A new coaxial flow-through probe for electromembrane extraction of methadone from clinical samples on-line coupled to capillary electrophoresis.

BACKGROUND: A new design of a flow-through coaxial electromembrane extraction (EME) probe that can be on-line coupled with CE instrument is described and tested. The supporting base of the probe is a PDMS microchip with T-shaped channels into which two coaxially arranged capillaries for inlet and outlet solutions are inserted. The extraction part of the probe is a porous polypropylene hollow fiber, sealed at one end and modified with nitrophenyloctyl ether (NPOE) extraction fluid. The internal volume of the extraction probe is 1.1 µL. RESULTS: The EME probe was tested on laboratory samples and methadone was extracted into 3.0 M AcOH as acceptor. The concentration dependence was linear in the range of 0.1-1.0 µg mL-1 at EME 300 s/150 V and in the range of 0.5-10.0 µg mL-1 at EME 100 s/150 V. The enrichment factor was greater than 30 and the LOD was 0.21 µg mL-1. The EME of methadone in clinical samples showed a linear concentration dependence in human urine and a nonlinear concentration dependence in serum. The distribution of methadone in each phase of the extraction system and the effect of extraction membrane thickness on the enrichment factor were studied. The EME probe can be applied repeatedly. SIGNIFICANCE: The supporting base of EME probe and flow gating interface (FGI) are realized by a microfluidic PDMS microchips cast in the laboratory without the use of lithography. A supporting PDMS chip with coaxially arranged capillaries and extraction membrane forms a compact analytical instrument. The entire EME/CE analysis process is performed on a laboratory-made instrument and automated by LabView.

Steady state microdialysis of microliter volumes of body fluids for monitoring of amino acids by capillary electrophoresis with contactless conductivity detection.

BACKGROUND: The availability of dialysis membranes in the form of hollow fibres with diameters compatible with the fused silica capillaries used in capillary electrophoresis is very limited. However, haemodialysis bicarbonate cartridges commonly used in human medicine containing polysulfone hollow fibres are available on the market and are used for the fabrication of coaxial microdialysis probes. The miniature probe design ensures that steady-state conditions are achieved during microdialysis of minimal volumes of body fluids. RESULTS: A coaxial microdialysis probe with a length of 5 cm and an inner diameter of 200 µm is used for microdialysis of 10 µL of body fluid collected into a sampling fused silica capillary with an inner diameter 430 µm. Microdialysis is performed into 0.01 M HCl as a perfusate at stopped flow and 2 µL of the resulting microdialysate are subjected to analysis by capillary electrophoresis with contactless conductivity detection. Microdialysis pre-treatment is verified by analysis of 11 common amino acids at a 100 µM concentration level, resulting in recoveries of 98.3-102.5%. The electrophoretic separation of amino acids is performed in 8.5 M acetic acid at pH 1.37 as a background electrolyte with analysis time up to 4.5 min and LOD in the range of 0.12-0.28 µM. The reproducibility of the developed technique determined for the peak area ranges from 1.2 to 4.5%. Applicability is tested in the quantification of valine and leucine in plasma during fasting and subsequent reconvalescence. SIGNIFICANCE: The fabrication of a coaxial microdialysis probe for the laboratory preparation of microliter volumes of various types of clinical samples is described, which is coupled off-line with capillary electrophoretic monitoring of amino acids in 2 µL volumes of microdialysate. The developed methodology is suitable for quantification of 20 amino acids in whole human blood, plasma, tears and has potential for analysis of dry blood spots captured on hollow fibre.

Severe COVID-19 associated hyperglycemia is caused by beta cell dysfunction: a prospective cohort study.

