Small Volumes Of Human Plasma Research Articles

Ultra-Sensitive Quantification of Coproporphyrin-I and -III in Human Plasma Using Ultra-Performance Liquid Chromatography Coupled to Quadrupole Time-of-Flight Mass Spectrometry.

Organic anion transporting polypeptides (OATP) 1B1 and 1B3 are expressed in liver cells and are involved in drug uptake in the liver. OATP1B activity varies due to polymorphisms and is decreased by OATP1B inhibitors. Variability of OATP1B activity impacts the pharmacokinetics of OATP1B substrate drugs through drug-drug interactions. Lately, coproporphyrin-I (CP-I) and -III (CP-III) have been featured as quantitative endogenous biomarkers for evaluating the activity of OATP1B. CP-III has been reported to be transported not only by OATP1B but also by OATP2B1 in vitro. We have established a highly sensitive assay for the simultaneous measurement of CP-I and CP-III using a small volume of human plasma. The sample was pretreated by solid-phase extraction and analyzed by ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS). This method that uses 100 μL of plasma met the acceptance criteria of the US Food and Drug Administration guidance for bioanalytical method validation, and the lower limit of quantification was 0.01 ng/mL for both coproporphyrins. The clinical application of the method was evaluated by measuring plasma CP-I and CP-III concentrations in healthy volunteers and rheumatoid arthritis (RA) patients. The measured concentrations were within the calibration range (0.01-50 ng/mL). Using this novel method to measure plasma concentrations of CP-I and CP-III may contribute to the evaluation of the activities of OATP1B1, OATP1B3, and OATB2B1 in healthy individuals and patients with various clinical conditions including RA.

A Simple UPLC-MS/MS Assay of Rifampin in a Small Volume of Human Plasma.

Rifampin (RIF) is a typical cytochrome P450 (CYP) 3A inducer and inhibitor of organic anion transporting polypeptide (OATP) 1B1 to assess drug-drug interaction (DDI) via CYP3A or OATP1B1 in clinical settings. To ensure sufficient exposure of RIF in DDI studies, it is important to determine plasma RIF concentrations. In this study, we developed a simple RIF assay in a small volume of human plasma by ultraperformance liquid chromatography with tandem mass spectrometry. RIF in 0.02 mL of plasma was extracted using protein precipitation and separated on a reverse phase column under gradient elution of three mobile phases, where the mobile phase C containing 1% formic acid was exclusively used to reduce the carryover of RIF. RIF and the internal standard were detected by multiple reaction monitoring in positive-ion electrospray ionization. RIF was quantifiable at 0.025-10 μg/mL without the carryover issue. The intra- and inter-run assays confirmed the reproducibility of the assay. Stability assessments ensured that RIF in human plasma was stable for 6 h at room temperature and for 409 days at -15 °C or below. The assay was successfully applied to a pharmacokinetic study with successful incurred sample reanalysis.

A Suitable Therapeutic Drug Monitoring Method for Amoxicillin in Plasma by High Performance Liquid Chromatography–UV (HPLC–UV) in Neonates

Amoxicillin, a broad-spectrum β-lactam antibiotic, is widely used for treatment of neonatal infections. Despite the unmet need in neonates, an adapted analytical method is still missing in clinical practice. The purpose of this study was to develop and test an easy and credible high performance liquid chromatography–UV (HPLC–UV) method to determine amoxicillin in small volumes of human plasma and use it in routine therapeutic drug monitoring (TDM) of neonates. After a protein precipitation, amoxicillin for standards, quality control samples, and patient samples were separated by LC and measured by UV detection, and tinidazole was used as the internal standard. The calibration range was 0.50–20.0 μg/mL. Intra- and inter-day precisions were less than 4.7%. The acceptance criteria of accuracy (between 85–115%) were met in all cases. A plasma volume of 50 μL was required to achieve the limit of quantification of 0.50 μg/mL. Thus, a simple, rapid, and accurate HPLC–UV method has been developed to detect the concentration of amoxicillin in human plasma. This method was adapted to do TDM of amoxicillin in neonates.

