Journal of Chromatography B (v.995-996, #C)
Editorial Board (i).
Pharmacokinetics of eupalinolide A, eupalinolide B and hyperoside from Eupatorium lindleyanum in rats by LC/MS/MS by Jun Zhang; Fei Zhao; Xiaoling Yu; Xiang Lu; Guofeng Zheng (1-7).
A simple, selective, and sensitive LC/MS/MS method was developed and validated for simultaneous determination of eupalinolide A, eupalinolide B, and hyperoside in rat plasma. Plasma samples were processed by protein precipitation with acetonitrile. The three analytes, together with internal standard (IS, lysionotin), were separated on a Venusil MP-C18 column (50 mm × 2.1 mm, 3 μm) using a mobile phase of methanol and 10 mM ammonium acetate (45:55, v/v) with isocratic elution. Mass spectrometric detection was performed by multiple-reaction monitoring mode via electrospray ionization source. Linear calibration curves were obtained for the following concentration range: 1.28–640 ng/mL for EA; 1.98–990 ng/mL for EB; and 2.00–1000 ng/mL for HYP. The intra- and inter-day precision was less than 10.25%, and the accuracy was between 89.16% and 110.63%. The extraction recovery of the analytes and IS from rat plasma was above 88.75%. The validated method has been successfully applied to pharmacokinetic studies of the three analytes following intragastric administration of Eupatorium lindleyanum extract at a single dose of 100, 250, and 625 mg/kg to Sprague-Dawley rats, respectively. The pharmacokinetic results may help to better understand the pharmacological actions of the herb E. lindleyanum.
Keywords: Eupalinolide; Hyperoside; Eupatorium lindleyanum; LC/MS/MS; Pharmacokinetic study;
Microemulsion liquid chromatographic method for simultaneous separation and determination of six flavonoids of Apocynum venetum leaf extract by Rui juan Song; Jun Zhou (8-14).
A simple, cost-effective, and efficient method was developed for the rapid simultaneous separation and determination of six flavonoids (rutin, hyperoside, quercetin-3-O-sophoroside, isoquercitrin, astragalin and quercetin) of Apocynum venetum leaf extract by reversed phase high performance liquid chromatography using a microemulsion system mixture as the mobile phase. Separations were performed on the Zorbax Extend-C18 column with UV detection at 360 nm. The flow rate was 0.8 mL min−1. The optimized microemulsion mobile phase consisted of 2.5% (v/v) n-butanol, 1.2% (v/v) of Genapol X-080, 0.5% (v/v) ethyl acetate and 95.8% (w/v) of aqueous 20 mM phosphoric acid, pH adjusted to 6.0 with 0.3% triethylamine. Under the optimized conditions, the calibration curve for six flavonoids was linear in the range of 5–1000 μg mL−1 with the correlation coefficients greater than 0.9994. The intra-day and inter-day precision (RSD) were below 8.11% and the limits of detection (LOD) for the six flavonoids were 1.7–6.0 μg mL−1 (S/N = 3). The microemulsion liquid chromatography (MELC) method was successfully applied to separate and determine the six flavonoids of A. venetum leaf extract.
Keywords: Apocynum venetum; Flavonoids; Microemulsion;
Simultaneous determination of amino acid and monoamine neurotransmitters in PC12 cells and rats models of Parkinson’s disease using a sensitizing derivatization reagent by UHPLC–MS/MS by Xian-En Zhao; Shuyun Zhu; Hongmei Yang; Jinmao You; Fengrui Song; Zhiqiang Liu; Shuying Liu (15-23).
