Internal Standard Research Articles

Bioanalytical method development and validation for the quantification of raloxifene hydrochloride from mice plasma by RP-HPLC

Raloxifene hydrochloride (RLX) belongs to the Selective estrogen receptor modulator (SERM) class of drugs, exhibiting diverse affinity to estrogen receptors. The drug is known for its anti-cancer and anti-osteoporosis activity. The current study employs a Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method that is sensitive and time-efficient for quantitative evaluation of RLX from mice plasma, using tamoxifen citrate as an internal standard (IS). The protein precipitation technique was used to extract the RLX and IS. RLX was separated using a C8 column with an isocratic elution of a mobile phase consisting of acetonitrile and Milli-Q water (pH 3) at a 1 mL/min flow rate. The ultraviolet (UV) detection of the analyte and IS were carried out at 287 nm. The linearity plot was constructed in the range of 100–3200 ng/mL with a correlation coefficient of r2 ≥ 0.9996. The inter-day and intra-day accuracy and precision were found to be within acceptable limits. Stability studies revealed that the analyte remained stable. The validated RP-HPLC method was successfully applied to quantify RLX after oral administration in mice, and the pharmacokinetic (PK) parameters were established. The present RP-HPLC method could be a beneficial tool for the fast and reliable estimation of future PK interactions, PK analysis of formulations, and therapeutic drug monitoring.

Integration of protein L-immobilized epoxy magnetic bead capture with LC-MS/MS for therapeutic monoclonal antibody quantification in serum.

In recent years, there has been a growing interest in the thriving monoclonal antibody (mAb) industry due to the wide utilization of mAbs in clinical therapies. Robust and accurate bioanalytical methods are required to enable fast quantification of mAbs in biological matrices, especially in the context of pharmacokinetics (PKs)/pharmacodynamics (PDs) and therapeutic drug monitoring (TDM) studies. In this investigation, we presented a novel immuno-magnetic capture coupled with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method designed for the quantification of immunoglobulin G-kappa-based mAbs in biological fluids. The immunoaffinity absorbent for mAb drug purification was meticulously crafted by immobilizing protein L onto monosize, magnetic poly(glycidyl methacrylate) (m-pGMA) beads, synthesized through dispersion polymerization. The microspheres were acquired with an average size of 1.6 μm, and the optimal binding of mAbs from the aqueous mAb solution was determined to be 45.82 mg g-1. The quantification of mAbs in 10 μL serum samples was achieved through affinity purification using m-pGMA@protein L beads (employing rituximab as an internal standard (IS)), on-bead reduction, and rapid tryptic digestion. Remarkably, the entire process, taking less than 2.5 hours, held significant potential for simplifying pretreatment procedures and minimizing analytical time. Furthermore, the developed method underwent validation in accordance with the European Medicines Agency (EMA) guidelines. The assay demonstrated commendable linearity within the 2-400 μg mL-1 range for both daratumumab and pembrolizumab. Intra- and inter-assay coefficients of variation fell within the range of 0.7% to 13.4%, meeting established acceptance criteria. Other validation parameters also conformed to regulatory standards. Ultimately, the efficacy of the method was substantiated in a pharmacokinetic study following a single-dose intravenous administration to mice, underscoring its applicability and reliability in real-world scenarios.

Development and validation of a high-performance liquid chromatography with tandem mass spectrometry method for quantification of tofacitinib in human plasma