Sensitive determination of gentamicin in plasma using ion-exchange solid-phase extraction followed by UHPLC-MS/MS analysis

BackgroundTherapeutic drug monitoring (TDM) of gentamicin sulfate (GEN) is usually recommended, particularly in critical patients. Only a few reports had described the determination of GEN in plasma or plasma using LC-MS/MS. ObjectiveThis study aimed to develop and validate a sensitive ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) assay for the quantification of GEN in small volumes of human plasma. ResultsThe use of a very low concentration of the ion-pairing agent HFBA allowed significant retention of the very polar GEN forms in a reversed phase UHPLC column. The solid-phase extraction (SPE) procedure allowed clean extracts, with no interferences detected in blank samples, and high sensitivity. The assay was linear on the range of 0.2–40 mg L−1 of GEN complex. The combined GEN complex had inter-assay CV of 8.8–10.0%, intra-assay CV of 10.2–11.0%, and accuracy of 96.8–104.0%. The assay was applied to 17 clinical samples obtained from neonate patients. Measured concentrations were in the range of 0.15–3.57 mg L−1 for GEN C1, 0.12–3.55 mg L−1 for GEN C1a, 0.20–5.77 mg L−1 for GEN C2, and 0.47–12.88 mg L−1 for the GEN complex, all within the linear range of the assay. ConclusionA sensitive assay for the quantification of gentamicin in plasma using anion-exchange SPE and UHPLC-MS/MS was validated. The assay can be used for TDM of gentamicin, particularly in centers with access to proper instrumentation and with a low demand for gentamicin measurements, where immunoassays are not cost-effective.

Sensitive detection of quinoline-derivatized sitagliptin in small volumes of human plasma by MALDI-TOF mass spectrometry

Dipeptidyl peptidase 4 (DPP-4) inhibitors are incretin-based medications used as oral antidiabetic agents for the treatment of type 2 diabetes. However, DPP-4 inhibitors produce side effects like acute pancreatitis, upper respiratory tract infection, nasopharyngitis, urinary tract infection, serious allergies, cardiovascular diseases, hemolysis, and retinopathy. Hence, the development of a fast and simple method to detect DPP-4 inhibitors in body fluids is important.In this study, we developed a derivatization-assisted microextraction method to enhance the detection sensitivity for trace levels of a DPP-4 inhibitor, sitagliptin, from a small volume (10 μL) of human plasma by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Subjecting the analyte to 100 W microwave irradiation after derivatization using a quinoline alkylating reagent (8-bromomethyl quinilone, BrMQ) shortened the reaction time to ~120 s and allowed the target analyte to be easily extracted to a small volume of the organic layer (20 μL). The analyte was then detected by MALDI-TOF MS using α-cyano-4-hydroxycinnamic acid as the matrix. The relative standard deviation and relative error were below 10% in intra- and inter-day assays. Using sitagliptin-d4 as an internal standard, the limits of quantitation and detection were found to be 0.03 μg/mL and 0.01 μg/mL, respectively. All the derivatization and extraction procedures described herein were of microliter grade. This method could effectively reduce the use of organic chemicals and solvents, thereby proving to be an eco-friendly strategy that will cause no harm to the environment.

Sequential phosphoproteomics and N-glycoproteomics of plasma-derived extracellular vesicles.

