Continuous chiral separation process for high-speed countercurrent chromatography established and scaled up: A case of Voriconazole enantioseparation.

An efficient method for the continuous separation of Voriconazole enantiomers was developed using sulfobutyl ether-ß-cyclodextrin (SBE-ß-CD) as a chiral selector in high-speed countercurrent chromatography (HSCCC) with different types. The separation was performed using a two-phase solvent system consisting of n-hexane/ethyl acetate/100 mmol/L phosphate buffer solution (pH = 3.0, containing 50 mmol/L SBE-ß-CD) (1.5:0.5:2, v/v/v). A fast and predictable scale-up process was achieved using an analytical DE HSCCC instrument. The optimized parameters were subsequently applied to a preparative Tauto HSCCC instrument, resulting in consistent separation time and enantiomeric purity, with throughput boosted by a remarkable 11-fold. Preparative HSCCC successfully separated 506 mg of the racemate, delivering enantiomers exceeding 99% purity as confirmed by high-performance liquid chromatography analysis. This investigation presents an effective methodology for forecasting the HSCCC scale-up process and attaining continuous separation of chiral drugs.

Recent trends in multidimensional countercurrent chromatography.

Countercurrent chromatography (CCC) is a potent separation approach known for its remarkable efficiency and capacity in preparation. It's applied as a substitute or combined with different chromatographic techniques, resulting in its rebranding as multidimensional CCC (MDCCC). Numerous essential mixtures from natural products contain hundreds or thousands of distinct components of importance. These mix types are too complicated to separate in any reasonable time using a single CCC dimension. However, if a multidimensional technique is utilized, where a complex mixture is separated by an initial dimension, smaller fractions of that separation are gathered. Each fraction is studied individually; complex mixes can be resolved relatively quickly. Thus, several MDCCC separation features have been studied to demonstrate their advantages, limitations, and prospective capacity to separate exceedingly complex mixtures. In this review, MDCCC aspects, including principles, multiple columns system, multilayer coil J-type, on-line monitoring system, and applications, have been thoroughly_explored.

An overview of recent progress in multiple dual-mode counter-current chromatography.

Counter-current chromatography is a chromatographic separation and purification technique being developed. The development of different elution modes has significantly contributed to this field. Multiple dual-mode elution is a method developed based on dual-mode elution, which consists of a series of changing cycles of the phase role and the direction by switching between normal and reverse elution modes of counter-current chromatography. This dual-mode elution method takes full advantage of the liquid nature of stationary and mobile phases of counter-current chromatography and effectively improves the separation efficiency. So, this unique elution mode has gained extensive attention for separating complex samples. This review mainly describes and summarizes in detail its development, applications, and characteristics in recent years. Meanwhile, its advantages, limitations, and future outlook also have been discussed in this paper.

A model for continuous sample feed and separation with counter-current chromatography based on elution-extrusion mode and its application.

A novel and meaningful theoretical model was established with counter-current chromatography based on the elution-extrusion mode for efficient continuous separation. For the experimental verification of the theory, the separation of the binary mixture luteolin/baicalein was studied. The velocity model and volume model of the chromatographic separation behavior of the target compounds in the separation process were given by theoretical analysis. The results showed that this method had obvious advantages in the separation of binary mixtures. In addition, the established model was used to predict and isolate oleuropein from olive leaves. A two-phase solvent system of n-butanol/ethyl acetate/methanol/water (1:19:1:19, v/v/v/v) was chosen for the continuous separation of oleuropein. After optimizing the conditions in this way, a large amount of sample loading was achieved; the volume of injections can reach 48 ml, approximately 35.29% of the volume of the counter-current chromatography column, and oleuropein with a purity of 86.42% was obtained within 80 min. The model provided technical support for the prediction of chromatographic behavior and operating parameters during continuous separation and preparation of counter-current chromatography. It has great application prospects and significance in separation preparation, especially in large-scale industrial preparation.

Primary lesion radiotherapy during first-line icotinib treatment in EGFR-mutated NSCLC patients with multiple metastases and no brain metastases: a single-center retrospective study.

