Biomedical Chromatography

Yang, Ying; Shen, Lisha; Wang, Ping; Tao, Yi

2023 Biomedical Chromatography

doi: 10.1002/bmc.5742pmid: 37674471

Achyranthes bidentata has been found to possess beneficial effects against osteoporosis, but there is still a lack of comprehensive studies on its anti‐osteoporotic compounds. Therefore, in this study, we established a zebrafish osteoporosis model to evaluate the anti‐osteoporotic effect of different fractions of raw and salt‐processed A. bidentata. Among these fractions, the dichloromethane fraction showed the most promising anti‐osteoporotic effect. To further investigate the active compounds responsible for the anti‐osteoporosis effects, we prepared and analyzed the dichloromethane fraction of 10 batches of raw and salt‐processed A. bidentata using liquid chromatography–mass spectrometry. As a result, we tentatively identified 19 compounds, including 11 saponins, three phenolic amides, three unsaturated fatty acids and two other compounds. To further narrow down the potential active compounds, we employed both orthogonal partial least squares discriminant analysis and gray relationship analysis. Through these analyses, we were able to identify eight compounds that showed a high correlation with the anti‐osteoporosis effects of the dichloromethane fraction. Furthermore, we validated the anti‐osteoporotic effects of β‐ecdysterone, wogonin, ginsenoside Ro, oleanolic acid, linoleic acid and palmitic acid using the zebrafish model. These compounds demonstrated significant anti‐osteoporotic effects, further supporting their potential as active compounds in A. bidentata.

Yu, Wentao; Luo, Man; Wu, Huan; Li, Junmao; Yang, Shilin; Zhang, Wugang; Yao, Min; Feng, Yunlin

2023 Biomedical Chromatography

doi: 10.1002/bmc.5752pmid: 37753581

Huaganjian decoction (HGJD) has been widely used clinically to treat liver injuries and gastritis. However, the quality evaluation system for HGJD is not perfect. In this study, paeoniflorin, hesperidin, geniposide, naringin, and quercetin were employed as quality markers. The quantitative analysis of these five components in HGJD was conducted using a high‐performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry method. This method underwent validation for linearity, precision, accuracy, repeatability, and recovery. In summary, a reliable quantitative method was successfully employed to establish a comprehensive quality evaluation of HGJD.

Huang, Hailing; Duan, Bailu; Zheng, Sili; Ye, Yan; Zhang, Dongning; Huang, Zhuang; Wang, Shanshan; Zhang, Fengyun; Huang, Ping; Huang, Fang; Han, Lintao

2023 Biomedical Chromatography

doi: 10.1002/bmc.5732

The current study utilizes a comprehensive network pharmacology and metabolomics analysis to investigate the mechanism of action of Ma‐Mu‐Ran Antidiarrheal Capsules (MMRAC) for the treatment of ulcerative colitis (UC). In this study, we established a mouse model of UC using dextran sulfate sodium. Colonic tissues were collected from mice and then subjected to hematoxylin and eosin staining, as well as histopathological analysis, to assess the therapeutic effect of MMRAC. Furthermore, we assessed the mechanisms through which MMRAC combats UC by employing integrated metabolomics and network pharmacology strategies. Lastly, we validated the key targets identified through western blot and molecular docking. An integrated network of metabolomics and network pharmacology was constructed using Cytoscape to identify eight endogenous metabolites involved in the therapeutic action of MMRAC on UC. Further comprehensive analyses were focused on four key targets and their associated core metabolites and pathways. The results of western blot and molecular docking demonstrated that MMRAC could modulate key targets and their expression levels. The cumulative results indicated that MMRAC restored intestinal function in UC, reduced inflammatory responses, and alleviated oxidative stress by influencing the methionine and cysteine metabolic pathways, as well as the urea cycle. In addition, it had an impact on arginine, proline, glutamate, aspartate, and asparagine metabolic pathways and their associated targets.

