Triple Quadrupole Research Articles

Comprehensive quality evaluation of different types of Gardeniae Fructus (Zhizi) and Shuizhizi based on LC-MS/MS.

Gardeniae Fructus (Zhizi) serves as both a medicinal and edible substance and finds widespread use in various industries. There are often two kinds of medicinal materials in the market: Zhizi and Shuizhizi. Typically, Zhizi with small, round fruit is used for medicinal purposes, while Shuizhizi, characterized by large, elongated fruit, is employed for dyeing. Market surveys have revealed a diverse range of Zhizi types, and modern research indicates that Shuizhizi contains rich chemical components and pharmacological activities. In this study, we collected 25 batches of Zhizi and Shuizhizi samples, categorizing them based on appearance into obovate and round fruits, with seven length grades (A-G). Using the ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) method, we simultaneously quantified 13 main chemical components in fruits of Gardenia species. In addition, we compared the weight percentage of the pericarp, flesh, and seeds parts of samples with different traits, and quantified 13 chemical components in different parts. Results indicated that, aside from a few instances of overlapping fruit size ranges, Shuizhizi generally exhibits larger and longer dimensions than Zhizi. The weight proportion of the Shuizhizi pericarp is often higher than that of the Zhizi pericarp. Quantitative results highlighted significant differences in the chemical component content between Zhizi and Shuizhizi, with Shuizhizi generally containing higher levels of iridoids. The PCA and OPLS-DA analysis distinctly divided Shuizhizi and Zhizi, among which three iridoids, two organic acids, and one flavonoid made significant contributions to their classification. Cluster heatmap analysis also demonstrated complete separation between Zhizi and Shuizhizi, with clear distinctions among Zhizi samples from different origins. The distribution of the 13 chemical components in different Zhizi and Shuizhizi parts remained consistent, with iridoids and pigments concentrated in the seeds and flesh, and two organic acids and one flavonoid enriched in the pericarp. In summary, this study contributes valuable insights for classifying Zhizi and offers guidance on the rational use of Shuizhizi and the different parts of Zhizi.

Employing 11-Ketotestosterone as a Target Analyte for Adrenosterone (11OXO) Administration in Doping Controls.

Adrenosterone (Androst-4-ene-3,11,17-trione, 11OXO) is forbidden in sports according to the Prohibited List of the World Anti-Doping Agency. The administration of 11OXO may be detected by monitoring the urinary concentrations of its main human metabolites 11β-hydroxy-androsterone and 11β-hydroxy-etiocholanolone. Preliminary urinary concentration and concentration ratio thresholds have been established for sports drug testing purposes, but adaptations are desirable as the suggested limits would result in numerous suspicious findings due to naturally elevated concentrations and ratios. Recently, the metabolism of 11-oxo-testosterone (KT) was investigated in the context of anti-doping research, resulting in a preliminary urinary concentration threshold and a confirmation procedure based on the determination of carbon isotope ratios (CIRs). Gas chromatography coupled to isotope ratio mass spectrometry was employed to investigate the CIRs of selected steroids. As KT is also a metabolite of 11OXO, the developed protocols for KT have been tested to elucidate their potential to detect the administration of 11OXO after a single oral dose of 100 mg. In order to further improve the analytical approach, the threshold for urinary concentrations of KT was re-investigated by employing a reference population of n = 5232 routine doping control samples. Quantification of urinary steroids was conducted by employing gas chromatography coupled to triple quadrupole mass spectrometry. Derived from these, a subset of n = 106 samples showing elevated concentrations of KT was investigated regarding their CIRs. By means of this, potentially positive samples due to the illicit administration of 11OXO or KT could be excluded, and the calculation of reference population-derived thresholds for the concentrations and CIR of KT was possible. Based on the results, the urinary concentration threshold for KT is suggested to be established at 130 ng/mL.