Display OmittedMulti-analytes simultaneous monitoring of amino acid and monoamine neurotransmitters (NTs) has important scientific significance for their related pathology, physiology and drug screening. In this work, in virtue of a mass spectrometry sensitizing reagent 10-ethyl-acridone-3-sulfonyl chloride (EASC) as derivatization reagent, an Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC–MS/MS) method was developed and validated for simultaneous determination of six amino acid NTs, two monoamine ones and its one metabolite. The simple and rapid derivatization reaction was innovatively combined with plasma preparation by using EASC acetonitrile solution as protein precipitant. This interesting combination brought the advantages of speediness, simpleness and high-throughput in a cost-effective way. Under the optimized conditions, LODs (0.004–3.80 nM) and LOQs (0.014–13.3 nM) of EASC derivatized-NTs were calculated and found to be significantly lower than those of direct UHPLC–MS/MS detection about 11.5–275.0 and 14.4–371.4 times, respectively. Moreover, EASC derivatization significantly improved chromatographic resolution and matrix effect when compared with direct UPLC–MS/MS detection method without derivatization. Meanwhile, it also brought acceptable precision (3.0–13.0%, peak area CVs%), accuracy (86.4–112.9%), recovery (88.3–107.8%) and stability (3.8–8.5%, peak area CVs%) results. This method was successfully applied for the antiparkinsonian effect evaluation of levodopa and Ginsenoside Rg1 using PC12 cells and rats models by measuring multiple NTs. This provided a new method for the NTs related studies in the future.
Keywords: Neurotransmitter; Derivatization; Parkinson’s disease; Plasma; PC12Cell; Ginsenoside Rg1;
Simultaneous determination of acetylpuerarin and puerarin in rat plasma by liquid chromatography–tandem mass spectrometry: Application to a pharmacokinetic study following intravenous and oral administration by Deqing Sun; Aiying Xue; Jing Wu; Bin Zhang; Jinlong Yu; Qiang Li; Chao Sun (24-30).
A rapid and sensitive liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed and validated for the simultaneous determination of acetylpuerarin (AP) and its major metabolite puerarin (PUE) in rat plasma using genistein as the internal standard (IS). Plasma samples were pretreated by protein precipitation with a mixture of methanol and acetonitrile. Chromatographic separation was performed on a CAPCELL PAK C18 MGШ column with a mixture of 0.1% formic acid in water and methanol (35:65, v/v) as the mobile phase. The analytes were detected using a tandem mass spectrometer in the positive ionization and multiple-reaction monitoring mode. The ion transition of m/z 669.4 → 627.3, 417.5 → 297.6 and 271.3 → 153.0 was utilized to quantify AP, PUE and the IS, respectively. The calibration curves showed good linearity over the plasma concentration range of 1–2000 ng/mL for AP and 2.5–5000 ng/mL for PUE. The intra- and inter-day precisions (RSD %) for each analyte were less than 6.91%, and the accuracies ranged from −2.17% to 2.93%. The validated LC–MS/MS method was further successfully applied to a pharmacokinetic study of AP and PUE in rats following intravenous and oral administration.
Keywords: Acetylpuerarin; Puerarin; LC–MS/MS; Pharmacokinetics;
Optimization of derivatization procedure and gas chromatography–mass spectrometry method for determination of bensulfuron-methyl herbicide residues in water by Yan Zhang; Jian Wang; Guoping Wang; Chuanyu Gao; Yan Yan; Bolong Wen (31-37).
A simple and efficient technique based on liquid phase extraction with CH2Cl2 solvent followed by derivatization with (C2H5)2O·BF3 solution and confirmation analysis with GC–MS analytical method was developed for detecting the bensulfuron-methyl (BSM) residues in water. Box-Behnken response surface methodology was employed for optimization of the derivatization efficiency. According to the optimization model, the derivatization time of 45 min, derivatization temperature at 55 °C and 0.2 mL (C2H5)2O·BF3 solvent were selected as the optimal derivatization condition for obtaining the maximum desirability of response. Method validation was performed at 6 working standard levels (0.05, 0.1, 0.2, 0.5, 1.0, 5.0 μg/mL) and the linearity of the calibration curve was linear well over the 6 fortification levels with the squared correlation coefficient of determination r 2 = 0.998 and the LOD was found to be 0.1 μg/L for BSM herbicide. The mean value of BSM was detected from 0.0414 to 4.7542 μg/mL at levels from 0.05 to 5 μg/mL with the recoveries remained at the acceptable level (42.8–95.0%) with the RSD values from 3.5% to 6.2%, which is more accptable and desirable than the results obtained by LC methods. Moreover, the method allowed the determination of BSM residue in real paddy field water samples at concentrations between 0.0902 and 3.4605 μg/L. Average recovery rates of the BSM spiked at levels 0.1, 0.2, 0.5, 1.0 μg/mL into thirty water samples ranged from 74.1% and 94.1% with the relative standard derivation (RSD) values from 1.9% to 6.7%.
