Stability of amyloid-β peptides in plasma and serum

Plasma amyloid‐β peptide (Aβ) levels have been suggested as a biomarker candidate for detecting incipient AD. Aβ peptides are known to be sensitive to distinct preanalytical sample handling, which calls for standardised preanalytical procedures. We investigated serum and plasma samples of 19 patients with no clinical signs of dementia for different preanalytical sample handlings. Both serum and plasma were analysed by the one‐dimensional Aβ‐SDS‐PAGE/immunoblot, either immediately or after storage at room temperature for 24 and 48 h, respectively. The panel of Aβ1–37/38/39/40/42 and Aβ2–40 was evaluated. In both analytical matrices, sample storage led to a significant loss of measurable peptide levels. This effect was most pronounced during the first 24 h of storage and stronger in serum than in plasma. There were no significant differences between the distinct analysed Aβ peptide species regarding these results. The ratios of peptides (e.g. Aβ1–42/Aβ1–40 and Aβ1–42/Aβ1–38) displayed a higher stability under the influence of storage than each single peptide. In conclusion, plasma may be more appropriate than serum for analysing Aβ peptides for routine application. At least, the analysis should be done within 24 h and peptide ratios should be created to minimise artificial results.     

Synthesis of zinc oxide powders in plasma–solution systems

A new method is proposed for the synthesis of powdered zinc oxide with the use of a plasma–solution system. The chemical and phase compositions and the morphology of the synthesized powders have been determined. It has been found that the calcination of powders obtained in the plasma–solution system leads to the formation of ZnO.     

Liquid chromatography–mass spectrometric determination of plasmalogens in human plasma

A new liquid chromatography–mass spectrometry (LC/MS) method has been developed for the quantitative analysis of plasmalogens in human plasma using a nonendogenous plasmalogen (1-O-1′-(Z)-tricosenyl-2-oleoyl-rac-glycero-3-phosphocholine, PLS 23:0/18:1) as an internal standard. 1-O-1′-(Z)-Tricosenyl glyceryl ether was prepared by reacting lithioalkoxyallyl with 1-iodoeicosane as the key intermediate in the formation of PLS 23:0/18:1. In LC/MS analyses, PLS 23:0/18:1 generated significant fragment ions in positive and negative modes. In positive ion mode, the [M+H]⁺ of PLS 23:0/18:1 yielded unique fragments with cleavages at the sn-1 and sn-2 positions of the glycerol backbone. In negative ion mode, the [M+CH₃COO]⁻ of PLS 23:0/18:1 resulted in characteristic fragmentation at the sn-2 and sn-3 positions. 1-O-1′-(Z)-Hexadecenyl-2-linoleoyl-rac-glycero-3-phosphocholine (PLS 16:0/18:2) and 2-arachidonoyl-O-1′-(Z)-hexadecenyl-rac-glycero-3-phosphocholine (PLS 16:0/20:4) were chemically synthesized as PLS 23:0/18:1. The calibration curves obtained for PLS 16:0/18:2 and PLS 16:0/20:4 were linear throughout the calibration range (0.04–1.60 pmol). The LOD (S/N = 5:1) was 0.008 pmol and the LOQ (S/N = 6:1) was 0.01 pmol for both PLS 16:0/18:2 and PLS 16:0/20:4. Plasma concentrations of PLS 16:0/18:2 and PLS 16:0/20:4 were 4.0 ± 1.3 μM and 3.5 ± 1.2 μM (mean ± SD), respectively, in five healthy volunteers.     

Clastogenic plasma factors in psoriasis--comparison of phototherapy and anti-TNF-α treatments

