Determination of the immunostimulatory drug—glucosoaminyl-muramyl-dipeptide—in human plasma using HPLC–MS/MS and its application to a pharmacokinetic study

GMDP (glucosoaminyl-muramyl-dipeptide), a synthetic analog of the peptidoglycan fragment of the bacterial cell wall, is an active component of the immunomodulatory drug Licopid. But the pharmacokinetic parameters of GMDP in humans after oral administration have not been investigated yet. The present study aimed at developing and validating a sensitive LC–MS/MS method for the analysis of GMDP in human plasma. The sample was prepared by solid-phase extraction using Strata-X 33 μm polymeric reversed-phase 60 mg/3 mL cartridges Phenomenex (Torrance, CA, USA). The analytes were separated using an Acquity UPLC BEN C18 column, 1.7 μm 2.1 × 50 mm Waters (Milford, USA). GMDP and internal standard growth hormone releasing peptide-2 (pralmorelin) were ionized in positive electrospray ionization mode and detected in multiple reaction monitoring mode. The developed method was validated within a linear range of 50–3000 pg/mL for GMDP. Accuracy for all analytes, given as the deviation between the nominal and measured concentration and assay variability , ranged from 1.61 to 3.02% and from 0.89 to 1.79%, respectively, for both within- and between-run variabilities. The developed and validated HPLC–MS/MS method was successfully used to obtain the plasma pharmacokinetic profiles of GMDP distribution in human plasma.     

UPLC–MS–MS Analysis of Baicalin in the Cerebrospinal Fluid of Rabbits: Application to a Pharmacokinetic Study

A sensitive and selective ultra-performance liquid chromatographic–tandem mass spectrometric method has been developed for analysis of baicalin in the cerebrospinal fluid of rabbits. Samples were separated on a C₁₈ column with a gradient prepared from acetonitrile and 0.3% aqueous formic acid solution as mobile phase. The target compounds were quantified by multiple reaction monitoring (MRM) using electrospray ionization (ESI). Intra-and inter-day accuracy, precision, and linear range were investigated in detail. The lower limit quantification (LOQ) was 0.108 ng mL^?1. The method has been successfully used for evaluation of the pharmacokinetics of baicalin in 12 rabbits.     

LC–MS–MS Analysis of Strictosamide in Rat Plasma, and Application of the Method to a Pharmacokinetic Study

A rapid and simple high-performance liquid chromatographic tandem mass spectrometric method has been developed and validated for analysis of strictosamide in rat plasma. Chromatographic separation was achieved on a C₁₈ column by gradient elution with mixtures of methanol, water, and acetonitrile containing 0.05% acetic acid. Digoxin was used as internal standard. Selected reaction monitoring (SRM) was used for MS quantitation. Linearity was good in the range 0.05–20 ng mL^?1 in rat plasma. The lower limit of quantitation was 0.04 ng mL^?1. The method is precise and reliable and can be applied to pharmacokinetic studies.     

Development of a UPLC–MS/MS method for the simultaneous determination of atorvastatin, 2-hydroxy atorvastatin,and naringenin in rat plasma and its application to pharmacokinetic interaction studies

Recent studies have revealed that the combination therapy of atorvastatin (ATV) with naringenin (NG) can offer meaningful benefits in the treatment of hypercholesterolemia, while decreasing adverse side effects. To investigate whether there are pharmacokinetic interactions among ATV, its metabolite 2-hydroxy atorvastatin (2-ATV), and NG, in the current study, we developed and validated a simple, rapid, and specific UPLC–MS/MS method to simultaneously determine the concentrations of these analytes in the rat plasma. Sample preparation was performed using simple protein precipitation. Chromatographic analysis was carried out on an Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm) using gradient elution mode, and these three analytes were detected using a Xevo® TQD triple quadrupole tandem mass spectrometer, in the positive ion electrospray ionization interface. The developed method showed good linearity over the following concentrations in rat plasma samples: 3–1200 ng/ml (r = 0.9965) for ATV, 1.5–600 ng/ml (r = 0.9934) for 2-ATV, and 3–1200 ng/ml (r = 0.9964) for NG. The assays were validated and satisfied the acceptance criteria recommended by U.S. Food and Drug Administration guidelines. Upon successful application of the method to a pharmacokinetic interaction study, the results indicated that NG significantly enhanced the bioavailability of ATV and 2-ATV.     

