Compounds In Human Plasma Research Articles

Novel electrospun-based extractive probes for rapid determination of clinically important compounds in human plasma

BackgroundCoated blade spray (CBS) represents an innovative approach that utilizes solid-phase microextraction principles for sampling and sample preparation. When combined with ambient mass spectrometry (MS), it can also serve as an electrospray ionization source. Therefore, it became a promising tool in analytical applications as it can significantly reduce the analysis time. However, the current CBS coatings are based on the immobilization of extractive particles in bulk polymeric glue, which constrains the diffusion of the analytes to reach the extractive phase; therefore, the full reward of the system cannot be taken at pre-equilibrium. This has sparked the notion of developing new CBS probes that exhibit enhanced kinetics. ResultsWith this aim, to generate a new extractive phase with improved extraction kinetics, poly(divinylbenzene) (PDVB) nanoparticles were synthesized by mini-emulsion polymerization and then immobilized into sub-micrometer (in diameter) sized polyacrylonitrile fibers which were obtained by electrospinning method. Following the optimization and characterization studies, the electrospun-coated blades were used to determine cholesterol, testosterone, and progesterone in plasma spots using the CBS-MS approach. For testosterone and progesterone, 10 ng mL−1 limits of quantification could be obtained, which was 200 ng mL−1 for cholesterol in spot-sized samples without including any pre-treatment steps to samples prior to extraction. SignificanceThe comparison of the initial kinetics for dip-coated and electrospun-coated CBS probes proved that the electrospinning process could enhance the extraction kinetics; therefore, it can be used for more sensitive analyses. The total analysis time with this method, from sample preparation to instrumental analysis, takes only 7 min, which suggests that the new probes are promising for fast diagnostic applications.

Ultrasensitive immunosensor for multiplex detection of cancer biomarkers carcinoembryonic antigen (CEA) and yamaguchi sarcoma viral oncogene homolog 1 (YES1) based on eco-friendly synthesized gold nanoparticles

Cancer is one of the most extensive diseases with the highest mortality rate, accounting for almost 10 million deaths in 2020. The most common cancers are breast, lung, colon and rectum and prostate cancers. Of these, lung cancer, accounted for about 1.8 million of all cancer deaths (25%) in 2020. Detection of cancer relies on presence of biomarkers such as DNA molecules, proteins and metabolites released by cancerous cells into the circulation. Carcinoembryonic antigen (CEA) is one of the biomarkers that has been used for the detection of lung cancer. However, CEA is not specific to lung cancer since it is also manifested in gastric cancer, pancreatic cancer, colorectal cancer, and breast cancer. Recently, v-YES1 Yamaguchi sarcoma viral oncogene homolog 1 (YES1) was described as a specific biomarker for lung cancer. The detection of both CEA and YES1 would give more precise and authentic information for detecting lung cancer. This is because detection of a single tumor marker usually limits the precision in tumor diagnosis, due to the fact that several cancers have more than one marker linked with their prevalence. Whereas traditional methods have been used for the detection of CEA, electrochemical immunosensors have attracted considerable attention owing to their profound advantages, including fast response, miniaturization, high selectivity, low sample requirements and magnificent sensitivity. The fabrication of a multiplex and simultaneous immunosensor is met with challenge of preparation of distinguishable immunoprobes with different redox activities. This can be addressed by incorporation of electroactive Nano metals into the sensing platform. In this study, gold nanoparticles were used for the fabrication of an ultrasensitive sandwich electrochemical multiplex immunosensor for simultaneous detection of CEA and YES1. Under optimized conditions, the electrochemical immunosensor detection limit for YES1 and CEA was found to be 0.0022 and 0.0034 ng/mL respectively within a linear range of 0.1–50 ng/mL. The proposed immunosensor proved to be stable for up to 2 weeks and had negligible cross reactivity towards various interfering compounds in human plasma. This study reports that gold nanoparticles can be bio synthesized using shade dried Mangifera indica leaves extract. The bio-synthesized gold nanoparticles coupled with thiolated protein G can be used for fabrication of a multiplex immunosensor for detection of CEA and YES1. The proposed immunosensor can provide a new approach for early diagnosis of circulating cancer biomarkers and holds great promise for application in clinical diagnosis.