As previously described, Psoralen plus UVA (PUVA) therapy induces chromosome damage in psoriatic patients. This study evaluates whether these effects are transitory or persistent. In addition, we studied these effects after narrowband UVB (nUVB) and anti-tumor necrosis factor (TNF)-α treatments. Among 40 responder patients, 10 received PUVA, 10 nUVB, 10 Infliximab and 10 Etanercept. Disease activity was determined with Psoriasis Area and Severity Index. Chromosomal breakage was evaluated by the clastogenic factor (CF) test. Potential clastogenic agents, malondialdehyde (MDA) and TNF-α were measured. Before treatment, the plasma-adjusted clastogenic scores (ACS) of patients were increased. During treatment, a further increase in ACS was observed in both phototherapy groups. Chromosome damage persisted for PUVA patients at week 32, while it diminished after nUVB to ACS values lower than before treatment. MDA and TNF-α values were also increased at baseline. MDA decreased during treatment in all groups, but without reaching normal levels. Plasma TNF-α remained unchanged in PUVA and nUVB but decreased in both anti-TNF-α treatment groups. Psoriasis is accompanied by CF-induced chromosomal breakage that increases during PUVA and nUVB treatments. Plasma clastogenic activity persisted in the follow-up after PUVA, while after nUVB ACS returned to values even lower than baseline. Clastogenic activity during the induction phase with anti-TNF-α remained unchanged.     

Dried plasma spot–based liquid chromatography–tandem mass spectrometry for the quantification of methotrexate in human plasma and its application in therapeutic drug monitoring

Methotrexate, a folic acid antitumor drug, is widely used to treat childhood acute lymphoblastic leukemia. Therapeutic drug monitoring is crucial for adjusting the dosage of methotrexate according to its plasma concentration and reducing adverse effects. Micro-sampling strategies, like dried plasma spot, is an attractive but underutilized method that has the desired features of easy collection, storage, and transport, and overcomes known hematocrit issues in dried blood spot analysis. This study describes a dried plasma spot–based liquid chromatography–tandem mass spectrometry method for quantification of methotrexate. The assay showed good linearity over 30–2000 ng/mL (R² ≥ 0.995) as well as excellent precision (0.6–9.3%) and accuracy (89.2–108.3%). Methotrexate was extracted from dried plasma spot and wet plasma samples with recoveries greater than 92.1%, and no significant matrix effect was observed. A comparison of dried plasma spot and wet plasma concentrations was assessed in 27 patients treated with methotrexate and Passing–Bablok regression coefficients showed that no significant difference between the two methods. The Bland–Altman plots showed similar agreement between the methods, indicating that the proposed dried plasma spot sampling method is an effective way to monitor the concentration of methotrexate in human plasma.     

Computerized simulation of solute–particle vaporization in an inductively coupled plasma

Recent experiments involving aerosol introduction into the inductively coupled plasma have shown that intact droplets and solute particles cause enormous fluctuations in analyte emission and mass-spectral signals. Here, particle-vaporization kinetics are simulated as a detailed function of the operating conditions, fundamental properties and spatial location in the inductively coupled plasma, and as a function of several of the properties of the particles themselves: diameter, chemical composition and size distribution. These simulations portray the particle vaporization as proceeding nominally linearly with respect to the particle radius when the particles are small, but roughly quadratically with radius when the particles are very large. Further, the heat- and mass-transfer-limited rates of vaporization are roughly equal for the typical gas-temperature range in the plasma tail flame, so that at any height either process might limit the rate of vaporization. This similarity gives rise to a dynamic, competitive picture of plasma vaporization kinetics.     

Quantitative behavior of vibrational excitation in AC plasma assisted dry reforming of methane

Quantitative behavior of non-equilibrium excitation by direct electron impact in low-temperature dry reforming of methane was investigated by integrated studies of experimental validation and kinetic modeling.A plasma chemistry kinetic mechanism incorporating the reactions involving vibrational excitation of CH4,CO2,H2 and CO molecules as well as the low temperature He/CH4/CO2 conversion pathways was developed and validated.The calculation results showed that at lower E/N values(<150 Td)large population of energized electrons generated in a He/CH4/CO2 discharge resulted in an intensification of vibrational excitation.Despite the large generation of vibration,the vibrationally excited molecules in a 0.5/0.25/0.25 of He/CH4/CO2 discharge mixture were easy to relax,due to the strong coupling of the vibration of different molecules in a gas mixture.The results showed that the moderate levels of the vibrational excitation,such as CO2(v10,11,...,18)and CO(v9,10),presented most efficient in the stimulation of species generation including CO,CH2 O,CH3 OH,C2 H4 and C2 H6.Specifically,under conditions of E/N of 108 Td,14.9%of CO formation was estimated from the recombination of CO2(v)with CH3 and H,CO2(v)+CH3→CH3 O+CO,CO2(v)+H→CO+OH.Also,4.8%of C2 H4 formation was from the recombination reaction CH4(v)+CH→C2 H4+H.These results highlight the strong roles of vibrational states in a complex plasma chemistry system.     

