Journal of Chromatography B (v.989, #C)

Micropreparative isolation and NMR structure elucidation of metabolites of the drug candidate 1-isopropyl-4-(4-isopropylphenyl)-6-(prop-2-yn-1-yloxy) quinazolin-2(1H)-one from rat bile and urine by Joachim Blanz; Thierry Délémonté; David Pearson; Alexandre Luneau; Michael Ritzau; Werner Gertsch; Philippe Ramstein; Jérôme Dayer; Sandrine Desrayaud; Elisabeth Braun; Reiner Aichholz (1-10).
LC-MS based drug metabolism studies are effective in the optimization stage of drug discovery for rapid partial structure identification of metabolites. However, these studies usually do not provide unambiguous structural characterization of all metabolites, due to the limitations of MS-based structure identification. LC-MS-SPE-NMR is a technique that allows complete structure identification, but is difficult to apply to complex in vivo samples (such as bile collected during in vivo drug metabolism studies) due to the presence, at high concentrations, of interfering endogenous components, and potentially also dosage excipient components (e.g. polyethylene glycols). Here, we describe the isolation and structure characterization of seven metabolites of the drug development candidate 1-isopropyl-4-(4-isopropylphenyl)-6-(prop-2-yn-1-yloxy) quinazolin-2(1H)-one from a routine metabolism study in a bile-duct cannulated rat by LC-MS-SPE. The metabolites were isolated from bile and urine by repeated automatic trapping of the chromatographic peak of each metabolite on separate Oasis HLB SPE columns. The micropreparative HPLC/MS was performed on an XBridge BEH130 C18 HPLC column using aqueous formic acid/acetonitrile/methanol as mobile phase for the gradient elution. Mass spectrometric detection was performed on a LTQ XL linear ion trap mass spectrometer using electrospray ionization. Desorption of each metabolite was performed after the separation sequence. NMR spectra (1H, 13C, 2D ROESY, HSQC and HMBC were measured on a Bruker AVANCE III spectrometer (600 MHz proton frequency) equipped with a 1.7 mm 1H{13C,15N} Bruker Biospin's TCI MicroCryoProbe™.
Keywords: Biotransformation; Drug metabolism; Metabolite identification; Micropreparative isolation; HPLC-MS-SPE-NMR;

A modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method using multi-walled carbon nanotubes (MWCNTs) as a reversed-dispersive solid phase extraction (r-dSPE) material combined with ultra-high liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) was developed for the simultaneous determination of 16 novel amide fungicides in vegetables and fruits. After extraction with acetonitrile, a dSPE cleanup procedure, which was developed after the optimization of the type and amount of MWCNTs, the pH value of the extract, the extraction time for MWCNTs, and the type of eluent with MWCNTs material, was conducted. The determination of the target compounds was conducted in less than 7.0 min while the specificity is ensured through the MRM acquisition mode. The linearity of the analytical response across the studied range of concentrations (0.25–500 μg/L) was excellent, obtaining correlation coefficients higher than 0.997. The samples were quantified with the matrix matched standard solutions. The average recoveries in cabbage, celery, strawberry, and grape at three spiked levels (0.01, 0.5, and 5.0 mg/kg) were ranged from 72.4 to 98.5% with all RSDs lower than 10%. The limits of detection were below 0.003 mg/kg and the limits of quantification did not exceed 0.01 mg/kg in all matrices. The method demonstrated to be suitable for the simultaneous determination of 16 novel amide fungicides in vegetables and fruits.
Keywords: Amide fungicides; DSPE; Vegetables; Fruits; LC–MS/MS; MWCNTs;