Introduction. The use of tofacitinib as a pharmacological treatment for rheumatoid arthritis remains relevant in the light of the predicted increase in prevalence of the disease. At the same time, due to the withdrawal of several foreign pharmaceutical companies from the Russian pharmaceutical market, there has been a heightened demand for domestically produced medications, including generic formulations of tofacitinib. Registration of generic drugs necessitates the conduct of bioanalytical studies. Development and validation of a method for quantifying the analyte in biosamples remains to be a crucial part of the bioequivalence studies.Aim. The aim of this research is to develop and validate a method for the quantitative determination of tofacitinib in human plasma using high-performance liquid chromatography as the separation system coupled with a tandem mass spectrometer for detection purposes.Materials and methods. Biosample preparation was based on plasma proteins precipitation using acetonitrile. Baricitinib was selected as an internal standard. The analytical range of the method was 1.00 to 200.00 ng/mL and was further expanded to 0.30 to 200.00 ng/mL during the analytical phase of the study. The mobile phase consisted of water and acetonitrile, both acidified with formic acid (0.1 % v/v). The stationary phase was a Phenomenex Kinetex C18 column [100 × 3.0 mm, with a particle size of 5 µm (Phenomenex, USA)]. Sample separation and detection were carried out using high-performance liquid chromatography coupled with a tandem mass spectrometry (HPLC-MS/MS), operating in positive ion mode. The multiple reaction monitoring (MRM) transitions selected for the analyte and the internal standard were 313.30 to 173.00 m/z and 371.90 to 186.00 m/z, respectively.Results and discussion. The developed assay was validated in accordance with the current requirements of regulatory documentation from the EAEU (Eurasian Economic Union), FDA (US Food and Drug Administration), and EMA (European Medicines Agency) with the following parameters being evaluated: selectivity, specificity, carry-over, matrix effect, recovery, calibration curve, lower limit of quantitation, accuracy, precision, stability. The validated method was applied in the analytical part of a bioequivalence study of domestically produced generic tofacitinib.Conclusion. A method for the quantitative determination of tofacitinib in human blood plasma with an analytical range of 0.30–200.00 ng/mL was developed and validated. Application of the assay during the analytical phase of bioequivalence study of generic tofacitinib confirms the possibility of using the method in similar bioanalytical investigations.

Integrated Synthesis of Calcitriol and 26,27-Hexadeutero Calcitriol

Calcitriol (1α,25-dihydroxyvitamin D3, 1), a classical vitamin D drug, is indicated primarily in the treatment of patients with postmenopausal osteoporosis and renal osteodystrophy. In this study, a practical synthesis of calcitriol (1), from readily available commercial vitamin D2 (5) via hub intermediate 18, has been accomplished in 9% overall yield. This semi-synthetic process embedded four prominent elements of vitamin D chemistry: (1) cheletropic sulfur dioxide (SO2) adduction for the isomerization of the characteristic triene from (5Z,7E) to (5E,7E), or for the protection of the triene for selective ozonolysis of the side chain, and cheletropic extrusion of SO2 from the adduct in ethanolic sodium bicarbonate to retrieve the triene; (2) direct, regio- and stereoselective 1α-hydroxylation of 3β-TBS-protected (5E)-calciferol intermediate 19 using selenium dioxide in the presence of N-methylmorpholine N-oxide as a re-oxidant in a hot mixture of methylene chloride and methanol; (3) nickel(0)-mediated conjugate addition of the 22-iodide 23 to electron-deficient ethyl acrylate followed by Grignard reaction with methylmagnesium bromide to construct the calcitriol side chain; and (4) triplet-sensitized photoisomerization of 26 to access the bioactive (5Z,7E)-triene in calcitriol (1). The high-performance liquid chromatography purities of batches of the synthesized calcitriol (1) were consistently more than 99.9%, with related substances listed in the USP 2023 and EP 11.0 well controlled. This robust process proved amenable to pilot scale-up and industrial production. 26,27-Hexadeutero calcitriol (4), a deuterium-labeled calcitriol derivative, is useful as the internal standard in the bioanalysis for the quantification of calcitriol in serum. 4 was efficiently synthesized in an integrated manner from hub intermediate 18 in 48% yield.