Liquid chromatographic–electrospray mass spectrometric determination of cyclosporin A in human plasma

A rapid, sensitive and selective liquid chromatography–mass spectrometry (LC–MS) assay has been developed for determination of cyclosporin A (CyA) in human plasma; cyclosporin B (CyB) was used as internal standard (IS). The method utilized a combination of a column-switching valve and a reversed-phase symmetry column. The mobile phase was a 25:75 (v/v) mixture of 10% aqueous glacial acetic acid and acetonitrile. Running time per single run was less than 10 min. Sample preparation included C₈ SPE of human plasma spiked with the analyte and internal standard, evaporation of the eluate to dryness at 50°C under N₂ gas, and finally reconstitution in the mobile phase. Detection of cyclosporin A and the IS was performed in selected ion-monitoring mode at m/z 601.3 and 594.4 Da for CyA and IS, respectively. Quantitation was achieved by use of the regression equation of relative peak area of cyclosporin to IS against concentration of cyclosporin. The method was validated according to FDA guideline requirements. The linearity of the assay in the range 5.0–400.0 ng mL^–1 was verified as characterized by the least-squares regression line Y=(0.00268±1.9×10^–4)X+(0.00078±1.8×10^–3), correlation coefficient, r=0.9986±1.1×10^–3 (n=48). Intra and inter-day quality-control measurements in the range 5.0–350.0 ng mL^–1 revealed almost 100% accuracy and 9% CV for precision. The mean absolute recovery of CyA was found to be 84.01±9.9% and the respective relative recovery was 100.3±9.19. The limit of quantitation (LOQ) achieved was 5 ng mL^–1. Eventually, stability testing of the analyte and IS in plasma or stock solution revealed that both chemicals were very stable when stored for long or short periods of time at room temperature or –20°C.     

Qualitative detection of desmopressin in plasma by liquid chromatography–tandem mass spectrometry

This work describes a liquid chromatography–electrospray tandem mass spectrometry method for detection of desmopressin in human plasma in the low femtomolar range. Desmopressin is a synthetic analogue of the antidiuretic hormone arginine vasopressin and it might be used by athletes as a masking agent in the framework of blood passport controls. Therefore, it was recently added by the World Anti-Doping Agency to the list of prohibited substances in sport as a masking agent. Mass spectrometry characterization of desmopressin was performed with a high-resolution Orbitrap-based mass spectrometer. Detection of the peptide in the biological matrix was achieved using a triple-quadrupole instrument with an electrospray ionization interface after protein precipitation, weak cation solid-phase extraction and high performance liquid chromatography separation with an octadecyl reverse-phase column. Identification of desmopressin was performed using three product ions, m/z 328.0, m/z 120.0, and m/z 214.0, from the parent ion, m/z 535.5. The extraction efficiency of the method at the limit of detection was estimated as 40% (n = 10), the ion suppression as 5% (n = 10), and the limit of detection was 50 pg/ml (signal-to-noise ratio greater than 3). The selectivity of the method was verified against several endogenous and synthetic desmopressin-related peptides. The performance and the applicability of the method were tested by analysis of clinical samples after administration of desmopressin via intravenous, oral, and intranasal routes. Only after intravenous administration could desmopressin be successfully detected.     

