Detection of trace components in Xiangdan injection of Dalbergia odorifera based on microextraction and back-extraction along with bar-form-diagram strategy.

Xiangdan Injection are commonly used traditional Chinese medicine formulations for the clinical treatment of cardiovascular diseases. However, the trace components of Dalbergia odorifera in Xiangdan Injection pose a challenge for evaluating its quality due to the difficulty of detection. This study proposes a technology combining dispersive liquid-liquid microextraction and back-extraction (DLLME-BE) along with Bar-Form-Diagram (BFD) to address this issue. The proposed combination method involves vortex-mixing tetradecane, which has a lower density than water, with the sample solution to facilitate the transfer of the target components. Subsequently, a new vortex-assisted liquid-liquid extraction step is performed to enrich the components of Dalbergia odorifera in acetonitrile. The sample analysis was performed on HPLC-DAD, and a clear overview of the chemical composition was obtained by integrating spectral and chromatographic information using BFD. The combination of BFD and CRITIC-TOPSIS strategies was used to optimize the process parameters of DLLME-BE. The determined optimal sample pre-treatment process parameters were as follows: 200 µL extraction solvent, 60 s extraction time, 50 µL back-extraction solvent, and 90 s back-extraction time. Based on the above strategy, a total of 29 trace components, including trans-nerolidol, were detected in the Xiangdan Injection. This combination technology provides valuable guidance for the enrichment analysis of trace components in traditional Chinese medicines.

Modeling pKa of the Brønsted Bases as an Approach to the Gibbs Energy of the Proton in Acetonitrile.

A simple but efficient computational approach to calculate pKa in acetonitrile for a set of phosphorus, nitrogen, and carbon bases was established. A linear function that describes relations between the calculated ΔG'a.sol(BH+) and pKa values was determined for each group of bases. The best model was obtained through the variations in the basis set, in the level of theory (density functionals or MP2), and in the continuum solvation model (IPCM, CPCM, or SMD). The combination of the IPCM/B3LYP/6-311+G(d,p) solvation approach with MP2/6-311+G(2df,p)//B3LYP/6-31G(d) gas-phase energies provided very good results for all three groups of bases with R2 values close to or above 0.99. Interestingly, the slopes and the intercepts of the obtained linear functions showed significant deviations from the theoretical values. We made a linear plot utilizing all the conducted calculations and all the structural variations and employed methods to prove the systematic nature of the intercept/slope dependence. The interpolation of the intercept to the ideal slope value enabled us to determine the Gibbs energy of the proton in acetonitrile, which amounted to -258.8 kcal mol-1. The obtained value was in excellent agreement with previously published results.

The separation regularity of the three-phase solvent system of counter-current chromatography based on polarity parameter modeling.

The three-phase solvent system of counter-current chromatography can separate compounds with a wide range of polarity, but there is no study of its separation regularity. Therefore, in this work, the separation regularity of the three-phase solvent system was initially investigated from the perspective of solvent polarities and compound polarities. The standard compounds covering a wide polarity range were selected, and three-phase solvent systems, n-hexane/methyl acetate/acetonitrile/water, and n-hexane/methyl tert-butyl ether/acetonitrile/water were used for modeling. The results showed that in the three-phase solvent system, the partition coefficient for the middle and lower phases (lgKM/L) increased with increasing logP values in three intervals logP < 0, 0 < logP < 4, and logP > 4. In addition, the partition coefficient for the upper and middle phases (lgKU/M) between the upper and middle phases of the small polarity compounds increases with increasing logP values. LogP vs lgKM/L of 7 solvent systems were employed for the smoothing spline fit through a predictive model design of the curve fitting toolbox in MATLAB software, and good results were achieved. LogP versus lgKM/L for n-hexane/methyl tert-butyl ether/acetonitrile/water solvent systems were used for the second-order power fit, and satisfactory results were obtained. The relationship between polarity parameters and separation case parameters was explored using a heat map approach. The separation regularity of the three-phase solvent system was preliminarily investigated. This regularity study gives hope of assistance to the chemists studying three-phase solvents and counter-current chromatography.

