Development and in-house validation of an LC-MS/MS method for the determination of stilbenes and resorcylic acid lactones in bovine urine

Stilbenes and zeranol are nonsteroidal estrogenic growth promoters which are banned in the European Union (EU) for use in food-producing animals by Council Directive 96/22/EC. A liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was developed for the screening and confirmation of stilbenes (diethylstilbestrol, dienestrol, hexestrol) and resorcylic acid lactones (zeranol and its metabolites taleranol and zearalanone as well as the mycotoxins α-zearalenol, β-zearalenol and zearalenone) in bovine urine. The method permits the confirmation and quantification of stilbenes and resorcylic acid lactones at levels below 1 μg L⁻¹ and 1.5 μg L⁻¹, respectively. The validation was carried out according to Commission Decision 2002/657/EC, Chap. 3.1.3 “alternative validation” by a matrix-comprehensive in-house validation concept. Decision limit CCα, detection capability CCβ, recovery, repeatabiliy, within-laboratory reproducibility and the uncertainty of measurement were calculated. Furthermore, a factorial effect analysis was carried out to identify factors that have a significant influence on the method. Factors considered to be relevant for the method in routine analysis (e.g. operator, matrix condition, storage duration of the extracts before measurement, different cartridge lots, hydrolysis conditions) were systematically varied on two levels. The factorial analysis showed that different cartridge lots, storage durations and matrix conditions can exert a relevant influence on the method.     

婴儿湿巾缓冲能力的测试——一个贴近生活的实验

为了有效提升学生在缓冲溶液学习过程中的学习兴趣及积极性,在此推荐一个将现实中的生活用品作为实验对象引入实验课教学过程中的案例。该实验设计将性质实验扩充到婴儿湿巾,将实验内容变为对婴儿湿巾工作原理的验证,而不再是过去简单的酸碱体系的配制及相关验证实验。通过该实验的实施,学生普遍反映能够有效激发其在学习缓冲溶液相关课程中的学习积极性与兴趣,并最终促使学生认知缓冲体系内pH变化的特点及缓冲机理。     

甲基丙烯酸甲酯悬浮聚合实验教学的改进

选用氢氧化镁为分散剂,甲基丙烯酸甲酯(MMA)为单体,进行悬浮聚合反应,研究了分散剂用量、引发剂用量、水和单体比、搅拌速度、反应温度及反应时间对聚合物珠粒大小、均匀程度和产率的影响。结果表明:与目前高分子实验教材普遍选用的以聚乙烯醇(PVA)为分散剂的悬浮聚合反应相比,以氢氧化镁为分散剂,具有操作简单、聚合时间短、体系稳定性高等优点,可代替原有的悬浮聚合反应实验,作为高分子化学实验的教学课程内容。     

Stability-indicating RP-HPLC method development and validation for determination of nine impurities in apixaban tablet dosage forms. Robustness study by quality by design approach

A quality by design (QbD) based high-resolution HPLC method is described for determination of impurities in apixaban (APX) in the tablet dosage form. Employing a simple and stability-indicating HPLC method, nine known impurities were quantified with good peak resolution. Mobile phase A (MP-A) was prepared with buffer and acetonitrile 90:10 v/v, while mobile phase B (MP-B) contained water and acetonitrile 10:90 v/v. The gradient program was 0 min, MP-A 75%, B 25%; 20 min, MP-A 65%, B 35%; 30 min, MP-A 40%, B 60%; 40min, MP-A 40%, B 60%; 42 min, MP-A 75%, B 25%; and 50 min, MP-A 75%, B 25%. The chromatographic separation was achieved using a Zorbax RX C₁₈ 250 × 4.6 mm column, 5 μm (1.0 ml min⁻¹, 280 nm, 50 μl) and a column temperature of 40°C. Several separation studies were carried out using design of experiments to optimize the method. Validation results confirm the applicability of the developed method for quality analysis and stability studies of the regular product on the manufacturing stream.     

Validated LC–MS/MS method for quantitation of a selective JAK1 inhibitor,filgotinib in rat plasma,and its application to a pharmacokinetic study in rats

Filgotinib is a selective JAK1 (Janus kinase) inhibitor, filed in Japan for the treatment of rheumatoid arthritis. In this paper, we report a validated liquid chromatography coupled with tandem mass spectrometry for the quantification of filgotinib in rat plasma using tofacitinib as an internal standard (IS) as per the Food and Drug Administration regulatory guidelines. Filgotinib and the IS were extracted from rat plasma using ethyl acetate as an extraction solvent and chromatographed using an isocratic mobile phase (0.2% formic acid:acetonitrile; 20:80, v/v) at a flow rate of 0.9 mL/min on a Gemini C₁₈ column. Filgotinib and the IS were eluted at ~1.31 and 0.89 min, respectively. The MS/MS ion transitions monitored were m/z 426.3 → 291.3 and m/z 313.2 → 149.2 for filgotinib and the IS, respectively. The calibration range was 0.78–1924 ng/mL. No matrix effect and carryover were observed. Intra- and inter-day accuracies and precisions were within the acceptance range. Filgotinib was stable for three freeze–thaw cycles: on bench-top up to 6 h, in an autosampler up to 21 h, and at −80 ° C for 1 month. This novel method has been applied to a pharmacokinetic study in rats.     