Extracellular vesicles (EVs) are increasingly being recognized as important vehicles for intercellular communication and as promising sources for biomarker discovery. Because the state of protein post-translational modifications (PTMs) such as phosphorylation and glycosylation can be a key determinant of cellular physiology, comprehensive characterization of protein PTMs in EVs can be particularly valuable for early-stage diagnostics and monitoring of disease status. However, the analysis of PTMs in EVs has been complicated by limited amounts of purified EVs, low-abundance PTM proteins, and interference from proteins and metabolites in biofluids. Recently, we developed an approach to isolate phosphoproteins and glycoproteins in EVs from small volumes of human plasma that enabled us to identify nearly 10,000 unique phosphopeptides and 1,500 unique N-glycopeptides. The approach demonstrated the feasibility of using these data to identify potential markers to differentiate disease from healthy states. Here we present an updated workflow to sequentially isolate phosphopeptides and N-glycopeptides, enabling multiple PTM analyses of the same clinical samples. In this updated workflow, we have improved the reproducibility and efficiency of EV isolation, protein extraction, and phosphopeptide/N-glycopeptide enrichment to achieve sensitive analyses of low-abundance PTMs in EVs isolated from 1 mL of plasma. The modularity of the workflow also allows for the characterization of phospho- or glycopeptides only and enables additional analysis of total proteomes and other PTMs of interest. After blood collection, the protocol takes 2 d, including EV isolation, PTM/peptide enrichment, mass spectrometry analysis, and data quantification.

A sensitive and improved throughput UPLC–MS/MS quantitation method of total cytochrome P450 mediated arachidonic acid metabolites that can separate regio-isomers and cis/trans-EETs from human plasma

A method for the detection and quantification of hydroxyl and epoxy arachidonic acid (AA) metabolites in human plasma was developed using liquid-liquid extraction, phospholipid saponification followed by derivatization of the acid moiety and liquid chromatographic tandem mass spectrometric detection. Derivatization with a pyridinium analog allowed for detection in the positive ion mode, greatly improving sensitivity and the stability of the more labile AA metabolites. The entire method utilizes a 96-well plate format, increasing sample throughput, and was optimized to measure 5-, 8-, 9-, 11-, 12-, 15-, 19-, and 20- hydroxyeicosatetraenoic acid (HETE), 5,6-, 8,9-, 11,12-, and 14,15- dihydroxyeicosatrienoic acid (DHET), and the regio- and cis-/ trans- isomers of 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid (EET). The method was validated for its applicability over the FA concentration range found in human plasma. Using 100 μL aliquots of pooled human plasma, EET levels, particularly 5,6-EET, were observed to be higher than previously reported, with measured concentrations of 23.6 ng/ml for 5,6-EET, 5.6 ng/mL for 5,6-trans-EET, 8.0 ng/mL for 8,9-EET, 1.9 ng/mL for 8,9-trans-EET, 8.8 ng/mL for 11,12-EET, 3.4 ng/mL for 11,12-trans-EET, 10.7 ng/mL for 14,15-EET, and 1.7 ng/mL 14,15-trans- EET. This method is suitable for large population studies to elucidate the complex interactions between the eicosanoids and various disease states and may be used for quantitation of a wide variety of fattyacids beyond eicosanoids from small volumes of human plasma.

Development of a UPLC-MS/MS method for quantitation of metronidazole and 2-hydroxy metronidazole in human plasma and its application to a pharmacokinetic study

An ultra-performance liquid-chromatography mass-spectrometry (UPLC-MS/MS) method for simultaneous quantitation of metronidazole and 2-hydroxymetronidazole in human plasma was developed and validated. Metronidazole and 2-hydroxymetronidazole were extracted from a small volume of human plasma (10 μL) by hydrophilic lipophilic balanced solid phase extraction on 96-well μ-elution plates. Chromatographic separation of analytes was achieved on an Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm) using gradient elution with a blend of 0.1% formic acid in water and 0.1% formic acid in methanol at a flow rate of 0.25 mL/min. Mass spectrometric detection was achieved using multiple reaction monitoring (MRM) in positive-ion electrospray-ionization (ESI) mode. Ion transitions were optimized at m/z 171.85->127.9 for metronidazole and m/z 187.9->125.9 for 2-hydroxymetronidazole. The assay was linear for both analytes over the concentration range of 0.1–300 μM; intra- and inter-assay precisions and accuracies were <13%. Recoveries for metronidazole and 2-hydroxymetronidazole ranged from 88 to 99% and 78 to 86%, respectively. Matrix effects for metronidazole and 2-hydroxymetronidazole in plasma ranged from 102 to 105% and 99 to 106%, respectively. The method was successfully applied to determine metronidazole and 2-hydroxymetronidazole plasma concentrations in a pharmacokinetic study conducted in adults administered an oral dose of 500 mg metronidazole. Pharmacokinetic parameters were comparable to previously reported values. By design, this method is amenable to high sample throughput and has the potential to be automated.

