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Concept: Chromatography in blood processing

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What is known and Objective:  Laevo-ornidazole is an enantiomer of ornidazole, a 5-nitroimidazole antimicrobial agent. It is not known whether chiral inversion of laevo-ornidazole occurs in humans. The objective of this study was to investigate the possible chiral inversion and pharmacokinetics of the drug in vivo. Methods:  We developed a stereo-specific high-performance liquid chromatographic method for investigating chiral inversion of the drug and a standard high-performance liquid chromatography (HPLC) for the routine assay of the drug in pharmacokinetic studies. We report on the pharmacokinetics of the drug following single dose and multiple doses and investigate the effect of food in healthy volunteers. Results and Discussion:  There was no chiral inversion of laevo-ornidazole in vivo. In the pharmacokinetic study of the drug in healthy Chinese volunteers, food intake affected the absorption rate of laevo-ornidazole but not the extent. What is new and Conclusion:  We present the first reported method for the chiral separation of ornidazole in human plasma. We demonstrate the absence of chiral inversion of laevo-ornidazole in vivo. Given the absence on in vivo chiral inversion, we also report and validate a simplified non-chiral method for the determination of laevo-ornidazole. We show that although food can affect the absorption rate of laevo-ornidazole, the extent was unaffected.

Concepts: Protein, Pharmacology, Nutrition, Chromatography, High performance liquid chromatography, Analytical chemistry, Pharmacokinetics, Chromatography in blood processing

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A sensitive, accurate and highly stereoselective assay for the simultaneous determination of venlafaxine (VEN) and its equipotent metabolite, O-desmethyl venlafaxine (ODV), in human plasma was developed and validated. Analytes were simultaneously extracted from plasma using solid-phase extraction and detected by tandem mass spectrometry in positive ion mode with a turbo ion spray interface. Deuterium-labeled VEN and ODV were used as internal standards. Chromatographic separation was performed on a Chiral AGP column, using a time programmed gradient flow with a total run time of 16 min. The method has a lower limit of quantitation of 0.60 ng/mL. The assay was linear over a range 0.60-300.00 ng/mL for both the enantiomers of VEN and ODV, respectively, with coefficient of correlation > 0.99. The extraction recoveries were >77.0% on an average for all the four analytes. The analytes were found stable in plasma through three freeze (-15 °C) and thaw cycles and under storage at room temperature for 8 h, and also in mobile phase at 10 °C for 54 h. The method has shown good reproducibility, with intra- and inter-day variation coefficients < 9%, for all the analytes, and has proved to be very reliable for analysis of VEN and its metabolite in clinical study samples. Copyright © 2012 John Wiley & Sons, Ltd.

Concepts: Mass spectrometry, Chromatography, High performance liquid chromatography, Analytical chemistry, Gas chromatography, Chromatography in blood processing, Tandem mass spectrometry, Separation process

28

Tamsulosin, a selective α(1)-adrenoceptor antagonist, is used for the treatment of benign prostatic hyperplasia (BPH). We developed and validated a rapid, sensitive, and simplified liquid chromatography analytical method utilizing tandem mass spectrometry (LC-MS/MS) for the determination of tamsulosin in human plasma. After liquid-liquid extraction with methyl t-butyl ether, chromatographic separation of tamsulosin was achieved using a reversed-phase Luna C(18) column (2.0mm×50mm, 5μm particles) with a mobile phase of 10mM ammonium formate buffer (pH 3.5)-methanol (25:75, v/v) and quantified by MS/MS detection in ESI positive ion mode. The flow rate of the mobile phase was 200μL/min and the retention times of tamsulosin and the internal standard (IS, diphenhydramine) were 0.8 and 0.9min, respectively. The calibration curves were linear over a range of 0.01-20ng/mL (r>0.999). The lower limit of quantification using 500μL of human plasma was 0.01ng/mL. The mean accuracy and precision for intra- and inter-day validation of tamsulosin were both within acceptable limits. The present LC-MS/MS method showed improved sensitivity for quantification of tamsulosin in human plasma compared with previously described analytical methods. The validated method was successfully applied to a pharmacokinetic study in humans.