BACKGROUND: COVID-19, an infectious disease caused by SARS-CoV-2, was shown to be associated with an increased risk of new-onset diabetes. Mechanisms contributing to the development of hyperglycemia are still unclear. We aimed to study whether hyperglycemia is related to insulin resistance and/or beta cell dysfunction. MATERIALS AND METHODS: Survivors of severe COVID-19 but without a known history of diabetes were examined at baseline (T0) and after 3 (T3) and 6 (T6) months: corticosteroids use, indirect calorimetry, and OGTT. Insulin response and sensitivity (IS) were expressed as insulinogenic (IGI), disposition (DI), and Matsuda insulin sensitivity index (ISI). Resting energy expenditure (REE) and respiratory quotient (RQ) was calculated from the gas exchange and nitrogen losses. RESULTS: 26 patients (out of 37) with complete outcome data were included in the analysis (age ~59.0 years; BMI ~ 30.4, 35% women). Patients were hypermetabolic at T0 (30.3 ± 4.0 kcal/kg lean mass/day, ~120% predicted) but REE declined over 6 months (ΔT6-T0 mean dif. T6-T0 (95% CI): -5.4 (-6.8, -4.1) kcal/kg FFM/day, p < 0.0001). 17 patients at T0 and 13 patients at T6 had hyperglycemia. None of the patients had positive islet autoantibodies. Insulin sensitivity in T0 was similarly low in hyperglycemic (H) and normoglycemic patients (N) (T0 ISIH = 3.12 ± 1.23, ISIN = 3.47 ± 1.78, p = 0.44), whereas insulin response was lower in the H group (DIH = 3.05 ± 1.79 vs DIN = 8.40 ± 5.42, p = 0.003). Over 6 months ISI (ΔT6-T0 mean dif. T6-T0 for ISI (95% CI): 1.84 (0.45, 3.24), p = 0.01)) increased in the H group only. CONCLUSIONS: Patients with severe COVID-19 had increased REE and insulin resistance during the acute phase due to the infection and corticosteroid use, but these effects do not persist during the follow-up period. Only patients with insufficient insulin response developed hyperglycemia, indicating that beta cell dysfunction, rather than insulin resistance, was responsible for its occurrence.

Microdialysis as a tool for antibiotic assessment in patients with diabetic foot: a review.

Diabetic foot is a serious late complication frequently caused by infection and ischaemia. Both require prompt and aggressive treatment to avoid lower limb amputation. The effectiveness of peripheral arterial disease therapy can be easily verified using triplex ultrasound, ankle-brachial/toe-brachial index examination, or transcutaneous oxygen pressure. However, the success of infection treatment is difficult to establish in patients with diabetic foot. Intravenous systemic antibiotics are recommended for the treatment of infectious complications in patients with moderate or serious stages of infection. Antibiotic therapy should be initiated promptly and aggressively to achieve sufficient serum and peripheral antibiotic concentrations. Antibiotic serum levels are easily evaluated by pharmacokinetic assessment. However, antibiotic concentrations in peripheral tissues, especially in diabetic foot, are not routinely detectable. This review describes microdialysis techniques that have shown promise in determining antibiotic levels in the surroundings of diabetic foot lesions.

Progress in on-line, at-line, and in-line coupling of sample treatment with capillary and microchip electrophoresis over the past 10 years: A review.

The review presents an evaluation of the development of on-line, at-line and in-line sample treatment coupled with capillary and microchip electrophoresis over the last 10 years. In the first part, it describes different types of flow-gating interfaces (FGI) such as cross-FGI, coaxial-FGI, sheet-flow-FGI, and air-assisted-FGI and their fabrication using molding into polydimethylsiloxane and commercially available fittings. The second part deals with the coupling of capillary and microchip electrophoresis with microdialysis, solid-phase, liquid-phase, and membrane based extraction techniques. It mainly focuses on modern techniques such as extraction across supported liquid membrane, electroextraction, single drop microextraction, head space microextraction, and microdialysis with high spatial and temporal resolution. Finally, the design of sequential electrophoretic analysers and fabrication of SPE microcartridges with monolithic and molecularly imprinted polymeric sorbents are discussed. Applications include the monitoring of metabolites, neurotransmitters, peptides and proteins in body fluids and tissues to study processes in living organisms, as well as the monitoring of nutrients, minerals and waste compounds in food, natural and wastewater.

