Abstract
Neferine, a natural alkaloid, is known for its multifaceted properties, including antioxidant, neuroprotective, anti-inflammatory, and autophagy-regulating effects. A simple, robust, specific, and sensitive analytical method is a key requirement for quantifying an analyte. Although a few methods have been reported for simultaneous estimation, none are available for the quantification of neferine alone in pharmaceutical or nutraceutical formulations. Whereas, the quantification of drugs alone is considered as a valuable requirement in the preformulation and formulation studies. In this research work, we report the development and validation of a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the accurate quantification of neferine in bulk and formulations. The chromatographic separation was achieved using a UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm) under an isocratic elution method. The mobile phase consisted of acetonitrile (80%) and 10 mM ammonium acetate with 0.1% formic acid (20%). The flow rate of the mobile phase was set at 0.3 mL/min. The method exhibited good linearity (R 2 = 0.9944) over a range of 25–600 ng/mL. High sensitivity of the method was demonstrated by the limit of detection (LOD) and limit of quantification (LOQ) values of 1.29 and 3.90 ng/mL, respectively. Results of the accuracy and precision study were in complete alignment with the ICH guidelines. The analytical method was found to be robust under varied analytical conditions. Furthermore, the method was successfully applied for the quantification of neferine in nanostructured lipid carriers in the diffusion studies. In conclusion, the newly developed and validated LC-MS/MS method is specific, sensitive, rapid, accurate, and precise for the quantification of neferine in bulk and formulated products.
1. Introduction
Neferine (NEF) is an alkaloid (bisbenzylisoquinoline) extracted from the embryos of Nelumbo nucifera, popularly known as the Indian lotus or sacred lotus. N. nucifera has been widely used across the Asian continent for food and nutrition, as well as a remedy for various disorders. , NEF (C38H44N2O6) is a bioactive moiety with a 624.778 g/mol molecular weight. NEF has garnered attention for its broad spectrum and diverse pharmacological properties in traditional medicine systems. , It demonstrates multifaceted therapeutic properties, including antioxidant, anti-inflammatory, cardioprotective, neuroprotective, antitumor, antidiabetic, and antithrombotic effects. It also modulates autophagy and has shown potential in reversing multidrug resistance. , On account of these diverse pharmacological activities, NEF has therapeutic potential for a variety of ailments, including Alzheimer’s disease, Huntington’s disease, lung carcinogenesis, aging-related skin disorders, diabetic vasculopathy, proliferative vitreoretinopathy, retinoblastoma, etc. −
A simple, rapid, highly sensitive, and reliable analytical method plays a valuable role in the quantification of active moieties across the pharmaceutical, nutraceutical, cosmeceutical, and food industries. , Liquid chromatography integrated with mass spectrometry (LC-MS/MS) is considered as a hyphenated analytical technique, offering myriad advantages such as high sensitivity, specificity, and rapid analysis. , At present, it is recognized as a gold standard in drug quantification due to its outstanding precision and ability to meet modern analytical demands. Furthermore, it allows high recovery, excellent reproducibility, and shorter chromatographic run times.
To the best of our knowledge, no analytical method is available in the literature, particularly for the assessment of NEF alone in bulk or formulations. It is noteworthy here that the quantification of drugs alone is considered a valuable requirement in the preformulation and formulation studies. To date, only three liquid chromatographic methods have been described for the assessment of NEF; two are bioanalytical methods developed for its quantification in biological matrices, and the other one is an analytical method intended for the simultaneous estimation of NEF along with other related alkaloids. − Hu et al. reported a gradient LC-MS/MS method for the simultaneous determination of neferine, liensinine, and isoliensinine in rat plasma. Another LC-MS/MS bioanalytical method was developed and validated using a gradient elution program for the quantification of NEF in rat plasma. The analytical technique was developed and validated using isocratic elution with a mobile phase of acetonitrile:water (16:84, v/v) containing 0.2% acetic acid and 0.1% trimethylamine for the simultaneous estimation of neferine, liensinine, and isoliensinine. Although these methods are useful, they fall short of delivering the efficiency required to meet the demand for estimating the NEF during preformulation and formulation development stages. Hence, there is a crucial need for a sensitive method to quantify the drug alone in various formulations.
In this research, we developed a new LC-MS/MS method that is highly sensitive, specific, rapid, and robust for the estimation of NEF alone. The developed method was validated for its specificity, linearity, precision, accuracy, stability, robustness, etc., according to ICH guidelines Q2(R2). The method was successfully applied to quantify NEF in the drug diffusion study of NEF-loaded NLC.
