Simultaneous analysis of multiple aminothiols in human plasma by high performance liquid chromatography with fluorescence detection

Aminothiols serve numerous vital functions in biochemistry, including detoxification and regulation of cellular metabolism, enzymatic activity, and protein trafficking and degradation. Plasma aminothiol concentrations are frequently measured for clinical and translational research investigating oxidative stress, and for routine clinical diagnosis and monitoring of vascular injury. Although a variety of techniques are available to measure aminothiol concentrations in plasma, high performance liquid chromatography with fluorescence detection (HPLC–FD) is the most widely used. This review summarizes HPLC–FD methods, including pre-analytical considerations, procedures for sample reduction, derivatization, and chromatographic separation of the primary biological aminothiols cysteine, homocysteine, cysteinylglycine, and glutathione in human plasma.     

Liquid chromatographic determination of total homocysteine in blood plasma with photometric detection

A rapid and sensitive method for quantification of homocysteine total forms and glutathione levels in blood plasma via HPLC was developed. Dithiotreitol as a water soluble agent has been used as a reductant for both protein and nonprotein disulphides. Dithiotreitol reacts with the mixed disulphides under 60 degrees C treatment within 10 min. Reduced aminothiols and homocystein were easily derivated with 5,5'-dithiobis-(2-nitrobenzoic acid) and the resultant ultraviolet absorbance within 330 nm was detected by the HPLC method. The concentration of total plasma homocysteine was significantly higher in groups of patients: with the end stage of renal disease: 45.5+/-40.9 micromol/l (n=79), with cerebral vascular disorders 12.3+/-7.0 micromol/l (n=65), and with coronary atherosclerosis 15.4+/-10.9 micromol/l (n=15) than that in healthy subjects (6.2+/-1.74 micromol/l, n=20). Some major advantages of the method include: simultaneous measurement of both total homocysteine and total glutathione, no loss of oxidized form during processing of blood plasma for aminothiols measurement, use of protein-bound aminothiols solution as a calibrator.     

Simultaneous determination of total homocysteine, cysteine, cysteinylglycine, and glutathione in human plasma by high-performance liquid chromatography: application to studies of oxidative stress

A sensitive, reproducible, and robust high-performance liquid chromatography (HPLC) method has been validated for simultaneously determining total concentrations of the aminothiols homocysteine, cysteine, cysteinylglycine, and glutathione in human plasma. Plasma aminothiols are reduced via incubation with tris-(2-carboxyethyl)-phosphine hydrochloride, followed by protein precipitation with trichloroacetic acid and derivatization with ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid. Separation of aminothiols and the internal standard mercaptopropionylglycine is achieved using reversed-phase HPLC conditions and fluorescence detection. Excellent linearity is observed for all analytes over their respective concentration ranges with correlation coefficients (r) > 0.99. The intra- and inter-day precision and accuracy were within +/-10%. This method utilizes an internal standard, employs phosphate buffered saline-based standards and quality controls, and demonstrates excellent plasma recovery and improved sensitivity. This assay is well suited for high-throughput quantitative determination of aminothiols in clinical studies, and is currently being used to support investigations of oxidative stress in patients with chronic kidney disease.     

Determination of total plasma homocysteine and related aminothiols by gas chromatography with flame photometric detection

A selective and sensitive method for the determination of total homocysteine (Hcy) and related aminothiols, such as cysteine (Cys) and cysteinylglycine (CysGly), in plasma samples by gas chromatography (GC) has been developed. After reduction of the sample with sodium borohydride, the liberated Hcy and other aminothiols were converted to their N,S-diisopropoxycarbonyl methyl ester derivatives and measured by GC with flame photometric detection using a DB-17 capillary column. The calibration curves were linear over the range 0.5–10 nmol for Hcy and CysGly, and over the range 5–100 nmol for Cys, and the correlation coefficients were above 0.996. Using this method, total plasma Hcy, Cys and CysGly could be directly analysed without prior clean-up of the sample and without any interference from coexisting substances. Overall recoveries of Hcy and other aminothiols added to plasma samples were 95–106%. Analytical results for the determination of total plasma Hcy, Cys and CysGly from normal subjects are presented.     

Determination of total aminothiols and neuroactive amino acids in plasma by high performance liquid chromatography with fluorescence detection

This paper describes a simple and sensitive reversed-phase HPLC method for the determination of total homocysteine, total cysteine, total glutathione (GSH + GSSG), and neuroactive amino acids (Asp, Glu, Tau, GABA) using precolumn derivatization with ortho-phtaldialdehyde and fluorimetric detection at 360 and 470 nm for emission and excitation, respectively. Derivatization was performed with ortho-phthaldialdehyde in the presence of 2-mercaptoethanol after alkylation of free sulfhydryl groups with iodoacetic acid. For determination of total aminothiols, the disulfide bonds were reduced and protein-bound thiols were released by addition of dithiothreitol to the plasma sample. The advantage of this method is the simultaneous determination of both homocysteine/cysteine/glutathione and neuroactive amino acids in the sample. The plasma levels of studied compounds were determined in 14 healthy volunteers (20–45 years old) and 55 patients with chronic hepatitis C (20–49 years old) and the resulting values were in a good agreement with results published earlier. The calibration curves were linear over a concentration range of 5–100 μM in plasma (r ² = 0.985−0.996). The intraday and interday coefficients of variation were 3–6% and 4–7%, respectively. The recovery of the standards added to the plasma samples ranged from 94 to 102%. The limits of detection (LOD) were 0.2–0.5 ng per 10 μl of the injection volume (signal-to-noise ratio of 3).     

