Publications by authors named "Rafal Głowacki"

62 Publications

Gas Chromatography-Mass Spectrometry Based Approach for the Determination of Methionine-Related Sulfur-Containing Compounds in Human Saliva.

Int J Mol Sci 2020 Dec 4;21(23). Epub 2020 Dec 4.

Department of Environmental Chemistry, Faculty of Chemistry, University of Lodz, 163 Pomorska Str., 90-236 Łódź, Poland.

Gas chromatography-mass spectrometry technique (GC-MS) is mainly recognized as a tool of first choice when volatile compounds are determined. Here, we provide the credible evidence that its application in analysis can be extended to non-volatile sulfur-containing compounds, to which methionine (Met), homocysteine (Hcy), homocysteine thiolactone (HTL), and cysteine (Cys) belong. To prove this point, the first method, based on GC-MS, for the identification and quantification of Met-related compounds in human saliva, has been elaborated. The assay involves simultaneous disulfides reduction with tris(2-carboxyethyl)phosphine (TCEP) and acetonitrile (MeCN) deproteinization, followed by preconcentration by drying under vacuum and treatment of the residue with a derivatizing mixture containing anhydrous pyridine, -trimethylsilyl--methyl trifluoroacetamide (MSTFA), and trimethylchlorosilane (TMCS). The validity of the method was demonstrated based upon US FDA recommendations. The assay linearity was observed over the range of 0.5-20 µmol L for Met, Hcy, Cys, and 1-20 µmol L for HTL in saliva. The limit of quantification (LOQ) equals 0.1 µmol L for Met, Hcy, Cys, while its value for HTL was 0.05 µmol L. The method was successfully applied to saliva samples donated by apparently healthy volunteers ( = 10).
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http://dx.doi.org/10.3390/ijms21239252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7729597PMC
December 2020

Application of High-Performance Liquid Chromatography for Simultaneous Determination of Tenofovir and Creatinine in Human Urine and Plasma Samples.

Pharmaceuticals (Basel) 2020 Nov 5;13(11). Epub 2020 Nov 5.

Department of Environmental Chemistry, Faculty of Chemistry, University of Lodz, 163 Pomorska Str., 90-236 Łódź, Poland.

Tenofovir disoproxil fumarate is widely used in the therapy of human immunodeficiency virus and hepatitis B virus; however, a high concentration of the prodrug effects kidney function damage. To control the effectiveness of kidney functions in treated patients, the level of creatinine in the body must be controlled. This work describes a simple, fast, and "plastic-waste" reducing method for the simultaneous determination of tenofovir and creatinine in human urine and plasma. In both assays, only 50 µL of body fluid was required. The tests were carried out by reversed phase high-performance liquid chromatography with UV detection. In urine samples, the limits of detection for tenofovir and creatinine were 4 µg mL and 0.03 µmol mL, respectively. In plasma samples, the limits of detection were 0.15 µg mL for tenofovir and 0.0003 µmol mL for creatinine. The method was applied for the determination of tenofovir and creatinine in human urine and plasma samples. The biggest advantage of the elaborated method is the possibility to determine tenofovir and creatinine in one analytical run in both urine and plasma sample collected from HIV and HBV patients. The possibility to reduce the level of laboratory waste in a sample preparation protocol is in the mainstream of a new trend of analytical chemistry which is based on green chemistry.
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http://dx.doi.org/10.3390/ph13110367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694483PMC
November 2020

Quantification of homocysteine thiolactone in human saliva and urine by gas chromatography-mass spectrometry.

J Chromatogr B Analyt Technol Biomed Life Sci 2020 Jul 15;1149:122155. Epub 2020 May 15.

Department of Environmental Chemistry, University of Lodz, Faculty of Chemistry, Łódź, Poland. Electronic address:

Homocysteine thiolactone (HTL) is a chemically reactive thioester that has been implicated in cardiovascular disease. So far, its presence has been documented in human and mouse plasma and urine. Here, using a new method, we show that HTL is present in human saliva. The assay involves chloroform-methanol extraction of HTL, lyophilization, and derivatization with N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA) and trimethylchlorosilane (TMCS). The method is based on a gas chromatography coupled with mass spectrometry (GC-MS) and quantifies HTL in a linear range from 0.05 to 1 µmol L saliva and urine. The limit of quantification (LOQ) was 0.05 µmol L. With respect to saliva specimen, the accuracy was 98.7-112.6%, and 90.2-100.5%, while the precision was 7.1-13.5% and 12.5-15.0% for the intra- and inter-day variation, respectively. In relation to urine samples, the accuracy was 91.9-110.9% and 91.2-103.3%, while the precision varied from 2.2% to 14.5% and 7.4% to 14.3% for intra- and inter-day measurements, respectively. Using this method, we show that in apparently healthy individuals (n = 18), HTL levels in saliva are not positively correlated with urinary HTL levels. Undoubtedly, larger population should be investigated to get more meaningful results.
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http://dx.doi.org/10.1016/j.jchromb.2020.122155DOI Listing
July 2020

2-(3-Hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic Acid, Novel Metabolite of Pyridoxal 5'-Phosphate and Cysteine Is Present in Human Plasma-Chromatographic Investigations.

Int J Mol Sci 2020 May 18;21(10). Epub 2020 May 18.

Department of Environmental Chemistry, Faculty of Chemistry, University of Lodz, 163 Pomorska Str., 90-236 Łódź, Poland.

