Liquid Chromatography-mass Spectrometry Method Research Articles

Urinary thymidine dimer excretion reflects personal ultraviolet radiation exposure levels

Exposure to ultraviolet radiation (UVR) leads to skin DNA damage, specifically in the form of cyclobutane pyrimidine dimers, with thymidine dimers being the most common. Quantifying these dimers can indicate the extent of DNA damage resulting from UVR exposure. Here, a new liquid chromatography-mass spectrometry (LC–MS) method was used to quantify thymidine dimers in the urine after a temporary increase in real-life UVR exposure. Healthy Danish volunteers (n = 27) experienced increased UVR exposure during a winter vacation. Individual exposure, assessed via personally worn electronic UVR dosimeters, revealed a mean exposure level of 32.9 standard erythema doses (SEDs) during the last week of vacation. Morning urine thymidine dimer concentrations were markedly elevated both 1 and 2 days post-vacation, and individual thymidine dimer levels correlated with UVR exposure during the last week of the vacation. The strongest correlation with erythema-weighted personal UVR exposure (Power model, r2 = 0.64, p < 0.001) was observed when both morning urine samples were combined to measure 48-h thymidine dimer excretion, whereas 24-h excretion based on a single sample provided a weaker correlation (Power model, r2 = 0.55, p < 0.001). Sex, age, and skin phototype had no significant effect on these correlations. For the first time, urinary thymidine dimer excretion was quantified by LC–MS to evaluate the effect of a temporary increase in personal UVR exposure in a real-life setting. The high sensitivity to elevated UVR exposure and correlation between urinary excretion and measured SED suggest that this approach may be used to quantify DNA damage and repair and to evaluate photoprevention strategies.Graphical abstract

Relative bioavailability study of ticagrelor in healthy subjects under fasting conditions

Objectives: This is a randomized, single-dose, two-period, two-sequence, and crossover study to evaluate the bioequivalence (BE) profiles of two formulations of ticagrelor 90 mg, under fasting conditions. Materials and Methods: The plasma concentrations of ticagrelor were measured using a validated liquid chromatography mass spectrometry method. Enrolled for the study were 30 healthy adult volunteers (21 males and 9 females). All completed the study. The two formulations of ticagrelor were considered bioequivalent if a 90% confidence interval (CI) fell within 80.00–125.00% for Cmax and AUC0-t. Tolerability and safety were assessed throughout the study. Results: The pharmacokinetic (PK) parameters were similar between the test product (T) Clenosan® and reference product (R) Brilinta® under fasting conditions (90% CI for all PK parameters fell within 80.00–125.00%). The 90% CI of the test/reference ratios of log-transformed PK parameter point estimates were Cmax: 95.49% (87.99– 103.62%) and AUC0-t: 103.20% (97.52–109.21%), respectively. Conclusion: Our results demonstrated BE between the test and reference drug products of oral ticagrelor in healthy subjects under fasting conditions.

Development and validation of a dried blood spots assay for metabolic profiling of ginsenosides using ultra-high performance liquid chromatography-mass spectrometry

