Effect of catechin and commercial preparation of green tea essence on the pharmacokinetics of l-dopa in rabbits.

The aim of this study was to investigate drug interactions of L-dopa/carbidopa with catechin and green tea essence in rabbits following the simultaneous administration via an intramuscular injection of catechin or via an intragastric route for green tea essence with L-dopa/carbidopa. The results indicated that catechin at doses of 10, 20 and 50 mg/kg increased the area under the plasma concentration-time curve from time zero to the time of the last quantifiable concentration (AUC0-t ) of L-dopa by about 69, 78 and 42%, respectively. The metabolic ratios of the AUC0-t for 3-O-methyldopa (3-OMD)/L-dopa significantly decreased by about 56, 68 and 76% (P < 0.05), respectively. In addition, a single dose of 5/1.25 mg/kg L-dopa/carbidopa was co-administrated with 150 mg/kg green tea essence via an intragastric route with an oral-gastric tube. Comparing the related pharmacokinetic parameters of L-dopa, the clearance and metabolic ratio of L-dopa decreased by 20 and 19% (P < 0.05), respectively. In conclusion, catechin and green tea essence can significantly affect the metabolism of L-dopa by the catechol-O-methyltransferase (COMT) metabolic pathway. Catechin can enhance L-dopa bioavailability, and both catechin and green tea essence decreased 3-OMD formation. Therefore, catechin and green tea essence may increase L-dopa efficacy for Parkinson's disease treatment.

Levodopa-carbidopa intestinal gel high concentration formulation is clinically bioequivalent to commercial formulation.

A new levodopa-carbidopa intestinal gel (LCIG) system featuring a higher levodopa/carbidopa (LD/CD) concentration and viscosity, LCIG-HV, is being developed to reduce the intrajejunal volume of LD/CD that is administered as compared to the current commercial formulation, LCIG-LV. This study characterizes the LCIG-HV formulation and compares it to the LCIG-LV formulation via dissolution testing and a clinical pharmacokinetic bioequivalence study. In vitro release profiles of LD/CD were determined using a USP Dissolution Apparatus 2 with 500 mL of phosphate buffer (pH 4.5) operating at 25 RPM. A single dose, open-label study was conducted according to a two-period, randomized, crossover design in 28 healthy subjects. The point estimate (PE) of the levodopa Cmax geometric mean for the LCIG-HV formulation was 4% higher than that of the LCIG-LV formulation. PEs of levodopa AUCt and AUCinf geometric means were comparable for both formulations. PEs of carbidopa Cmax , AUCt and AUCinf geometric means for the LCIG-HV formulation were 3%-5% higher than those of the LCIG-LV formulation. For both formulations, the median Tmax for levodopa was 1.0 and 3.0 hours for carbidopa. The levodopa half-life harmonic mean was 1.6 hour for both formulations. The carbidopa half-life harmonic mean was 1.9 and 2.0 hour, respectively, for the LCIG-HV and LCIG-LV formulations. Cmax , AUCt and AUCinf of LD/CD carbidopa were comparable for both formulations. The current study demonstrates that the LCIG-LV and LCIG-HV formulations are clinically bioequivalent for LD/CD according to FDA guidance. However, the dissolution method was over discriminatory of formulation differences.

Effects of magnesium oxide on pharmacokinetics of L-dopa/carbidopa and assessment of pharmacodynamic changes by a model-based simulation.

BACKGROUND: Magnesium oxide (MgO) is often co-prescribed with L-dopa/carbidopa (LDCD) to improve constipation in Parkinson's disease patients. The mixing of L-dopa and MgO has been shown to degrade L-dopa; however, there is no interaction study on humans. We proposed mechanisms for the interaction between LDCD and MgO and conducted pharmacokinetic studies on rats and humans. To assess pharmacodynamic changes with the MgO treatment, we applied a model-based meta-analysis (MBMA). METHODS: The effects of MgO on the stabilities of L-dopa and carbidopa were evaluated in in vitro studies. We conducted pharmacokinetic interaction studies of MgO and LDCD on rats and healthy volunteers. A clinical study was conducted with an open-label, non-randomized, single-arm, and two-phase study. In MBMA, we constructed a population pharmacokinetic/pharmacodynamic model of L-dopa and predicted the effects of the MgO treatment on the pharmacodynamics of L-dopa. RESULTS: In vitro results suggested that carbidopa was unstable under alkaline pH conditions. Reductions in plasma LDCD concentrations were observed after oral-MgO/oral-LDCD, but not in oral-MgO/i.v.-LDCD treatments in rats, suggesting that the gastrointestinal tract is an interaction site. A healthy volunteer study showed that MgO was also associated with significant decreases of 35.3 and 80.9% in the AUC0-12 of L-dopa and carbidopa, respectively. A model-based simulation suggested that the MgO treatment was undesirable for the effectiveness of L-dopa. CONCLUSIONS: This is the first study to show a clear pharmacokinetic interaction between LDCD and MgO in humans. Further investigations to confirm the effects of MgO on the pharmacodynamics of L-dopa are required.