Quantitative Analysis of Isoniazid and Its Four Primary Metabolites in Plasma of Tuberculosis Patients Using LC-MS/MS

Isoniazid and its metabolites are potentially associated with hepatotoxicity and treatment outcomes in patients who receive antituberculosis (TB) therapy. To further understand the pharmacokinetic profiles of these molecules, a method based on LC-MS/MS was developed to determine the concentration of these compounds in human plasma. Isoniazid, acetylisoniazid, and isonicotinic acid were directly analyzed, whereas hydrazine and acetylhydrazine were determined after derivatization using p-tolualdehyde. Chromatographic separation was conducted on reversed-phase C18 columns with gradient elution, and detection was carried out in multiple reaction monitoring mode. The calibration curves were linear with correlation coefficients (r) greater than 0.9947 for all analytes. The intra- and inter-day precision was less than 13.43%, and the accuracy ranged between 91.63 and 114.00%. The recovery and matrix effect of the analytes were also consistent (coefficient of variation was less than 9.36%). The developed method successfully quantified isoniazid and its metabolites in TB patients. The method has broad applications in clinical research, including isoniazid one-point-based therapeutic drug monitoring, genotype-phenotype association studies of isoniazid metabolic profile and isoniazid-induced hepatotoxicity, and the initial dose prediction of isoniazid using population pharmacokinetic modeling.

Quantitation of cystic fibrosis triple combination therapy, elexacaftor/tezacaftor/ivacaftor, in human plasma and cellular lysate

The triple combination modulator therapy (ETI, elexacaftor (ELX), tezacaftor (TEZ), and ivacaftor (IVA)) is a recent advancement for the care of patients with cystic fibrosis. To aid in the development of clinical pharmacokinetics studies of this treatment, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for quantifying the component compounds in human plasma and cell lysate. This assay was optimized for small volumes (10 µL), uses stably labeled isotopes of the ETI compounds as internal standards, and employs a simple methanol protein precipitation method. Chromatography was performed on an ACE Excel C18, 2.1 × 50 mm, reversed phase analytical column, using a step or bump isocratic method, with mobile phases consisting of 0.1% formic acid in water for A, and 0.1% formic acid in acetonitrile for B. Analyte and internal standard detection was conducted with ESI positive ionization tandem mass spectrometry. The precursor/product transitions (m/z) monitored were 598.0/422.0 for ELX, 521.0/449.0 for TEZ, 393.0/172.0 for IVA, 601.0/422.0 for IS-ELX, 525.0/453.0 for IS-TEZ, and 399.0/178.0 for IS-IVA, respectively. The assay has a dynamic range of 10 to 10,000 ng/mL, with a mean coefficient of determination (r2, mean ± SD) of 0.9970 ± 0.0027 (ELX), 0.9989 ± 0.0004 (TEZ), 0.9981 ± 0.0003 (IVA), regardless of specimen matrix. The mean precision values for all calibration standards ranged from 0.0 to 10.8% (ELX), 0.0 to 6.7% (TEZ), and 0.2 to 5.6% (IVA), while the accuracy for calibration standards was within the range of −5.7 to 3.5% (ELX), −3.2 to 6.0% (TEZ), and −3.8 to 5.2% (IVA). Validation results demonstrated high accuracy (≤7.3, ≤9.8, ≤10.6% deviation) and high precision (≤11.5, ≤6.3, ≤11.0% CV) for the respective ETI quality control samples. This method provides a fully validated assay for ETI quantitation for use in clinical research.