Determination of lesinurad in rat plasma by a UHPLC–MS/MS assay

Lesinurad is an oral inhibitor of urate-anion exchanger transporter 1 and has been approved by the US Food and Drug Administration for combination therapy with a xanthine oxidase inhibitor for the treatment of hyperuricemia associated with refractory gout. In the present study, a sensitive and specific ultra high-performance liquid chromatography with tandem mass spectrometry assay was established and verified for the determination of lesinurad in rat plasma and was described in details for the first time. Chromatographic separation of lesinurad and diazepam (internal standard, IS) was performed on a Rapid Resolution HT C18 column (3.0 × 100 mm, 1.8 µm) using methanol–water (70:30, v/v) as the mobile phase at a flow rate of 0.3 mL/min. Lesinurad and IS were extracted from plasma by liquid–liquid extraction using ethyl acetate. The mass spectrometric detection was carried out using an electrospray ionization source in positive mode. Multiple reaction monitoring was used for quantification of the precursor to product ion at m/z 405.6 → 220.9 for lesinurad and m/z 285.1 → 192.8 for IS. The assay was well validated for selectivity, accuracy, precision, recovery, linearity, matrix effects, and stability. The verified method was applied to obtain the pharmacokinetic parameters and concentration–time profiles for lesinurad after oral/intravenous administration in rats. The study might provide an important reference and a necessary complement for the qualitative and quantitative evaluation of lesinurad.     

Simulation of droplet heating and desolvation in an inductively coupled plasma — Part I

The total desolvation rate of sample droplets in an argon inductively coupled plasma (Ar ICP) is investigated through the development of a two-phase continuum flow computer model. The desolvation model is supplemented by equations used to determine the trajectories of particles through the plasma. The model is used to calculate the behavior of aerosol droplets from a direct injection high efficiency nebulizer (DIHEN), a micronebulizer used to inject microliter quantities of samples that are toxic, expensive, or of limited volume. We use the combination of desolvation and transport models to present the first predicted spatial distribution of droplet concentrations and evaporation rates in an ICP flow. These data are compared with the behavior of a DIHEN spray in an environment with no net argon gas flow to determine the importance of gas flow rates to overall droplet concentration profiles in the ICP. In addition, two separate techniques (Stokes’ equation and the direct simulation Monte Carlo treatment) for determining droplet trajectories are contrasted.     

Validated HPLC–MS–MS method for determination of azithromycin in human plasma

A validated, highly sensitive, and selective HPLC method with MS–MS detection has been developed for quantitative determination of azithromycin (AZI) in human Na₂EDTA plasma. Roxithromycin (ROX) was used as internal standard. Human plasma containing AZI and internal standard was ultrafiltered through Centrifree Micropartition devices and the concentration of AZI was determined by isocratic HPLC–MS–MS. Multiple reaction monitoring mode (MRM) was used for MS–MS detection. The calibration plot was linear in the concentration range 2.55–551.43 ng mL⁻¹. Inter-day and Intra-day precision and accuracy of the proposed method were characterized by R.S.D and percentage deviation, respectively; both were less than 8%. Limit of quantification was 2.55 ng mL⁻¹. The proposed method was used to determine the pharmacokinetic profile of AZI (250-mg tablets).     

A simple method for methylmercury,inorganic mercury and ethylmercury determination in plasma samples by high performance liquid chromatography–cold-vapor-inductively coupled plasma mass spectrometry

A simple and sensitive method with a fast sample preparation procedure is proposed for the determination of mercury species in plasma/serum. The method combines online high-performance liquid chromatography separation, Hg cold-vapor formation and inductively coupled plasma mass spectrometry detection. Prior to analysis, plasma (250 μL) was accurately pipetted into 15 mL conical tubes. Then, an extractant solution containing mercaptoethanol, L-cysteine and HCl was added to the samples following sonication for 10 min. Quantitative mercury extraction was achieved with the proposed procedure. Separation of mercury species was accomplished in less than 8 min on a C8 reverse phase column with a mobile phase containing 3% v/v methanol + 97% v/v (0.5% v/v 2-mercaptoethanol + 0.05% v/v formic acid). The method detection limits were found to be 12 ng L⁻¹, 5 ng L⁻¹ and 4 ng L⁻¹ for inorganic mercury, ethylmercury and methylmercury, respectively. Method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from NIST. Additional validation was provided by the analysis of a secondary reference serum sample from the INSQ-Canada. Finally, the method was successfully applied for the speciation of mercury in plasma samples collected from volunteers exposed to methylmercury through fish consumption. For the first time to our knowledge, levels of different species of Hg in plasma samples from riverside populations exposed to MeHg were determined.     