Sensitive LC-MS/MS method for quantitation of levodopa and carbidopa in plasma: application to a pharmacokinetic study.

AIM: The combination of levodopa with carbidopa has been used for treatment of Parkinson's disease being an important therapy in dopamine level control in the brain. Both are very polar compounds becoming a challenge for analysis by LC-MS/MS. MATERIALS & METHODS: In this work, it was developed and validated a sensitive bioanalytical method by UHPLC-MS/MS for simultaneous levodopa and carbidopa quantification in human plasma using a fast protein precipitation method. Moreover, the bioanalytical method was applied to a pharmacokinetic study in healthy volunteers. RESULTS/CONCLUSION: The results demonstrated a sensitive and adequate method for application to pharmacokinetic/bioequivalence studies.

Micellar HPLC-UV method for the simultaneous determination of levodopa, carbidopa and entacapone in pharmaceuticals and human plasma.

A method based on micellar liquid chromatography to quantify levodopa, carbidopa and entacapone in plasma is reported. The sample pretreatment was a simple dilution in a pure micellar solution then filtration and direct injection, without requiring extraction or purification steps. The three drugs were resolved from the matrix in 7 min, using an aqueous solution of 0.1 M sodium dodecyl sulphate-10% n-propanol-0.3 tiethylamine, adjusted at pH 2.8 with 0.02 M orthophosphoric acid as mobile phase, running under isocratic mode at 1.0 mL/ min through VP-ODS column. The detection was done by UV (ultraviolet) absorbance at 225 nm. The method was successfully validated by the International Conference Harmonization guidelines in terms of: selectivity, linearity (r2 > 0.998) over the concentration ranges of 0.025-1.2, 0.05-1.0 and 0.3-2.0 µg mL-1 with limits of detection of 0.01, 6.16 × 10-3 and 0.02 µg mL-1 and limits of quantification of 0.03, 0.02 and 0.07 µg mL-1 for levodopa, carbidopa and entacapone, respectively. The proposed method was applied successfully for quantification of the studied drugs in their different dosage forms. Moreover, the method was further extensive to the quantification of the studied drugs in spiked human plasma and was successfully validated by the guidelines of the European Medicines Agency. The proposed procedures were successfully evaluated to determine the studied drugs in real human plasma. The procedure was found reliable, practical, cost-effective, available, short period, easy-to-handle, low-cost, environmental-friendly, secure, useful for the analysis of numerous samples per day. Lastly, the method was performed to the analysis of incurred, using quality control samples in the same analytical run, with adequate results. Therefore, it can be implementable for custom analysis in clinical laboratories.

Levodopa-carbidopa intestinal gel: is the naso-jejunal phase a redundant convention?

Levodopa-carbidopa intestinal gel (LCIG) is an effective treatment for Parkinson disease. Initiating therapy involves an initial naso-jejunal (NJ) titration phase. The NJ phase is prolonged with significant morbidity. The aim of this study is to assess the impact of proceeding without the NJ phase on resource utilisation and the outcomes of patients. Twenty-five patients were started on LCIG using the patients existing levodopa equivalent dose (LED). We recorded change in LED, length of hospital stay, readmission rates and use of outpatient services and clinical outcomes within 6 months. The median length of stay was 4.5 days. Patients had four outpatient clinic reviews and 2.5 community nurse contacts within 6 months. There was no significant change in daily LED on discharge (P = 0.56). There were significant improvements in all Unified Parkinson Disease Rating Scale subscores (P < 0.05), the Freezing of Gait scale (P < 0.01) and Parkinson Disease Quality Of Life 39 score (P < 0.01). Initiating LCIG without the NJ phase resulted in short admissions, a minimal outpatient burden and no significant requirement for dose titration while producing good clinical outcomes.

Sensitivity improvement of the LC-MS/MS quantification of carbidopa in human plasma and urine by derivatization with 2,4-pentanedione.

