Optimization of Taxol Extraction Process Using Response Surface Methodology and Investigation of Temporal and Spatial Distribution of Taxol in Taxus mairei.

Taxus mairei is an important source for industrial extraction of taxol in China. However, the standard and steps of extraction are currently not uniform, which seriously affects the taxol yield. In the present study, the influence of four factors (methanol concentration, solid-liquid ratio, ultrasonic extraction temperature, and ultrasonic extraction time) on the taxol yield was successively explored in T. mairei. A response surface methodology (RSM) was used to optimize the extraction process based on the single-factor experiments above. The optimal conditions were as follows: methanol concentration was 90%, solid-liquid ratio was 1:15 (g/mL), ultrasonic extraction temperature was 40 °C and ultrasonic extraction time was 60 min. Moreover, the twigs and needles from T. mairei with different tree ages were treated by the optimum extraction process, which further revealed temporal and spatial distribution of taxol in the reproducible tissues. Interestingly, the taxol content was relatively higher in needles of T. 'Jinxishan' (a cultivar from T. mairei with yellow aril, FY), but was less in FY twigs. The accumulation of taxol in twigs and leaves of females (with red aril, FR) was significantly higher than that of males (M); however, the content showed a decreasing trend with the increasing tree ages. Therefore, it is suitable to increase the proportion of female trees especially the FY leaves as raw materials for the industrial production of taxol from T. mairei, and the tree ages should be better controlled at 3-7 years.

Pathways Related to the Anti-Cancer Effects of Metabolites Derived from Cerrado Biome Native Plants: An Update and Bioinformatics Analysis on Oral Squamous Cell Carcinoma.

BACKGROUND: Oral cancer is a significant health problem worldwide. Oral squamous cell carcinoma (OSCC) is a malignant neoplasm of epithelial cells that mostly affects different anatomical sites in the head and neck and derives from the squamous epithelium or displays similar morphological characteristics. Generally, OSCC is often the end stage of several changes in the stratified squamous epithelium, which begin as epithelial dysplasia and progress by breaking the basement membrane and invading adjacent tissues. Several plant-based drugs with potent anti-cancer effects are considered inexpensive treatments with limited side effects for cancer and other diseases. OBJECTIVE: The aim of this review is to explore whether some Brazilian plant extracts or constituents exhibit anti-tumorigenic activity or have a cytotoxic effect on human oral carcinoma cells. METHODS: Briefly, OSCC and several metabolites derived from Brazilian plants (i.e., flavonoids, vinblastine, irinotecan, etoposide and paclitaxel) were used as keywords to search the literature on PubMed, GenBank and GeneCards. RESULTS: The results showed that these five chemical compounds found in Cerrado Biome plants exhibit anti-neoplastic effects. Evaluating the compounds revealed that they play a main role in the regulation of cell proliferation. CONCLUSION: Preserving and utilising the biodiversity of our planet, especially in unique ecosystems, such as the Cerrado Biome, may prove essential to preserving and promoting human health in modern contexts.

Research progress on the source, production, and anti-cancer mechanisms of paclitaxel.

Paclitaxel, a tetracyclic diterpenoid compounds, was firstly isolated from the bark of the Pacific yew trees. Currently, as a low toxicity, high efficiency, and broad-spectrum natural anti-cancer drug, paclitaxel has been widely used against ovarian cancer, breast cancer, uterine cancer, and other cancers. As the matter of fact, natural paclitaxel from Taxus species has been proved to be environmentally unsustainable and economically unfeasible. For this reason, researchers from all over the world are devoted to searching for new ways of obtaining paclitaxel. At present, other methods, including artificial cultivation of Taxus plants, microbial fermentation, chemical synthesis, tissue and cell culture have been sought and developed subsequently. Meanwhile, the biosynthesis of paclitaxel is also an extremely attractive method. Unlike other anti-cancer drugs, paclitaxel has its unique anti-cancer mechanisms. Here, the source, production, and anti-cancer mechanisms of paclitaxel were summarized and reviewed, which can provide theoretical basis and reference for further research on the production, anti-cancer mechanisms and utilization of paclitaxel.

Biotechnological approaches to the production of plant-derived promising anticancer agents: An update and overview.