A sensitive and accurate quantitative method to determine N-arachidonoyldopamine and N-oleoyldopamine in the mouse striatum using column-switching LC–MS–MS: use of a surrogate matrix to quantify endogenous compounds

The transient receptor potential vanilloid 1 (TRPV1) channel, a nonselective Ca²⁺ and Na⁺ channel, is a molecular transducer of nociceptive stimuli. N-Arachidonoyl dopamine (NADA) and N-oleoyldopamine (OLDA), two unsaturated N-acyldopamines, are major activating endogenous TRPV1 ligands and their presence in mammalian brain tissue has been reported. However, the biological significance of NADA and OLDA remains unknown. To investigate their biological function in the nervous system, a sensitive and accurate quantitative method for determining endogenous NADA and OLDA in the brain is necessary. Thus, a column-switching liquid chromatography–tandem mass spectrometry (LC–MS–MS) method was developed to quantify NADA and OLDA in mouse striatum. Mouse cerebellum tissue in which neither NADA nor OLDA were detected was used as a surrogate matrix to prepare calibrators. NADA and OLDA were extracted from mouse brain tissue by solid-phase extraction and then filtered and analyzed by LC–MS–MS with electrospray ionization in the positive ion mode. The selectivity results and comparison of calibration curves prepared with mouse cerebellum and striatum established that the former was acceptable as the surrogate matrix of the latter for analyzing NADA and OLDA. The validation results of the matrix effect, linearity, precision, accuracy, and stability were satisfactory. The limits of detection and limits of quantification were 0.125 pg mg^?1 for both analytes. This method was sensitive and accurate enough to determine endogenous concentrations of these compounds in mouse striatum and will be very useful for further study of the biological functions of NADA and OLDA and other related factors in vivo.     

Highly efficient proteome analysis with combination of protein pre-fractionation by preparative microscale solution isoelectric focusing and identification by μRPLC-MS/MS with serially coupled long microcolumn

To improve the efficiency of proteome analysis, a strategy with the combination of protein pre-fractionation by preparative microscale solution isoelectric focusing, peptide separation by μRPLC with serially coupled long microcolumn and protein identification by ESI-MS/MS was proposed. By preparative microscale solution isoelectric focusing technique, proteins extracted from whole cell lysates of Escherichia coli were fractionated into five chambers divided by isoelectric membranes, respectively with pH range from 3.0 to 4.6, 4.6 to 5.4, 5.4 to 6.2, 6.2 to 7.0 and 7.0 to 10.0. Compared to the traditional on-gel IFF, the protein recovery could be obviously improved to over 95%. Subsequently, the enriched and fractionated proteins in each chamber were digested, and further separated by a 30-cm long serially coupled RP microcolumn. Through the detection by ESI-MS/MS, about 200 proteins were identified in each fraction, and in total 835 proteins were identified even with one-dimensional μRPLC-MS/MS system. All these results demonstrate that by such a combination strategy, highly efficient proteome analysis could be achieved, not only due to the in-solution protein enrichment and pre-fractionation with improved protein recovery but also owing to the increased separation capacity of serially coupled long μRPLC columns.     

LC–MS/MS method for the determination of erianin in rat plasma: Application to a pharmacokinetic study

Erianin is one of the bibenzyl ingredients isolated from Dctidrobium chrysotoxum Lindl. In recent years, erianin has attracted attention owing to its antitumor activity. In this study, an LC–MS/MS method was established to measure erianin in rat plasma. Gigantol was used as the internal standard. A Waters Acquity UPLC BEH C₁₈ column was employed for chromatographic separation. The mobile phase consisted of water containing 0.1% formic acid and acetonitrile with a gradient elution at the flow rate of 0.4 ml/min. Selective reaction monitoring mode was used for quantitative analysis of erianin in positive electrospray ionization. In the concentration range of 0.1–1200 ng/ml, erianin in rat plasma was linear with correlation coefficient >0.999. The lowest limit of quantification was 0.1 ng/ml. The intra- and inter-day RSDs were <9.69%, while the RE was in the range of −8.59–11.24%. The mean recovery was >85.37%. Erianin was stable in rat plasma after storage under certain conditions. The validated method was demonstrated to be selective, sensitive and reliable, and has been successfully applied to pharmacokinetic study of erianin in rat plasma. Erianin was rapidly eliminated from rat plasma with a short half-life (〜1.5 h) and low oral bioavailability (8.7%).     