A new electromembrane extraction probe for on-line connection with capillary electrophoresis for determination of substances in biological matrices.

A miniature probe for electromembrane extraction is developed and constructed. The tubular probe with an internal volume of 1.1 µL is made of polypropylene hollow fiber with a supported liquid membrane of 85% nitrophenyloctyl ether (NPOE) with 15% bis(2-ethylhexyl)phosphonic acid (DEHP). The probe is connected on-line to the electrophoresis with short separation capillary via an air assisted flow gating interface cast from poly (dimethylsiloxane). The compact instrument is computer controlled via LabView. The probe parameters are tested for extraction of creatinine and basic amino acids from artificial solution and human urine. The sensitivity of the electrophoretic determination after 300 s extraction at 150 V compared to the sensitivity without extraction is 4.9-fold and 2.6-fold higher for creatinine and arginine, respectively. The RSDs for peak area measured from 5 repeated extractions of 50 µM solutions are 7.5%, 7.2%, 8.6% and 9.2% for Crea, Lys, Arg and His, respectively. The probe can be used for all-day measurements. The preparation of the probe is simple and requires no special tool.

Covalent anionic copolymer coatings with tunable electroosmotic flow for optimization of capillary electrophoretic separations.

We present a method for finely adjustable electroosmotic flow (EOF) velocity in cathodic direction for the optimization of separations in capillary electrophoresis. To this end, we use surface modification of the separation fused silica capillary by the covalently attached copolymer of acrylamide (AM) and 2-acrylamido-2-methyl-1-propanesulfonate (AMPS), that is, poly(AM-co-AMPS) or PAMAMPS. Coatings were formed by the in-capillary polymerization of a mixture of the neutral AM and anionic AMPS monomers premixed in various ratios in order to control the charge density of the copolymer. EOF mobility varies in the 0 to ∼40 × 10-9  m2 V-1 s-1 interval for PAMAMPS coatings ranging from 0 to 60 mol.% of charged AMPS monomer. For EOF in PAMAMPS-treated capillaries, we observed (i) a negligible dependence on pH in the 2-10 interval, (ii) a minor variance among background electrolytes (BGEs) in function of their components and (iii) its standard decrease with increasing ionic strength of the BGE. Interest in variable cathodic EOF was demonstrated by the amelioration of separation of two kinds of isomeric anionic analytes, that is, monosaccharides phosphates and helquat enantiomers, in counter-EOF mode.

Monitoring of biologically active substances in clinical samples by capillary and microchip electrophoresis with contactless conductivity detection: A review.

Contactless conductivity detection (C4D) as a universal detection technique plays an important role in combination with efficient electrophoretic separation carried out in capillaries (CE) or on microchips (ME) in the analysis of clinical samples. C4D is particularly sensitive in the quantification of low molecular weight biogenic substances such as inorganic cations and anions, amino acids, amines, low molecular weight organic acids, saccharides and many drugs such as antibiotics, analgesics, anaesthetics or antiepileptics. Biogenic substances are determined in CE/C4D or ME/C4D directly in their native form without derivatization and sample matrix treatment is often based only on dilution or addition of an organic solvent. The limit of detection for most CE/C4D determinations is at the micromolar concentration level, which is sufficient to monitor physiological or therapeutic levels of most of low molecular weight biogenic substances. Therefore, CE/C4D and ME/C4D are widely used for sequential monitoring of nutrients, metabolites and waste products at the level of individual tissues and organs, low-invasive detection of inborn errors of metabolism and cystic fibrosis, pharmacokinetic monitoring and therapeutic drug monitoring. Innovative trends such as electrophoretic stacking, microdialysis, electromembrane extraction, portable and disposable CE instruments and minimally invasive clinical sampling techniques are mentioned. A critical evaluation of the positives and negatives of this technique is presented, covering the main applications published over the last 10 years.