Enantioselective LC–ESI–MS/MS method for quantitation of (−)-lumefantrine and (+)-lumefantrine in mice plasma and application to a pharmacokinetic study

We developed and validated a simple, sensitive, selective, and reliable LC–MS/MS–ESI method for the direct quantitation of lumefantrine (LFN) enantiomers [(−)-LFN and (+)-LFN] in mice plasma as per regulatory guideline. LFN enantiomers and carbamazepine (internal standard) were extracted from mice plasma using Strata X SPE (solid-phase extraction) cartridges. Good resolution between enantiomers was achieved on a Chiralpak IA-3 column using an isocratic mobile phase (0.1% of diethyl amine in methanol), which was delivered at a flow rate of 0.8 mL/min. Detection and quantitation were performed using multiple reaction monitoring mode following the transitions m/z 530.27 → 512.30 and 237.00 → 194.00 for LFN enantiomers and the internal standard, respectively, in the positive-ionization mode. The proposed method provided accurate and reproducible results over the linearity range of 2.39–895 ng/mL for each enantiomer. The intra- and inter-day precisions were in the range of 1.03–6.14 and 6.36–8.70 and 2.03–4.88 and 5.82–11.5 for (−)-LFN and (+)-LFN, respectively. Both (−)-LFN and (+)-LFN were found to be stable under different stability conditions. The method was successfully used to delineate stereoselective pharmacokinetics of LFN enantiomers in mice after an oral administration of rac-LFN (20 mg/kg). The pharmacokinetic results indicated that the disposition of LFN enantiomers was stereoselective in mice.     

Simultaneous determination of colchicine and febuxostat in rat plasma: Application in a rat pharmacokinetic study

A selective, sensitive and rapid LC–MS/MS method has been developed and validated as per US Food and Drug Administration regulatory guidelines for the simultaneous quantitation of colchicine and febuxostat in rat plasma. Colchicine and febuxostat were extracted from the rat plasma using 10% tert-butyl methyl ether in ethyl acetate using colchicine-d₆ as an internal standard (IS). The chromatographic separation of colchicine, febuxostat and the IS was achieved using a mobile phase comprising 5 mm ammonium formate and 0.025% formic acid in acetonitrile (20:80, v/v) in isocratic mode on an Eclipse XDB-C₁₈ column. The injection volume and flow rate were 5.0 μl and 0.9 ml/min, respectively. Colchicine and febuxostat were detected by positive electrospray ionization in multiple reaction monitoring mode using transition pairs (Q1 → Q3) of m/z 400.10 → 358.10 and 317.05 → 261.00, respectively. The assay was linear in the ranges of 0.25–254 and 2.60–622 ng/ml for colchicine and febuxostat, respectively. The inter- and intra-day precision values were 0.58–13.0 and 1.03–4.88% for colchicine and febuxostat, respectively. No matrix or carryover effects were observed during the validation. Both analytes were stable on the bench-top, in the autosampler and in storage (freeze–thaw cycles and long-term storage at −80 ° C). A pharmacokinetic study in rats was performed to show the applicability of the validated method.     

A dried blood spot assay with HPLC–MS/MS for the determination of larotrectinib in mouse blood and its application to a pharmacokinetic study

Larotrectinib is a first-generation tropomyosin kinase inhibitor, approved for the treatment of solid tumors. In this paper, we present a validated dried blood spot (DBS) method for the quantitation of larotrectinib from mouse blood using HPLC–MS/MS, which was operated under multiple reaction monitoring mode. To the DBS disc cards, acidified methanol enriched with internal standard (IS; enasidenib) was added and extracted using tert-butyl methyl ether as an extraction solvent with sonication. Chromatographic separation of larotrectinib and the IS was achieved on an Atlantis dC₁₈ column using 10 mm ammonium formate–acetonitrile (30:70, v/v) delivered at a flow-rate of 0.80 ml/min. Under these optimized conditions, the retention times of larotrectinib and the IS were ~0.93 and 1.37 min, respectively. The total run time was 2.50 min. Larotrectinib and the IS were analyzed using positive ion scan mode and parent–daughter mass to charge ion (m/z) transitions of 429.1 → 342.1 and 474.1 → 267.1, respectively, were used for the quantitation. The calibration range was 1.06–5,080 ng/ml. No matrix effect or carryover was observed. Hematocrit did not influence DBS larotrectinib concentrations. All of the validation parameters met the acceptance criteria. The applicability of the validated method was shown in a mouse pharmacokinetic study.