Positive Electrospray Research Articles

Non-targeted metabolomics for the identification of plasma metabolites associated with organic anion transporting polypeptide 1B1 function.

Our aim was to evaluate biomarkers for organic anion transporting polypeptide 1B1 (OATP1B1) function using a hypothesis-free metabolomics approach. We analyzed fasting plasma samples from 356 healthy volunteers using non-targeted metabolite profiling by liquid chromatography high-resolution mass spectrometry. Based on SLCO1B1 genotypes, we stratified the volunteers to poor, decreased, normal, increased, and highly increased OATP1B1 function groups. Linear regression analysis, and random forest (RF) and gradient boosted decision tree (GBDT) regressors were used to investigate associations of plasma metabolite features with OATP1B1 function. Of the 9152 molecular features found, 39 associated with OATP1B1 function either in the linear regression analysis (p < 10-5) or the RF or GBDT regressors (Gini impurity decrease > 0.01). Linear regression analysis showed the strongest associations with two features identified as glycodeoxycholate 3-O-glucuronide (GDCA-3G; p = 1.2 × 10-20 for negative and p = 1.7 × 10-19 for positive electrospray ionization) and one identified as glycochenodeoxycholate 3-O-glucuronide (GCDCA-3G; p = 2.7 × 10-16). In both the RF and GBDT models, the GCDCA-3G feature showed the strongest association with OATP1B1 function, with Gini impurity decreases of 0.40 and 0.17. In RF, this was followed by one GDCA-3G feature, an unidentified feature with a molecular weight of 809.3521, and the second GDCA-3G feature. In GBDT, the second and third strongest associations were observed with the GDCA-3G features. Of the other associated features, we identified with confidence two representing lysophosphatidylethanolamine 22:5. In addition, one feature was putatively identified as pregnanolone sulfate and one as pregnenolone sulfate. These results confirm GCDCA-3G and GDCA-3G as robust OATP1B1 biomarkers in human plasma.

Analytical Method for Melengestrol Acetate in Livestock Products Using LC-MS/MS

The present study verified that it is possible to analyze melengesterol acetate using the existing multi-residue method. Melengestrol acetate was extracted from livestock products using acidic acetonitrile acidified with acetic acid in the presence of n-hexane and anhydrous sodium sulfate. The crude extracts were cleaned up using an octadecylsilanized silica gel cartridge column. Separation by HPLC was performed using an octadecylsilanized silica gel column with linear gradient elution of 0.1 vol% formic acid and acetonitrile containing 0.1 vol% formic acid. For the determination of the analyte, tandem mass spectrometry with positive ion electrospray ionization was used. In recovery tests using four livestock products fortified with maximum residue limits levels of melengestrol acetate (0.001-0.02 mg/kg), the truenesses ranged from 82% to 100%, and the repeatabilities for the entire procedure ranged from 0.5 RSD% to 5.6 RSD%. In recovery tests using 11 livestock products fortified with 0.0005 mg/kg of melengestrol acetate, the truenesses ranged from 88% to 99%, and the repeatabilities ranged from 1.3 RSD% to 5.4 RSD%. The limit of quantification for melengestrol acetate in livestock products was 0.0005 mg/kg.

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 a novel anti‐influenza pinanamine‐based analog in rat plasma by liquid chromatography‐tandem mass spectrometry and its application in pharmacokinetic evaluations

AbstractA pinanamine‐based analog, (1R,2R,3R,5S)‐N‐((3‐cyclopropylthiophen‐2‐yl)methyl)‐2,6,6‐trimethylbicyclo[3.1.1]heptan‐3‐amine (M090) was synthesized and demonstrated with anti‐influenza activity to overcome the multi‐drug resistance. Herein, a rapid and robust liquid chromatography‐tandem mass spectrometry method was developed and validated to quantify M090 in rat plasma. With simple protein precipitation by methanol, the separation of M090 was performed on a Waters BEH‐C18 column (2.1 × 100 mm, 1.7 μm) by gradient elution at 0.4 mL/min with mobile phases consisting of water containing 0.1 % formic acid (phase A) and methanol (phase B). M090 and clenbuterol (internal standard) were detected using multiple reaction monitoring in positive electrospray ionization mode with transitions of m/z 290.3→137.0 and m/z 277.0→203.0, respectively. The method was validated over a linear range of 1.0–2400 ng/mL with a regression coefficient of 0.9974 and no endogenous interference. The intra‐ and inter‐batch precisions were within 8.29% and accuracy ranged from 100.3% to 108.1% for M090. The validated method was utilized to evaluate the in vitro metabolic stability and in vivo pharmacokinetics of M090 in rats.