Sensitive monitoring of 3-hydroxybutyrate as an indicator of human fasting by capillary electrophoresis in a PAMAMPS coated capillary.

Sensitive electrophoretic determination of 3-hydroxybutyrate (3HB) as an indicator of human ketogenesis is performed in fused silica capillary covalently coated by an anionic copolymer of poly(acrylamide-co-sodium-2-acrylamido-2-methylpropanesulphonate) (PAMAMPS). Baseline separation of 3HB from other components of human serum is achieved in a 20 µm capillary with an effective length of 17 cm covered by 4% PAMAMPS, which generates a cathodic EOF with a mobility of 8.30 ± 0.00 · 10-9 m2/V.s in 80 mM MES/His as background electrolyte. 3HB migrates in counter-current electrophoretic mode against EOF, that effectively improving electrophoretic resolution. Sample pre-treatment is based on adding of 45 µL acetonitrile to 15 µL serum and, after shaking, a 28 mm long zone of supernatant is injected into the capillary, and sharpened after turning on a separation voltage of 20 kV using the technique of large volume sample stacking, where the EOF forces the residual acetonitrile from the capillary. When combined with universal contactless conductivity detection, the achieved LOD and LOQ are 0.43 µM and 1.44 µM, respectively, that are sufficiently low for monitoring the physiological 3HB level. The performed clinical study subsequently showed that serum 3HB increases from a concentration of 71 µM, corresponding to normal food, to level of 1924 µM after 60 h of fasting and returns to the normal physiological concentration 48 h after commencing consumption of high-saccharide food.

Monitoring of amoxicilline and ceftazidime in the microdialysate of diabetic foot and serum by capillary electrophoresis with contactless conductivity detection.

Determination of the broad-spectrum antibiotics amoxicilline (AMX) and ceftazidime (CTZ) in blood serum and microdialysates of the subcutaneous tissue of the lower limbs is performed using CE with contactless conductivity detection (C4 D). Baseline separation of AMX is achieved in 0.5 M acetic acid as the background electrolyte and separation of CTZ in 3.2 M acetic acid with addition of 13% v/v methanol. The CE-C4 D determination is performed in a 25 µm capillary with suppression of the EOF using INST-coating on an effective length of 18 cm and the attained migration time is 4.2 min for AMX and 4.4 min for CTZ. The analysis was performed using 20 µl of serum and 15 µl of microdialysate, treated by the addition of acetonitrile in a ratio of 1/3 v/v and the sample is injected into the capillary using the large volume sample stacking technique. The LOQ attained in the microdialysate is 148 ng/ml for AMX and 339 ng/ml for CTZ, and in serum 143 ng/ml for AMX and 318 ng/ml for CTZ. The CE-C4 D method is employed for monitoring the passage of AMX and CTZ from the blood circulatory system into the subcutaneous tissue at the sites of diabetic ulceration in patients suffering from diabetic foot syndrome and also for measuring the pharmacokinetics following intravenous application of bolus antibiotic doses.

Capillary and microchip electrophoresis with contactless conductivity detection for analysis of foodstuffs and beverages.

The paper provides a comprehensive survey of the use of capillary and microchip electrophoresis in combination with contactless conductivity detection (C4D) for the analysis of drinking water, beverages and foodstuffs. The introduction sets forth the fundamentals of conductivity detection anddescribes an axialC4Dversion. There is also a detailed discussion of the determination of inorganic ions, organic acids, fatty acids, amino acids, amines, carbohydrates, foreign substances and poisons from the standpoint of separation conditions, sample treatment and detection limits. Special attention is paid to the analysis of foodstuffs at microchips with emphasis on the employed material and connection of the microchip with the C4D. The review attempts to draw attention to modern trends, such as dual-opposite injection, field-enhanced sample injection, electromembrane extraction and on-line combination of microdialysis with CE. CE/C4D is characterised by high universality, high speed of analysis, simple sample preparation, small consumption of sample and other chemicals.