The reliable quantification of carbidopa in biological samples at low concentrations is challenging because of the polar and highly unstable nature of the compound. In this paper, LC-MS/MS methods are described for the determination of carbidopa in 50µL of human plasma and 25µL of human urine in the concentration ranges 1-1,000ng/mL and 100-50,000ng/mL, respectively. After a simple protein precipitation (plasma) or dilution (urine) step, carbidopa is derivatized at its hydrazine moiety by reaction for one hour with 2,4-pentanedione under acidic conditions and at 40°C. The product is a relatively non-polar molecule that is suitable for reversed-phase liquid chromatography (3.5min run time) with detection by tandem mass spectrometry with electrospray ionization. A stable-isotope labeled internal standard is used for response normalization. Precision, accuracy and selectivity of the methods meet the criteria of international guidelines for bioanalytical method validation. Acidification of urine to pH 1.5 and the addition of two anti-oxidants (5mg/mL sodium metabisulfite and 1mg/mL butylated hydroxytoluene) to plasma, in combination with sampling and analysis on ice and under yellow light, ensure sufficient stability of carbidopa. The methods were successfully used to determine plasma pharmacokinetics and urinary excretion of carbidopa in healthy volunteers after a single 37.5mg oral dose.

Ninety-day Local Tolerability and Toxicity Study of ND0612, a Novel Formulation of Levodopa/Carbidopa, Administered by Subcutaneous Continuous Infusion in Minipigs.

A 90-day study in Göttingen minipigs was conducted to test the local tolerability and systemic toxicity of ND0612, a novel aqueous solution of carbidopa (CD)/levodopa (LD) intended for the treatment of Parkinson's disease by continuous subcutaneous administration using a discrete infusion pump. To evaluate tissue site reactions, we used a unique study design involving multiple infusion sites to evaluate the effect of dose per site (270/63, 360/45, and 360/84 mg LD/CD), volume of infusion per site (4.5 and 6 ml per site), formulation concentration (60/14 and 60/7.5 mg/ml LD/CD), daily rate of infusion per site (240 µl/hr for16 hr and 80 µl/hr for 8 hr, 320 µl/hr for 16 hr and 100 µl/hr for 8 hr, or 750 µl/hr for 8 hr), frequency (once every 5, 10, 15, or 20 days), and number of infusions (4, 6, or 9) to the same infusion site. No systemic adverse effects were observed. Histopathological changes at infusion sites started with localized minimal necrosis and acute inflammation that progressed to subacute and chronic inflammatory and reparative changes with evidence of progressive recovery following the final infusion. None of the infusion site effects were judged to be adverse, and clinical exposures to ND0612 are not expected to result in adverse responses.

Quantitation of levodopa and carbidopa in rat plasma by LC-MS/MS: The key role of ion-pairing reversed-phase chromatography.

A simple and selective bioanalytical method was developed for simultaneous determination of levodopa and carbidopa in rat plasma by LC-MS/MS. Levodopa and carbidopa are small polar molecules, posing challenges in the development of selective and efficient chromatography conditions. Perfluoropentanoic acid (PFPA), a volatile ion-pairing agent, was utilized to enhance chromatographic characteristics of both compounds in the reversed-phase mechanism. The ion-pairing chromatography played an essential role in mitigating matrix effects and achieving adequate separation between interfering background peaks and those of the analytes of interest, especially for levodopa. A 96-well based, automated liquid-liquid extraction, via the use Hamilton NIMBUS liquid handlers, was developed. Butyl alcohol, when mixed with ethyl acetate, greatly increased the recovery of both levodopa and carbidopa. The addition of PFPA further enhanced recovery for both analytes. Sodium metabisulfite, an antioxidant, was used to stabilize levodopa and carbidopa in rat plasma. The method was validated in the ranges of 50-10,000ng/mL and 25-5000ng/mL for levodopa and carbidopa, respectively, using levodopa-d3 and carbidopa-d3 as internal standards. The validated method was successfully applied to analyze rat plasma samples from in-life studies.

Levodopa-Carbidopa Intestinal Gel Pharmacokinetics: Lower Variability than Oral Levodopa-Carbidopa.

In a double-blind, double-dummy, double-titration Phase 3 trial in advanced Parkinson's disease (PD) patients, the efficacy and safety of Levodopa-carbidopa intestinal gel (LCIG) infusion were characterized relative to immediate-release oral levodopa-carbidopa (LC-oral) treatment. We present in this report the comparative pharmacokinetic profiles of LCIG and LC-oral from this pivotal study. The results presented in this report clearly demonstrate that LCIG results in lower variability and fluctuations in levodopa and carbidopa plasma concentrations compared to LC-oral. The superior pharmacokinetic profiles with LCIG were consistent with its improved efficacy compared to LC-oral as demonstrated in this study.