A new method combining dispersive-solid phase purification procedure with ultra high performance liquid chromatography–triple quadrupole/linear ion trap mass spectrometry was developed for simultaneous determination of 18 preservative residues in vegetables. The new method not only had the advantages of dispersive-solid phase purification procedure such as high recoveries, easy operation, rapid analysis, little solvent usage and wide analysis range of preservatives, but also had the advantages of ultra high performance liquid chromatography–triple quadrupole/linear ion trap mass spectrometry to be operated in positive mode and negative mode simultaneously. The method was validated for the following representative matrices: radish (tuber), tomato (eggplant fruit), cabbage (leafy), cowpea (bean), cucumber (melon) and so on. Samples were extracted with hexane–ethyl acetate (1:2, v/v), and then detected by ultra high performance liquid chromatography–triple quadrupole/linear ion trap mass spectrometry after being cleaned up with dispersive-solid phase purification procedure. Significant matrix effects were compensated by using the matrix-matched calibration curves. 18 preservatives showed good linearity over the range of 5.0–100.0 μg/L with correlation coefficients of 0.9904–1.000. The limits of detections were in the range of 0.04–4.16 μg/kg and the limits of quantity were in the range of 0.13–13.85 μg/kg. The recoveries of 18 preservatives ranged from 76.0% to 120.0% with the spiked levels of 2, 4 and 10 μg/kg into homogenized vegetables, and the relative standard deviations (RSDs) ranged from 0.3% to 14.8%. Compared with the reported literatures, the method is more rapid, simple, highly sensitive, reliable and can meet testing requirements of 18 preservative residues in vegetables.
Keywords: UHPLC–QTRAP; Vegetables; Preservatives; Dispersive-SPE;

A rapid selective and sensitive liquid chromatography/tandem mass spectrometry (LC–MS/MS) method was developed for the quantitative determination of derivatised cytochrome P450-2C19 oxidation product (dansyl-4-OH mephenytoin) and its underivatised form (4-OH mephenytoin). Samples were anaysed on C18 column (Waters Xbridge, 50 mm × 4.6 mm, 3.5 μm particle size) with the mobile phase consisting of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. A gradient method with a short run time of 2.5 min and 3.5 min was developed for the analysis of dansyl-4-OH mephenytoin and 4-OH mephenytoin, respectively. The standard curve was linear (r 2  = 0.9972 for 4-OH mephenytoin; r 2  = 0.9946 for dansyl-4-OH mephenytoin) over the concentration range of 0.16 to 40 ng/mL for both derivatised and underivatised forms. The CV (%) and relative error (RE) for inter and intraassay at three QC levels for dansyl-4-OH mephenytoin was 0.97–5.85% and −9.80 to 2.51%, respectively. Whereas, for 4-OH mephenytoin the CV (%) and RE (%) at three QC levels was 0.82–3.47% and −6.69 to −0.01%, respectively. The developed method was validated for various parameters such as linearity, precision & accuracy, extraction recovery, matrix effect, autosampler stability and was proved to be consistent across three QC levels with overall CV (%) less than 15. Dansylation helped in increasing the sensitivity of hydroxy mephenytoin by 100–200 fold. Given the simplicity involved in derivatisation process, we believe that this novel methodology will change the current approaches used for the enhancing the detection sensitivity of 4-OH mephenytoin.
Keywords: 4-OH mephenytoin; CYP2C19; Dansyl chloride; LC–MS/MS; IC50; Method validation;

Dabigatran etexilate (DABE) is a low-molecular-weight prodrug that is converted after oral administration to dabigatran (DAB)—a directly acting oral anticoagulant. In this study, an LC–MSMS assay was developed and validated for the determination of DABE, free DAB and its equipotent O-glucuronide conjugates in plasma. Owing to the susceptibility of DABE and DAB to chemical hydrolysis, cleavage of the O-glucuronide moiety was carried out using β-glucuronidase enzyme. Free and total plasma concentrations of DAB were determined in incurred plasma samples before and after enzymatic cleavage (50 °C and 3 h), respectively. RP-HPLC separation was carried out using acetonitrile: water (30:70, v/v), adjusted to pH 3.0 using formic acid. Tandem mass spectrometric detection at positive electrospray ionization in the MRM mode was then employed for the determination of DABE and DAB. The analysis was carried out within 5.0 min over a linear concentration range of 1.00–600.00 ng/mL for the prodrug and its active metabolite. Validation was carried out according to FDA guidelines for bioanalytical method. The recoveries were higher than 89.48%, the accuracy was within 98.33–110.12% and the RSD was below 10% for the studied compounds in both incurred plasma and quality control samples. Results of incurred sample re-analysis and incurred sample stability revealed less than 10% variability. This indicated good assay precision and sufficient stability of target analytes in their real matrix at the employed experimental conditions. The applicability of the assay for therapeutic drug monitoring and the determination of the pharmacokinetic parameters were demonstrated.
Keywords: LC–MSMS; Enzymatic cleavage; Dabigatran; Dabigatran etexilate; β-glucuronidase;