Ethnopharmacological relevancePanax ginseng C.A. Meyer., a famous and valuable traditional Chinese medicine with thousand years of history for its healthcare and therapeutic effects. It is necessary and meaningful to study the pharmacokinetic behavior of ginsenosides in vivo as they are the most active components. Dried blood spots (DBS) are a mature and advanced blood collection method with meet the needs for the measurement of numerous analytes. Aim of the studyThis study aimed to explore the feasibility on DBS in the metabolic profile analysis of complex herbal products. Materials and methodsAn ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of ginsenosides. The preparation of DBS samples was conducted by spiking the whole blood with analytes to obtain 20 μL of blood spots on Whatman 903 collection card. A punched dish of 10 mm in diameter was extracted with 70 % methanol aqueous solution, digoxin was used as an internal standard. Target compounds were separated on a Waters T3 column (2.1 × 100 mm, 1.8 μm) with acetonitrile and water (0.1 % formic acid) at a flow rate of 0.4 mL/min. ResultsThe various ginsenosides showed good linearity in the range of 1–2000 ng/mL. The extraction recoveries and matrix effects of the target analytes were above 82.2%. The intra- and inter-batch accuracy and precision were within the limits of ≤15% for all tested concentrations. Moreover, the collected dried blood spot samples could be stably stored at room temperature for 14 days and 4 °C for 1 month without being affected. And it is delightful that the DBS-based analysis is compatible or even superior to the conventional protein precipitation in terms of sensitivity, linearity, and stability. In particular, the target analytes are stable in the DBS sampling under normal storing condition and the sensitivity for some trace metabolites of ginsenosides, such as 20(S)-Rg3, 20(R)-Rg3, F1, Rk1, Rg5, etc. increases 3–4 folds as evaluated by LLOQ. ConclusionsThe established method was successfully applied to pharmacokinetic studies of ginseng extract in mice, this suggests a more feasible strategy for pharmacokinetic study of traditional and natural medicines both in animal tests and clinical trials.

Spherical fluorinated covalent organic polymer for highly efficient and selective extraction of fipronil and its metabolites in soil

Covalent organic polymers (COPs) have garnered considerable attention as promising adsorbents of online solid phase extraction (online SPE). Morphology modulation provides an appealing solution to enhance adsorption efficiency and reduce back-pressure in the absorbent. However, the synthesis of COPs with regular geometric shapes and specific adsorption selectivity remains challenging. In this study, a uniform spherical fluorinated COP (F-sCOP, average diameter: 2.14 μm) was successfully synthesized by Schiff base reaction of 1,3,5-triformylphoroglucinol (TP) and 2,2’-bis(trifluoromethyl)benzidine (TFMB). The F-sCOP had a large surface area (BET: 346.2 m2 g−1), remarkable enrichment capacity (enrichment factors: 186–782), high selectivity toward fipronil and its metabolites (adsorption efficiency >93.1%), and admirable service life (>60 times). Based on the adsorbent, a novel μ-matrix cartridge extraction-online-μ-solid phase extraction-high performance liquid chromatography-mass spectrometry (μ-MCE-online-μ-SPE-HPLC-MS) method was constructed and used to track trace fipronil and its metabolites in soil. The proposed method exhibited a wide linear range (0.05–1000 ng g−1), low quantitation limits (LOQs: 0.0027–0.011 ng g−1), high recoveries (90.1–119.6%) and good repeatability (RSD ≤10.5%, n = 3) for fipronil analysis. This study paves the way for pesticide analysis in soil risk assessment.

Changes in the Serum and Tissue Levels of Free and Conjugated Sialic Acids, Neu5Ac, Neu5Gc, and KDN in Mice after the Oral Administration of Edible Bird’s Nests: An LC–MS/MS Quantitative Analysis