Double-Blind, Placebo-Controlled, Randomized Phase I/IIa Study (Safety and Efficacy) with Buspirone/Levodopa/Carbidopa (SpinalonTM) in Subjects with Complete AIS A or Motor-Complete AIS B Spinal Cord Injury.

BACKGROUND: No drug treatment capable of restoring locomotor capabilities in patients suffering a motor-complete spinal cord injury (SCI) has ever been developed. We assessed the safety and efficacy of an activator of spinal locomotor neurons in humans, which were shown in paraplegic animals to elicit temporary episodes of involuntary walking. METHODS: Single administration of buspirone/levodopa/carbidopa (SpinalonTM), levodopa/carbidopa (ratio 4: 1), and buspirone or placebo was performed using a dose-escalation design in 45 subjects placed in supine position who had had an SCI classified as complete (AIS A) or motor-complete/sensory incomplete (AIS B) for at least 3 months. Blood samples before and at regular intervals (15, 30, 60, 120, 240 min) after treatment were collected for hematological and pharmacokinetic (PK) analyses. Electromyographic (EMG) activity of eight muscles (four per leg) was monitored prior to and at several time points after drug administration. RESULTS: SpinalonTM (10-35 mg buspirone/100-350 mg levodopa/25-85 mg carbidopa) displayed no sign of safety concerns - only mild nausea was found in 3 cases. At higher doses, 50 mg/500 mg/125 mg SpinalonTM was considered to have reached maximum tolerated dose (MTD) since 3 out of 4 subjects experienced related adverse events including vomiting. PK analyses showed comparable data between groups suggesting no significant drugdrug interaction with SpinalonTM. Only the SpinalonTM-treated groups displayed significant EMG activity accompanied by locomotor-like characteristics - that is with rhythmic and bilaterally alternating bursts. CONCLUSION: Therefore, this study provides evidence of safety and preliminary efficacy following a single administration of SpinalonTM in subjects with SCI.

Simultaneous estimation of levodopa, carbidopa and 3-oxymethyldopa in rat plasma using HPLC-ECD.

The aim of study was to develop a suitable analytical method for simultaneous estimation of levodopa, carbidopa and 3-O-methyl dopa in rat plasma. Chromatographic separation of plasma samples was achieved using a reverse-phase C18 column. The mobile phase used consisted of a mixture of methanol and phosphate buffer (10 mM, pH 3.50) in the ratio of 90:10 v/v. All analytes were estimated by electrochemical detection at +800 mV. The developed method has been validated as per the standard guidelines. Precision study results were found to be satisfactory, with percentage relative standard deviation for repeatability and intermediate precision <3.96 and 6.56%, respectively, for all analytes detected in rat plasma. The developed method in rat plasma was found to be simple, rapid, accurate, precise and specific. The proposed method has been successfully applied for analysis of rat plasma samples obtained during an oral pharmacokinetic study of sustained release pellets of levodopa and carbidopa in rats. Copyright © 2016 John Wiley & Sons, Ltd.

A Catecholamine Precursor Does Not Influence Exercise Performance in Warm Conditions.

PURPOSE: Acute doses of Sinemet® (L-DOPA combined with carbidopa) previously failed to influence prolonged exercise performance in a temperate environment, but it is not known whether acute doses of L-DOPA timed to reach maximum plasma concentrations (Cmax) during exercise will improve prolonged cycling performance in warm conditions (30.2°C ± 0.2°C, 50% ± 1%). METHODS: Ten physically active men (age, 26 ± 4 yr; height, 1.76 ± 0.08 m; body mass, 76.3 ± 10.6 kg; V˙O2peak, 57 ± 8 mL·kg(-1)·min(-1)) were recruited for this study. Participants cycled for 1 h at 60% V˙O2peak followed by a 30-min exercise test, during which they were instructed to complete as much work as possible. Heart rate, skin and core temperatures, as well as RPE and thermal stress were recorded throughout the exercise, and blood samples were collected at rest, at 15-min intervals during the first hour of exercise, and at the end of the exercise test. Finger tapping tests at the beginning and end of the exercise were conducted to examine fine motor control. RESULTS: There was no significant difference in the work done on the placebo (314 ± 43 kJ) and L-DOPA trials (326 ± 48 kJ, P = 0.276). Prolactin concentrations were increased at the end of the exercise in all trials (P < 0.001), but this response was attenuated at the end of the exercise for the L-DOPA trial (11.4 ± 5.5 ng·mL(-1)) and placebo trials (20.8 ± 3.3 ng·mL(-1), P = 0.003). No differences between trials were found for any other measure. CONCLUSIONS: The results suggest that increasing central catecholamine availability inhibits the normal prolactin response to exercise in the heat but does not alter performance, thermoregulation, or sympathetic outflow.

