Retention dependences support highly confident identification of lipid species in human plasma by reversed-phase UHPLC/MS

Reversed-phase ultrahigh-performance liquid chromatography - mass spectrometry (RP-UHPLC/MS) method was developed with the aim to unambiguously identify a large number of lipid species from multiple lipid classes in human plasma. The optimized RP-UHPLC/MS method employed the C18 column with sub-2 micrometer particles with the total run time of 25 min. The chromatographic resolution was investigated with 42 standards from 18 lipid classes. The UHPLC system was coupled to high-resolution quadrupole - time-of-flight (QTOF) mass analyzer using electrospray ionization (ESI) measuring full scan and tandem mass spectra (MS/MS) in positive- and negative-ion modes with high mass accuracy. Our identification approach was based on m/z values measured with mass accuracy within 5 ppm tolerance in the full scan mode, characteristic fragment ions in MS/MS, and regularity in chromatographic retention dependences for individual lipid species, which provides the highest level of confidence for reported identifications of lipid species including regioisomeric and other isobaric forms. The graphs of dependences of retention times on the carbon number or on the number of double bond(s) in fatty acyl chains were constructed to support the identification of lipid species in homologous lipid series. Our list of identified lipid species is also compared with previous publications investigating human blood samples by various MS based approaches. In total, we have reported more than 500 lipid species representing 26 polar and nonpolar lipid classes detected in NIST Standard reference material 1950 human plasma.

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Classification and Authentication of Paprika by UHPLC-HRMS Fingerprinting and Multivariate Calibration Methods (PCA and PLS-DA)

Foods ◽

10.3390/foods9040486 ◽

2020 ◽

Vol 9 (4) ◽

pp. 486 ◽

Cited By ~ 2

Author(s):

Sergio Barbosa ◽

Javier Saurina ◽

Lluís Puignou ◽

Oscar Núñez

Keyword(s):

Multivariate Calibration ◽

Principal Component ◽

Small Region ◽

Reversed Phase ◽

Negative Ion ◽

Mass Analyzer ◽

Least Squares Regression ◽

Calibration Methods ◽

Chemical Descriptors ◽

Negative Ion Mode

In this study, the feasibility of non-targeted UHPLC-HRMS fingerprints as chemical descriptors to address the classification and authentication of paprika samples was evaluated. Non-targeted UHPLC-HRMS fingerprints were obtained after a simple sample extraction method and C18 reversed-phase separation. Fingerprinting data based on signal intensities as a function of m/z values and retention times were registered in negative ion mode using a q-Orbitrap high-resolution mass analyzer, and the obtained non-targeted UHPLC-HRMS fingerprints subjected to unsupervised principal component analysis (PCA) and supervised partial least squares regression-discriminant analysis (PLS-DA) to study sample discrimination and classification. A total of 105 paprika samples produced in three different regions, La Vera PDO and Murcia PDO, in Spain, and the Czech Republic, and all of them composed of samples of at least two different taste varieties, were analyzed. Non-targeted UHPLC-HRMS fingerprints demonstrated to be excellent sample chemical descriptors to achieve the authentication of paprika production regions with 100% sample classification rates by PLS-DA. Besides, the obtained fingerprints were also able to perfectly discriminate among the different paprika taste varieties in all the studied cases, even in the case of the different La Vera PDO paprika tastes (sweet, bittersweet, and spicy) which are produced in a very small region.