A sandwich enzyme-linked immunosorbent assay for the quantitation of human plasma ferritin

There is a lack of published enzyme linked immunosorbent assay (ELISA) protocols which use commercially available reagents for the measurement of ferritin in human plasma for research purposes. ELISA kits are often expensive and do not always provide detailed information about reagents included. A commercially available antibody pair was used to develop an in-house ELISA to measure ferritin in small (25?µl) volumes of human plasma. ELISA results were compared to ferritin levels measured using a commercial immune-assay system. The sensitivity, intra and inter assay variation of the ELISA assay were also determined. ELISA measurements of plasma ferritin ranged between 3.2–232?ng/mL and were comparable to those measured by a commercial immunoassay system (Pearson correlation r?=?0.93 P?<?0.0001). Ferritin levels as low as 0.5?ng/mL were detectable and samples with both low and normal ferritin levels showed low inter and intra-assay variation. This ELISA protocol allows the accurate, reliable and cost-effective quantitative determination of plasma ferritin levels from small volumes of human plasma.•No published protocols detail how to measure ferritin by ELISA using commercially available antibodies.•ELISA kits are expensive and information on antibodies included are often lacking.•We have identified a commercially available antibody pair to measure plasma ferritin using a cost-effective ELISA.

Simultaneous quantification of acetaminophen and five acetaminophen metabolites in human plasma and urine by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry: Method validation and application to a neonatal pharmacokinetic study.

Drug metabolism plays a key role in acetaminophen (paracetamol)-induced hepatotoxicity, and quantification of acetaminophen metabolites provides critical information about factors influencing susceptibility to acetaminophen-induced hepatotoxicity in clinical and experimental settings. The aims of this study were to develop, validate, and apply high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) methods for simultaneous quantification of acetaminophen, acetaminophen-glucuronide, acetaminophen-sulfate, acetaminophen-glutathione, acetaminophen-cysteine, and acetaminophen-N-acetylcysteine in small volumes of human plasma and urine. In the reported procedures, acetaminophen-d4 and acetaminophen-d3-sulfate were utilized as internal standards (IS). Analytes and IS were recovered from human plasma (10μL) by protein precipitation with acetonitrile. Human urine (10μL) was prepared by fortification with IS followed only by sample dilution. Calibration concentration ranges were tailored to literature values for each analyte in each biological matrix. Prepared samples from plasma and urine were analyzed under the same HPLC-ESI-MS/MS conditions, and chromatographic separation was achieved through use of an Agilent Poroshell 120 EC-C18 column with a 20-min run time per injected sample. The analytes could be accurately and precisely quantified over 2.0-3.5 orders of magnitude. Across both matrices, mean intra- and inter-assay accuracies ranged from 85% to 112%, and intra- and inter-assay imprecision did not exceed 15%. Validation experiments included tests for specificity, recovery and ionization efficiency, inter-individual variability in matrix effects, stock solution stability, and sample stability under a variety of storage and handling conditions (room temperature, freezer, freeze-thaw, and post-preparative). The utility and suitability of the reported procedures were illustrated by analysis of pharmacokinetic samples collected from neonates receiving intravenous acetaminophen.