Two microextraction techniques based on hollow fiber liquid-phase microextraction (HF-LPME) and ultrasound-assisted low-density solvent dispersive liquid–liquid microextraction (UA-LDS-DLLME) had been applied for the determination of drugs of abuse (methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, methcathinone, ketamine, meperidine, and methadone) in urine and blood samples by gas chromatography–mass spectrometry. Parameters affecting extraction efficiency have been investigated and optimized for both methods. Under the optimum conditions, linearities were observed for all analytes in the range 0.0030–10 μg/ml with the correlation coefficient (R) ranging from 0.9985 to 0.9995 for HF-LPME and in the range 0.0030–10 μg/ml with the R ranging from 0.9985 to 0.9994 for DLLME. The recovery of 79.3–98.6% with RSDs of 1.2–4.5% was obtained for HF-LPME, and the recovery of 79.3–103.4% with RSDs of 2.4–5.7% was obtained for DLLME. The LODs (S/N = 3) were estimated to be in the range from 0.5 to 5 ng/ml and 0.5 to 4 ng/ml, respectively. Compared with HF-LPME, the UA-LDS-DLLME technique had the advantages of less extraction time, suitability for batches of sample pretreatment simultaneously, and higher extraction efficiency, while HF-LPME has excellent sample clean-up effect, and is a robust and suitable technique for various sample matrices with better repeatability. Both methods were successfully applied to the analysis of drugs of abuse in real human blood sample.
Keywords: Hollow fiber liquid-phase microextraction; Ultrasound-assisted dispersive liquid–liquid microextraction; GC–MS; Drugs of abuse; Biological samples;

Four years of LC–MS/MS method for quantification of 25-hydroxyvitamin D (D2+D3) for clinical practice by Cécile Meunier; Julien Montérémal; Patrice Faure; Véronique Ducros (54-61).
Many methods for routine total plasma/serum 25-hydroxyvitamin D (25OHD) measurements are available from automated immunoassays to the most specific LC–MS/MS techniques. These last ones are nowadays numerous, still perfectible but more powerful than immunoassays in specific illnesses. We presented a robust method for simple quantification of 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) in human plasma by LC–APCI-MS/MS. This method is reliable and easy to perform for clinical measurements as we report a 4-year of clinical laboratory use. A brief off-line sample pretreatment (protein precipitation with addition of the internal standard) was realized then the supernatant was loaded into 96-well plates and analyzed by an online SPE-LC/MS/MS method on an APCI mode. 25OHD2 and 25OHD3 were both measured. The chromatographic system was thought and optimized for providing a dedicated line for this measurement on a shared instrument. The linearity was tested up to 380 nmol/L for both 25OHD2 and 25OHD3. The limit of quantification (LOQ) was 7 and 8 nmol/L for 25OHD3 and 25OHD2, respectively. Routine imprecision and bias were found in agreement with recommended limits for routine testing, CV ≤ 10% and bias ≤ 5%. Since July 2010, our participation to DEQAS was successfully validated. This simple robust online SPE-LC/MS/MS method is suitable for routine measurement of 25OHD2 and 25OHD3 in human adult plasma. The assay operates for 4 years and has performed more than 40,000 patient samples on a shared instrument.
Keywords: Vitamin D; Mass spectrometry; High throughput; Clinical practice;

LC–MS guided isolation, quantification and antioxidant evaluation of bioactive principles from Epimedium elatum by Syed Naseer; Shabir H. Lone; Javeed A. Lone; Mohd A. Khuroo; Khursheed A. Bhat (62-70).
This article presents the isolation, quantification and antioxidant evaluation of bioactive principles from Epimedium elatum. LC–MS guided isolation technique was applied for the separation of target constituents. Three isolates; magnoflorine, chrysin and dibenzylideneacetone (DBA) were isolated for the first time from E. elatum using LC–MS guided isolation method. Nine natural products, viz. icariin, epimedoside A, epimedin A, epimedin B, epimedin C, ikarisoside C, baohuoside II, magnoflorine and chrysin were simultaneously quantified by reverse phase HPLC-UV-DAD method. The HPLC method was validated in terms of precision and accuracy. Excellent specificity and linearity within test ranges for all standard calibration curves having regression coefficient of different linear equations in the range of 0.9966–0.9999 were observed. Relative recovery rates varied between 98.09 ± 0.44 and 105.34 ± 1.89% with relative standard deviation of less than 3%. This modified HPLC method is in accordance with yinyanghuo. All the 10 isolated constituents were screened for DPPH radical scavenging activity. Dibenzylideneacetone (DBA) turned out to be the most potent isolate with IC50 of 4.32 μM.
Keywords: Epimedium elatum; LC–MS; HPLC; Magnoflorine; Flavonoids; DPPH;

