Internal Standard Research Articles

Simultaneous estimation of paclitaxel and bortezomib via LC-MS/MS: pharmaceutical and pharmacokinetic applications.

Aim & Objective: This study evaluates the potential of combining paclitaxel (PTX) and bortezomib (BTZ) for breast cancer therapy. Materials & Methods: The nanoformulation was optimized via Box-Behnken Design (BBD), with method validation adhering to US-FDA guidelines. Results: Multiple reaction monitoring transitions for PTX, BTZ and internal standard were m/z 855.80→286.60, 366.80→226.00 and 179.80→110.00, respectively. Elution done on C18 Luna column with 0.1% FA in MeOH:10mM ammonium acetate. The size of nanoformulation was 133.9±1.97nm, PDI 0.19±0.01 and zeta potential -19.20±1.36mV. Pharmacokinetics showed higher Cmax for PTX-BTZ-NE (313.75±10.71ng/ml PTX, 11.92±0.53ng/ml BTZ) versus free PTX-BTZ (104±13.06ng/ml PTX, 1.9±0.08ng/ml BTZ). Conclusion: Future findings will contribute to the treatment of breast cancer using PTX and BTZ.

Determination of Gb3 and Lyso-Gb3 in Fabry Disease-Affected Patients by LC-MRM/MS

Limited or absent activity of the enzyme α-galactosidase A (α-Gal A), due to mutation in the related gene on the X chromosome, leads to the development of a rare hereditary and genetic disease known as Fabry disease (FD). This pathology involves a progressive accumulation in various organs of the substrates of the enzyme e.g., globotriaosylceramide (Gb3) and its deacylated form, globotriaosylsphingosine (Lyso-Gb3), suggesting these molecules as biomarkers of Fabry disease. The present paper describes the development of an analytical strategy for the identification and quantification of Gb3 and Lyso-Gb3, in serum and blood samples by using liquid chromatography (LC) coupled to mass spectrometry in multiple reaction monitoring (MRM/MS) ion mode. The best experimental conditions were obtained by extracting the glycolipids with chloroform/methanol/H2O (2/1/0.3) and by separating them on a C4 column with a linear gradient (A: H2O with 2 mM ammonium formate. B: methanol with 1 mM ammonium formate, both acidified with 0.2% formic acid). The best transitions (a combination of precursor and fragment ions—m/z) were 786.8 m/z > 268.3 m/z for Lyso-GB3, 1137.3 m/z > 264.3 m/z for Gb3, 1039.3 m/z > 264.4 m/z for N-heptadecanoyl-ceramide trihexoside, and 843.5 m/z > 264.3 m/z for N-glycinated lyso-ceramide trihexoside, the latter being used as an internal standard. The developed method provided a reliable, fast, and effective procedure for direct measurements of GB3 and Lyso-GB3 in serum and blood for diagnosis of Fabry disease, suggesting this method as a complementary assay to the current enzymatic test. Therefore, this approach could open new insights into the clinical diagnostics of lysosomal storage disorders.

Analytical Method Development and Validation of Simultaneous Estimation of Pure and Tablet Dosage Form by RP-HPLC

The aim of this analytical research is to establish and validate the RP-HPLC (Reverse Phase High Performance Liquid Chromatography) method for concurrently quantifying Montelukast Sodium, Fexofenadine Hydrochloride, and Acebrophylline in a pharmaceutical formulation. The method utilized Rosagiline mesylate as an internal standard, employing a Shim-Pack Solar C18 Column (4.6 x 150 mm, 5 µm) as the stationary phase and a mobile phase composed of acetonitrile, methanol, and 10 mM Na2HPO4 buffer (50:30:20 % v/v/v, pH 5.5). The flow rate was maintained at 1.0 mL/min, and detection occurred at 210 nm. Validation followed ICH guidelines. The linear concentration ranges were 5-30 µg/ml, 10-60 µg/ml, and 10-100 µg/ml at 210 nm for Montelukast Sodium, Fexofenadine Hydrochloride, and Acebrophylline, respectively. Retention times were 7.643 min, 2.117 min, 3.863 min, and 3.050 min for Montelukast sodium, Fexofenadine Hydrochloride, Acebrophylline, and Rosagiline mesylate respectively. The RP-HPLC analysis of marketed formulations yielded concentrations within the ranges of 98.3–100.9%, 99.46–101.23%, and 99.82–101.74% for Montelukast sodium, Fexofenadine Hydrochloride, and Acebrophylline, respectively. Recovery fell within the range of 95.81-101.35% for both drugs. The proposed method has been validated as per ICH guidelines and successfully applied to the estimation of Montelukast, Fexofenadine and Acebrophylline in their combined tablet dosage form.