It is well-established that aminothiols, to which cysteine (Cys) belongs, are highly reactive towards aldehydes in an aqueous environment, forming substituted thiazolidine carboxylic acids. This report provides evidence that formation of the product containing a thiazolidine ring through non-enzymatic condensation of Cys and an active form of vitamin B6 pyridoxal 5'-phosphate (PLP) occurs in vivo in humans. To prove this point, a new method, based on a gas chromatography coupled with mass spectrometry (GC-MS), has been designed to identify and quantify Cys and PLP adduct, 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid (HPPTCA) in human plasma. The GC-MS assay relies on sample deproteinization by ultrafiltration over cut-off membranes and preconcentration by drying under vacuum, followed by treatment of the residue with derivatization mixture containing anhydrous pyridine, N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA) and trimethylchlorosilane (TMCS). The method quantifies HPPTCA in a linear range from 1 to 20 µmol L, where the lowest standard on the calibration curve refers to the limit of quantification (LOQ). The validity of the method was demonstrated. Furthermore, the method was successfully applied to plasma samples donated by apparently healthy volunteers and breast cancer patients. The GC-MS assay provides a new tool that will hopefully facilitate studies on the role of HPPTCA in living systems.
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http://dx.doi.org/10.3390/ijms21103548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278924PMC
May 2020

Higher Levels of Low Molecular Weight Sulfur Compounds and Homocysteine Thiolactone in the Urine of Autistic Children.

Molecules 2020 Feb 21;25(4). Epub 2020 Feb 21.

Institute of General and Ecological Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland.

In this study, the levels of concentration of homocysteine thiolactone (HTL), cysteine (Cys), and cysteinylglycine (CysGly) in the urine of autistic and non-autistic children were investigated and compared. HTL has never been analyzed in autistic children. The levels of low molecular weight sulfur compounds in the urine of both groups were determined by validated methods based on high-performance liquid chromatography with spectrofluorometric and diode-array detectors. The statistical data show a significant difference between the examined groups. Children with autism were characterized by a significantly higher level of HTL (p = 5.86 × 10), Cys (p = 1.49 × 10) and CysGly (p = 1.06 × 10) in urine compared with the control group. A difference in the p-value of <0.05 is statistically significant. Higher levels of HTL, Cys, and CysGly in the urine of 41 children with autism, aged 3 to 17, were observed. The obtained results may indicate disturbances in the metabolism of methionine, Cys, and glutathione in some autistic patients. These preliminary results suggest that further research with more rigorous designs and a large number of subjects is needed.
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http://dx.doi.org/10.3390/molecules25040973DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7070266PMC
February 2020

Determination of homocysteine thiolactone in human urine by capillary zone electrophoresis and single drop microextraction.

Anal Biochem 2020 05 21;596:113640. Epub 2020 Feb 21.

University of Lodz, Faculty of Chemistry, Department of Environmental Chemistry, Poland. Electronic address:

A simple, fast, sensitive and reproducible capillary zone electrophoresis (CZE) method with single drop microextraction (SDME) for determination of homocysteine thiolactone (HTL) in human urine has been developed and validated. The method is characterized by good precision, high accuracy, short analysis time and low consumption of reagents. The procedure consists only of few steps: urine sample centrifugation, dilution with phosphate buffer and methanol, chloroform addition onto the top of donor phase, on-line SDME in CE system, sample separation by CZE and ultraviolet detection of HTL at 240 nm. The background electrolyte was 0.1 M pH 4.75 phosphate buffer. Effective separation was achieved within 6.04 min under the separation voltage of 24 kV (~110 μA). The LOQ and LOD for HTL were 50 and 25 nM urine, respectively. The calibration curve in urine showed linearity in the range of 50-200 nM, with R 0.9995. The intra- and inter-day precision and recovery were 4.0-14.5% (average 8.7% and 9.3%) and 92.7-115.5% (average 103.6% and 104.8%), respectively. The procedure was successfully applied to analysis of urine samples.
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http://dx.doi.org/10.1016/j.ab.2020.113640DOI Listing
May 2020

A Simplified Method for Simultaneous Determination of α-Lipoic Acid and Low-Molecular-Mass Thiols in Human Plasma.

Int J Mol Sci 2020 Feb 5;21(3). Epub 2020 Feb 5.

Department of Environmental Chemistry, Faculty of Chemistry, University of Lodz, Pomorska 163, 90-236 Łódź, Poland.

α-Lipoic acid, glutathione, cysteine, and cysteinylglycine can be applied as therapeutic agents in civilization diseases such as diabetes mellitus, cardiovascular diseases, and cancers. On the other hand, a higher concentration of homocysteine can result in health problems and has been indicated as an independent risk factor for cardiovascular disease and accelerated atherosclerosis. Here, the first simplified HPLC-UV assay that enables simultaneous determination of α-lipoic acid and low-molecular-mass thiols in plasma, reduces the number of steps, shortens the total time of sample preparation, and limits the amount of single-use polypropylene laboratory materials is described. The assay is based on reversed-phase high performance liquid chromatography with UV detection and simultaneous reduction of disulfide bound with (2-carboxyethyl)phosphine and the selective pre-column derivatization of the thiol group with 1-benzyl-2-chloropyridinium bromide. Linearity in the detector responses for plasma samples were observed in ranges: 0.12-5.0 nmol mL for α-lipoic acid; 2.0-20.0 nmol mL for glutathione, cysteinylglycine, and homocysteine; and 40.0-400.0 for cysteine. The LODs for α-lipoic acid and low-molecular-mass thiols were 0.08 and 0.12 nmol mL, respectively, while LOQs were 0.12 and 0.16 nmol mL, respectively. The usefulness of the proposed method has been proven by its application to real samples.
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http://dx.doi.org/10.3390/ijms21031049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037620PMC
February 2020

Rapid electroanalytical procedure for sesamol determination in real samples.

Food Chem 2020 Mar 25;309:125789. Epub 2019 Oct 25.

University of Lodz, Department of Inorganic and Analytical Chemistry, 12 Tamka Str, Lodz 91-403, Poland. Electronic address:

In this study, the development of an electroanalytical assay based on square wave voltammetry technique for determining sesamol (Ses) in sesame oil samples is described. The influence of various factors such as pH of the supporting electrolyte, its composition, and SW (square wave) parameters was studied. Linearity of the peak current depended on the concentration of Ses in the range from 3.0 to 140.0 μmol L with a limit of detection of 0.71 μmol L. Furthermore, the cyclic voltammetric behavior of Ses and the effects of scan rate and pH on the peak current and peak potential of Ses were determined. Moreover, the electrode process was found to be diffusion-controlled. The proposed methodology was successfully applied for determining Ses in commercial sesame oil samples. The obtained results were in good agreement with the results from the HPLC-UV reference method.
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http://dx.doi.org/10.1016/j.foodchem.2019.125789DOI Listing
March 2020

The first method for determination of lipoyllysine in human urine after oral lipoic acid supplementation.