Optimisation and application of the multi-residue analysis method for detection of 50 pesticides in cabbage by using LC-MS/MS- QuEChERS

ABSTRACT A multi-residue method for simultaneous determination of 50 pesticides in cabbage, belonging to different chemical classes has been developed by LC-MS/MS. The pesticides were analysed simultaneously in a single run using positive electrospray ionisation with multiple reaction monitoring (MRM) after extraction with modified QuEChERS method. The calibration curve for each analyte was linear with the concentration of 0.0025, 0.005 0.01, 0.025, 0.05 and 0.10 µg mL−1 with a correlation coefficient which ranged between 0.993 and 0.999. The mean recoveries obtained for four fortification levels (0.01, 0.025, 0.050 and 0.10 μg/g) were 77.96–119.67% with satisfactory precision (RSD < 20%). The matrix effects were found in range of 6.42 to 19.52% with optimised cleanup process. The developed method was used to investigate the occurrence of pesticides residue in cabbage head samples collected from different markets of the Varanasi region, India. All the 50 compounds were eluted from the column of the LC-MS/MS within the 7.961 min. The limit of quantification of the optimised method was 0.01 mg kg−1 for all the 50 pesticides. Therefore, it would be recommended as a very sensitive and quick method for estimation of multi-residue in cabbage. The developed method was successfully utilised for the analysis of the market samples collected from four different market of the Varanasi, India. Total five compounds namely thiamethoxam, carbendazim, imidacloprid, acetamiprid and dimethoate were detected in cabbage samples. Out of five compounds, only two compounds i.e. acetamiprid and dimethoate are recommended by Central Insecticide Board and Registration Committee (CIB&RC), Govt. of India.

Integrative analysis of tepotinib forced degradation: Combining in‐silico and liquid chromatography‐quadrupole time‐of‐flight‐tandem mass spectrometry approaches for structural elucidation

AbstractThe present research aimed to comprehensively investigate the forced degradation of tepotinib through a combined in‐silico and experimental analysis. Tepotinib is a recently approved drug for metastatic non‐small cell lung cancer. A short and precise ultra‐high‐performance liquid chromatography (UHPLC) method utilizing a CSH C18 column (100 × 2.1 mm, 1.7 μm) was developed, using a mobile phase A as 0.1% formic acid and mobile phase B as HPLC‐grade acetonitrile. The flow rate was set at 0.2 mL/min, and detection was carried out at 260 nm. Forced degradation was carried out under acidic, basic, oxidative, and photolytic environments and also virtually with Zeneth software. Structural characterization of degradation products (DPs) was carried out using hyphenated techniques such as LC‐quadrupole time‐of‐flight‐tandem mass spectrometry in positive electrospray ionization mode. Further, toxicity was assessed with Derek and Sarah software. Analysis revealed the formation of five distinct forced DPs: DP‐1 (m/z 511), DP‐2 (m/z 527), DP‐3 (m/z 525), DP‐4 (m/z 512), and DP‐5 (m/z 509) under experimental conditions and eight in Zeneth prediction. Furthermore, nephrotoxicity alerts were observed for DP‐3 and DP‐5, whereas DP‐3 also showed mutagenicity alerts. This integrated blend of in‐silico and real experimental analyses was found to be effective in quality control for routing monitoring of degradation products of tepotinib.