2. Materials and Methods
2.1. Materials
NEF was purchased from Naturewill Biotechnology (Sichuan Province, China). The LC/MS-grade solvents acetonitrile and methanol were procured from Thermo Fisher Scientific (Mumbai, India). Formic acid (LiChropur grade) and ammonium acetate were purchased from Merck Life Science Pvt., Ltd. (Mumbai, India). Glycerol monostearate (GMS) and oleic acid were purchased from Central Drug House (P) Ltd. (New Delhi, India) and Thermo Fisher Scientific (Mumbai, India), respectively. Milli-Q water (Millipore Corp., Bedford) was used to prepare buffers.
2.2. Instrumentation
The high-performance liquid chromatography triple quadrupole mass spectrometer (LC-8045, Shimadzu Corporation, Japan) was equipped with a binary pump (Nexera LC-40B X3), a 2-line degasser, and an autosampler temperature controller. The LC is hyphenated with a mass spectrometer that comprises an electrospray ionization (ESI) source and an electron multiplier detector. An Acquity BEH UPLC (2.1 × 50 mm, 1.7 μm) column from Waters Corporation (Milford) was used for chromatographic separation. The LC-MS/MS system was controlled by LC Solution software (version 5.123) for data acquisition and processing.
2.3. Method Optimization
The method development was focused on optimizing a sensitive, rapid, and robust LC-MS/MS method for the quantification of NEF. In the multiple reaction monitoring (MRM) mode, a nebulizing gas flow of 3 L/min was used to convert the liquid sample into a fine aerosol spray, thereby enabling efficient ionization at the source. A heating gas flow of 10 L/min was maintained to facilitate rapid solvent evaporation from the aerosolized droplets. The interface temperature and desolvation temperature were regulated at 300 and 526 °C, respectively. This ensures the efficient transfer of analyte ions from atmospheric pressure into the mass spectrometer’s vacuum system and accelerates solvent removal. The desolvation line (DL) temperature was set at 250 °C to prevent condensation. Additionally, the heat block temperature was optimized to 400 °C, and a drying gas flow of 1 L/min was used to support desolvation, ensuring stable signals suitable for accurate quantification. Argon was used as a collision gas to break the precursor ion, and the collision energy in the cell was analyzed by supplying it in a range from −45 to −5 V. The subsequent LC-MS/MS study was designed to monitor precursor–product-ion transitions.
Various chromatographic parameters were systematically evaluated to achieve optimal peak shape, resolution, and short run time. The liquid chromatography was operated using a column temperature of 40 °C and an autosampler temperature of 15 °C. Distinct chromatographic conditions were examined by varying the mobile phase composition and flow rate to optimize separation efficiency and resolution. Acetonitrile (ACN), methanol, and Milli-Q water, along with formic acid, were examined during the optimization process. Formic acid was used to set the optimum pH of the mobile phase. To establish the most suitable and standard analytical method, different flow rates within the range of 0.1–0.5 mL/min were systematically evaluated to determine the appropriate operating conditions. An injection volume of 10 μL was maintained throughout the analysis for all samples. The retention time, molecular mass, molecular formula, and the mass obtained for all product ions are mentioned in the Supporting Information (Table S1).
2.4. Preparation of Calibration Curve Standards
The primary stock solution was prepared by dissolving NEF (5 mg) in 5 mL of LC-MS-grade ACN. It was further diluted stepwise to prepare a working standard of 1000 ng/mL. For preparing the calibration curve standards, the working stock was appropriately diluted to make concentrations of 25, 50, 100, 200, 400, and 600 ng/mL. The quality control (QC) samples, including low quality control (LQC) at 30 ng/mL, medium quality control (MQC) at 250 ng/mL, and high quality control (HQC) at 500 ng/mL, were prepared independently from the working standard. These QC samples were further stored at 4 °C until employed in the accuracy, precision, robustness, and stability studies.
2.5. Method Validation
Specificity is the parameter of a method used to demonstrate its ability to estimate the analyte in the presence of excipients, solvents, and matrix components. The specificity was performed to ensure the absence of any intruding peaks at the elution time of the NEF. For this, the blank and a standard NEF sample (200 ng/mL) were analyzed and compared.