The plant kingdom is a rich source of bioactive compounds, many of which have been used since pre-history for their therapeutic properties to treat a range of illnesses. These metabolites have recently attracted attention to their antineoplastic activities to treat various cancers relying on different mechanisms. Some of these molecules are glycosides, which have proven useful as anti-cancer agents, namely podophyllotoxin (PPT) anaryltetralin lignan or alkaloids. There are three primary forms of alkaloids, such as indole alkaloids (vincristine and vinblastine from Catharanthus roseus), quinoline alkaloid (camptothecin from Camptotheca acuminata), and diterpenoid alkaloid (taxol and it's analogous from Taxus and Corylus species). This review considers various plant biotechnology approaches used to enhance the production of these anticancer molecules in different species. In this regard, many in vitro culture techniques such as stimulation of suspension culture and hairy roots are being used to investigate the effects of plant growth regulators and elicitors on various explants.

Detection, Purification and Elucidation of Chemical Structure and Antiproliferative Activity of Taxol Produced by Penicillium chrysogenum.

Penicillium chrysogenum has been reported as a potent taxol producer based on quantitative analysis by TLC and HPLC. The biosynthetic potency of taxol has been validated from PCR detection of rate-limiting genes of taxol synthesis such as taxadienesynthase and 10-de-acetylbaccatin III-O-acetyltransferase (DBAT), which catalyzes the immediate diterpenoid precursor of the taxol substance, as detected by PCR. Taxol production by P. chrysogenum was assessed by growing the fungus on different media. Potato dextrose broth (PDB) was shown to be the best medium for obtaining the higher amount of taxol (170 µg/L). A stepwise optimization of culture conditions necessary for production of higher amounts of taxol was investigated. The substance taxol was produced optimally after 18 d of incubation at 30 °C in PDB adjusted initially at pH 8.0 with shaking (120 rpm) (250 µg/L). The P. chrysogenum taxol was purified successfully by HPLC. Instrumental analyzes such as Fourier transform infrared spectroscopy (FTIR), ultraviolet (UV) spectroscopy, 1HNMR and 13C NMR approved the structural formula of taxol (C47H51NO14), as constructed by ChemDraw. The P. chrysogenum taxol showed promising anticancer activity.

Assessment of efficacy of postinfusion tubing flushing in reducing risk of cytotoxic contamination.

PURPOSE: Infusion of cytotoxic drugs carries the risk of occupational exposure of healthcare workers. Since disconnecting an infusion line is a source of contamination, flushing of tubing after infusion of cytotoxic agents is recommended, but the optimal volume of rinsing solution is unknown. The objective of this study was to assess whether postinfusion line flushing completely eliminates cytotoxics. METHODS: Infusions were simulated with 3 cytotoxics (gemcitabine, cytarabine, and paclitaxel) diluted in 5% dextrose injection or 0.9% sodium chloride injection in 250-mL infusion bags. Infusion lines were flushed using 5% dextrose injection or 0.9% sodium chloride solution at 2 different flow rates. The remaining concentration of cytotoxics in the infusion line was measured by a validated high-performance liquid chromatography (HPLC) method after passage of every 10 mL of flushing volume until a total of 100 mL had been flushed through. RESULTS: All cytotoxics remained detectable even after line flushing with 80 mL of flushing solution (a volume 3-fold greater than the dead space volume within the infusion set). Gemcitabine and cytarabine were still quantifiable via HPLC even after flushing with 100 mL of solution. Efficacy of flushing was influenced by the lipophilicity of drugs but not by either the flushing solvent used or the flushing flow rate. After 2-fold dead space volume flushing, the estimated amount of drug remaining in the infusion set was within 0.19% to 0.56% of the prescribed dose for all 3 cytotoxics evaluated. CONCLUSION: Complete elimination of cytotoxics from an infusion line is an unrealistic objective. Two-fold dead space volume flushing could be considered optimal in terms of administered dose but not from an environmental contamination point of view. Even when flushed, the infusion set should still be considered a source of cytotoxic contamination.

Comparative transcriptome analysis of a taxol-producing endophytic fungus, Aspergillus aculeatinus Tax-6, and its mutant strain.