González‐López, Nicolás Mateo; Guerra‐Acero‐Turizo, Luisa María; Blanco‐Medina, Isabella; Barragán‐Cárdenas, Andrea Carolina; Ramírez‐Celis, David Augusto; Martínez‐Ramírez, Jorge Ariel; Fierro‐Medina, Ricardo; García‐Castañeda, Javier Eduardo; Rivera‐Monroy, Zuly Jenny

2023 Biomedical Chromatography

doi: 10.1002/bmc.5741pmid: 37688464

Matrix effect and sample pretreatment significantly affect the percentage recovery of peptides in biological matrices, affecting the method robustness and accuracy. To counteract this effect, an internal standard (IS) is used; however, in most cases this is not available, which limits the analytical method. It is important to identify short peptides that can be used as ISs in the quantification of peptides in biological matrices. In this study, doping peptides GHRP‐4, GHRP‐5, GHRP‐6, Sermorelin (1–11), Sermorelin (13–20) and Sermorelin (22–29) were synthesized using solid‐phase peptide synthesis. Treatment with human blood, trypsin and chymotrypsin was used to determine the stability of the peptides. Products were evaluated using the high‐performance liquid chromatography‐diode array detector (HPLC‐DAD) method. The analytical methodology and sample pretreatment were effective for the analysis of these molecules. A unique profile related to protein binding and enzymatic stability of each peptide was established. GHRP‐4, GHRP‐6 and Sermorelin (22–29) can be considered as in‐house ISs as they were stable to enzyme and blood treatment and can be used for the quantification of peptides in biological samples. Peptides GHRP‐6 and Sermorelin (22–29) were used to analyse a dimeric peptide (26[F] LfcinB (20–30)2) in four different matrices to test these peptides as in‐house IS.

Xia, Demeng; Wu, Rui; Xue, Qian; Jiang, Gehan; Xu, Shuogui

2023 Biomedical Chromatography

doi: 10.1002/bmc.5733pmid: 37705144

While clinical surveys have frequently reported that patients with traumatic brain injuries (TBIs) and comorbidities experience faster healing, the underlying mechanisms have been investigated but remain unclear. As a comprehensive comparison and analysis of the metabolic characteristics of these two pathologies have not been undertaken, we developed a rat model of fracture and TBI and collected serum samples for metabolomic analysis using ultra‐high performance liquid chromatography–quadrupole time‐of‐flight MS (UHPLC–Q‐TOF/MS). In total, we identified 40 differential metabolites and uncovered related pathways and potential mechanisms, including aminoacyl‐transfer RNA biosynthesis; differential amino acids such as leucine, cholylhistidine, aspartyl‐lysine; and related lipid metabolism, and discussed their impacts on bone formation in detail. This study highlights that the UHPLC–Q‐TOF/MS–based metabolomics approach offers a better understanding of the metabolic links between TBI and accelerated bone recovery.

Wang, Shuai; Yang, Xin Xin; Li, Tian Jiao; Tian, Xiang Mu; Wang, Ying Li; Bai, Gang; Bao, Yong Rui; Meng, Xian Sheng

2023 Biomedical Chromatography

doi: 10.1002/bmc.5740pmid: 37670539

Bufei Jianpi granule (BJG) is clinically effective for treating chronic obstructive pulmonary disease (COPD). At present, there is no report regarding the drug metabolism of BJG in vivo. This work developed an ultra‐high‐performance liquid chromatography coupled with triple quadrupole mass spectrometry method with high accuracy and sensitivity to determine drug metabolism of this compound in vivo. After continuous administration of BJG, the concentrations of 10 components in rat plasma, namely betaine, peimine, peiminine, astragaloside A, sinensetin, nobiletin, naringin, calycosin, formononetin, and magnolol, were determined at different time points. Meanwhile, the pharmacokinetic parameters and metabolic rules of these 10 components were evaluated: Cmax, 8.624–574.645 ng/mL; Tmax, 0.250–8.667 h; AUC0–t, 17.640–8947.393 ng h/mL; T1/2, 3.405–66.014 h; mean residence time (MRT), 6.893–11.223 h. All these components possessed anti‐inflammatory, antioxidant, and other biological activities to varying degrees, contributing to improving lung function, mitigating pneumonia and pulmonary fibrosis, and preventing and treating chronic obstructive pulmonary disease. Exploring the pharmacokinetic parameters and the laws of chemical components in BJG forms the scientific basis for applying the compound clinically and identifying quality markers for the control of the compound.