Improved microwave-assisted saponification to reduce the variability of MOAH determination in edible oils

BackgroundMineral oil aromatic hydrocarbon (MOAH) analysis in foods is a major analytical challenge. Quantification is associated with a high uncertainty. The sources of uncertainty are multiple, but the major one is related to data interpretation and integration, which is partially derived from insufficiently efficient sample preparation. Recently, an updated ISO method for the analysis of mineral oil in fats and oils and a standard operating procedure for infant formula analysis have been published. Both methods reported significantly different (up to 1.25) distributions of the internal standards used for quantification (i.e., tri-tert-butyl benzene (TBB) and 2-methyl naphthalene (2-MN)) over the different solvent phases used in the saponification step. ResultsIn this work, a microwave-assisted saponification and extraction method was optimized for MOAH analysis to solve the problem related to the MOAH internal standards partition. The paper examines the impact of the solvent mixture used, the concentration of KOH on the partition of TBB and 2-MN, and the effect of the matrix and the washing step to extract the unsaponifiable fraction containing the mineral oils. SignificanceThe optimized procedure achieved a TBB/2-MN ratio of 1.05 ± 0.01 tested in five different fats and oils, namely, sunflower, rapeseed, coconut, palm, and extra virgin olive oils. The method can significantly contribute to reducing the uncertainty of the MOAH quantification when saponification is applied.

Rapid determination of four flavonoids from hawthorn leaves in plasma by ultra‐high performance liquid chromatography‐tandem mass spectrometry and study on its pharmacokinetics

AbstractIn this study, a rapid and sensitive ultra‐high performance liquid chromatography‐tandem mass spectrometry method was established for the simultaneous determination of four Vitexin‐4“‐O‐glucoside (VOG), Vitexin‐2”‐O‐rhamnoside (VOR), vitexin (VIT), and hyperoside (HP) in plasma, which are representative flavonoids constituents of hawthorn leaves. The four components were separated by CORTECS UPLC C18 column (100 × 3.0 mm, 1.6 µm), using acetonitrile‐water containing 0.02% formic acid and 2 mmol/L ammonium formate as the mobile phase, the gradient elution flow rate was 0.30 mL/min. The analyte was quantified using an electrospray ionization triple quadrupole mass spectrometer. The analytes and baicalin internal standard were performed at m/z 593.2/413.2 for VOG, m/z 577.2/413.1 for VOR, m/z 431.1/310.9 for VIT, m/z 463.2/300 for HP and m/z 445.1/269 for internal standard. Among the four flavonoids, the correlation coefficient of the standard curve of each component is between 0.9939 and 0.9987. After oral administration of hawthorn leaf extract, VOG and VOR showed similar pharmacokinetic characteristics; VIT was eliminated in the body more slowly than the other three flavonoids and distributed more in the tissues; HP can be quickly absorbed in the blood. The developed analytical method has high sensitivity, precision, and accuracy with no interference from plasma components and has been successfully used in pharmacokinetic studies.

Electric Field-Modulated Electrospray Ionization Mass Spectrometry for Quantity Calibration and Mass Tracking.

Analyses conducted by electrospray ionization (ESI) mass spectrometry (MS) typically entail performing a number of preparatory steps, which include quantity calibration and mass calibration. Quantity calibration can be affected by signal noise, while mass calibration can be affected by instrumental drift if analyses are performed over an extended period of time. Here, we present two methods for achieving these calibrations using modulation of electrospray plume by alternating electric fields and demodulating the resulting MS ion currents. For this purpose, we use an ESI source fitted with three ring electrodes between the electrospray emitter and the mass spectrometer's inlet. One of these electrodes is supplied with a sine electric signal. Optionally, a nanoESI emitter is also placed between the ring electrodes and the mass spectrometer's orifice to supply calibrant ions. The ion currents, recorded with this setup, present wave-like features. In the first variant, using a triple quadrupole mass analyzer, the ion currents are subjected to data treatment by fast Fourier transform (FFT), and the resulting FFT magnitudes are correlated with analyte concentrations to produce a calibration plot. In the second variant, using a quadrupole time-of-flight mass analyzer, the mass spectra recorded at the analyte ion current maxima are mass-checked using the m/z value of the internal standard (injected via nanoESI emitter), which appears predominantly in the time intervals corresponding to the analyte ion current minima. The setup has been characterized using simulation software and optimized. Overall, the method enables the preparation of quantity calibration plots and monitoring (minor) m/z drifts during prolonged analyses.