Molecular transformations of heteroatomic organic compounds in crude oils caused by biodegradation and subsequent thermal maturation: Insights from ESI FT-ICR MS

The Tarim and Junggar basins are major petroliferous sedimentary deposits in China. Some oil reservoirs in these basins experienced superimposed secondary alterations including early biodegradation followed by thermal maturation/alteration (SSA-B&M). Heteroatomic polar organic compounds in crude oil are significantly influenced by SSA-B&M, which play a vital role in understanding the evolutionary history of oil reservoir. However, research on such alterations is limited. The present study employed negative and positive ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) to investigate the molecular transformations of heteroatomic organic compounds in crude oils resulting from SSA-B&M. The findings indicate that biodegradation leads to an enrichment of naphthenic acids, while subsequent cyclization, aromatization and thermal cracking reactions enhances the aromaticity of the pyrolysis products of all the oil samples with varying degree of biodegradation. Notably, highest aromaticity was observed for the crude oil that has been most severely altered by biodegradation. During early and moderate stages of biodegradation, the abundances of O2 compounds (molecules contain two oxygen atoms) representing carboxylic acids were continuously increasing. However, at severe biodegradation levels, fatty acids with 1 double bond equivalents (DBE) underwent degradation, while the naphthenic acids were further enriched. Subsequently, during maturation, O2 compounds with 1 to 5 DBE experienced a steady decline in abundance. The naphthenic acids mainly experienced aromatization, while the fatty acids were partly decarboxylized and cracked thereby forming new n-alkanes. Biodegradation led to modification in neutral nitrogen compounds with 3 to 4 condensed rings (DBE = 9–12) and long alkyl side chains, while the impact on basic nitrogen compounds was relatively minimal. After subsequent thermal maturation, both neutral and basic nitrogen compounds with long alkyl side chains underwent cracking, while those with shorter alkyl side chains and higher degrees of condensation tended to remain relatively unchanged. Finally, two new indices were defined and tested to correctly evaluate the maturation and biodegradation level for crude oils that have experienced SSA-B&M.

A simple and sensitive liquid chromatography triple quadrupole mass spectrometry method for the determination of XL092 in monkey plasma and its application to pharmacokinetic study.

XL092 is a potent ATP-competitive inhibitor of multiple receptor tyrosine kinases that is undergoing clinical development for the treatment of lung cancer. In this study, an LC triple quadrupole mass spectrometry method was established to measure XL092 in monkey plasma. A Waters ACQUITY UPLC BEH C18 column was used for chromatographic separation. The mobile phase consisted of water containing 0.1% formic acid and acetonitrile with a gradient elution at the flow rate of 0.4 mL/min. Multiple reaction monitoring mode was used for quantitative analysis of XL092 in positive electrospray ionization. In the concentration range of 0.5-1000 ng/mL, XL092 showed excellent linearity in monkey plasma with a correlation coefficient greater than 0.995 (r > 0.995). The lowest limit of quantification was 0.5 ng/mL. The intra- and inter-day relative standard deviations were <9.99%, while the relative error ranged from -12.50% to 8.10%. The mean recovery was over 82.51%. XL092 was stable in monkey plasma after storage under certain conditions. The validated method was demonstrated to be selective, sensitive, and reliable, and has been successfully applied to the pharmacokinetic study of XL092 in monkey plasma. XL092 showed moderate short half-life (~3.81 h) and good oral bioavailability (80%).

An Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) Method for Qualifying DAPB in Rat Plasma and Application to Pharmacokinetic Studies.

DAPB, a new molecule including danshensu, borneol, and a mother nucleus of ACEI (Angiotensin-converting enzyme inhibitors), is being developed as an antihypertensive candidate compound. A rapid, accurate, and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established and validated for the determination of DAPB in rat plasma. Chromatographic separation was performed on an Agilent SB-C18 column after protein precipitation by acetonitrile with a mobile phase consisting of acetonitrile and deionized water with 0.02% formic acid and 5 mM NH4F (v/v) at a flow rate of 0.2 mL/min. Quantification was performed using electrospray positive ionization mass spectrometry in the multiple reaction monitoring (MRM) mode. The method was linear over the range of 2-1000 ng/mL. The intra- and inter-day precision was within 12%, with accuracies less than 7%. Stability was within the acceptable limits under various storage and processing conditions. No apparent matrix effect was detected. The validated method was applied to the pre-clinical pharmacokinetic study of DAPB after oral administration of 30 mg/kg and intravenous administration of 6 mg/kg in rats.

