoxetine, a higher Cmax and a shorter tmax were observed postoperatively. Bariatric surgery may increase the systemic exposure of dexamphetamine after intake of lisdexamphetamine. Patients using lisdexamphetamine should be followed with regard to adverse drug reactions after bariatric surgery, and, if available, therapeutic drug monitoring should be considered.

Inhibitory Effects of Powdered Liquid Smoke from Mangrove (Avicennia marina) Twigs on Spoilage Bacteria

This study investigated the inhibitory effects of powdered liquid smoke derived from Avicennia marina (mangrove) twigs on spoilage bacteria. The research method used was experimental. Branches of mangrove (A. marina) were burned and distilled to produce liquid smoke. The powdered liquid smoke was subsequently tested for its antibacterial capabilities against foodborne pathogen bacteria. The damage to the bacterial formation was further illustrated using a Scanning electron micrograph. The components of liquid smoke were analysed using GC-MS. GC-MS test results on liquid smoke from mangrove twigs revealed several components including groups of acids such as butanoic acid, 1H-pyrrole-3-propanoic acid, 2-(4-methoxy-3-methylphenyl)-thiazolidine-4-carboxylic acid, hexanedioic acid, 2-bromo propionic acid, carbamic acid, oxalic acid, ritalinic acid and 7-dimethyl-6-octenyl acetate (acetic acid), as well as oxalic acid decyl propyl ester and 1,2-benzene dicarboxylic acid. The phenol group includes cyclohexanol, 2-cyclohexen-1-ol, 2-methyl-5-(1-methylethenyl)-, cis, 1,4-dihydroxy-p-menth-2-ene, phenol, 3-methyl, phenol, 2-methoxy-4-(1-propenyl)-, acetate, 1,2-diphenyl-2-ethanediol and 1,2-ethanediol. The ketone group includes beta-piperidino propiophenone, pyrrolidine-2-one, 5-[2-benzoylethyl]-,benzoyl amino-1-(4-methylphenyl)-3-piperidinyl propane, 3-nonen-5-one, 1-(1-butoxypropan-2-yloxy) propan-2-yl 2-methylbut-2-enoate, 6-octen-1-ol, 3,7-dimethyl, 4-(2,2,6-trimethyl cyclohexyl)-2-butanone and the carbonyl group includes N2-[(phenylmethoxy) carbonyl].

Prevalence of Drugs of Abuse and Cognitive Enhancer Consumption Monitored in Grab Samples and Composite Wastewater via Orbitrap Mass Spectrometry Analysis.

Wastewater (WW)-based epidemiology is an approach for the objective surveillance of the consumption of (illicit) drugs in populations. The aims of this study were to monitor drugs of abuse, cognitive enhancers, and their metabolites as biomarkers in influent WW. Data obtained from different sampling points and mean daily loads were compared with previously published data. The prevalence of analytes was monitored in WW grab samples collected monthly over 22 months at two sampling points and 24 h composite WW samples collected over 2 weeks at a WW treatment plant in the same city. Quantification was performed using a previously validated and published method based on solid-phase extraction followed by liquid chromatography coupled with high-resolution tandem mass spectrometry. Grab samples allowed for frequent detection of ritalinic acid and sporadic detection of drugs of abuse. The daily mean loads calculated for 24 h WW composite samples were in accordance with data published in an international study. Furthermore, loads of amphetamine and methamphetamine increased compared with those observed in a previously published study from 2014. This study showed frequent quantification of ritalinic acid in the grab samples, while drugs of abuse were commonly quantified in the composite WW samples. Daily mean loads were in accordance with trends reported for Germany.

Quantitative analysis of drugs of abuse and cognitive enhancers in influent wastewater by means of two chromatographic methods.

