Multicolorimetric Sensor Array Based on Silver Metallization of Gold Nanorods for Discriminating Dopaminergic Agents.

Dopaminergic agents are compounds that modulate dopamine-related activity in the brain and peripheral nerves within the pathways on both sides of the blood-brain barrier. Atypical levels of them can precipitate a multitude of neurological disorders, whose timely diagnosis signifies not only stopping the advancement of the illness but also surmounting it. A silver metallized gold nanorod (AuNRs) conditional sensor array, designed to detect dopaminergic agents for assessing nervous system disorders, yielded significant results in simultaneous detection and discrimination of Benserazide (Benz), Levodopa (L-DOPA), and Carbidopa (Carb). The array was composed of two different concentrations of silver ions as sensor elements (SEs), which generated unique signatures indicative of the presence of reductive target analytes, triggered by the incongruent formation of the Au@Ag core-shell, causing visual and fingerprint colorimetric patterns. Generating diverse responses is the key to the functionality of array-based sensing, which facilitated achieving spectral and color variation originating from the blue shift of AuNRs longitudinal localized surface plasmon resonance (LLSPR) in the extinction spectrum. Also, employing a smartphone camera enables clear visual discrimination across an extensive concentration span. Pattern recognition through linear discriminant analysis (LDA) underscored the robust discrimination accuracies of this sensor, along with quantification by means of partial least-squares regression (PLSR), affirming its potential for practical applications. Notably, the array demonstrated high sensitivity in detecting varied concentrations of target analytes, even in commercial drug samples. The sensor responses exhibited a linear correlation with the concentrations of Benz, L-DOPA, and Carb ranging from 1.59 to 100.0, 5.26 to 100.0, and 5.32 to 100.0 µmol L-1, respectively, and the minimum detectable concentrations for Benz, L-DOPA, and Carb were measured at 0.53, 1.75, and 1.77 µmol L-1, respectively. The implemented machine-learning-empowered array-based sensor represents advancements in dopaminergic agent tracing and naked eye detection.

Levodopa: From Biological Significance to Continuous Monitoring.

A continuous levodopa sensor can improve the quality of life for patients suffering with Parkinson's disease by enhancing levodopa titration and treatment effectiveness; however, its development is currently hindered by the absence of a specific levodopa molecular recognition element and limited insights into how real-time monitoring might affect clinical outcomes. This gap in research contributes to clinician uncertainty regarding the practical value of continuous levodopa monitoring data. This paper examines the current state of levodopa sensing and the inherent limitations in today's methods. Further, these challenges are described, including aspects such as interference from the metabolic pathway and adjunct medications, temporal resolution, and clinical questions, with a specific focus on a comprehensive selection of molecules, such as adjunct medications and structural isomers, as an interferent panel designed to assess and validate future levodopa sensors. We review insights and lessons from previously reported levodopa sensors and present a comparative analysis of potential molecular recognition elements, discussing their advantages and drawbacks.

Tapered cross-linked ZnO nanowire bundle arrays on three-dimensional graphene foam for highly sensitive electrochemical detection of levodopa.

It is crucial to accurately and rapidly monitor the levodopa (LD) concentration for accurate classification and treatment of dyskinesia in Parkinson's disease. In this paper, 3D graphene foam (GF) with a highly conductive network is obtained by chemical vapor deposition. 3D GF serves as the substrate for hydrothermal in situ growth of tapered cross-linked ZnO nanowire bundle arrays (ZnO NWBAs), enabling the development of a highly sensitive detection platform for LD. The formation mechanism of a tapered cross-linked ZnO nanowire bundle arrays on 3D GF is put forward. The integration of 3D GF and ZnO NWBAs can accelerate the electron transfer rate and increase the contact area with biomolecules, resulting in high electrochemical properties. The electrode composed of ZnO NWBAs on 3D GF exhibits significant sensitivity (1.66 µA·µM-1·cm-2) for LD detection in the concentration range 0-60 µM. The electrode is able to rapidly and specifically determine LD in mixed AA or UA solution. The selectivity mechanism of the electrode is also explained by the bandgap model. Furthermore, the successful detection of LD in serum demonstrates the practicality of the electrode and its great potential for clinical application.