Synthesis of n-isomers: Native and deuterium-labelled short-chain perfluoroalkane sulfonamide derivatives

Perfluorooctane sulfonamide derivatives have been extensively used by industry for their surfactant properties and as building blocks for other perfluoroalkyl substances (PFAS). Due to their environmental impact and their potential degradation to other harmful PFAS, short-chain sulfonamide derivatives alternatives were introduced. However, these are now also suspected to be present in different environmental matrices and there is a lack of labelled and unlabelled reference standards to perform analyses. To address this gap, 40 native and deuterium labelled short-chain perfluoroalkane sulfonamide derivatives were synthesized, utilizing commercial perfluoroalkane sulfonyl fluoride as starting materials. All products were synthesized and then purified to obtain linear n-isomer reference standards. NMR, GC-FID-MS and LC-MS techniques were used for product identification and purity assessment. Recrystallization method was developed to selectively isolate the n-isomer from the isomer mixtures, thereby providing valuable reference and internal standards for environmental and toxicological investigations.

Self-Referenced Au Nanoparticles-Coated Glass Wafers for In Situ SERS Monitoring of Cell Secretion.

Surface-enhanced Raman spectroscopy (SERS) is a powerful technique for discrimination of bimolecules in complex systems. However, its practical applications face challenges such as complicated manufacturing procedures and limited scalability of SERS substrates, as well as poor reproducibility during detection which compromises the reliability of SERS-based analysis. In this study, we developed a convenient method for simultaneous fabrication of massive SERS substrates with an internal standard to eliminate the substrate-to-substrate differences. We first synthesized Au@CN@Au nanoparticles (NPs) which contain embedded internal standard molecules with a single characteristic peak in the Raman-silent region, and then deposited the NPs on 6 mm glass wafers in a 96-well plate simply by centrifugation for 3 min. The one-time obtained 96 SERS substrates have excellent intrasubstrate uniformity and intersubstrate repeatability for SERS detection by using the internal standard (relative standard deviation = 10.47%), and were able to detect both charged and neutral molecules (crystal violet and triphenylphosphine) at a concentration of 10-9 M. Importantly, cells can be directly cultured on glass wafers in the 96-well plate, enabling real time monitoring of the secretes and metabolism change in response to external stimulation. We found that the release of nucleic acids, amino acids and lipids by MDA-MB-231 cells significantly increased under hypoxic conditions. Overall, our approach enables fast and large-scale production of Au@CN@Au NPs-coated glass wafers as SERS substrates, which are homogeneous and highly sensitive for monitoring trace changes of biomolecules.

Determination of ivermectin in plasma and whole blood using LC-MS/MS

Background Ivermectin is a widely used drug for the treatment of helminthiasis and filariasis worldwide, and it has also shown promise for malaria elimination through its potent mosquito-lethal activity. The objective of this study was to develop and validate a high-throughput and sensitive method to quantify ivermectin in plasma and whole blood samples, using automated sample extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Methods Phospholipids were removed in patient whole blood (100 µl) and plasma (100 µl) samples using a 96-well plate Hybrid-solid phase extraction technique. Ivermectin and its isotope-labelled internal standard (ivermectin-D2) were separated on an Agilent Poroshell 120 EC-C18 50mm × 3.0mm I.D. 2.7µm, using a mobile phase of acetonitrile: ammonium formate 2 mM containing 0.5% formic acid (90: 10, v/v). Detection was performed using a triple quadrupole mass spectrometer in the positive ionization mode. Results The method was validated in the concentration range 0.970 - 384 ng/ml in both plasma and whole blood matrices. Intra- and inter-batch precisions during the validation were below 15%. There was no carryover or matrix effects detected. Ivermectin is a stable compound and results showed no degradation in the different stability tests. Conclusions The validated method proved to have high sensitivity and precision, good selectivity and to be suitable for clinical application or laboratory quantification of ivermectin in plasma or whole blood samples.