Bioanalysis 2019 Jul 1;11(14):1359-1373. Epub 2019 Aug 1.

University of Lodz, Faculty of Chemistry, Department of Environmental Chemistry, 163 Pomorska Str., 90-236 Łódź, Poland.

The first method on urinary excreted amounts of lipoyllysine (LLys) after lipoic acid (LA) supplementation was developed and validated. The suggested procedure allowed simultaneous determination of LLys and LA. After the conversion of analytes into their reduced forms with tris(2-carboxyethyl)phosphine and derivatization via thiol group with 1-benzyl-2-chloropyridinium bromide, separation of analytes derivatives was performed on C18 column using a gradient mobile phase consisting of acetic acid and acetonitrile. The calibration curves for LA and LLys were linear (R > 0.999) in the range of 0.4-12 μM concentration and all validation results were acceptable, according to the US FDA bioanalytical method guidelines. This method was effectively applied for LA and LLys quantification in human urine after oral LA supplementation.
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http://dx.doi.org/10.4155/bio-2019-0011DOI Listing
July 2019

Application of Butylamine as a Conjugative Reagent to On-Column Derivatization for the Determination of Antioxidant Amino Acids in Brain Tissue, Plasma, and Urine Samples.

Int J Mol Sci 2019 Jul 7;20(13). Epub 2019 Jul 7.

Department of Environmental Chemistry, Faculty of Chemistry, University of Łódź, 163 Pomorska Street, 90-236 Łódź, Poland.

(1) Antioxidants are involved in body protection mechanisms against reactive oxygen species. Amino acids such as glutathione (GSH) and -acetylcysteine (NAC) are known to be involved in providing protection against oxidative lethality. A quick and simple method for the determination of NAC and GSH in various biological matrices such as urine, plasma, and homogenates of brain tissues has been developed and described in this work. (2) The assay is based on reversed phase high performance liquid chromatography with spectrofluorimetric detection and on-column derivatization. Butylamine and -phthaldialdehyde have been used as derivatization reagents. Since -phthaldialdehyde constitutes a part of the mobile phase, the derivatization reaction and chromatographic separation occur simultaneously. (3) Linearity in the detector response for NAC in human urine was observed in the range of 5-200 nmol mL, and NAC and GSH in the brain tissue homogenates were observed in the range of 0.5-5 nmol mL and 0.5-15 nmol mL, respectively. Human plasma linearity ranges covered 0.25-5.00 nmol mL and 0.5-15 nmol mL for NAC and GSH, respectively. The LODs for NAC and GSH were 0.01 and 0.02 nmol mL while the LOQs were 0.02 and 0.05 nmol mL, respectively. The usefulness of the proposed method was proven through its application to real samples.
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http://dx.doi.org/10.3390/ijms20133340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6651812PMC
July 2019

Determination of nikethamide by micellar electrokinetic chromatography.

Biomed Chromatogr 2019 Oct 25;33(10):e4571. Epub 2019 Jun 25.

Department of Environmental Chemistry, Faculty of Łódź, University of Łódź, Łódź, Poland.

A simple, fast, sensitive and reproducible micellar electrokinetic chromatography (MEKC)-UV method for the determination of nikethamide (NKD) in human urine and pharmaceutical formulation has been developed and validated. The method exhibits high trueness, good precision, short analysis time and low reagent consumption. NKD is an organic compound belonging to the psychoactive stimulants used as an analeptic drugs. The proposed analytical procedure consists of few steps: dilution of urine or drug in distilled water, centrifugation for 2 min (12,000g), separation by MEKC and ultraviolet-absorbance detection of NKD at 260 nm. The background electrolyte used was 0.035 mol/L pH 9 borate buffer with the addition of 0.05 mol/L sodium dodecyl sulfate and 6.5% ACN. Effective separation was achieved within 5.5 min under a voltage of 21 kV (~90 μA) using a standard fused-silica capillary (effective length 51 cm, 75 μm i.d.). The determined limit of detection for NKD in urine was 1 μmol/L (0.18 μg/mL). The calibration curve obtained for NKD in urine showed linearity in the range 4-280 μmol/L (0.71-49.90 μg/mL), with R 0.9998. The RSD of the points of the calibration curve varied from 5.4 to 9.5%. The analytical procedure was successfully applied to analysis of pharmaceutical formulation and spiked urine samples from healthy volunteers.
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http://dx.doi.org/10.1002/bmc.4571DOI Listing
October 2019

Microvascular circulatory dysregulation driven in part by cystathionine gamma-lyase: A new paradigm for cardiovascular compromise in the preterm newborn.

Microcirculation 2019 02 30;26(2):e12507. Epub 2018 Oct 30.

Illawarra Health and Medical Research Institute, Wollongong, New South Wales, Australia.

Objective: H S may explain the dysregulation of microvascular tone associated with poor outcome following preterm birth. In adult vasculature, H S is predominantly produced by CSE. We hypothesized that vascular CSE activity contributes to microvascular tone regulation during circulatory transition.

Methods: Preterm (GA62) and full-term (GA69) guinea pig fetuses and neonates were studied. Microvascular blood flow was assessed by laser Doppler flowmetry. Thiosulfate, primary urinary metabolite of H S, was determined by high-performance liquid chromatography. Real-time H S production was assessed using a microrespiration system in fetal and postnatal (10, 24 hours) skin and heart samples. CSE contribution was investigated by inhibition via propargylglycine.

Results: In preterm animals, postnatal H S production capacity in peripheral vasculature increased significantly and was significantly reduced by the inhibition of CSE. Urinary thiosulfate correlated with both microvascular blood flow and capacity of the vasculature to produce H S. H S produced via CSE did not correlate directly with microvascular blood flow.