Monitoring of circulating amino acids in patients with pancreatic cancer and cancer cachexia using capillary electrophoresis and contactless conductivity detection.

Branched chain amino acids (BCAAs), alanine and glutamine are determined in human plasma by capillary electrophoresis with contactless conductivity detection (CE/C4 D). The baseline separation of five amino acids from other plasma components is achieved on the short capillary effective length of 18 cm in 3.2 mol/L acetic acid with addition of 13% v/v methanol as background electrolyte. Migration times range from 2.01 min for valine to 2.84 min for glutamine, and LODs for untreated plasma are in the interval 0.7-0.9 µmol/L. Sample treatment is based on the addition of acetonitrile to only 15 µL of plasma and supernatant is directly subjected to CE/C4 D. Circulating amino acids are measured in patients with pancreatic cancer and cancer cachexia during oral glucose tolerance test. It is shown that patients with pancreatic cancer and cancer cachexia syndrome exhibit low basal circulating BCAAs and glutamine levels and loss of their insulin-dependent suppression.

Electrophoretic Determination of Symmetric and Asymmetric Dimethylarginine in Human Blood Plasma with Whole Capillary Sample Injection.

Asymmetric and symmetric dimethylarginines are toxic non-coded amino acids. They are formed by post-translational modifications and play multifunctional roles in some human diseases. Their determination in human blood plasma is performed using capillary electrophoresis with contactless conductivity detection. The separations are performed in a capillary covered with covalently bonded PAMAPTAC polymer, which generates anionic electroosmotic flow and the separation takes place in the counter-current regime. The background electrolyte is a 750 mM aqueous solution of acetic acid with pH 2.45. The plasma samples for analysis are treated by the addition of acetonitrile and injected into the capillary in a large volume, reaching 94.5% of the total volume of the capillary, and subsequently subjected to electrophoretic stacking. The attained LODs are 16 nm for ADMA and 22 nM for SDMA. The electrophoretic resolution of both isomers has a value of 5.3. The developed method is sufficiently sensitive for the determination of plasmatic levels of ADMA and SDMA. The determination does not require derivatization and the individual steps in the electrophoretic stacking are fully automated. The determined plasmatic levels for healthy individuals vary in the range 0.36-0.62 µM for ADMA and 0.32-0.70 µM for SDMA.

Characterization of various geometric arrangements of "air-assisted" flow gating interfaces for capillary electrophoresis.

For connecting flow-through analytical methods with capillary electrophoresis, a chip working in the air-assisted flow gating interface regime is cast from poly(dimethylsiloxane). In the injection space, the exit from the delivery capillary is placed close to the entrance to the separation capillary. Prior to injecting the sample into the separation capillary, the background electrolyte is forced out of the injection space by a stream of air. In the empty space, a drop of the sample with a volume of <100 nL is formed between the exit from the delivery capillary and the entrance into the separation capillary, from which the sample is injected hydrodynamically into the separation capillary. After injection, the injection space is filled with BGE, and the separation can be begun. Three geometric variants for the mutual geometric arrangement of the delivery and separation capillaries were tested: the delivery capillary is placed perpendicular to the separation capillary, from either above or below, or the capillaries are placed axially, that is, directly opposite one another. All of the variants are equivalent from the analytical and separation efficiency viewpoints. The repeatability expressed by RSD is up to 5%. The tested flow gating interface variants are also suitable for continuous and discontinuous sampling at flow rates of the order of units of µL/min. The developed instrument for sequential electrophoretic analysis operates fully automatically and is suitable for rapid sequential monitoring of dynamic processes.

Large volume sample stacking of antiepileptic drugs in counter current electrophoresis performed in PAMAPTAC coated capillary.