Taxol is a rare but extremely effective antitumor agent extracted from Taxus yew barks. Taxus plants are valuable and rare species, and the production of taxol from them is a complex process. Therefore, taxol-producing endophytic fungi seem to be a promising alternative because of their high practical value and convenient progress. In this study, the transcriptome of an endophytic fungus, Aspergillus aculeatinus Tax-6 was analyzed in order to understand the molecular mechanisms of producing fungal taxol. The results showed that genes involved in the mevalonate (MVA) pathway and non-mevalonate (MEP) pathway were expressed, including isopentenyl pyrophosphate transferase, geranyl pyrophosphate transferase, and geranylgeranyl pyrophosphate synthetase. However, those downstream genes involved in the conversion of taxa-4(5)-11(12)-diene from geranylgeranyl pyrophosphate were not expressed except for taxane 10-beta-hydroxylase. Additionally, a mutant strain, A. aculeatinus BT-2 was obtained from the original strain, A. aculeatinus Tax-6, using fungicidin as the mutagenic agent. The taxol yield of BT-2 was 560 µg L-1, which was higher than that of Tax-6. To identify the mechanism of the difference in taxol production, we compared the transcriptomes of the two fungi and explored the changes in the gene expression between them. When compared with the original strain, Tax-6, most genes related to the MVA pathway in the mutant strain BT-2 showed upregulation, including GGPPS. Moreover, most of the downstream genes were not expressed in the mutant fungi as well. Overall, the results revealed the pathway and mechanism of taxol synthesis in endophytic fungi and the potential for the construction of taxol-producing genetic engineering strains.

Conjugation of Aspergillus flavipes Taxol with Porphyrin Increases the Anticancer Activity of Taxol and Ameliorates Its Cytotoxic Effects.

Taxol is one of the potential anticancer drugs; however, the yield of Taxol and its cytotoxicity are common challenges. Thus, manipulating the Taxol biosynthetic pathway from endophytic fungi, in addition to chemical modification with biocompatible polymers, is the challenge. Four fungal isolates, namely, Aspergillus flavipes, A. terreus, A. flavus, and A. parasiticus, were selected from our previous study as potential Taxol producers, and their potency for Taxol production was evaluated in response to fluconazole and silver nitrate. A higher Taxol yield was reported in the cultures of A. flavipes (185 µg/L) and A. terreus (66 µg/L). With addition of fluconazole, the yield of Taxol was increased 1.8 and 1.2-fold for A. flavipes and A. terreus, respectively, confirming the inhibition of sterol biosynthesis and redirecting the geranyl phosphate pool to terpenoids synthesis. A significant inhibition of ergosterol biosynthesis by A. flavipes with addition of fluconazole was observed, correlating with the increase on Taxol yield. To increase the Taxol solubility and to reduce its cytotoxicity, Taxol was modified via chemical conjugation with porphyrin, and the degree of conjugation was checked from the Thin layer chromatography and UV spectral analysis. The antiproliferative activity of native and modified Taxol conjugates was evaluated; upon porphyrin conjugation, the activity of Taxol towards HepG2 was increased 1.5-fold, while its cytotoxicity to VERO cells was reduced 3-fold.

Isolation of taxol producing endophytic fungus Alternaria brassicicola from non-Taxus medicinal plant Terminalia arjuna.

In the present study, an endophytic fungal strain was isolated from its non-Taxus host plant Terminalia arjuna and identified as Alternaria brassicicola based on its morphological characteristics and internal transcribed spacer sequence analysis. This fungus was grown in potato dextrose broth and analyzed for the presence of taxol by using chromatographic and spectrometric techniques. The ethyl acetate extract of A.brassicicola was subjected to column chromatography. Among the different fractions, the fraction 7 showed positive to taxol, which was further confirmed by UV absorption, HPLC, FTIR spectra and LC-ESI-MS by comparing with the authentic taxol (Paclitaxel). The peaks of fraction 7 obtained by UV spectroscopy, FTIR and HPLC analysis were quite similar to that of standard taxol confirming the presence of taxol. A parent ion peak of m/z 854.95 was observed in the LC-ESI-MS spectrum which was similar to paclitaxel with reported m/z of 854 [M+H]+ ion. A. brassicicola produced about 140.8 µg/l taxol as quantified through HPLC. Present study results suggest that the endophytic fungus A.brassicicola serves as a potential source for the production of taxol isolated from non-Taxus plant.

Are Microbial Endophytes the 'Actual' Producers of Bioactive Antitumor Agents?

For millenia, plants have been a major source of medications against human and animal diseases. In the case of anticancer agents, a significant number of current agents can trace their source back to nominally plant secondary metabolites, with examples being taxol, vinca alkaloids, camptothecin (CPT), and their modified derivatives. However, it is now becoming apparent that these and other plant-derived materials, plus similar agents from marine sources may well have a microbe in their background. In this short Opinion, evidence for such claims are presented for some of the agents currently in use or in preclinical and clinical trials against cancer.