A simple and sensitive ultra-high performance liquid chromatography tandem mass spectrometry method for the quantitative analysis of VX-548 in monkey plasma: Method validation and application to pharmacokinetic study.

VX-548 is an orally active and highly selective NaV 1.8 inhibitor that is undergoing development for the treatment of acute pain. The aim of this study was to develop a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the measurement of VX-548 in monkey plasma. VX-548 was extracted from the plasma using acetonitrile-mediated protein precipitation. The quantitative analysis was performed on a Thermo Vantage TSQ mass spectrometer with ibrutinib as an internal standard. Chromatography was performed on a Waters ACQUITY UPLC BEH C18 column with 0.1% aqueous formic acid and acetonitrile as mobile phase. The precursor-to-product ion transitions were m/z 474.2 > 165.0 and m/z 441.2 > 138.1 for VX-548 and internal standard, respectively. This developed method was successfully validated in the concentration range of 1-1000 ng/mL. The calibration curve showed excellent linearity with a correlation coefficient of >0.999. The precision expressed as relative standard deviation (RSD) was <8.4%, whereas the accuracy denoted as relative error (RE) ranged from -5.0% to 9.1%. The mean recovery was >84%. VX-548 was stable in monkey plasma after storage under certain conditions. The validated method was successfully applied to the pharmacokinetic study of VX-548 in monkey plasma after single oral (2 mg/kg) and intravenous (1 mg/kg) administrations.

Pharmacokinetic assessment of tacrolimus in combination with deoxyschizandrin in rats

BackgroundTacrolimus (Tac) is commonly used for postoperative immunosuppressive therapy in transplant patients. However, problems, for example, low bioavailability and unstable plasma concentration, persist for a long time, Studies have reported that the deoxyschizandrin could effectively improve these problems, but the pharmacokinetic parameters (PKs) of Tac combined with deoxyschizandrin are still unknown.MethodIn this study, an UHPLC-MS/MS method has been established for simultaneous quantitation of Tac and deoxyschizandrin. The PKs of Tac influenced by different doses of deoxyschizandrin after single and multiple administrations were analyzed, and the different impact of deoxyschizandrin and Wuzhi capsule on PKs of Tac were compared.ResultThe modified UHPLC-MS/MS method could rapid quantification of Tac and deoxyschizandrin within 2 min using bifendatatum as the internal standard (IS). All items were successfully validated. The Cmax of deoxyschizandrin increased from 148.27 ± 23.20 to 229.13 ± 54.77 ng/mL in rats after multiple administrations for 12 days. After co-administration of 150 mg/mL deoxyschizandrin, Tac had an earlier Tmax and greater Cmax and AUC0–t, and the Cmax and AUC0–t of Tac increased from 14.26 ± 4.73 to 54.48 ± 14.37 ng/mL and from 95.10 ± 32.61 to 315.23 ± 92.22 h/ng/mL, respectively; this relationship was positively proportional to the dosage of deoxyschizandrin. In addition, compared with Wuzhi capsule, the same dose of deoxyschizandrin has a better effective on Tac along with more stable overall PKs.ConclusionAn UHPLC-MS/MS method was established and validated for simultaneous detection of deoxyschizandrin and Tac. Deoxyschizandrin could improve the in vivo exposure level and stability of Tac, besides, this effect is better than Wuzhi capsule in same dose.