Impacts of Agricultural Practices (Pesticides and Fertilizers) and Amalnala Stream on Amalnala Lake in Gadchandur, India

Amalnala lake is situated in the Gadchandur town of Chandrapur district in Maharashtra, India. The lake water was turning green in colour in the month of september. This study gives an insight into the causes and factors responsible for the greening of the lake adjacent to Manikgarh limestone mine in the month of September. It was noted that, phosphate concentration in lake water showed rise after July (2020, 2021) and peaked in August (2020, 2021). The phosphates were mainly contributed by organophosphate pesticides such as monocrotophos, profenofos, and chlorpyrifos, which were identified by liquid chromatography triple quadrupole mass spectrometry (LC MS/MS). The rise in phosphates showed complete correlation with the rainfall and chlorophyl a (chl a) content in September (2020, 2021). Similarly, the nitrate concentration of the lake showed peak in August (2020, 2021) followed by the rise in chl a content in September (2020, 2021). Chemical oxygen demand (COD) and biological oxygen demand (BOD) of the lake was calculated and represented in terms of COD/BOD. The COD/BOD were highest in September (2020, 2021). Positive correlation was observed between the pH, total dissolved solids, and electrical conductivity of Amalnala Lake and Amalnala Stream which flows into the lake. This stream mainly carries the contaminants from open cast Manikgarh limestone mines to Amalnala Lake. Chl a was mainly contributed by two dominant species of cyanobacteria, Phormidium species CCALA 726 which was identified by 16S rRNA sequencing and Microcystis aeruginosa which was identified by using the keys given by Desikachary and Komarek and Anagnostidis. This study thus concluded that the pesticides, nitrates, and Amalnala Stream contributed in the growth of cyanobacteria which causes the greening of the lake.

Antimicrobials in Eisenia fetida earthworms: A comprehensive study from method development to the assessment of uptake and degradation

In this work, an accurate analytical method was developed for the simultaneous analysis of twenty-seven antimicrobials (AMs) in earthworms using liquid chromatography coupled to a triple quadrupole mass spectrometry detector (UHPLC-MS/MS). Adequate apparent recoveries (80–120 %) and limits of quantification (LOQ) (1 μg·kg−1 - 10 μg·kg−1) were obtained, with the exception of norfloxacin (34 μg·kg−1). The method was applied to evaluate the accumulation of sulfamethazine (SMZ) and tetracycline (TC) in earthworms after performing OECD-207 toxicity test, in which Eisenia fetida (E. fetida) organisms were exposed to soils spiked with 10 mg·kg−1, 100 mg·kg−1 or 1000 mg·kg−1 of SMZ and TC, individually. The results confirmed the bioaccumulation of both AMs in the organisms, showing a greater tendency to accumulate SMZ since higher bioconcentration factor values were obtained for this compound at the exposure concentrations tested. In addition, the degradation of both AMs in both matrices, soils and earthworms was studied using liquid chromatography coupled to a q-Orbitrap high resolution mass spectrometry detector. Thirteen transformation products (TPs) were successfully identified, eight of them being identified for the first time in soil/earthworm (such as 4-Amino-3-chloro-n-(4,6-dimethylpyrimidin-2-yl)benzenesulfonamide or 4-(dimethylamino)-1,11,12a-trihydroxy-6,6-dimethyl-3,7,10,12-tetraoxo-3,4,4a,5,5a,6,7,10,12,12a-decahydrotetracene-2-carboxamide, among others) and their formation/degradation trend over time was also studied. Regarding the biological effects, only SMZ caused changes in earthworm growth, evidenced by weight loss in earthworms exposed to concentrations of 100 mg·kg−1 and 1000 mg·kg−1. Riboflavin decreased at all concentrations of SMZ, as well as at the highest concentration of TC. This indicates that these antibiotics can potentially alter the immune system of E. fetida. This research represents a significant advance in improving our knowledge about the contamination of soil by AM over time. It investigates the various ways in which earthworms are exposed to AMs, either by skin contact or ingestion. Furthermore, it explores how these substances accumulate in earthworms, the processes by which earthworms break them down or metabolise them, as well as the resulting TPs. Finally, it examines the potential effects of these substances on the environment.

Pharmacokinetic and tissue distribution analysis of bioactive compounds from Fuke Qianjin capsules in rats by a validated UPLC[sbnd]MS/MS method