Concepts: Mass spectrometry, Chromatography, Analytical chemistry, Gas chromatography, Benign prostatic hyperplasia, Chromatography in blood processing, Tandem mass spectrometry, Tamsulosin

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Diosmin is a flavonoid often administered in the treatment of chronic venous insufficiency, hemorrhoids, and related affections. Diosmin is rapidly hydrolized in the intestine to its aglicone, diosmetin, which is further metabolized to conjugates. In this study, the development and validations of three new methods for the determination of diosmetin, free and after enzymatic deconjugation, and of its potential glucuronide metabolites, diosmetin-3-O-glucuronide, diosmetin-7-O-glucuronide, and diosmetin-3,7-O-glucuronide from human plasma and urine are presented. First, the quantification of diosmetin, free and after deconjugation, was carried out by high-performance liquid chromatography coupled with tandem mass spectrometry, on an Ascentis RP-Amide column (150 × 2.1 mm, 5 μm), in reversed-phase conditions, after enzymatic digestion. Then glucuronide metabolites from plasma were separated by micro-liquid chromatography coupled with tandem mass spectrometry on a HALO C18 (50 × 0.3 mm, 2.7 μm, 90 Å) column, after solid-phase extraction. Finally, glucuronides from urine were measured using a Discovery HSF5 (100 × 2.1 mm, 5 μm) column, after simple dilution with mobile phase. The methods were validated by assessing linearity, accuracy, precision, low limit of quantification, selectivity, extraction recovery, stability, and matrix effects; results in agreement with regulatory (Food and Drug Administration and European Medicines Agency) guidelines acceptance criteria were obtained in all cases. The methods were applied to a pharmacokinetic study with diosmin (450 mg orally administered tablets). The mean C max of diosmetin in plasma was 6,049.3 ± 5,548.6 pg/mL. A very good correlation between measured diosmetin and glucuronide metabolites concentrations was obtained. Diosmetin-3-O-glucuronide was identified as a major circulating metabolite of diosmetin in plasma and in urine, and this finding was confirmed by supplementary experiments with differential ion-mobility mass spectrometry.

Concepts: Protein, Pharmacology, Metabolism, Mass spectrometry, Chromatography, High performance liquid chromatography, Analytical chemistry, Chromatography in blood processing

27

A sensitive method for the simultaneous determination of isosorbide dinitrate (ISDN) and its mononitrate metabolites, isosorbide 2-mononitrate and isosorbide 5-mononitrate (IS-2-MN and IS-5-MN), in human plasma was developed using capillary gas chromatography with electron-capture detection, whereas 1,2,4-butanetriol trinitrate was used as internal standard. The analytes were extracted with a simple liquid-liquid extraction from plasma and separated on a DB-1 column. The results of method validation demonstrated that the calibration curves were linear in range of 2-60 ng/mL for ISDN and IS-5-MN, 1-20 ng/mL for IS-2-MN, respectively. The precision (RSD%) was less than 15%, and the lower limit of quantitation was identifiable and reproducible at 2 ng/mL for ISDN and IS-5-MN, 1 ng/mL for IS-2-MN. The analytes in plasma were stable after being stored for more than 30 days and after 2 freeze-thaw cycles (-20 to 25°C). And then this method was successfully applied to a pharmacokinetic investigation on isosorbide dinitrate oral spray in healthy volunteers.

Concepts: Scientific method, Blood, Analytical chemistry, Pharmacokinetics, Standard, Chromatography in blood processing, Limit superior and limit inferior

25

A high-throughput ultra-performance liquid chromatography coupled to tandem mass spectrometry (LC-ESI-MS-MS) method was developed for the determination of pinaverium bromide in human plasma. Protein precipitation with acetonitrile was used to extract pinaverium and itraconazole (as internal standard) from 500 µL plasma samples. The chromatographic separation was achieved with an Acquity UPLC BEH C18 column (1.7 µm, 2.1 × 100 mm) using a mixture of acetonitrile-5 mM ammonium formate (80:20, v/v) as mobile phase. Isocratic elution at 0.3 mL/min was used. Detection was performed by positive ion electrospray tandem mass spectrometry on a XEVO TQ-S by multiple reaction monitoring mode. The mass transitions monitorized were as follows: m/z 511.2 → 230 for pinaverium bromide, and m/z 705.29 → 392.18 for the itraconazole. The method was validated over a concentration range of 12-12,000 pg/mL. The chromatographic method runtime is 2.5 min and was applied to characterize the pharmacokinetics of pinaverium bromide after the oral administration of 100 mg to healthy Mexican subjects.

Concepts: Protein, Mass spectrometry, Chromatography, High performance liquid chromatography, Analytical chemistry, Gas chromatography, Chromatography in blood processing, Separation process

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Plasma extracellular vesicles (EVs), especially exosome-like vesicles (ELVs), are being increasingly explored as a source of potential noninvasive disease biomarkers. The discovery of blood-based biomarkers associated with ELVs requires methods that isolate high yields of these EVs without significant contamination with highly abundant plasma proteins and lipoproteins. The rising interest in blood-based EV-associated biomarkers has led to the rapid development of novel EV isolation methods. However, the field suffers from a lack of standardization and often, new techniques are used without critical evaluation. Size exclusion chromatography (SEC) has become the method of choice for rapid isolation of relatively pure EVs from plasma, yet it has technical limitations for certain downstream applications. The recently released exoEasy kit (Qiagen) is a new membrane affinity spin column method for the isolation of highly pure EVs from biofluids with the potential to overcome most of the limitations of SEC.