Keywords: Bensulfuron-methyl; Water; Derivatization; Response surface methodology; GC–MS analysis;
Three-phase molecularly imprinted sol–gel based hollow fiber liquid-phase microextraction combined with liquid chromatography–tandem mass spectrometry for enrichment and selective determination of a tentative lung cancer biomarker by Mohammad Mahdi Moein; Mehran Javanbakht; Mohammad Karimi; Behrouz Akbari-adergani; Mohamed Abdel-Rehim (38-45).
In the present study, the modification of a polysulfone hollow fiber membrane with in situ molecularly imprinted sol–gel process (as a novel and one-step method) was prepared and investigated. 3-(propylmethacrylate)trimethoxysilane (3PMTMOS) as an inorganic precursor was used for preparation of molecularly imprinted sol–gel. The modified molecularly imprinted sol–gel hollow fiber membrane (MSHM) was used for the liquid-phase microextraction (LPME) of hippuric acid (HA) in human plasma and urine samples. MSHM as a selective, robust, and durable tool was used for at least 50 extractions without significant decrease in the extraction efficiency. The non-molecularly imprinted sol–gel hollow fiber membrane (NSHM) as blank hollow fiber membrane was prepared by the same process, only without HA. To achieve the best condition, influential parameters on the extraction efficiency were thoroughly investigated. The capability of this robust, green, and simple method for extraction of HA was successfully accomplished with LC/MS/MS. The limits of detection (LOD) and quantification (LOQ) in human plasma and urine samples were 0.3 and 1.0 nmol L−1, respectively. The standard calibration curves were obtained within the concentration range 1–2000 nmol L−1 for HA in human plasma and urine. The coefficients of determination (r 2) were ≥0.998. The obtained data exhibited recoveries were higher than 89% for the extraction of HA in human plasma and urine samples.
Keywords: Molecularly imprinted polymers; Sol–gel; Hollow fiber membrane; Hippuric acid; Liquid phase microextraction; Mass spectrometry;
Development and validation of an UFLC–MS/MS assay for the absolute quantitation of nine notoginsenosides in rat plasma: Application to the pharmacokinetic study of Panax Notoginseng Extract by Lijun Zhou; Rong Xing; Lin Xie; Tai Rao; Qian Wang; Wei Ye; Hanxu Fu; Jingcheng Xiao; Yuhao Shao; Dian Kang; Guangji Wang; Yan Liang (46-53).
Notoginsenosides, the main active gradients of Chinese traditional medicine Panax notoginseng, possesses a variety of biological activities including antioxidant property, anti-hyperglycemic, anti-obese, etc. However, pharmacokinetic evaluation for notoginsenosides is still a formidable task due to their low concentrations and complex components in vivo. The summation of this work generated a rapid and sensitive method for quantitative analysis of multi-notoginsenoside in rat plasma based on ultra fast liquid chromatographic-tandem mass spectrometric. After liquid–liquid extraction by n-butanol, notoginsenoside R1, Rg3, Rd, Rg2, Rb2, Rf, Rg1, Rb1 and Re were simultaneously monitored in negative ionization mode after separating on a Thermo ODS C18 column (5 mm 50 mm × 2.1 mm) by a binary gradient elution, and all compounds were analyzed within 9 min. Multiple reaction monitoring (MRM) was performed as follows: R1 (m/z 967.7 → 637.4), Rg3 (m/z 819.6 → 621.4), Rd (m/z 981.6 → 783.5), Rg2 (m/z 819.6 → 475.4), Rb2 (m/z 1113.4 → 783.4), Rf (m/z 835.6 → 475.4), Rg1 (m/z 835.6 → 637.6), Rb1 (m/z 1143.7 → 945.6), Re (m/z 981.6 → 637.4), internal standard (digoxin, m/z 815.5 → 779.4). Validation parameters (linearity, sensitivity, intra-and inter-assay precision and accuracy, recovery and matrix effect) were within acceptable ranges and biological extracts were stable during the entire storing and preparing process. This UFLC–MS/MS approach was further validated by being applied to the pharmacokinetic study for P. Notoginseng extract in rats, and the pharmacokinetic parameters were calculated by Winolin software. Thus, the presently developed methodology was simple, robust, accurate, precise, and would be useful for the pharmacokinetic studies for all kinds of notoginsenosides and other herbal saponins.