A practical and fast isolation of 12 cyclolinopeptides (linusorbs) from flaxseed oil via preparative HPLC with phenyl-hexyl column.

Linusorbs, known as cyclolinopeptides, are a group of cyclic hydrophobic peptides derived from flaxseed oil with various health benefits. However, the current research efforts on both the biological activities and antioxidant capacities of linusorbs are limited because of existing issues with their purification and characterization. A practical method based on preparative HPLC for isolating 12 linusorbs simultaneously was developed and factors such as the solvent selection, gradient elution program, flow rate, loaded mass, and loading concentration, were optimized. The optimum conditions were an initial acetonitrile (ACN) to water ratio of 40%, final ACN ratio of 80%, eluting time of 21 min, a flow rate of 16 mL/min, sample load of 12.5 mg, and concentration of 80 mg/mL (in methanol). The 12 linusorbs obtained were verified using off-line MS/MS, recording purities of above 95.5%. The method could serve as a practical and fast isolation method enabling further investigation of minor linusorbs.

A novel sample-preparation method for the generic and rapid determination of pesticides and mycotoxins in tea by ultra-performance liquid chromatography-tandem mass spectrometry.

A rapid, simple, and generic analytical method for the simultaneous determination of 140 undesirable low-weight pesticides and mycotoxins from different chemical classes in black tea was developed. The method involved swelling the sample in ammonium acetate buffer, extraction with acetonitrile-dimethyl sulfoxide, cleanup by dual dispersive solid-phase extraction (D-SPE) with the assistance of low-temperature centrifugation, and analysis by ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry using multiple reaction monitoring mode. The interferences in the extract were eliminated by the combination of dual d-SPE using only C18 sorbent and anhydrous magnesium sulfate, which maintained the chromatographic column under the ideal condition for a long time and enabled satisfactory recoveries of hydrophobic and hydrophilic analytes simultaneously. Matrix-matched calibration curves were obtained for most target compounds with linear regression coefficients above 0.9900. The limits of quantification (LOQs) ranged within 0.5-10.0 µg/kg, which were usually sufficient to verify the compliance of products with legal tolerances. Satisfactory recoveries of 64.5%-138.1% were obtained in black ta samples with the relative standard deviation (RSD) values between 1.8 and 25.9%. The inter-day precision ranged within 2.2%-24.9%. For over 90% of the analytes, the recoveries were between 70% and 120%, with RSD values below 15.0%. The application of this method in routine monitoring programs can drastically reduce effort and time.

Establishment of a UPLC-PDA/ESI-Q-TOF/MS-Based Approach for the Simultaneous Analysis of Multiple Phenolic Compounds in Amaranth (A. cruentus and A. tricolor).

We used ultraperformance liquid chromatography coupled with a photodiode-array detector and electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-PDA/ESI-Q-TOF/MS) to rapidly and accurately quantify 17 phenolic compounds. Then, we applied this method to the seed and leaf extracts of two Amaranthus species to identify and quantify phenolic compounds other than the 17 compounds mentioned above. Compounds were eluted within 30 min on a C18 column using a mobile phase (water and acetonitrile) containing 0.1% formic acid, and the specific wavelength and ion information of the compounds obtained by PDA and ESI-Q-TOF/MS were confirmed. The proposed method showed good linearity (r2 > 0.990). Limits of detection and quantification were less than 0.1 and 0.1 µg/mL, respectively. Intra- and interday precision were less than 2.4% and 1.8%, respectively. Analysis of amaranth seed and leaf extracts using the established method showed that the seeds contained high amounts of 2,4-dihydroxybenzoic acid and kaempferol, and leaves contained diverse phenolic compounds. In addition, six tentatively new phenolic compounds were identified. Moreover, seeds potentially contained 2,3-dihydroxybenzaldehyde, a beneficial bioactive compound. Thus, our method was an efficient approach for the qualitative and quantitative analysis of phenolic compounds, and could be used to investigate phenolic compounds in plants.

Combination of dispersive solid phase extraction with solidification organic drop-dispersive liquid-liquid microextraction based on deep eutectic solvent for extraction of organophosphorous pesticides from edible oil samples.