Conclusions: In preterm neonates, H S production increases during fetal-to-neonatal transition and CSE contribution to total H S increases postnatally. CSE-dependent mechanisms may therefore underpin the increase in H S production over the first 72 hours of life in preterm human neonates, associated with both central and peripheral cardiovascular instability.
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http://dx.doi.org/10.1111/micc.12507DOI Listing
February 2019

Application of GC-MS technique for the determination of homocysteine thiolactone in human urine.

J Chromatogr B Analyt Technol Biomed Life Sci 2018 Nov 12;1099:18-24. Epub 2018 Sep 12.

University of Łódź, Faculty of Chemistry, Department of Environmental Chemistry, 163 Pomorska Str., 90-236 Łódź, Poland. Electronic address:

It is well established that homocysteine thiolactone (HTL) is associated with some health disorders, including cardiovascular diseases. HTL is a by-product of sulfur metabolic cycle. So far, its presence has been confirmed in human plasma and urine. It has been also shown that a vast majority of HTL is removed from human body through kidney. Thus, the aim of the current investigations has been the identification, separation and quantification of HTL in urine samples. For the first time a cheap, reliable and robust GC-MS method was developed for the determination of HTL in human urine in the form of its volatile isobutyl chloroformate derivative. Separation of the analyte and internal standard (homoserine lactone (HSL)) was achieved in 15 min followed by mass spectrometry detection (MS). Isocratic elution was accomplished with helium at a flow rate of 1 mL min and a gradient of the column temperature was concomitant with the analysis. The mass spectrometer was set to the electron impact mode at 70 eV. The ion source, quadrupole and MS interface temperatures were set to 230 °C, 150 °C and 250 °C, respectively. Elaborated analytical procedure allows quantification of analyte in a linear range of 0.01-0.20 nmol mL urine. The LOQ and LOD values were 0.01 and 0.005 nmol mL, respectively. The method accuracy ranged from 98.0% to 103.2%, while precision varied from 6.4% to 9.5% and from 10.7% to 16.9% for intra- and inter-day measurements, respectively. Finally, the method has been successfully implemented in the analysis of 12 urine samples donated by apparently healthy volunteers. Concentration of HTL ranged from
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http://dx.doi.org/10.1016/j.jchromb.2018.09.009DOI Listing
November 2018

Simultaneous determination of total homocysteine, cysteine, glutathione, and N-acetylcysteine in brain homogenates by HPLC.

J Sep Sci 2018 Aug 29;41(16):3241-3249. Epub 2018 Jul 29.

Faculty of Chemistry, Department of Environmental Chemistry, University of Łódź, Łódź, Poland.

We have developed a simple, fast, accurate, and cheap method for the simultaneous determination of total cysteine, homocysteine, glutathione, and N-acetylcysteine in brain homogenates based on the reduction of disulfide bonds by tris(2-carboxyethyl) phosphine, pre-column derivatization of free thiol groups with 2-chloro-1-methylquinolinium tetrafluoroborate followed by ion-pair reversed-phase high-performance liquid chromatography separation with ultraviolet detection. The separation of thiol derivatives was achieved in 10 min. Linearity was observed in the range of 10-300, 0.7-10, 2-30, and 3-20 μmol/L homogenate with a limit of detection of 3.7, 0.2, 0.8, and 1.2 μmol/L homogenate for cysteine, homocysteine, glutathione, and N-acetylcysteine, respectively. The precision, calculated as relative standard deviation, was in the range of 1.21-4.77, 1.53-14.35, 0.47-1.92, and 1.61-8.95% for cysteine, homocysteine, glutathione, and N-acetylcysteine, respectively. The presented method was successfully applied to the selective determination of total amino thiols in pig brain tissue samples.
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http://dx.doi.org/10.1002/jssc.201800381DOI Listing
August 2018

Paraoxonase 1 Q192R genotype and activity affect homocysteine thiolactone levels in humans.

FASEB J 2018 May 21:fj201800346R. Epub 2018 May 21.

Department of Biochemistry and Biotechnology, University of Life Sciences, Poznań, Poland.

Genetic or nutritional deficiencies in 1 carbon and homocysteine (Hcy) metabolism elevate Hcy-thiolactone levels and are associated with cardiovascular and neurologic diseases. Hcy-thiolactone causes protein damage, cellular toxicity, and proatherogenic changes in gene expression in human cells and tissues. A polymorphic cardio-protective enzyme, paraoxonase 1 (PON1), hydrolyzes Hcy-thiolactone in vitro. However, whether Hcy-thiolactone hydrolysis is a physiologic function of the PON1 protein and whether polymorphisms in the PON1 gene affect Hcy-thiolactone levels in humans was unknown. Here we show that the PON1-192 genotype, which affects the enzymatic activity of the PON1 protein, also affected urinary Hcy-thiolactone levels, normalized to creatinine. Carriers of the PON1-192R allele had significantly lower Hcy-thiolactone/creatinine levels than individuals carrying the PON1-192Q allele. Individuals with low serum PON1 paraoxonase activity had significantly higher Hcy-thiolactone/creatinine levels compared with individuals with high paraoxonase activity. In contrast, Hcy-thiolactone/creatinine levels were unaffected by serum PON1 arylesterase activity or by PON1 protein levels. Taken together, these findings suggest that PON1 hydrolyzes Hcy-thiolactone in humans and that the interindividual variations in PON1 genotype/activity can modulate the pathology of hyperhomocysteinemia.-Perła-Kaján, J., Borowczyk, K., Głowacki, R., Nygård, O., Jakubowski, H. Paraoxonase 1 Q192r genotype and activity affect homocysteine thiolactone levels in humans.
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http://dx.doi.org/10.1096/fj.201800346RDOI Listing
May 2018

An inverse relationship between plasma glutathione concentration and fasting glycemia in patients with coronary artery disease and concomitant type 2 diabetes: A pilot study.

Adv Clin Exp Med 2017 Dec;26(9):1359-1366

Department of Hemostatic Disorders, Medical University of Lodz, Poland.