Impact of internal standard selection on measurement results for long chain fatty acids in blood

IntroductionInternal standards correct for measurement variation due to sample loss. Isotope labeled analytes are ideal internal standards for the measurement of fatty acids in human plasma but are not always readily available. For this reason, quantification of multiple analytes at once is most often done using only a single or few internal standards. The magnitude of the impact this has on method accuracy and precision is not well studied for gas chromatography-mass spectrometry systems. ObjectiveThis study aims to estimate bias and changes in uncertainty associated with using alternative fatty acid isotopologue internal standards for the estimation of similar or dissimilar long chain fatty acids. MethodUsing a previously reported method for the quantification of 27 fatty acids in human plasma using 18 internal standards we obtained estimates of bias and uncertainty at up to three levels of fatty acid concentration. ResultsWith some notable exceptions, method accuracy remained relatively stable when using an alternative internal standard (Median Relative Absolute Percent Bias: 1.76%, Median Spike-Recovery Absolute Percent Bias: 8.82%), with larger changes in method precision (Median Increase in Variance: 141%). Additionally, the degree of difference between analyte and internal standard structure was related to the magnitude of bias and uncertainty of the measurement. ConclusionThe data presented here show that the choice of internal standard used to estimate fatty acid concentration can affect the accuracy and reliability of measurement results and, therefore, needs to be assessed carefully when developing analytical methods for the measurement of fatty acid profiles.Disclaimer: The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry. Use of trade names is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention, the Public Health Service, and the US Department of Health and Human Services.

Green analytical chemistry and quality by design: A combined approach towards simultaneous determination of Letrozole with its co-administered Zoledronic Acid for cancer patients

Nowadays, breast cancer is the most affecting and globally diagnosed malignancy among women, yet Letrozole (LTZ) was considered the first-line treatment as hormonal anticancer drug. Unfortunately, LTZ develops osteoporosis as a main side effect which was overcome by using the co-administered; Zoledronic Acid (ZDA). Thus, there was a crucial need for this simultaneous quantification innovation, especially there were no any previously reported methods regarding both drugs together. In this study, an integrated framework was conducted between the experimental analytical quality-by-design (AQbD) approach and green analytical chemistry (GAC), emerging sensitive and robust RP-HPLC method. Box-Behnken Design was the developed model for optimizing an isocratic chromatographic separation on C18 Equisil® ODS (4.6×250mm, 5.0 μm) column at ambient temperature, using the mobile phase of 0.1% aqueous trifluroacetic acid (pH 2.8): acetonitrile (54.5:45.5, v/v), at 1.0mL/min flow rate with PDA detection at 254.0nm and 210.0nm for LTZ and ZDA, respectively. Model statistical and residual plots analysis was significant and normally distributed. Method was fully validated as per ICH guidelines, where good linearity was 0.20-10.00µg/mL for both drugs in presence of Tadalafil (TDF) as an internal standard, obtaining adequate correlation coefficients (r) values. Calculated LOD results were 0.058 and 0.040µg/mL while calculated LOQ results were 0.175 and 0.122µg/mL for LTZ and ZDA, respectively. The proposed method was effectively applied on bulk, pharmaceutical dosage forms, and spiked human plasma. Statistical comparison of the anticipated results with the reported ones was performed. Greenness assessment was evaluated using Green Analytical Procedure Index (GAPI) and Analytical Greenness (AGREE) tools; where superiority results were achieved relative to other reported methods. Finally, an EVG method evaluation tool was assessed, and the attained results were represented through its radar chart.