Capecitabine (Cape) is a prodrug that is metabolized into 5′-deoxy-5-fluorocytidine (DFCR), 5′-deoxy-5-fluorouridine (DFUR), and 5-fluorouracil (5-FU) after oral administration. A liquid chromatography–tandem mass spectrometry method for the simultaneous determination of capecitabine and its three metabolites in human plasma was developed and validated. The ex vivo conversion of DFCR to DFUR in human blood was investigated and an appropriate blood sample handling condition was recommended. Capecitabine and its metabolites were extracted from 100 μL of plasma by protein precipitation. Adequate chromatographic retention and efficient separation were achieved on an Atlantis dC18 column under gradient elution. Interferences from endogenous matrix and the naturally occurring heavy isotopic species were avoided. Detection was performed in electrospray ionization mode using a polarity-switching strategy. The method was linear in the range of 10.0–5000 ng/mL for Cape, DFCR, and DFUR, and 2.00–200 ng/mL for 5-FU. The LLOQ was established at 10.0 ng/mL for Cape, DFCR, and DFUR, and 2.00 ng/mL for 5-FU. The inter- and intra-day precisions were less than 13.5%, 11.1%, 9.7%, and 11.4%, and the accuracy was in the range of −13.2% to 1.6%, −2.4% to 2.5%, −7.1% to 8.2%, and −2.0% to 3.8% for Cape, DFCR, DFUR, and 5-FU, respectively. The matrix effect was negligible under the current conditions. The mean extraction recoveries were within 105–115%, 92.6–101%, 94.0–100%, and 85.1–99.9% for Cape, DFCR, DFUR, and 5-FU, respectively. Stability testing showed that the four analytes remained stable under all relevant analytical conditions. This method has been applied to a clinical bioequivalence study.
Keywords: Capecitabine; 5-Fluorouracil; Nucleoside metabolites; LC–MS/MS;

A simple HPLC–UV method for the determination of ciprofloxacin in human plasma by Janis Vella; Francesca Busuttil; Nicolette Sammut Bartolo; Carmel Sammut; Victor Ferrito; Anthony Serracino-Inglott; Lilian M. Azzopardi; Godfrey LaFerla (80-85).
A rapid and sensitive HPLC–UV method for the determination of ciprofloxacin in human plasma is described. Protein precipitation with acetonitrile was used to separate the drug from plasma protein. An ACE® 5 C18 column (250 mm × 4.6 mm, 5 μm) with an isocratic mobile phase consisting of phosphate buffer (pH 2.7) and acetonitrile (77:23, v/v) was used for separation. The UV detector was set at 277 nm. The method was validated in the linear range of 0.05–8 μg/ml with acceptable inter- and intra-assay precision, accuracy and stability. The method is simple and rapid and can be used to quantify this widely used antibiotic in the plasma of patients suffering from Peripheral Arterial Disease.
Keywords: HPLC–UV; Ciprofloxacin; Method development; Protein precipitation; Validation;

A HPLC-fluorescence method for the quantification of abiraterone in plasma from patients with metastatic castration-resistant prostate cancer by Tiphaine Belleville; Gaëlle Noé; Olivier Huillard; Audrey Thomas-Schoemann; Michel Vidal; François Goldwasser; Jerome Alexandre; Benoit Blanchet (86-90).
Abiraterone acetate is an oral prodrug of abiraterone, a selective inhibitor of CYP17, used for patients with metastatic castration-resistant prostate cancer (mCRPC). To date, a single liquid chromatographic–tandem mass spectroscopy method has been reported to assay abiraterone concentration in plasma from mCRPC patients. The aim of this study was to develop a simple and sensitive high performance liquid chromatographic (HPLC) method with fluorescence detection for quantification of abiraterone in plasma from mCRPC patients. After protein precipitation with acetonitrile and a liquid–liquid extraction with diethyl ether, abiraterone, and hydroxy-itraconazole (internal standard) were separated on a C8 Xterra® MS column using a mobile phase of acetonitrile and glycine buffer 88.4 mM (pH 9.0) (60:40, v/v). Samples were eluted isocratically at a flow rate of 0.9 ml/min throughout an 11-min run. Fluorescence wavelengths’ excitation and emission were 255 and 373 nm, respectively. The calibration was linear in the range 1.75–50 ng/ml. Inter- and intraday imprecision were less than 3.5 and 7%, respectively. This method is simple, sensitive, and selective. This analytical method was successfully applied to determine the steady-state plasma exposure to abiraterone in mCRPC patients. This method can be used in routine clinical practice to monitor plasma abiraterone concentrations in mCRPC patients.
Keywords: Abiraterone; HPLC; Fluorescence detection; Prostate cancer; Therapeutic drug monitoring;