Background: There have been occasional reports indicating that plasma concentrations of reduced glutathione (GSH) may be associated in some way with blood glucose. This relationship, however, has not hitherto been explored in the blood plasma of patients with coronary artery disease (CAD).

Objectives: The aim of this study was to evaluate potential associations of fasting glycemia and peripheral blood plasma GSH concentrations in CAD-free and CAD-affected subjects.

Material And Methods: In blood samples obtained from patients with CAD, defined by coronary angiography and/or echocardiography, and from an age-matched control group of patients with a confirmation of no coronary artery occlusion and with no history of cardiovascular events, plasma concentrations of glucose and reduced glutathione were analyzed by routine laboratory diagnostic methods and high performance liquid chromatography (HPLC), respectively.

Results: The results showed that in the CAD patients, but not in the non-CAD controls, fasting glycemia is negatively associated with plasma levels of GSH (r = -0.328; p = 0.011). Moreover, in the CAD-affected subjects (but not in the controls) the presence of type 2 diabetes mellitus significantly discriminated plasma levels of GSH (rP = -0.125; p = 0.350, between GSH and glucose adjusted for the occurrence of diabetes).

Conclusions: The study suggests that GSH may be an important factor contributing to glucose metabolism in CAD patients. Hence, it may be considered a significant therapeutic target in strategies aimed at improving glycemic control in CAD-affected subjects.
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http://dx.doi.org/10.17219/acem/65441DOI Listing
December 2017

Developmental changes in the levels and redox potentials of main hemolymph thiols/disulfides in the Jamaican field cricket Gryllus assimilis.

Acta Biochim Pol 2017 27;64(3):503-506. Epub 2017 Jul 27.

Department of Environmental Chemistry, Faculty of Chemistry, University of Łodz, Łódź, Poland.

Main thiols and disulfides were determined in the hemolymph of the Jamaican field cricket Gryllus assimilis at various developmental stages. On the basis of these data, redox potentials of the glutathione, cysteine and homocysteine redox systems were calculated. The concentrations of all thiols studied decreased during development (at a stage of 6 molts) with respect to young crickets, and increased again in adult insects. Redox potentials of the glutathione and cysteine systems increased from values of -131.0±5.6 mV and -86.9±17.1 mV, respectively in young crickets to -58.0±3.6 mV and -36.1±4.2 mV, respectively, at the stage of 6 molts and decreased to values of -110.4±24.8 mV and -66.3±12.2 mV, respectively, in adult insects. Redox potentials of the glutathione and cysteine systems in the hemolymph of young and adult insects were similar to those reported for human plasma.
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http://dx.doi.org/10.18388/abp.2017_1510DOI Listing
February 2018

Determination of lipoic acid in human urine by capillary zone electrophoresis.

Electrophoresis 2017 07 11;38(13-14):1800-1805. Epub 2017 May 11.

Department of Environmental Chemistry, Faculty of Chemistry, University of Łódź, Łódź, Poland.

Fast, simple, and accurate CE method enabling determination of lipoic acid (LA) in human urine has been developed and validated. LA is a disulfide-containing natural compound absorbed from the organism's diet. Due to powerful antioxidant activity, LA has been used for prevention and treatment of various diseases and disorders, e.g. cardiovascular diseases, neurodegenerative disorders, and cancer. The proposed analytical procedure consists of liquid-liquid sample extraction, reduction of LA with tris(2-carboxyethyl)phosphine, derivatization with 1-benzyl-2-chloropyridinium bromide (BCPB) followed by field amplified sample injection stacking, capillary zone electrophoresis separation, and ultraviolet-absorbance detection of LA-BCPB derivative at 322 nm. Effective baseline electrophoretic separation was achieved within 6 min under the separation voltage of 20 kV (∼80 μA) using a standard fused-silica capillary (effective length 51.5 cm, 75 μm id) and BGE consisted of 0.05 mol/L borate buffer adjusted to pH 9. The experimentally determined limit of detection for LA in urine was 1.2 μmol/L. The calibration curve obtained for LA in urine showed linearity in the range 2.5-80 μmol/L, with R 0.9998. The relative standard deviation of the points of the calibration curve was lower than 10%. The analytical procedure was successfully applied to analysis of real urine samples from seven healthy volunteers who received single 100 mg dose of LA.
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http://dx.doi.org/10.1002/elps.201700002DOI Listing
July 2017

Simultaneous Determination of Methionine and Homocysteine by on-column derivatization with o-phtaldialdehyde.

Talanta 2016 Dec 15;161:917-924. Epub 2016 Sep 15.

University of Łódź, Faculty of Chemistry, Department of Environmental Chemistry, 163 Pomorska Str., 90-236 Łódź, Poland.

A fast and simple HPLC-based assay has been developed for the simultaneous determination of homocysteine (Hcy) and methionine (Met) in plasma and urine samples, utilizing as small volume of sample as 10μL. The assay uses on-column derivatization with o-phthaldialdehyde. The separation of Hcy and Met was achieved in 14min on a reversed phase C-18 column, followed by fluorescence detection (excitation at 348nm and emission at 438nm for Met; excitation at 370nm and emission at 480nm for Hcy). Linearity of the detector response was observed in the range of 2-60 μmol L for Met and 2-40 μmol L for Hcy. The method was successfully applied for Met and Hcy quantification in human and mouse plasma and urine samples from cystathionine β-synthase deficient and unaffected individuals.
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http://dx.doi.org/10.1016/j.talanta.2016.09.039DOI Listing
December 2016

Antioxidant efficacy of Kalanchoe daigremontiana bufadienolide-rich fraction in blood plasma in vitro.

Pharm Biol 2016 Dec 3;54(12):3182-3188. Epub 2016 Aug 3.

c Department of Biochemistry , Institute of Soil Science and Plant Cultivation, State Research Institute , Pulawy , Poland.

Context: The main source of bufadienolides is toad venom; however, plants such as members of Kalanchoe Adans. (Crassulaceae) genus may also synthesize these bioactive substances.

Objective: This is the first study on antioxidant effects and cytotoxicity of bufadienolide-rich fraction isolated from Kalanchoe daigremontiana Raym.-Hamet & H. Perrier.