Fuke Qianjin capsules (FKQJ) exhibit obvious advantages and characteristics in the treatment of pelvic inflammatory disease. At present, information regarding the in vivo process of FKQJ is lacking, which has become a bottleneck in further determining the therapeutic effect of this traditional Chinese medicine. In the present study, a sensitive, simple and reliable method was developed and validated for the simultaneous quantification of 12 main components (4 flavonoids, 4 alkaloids, 2 phthalides and 2 diterpene lactones) in plasma and seven tissues of rats to study the pharmacokinetic and distribution characteristics of these components in vivo by using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the first time. Plasma and tissue were prepared by protein precipitation with acetonitrile and methanol, followed by its separation on a Waters Acquity UPLC BEH C18 column. The quantification was performed via multiple reaction monitoring (MRM) by a triple quadrupole mass spectrometer under positive electrospray ionization (ESI) mode. The method was validated to demonstrate its selectivity, linearity, accuracy, precision, recovery, matrix effect and stability. For 12 analytes, the low limit of quantification (LLOQs) reached 0.005–2.44 ng/mL, and all calibration curves showed good linearity (r2 ≥ 0.990) in linear ranges. The intra-day and inter-day precision (relative standard deviation) for all analytes was less than 14.96%, and the accuracies were in the range of 85.29%−114.97%. Extraction recoveries and matrix effects of analytes were acceptable. The pharmacokinetic results showed that the main components could be absorbed quickly, had a short residence time, and were eliminated quickly in vivo. At different time points, the 12 components were widely distributed with uneven characteristics in the body, which tended to be distributed in the liver, kidney and lung and to a lesser extent in the uterus, brain and heart. The pharmacokinetic process and tissue distribution characteristics of FKQJ were expounded in this study, which can provide a scientific theory for in-depth development of FKQJ and guide FKQJ use in the clinic.

Determination of fentanyl contamination on United States paper currency by LC-QQQ-MS.

Previous research has evaluated the extent to which cocaine and other drugs were detectable on currency in the USA. The literature was in agreement that the majority of bills exhibited some degree of contamination. With the increase of fentanyl in the illicit drug supply, this study was designed to evaluate the extent that fentanyl, cocaine, methamphetamine and other substances were present on circulating currency in 2022. A quantitative assay using liquid chromatography-triple quadrupole mass spectrometry was developed and validated to detect six analytes: fentanyl, 4-anilino-N-phenethylpiperidine, acetylfentanyl, benzylfentanyl, cocaine and methamphetamine. One-dollar bills were collected from 13 cities across the country. Sample preparation consisted of soaking the bills in methanol followed by liquid-liquid extraction. Chromatographic separation was achieved using a C18 analytical column and gradient elution with ammonium formate in water (5 mM, pH 3) and 0.1% formic acid in acetonitrile. The quantitative working range for this assay was 0.1 μg to 1.0 μg per bill (equivalent to 1 ng/mL to 100 ng/mL of extract). Fentanyl was detected on the majority (63%) of samples, with 61% of samples having ≥0.1 μg of fentanyl and 4% of samples having ≥1.0 μg. Cocaine and methamphetamine were detected on 100% and 98% of bills, respectively, typically in amounts >1.0 μg. The remaining fentanyl-related substances were detected in 15% of samples in amounts no >0.69 μg per bill and exclusively in the presence of fentanyl. Unsurprisingly, areas of the country with higher incidence of fentanyl use yielded higher frequency of contaminated bills and higher concentrations. Human exposure to drugs on currency is unlikely to have any significant impacts toxicologically or pharmacologically; however, our research findings suggest that paper currency could serve as a useful substrate for surveillance of drug trends regionally, nationally and/or internationally.

Studies of Potential Migration of Hazardous Chemicals from Sustainable Food Contact Materials.

In recent years, due to modern techniques for the distribution, transport, and retail sale of food, the production of large amounts of non-biodegradable and bioaccumulative packaging waste has become a major environmental issue. To address this issue, new food packaging materials based on renewable biomass have been studied as eco-friendly, biodegradable, and biocompatible alternatives to synthetic materials. However, although these materials are not petrochemical derivatives, the presence of contaminants cannot be excluded. This work aims to extend the knowledge on bio-based packaging materials, researching the presence of contaminants potentially able to migrate to food at concentrations of concern. In this study, we focus on two classes of contaminants, organophosphate esters (OPEs) and perfluoroalkyl substances (PFASs), carrying out migration tests toward different simulants, according to the current European regulation. PFAS analysis was performed using high-resolution liquid chromatography coupled to ion trap-tandem mass spectrometry (QTrap). OPE analyses were performed both by gas chromatography-mass spectrometry (GC-MS) and high-resolution liquid chromatography coupled to triple quadrupole mass spectrometry (TQMS). Preliminary findings demonstrate the release of toxic OPEs and PFASs from bio-based food packaging, highlighting the need to investigate the presence of potentially harmful chemicals in these materials.