A sensitive and rapid HPLC–MS/MS method for the quantitative determination of trace amount of bromocriptine in small clinical prolactinoma tissue by Qingce Zang; Yang Liu; Jiuming He; Xiaofei Yue; Ruiping Zhang; Renzhi Wang; Zeper Abliz (91-97).
Usually, insufficient intratumoral concentration of therapeutic drugs is one of the reasons for tumor treatment failure. However, little is known about intratumoral distribution of bromocriptine in non-responding prolactinomas because of extremely low drug concentration and small prolactinoma tissue samples. In this study, a sensitive, rapid and high-throughput quantitative bioanalytical method has been established by using high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) for the determination of bromocriptine at trace level in human prolactinoma tissue. As little as 20 mg (wet weight) tissue sample was required and total analysis time was 6 min in this method. The assay quantifies over a linear range of 50 fg/mg to 5 pg/mg, and has a 25 fg/mg limit of detection at a signal/noise ratio of 3. This validated method was successfully used to quantitatively determine bromocriptine in clinical post-operative bromocriptine-sensitive and -resistant prolactinomas. The results revealed bromocriptine concentration in resistant prolactinomas (0.49–1.25 pg/mg) was significantly higher than that in sensitive prolactinomas (0.057–0.47 pg/mg). These results provided direct evidence to demonstrate the reseaon for failure of bromocriptine treatment in some patients with prolactinoma was “intrinsic” tumor (cell) resistence, rather than insufficient drug concentration in tumor tissue. Additionaly, this HPLC–MS/MS method has been shown to be suitable for bromocriptine analysis in small amount tissue sample and could be adapted for therapeutic drug monitoring of other clinical medicine.
Keywords: Quantitative analytical method; HPLC–MS/MS; Bromocriptine; Prolactinoma; Drug resistance;

Determination of the metabolic profile of gentianine after oral administration to rats by high performance liquid chromatography/electrospray ionization-trap mass spectrometry by Xiuhong Wu; Shuhan Tang; Yan Jin; Shanshan Wang; Xijun Wang; Masao Hattori; Hailong Zhang; Zhigang Wang (98-103).
We investigated the metabolic fate of gentianine after oral administration to Wistar rats for the first time. Liquid chromatography/ion trap mass spectrometry detected four metabolites secogentianoxide, gentiandiol, gentianepoxide and gentianoxide in rat plasma together with the original compound gentianine. The structures of the metabolites were identified by comparing the retention times, as well as MS (mass) and MS/MS (tandem mass) spectra with those of authentic compounds, which were synthesized from gentianine or isolated from the urine. Three of the metabolites, secogentianoxide, gentianepoxide and gentianoxide, are novel compounds. The major in vivo metabolic processes associated with gentianine include N-oxide, epoxidation, dihydroxylation of double bond and hydrolysis of lactone. Gentianine together with the metabolites in plasma were quantified using gentianone as the internal standard. The mean C max of G0, G1, G2 and G3 are 425.76, 287.56, 188.45 and 85.05 ng/mL, respectively. The mean T max of G0, G1, G2 and G3 are 1.16, 3.87, 6.23 and 4.28 h, respectively. The mean T 1/2 of G0, G1, G2 and G3 are 5.23, 12.34, 7.78 and 5.64 h, respectively. A comprehensive metabolic pathway was proposed. The new metabolites may shed light on clinical efficacy of gentianine.
Keywords: Gentianine; Metabolite; LC–MS;