Determination of Patulin in Apple Juice and Apple-Derived Products Using a Robotic Sample Preparation System and LC-APCI-MS/MS

Patulin, a toxic mycotoxin, can contaminate apple-derived products. The FDA has established an action level of 50 ppb (ng/g) for patulin in apple juice and apple juice products. To effectively monitor this mycotoxin, there is a need for adequate analytical methods that can reliably and efficiently determine patulin levels. In this work, we developed an automated sample preparation workflow followed by liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS) detection to identify and quantify patulin in a single method, further expanding testing capabilities for monitoring patulin in foods compared to traditional optical methods. Using a robotic sample preparation system, apple juice, apple cider, apple puree, apple-based baby food, applesauce, fruit rolls, and fruit jam were fortified with 13C-patulin and extracted using dichloromethane (DCM) without human intervention, followed by an LC-APCI-MS/MS analysis in negative ionization mode. The method achieved a limit of quantification of 4.0 ng/g and linearity ranging from 2 to 1000 ng/mL (r2 &gt; 0.99). Quantitation was performed with isotope dilution using 13C-patulin as an internal standard and solvent calibration standards. Average recoveries (relative standard deviations, RSD%) in seven spike matrices were 95% (9%) at 10 ng/g, 110% (5%) at 50 ng/g, 101% (7%) at 200 ng/g, and 104% (4%) at 1000 ng/g (n = 28). The ranges of within-matrix and between-matrix variability (RSD) were 3–8% and 4–9%, respectively. In incurred samples, the identity of patulin was further confirmed with a comparison of the information-dependent acquisition-enhanced product ion (IDA-EPI) MS/MS spectra to a reference standard. The metrological traceability of the patulin measurements in an incurred apple cider (21.1 ± 8.0 µg/g) and apple juice concentrate (56.6 ± 15.6 µg/g) was established using a certified reference material and calibration data to demonstrate data confidence intervals (k = 2, 95% confidence interval).

Quantitation of tazemetostat in human plasma using liquid chromatography-tandem mass spectrometry.

To support a phase 1 trial in patients with lymphomas, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for tazemetostat quantitation in 20 μL of human plasma. After protein precipitation, chromatographic separation employed a Kinetex C18 column and a gradient of 0.1% formic acid in both water and acetonitrile, during a 3-min run time. Detection was achieved using a SCIEX 6500+ tandem mass spectrometer with electrospray positive-mode ionization. Validation was based on the latest Food and Drug Administration guidance. With a stable isotopic internal standard, the assay was linear within the range of 10-5000 ng/mL and proved to be accurate (91.9%-103.7%) and precise (<4.4% imprecision). Recovery varied between 93.3% and 121.1%, and matrix effect ranged from -25.5% to -4.9%. Hemolysis, lipemia, and dilution did not impact quantitation. Plasma stability was confirmed after three freeze-thaw cycles, 24 h at room temperature, and 4months at -80°C. Incurred sample reanalysis yielded 94.4% samples within 20% difference (n = 36). External validation showed a mean bias of -11.1%. Pharmacokinetic (PK) data obtained from three patients suggested variable concentration time profiles, warranting collection of further data. The assay proved to be suitable for tazemetostat quantitation in human plasma and will support clinical studies by defining tazemetostat PKs.

Pharmacokinetic and oral bioavailability study of schaftoside in rats.

A simple and sensitive LC-tandem mass spectrometry method was established and validated for the determination of schaftoside in rat plasma. After prepared by protein precipitation with acetonitrile, schaftoside and internal standard were separated on a Waters HSS T3 column using acetonitrile containing 0.1% formic acid and 0.1% formic acid in water as the mobile phase by gradient elution. The method showed excellent linearity over the range of 0.5-500 ng/mL with acceptable intra- and inter-day precision, accuracy, matrix effect, and recovery. The stability assay indicated that schaftoside was stable during the sample acquisition, preparation, and storage. The method was applied to a pharmacokinetic study of schaftoside in rats. The result suggested that after intravenous administration at a dose of 1 mg/kg, schaftoside was quickly eliminated from the plasma with an elimination half-life of 0.58 h. After oral administration at doses of 5, 10, and 20 mg/kg, schaftoside was quickly absorbed into the plasma and reached the peak concentration (Cmax) of 45.1-104.99 ng/mL at 0.67-1.17 h. The increase of exposure (area under the curve) was linear with the increase of dose. The oral bioavailability was 0.42%-0.71% in the range of 5-20 mg/kg.