Development and Validation of High Performance Liquid Chromatography Tandem Mass Spectrometry (HPLC-MS/MS) Method for Determination of Tenofovir in Small Volumes of Human Plasma

Determination of plasma tenofovir (TFV) concentrations in small plasma volumes and in a short time period is most desirable in a clinical setting. The HPLC-MS/MS method was developed and validated for the determination of TFV. Plasma sample volumes of 10 μL were extracted by protein precipitation. Cimetidine, used as internal standard (ISTD) and TFV were separated on a C18 (Phenomenex Kinetex TM 30 mm × 2.1 mm, 2.6 μm) reversed phase column with a pre-column. The gradient mobile phase consisted of 10 mmol/L ammonium acetate in water and acetonitrile/methanol (50:50, v/v). TFV and ISTD retentiontimes were 0.27 and 0.90 minutes, respectively, with a run time of 2 minutes. Transition of the parent to the product ion of TFV (m/z 288.072→176.038) and ISTD (m/z 253.13→158.987) were monitored in positive ionization mode. Calibration curves were linear (average r2, 0.9958) over a TFV concentration range of 12.5-600 ng/mL. The mean recovery was 96.9%. Accuracy, inter and intra-assay of three quality controls and lower limit of quantification (12.5 ng/mL) were within the acceptable limit of <15% relative standard error. TFV was stable at studied conditions and neither matrix effect nor significant carry-over was observed. A distinct, reliable and robust method for determination of TFV in a small volume (10 μL) of human plasma was developed, validated and incorporated to determine the plasma TFV levels in 30 HIV-infected women on antiretroviral treatment.

Simultaneous determination of primaquine and carboxyprimaquine in plasma using solid phase extraction and LC–MS assay

Sensitive bioanalytical methods are required for pharmacokinetic studies in children, due to the small volume and modest number of samples that can be obtained. We sought to develop a LC–MS assay for primaquine and its active metabolite, carboxyprimaquine, following simultaneous, solid phase extraction of both analytes from human plasma. The analysis was conducted on a single-quad LC–MS system (Shimadzu Model 2020) in ESI+ mode, with quantitation by selected ion monitoring. Primaquine, carboxyprimaquine and 8-aminoquinoline (internal standard) were separated using a mobile phase of 80:20 methanol:water with 0.1% (v/v) formic acid and a Luna C18 HPLC column, at ambient temperature. Solid phase extraction of the analytes from plasma (0.5mL) was achieved with Oasis® HLB cartridges. The retention times for primaquine, 8-aminoquinoline and carboxyprimaquine were 3.3, 5.7 and 8.5min, respectively. The calibration curve range (2–1500μg/L) was appropriate for the limits of quantification and detection for primaquine (2μg/L and 1μg/L, respectively) and carboxyprimaquine (2.5μg/L and 1μg/L) and the anticipated plasma concentrations of the analytes. Intra- and inter-day precision for both primaquine and carboxyprimaquine was <10% across the concentration range 5–1000μg/L. Accuracy for both analytes was <15% (5–500μg/L). This validated LC–MS method with solid phase extraction facilitates the simultaneous analysis of primaquine and carboxyprimaquine from small volumes of human plasma, with run time <10min, recovery >85% and sensitivity of 1–2μg/L.

Quantitation of melatonin and n‐acetylserotonin in human plasma by nanoflow LC‐MS/MS and electrospray LC‐MS/MS

Melatonin (MEL) and its chemical precursor N-acetylserotonin (NAS) are believed to be potential biomarkers for sleep-related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC-MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies, and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays, plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1 × 100 mm, 3.5 µm) or on a polyimide-coated, fused-silica capillary self-packed with 17 cm AquaC18 (3 µm, 125 Å). Quantitation was done using the SRM transitions m/z 233 → 174 and m/z 219 → 160 for MEL and NAS, respectively. The analytical response ratio versus concentration curves were linear for MEL (nanoflow LC: 11.7-1165 pg/mL, LC: 1165-116,500 pg/mL) and for NAS (nanoflow LC: 11.0-1095 pg/mL).