Development and validation of simultaneous assay of simvastatin, beta-hydroxy simvastatin as metabolite in human plasma using liquid chromatography-tandem mass spectrometry

Several generic products containing simvastatin are circulating on the Vietnamese market at a more inexpensive price than that of a brand-name one. These formulations, however, have not been assessed for in vivo bioequivalence to the reference product. After oral administration, simvastatin (SIM) is extensively converted into an active metabolite, beta-hydroxy simvastatin acid (SIM-A) and a very low concentration of simvastatin can be found in plasma. Therefore, a method for quantification of simvastatin and its metabolite needs to be developed with a high specificity and sensitivity to detect these analytes in human plasma at such low concentrations. Our purpose was to develop a reliable LC-MS/MS (liquid chromatography-tandem mass spectrometry) method for simultaneous determination of simvastatin and metabolite of simvastatin, beta-hydroxy simvastatin acid, in human plasma and to apply this method to evaluate the bioequivalence of a test product in comparison with the reference product. Mass spectrometry, internal standard (IS), and chromatographic conditions were investigated to find out the suitable IS and conditions. Human plasma samples were treated by liquid-liquid extraction (LLE). The assay was validated in compliance with US-FDA (United States-Food and Drug Administration), and EMA (European Medicines Agency) guidelines. LC-MS/MS with electrospray ionization interface in positive (for SIM and lovastatin as IS) and negative (for SIM-A) ionization mode performed under the multiple reaction monitoring mode was used for detection of the analytes. The transition of m/z is 436.00 → 285.15, 435.10 → 319.15, and 404.95 → 199.10 for SIM, SIM-A, and IS, respectively. Tert-buthyl methyl ether was used for extraction of analytes from human plasma by a simple LLE followed by addition of an ammonium acetate buffer. The developed method was fully validated with acceptable selectivity, linearity and linear range, matrix effect, lower limit of quantitation (LLOQ), carryover, dilution integrity, and intra-and inter-day accuracy and precision, free-thaw stability. The method can be applied for quantification of these compounds in human plasma for in vivo bioavailability and bioequivalence studies.

Analysis and prediction pathways of natural products and their cytotoxicity against HeLa cell line protein using docking, molecular dynamics and ADMET

Natural product such as flavonoids and their derivatives have a discernible capability to inhibit tumor formation and the growth of cancer cell, which have a vital link between diet and chronic disease prevention. Several plants and spices that contain flavonoid derivatives have been used in traditional medicine as disease preventative and therapeutic agents. Therefore, flavonoids could be used as chemotherapeutic drugs, indicating their potential clinical utility in cancer treatment. The purpose of this research was to discover and produce innovative pharmaceuticals from natural sources by introducing structural changes into flavonoids' backbones and changing their structures to improve biological activity and anticancer effects. In the current study, it was expected that the percent unbound values for the 15 compounds in human plasma would be low, ranging between 0.188 and 0.391. However, all compounds have a safe range and are not toxic to the brain. Compounds 2, 10, and 13 were shown to be permeable to the CNS (log PS > −3), but all other compounds had difficulty penetrating the CNS. Furthermore, all compounds had a low total clearance, ranging from 0.038 to 1.216 ml/min/kg, indicating that these compounds have a long half-life. None of the compounds caused skin sensitization (SS), and only compounds 1, 11, and 12 are expected to be AMES-positive, suggesting that the other compounds are not mutagenic. The result of the study showed based on the Drug-likeness and ADMET studies, only 3 compounds, including 3, 4, and 15, have a good pharmacokinetics propriety, the lowest toxicity, and good binding affinity towards Caspase 3 V266APDB (ID: 5I9B) as potential inhibitor candidates for the HeLa cell line, they have a low total clearance property and no AMES mutagenicity or hERG inhibition properties. These compounds (3,4,15) were examined to act as new cytotoxic drug candidates and would have an interest as starting point for designing compounds against the HeLa cell line. Communicated by Ramaswamy H. Sarma

Ultra-performance liquid chromatography-tandem mass spectrometric method for quantitation of the recently Food and Drug Administration approved combination of vaborbactam and meropenem in human plasma.