Therapeutic drug monitoring of free perampanel concentrations in practice: A practical analytical technique based on centrifugal ultrafiltration sample separation

ObjectivesThe centrifugal ultrafiltration–high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was established to determine the free perampanel (PER) concentration in children with epilepsy. MethodsFree PER concentration was obtained using centrifugal ultrafiltration devices. The internal standard was PER-D5. The method was investigated for selectivity, carryover, lower limit of quantification, calibration curve, accuracy, precision, matrix effects, recovery, and stability. The Spearman's correlation coefficient was used to evaluate the correlation between the free and total PER concentrations. A nonparametric test was used to estimate the effects of PER along with other antiepileptic drugs on the total and free PER concentrations. ResultsThe free PER concentration was positively correlated with the total PER concentration in the 57 plasma samples (r = 0.793 > 0, P < 0.001). Additionally, the free PER concentrations were significantly (P < 0.05) increased in valproic acid (VPA) co-therapy (9.87 ± 5.83) compared with non-VPA co-therapy (5.03 ± 4.57). ConclusionsThe proposed method is efficient, sensitive, and suitable for detecting free PER concentrations in children with epilepsy. Simultaneously, the free PER concentration response to clinical outcomes in children with epilepsy was more clinically significant, particularly when combined with VPA.

An ultra-fast ultra-high-performance liquid chromatography-tandem mass spectrometry method for estimating the in vitro metabolic stability of palbociclib in human liver microsomes: In silico study for metabolic lability, absorption, distribution, metabolism, and excretion features, and DEREK alerts screening.

Palbociclib (Ibrance; Pfizer) was approved for the management of metastatic breast cancer characterized by hormone receptor-positive/human epidermal growth factor receptor 2 negative status. The objective of this study was to create a fast, precise, environmentally friendly, and highly sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry approach for quantifying palbociclib (PAB) in human liver microsomes with the application for assessing metabolic stability. The validation features were performed in agreement with the bioanalytical method validation standards outlined by the US Food and Drug Administration. The StarDrop software (WhichP450 and DEREK modules) was used in screening the metabolic lability and structural alerts of PAB. The separation of PAB and encorafenib (as an internal standard) was achieved on a C8 column, employing an isocratic mobile phase. The inter-day and intra-day accuracy and precision ranged from -6.00% to 4.64% and from -2.33% to 3.13%, respectively. The constructed calibration curve displayed a linearity in the range of 1-3000ng/mL. The sensitivity of the established approach was proven by the lower limit of quantification of 0.73ng/mL. The Analytical GREEness calculator results revealed the high level of greenness of the developed method. The PAB's metabolic stability (t1/2 of 18.5 min and a moderate clearance (Clint) of 44.8mL/min/kg) suggests a high extraction ratio medication that matched the WhichP450 software results.

Interest of hair tests to discriminate a tail end of a doping regimen from a possible contamination in case of challenging an anti-doping rule violation. V. Case reports involving trimetazidine, a drug where the concentration after a single 20 mg dose has been established.

The identification of trimetazidine, a medicine used for treating stable angina pectoris and for preventing angina attacks, has been recently observed in doping cases involving high profile athletes from various countries over the world. In all the files where the authors have been involved, the urine concentration of trimetazidine was low (<2 ng/mL), and the athletes argued that contamination was the source of their adverse analytical finding. It is possible to challenge imposed sanctions in relation to an adverse analytical finding, but it is the responsibility of the athlete to demonstrate he/she is innocent and can qualify for no fault or negligence. When the delay between the urine collection and the notification of the violation was not too long (less than 6months), these athletes requested a head hair test. Trimetazidine was analyzed by an original LC-MS/MS method involving pH 9.5 borate buffer overnight incubation of 20 mg and subsequent solvents extraction in presence of trimetazidine-D8 used as internal standard. Linearity was verified from 1 to 200 pg/mg (R2 = 0.9987). Limit of detection of the method was 0.1 pg/mg. The hair specimen of a male subject, collected 4 weeks after single oral ingestion of 20 mg trimetazidine, tested positive at 146 pg/mg in the corresponding segment. Concentrations of trimetazidine measured in several hair specimens (n = 5) collected from athletes challenging their anti-doping rule violation were below 1 pg/mg, which is consistent with incidental exposure due to contamination. This is the first evidence that trimetazidine is incorporated in human hair after a single therapeutic dose administration.

A quantitative method for the analysis of total and unbound concentrations of amoxicillin, ampicillin, ceftazidime, ceftriaxone, ertapenem, fosfomycin and penicillin G in human plasma with liquid chromatography-tandem mass spectrometry assay.