Determination of ganciclovir in human plasma by ultra performance liquid chromatography–UV detection

Implement a sensitive UHPLC method for the assay of ganciclovir in human plasma. We developed and validated a chromatographic method coupled to ultraviolet detection for quantification of ganciclovir, with a short run time using a small volume of human plasma. Comparison of system performance was made with respect to analysis time, efficiency and sensitivity. Correlation coefficients (r) of the calibration curves ranged from 0.999744 to 0.999784. Within-day and between-day imprecision and inaccuracy, specificity and recovery were also evaluated for validation. The method was precise and accurate and the retention time was 0.7 min. The calibration curves were linear between 0.5 and 30 μg/mL. There was a good correlation between HPLC and UHPLC techniques. We developed a method that is currently applied in a clinical study assessing GCV plasma concentration variability after ganciclovir and valganciclovir administration.

Determination of vitamins A, D and E in a small volume of human plasma by a high‐throughput method based on liquid chromatography/tandem mass spectrometry

We have developed an automated high-throughput assay for the determination of vitamin A (retinol), ergocalciferol (25-OH D2), cholecalciferol (25-OH D3) and vitamin E (α-tocopherol) in a small volume of human plasma. Sample preparation involved mixing 50 μL of plasma with 100 μL of ethanol containing isotope-labelled internal standards, followed by mixing with isooctane/chloroform (3:1, 300 μL). The organic phase was evaporated, and the sample reconstituted in 50 μL methanol. The analysis was performed using reversed-phase liquid chromatography with a gradient mobile phase containing water, methanol and ammonium formate. Chromatographic run-time was 5 min, and positive mode electrospray tandem mass spectrometry (MS/MS) was used for detection. The limits of detection were 0.10 μM for all-trans retinol and 3.3 nM for 25-OH D2 and 25-OH D3. Recoveries were 91.9-105.0%, and within- and between-day coefficients of variance (CVs) 2.4-5.3 and 3.1-8.2, respectively. The assay is presently being used in large-scale studies.

A gel-based method for purification of apolipoprotein A-I from small volumes of plasma

We present here a gel-based method for rapid purification of apolipoprotein A-I (apoA-I) from small volumes of human plasma. After isolation of high density lipoprotein from plasma, the apoA-I protein was separated by electrophoresis and the apoA-I band excised from the gel. The apoA-I was then eluted from the gel strip, concentrated, and delipidated ready for use. The structure and function of the gel-purified apoA-I protein was compared against apoA-I purified by the traditional size-exclusion chromatography method. The α-helical content of the gel-purified apoA-I as determined by circular dichroism was similar to chromatography-purified apoA-I. The functional activity of gel-purified apoA-I, as determined by cholesterol efflux assays in primary human fibroblasts and RAW264.7 macrophages, was also comparable with chromatography-purified apoA-I. This method is a valid alternative for apoA-I purification with some advantages over traditional chromatography purification including a much reduced plasma volume requirement, less time and cost, and a higher percentage protein recovery. The method is particularly suitable for applications requiring the purification of apoA-I from multiple human or animal samples of interest.

Analysis of human plasma lipids and soybean lecithin by means of high‐performance thin‐layer chromatography and matrix‐assisted laser desorption/ionization mass spectrometry

An improved analytical strategy for the analysis of complex lipid mixtures using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in combination with high-performance thin-layer chromatography (HPTLC) is reported. Positive ion MALDI RTOF MS was applied as a rapid screening tool for the various neutral (e.g. triacylglycerols) and polar (e.g. glycerophospholipids and -sphingolipids) lipid classes derived from crude lipid extracts of e.g. human plasma as well as soybean lecithin. Finally, MALDI seamless post-source decay (PSD) product ion analysis was performed in order to obtain further structural information (head- and acyl-group identification) of selected lipid species and structure verification. A Coomassie Brilliant Blue R-250 staining protocol for lipids on HPTLC plates was evaluated and was found to be fully compatible with subsequent MALDI-MS. Lipids were analyzed after elution from the HPTLC phase material of the selected band (corresponding to certain lipid classes) by using the proper organic solvent mixture or in few cases directly from the HPTLC plates (a type of on-line HPTLC/MALDI-MS coupling). More than 70 distinct lipid species from seven different lipid classes in the range between m/z 500 and 1500 could be identified from the lipid extracts of human plasma and soybean lecithin, respectively. The general high sensitivity of MALDI-MS detection allowed the analysis of even minor lipid classes from only very small volumes of human plasma (50 microL). The combination of HPTLC, Coomassie staining and positive ion MALDI curved field RTOF-MS represents a straightforward strategy during lipidomics studies of food and clinically relevant human lipid samples.