BACKGROUND: The most frequent mode of progression in the majority of non-small cell lung cancer (NSCLC) patients treated with  Epidermal growth factor - receptor tyrosine kinase inhibitors (EGFR-TKIs) is failure to respond to treatment at the primary lesion, suggesting that concurrent radiotherapy (CRT) to the primary lesion (CPRT) during first-line treatment with EGFR-TKI may be a novel therapeutic approach with a potential of additional benefit for metastatic NSCLC. Therefore, this study investigated the progression-free survival (PFS) and safety of CPRT during first-line icotinib treatment in NSCLC patients with EGFR mutations. METHODS: EGFR-mutant NSCLC patients diagnosed with limited multiple metastases were treated with first-line icotinib. The decision to treat the primary lesions with radiation largely depended on the patient's preference. The study endpoints included PFS, toxicity, progression pattern, and acquisition of the T790M mutation. RESULTS: The median PFS in the CPRT and Non-CPRT groups was 13.6 and 10.6 months (hazard ratio [HR] 0.23, 95% confidence interval [CI] 0.15-0.37, P < 0.001). Subgroup analysis showed that the results were statistically significant with 14.7 and 11.5 months for the 19del mutation (HR 0.20, 95% CI 0.10-0.40, P < 0.001) and 12.9 and 9.9 months for the L858R mutation (HR 0.25, 95% CI 0.13-0.48, P < 0.001). There were no reports of interstitial pneumonia associated with treatment at grade 4 or above. Patients who received CPRT during first-line icotinib treatment had the potential to decrease the primary lesion progression (P < 0.05) without increasing newly metastatic lesions (P > 0.05). The proportion of acquired T790M mutations was 26.7% and 45.7% in both groups (P > 0.05). CONCLUSION: This study suggests that CPRT is a viable option for patients with EGFR-sensitive mutations in NSCLC with limited multiple metastases during first-line icotinib treatment, which can significantly improve PFS with acceptable toxicities. Data on progression patterns and T790M mutations suggest the need to further investigate the benefits of radiation treatment from a molecular perspective.

Comprehensive separation of a wide variety of compounds from olive leaves by counter-current chromatography with three-phase solvent system.

The three-phase solvent system counter-current chromatography has been of great research interest, because it can separate compounds with a wide range of polarity. The solvent system of n-hexane/methyl tert-butyl ether/acetonitrile/water (5:5:7:5, v/v) was used for counter-current chromatographic comprehensive separation of olive leaves. The study adopted the normal elution mode. The middle phase and the lower phase (at a volume ratio of 7:3) were pumped into the column simultaneously, followed by eluting with the upper, middle, and lower phases in sequence. The retention rate of the stationary phase measured by the experiment was 73.5%. The upper phase was used to elute the nonpolar compounds, then the mobile phase was switched to the middle phase to elute the moderately hydrophobic compounds, finally, the polar compounds were eluted by the lower phase remaining in the chromatographic column. This method successfully separated eight compounds in one step within 270 min and five compounds were identified. The logP values of these five compounds were 7.44, 7.86, 4.16, -0.11, and 0.96, respectively, covering a wide range of polarities. The present study demonstrated that the three-phase solvent has a strong extraction capacity for ingredients from extremely hydrophilic compounds to extremely hydrophobic compounds.

Role of three-phase solvent system in the counter-current chromatography.

In counter-current chromatography, the separation efficiency greatly depends on the partitioning ability of the separated substance between the stationary phase and the mobile phase. Partitioning ability is mainly represented by the parameter partition coefficient which is one of the important parameters to evaluate the separation effect of counter-current chromatography. The scope of the partition coefficient value mainly depends on the solvent system. A suitable solvent system election is, therefore, a critical role in the separation of counter-current chromatography. The existing solvent systems that are widely used are mainly two-phase solvent systems. It is difficult to decide on an appropriate solvent system for the separation of compounds with a wide polarity range, which promotes the development of the three-phase solvent system in counter-current chromatography. This review mainly described the origin, development history of three-phase solvent system, summarized the volume ratios and volume fractions of the upper, middle, and lower phases of nearly 50 three-phase solvent systems, their advantages, and disadvantages in counter-current chromatography. In addition, the challenges and future perspectives on three-phase solvent systems in counter-current chromatography also are discussed in this review.

Establishment and verification of a nomogram to predict tumor-specific mortality risk in triple-negative breast cancer: a competing risk model based on the SEER cohort study.