A dispersive solid phase extraction method was combined with deep eutectic solvent-based solidification of floating organic drop-dispersive liquid-liquid microextraction and used for the extraction/preconcentration of some organophosphorus pesticides residues from edible oil samples. The extracted analytes were quantified with gas chromatography-nitrogen phosphorous detector. In this procedure, the sample lipids are saponified with a sodium hydroxide solution and then the analytes are adsorbed onto a primary secondary amine sorbent. After that the analytes are desorbed with acetone as an elution/dispersive solvent and mixed with choline chloride: 3,3-dimethyl butyric acid deep eutectic solvent and the mixture is rapidly dispersed into deionized water. Then, the obtained cloudy solution is centrifuged and placed into an ice bath. The extraction solvent is solidified on the top of the solution. Finally, it is removed and dissolved in acetonitrile, and 1 µL of the solution is injected into the separation system. Validation of the method showed that limits of detection and quantification were in the ranges of 0.06-0.24 and 0.20-0.56 ng mL-1, respectively. Enrichment factors and extraction recoveries of the analytes ranged from 170-192 and 68-77%, respectively. The method had an acceptable precision with relative standard deviations less than ≤9.2% for intra- (n=6) and inter-day (n=6) precisions at four concentrations (3, 10, 50, and 250 ng mL-1, each analyte). Finally the method was used for determination of the analytes in five edible oil samples.

Bioassay-Guided Separation of Centipeda minima Using Comprehensive Linear Gradient Centrifugal Partition Chromatography.

A comprehensive linear gradient solvent system for centrifugal partition chromatography (CPC) was developed for the bioassay-guided isolation of natural compounds. The gradient solvent system consisted of three different ternary biphasic solvents types: n-hexane-acetonitrile-water (10:2:8, v/v), ethyl acetate-acetonitrile-water (10:2:8, v/v), and water-saturated n-butanol-acetonitrile-water (10:2:8, v/v). The lower phase of the n-hexane-acetonitrile-water (10:2:8, v/v) was used as the stationary phase, while its upper phase, as well as ethyl acetate-acetonitrile-water (10:2:8), and water-saturated n-butanol-acetonitrile-water (10:2:8, v/v) were pumped to generate a linear gradient elution, increasing the mobile phase polarity. We used the gradient CPC to identify antioxidant response elements (AREs), inducing compounds from Centipeda minima, using an ARE-luciferase assay in HepG2 cells, which led to the purification of the active molecules 3-methoxyquercetin and brevilin A. The developed CPC solvent systems allow the separation and isolation of compounds with a wide polarity range, allowing active molecule identification in the complex crude extract of natural products.

Development and Validation of Liquid Chromatography-Tandem Mass Spectrometry Method for Pharmacokinetic Evaluation of 7ß-(3-Ethyl-cis-crotonoyloxy)-1α-(2-methylbutyryloxy)-3,14-dehydro-Z-notonipetranon in Rats.

Recently, potent neuroprotective and anti-diabetic effects of 7ß-(3-Ethyl-cis-crotonoyloxy)-1α-(2-methylbutyryloxy)-3,14-dehydro-Z-notonipetranone (ECN), a sesquiterpenoid isolated from Tussilago farfara Linnaeus, have been elucidated. To facilitate further pre-clinical evaluation in rats, an analytical method for the determination of ECN in rat plasma was developed and optimized by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasma samples were pretreated by the protein precipitation method with an acetonitrile solution of losartan (LST) as the internal standard. Chromatographic separation was performed using a an Octadecyl-silica (ODS) column (2.6 µm, 100 x 4.6 mm) in the isocratic mode. The mobile phase, comprising 10 mM ammonium formate in water pH 5.75) and acetonitrile (11:89, v/v), was eluted at a flow rate of 0.4 mL/min. Mass spectrometric detection was performed in the multiple reaction monitoring mode with positive electrospray ionization, and the mass transitions of ECN and LST were m/z 431.3 to 97.3 and m/z 423.1 to 207.2, respectively. The calibration curves of spiked plasma samples were linear in the 10.0-10,000 ng/mL range (r2 > 0.996). The lower limit of quantification (LLOQ) was determined as 10.0 ng/mL. Validation was conducted in the LLOQ, and three quality control (QC) sample levels (10.0, 25.0, 3750, and 7500 ng/mL) were studied. Among them, the relative standard deviation for the within- and between-run precisions was under 9.90%, and the relative error of the accuracies was within the -8.13% to 0.42% range. The validated method was successfully employed to investigate the pharmacokinetic properties of ECN in rats, which revealed the linear pharmacokinetic behavior of ECN for the first time.