Flexible Wearable Electrochemical Sensors Based on AuNR/PEDOT:PSS for Simultaneous Monitoring of Levodopa and Uric Acid in Sweat.

As a facile substitute for the invasive technique of blood testing, wearable electrochemical sensors exhibit high potential for the noninvasive and real-time monitoring of biomarkers in human sweat. However, owing to enzyme specificity, the simultaneous detection of multiple biomarkers by enzymatic analysis is challenging. Moreover, sweat accumulation under sensors causes sweat contamination, which hinders real-time biomarker detection from sweat. This study reports the design and fabrication of flexible wearable electrochemical sensors containing a composite comprising Au nanorods (AuNRs) and poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) for the nonenzymatic detection of levodopa (LD) and uric acid (UA) in sweat. Each sensor was integrated with a flexible three-electrode system and a microfluidic patch for sweat sampling. AuNRs immobilized by PEG-doped PEDOT:PSS showed excellent analytical performance for LD and UA at different potentials. Thus, the newly fabricated sensors could detect LD and UA over a broad detection range with high sensitivity and showed a low limit of detection for both species. On-body assessments confirmed the ability of these sensors to simultaneously detect LD and UA in real time. Therefore, this study could open new frontiers in the fabrication of wearable electrochemical sensors for the pharmacokinetic profile tracking of LD and gout management.

Simultaneous sensing of carbidopa and levodopa by a novel strategy based on dual-emission ratiometric assay of modified carbon dots.

Rapid control of the content of Parkinson's drugs in biological fluids and pharmaceutical formulations is of great importance because changes in the concentration of these drugs affect their bioavailability and biopharmaceutical properties. Therefore, we presented a simple and convenient method for the ratiometric detection of carbidopa and levodopa for carbon dots (CDs) dual-fluorescent emission. Dual-emission CDs were prepared from chitosan using a microwave method, following which the surface was chemically modified with terephthalaldehyde. CDs had two strong well-separated peaks at 445 and 510 nm. The relative measurement of carbidopa and levodopa was based on the static extinction of CDs at 445 nm and increase at 510 nm, respectively. The linear range for carbidopa measurement was 2.5-300 nM, with a limit of detection (LOD) of 2.1 nM, and a relative standard deviation (RSD) of 1.68%. Further, the linear range for levodopa measurement was equal to 3.0-400 nM, with LOD and RSD% of 2.8 nM and 3.5%, respectively. Also, selectivity of ratiometric sensor in the presence of interferences was investigated, which showed that the recovery of carbidopa and levodopa in serum and urine samples has changed between 96.80% and 116.24% with RSD% 0.11-0.77. CDs also provided good results for the determination of carbidopa and levodopa in real samples, and had high selectivity in the presence of possible interferences.

Reusable graphite-based electrochemical sensors for L-dopa and dopamine detection.

A fully reusable electrochemical device is proposed for the first time made from laser cutting and a homemade conductive ink composed of carbon and nail polish. As a sensor substrate, we applied polymethyl methacrylate, which allows the surface to be renewed by simply removing and reapplying a new layer of ink. In addition to the ease of renewing the sensor's conductive surface, the design of the device has allowed for the integration of different forms of analysis. The determination of L-Dopa was performed using DPV, which presented a linear response range between 5.0 and 1000.0 µmol L-1, and a LOD of 0.11 µmol L-1. For dopamine, a flow injection analysis system was employed, and using the amperometric technique measurements were performed with a linear ranging from 2.0 to 100.0 µmol L-1 and a LOD of 0.26 µmol L-1. To demonstrate its applicability, the device was used in the quantification of analytes in pharmaceutical drug and synthetic urine samples.

Evaluation of haemolymph phenoloxidase activity from the grub of Zophobas morio as a predictor of immune response.