A parenteral medical combination containing vaborbactam and meropenem is used mainly to treat complicated urinary tract infections. A novel ultra-performance liquid chromatography tandem mass spectrometric method was developed for the sensitive determination of both compounds in human plasma. Sample preparation was performed by precipitation technique. The chromatographic separation was accomplished using the Acquity C18-BEH column, 0.01 M ammonium formate: acetonitrile (47 : 53, v/v) as a mobile phase with a flow rate of 0.2 ml min−1. Analytes were monitored by applying multiple reaction monitoring. The bioanalytical validation criteria were conducted following the Food and Drug Administration recommendations. The method was linear within range 0.5 to 50 µg ml−1, for both drugs. The intra-day and inter-day precision, as coefficient variation (% CV) and the accuracy, as % bias did not exceed 15% for both drugs. The percentage recovery of targeted analytes was not less than 77%, calculated at three quality control levels. The proposed method showed a suitable lower level of quantification value of 0.50 µg ml−1 for both analytes, which is far lower than the expected Cmax, which permits the use of this method for pharmacokinetic studies. The proposed method proved to be useful for the evaluation of this combination in both human plasma and pharmaceutical formulation.

Effects of Collard Green Consumption on the Human Plasma and Urine Metabolome: An Untargeted Analysis

Abstract Objectives Whereas phytochemicals derived from cruciferous vegetables have demonstrated health benefits, the data linking intake of these vegetables to health outcomes are inconsistent. These inconsistencies may stem from methodological limitations in accurately assessing cruciferous vegetable exposure. Goals of this study were to explore the use of endogenously deuterium-labeled collard greens combined with untargeted metabolomics to identify unique plant-derived and host-derived metabolites following vegetable consumption as potential biomarkers of cruciferous vegetable intake in humans. Methods 26 participants (16 women, 10 men) were fed a breakfast including 100 g of collard greens grown with or without deuterium-labeled water. Plasma was sampled prior to ingestion and 4 h post-ingestion; 24 h urine samples were also collected. High-pressure liquid chromatography Triple Q-ToF mass spectrometry-based untargeted metabolomics was performed. Results Analysis of collard greens confirmed deuterium-labeled compounds, including glucosinolates (glucobrassicin, sinigrin), and flavonols (quercetin). The maximum abundance of stable isotopologues was 2H4 (M4) for glucobrassicin and 2H12 (M12) for 1,2,2'-trisinapoylgentiobiose. Consumption of collard greens was associated with a significant increase in 199 compounds in plasma, including a 5.26-fold increase in the antioxidant hydroferulic acid (q < 0.05). A significant decrease in 144 compounds in plasma was also found with collard green consumption. Likewise, a significant increase in 819 compounds and decrease in 1209 compounds was found in urine following collard green consumption. A deuterium labeled 18-carbon fatty acid was significantly increased in plasma, indicating it was derived from the collard greens. Work is ongoing to further identify deuterium-labeled plant derived compounds in human plasma and urine. Conclusions Consumption of a cruciferous vegetable changed the urine and plasma metabolome. Ongoing research is required to identify these metabolites in order to develop novel signatures of food intake. Funding Sources National Institutes of Health, United States Department of Agriculture Agricultural Research Service, National Institute of Food and Agriculture.

Human bioavailability of phenolic compounds found in common beans: the use of high-resolution MS to evaluate inter-individual variability.

Although common beans (Phaseolus vulgaris L.) are consumed worldwide, studies on the metabolic fate of phenolic compounds from common beans are still very scarce. The present work aimed to study the bioavailability of phenolic compounds in human plasma and urine, after acute consumption of a single meal of cooked common beans. Blood and urine of seven volunteers were collected before (0 h) and at different time points (1, 2, 4, 6 and 8 h for plasma and 0-2, 2-4, 4-6, 6-8 and 8-24 h for urine) after beans' intake. Ultra-high performance liquid chromatography-quadrupole-time of flight-MS (UPLC-Q-TOF-MS) was used for quantification. After beans' intake, 405 (sd 3) g, containing 188 mg of phenolic compounds (expressed as gallic acid equivalents), there was a significant increase (P < 0·05) in the plasma concentration of six metabolites and in the urinary excretion of eleven metabolites. After 1 h post-consumption, metabolites, such as kaempferol-3-O-glucuronide, showed a significant increase in plasma concentration, suggesting kaempferol's glucuronidation in the upper gastrointestinal tract. More than 50 % of the total amount of metabolites, such as 4-methylcatechol-O-sulphate and dihydrocaffeic acid-3-O-sulphate, were excreted after 8 h post-consumption, indicating colonic bacterial metabolism of the phenolic compounds. Partial least square-discriminant analysis models clearly showed clusters of metabolites, which contributed to extend the list of compounds related to cooked common beans' human intake at different time points and showed the human inter-individual variability in plasma concentration as well as in urinary excreted metabolites, after cooked common beans' intake.