Hassan, Mohammed H.; Galal, Omyma; Sakhr, Hala M.; Kamaleldeen, Eman B.; Zekry, Nadia Farouk; Fateen, Ekram; Toghan, Rana

2023 Biomedical Chromatography

doi: 10.1002/bmc.5747

Fifty diabetic nephropathy (DN) children with type 1 diabetes mellitus (T1DM) and 50 healthy matched controls were included. Chromatographic assays of 14 amino acids, free carnitine and 27 carnitine esters using high‐performance liquid chromatography/electrospray ionization–mass spectroscopy, and genetic testing for JAK2v617f mutation using real‐time PCR were performed. Patients had significantly lower levels of tyrosine, branched‐chain amino acids (BCAAs), and BCAA/AAA (aromatic chain amino acids) ratios, glycine, arginine, ornithine, free carnitine and some carnitine esters (C5, 6, 12 and 16) and higher phenylalanine, phenylalanine/tyrosine ratio and C18 compared with the controls and in the macro‐albuminuria vs. the microalbuminuria group (p < 0.05 for all) except for free carnitine. Plasma carnitine was negatively correlated with eGFR (r = −0.488, p = 0.000). There were significant positive correlations between tyrosine with UACR ratio (r = 0.296, p = 0.037). The plasma BCAA/AAA ratio showed significant negative correlations with UACR (r = −0.484, p = 0.000). There was a significantly higher frequency of the JAK2V617F gene mutation in diabetic nephropathy patients compared with the control group and in macro‐albuminuria than the microalbuminuria group (p = 0.000) for both. When monitoring children with T1DM, plasma free amino acids and acylcarnitine profiles should be considered, especially if they have tested positive for JAK2V617F for the early diagnosis of DN.

Fu, Xiaojie; Zheng, Ting; Li, Zegeng; Wu, Huan

2023 Biomedical Chromatography

doi: 10.1002/bmc.5748pmid: 37750002

Research into traditional Chinese medicine metabolism in feces is one of the key avenues to understanding the fate of traditional Chinese medicines in vivo. In this study, we used ultraperformance liquid chromatography–quadrupole time‐of‐flight MS in combination with a post‐targeted screening strategy to identify the prototype components and metabolites in rat feces after oral administration. Based on our group's previous research, the component database of Qi‐Yu‐San‐Long decoction (QYSLD) was established. Prototype components were screened from the fecal samples based on summarized chromatographic and MS behaviors. According to the chemical structure characteristics of related compounds, the possible metabolic pathways were inferred, and the metabolites related to QYSLD were predicted. We extracted ion chromatograms by predicting the m/z values of metabolite excimer ions and identified related metabolites based on their retention time and fragmentation behavior. A total of 93 QYSLD‐related xenobiotics were confirmed or tentatively identified in rat fecal samples, and the results indicated that the main metabolic pathways of QYSLD were hydrolysis, deglycosylation, oxidation, reduction, decarboxylation, methylation and acetylation. This study presents a rapid method for identifying the prototype components and metabolites, and offers valuable insights into the biotransformation profiling of QYSLD in rat feces.

Dong, Bizhang; Hu, Jiye

2023 Biomedical Chromatography

doi: 10.1002/bmc.5728pmid: 37700621

Acetamiprid and pyridaben are highly efficient insecticides widely used to protect leafy vegetables against various pests, such as Phyllotreta striolata, but analyses of their residual behaviors applied in mixtures in cabbage fields are primarily lacking. Herein, field trials were performed by spraying 50% acetamiprid–pyridaben wettable powder (50% WP) once at a dose of 150 g of active ingredient per hectare in 12 representative provinces of China under Good Agricultural Practices. The residues of acetamiprid and pyridaben were detected using modified Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) and liquid chromatography–tandem mass spectrometry, together with an assessment of their dietary risks. The average recoveries of the two insecticides were 84.6–104%, and the relative standard deviations were 0.898–10.1%. The residual concentrations of acetamiprid and pyridaben at the preharvest interval of 7 days were <0.364 and 0.972 mg/kg, respectively, and less than their maximum residue limits in cabbage (0.5 mg/kg for acetamiprid and 2 mg/kg for pyridaben) in China. The chronic and acute risk values of acetamiprid and pyridaben were 0.0787–33.3%, implying acceptable health hazards to Chinese consumers. In conclusion, applying 50% WP in cabbage fields under Good Agricultural Practices is acceptable. These results provide essential data for using mixtures of acetamiprid and pyridaben in cabbage fields.