Background: Triple-negative breast cancer (TNBC) is the subtype of breast cancer with the worst prognosis, and traditional survival analysis methods are biased when predicting mortality. To predict the risk of death in patients with triple-negative breast cancer more precisely, a competing risk model was developed. Methods: The clinicopathological data of the TNBC patients from 2010 to 2015 were collected from the Surveillance, Epidemiology, and End Results (SEER) database. The data were assigned into a training set and testing set at a ratio of 7:3 in a randomized pattern. Univariate and multivariate competing risk models were applied to find the independent prognostic factors. A prediction nomogram for cancer-specific mortality (CSM) risk was constructed. The accuracy and discrimination of the nomogram were assessed using receiver operating characteristic (ROC) area under the curve (AUC), concordance index (C-index), and a calibration curve using the training and testing sets, respectively. Results: A total of 28,430 TNBC patients were randomly grouped into the training set (n=19,900) and the testing set (n=8,530). The median time for follow-up was 59 [1-107] months. A total of 7,014 (24.67%) patients died, among whom 4,801 (68.45%) died from breast cancer and 2,213 (31.55%) due to non-breast cancer events. The independent risk factors were primary site of tumor, grade, tumor-node-metastasis (TNM) stage, T stage, approach of surgery, chemotherapy, axillary lymph node metastases, brain metastases, and liver metastases. The prediction nomogram was constructed by using the aforementioned variables. The 1-, 3-, and 5-year AUC of CSM were predicted to be 0.856, 0.81, and 0.782, respectively, in the training set, and 0.856, 0.81, and 0.782 in the testing set, respectively. The C-index of the nomogram was 0.801 and 0.799 in the training and testing sets, respectively. As confirmed by the validation and training calibration curves, the nomogram was consistent with the results. Conclusions: We used competing risk models to identify risk factors for CSM and constructed a CSM risk prediction nomogram for TNBC patients, that may be utilized to predict CSM risk in TNBC patients clinically and assist in the creation of individualised clinical treatment options.

Development of an effective method based upon second-order overlapping repeated sample injections for isolation of carotenoids from Lycium barbarum L. fruits with elution-extrusion counter-current chromatography.

Carotenoids are one of the main active components in Lycium barbarum L. fruit, which has a wide range of excellent biological activities. In this study, a novel second-order overlapping repeated injection method with elution-extrusion counter-current chromatography was developed for isolation and preparation of carotenoids from L. barbarum fruits. And three carotenoids were successfully separated using the solvent system composed of n-hexane/dichloromethane/acetonitrile (10:3.5:6.5, v/v) with the injection before equilibrium method. The entire separation process consisted of three complete elution-extrusion cycles with a total of 9 injections (80 mg crude extract per injection). Finally, three target compounds including zeaxanthin (28.5 mg), zeaxanthin monopalmitate (45.8 mg), and zeaxanthin dipalmitate (161.5 mg) with average purities of 87.9%, 88.9%, and 91.2% were successfully obtained in one complete second-order overlapping repeated elution-extrusion CCC process within 651 min. The result indicated that this second-order overlapping repeated method is efficient for large-scale preparation of carotenoids based on its advantages of large amount of sample injection and low solvent consumption. So this novel second-order overlapping repeated elution-extrusion counter-current chromatography separation method has enormous potential for largely preparative separation of natural bioactive compounds, such as carotenoids, which have good biological activity but possess unstable or other special chemical structure. It is worth noting that this overlapping repeated injections method requires target compounds to meet the requirements of elution-extrusion counter-current chromatography, and the normal implementation of this method is closely related to the sufficient interval of elution time between the target compounds.

Recovery and recycling of solvent of counter-current chromatography: The sample of isolation of zeaxanthin in the Lycium barbarum L. fruits.

An efficient method of recovering and recycling solvent for counter-current chromatography was established by which zeaxanthin was separated from Lycium barbarum L. fruits. A column with activated carbon combined with high performance counter-current chromatography formed the recovering and recycling solvent system. Using the solvent system of n-hexane-ethyl acetate-ethanol-water (8:2:7:3, v/v) from the references, five injections were performed with an almost unchanged purity of zeaxanthin (80.9, 81.2, 81.5, 81.3, and 80.2% respectively) in counter-current chromatography separation. Meanwhile, the mobile phase reduced by half than conventional counter-current chromatography. By this present method, an effective improvement of counter-current chromatography solvent utilization was achieved.

Chiral counter-current chromatography: A survey of its instrument, mechanism, procedure, and applications.

The chiral separation by counter-current chromatography has made great progress in the past three decades. It has become increasingly popular in the field of chiral separation, and many applications have been introduced during the last years. This review mainly focuses on the current topics, applications, and trends in chiral separation by counter-current chromatography. It contains the development of modern counter-current chromatography apparatus, theory of counter-current chromatography, overview of applications of chiral counter-current chromatography enantioseparation, its current situation, and challenges. At last, some conclusions and perspectives also have been discussed in this review.