Monitoring antibiotic plasma levels is critical in populations with altered pharmacokinetics, such as critically ill patients in neonatal or adult intensive care units. This study aimed to develop and validate a rapid, reproducible and sensitive liquid chromatography-tandem mass spectrometry assay (LC-MS/MS) for measuring total and unbound concentrations of amoxicillin, ampicillin, ceftazidime, ceftriaxone, ertapenem, fosfomycin and penicillin G in human plasma. The method required 20 and 250 μl sample volumes for measuring total and unbound concentrations, respectively. Sample preparation involved protein precipitation and the addition of an internal standard. Ultrafiltration separated unbound drugs. Method validation covered selectivity, carryover, linearity, accuracy, precision, dilution effects, matrix effects and stability. The LC-MS/MS was performed within a run time of 7.5min. Calibration curves were linear for ceftazidime and ertapenem (ranges 0.1-50 and 0.05-100 mg/l, respectively) and quadratic for other analytes (0.1-50 mg/l, except for ampicillin: 0.1-20 mg/l; R2 > 0.990). Accuracy was within ±15% of the nominal concentration, and precision did not exceed ±15% (relative standard deviation). Samples showed no significant degradation at the tested temperatures and time points. Clinical applicability was demonstrated in a critically ill neonate. This method with minimal sample volume and short analysis time enables the measurement of total and unbound concentrations of selected antibiotics, and is suitable for routine clinical care and studies.

Matrix effects in ultra-high performance supercritical fluid chromatography-mass spectrometry analysis of Vitamin E in plasma: the effect of sample preparation and data processing

The approaches to matrix effects determination and reduction in ultra-high performance supercritical fluid chromatography with mass spectrometry detection have been evaluated in this study using different sample preparation methods and investigation of different calibration models. Five sample preparation methods, including protein precipitation, liquid-liquid extraction, supported liquid extraction, and solid phase extraction based on both “bind and elute” and “interference removal” modes, were optimized with an emphasis on the matrix effects and recovery of 8 forms of vitamin E, including α-, β-, γ-, and δ- tocopherols and tocotrienols, from plasma. The matrix effect evaluation included the use and comparison of external and internal calibration using three models, i.e., least square with no transformation and no weighting (1/x0), with 1/x2 weighting, and with logarithmic transformation. The calibration model with logarithmic transformation provided the lowest %-errors and the best fits. Moreover, the type of the calibration model significantly affected not only the fit of the data but also the matrix effects when evaluating them based on the comparison of calibration curve slopes. Indeed, based on the used calibration model, the matrix effects calculated from calibration slopes ranged from +92% to – 72% for α-tocopherol and from -77% to +19% in the case of δ-tocotrienol. Thus, it was crucial to calculate the matrix effect by Matuszewski's post-extraction approach at six concentration levels, as a strong concentration dependence was observed for all optimized sample preparation methods, even if the stable isotopically labelled internal standard (SIL-IS) was used for the compensation. The significant differences between individual concentration levels and compounds were observed, even if the tested calibration range covered only one order of magnitude. In methods with wider calibration ranges, the inappropriate use of calibration slope comparison instead of the post-extraction addition approach could result in false negative results of matrix effects. For the selected example of vitamin E, solid-phase extraction was least affected by matrix effects when used in interference removal mode, but supported liquid extraction resulted in the highest recoveries. We showed that the calibration model, the use of a SIL-IS, and the analyte concentration level played a crucial role in the matrix effects. Moreover, the matrix effects can significantly differ for compounds with similar physicochemical properties and close retention times. Thus, in all bioanalytical applications, where different analytes are typically determined in one analytical run, it is necessary to carefully select the data processing in addition to the method for the sample preparation, SIL-IS, and chromatography.