Dawoud, Turki; Ameen, Fuad

2023 Biomedical Chromatography

doi: 10.1002/bmc.5739pmid: 37674346

In various countries, Pimpinella has been used to cure several diseases for centuries. Therefore, we focus on one of its potent species in this research. The aim of this experimental study was to document the various extracts derived from Pimpinella anisum that can effectively eradicate oral pathogens. In addition, the presence of antioxidants, antimicrobials, and cytotoxicity was determined using chromatographic testing methods. The alkaloid range was from 22.34 ± 043 mg/g, and the saponin range was from 15.1 ± 1.07 mg/g. HPLC analysis showed that the samples contained eight identified phenolic compounds. The antibacterial activity of ethanolic extract exhibited the highest inhibition region against Streptococcus iniae (43 ± 0.6 mm) and the lowest inhibition region against Staphylococcus haemolyticus (19 ± 0.2 mm) in 200 mg/mL of leaf ethanolic extracts. The antifungal activity revealed that ethanol showed the maximum inhibition zone against Aspergillus luchuensis (42.5 ± 0.19 mm) and the minimum inhibition zone against Aspergillus kawachii (15 ± 0.13 mm) in 200 mg/mL. The current study suggested that, after the isolation of individual components, P. anisum be investigated for assessing biological activity. The mixture and various combinations of these compounds may indicate a truly potent agent that is novel in its ability to combat a wide range of bacteria and oral pathogens.

Neves, Ana Paula; Rosa, Ana Cristina Simões; Larentis, Ariane Leites; Silva Rodrigues Vidal, Priscila Jeronimo; Gonçalves, Eline Simões; Geraldino, Barbara Rodrigues; Silveira, Gabriel Rodrigues; Carvalho, Leandro Vargas Barreto; Alves, Sergio Rabello

2023 Biomedical Chromatography

doi: 10.1002/bmc.5746

The general population and workers are exposed to organophosphate insecticides, one of the leading chemical classes of pesticides used in rural and urban areas. This paper aims to conduct an integrative review of the most used analytical methods for identifying and quantifying dialkylphosphate—which are metabolites of organophosphate insecticides—in the urine of exposed workers, discussing their advantages, limitations and applicability. Searches utilized the PubMed, the Scientific Electronic Library Online and the Brazilian Digital Library of Theses and Dissertations databases between 2000 and 2021. Twenty‐five studies were selected. The extraction methods most used were liquid–liquid extraction (LLE) (36%) and solid‐phase extraction (SPE) (36%), with the SPE being more economical in terms of time and amount of solvents needed, and presenting the best percentage of recovery of analytes, when compared with LLE. Nineteen studies (76%) used the gas chromatography method of separation, and among these, 12 records (63%) indicated mass spectrometry used as a detection technology (analyzer). Studies demonstrate that dialkylphosphates are sensitive and representative exposure biomarkers for environmental and occupational organophosphate exposure.

Kul, Aykut; Sagirli, Olcay

2023 Biomedical Chromatography

doi: 10.1002/bmc.5744pmid: 37698043

The World Health Organization recommends that infants be exclusively breastfed for the first 6 months. Antibiotics are among the most commonly prescribed drugs for pregnant and lactating women. The vast majority of drugs pass into breast milk, which may create a risk for the infant. In cases where drug exposure may pose a risk, breastfeeding should be discontinued. Therefore, the mother's drug use should be decided by considering the most accurate and recent data. Cefuroxime is a second‐generation cephalosporin antibiotic with a broad spectrum of activity against Gram‐negative and ‐positive microorganisms. In this study, we aimed to develop the LC–MS/MS method using salt‐assisted liquid–liquid micro‐extraction (SALLME) for the determination of cefuroxime in breast milk. The method was validated according to the European Medicines Agency (EMA) guidelines. Cefuroxime and the internal standard cefixime were extracted from plasma by a SALLME technique. The results obtained from the entire validation study are at an acceptable level according to the EMA criteria. The calibration curve of cefuroxime was between 25 and 1000 ng/ml, with correlation coefficients of >0.99. The lower limit of quantitation was 25 ng/ml for cefuroxime. Furthermore, the developed method was applied for the determination of cefuroxime in real patient breast milk.

Moldoveanu, Serban C.