Quantitative assessment of betainized compounds and associations with dietary and metabolic biomarkers in the randomized study of the healthy Nordic diet (SYSDIET)

Recently, a group of betainized compounds have been suggested to play a role in health effects in relation to a whole-grain-rich diet. The aims of this study were to develop a quantitative mass spectrometric method for selected betainized compounds in human plasma, and to investigate their association with nutrient intake and measures of metabolic health in participants of the SYSDIET study. The SYSDIET study was a controlled randomized intervention including individuals with metabolic syndrome, where the healthy Nordic diet (HND) group increased intakes of whole grains, canola oil, berries, and fish, whereas the control diet (CD) group consumed low-fiber cereal products, milk fat, and restricted amounts of fish and berries. A quantitative LC combined with triple quadrupole MS method for betainized compounds was developed and applied to fasting plasma samples from baseline (week 0) and the end of the intervention (week 18 or 24). Concentrations of betainized compounds were correlated with intakes of selected nutrients and fiber and measures of metabolic health. Pipecolic acid betaine (PAB) concentrations were significantly higher in the HND group than in the CD group (P=0.00032) at the end of the intervention and correlated directly (P<0.0001) with intakes of dietary fiber (r=0.376) and a biomarker related to whole-grain rye intake, namely the ratio of alkylresorcinol C17:0 to C21:0 (r=0.442). PAB was associated inversely with fasting plasma insulin consistently at the beginning and at the end of the intervention (P<0.001, r=-0.300; P<0.01, r=-0.250, respectively), as well as IL-1 receptor antagonist (P<0.01, r=-0.232 at the beginning; P<0.01, r=-0.236 at the end) and serum LDL/HDL cholesterol (P<0.01, r=-0.239 at the beginning; P<0.01, r=-0.241 at the end). Among adults with the metabolic syndrome, PAB plasma concentrations were associated with fasting insulin, inflammation, and lipids and were significantly increased with adoption of the HND. Further studies are needed to clarify the biological functions of betainized compounds. This trial was registered at clinicaltrials.gov as NCT00992641.

Simultaneous quantitative analysis of polyphenolic compounds in human plasma by liquid chromatography tandem mass spectrometry.

A diet rich in polyphenolic compounds has recognized health benefits, and as such is routinely monitored as part of dietary intervention studies. A method for the simultaneous determination of 36 phenolic compounds, including phenolic acids and flavonoids, using liquid chromatography and tandem mass spectrometry is described here. The target analytes were quantified based on their specific mass spectral fragments using a selected reaction monitoring approach. A C18 column with embedded aromatic functionality ensured separation of all phenolic compounds studied which included several pairs of isomers. Sample preparation involved the use of β-glucuronidase to release the phenolic compounds from their conjugated forms. The intra-day and inter-day precision and accuracy was less than 7% for all phenolic compounds studied. Recoveries, where plasma was spiked with three different concentrations of the analytes, ranged from 95-115%. The limits of detection and quantification were 0.23-3.89 and 1.15-7.79nM, respectively. The method was successfully applied to real samples and the range reported for each phenolic compound, with the exception of hydroferulic acid, nordihydroguaiaretic acid, methylgallate, and m-coumaric acid, was at least an order of magnitude higher than the limit of quantification for the method.

Simultaneous Quantification of 13 Cannabinoids and Metabolites in Human Plasma by Liquid Chromatography Tandem Mass Spectrometry in Adult Epilepsy Patients.