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Separation and Quantification of Superwarfarin Rodenticide Diastereomers—Bromadiolone, Difenacoum, Flocoumafen, Brodifacoum, and Difethialone—in Human Plasma

Journal of AOAC International ◽

10.1093/jaoacint/qsaa007 ◽

2020 ◽

Vol 103 (3) ◽

pp. 770-778

Author(s):

Daniel G Nosal ◽

Douglas L Feinstein ◽

Luying Chen ◽

Richard B van Breemen

Keyword(s):

Human Plasma ◽

Clinical Care ◽

Reversed Phase ◽

Negative Ion ◽

Ultrahigh Pressure ◽

Selected Reaction Monitoring ◽

Triple Quadrupole Mass Spectrometer ◽

Accuracy And Precision ◽

Liquid Chromatography Tandem Mass ◽

Long Acting

Abstract Background Superwarfarins, second-generation long-acting anticoagulant rodenticides, are 4-hydroxycoumarin analogues of warfarin that contain a large hydrophobic side chain. These compounds contain two chiral centers and are synthesized for commercial use as two pairs of diastereomer. Objective To support studies of superwarfarin pharmacokinetics and other efforts to improve clinical care for poisoning victims, a quantitative assay was developed for the measurement of diastereomer of bromadiolone, difenacoum, flocoumafen, brodifacoum, and difethialone in human plasma. Method Based on ultrahigh-pressure liquid chromatography-tandem mass-spectrometry (UHPLC-MS/MS), this method was validated according to U.S. Food and Drug Administration (FDA) guidelines. Sample preparation involved simple protein precipitation followed by reversed phase UHPLC, which resolved all five pairs of cis/trans diastereomer in less than 10 min. Superwarfarins were measured using negative ion electrospray followed by selected-reaction monitoring on a triple quadrupole mass spectrometer. Results Calibration curves covered 3–4 orders of magnitude with linear regression coefficients of >0.999. The lower limits of quantitation were from 0.013 to 2.41 ng/mL, and intra-day and inter-day accuracy and precision coefficients of variation were <12%. Conclusions A 10-min UHPLC-MS/MS assay was developed and validated for the separation and quantitative analysis of the pairs of diastereomer of five superwarfarins in human plasma. Highlights This method was used to identify and measure superwarfarins and their cis/trans diastereomers in plasma obtained from patients treated for coagulopathy following consumption of contaminated synthetic cannabinoid products.

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Retention dependences support highly confident identification of lipid species in human plasma by reversed-phase UHPLC/MS

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-021-03492-4 ◽

2021 ◽

Author(s):

Zuzana Vaňková ◽

Ondřej Peterka ◽

Michaela Chocholoušková ◽

Denise Wolrab ◽

Robert Jirásko ◽

...

Keyword(s):

Human Plasma ◽

Reversed Phase ◽

Lipid Species

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Evaluation of 6 MALDI-Matrices for 10 μm lipid imaging and on-tissue MSn with AP-MALDI-Orbitrap

10.1101/2021.10.27.466111 ◽

2021 ◽

Author(s):

Tina B. Angerer ◽

Jerome Bour ◽

Jean-Luc Biagi ◽

Eugene Moskovets ◽

Gilles Frache

Keyword(s):

Spatial Resolution ◽

Tissue Sample ◽

Additional Treatment ◽

Negative Ion ◽

High Mass ◽

Lipid Species ◽

High Mass Resolution ◽

Matrix Additives ◽

Negative Ion Mode ◽

Positive Ion

Mass spectrometry imaging (MSI) is a technique uniquely suited to localize and identify lipids in a tissue sample. Using an AP-MALDI UHR source coupled to an Orbitrap Elite, numerous lipid locations and structures can be determined in high mass resolution spectra and at cellular spatial resolution, but careful sample preparation is necessary. We tested 11 protocols on serial brain sections for the commonly used MALDI matrices, CHCA, Norharmane, DHB, DHAP, THAP, and DAN, in combination with tissue washing and matrix additives, to determine the lipid coverage, signal intensity, and spatial resolution achievable with AP-MALDI. In positive ion mode, the most lipids could be detected with CHCA and THAP, while THAP and DAN without additional treatment offered the best signal intensities. In negative ion mode, DAN showed the best lipid coverage and DHAP performed superior for Gangliosides. DHB produced intense cholesterol signals in the white matter. 155 lipids were assigned in positive (THAP), 137 in negative ion mode (DAN) and 76 lipids were identified using on tissue tandem-MS. The spatial resolution achievable with DAN was 10 μm, confirmed with on tissue line-scans. This enabled the association of lipid species to single neurons in AP-MALDI images. The results show that the performance of AP-MALDI is comparable to vacuum MALDI techniques for lipid imaging.