Sewage-based epidemiology using influent wastewater is used to estimate the consumption trends of (illicit) drugs over a short or long period of time in a subpopulation. The current study aimed to develop two separate methods for the quantitative analysis of selected drugs of abuse (DOA) and cognitive enhancers in influent wastewater using reversed-phase (RP) or hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS). The performance of RP and HILIC column was evaluated. A simple solid phase extraction was used for sample preparation. Short runtimes of 10 and 15 min on the RP and the HILIC column, respectively, allowed sufficient throughput. A six-point calibration was used for quantification with calibration ranges between 10 and 100 ng/L for all analytes except for benzoylecgonine (BZE, 30-300 ng/L). Method validation was performed according to ICH guideline M10. Analytes such as amphetamine (AMPH), BZE, cocaethylene (CE), cocaine (COC), ethyl sulfate, 4-hydroxy-3-methoxymethamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine, methylphenidate (MPH), and ritalinic acid (RA) were included in method development and validation. Two different column types were necessary for sufficient chromatographic resolution. The analytical setup allowed detection of all other analytes at concentration levels between 1ng/L for methylphenidate to 10ng/L for amphetamine. A method for the detection and quantification of DOA, cognitive enhancers, and their biomarkers in wastewater was successfully developed and validated. Moreover, six proof-of-concept samples were analyzed in which AMPH, BZE, COC, MDMA, MPH, and RA were identified and further quantified.

Target and suspect screening of (new) psychoactive substances in South Korean wastewater by LC-HRMS

New psychoactive substances (NPS) are a type of abused drug designed to mimic the effects of the currently known illicit drugs, whose structures are constantly changing to escape surveillance. The quick identification of NPS use in the community therefore demands immediate action. This study aimed to develop a target and suspect screening method using LC-HRMS to identify NPS in wastewater samples. An in-house database of 95 traditional and NPS was built using the reference standards, and an analytical method was developed. Wastewater samples were collected from 29 wastewater treatment plants (WWTP) across South Korea, representing 50 % of the total population. The psychoactive substances in waste water samples were screened using in-house database and developed analytical methods. A total of 14 substances were detected in the target analysis, including three NPS (N-methyl-2-AI, 25E-NBOMe, and 25D-NBOMe) and 11 traditional psychoactive substances and their metabolites (zolpidem phenyl-4-COOH, ephedrine, ritalinic acid, tramadol, phenmetrazine, phendimetrazine, phentermine, methamphetamine, codeine, morphine, and ketamine). Out of these, N-methyl-2-AI, zolpidem phenyl-4-COOH, ephedrine, ritalinic acid, tramadol, phenmetrazine, and phendimetrazine were detected with a detection frequency of over 50 %. Primarily, N-methyl-2-Al was detected in all the wastewater samples. Additionally, four NPSs (amphetamine-N-propyl, benzydamine, isoethcathinone, methoxyphenamine) were tentatively identified at level 2b in a suspect screening analysis. This is the most comprehensive study to investigate NPS using target and suspect analysis methods at the national level. This study raises a need for continuous monitoring of NPS in South Korea.

Chiral separation and quantitation of methylphenidate, ethylphenidate, and ritalinic acid in blood using supercritical fluid chromatography.

Supercritical fluid chromatography (SFC)is a technique that analyzes compounds that are temperature-labile, have moderately low weight, or are chiral compounds. Methylphenidate (MPH) is a chiral compound with two chiral centers. MPH has two chiral metabolites, ethylphenidate (EPH) and ritalinic acid (RA). MPH is sold as a racemic mixture. The d-enantiomer of threo-MPH is responsible for medicinal effects. Due to the differing effects of the enantiomers, it is important to analyze the enantiomers individually to better understand their effects. This method utilizes SFCand solid-phase extraction (SPE) to separate and analyze the enantiomers of MPH, EPH, and RA in postmortem blood. The objective of this method was to assess a unique approach with SFC for enantiomeric separation of MPH, EPH, and RA. A SPE method was developed and optimized to isolate the analytes in blood and validated as fit-for-purpose following international guidelines. The linear range for MPH and EPH was 0.25-25 and 10-1000 ng/mL for RA in blood. Bias was -8.6% to 0.8%, and precision was within 15.4% for all analytes. Following method validation, this technique was applied to the analysis of 49 authentic samples previously analyzed with an achiral method. Quantitative results for RA were comparable to achiral technique, whereas there was loss of MPH and EPH over time. The l:d enantiomer ratio was calculated, and MPH demonstrated greater abundance of the d-enantiomer. This is the first known method to separate and quantify the enantiomers of all three analytes utilizing SFC and SPE.