In insects, enzyme phenoloxidase plays a critical role in cuticular sclerotisation and defensive functions. In the present investigation, haemolymph phenoloxidase activity from the grub of Zophobas morio was attempted to evaluate as a reliable predictor of insect's immunological response. Among the various substrates tested, L-DOPA was chosen as an appropriate substrate due to its high oxidation. The optimum pH and temperature for haemolymph PO activity was found to be 8 and 30 °C, respectively. The optimum substrate concentration of L-DOPA was found to be 7.5 mM for subsequent PO enzymatic characterisation. Among the various chemical inhibitors and copper chelators, PO activity was significantly reduced in the case of PMSF and thiourea. Preincubation of haemolymph with non-self-molecules showed enhancement of PO activity in the case of LPS from Serratia marcescens. In addition, exogenous proteases like α-chymotrypsin enhanced the PO activity of haemolymph and an increase in PO activity was demonstrated when haemolymph was preincubated with the anionic detergent, SDS and cationic detergent, cetyl pyridium chloride. Alteration of PO activity was observed under agonising conditions of starvation, ligation and microplastics injection at different time intervals. Interestingly, there were no correlation between PO and insect defence under live challenge of microbes. SDS protein profile revealed a significant increase in the 85 kDa and 55 kDa polypeptides in all the experiments over control after 24 h, 48 h and 96 h. Mass spectrophotometric analysis of the polypeptides revealed their homology to antimicrobial peptides for 55 kDa protein and 85 kDa protein. A significant increase in 85 kDa polypeptide was observed in the haemolymph of the grubs after 72 h in the case of starved and microplastics injected groups only. These results demonstrated that PO may not be a reliable benchmark of immunological response in this insect.

Comparison of betalain compounds in two Beta vulgaris var. cicla and BvCYP76AD27 function identification in betalain biosynthesis.

Beta vulgaris var. cicla is an edible, ornamental and horticultural plant. However, the difference of components and contents of betalain in beets with different leaf color are not well understood. Here, the stress resistance and metabolites of two B. vulgaris var. cicla cultivars were determined. The differences in stress resistance between red leaf-colored chard (RC) and yellow leaf-colored chard (YC) were positively related to betacyanins (BC) and betaxathins (BX) content in the leaves. Furthermore, a total of 3615 distinct metabolites were identified by UPLC-QTOF-MS in two cultivars, including 70 alkaloids and their derivatives, 249 flavonoids, and 264 terpenoids. There were 17 metabolites attributed to betalain biosynthesis pathway, seven of nine BC were up-regulated, and eight BX showed no significant difference in RC compared with YC. The contents of celosianin II and betanin were the highest BC in RC, at approximately 84.38 and 19.97 times that of YC, respectively. The content of portulacaxanthin II was the highest BX in two beets. Additionally, the BvCYP450 genes were identified based on genome, and the members that might be involved in betalain biosynthesis were screened. BvCYP76AD27, a member of the BvCYP76AD subfamily, had a higher expression level in RC than YC under freezing, drought and shading stress. In yeast Saccharomyces cerevisiae, BvCYP76AD5 and BvCYP76AD27 only hydroxylated tyrosine to L-DOPA, which was transformed into portulacaxanthin II by 4,5-DOPA extradiol dioxygenase. The results contribute to illustrating the molecular mechanism of betalain biosynthesis and provide useful information for further investigation of beet chemistry and sufficient utilization of this species.

Enhanced Physicochemical Stability of the L-DOPA Extract of Mucuna pruriens Seeds by Adding Phyllanthus emblica.