A sensitive, robust method was developed and validated to quantitate 13 major natural cannabinoid parent and metabolite compounds in human plasma at or below 0.5 ng/mL. A liquid chromatography tandem mass spectrometry method was developed and validated to measure 13 cannabinoid compounds: cannabidiol (CBD), cannabidiolic acid, cannabidivarin, cannabinol, cannabigerol, cannabigerolic acid, cannabichromene, Δ-tetrahydocannabinol (THC), Δ-tetrahydrocannabinolic acid A (THCA), Δ-tetrahydrocannabivarin (THCV), 11-hydroxy-Δ-tetrahydrocannbinol (11-OH-THC), 11-nor-9-carboxy-Δ-tetrahydrocannbinol (THC-COOH), and 11-nor-9-carboxy-Δ-tetrahydrocannabinol glucuronide (THC-COOH-glu). Samples (200 µL) were extracted through protein precipitation and separated with a Kinetex EVO C18 column and a 65%-95% gradient of methanol and 0.2% ammonium hydroxide/H2O at a flow rate of 0.4 mL/min. Samples were obtained from patients with epilepsy receiving cannabis for the treatment of seizures. The extracted lower limit of quantification was 0.05 ng/mL for CBD, cannabidivarin, cannabinol, and 11-OH-THC; 0.10 ng/mL for cannabidiolic acid, cannabigerol, cannabichromene, cannabigerolic acid, THC, THCA, and THCV; and 0.50 ng/mL for THC-COOH and THC-COOH-glu. Mean quality control intraday accuracy and precision for all analytes ranged 96.5%-104% and 2.7%-4.9%, respectively, whereas interday accuracy and precision ranged 98%-103.3% and 0.2%-3.6%, respectively. An absolute matrix effect was observed for some analytes, however, with minimal relative matrix effect. Lack of nonspecific drug binding to extraction glass and plasticware was verified. Patient CBD levels ranged from 0.135 to 11.13 ng/mL. The validated method met FDA guidelines for bioanalytical assays precision and accuracy criteria. The assay reliably confirmed the use of particular medical cannabis formulations in patient samples as well as reliably measured low CBD concentrations from single-dose CBD exposure.

Extraction and determination of tricyclic antidepressants in real samples using air-dispersed liquid-liquid microextraction prior to gas chromatography and flame ionization detection.

In the present research, a novel microextraction method was used based on dispersive liquid-liquid microextraction using low-density solvent for the determination of trace amounts of amitriptyline and doxepin as model compounds in human plasma and spring water samples before gas chromatography and flame ionization detection. The parameters influencing the extraction efficiencies including type of the extracting solvent, volume of the aqueous sample solution (donor phase), volume of the extraction solvent (acceptor phase), the number of air-injection, and the effects of pH and ionic strength were optimized. Under the optimized conditions, the obtained enrichment factors were >80. By plotting peak areas of the sample solutions versus various concentrations of the analytes, calibration curves were obtained which show the linear ranges of 10-3000ng/mL with correlation coefficients of >0.996. The precision of the method was calculated <4.2 and the limit of detection was 1ng/mL for two analytes. The proposed microextraction method was used for the extraction of the analytes in environmental water and plasma samples and the calculated relative recoveries were all >91%.

A LC–MS-MS assay for simultaneous determination of two glycopeptides and two small molecule compounds in human plasma