Edible bird’s nests have a variety of biological activities, the main components of which are sialic acids. Sialic acids are a group of nine-carbon N-acetylated derivatives of neuraminic acid containing a keto group at position C2 and play important roles in many biological processes. To verify whether the oral administration of edible bird’s nests would change the content and distribution of sialic acid components in vivo, a liquid chromatography–mass spectrometry method for the quantitative analysis of sialic acid levels in serum and tissues was developed. In the negative ion mode, the mobile phases consist of 0.1% formic acid in water (A) and acetonitrile (v/v) (B). Isocratic elution was performed with 60% B for 0−15 min. The chromatographic separation was performed on a Morphling HILIC Amide column (2.1 mm × 150 mm, 5 μm) at a flow rate of 0.5 mL min−1. The results showed that the correlation coefficients of the typical calibration curves were all higher than 0.995, exhibiting good linearity. The levels of free and conjugated forms of N-glycolylneuraminic acid (Neu5Gc), N-acetylneuraminic acid (Neu5Ac), and 2-keto-3-deoxy-D-glycero-D-galactonononic acid (KDN) in the serum and different tissues were simultaneously detected after the oral administration of the edible bird’s nests at a daily dose of 300 and 700 mg Kg−1 for seven days in mice. Our study found that the oral administration of edible bird’s nests can significantly increase the concentration of total sialic acids (Neu5Gc + Neu5Ac + KDN) in serum and spleen and lungs tissues, which may be related to the anti-inflammatory and immune function of edible bird’s nest, but further studies are needed to verify this. Neu5Ac was the dominant sialic acid in brain tissue, and Neu5Gc was the dominant sialic acid in serum and other tissues, including heart, liver, spleen, lungs, and kidney. Moreover, we found that the forms of Neu5Ac and Neu5Gc were mainly conjugated in all groups except liver tissue. In conclusion, the method we established had good linearity and accuracy; it allowed the analytes to be effectively separated from the matrix and endogenous substances in serum or tissues, so it could effectively detect the distribution and concentration of free and conjugated forms of sialic acids in serum and tissues, which was beneficial to the research and exploitation of edible bird’s nests and sialic acids.

First trimester maternal tryptophan metabolism and embryonic and fetal growth: the Rotterdam Periconceptional Cohort (Predict Study).

What is the association between first trimester maternal tryptophan (TRP) metabolites and embryonic and fetal growth? Higher 5-hydroxytryptophan (5-HTP) concentrations are associated with reduced embryonic growth and fetal growth and with an increased risk of small-for-gestational age (SGA), while higher kynurenine (KYN) concentrations are associated with a reduced risk of SGA. The maternal TRP metabolism is involved in many critical processes for embryonic and fetal growth, including immune modulation and regulation of vascular tone. Disturbances in TRP metabolism are associated with adverse maternal and fetal outcomes. This study was embedded within the Rotterdam Periconceptional Cohort (Predict Study), an ongoing prospective observational cohort conducted at a tertiary hospital from November 2010 onwards. A total of 1115 women were included before 11 weeks of gestation between November 2010 and December 2020. Maternal serum samples were collected between 7 and 11 weeks of gestation, and TRP metabolites (TRP, KYN, 5-HTP, 5-hydroxytryptamine, and 5-hydroxyindoleacetic acid) were determined using a validated liquid chromatography (tandem) mass spectrometry method. Serial 3D ultrasound scans were performed at 7, 9, and 11 weeks of gestation to accurately assess features of embryonic growth, including crown-rump length (CRL) and embryonic volume (EV) offline using virtual reality systems. Fetal growth parameters were retrieved from medical records and standardized according to Dutch reference curves. Mixed models were used to assess associations between maternal TRP metabolites and CRL and EV trajectories. Linear and logistic regression models were utilized to investigate associations with estimated fetal weight (EFW) and birthweight, and with SGA, respectively. All analyses were adjusted for potential confounders. Maternal 5-HTP concentrations and the maternal 5-HTP/TRP ratio were inversely associated with embryonic growth (5-HTP, √CRL: β = -0.015, 95% CI = -0.028 to -0.001; 5-HTP 3√EV: β = -0.009, 95% CI = -0.016 to -0.003). An increased maternal 5-HTP/TRP ratio was also associated with lower EFW and birthweight, and with an increased risk of SGA (odds ratio (OR) = 1.006, 95% CI = 1.00-1.013). In contrast, higher maternal KYN concentrations were associated with a reduced risk of SGA in the unadjusted models (OR = 0.548, 95% CI = 0.320-0.921). Residual confounding cannot be ruled out because of the observational design of this study. Moreover, this study was conducted in a single tertiary hospital, which assures high internal validity but may limit external validity. The novel finding that maternal 5-HTP concentrations are associated with a smaller embryo and fetus implies that disturbances of the maternal serotonin pathway in the first trimester of pregnancy are potentially involved in the pathophysiology of fetal growth restriction. The association between higher maternal KYN concentrations and a reduced risk of SGA substantiate the evidence that the KYN pathway has an important role in fetal growth. More research is needed to delve deeper into the potential role of the maternal TRP metabolism during the periconception period and pregnancy outcome for mother and offspring. This study was funded by the Department of Obstetrics and Gynecology and the Department of Clinical Chemistry of the Erasmus MC, University Medical Center, Rotterdam, the Netherlands. The authors have no competing interests to disclose. N/A.