A Sensitive Liquid Chromatography-Tandem Mass Spectrometry Method for Measuring Fosfomycin Concentrations in Human Prostatic Tissue.

The aim of this study was to develop and validate a fast and sensitive bioanalytical method for the accurate quantification of fosfomycin concentrations in human prostatic tissue. The sample preparation method only required milligrams of tissue sample. Each sample was mixed with two times its weight of water and homogenized. A methanol solution that was three times the volume of the internal standard (fosfomycin-13C3) was added, followed by vortex mixing and centrifugation. After its extraction from the homogenized prostatic tissue, fosfomycin was quantified by means of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) triple quadrupole system operating in negative electrospray ionization and multiple reaction monitoring detection mode. The analytical procedure was successfully validated in terms of specificity, sensitivity, linearity, precision, accuracy, matrix effect, extraction recovery, limit of quantification, and stability, according to EMA guidelines. The validation results, relative to three QC levels, were 9.9% for both the within-day and inter-day accuracy (BIAS%); 9.8% for within-day precision; and 9.9 for between-day precision. A marked matrix effect was observed in the measurements but was corrected by normalization with the internal standard. The average total recovery was high (approximatively 97% at the three control levels). The dynamic range of the method was 0.1-20 μg/g (R2 of 0.999). Negligible carry-over was observed after the injection of highly concentrated samples. F in the sample homogenate extracts was stable at 10 °C and 4 °C for at least 24 h. In the tissue sample freeze-thaw experiments, a significant decrease in F concentrations was observed after only two cycles from -80 °C to room temperature. The novel method was successfully applied to measure fosfomycin in prostatic tissue samples collected from 105 patients undergoing prostatectomy.

An Inhibitor-Monitorable Single-Tube Duplex Quantitative Real-Time PCR Assay for the Detection of 'Candidatus Liberibacter asiaticus'.

Huanglongbing (HLB) is a citrus infectious disease caused by 'Candidatus Liberibacter' spp. Recently, it has begun to spread rapidly worldwide, causing significant losses to the citrus industry. Early diagnosis of HLB relies on quantitative real-time PCR assays. However, the PCR inhibitors found in the nucleic acid extracted from plant materials pose challenges for PCR assays because they may result in false-negative results. Internal standard (IS) can be introduced to establish a single-tube duplex PCR for monitoring the influence of the PCR inhibitor, but it also brings the risk of false-negative results because the amplification of IS may compete with the target. To solve this problem, we proposed a mutation-enhanced single-tube duplex PCR (mSTD-PCR) containing IS with mutant-type primers. By introducing the 3'-terminal mutation in the primer of IS to weaken its amplification reaction and its inhibition of 'Candidatus Liberibacter asiaticus' (CLas) detection, the sensitivity and quantitative accuracy of CLas detection will not be affected by IS. In evaluating the sensitivity of CLas detection using simulation samples, the mSTD-PCR showed consistent sensitivity at 25 copies per test compared with the single-plex CLas assay. The detection result of 30 leaves and 30 root samples showed that the mSTD-PCR could recognize false-negative results caused by the PCR inhibitors and reduce workload by 48% compared with the single-plex CLas assay. Generally, the proposed mSTD-PCR provides a reliable, efficient, inhibitor-monitorable, quantitative screening method for accurately controlling HLB and a universal method for establishing a PCR assay for various pathogens.