Doping control in horses in the Czech Republic in 2010-2019

The aim of this study was to evaluate data on doping controls in racehorses over a given ten-year period, primarily to identify positive findings and to summarise recommendations for the prevention of accidental contamination with prohibited substances, where appropriate. Data on doping controls of racehorses in the Czech Republic from 2010 to 2019 were obtained from the archives of the Jockey Club of the Czech Republic. For each year, the total number of horses starting at races held in the Czech Republic, the number of horses tested, and the results of the doping controls were determined. Data on the type of samples, positive findings and statements from responsible persons about the cause of the positive finding were recorded. During the monitoring period, 11,852 horses competed in races in the Czech Republic and 641 of them underwent a doping control. Blood was taken from 356 horses as the sample for testing and urine was collected from 285 horses. A total of 13 positive findings (2.03% of the 641 tested) were found during the period, namely of morphine, caffeine, theobromine, omeprazole sulphide, furosemide, clenbuterol, norketamine, ritalinic acid, dexamethasone, flunixin, hydroxylidocaine and oripavine. The most common cause, in a total of seven horses, was confirmed as suspected feed contamination. Prevention of positive doping results in our circumstances should therefore be directed primarily towards compliance with proper feed and stable management.

What is the added value of liquid chromatography-tandem mass spectrometry for routine urine drug screening in clinical situations?

Replacing the common two-step process of urine drug screening by a single-step methodology based on selective and quantitative liquid chromatography-tandem mass spectrometry (LC-MSMS) seems quite attractive. With this approach, it may be more appropriate to use the limit of analytical quantification instead of a cutoff concentration to report positive results. In this study, we want to evaluate the impact of this modification on the results of urine samples received in clinical situations. Results of all urine drug screens and associated confirmations performed between July 2021 and March 2022 for amphetamine, benzodiazepine and opiate groups, cannabis and cocaine metabolites and methadone were retrieved from the laboratory information system of the Medical Biology Department of Laboratoire National de Santé in Luxembourg. The samples came from hospitals, addiction centers, prisons and occupational health clinics. They were analyzed by an automated immunoassays, Cobas (Roche Diagnostics) and by a targeted quantitative screening assay, MassTox Drugs of Abuse testing, (Chromsystems) on a 6500QTRAP LC-MSMS (Sciex). We calculated the number of samples where results were discordant. Cases where no drugs were identified by LC-MSMS but results of the immunoassay were above the cut-off concentration (based on the screen test cut-off concentration of European guidelines) were considered as false positive (FP) results. Whereas cases where drugs were quantified by LC-MSMS but results of the immunoassay were below the cut-off concentration were considered as false negative (FN) results. The FP and FN results were 1 and 5% for cannabis metabolite ( n = 477), 0 and 14% for cocaine metabolite ( n = 189), 2 and 20% for methadone ( n = 211), 1 and 10% for benzodiazepines ( n = 489), 1 and 20% for opiates ( n = 149) and 48 and 2% for Amphetamines ( n = 92). The median concentrations of FN were 18 ng/ml (range from 15 to 31) for THC-COOH, 62 ng/ml (range from 24 to 121) for benzoylecgonin, 221 ng/ml (from 63 to 785) for methadone and 367 ng/ml (from 66 to 3174) for EDDP. In the benzodiazepine FN results (median concentration 69 ng/ml, range from 26-318), oxazepam, lorazepam and OH-alprazolam were the most frequently detected drugs. Among the opiate FN results, morphine associated or not with codeine was the most frequently quantified drug (median 81 ng/ml, range from 27 to 519). Four out of the 29 FN samples contained 6-monoacetylmorphine (from 17 to 136 ng/ml) in the absence of morphine, 2 samples were positive for pholcodine and one sample contained high concentration of meperidine. The two FN samples for amphetamines contained amphetamine (500 and 800 ng/ml). Nine out of the 42 amphetamines FP samples contained ritalinic acid (from 10,000 to 130,000 ng/ml). This targeted method improves the sensitivity but is restricted to a pre-defined list of drugs, although it covers a wider panel than commercial immunoassays, some relevant molecules may be missing. The hydrolysis protocol used before LC-MSMS analysis also explains the increase in sensitivity for benzodiazepines and opiates (allowing the detection of conjugated metabolites). Decreasing the threshold of detection for urine drug screening allowed for the extension of the drug detection window, improving the detection of drugs administered at low dose or those reacting poorly in immunoassay. This single step approach bypasses the main pitfalls of the immunoassays, namely the lack of analytical specificity (mainly for amphetamines) and a screening limited to well-established (parent) drugs, with consequences on patient management still only partially objectified. This targeted quantitative screening LC-MSMS method with its high sensitivity, accurate quantification, and associated internal quality control, achieves the required analytical performances expected in a clinical setting. The implementation of this approach will largely be conditioned by the optimization of the sample throughput to ensure a turn-around-times compatible with various clinical situations and the availability of the equipment and highly trained staff.