2023 Biomedical Chromatography

doi: 10.1002/bmc.5753pmid: 37750455

Ascorbic acid is a water‐soluble vitamin common in food and dietary supplements. A usual problem with ascorbic acid analysis is the lack of stability of its samples and standard solutions owing to oxidation. A procedure to protect ascorbic acid from oxidation using mercaptoethanol is described in this study in connection with the comparison of three HPLC measuring methods. Two reversed‐phase columns were evaluated for the separation. One technique uses UV detection, and two others use MS/MS detection. The methods were calibrated for quantitation on different ranges of concentrations. The LC–UV method covers the range 3.9 μg/ml to 500 μg/ml, one LC–MS/MS the range 80 ng/ml to 20 μg/ml, and the other 0.1 ng/ml to 20 μg/ml. As a proof of functionality all three methods were utilized for measuring vitamin C in energy drinks and chews (gummies). The sensitivity of LC–MS/MS methods was not necessary for the analysis of those samples, but the high sensitivity can be beneficial for other types of sample such as environmental or biological, where the levels of ascorbic acid are very low. The study showed that the formation of 2,3‐diketogulonic acid is not a likely path for ascorbic acid oxidation following hydrolysis as reported in some studies.

Dong, Fengyu; Xie, Mengdi; Xu, Manwen; Lu, Lu; Miao, Yan; Zhang, Panpan; Li, Xiaopeng; Gui, Xinjing; Liu, Ruixin

2023 Biomedical Chromatography

doi: 10.1002/bmc.5745pmid: 37736670

Dispensing granules of Chinese medicine (DGCM) have emerged as a more convenient alternative to traditional decoction (TD) of Chinese medicine, gaining popularity in recent years. However, the debate surrounding the consistency of DGCM compared to TD remains unresolved. In this study, three batches of Baishao and Gancao DGCM were obtained from manufacturers A, B, and C, and 15 batches of crude drugs were procured from hospital pharmacies for the preparation of dispensing granule decoction (DGD) and TD of Shaoyao‐Gancao decoction (SGD). The HPLC‐UV method was employed to determine the levels of gallic acid, paeoniflorin, albiflorin, liquiritin, liquiritin apioside, isoliquiritin apioside, isoliquiritin, glycyrrhizic acid, and isoliquiritigenin. The analgesic and antispasmodic effects were assessed using the hot plate and acetic acid writhing test in mice. To evaluate the consistency of chemical constituents and pharmacological effects between the two decoctions, the Criteria Importance Though Intercriteria Correlation (CRITIC) method combined with chemometrics was employed. Grey relation analysis (GRA) was used to assess the comprehensive quality consistency of the two decoctions. The CRITIC results revealed certain differences in chemical constituents and pharmacological effects between the selected DGCM and TD. Notably, DGD‐A/C exhibited a significant difference from TD (p > 0.05), whereas DGD‐B demonstrated no significant difference from TD (p > 0.05). The GRA analysis demonstrated that the overall quality consistency between DGD‐B and TD was the highest among the three manufacturers. This study presents a method for evaluating the quality consistency of DGCM and TD of SGD, offering novel insights into the evaluation of consistency between DGCM and TD.

Shi, Wei; Han, Yue

2023 Biomedical Chromatography

doi: 10.1002/bmc.5736pmid: 37668238

Rheumatoid arthritis (RA) is a systemic autoimmune disease dominated by chronic inflammatory lesions of peripheral synovial joints. Growing evidence suggests that abnormal lipid metabolism levels contribute to the progression of RA. Although several metabolomics studies have shown abnormality in the RA lipidome, the relationship between the overall lipid metabolites and RA has not been systematically evaluated. In this study, an untargeted lipidomics method based on ultra performance liquid chromatography‐mass spectrometry (UPLC–MS) was used to analyze the serum and urine lipidomes of adjuvant‐induced arthritis rats to study the characteristics of lipid metabolism changes in the rats and search lipid markers for diagnosing RA. By combining with orthogonal partial least squares discriminant analysis, a total of 52 potential lipid markers were identified, mainly involved in sphingolipid metabolism, glycerophospholipid metabolism, sterol lipid metabolism, glycerolipid metabolism and fatty acid metabolism, which provided crucial insight into lipid metabolism disturbances in RA. Further receiver operating characteristic analysis revealed that the areas under the curve of PC(22:4/16:0), PI(18:1/16:0) and LacCer(d18:1/12:0) from serum and 25‐hydroxycholesterol from urine were 0.94, 1.00, 1.00 and 1.00, respectively, indicating the high predictive ability of this method for RA. In this study, our results indicated that a combination of serum and urine analysis can provide a more comprehensive and reliable assessment of RA, and a UPLC–MS‐based lipidomics strategy is a powerful tool to search for potential lipid markers associated with RA and explore the pathogenesis of RA.

