At-line coupling of hollow fiber liquid-phase microextraction to capillary electrophoresis for trace determination of acidic drugs in complex samples.

Direct analysis of complex samples is demonstrated by the at-line coupling of hollow fiber liquid-phase microextraction (HF-LPME) to capillary electrophoresis (CE). The hyphenation of the preparative and the analytical technique is achieved through a 3D-printed microextraction device with an HF located in a sample vial of a commercial CE instrument. The internal geometry of the device guides the CE separation capillary into the HF and the CE injection of the HF-LPME extract is performed directly from the HF lumen. The 3D-printing process ensures uniform dimensions of the devices, their constant position inside the sample vial, and excellent repeatability of the HF-LPME as well as the CE injection. The devices are cheap (∼0.01 €) and disposable, thus eliminating any possible sample-carryover, moreover, the at-line CE analysis of the extract is performed fully autonomously with no need for operator's intervention. The developed HF-LPME/CE-UV method is applied to the determination of acidic drugs in dried blood spot and wastewater samples and is characterized by excellent repeatability (RSD, 0.6-9.6%), linearity (r2, 0.9991-0.9999), enrichment (EF, 29-97), sensitivity (LOD, 0.2-3.4 µg/L), and sample throughput (7 samples/h). A further improvement of selected characteristics of the analytical method is achieved by the at-line coupling of HF-LPME to capillary isotachophoresis (ITP) with electrospray ionization-mass spectrometry (ESI-MS). The HF-LPME/ITP-ESI-MS system facilitates enhanced selectivity, matrix-free analytical signals, and up to 34-fold better sensitivity due to the use of ESI-MS detection and additional on-capillary ITP preconcentration of the HF-LPME extracts.

Hollow Fiber Liquid-Phase Microextraction At-Line Coupled to Capillary Electrophoresis for Direct Analysis of Human Body Fluids.

A simple and cheap all-in-one concept for at-line coupling of hollow fiber liquid-phase microextraction (HF-LPME) to commercial capillary electrophoresis (CE) is demonstrated, which enables the direct analysis of complex samples. A disposable microextraction device compatible with injection systems of Agilent CE instruments is proposed, which consists of a short segment of a porous HF attached to a tapered polypropylene holder. The holder maintains a constant position of the HF in a CE vial during extraction and simultaneously guides the injection end of a separation capillary into the HF lumen for automated CE injection and analysis. In a typical analytical procedure, the HF is impregnated with a water-immiscible solvent, its lumen is filled with 5 µL of an aqueous acceptor solution, and the microextraction device is placed in a 2 mL glass CE vial containing 550 µL of a donor solution. The vial is agitated at 750 rpm for 10 min, and the resulting acceptor solution is injected directly from the HF lumen into the commercial CE. No additional manual handling is required, except for the transfer of the CE vial to the CE autosampler. Multiple complex samples can be simultaneously pretreated in a multiple-well plate format, thus significantly reducing the total analysis time. Suitability of the analytical method is demonstrated by the direct determination of model basic drugs (nortriptyline, haloperidol, loperamide, and papaverine) in physiological solutions, urine, and dried blood spot (DBS) samples. Repeatability of the method is better than 12.8% (%RSD), extraction recoveries range between 34 and 76%, and enrichment factors are 37-84. The method is linear in a range of 2 orders of magnitude (R2 ≥ 0.9977) with limits of detection of 0.7-1.55 µg/L. The method has a high potential for the direct analysis of DBS samples since DBS elution and HF-LPME are performed simultaneously during the 10 min agitation. The manual DBS handling is thus reduced to inserting the DBS punch into the CE vial only. Moreover, the universal character of the HF-LPME might extend the applicability of the method to a wide range of analytes/matrices, and combination with other commercial detectors might improve the selectivity/sensitivity of the CE analysis.

Evaluation of IFN-γ Enzyme-linked Immunospot Assay (ELISPOT) as a First-line Test in the Diagnosis of Non-Immediate Hypersensitivity to Amoxicillin and Penicillin.

The current diagnostic algorithm for beta-lactam allergy is based on skin and provocation tests, both of which carry a certain risk of inducing hypersensitivity reactions. Thus, non-invasive in vitro tests reliable enough to replace skin and provocation tests at least in a portion of patients are desirable. We aimed to verify the utility of IFN-γ ELISPOT as a first-line test in patients with suspected non-immediate hypersensitivity reaction to amoxicillin (AMX) and penicillin (PNC). The prospective observational study included 24 patients with recent, suspected non-immediate hypersensitivity reaction to AMX or PNC and 6 recently-exposed healthy subjects. In vitro tests were performed in all patients and healthy subjects: a) IFN-γ ELISPOT with PNC, AMX and amoxicillin plus clavulanic acid (AMX-CL); b) penicillin specific IgE; c) basophil activation test (BAT). Skin and provocation tests followed only in certain patients. IFN-γ ELISPOT results with PNC and AMX stimulation did not differ from the unstimulated condition. The highest IFN-γ responses to AMX-CL were close to previously published criteria in three patients; one of which had true hypersensitivity according to drug provocation tests. Five patients with confirmed hypersensitivity by skin tests showed no response to the culprit antibiotic on IFN-γ ELISPOT assay. Our results did not support the utility of IFN-γ ELISPOT in the diagnosis of mild, non-immediate hypersensitivity to amoxicillin and penicillin.