Characterization of a new Teflon chamber and on-line analysis of isomeric multifunctional photooxidation products

Abstract. The photooxidation of volatile organic compounds (VOCs) in the troposphere has important implications for air quality, weather, and climate. A deeper understanding of the underlying mechanisms can be achieved by studying these reactions under controlled conditions and analysing the emerging photooxidation products. This requires dedicated laboratory infrastructure as well as sensitive and selective analytical techniques. Here, we constructed a new 300 L indoor Teflon atmospheric simulation chamber as part of the Bayreuth ATmospheric simulation CHambers (BATCH) infrastructure. The chamber was irradiated by a bandpass-filtered solar simulator that enabled experiments with realistic photon fluxes and OH radical concentrations. It was coupled to a proton-transfer-reaction time-of-flight mass spectrometer (PTR-ToF-MS) and a solid-phase microextraction–gas chromatography–mass spectrometry (SPME-GC-MS) system for the on-line analysis of the precursor VOC and its oxidation products in the gas phase. As part of the SPME-GC-MS method, multifunctional oxygenated compounds (carbonyls, alcohols, carboxylic acids) were derivatized with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBHA) and N-trimethylsilyl-N-methyltrifluoroacetamide (MSTFA). We designed a permeation source for the on-line addition of internal standards to improve method reproducibility. The joint setup was tested and validated by studying the OH-radical-induced photooxidation of toluene, one of the most abundant aromatic hydrocarbons in the atmosphere. For chamber characterization, we first derived the photolysis rates for several typical toluene products in the irradiated BATCH Teflon chamber (1.77 × 10−8–3.02 × 10−4 s−1). Additionally, wall loss rates were determined empirically (4.54 × 10−6–8.53 × 10−5 s−1) and then parameterized according to fundamental molecular properties. For the cresols, we compiled a weighted calibration factor for the PTR-ToF-MS, taking into account isomer-specific sensitivities as well as the relative distribution as determined by the SPME-GC-MS. The weighted calibration improved the instrumental agreement to 14 %, whereas the PTR-ToF-MS overestimated the sum of the isomers by 31 % compared to the SPME-GC-MS concentrations when using the averaged calibration factor. Thus, the combined data set offered insight into both temporal trends and the isomeric composition. Finally, we conducted six toluene photooxidation experiments to evaluate the ring-retaining first-generation products. Based on the loss-corrected concentrations, we derived formation yields for o-cresol (8.0 ± 1.8 %), m-cresol (0.4 ± 0.1 %), p-cresol (2.4 ± 0.6 %), benzyl alcohol (0.5 ± 0.1 %), and benzaldehyde (4.6 ± 1.7 %) under NOx-free conditions at T = 298 ± 1 K. These yields are consistent with previous studies and therefore serve as proof of concept for our applied methods.

The Prognostic Significance of Pontine-White Matter Score in Primary Central Nervous System Lymphoma Patients

Background: Limited data exist on the significance of PET imaging and quantitative PET parameters in primary central nervous system (CNS) lymphoma due to its relative rarity. This study was conducted to investigate the prognostic value of a novel internal standardization indicator, the pontine-white matter (PW) score, in primary CNS lymphoma patients undergoing post-treatment 18F-FDG PET/CT and PET/MR imaging. Methods: From January 2014 to December 2022, eligible patients with primary CNS lymphoma who underwent post-treatment PET imaging were enrolled. Using the FDG uptake of the pons and white matter as an internal reference, the PW score was graded based on the metabolism of the post-therapeutic lesion for each patient, and its associations with patients’ prognosis were investigated. Results: In total, 41 patients with post-treatment PET/CT and 49 patients with post-treatment PET/MR imaging were enrolled. ROC curve analysis indicated that the PW score possessed robust discriminative ability in distinguishing patients with worse outcomes. Furthermore, a higher PW score was significantly correlated with and identified as an independent prognostic indicator for, worse prognosis in both the PET/CT and PET/MR cohorts. Conclusion: The study demonstrated that the PW score was an effective prognostic indicator for identifying post-treatment primary CNS lymphoma patients with worse outcomes.