A highly selective, sensitive, and rapid microemulsion liquid chromatography (MELC) method was developed and validated for the simultaneous determination of a novel type of dopamine receptor antagonist LE300 and its N-methyl metabolite in mouse sera. LE300, its N-methyl metabolite, and pindolol (an internal standard) were detected using excitation and emission wavelengths of 275 and 340 nm, respectively. HPLC analysis by using a monolithic column was performed by directly injecting the sample after appropriate dilution with the microemulsion mobile phase. The chromatographic behaviour of these compounds was studied to demonstrate their chromatographic efficiency, retention, and peak symmetry. The MELC method was validated for its specificity, linearity, accuracy, precision, robustness and stability. An experimental design was used during validation to evaluate method robustness. The calibration curves in serum showed excellent linearity (r = 0.997) over concentrations ranging from 10 to 400 ng mL−1 for LE300 and 15 to 500 ng mL−1 for its N-methyl metabolite. The mean relative standard deviation (RSD) of the results of inter- and intra-day precision and accuracy of LE300 and its N-methyl metabolite were ≤5%. The overall recoveries of LE300 and its N-methyl metabolite from mouse sera were in the range 97.9–101.5% with %RSD ranging from 0.98% to 3.63%, which were in line with ICH guidelines. The assay was successfully applied in a pharmacokinetic study.
Keywords: Microemulsion liquid chromatography; Monolithic column; LE300; Serum Pharmacokinetics;

This research reports a proof-of-concept that describes an instrumental approach that is gel free and label free at both the separation and mass spectrometry ends for the capturing and identification of differentially expressed proteins (DEPs) in diseases, e.g., cancers. The research consists of subjecting/processing equalized and non-equalized (i.e., untreated) disease-free and hepatocellular carcinoma (HCC) human sera via a multicolumn platform for capturing/fractionating human serum fucome. The equalization was performed via the combinatorial peptide ligand library (CPLL) beads technology that ensured narrowing the protein concentration range, thus allowing the detection of low abundance proteins. The equalized and non-equalized disease-free and HCC sera were first fractionated online onto two lectin columns specific to fucose, namely Aleuria aurantia lectin (AAL) and Lotus tetragonolobus agglutinin (LTA) followed by the online fractionation of the lectin captured fucome by reversed phase chromatography. The online desalted fractions were first subjected to trypsinolysis and then to liquid chromatography–mass spectrometry (LC–MS/MS) analysis. In comparison with untreated serum, the CPLL treated serum is superior in terms of the total number of identified DEPs, which reflected an increased number of DEPs in a wide abundance range. The DEPs in HCC serum were found to be 70 and 40 in both LTA and AAL fractions for the serum treated by CPLL and untreated serum, respectively. In addition, the platform combined with the CPLL treatment was accomplished with virtually no sample loss and dilution as well as with no experimental biases and sample labeling when comparing the diseased-free and cancer sera using LC–MS/MS.
Keywords: Fucosylation; Glycoproteins; Hepatocellular carcinoma; Lectin affinity chromatography; Monolithic columns; ProteoMiner™;

Preparative separation and purification of fumigaclavine C from fermented mycelia of Aspergillus fumigatus CY018 by macroporous adsorption resin by Ling-Yun Yao; Yi-Xiang Zhu; Chang-Qing Liu; Rui-Hua Jiao; Yan-Hua Lu; Ren-Xiang Tan (122-128).
In this work, the separation and purification of fumigaclavine C (FC), an ergot alkaloid with strong anti-inflammatory activity from fermented mycelia of Aspergillus fumigatus was systematically evaluated. Among the eight tested resins, the non-polar resin D101 displayed the best adsorption and desorption based on of static adsorption and desorption tests. Adsorption isotherms were constructed on D101 resin and fitted well to the Freundlich model. Dynamic adsorption and desorption tests on a column packed with D101 resin have been investigated for optimization of chromatographic parameters. Under optimized conditions, the contents of FC increased from 7.32% (w/w) in the crude extract to 67.54% in the final product with a recovery yield of 90.35% (w/w) via one run. Furthermore, a lab scale-up separation was carried out, in which the FC content and recovery yield were 65.83% and 90.13%, respectively. These results demonstrated that this adsorption–desorption strategy by using D101 resin was simple and efficient, thus showing potential for large scale purification and preparation of FC in the future.
Keywords: Adsorption macroporous resin; Aspergillus fumigatus; Fumigaclavine C; Purification; Thermodynamics;