Determination of Nɛ-homocysteinyl-lysine and γ-glutamylcysteine in plasma by liquid chromatography with UV-detection

Homocysteine (Hcy) is a sulfur-containing nonprotein amino acid, a metabolite of methionine. Mechanisms by which Hcy is involved in the pathogenesis of vascular diseases remain unclear. One of the potential mechanisms underlying harmful effects of Hcy is the protein N-homocysteinylation induced by Hcythiolactone. Proteolytic degradation of N-homocysteinylated protein yields N?-homocysteinyl-lysine, a novel and important component of Hcy metabolism. Here we describe new high-performance liquid chromatography assay for the determination of N?-homocysteinyl-lysine and γ-glutamylcysteine in plasma, based on a derivatization with 2-chloro-1-methyllepidinium tetrafluoroborate and UV detection. Baseline separation was achieved on an analytical column from Phenomenex (Kinetex C18, 100 × 4.6 mm, 2.6 μm) using gradient elution, with a mobile phase consisting 0.1 M trichloroacetic acid (pH 2.3) — acetonitrile. The quantification limits for N?-homocysteinyl-lysine and γ-glutamylcysteine in plasma were 0.1 and 0.2 μM, respectively. Other main endogenous thiols can also be measured during the same analytical run. The proposed method was applied for the analysis of 15 plasma samples for total form of N?-homocysteinyl-lysine and γ-glutamylcysteine.     

Determination of tandospirone in human plasma by a liquid chromatography–tandem mass spectrometry method

A rapid, sensitive, and selective liquid chromatography–tandem mass spectrometry method for the detection of tandospirone in human plasma is described. It was employed in a pharmacokinetic study. The analyte and internal standard diphenhydramine were extracted from plasma using liquid–liquid extraction, then separated on a Zorbax XDB C₁₈ column using a mobile phase of methanol–water–formic acid (80:20:0.5, v/v/v). The detection was performed with a tandem mass spectrometer equipped with an electrospray ionization source. Linearity was established in the concentration range of 10.0-5,000 pg/ml. The lower limit of quantification was 10.0 pg/ml. The intraday and interday relative standard deviation across three validation runs over the entire concentration range was less than 13%. Accuracy determined at three concentrations (25.0, 200, and 4,000 pg/ml for tandospirone) ranged from 94.4 to 102.1%. Each plasma sample was chromatographed within 3.4 min. The method proved to be highly selective and suitable for bioequivalence evaluation of different formulations containing tandospirone and clinical pharmacokinetic investigation of tandospirone.     

The calculation of Cs and Rb conductivities in the region of liquid–plasma transition

The conductivity and thermal conductivity of Cs and Rb are calculated in the liquid phase and in the region between the plasma (gas) and the liquid states. The last area is located at the temperatures higher than the critical one, near the critical point. The Ziman formalism originated from the liquid metal theory was used for the calculations. The results of present calculations were compared with available experiments and calculations of other researchers. It was found that the liquid state formalism can be applied to expanded liquid Cs and Rb at densities higher than the critical one, but another type of models is necessary at lower densities.     

Solid phase extraction with electrospun nanofibers for determination of retinol and α-tocopherol in plasma

A novel packed-fiber solid phase extraction procedure based on electrospun nanofibers for simultaneous determination of vitamins A (retinol) and E (α-tocopherol) in human plasma has been developed. Parameters affecting extraction efficiency were investigated in detail. The limit of detection is 0.01 μg mL^?1 for retinol, and 0.3 μg mL^?1 for α-tocopherol. The linear range is from 0.05 to 2.0 μg mL^?1 for retinol, and from 0.5 to 30 μg mL^?1 for α-tocopherol. The precision (RSD) is <6%, and the relative recovery >90%. The method was applied to analysis of retinol and α-tocopherol in human plasma with satisfactory results.     