Phase I Pharmacokinetics Study of Oral Administration of Esperavir® (INN: Molnupiravir) (LLC "PROMOMED RUS", Russia)

Introduction. The pandemic of the new coronavirus infection COVID-19 (Coronavirus Disease 2019) was caused by a single-stranded RNA virus SARS-CoV-2 (Severe acute respiratory syndrome-related coronavirus 2). Molnupiravir is an antiviral drug with activity against RNA viruses including SARS-CoV-2. Molnupiravir exerts the antiviral effect by introducing copy errors during viral RNA replication – by that the replication of SARS-CoV-2 inhibits. For oral administration of molnupiravir the drug Esperavir® has been registered in Russia.Aim. The aim is pharmacokinetics study of Esperavir®, capsules 200 mg (the manufacturer is JSC "Biokhimic", LLC "PROMOMED RUS", Russia, as registration certificate holder) by a single dose (800 mg) and multiple doses oral administration (800 mg twice a day with a gap of 12 hours between doses) in healthy volunteers in a phase I pharmacokinetics study, comparison the obtained data of pharmacokinetic parameters with the literature data.Materials and methods. The clinical and analytical phases of the pharmacokinetic study as well as pharmacokinetic analyses have been performed as a part of a clinical trial of the drug Esperavir®, capsules 200 mg (the manufacturer is JSC "Biokhimic", LLC "PROMOMED RUS", Russia, as registration certificate holder). Chromatographic separation and detection by Nexera XR high-performance liquid chromatograph with triple quadrupole tandem mass spectrometry LCMS-8040 (Shimadzu Corporation, Japan). The pharmacokinetic parameters were calculated with the Boomer pharmacokinetic analysis add-in for Microsoft Excel (Department of Pharmacokinetics and Drug Metabolism, Allergan, Irvine, CA 92606, USA). Descriptive pharmacokinetic statistics were calculated with Microsoft Excel (Microsoft Corporation, USA).Results and discussion. Pharmacokinetic parameters for cohort 1 (800 mg single dose of Esperavir®) and for cohort 2 (800 mg of Esperavir® twice a day with a gap of 12 hours between doses) were calculated. Averaged pharmacokinetic profiles of mean NHC concentrations over time in linear and log-linear scales were plotted. The geometric mean of Cmax and T1/2, the median, minimum and maximum of Tmax showed the comparability of the obtained data after a single dose administration of 800 mg of Esperavir® and the available literature data.Conclusion. According to the concentrations from the analytical phase of the pharmacokinetic study the pharmacokinetic parameters were calculated, averaged pharmacokinetic profiles in linear and log-linear scales were plotted after a single dose and multiple doses of the drug Esperavir®, capsules (the manufacturer is JSC "Biokhimic", LLC "PROMOMED RUS", Russia, as registration certificate holder). The comparability of the obtained data and the available literature data was shown. The results justified the study of the subsequent phases of clinical trials of Esperavir®.

Elemental Analysis of Tree Nuts and Their Pressed Oils by Triple Quadrupole Inductively Coupled Plasma-Mass Spectrometry (ICP-MS/MS)

Trace elements in tree nut oils may be toxic for humans and catalyze the auto-oxidation of the oils, affecting their stability. These elements have not been comprehensively characterized. In this study, we prepared tree nut oils from tree nuts (camellia, acer truncatum, shiny leaf yellow horn, and pecan seeds, as well as walnuts and Brazil nuts) by cold-pressing and determined As, Cd, Cu, Fe, Mn, Pb, Se, and Zn in these seeds and their oils and residues. Moreover, we investigated the elemental migration from seed to oil. The migration of all measured elements from seed to oil was <2%, where Cu exhibited the lowest migration from 0.09 to 0.31%. Moreover, we studied the effect of preheating (25–150 °C) the seeds upon their elemental migration. The Cu, Fe, Mn, and Zn contents increased with the temperature in all tree nut oils. The migration of toxic As, Cd, and Pb did not significantly differ with temperature, suggesting that temperature does not affect the release of these elements from nuts to oil.

