Biomedical Chromatography

Li, Lin‐ling; Chen, Cai‐li; Cai, Ning‐fang; Yi, Jin‐ling; Zheng, Chao; Feng, Yan; Xiong, Wen‐gang; Luo, Xi; Li, Wei‐hua; Cheng, Ze‐neng

doi: 10.1002/bmc.4631pmid: 31254484

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of cinacalcet in human plasma was developed and validated. This assay was based on liquid–liquid extraction and cinacalcet‐d4 was used as an internal standard (IS). Separation was achieved on a C18 column by the mobile phase A of water (containing 0.1% formic acid) and the mobile phase B of acetonitrile–water (95:5, v/v) (containing 0.2% formic acid) with gradient elution. Quantification was done using multiple reaction monitoring mode to monitor the precursor‐to‐product ion transitions of m/z 358.2 → m/z 155.2 for cinacalcet and m/z 362.3 → m/z 155.0 for IS at positive ionization mode. The calibration curve was established over the range 0.05–20.0 ng/mL and the correlation coefficient was >0.99. The intra‐ and inter‐day relative standard deviations were <5.8%. Accuracy determined at four concentrations ranged between 96.0 and 106.0%. This method was successfully applied to a pharmacokinetic description of oral dose of cinacalcet and the significant effect of food intake on the pharmacokinetics of cinacalcet was first demonstrated in Chinese healthy volunteers.

Wang, Huijun; Guan, Yunyun; Wu, Ruoming; Lv, Xing; Shen, Xiaoyan; Ye, Guan

doi: 10.1002/bmc.4628pmid: 31243781

Wang‐Bi tablet (WB) is popularly used for the treatment of rheumatoid arthritis. However, few studies have been carried out on its active ingredients and mechanism. In this study, the effect of WB medicated serum on the changes in differentiation and function in osteoblast was investigated, the results showed that WB induced the production of ALP and mineralized nodules to promote the final maturation of osteoblasts and enhance the function of osteoblasts. The potential mechanism may that WB significantly inhibits gene expressions of RANKL and miR‐141, up‐regulates the gene expressions of RUNX2 and OPG, decreases expression of DKK‐1 and increases levels of β‐catenin protein to promote the activation of Wnt/β‐catenin signaling pathways, which enhances osteogenesis and bone repair function. To investigate which compounds contributed to the activity and mechanisms, a total of 138 compounds were characterized from WB, and 13 parent molecules and eight metabolites in rat serum were rapidly characterized by UPLC–Q‐TOF/MS. Total glycosides of paeony, loganin, α‐linolenic acid, linoleic acid and naringin from WB may contribute to the actions on osteoblasts according to our study and literature review. Our research provides a method to explore the bioactive ingredients and action mechanisms of WB.

Emam, Aml A.; Abdelwahab, Nada S.

doi: 10.1002/bmc.4612pmid: 31152603

Recently, concepts of sustainable developments, like considering the environmental effect of chemicals used and the amount of hazardous wastes produced, has gained much interest. In this work, a recently approved treatment for type II diabetes mellitus, canagliflozin, was quantified along with its degradation product by two eco‐friendly methods. The first was a specific green HPLC method using a C18 column as a stationary phase and a mobile phase consisting of methanol–water (98:2, v/v) pumped at a flow rate of 1 mL/min with UV detection at 225 nm, and using ibuprofen as an internal standard. The second method was a partial least square chemometric method with the wavelength range 220–320 nm and the data was autoscaled as a preprocessing step for determination of canagliflozin and its degradation product. The greenness profile of the developed methods was studied and compared with the reported methods. The proposed methods were suitable alternatives for the environmentally harmful reported methods for quality control analyses of canagliflozin‐containing samples, analysis of pharmaceutical formulations and sensitive tracing of its possible degradation product. The methods were validated as per International Conference on Harmonization guidelines and statistically compared with the reported HPLC method.