Characterization and Quantitation of Anthocyanins of the Pigmented Tea Cultivar TRI 2043 (Camellia sinensis L.) from Sri Lanka

Tea leaves are rich in diverse bioactive compounds. The tea accession TRI 2043 is unique due to its pigmented leaves caused by anthocyanins, clonal origin, resistance to blister blight disease, and high pubescence density. Because of its peculiarity, TRI 2043 is used to produce high-quality silver tip tea, a premium type of tea that commands high prices. This study was carried out to clarify and elucidate the types of anthocyanins in this particular accession. Four different anthocyanin species were identified and quantitated as cyanidin-3-O-β-d-galactoside and delphinidin-3-O-β-d-galactoside equivalents for leaf blades and stems of the cultivar TRI 2043. The characterization was performed by comparison with commercially available reference substances and further confirmed using ion mobility high-resolution time-of-flight-mass spectrometry (IMS-HRTOF-MS). Quantitation was carried out using ultra-high-performance liquid chromatography ultraviolet–visible detection (UHPLC-UV-vis) with cyanidin-3-O-β-d-glucoside as an internal standard. E- and Z-geometric isomers of 6-p-coumaroyl derivates of delphinidin and cyanidin-3-O-β-d-galactopyranosides were observed, and collision cross section (CCS) values were determined for all four different anthocyanidin species. The content of anthocyanins in leaf blades of cultivar TRI 2043 was 856.32 ± 41.56 µg/g dry weight, with cyanidin being the more abundant anthocyanin (69.8%). Conversely, the stem material contained an anthocyanin amount of 459.5 ± 44.7 µg/g dry weight, with a higher content of delphinidin (69.6%). In summary, an enrichment strategy using analytical membrane chromatography was established to fully elucidate and quantify the anthocyanin profile of plant samples such as the special tea variety TRI 2043.

A validated ultra-performance liquid chromatography with tandem mass spectrometry method for the quantification of Brazilian green propolis main compounds

Brazilian green propolis is used in folk medicine because of its various biological properties. The hydroalcoholic extract of Brazilian green propolis is characteristic for possessing several pharmacological properties. Phytochemical investigations have attributed some of these properties to the presence of compounds, which were chosen as analytical markers. This paper reports the development and analytical validation using UPLC-MS/MS in MRM mode. Veratraldehyde was used as an internal standard in qualitative and quantitative analyses of the extracts. Relative standard deviation values obtained for intra-day and inter-day precision were lower than 4%. Of the five parameters for robustness, wavelength detection and flow rate were the critical ones. Limits of detection and quantification ranged from 0.300 to 39.500 ng.mL−1 and from 1.400 to 85.00 ng.mL−1, respectively. The recoveries were between 94.00 and 119.00%, with relative standard deviation values around 5.0%. The developed method is precise, sensitive, and reliable for analysing Brazilian green propolis.

Post column infusion of an internal standard into LC-FT-ICR MS enables semi-quantitative comparison of dissolved organic matter in original samples.

Ultrahigh resolution mass spectrometry hyphenated with liquid chromatography (LC) is an emerging tool to explore the isomeric composition of dissolved organic matter (DOM). However, matrix effects limit the potential for semi-quantitative comparison of DOM molecule abundances across samples. We introduce a post-column infused internal standard (PCI-IS) for reversed-phase LC-FT-ICR MS measurements of DOM and systematically evaluate matrix effects, detector linearity and the precision of mass peak intensities. Matrix effects for model compounds spiked into freshwater DOM samples ranging from a headwater stream to a major river were reduced by 5-10% for PCI-IS corrected mass peak intensities as compared to raw (i.e., untransformed) intensities. A linear regression of PCI-IS corrected DOM mass peak intensities across a typical DOM concentration range (2-15 mg dissolved organic carbon L-1) in original, non-extracted freshwater samples demonstrates excellent linearity of the detector response (r2 > 0.9 for 98% of detected molecular formulas across retention times). Importantly, PCI-IS could compensate for 80% of matrix effects across an environmental gradient of DOM composition from groundwater to surface water. This enabled studying the ionization efficiency of DOM isomers and linking the observed differences to the biogeochemical sources. With PCI-IS original, non-extracted DOM samples can be analysed by LC-FT-ICR MS without carbon load adjustment, and mass peak intensities can be reliably used to semi-quantitatively compare isomer abundances between compositionally similar DOM samples.