The linearity of the calibration curve for NEF was demonstrated using six calibration standards in the range 25–600 ng/mL. The calibration curve (n = 3) was constructed by plotting analyte concentration on the x-axis against the corresponding peak area on the y-axis. The limit of detection (LOD) is the lowest concentration of the analyte that can be detected with the developed method. The limit of quantification (LOQ) indicates the lowest concentration that can be measured with an acceptable precision and accuracy. Both of these parameters were calculated using the standard deviation of the intercept and the mean of the slope derived from the calibration curves.
The three QC levels (n = 6) were used to calculate the accuracy. It is demonstrated as a percentage bias, showing the difference between the observed concentration and the nominal concentration. The intraday and interday precision (n = 3) was assessed across all three QC levels. For intraday precision, the samples were analyzed at three distinct times on the same day, while interday precision was assessed over three consecutive days. Data from the precision study were presented as percent relative standard deviation (% RSD). Robustness of the method was evaluated to determine its ability to withstand small variations in the chromatographic conditions. It was performed by deliberately implementing slight modifications to the suggested chromatographic parameters. The parameters include buffer pH (±0.2), column temperature (±2 °C), and buffer concentration (±2 mM). Additionally, the percentage recovery was estimated by comparing the chromatographic response to that of the standard QC sample.
The stability study of the samples was conducted over 72 h. For this, the samples were placed in the autosampler, set at 15 °C, during the analyses. Furthermore, the LQC, MQC, and HQC samples were used for performing the stability study, and the results were expressed as percent recovery.
2.6. Application of the Validated Method
2.6.1. Preparation of NEF-Loaded Nanostructured Lipid Carriers
NEF-loaded NLCs were prepared by the hot homogenization method. Briefly, glycerol monostearate (70 mg) and oleic acid (30 mg) were weighed precisely and used as solid lipids and liquid lipids, respectively. The lipid mixture was melted at 75 °C in 15 mL glass vials. NEF (5 mg) was added to the melted lipid solution. The aqueous phase solution (1% w/v) was prepared by adding 100 mg of Tween 80 to 10 mL of Milli-Q water and maintaining it at the same temperature. The aqueous phase was added dropwise to the drug-containing lipid solution under continuous stirring (700 rpm) at 75 °C. Afterward, the suspension was stirred at 700 rpm for 15 min to facilitate self-assembly. Further, the prepared NLC was ultrasonicated (VibraCell, Sonics and Materials Inc., USA) for 4 min. The probe was kept at 25% amplitude under a pulse of 30 s on and 10 s off.
2.6.2. Particle Size Analysis
Particle size and polydispersity index (PDI) of the NEF-loaded NLC were analyzed using Zetasizer Nano ZS (Malvern Instrument Ltd., U.K.) at 25 °C. The samples were diluted with water (Milli-Q) in a 1:50 ratio for particle size analysis (n = 3). The measurement was performed in automatic mode at 173° backscattering detection angle, which enhances sensitivity and reduces multiple-scattering effects, enabling accurate and reproducible particle-size determination for both dilute and concentrated samples.
2.6.3. In Vitro Drug Diffusion
An in vitro diffusion study of NEF from NLC and pure drug was performed using the Franz diffusion cell apparatus (Orchid Scientific & Innovative India Pvt Ltd., Nashik, India). A dialysis membrane with a specific molecular weight cutoff (12–14 kDa) was used for the diffusion study. NEF-loaded NLC/pure drug (equivalent to 50 μg NEF) were added to the donor compartment of the apparatus, and 6 mL of 40% ethanolic media was added to the receptor compartment of the apparatus. The diffusion medium was maintained at 37 ± 0.5 °C and stirred at 250 rpm. At 30, 60, 180, and 360 min, 0.8 mL samples were withdrawn and accordingly replaced with an equal volume of the diffusion media. Samples (n = 3) were analyzed using the validated LC-MS/MS method, and the percent cumulative drug diffused at the respective time point was estimated.
3. Results and Discussion
3.1. Method Optimization
In MS, the positive ESI mode was selected due to efficient protonation of NEF. The full mass spectra of NEF are illustrated in Figure . The MS/MS optimization using MRM mode led to the detection of the precursor ion at m/z 625.25, and three product ions at m/z 625.25 > 489.15, 625.25 > 206.15, and 625.25 > 121.25. The selected collision energy for efficient fragmentation of the NEF precursor ion into the product ion was −35 V. The product-ion profile is shown in Figure . For quantification, the product ion with m/z 625.25 > 206.15 was selected based on its high response. This selected product ion was used to develop and validate the method.