Optimized LC-MS/MS method for quantifying insulin degludec and liraglutide in rat plasma and Tissues: Application in pharmacokinetics and biodistribution

We present an ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous detection of insulin degludec (I-Deg) and liraglutide (LIRA) in rat plasma and tissues, characterized by its sensitivity and selectivity. Chromatographic separation was achieved using an Acquity UPLC BEH C18 column, leveraging a mobile phase of acetonitrile and water (both with 0.1 % formic acid) under gradient elution over a run time of 7.5 min. The mass spectrometer operated in positive electrospray ionization multiple reaction monitoring (MRM) mode, tracking transitions of m/z 1221.6 → 641.6 for I-Deg, m/z 938.7 → 1064.1 for LIRA, and m/z 1184.7 → 454.4 for the internal standard. Validation ranged from 5 to 100 ng/mL, exhibiting robust linearity (r2 > 0.99) and limits of detection (LOD) of 1.63–2.02 ng/mL for I-Deg and 0.96–1.62 ng/mL for LIRA. Limits of quantification (LOQ) were 2.38–4.76 ng/mL for I-Deg and 3.22–4.40 ng/mL for LIRA. Notably, no significant matrix effects were detected. Stability was confirmed under various conditions, and precision metrics (intraday RSD 1.68–8.05 % for I-Deg and 1.11–7.69 % for LIRA; interday RSD 1.39–8.61 % for I-Deg and 1.06–8.83 % for LIRA) alongside accuracy (90.5–114.9 % for I-Deg and 92.7–113.7 % for LIRA) were within acceptable ranges. The method was successfully applied to pharmacokinetic and biodistribution studies following simultaneous subcutaneous administration of LIRA and I-Deg in rats.

Polyphenols, Antioxidants, and Wound Healing of Lecythis pisonis Seed Coats.

To better use the Lecythis pisonis Cambess. biomass, this study investigates whether Sapucaia seed coats present wound healing properties. We analyzed the antibacterial, antioxidant, and wound healing-promoting potentials, plus cytotoxicity and stimulation of vascular endothelial growth factor-A. The chemical composition was analyzed by positive ion mode electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. A total of 19 compounds were identified, such as proanthocyanidin A1, procyanidins A1, B2, and C1, epigallocatechin, and kaempferol (p-coumaroyl) glycoside. Potent antioxidant strength/index was verified for 2,2-diphenyl-1-picrylhydrazyl radical (IC50 = 0.99 µg/mL) and ferric reducing antioxidant power (IC50 = 1.09 µg/mL). The extract did not present cytotoxicity and promoted significant cell migration and/or proliferation of fibroblasts (p < 0.05). Vascular endothelial growth factor-A was stimulated dose-dependently at 6 µg/mL (167.13 ± 8.30 pg/mL), 12.5 µg/mL (210.3 ± 14.2 pg/mL), and 25 µg/mL (411.6 ± 29.4 pg/mL). Platelet-derived growth factor (PDGF) (0.002 µg/mL) was stimulated at 215.98 pg/mL. Staphylococcus aureus was susceptible to the extract, with a minimum inhibitory concentration of 31.25 µg/mL. The identified compounds benefit the antioxidant activity, promoting hemostasis for the wound healing process, indicating that this extract has the potential for use in dermatological cosmetics.

Development and validation of an UPLC-MS/MS method for the determination of irinotecan (CPT-11), SN-38 and SN-38 glucuronide in human plasma and peritoneal tumor tissue from patients with peritoneal carcinomatosis