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Unambiguous determination of isobaric histone modifications by reversed-phase retention time and high-mass accuracy

Analytical Biochemistry ◽

10.1016/j.ab.2009.08.027 ◽

2010 ◽

Vol 396 (1) ◽

pp. 13-22 ◽

Cited By ~ 19

Author(s):

Lanhao Yang ◽

Shengjiang Tu ◽

Chen Ren ◽

Esther M.M. Bulloch ◽

Chung-Lin Liao ◽

...

Keyword(s):

Histone Modifications ◽

Retention Time ◽

Reversed Phase ◽

Mass Accuracy ◽

High Mass ◽

High Mass Accuracy ◽

Unambiguous Determination ◽

Phase Retention

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Simultaneous isotope dilution quantification and metabolic tracing of deoxyribonucleotides by liquid chromatography high resolution mass spectrometry

10.1101/454322 ◽

2018 ◽

Cited By ~ 1

Author(s):

Rostislav Kuskovsky ◽

Raquel Buj ◽

Peining Xu ◽

Samuel Hofbauer ◽

Mary T Doan ◽

...

Keyword(s):

Mass Spectrometry ◽

Stable Isotope ◽

Isotope Dilution ◽

High Resolution Mass Spectrometry ◽

Reversed Phase ◽

Pentose Phosphate ◽

High Mass ◽

Mass Analyzer ◽

Isotope Tracing ◽

Metabolic Tracing

AbstractQuantification of cellular deoxyribonucleoside mono-(dNMP), di-(dNDP), triphosphates (dNTPs) and related nucleoside metabolites are difficult due to their physiochemical properties and widely varying abundance. Involvement of dNTP metabolism in cellular processes including senescence and pathophysiological processes including cancer and viral infection make dNTP metabolism an important bioanalytical target. We modified a previously developed ion pairing reversed phase chromatography-mass spectrometry method for the simultaneous quantification and 13C isotope tracing of dNTP metabolites. dNMPs, dNDPs, and dNTPs were chromatographically resolved to avoid mis-annotation of in-source fragmentation. We used commercially available 13C15N-stable isotope labeled analogs as internal standards and show that this isotope dilution approach improves analytical figures of merit. At sufficiently high mass resolution achievable on an Orbitrap mass analyzer, stable isotope resolved metabolomics allows simultaneous isotope dilution quantification and 13C isotope tracing from major substrates including 13C-glucose. As a proof of principle, we quantified dNMP, dNDP and dNTP pools from multiple cell lines. We also identified isotopologue enrichment from glucose corresponding to ribose from the pentose-phosphate pathway in dNTP metabolites.

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Developing a High-performance Liquid Chromatography Method for Simultaneous Determination of Loratadine and its Metabolite Desloratadine in Human Plasma

Current Drug Metabolism ◽

10.2174/1389200220666191125095648 ◽

2020 ◽

Vol 20 (13) ◽

pp. 1053-1059

Author(s):

Mahmoud M. Sebaiy ◽

Noha I. Ziedan

Keyword(s):