Full use of the liquid nature of the stationary phase: The development of elution-extrusion counter current chromatography.

Elution-extrusion counter current chromatography extrudes the most solute retained in the column with the highest possible peak resolution. It can greatly improve the hydrophobic window. In recent years, elution-extrusion counter current chromatography has received extensive attention in the separation of complex samples. This article first reviews the development and application of elution-extrusion counter current chromatography, including its origin, mechanism, advantages and disadvantages, and some representative applications. At the same time, this review also shared our visions and ideas on how to improve the elution-extrusion mode. This article aims to provide certain reference for the research of this technology.

The applicability of high-speed counter current chromatography to the separation of natural antioxidants.

Antioxidants play an essential role in human health, as they have been found to be capable of lowering the incidence of many diseases, such as cancer and angiocardiopathy. Currently, more attention is paid to natural antioxidants because of the possible insecurity of synthetic antioxidants. Thus, the development of efficient techniques or methods to separate antioxidants from natural sources is requested urgently. High-speed counter current chromatography (HSCCC) is a unique support-free liquid-liquid chromatographic technique and has been widely applied in the field of separation of natural products. In this review, we summarize and analyze the related researches on the application of HSCCC in the separation of various natural antioxidants so far. The purpose of the article is to provide a certain theoretical support for the separation of natural antioxidants by HSCCC, and to make full use of advantages of HSCCC in the separation of bioactive components. In particular, some key problems associated with the separation strategies, the structural categories of natural antioxidants, solvent system choices, and the application of different elution modes in HSCCC separation, are summarized and commented. We expect that the content reviewed can offer more evidence for the development of the field of natural antioxidants separation, so as to achieve large-scale preparation of natural antioxidants.

Effective on-line high-speed shear dispersing emulsifier technique coupled with high-performance countercurrent chromatography method for simultaneous extraction and isolation of carotenoids from Lycium barbarum L. fruits.

An efficient combination strategy based on high-speed shear dispersing emulsifier technique and high-performance countercurrent chromatography was developed for on-line extraction and isolation of carotenoids from the fruits of Lycium barbarum. In this work, the high-speed shear dispersing emulsifier technique has been employed to extract crude extracts using the upper phase of high-performance countercurrent chromatography solvent system composed of n-hexane-dichloromethane-acetonitrile (10:4:6.5, v/v) as the extraction solvent. At the separation stage, the high-performance counter-current chromatography process adopts elution-extrusion mode and the upper phase of the solvent system as stationary phase (reverse-phase mode). As a result, three compounds including zeaxanthin, zeaxanthin monopalmitate, and zeaxanthin dipalmitate with purities of 89, 90, and 93% were successfully obtained in one extraction-separation operation within 120 min. The targeted compounds were analyzed and identified by high-performance liquid chromatography, mass spectrometry, and NMR spectroscopy. The results indicated that the present on-line combination method could serve as a simple, rapid, and effective way to achieve weak polar and unstable compounds from natural products.

Aqueous two-phase systems based on deep eutectic solvents and their application in green separation processes.

As a new environmentally friendly separation technology, deep eutectic solvent based aqueous two-phase systems are extensively applied in various fields. Herein, we review recent advances in this field and highlight the possible directions of future developments. This article focuses on the effects of deep eutectic solvent and inorganic salts on the phase equilibrium, the microstructure of deep eutectic solvent based aqueous two-phase systems, the applications of deep eutectic solvent based aqueous two-phase systems in separation (proteins, biopolymers, saponins, and organic acids), and removal and recovery technologies for deep eutectic solvent from aqueous two-phase systems.

Continuous separation of maslinic and oleanolic acids from olive pulp by high-speed countercurrent chromatography with elution-extrusion mode.

In this work, a continuous high-speed countercurrent chromatography method has been developed on the basis of elution-extrusion mode and this method was successfully applied to the separation of maslinic and oleanolic acid from the extract of olive pulp. In the process of 'elution', the sample solution was continuously loaded into the column and the maslinic acid was steadily eluted out in this step while highly retained oleanlic acid always stayed in the column. In the process of 'extrusion', the oleanlic acid was pushed out of the column with the stationary phase. In this way, we achieved a large sample loading. A total of 120 mL sample solution (about 89.55% of the column volume) which contains 600 mg olive pulp extract was pumped in the apparatus by a constant-flow pump and the maslinic and oleanolic acids were largely separated within 120 min. Both of these two compounds presented high yields and high purities (271.6 mg for maslinic acid with 86.7% and 83.9 mg oleanolic acids with 83.4%).