Simultaneous quantification of baloxavir marboxil and its active metabolite in human plasma using UHPLC-MS/MS: Application to a human pharmacokinetic study with different anticoagulants

Baloxavir marboxil (BXM) is a cap-dependent nucleic acid endonuclease inhibitor, which exerts its antiviral effects after being metabolized to its active form baloxavir acid (BXA). Ethylenediamine tetra-acetic acid (EDTA) and heparin are the two most used anticoagulants in clinical blood sample collection to estimate drug levels in plasma. However, compared to heparin plasma, there is a lack of clinical pharmacokinetic data of BXA using EDTA anticoagulant tubes for blood collection. In the present study, an efficient, rapid, and sensitive ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for simultaneous quantification of BXM and its active metabolite BXA in human plasma with its isotopic baloxavir-d5 (BXA-d5) as internal standard (IS). Plasma samples (50 μL) were undergone using acetonitrile containing 0.1 % formic acid a precipitant. Chromatographic separation was achieved by a Waters XBridge®C8 (2.1 mm × 50 mm, 2.5 µm) column. The gradient mobile phase was 0.1 % formic acid in water (A, pH 2.8) and 0.1 % formic acid in acetonitrile (B) and delivered at a flow rate of 0.6 mL/min for 4.5 min. BXM and BXA were monitored using a positive electrospray triple quadrupole mass spectrometer (TRIPLE QUAD™ 6500+) via multiple reaction monitoring mode. The mass-to-charge ratios (m/z) were 572.2→247.0, 484.2→247.0 and 489.2→252.0 for BXM, BXA, and BXA-d5 (IS). Calibration curves exhibited excellent linearity in the range of 0.1–10 ng/mL for BXM (r2 > 0.996), and 0.3–300 ng/mL for BXA (r2 > 0.998). Within-run and between-run precisions in coefficients of variations were less than 11.62 % for BXM and less than 7.47 % for BXA, and accuracies in relative error were determined to be within −7.78 % to 5.70 % for BXM and −6.67 % to 8.56 % for BXA. Extraction recovery efficiency was 92.76 % for BXM, 95.32 % for BXA, and 99.26 % for BXA-d5, respectively. The matrix effect of BXM and BXA was in line with the requirements, where the relative deviation of the accuracy was less than 6.67 % and the precision was less than 6.69 %. The validated efficient and simple UHPLC-MS/MS method was successfully used in the pharmacokinetic study of BXM and BXA in healthy human volunteers with K2EDTA and heparin tubes for blood collection. EDTA might compete with BXA for chelating metal ions and thereby decrease the plasma ratio in whole blood, leading to approximately 50 % lower measurement of pharmacokinetic parameters as compared with those obtained from heparin plasma anticoagulant tubes.

Validation of the method for determining priority phthalates by GC-MS at trace concentrations in surface water with a background pollution level

A methodology for determining priority phthalates (PP) at trace concentrations in surface waters with a background pollution level has been validated. Lake Baikal, the largest natural reservoir that retains up to 20% of the world’s fresh surface waters, was chosen as a natural model for the study. Baikal is characterized by a minimal content of suspended organic matter, a low degree of mineralization and a background level of organic pollutants. Four priority phthalates were found in Baikal water: dimethyl phthalate, diethyl phthalate di-n-butyl phthalate and di-(2-ethylhexyl) phthalate present in the concentration range from 0.01 to 0.66 μg/liter. The method for phthalate determination includes a single liquid-liquid extraction of phthalates (Vsample = 1 liter) and direct analysis of the extracts by GC-MS. Considering the minimum content of suspended particles in water and the use of high sensitivity of the mass spectrometry, the stages of sample filtration and concentration of extracts are excluded from the procedure. Deuterated phthalates are used as surrogate internal standards for the quantitative determination of priority phthalates. The laboratory background of phthalates was assessed using reagent-blank methods, exhaustive multiple extraction and subsequent exclusion of the resulting value from the determination result as a systematic error. The limits of phthalate determination (0.01 – 0.17 μg/liter) and the error of determination (±δ from 12 to 38%) were assessed in the concentration range from 0.01 to 0.66 μg/liter. The method was validated during monitoring of persistent organic pollutants in water of Lake Baikal for the period 2015 – 2023.