Irinotecan (CPT-11), an antineoplastic drug, is used for the treatment of colorectal and pancreatic cancer due to its topoisomerase I inhibitory activity. CPT-11 is a prodrug which is converted to its active metabolite SN-38 by carboxylesterases. SN-38 is further metabolized to its inactive metabolite SN-38 glucuronide. When evaluating the pharmacokinetic properties of CPT-11 and its metabolites, it is important to accurately assess the concentrations in both plasma as well as tumor tissues. Therefore, the aim of the current study was to develop and validate a robust and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method to quantify the concentration of CPT-11 and its metabolites (SN-38 and SN-38 glucuronide) in human plasma and peritoneal tumor tissue. The sample preparation of plasma and tumor tissue consisted of protein precipitation and enzymatic digestion/liquid–liquid extraction, respectively. Chromatographic separation was achieved with an Acquity UPLC BEH C18 column combined with a VanGuard pre-column. The mobile phases consisted of water +0.1 % formic acid (mobile phase A) and acetonitrile +0.1 % formic acid (mobile phase B). Mass analysis was performed using a Xevo TQS tandem mass spectrometer in the positive electrospray ionization mode. Method validation was successfully performed by assessing linearity, precision and accuracy, lower limit of quantification, carry over, selectivity, matrix effect and stability according to the following guidelines: “Committee for Medicinal Products for Human use, Guideline on Bioanalytical Method Validation”. A cross-validation of the developed method was performed in a pilot pharmacokinetic study, demonstrating the usefulness of the current method to quantify CPT-11 and its metabolites in the different matrices.

Identification of Serine-Containing Microcystins by UHPLC-MS/MS Using Thiol and Sulfoxide Derivatizations and Detection of Novel Neutral Losses.

Microcystins (MCs) are hepatotoxic cyclic heptapeptides produced by cyanobacteria, and their structural diversity has led to the discovery of more than 300 congeners to date. However, with known amino acid combinations, many more MC congeners are theoretically possible, suggesting many remain unidentified. Herein, two novel serine (Ser)-containing MCs were putatively identified in a Lake Erie cyanobacterial harmful algal bloom (cyanoHAB), using high-resolution UHPLC-MS as well as thiol and sulfoxide derivatization procedures. These MCs contain an α,β-unsaturated carbonyl on methyl dehydroalanine (Mdha) residue that undergoes Michael addition to produce a thiol-derivatized MC. Derivatization reactions using various thiolation reagents were followed by MS/MS, and two Python codes were used for data analysis and structural elucidation of MCs. Two novel MCs containing Ser at position 1 (i.e., next to Mdha) were putatively identified as [Ser1]MC-RR and [Ser1]MC-YR. Using thiol- and sulfoxide-modified [Ser1]MCs, identifications were confirmed by the observation of specific neutral losses of the oxidized thiols or sulfoxides in CID-MS/MS spectra in both positive and negative electrospray ionization (ESI) modes. These novel neutral losses are unique for MCs with Mdha and an adjacent Ser residue. Data suggest that a gas-phase reaction occurs between oxygen from adjacent Ser residue and sulfur of the Mdha-bonded thiol or sulfoxide, which leads to the formation and detection of stable cyclic MC ions in MS/MS spectra at m/z values corresponding to the loss of oxidized thiols or oxidized sulfoxides from Ser1-containing MCs.

Spatial and temporal variability of micropollutants within a wastewater catchment system.

Treated wastewater effluent is a major contributor to concentrations of many anthropogenic chemicals in the environment. Examining patterns of these compounds measured from different catchment areas comprising the influent to a wastewater treatment plant, across many months, may reveal patterns in compound sources and seasonality helpful to management efforts. This study considers a wastewater catchment system that was sampled at six sub-catchment sites plus the treatment plant influent and effluent at seven time points spanning nine months. Wastewater samples were analyzed with LC-QTOF-MS using positive electrospray ionization and GC-QTOF-MS using negative chemical ionization and electron ionization. MS data were screened against spectral libraries to identify micropollutants. As expected, multiple classes of chemicals were represented, including pharmaceuticals, plasticizers, personal care products, and flame retardants. Patterns in the compounds seen at different sampling sites and dates reflect the varying uses and down-the-drain routes that influence micropollutant loading in sewer systems. Patterns in examined compounds revealed little spatial variation, and greater temporal variation. For example, the greatest loads of DEET were found to occur in the summer months. Additionally, groups of compounds exhibited strong correlation with each other, which could be indicative of similar down-the-drain routes (such as a group intercorrelated chemicals that are components of cleaning products) or the influence of similar physicochemical processes within the sewer system. This study contributes to the understanding of dynamics of micropollutants in sewer systems.