Metallomics in drug development: characterization of a liposomal cisplatin drug formulation in human plasma by CE–ICP–MS

A capillary electrophoresis inductively coupled plasma mass spectrometry method for separation of free cisplatin from liposome-encapsulated cisplatin and protein-bound cisplatin was developed. A liposomal formulation of cisplatin based on PEGylated liposomes was used as model drug formulation. The effect of human plasma matrix on the analysis of liposome-encapsulated cisplatin and intact cisplatin was studied. The presence of 1 % of dextran and 4 mM of sodium dodecyl sulfate in HEPES buffer was demonstrated to be effective in improving the separation of liposomes and cisplatin bound to proteins in plasma. A detection limit of 41 ng/mL of platinum and a precision of 2.1 % (for 10 μg/mL of cisplatin standard) were obtained. Simultaneous measurements of phosphorous and platinum allows the simultaneous monitoring of the liposomes, liposome-encapsulated cisplatin, free cisplatin and cisplatin bound to plasma constituents in plasma samples. It was demonstrated that this approach is suitable for studies of the stability of liposome formulations as leakage of active drug from the liposomes and subsequent binding to biomolecules in plasma can be monitored. This methodology has not been reported before and will improve characterization of liposomal drugs during drug development and in studies on kinetics.

Determination of seven selected antipsychotic drugs in human plasma using microextraction in packed sorbent and gas chromatography–tandem mass spectrometry

A method using microextraction by packed sorbent (MEPS) and gas chromatography–tandem mass spectrometry (GC-MS/MS) is described for the determination of seven antipsychotic drugs in human plasma. The studied compounds were chlorpromazine (CPZ), haloperidol (HAL), cyamemazine, quetiapine, clozapine, olanzapine (OLZ), and levomepromazine; promazine, protriptyline, and deuterated CPZ were used as internal standards. The validation parameters included selectivity, linearity and limits of detection and quantitation, intra- and interday precision and trueness, recovery, and stability and were studied according to internationally accepted guidelines. The method was found to be linear between the lower limit of quantitation and 1000 ng/mL, except for OLZ and HAL (200 ng/mL), with determination coefficients higher than 0.99 for all analytes, and extraction efficiencies ranged from 62 to 92 %. Intra- and interday precision ranged from 0.24 to 10.67 %, while trueness was within a ±15 % interval from the nominal concentration for all analytes at all studied levels. MEPS has shown to be a rapid procedure for the determination of the selected antipsychotic drugs in human plasma, allowing reducing the handling time and the costs of analysis. Furthermore, GC-MS/MS has demonstrated to be a powerful tool for the simultaneous quantitation of the studied compounds, enabling obtaining adequate selectivity and sensitivity using a sample volume of as low as 0.25 mL.     

Doping control analysis of seven bioactive peptides in horse plasma by liquid chromatography–mass spectrometry

In recent years, there has been an ongoing focus for both human and equine doping control laboratories on developing detection methods to control the misuse of peptide therapeutics. Immunoaffinity purification is a common extraction method to isolate peptides from biological matrices and obtain sufficient detectability in subsequent instrumental analysis. However, monoclonal or polyclonal antibodies for immunoaffinity purification may not be commercially available, and even if available, such antibodies are usually very costly. In our study, a simple mixed-mode anion exchange solid-phase extraction cartridge was employed for the extraction of seven target peptides (GHRP-1, GHRP-2, GHRP-6, ipamorelin, hexarelin, CJC-1295, and N-acetylated LKKTETQ (active ingredient of TB-500)) and their in vitro metabolites from horse plasma. The final extract was subject to ultra-high-performance liquid chromatographic separation and analysed with a hybrid high-resolution mass spectrometer. The limits of detection for all seven peptides were estimated to be less than 50 pg/mL. Method validation was performed with respect to specificity, precision, and recovery. The applicability of this multi-analyte method was demonstrated by the detection of N-acetylated LKKTETQ and its metabolite N-acetylated LK from plasma samples obtained after subcutaneous administration of TB-500 (10 mg N-acetylated LKKTETQ) to two thoroughbred geldings. This method could easily be modified to cover more bioactive peptides, such as dermorphin, β-casomorphin, and desmopressin. With the use of high-resolution mass spectrometry, the full-scan data acquired can also be re-processed retrospectively to search for peptides and their metabolites that have not been targeted at the time of analysis. To our knowledge, this is the first identification of in vitro metabolites of all the studied peptides other than TB-500 in horses.