Clinical Pharmacokinetics of IPX066: Evaluation of Dose Proportionality and Effect of Food in Healthy Volunteers.

OBJECTIVES: IPX066 is an oral, extended-release capsule formulation of carbidopa-levodopa (CD-LD) available in 4 strengths. The goals of this investigation were to assess the dose proportionality of IPX066 and to study the effects of a high-fat, high-calorie meal and of sprinkling the capsule contents on applesauce on the pharmacokinetics of IPX066 in healthy volunteers. METHODS: Three open-label studies were conducted. In the first study, subjects received 1 capsule of each IPX066 strength (23.75-95, 36.25-145, 48.75-195, and 61.25-245 mg of CD-LD). In the second study, subjects received 1 and 2 capsules of IPX066 245-mg LD under fasting conditions. In the third study, subjects received 2 capsules of IPX066 245-mg LD under 3 conditions: fasting; following a high-fat, high-calorie breakfast; and with the capsule contents sprinkled on applesauce under fasting conditions. RESULTS: Peak plasma concentrations (Cmax) and systemic exposure (AUCt, AUCinf) for LD and CD increased dose-proportionally over the range of the IPX066 capsule strengths. Comparison of 1 and 2 IPX066 245-mg LD capsules showed dose-proportional pharmacokinetics for Cmax and AUCt. Sprinkling the capsule contents on applesauce did not affect the pharmacokinetics. A high-fat, high-calorie meal delayed the initial increase in LD concentration by approximately 1 to 2 hours, reduced Cmax by 21%, and increased AUCinf by 13% compared with the fasted state. CONCLUSIONS: IPX066 shows dose-proportional pharmacokinetics. Sprinkling the capsule contents on applesauce does not affect the pharmacokinetics; a high-fat, high-calorie meal delayed absorption by 1 to 2 hours, slightly reduced Cmax, and slightly increased extent of absorption.

Comparison of the pharmacokinetics of an oral extended-release capsule formulation of carbidopa-levodopa (IPX066) with immediate-release carbidopa-levodopa (Sinemet(®)), sustained-release carbidopa-levodopa (Sinemet(®) CR), and carbidopa-levodopa-entacapone (Stalevo(®)).

IPX066 (extended-release carbidopa-levodopa [ER CD-LD]) is an oral extended-release capsule formulation of carbidopa and levodopa. The single-dose pharmacokinetics of ER CD-LD (as 2 capsules; total dose, 97.5 mg-390 mg CD-LD) versus immediate-release (IR) CD-LD (25 mg-100 mg), sustained-release (CR) CD-LD (25 mg-100 mg), and CD-LD-entacapone (25 mg-100 mg-200 mg) was evaluated in healthy subjects. Following IR dosing, LD reached peak concentrations (Cmax ) at 1 hour; LD concentrations then decreased rapidly and were less than 10% of peak by 5 hours. With CR CD-LD and CD-LD-entacapone, LD Cmax occurred at 1.5 hours, and concentrations were less than 10% of peak by 6.3 and 7.5 hours, respectively. The initial increase in LD concentration was similar between ER CD-LD and IR CD-LD and faster than for CR CD-LD and CD-LD-entacapone. LD concentrations from ER CD---LD were sustained for approximately 5 hours and did not decrease to 10% of peak until 10.1 hours. Dose-normalized LD Cmax values for ER CD-LD were significantly lower (P< .05) than for the other CD-LD products. Bioavailability of LD from ER CD-LD was 83.5%, 78.3%, and 58.8% relative to IR CD-LD, CR CD-LD, and CD-LD-entacapone, respectively.

Long-Term Treatment with Extended-Release Carbidopa-Levodopa (IPX066) in Early and Advanced Parkinson's Disease: A 9-Month Open-Label Extension Trial.