Mass Spectrometry-Based Method to Measure Aflatoxin B1 DNA Adducts in Formalin-Fixed Paraffin-Embedded Tissues.

Aflatoxin B1 (AFB1) is a potent human liver carcinogen produced by certain molds, particularly Aspergillus flavus and Aspergillus parasiticus, which contaminate peanuts, corn, rice, cottonseed, and ground and tree nuts, principally in warm and humid climates. AFB1 undergoes bioactivation in the liver to produce AFB1-exo-8,9-epoxide, which forms the covalently bound cationic AFB1-N7-guanine (AFB1-N7-Gua) DNA adduct. This adduct is unstable and undergoes base-catalyzed opening of the guanine imidazolium ring to form two ring-opened diastereomeric 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy-aflatoxin B1 (AFB1-FapyGua) adducts. The AFB1 formamidopyrimidine (Fapy) adducts induce G → T transversion mutations and are likely responsible for the carcinogenic effects of AFB1. Quantitative liquid chromatography-mass spectrometry (LC-MS) methods have shown that AFB1-N7-Gua is eliminated in rodent and human urine, whereas ring-opened AFB1-FapyGua adducts persist in rodent liver. However, fresh frozen biopsy tissues are seldom available for biomonitoring AFB1 DNA adducts in humans, impeding research advances in this potent liver carcinogen. In contrast, formalin-fixed paraffin-embedded (FFPE) specimens used for histopathological analysis are often accessible for molecular studies. However, ensuring nucleic acid quality presents a challenge due to incomplete reversal of formalin-mediated DNA cross-links, which can preclude accurate quantitative measurements of DNA adducts. In this study, employing ion trap or high-resolution accurate Orbitrap mass spectrometry, we demonstrate that ring-opened AFB1-FapyGua adducts formed in AFB1-exposed newborn mice are stable to the formalin fixation and DNA de-cross-linking retrieval processes. The AFB1-FapyGua adducts can be detected at levels comparable to those in a match of fresh frozen liver. Orbitrap MS2 measurements can detect AFB1-FapyGua at a quantification limit of 4.0 adducts per 108 bases when only 0.8 μg of DNA is assayed on the column. Thus, our breakthrough DNA retrieval technology can be adapted to screen for AFB1 DNA adducts in FFPE human liver specimens from cohorts at risk of this potent liver carcinogen.

LC-MS based untargeted metabolomics studies of the metabolic response of Ginkgo biloba extract on arsenism patients

Arsenic is an environmentally ubiquitous toxic metalloid. Chronic exposure to arsenic may lead to arsenicosis, while no specific therapeutic strategies are available for the arsenism patients. And Ginkgo biloba extract (GBE) exhibited protective effect in our previous study. However, the mechanisms by which GBE protects the arsenism patients remain poorly understood. A liquid chromatography-mass spectrometry (LC-MS) based untargeted metabolomics analysis was used to study metabolic response in arsenism patients upon GBE intervention. In total, 39 coal-burning type of arsenism patients and 50 healthy residents were enrolled from Guizhou province of China. The intervention group (n = 39) were arsenism patients orally administered with GBE (three times per day) for continuous 90 days. Plasma samples from 50 healthy controls (HC) and 39 arsenism patients before and after GBE intervention were collected and analyzed by established LC-MS method. Statistical analysis was performed by MetaboAnalyst 5.0 to identify differential metabolites. Multivariate analysis revealed a separation in arsenism patients between before (BG) and after GBE intervention (AG) group. It was observed that 35 differential metabolites were identified between BG and AG group, and 30 of them were completely or partially reversed by GBE intervention, with 14 differential metabolites significantly up-regulated and 16 differential metabolites considerably down-regulated. These metabolites were involved in promoting immune response and anti-inflammatory functions, and alleviating oxidative stress. Taken together, these findings indicate that the GBE intervention could probably exert its protective effects by reversing disordered metabolites modulating these functions in arsenism patients, and provide insights into further exploration of mechanistic studies.