Novel resources of Taxol from endophytic and entomopathogenic fungi: Isolation, characterization and LC-Triple mass spectrometric quantification.

This work presents a second-to-none method for Taxol isolation from the Endophytic fungus Cladosporium sphaerospermum (AUMC 6896) and the Entomopathogenic fungus Metarizium anisopliae (AUMC 5130). The extracts were analyzed by high performance liquid chromatography (HPLC) and Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using positive electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. This is rapid, consistent, reproducible, accurate, and sensitive for quantifying Taxol across multiple samples. The yield of crude Taxol product obtained from Potato Dextrose broth (PDB) medium inoculated with Cladosporium sphaerospermu and Metarizium anisopliae was found to be 3.732, and 0.0023 µg L-1 respectively. The yield can be improved by adding ammonium acetate or salicylic acid to the culture broth. Addition of ammonium acetate (AA) (20 mg L-1) to culture media resulted in an increase of Taxol yield to 30.365 and 27.289 µg L-1 respectively. Production of Taxol was 29.844 and 67.254 µg L-1 for the two fungus species when ammonium acetate was substituted by 90 mg L-1 salicylic acid (SA). Adding both AA (20 mg L-1) and SA (90 mg L-1) to the culture media resulted in an increase of the Taxol yield to 4.054 and 116.373 µg L-1 respectively. Our proposed analytical method offers very fast (3 min) quantitation of Taxol in comparison with other published methods. These findings represent a new bioprospecting of the endophytic fungi that may serve as a potential material for the production of Taxol for anticancer treatment.

Fast and accurate separation of the paclitaxel from yew extracum by a pseudo simulated moving bed with solvent gradient.

A pseudo simulated moving bed (SMB) with solvent gradient was used to trap and separate paclitaxel from yew extracum. This SMB process consisted of three steps: feeding, purification and recovery. In comparison with methanol/water as an eluent, acetonitrile/water could give a better separation but had a poor dissolubility of the yew extracum, and therefore methanol/water was used in the feeding followed by acetonitrile/water in the purification. In the first two steps, water was deliberately added into zone III to modulate the eluotropic strength of the liquid entering zone III, so as to make paclitaxel separation from impurities be more efficient. Once most of impurities discarded, the columns were in turn eluted to recover the trapped paclitaxel of 98% yield with a purity of 78% from the yew extracum containing 1.5% paclitaxel. Afterward, an additional operation of crystallization improved the purity further to 97.8% with the yield of 95%.

Extraction and determination of trace amounts of three anticancer pharmaceuticals in urine by three-phase hollow fiber liquid-phase microextraction based on two immiscible organic solvents followed by high-performance liquid chromatography.

An automated three-phase hollow fiber liquid-phase microextraction based on two immiscible organic solvents followed by high-performance liquid chromatography with UV-Vis detection method was applied for the extraction and determination of exemestane, letrozole, and paclitaxel in water and urine samples. n-Dodecane was selected as the supported liquid membrane and its polarity was justified by trioctylphosphine oxide. Acetonitrile was used as an organic acceptor phase with desirable immiscibility having n-dodecane. All the effective parameters of the microextraction procedure such as type of the organic acceptor phase, the supported liquid membrane composition, extraction time, pH of the donor phase, hollow fiber length, stirring rate, and ionic strength were evaluated and optimized separately by a one variable at-a-time method. Under the optimal conditions, the linear dynamic ranges were 1.8-200 (R2  = 0.9991), 0.9-200 (R2  = 0.9987) and 1.2-200 µg/L (R2  = 0.9983), and the limits of detection were 0.6, 0.3, and 0.4 µg/L for exemestane, letrozole, and paclitaxel, respectively. To evaluate the capability of the proposed method in the analysis of biological samples, three different urinary samples were analyzed under the optimal conditions. The relative recoveries of the three pharmaceuticals were in the range of 91-107.3% for these three analytes.

Specialized Plant Metabolism Characteristics and Impact on Target Molecule Biotechnological Production.