Materials And Methods: The methanolic fraction was extracted from the plant roots and contained 0.48 mg bufadienolides/mg of dry mass (11α,19-dihydroksytelocinobufagin, bersaldegenin-1-acetate, bersaldegenin-1,3,5-orthoacetate, 19-(acetyloxy)-3β,5β,11α,14-tetrahydroxyl-12-oxo-bufa-20,22-dienolide and 19-(acetyloxy)-1β,3β,5β,14-tetrahydroxyl-bufa-20,22-dienolide, mainly). The cytotoxicity of K. daigremontiana fraction was evaluated in an in vitro experimental model of blood platelets. The viability of blood platelets was determined on the basis of a release of lactate dehydrogenase.

Results: The fraction scavenged DPPH radicals, with EC of 21.80 μg/mL. Studies on an experimental model of blood plasma under peroxynitrite-induced oxidative stress revealed that the plant preparation had moderate antioxidant properties. Levels of 3-nitrotyrosine and thiol groups indicated that the protective effect of K. daigremontiana was significant mainly for its concentration of 50 μg/mL. No effect was found in prevention of oxidation of low-molecular plasma thiols (glutathione, cysteine and cysteinylglycine). Simultaneously, measurements of lipid hydroperoxides and thiobarbituric acid-reactive substances (TBARS) indicated that the examined fraction might be effective antioxidant at broader concentration range, that is 1-5 and 25-50 μg/mL for hydroperoxides and TBARS generation, respectively. No cytotoxicity was observed at the concentration range of 1-50 μg/mL.

Conclusions: Based on the obtained results, we suggest that antioxidant activity may additionally contribute to beneficial properties of K. daigremontiana-derived extracts.
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http://dx.doi.org/10.1080/13880209.2016.1214740DOI Listing
December 2016

Determination of Total Apigenin in Herbs by Micellar Electrokinetic Chromatography with UV Detection.

J Anal Methods Chem 2016 29;2016:3827832. Epub 2016 Jun 29.

Department of Environmental Chemistry, Faculty of Chemistry, University of Łódź, 163 Pomorska Street, 90-236 Łódź, Poland.

Apigenin is a naturally occurring plant flavone that exhibits strong antioxidant, anti-inflammatory, and antitumor properties. A MEKC-UV based method was developed for the determination of total apigenin in selected herbs. Application of pseudostationary phase in the form of SDS micelles resulted in great repeatability of retention times and peak areas. A buffer solution consisting of 30 mmol/L sodium borate (pH 10.2), 10% acetonitrile, and 10 mmol/L sodium dodecyl sulfate was found to be the most suitable BGE for the separation. The method was validated and calibrated for total apigenin in the range of 1.0-100 μmol/L (R (2) = 0.9994). The limits of detection and quantification were 0.48 μmol/L and 0.92 μmol/L, respectively. This precise and robust method was successfully applied to the analysis of plant samples for total apigenin content.
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http://dx.doi.org/10.1155/2016/3827832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4942635PMC
July 2016

Novel MEKC method for determination of sodium 2-mercaptoethanesulfonate in human plasma with in-capillary derivatization and UV detection.

J Chromatogr B Analyt Technol Biomed Life Sci 2016 Aug 24;1027:88-95. Epub 2016 May 24.

University of Łódź, Faculty of Chemistry, Department of Environmental Chemistry, Poland. Electronic address:

Sensitive electrophoretic method for determination of total sodium 2-mercaptoethanesulfonate (mesna) in human plasma, based on the stacking with high salt concentration in MEKC and in-capillary derivatization with 2-chloro-1-methyllepidinium tetrafluoroborate followed by UV detection was developed. In the method 0.03molL(-1)pH 7 phosphate buffer with the addition of 0.01molL(-1) SDS, and 10% ACN was used as a BGE. The limit of quantification (LOQ) of the method was 0.5μmolL(-1). Linearity in detector response was observed over the range of 0.5-10μmolL(-1) with the correlation coefficient 0.9971. The intra- and inter-day accuracy (three concentration levels, 5 days, n=3) of the method ranged from 97.2 to 110.0% and from 94.0 to 101.2%, respectively. The novel MEKC method with UV detection proved to be suitable for determination of total mesna in human plasma.
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http://dx.doi.org/10.1016/j.jchromb.2016.05.032DOI Listing
August 2016

A versatile method for analysis of saliva, plasma and urine for total thiols using HPLC with UV detection.

Talanta 2016 08 22;155:70-7. Epub 2016 Apr 22.

University of Łódź, Faculty of Chemistry, Department of Environmental Chemistry, 163 Pomorska Str., 90-236 Łódź, Poland. Electronic address:

A simple and rapid HPLC method using 2-chloro-1-methyllepidinium tetrafluoroborate (CMLT) as a derivatization reagent was developed for simultaneous determination of homocysteine (Hcy), glutathione (GSH), γ-glutamylcysteine (γ-GluCys), cysteinylglycine (CysGly), N-acetylcysteine (NACys) and cysteine (Cys) in human saliva, plasma and urine. Separation of the analytes was achieved in just 7min using an HPLC, followed by UV detection at 355nm. Chromatographic separation was accomplished on Aeris PEPTIDE XB-C18 (150mm×4.6mm, 3.6µm) column from Phenomenex with a gradient elution: 0-4.0min, 7-30% B; 4.0-5.5min, 30-7% B; 5.5-7.5min, 7% B; (A: B, v/v); (A) 0.5% CH3COOH and (B) EtOH. Mobile phase was delivered at a flow rate 1.0mLmin(-1). Linearity in detector response for total thiols was observed over the range of 0.1-20μmolL(-1) for Hcy, GSH and γ-GluCys, 0.25-50μmolL(-1) for NACys and CysGly and 5-300 for Cys. The LOQ values for Hcy, GSH, γ-GluCys, NACys, CysGly and Cys were 0.05, 0.05, 0.10, 0.06, 0.12 and 0.08μmolL(-1), respectively. The method was successfully implemented to analysis of the samples donated by 15 apparently healthy volunteers and 10 patients.
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http://dx.doi.org/10.1016/j.talanta.2016.04.031DOI Listing
August 2016

Quantification of homocysteine and cysteine by derivatization with pyridoxal 5'-phosphate and hydrophilic interaction liquid chromatography.