DEVELOPMENT AND VALIDATION OF AN LC-MS/MS METHOD FOR THE DETERMINATION OF TENOFOVIR AND EMTRICITABINE

Objective: Therapeutic drug monitoring of tenofovir and emtricitabine, two commonly used antiretroviral drugs, is important to maximize effectiveness while minimizing side effects. Materials: A Liquid Chromatography-Mass spectrometry (LC-MS/MS) method was developed to quantify tenofovir and emtricitabine in human plasma samples. The method involves a simple solid phase extraction procedure followed by liquid chromatography separation using a Penta Fluoro Phenyl (PFP) column with a Phenomenex C18 column and a mobile phase of ammonium formate, acetonitrile, and methanol, achieving separation in under 4 min. Results: The method showed good accuracy, low limits of quantification, adequate recovery, minimal matrix effects, and specificity. Analyte stability under multiple storage conditions was demonstrated. Conclusion: The validated LC-MS/MS method provides a reliable tool for therapeutic drug monitoring and pharmacokinetic studies of anti-Human Immuno-deficiency Virus (HIV) regimens. The assay can be applied to large populations, especially in resource-poor settings, to help individualize dosing and improve clinical outcomes while reducing toxicity.

Effect of liposomal complexes of quercetin-rich flavonoids from French Marigold (Tagetes patula L.) on Jurkat cell viability

Background: Natural polyphenols are naturally used in traditional medicine to treat various diseases. Despite their healthful properties, ingesting phenolic compounds in food form does not provide a sufficient concentration for systemic therapeutic effects due to their low solubility in water, poor absorption, and fast metabolism. This problem has been solved by creating various composite pharmaceuticals from phenolic compounds using different methods to stabilize polyphenols. Objective: The purpose of this study was to assess the effects of the DPPA (1,2-palmitoyl phosphatidic acid) and DPPC (dipalmitoyl phosphatidylcholine) liposomes on the protective effects of a quercetin-rich flavonoid fraction extracted from French Marigold (Tagetes patula L.) on the viability of Jurkat cells. The study will examine both intact cells and cells that have been incubated under oxidative stress conditions. Materials and Methods: Quercetin-rich flavonoid fraction was extracted from a French Marigold (Tagetes patula L.) by thin-layer chromatography (TLC), High-pressure liquid chromatography (HPLC), and Liquid Chromatography-Mass Spectrometry (LC-MS) methods. Extract alone and in complex with dipalmitoyl phosphatidylcholine (DPPC) and 1,2- dipalmitoyl phosphatidic acid (DPPA) liposomes were added to the Jurkat cells culture at a rate of 2 mg/mL−1.The 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) test assayed Cell viability by evaluating cellular dehydrogenase activity. Results: Flavonoids were separated and identified in the marigold extracts by TLC, HPLC, and LC-MS methods. The spectrophotometric absorption spectrum of the quercetin-rich flavonoid fraction extracted from French Marigold (Tagetes patula L.) shows two peaks corresponding to benzoyl (254nm) and cinnamyl (375nm) aromatic rings. In the complex of quercetin-rich flavonoid fraction with DPPC and DPPA liposomes, the spectrophotometric absorption peak at 254nm was not detected, while the absorption intensity of the peak at 375nm was sharply reduced. The quercetin-rich flavonoid fraction alone and in combination with DPPC liposome increased intact and incubated under low- and high-intensity oxidative stress conditions Jurkat cells’ viability but did not reveal effect in combination with DPPA liposome. Conclusions: The quercetin-rich flavonoid fraction extracted from French Marigold (Tagetes patula L.) forms stable complexes with DPPC and DPPA liposomes that allow the storage of high content of phenolic compounds in lipid nanocapsules. The use of the liposomal system in the pharmaceutical and food industry allows for carried and controlled bioactive-compound release, which is considered one of the main strategies to improve and enhance the quality of food, providing preventative healthcare for the population and decreasing the risk of disease. Keywords: bioactive-compound, polyphenols, Quercetin, Liposomes, Jurkat cells, therapeutic effects, pharmaceuticals