Levodopa (L-DOPA) is an essential drug for the treatment of Parkinson's disease. Currently, L-DOPA can be produced by chemical synthesis and can also be found naturally in many herbs, especially Mucuna Pruriens (MP). According to clinical research, the MP extract containing L-DOPA for the treatment of Parkinson's disease could reduce side effects more than the synthetic one. Unfortunately, MP extracts can be easily degraded. Changes in physical and chemical properties such as the appearance (color, melt, solid lump) and the reduction of L-DOPA content in the extract were commonly observed. Therefore, it is necessary to develop an extraction procedure to stabilize the extract of L-DOPA. This study attempted to enhance the extraction process by modifying the traditional acidification approach using hydrochloric acid, citric acid, or ascorbic acid. According to the stability test results, using Phyllanthus emblica water (PEW) as a solvent improved the preservative properties more than other solvents. The color of the PEW-MP powder changed slightly after 12 months of accelerated storage, but the amount of L-DOPA remained the highest (73.55%). Moreover, L-DOPA was only detected in MP and PEW-MP, but not PEW alone (the HPTLC chromatogram at Rf 0.48 and the HPLC chromatogram at Rt 6.0 min). The chemical profiles of PEW and L-DOPA observed in the chromatograms indicated that they are independently separated. As a result, they can be applied to a quality control process. Therefore, PEW was proven to be a powerful solvent for L-DOPA herbal extract that could be readily used as a raw material for herbal products.

Application of net analyte signal and principal component regression for rapid simultaneous determination of Levodopa and carbidopa in commercial pharmaceutical formulation and breast (human) milk sample using spectrophotometric method.

In this study, a UV-vis spectrophotometric method coupled with net analyte signal (NAS) and principal component regression (PCR) as multivariate calibration methods were used for the simultaneous determination of levodopa (LEV) and carbidopa (CBD) in prepared mixtures, pharmaceutical formulation, and breast milk sample. The mean recovery of the NAS model was 98.10% and 99.60% for LEV and CBD, respectively. Also, the relative standard deviation (RSD%) values were found to be lower than 5.5% and 4% for LEV and CBD, respectively. On the other hand, the mean recovery of LEV and CBD related to the PCR method was obtained at 96.86% and 92.43%, respectively. K-Fold cross-validation was used to estimate the number of components, which was 7 and 3 with a mean square error prediction (MSEP) of 1.50 and 7.14 for LEV and CBD, respectively. The results revealed that the NAS model was better than the PCR model. Additionally, the proposed NAS-based calibration method was successfully developed for the simultaneous analyses of LEV and CBD in a commercial tablet and breast milk.

Mucuna pruriens Seed Aqueous Extract Improved Neuroprotective and Acetylcholinesterase Inhibitory Effects Compared with Synthetic L-Dopa.

L-dopa, a dopaminergic agonist, is the gold standard for the treatment of Parkinson's disease. However, due to the long-term toxicity and adverse effects of using L-dopa as the first-line therapy for Parkinson's disease, a search for alternative medications is an important current challenge. Traditional Ayurvedic medicine has suggested the use of Mucuna pruriens Linn. (Fabaceae) as an anti-Parkinson's agent. The present study aimed to quantify the amount of L-dopa in M. pruriens seed extract by HPLC analysis. The cytotoxicity and neuroprotective properties of M. pruriens aqueous extract were investigated by two in vitro models including the serum deprivation method and co-administration of hydrogen peroxide assay. The results showed the significant neuroprotective activities of M. pruriens seed extracts at a concentration of 10 ng/mL. In addition, the effects of L-dopa and M. pruriens seed extract on in vitro acetylcholinesterase activities were studied. M. pruriens seed extract demonstrated acetylcholinesterase inhibitory activity, while synthetic L-dopa enhanced the activity of the enzyme. It can be concluded that the administration of M. pruriens seed might be effective in protecting the brain against neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. M. prurience seed extract containing L-dopa has shown less acetylcholinesterase activity stimulation compared with L-dopa, suggesting that the extract might have a superior benefit for use in the treatment of Parkinson's disease.