Concepts: Molecular biology, Blood, Cell membrane, Chromatography, High performance liquid chromatography, Quaternary structure, Chromatography in blood processing, Size exclusion chromatography

2

The comparison of extraction methods for global metabolomics is usually executed in biofluids only and focuses on metabolite coverage and method repeatability. This limits our detailed understanding of extraction parameters such as recovery and matrix effects and prevents side-by-side comparison of different sample preparation strategies. To address this gap in knowledge, seven solvent-based and solid-phase extraction methods were systematically evaluated using standard analytes spiked into both buffer and human plasma. We compared recovery, coverage, repeatability, matrix effects, selectivity and orthogonality of all methods tested for non-lipid metabolome in combination with reversed-phased and mixed-mode liquid chromatography mass spectrometry analysis (LC-MS). Our results confirmed wide selectivity and excellent precision of solvent precipitations, but revealed their high susceptibility to matrix effects. The use of all seven methods showed high overlap and redundancy which resulted in metabolite coverage increases of 34-80% depending on LC-MS method employed as compared to the best single extraction protocol (methanol/ethanol precipitation) despite 7x increase in MS analysis time and sample consumption. The most orthogonal methods to methanol-based precipitation were ion-exchange solid-phase extraction and liquid-liquid extraction using methyl-tertbutyl ether. Our results help facilitate rational design and selection of sample preparation methods and internal standards for global metabolomics.

Concepts: Metabolism, Mass spectrometry, Analytical chemistry, Metabolite, Chromatography in blood processing, Metabolomics, Diethyl ether, Metabolome

1

Profiling of body fluids is crucial for monitoring and discovering metabolic markers of health and disease and for providing insights into human physiology. Since human urine and plasma each contain an extreme diversity of metabolites, a single liquid chromatographic system when coupled to mass spectrometry (MS) is not sufficient to achieve reasonable metabolome coverage. Hydrophilic interaction liquid chromatography (HILIC) offers complementary information to reverse-phase liquid chromatography (RPLC) by retaining polar metabolites. With the objective of finding the optimal combined chromatographic solution to profile urine and plasma, we systematically investigated the performance of five HILIC columns with different chemistries operated at three different pH (acidic, neutral, basic) and five C18-silica RPLC columns. The zwitterionic column ZIC-HILIC operated at neutral pH provided optimal performances on a large set of hydrophilic metabolites. The RPLC columns Hypersil GOLD and Zorbax SB aq were proven to be best suited for the metabolic profiling of urine and plasma, respectively. Importantly, the optimized HILIC-MS method showed excellent intra-batch peak area reproducibility (CV < 12%) and good long-term inter-batch (40 days) peak area reproducibility (CV < 22%) that were similar to those of RPLC-MS procedures. Finally, combining the optimal HILIC- and RPLC-MS approaches greatly expanded metabolome coverage with 44% and 108% new metabolic features detected compared to RPLC-MS alone for urine and plasma, respectively. The proposed combined LC-MS approaches improve the comprehensiveness of global metabolic profiling of body fluids and thus are valuable for monitoring and discovering metabolic changes associated with health and disease in clinical research studies.

Concepts: Protein, Metabolism, Blood, High performance liquid chromatography, Analytical chemistry, PH, Chromatography in blood processing, Metabolomics

0

Ultra-performance liquid chromatography (UPLC) interfaced with atmospheric pressure chemical ionization mass-spectrometry was used to separate and quantify ursolic acid (UA) and oleanolic acid (OA) in human plasma. UA and OA were extracted from 0.5 mL human plasma using supported liquid extraction and separated utilizing an Acquity UPLC HSS column. The method has been validated for both UA and OA quantitation with a limit of detection of 0.5 ng/mL. The UPLC separations are carried out with isocratic elution with methanol and 5 mM ammonium acetate in water (85:15) as a mobile phase at a flow rate of 0.4 mL/min. The assay was linear from 1 ng/mL to 100 ng/mL for both analytes. The total analysis time was 7 min with the retention times of 3.25 (internal standard), 3.65 (UA) and 3.85 min (OA). Recovery of drug from plasma ranged from 70% to 115%. Analysis of quality control samples at 3, 30 and 80 ng/mL (n = 14) had an intra-day coefficient of variation of 9.9%, 4.3% and 5.5%, respectively. A proof-of-concept study in human patients who consumed apple peels indicates that this analytical method could be applied to clinical studies of UA and/or OA in human subjects.

Concepts: Mass spectrometry, Chromatography, High performance liquid chromatography, Analytical chemistry, Gas chromatography, Chromatography in blood processing, Ursolic acid, Oleanolic acid