Biomedical Chromatography

Leng, Guangyi; Zuo, Yanhua; Hu, Jiahui; Yu, Fei; Liu, Wanhui

doi: 10.1002/bmc.4912pmid: 32496589

Fulvestrant (‘Faslodex’), an estrogen receptor antagonist, is available for the treatment of advanced breast cancer. The oil‐based vehicle of Faslodex can lead to various adverse effects. A novel fulvestrant microcrystal (aqueous suspension) was developed in this study to eliminate these adverse effects. A sensitive and robust liquid chromatography tandem mass spectrometry method was developed and validated for the determination of fulvestrant in rat plasma using supported‐liquid extraction. The separation of fulvestrant was achieved on an Agilent SB‐C18 column (2.1 × 50 mm, 3.5 μm) with isocratic elution using fulvestrant‐d3 as internal standard. Mass spectrometric detection was conducted in negative multiple reaction monitoring mode. Ion transitions were at m/z 605.5 → 427.5 for fulvestrant and m/z 608.5 → 430.5 for fulvestrant‐d3. The excellent linearity was demonstrated over the range 0.05–100.0 ng/ml (r2 = 0.99). The lower limit of quantitation was 0.05 ng/ml, which was superior to that reported in literature The method validation was evaluated by selectivity, accuracy, precision, recovery and matrix effect in agreement with the US Food and Drug Administration guidance. The method was successfully applied to a pharmacokinetic study of a novel fulvestrant microcrystal in rats after intramuscular administration. It revealed that the rate of absorption increases and the extent of absorption is constant with a decrease in microcrystal diameter.

Ghafari, Javad; Vahabi, Masoomeh; Dehghan, Somayeh Farhang; Zendehdel, Rezvan

doi: 10.1002/bmc.4924pmid: 32559819

Monitoring the trace amount of chemicals in various samples remains a challenge. This study was conducted to develop a new solid‐phase microextraction (SPME) system (inside‐tube SPME) for trace analysis of n‐hexane in air and urine matrix. The inside‐tube SPME system was prepared based on the phase separation technique. A mixture of carbon aerogel and polystyrene was loaded inside the needle using methanol as the anti‐solvent. The air matrix of n‐hexane was prepared in a Tedlar bag, and n‐hexane vapor was sampled at a flow rate of 0.1 L/min. Urine samples spiked with n‐hexane were used to simulate the sampling method. The limit of detection using the inside‐tube SPME was 0.0003 μg/sample with 2.5 mg of adsorbent, whereas that using the packed needle was 0.004 μg/sample with 5 mg of carbon aerogel. For n‐hexane analysis, the day‐to‐day and within‐day coefficient variation were lower than 1.37%, with recoveries over 98.41% achieved. The inside‐tube SPME is an inter‐link device between two sample preparation methods, namely, a needle trap device and an SPME system. The result of this study suggested the use of the inside‐tube SPME containing carbon aerogel (adsorbent) as a simple and fast method with low cost for n‐hexane evaluation.

Yikilmaz, Yeliz; Kuzukiran, Ozgur; Erdogan, Ekrem; Sen, Filiz; Kirmizibayrak, Ozlem; Filazi, Ayhan

doi: 10.1002/bmc.4926pmid: 32558952

We aimed to develop a rapid, simple and reproducible method based on LC–tandem mass spectrometry (LC–MS/MS) to analyze β‐agonist residues (clenbuterol, zilpaterol, ractopamine and isoxsuprine) in bovine tissues. The method was validated in accordance with the European Council Decision 2002/657/EC. The samples were homogenized, and then 10 mL of an acetate buffer was added to a 5‐g sample. The sample was then centrifuged at 12,000 rpm and filtered. Sodium hydroxide (2 m) was added to adjust pH of the sample that was centrifuged again. The extract was filtered through a solid‐phase extraction column. The residue was re‐dissolved in 250 μL acetonitrile and then subjected to LC–MS/MS. The separation was done on a C18 column. The mobile phase consisted of 0.1% formic acid in deionized water and 0.1% formic acid in methanol. The mean recoveries of β‐agonists were in the range of 84.3%–119.1% with relative standard deviations (%RSDs) of 0.683%–4.05%. Decision limits and detection capabilities of the analytes ranged from 0.0960 to 4.9349 μg/kg and from 0.0983 to 5.0715, respectively. This method was used to detect four β‐agonists in 100 bovine muscle, 100 liver and 100 kidney tissues from a slaughterhouse. No residue was found above the maximum residue limit level.