Plant secondary metabolism evolved in the context of highly organized and differentiated cells and tissues, featuring massive chemical complexity operating under tight environmental, developmental and genetic control. Biotechnological demand for natural products has been continuously increasing because of their significant value and new applications, mainly as pharmaceuticals. Aseptic production systems of plant secondary metabolites have improved considerably, constituting an attractive tool for increased, stable and large-scale supply of valuable molecules. Surprisingly, to date, only a few examples including taxol, shikonin, berberine and artemisinin have emerged as success cases of commercial production using this strategy. The present review focuses on the main characteristics of plant specialized metabolism and their implications for current strategies used to produce secondary compounds in axenic cultivation systems. The search for consonance between plant secondary metabolism unique features and various in vitro culture systems, including cell, tissue, organ, and engineered cultures, as well as heterologous expression in microbial platforms, is discussed. Data to date strongly suggest that attaining full potential of these biotechnology production strategies requires being able to take advantage of plant specialized metabolism singularities for improved target molecule yields and for bypassing inherent difficulties in its rational manipulation.

Extraction protocol and liquid chromatography/tandem mass spectrometry method for determining micelle-entrapped paclitaxel at the cellular and subcellular levels: Application to a cellular uptake and distribution study.

Paclitaxel-loaded polymeric micelles (PTX-PM) are commonly used as tumor-targeted nanocarriers and display outstanding antitumor features in clinic, but its accumulation and distribution in vitro are lack of investigation. It is probably due to the complex micellar system and its low concentration at the cellular or subcellular levels. In this study, we developed an improved extraction method, which was a combination of mechanical disruption and liquid-liquid extraction (LLE), to extract the total PTX from micelles in the cell lysate and subcellular compartments. An ultra-performance liquid chromatography tandem mass spectroscopy (UPLC-MS/MS) method was optimized to detect the low concentration of PTX at cellular and subcellular levels simultaneously, using docetaxel as internal standard (IS). The method was proved to release PTX totally from micelles (≥95.93%) with a consistent and reproducible extraction recovery (≥75.04%). Good linearity was obtained at concentrations ranging from 0.2 to 20ng/mL. The relative error (RE%) for accuracy varied from 0.68 to 7.56%, and the intra- and inter-precision (relative standard deviation, RSD%) was less than 8.64% and 13.14%, respectively. This method was fully validated and successfully applied to the cellular uptake and distribution study of PTX-loaded PLGA-PEG micelles in human breast cancer cells (MCF-7).

Ultrasonic Assisted Extraction of Paclitaxel from Taxus x media Using Ionic Liquids as Adjuvants: Optimization of the Process by Response Surface Methodology.

(1) Background: Ionic liquids (ILs) are considered "green" solvents and have been widely used in the extraction and separation field in recent years; (2) Methods: In this study, some common ILs and functionalized magnetic ionic liquids (MILs) were used as adjuvants for the solvent extraction of paclitaxel from Taxus x media (T. x media) using methanol solution. The extraction conditions of methanol concentration, IL type and amount, solid-liquid ratio, extraction temperature, and ultrasonic irradiation time were investigated in single factor experiments. Then, three factors of IL amount, solid-liquid ratio, and ultrasonic irradiation time were optimized by response surface methodology (RSM); (3) Results: The MIL [C4MIM]FeCl3Br was screened as the optimal adjuvant. Under the optimization conditions of 1.2% IL amount, 1:10.5 solid-liquid ratio, and 30 min ultrasonic irradiation time, the extraction yield reached 0.224 mg/g; and (4) Conclusions: Compared with the conventional solvent extraction, this ultrasonic assisted extraction (UAE) using methanol and MIL as adjuvants can significantly improve the extraction yield, reduce the use of methanol, and shorten the extraction time, which has the potentiality of being used in the extraction of some other important bioactive compounds from natural plant resources.

Isolation, Purification, and Identification of Taxol and Related Taxanes from Taxol-Producing Fungus Aspergillus niger subsp. taxi.