Rapid simultaneous determination of pentacyclic triterpenoids by mixed-mode liquid chromatography-tandem mass spectrometry.

Pentacyclic triterpenoids (PCTs) possess high biological activity, including antitumor, anti-inflammatory, antiviral and hepatoprotective properties and are widespread in a plant biomass. Due to significant differences in polarity and other physicochemical properties, the simultaneous determination of different classes of PCTs by the methods of reversed phase liquid chromatography is difficult. In the present study, we proposed a new approach to chromatographic separation of such compounds based on the use of a stationary phase with a mixed retention mechanism combining hydrophobic, weak anion exchange and hydrophilic interactions. The use of the Acclaim Mixed-Mode WAX-1 column and tuning the selectivity by changing the contributions of different types of analyte-stationary phase interactions allowed the separation of 10 PCTs (betulin, erythrodiol, uvaol, friedelin, lupeol, ß-amyrin, α-amyrin, betulinic, oleanolic and ursolic acids) belonging to four different classes (monools, diols, ketones and triterpenic acids) during 7.5 min in isocratic elution mode. The combination of this approach with atmospheric pressure chemical ionization tandem mass spectrometric detection and pressurized liquid extraction of analytes with methanol allowed to develop a rapid, accurate and highly sensitive method for analyzing PCTs in plant tissues with a total duration of the analytical cycle (including sample preparation steps) of not more than 40 min. It provides the detection limits in plant biomass extracts of 3-12 µg L-1 (44 µg L-1 for friedelin). The developed method was validated and successfully tested in the analyses of real birch bark and lingonberry peels.

A novel and sensitive HILIC-CAD method for glucosamine quantification in plasma and its application to a human pharmacokinetic study.

The clinical effect of glucosamine, the most widely used supplement in patients with osteoarthritis, on joint pain and function improvement, is reported to be inconsistent. Inter-patient variability in the pharmacokinetics of glucosamine, especially its oral absorption, could contribute to the inconsistent clinical outcomes. To test this hypothesis, a novel but simple Hydrophilic Interaction Liquid Chromatography coupled with Charged Aerosol Detector method was developed and validated. The sample was prepared by simple protein precipitation and analysed using an amino column and acetonitrile:100 mM ammonium formate with gradient elution. The developed method was linear (12.5-800 ng/mL, r2 = 0.999) and the relative standard deviations for intra- and inter-day accuracy, precision and repeatability were all less than 6%. The sensitivity of the method (lower limit of quantitation; 12.5 ng/mL) allowed the quantification of endogenous and exogenous glucosamine levels in 12 patients with osteoarthritis, taking 1500 mg glucosamine daily. The analysis showed 120-fold variation (81.7% variance) in exogenous glucosamine levels among the patients, indicating that substantial variability in the extent of absorption and/or rate of elimination could be a possible cause for the reported inconsistent clinical outcomes. The newly-developed method was sensitive and can be used to study the pharmacokinetics of glucosamine.

Determination of Cyanotoxins and Phycotoxins in Seawater and Algae-Based Food Supplements Using Ionic Liquids and Liquid Chromatography with Time-Of-Flight Mass Spectrometry.