Therapeutic drug monitoring of clozapine in human serum by high-throughput paper spray mass spectrometry

IntroductionMonitoring the atypical antipsychotic drug clozapine is crucial to ensure patient safety. This article showcases a high-throughput analytical method for measuring clozapine and its primary metabolite norclozapine (N-desmethylclozapine) in serum using paper spray mass spectrometry (PS-MS). ObjectivesThis study aimed to assess the viability of a PS-MS method for the rapid measurement of clozapine and norclozapine in human serum samples as an alternative to liquid chromatography mass spectrometry (LC-MS). MethodsSerum samples were processed by protein precipitation followed by deposition of the supernatant containing labelled internal standards onto paper spray substrates mounted in cartridges. Analytes were then analyzed using a triple quadrupole mass spectrometer equipped with a commercial paper spray ionization source. The results obtained from the patient samples were compared to those from a validated LC-MS assay. ResultsPS-MS calibrations for clozapine and norclozapine were linear (R2 > 0.99) over five days. Between-run precision was below 8 %, and within-run precision did not exceed 10 %. When compared to a validated LC-MS method, the mean bias for 39 patient samples was −9% for clozapine and −1% for norclozapine, with no outliers. Mass spectrometry ion ratio comparisons indicated no interference for patient samples above the lower limit of quantification. There was less than 7 % change in the measured concentrations of both analytes over five days for samples dried on paper substrates. Notably, virtually no maintenance of the MS source was required during this study. ConclusionThis study illustrates the potential of PS-MS for serum drug monitoring in the clinical laboratory.

Synthesis and Trace-Level Quantification of Mutagenic and Cohort-of-Concern Ciprofloxacin Nitroso Drug Substance-Related Impurities (NDSRIs) and Other Nitroso Impurities Using UPLC-ESI-MS/MS-Method Optimization Using I-Optimal Mixture Design.

Globally, the pharmaceutical industry has been facing challenges from nitroso drug substance-related impurities (NDSRIs). In the current study, we synthesized and developed a rapid new UPLC-MS/MS method for the trace-level quantification of ciprofloxacin NDSRIs and a couple of N-nitroso impurities simultaneously. (Q)-SAR methodology was employed to assess and categorize the genotoxicity of all ciprofloxacin N-nitroso impurities. The projected results were positive, and the cohort of concern (CoC) for all three N-nitroso impurities indicates potential genotoxicity. AQbD-driven I-optimal mixture design was used to optimize the mixture of solvents in the method. The chromatographic resolution was accomplished using an Agilent Poroshell 120 Aq-C18 column (150 mm × 4.6 mm, 2.7 μm) in isocratic elution mode with 0.1% formic acid in a mixture of water, acetonitrile, and methanol in the ratio of 475:500:25 v/v/v at a flow rate of 0.5 mL/min. Quantification was carried out using triple quadrupole mass detection with electrospray ionization (ESI) in a multiple reaction monitoring technique. The finalized method was validated successfully, affording ICH guidelines. All N-nitroso impurities revealed excellent linearity over the concentration range of 0.00125-0.0250 ppm. The Pearson correlation coefficient of each N-nitroso impurity was >0.999. The method accuracy recoveries ranged from 93.98 to 108.08% for the aforementioned N-nitrosamine impurities. Furthermore, the method was effectively applied to quantify N-nitrosamine impurities simultaneously in commercially available formulated samples, with its efficiency recurring at trace levels. Thus, the current method is capable of determining the trace levels of three N-nitroso ciprofloxacin impurities simultaneously from the marketed tablet dosage forms for commercial release and stability testing.

Simultaneous quantification of four hormone therapy drugs by LC-MS/MS: Clinical applications in breast cancer patients

IntroductionAromatase inhibitors such as anastrozole, letrozole, exemestane and selective estrogen down-regulator (SERD) fulvestrant are used mostly to treat breast cancer estrogen receptor positive in post-menopausal women. These drugs are given either through the oral route or by intramuscular injection. They have shown great inter-individual variability with a risk of cardiometabolic disorders. Hence the importance of their therapeutic drug monitoring not only for exposure-efficacy but also exposure-toxicity. We describe here a LC-MS/MS method for the simultaneous quantification of anastrozole, letrozole, exemestane and fulvestrant in human plasma. Material and methodsPlasma samples were prepared by a single-step protein precipitation. The liquid chromatography system was paired with a triple quadrupole mass spectrometer. Quantification were achieved in Multiple Reactions Monitoring mode and the electrospray ionization was in positive mode. ResultsThe method demonstrated consistent analytical performance across various parameters, including linearity, specificity, sensitivity, matrix effect, upper and lower limits of quantification, extraction recovery, precision, accuracy, hemolysis effect, dilution integrity, and stability under different storage conditions, in accordance with established guidelines. The analysis time for each run was 4 min. Calibration curves exhibited linearity within the 1–100 ng/mL range, with correlation coefficients > 0.99 for the four analytes. Plasma concentrations from 42 patients were integrated into the selected calibration. Stability assessments indicated that the four drugs remained stable at − 20 °C for three months, 15 days under refrigeration, up to 7 days at room temperature, and after three freeze-thaw cycles. ConclusionWe have developed and validated this quantitative method for therapeutic drug monitoring of those four hormone therapy drugs:anastrozole, letrozole, fulvestrant and exemestane. This method can be also used for future clinical pharmacokinetics /pharmacodynamics studies.