High Performance Liquid Chromatography ◽

Liquid Chromatography ◽

Human Plasma ◽

High Performance ◽

Allergic Diseases ◽

Reversed Phase ◽

Hplc Method ◽

H1 Receptor ◽

Chromatography Method

Background: Allergic diseases are considered as the major burden on public health with increased prevalence globally. Histamine H1-receptor antagonists are the foremost commonly used drugs in the treatment of allergic disorders. The target drug in this study, loratadine, belongs to this class of drugs and its biometabolite desloratadine which is also a non-sedating H1 receptor antagonist with anti-histaminic activity being 2.5 to 4 times greater than loratadine. This study aimed to develop and validate a novel isocratic Reversed-phase High-Performance Liquid Chromatography (RP-HPLC) method for rapid and simultaneous separation and determination of loratadine and its metabolite, desloratadine in human plasma. Methods: The drug extraction method from plasma was based on protein precipitation technique. The separation was carried out on a Thermo Scientific BDS Hypersil C18 column (5μm, 250 x 4.60 mm) in a mobile phase of MeOH: 0.025M KH2PO4 adjusted to pH 3.50 using orthophosphoric acid (85: 15, v/v) at an ambient temperature. The flow rate was maintained at 1 mL/min and maximum absorption was measured using the PDA detector at 248 nm. Results: The retention times of loratadine and desloratadine in plasma samples were recorded to be 4.10 and 5.08 minutes, respectively, indicating a short analysis time. Limits of detection were found to be 1.80 and 1.97 ng/mL for loratadine and desloratadine, respectively, showing a high degree of sensitivity of the method. The method was then validated according to FDA guidelines for the determination of the two analytes in human plasma. Conclusion: The results obtained indicate that the proposed method is rapid, sensitive in the nanogram range, accurate, selective, robust and reproducible compared to other reported methods.

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Simultaneous Quantification of Pantoprazole and Levosulpiride in Spiked Human Plasma Using High Performance Liquid Chromatography Tandem Mass Spectrometry

Current Pharmaceutical Analysis ◽

10.2174/1573412914666180101142646 ◽

2018 ◽

Vol 15 (1) ◽

pp. 17-23

Author(s):

Vulli Srinandan ◽

Krishnaveni Nagappan ◽

Sonam Patel ◽

Karthik Yamjala ◽

Gowramma Byran ◽

...

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Human Plasma ◽

Mass Analyzer ◽

Tandem Mass ◽

Preparation Technique ◽

Spiked Human Plasma ◽

Simultaneous Quantification ◽

Electro Spray Ionization

Background: Pantoprazole (PTZ) and Levosulpiride (LS) were proven as effective agents for the treatment of Gastro-Esophageal Reflux Disease (GERD). It is a complex motor disorder that results in regurgitation of the gastric contents into the lower esophagus with consequent symptoms such as heart burn, retrosternal pain, dysphagia and belching. Methods: A rapid, sensitive, selective and specific liquid chromatography- electro spray ionization tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of Pantoprazole (PTZ) and Levosulpiride (LS) in spiked Human Plasma. The method utilized SPE as sample preparation technique and the analysis was carried out on a HPLC system utilizing electro spray ionization as interface and triple quadrupole mass analyzer for quantification in MRM possitive mode. Iloperidone was used as internal standard (IS). Chromatographic separation was performed on a Phenomenex C-18 Column (4.6 mm x 50 mm, 5µ) with an isocratic elution mode utilizing a mobile phase composition of Solution containing a mixture of 70 volumes of acetonitrile: 30 volumes of methanol and 10mM ammonium formate (pH 4.0) at the ratio of 80:20 % v/v. The flow rate was maintained at 0.3 mL/min. Results: PTZ, LS and IS were detected and quantified with proton adducts at m/z 383.37→200.00, m/z 341.42→112.15 and 426.48→261.00 respectively. The linearity and range was established by fortifying blank plasma samples in the concentration range of 3.5-2000 ng/mL for PTZ and 3.0-2400 ng/mL for LS. The correlation coefficient (r2) was found to be ≥ 0.993 for PTZ and (r2) ≥ 0.990 for LS. The lower limit of quantification for PTZ was 3.5 ng/mL and LS was 3.0 ng/mL. The intra and inter day precision and accuracy for PTZ and LS were within the limits fulfilling the international acceptance criteria. PTZ and LS were found to be stable throughout three freeze-thaw cycles, bench top and short term stability studies. Conclusion: The proposed validated LC-MS/MS method offers a sensitive quantification of PTZ and LS in spiked human plasma and can be utilized for the quantification of PTZ and LS in real-time samples.