Jiannao pills mitigate chronic restraint stress-induced anxiety in mice through the regulation of intestinal microflora.

To investigate the effects of Jiannao pills on mice with chronic restraint stress-induced anxiety and its mechanisms. Anxiety-like behaviors were induced in mice by exposing them to chronic restraint stress (8 h/day for 21 days). Subsequently, Jiannao pills were given to these mice for the treatment of the induced anxiety. Following treatment, the intestinal microflora of the mice were analyzed using the 16S rRNA sequencing method. Under positive electrospray ionization mode, a total of 68 chemical compositions were found in Jiannao pills, and under negative electrospray ionization mode, the number was 18. With these chemical compositions in effect, it was observed that Jiannao pills alleviated chronic restraint induced anxiety-like behaviors in mice by extending their dwelling time, standing time, and grooming time in the central area, as well as the percentage of entries and time spent in the open arms. This effect was similar to that of alprazolam. In addition, Jiannao pills significantly improved neural functions in mice with chronic restraint-induced anxiety, diminished the levels of 5-hydroxytryptamine and glutamate, and increased the levels of γ-aminobutyric acid. Furthermore, Jiannao pills decreased the expressions of the corticotropin-releasing factor and cholecystokinin protein, while elevating neuropeptide Y protein levels. The results of 16S rRNA sequencing analysis revealed both Jiannao pills and alprazolam altered the composition of intestinal microflora in mice, with Jiannao pills exhibiting a more pronounced effect. Specifically, there was a significant increase in the abundance of S24-7 in mice following treatment. Besides, significant differences were observed in a total of 632 operational taxonomic units in mice after Jiannao pill treatment. The functions of the intestinal microflora of mice were primarily associated with their betalain biosynthesis and classification levels. Jiannao pills effectively ameliorated chronic restraint anxiety-like behaviors in mice and enhanced their neural functions potentially through the regulation of their intestinal microflora.

Evaluating the Presence and Depletion of Residual Ivermectin in Milk Following Subcutaneous Injections on Lactating Cows

Dairy farming industry in Kenya has experienced significant growth due to the rising consumption of milk and dairy products. Consequently, to improve output,veterinary drugs were administered prophylactically and/or therapeutically to dairy cattle. Commonly used drugs included beta-agonists, anthelmintics, antibiotics, and steroid hormones. Parasitic diseases more so tick infestations hindered production. Chemical methods were primarily used in tick management however, the technique proved expensive and developed resistance. Conversely, ivermectin effectively controlled ecto- and endoparasites in livestock, hence gaining popularity in tick management. Nonetheless, its residues accumulated in animal tissues, rendering it unacceptable on animals that produced milk for human use. Apparently, farmers in tick-infested areas in Nyandarua County, Kenya used ivermectin, but there lacked adequate evidence to show that it occurred in milk. Thus, this study examined the performance of Liquid Chromatography tandem mass spectrometry (LC-MS-MS) in order to analyse the presence and depletion pattern of ivermectin residues after it excreted in cow milk following subcutaneous treatment of dairy cows. Between day 0.5 and day 57 following treatment, 24 milk samples were purposefully collected and stored at -20°C.. The samples were prepared using QuEChERS, a solid-phase extraction technique.Further, positive Electrospray ionization (+ESI) LC-MSMS qualitative analysis identified a precursor ion with the mass number of m/z 897.5 and corresponding product ions—m/z 897.5 &gt; 329, m/z 897.5 &gt; 240, and m/z 897.5 &gt; 183—within a retention time of 8.782-8.858 (mean = 8.362±0.002) minutes. The solvent and matrixmatched calibration curves displayed satisfactory linearity with r=0.998 and 0.993 as their respective correlation coefficients. Additionally, the limit of detection was 2.5 ng/ml and limit of quantification reached 10 ng/ml. The analyte recovery ranged from 74.977% to 101.435%, within satisfactory limits. Hence, ivermectin residues were quantified in milk samples from the 0.5th to 17th day. The highest concentration (60.90±0.98 ng/ml) was observed on 2nd day. No traces of the residue were detected beyond day 18 and as a result, the study recommends a withdrawal period of 18days after subcutaneous ivermectin treatment. It is therefore important to perform further research on other dairy and animal products that people consume.