1.
Full mass spectra of neferine.
2.
Overlay of multiple reaction monitoring chromatograms of the product ions of neferine.
The ACN was preferred over methanol as the organic solvent of the mobile phase due to its lower baseline noise, superior elution strength, and lower viscosity, which improved signal response and peak symmetry. The optimized mobile phase consisted of ACN and ammonium acetate buffer and operated under an isocratic mode. To achieve better elution, 10 mM ammonium acetate buffer (pH 3.5) and ACN at a 20:80 (v/v) ratio were selected. A mobile-phase flow rate of 0.3 mL/min yielded a sharp and symmetric peak with a retention time of 0.59 min for the analyte. The total run time of the chromatograph was set at 3 min.
The method offers a valuable approach due to its simplicity, high sensitivity, and rapid analysis for routine analysis of NEF using LC-MS/MS.
3.2. Method Validation
3.2.1. Specificity
The developed LC-MS/MS method was checked for specificity by comparing blank and drug samples (200 ng/mL). The mobile phase (ACN/acetate buffer 10 mM; 80:20) was used as the blank. From a primary neferine stock solution, a 1000 ng/mL working standard was prepared, which was subsequently diluted to obtain a 200 ng/mL standard. The specificity of the developed method was demonstrated by the absence of any other interfering peaks in the chromatogram. Figure confirms the absence of any other peaks at a retention time of NEF.
3.
Comparative LC-MS/MS chromatograms of neferine and the blank.
3.2.2. Calibration Curve
The final calibration curve was constructed using selected standard concentrations of 25, 50, 100, 200, 400, and 600 ng/mL. Figure exhibits the overlay of multiple reaction monitoring chromatograms of all calibration standards. The regression coefficient (R 2) of the calibration curve was found to be 0.9944, as shown in Figure S1. Following is the regression equation for NEF (n = 3)
where “y” represents the peak area for the respective concentrations.
4.
Overlay of multiple reaction monitoring chromatograms of all calibration standards.
The multiple reaction monitoring chromatograms for the calibration curve are mentioned in Figure S2 of the Supporting Information.
The method depicted good linearity across the concentration ranges of 25–600 ng/mL. The quality control samples, that is, 30 ng/mL (LQC), 250 ng/mL (MQC), and 500 ng/mL (HQC), were selected on the basis of standard criteria and used for further validation studies.
3.2.3. Limit of Detection and Limit of Quantification
The calculated LOD and LOQ values represent the sensitivities of the developed method. The LODs and LOQ of the method were observed to be 1.29 and 3.90 ng/mL. This result indicates the high sensitivity of the LC-MS/MS method for estimating the NEF.
3.2.4. Accuracy and Precision
Accuracy of the analytical method was evaluated at all three QC levels (LQC, MQC, and HQC), and the results are presented in Table . For the LQC, the measured concentration ranged from 29.67 to 30.59 ng/mL. The mean concentration was found to be 30.28 ± 0.30 ng/mL, with a % RSD of 1.00. A % bias value of 0.93 indicates only a slight deviation between the measured and the true values. The percent recovery was 100.93 ± 1.01%, implying that the method is highly accurate at lower concentrations. For the MQC, the measured concentration ranged from 249.88 to 253.86 ng/mL, with a mean measured concentration of 251.88 ± 1.29 ng/mL and a % RSD of 0.51. The percent recovery (%) of 100.75 ± 0.52 confirms the method’s ability to accurately quantify NEF at medium concentrations. For the HQC, the measured concentration ranged from 502.50 to 510.00 ng/mL. The mean concentration was 506.45 ± 2.60 ng/mL, with a % RSD of 0.51. The % bias value of 1.29 and the percent recovery of 101.29 ± 0.52% confirm the method’s ability to accurately quantify neferine at higher concentrations.
1. Accuracy Study.
| measured
concentration (ng/mL) |
% recovery |
||||
|---|---|---|---|---|---|
| QC levels | range | mean ± SD | % RSD | (mean ± SD) | (% bias) |
| LQC | 29.67–30.59 | 30.28 ± 0.30 | 1.00 | 100.93 ± 1.01 | 0.93 |
| MQC | 249.88–253.86 | 251.88 ± 1.29 | 0.51 | 100.75 ± 0.52 | 0.75 |
| HQC | 502.50–510.00 | 506.45 ± 2.60 | 0.51 | 101.29 ± 0.52 | 1.29 |
SD: standard deviation; % RSD: percent relative standard deviation.