Electrophoretic stacking is developed for sensitive determination of three zwitterionic antiepileptics, namely vigabatrin, pregabalin and gabapentin, in human serum. CE separation is performed in a 25 µm fused silica capillary covalently coated with the copolymer of acrylamide with 5% content of permanently charged 3-acrylamidopropyl trimethylammonium chloride (PAMAPTAC). In background electrolyte of 500 mM acetic acid, the 5% PAMAPTAC generates an anodic electro-osmotic flow with a magnitude of (-18.6 ± 0.5) · 10-9 m2V-1s-1, which acts against the direction of the electrophoretic migration of the analytes. A sample of the antiepileptic prepared in a 25% v/v infusion solution and 75% v/v acetonitrile is injected into the capillary in a large volume attaining a zone length of up to 270 mm. After turning on the separation voltage, the antiepileptics are isotachophoretically focussed behind the zone of Na+ ions with a sensitivity enhancement factor of 78. For the clinical determination of antiepileptics, the human serum is diluted with acetonitrile in a ratio of 1:3 v/v and a zone with a length of 90 mm is injected into the capillary. The method is linear in the 0.025-2.5 µg/mL concentration range; the attained limit of quantification is in the range 18.3-22.8 nmol/L; the within-day precision for the migration time is 0.8-1.2% and for the peak area 1.5-2.4%.

Determination of amino acids by capillary and microchip electrophoresis with contactless conductivity detection - Theory, instrumentation and applications.

This review article summarises aspects of the determination of amino acids using capillary and chip electrophoresis in combination with contactless conductivity detection from their historical beginnings to the present time. Discussion is included of the theory of conductivity detection in electromigration techniques, the design of contactless conductivity cells for detection in capillaries and on microchips, including the use of computer programs for simulation of the conductivity response and the process of the electrophoretic separation of amino acids. Emphasis is placed on optimisation of the background electrolyte composition, chiral separation, multidimensional separation, stacking techniques and the use of multidetection systems. There is also a description of clinical applications, the determination of amino acids in foodstuffs, waters, soils and composts with emphasis on modern techniques of sample treatment, such as microdialysis, liquid membrane extraction and many other techniques.

Modified Strategies for Invasive Management of Acute Coronary Syndrome during the COVID-19 Pandemic.

The COVID-19 pandemic presents several challenges for managing patients with acute coronary syndrome (ACS). Modified treatment algorithms have been proposed for the pandemic. We assessed new algorithms proposed by The European Association of Percutaneous Cardiovascular Interventions (EAPCI) and the Acute Cardiovascular Care Association (ACCA) on patients with ACS admitted to the hospital during the COVID-19 pandemic. The COVID-19 period group (CPG) consisted of patients admitted into a high-volume centre in Prague between 1 February 2020 and 30 May 2020 (n = 181). The reference group (RG) included patients who had been admitted between 1 October 2018 and 31 January 2020 (n = 834). The proportions of patients with different types of ACS admitted before and during the pandemic did not differ significantly: in all ACS patients, KILLIP III-IV class was present in 13.9% in RG and in 9.4% of patients in CPG (p = 0.082). In NSTE-ACS patients, the ejection fraction was lower in the CPG than in the RG (44.7% vs. 50.7%, respectively; p < 0.001). The time from symptom onset to first medical contact did not differ between CPG and RG patients in the respective NSTE-ACS and STEMI groups. The time to early invasive treatment in NSTE-ACS patients and the time to reperfusion in STEMI patients were not significantly different between the RG and the CPG. In-hospital mortality did not differ between the groups in NSTE-ACS patients (odds ratio in the CPG 0.853, 95% confidence interval (CI) 0.247 to 2.951; p = 0.960) nor in STEMI patients (odds ratio in CPG 1.248, 95% CI 0.566 to 2.749; p = 0.735). Modified treatment strategies for ACS during the COVID-19 pandemic did not cause treatment delays. Hospital mortality did not differ.

Dialysis of one sample drop on-line connected with electrophoresis in short capillary.