Quantitative and Qualitative Analysis of CKD-501, Lobeglitazone,in Human Plasma and Urine Using LC–MS/MS and Its Application to a Pharmacokinetic Study

A simple, rapid and sensitive LC–MS/MS method in positive ion mode was developed and validated to determine CKD-501, lobeglitazone, in human plasma and urine using glipizide as an internal standard (IS). Lobeglitazone is a novel thiazolidinedione (TZDs)-based peroxisome proliferator-activated receptor (PPAR) agonist, used for the management of type-2 diabetes. After mixing the IS, dissolved in acetonitrile, with a plasma or urine sample containing lobeglitazone, 10 μL of supernatant was injected into the LC–MS/MS system. Quantification was performed in the multiple reaction monitoring (MRM) mode using transition of 481.5 → 152.2 (m/z) for lobeglitazone and 446.1 → 321.2 (m/z) for the IS. The method showed good linearity over concentration ranges of 0.5–1,000 ng mL⁻¹ for plasma and 0.2–250 ng mL⁻¹ for urine (r ² ≥ 0.9996). The mean percent extraction recovery of lobeglitazone was 90.8 % for plasma and 87.3 % for urine, while the recoveries of the IS were greater than 86.4 % for both. The intra-day and inter-day precision of plasma ranged from 1.1 to 3.7 and 2.5 to 3.3 % (RSD), respectively, and the intra- and inter-day precision of urine ranged from 1.5 to 2.7 and 3.2 to 3.5 %, respectively. This method is simple, sensitive, and applicable for the pharmacokinetic study of lobeglitazone in human plasma. Most of the urine concentrations of lobeglitazone were below the LLOQ because the lobeglitazone is extensively metabolized.

     

Validation of a sensitive and specific LC–MS/MS method and application to evaluate the systemic exposure and bioavailability of glycopyrrolate via different drug delivery approaches/devices

A specific and sensitive LC–MS/MS method using d₃-glycopyrrolate as the internal standard (IS) was developed for the quantitative determination of glycopyrrolate (GLY) in human plasma over a concentration range of 4.00–2000 pg/ml. The GLY and IS were extracted using a solid-phase extraction cartridge, then eluted with 0.5% formic acid in 70:30 acetonitrile–water. The eluate was directly injected into an Agilent Pursuit 5PFP column for analysis using an isocratic mobile phase of 50:50 A:B at a flow-rate of 0.500 ml/min (A, 10 mm ammonium acetate in 1% formic acid; B, methanol). The MS detection was in positive mode by monitoring m/z 318.3 → 116.1 (GLY) and 321.3 → 119.1 (IS). The method validation showed the linearity of r² ≥ 0.9960, intra-/inter-run precisions of ≤11.1% coefficient of variation and accuracies ranging from −2.5 to 12.8% relative error for all levels of quality control samples. This method was successfully employed to support a clinical study to compare absorption and bioavailability of GLY administered by a Magnair^® eFlow nebulizer to a Seebri^® Breezhaler^® with/without a charcoal blockade of gastric absorption. By comparison with intravenous administration, respective bioavailabilities of ~15% for GLY/Magnair and ~22% for the Seebri Breezhaler were found. The bioanalytical reliability was also demonstrated by satisfactory incurred sample reanalysis performance.     

ZrCl4/Hantzsch 1,4-dihydropyridine as a new and efficient reagent combination for the direct reductive amination of aldehydes and ketones with weakly basic amines

ZrCl4/Hantzsch 1,4-dihydropyridine is a mild and highly efficient reagent combination for the direct reductive amination.Weakly basic amines such as anilines substituted by electron-withdrawing group and heteroaromatic amines can be reductivelyalkylated with electron rich aldehydes and ketones under mild conditions to form the secondary amines in excellent yields.     

Charge transfer spectra of organometallic complexes—VIII. Energy contributions to the formation of a donor—acceptor complex and to the electron transfer in the interaction between trialkyltindonors R3SnX and (X = NCS,Br) the acceptors,I2 and TCNE

The enthalpies of formation for charge transfer complexes of the type [R₃SnNCS-I₂] (R = Et, iPr), [Et₃SnNCSTCNE] and [R₃SnBrI₂] (R = Me, Et) have been determined by calorimetric measurements. The data are analyzed using Mulliken's resonance structure theory, to produce the different energy contributions to the charge transfer interaction in these complexes.     

Adding a new separation dimension to MS and LC–MS: What is the utility of ion mobility spectrometry?

Ion mobility spectrometry is an analytical technique known for more than 100 years, which entails separating ions in the gas phase based on their size, shape, and charge. While ion mobility spectrometry alone can be useful for some applications (mostly security analysis for detecting certain classes of narcotics and explosives), it becomes even more powerful in combination with mass spectrometry and high‐performance liquid chromatography. Indeed, the limited resolving power of ion mobility spectrometry alone can be tackled when combining this analytical strategy with mass spectrometry or liquid chromatography with mass spectrometry. Over the last few years, the hyphenation of ion mobility spectrometry to mass spectrometry or liquid chromatography with mass spectrometry has attracted more and more interest, with significant progresses in both technical advances and pioneering applications. This review describes the theoretical background, available technologies, and future capabilities of these techniques. It also highlights a wide range of applications, from small molecules (natural products, metabolites, glycans, lipids) to large biomolecules (proteins, protein complexes, biopharmaceuticals, oligonucleotides).     