Dar, Alamgir A.; Jan, Ishrat; Shah, Mehraj D.; Sofi, Javid A.; Hassan, G. I.; Dar, Shahnawaz R.

2023 Biomedical Chromatography

doi: 10.1002/bmc.5756pmid: 37750442

In this study, an analytical method was developed and validated for the assessment of pesticide residues in commonly consumed vegetables and fruits. Fresh samples of apple, green peas, tomatoes, and cucumbers were processed and subjected to analysis using a modified QuEChERS (quick, easy, cheap, effective, rugged, safe) extraction technique. Subsequently, quantification of pesticide residues was conducted utilizing gas chromatography (GC)‐electron capture detector. Extraction and cleanup parameters were meticulously optimized, resulting in a modification of the original QuEChERS method. This modification aimed to reduce solvent consumption, making the study more environmentally friendly. The developed method was validated in terms of selectivity, specificity, linearity, precision, and accuracy by following the SANTE guidelines. Calibration curves showed good linearity (r > 0.99) within the test range. Precision was evaluated by intra‐ and inter‐day experiments with an acceptable relative standard deviation (<20.0%). Recovery was assessed at the limit of quantification level and was observed to fall within the range of 70%–120%, with relative standard deviations below 5.45%. The validated method presented here can be applied to analyze pesticide residues in various other vegetables, fruits, and cereals. It is essential for ongoing monitoring of pesticide residues to ensure public safety.

Yang, Wei‐Han; Hao, Jing‐Wen; Chen, Nai‐Dong; Li, Jiao

2023 Biomedical Chromatography

doi: 10.1002/bmc.5743pmid: 37700561

The determination of monosaccharides is crucial for studying the structure of polysaccharides and the composition of free monosaccharides in living organisms. Based on previous derivatization gas chromatography–mass spectrometry (GC–MS) methods, we aimed to develop a novel analytical protocol for better quantifying monosaccharides. In this study, sugar alcohol acetylation, saccharonitrile acetylation, silylation and a combination of sugar alcohols acetylation and saccharonitrile acetylation were compared. The optimal method was verified with the monosaccharide determination of four polysaccharides and four free monosaccharides from Dendrobium. The results showed that the novel combined derivatization method was superior to the other three methods in terms of content analysis of monosaccharides. Furthermore, it possessed good linearity (all calibration curves showed relative coefficients ≥ 0.999), sensitivity, precision (relative standard deviation < 2%), and accuracy (recovery, 95.7–105%). Finally, the novel method established in this study was successfully employed in determining the monosaccharide composition of four polysaccharides and four free monosaccharide samples from Dendrobium.

Yang, Yan‐Ling; Qian, Zhou‐Yi; Zhao, Yang; Chen, Xiang‐Long; Huang, Qiong‐Ye; Guo, Yu‐Jiao; Sun, Lu‐Ning; Wang, Yong‐Qing

2023 Biomedical Chromatography

doi: 10.1002/bmc.5738

We developed and validated sensitive MS/MS methods for the determination of venetoclax, an oral selective B‐cell lymphoma‐2 inhibitor, in human plasma and cerebrospinal fluid (CSF). Acetonitrile was used as protein precipitant. The mobile phase was 10 mM ammonium formate consisting of 0.1% formic acid and acetonitrile (40:60, v/v). The analytes were separated on an ACQUITY UPLC HSS T3 column (2.1 × 50 mm, 1.8 μm) in 5 min. An API 4000 mass spectrometer was selected to quantify venetoclax and internal standard using m/z 868.3 → 636.3 and 876.3 → 644.3 under multiple response monitoring mode. In plasma, the calibration curve exhibited good linearity ranging from 20.0 to 5000 ng/mL, whereas in the CSF, the linear range was 0.500–100 ng/mL. The matrix effect of venetoclax and internal standard (venetoclax‐d8) was not obvious in both plasma and CSF. The inter‐ and intra‐run accuracy was within ±11.9%, and the inter‐ and intra‐run precision was below 13.6%. Both methods had no carryover, and the recovery was close to 100%. The validated methods were employed to quantify the concentrations of venetoclax in the plasma and CSF of patients diagnosed with chronic lymphocytic leukemia or acute myelogenous leukemia.