Dried blood spot sampling of testosterone microdosing in healthy females

Capillary dried blood spot (DBS) analysis coupled with multi-analyte steroid liquid chromatography mass spectrometry (LCMS) is attractive for field studies, home-based self-sampling as well as clinical trials by eliminating costly and laborious sample processing involving venipuncture and frozen storage/shipping while providing multiple steroid measurements from a single small sample. We investigated steroid measurements in DBS samples stored for four years at room temperature prior to analysis compared with the original venipuncture serum samples. Healthy women (n=12) provided paired DBS and blood samples over two weeks run-in before seven days treatment with daily transdermal T gel (12.5 mg) and after the end of treatment on days 0, 1, 2, 4, 7 and 14. Compliance with treatment and sampling was high and no adverse effects were reported. Testosterone (T), androstenedione (A4), 17 hydroxyprogesterone (17OHP) and progesterone (P4) were measured in extracted DBS samples as whole blood concentrations with and without adjustment for hematocrit. Using the same LCMS methods, DBS T and A4 measurements had high correlation with minimal bias from prior serum measurements with DBS T displaying the same pattern as serum, with or without hematocrit adjustment. However, serial whole blood measurements of T without hematocrit adjustment provided the best fitting model compared with serum, urine, or hematocrit-adjusted whole blood T measurements. These finding facilitate and simplify DBS methodology for wider field and home-based self-sampling studies of reproductive steroids indicating the need for hematocrit adjustment may be superfluous.

Application of solid-phase microextraction and LCMS/MS for the detection of pesticide residues

Aiming at the organophosphorus pesticide residues in tea, a method integrating solid-phase microextraction and liquid chromatography-mass spectrometry was investigated for the detection of organophosphorus pesticide residues in tea. Firstly, the conditions in the process were optimized; then the method was established to analyze the organophosphorus pesticides in tea; finally, the method feasibility was verified by using actual sample determination. In terms of the results, the standard curve correlation coefficients of the solid-phase microextraction and liquid chromatography-mass spectrometry methods used in the study were greater than 0.99, while the average recoveries ranged from 72 to 109% with the relative standard deviations (RSDs) of less than 9.0%. This indicates that the method has a good linear range, low detection limit and high recovery. In the application validation in real samples, the concentrations of organophosphorus pesticide residues in tea were within the safe range. The method was validated to be suitable for the monitoring and control of organophosphorus pesticide residues in tea with low cost and simplified sample preparation.

Mitigating matrix effects for LC-MS/MS quantification of nitrosamine impurities in rifampin and rifapentine