Reaction of Chlorine Dioxide with Saturated Nitrogen-Containing Heterocycles and Comparison with the Micropollutant Behavior in a Real Water Matrix.

Chlorine dioxide (ClO2) is a very selective oxidant that reacts with electron-rich moieties such as activated amines and thus can degrade specific N-containing micropollutants. N-containing heterocycles (NCHs) are among the most frequent moieties of pharmaceuticals. In this study, the reactions of ClO2 with ritalinic acid and cetirizine, two abundant micropollutants, and model compounds representing their NCH moiety were investigated. The pH-dependent apparent reaction rates of all NCHs with ClO2 were measured and modeled. This model showed that neutral amines are the most important species having reaction rates between 800 and 3200 M-1 s-1, while cationic amines are not reactive. Ritalinic acid, cetirizine, and their representative model compounds showed a high stoichiometric ratio of ≈5 moles ClO2 consumption per degraded ritalinic acid and ≈4 moles ClO2 consumption per degraded cetirizine, respectively. Investigation of chlorine-containing byproducts of ClO2 showed that all investigated NCHs mostly react by electron transfer and form above 80% chlorite. The reactions of the model compounds were well comparable with cetirizine and ritalinic acid, indicating that the model compounds indeed represented the reaction centers of cetirizine and ritalinic acid. Using the calculated apparent reaction rate constants, micropollutant degradation during ClO2 treatment of surface water was predicted for ritalinic acid and cetirizine with -8 to -15% and 13 to -22% error, respectively. The results indicate that in ClO2-based treatment, piperidine-containing micropollutants such as ritalinic acid can be considered not degradable, while piperazine-containing compounds such as cetirizine can be moderately degraded. This shows that NCH model compounds could be used to predict micropollutant degradation.

The Pharmacokinetics and Pharmacogenomics of Psychostimulants.

The psychostimulants-amphetamine and methylphenidate-have been in clinical use for well more than 60 years. In general, both stimulants are rapidly absorbed with relatively poor bioavailability and short half-lives. The pharmacokinetics of both stimulants are generally linear and dose proportional although substantial interindividual variability in pharmacokinetics is in evidence. Amphetamine (AMP) is highly metabolized by several oxidative enzymes forming multiple metabolites while methylphenidate (MPH) is primarily metabolized by hydrolysis to the inactive metabolite ritalinic acid. At present, pharmacogenomic testing as an aid to guide dosing and personalized treatment cannot be recommended for either agent. Few pharmacokinetically based drug-drug interactions (DDIs) have been documented for either stimulant.