Determination of pancratistatin in mouse blood by ultra-liquid chromatography-tandem quadrupole mass spectrometry and its application

AbstractIn this study, a UPLC-MS/MS method was developed for determination of pancratistatin in the mouse blood, and the pharmacokinetics of pancratistatin in mice after intravenous (5 mg kg−1) and intragastric (15 mg kg−1) administration was studied. HSS T3 column was used for separation with mobile phases of acetonitrile and 0.1% formic acid using gradient elution procedure. The blood sample was treated by protein precipitant with acetonitrile, midazolam was used as internal standard (IS). Multiple reaction monitoring mode (MRM) was used for quantitative analysis, m/z 326.2→83.8 for pancratistatin and m/z 326.2→291.4 for IS in electrospray (ESI) positive interface. It showed a good linear in the range of 10–4,000 ng mL−1 (r &gt; 0.998); the intra-day and inter-day precision was &lt;15%, and the accuracy was 93%–105%. The recovery was better than 82%, and the matrix effect was 94%–105%. The developed UPLC-MS/MS method was fast, selective, and suitable for the pharmacokinetics of pancratistatin in mice.

Olaris Global Panel (OGP): A Highly Accurate and Reproducible Triple Quadrupole Mass Spectrometry-Based Metabolomics Method for Clinical Biomarker Discovery.

Mass spectrometry (MS)-based clinical metabolomics is very promising for the discovery of new biomarkers and diagnostics. However, poor data accuracy and reproducibility limit its true potential, especially when performing data analysis across multiple sample sets. While high-resolution mass spectrometry has gained considerable popularity for discovery metabolomics, triple quadrupole (QqQ) instruments offer several benefits for the measurement of known metabolites in clinical samples. These benefits include high sensitivity and a wide dynamic range. Here, we present the Olaris Global Panel (OGP), a HILIC LC-QqQ MS method for the comprehensive analysis of ~250 metabolites from all major metabolic pathways in clinical samples. For the development of this method, multiple HILIC columns and mobile phase conditions were compared, the robustness of the leading LC method assessed, and MS acquisition settings optimized for optimal data quality. Next, the effect of U-13C metabolite yeast extract spike-ins was assessed based on data accuracy and precision. The use of these U-13C-metabolites as internal standards improved the goodness of fit to a linear calibration curve from r2 < 0.75 for raw data to >0.90 for most metabolites across the entire clinical concentration range of urine samples. Median within-batch CVs for all metabolite ratios to internal standards were consistently lower than 7% and less than 10% across batches that were acquired over a six-month period. Finally, the robustness of the OGP method, and its ability to identify biomarkers, was confirmed using a large sample set.

Development and validation of HPLC-ultraviolet method for quantitative determination of pritelivir in human placental perfusion medium.

A simple and reliable HPLC-ultraviolet (HPLC-UV) method was developed and validated for the quantification of pritelivir in the samples of medium from the experiments utilizing the ex vivo technique of dual perfusion of the human placental lobule. Phenacetin was used as an internal standard (IS) in our HPLC-UV method. Chromatographic separation of pritelivir and phenacetin was achieved on a Waters Symmetry C18 HPLC column (100 × 2.1 mm, 3.5 μm) at ambient temperature (22-25°C). The mobile phase was composed of 50% methanol in deionized water (v/v), the flow rate for isocratic elution was established at 0.25 mL/min, and the detection wavelength for pritelivir and IS was set at 254 nm. Pritelivir and IS were extracted with the protein precipitation method using methanol as a solvent. The calibration curve for pritelivir exhibited linearity (r2 > 0.99) within the concentration range from 0.155 to 6.62 μg/mL. Within- and between-day accuracy ranged from 97% to 110% with relative standard deviation (RSD) values not exceeding 10%. The extraction recovery of pritelivir and IS ranged from 89% to 91% with RSD not exceeding 7%. Pritelivir was stable under the storage and sample handling conditions. This validated HPLC-UV method was utilized to quantify pritelivir in the placental perfusion medium samples, and the resulting concentrations were authenticated with incurred sample reanalysis to confirm the reliability of the method.