Counter-current chromatography melamine-modified column and its separation mechanism.

In this work, to further verify and develop the novel counter-current chromatography modified column separate mode, a melamine modified counter-current chromatography column was prepared. Meanwhile, the modified counter-current chromatography column was used to separate stevioside and rebaudioside A with the same partition coefficient in chosen solvent system to evaluate its separation efficiency. The results show that because of the presence of intermolecular forces between melamine and model compounds, better separation could be achieved on the modified column while it's almost impossible to be separated on the unmodified column. So the results of this research further show that column modified method is a possible approach to further increase the separation ability of counter-current chromatography. Take advantage of large sample handing capacity of counter-current chromatography, the mothed may have great potential to be an efficient method of separation and preparation enantiomer compounds.

Isolation of high-purity peptide Val-Val-Tyr-Pro from Globin Peptide using MCI gel column combined with high-speed counter-current chromatography.

Peptides have gained increased interest over the past several decades because of their therapeutics. In this research, a strategy combining MCI gel column chromatography and high-speed countercurrent chromatography was developed for the separation of high-purity peptide Val-Val-Tyr-Pro from Globin Peptide. First, the fraction of Val-Val-Tyr-Pro mixtures with a purity of 15.8% was obtained by using MCI gel column with a mixture of ethanol/water (20:80, v/v/v). Then, the high-purity Val-Val-Tyr-Pro was separated by high-speed countercurrent chromatography with a aqueous two phase systems of ethanol/acetonitrile/iso-propyl alcohol/(NH4 )2 SO4 Saturated solution /H2 O (0.5:0.5:0.25:1.5:0.7,v/v). The ammonium sulfate from high-speed countercurrent chromatography fractions was removed from target compound by MCI gel column chromatography using ethanol/water in stepwise elution mode. A 78 mg of Val-Val-Tyr-Pro was successfully purified with the purities of 98.80% from 30 g crude Globin Peptide. The amino acid sequence of the Val-Val-Tyr-Pro was determined by electrospray ionization high resolution tandem mass spectrometry. The method presents a practical strategy for the large-scale separation of pure peptide Val-Val-Tyr-Pro from Globin Peptide, and provides a reference method for obtaining high-purity peptide from other polypeptide mixtures.

Hydroxychloroquine induced lung cancer suppression by enhancing chemo-sensitization and promoting the transition of M2-TAMs to M1-like macrophages.

BACKGROUND: Lysosome-associated agents have been implicated as possible chemo-sensitizers and immune regulators for cancer chemotherapy. We investigated the potential roles and mechanisms of hydroxychloroquine (HCQ) in combination with chemotherapy in lung cancer treatment. METHODS: The effects of combined treatment on non-small cell lung cancer (NSCLC) were investigated using cell viability assays and animal models. The influence of HCQ on lysosomal pH was evaluated by lysosomal sensors and confocal microscopy. The effects of HCQ on the tumour immune microenvironment were analysed by flow cytometry. RESULTS: HCQ elevates the lysosomal pH of cancer cells to inactivate P-gp while increasing drug release from the lysosome into the nucleus. Furthermore, single HCQ therapy inhibits lung cancer by inducing macrophage-modulated anti-tumour CD8+ T cell immunity. Moreover, HCQ could promote the transition of M2 tumour-associated macrophages (TAMs) into M1-like macrophages, leading to CD8+ T cell infiltration into the tumour microenvironment. CONCLUSIONS: HCQ exerts anti-NSCLC cells effects by reversing the drug sequestration in lysosomes and enhancing the CD8+ T cell immune response. These findings suggest that HCQ could act as a promising chemo-sensitizer and immune regulator for lung cancer chemotherapy in the clinic.

A simple gradient equilibrium method for better separation in countercurrent chromatography.

The stationary phase retention is one of the most important parameters in countercurrent chromatography. In this work, a simple gradient equilibrium method was developed to further improve the stationary phase retention based on the optimized condition in the traditional equilibrium model. Meanwhile, this novel gradient equilibrium method was used to separate three flavone model compounds and compared with the conventional isocratic equilibrium method to evaluate the separation efficiency. The results show that better resolution or shorter separation time could be achieved with gradient equilibrium compared to isocratic equilibrium. So this novel equilibrium method has enormous potential for obtaining a better separation or saving the separating time in the preparative separation of target compounds.