Development of analytical method for the determination of methylphenidate, the analog ethylphenidate and their metabolite ritalinic acid in oral fluid samples by micro-QuEChERS and liquid chromatography–tandem mass spectrometry

The aim of this study was to develop a quantitative method for the analysis of methylphenidate, the analog ethylphenidate and their metabolite ritalinic acid in oral fluid, using micro-QuEChERS extraction and liquid chromatography–tandem mass spectrometry (LC–MS/MS). Oral fluid samples were collected with Quantisal™ device, extracted by micro-QuEChERS technique and analyzed by LC–MS/MS. The developed method met the validation criteria of Academy Standards Board (ASB) Standard Practices for Method Validation in Forensic Toxicology (Standard 036, 2019) with limits of detection and quantification of 0.5 ng/mL and calibration curve from 0.5 to 50 ng/mL. Within-run imprecision was greater than 18.7% while between-run imprecision was greater than 17.0 % for all analytes. Bias did not vary more than 7.7 %. No evidence of carryover was found. Stability studies presented satisfactory results for 24 h on autosampler (10 °C), after 3 cycles of freeze/thaw, 7 days on freezer (−20 °C) and until 7 days on refrigerator (4 °C) for methylphenidate. The validated method was further successfully applied to the analysis of 5 authentic oral fluid samples collected from volunteers at parties and music festivals from different cities in Brazil. Four samples had positive results for methylphenidate and ritalinic acid, and only one sample was positive for methylphenidate. Ethylphenidate was not detected in the samples. The method showed acceptable analytical performance and is environmentally friendly, requiring reduced use of solvents and reagents, with potential to be applied to clinical and forensic analyses.

Plasma concentrations of methylphenidate enantiomers in adults with ADHD and substance use disorder, with focus on high doses and relationship to carboxylesterase activity.

Scarce data are available on methylphenidate (MPH) plasma concentrations reached after doses higher than 180 mg. The interindividual and intraindividual variability in the exposure of MPH and ritalinic acid (RA) enantiomers was examined in 28 patients with ADHD and substance use disorders, with MPH daily doses between 30 and 600 mg (median 160 mg). MPH and RA plasma concentrations were analysed with an enantioselective LC–MS/MS method. d‐MPH plasma concentration/dose varied 25‐fold between subjects but was reasonably stable within an individual. Twelve subjects had quantifiable l‐MPH plasma concentrations, which accounted for up to 48% of the total MPH plasma concentration. The less active l‐MPH enantiomer could, in individuals with low carboxylesterase 1 (CES1) activity, contribute significantly to the total MPH plasma drug concentration and hamper the estimation of the exposure to the more active d‐MPH enantiomer. However, the high correlation between the total (d + l) RA/MPH metabolic ratio and the d‐RA/d‐MPH metabolic ratio (r s = 0.94) indicates that the ratio based on non‐enantioselective analysis could be used as a marker of CES1 activity. Whether this holds true for subjects with aberrant metabolism due to genetic variants or during concomitant treatment with inhibitors or inducers of the enzyme remains to be studied.

Chiral separation and quantification of d,l-methylphenidate, d,l-ethylphenidate and ritalinic acid in blood by LC-MS/MS

Drugs such as methylphenidate (MPH) are commonly prescribed for Attention-Deficit/Hyperactivity Disorder but may be abused recreationally. Threo-MPH is associated with pharmacological effects and is present as dextro (d) or levo (l) configuration, with the d configuration being more potent. However, many medications are sold as a racemic mixture and thus analytic separation of isomers is essential. Additionally, enantiomeric separation can be used to potentially determine illegal production. MPH metabolizes into ritalinic acid (RA) as well as ethylphenidate (EPH) in the presence of ethanol. Chiral analysis poses challenges to researchers. Due to limited assays, this project aimed to develop a method that separates and quantifies the enantiomers of MPH and EPH as well as RA in blood. Methods such as this are critical to understanding the pharmacokinetics of chiral cognitive stimulants. This method uses solid phase extraction and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and was fully validated. The linear range for MPH and EPH was 0.5–200 ng/mL and 0.5–500 ng/mL for RA. Limit of detection was 0.1 ng/mL (with the exception of RA with a LOD of 0.5 ng/mL) and the limit of quantification was 0.5 ng/mL, despite significant matrix effects. Extraction recovery was > 79%. Bias was −4.8 to −12.7% and maximum within-run precision was ± 12.5% for all analytes. The optimized and validated technique offers chiral separation of the threo-enantiomers of d,l-MPH, d,l-EPH and RA and quantification in blood utilizing LC-MS/MS.