Anal Bioanal Chem 2016 Mar 21;408(7):1935-41. Epub 2016 Jan 21.

Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers New Jersey Medical School, Newark, NJ, 07103, USA.

A simple and rapid assay using pyridoxal 5'-phosphate (PLP) as a derivatizing reagent was developed for the simultaneous determination of homocysteine (Hcy) and cysteine (Cys) in human plasma. Derivatization with PLP affords UV-absorbing tetrahydrothiazine and thiazolidine derivatives of Hcy and Cys, respectively. Separation of these derivatives was achieved in 5 min using a hydrophilic interaction liquid chromatography, followed by UV detection at 330 nm. Linearity in detector response was observed over the range of 0.25-20 μM for Hcy and 10-300 μM for Cys. The limit of quantification (LOQ) values for Hcy and Cys were 0.25 and 2.5 μM, respectively. The method was successfully applied to plasma samples donated by apparently healthy volunteers.
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http://dx.doi.org/10.1007/s00216-016-9308-3DOI Listing
March 2016

Fast and simple MEKC sweeping method for determination of thiosulfate in urine.

Electrophoresis 2016 05 16;37(9):1155-60. Epub 2015 Nov 16.

Department of Environmental Chemistry, Faculty of Chemistry, University of Łódź, Poland.

A new method for determination of thiosulfate in human urine has been developed and validated. Analytical procedure is very simple and consists of only few steps: derivatization of thiosulfate with 2-chloro-1-methylquinolinium tetrafluoroborate, centrifugation of a mixture, separation of so-formed derivative by micellar electrokinetic chromatography with sweeping and UV detection at 375 nm. A fused-silica capillary with an inlet to detector length of 51.5 cm and a total length of 60 cm (75 μm id) was served as a separation column. The separation voltage of 20.5 kV (∼160 mA) and buffer solution consisting of 0.055 mol/L sodium phosphate (pH 8), 25% acetonitrile, and 0.035 mol/L sodium dodecyl sulfate were found to be the most suitable conditions for the effective separation. The limit of quantification for thiosulfate was 4 μmol/L urine. The method was validated and calibrated for thiosulfate in the range of 4-64 μmol/L (R(2) = 0.9997). The relative standard deviation of the points of the calibration curve varied from 1.2 to 4.8% RSD.
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http://dx.doi.org/10.1002/elps.201500411DOI Listing
May 2016

Protein N-linked homocysteine is associated with recurrence of venous thromboembolism.

Thromb Res 2015 Nov 5;136(5):911-6. Epub 2015 Sep 5.

Institute of Cardiology, Jagiellonian University Medical College, John Paul II Hospital, Krakow, Poland. Electronic address:

Background: Recently, protein N-linked homocysteine (Hcy) has been measured in healthy subjects and patients with marked hyperhomocysteinemia. Since elevated total Hcy (tHcy) levels are associated with increased risk of venous thromboembolism (VTE), we aimed to investigate protein N-linked Hcy levels in patients with VTE.

Methods: We studied 200 consecutive patients with VTE (89 men, 111 women, aged from 17 to 83 years), including 57 subjects with a subsequent episode of VTE (recurrent VTE) during 24 months of follow-up. Protein N-linked Hcy was assayed using high-performance liquid chromatography with an on-column derivatization with o-phthaldialdehyde and fluorescence detection.

Results: The median protein N-linked Hcy was 1.404 μM (interquartile range [IQR] 0.859-2.066), while the median tHcy (IQR) was 9.1 μM (6.8-11.2). In the whole group protein N-linked Hcy correlated only with C-reactive protein (CRP; r = 0.44, p < 0.0001). In patients with recurrent VTE protein N-linked Hcy correlated with C-reactive protein (r = 0.43, p < 0.0001), tHcy (r = 0.42, p = 0.001) and age (r = 0.32, p = 0.014), but not with thrombophilia, unprovoked VTE or the current anticoagulation. Hyperhomocysteinemia, defined as tHcy ≥ 15 μM (n = 14.7%), was not associated with higher protein N-linked Hcy. Patients with recurrent VTE had higher levels of protein N-linked Hcy compared to those who experienced a single episode of VTE (1.553 μM, 1.157-2.445 vs. 1.27 μM, 0.826-1.884; p = 0.002). Multiple regression adjusted for potential confounders showed that the only independent predictor of protein N-linked Hcy in the upper quartile was CRP > 3mg/L (odds ratio 3.04, 95% confidence interval 2.12-4.36, p < 0.0001).

Conclusion: Elevated protein N-linked Hcy concentrations, indicating enhanced protein homocysteinylation in vivo, characterize patients with recurrent VTE and this phenomenon is associated with enhanced inflammatory state.
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http://dx.doi.org/10.1016/j.thromres.2015.09.002DOI Listing
November 2015

Influence of Pre-Storage Irradiation on the Oxidative Stress Markers, Membrane Integrity, Size and Shape of the Cold Stored Red Blood Cells.

Transfus Med Hemother 2015 May 29;42(3):140-8. Epub 2015 Jan 29.

Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland.

Background: To investigate the extent of oxidative damage and changes in morphology of manually isolated red blood cells (RBCs) from whole blood, cold stored (up to 20 days) in polystyrene tubes and subjected to pre-storage irradiation (50 Gy) and to compare the properties of SAGM-preserved RBCs stored under experimental conditions (polystyrene tubes) with RBCs from standard blood bag storage.

Methods: The percentage of hemolysis as well as the extracellular activity of LDH, thiobarbituric acid-reactive substances, reduced glutathione (GSH), and total antioxidant capacity (TAC) were measured. Changes in the topology of RBC membrane, shape, and size were evaluated by flow cytometry and judged against microscopy images.