Analysis of angiotensin II receptor antagonist and protein markers at microliter level plasma by LC–MS/MS

An analytical method based on a green approach is proposed for clinical analysis. The proposed procedure involves the reduction of the sample preparation steps, the amounts of reagents and organic solvents. This simple and sensitive method for the analysis of clinical drug and biomarkers in human plasma was developed using LC connected to tandem mass spectrometry (LC–MS/MS) with a nanospray ion source. In this study, the desired drug and proteins were separated on a 5 and 10 cm RP C18 nano-flow column. Undesired polar substances in human plasma were washed out by using ACN:1% FA = 20:80 (v/v) as the loading mobile phase for drug analysis and good linearity was attainable. Only a small volume of human plasma (10 μL) was utilized for the monitoring of drug plasma concentration and significant proteins under clinical studies. All the sample preparation procedures and the analytical scheme were at microliter level. This strategy would lower the consumption of reagents and organic solvents and make a contribution toward the goal of reduction of pollution from analytical methods in general.

Determination of Losartan in Human Plasma by Liquid Chromatography Electrospray Ionization Mass Spectrometry (LC-ESI-MS): Application to Bioequivalence Study

A sensitive, simple, rapid, and specific liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) method was developed and validated for the identification and quantification of losartan in a small volume of human plasma. Losartan and I.S. were successfully separated on a CN column with a mobile phase of acetonitrile-0.2% formic acid solution (68:32, v/v). Detection was performed on a single quadrupole mass spectrometer by a selected ion monitoring (SIM) mode via electrospray ionization (ESI) source. The ESI source was set at positive ionization mode. The ion signal of m/z 422.79 and 194.81 were measured for losartan and I.S., respectively. The limit of detection (LOD) was 0.5 ng/mL (signal-to-noise ratio of 10.03) using only 200 μL of human plasma samples. The calibration curve was an excellent linear fit over the range of concentrations 1.0–1000 ng/mL (R2 = 0.9987) of losartan in human plasma. Consequently, all of our results fulfilled the common standard criteria of bioequivalence, 0.80 to 1.25 by the Korean and US Food and Drug Administration. In addition to the confidence intervals (C.I.) 90% of the pharmaceutical parameters, a two-way ANOVA showed no significant difference between the two formulations. This method was successfully applied to bioequivalence study of two brands of losartan potassium tablet (100 mg) formulations after a single oral administration.

Development and validation of a fast quantitative method for plasma dimethylarginines analysis using liquid chromatography–tandem mass spectrometry

Objectives The aim of this work was to implement a fast, accurate and simple method to quantify plasma ADMA and SDMA, in a run time suitable for routine analysis. Design and methods We developed and validated a hydrophilic interaction chromatographic method coupled to tandem mass spectrometry (HILIC–MS/MS) for separation and simultaneous quantification of Arginine (Arg) and its dimethylarginines, ADMA and SDMA, with a short run time (less than 5 min) using a small volume of human plasma (0.02 mL). Results Correlation coefficients ( r) of the calibration curves ranged from 0.9926 to 0.9984. Within-day and between-day imprecision (CV%) and inaccuracy (%), carry-over and recovery were also evaluated for validation. Preliminary data of Arg, ADMA and SDMA from 30 apparently healthy subjects and type 2 diabetic patients ( n = 33) with and without kidney dysfunction were calculated and some statistical differences occurred among them ( p < 0.05). Conclusions Data from calibration curves and quality controls reveal that the method is accurate and precise. Healthy subjects and diabetic patients' values are in agreement with those reported in other studies.