In this study, a novel and high-throughput liquid chromatography-tandem mass spectrometric (LC-MS-MS) assay was developed and validated for simultaneous determination of two glycopeptides (vancomycin, teicoplanin) and two small molecule compounds (meropenem, voriconazole) in human plasma. Only 50 μL of human plasma is used to quantify these four drugs simultaneously at clinical concentration levels. After a relative simple protein precipitation, the supernatant was then diluted with mobile phase acetonitrile: 0.1% formic acid (5:95, v/v) to avoid solvent effect and reduce the matrix effect. Then, the target compounds were separated on an Agilent Zorbax SB-C18 column (4.6 × 50 mm, 2.7 μm). All the target compounds were detected by positive ion mode. The teicoplanin concentration was determined as the sum of six components (A2-1, A2-2, A2-3, A2-4, A2-5 and A3-1). The method was linear in the concentration range 0.3-30 μg/mL for meropenem; 1-100 μg/mL for teicoplanin and vancomycin; 0.3-10 μg/mL for voriconazole. The lower limit of quantitation (LLOQ) of meropenem and voriconazole were 0.30 μg/mL; and the LLOQ of teicoplanin and vancomycin were 1.0 μg/mL. The intra- and inter-day accuracies were <9.67% and 13.0%, and the precisions were <14.5% at all tested concentrations. The entire analysis time for the four drugs was only 5 min for each sample. The currently developed assay was successfully applied for the therapeutic drug monitoring of 85 patients administered with standard drug treatments, demonstrating its high-throughput clinical usage for the therapeutic drug monitoring.

A Simple and Sensitive LC-MS/MS Method for the Simultaneous Determination of Cyclophosphamide and Doxorubicin Concentrations in Human Plasma

Background: The combination of cyclophosphamide and doxorubicin is commonly used in the adjuvant treatment of breast cancer in women with a high risk of recurrent disease. It is essential to devise dosing strategies for such drugs in order to minimise their in-use toxicity and to maximise their efficacy. Data have shown that concentration directed rather than dose targeted therapy provides better clinical outcomes. Therefore, analytical methods that permit the simple, sensitive and accurate determination of such compounds in human plasma will assist in drug dosing strategies Methods: We report herein a method for the LC-MS/MS determination of cyclophosphamide and doxorubicin concentrations in patient plasma, following intravenous chemotherapy using protein precipitation without time-consuming supernatant drying and reconstitution steps, at the same time using a HILIC pre-column to further remove residual phospholipids. Results: The method has a less than 15% RE and less than 5% RSD both intra- and inter- day. It has following principal advantages over recently published LC-MS/MS methods for cyclophosphamide and doxorubicin in human plasma: 1) the sample preparation method is very simple and rapid; 2) the method has a very low LLOQ (cyclophosphamide 0.5 ng/mL and doxorubicin 1.0 ng/mL) at the same time the precision and accuracy meeting the primary requirements established by the FDA; and 3) the procedure only requires a very small plasma volume (30 µL). Conclusion: Such a methodology, when applied in close temporal and geographical proximity to patients will permit a short turn-around time and thereby facilitate the clinical interpretation and feedback to improve patient outcomes. Keywords: Cyclophosphamide, doxorubicin, LC-MS/MS, human, plasma, protein.

LC-MS/MS simultaneous analysis of allopregnanolone, epiallopregnanolone, pregnanolone, dehydroepiandrosterone and dehydroepiandrosterone 3-sulfate in human plasma.

Several neuropsychopharmacological properties have been attributed to the 3α-reduced pregnane steroids, allopregnanolone and pregnanolone, as well as to dehydroepiandrosterone sulfate because of their ability to modulate γ-aminobutyric acid (GABAA) receptors in the CNS. In order to understand better their role in several mechanisms in CNS, a new methodology is proposed to monitor these compounds in human plasma. Methodology & results: The analytes were first derivatized with 2-hydrazinopyridine and extracted from plasma using SPE. Then, the compounds were separated and detected by LC-MS/MS. A mobile phase of formic acid (0.1%) in water and methanol through a gradient of composition and a flow rate of 0.3 ml min-1 resulted in good separations of the analytes. Linear responses in wide range of concentrations and LOQs ranging from 10 (dehydroepiandrosterone 3-sulfate) to 40 pg ml-1 (dehydroepiandrosterone) were obtained in <9 min. The method proposed has been validated and then applied to monitor these neurosteroids in plasma samples from ten volunteers. For the first time, a straightforward and reliable method for the chromatographic separation of allopregnanolone, epiallopregnanolone and pregnanolone, as well as of dehydroepiandrosterone and dehydroepiandrosterone 3-sulfate was carried out, with optimal accuracy, sensitivity and specificity.