Similarly, a precision study of the developed method was conducted at all three QC levels by assessing intraday and interday repeatability. The results are presented in Table . Interday precision was determined separately for three consecutive days, and the percent RSD values ranged from 0.10 to 1.53% for LQC, 0.14 to 1.01% for MQC, and 0.45 to 1.68% for HQC. Intraday precision was assessed by analyzing all three QC samples within the same day, yielding % RSD values of 1.22% (LQC), 1.16% (MQC), and 1.50% (HQC). The results of the study were found within the acceptable limit, confirming the high precision of the method.
2. Precision Study.
| interday
repeatability (% RSD) |
||||
|---|---|---|---|---|
| QC level | day 1 | day 2 | day 3 | intraday repeatability (% RSD) |
| LQC | 0.10 | 0.98 | 1.53 | 1.22 |
| MQC | 0.14 | 1.01 | 0.95 | 1.16 |
| HQC | 0.70 | 0.45 | 1.68 | 1.50 |
% RSD: Percent relative standard deviation.
The results of the accuracy and precision studies were found to be in complete alignment with the guidelines provided by ICH. These findings reveal that the LC-MS/MS method exhibits acceptable accuracy and precision across all QC levels, ensuring its suitability for quantitative measurements in the relevant applications.
3.2.5. Robustness
For a robustness study, standard QC samples were injected under various modified chromatographic conditions, specifically by changing the column oven temperature from 40 to 38 °C and 42 °C, and adjusting the buffer pH from 3.5 to 3.3 and 3.7. Further buffer concentration was also deliberately changed by 10 ± 2 mM from the actual concentration (10 mM). The percentage recovery of all the QC samples ranged from 97.13 ± 4.62 to 103.96 ± 3.71. The acquired result was satisfactory, and the developed method proved to be robust. Results of the robustness study are provided in Table .
3. Robustness Parameter.
| % recovery
(±SD) |
||||
|---|---|---|---|---|
| parameters | level | LQC | MQC | HQC |
| mobile phase pH (3.5 ± 0.2) | 3.3 | 101.41 ± 1.92 | 102.34 ± 2.81 | 99.75 ± 2.26 |
| 3.7 | 97.13 ± 4.62 | 101.34 ± 2.09 | 98.96 ± 2.65 | |
| column temperature (40 ± 2 °C) | 38 | 97.87 ± 2.79 | 98.89 ± 0.15 | 102.11 ± 0.71 |
| 42 | 103.96 ± 3.71 | 100.67 ± 1.03 | 101.09 ± 1.46 | |
| buffer concentration (10 ± 2 mM) | 08 | 102.01 ± 2.95 | 102.13 ± 1.26 | 101.40 ± 1.04 |
| 12 | 101.14 ± 1.94 | 102.04 ± 2.87 | 102.15 ± 1.90 | |
SD: Standard deviation; % recovery = [nominal concentration/mean measured concentration] × 100.
3.2.6. Stability Studies
Stability studies of QC samples were carried out under both short-term and long-term conditions. In benchtop stability assessment at 24, 48, and 72 h, no significant variation was found between the concentration of all three QC samples. The % RSD was found within 2% for all QC samples, meeting the acceptance criteria. A summary of the stability profiles is provided in Table . Hence, the results confirm that the samples remained stable for up to 72 h at room temperature.
4. Stability Study.
| time (h) | QC level | mean measured concentration | % RSD | % recovery |
|---|---|---|---|---|
| 24 | LQC | 29.70 | 1.17 | 99.00 |
| MQC | 249.87 | 0.35 | 99.95 | |
| HQC | 499.77 | 0.58 | 99.95 | |
| 48 | LQC | 30.15 | 1.05 | 100.49 |
| MQC | 249.41 | 1.71 | 99.77 | |
| HQC | 508.55 | 1.88 | 101.71 | |
| 72 | LQC | 29.76 | 1.02 | 98.70 |
| MQC | 248.92 | 0.86 | 99.55 | |
| HQC | 510.66 | 1.84 | 98.82 |
3.3. Application of the Validated Method
3.3.1. Determination of Particle Size and PDI of NLC
The average particle size and PDI of NEF-loaded NLC were found to be 82 ± 2.58 nm and 0.283 ± 0.007 (n = 3), respectively. Figure depicts the particle size and PDI of the prepared NLC. The D10, D50, and D90 of NLC were found to be 43 ± 2.15, 103 ± 4.61, and 213 ± 7.51 nm, respectively.