Silva, Jessica; Basso, João; Sousa, João; Fortuna, Ana; Vitorino, Carla

doi: 10.1002/bmc.4621pmid: 31215044

There is increasing interest in atorvastatin and curcumin owing to their potential anticancer activity. A new, accurate and sensitive HPLC method was developed, for the first time, to simultaneously quantify atorvastatin and curcumin in mouse plasma and brain, liver, lung and spleen tissues following protein precipitation sample preparation. The chromatographic separation was achieved in 13 min on a C18 column, at 35°C, using a mobile phase composed of acetonitrile–methanol–2% (v/v) acetic acid (37.5:2.5:60, v/v/v) at a flow rate of 1.0 mL/min. The detection of analytes and internal standard was carried out at 247, 425 and 250 nm, respectively. According to international guidelines, the method was shown to be selective, with lower limits of quantification ranging from 10 to 500 ng/mL for curcumin, and from 100 to 600 ng/mL for atorvastatin, linear over a wide concentration range (r2 ≥ 0.9971) and with acceptable accuracy (bias ± 12.29%) and precision (coefficient of variation ≤13.15%). The analytes were reproducibly recovered at a percentage >81.10% and demonstrated to be stable under various experimental conditions in all biological matrices. This method can be easily applied to in vivo biodistribution studies related to the intranasal administration of atorvastatin and curcumin, separately or simultaneously.

Kertys, Martin; Kosutova, Petra; Mokra, Daniela; Mokry, Juraj

doi: 10.1002/bmc.4620pmid: 31215048

A simple, highly sensitive and rapid method for quantification of olprinone (phosphodiesterase 3 inhibitor) in rabbit plasma using liquid chromatography–tandem mass spectrometry with electrospray was developed. An aliquot of 50 μL of plasma sample was cleaned up and extracted using Ostro™ 96‐well plate followed by dilution. Chromatographic separation of olprinone and olprinone‐d3 was carried out on a CORTECS® T3 column within 3 min. Detection was achieved using a triple quadrupole mass spectrometer employing electrospray ionization operated in positive ion multiple reaction monitoring mode using the transitions m/z 251.07 → m/z 155.06 and m/z 254.21 → m/z 158.10 for olprinone and olprinone‐d3, respectively. The method was validated according to US Food and Drug Administration guideline for bioanalytical methods, and showed excellent linearity in the range 10.0–2000.0 ng/mL with coefficient of determination >0.99. The intra‐ and inter‐day precisions (CV) were <5.1% and the accuracies were within the range 99.7–103.2% at all quality control concentrations. Furthermore, olprinone was stable under various stability conditions. The developed method was used for quantification of olprinone in rabbit plasma after its intravenous administration at the dose of 1 mg/kg in order to better understand the metabolism of olprinone in a rabbit model of lung injury.

Zhang, Qibo; Adam, Klaus‐Peter

doi: 10.1002/bmc.4636pmid: 31256428

An LC–MS/MS‐based bioanalytical method has been developed to measure the concentration of L‐threonate at its endogenous level in human plasma. Following isotope dilution and protein precipitation, the samples were acetylated and chromatographed under reversed‐phase conditions for baseline separation of the derivatized L‐threonate and its stereoisomer D‐erythronate. The method was assessed by a fit‐for‐purpose validation with a calibration range from 100 to 10,000 ng/mL. The intra‐run coefficients of variation (CVs) were <3.6% and the inter‐run CV was 3.2% for the QC samples at endogenous level. At the lower limit of quantitation, the intra‐run CV was 6.1% and the average inaccuracy was −1.4%. This method provides an efficient and reliable quantitation of L‐threonate and could be useful to certain biomarker investigators.

Dongala, Thirupathi; Katari, Naresh Kumar; Palakurthi, Ashok Kumar; Jonnalagadda, Sreekantha B.

doi: 10.1002/bmc.4608pmid: 31145484

We developed novel stability‐indicating HPLC method for simultaneous estimation of 14 impurities in excedrin tablet, a formulation with a combination of acetaminophen, aspirin, and caffeine. In addition, a new impurity that was generated through degradation of aspirin at high temperatures during the accelerated stability conditions was positively identified and confirmed, using liquid chromatography–mass spectrometry technique. The HPLC method was optimized using the Inertsustain C18, 250 × 4.6 mm, 5.0 μm column, employing simple gradient method. Forced degradation studies were performed under acidic, basic, oxidative and thermal conditions to prove the scope and stability‐indicating the nature of the method. The optimized method was validated as per the International Conference on Harmonization guidelines. The HPLC method showed linearity from LOQ concentration to 21 μg mL−1. Precision and intermediate precision values were <5% RSD. The validated HPLC method is currently applied for the routine testing of excedrin tablet formulations in quality control laboratories.