An analytical procedure is proposed for determining three cyanotoxins (microcystin RR, microcystin LR, and nodularin) and two phycotoxins (domoic and okadaic acids) in seawater and algae-based food supplements. The toxins were first isolated by a salting out liquid extraction procedure. Since the concentration expected in the samples was very low, a dispersive liquid-liquid microextraction procedure was included for preconcentration. The ionic liquid 1-hexyl-3-methylimidazolium hexafluorophosphate (80 mg) was used as green extractant solvent and acetonitrile as disperser solvent (0.5 mL) for a 10 mL sample volume at pH 1.5, following the principles of green analytical chemistry. Liquid chromatography with electrospray ionization and quadrupole time of flight-mass spectrometry (LC-Q-TOF-MS) was used. The selectivity of the detection system, based on accurate mass measurements, allowed the toxins to be unequivocally identified. Mass spectra for quadrupole time of flight-mass spectrometry (Q-TOF-MS) and Q-TOF-MS/MS were recorded in the positive ion mode and quantification was based on the protonated molecule. Retention times ranged between 6.2 and 17.9 min using a mobile phase composed by a mixture of methanol and formic acid (0.1%). None of the target toxins were detected in any of the seawater samples analyzed, above their corresponding detection limits. However, microcystin LR was detected in the blue green alga sample.

Analysis of Evodiae Fructus by capillary electrochromatography-mass spectrometry with methyl-vinylimidazole functionalized organic polymer monolilth as stationary phases.

Evodiae Fructus is used as a traditional Chinese medicine for the treatment of several kinds of diseases with its bioactive constituents. In this study, a capillary electrochromatography-mass spectrometry (CEC-MS) method was developed to determine three bioactive compounds including evodiamine, rutaecarpine and limonin in Evodiae Fructus fruit. Home-developed monolithic columns with methyl-vinylimidazole functionalized organic polymer monolilth as stationary phases were used in CEC-MS with excellent separation selectivity and high efficiency. The CEC-MS methods provided 4-16 folds improvement of LODs when compared with CEC-UV method. The conditions, which could affect separation efficiency and detection sensitivity, were optimized. Under optimum conditions, baseline separation with high detection sensitivity was obtained. The method showed good linearity (R2 >0.99) of 0.8-160 µg mL-1 with low limits of detection of 0.15-0.31 µg mL-1. Relative standard deviations of migration time and relative peak areas were <13.89%. Recoveries of evodiamine, rutaecarpine and limonin in Evodiae Fructus fruit were tested and calculated, which ranged from 102% to 113%. Finally, the three bioactive compounds in Evodiae Fructus herb samples from different regions were analyzed and studied. It has been demonstrated that the developed method has great potential for quality control of Evodiae Fructus herb.

Reversed-phase liquid chromatography system constant database over an extended mobile phase composition range for 25 siloxane-bonded silica-based columns.

The solvation parameter model is used to characterize the retention properties of 25 siloxane-bonded type-B silica columns under typical reversed-phase liquid chromatography conditions with binary aqueous mobile phases containing 10-70% (v/v) methanol and acetonitrile. To further explore the affect of solvent type on column selectivity complementary information is presented for aqueous phases containing 10-70% (v/v) tetrahydrofuran for two columns. Columns were selected to include examples of all common column packing morphologies for small molecule separations (totally porous particles, superficially porous particles, organic-inorganic hybrid particles, and a monolith) and common topologies octadecylsiloxane-bonded (including phases with a polar-embedded group, mixed-mode phases with a short-chain siloxane-bonded polar functional group, sterically crowded, positive shield, and fluorine-containing phases); octylsiloxane-bonded; and various phenyl-containing stationary phases with different linker arms, pentafluorophenylalkyl and biphenyl groups. The column properties are reported as a system constant database and as system maps for the full range of mobile phase compositions. Selectivity differences are evaluated using principal component factor analysis to classify the columns into selectivity groups and correlation plots of the system constants for compared columns at all mobile phase compositions. The later is shown to be a suitable technique to identify (near) selectivity equivalent columns and varied columns suitable for method development. Contributions to retention from steric resistance and cation-exchange interactions not parameterized in the solvation parameter model are identified for columns and conditions where they may be important. It is shown that the interaction parameters employed in the hydrophobic-subtraction model have little overlap with those of the solvation parameter model and a quantitative comparison of column properties delineated by both models is not possible.

Efficient separation determination of protopanaxatriol ginsenosides Rg1, Re, Rf, Rh1, Rg2 by HPLC.