Keywords: Panax Notoginseng extract; Notoginsenosides; UFLC–MS/MS; Pharmacokinetics;
Quantification of β-hydroxymethylbutyrate and leucine by ultrahigh performance liquid chromatography tandem mass spectrometry at different situations and stages of a rodent life by A. Santos-Fandila; P. Bueno-Vargas; A. Zafra-Gómez; J.M. López-Pedrosa; M. Ramírez (54-63).
The main objective of this work was to develop a method to measure Leucine (Leu) and β-hydroxymethylbutyrate (HMB) at basal levels in serum, urine, milk and brain microdialysates in rats. Ultrahigh performance liquid chromatography–electrospray-tandem mass spectrometry (UHPLC-ESI-MS/MS) was used as analytical technique. The sample treatment was simple and consisted of dilution with methanol and centrifugation for serum and urine, dilution with water and filtration with an Amicon filter for milk, and treatment with formic acid with no further dilution for microdialyzates. The procedures for sampling and the UHPLC-MS/MS parameters were accurately optimized to achieve the highest recoveries and to enhance the analytical characteristics of the method. For chromatographic separation, an Acquity UPLC BEH Amide column using acetonitrile-water gradient with formic acid as additive was used. The total run time was 4 min. The analytical characteristics (accuracy, selectivity and sensitivity) of the proposed method were evaluated. The limits of detection (LODs) obtained ranged from 0.4 to 7 ng mL−1 and the limits of quantification (LOQs) from 1 to 22 ng mL−1. Precision, expressed as relative standard deviation (% RSD), was lower than 15% in all cases, and the determination coefficient (R 2) was equal or higher than 99.0% with a residual deviation for each calibration point lower than ±25%. Mean recoveries were between 85 and 115%. The method was successfully applied to these matrices being able to detect significant differences between physiological situations, strains and stages of life.
Keywords: UHPLC–ESI–MS/MS; β-Hydroxymethylbutyrate; Leucine; Biological fluids;
Analysis of α-amylase inhibitor from corni fructus by coupling magnetic cross-linked enzyme aggregates of α-amylase with HPLC–MS by Liangliang Liu; Yin Cen; Fang Liu; Jingang Yu; Xinyu Jiang; Xiaoqing Chen (64-69).
As a carrier-free immobilization strategy, magnetic cross-linked enzyme aggregates (MCLEAs) showed improved enzyme activity, stability and magnetic response. In this study, MCLEAs of α-amylase (MCLEAs–amylase) was prepared under optimized conditions and characterized with scanning electron microscope and vibrating sample magnetometer. The prepared MCLEAs–amylase showed an amorphous structure and the saturation magnetization was 33.5 emu/g, which was sufficient for magnetic separation. Then MCLEAs–amylase coupled with high performance liquid chromatography–mass spectrometry (HPLC–MS) was utilized to screen and identify α-amylase inhibitors from ethyl acetate extract of corni fructus. The experiment conditions were optimized. At the optimum conditions (incubation time: 10 min, pH: 7.0 and temperature: 20 °C), querciturone was successfully screened and identified with weak non-specific binding. The screening result was verified by inhibition assays and the IC50 value of querciturone was 22.5 μg/mL. This method provided a rapid way to screen active compounds from natural products.
Keywords: α-Amylase; Corni fructus; Cross-linked enzyme aggregates; HPLC–MS; Magnetic;
Analysis of caffeine and paraxanthine in human saliva with ultra-high-performance liquid chromatography for CYP1A2 phenotyping by Nan Yeun Jordan; Jolet Y. Mimpen; Willie J.M. van den Bogaard; Frits M. Flesch; Michiel H.M. van de Meent; Javier Sastre Torano (70-73).
Cytochrome P450 1A2 (CYP1A2) plays an important role in drug metabolism. Caffeine (CAF) is converted into paraxanthine (PX) by this enzyme and is used as a xenobiotic substrate to determine the CYP1A2 phenotype in humans. A method for the quantification of CAF and PX in saliva was developed using liquid–liquid extraction with ethyl acetate and analysis with ultra-high-performance liquid chromatography. Peaks from CAF, PX and internal standard were resolved within 6 min. The method was validated from 0.05 to 5 μg mL−1 CAF and 0.025–2.5 μg mL−1 PX. Inter- and intra-day accuracies ranged from 91.2 to 107.2% with precisions <13.5%. The limits of detection were 0.16 and 0.63 ng mL−1 for PX and CAF, respectively. PX/CAF concentration ratios from volunteers were 0.26–1.09 with mean ratios of 0.78 ± 0.26 and 0.38 ± 0.10 for regular and light/non-coffee drinkers, respectively.