Xi, Wang; Zou, Qiaogen; Ouyang, Pingkai

doi: 10.1002/bmc.4611pmid: 31145820

In this study, a simple and reliable liquid chromatography coupled with Q‐Exactive‐Orbitrap–MS was developed and validated for detecting and quantifying cligosiban and its metabolites in dog plasma after oral administration. The plasma samples were pretreated with acetonitrile and separated on a Diamonsil C18 column (4.6 × 100 mm, i.d. 3 μm) with 0.1% formic acid in water and acetonitrile as mobile phase. The method was validated according to the guidance of the US Food and Drug Administration. The assay was linear over the tested concentration ranges with coefficients of correlation >0.995. The extraction recovery was >83.23% with RSD <15%. Precision was <9.31% and accuracy ranged from −4.40 to 10.20%. The method was free of matrix effects. Under the conditions used, four metabolites were detected and their identities were identified by accurate masses and fragment ions. M1 and M3 were further confirmed by reference standards. The biotransformation pathways included demethylation and glucuronidation. The validated method was further applied to quantify cligosiban, M1 and M3 in dog plasma. After oral administration, cligosiban was detectable in dog plasma and reached the maximum concentration at ~1.67 ± 0.58 h post‐dose. It was rapidly eliminated with a half‐life of 3.48 ± 0.80 h. M1 showed high plasma exposure with its area under the curve being 23.31% of that of cligosiban.

Chen, Weikang; Zheng, Yangbin; Yan, Liang; Yuan, Mingming; Ouyang, Lan; Li, Jun; Zhou, Guoping; Zhong, Ruijian

doi: 10.1002/bmc.4585pmid: 31099065

Saponin PH, akemisaponins E, saponin PJ1 and scheffoleoside A, the main bioactive triterpene saponins of Chinese traditional medicine Akebia trifoliata, contribute to its diuretic pharmacological activity. Because of interactions of the multiple ingredients in vivo, pharmacokinetic studies of multiple triterpenes after administration of A. trifoliata extract are essential to clarify their pharmacological effects. The purpose of this study was to develop an efficient and sensitive UHPLC–MS/MS method for simultaneous determination of these four triterpene saponins in rat plasma. The biosamples were prepared by liquid–liquid extraction with n‐butanol. The chromatographic separation was performed on a Phenomenex Luna® C18 (150 × 2 mm, 3 μm) with a mobile phase consisting of acetonitrile and water at a flow rate of 0.5 mL/min. The MS/MS system was operated in a negative multiple reaction monitoring mode, and the precursor–product ion transitions were optimized as m/z 941.6 → 471.1 for saponin PH, 941.7 → 471.2 for akemisaponins E, 1089.7 → 601.1 for saponin PJ1, 957.6 → 487.4 for scheffoleoside A and 799.5 → 637.3 for ginsenoside Rg1 (Rg1, internal standard). Method validation parameters (calibration curve linearity, lower limit of detection, recovery, matrix effect, intra‐ and inter‐day precision) were within the acceptable ranges. This is the first reported on the UHPLC–MS/MS detection of saponin PH, akemisaponins E, saponin PJ1 and scheffoleoside A, and applied to a preclinical pharmacokinetic study after oral administration of A. trifoliata extract in rats. This study provides a basis for clinical application and further development of A. trifoliata extract.

Liu, Yang; Zhang, Dongjie; Du, Juan; Qin, Ying; Zhao, Zhigang; Shi, Yanjun; Mei, Shenghui; Liu, Yi

doi: 10.1002/bmc.4634pmid: 31257625

Plasma concentrations of nicotine and its active metabolite cotinine are highly correlated with its biological effects. A UHPLC–MS/MS method was developed, validated and applied for nicotine and cotinine analysis in mice plasma. Chromatographic separation was achieved on a BEH HILIC column using acetonitrile (0.1% formic acid) and 10 mm ammonium formate as mobile phase. The gradient elution was performed at 0.4 mL/min with a run time of 3.6 min. The quantitative ion transition was m/z 163.1 > 130.0 for nicotine, m/z 177.1 > 80.0 for cotinine and m/z 167.1 > 134.0 for nicotine‐D4 (internal standard, IS). For both nicotine and cotinine, the calibration range was 5–500 ng/mL with 5 ng/mL as the lower limit of quantitation, and the intra‐ and inter‐day bias and imprecision were −4.61–12.00% and <11.12%. The IS normalized recovery was 90.62–98.95% for nicotine and 89.18–101.53% for cotinine, and the IS normalized matrix factor was 106.00–116.44% for nicotine and 100.34–109.85% for cotinine. Both nicotine and cotinine were stable under conventional storage conditions. The validated method has been applied to a pharmacokinetic study in mice to calculate the pharmacokinetic parameters for both analytes.