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Predicting Pharmacokinetic Properties of Potential Anticancer Agents via Their Chromatographic Behavior on Different Reversed Phase Materials

International Journal of Molecular Sciences ◽

10.3390/ijms22084257 ◽

2021 ◽

Vol 22 (8) ◽

pp. 4257

Author(s):

Małgorzata Janicka ◽

Anna Mycka ◽

Małgorzata Sztanke ◽

Krzysztof Sztanke

Keyword(s):

Liquid Chromatography ◽

Anticancer Agents ◽

Skin Permeation ◽

Reversed Phase ◽

Chromatographic Behavior ◽

Chromatographic Retention ◽

Quantitative Structure ◽

Animal Testing ◽

Structure Activity ◽

Pharmacokinetic Properties

The Quantitative Structure-Activity Relationship (QSAR) methodology was used to predict biological properties, i.e., the blood–brain distribution (log BB), fraction unbounded in the brain (fu,brain), water-skin permeation (log Kp), binding to human plasma proteins (log Ka,HSA), and intestinal permeability (Caco-2), for three classes of fused azaisocytosine-containing congeners that were considered and tested as promising drug candidates. The compounds were characterized by lipophilic, structural, and electronic descriptors, i.e., chromatographic retention, topological polar surface area, polarizability, and molecular weight. Different reversed-phase liquid chromatography techniques were used to determine the chromatographic lipophilicity of the compounds that were tested, i.e., micellar liquid chromatography (MLC) with the ODS-2 column and polyoxyethylene lauryl ether (Brij 35) as the effluent component, an immobilized artificial membrane (IAM) chromatography with phosphatidylcholine column (IAM.PC.DD2) and chromatography with end-capped octadecylsilyl (ODS) column using aqueous solutions of acetonitrile as the mobile phases. Using multiple linear regression, we derived the statistically significant quantitative structure-activity relationships. All these QSAR equations were validated and were found to be very good. The investigations highlight the significance and possibilities of liquid chromatographic techniques with three different reversed-phase materials and QSARs methods in predicting the pharmacokinetic properties of our important organic compounds and reducing unethical animal testing.

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Development and Validation of an Up-to-Date Highly Sensitive UHPLC-MS/MS Method for the Simultaneous Quantification of Current Anti-HIV Nucleoside Analogues in Human Plasma

Pharmaceuticals ◽

10.3390/ph14050460 ◽

2021 ◽

Vol 14 (5) ◽

pp. 460

Author(s):

Amedeo De Nicolò ◽

Alessandra Manca ◽

Alice Ianniello ◽

Alice Palermiti ◽

Andrea Calcagno ◽

...

Keyword(s):

Human Plasma ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Reversed Phase ◽

Therapeutic Drug ◽

People Living With Hiv ◽

Simultaneous Quantification ◽

Working Solution ◽

Tenofovir Alafenamide ◽

Combination Regimens

Therapeutic options to treat HIV infection have widened in the past years, improving both effectiveness and tolerability, but nucleoside reverse transcriptase inhibitors (NRTIs) are still considered the standard backbone of the combination regimens. Therapeutic drug monitoring (TDM) can be useful for these drugs, due to concentration–effect relationship, with risk of ineffectiveness, toxicity or adherence concerns: in this scenario, robust and multiplexed methods are needed for an effective TDM activity. In this work, the first validated ultra-high spectrometry liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method is described for the high-sensitive simultaneous quantification of all the currently used NRTIs in human plasma, including tenofovir alafenamide (TAF), following FDA and EMA guidelines. The automated sample preparation consisted in the addition of an internal standard (IS) working solution, containing stable-isotope-linked drugs, protein precipitation and drying. Dry extracts were reconstituted with water, then, these underwent reversed phase chromatographic separation: compounds were detected through electrospray ionization and multiple reaction monitoring. Accuracy, precision, recovery and IS-normalized matrix effect fulfilled guidelines’ requirements. The application of this method on samples from people living with HIV (PLWH) showed satisfactory performance, being capable of quantifying the very low concentrations of tenofovir (TFV) in patients treated with TAF.

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