Biomedical Chromatography

Chen, Xinyan; Liu, Gege; Wang, Changmao; Liu, Ran; Wang, Minhui; Huang, Yunzhe; Shen, Jie; Jia, Yuanwei

doi: 10.1002/bmc.5638pmid: 37002731

A steady, high‐efficiency, and precise liquid chromatography‐electrospray ionization‐tandem mass spectrometry method was established and validated using cefaclor‐d5 as the stable isotope‐labeled internal standard for quantification of cefaclor in human plasma. One‐step protein precipitation was applied to extract human plasma samples using methanol as precipitant. An Ultimate XB C18 column (2.1 × 50.0 mm, 5.0 μm) was used to achieve chromatographic separation. Mobile phases of gradient elution were an aqueous solution containing 0.1% formic acid (mobile phase A) and an acetonitrile solution containing 0.1% formic acid (mobile phase B). Electrospray ionization in positive‐ion mode was applied to detect under multiple reaction monitoring mode. Target fragment ion pairs of cefaclor and stable isotope‐labeled internal standard, respectively, were m/z 368.2 → 191.1 and m/z 373.2 → 196.1. Linear range of this method was between 20.0 and 10,000.0 ng/ml, with coefficient of determination (R2) >0.9900. Seven concentrations of quality control samples were used: 20.0 ng/ml (lower limit of quantitation), 60.0 ng/ml (low quality control), 650 ng/ml (middle quality control), 5000 ng/ml (arithmetic average middle quality control [AMQC]), 7500 ng/ml (high quality control), 10,000 ng/ml (upper limit of quantification), and 40,000 ng/ml (dilution quality control [DQC]). The method was validated for selectivity, lower limit of quantitation, linearity, accuracy, precision, recovery, matrix effect, dilution reliability, stability, carryover, and incurred sample reanalysis. This stable isotope‐labeled internal standard liquid chromatography‐electrospray ionization‐tandem mass spectrometry approach has been successfully applied to study the pharmacokinetics of cefaclor dry suspension among healthy Chinese volunteers.

Bala, Veenu; Chhonker, Yashpal S.; Morales, Guillermo A.; Maddeboina, Krishnaiah; Pal, Dhananjaya; Durden, Donald L.; Murry, Daryl J.

doi: 10.1002/bmc.5643pmid: 37042063

A sensitive and selective liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantitation of dual PI3K/BRD4 inhibitor SF2523 in mouse plasma. The analysis was performed on a UPLC system connected to a Shimadzu 8060 mass spectrometer by electrospray ionization in positive multiple reaction monitoring mode. Chromatographic separation was carried out on an ACE Excel C18 column with a gradient elution containing 0.1% formic acid and methanol as the mobile phase. The linearity was conducted in the concentration range 0.1–500 ng/ml for SF2523 in 100 μl of plasma. The inter‐ and intra‐batch precision (RSD) were both lower than 13.5%, with the accuracy (percentage bias) ranging from −10.03 to 11.56%. The validated method was successfully applied to plasma protein binding and in vitro metabolism studies. SF2523 was highly bound to mouse plasma proteins (>95% bound). Utilizing mouse S9 fractions, a total of seven phase I and II metabolites were identified with hydroxylation found to be the major metabolic pathway. Metabolite identification included analysis of retention behaviors, molecular weight changes and MS/MS fragment patterns of SF2523 and the metabolites. This newly developed and validated method allows the rapid and easy determination of the SF2523 concentration with high sensitivity in a low sample volume and can be applied to future pre‐clinical studies.

Wani, Taha U.; Mir, Khalid B.; Raina, Arun; Dar, Alamgir A.; Jan, Ishrat; Khan, Nisar A.; Wani, Taseen A.; Sofi, Javid A.; Hassan, G. I.; Almoallim, Hesham S.; Alharbi, Sulaiman Ali; Ansari, Mohammad Javed; Alfarraj, Saleh; Tarique, Mohammed; Dar, Showket A.