Determination of Mo-93 inventory in irradiated BWR tie plate using triple quadrupole ICP-MS

AbstractThe inventory of radionuclides is a crucial input parameter for the safety assessment of irradiated metal waste. Among the radionuclides, Mo-93 is remarkable radionuclide owing to its long half-life (4,000 years) and poor sorptive property (anionic species). A method for determining Mo-93 by measuring characteristic X-rays was proposed previously; however, this method requires complex sequential and/or chromatographic separation of Mo-93. In the present study, triple quadrupole inductively coupled plasma mass spectrometry (ICP-MS/MS)—a leading-edge method—was used to determine the inventory of Mo-93 in an irradiated boiling water reactor (BWR) tie plate (stainless steel) for the first time. Furthermore, an activation calculation for Mo-93 was conducted using ORIGEN2. The results indicated that the inventory of Mo-93 in the BWR tie plate was 690 ± 83 Bq/g. This study demonstrates that triple quadrupole ICP-MS allows the simple quantitative analysis of Mo-93 in irradiated stainless steel without complex chemical separation. Graphical abstract

Electrospray triple quadrupole mass spectrometry guides pathologists to suggest appropriate molecular testing in the identification of rare hemoglobin variants

BackgroundThe presumptive diagnosis of hemoglobinopathies relies on routine tests such as Complete Blood Count (CBC), peripheral blood smear, Liquid Chromatography (LC), and Capillary Electrophoresis (CE), along with clinical findings. Pathologists suggest molecular sequencing of HBA and HBB genes to correlate blood picture with clinical findings in order to identify unknown rare haemoglobin (Hb) variants or variants that coelute with Hb. This paper presents a low-resolution mass spectrometry (MS)-based method for presumptive identification of variants that eluted in zone 12 of CE, followed by molecular sequencing of the HBB gene for a definitive diagnosis of hemoglobinopathies. MethodsEight patient samples with a variant peak in zone 12 of CE (Sebia) were analyzed using MS. The mass-to-charge ratio (m/z) observed was deconvoluted to determine the mass of Hb variants. The β variants were subsequently confirmed through molecular sequencing. ResultsBased on the intact mass of the variants, there were two samples of the α variant (α + 58 Da and α + 44 Da), and six samples of the β variant. Out of these six β variant samples, three were the β + 58 Da variant, and three were the β + 30 Da variant. By correlating the intact mass information with the CE pattern and considering the ethnicity of the patients, it was presumed that the α variants were HbJ Meerut (α + 58 Da, x-axis 102) and HbJ Paris-I (α + 44 Da, x-axis 80). Molecular analysis confirmed the identity of β variants as Hb Rambam/HbJ Cambridge, HbJ Bangkok (+58 Da), and Hb Hofu (+30 Da). ConclusionThe mass information of Hb variants obtained using Electrospray triple quadrupole MS assists pathologists in recommending the appropriate molecular sequencing for identifying unknown variants.

Assessing human exposure to pesticides and mycotoxins: optimization and validation of a method for multianalyte determination in urine samples

Humans are exposed to an increasing number of contaminants, with diet being one of the most important exposure routes. In this framework, human biomonitoring is considered the gold standard for evaluating human exposure to chemicals. Pesticides and mycotoxins are chemicals of special concern due to their health implications. They constitute the predominant border rejection notifications for food and feed in Europe and the USA. However, current biomonitoring studies are focused on a limited number of compounds and do not evaluate mycotoxins and pesticides together. In this study, an analytical method has been developed for the determination of 30 pesticides and 23 mycotoxins of concern in urine samples. A salting-out liquid–liquid extraction (SALLE) procedure was optimized achieving recoveries between 70 and 120% for almost all the compounds and limits as lower as when QuEChERS was applied. The compounds were then determined by liquid chromatography coupled to triple quadrupole mass spectrometry. Different chromatographic conditions and analytical columns were tested, selecting a Hypersild gold aQ column as the best option. Finally, the method was applied to the analysis of 45 urine samples, in which organophosphate and pyrethroid pesticides (detection rates (DR) of 82% and 42%, respectively) and ochratoxin A and deoxynivalenol (DR of 51% and 33%, respectively) were the most detected compounds. The proposed analytical method involves the simultaneous determination of a diverse set of pesticides and mycotoxins, including their most relevant metabolites, in human urine. It serves as an essential tool for biomonitoring the presence of highly prevalent contaminants in modern society.Graphical

Therapeutic Monitoring of Palbociclib, Ribociclib, Abemaciclib, M2, M20, and Letrozole in Human Plasma: A Novel LC-MS/MS Method.