5.
Particle size distribution of neferine-loaded NLC.
3.3.2. In Vitro Drug Diffusion
The in vitro diffusion study of formulated NLC was performed using Franz diffusion cells to demonstrate the practical application of the validated LC-MS/MS method. The diffusion profile illustrated in Figure shows that NEF released from NLC exhibited a faster and higher cumulative diffusion as compared to the pure NEF. After 6 h, more than 7 μg of NEF was diffused from the NLC formulation, whereas in the same interval of time, pure NEF showed only about 5 μg diffusion.
6.
Amount of drug diffused from the neferine suspension and neferine-loaded NLC.
The enhanced diffusion observed with NEF-loaded NLCs could be linked to multiple formulation-related factors. Tween 80 present in the NLC may enhance the drug’s diffusion across the membrane and increase the NEF permeability in the diffusion medium. These results endorse the utility of the developed and validated LC-MS/MS method for estimating the NEF in formulations.
4. Conclusion
In this research work, a new LC-MS/MS method was developed and validated for the quantification of NEF in bulk and formulations. The method exhibited excellent linearity along with high precision and accuracy for the estimation of the active moiety. The validated method was found to be simple, sensitive, rapid, and robust. Furthermore, the method was successfully applied in the drug diffusion study of the prepared nanoformulations. The method extends a reliable and efficient approach for the routine quantification of NEF in pharmaceutical and nutraceutical formulations.
Supplementary Material
Acknowledgments
The authors would like to extend gratitude to BITS Pilani, Pilani Campus, for the research fellowship to Y.P. We would also like to acknowledge the BITS BioCityH foundation, Pilani campus, for providing the LC-MS/MS facility.
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c00933.
Summary of neferine and method parameters (Table S1); calibration curve of neferine (Figure S1); multiple reaction monitoring chromatogram of calibration curve of NEF (A) 25 ng/mL, (B) 50 ng/mL, (C) 100 ng/mL, (D) 200 ng/mL, (E) 400 ng/mL, and (F) 600 ng/mL (Figure S2) (PDF)
The authors declare no competing financial interest.
References
- Bharathi Priya L., Huang C. Y., Hu R. M., Balasubramanian B., Baskaran R.. An updated review on pharmacological properties of neferineA bisbenzylisoquinoline alkaloid from Nelumbo nucifera. J. Food Biochem. 2021;45(12):e13986. doi: 10.1111/jfbc.13986. [DOI] [PubMed] [Google Scholar]
- Marthandam Asokan S., Mariappan R., Muthusamy S., Velmurugan B. K.. Pharmacological benefits of neferine - A comprehensive review. Life Sci. 2018;199:60–70. doi: 10.1016/j.lfs.2018.02.032. [DOI] [PubMed] [Google Scholar]
- Bi Z., Liang Y., Tang X., Shu Y., Xie Z., Xu G.. et al. Identification of Neferine as a DOR Agonist Activating Gi and Gz Signaling: In Silico and In Vitro Studies. Int. J. Mol. Sci. 2026;27(4):2058. doi: 10.3390/ijms27042058. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhang Q., Zhou Q., Li H.. Action and mechanisms of neferine in inflammatory diseases (Review) Mol. Med. Rep. 2025;32(1):1–8. doi: 10.3892/mmr.2025.13539. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lin T. Y., Hung C. Y., Chiu K. M., Wang S. J., Lee M. Y., Lu C. W.. Neferine, an Alkaloid from Lotus Seed Embryos, Exerts Antiseizure and Neuroprotective Effects in a Kainic Acid-Induced Seizure Model in Rats. Int. J. Mol. Sci. 2022;23(8):4130. doi: 10.3390/ijms23084130. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ozal S. A., Vuslat G., Kader T., Hande G., Suat E.. Neferine inhibits epidermal growth factor-induced proliferation and migration of retinal pigment epithelial cells through downregulating p38 MAPK and PI3K/AKT signalling. Cutaneous Ocul. Toxicol. 2020;39:97. doi: 10.1080/15569527.2020.1730882. [DOI] [PubMed] [Google Scholar]
