Parkinson's disease (PD) is one of the most prevalent neurodegenerative disorders, the pathological depletion of dopamine in the basal ganglia leads to the motor deficits classically associated with PD, including rigidity, bradykinesia, and resting tremor [1]. PD has become a public health concern with global aging, demonstrating a pronounced age-dependent epidemiology. The incidence culminating in peak rates between 85–89 years, notably, nearly 25 % of affected individuals exhibit symptom onset before 65 years, while only 5–10 % develop the disease prior to 50 years. Cases with onset under 40 years (or alternatively < 50 years) are clinically designated as early-onset Parkinson's disease (EOPD), which is mostly related to genetic factors [1,2]. The World Health Organization (WHO) reported that in 2019, PD led to 5.8 million disability-adjusted life years (DALYs), and caused approximately 329,000 deaths since 2000 [3]. As a dopamine precursor, l-Dopa can effectively cross the blood brain barrier (BBB) and is converted into dopamine via decarboxylase enzymes in the brain, making it a well-tolerated and potent dopamine replacement agent [4,5]. Long-term clinical evidence, including the PD MED trial with 7-year follow-up data, has shown sustained (albeit modest) motor benefits in patients initiated on l-Dopa compared to those receiving MAO-B inhibitors or dopamine agonists [6]. With over five decades of widespread use, l-Dopa remains the primary medication for the treatment of PD [7,8], in China alone, its total sales reached approximately 4.5 billion CNY in 2023. As an enantiomer of l-Dopa, d-Dopa is associated with the side effect of granulocytopenia, and exhibits extremely only 3 % bioavailability in the brain [9,10]. Moreover, d-Dopa possesses irrelevant pharmacological activities, such as the inhibition of Glutamate Carboxypeptidase II activity [11]. Implementation of rigorous quality control measures is essential for monitoring and controlling pharmacologically inactive impurities, encompassing both stereoisomeric contaminants (e.g., enantiomers, diastereomers) and other byproducts. The United States Pharmacopeia (USP) [12] lists three related substances associated with l-Dopa, namely l-Tyrosine, 3-Methoxytyrosine, and 1-Veratrylglycine, and stipulates and specifies their allowable limits. The European Pharmacopoeia and Pharmacopoeia Bohemoslovaca specify polarimetry for controlling the d-dopa enantiomer, but this method lacks the sensitivity to accurately detect trace impurities [13].

The structure of l-Dopa contains a chiral center (Fig. 1), which is similar to amino acids with high polarity, making them difficult to retain and separate on C18 column. The relative content of Dopa enantiomers can be quantified by capillary electrophoresis (CE), employing chiral selectors such as cyclodextrins or chiral crown ethers. Most of reported methods typically achieving detection limits (LOD) at the mg l-1 level or higher [[14], [15], [16]]. To date, one CE-based method developed by Zhao et al. has been reported for the optical purity analysis of levodopa, allowing the detection of 0.14 % d-Dopa by incorporating hydroxypropyl-β-cyclodextrin (HP-β-CD) and 120 mM sodium dodecyl sulfate (SDS) [17]. HPLC has also been applied for the separation of D/L-Dopa, mostly involved chiral ligand exchange chromatography [14,18,19]. Additionally, Takashi H et al. [20] developed a chiral column-based method for separating D/L-Dopa and applied it to detect the enantiomers in dietary supplements containing Mucuna pruriens, and the result indicated that d-Dopa was not detected. Derivatization techniques have also been developed using 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate as a pre-column derivatization reagent to analyze d-Dopa, with quantification achieved through an internal standard calibration curve [21]. Furthermore, emerging technologies for the chiral separation of D/L-Dopa are constantly emerging. For example, chiral selectivity can be achieved by improving electrochemical electrodes (the glassy carbon electrode and graphene nanohybrid (γGO/x-CDs) and designing stereoselective oxidases (in-situ growth of cerium oxide nanoparticles (CeO2) and dipeptide-capped copper nanoparticles) [[22], [23], [24], [25]]. The summary of the methods is shown in Table S1. During drug development and manufacturing, accurate quantification of enantiomeric impurities is critical. That not only ensure the active pharmaceutical ingredient (API) exerts its desired therapeutic efficacy, but also minimize the potential risks posed by inactive or harmful enantiomers.

In our previous work, we successfully separated chiral amino acid isomers using chiral aldehyde probes, which can enhance the detection signals for d-amino acids [[26], [27], [28]]. Given that D/L-Dopa shares a structural similarity with amino acids, we developed a derivatization and analytical method for D/L-Dopa and its impurities using a chlorine-labeled chiral probe (D-BPCl). d-BPCl undergoes a derivatization reaction with the amino group of amino acids via its aldehyde group, by introducing a chiral molecular moiety to alter polarity and convert enantiomers into diastereomer. This facilitates the retention and separation of D/L-Dopa on a conventional C18 column. The method demonstrates relatively low limits of detection (LOD) and quantification (LOQ) through derivatization compared to previously reported techniques. Notably, it successfully detected d-Dopa in pharmaceutical tablets from five different manufacturers, suggesting a potential gap in current quality control processes.