Therapeutic drug monitoring (TDM) using cyclin-dependent kinase inhibitors (CDK4/6is) is a novel approach for optimizing treatment outcomes. Currently, palbociclib, ribociclib, and abemaciclib are the available CDK4/6is and are primarily coadministered with letrozole. This study aimed to develop and validate an LC-MS/MS method for the simultaneous analysis of CDK4/6is, 2 active metabolites of abemaciclib (M2 and M20), and letrozole in human plasma for use in TDM studies. Sample pretreatment comprised protein precipitation with methanol and dilution of the supernatant with an aqueous mobile phase. Chromatographic separation was achieved using a reversed-phase XBridge BEH C18 column (2.5 μm, 3.0 × 75 mm XP), with methanol serving as the organic mobile phase and pyrrolidine-pyrrolidinium formate (0.005:0.005 mol/L) buffer (pH 11.3) as the aqueous mobile phase. A triple quadrupole mass spectrometer was used for the detection, with the ESI source switched from negative to positive ionization mode and the acquisition performed in multiple reaction monitoring mode. The complete validation procedure was successfully performed in accordance with the latest regulatory guidelines. The following analytical ranges (ng/mL) were established for the tested compounds: 6-300, palbociclib and letrozole; 120-6000, ribociclib; 40-800, abemaciclib; and 20-400, M2 and M20. All results met the acceptance criteria for linearity, accuracy, precision, selectivity, sensitivity, matrix effects, and carryover. A total of 85 patient samples were analyzed, and all measured concentrations were within the validated ranges. The percent difference for the reanalyzed samples ranged from -11.2% to 7.0%. A simple and robust LC-MS/MS method was successfully validated for the simultaneous quantification of CDK4/6is, M2, M20, and letrozole in human plasma. The assay was found to be suitable for measuring steady-state trough concentrations of the analytes in patient samples.

Lipid Isobar and Isomer Imaging Using Nanospray Desorption Electrospray Ionization Combined with Triple Quadrupole Mass Spectrometry.

Mass spectrometry imaging (MSI) is widely used for examining the spatial distributions of molecules in biological samples. Conventional MSI approaches, in which molecules extracted from the sample are distinguished based on their mass-to-charge ratio, cannot distinguish between isomeric species and some closely spaced isobars. To facilitate isobar separation, MSI is typically performed using high-resolution mass spectrometers. Nevertheless, the complexity of the mixture of biomolecules observed in each pixel of the image presents a challenge, even for modern mass spectrometers with the highest resolving power. Herein, we implement nanospray desorption electrospray ionization (nano-DESI) MSI on a triple quadrupole (QqQ) mass spectrometer for the spatial mapping of isobaric and isomeric species in biological tissues. We use multiple reaction monitoring acquisition mode (MRM) with unit mass resolution to demonstrate the performance of this new platform by imaging lipids in mouse brain and rat kidney tissues. We demonstrate that imaging in MRM mode may be used to distinguish between isobaric phospholipids requiring a mass resolving power of 3,800,000. Additionally, we have been able to image eicosanoid isomers, a largely unexplored class of signaling molecules present in tissues at low concentrations, in rat kidney tissue. This new capability substantially enhances the specificity and selectivity of MSI, enabling spatial localization of species that remain unresolved in conventional MSI experiments.

Quantification of parabens in marine fish samples by a rapid, simple, effective sample preparation method.