A rapid UHPLC–MS/MS method for the quantification of ARQ531 in rat plasma: Validation and its application to a pharmacokinetic study

A simple and sensitive ultra-high performance liquid chromatography–tandem mass spectrometric (UHPLC–MS/MS) method was developed and validated for the determination of ARQ531, a Bruton’s tyrosine kinase inhibitor in rat plasma. After protein precipitation with acetonitrile, the samples were separated on a UPLC BEH C₁₈ column with 0.1% formic acid in water and acetonitrile as mobile phase at a flow rate of 0.4 ml/min. The mass detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring with precursor-to-product ion transitions of m/z 479.1 > 365.1 and m/z 441.2 > 138.1 for ARQ531 and internal standard, respectively. Good linearity (correlation coefficient > 0.9988) was achieved over the concentration range of 0.5–1,000 ng/ml and the lower limit of quantitation was 0.5 ng/ml. The accuracy ranged from −13.50 to 11.35% and the precision was <8.87%. The extraction recovery was >85.56%. ARQ531 was demonstrated to be stable under the tested conditions. The validated method was further applied to a pharmacokinetic study of ARQ531 in rats after intravenous (1 mg/kg) and oral (1, 3 and 10 mg/kg) administration. The results demonstrated that ARQ531 displayed linear pharmacokinetic profiles over the oral dose range of 1–10 mg/kg and good oral bioavailability (>50%).     

Quantification of low molecular weight fatty acids in cave drip water and speleothems using HPLC-ESI-IT/MS — development and validation of a selective method

This study presents a novel method for the analysis of low molecular weight (LMW) fatty acids in cave drip water and speleothems. The method development included optimization of sample preparation procedures, e.g., blank reduction, solid phase extraction, concentration of extracts as well as liquid chromatography coupled to electrospray ion-trap mass spectrometry (HPLC-ESI-IT/MS) measurement parameters. Retention times for five analytes (lauric acid, myristic acid, palmitic acid, stearic acid and arachidic acid) were between 5 and 13.5 min. Spiking experiments were performed to accomplish external calibrations which ranged from 12.5 to 75 ng per spiked water sample. The correlation coefficient ranged from 0.9558 to 0.9989. Inter-batch precision, expressed as the relative standard deviation of three replicates, was <7 %. Limits of detection ranged from 0.77 to 55.97 ng for the diverse analytes; obtained recoveries varied from 30 to 103 %. For a first application, cave drip water and stalagmite samples from Herbstlabyrinth-Adventhöhle cave system were analyzed. Concentrations ranged from 38.37 to 9,982.54 ng L^?1 for water samples and 2.52 to 1,344.96 ng g^?1 for the stalagmite. Thereby, the different fatty acids showed a distinctive variation. Whereas shorter-chained fatty acids exhibited similarities, arachidic acid showed opposite trends. Diverse correlations were found, which could improve the understanding of different organic sources of the lipids transported by drip water and preserved in speleothems. This new method provides a more selective extraction process, particularly adjusted to LMW fatty acids and therefore reduces the required sample size. Furthermore, it is applicable to stalagmite as well as cave drip water samples.     

Dried blood spot UHPLC-MS/MS analysis of oseltamivir and oseltamivircarboxylate—a validated assay for the clinic

The neuraminidase inhibitor oseltamivir (Tamiflu®) is currently the first-line therapy for patients with influenza virus infection. Common analysis of the prodrug and its active metabolite oseltamivircarboxylate is determined via extraction from plasma. Compared with these assays, dried blood spot (DBS) analysis provides several advantages, including a minimum sample volume required for the measurement of drugs in whole blood. Samples can easily be obtained via a simple, non-invasive finger or heel prick. Mainly, these characteristics make DBS an ideal tool for pediatrics and to measure multiple time points such as those needed in therapeutic drug monitoring or pharmacokinetic studies. Additionally, DBS sample preparation, stability, and storage are usually most convenient. In the present work, we developed and fully validated a DBS assay for the simultaneous determination of oseltamivir and oseltamivircarboxylate concentrations in human whole blood. We demonstrate the simplicity of DBS sample preparation, and a fast, accurate and reproducible analysis using ultra high-performance liquid chromatography coupled to a triple quadrupole mass spectrometer. A thorough validation on the basis of the most recent FDA guidelines for bioanalytical method validation showed that the method is selective, precise, and accurate (≤15% RSD), and sensitive over the relevant clinical range of 5–1,500 ng/mL for oseltamivir and 20–1,500 ng/mL for the oseltamivircarboxylate metabolite. As a proof of concept, oseltamivir and oseltamivircarboxylate levels were determined in DBS obtained from healthy volunteers who received a single oral dose of Tamiflu®.