The content of taxol in the bark of yews is very low, and this is not affordable from the environmental point of view. Thus, it is a necessity to look for alternative sources of taxol production to solve its supply. Currently, a large portion of the taxol in the market comes from chemical semi-synthesis, but the semi-synthetic precursors such as baccatin III and 10-deacetyl-baccatin III are extracted from needles and twigs of yew trees. Taxol-producing fungi as a renewable resource is a very promising way to increase the scale of taxol production. Our group has obtained a taxol-producing endophytic fungus, Aspergillus niger subsp. taxi HD86-9, to examine if A. niger can produce the taxanes. Six compounds from the fermentation broth of strain HD86-9 were isolated and identified by 1H NMR, 13C NMR, and ESI-MS. The results showed that the six compounds included four taxane diterpenoids (taxol, cephalomannine, baccatin III, and 10-deacetyl-baccatin III) and two non-taxane compounds (ß-sitosterol and flavonoid isovitexin). The study verified that the taxanes can be produced by the A. niger, which is very important to taxol production via chemical semi-synthesis. Additionally, the finding is potentially very significant to solve the taxol semi-synthetic precursors extracted from needles and twigs of yew trees, and the precursor production can be easily increased through the culture condition optimization, genetic breeding, and metabolic engineering of the A. niger.

Fluorescent Immortalized Human Adipose Derived Stromal Cells (hASCs-TS/GFP+) for Studying Cell Drug Delivery Mediated by Microvesicles.

BACKGROUND: A new tool for the drug delivery is based on the use of Mesenchymal Stromal Cells (MSCs) loaded in vitro with anti-cancer drugs. Unfortunately, the restricted lifespan of MSCs represents a significant limitation to produce them in high amounts and for long time studies. Immortalized MSCs from adipose tissue (hASCs) have been generated as good source of cells with stable features. These cells could improve the development of standardized procedures for both in vitro and preclinical studies. Furthermore they facilitate procedures for preparing large amounts of secretome containing microvesicles (MVs). METHOD: We used human adipose tissue derived MSCs immortalized with hTERT+SV40 (TS) genes and transfected with GFP (hASCs-TS/GFP+). This line was investigated for its ability to uptake and release anticancer drugs. Microvesicles associated to paclitaxel (MVs/PTX) were isolated, quantified, and tested on pancreatic cancer cells. RESULTS: The line hASCs-TS/GFP+ maintained the main mesenchymal characters and was able to uptake and release, in active form, both paclitaxel and gemcitabine. From paclitaxel loaded hASCs-TS/GFP+ cells were isolated microvesicles in sufficient amount to inhibit "in vitro" the proliferation of pancreatic tumor cells. CONCLUSION: Our study suggests that human immortalized MSCs could be used for a large scale production of cells for mediated drug delivery. Moreover, the secretion of drug-associated MVs could represent a new way for producing new drug formulation by "biogenesis". In the context of the "advanced cell therapy procedure", the MVs/PTX production would use less resource and time and it could possibly contribute to simplification of GMP procedures.

COF-1-modified magnetic nanoparticles for highly selective and efficient solid-phase microextraction of paclitaxel.

Novel covalent organic framework-1 modified magnetic nanoparticles (M-COF-1) were prepared for extraction of paclitaxel from rat plasma samples. Paclitaxel is an important anti-cancer drug in clinical chemotherapy. COF-1 was immobilized on the surface of magnetic nanoparticles by bio-inspired polydopamine functionalization method because polydopamine can supply catechol groups for supporting the immobilization of COF-1. The formation of M-COF-1 has been confirmed by Fourier transform infrared spectroscopy and transmission electron microscopy. A M-COF-1-based extraction method was developed for selective extraction of paclitaxel in aqueous solutions. Extraction conditions were investigated, including sample pH value, acetonitrile content, extraction time and sample volume. By combining with HPLC, this method showed a good linear range of 0.1-200ng/mL with a low limit of detection of 0.02ng/mL. The method was also applied for the pretreatment of paclitaxel in rat plasma samples, which showed effective enrichment efficiency and good clean-up capacity. Recoveries were calculated to be 99.4-103.7% with relative standard deviations less than 2.3%.

Characterization of matrix effects in developing rugged high-throughput LC-MS/MS methods for bioanalysis.

AIM: There is an ever-increasing demand for high-throughput LC-MS/MS bioanalytical assays to support drug discovery and development. RESULTS: Matrix effects of sofosbuvir (protonated) and paclitaxel (sodiated) were thoroughly evaluated using high-throughput chromatography (defined as having a run time ≤1 min) under 14 elution conditions with extracts from protein precipitation, liquid-liquid extraction and solid-phase extraction. A slight separation, in terms of retention time, between underlying matrix components and sofosbuvir/paclitaxel can greatly alleviate matrix effects. CONCLUSION: High-throughput chromatography, with proper optimization, can provide rapid and effective chromatographic separation under 1 min to alleviate matrix effects and enhance assay ruggedness for regulated bioanalysis.