High-Speed Quantitative UPLC-MS Analysis of Multiple Amines in Human Plasma and Serum via Precolumn Derivatization with 6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate: Application to Acetaminophen-Induced Liver Failure.

A targeted reversed-phase gradient UPLC-MS/MS assay has been developed for the quantification /monitoring of 66 amino acids and amino-containing compounds in human plasma and serum using precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQTag Ultra). Derivatization of the target amines required minimal sample preparation and resulted in analytes with excellent chromatographic and mass spectrometric detection properties. The resulting method, which requires only 10 μL of sample, provides the reproducible and robust separation of 66 analytes in 7.5 min, including baseline resolution of isomers such as leucine and isoleucine. The assay has been validated for the quantification of 33 amino compounds (predominantly amino acids) over a concentration range from 2 to 20 and 800 μM. Intra- and interday accuracy of between 0.05 and 15.6 and 0.78-13.7% and precision between 0.91 and 16.9% and 2.12-15.9% were obtained. A further 33 biogenic amines can be monitored in samples for relative changes in concentration rather than quantification. Application of the assay to samples derived from healthy controls and patients suffering from acetaminophen (APAP, paracetamol)-induced acute liver failure (ALF) showed significant differences in the amounts of aromatic and branched chain amino acids between the groups as well as a number of other analytes, including the novel observation of increased concentrations of sarcosine in ALF patients. The properties of the developed assay, including short analysis time, make it suitable for high-throughput targeted UPLC-ESI-MS/MS metabonomic analysis in clinical and epidemiological environments.

High sensitivity bioanalysis for small drug-like compounds in human plasma using microflow LC and high resolution mass spectrometry

High sensitivity bioanalysis for small drug-like compounds in human plasma using microflow LC and high resolution mass spectrometry

Separation of the Structural Isomers of Monomethylarginine in Human Plasma by 2-D-HPLC and MS–MS Detection

Elevated plasma concentrations of the methylated metabolites of the amino acid l-arginine, especially asymmetric and symmetric dimethylarginine, are long established cardiovascular risk markers. The role of the two structural isomers of monomethylated l-arginine, N G-monomethyl-l-arginine and N δ-monomethyl-l-arginine, is not so well understood, probably because of the low concentrations of these compounds in human plasma and difficulties in separating them by chromatographic or mass spectrometric means. In this work, we provided a method for the interference-free baseline separation of the two substances, together with l-arginine, by derivatization with o-phthalaldehyde/2-mercaptoethanol and 2-D chromatographic separation on C18- and phenyl-modified stationary phases followed by MS–MS detection. Stable isotope labeled arginine served as an internal standard. Linear calibration functions were obtained in the ranges of 7.5–150 µmol L−1 for l-arginine, 25–1000 nmol L−1 for N G-monomethyl-l-arginine, and 5–350 nmol L−1 for N δ-monomethyl-l-arginine. Relative standard deviations were less than 9% (precision) for all compounds in all concentration levels and the accuracy was 97.48–106.53% of the theoretical value.

LC–ESI-MS/MS profile of phenolic and glucosinolate compounds in samh flour (Mesembryanthemum forsskalei Hochst. ex Boiss) and the inhibition of oxidative stress by these compounds in human plasma

Samh flour (Mesembryanthemum forsskalei) is a foodstuff with high protein content, which can be used as a replacement for wheat flour. It is often consumed by Bedouin tribes of northern Saudi Arabia. Very little is known about bioactive molecules present in samh flour, therefore we analyzed its extracts to evaluate the contents of secondary metabolites. A total of 43 secondary metabolites present in 60% MeOH extract of samh flour were tentatively identified using LC–ESI-MS/MS. These compounds represented five major categories: glucosinolates, sinapic acid and sinapoylglycosides, acylated flavonoids, flavonoids, and amide derivatives. Their effect on oxidative damage of proteins and lipids was determined in vitro by assessing levels of protein thiol groups and concentrations of thiobarbituric acid reactive species (TBARS) in human plasma. Obtained results indicated that samh flour is a rich source of compounds with antioxidant activity.