Development of Analytical Method for the Determination of Methylphenidate, the Analog Ethylphenidate and Their Metabolite Ritalinic Acid in Oral Fluid Samples by Micro-Quechers and Liquid Chromatography−Tandem Mass Spectrometry

Development of Analytical Method for the Determination of Methylphenidate, the Analog Ethylphenidate and Their Metabolite Ritalinic Acid in Oral Fluid Samples by Micro-Quechers and Liquid Chromatography−Tandem Mass Spectrometry

Ritalinic acid in urine: Impact of age and dose

ObjectivesThe objective of this work was to study the results of urine drug testing for ritalinic acid (RA), the major urinary metabolite of methylphenidate (MP) (e.g., Ritalin®). The impact of age from 4 to 65 years old and older on median levels of RA was investigated as well as potential variations in pH, specific gravity and creatinine content of the patient urine samples. Design and MethodsSamples from patients who were 1) prescribed MP and found to be positive for RA, 2) prescribed MP but found to be negative for RA and 3) not prescribed MP but tested positive for RA were examined by liquid chromatography – mass spectrometry/mass spectrometry (LC-MS/MS) for RA concentration. The levels of RA were examined for median and average levels and further normalized and transformed to reveal a near gaussian distribution. ResultsOver 20,000 samples from patients who were prescribed MP were examined for this work. Analysis of these data for a subset of patients prescribed MP and testing positive for RA revealed statistically different median values of RA for school age patients of 6 years old through 17 years old from adult patients 18 through 64 years old. Another 6751 samples were positive for RA without a prescription but were not included in the overall assessment of these data. ConclusionsWhile not clear as to the reason, these data indicate that school age children under the age of 18 have much higher levels of RA than adult patients. These results can be used to estimate “normal” levels of RA in these chronically dosed populations.

Stability of Methylphenidate under Various pH Conditions in the Presence or Absence of Gut Microbiota.

Methylphenidate is one of the most widely used oral treatments for attention-deficit/hyperactivity disorder (ADHD). The drug is mainly absorbed in the small intestine and has low bioavailability. Accordingly, a high interindividual variability in terms of response to the treatment is known among ADHD patients treated with methylphenidate. Nonetheless, very little is known about the factors that influence the drug’s absorption and bioavailability. Gut microbiota has been shown to reduce the bioavailability of a wide variety of orally administered drugs. Here, we tested the ability of small intestinal bacteria to metabolize methylphenidate. In silico analysis identified several small intestinal bacteria to harbor homologues of the human carboxylesterase 1 enzyme responsible for the hydrolysis of methylphenidate in the liver into the inactive form, ritalinic acid. Despite our initial results hinting towards possible bacterial hydrolysis of the drug, up to 60% of methylphenidate is spontaneously hydrolyzed in the absence of bacteria and this hydrolysis is pH-dependent. Overall, our results indicate that the stability of methylphenidate is compromised under certain pH conditions in the presence or absence of gut microbiota.

Short- and Long-Term Stability of Methylphenidate and Its Metabolites in Blood.

Methylphenidate (MPH) is a medication used to combat attention-deficit/hyperactivity disorder by speeding up brain activity. MPH has two chiral centers; however, d-threo-MPH is responsible for its effects. Few studies have analyzed MPH and its metabolites, ritalinic acid (RA) and ethylphenidate (EPH), in blood. Stability studies are crucial in a forensic setting to provide insight on ideal storage conditions and analysis time. In this study, d,l-MPH, d,l-EPH and RA were analyzed at two concentrations (15 and 150 ng/mL) over 5 months at room temperature (∼25°C), refrigerated (4°C), frozen (-20°C) and elevated (35°C) temperatures. Analytes were analyzed using a validated liquid chromatography--mass spectrometry method. RA concentrations increased 53% at 25°C after 24 h, while d- and l-MPH concentrations dropped 18.1 and 20.6%, respectively. Additionally, d- and l-EPH concentrations decreased 22.3 and 28.8%, respectively. All analytes were stable at 4°C for 1 week (±17% change). At -20°C, all analytes were stable for 5 months. At 35°C, l-EPH remained stable for 24 h (14.4% loss) at the high concentration, while RA increased 244%. Losses of 64.1, 68.7 and 27.2% were observed for d- MPH, l-MPH and d-EPH, respectively. Due to this, a follow-up study was designed to assess the breakdown of MPH. The short-term experiment assessed d,l-MPH at two concentrations for 1 month in the same conditions. As MPH decreased, RA concentrations rose. At 25°C, it took 2 weeks for MPH to metabolize completely into RA. In refrigerated and frozen temperatures, MPH did not completely metabolize to RA. In elevated temperatures, MPH broke down to RA within 2 weeks. Due to this, it was concluded that d,l-MPH breaks down in the blood to its metabolite RA and may make data interpretation difficult if samples are not properly stored. The optimal storage for these analytes is recommended at -20°C.