An analytical apparatus is described, based on on-line connection of electrophoresis in a short capillary with a dialysis unit enabling dialysis in micro-litre sample volumes into submicro-litre volumes of an acceptor solution in a dialysing fibre. After a defined dialysis time, the dialysate from the dialysing fibre is injected into a separation capillary through an air-assisted flow-gating interface cast from PDMS. In the flow-gating injection space, the exit from the delivery capillary bringing the dialysate is placed directly opposite the entrance into the separation capillary at a distance of 380 µm. In order to enable injection of a very small volume of dialysate, the background electrolyte is forced out of the injection space with air before the injection, so that a drop of dialysate with a volume of about 0.1 µL is formed between the exit from the delivery and the entrance into the separation capillary; the dialysate is injected hydrodynamically from this dialysate drop. Then the injection space is filled with the background electrolyte and the separation is commenced. The basic properties of the apparatus were tested on model mixtures of inorganic cations (K+, Ba2+ and Na+) and organic molecules (creatinine, histidine and arginine). The applicability to real samples was tested on the determination of basic amino acids (histidine, lysine and arginine) in a blood serum sample.

Effect of Food with Low Enrichment of N-3 Fatty Acids in a Two-Month Diet on the Fatty Acid Content in the Plasma and Erythrocytes and on Cardiovascular Risk Markers in Healthy Young Men.

Polyunsaturated fatty acids of the n-3 series (n-3 PUFA) exhibit a number of favorable effects on the human organism and it is desirable to increase their intake in the diet. For this purpose, flaxseed oil was added to a chicken-feed mixture for the production of meat and eggs. The content of n-3 PUFA in the obtained meat was increased from 250 mg (reference value) to 900 mg in 100 g of meat and from 110 mg (reference value) to 190 mg in 100 g of whole egg; the enriched products are designated as omega-3 meat and omega-3 eggs. Omega-3 meat and eggs were subsequently fed for a period of eight weeks in an amount of 480 g of meat and four eggs (228 g netto) a week to a group of 14 healthy volunteers, whose body composition parameters were measured and blood was analyzed biochemically to determine blood lipids, coagulation parameters, plasma, and erythrocyte fatty acid spectrum composition. A control group of 14 volunteers was fed normal chicken and eggs in the same regime. The performed dietary intervention increases the intake of long-chain PUFA (LC-PUFA) by 37 mg per day, which represents 7-15% of the recommended daily dose. The performed tests demonstrated that the consumption of omega-3 enriched meat and eggs significantly increases the content of n-3 PUFA in the erythrocytes, which are a long-term indicator of fatty acid intake. This intervention has no demonstrable effect on the basic body parameters, such as body weight, fat content, Body Mass Index (BMI), and also on the plasma cholesterol level, high-density lipoprotein (HDL), low-density lipoprotein (LDL), blood clotting and inflammation markers, and omega-3 index.

Sensitive CE-MS method for monitoring of riociguat and desmethylriociguat levels in human serum.

Riociguat is novel antihypertensive drug for treatment of pulmonary hypertension. As such, it is still being tested in many clinical and pharmacokinetic trials. Existing methods that determine serum riociguat and desmethylriociguat (DMR) are based solely on liquid chromatography with mass spectrometry. Therefore, we present a novel capillary electrophoresis with mass spectrometry method (CE-MS) for their determination in human serum as alternative method for ongoing trials. Complete resolution of both analytes was achieved by means of pH optimization of ammonium formate background electrolytes that are fully compatible with ESI/MS detection. Simple liquid-liquid extraction was used as sample pretreatment. The calibration dependence of the method was linear (in the range of 10-1000 ng/mL), with adequate accuracy (90.1-114.9%) and precision (13.4%). LOD and LOQ were arbitrarily set at 10 ng/mL for both analytes. Clinical applicability was validated using serum samples from patients treated with riociguat in pharmacokinetic study and the results corresponded with reference HPLC-MS/MS values. Capillary electrophoresis proved to be sensitive and selective tool for the analysis of riociguat and DMR.