Administration of mucuna beans (Mucuna pruriences (L.) DC. var. utilis) improves cognition and neuropathology of 3 × Tg-AD mice.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of extracellular amyloid-beta peptides (Aß) resulting in senile plaques and intracellular hyperphosphorylated tau protein resulting in neurofibrillary tangles (NFTs). Mucuna beans (Mucuna pruriences (L.) DC. var. utilis) are unique plants containing 3-9% L-3,4-dihydroxyphenylalanine (L-DOPA). Here we investigated the effect of the administration of Mucuna beans on AD prevention by feeding triple-transgenic mice (3 × Tg-AD mice) with a diet containing Mucuna beans for 13 months. The levels of Aß oligomers and detergent-insoluble phosphorylated tau decreased in the brain of mice fed with Mucuna beans (Mucuna group) compared to those of the Control group. Aß accumulation and phosphorylated tau accumulation in the brain in the Mucuna group were also reduced. In addition, administration of Mucuna beans improved cognitive function. These results suggest that administration of Mucuna beans may have a preventive effect on AD development in 3 × Tg-AD mice.

Melanochrome-based colorimetric assay for quantitative detection of levodopa in co-presence of carbidopa and its application to relevant anti-Parkinson drugs.

In this paper is reported the selective detection and quantification of levodopa in co-presence of carbidopa. The method took advantage of the spontaneous oxidation and color development of levodopa at basic pH here driven by alkaline earth cations and co-solvent in solution. We have shown for the first time the generation and stabilization of the purple melanochrome from levodopa, by using magnesium acetate and dimethyl sulfoxide, which was here exploited for the development of a quantitative colorimetric assay for the active principle ingredient in commercial drugs for the treatment of Parkinson's disease. The calibration curves of levodopa in the two tablet formulations, containing carbidopa as decarboxylase inhibitor, showed a common linear trend between 10 mg L-1 and 40 mg L-1 with levodopa alone or in combination with carbidopa in standard solutions, with very good reproducibility (CVav%, 3.3% for both brand and generic drug) and very good sensitivity, with limit of quantification about 0.6 mg L-1 in any case. The colorimetric method here developed is very simple and effective, appearing as a rapid and low-cost alternative to other methodologies, involving large and expensive instrumentations, for drug estimation and quality control of pharmaceutical formulations.

Voluntary oral dosing for precise experimental compound delivery in adult rats.

Preclinical drug studies routinely administer experimental compounds to animal models with the goal of minimizing potential adverse events from the procedure. In this study, we assessed the ability to train adult male Long Evans rats to accept daily voluntarily syringe feedings of l-3,4-dihydroxyphenylalanine (L-DOPA) compared to intraperitoneal (IP) injections. Rats were trained to become familiar with the syringe and then fed a training solution that did not contain the experimental compound. If the rat was compliant during the training phase, the dilution of training solution was continuously decreased and replaced with the experimental solution. Voluntary oral dosing compliance was recorded and quantified throughout the study. To assess drug activity within the drug-targeted tissues, the striatum and retina were collected and analyzed for L-DOPA, dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels by high performance liquid chromatography (HPLC). Drug delivery efficiency by oral dosing was directly compared to IP injection by collecting plasma and analyzing L-DOPA levels with HPLC. Adult male rats had high compliance for voluntary oral dosing. HPLC showed that oral administration of the compound at the same dose as IP injection yielded significantly lower plasma levels, and that higher oral L-DOPA doses yield higher plasma L-DOPA content. This study describes detailed methodology to train adult rats to syringe feed experimental compounds and provides important preclinical research on drug dosing and drug delivery to the striatum and retina.

Highly Sensitive Levodopa Determination by Means of Adsorptive Stripping Voltammetry on Ruthenium Dioxide-Carbon Black-Nafion Modified Glassy Carbon Electrode.