Simple plasma work-up for a fast chromatographic analysis of homocysteine,cysteine, methionine and aromatic amino acids

Simplified sample workup obviating protein precipitation and eluent evaporation commonly employed in earlier reports using chloroformate-mediated derivatization of aminothiols prior to mass spectrometric (MS) detection is presented. The reduction of disulfides in plasma is accomplished with dithiothreitol within minutes. A simultaneous derivatization with ethyl chloroformate (ECF) and extraction of derivatives into organic phase takes place within seconds. Along with S-amino acids, also aromatic amino acids can be determined during a 5-min run. Gas chromatography with flame ionization detection (GC-FID) proved to be sensitive enough to reach plasma homocysteine levels. A prerequisite for a reliable quantitation was fulfilled under the given conditions. Intra-assay precision was <5%, recoveries from spiked plasma complete (101.2%), detection and quantitation limits for homocysteine came to <1 and 3 micro mol/l. Our results were in full agreement with those obtained by liquid chromatography (r=0.999 for homocysteine and 0.987 for cysteine), and were close to two homocysteine immunoassays (r=0.991 and 0.939, respectively).     

Simultaneous determination of ascorbic acid, aminothiols, and methionine in biological matrices using ion-pairing RP-HPLC coupled with electrochemical detector

A novel highly sensitive ion-pairing reversed-phase high performance liquid-chromatography/electrochemical detection method for simultaneous determination of l-ascorbic acid, aminothiols, and methionine in biological matrices was developed, optimized, and validated. Reduced forms of the analytes were extracted from the sample matrices with 10% meta-phosphoric acid solution((aqueous)). To determine the total vitamin C, the total aminothiols, and the total methionine, samples were treated with tris(2-carboxyethyl)phosphine solution in 0.05% trifluoroacetic acid solution((aqueous)) subsequent to deproteination to reduce the oxidized forms of these compounds. Various analytes were separated on a C(18) (250 × 4.6 mm, 5 μm) analytical column using methanol-0.05% trifluoroacetic acid solution((aqueous)) (05/95, v/v), containing 0.1mM 1-octane sulphonic acid as the ion-pairing agent) as the isocratic mobile phase pumped at a flow rate of 1.5 mL min(-1) at room temperature. The column eluents were monitored at a voltage of 0.85 V. These analytes were efficiently resolved in less than 20 min using n-acetyl cysteine as the internal standard. The present method was specific for the analysis of these analytes and demonstrated acceptable values for linearity (r(2)>0.999 in the range of 0.2-10,000 ng mL(-1) for all the analytes), recovery (>96%), precision (%RSD ≤ 2.0), and sensitivity (on column limit of detection: 250-400 fg and limit of quantification: 0.8-1.25 pg), indicating that the proposed method could be efficiently used for determination of these analytes in the context of clinical research.     

Capillary electrophoresis in the evaluation of aminothiols in body fluids

Thiols play a fundamental role in cell biology, biochemistry and pharmacology. Altered thiol levels in body fluids are linked to specific pathological conditions. Glutathione is the most abundant intracellular low-molecular-mass thiol, playing an essential role in protecting cells from toxic species; other relevant thiol-containing compounds are homocysteine (Hcy), cysteine (Cys), cysteinylglycine (CysGly). Plasma aminothiols can be bound to proteins but they also occur free in the disulfide (symmetrical and mixed) and in the reduced forms. The simultaneous determination of these aminothiols, their precursor and metabolites is a useful tool in studying oxidative stress, metabolic and redox regulation. Many capillary electrophoresis methods have been proposed for this purpose, the aim of the present review is to support researchers in the choice of suitable methods for the determination of thiols in body fluids evaluating the different approaches and technologies proposed from the literature.     

Simultaneous quantification of tracheloside and trachelogenin in rat plasma using liquid chromatography/tandem mass spectrometry

We developed and validated a quantitative method for simultaneously determining the concentrations of tracheloside and trachelogenin in rat plasma. Plasma samples were prepared by liquid-liquid extraction with ethyl acetate. Isocratic chromatographic separation was performed on a reversed-phase Diamonsil C(18) column (4.6×200 mm, 5 μm). The mobile phase consisted of methanol and 10mM aqueous ammonium formate (80:20, v/v). Analyte detection was achieved by positive electrospray ionization (ESI) tandem mass spectrometry. Calibration was performed by internal standardization with glipizide, and regression curves ranging from 0.625 to 625 ng/mL were constructed for both the analytes. The intra- and inter-day precision values were below 8%, and accuracy ranged from -5.33% to 2.53% in all quality control samples. In this study, the validated method was successfully applied to determine the pharmacokinetic profile of tracheloside and trachelogenin in rat plasma after oral and intravenous administration of trachelospermi total lignans.     