Keywords: Caffeine; Paraxanthine; CYP1A2; Phenotyping; Saliva; UHPLC;
A comprehensive quantification method for eicosanoids and related compounds by using liquid chromatography/mass spectrometry with high speed continuous ionization polarity switching by Masaki Yamada; Yoshihiro Kita; Takahiro Kohira; Kenji Yoshida; Fumie Hamano; Suzumi M. Tokuoka; Takao Shimizu (74-84).
Fatty acids and related metabolites, comprising several hundreds of molecular species, are an important target in disease metabolomics, as they are involved in various mammalian pathologies and physiologies. Selected reaction monitoring (SRM) analysis, which is capable of monitoring hundreds of compounds in a single run, has been widely used for comprehensive quantification. However, it is difficult to monitor a large number of compounds with different ionization polarity, as polarity switching requires a sub-second period per cycle in classical mass spectrometers. In the present study, we developed and evaluated a comprehensive quantification method for eicosanoids and related compounds by using LC/MS with high-speed continuous ionization polarity switching. The new method employs a fast (30 ms/cycle) continuous ionization polarity switching, and differentiates 137 targets either by chromatography or by SRM transition. Polarity switching did not affect the lower limits of quantification, which ranged similarly from 0.5 to 200 pg on column. Lipid extracts from mouse tissues were analyzed by this method, and 65 targets were quantitatively detected in the brain, including 6 compounds analyzed in the positive ion mode. We demonstrated that a fast continuous ionization polarity switching enables the quantification of a wide variety of lipid mediator species without compromising the sensitivity and reliability.
Keywords: Eicosanoid; Lipid mediator; Quantification; LC–MS; Lipidomics;
Tentative identification of new metabolites of cnidilin by liquid chromatography–mass spectrometry by Haoli Huo; Peipei Jia; Xiaoxu Zhang; Zhiyong Zhang; Haotian Yang; Qiaoyue Zhang; He Shi; Lantong Zhang (85-92).
Cnidilin is one of the major bioactive constituents of Radix Angelicae dahuricae. The present study was designed to characterize and interpret the structures of metabolites in rats after oral administration of cnidilin at a dose of 48 mg/kg body weight. Metabolite identification was accomplished using a predictive multiple reaction monitoring-information-dependent acquisition-enhanced product ion (pMRM-IDA-EPI) scan and precursor ion scan-information-dependent acquisition-enhanced product ion (PREC-IDA-EPI) scan in positive ion mode. Comparing the changes in protonated molecular masses, MS/MS spectra and retention times with the parent drug, 18 metabolites were identified. The result shows that the metabolic pathways contain deisopentenyl, combination with glucose, hydroxylation, oxidation, demethylation and addition reaction. The study identified 18 metabolites, analyzed and summarized the fragmentation regularities of mass spectra of 8-methoxy-5-hydroxy psoralen. The study provides a new pathway to discovery new compounds, new fragmentation regularities and the mode of metabolites.
Keywords: Cnidilin; HPLC–ESI-MS; Identification; Metabolites;
Simple and sensitive GC/MS-method for the quantification of urinary phenol, o- and m-cresol and ethylphenols as biomarkers of exposure to industrial solvents by T. Schettgen; A. Alt; P. Dewes; T. Kraus (93-100).