BACKGROUND AND OBJECTIVE: IPX066 is a multiparticulate extended-release formulation of carbidopa-levodopa, designed to produce prolonged therapeutic levodopa plasma concentrations. This 9-month open-label extension study assessed its long-term safety and clinical utility in early and advanced Parkinson's disease (PD). METHODS: Participants were enrolled from two phase III IPX066 studies and one open-label phase II study. Early PD patients were titrated to an appropriate dosing regimen while advanced patients started with regimens established in the antecedent studies. Adjustment was allowed throughout the extension. Clinical utility measures included the Unified Parkinson's Disease Rating Scale (UPDRS) and Patient Global Impression (PGI) ratings. RESULTS: Among 268 early PD patients, 53.4 % reported adverse events (AEs) and 1.1 % (three patients) discontinued due to AEs; the most frequent AEs were nausea (5.6 %) and insomnia (5.6 %). Among 349 advanced patients, 60.2 % reported AEs and 3.7 % (13 patients) discontinued due to AEs; the most frequent AEs were dyskinesia (6.9 %) and fall (6.6 %). At month 9 (or early termination), 78.3 % of early patients were taking IPX066 three times daily (median: 720 mg/day) and 87.7 % of advanced patients were taking IPX066 three or four times daily (median: 1450 mg/day). Adjusting for 70 % bioavailability relative to immediate-release (IR) carbidopa-levodopa, the median dosages correspond to ~500 and ~1015 mg/day of IR levodopa in early and advanced PD, respectively. Based on the plasma profiles previously observed in PD patients, the IPX066 regimens in the extension can be estimated to provide a levodopa Cmax (maximum plasma drug concentration) similar to or lower than that provided by IR regimens during the antecedent trials. UPDRS and PGI findings showed sustained treatment effects throughout the extension. CONCLUSION: During 9 months of extended use, IPX066 exhibited a safety/tolerability profile consistent with dopaminergic PD therapy.

Simultaneous determination of levodopa, carbidopa, entacapone, tolcapone, 3-O-methyldopa and dopamine in human plasma by an HPLC-MS/MS method.

BACKGROUND: In this study, we developed and validated a HPLC-MS/MS method capable of simultaneously determining levodopa, carbidopa, entacapone, tolcapone, 3-O-methyldopa and dopamine in human plasma. RESULTS & METHODOLOGY: Chromatographic separation was achieved using a C8 column with a mobile phase consisting of a gradient of water and acetonitrile:methanol (90:10 v/v), both containing 0.1% formic acid. The developed method was selective, sensitive (LD<7.0 ng ml(-1)), linear (r>0.99), precise (RSD<11.3%), accurate (RE<11.8%) and free of residual and matrix effects. The developed method was successfully applied in plasma patients with Parkinson's disease using Stalevo®. CONCLUSION: The new method can be used for the clinical monitoring of these substances and applied to adjustments in drug dosages.

A new HILIC-MS/MS method for the simultaneous analysis of carbidopa, levodopa, and its metabolites in human plasma.

Monitoring of the plasmatic levels of levodopa (LEV) and carbidopa (CAR) is necessary to adjust the dose of these drugs according to the individual needs of Parkinson's disease patients. To support drug therapeutic monitoring, a method using HILIC mode and LC-MS/MS was developed for the simultaneous determination of carbidopa, levodopa, and its metabolites (3-o-methyldopa (3-OMD) and dopamine (DOPA)) in human plasma. A triple quadrupole mass spectrometry was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. After straightforward sample preparation via protein precipitation, an Atlantis HILIC (150 × 2.1 mm, 3 µm, Waters, USA) column were used for separation under the isocratic condition of acetonitrile/water (79:21, v/v) containing 0.05% formic acid and 3 mmol/L ammonium formate and the total run time was 7 min. Deuterated LEV was used as internal standard for quantification. The developed method was validated in human plasma with a lower limit of quantitation of 75 ng/mL for LEV, 65 ng/mL for CAR and 3-OMD, and 20 ng/mL for DOPA. The calibration curve was linear within the concentration range of 75-800 ng/mL for LEV, 65-800 ng/mL for CAR and 3-OMD, and 20-400 ng/mL for DOPA (r>0.99). The assay was accurate and precise, with inter-assay and intra-assay accuracies within ±13.44% of nominal and inter-assay and intra-assay precision≤13.99%. All results were within the acceptance criteria of the US FDA and ANVISA guidelines for method validation. LEV, CAR, 3-OMD and DOPA were stable in the battery of stability studies, long-term, bench-top, autosampler, and freeze/thaw cycles. Samples from patients undergoing treatment were analyzed, and the results indicated that this new method is suitable for therapeutic drug monitoring in Parkinson's disease patients.