Rajaratnam, Vilashini; Islam, Mohammad Mohiminul; Kub, Ethan F.; Rajaratnam, Shaarwin; Kim, Kyu Bum; Rahman, Md Toufiqur; Rashid, Farjana; Benko, Anna M.; Cook, James M.; Arnold, Leggy A.; Mirza, Shama P.

2023 Biomedical Chromatography

doi: 10.1002/bmc.5754pmid: 37750452

Despite aggressive treatment approaches, the overall survival of glioblastoma (GBM) patients remained poor with a strong need for more effective chemotherapeutic agents. A previous study has shown that ARN14988 is more cytotoxic to GBM cells compared to US Food and Drug Administration‐approved temozolomide. This finding makes ARN14988 a desirable candidate for further pharmacological assessment. Therefore, an efficient analytical method is needed to quantify ARN14988. Herein, we have developed and validated sample preparation and LC–MS/MS triple quadrupole (QQQ) method for quantification of ARN14988 in mouse plasma. In this method, the liquid–liquid extraction of ARN14988 from mouse plasma was performed using 5% ethyl acetate in hexane. The chromatographic separation was achieved using a C18‐column with mobile phases of 10 mm ammonium acetate (pH 5) and 0.1% formic acid in methanol, within a runtime of 10 min. The monitored transitions were m/z 391.20 → m/z 147.00 for ARN14988, and m/z 455.30 → m/z 165.00 for verapamil (internal standard) in positive electrospray ionization. The developed method for ARN14988 showed linearity over the range of 10–5,000 ng/ml (r2 > 0.99). The selectivity, sensitivity, matrix effect, recovery, stability, inter‐day and intraday accuracy and precision were determined using four quality control samples. This validated method was successfully applied to the pharmacokinetic study of ARN14988 in mice.

Chen, Feng; Yang, Xiaoxia; Li, Huanhuan; Zeng, Xiaodan; Deng, Ziwei; Wang, Hongqiang; Jin, Yuanxiang; Qiu, Chengfeng; Shi, Zhihua

2023 Biomedical Chromatography

doi: 10.1002/bmc.5751

In order to facilitate therapeutic drug monitoring of tacrolimus and cyclosporine A in clinical practice, a simple, rapid, robust, sensitive and specific LC–MS/MS assay was developed and validated for the simultaneous determination of tacrolimus and cyclosporine A in human whole blood. Erythrocytes were destroyed using internal standard solution with 10% (w/v) zinc sulfate in water. The analytes were extracted from 100 μl of whole blood by protein precipitation with acetonitrile. Chromatographic separation was conducted on a Kinetex PFP column (60°C) by a gradient elution with a flow rate of 0.450 ml/min in 2.5 min. Quantitative analysis was performed using electrospray ionization and multiple reaction monitoring in positive ionization mode. The method was fully validated as per current guidelines on bioanalytical methodologies of the US Food and Drug Administration and European Medicines Agency. The method developed was applied successfully in analyzing clinical samples from patients administered tacrolimus or cyclosporine A. The sample treatment procedure was rationalized and improved to fulfill the complete target extraction. The chromatography conditions were optimized to achieve rapid and accurate quantification of both analytes. This method may be beneficial as a constructive input for the therapeutic drug monitoring of tacrolimus and cyclosporine A in obtaining individualized therapy.

Ezquer‐Garin, Carlos; Aguilar, Gerardo; Ferriols‐Lisart, Rafael; Alos‐Almiñana, Manuel

2023 Biomedical Chromatography

doi: 10.1002/bmc.5749

Amphotericin B (AMB) is a polyene macrolide antifungal agent used for treating invasive fungal infections. Liposomal AMB is a lipid dosage form, available as AmBisome, which reduces the toxicity of the drug. A simple HPLC‐UV method was developed for the determination of AMB in plasma to study its pharmacokinetic profile in a critical patient receiving AmBisome and treated with extracorporeal replacement therapies. Sample preparation was performed using plasma deproteinization and drug release from liposome by the addition of acetonitrile (ACN)/zinc sulfate and ultrasonication. Chromatographic separation was performed using a C18 column and a mobile phase consisting of phosphate buffer (pH 3.0)/ACN (65/35, v/v). The UV detector was set at 407 nm. The total run time analysis was 23 min. The method was validated according to the standard guidelines and applied to study the pharmacokinetics of AMB in a critical patient. The total run time analysis obtained was shorter than that of the previously reported methods, being useful for therapeutic drug monitoring or pharmacokinetic profile research.