Determination of S, Ni, Cu in copper-nickel sulfide ores by total reflection X-ray fluorescence analysis: experience of participation in the interlaboratory comparisons

The quality of the determination of S, Ni, Cu in copper-nickel sulfide ores was assessed in the framework of the program for interlaboratory comparative tests (ICT) using total reflection X-ray fluorescence analysis (TXRF) taking into account the criteria for methods for determining the chemical composition of mineral raw materials. Certified TXRF techniques for the analysis of mineral materials are absent, so the interlaboratory comparison test was used to control and improve the accuracy of measurements by TXRF. Powdered ore samples were prepared as suspensions by wet grinding: 100 mg of the powder, 4 ml of ultra-pure water and 250 μl of the Ga standard solution were milled using 20 g of ZrO2 balls 1 mm in diameter in the mixing mill for 10 minutes at a frequency of 20 Hz. The ratio of the intensities of the characteristic lines of the analyzed elements and the Ga internal standard was chosen as an analytical signal. To plot calibration curves, a set of ore reference samples was analyzed using certified procedures (atomic absorption spectrometry for Cu, Ni, gravimetry for S). The quality of the TXRF analysis was assessed by the value of Z-criterion. The measurement error did not exceed 5 rel. %. The relative percentage differences between the TXRF results and the results obtained in the interlaboratory study was less than 10%. The described method can be used for rapid analysis of copper-nickel sulfide ores.

Determination of doxorubicin in plasma and tissues of mice by UPLC-MS/MS and its application to pharmacokinetic study

A rapid and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established for the simultaneous determination of doxorubicin (DOX) in mouse plasma and tissues, including the heart, liver, spleen, lung, kidney and tumor, and to investigate the pharmacokinetics and distribution in mice. In this study, daunorubicin (DNR) was used as an internal standard, and the mobile phase consisted of ammonium formate 2 mM containing 0.1 % formic acid (A) and acetonitrile (B), the chromatographic column was ACQUITY UPLC BEHTM C18 with a gradient elution at a flow rate of 0.2 mL/min. Electrospray ionization (ESI) in positive ion pattern was utilized for the ion separation of DOX, with the ions used for quantitative analysis being DOX m/z 544.28 → 397.10 and DNR m/z 528.35 → 321.08, respectively. The results showed that a good linear relationship in the calibration curve range of 1–800 ng/mL in mouse plasma and 1–2500 ng/g in tissues (R2 > 0.99) with the limits of quantification of 1 ng/mL in plasma and tissues. The method exhibited good matrix effect and extraction recovery, with the intra-day and inter-day precision of plasma and tissue were less than 10.3 % and 15.4 %, and the relative error (RE) were both less than ±14.8 % and ±18.9 %, respectively. The stability results under different conditions were found to be accurate. It also revealed the distribution of DOX in various tissues of mice, with the concentration ranking as liver > heart > kidney > spleen > lung > tumor. This method was successfully used to the study for the pharmacokinetics in plasma and drug distribution in tissues of BALB/c mice.

Is the Epworth Sleepiness scale a valid outcome measure to evaluate the effectiveness of positive airway pressure treatments on daytime sleepiness? Psychometric insights from measurement invariance and response shifts.

Although the validity of the Epworth Sleepiness Scale (ESS) as an effectiveness measure for sleep apnea treatments such as continuous positive airway pressure (CPAP) has been supported by multiple studies, some researchers continue to challenge it. They suggest that in addition to its impact on relieving patients' daytime sleepiness, CPAP also alters the internal standards patients use to evaluate their sleepiness (i.e., response shift; RS), confounding the meaning of the difference in the ESS scores. We believe an issue yet to be addressed in this debate is that all existing evidence of RS has been obtained through the then-test approach, a retrospective method sensitive to various cognitive mechanisms. Thus, in the current study, we re-examined this issue using the structural equation modeling (SEM) approach, a method that can be directly applied to randomized clinical trial (RCT) data without retrospective measures. With the ESS data from two independent RCTs, we conducted cross-sectional and longitudinal measure invariance tests in SEM to examine whether CPAP would lead to RS. The ESS demonstrated cross-sectional and longitudinal scalar invariance against CPAP treatments. Its factorial pattern, loadings, and thresholds were invariant between the treatment and control groups and pre- and post-treatment, supporting the comparability of the observed mean ESS scores across time and groups. Our results support the validity of the average difference scores of the ESS for quantifying the effectiveness of CPAP on group-level daytime sleepiness in RCTs with relatively large sample sizes.