A new, highly sensitive Adsorptive Stripping Voltammetric method for levodopa determination was developed. As a working electrode, the glassy carbon electrode (GCE) modified with carbon black (CB), RuO2·xH2O (RuO2) and Nafion was used (CB-RuO2-Nafion GCE). Levodopa signal obtained on the modified electrode was 12 times higher compared to GCE. During research, instrumental parameters were optimized: sampling time ts = 10 ms, waiting time tw = 10 ms, step potential Es = 5 mV and pulse amplitude ΔE = 50 mV. Preconcentration potential Eprec was equal to 0 mV. The best results were obtained in 0.025 M perchloric acid (approx. pH 1.4). Signal repeatability measured on the CB-RuO2-Nafion modified electrode for 0.2 µM of levodopa was equal to 2.1% (levodopa concentration 1 µM, n = 5). Linearity of the method was achieved in the concentration range from 1 to 8 µM. Limit of detection was equal to 17 nM. Recoveries calculated for pharmaceutical products and tap water measurements were in the range 102-105%, which confirms the accuracy of the developed. The applicability of the method was confirmed by analysis of pharmaceutical products and tap water samples. Based on obtained results, it might be concluded that the developed voltammetric method could be a useful tool in routine drug analysis.

Polypyrrole merged zirconium-based metal-organic framework NU-1000 for detection of levodopa.

A post-synthetic integration of polypyrrole onto NU-1000 MOF (PPy@NU-1000) was done by pyrrole adsorption, followed by oxidative polymerization. The synthesized materials were characterized by XRD, SEM, BET, and FTIR. The ultra-high specific surface area with high-density catalytic sites of NU-1000 (2223 m2 g-1) was combined with the electrical conductivity of PPy (2-100 S cm-1). PPy@NU-1000 provides superior electrocatalytic activity and charge transfer properties compared to an individual component. The PPy@NU-1000-modified GCE was applied to detect the biomolecule Levodopa (LD). The DPV oxidation peak of LD was strongest at 272 ± 10 mV vs. Ag/AgCl reference electrode. Under the optimized experimental condition, the fabricated electrochemical sensor exhibited a wide quantification range of 0.005-70 µM with a sub-nanomolar detection limit of 0.0001 µM (S/N 3). The described sensor exhibits high sensitivity (2.08 µA µM-1 cm-2) with reasonable stability, reproducibility, and selectivity for the detection LD in the presence of potentially interfering compounds. Furthermore, human serum analysis showed excellent recovery values within the range 99.3-101.6%. Validation of the method was performed against HPLC.Graphical abstract.

Differential coulometry based on dual screen-printed strips for high accuracy levodopa determination towards Parkinson's disease management.

As a vital therapeutic agent for Parkinson's disease, dosage control of levodopa has always been a major obstacle in ensuring efficacy and curbing side effects. Simple and fast electrochemical detection methods are the main force in this field. Here, we presented a differential dual-strip method based on the coulometry for high accuracy determination of levodopa. The difference between the two strip signals with or without the tyrosinase extracted the levodopa signal from the samples. The Prussian Blue modified carbon screen-printed electrode was used to convert and amplify the electrochemical signal upon the presence of levodopa. The system exhibited a linear behavior in the 0-10 µM concentration range and a detection limit of 0.25 µM. Furthermore, it was proved to be stable in effectively distinguishing levodopa from complex samples through anti-interference experiments and serum tests. We demonstrated the superiority of dual-strip differential coulometry for the determination of levodopa towards Parkinson's disease clinical management.

Regulating the biosynthesis of pyridoxal 5'-phosphate with riboswitch to enhance L-DOPA production by Escherichia coli whole-cell biotransformation.

Pyridoxal 5'-phosphate (PLP) is an essential cofactor that participates in ∼4% enzymatic activities cataloged by the Enzyme Commission. The intracellular level of PLP is usually lower than that demanded in industrial catalysis. To realize the self-supply of PLP cofactor in whole-cell biotransformation, the de novo ribose 5-phosphate (R5P)-dependent PLP synthesis pathway was constructed. The pdxST genes from Bacillus subtilis 168 were introduced into the tyrosine phenol-lyase (TPL)-overexpressing Escherichia coli BL21(DE3) strain. TPL and PdxST were co-expressed with a double-promoter or a compatible double-plasmid system. The 3,4-dihydroxyphenylacetate-L-alanine (L-DOPA) titer did not increase with the increase in the intracellular PLP concentration in these strains with TPL and PdxST co-expression. Therefore, it is necessary to optimize the intracellular PLP metabolism level so as to achieve a higher L-DOPA titer and avoid the formation of L-DOPA-PLP cyclic adducts. The thi riboswitch binds to PLP and forms a complex such that the ribosome cannot have access to the Shine-Dalgarno (SD) sequence. Therefore, this metabolite-sensing regulation system was applied to regulate the translation of pdxST mRNA. Riboswitch was introduced into pET-TPL-pdxST-2 to downregulate the expression of PdxST and biosynthesis of PLP at the translation level by sequestering the ribosome-binding site. As a result, the titer and productivity of L-DOPA using the strain BL21-TPLST-Ribo1 improved to 69.8 g/L and 13.96 g/L/h, respectively, with a catechol conversion of 95.9% and intracellular PLP accumulation of 24.8 µM.