Placental growth factor may predict increased left ventricular mass index in patients with mild to moderate chronic kidney disease--a prospective observational study.

BACKGROUND: Placental growth factor [PlGF) is a cardiovascular (CV) risk marker, which is related to left ventricle hypertrophy (LVH) in animal models. Currently there are no data available regarding the possible relationship of PlGF and the development of LVH or diastolic dysfunction in patients with chronic kidney disease (CKD) and the relationship of PlGF to other CV risk factors in CKD patients. The aim of our study was to determine the possible association of PlGF and several other CV risk markers to echocardiographic parameters in CKD population. METHODS: We prospectively examined selected laboratory (PlGF, fibroblast growth factor-23 -FGF23, vitamin D, parathyroid hormone, extracellular newly identified RAGE-binding protein - EN-RAGE, B-type natriuretic peptide - BNP) and echocardiographic parameters in 62 patients with CKD 2-4. Mean follow-up was 36 ±10 months. Laboratory and echocardiographic data were collected 2-3 times, at the shortest interval of 12 months apart. Multivariate regression analysis was used to detect independent correlations of variables. RESULTS: Increased left ventricular mass index (LVMI, g/m2.7) was found in 29% patients with CKD 2-4, left ventricular (LV) diastolic dysfunction was detected in 74.1% patients (impaired LV relaxation in 43.5% patients and pseudonormal pattern in 30.6% patients). After 36 ± 10 months increased LVMI was found in 37.1% patients with CKD 2-4, LV diastolic dysfunction was detected in 75.8% patients (impaired LV relaxation in 43.5% patients and pseudonormal pattern in 32.3% patients). Following independent correlations were found: LVMI was related to PlGF, cholesterol, BNP, systolic blood pressure and serum creatinine. EN-RAGE correlated positively with left atrial diameter and inversely with E/A ratio. During the follow-up we found a significant increase in LVMI and left atrial diameter, whereas a significant decrease in LVEF was noted. CONCLUSION: According to our data, PlGF is independently related to increased LV mass in CKD, whereas EN-RAGE is more likely related to diastolic dysfunction in this population.

Pregnancy-associated plasma protein A (PAPP-A) and soluble receptor for advanced glycation end products (sRAGE)--intra- and inter-individual variability in chronic hemodialysis patients.

BACKGROUND: Dialysis patients are at high risk of cardiovascular complications. Pregnancy-associated plasma protein A (PAPP-A) as well as sRAGE (soluble receptor for advanced glycation end products) are new biomarkers related to cardiovascular disease. The aim of our study was to describe their intra- and inter-individual variability. METHODS: The studied group consisted of 21 chronic hemodialysis patients. PAPP-A, sRAGE and selected routine parameters were measured monthly during a 1-year prospective study. RESULTS: Our results show high intra-individual variability of both PAPP-A and sRAGE. Both PAPP-A and sRAGE were closely linked to serum transferrin levels. Additionally, sRAGE was significantly associated with leukocyte count and haemoglobin. CONCLUSION: Our study demonstrates high intra-individual variability of PAPP-A and sRAGE in stable clinical status. This finding could be helpful for further evaluation of the significance of PAPP-A and sRAGE in chronic kidney disease.

Genetic analysis of metabolic defects in the spontaneously hypertensive rat.

Abnormalities in carbohydrate and lipid metabolism are common in patients with essential hypertension and in the spontaneously hypertensive rat (SHR). To identify chromosome regions contributing to this clustering of cardiovascular risk factors in the SHR, we searched for quantitative trait loci (QTL) associated with insulin resistance, glucose intolerance, and dyslipidemia by using the HXB/BXH recombinant inbred (RI) strains. Analysis of variance in RI strains suggested significant effects of genetic factors. A genome screening of the RI strains with more than 700 markers revealed QTL significantly associated with insulin resistance on Chromosomes (Chrs) 3 and 19. The Chr 19 QTL was confirmed by testing a previously derived SHR-19 congenic strain: transfer of a Chr 19 segment delineated by markers D19Rat57 and D19Mit7 from the Brown Norway (BN/Cr) strain onto the genetic background of the SHR/Ola was associated with decreased insulin and glucose concentrations and ameliorated insulin resistance at the tissue level. These findings suggest that closely linked genes on Chr 19, or perhaps even a single gene with pleiotropic effects, influence the clustering of metabolic disturbances in the SHR-BN model.