Hofmann, Sabrina; Gebauer, Julian; Krnáč, Dušan; Prantz, Anja; Reiffová, Katarína; Rolinski, Boris; Serbin, Rastislav

doi: 10.1002/bmc.5657pmid: 37070208

A simple and rapid HPLC‐MS/MS analytical method was developed and validated for the determination of methylmalonic acid (MMA) in human serum without a derivatization step. Serum samples (200 μl) were pretreated using a simple method based on ultrafiltration using a VIVASPIN 500 ultrafiltration column. Chromatographic separation was achieved on a Luna Omega C18 column with a PS C18 precolumn guard by gradient elution using 0.1% (v/v) formic acid in water (mobile phase A) and 0.5% (v/v) formic acid in acetonitrile (mobile phase B) at a flow rate of 0.2 ml min−1. The total run time of the analysis was 4.5 min. Negative electrospray ionization and multiple reaction monitoring mode were used. The lower limit of detection and lower limit of quantification for MMA were determined to be 13.6 and 42.3 nmol L−1, respectively. The developed method enabled the quantification of MMA in a wide linear range of 42.3–4230 nmol L−1 with a correlation coefficient of 0.9991.

Cui, Yue; Tang, Xinmiao; Feng, Bo

doi: 10.1002/bmc.5653pmid: 37052131

Gansuibanxia decoction (GSBXD), a traditional Chinese medicine (TCM) formula with 2000 years of history, has good efficacies on treating cancerous ascites, pleural effusion, etc. However, little is known about its metabolite profiles owing to the lack of in vivo studies. In this study, we explored the prototypes and metabolites of GSBXD in rat plasma and urine using UHPLC–Q‐TOF/MS technology. A total of 82 GSBXD‐related xenobiotic bioactive components (38 prototypes and 44 metabolites) were confirmed or tentatively characterized, including 32 prototypes and 29 metabolites in plasma, and 25 prototypes and 29 metabolites in urine. The results showed that the bioactive components absorbed in vivo mainly contained diterpenoids, triterpenoids, flavonoids and monoterpene glycosides. Both phase I reactions (methylation, reduction, demethylation, hydrolysis, hydroxylation and oxidation) and phase II reactions (glucuronidation and sulfation) were involved in the metabolism of GSBXD in vivo. This study will provide a foundation for the quality control, pharmacological study and clinical application of GSBXD.

Seo, Yerim; Chung, Eun Kyoung; Jung, Byung Hwa

doi: 10.1002/bmc.5642pmid: 37016500

The hexosamine biosynthesis pathway (HBP) is a glucose metabolism pathway that produces uridine diphosphate N‐acetyl glucosamine (UDP‐GlcNAc). Substantial changes in HBP, including elevated HBP flux and UDP‐GlcNAc levels, are associated with cancer pathogenesis. Particularly, cancer cells expressing oncogenic Kirsten rat sarcoma virus (KRAS) are highly dependent on HBP for growth and survival. To differentiate between HBP metabolites in KRAS wild‐type (WT) and mutant (MT) lung cancer cells, a simultaneous quantitative method for analyzing seven HPB metabolites was developed using ultra‐high‐performance liquid chromatography‐tandem mass spectrometry. A simple method without complicated preparation steps, such as derivatization or isotope labeling, was optimized for the simultaneous analysis of highly hydrophilic HBP metabolites, and the developed method was successfully verified. The intra‐ and inter‐day coefficients of variation were less than 15% for all HBP metabolites, and the recovery was 89.67–114.5%. All results of the validation list were in accordance with ICM M10 guidelines. Through this method, HBP metabolites in lung cancer cells were accurately quantified, and it was confirmed that all HBP metabolites were upregulated in KRAS MT cells compared with KRAS WT lung cancer cells. We expect that this will be a useful tool for metabolic research on cancer and for the development of new drugs for cancer treatment.

Garzinsky, Ann‐Marie; Thomas, Andreas; Guddat, Sven; Görgens, Christian; Dib, Josef; Thevis, Mario

doi: 10.1002/bmc.5633pmid: 36974028

Currently, primarily urine, whole blood and serum samples are analyzed for doping‐relevant substances in professional sports, but recently dried blood spots (DBS) have been introduced as complementary matrix, offering advantageous features, e.g. a minimally invasive sampling procedure. In order to cope with the increased application of DBS, a comprehensive initial testing procedure (ITP) was developed, optimized and validated, comprising a total of 233 substances representing all groups on the World Anti‐Doping Agency's (WADA's) Prohibited List. The sample preparation was conducted by employing a fully automated system using an efficient flow‐through extraction of a 4 mm diameter spot followed by LC–HRMS/MS analysis. The procedure was successfully validated in terms of selectivity, limit of detection, reproducibility, carryover and robustness with respect to an alternative manual sample preparation, an alternative dried blood collection device and the sample extract stability, and was thus found to meet the required criteria of the relevant guidelines published by WADA for routine application. As a proof‐of‐concept, DBS samples were analyzed after the administration of the glucocorticoids prednisone and dexamethasone, as well as the stimulant pseudoephedrine and the beta‐blocker propranolol. All substances were detected in post‐administration samples for at least 4 h and up to 24 h after intake, depending on the collection time period, using the developed testing procedure. In particular, for substances that are only banned in‐competition, data obtained from DBS samples can be useful for the interpretation of adverse analytical findings. In conclusion, the developed ITP accounts for the anticipated increasing relevance of DBS in anti‐doping analysis in the future and provides a foundation for optimized approaches for specific substance classes.