A rapid liquid chromatography/tandem mass spectrometry method for simultaneous determination of levodopa, carbidopa, entacapone and their six related compounds in film-coated tablets.

RATIONALE: A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated to determine levodopa, carbidopa, entacapone, and corresponding six related substances - levodopa impurity B, levodopa impurity C, methyldopa, methylcarbidopa, entacapone impurity C, and entacapone impurity A - in film-coated tablets for the first time. METHODS: Chromatographic separation was achieved with a gradient elution by using a C18 column, a mobile phase containing 0.5% formic acid in water and 0.5% formic acid in methanol. The mobile phase flow rate was 0.5 mL min-1 . The UV detector was set at 280 nm and the triple quadrupole mass spectrometer was used in multiple reaction monitoring (MRM) mode. RESULTS: The limit of detection (LOD) and limit of quantification (LOQ) results were 1.3 and 3.94 ng mL-1 for levodopa impurity B; 5.26 and 15.9 ng mL-1 for levodopa impurity C; 0.833 and 2.53 ng mL-1 for methyldopa; 3.31 and 10.0 ng mL-1 for methylcarbidopa; 1.67 and 5.06 ng mL-1 for entacapone impurity C; and 0.61 and 1.86 ng mL-1 for entacapone impurity A. CONCLUSIONS: The method was rapid, linear, accurate, and reproducible. The LC/MS/MS method that was developed to determine the related substances and assay of levodopa, carbidopa, and entacapone can be used to evaluate the quality of regular samples in the pharmaceutical industry. It can be also used to test the stability of samples.

Determining the Distribution of Active Pharmaceutical Ingredients in Combination Tablets Using Near IR and Low-Frequency Raman Spectroscopy Imaging.

Combination tablets containing multiple active pharmaceutical ingredients (APIs) are expected to improve patient convenience by decreasing the number of tablets to be taken; thus, numerous formulations containing multiple APIs have recently been developed. To allow for dose adjustments based on patient conditions, many tablets have a bisection line to allow equal division of tablets. However, there have been no investigations regarding content uniformity among divided combination tablets. Therefore, in this study, the content uniformity of combination tablets after division was investigated using near IR and low-frequency (LF) Raman spectroscopy imaging as well as the Japanese Pharmacopoeia (JP) content uniformity tests. As model drugs, five tablets of three combination drugs containing 3-(3,4-dihydroxyphenyl)-L-alanine (L-DOPA) and benserazide hydrochloride (BNS) as APIs for treating Parkinson's disease were bisected; the resultant 10 samples were subjected to the JP content uniformity tests. We found that acceptance values of L-DOPA and BNS were 11.0-21.9% and 13.3-17.5%, respectively, with some non-conformity to the maximum allowed acceptance value (15.0%) as per the current JP. Image analyses by near IR showed that L-DOPA, BNS, lactose, and corn starch were uniformly distributed in each tablet; moreover, LF Raman spectroscopy imaging also supported the result that L-DOPA, BNS, and lactose were evenly distributed. Therefore, drug content in the tablets was uniform; thus, careful manipulation was recommended in the tablet bisection. However, the results of bisection line specifications and hardness tests revealed that the ease of division differed depending on the tablets, which warrants attention.