Since the discovery of potentially mutagenic nitrosamine impurities in pharmaceuticals in 2018, liquid chromatography-mass spectrometry (LC-MS) methods have been widely applied for the trace level quantification of nitrosamines. As more nitrosamines and drug samples are analyzed, method accuracy is increasingly recognized as a major analytical challenge. Matrix effects can have a significant adverse impact on method accuracy but have not attracted much attention in nitrosamine quantification literature. In this study, we observed severe matrix effects when adapting LC-MS method conditions from a published and validated method for the analysis of nitrosamines in tuberculosis drugs. Several approaches were explored to mitigate these matrix effects, including using stable isotope-labeled internal standards, modifying LC conditions and sample concentration, and quantifying with standard addition instead of an external calibration method. The results underscore the importance of monitoring matrix effects and validating nitrosamine quantification procedures for specific sample matrices. Finally, two LC-MS/MS methods are presented with validation data for the quantification of 1-methyl-4-nitrosopiperazine (MNP) in rifampin and 1-cyclopentyl-4-nitrosopiperazine (CPNP) in rifapentine and revealed high nitrosamine levels in the tested samples that far exceeded recommended acceptable intake limits.

Biomonitoring of parabens in wild boars through hair samples analysis.

Parabens are compounds widely utilized in the industry as preservative additives to personal care products, cosmetics and food. They pollute the environment and penetrate to the living organisms through the digestive tract, respiratory system and skin. Till now the knowledge about exposure of terrestrial wild mammals to parabens is extremely scarce. Therefore, this study for the first time assessed the concentration levels of five parabens commonly used in industry (methylparaben-MeP, ethylparaben-EtP propylparaben-PrP, benzylparaben -BeP and butylparaben-BuP). Substances have been analyzed in hair samples collected from wild boars using liquid chromatography-mass spectrometry (LC-MS) method. The hair is a matrix, which allows to study long-term exposure of organisms to parabens. During this study MeP was noted in 96.3% of samples with mean 88.3±72.9 pg/mg, PrP in 87.0% of samples with mean 8.5±3.3 pg/mg, BeP in 44.4% of samples with mean 17.2±12.3 pg/mg and EtP in 11.1% of samples with mean 17.2±4.8 pg/mg. In turn BuP was noted only in 3.7% of samples with concentration levels below limit of quantification (2.6 pg/mg). Statistically significant intragender differences in parabens levels have not been noted. Only BeP concentration levels depended on industrialization and density of human population of area, where the animals lived. This study indicates that wild boars are exposed to parabens, especially to MeP and PrP, and analysis of the hair seems to be a useful tool of biomonitoring of parabens in wild mammals.

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.

Fast Label-Free Metabolic Profile Recognition Identifies Phenylketonuria and Subtypes.

Phenylketonuria (PKU) is the most common inherited metabolic disease in humans. Clinical screening of newborn heel blood samples for PKU is costly and time-consuming because it requires multiple procedures, like isotope labeling and derivatization, and PKU subtype identification requires an additional urine sample. Delayed diagnosis of PKU, or subtype identification can result in mental disability. Here, plasmonic silver nanoshells are used for laser desorption/ionization mass spectrometry (MS) detection of PKU with label-free assay by recognizing metabolic profile in dried blood spot (DBS) samples. A total of 1100 subjects are recruited and each DBS sample can be processed in seconds. This platform achieves PKU screening with a sensitivity of 0.985 and specificity of 0.995, which is comparable to existing clinical liquid chromatography MS (LC-MS) methods. This method can process 360 samples per hour, compared with the LC-MS method which processes only 30 samples per hour. Moreover, this assay enables precise identification of PKU subtypes without the need for a urine sample. It is demonstrated that this platform enables high-performance and fast, low-cost PKU screening and subtype identification. This approach might be suitable for the detection of other clinically relevant biomarkers in blood or other clinical samples.

Assessment of antioxidant potential, cytotoxicity, and anticancer activity of methanolic extracts from selected wild medicinal plants