Results: Irradiation caused significant LDH release as well as increased hemolysis and lipid peroxidation, GSH depletion, and reduction of TAC. Prolonged storage of irradiated RBCs resulted in phosphatidylserine exposure on the cell surface. By day 20, approximately 60% of RBCs displayed non-discoid shape. We did not notice significant differences in percentage of altered cells and cell volume between RBCs exposed to irradiation and those not exposed.

Conclusion: Irradiation of RBC transfusion units with a dose of 50 Gy should be avoided. For research purposes such as studying the role of antioxidants, storage of small volumes of RBCs derived from the same donor would be more useful, cheaper, and blood-saving.
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http://dx.doi.org/10.1159/000371596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4483300PMC
May 2015

Salicylic acid and cysteine contribute to arbutin-induced alleviation of angular leaf spot disease development in cucumber.

J Plant Physiol 2015 Jun 17;181:9-13. Epub 2015 Apr 17.

Department of Plant Physiology and Biochemistry, Faculty of Biology and Environmental Protection, University of Łódź, Banacha 12/16, 90-237, Łódź, Poland.

Arbutin induced suppression of angular leaf spot disease in cucumber resulting from lower populations of Pseudomonas syringae pv lachrymans in the infected tissues. This study provides insight into mechanisms that may potentially account for this effect. In the absence of the pathogen, exogenous arbutin-induced expression of PR1, the marker of salicylic acid signaling, increased the content of salicylic acid and modulated the cysteine pool. This suggested that arbutin promoted cucumber plants to a "primed" state. When challenged with the pathogen, the arbutin-treated plants showed strongly reduced infection symptoms 7 days after inoculation. At this time point, they were characterized by higher contents of free and protein-bound cysteine due to higher cysteine biosynthetic capacity related to increased activities of serine acetyltransferase and cysteine synthase when compared with plants infected without arbutin treatment. Moreover, in the arbutin-treated and infected plants the contents of free salicylic acid and its conjugates were also increased, partly owing to its biosynthesis via the phenylpropanoid pathway. We suggest that arbutin-induced abrogation of angular leaf spot disease in cucumber could be mediated by salicylic acid and cysteine-based signaling.
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http://dx.doi.org/10.1016/j.jplph.2015.03.017DOI Listing
June 2015

Interactions of the gasotransmitters contribute to microvascular tone (dys)regulation in the preterm neonate.

PLoS One 2015 25;10(3):e0121621. Epub 2015 Mar 25.

Mothers and Babies Research Centre, Hunter Medical Research Institute, New Lambton Heights, NSW, 2305, Australia; School of Medicine and Public Health, University of Newcastle, Callaghan, NSW, 2308, Australia; Illawarra Health and Medical Research Institute and Graduate School of Medicine, University of Wollongong, NSW, 2522, Australia; Kaleidoscope Neonatal Intensive Care Unit, John Hunter Children's Hospital, New Lambton Heights, NSW, 2305, Australia.

Background & Aims: Hydrogen sulphide (H2S), nitric oxide (NO), and carbon monoxide (CO) are involved in transitional microvascular tone dysregulation in the preterm infant; however there is conflicting evidence on the interaction of these gasotransmitters, and their overall contribution to the microcirculation in newborns is not known. The aim of this study was to measure the levels of all 3 gasotransmitters, characterise their interrelationships and elucidate their combined effects on microvascular blood flow.

Methods: 90 preterm neonates were studied at 24h postnatal age. Microvascular studies were performed by laser Doppler. Arterial COHb levels (a measure of CO) were determined through co-oximetry. NO was measured as nitrate and nitrite in urine. H2S was measured as thiosulphate by liquid chromatography. Relationships between levels of the gasotransmitters and microvascular blood flow were assessed through partial correlation controlling for the influence of gestational age. Structural equation modelling was used to examine the combination of these effects on microvascular blood flow and derive a theoretical model of their interactions.

Results: No relationship was observed between NO and CO (p = 0.18, r = 0.18). A positive relationship between NO and H2S (p = 0.008, r = 0.28) and an inverse relationship between CO and H2S (p = 0.01, r = -0.33) exists. Structural equation modelling was used to examine the combination of these effects on microvascular blood flow. The model with the best fit is presented.

Conclusions: The relationships between NO and H2S, and CO and H2S may be of importance in the preterm newborn, particularly as NO levels in males are associated with higher H2S levels and higher microvascular blood flow and CO in females appears to convey protection against vascular dysregulation. Here we present a theoretical model of these interactions and their overall effects on microvascular flow in the preterm newborn, upon which future mechanistic studies may be based.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0121621PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4373676PMC
February 2016

Simple micellar electrokinetic chromatography method for the determination of hydrogen sulfide in hen tissues.

Electrophoresis 2015 Apr 24;36(7-8):1028-32. Epub 2015 Mar 24.

Department of Environmental Chemistry, Faculty of Chemistry, University of Łódź, Poland.

A new method for the determination of hydrogen sulfide in hen tissues has been developed and validated. For estimation of hydrogen sulfide content, a sample (0.1 g) of hen tissue was treated according to the procedure consisted of some essential steps: simultaneous homogenization of a tissue and derivatization of hydrogen sulfide to its S-quinolinium derivative with 2-chloro-1-methylquinolinium tetrafluoroborate, separation of so-formed derivative by micellar electrokinetic chromatography with sweeping, and detection and quantitation with the use of UV detector set to measure analytical signals at 375 nm. Effective electrophoretic separation was achieved using fused silica capillary (effective length 41.5 cm, 75 μm id) and 0.05 mol/L, pH 8 phosphate buffer with the addition of 0.04 mol/L SDS and 26% ACN. The lower limit of quantification was 0.12 μmol hydrogen sulfide in 1 g of tissue. The calibration curve prepared in tissue homogenate for hydrogen sulfide showed linearity in the range from 0.15 to 2.0 μmol/g, with the coefficient of correlation 0.9978. The relative standard deviation of the points of the calibration curve varied from 8.3 to 3.2% RSD.
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http://dx.doi.org/10.1002/elps.201400518DOI Listing
April 2015