Parabens (p-hydroxybenzoic acid esters) commonly used preservatives (in cosmetics, pharmaceuticals, and foods) can pose potential effects on environmental health. In this study, seven parabens were quantified in marine fish samples using an ultra-high performance liquid chromatography triple quadrupole mass spectrometer (UHPLC-MS/MS) system. Parabens in the fish samples were extracted and purified by a rapid, simple, and effective procedure comprising sample homogenization with solvent, solid-phase extraction clean-up, and solvent evaporation. Results demonstrated that the recoveries of seven compounds (with relative standard deviation < 15%) were 88-103% in matrix-spike samples and 86-105% in surrogate standards. The method detection limits and method quantification limits of seven parabens were 0.015-0.030 and 0.045-0.090ng/g-ww (wet weight), respectively. The optimized method was applied to measure the concentration of parabens in the 37 marine fish samples collected from Vietnam coastal waters. The concentration ranges of seven parabens found in round scad and greater lizardfish samples were 6.82-25.3ng/g ww and 6.21-17.2ng/g-ww, respectively. Among parabens, methylparaben accounted for the highest contribution in both fish species (43.2 and 44.9%, respectively). Based on the measured concentrations of parabens in marine fish samples, the estimated daily intake was calculated for children and adults with the corresponding values of 0.0477µg/kg/day and 0.0119µg/kg/day, respectively. However, the presence of parabens in Vietnamese marine fish may not pose a significant risk to human health.

Ultra‐high‐performance liquid chromatography‐tandem mass spectrometry analytical method for the determination of nitrosamine drug substance‐related impurity in sitagliptin base and salts

AbstractA reversed‐phase ultra‐high‐performance liquid chromatography‐tandem mass spectrometry method is presented for the quantification of the mutagenic impurity 7‐nitroso‐3‐(trifluoromethyl)‐5,6,7,8‐tetrahydro‐[1,2,4]triazolo[4,3‐a]pyrazine found in three sitagliptin drug substances (sitagliptin base [SG], sitagliptin hydrochloride monohydrate [SG HCl{H}] salt, and sitagliptin phosphate monohydrate [SG P{H}] salt). A simple and highly sensitive method was developed for SG, SG HCl(H) salt, and SG P(H) salt. Sample preparation was adapted to each product considering solubility, sensitivity, and accuracy issues. Chromatographic separation was achieved using an Acquity HSS T3 column (3.0 × 100 mm, 1.8 μm) and a mobile phase consisting of formic acid 0.1% in water combined with methanol. Detection and quantification of the impurity were carried out using triple quadrupole mass spectrometry detection with electrospray ionization in the multiple reaction monitoring mode. The limit of detection and limit of quantification was found to be 0.1–0.3 and 10 ppb, respectively. The accuracy and precision of the method were satisfactorily determined with recovery values between 74.1% and 119.4%. Linearity is demonstrated in the range of 10 and 2000 ppb with regression coefficients (R) within the range of 0.9918–0.9972. The method is currently used for the analysis of the mutagenic impurity in the three‐drug substances in the Moehs Group.

A quantitative LC-MS/MS analysis of Xylazine, p-Fluorofentanyl, Fentanyl and Fentanyl-Related compounds in postmortem blood

The purpose of this study was to develop and validate a method to quantitate the veterinary sedative xylazine as well as 4-anilino-N-phenethylpiperidine (4-ANPP), acetyl fentanyl, fentanyl, norfentanyl, and p-fluorofentanyl in blood utilizing liquid chromatography tandem mass spectrometry. This method also qualitatively monitors for the presence of o-fluorofentanyl and m-fluorofentanyl isomers. UCT Clean Screen® DAU extraction columns were utilized to isolate the analytes in postmortem blood samples. The extracts were eluted, evaporated, reconstituted, and then analyzed using a Waters Acquity™ UPLC coupled a triple quadrupole mass spectrometer. The lower limit of quantitation was determined to be 0.1 ng/mL for all analytes, except for xylazine (0.2 ng/mL). The upper limit of quantitation for all analytes was 100 ng/mL. No interferences from matrix, internal standard, or common drug analytes were observed. Bias (-13.1–4.6 %) and precision (-13.14–10.3 %) fell within the acceptable ± 20 % criteria range. Dilution integrity at x2, x10, and x100 was evaluated and all results were within ± 20 % of the target concentration. Processed extract stability was evaluated after 72 h and all results were within ± 20 % of the analyte initial concentration. Matrix effects were the most prominent with xylazine, but deemed acceptable as the deuterated internal standard also observed comparable enhancement. Analysis of 89 postmortem blood samples by this method resulted in positive results for fentanyl (0.27–66 ng/mL, n = 82), xylazine (0.24–958 ng/mL, n = 21), 4-ANPP (0.10–38 ng/mL, n = 72), acetyl fentanyl (0.18–1.5 ng/mL, n = 3), p-fluorofentanyl (0.11–33 ng/mL, n = 30), and norfentanyl (0.10–98 ng/mL, n = 73).