An efficient and flexible protocol was developed for simultaneous detection of the ginsenosides Rg1, Re, Rf, Rh1, and Rg2 in ginseng extract. In the analysis of white ginseng that contains no ginsenoside Rf, separation of the remaining ginsenosides was achieved within just 10 min under isocratic chromatographic conditions. For the analysis of red ginseng that contains ginsenoside Rf as a characteristic constituent, the gradient elution conditions were optimized. The method is based on high-pressure liquid chromatography employing a Shiseido UG 80 Capcell Pak NH2(4.6 mm I.D. × 250 mm, 5 µm)column and isocratic elution using acetonitrile (A) and water(B) in a ratio of 76: 24 (v/v), The optimal gradient elution conditions are as follows: 0-3 min, 89% A, 3-25 min, 89-84% A, 25-30 min, 84-82% A, 30-35 min, 82-76% A, then returning to 89% solvent A in 5 min. The flow rate was 0.80 mL min-1. The column temperature was set at 25℃ and the detection wavelength was at 203 nm. The working concentration ranges for ginsenoside Re, Rh1, Rg2, Rg1, and Rf were 0.23-1450.0 mg• L-1, 0.05-1130.0 mg• L-1, 0.11-687.0 mg• L-1, 0.051-1325 mg• L-1, and 0.55-800.0 mg• L-1, respectively. The method was validated for linearity, precision, and accuracy. And the further confirmation of five ginsenosides was conducted by QTOF-MS. Analysis of raw extracts of ginseng, white ginseng, and red ginseng for the five components showed satisfactory recovery.

An enhanced sensitivity and cleanup strategy for the nontargeted screening and targeted determination of pesticides in tea using modified dispersive solid-phase extraction and cold-induced acetonitrile aqueous two-phase systems coupled with liquid chromatography-high resolution mass spectrometry.

At present, matrix interferences in tea are still a great challenge for analysis of multi-pesticide residues. Herein, a simple sample preparation method was developed based on the modified dispersive solid-phase extraction (DSPE) procedure and cold-induced acetonitrile aqueous two-phase systems (ATPS). In modified DSPE procedure, polyvinylpolypyrrolidone (PVPP) was selected as the matrix dispersive adsorbent to remove polyphenols from the tea with the removal rate of >98.3%. Using cold-induced acetonitrile ATPS, caffeine of the tea extract was sharply reduced by 81.2%, and a 0.798-3.167 of enrichment factor for the representative pesticides was achieved. In liquid chromatography-high resolution mass spectrometry analysis, a full scan/data independent acquisition approach was used for the nontargeted screening and targeted determination of pesticides. In data analysis, an in-house database of pesticides was constructed, and a simple accurate mass calibration method was used to correct the accurate mass variation, which was conducive to reduce the number of false detects.

Determination of kaurenoic acid in rat plasma using UPLC-MS/MS and its application to a pharmacokinetic study.

Kaurenoic acid (KA), a kaurane diterpene found in several medicinal plants, is an active ingredient with potential anti-inflammatory, anticonvulsant, antibacterial and antitumor activities. In this work, an ultra-performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) was firstly developed and validated to quantify kaurenoic acid in rat plasma. Rhein was chosen as the internal standard (IS) and the plasma was processed with one-step acetonitrile protein precipitation; the chromatographic separation was achieved on a HSS T3 (2.1 × 50 mm, 1.8 µm) column with the mobile phase consisting of acetonitrile and water containing 0.1% formic acid via gradient elution. An electrospray ionization source was applied and operated in the negative ion and multiple reaction monitoring (MRM) modes. Kaurenoic acid and IS were quantified using the transitions of m/z 301.2→301.2 (pseudo MRM) and m/z 283.2 → 238.9, respectively. The calibration curves were linear over the range of 5∼ 100 ng/mL (R2 = 0.990). The lower limit of quantification (LLOQ) was 5 ng/mL. The intra- and inter- day precision (RSD) ranged from 3.0% to 11.4%. The matrix effect and extraction recovery were within acceptable limits. The validated method was successfully applied to the pharmacokinetic study of kaurenoic acid in rats after oral administration at three dosages.

Development and validation of RP-HPLC method for the determination of stigmasterol in the botanical extract of Ficus deltoidea.