Li, Le; Xia, Ying; Zhao, Shunbo; Ding, Li; Ji, Shunli

doi: 10.1002/bmc.4905pmid: 32449539

A simple and specific LC–MS/MS method was developed and validated for the determination of ethyl ester of eicosapentaenoic acid (EPAEE) and ethyl ester of docosahexaenoic acid (DHAEE). After deproteinized with acetonitrile, the plasma samples were separated on a C18 column using a gradient elution system consisted of methanol and 1.0 mM ammonium acetate in water. The detection used an atmospheric‐pressure chemical ionization ion source in positive mode with multiple reaction monitoring for the quantitation of EPAEE and DHAEE. The acceptable linearity was achieved over the concentration ranges of 1.00~1000 ng/mL for EPAEE and 2.50~2500 ng/mL for DHAEE. The method was successfully applied to a pharmacokinetic study of EPAEE and DHAEE in healthy Chinese volunteers after the oral administration of 4 g omega‐3‐acid ethyl esters 90 soft capsule. The pharmacokinetic profiles of EPAEE and DHAEE were observed for the first time in Chinese volunteers, which reached a maximum concentration of 499 ± 243 ng/mL and 1596 ± 476 ng/mL for EPAEE and DHAEE, respectively. The areas under the plasma concentration–time curve were 1290 ± 765 ng/mL·h for EPAEE and 4369 ± 1680 ng/mL·h for DHAEE, respectively.

Zhai, Jianping; Li, Li; Dong, Lihou; Dong, Kelly; Xiang, Shensi; Gui, Luolan; Zhang, Jiaying; Song, Haifeng; Ge, Zhiqiang

doi: 10.1002/bmc.4921pmid: 32537846

A simple, fast and high‐throughput LC–tandem mass spectrometry method was developed and validated to simultaneously measure liraglutide and insulin degludec in rat plasma. After protein precipitation, plasma samples were subjected to gradient elution using an InertSustain Bio C18 column with 1000/20/1 water/acetonitrile/formic acid (v/v/v) and 1000/1 acetonitrile/formic acid (v/v) as the mobile phase. The method was validated from 1.00 to 500 ng/mL of liraglutide and insulin degludec. Further, the extraction recovery from the plasma was 41.8%–49.2% for liraglutide and 56.5%–69.7% for insulin degludec. Intra‐ and inter‐day precision of liraglutide was 3.5%–9.4% and 8.4%–9.8%, respectively, whereas its accuracy was between −12.6% and −1.3%. Intra‐ and inter‐day precision of insulin degludec was 5.2%–13.6% and 11.8%–19.1%, respectively, showing an accuracy between −3.0% and 9.9%. As a result, the method was successfully applied to a pharmacokinetics study of liraglutide and insulin degludec following a single‐dose subcutaneous administration to rats.

Kręcisz, Paweł; Czarnecka, Kamila; Szymański, Paweł

doi: 10.1002/bmc.4906pmid: 32449534

Tacrine derivatives containing iodobenzoic acid were developed as a novel multitarget‐directed ligand and find potential application in the treatment of Alzheimer's disease. The aim of this study is to perform a physicochemical profile of this series. Experimental log P and pKa values were determined and compared with those already calculated. The results indicated better values of the tested compounds than the values predicted using computer software. The stability report was obtained using the developed HPLC method. The stability assay in different environment conditions provided information about the photosensitivity of these compounds and a proper method for the storage of this series of compounds.

Gong, Jing; Li, Lin; Lin, Yi‐xuan; Xiao, Dan; Liu, Wen; Zou, Bao‐rong; Tian, Xing; Han, Bo; Zhang, Shao‐bao; Lin, Lei; Li, Pei; Xie, Zhi‐yong; Liao, Qiong‐feng