IntroductionMedicinal plants are considered an important source of human health because of their therapeutic capabilities in treating various diseases. This study aimed to evaluate the phytochemical profile, antioxidant, and cytotoxic activities of the methanolic extracts of three wild medicinal plants (Ruta graveolens, Peganum harmala and Citrullus colocynthis). MethodsThe total phenolic content was determined according to the Folin–Ciocalteu method. The total flavonoid contents were measured by the aluminum chloride colorimetric method. The antioxidant activity was estimated based on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and ferric reducing antioxidant power (FRAP) assays. The phytochemical analysis was performed using liquid chromatography-mass spectrometry method. The cytotoxicity and anticancer activity of each plant extract was investigated against fibroblast and MDA-MB-231 breast cancer cell lines, respectively, using Giemsa staining assay. ResultsR. graveolens extract exhibited the highest concentrations of total phenols (25.5 ± 0.5 mg GA/g dry extract) and flavonoids (9.8 ± 0.5 mg Rutin/g dry extract). Additionally, it demonstrated the most robust antioxidant capacity, as demonstrated by both DPPH (100.8 ± 0.56 mg GA/g dry extract) and FRAP (91.2 ± 1.33 mg GA/g dry extract) assays. Within this plant, Rutin constituted 13.7 % of the total compounds. Furthermore, R. graveolens displayed the most significant anticancer activity, with statistical significance at p< 0.01. Notably, it exhibited the lowest cytotoxicity against the fibroblast cell line, even at the highest tested concentration of 100 μg/ml. In contrast, both P. harmala and C. colocynthis demonstrated substantial anticancer potential (p< 0.01); however, they both exhibited significant cytotoxicity against the fibroblast cell line (p<0.01). ConclusionR. graveolens extract holds promise as a potential reservoir of safe and effective antioxidant and anticancer compounds for human use.

Determination of glutathione in ready-to-eat edible bird's nest using a novel LC-MS method

Glutathione (GSH) is a strong antioxidant. However, there was no method to determine the GSH in ready-to-eat edible bird’s nest (EBN). A liquid chromatography-mass spectrometry (LC-MS) method was developed for the determination of GSH in ready-to-eat EBN in this study. The method involves extraction by formic acid and methanol, vortex, ultra sonication and analysis using HPLC-MS/MS. The results showed that the linearity was good in the concentration range of 0.05–200 μg/L. The limit of detection and the limit of quantification of the method were 0.1 μg/kg and 1.0 μg/kg. In addition, the recovery was 90.6∼100.4% and the relative standard deviation was 1.2∼2.5%. Moreover, the content of GSH in ready-to-eat EBN produced by different stewing treatment was investigated. It was found that the content of GSH in ready-to-eat EBN increased with the increasing stewing time and temperature. When the stewing temperature was above 110 °C and the stewing time was above 15 min, it was more conducive to the formation of GSH in ready-to-eat EBN. In conclusion, this study provided a new method for the determination of GSH in ready-to-eat EBN, and also opened up a new direction for exploring the nutritional value of ready-to-eat EBN.

A novel liquid chromatography-mass spectrometry method for transthyretin variant detection

A novel liquid chromatography-mass spectrometry method for transthyretin variant detection

An Automated Micro Solid-Phase Extraction (μSPE) Liquid Chromatography-Mass Spectrometry Method for Cyclophosphamide and Iphosphamide: Biological Monitoring in Antineoplastic Drug (AD) Occupational Exposure.

Despite the considerable steps taken in the last decade in the context of antineoplastic drug (AD) handling procedures, their mutagenic effect still poses a threat to healthcare personnel actively involved in compounding and administration units. Biological monitoring procedures usually require large volumes of sample and extraction solvents, or do not provide adequate sensitivity. It is here proposed a fast and automated method to evaluate the urinary levels of cyclophosphamide and iphosphamide, composed of a miniaturized solid phase extraction (µSPE) followed by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The extraction procedure, developed through design of experiments (DoE) on the ePrep One Workstation, required a total time of 9.5 min per sample, with recoveries of 77-79% and a solvent consumption lower than 1.5 mL per 1 mL of urine sample. Thanks to the UHPLC-MS/MS method, the limits of quantification (LOQ) obtained were lower than 10 pg/mL. The analytical procedure was successfully applied to 23 urine samples from compounding wards of four Italian hospitals, which resulted in contaminations between 27 and 182 pg/mL.