- Cevik M., Gobeka H. H., Aydemir O.. Effects of neferine on retinal tissue in experimental diabetic rat model. Int. Ophthalmol. 2023;43(1):249–260. doi: 10.1007/s10792-022-02424-0. [DOI] [PubMed] [Google Scholar]
- Sivalingam K., Amirthalingam V., Ganasan K., Huang C. Y., Viswanadha V. P.. Neferine suppresses diethylnitrosamine-induced lung carcinogenesis in Wistar rats. Food Chem. Toxicol. 2019;123:385–398. doi: 10.1016/j.fct.2018.11.014. [DOI] [PubMed] [Google Scholar]
- Wong V. K. W., Wu A. G., Wang J. R., Liu L., Law B. Y. K.. Neferine attenuates the protein level and toxicity of mutant huntingtin in PC-12 cells via induction of autophagy. Molecules. 2015;20(3):3496–3514. doi: 10.3390/molecules20033496. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mohite P., Nimse S. B., Joy J. G., Kulkarni R., Pandhare R. B., Pawar A.. New analytical LC – MS/MS method for fluconazole and ivermectin estimation in combined pharmaceutical dosage form: development and validation. Future J. Pharm. Sci. 2023;9:1–10. doi: 10.1186/s43094-023-00497-x. [DOI] [Google Scholar]
- Kılıç H., Kandemir L.. Method development and validation for determination of N-Nitrosamines in pharmaceutical preparations by LC-MS/MS: Application to extractables and leachables studies. J. Chromatogr. A. 2025;1745:465741. doi: 10.1016/j.chroma.2025.465741. [DOI] [PubMed] [Google Scholar]
- Ragab M., Khaled O., Eissa F., Medhat M.. Optimization and validation of a rapid LC-MS/MS method for the detection and quantification of illegal dyes in olive oil from Egyptian markets. Food Chem. 2025;488:144924. doi: 10.1016/j.foodchem.2025.144924. [DOI] [PubMed] [Google Scholar]
- Tan J., Xiao H., Mei C., Wu X., Temerdashev A., Zhu Q., Feng Y.. Rapid and Sensitive Determination of Alkynyl Steroid Hormones in Milk Samples Using Magnetic Solid-Phase Extraction Coupling Click Derivatization-Based LC–MS Method. J. Anal. Test. 2025;9(4):584–594. doi: 10.1007/s41664-025-00372-z. [DOI] [Google Scholar]
- Takyi-Williams J., Erasmus L., Hayeshi R., Grobler A.. Development and validation of an LC-MS/MS method for the quantification of goserelin in a Pheroid formulation, in simulated intestinal fluid. J. Pharm. Biomed. Anal. 2020;180:113044. doi: 10.1016/j.jpba.2019.113044. [DOI] [PubMed] [Google Scholar]
- Hu L., Wang Y., Shu C., Yu J., Tao R., Yang H.. et al. Pharmacokinetics, bioavailability and metabolism of neferine in rat by LC – MS/MS and LC – HRMS. Biomed. Chromatogr. 2021;35:e5193. doi: 10.1002/bmc.5193. [DOI] [PubMed] [Google Scholar]
- Hu G., Xu R. a., Dong Y. y., Wang Y. y., Yao W. w., Chen Z. c.. et al. Simultaneous determination of liensinine, isoliensinine and neferine in rat plasma by UPLC – MS/MS and application of the technique to pharmacokinetic studies. J. Ethnopharmacol. 2015;163:94–98. doi: 10.1016/j.jep.2015.01.020. [DOI] [PubMed] [Google Scholar]
- Chen Y., Fan G., Wu H., Wu Y., Mitchell A.. Separation, identification and rapid determination of liensine, isoliensinine and neferine from embryo of the seed of Nelumbo nucifera GAERTN. by liquid chromatography coupled to diode array detector and tandem mass spectrometry. J. Pharm. Biomed. Anal. 2007;43:99–104. doi: 10.1016/j.jpba.2006.06.016. [DOI] [PubMed] [Google Scholar]
- International Conference Harmonisation. ICH guideline Q2(R2) on validation of analytical procedures. Europea. 1994;50(4):97–105. [Google Scholar]
- Taha I. E., Elsohly M. A., Radwan M. M., Elkanayati R. M., Wanas A.. et al. Enhancement of cannabidiol oral bioavailability through the development of nanostructured lipid carriers: In vitro and in vivo evaluation studies. Drug Delivery Transl. Res. 2025;15:2722–2732. doi: 10.1007/s13346-024-01766-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.