Determination of methamphetamine and methylphenidate in urine by liquid chromatography/time-of-flight mass spectrometry coupled with solid-phase dispersive extraction, and its pharmacokinetic application

A simple and rapid analytical method for the determination of methamphetamine (MA) and methylphenidate (MPD) in urine was developed, which involves liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS) and solid-phase dispersive extraction (SPDE). The developed method could be used in pharmacokinetic studies. Oasis® WCX gel was used in SPDE. LC separation was carried out on a Poroshell 120 EC-C18 column (100 mm × 4.6 mm I.D., 2.7 µm) using a mixture (30:70) of methanol/10 mM ammonium formate aqueous solution (pH 3.0). The calibration curves showed good linearity in the range of 2 to 100 ng/mL for MA and 0.5 to 200 ng/mL for MPD. The limits of detection were 0.5 ng/mL and 0.1 ng/mL, and the limits of quantification were 2 ng/mL and 0.5 ng/mL for MA and MPD, respectively. The average recoveries of MA and MPD from urine samples spiked with MA at 5 and 50 ng/mL and MPD at 1 and 100 ng/mL were 93.9–94.8% (RSD: 1.6–2.1%) and 91.5–106.5% (RSD: 1.3–3.0%), respectively. Periodic analyses of urine samples collected from a volunteer receiving clinical doses of MPD revealed the presence of ritalinic acid (RA), an inactive major metabolite of MPD. Furthermore, when the RA/MPD ratios corrected by the urinary creatinine level were plotted against time, the regression curve showed linearity after 4 h. Pharmacokinetic study revealed that the extrapolated line of the regression line reached the origin in the graph, suggesting that the regression line could be used to calculate the dosage and timing of administration.

Wastewater-based epidemiology pilot study to examine drug use in the Western United States

The extent of prescription and illicit drug abuse in geographically isolated rural and micropolitan communities in the intermountain western United States (US) has not been well tracked. The goal of this pilot study was to accurately measure drug dose consumption rates (DCR) between two select populations, normalize the data and compare the DCRs to similar communities. To learn about patterns of drug abuse between the two disparate communities, we used the emergent field of wastewater-based epidemiology (WBE). A rapid, quantitative and systematic process for the determination of multiple classes of prescribed and illicit drugs was applied to influent wastewater samples. Influent samples were collected over the course of three months (April to June 2019) at two wastewater treatment plants representing a small urban and a rural community. Collection of sewage influent included 24-h composite samples and the use of polar organic chemical integrative samplers (POCIS), time-weighted samplers. Using the results from the composite sampling data, DCRs per 1000 population could be calculated from the concentration data and the use of excretion correction factors. The following 18 compounds: amphetamine, methamphetamine, MDA, MDMA, morphine, 6-acetylmorphine, methadone, EDDP, codeine, benzoylecgonine, hydrocodone, hydromorphone, oxycodone, noroxycodone, ketamine, fluoxetine, tramadol, and ritalinic acid; represent a subset of the targeted analytes that were consistently measured at detectable concentration levels, and present at both sites. Following normalization of the drug measurements to influent flow rates and per capita, the small urban community demonstrated greater collective excretion rates (CER) than the rural community, with the exceptions of amphetamine and methamphetamine.