Feng, Xinchi; Wang, Kun; Hu, Xintong; Chai, Liwei; Cao, Shijie; Ding, Liqin; Qiu, Feng

doi: 10.1002/bmc.4919pmid: 32533560

Rhizoma coptidis has been used for a long time in China owing to its anti‐bacterial, anti‐diabetes, anti‐hyperlipidemia and anti‐obesity activities. However, the in vivo biotransformation of Rhizoma coptidis is still unclear to date. In this study, a three‐step strategy using UPLC‐Q‐TOF/MS was applied to clarify the in vivo absorbed constituents and metabolites in rats after oral administration of Rhizoma coptidis. First, alkaloids in Rhizoma coptidis extract were identified. Second, six abundant alkaloids (berberine, palmatine, coptisine, epiberberine, jatrorrhizine, and columbamine) were selected as representative prototypes and the metabolic fates of them in rats were investigated to obtain a database of Rhizoma coptidis‐derived metabolites. Finally, the metabolic profiles of Rhizoma coptidis were fully elucidated based on the above‐mentioned results. In summary, 29 alkaloids were identified in Rhizoma coptidis, and a database of Rhizoma coptidis‐derived metabolites was obtained with 144 characterized metabolites. A total of 89 xenobiotics including 12 absorbed constituents and 77 metabolites were identified in dosed rat biosamples. Major metabolic pathways of Rhizoma coptidis were hydroxylation, reduction, methylation, demethylation, demethylenation, desaturation, glucuronidation and sulfation. This is the first systematic study on the in vivo absorbed constituents and metabolic profiling of Rhizoma coptidis and will be beneficial for its further studies.

Yıldırım, Sercan; Karakoç, Hanife Nur; Yaşar, Ahmet; Köksal, İftihar

doi: 10.1002/bmc.4925pmid: 32557742

Concentrations of fluoroquinolones, which are used in the treatment of many bacterial infections, should be monitored in biological fluids as they exhibit concentration‐dependent bactericidal activity. In this study, a liquid chromatography method for the determination of levofloxacin, ciprofloxacin, moxifloxacin and gemifloxacin in human urine and plasma was developed for the first time. The efficiency of five different columns for the separation of these fluoroquinolones was compared. Experimental parameters that affect the separation, such as percentage of organic solvent, pH, temperature, gradient shape and detector wavelength, were optimized by a step‐by‐step approach. Using a pentafluorophenyl core–shell column (100 × 4.6 mm, 2.7 μm), the separation of four analytes was accomplished in <7.5 min. The developed method was validated for the determination of analytes in both urine and plasma with respect to sensitivity, specificity, linearity (r ≥ 0.9989), recovery (79.46–102.69%), accuracy, precision and stability (85.79–111.07%). The intra‐ and inter‐day accuracies were within 89.55–111.94% with relative standard deviations of 0.35–8.05%. The feasibility of method was demonstrated by analyzing urine and plasma samples of patients orally receiving levofloxacin, ciprofloxacin or moxifloxacin. The developed method is suitable for therapeutic drug monitoring of these fluoroquinolones and can be applied to pharmacokinetic and toxicological studies.

Piestansky, Juraj; Galba, Jaroslav; Kovacech, Branislav; Parrak, Vojtech; Kovac, Andrej; Mikuš, Peter

doi: 10.1002/bmc.4907pmid: 32460377

Creatinine is an important diagnostic marker and is also used as a standardization tool for the quantitative evaluation of exogenous/endogenous substances in urine. This study aimed at evaluating and comparing three analytical approaches, based on hyphenations of different separation [two‐dimensional capillary isotachophoresis (CITP–CITP), capillary zone electrophoresis (CZE), ultra‐high‐performance liquid chromatography (UHPLC)] and detection [conductivity (CD), ultraviolet (UV), tandem mass spectrometry (MS/MS)] techniques, for their ability to provide reliable clinical data along with their suitability for the routine clinical use (cost, simplicity, sample throughput). The developed UHPLC–MS/MS, CITP–CITP–CD, and CZE–UV methods were characterized by favorable performance parameters, such as linearity (r ˃ 0.99), precision (relative standard deviation, 0.22–2.97% for the creatinine position in analytical profiles), and recovery (87.1–115.1%). Clinical data, obtained from the analysis of 24 human urine samples by a reference enzymatic method, were comparable with those obtained by the tested methods (Passing–Bablok regression and Bland–Altman analysis), approving their usefulness for the routine clinical use. In this context, the UHPLC–MS/MS method provides benefits of enhanced orthogonality/accuracy and high sample throughput (threefold shorter total analysis times than the CE methods), whereas advantages of the CE methods for routine labs are simplicity and low cost of both the instrumentation and measurements.