We have developed and validated a simple and sensitive method for the determination of urinary phenol as well as the urinary metabolites of toluene and ethylbenzene in one analytical run. After enzymatic hydrolysis for the cleavage of conjugates overnight, the analytes are extracted from the matrix with a liquid–liquid extraction procedure using toluene as solvent under acidic conditions. The analytes are then derivatised to volatile ethers using N,O-bis(trimethylsilyl) trifluoroacetamid (BSTFA) for cresols and ethylphenols as well as N-tert-butyldimethylsilyl-N-methyltrifluoroacetamid (MTBSTFA) for the determination of phenol. Separation and detection was carried out using capillary gas chromatography with mass-selective detection (GC–MS). Deuterium-labeled o-cresol served as internal standard for the quantification of all metabolites and guaranteed good accuracy of the results. No matrix effects were observed in the quantification of the analytes. The limit of detection for o- and m-cresol and 2- and 4-ethylphenol was 10 and 20 μg/l urine and linearity ranged up to 3 and 12 mg/L urine, respectively. The limit of detection for urinary phenol was 0.5 mg/L with a linear range up to 200 mg/L. The relative standard deviation of the within-series imprecision ranged between 3.0 and 7.2% at two spiked concentrations of 60 and 400 μg/l and the relative recovery was between 84 and 104%, depending on the analyte. The method was successfully applied in proficiency testing for urinary o-cresol and phenol.This method was used for the analysis of urine samples of 17 non-smoking and 13 smoking persons from the general population without known exposure to solvents. Smokers showed a significantly higher excretion of o-cresol (median: 23 vs. 33 μg/l), m-cresol (median: 43 vs. 129 μg/l) as well as 4-ethylphenol (median: 25 vs. 124 μg/l). Especially excretion of 4-ethylphenol was significantly correlated to smoking habits. The method seems to be suitable for biological monitoring of low-level solvent exposures and allows determination of background values in the general population.
Keywords: Biological monitoring; Solvents; General population; Urine; Toluene; Ethylbenzene;
Determination of phytochemicals, antioxidant activity and total phenolic content in Andrographis paniculata using chromatographic methods by Marzanna Kurzawa; Anna Filipiak-Szok; Ewa Kłodzińska; Edward Szłyk (101-106).
Antioxidant activity, total phenolics content and selected phytochemicals (alkaloids and andrographolides) were determined in Andrographis paniculata and in dietary supplements containing this plant. Antioxidant activity was measured by FRAP, CUPRAC and DPPH procedures and ranged from 503.36 to 6164.09 μmol TE/100 g d.m. depending on methods, part of plant and kind of dietary supplement. The total phenolics (175.13–1723.79 mg GAE/100 g) and andrographolides content (19.44–85.13 mg/g) in the studied samples were correlated with antioxidant activities determined by CUPRAC, FRAP and DPPH (r > 0.95, p < 0.05 level). Purine alkaloids: caffeine, theobromine, theophylline and indole alkaloids: harmine, harmane, harmol, yohimbine, brucine and strychnine were detected in the studied samples by different chromatographic techniques (HPLC–DAD, LC–MS/MS, GC–MS). The total alkaloids content in APs-roots and APs-leaves varies from 50.71 ± 0.36 mg/g d.m. to 78.71 ± 0.48 mg/g d.m., respectively, whereas for dietary supplements (Pn and DK) TAC was found between 19.52 ± 0.15 mg/g and 22.18 ± 0.15 mg/g d.m.. The highest concentration of andrographolides was found in A. paniculata leaves, whereas the lowest in dietary supplement Pn. Moreover principal component analysis, cluster analysis and one-way ANOVA follow by Duncan’s tests were also performed.
Keywords: Andrographis paniculata; Phytochemicals; Antioxidants; Alkaloids; Andrographolides; LC–MS/MS; GC–MS; HPLC–DAD;
Improving impurities clearance by amino acids addition to buffer solutions for chromatographic purifications of monoclonal antibodies by Takashi Ishihara; Mareto Hosono (107-114).
The performance of amino acids in Protein A affinity chromatography, anion exchange chromatography and cation exchange chromatography for monoclonal antibody purification was investigated. Glycine, threonine, arginine, glutamate, and histidine were used as buffer components in the equilibration, washing, and elution steps of these chromatographies. Improved clearance of impurity, high molecular weight species (HMW) and host cell proteins (HCP) was observed in the purification processes when using the amino acids as base-buffer constituents, additives or eluents compared with that of buffers without these amino acids. In addition, we designed a buffer system in which the mobile phases were composed of only a single amino acid, histidine, and applied it to the above three chromatographies. Effective HMW and HCP clearance was also obtained in this manner. These results suggest that amino acids may enhance impurity clearance during the purification of monoclonal antibodies.
Keywords: Protein separation; Biopharmaceutical; Cell cultures; Chromatography resins;