A simple, rapid, accurate and precise RP-HPLC method was developed for the determination of stigmasterol in botanical extract of Ficus deltoidea. Separation was achieved with acetonitrile and acetic acid in water (75:25% v/v) in isocratic mode at 210 nm. Single sharp peak of standard stigmasterol was detected at retention time 3.17 min which overlay with the peak of plant extract at 3.14 min. The calibration curve was found to be linear in a concentration range of 2-10 µg/ml with correlation coefficient of 0.998. The LOD and LOQ were found to be 1.50 µg/ml and 4.55 µg/ml respectively. Accuracy and precision was determined with overall recovery of 99.6-100.1% for stigmasterol and RSD values in both intra-day and inter-day repeatability assay lesser than 0.340%, respectively. The robustness study also indicated that there is no influence of minor changes in detecting wavelength and flow rate of mobile phase on the response.

Effect of inorganic salt on partition of high-polarity parishins in two-phase solvent systems and separation by high-speed counter-current chromatography from Gastrodia elata Blume.

Parishins are high-polarity and major bioactive constituents in Gastrodia elata Blume. In this study, the effect of several inorganic salts on the partition of parishins in two-phase solvent systems was investigated. Adding ammonium sulfate, which has a higher solubility in water, was found to significantly promote the partition of parishins in the upper organic polar solvents. Based on the results, a two-phase solvent system composed of butyl alcohol/acetonitrile/near-saturated ammonium sulfate solution/water (1.5:0.5:1.2:1, v/v/v/v) was used for the purification of parishins by high-speed counter-current chromatography. Fractions obtained from high-speed counter-current chromatography were subjected to semi-preparative high-performance liquid chromatography to remove salt and impurities. As a result, parishin E (6.0 mg), parishin B (7.8 mg), parishin C (3.2 mg), gastrodin (15.3 mg), and parishin A (7.3 mg) were isolated from water extract of Gastrodia elata Blume (400 mg). These results demonstrated that adding inorganic salt that has high solubility in water to the two-phase solvent system in high-speed counter-current chromatography was a suitable approach for the purification of high-polarity compounds.

The Salt-Stress Response of the Transgenic Plum Line J8-1 and Its Interaction with the Salicylic Acid Biosynthetic Pathway from Mandelonitrile.

Salinity is considered as one of the most important abiotic challenges that affect crop productivity. Plant hormones, including salicylic acid (SA), are key factors in the defence signalling output triggered during plant responses against environmental stresses. We have previously reported in peach a new SA biosynthetic pathway from mandelonitrile (MD), the molecule at the hub of the cyanogenic glucoside turnover in Prunus sp. In this work, we have studied whether this new SA biosynthetic pathway is also present in plum and the possible role this pathway plays in plant plasticity under salinity, focusing on the transgenic plum line J8-1, which displays stress tolerance via an enhanced antioxidant capacity. The SA biosynthesis from MD in non-transgenic and J8-1 micropropagated plum shoots was studied by metabolomics. Then the response of J8-1 to salt stress in presence of MD or Phe (MD precursor) was assayed by measuring: chlorophyll content and fluorescence parameters, stress related hormones, levels of non-enzymatic antioxidants, the expression of two genes coding redox-related proteins, and the content of soluble nutrients. The results from in vitro assays suggest that the SA synthesis from the MD pathway demonstrated in peach is not clearly present in plum, at least under the tested conditions. Nevertheless, in J8-1 NaCl-stressed seedlings, an increase in SA was recorded as a result of the MD treatment, suggesting that MD could be involved in the SA biosynthesis under NaCl stress conditions in plum plants. We have also shown that the plum line J8-1 was tolerant to NaCl under greenhouse conditions, and this response was quite similar in MD-treated plants. Nevertheless, the MD treatment produced an increase in SA, jasmonic acid (JA) and reduced ascorbate (ASC) contents, as well as in the coefficient of non-photochemical quenching (qN) and the gene expression of Non-Expressor of Pathogenesis-Related 1 (NPR1) and thioredoxin H (TrxH) under salinity conditions. This response suggested a crosstalk between different signalling pathways (NPR1/Trx and SA/JA) leading to salinity tolerance in the transgenic plum line J8-1.