Xiong, Yun; Ma, Peijie; Yan, Yitong; Huang, Limei; Li, Yajiao; Wang, Xiaoli

doi: 10.1002/bmc.5620pmid: 36942894

Citronella is used as a spice and a traditional herbal medicine. Dried citronella is easy to store and transport, and it is unclear whether dried citronella has more or fewer medicinal components compared to fresh citronella. In the present study, various metabolites in fresh and dry citronella were detected using a widely targeted metabolomics strategy. We identified 712 metabolites and classified them into 31 categories, and we identified 132 flavonoids. After citronella was dried, the quantities of most kinds of flavonoids increased, but the quantities of amino acids, organic acids, and vitamins decreased, and the quantity of quercetin increased significantly. Therefore, the medicinal value of dry citronella may have increased, and the nutritional value of amino acids and vitamins may have decreased. The results of this study can serve as a new theoretical reference to study citronella and promote its nutrition and medicinal chemical composition.

Cai, Jin; Zhao, Linfang; Li, Lingdi; Lou, Jun; Tang, Rongjun; Zhang, Ke; Yu, Qingqing; Mo, Weixing

doi: 10.1002/bmc.5658pmid: 37080899

Colon cancer (CC) is a malignancy of the digestive tract, and computed tomography (CT)‐guided radiofrequency ablation (RFA) has been extensively adopted in cancer treatment. We aimed to explore the changes in fecal metabolism after CT‐guided RFA in CC mice. The orthotopic CC mice received CT‐guided RFA upon modeling. Subsequently, we quantified tumor volumes and weights to assess treatment efficacy. Next, because metabolomics is useful for evaluating therapeutic validity, feces were collected for metabolomics analysis. CT‐guided RFA inhibited tumor growth effectively. Additionally, metabolomics results showed that the contents of bile acids and fatty acids were downregulated in CC mouse feces. Moreover, the levels of amino acids and carbohydrates were decreased while the levels of fatty acids, organic acids, phenols, pyridines and short‐chain fatty acids were elevated in feces after CC mice received CT‐guided RFA. Pathway enrichment analysis revealed that those differential metabolites were closely related to fatty acids degradation and synthesis. CT‐guided RFA possesses a strong ability to suppress CC development in mice, accompanied by a significant increase of fatty acid content in feces. This study proposes a novel approach and target for CC treatment, which provides hope for CC patients and establishes a solid basis for future in‐depth studies.

Nagulancha, Bhujanga Rao; Lakka, Narasimha Swamy; Vandavasi, Koteswara Rao

doi: 10.1002/bmc.5644pmid: 37052118

Baloxavir marboxil (BXM) is a polymerase acidic endonuclease inhibitor used as an antiviral drug. A simple, reliable, and robust liquid chromatographic method was developed and validated per International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2(R1) for estimating the assay and impurities of BXM in drug substance and pharmaceutical formulations. The chromatographic separation was carried out on C18 (100 × 4.6 mm, 5 μm) with binary solvent delivery system (A:0.1% trifluoroacetic acid in water; B:0.1% trifluoroacetic‐acid in acetonitrile) along with detection wavelength of 260 nm, column temperature of 57°C, flow of 1.2 mL/min and injection volume of 10 μL. All five known impurities and unknown impurities were separated well with resolution >1.7 and were estimated accurately without any interference. Recovered values and regression value were 99.5%–101.2% and R2 > 0.999, respectively. The recovery and linearity studies covered from 50% to 150% for assay, and quantitation limit, 120% for five BXM impurities. Stability‐indicating property of the HPLC developed method was assessed from the forced degradation studies. The mass spectral data of unknown impurity formed under oxidation stress condition were discussed. The developed method was also successfully utilized for stability sample analysis of drug substance and tablet dosage form.