Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of polymixin B1, B2, ile-B1, E1, and E2 in human plasma and its clinical pharmacokinetic application.

Polymyxin B (PB) and Polymyxin E (PE, also called colistin) are used as the last treatment resort for multidrug-resistant Gram-negative bacterial infections. The nephrotoxicity and neurotoxicity of polymyxins limit their clinical use, and guidelines recommend therapeutic drug monitoring (TDM) to optimize efficacy and reduce toxicity. However, there are limited analytical methods available for the determination of PB and PE. This study aimed to develop a simple and robust liquid chromatography with tandem mass spectrometry (LC-MS/MS) analytical method for determining the main compounds of PB and PE, namely PB1, PB2, ile-PB1, PE1, and PE2, in human plasma and to investigate of their pharmacokinetics in critically ill patients with the use of PB and PE, respectively. Plasma PB1, PB2, ile-PB1, PE1, and PE2 were chromatographically separated on a Welch LP-C18 column and detected using electrospray ionization mode coupled with multiple reaction monitoring. The calibration curve showed acceptable linearity over 20-10,000 ng/mL for PB1, PE1, and PE2 and 10-5000 ng/mL for PB2 and ile-PB1 in the plasma, respectively. After validation following approved guidelines, this method was successfully applied for PB and PE pharmacokinetic analysis and TDM in critically ill patients. Additionally, the composition of PB1, PB2, ile-PB1, PE1, and PE2 remains unchanged from 0 to 12 h after entering the patient's body.

Development and validation of enzyme-linked immunosorbent assays for the measurement of infliximab and anti-drug antibody levels.

Infliximab and its anti-drug antibody (ADA) serum concentrations exhibit a strong correlation with clinical response and loss of response. The use of therapeutic drug monitoring to measure the concentration of infliximab and ADA can facilitate clinical decision-making, helping patients attain optimal therapeutic effects. However, there are still limitations to the existing infliximab and its ADA detection methods. Therefore, this study aimed to develop and validate enzyme-linked immunosorbent assay (ELISA)-based methods for measuring infliximab and its ADA levels in human plasma according to the general recommendations for immunoassays. Free infliximab is bound by recombinant TNF-α and detected using HRP-labeled anti-human antibody. The ADA is captured by on-plate-coated infliximab and recognized by biotin-labeled infliximab. Two bridging ELISA assays were developed and after assay optimization and validation, these assays have been applied in ten patients with inflammatory bowel disease (IBD). In infliximab detection assay, a standard curve ranging from 0.10 µg/mL to 8.0 µg/mL with great precision and accuracy has been established. Drug tolerance of the ADA assay was that 100 ng/mL ADA could tolerate at least 5.0 µg/mL infliximab in the plasma using a commercially available monoclonal anti-infliximab antibody as the positive control. The ADA screening and confirmatory assays achieved a sensitivity of 36.74 ng/mL and 37.15 ng/mL, respectively. All other assay characteristics met the requirements. The mean concentration of infliximab in eight patients with IBD was 7.88 (1.87-21.1) µg/mL, and the ADA levels were all negative. Moreover, the concentrations of infliximab in the remaining two patients were below the LLOQ and the ADAs were positive. Thus, accurate and sensitive ELISA methods have been developed and validated for the detection of infliximab and its ADA concentrations and have been successfully applied to clinical therapeutic drug monitoring.

Determination of olverembatinib in human plasma and cerebrospinal fluid by an LC-MS/MS method: Validation and clinical application.

A sensitive and robust LC-MS/MS method has been developed and validated for olverembatinib quantification in human plasma and cerebrospinal fluid (CSF). The method involved liquid-liquid extraction with methyl tertiary butyl ether for plasma pretreatment and precipitation enrichment with methanol for CSF pretreatment. Separation was achieved on the C18 column with gradient elutions of 10 mM ammonium formate in water and methanol-acetonitrile (50:50,v/v). Analyte detection was conducted by electrospray ionization (ESI) in a positive ion mode using multiple reaction monitoring (MRM). The m/z transitions were 533.4→433.2 for olverembatinib and m/z 502.4→394.2 for the internal standard (IS, Imatinib-d8). Calibration curves ranged from 0.500 to 50.0 ng/mL for plasma and from 0.0100 to 1.00 ng/mL for CSF. The intra- and inter-day precision and accuracy were < 15% for both plasma and CSF with four different quality control concentrations. The relative matrix effect was < 10% in plasma and artificial CSF. This method was successfully utilized for the measurement of olverembatinib concentrations in plasma and CSF from chronic myeloid leukemia patients.

Combined Analysis of Volatile Terpenoid Metabolism and Transcriptome Reveals Transcription Factors Related to Terpene Synthase in Two Cultivars of Dendrobium officinale Flowers.

Dendrobium officinale is a kind of traditional Chinese herbal medicine. Its flowers could be used as health care tea for its aroma flavor and medicinal value. Most recent studies demonstrated that terpenoids are the main components of the aromatic compounds in the flowers, but the biosynthesis of terpenoids is poorly understood in D. officinale. In the experiment, the flowers from two cultivars of D. officinale with different smells were collected. The transcriptome analysis and combined volatile terpenoids determination were performed to identify the genes related to the biosynthesis of the terpenoids. The results showed that the different products of volatile terpenoids are α-thujene, linalool, α-terpineol, α-phellandrene, γ-muurolene, α-patchoulene, and δ-elemene in two cultivar flowers. The transcriptome analysis detected 25,484 genes in the flowers. And 18,650 differentially expressed genes were identified between the two cultivars. Of these genes, 253 genes were mapped to the terpenoid metabolism pathway. Among these genes, 13 terpene synthase (TPS) genes may have correlations with AP2/ERF, WRKY, MYB, bHLH, and bZIP transcription factors by weighted gene co-expression network analysis (WGCNA). The transcription factors have regulatory effects on TPS genes. These results may provide ideas for the terpenoid biosynthesis and regulatory network of D. officinale flowers.

Genome-wide identification and expression analyses of R2R3-MYB transcription factor genes from two Orchid species.

MYB transcription factors play important roles in different plant biological processes during plant growth, development and stress response. In this study, 101 (DoMYB1-101) and 99 (PaMYB1-99) R2R3-MYB genes were identified in the genomes of Dendrobium officinale and Phalaenopsis aphrodite, respectively. To classify the isolated candidate genes, the R2R3-MYB genes from A. thaliana were selected as references. As a result, all identified DoMYB and PaMYB genes were classified into 22 subfamilies. Phylogenetic analysis revealed that S21 had the largest number of members of all the subfamilies. The numbers of introns, exons and conserved sequences in all of the identified genes are different. In addition, 20 DoMYB genes from six subfamilies were selected for further analysis of tissue-specific expression and responses to various abiotic stresses treatments. The results showed that all of the DoMYB genes in S4 and S19 subfamilies exhibited the highest relative expression levels in flowers. And five DoMYB genes including DoMYB31, DoMYB40, DoMYB49, DoMYB52 and DoMYB54, responded to the stress response. These results may provide useful information for further studies of the R2R3-MYB gene family.