Quantitative analysis of tetrahydrocannabinol isomers and other toxicologically relevant drugs in blood.

A multi-analyte liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described, involving the separation of delta-9-tetrahydrocannabinol (delta-9-THC) and delta-8-THC in addition to other commonly encountered drugs and metabolites. Briefly, sample preparation involved an alkaline liquid-liquid extraction (methyl tert-butyl ether) of blood (100 μl). The solvent layer was transferred, evaporated to dryness, reconstituted, and samples then separated on an Agilent Poroshell 120 EC-C18 100 Å (50 mm × 3.0mm, 2.7μm) analytical column using a multi-step gradient elution of 50 mM ammonium formate in water (pH3.5) and 0.1% formic acid in methanol over 14 min. A SCIEX Triple Quad 6500+ system operating in scheduled multiple reaction monitoring and positive electrospray ionization was used for detection. There were no interferences, and matrix effects were generally acceptable (±20% of neat response). Linearity was achieved within the calibration range, including methylamphetamine (MA) (10-1000 ng/ml), 3,4-methylenedioxy-N-methylamphetamine (MDMA) (10-1,000 ng/ml), cocaine (10-1000 ng/ml), and two THC isomers (1-100 ng/ml). Accuracies of MA, MDMA, cocaine, and two THC isomers were 3.6 to 8.9%, -1.2 to 4%, -5.3 to 5.8%, and -11 to 14%, respectively; while precision estimates of the same were 1.6 to 5.4%, 1.7 to 5.3%, 1.2 to 4.5%, and 2 to 10%, respectively. Autosamplerstability and dilution integrity were within acceptable limits, and no carryover was detected at the limit of detection. This validated LC-MS/MS method made the routine identification of both delta-9-THC and delta-8-THC in blood possible.

Dioxin(-like)-Related Biological Effects through Integrated Chemical-wide and Metabolome-wide Analyses.

Dioxin(-like) exposures are linked to adverse health effects, including cancer. However, metabolic alterations induced by these chemicals remain largely unknown. Beyond known dioxin(-like) compounds, we leveraged a chemical-wide approach to assess chlorinated co-exposures and parent compound products [termed dioxin(-like)-related compounds] among 137 occupational workers. Endogenous metabolites were profiled by untargeted metabolomics, namely, reversed-phase chromatography with negative electrospray ionization (C18-negative) and hydrophilic interaction liquid chromatography with positive electrospray ionization (HILIC-positive). We performed a metabolome-wide association study to select dioxin(-like) associated metabolic features using a 20% false discovery rate threshold. Metabolic features were then characterized by pathway enrichment analyses. There are no significant features associated with polychlorinated dibenzo-p-dioxins (PCDDs), a subgroup of known dioxin(-like) compounds. However, 3,110 C18-negative and 2,894 HILIC-positive features were associated with at least one of the PCDD-related compounds. Abundant metabolic changes were also observed for polychlorinated dibenzofuran-related and polychlorinated biphenyl-related compounds. These metabolic features were primarily enriched in pathways of amino acids, lipid and fatty acids, carbohydrates, cofactors, and nucleotides. Our study highlights the potential of chemical-wide analysis for comprehensive exposure assessment beyond targeted chemicals. Coupled with advanced endogenous metabolomics, this approach allows for an in-depth exploration of metabolic alterations induced by environmental chemicals.

Applicable Pharmacokinetic Study: Development and Validation of Bioanalytical LC-MS/MS Method for Simultaneous Determination of Tipiracil, Trifluridine and Its Two Metabolites 5-Trifluoromethyluracil, 5-Carboxy 2′-Deoxyuridine in Rat Plasma

A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous determination of tipiracil (TIP), trifluridine (FTD), and their metabolites, 5-trifluoromethyluracil (FTY) and 5-carboxy-2′-deoxyuridine (5CDU), in rat plasma. This method is highly sensitive, specific, and fast. Paracetamol (PAR) is used as an internal standard (IS). Using acetonitrile-induced protein precipitation, the analytes were extracted from a plasma sample and separated on a Waters BEH C18 (1.7 μm particle size, 50 mm × 2.1 mm ID) column protected by a security guard cartridge (C18, 4 × 2.0 mm). The isocratic mobile phase was made up of methanol and water containing 0.1% formic acid (80:20, v/v) at a flow rate of 0.5 mL/min for 4 min. The quantification was performed using a positive electrospray ionization (ESI) interface and a multiple-reaction monitoring (MRM) mode. The MRM transitions employed were m/z 242.96 → 182.88 for TIP, 296.96 → 116.86 for FTD, 180.98 → 139.85 for FTY, 272.96 → 156.86 for 5CDU, and 151.97 → 92.68 for IS. The validated method complied with the guidelines set by the US-FDA over on a linear concentration range of 5–4000 ng/mL for FTD, FTY, and 5CDU, and 5–1000 ng/mL for TIP. The coefficient of determination (r2) was equal to or greater than 0.997. The corresponding lower limits of detection (LLOD) were 1.5 ng/mL for FTD, FTY, and 5CDU and 1.0 ng/mL for TIP. The recoveries of all analytes from rat plasma ranged from 88.67% to 112.18%, and the mean relative standard deviation (RSD) of accuracy and precision result was less than or equal to 6.84%. FTD, FTY, 5CDU, and TIP demonstrated adequate stability throughout the various circumstances examined. Additionally, no matrix effects were identified for any of the analytes. The assay was effectively utilized to conduct a pharmacokinetic study in rats following the oral administration of FTD and TIP at a dosage of 5.6 mg/kg, with a ratio of 1:0.5 for FTD and TIP, respectively. This indicates that the suggested approach is suitable for future clinical research. The pharmacokinetic parameters Cmax (maximum concentration), Tmax (time to reach maximum concentration), t1/2 (half-life), AUC0-24 (area under the concentration–time curve from 0 to 24 h), AUC total (total area under the concentration–time curve), Ke (elimination rate constant), Vd (volume of distribution), and CL (clearance) of all analytes were assessed. The assay developed exhibits significant advancements compared to earlier bioanalytical methods documented in the literature. These improvements include high sensitivity, specificity, and efficacy in high throughput analysis of complex matrices. Additionally, the assay offers a shorter run time and smaller sample volume (50 μL).

High‐throughput mass spectrometry analysis using immediate drop‐on‐demand technology coupled with an open port sampling interface

RationaleThe sampling throughput of immediate drop‐on‐demand technology (I.DOT) coupled with an open port sampling interface (OPSI) is limited by software communication. To enable much‐needed high‐throughput mass spectrometry (MS) analysis capabilities, a novel software was developed that allows for flexible sample selection from a 96‐well plate and for maximized analysis throughput using I.DOT/OPSI‐MS coupling.MethodsWells of a 96‐well I.DOT plate were filled with propranolol solution and were used to test maximum sampling throughput strategies to minimize analysis time. Demonstration of chemical reaction monitoring was done using acid‐catalyzed ring closure of 2,3‐diaminonaphthalene (DAN) with nitrite to form 2,3‐naphthotriazole (NAT). Analytes were detected in positive electrospray ionization mode using selected reaction monitoring.ResultsA maximum throughput of 1.54 s/sample (7.41 min/96‐well plate with three technical replicates) was achieved, and it was limited by the peak width of the MS signal resulting in an occasional slight overlap between the peaks. Relative standard deviation was 10 ± 1% with all tested sampling strategies. Chemical reaction monitoring of DAN to NAT using nitrite was successfully accomplished with 2 s/sample throughout showing almost complete transformation in 10 min with no signal overlap.ConclusionsThis work illustrates the development of a noncontact, automated I.DOT/OPSI‐MS system with improved throughput achieved through an optimized software interface. Its achievable analysis time and precision make it a viable approach for drug discovery and in situ reaction monitoring studies.