Determination of plasma homocysteine by high-performance liquid chromatography with fluorescence detection

Severe homocystinemia is frequently associated with vascular disease while the pathological consequences of moderate or slightly elevated plasma homocysteine are unknown. Cobalamin and folate deficiencies may result in an elevation of plasma homocysteine. A sensitive and reproducible assay for total plasma homocysteine has been developed. The essential steps in the assay include (i) conversion of homocysteine disulfides to free homocysteine with borohydride reduction; (ii) conjugation of homocysteine with monobromobimane; (iii) separation of homocysteine-bimane from other plasma thiol-bimane adducts by reverse-phase high-performance liquid chromatography; and (iv) detection and quantitation of homocysteine-bimane by fluorometry. The method has a sensitivity of 4.4 pmol of homocysteine and is highly reproducible (intra- and interassay coefficients of variation = 4.97 and 4.53%, respectively). The mean concentration of total plasma homocysteine in nonfasting adult males (n = 12) and females (n = 12) was 15.8 (range, 7.0-23.7) and 16.5 nmol/ml (range, 8.6-20.7), respectively. Markedly elevated levels of homocysteine were found in patients with cobalamin and folate deficiency. Total plasma homocysteine represents approximately 4% of borohydride-generated thiol reactivity in the plasma of normal individuals.     

High performance liquid chromatography analysis of 2-mercaptoethylamine (cysteamine) in biological samples by derivatization with N-(1-pyrenyl) maleimide (NPM) using fluorescence detection

2-Mercaptoethylamine (cysteamine) is an aminothiol compound used as a drug for the treatment of cystinosis, an autosomal recessive lysosomal storage disorder. Because of cysteamine's important role in clinical settings, its analysis by sensitive techniques has become pivotal. Unfortunately, the available methods are either complex or labor intensive. Therefore, we have developed a new rapid, sensitive, and simple method for determining cysteamine in biological samples (brain, kidney, liver, and plasma), using N-(1-pyrenyl) maleimide (NPM) as the derivatizing agent and reversed-phase high performance liquid chromatography (HPLC) with a fluorescence detection method (lambda(ex)=330 nm, lambda(em)=376 nm). The mobile phase was acetonitrile and water (70:30) with acetic acid and o-phosphoric acid (1 mL/L). The calibration curve for cysteamine in serine borate buffer (SBB) was found to be linear over a range of 0-1200 nM (r(2)=0.9993), and in plasma and liver matrix, the r(2) values were 0.9968 and 0.9965, respectively. The coefficients of the variation for the within-run and between-run precisions ranged from 0.68 to 9.90% and 0.63 to 4.17%, respectively. The percentage of relative recovery ranged from 94.1 to 98.6%.     

Determination of homocysteine thiolactone and homocysteine in cell cultures using high-performance liquid chromatography with fluorescence detection

A sensitive and simple method utilising fluorometric detection for the simultaneous routine monitoring of homocysteine thiolactone (HTL) and homocysteine (Hcy) in biological samples has been developed. Separation relies on isocratic ion-pairing and reversed-phase chromatography while the principle of the detection is that the lactone ring in HTL molecule is cleaved with an alkali to produce Hcy, which reacts with ortho-phthalaldehyde (OPA) in the absence of an added thiol reagent to form a stable fluorescent derivative. The method has a sensitivity of 200 fmol of HTL and 100 fmol for Hcy in the sample. The present method was applied to the determination of HTL and Hcy in Hep G2 cell.     

Neural tube defects and maternal biomarkers of folate, homocysteine, and glutathione metabolism

BACKGROUND: Alterations in maternal folate and homocysteine metabolism are associated with neural tube defects (NTDs). The role played by specific micronutrients and metabolites in the causal pathway leading to NTDs is not fully understood. METHODS: We conducted a case-control study to investigate the association between NTDs and maternal alterations in plasma micronutrients and metabolites in two metabolic pathways: methionine remethylation and glutathione transsulfuration. Biomarkers were measured in a population-based sample of women who had NTD-affected pregnancies (n = 43) and a control group of women who had a pregnancy unaffected by a birth defect (n = 160). We compared plasma concentrations of folate, vitamin B(12), vitamin B(6), methionine, S-adenosylmethionine (SAM), s-adenosylhomocysteine (SAH), adenosine, homocysteine, cysteine, and reduced and oxidized glutathione between cases and controls after adjusting for lifestyle and sociodemographic factors. RESULTS: Women with NTD-affected pregnancies had significantly higher plasma concentrations of SAH (29.12 vs. 23.13 nmol/liter, P = .0011), adenosine (0.323 vs. 0.255 mumol/liter; P = .0269), homocysteine (9.40 vs. 7.56 micromol/liter; P < .001), and oxidized glutathione (0.379 vs. 0.262 micromol/liter; P = .0001), but lower plasma SAM concentrations (78.99 vs. 83.16 nmol/liter; P = .0172) than controls. This metabolic profile is consistent with reduced methylation capacity and increased oxidative stress in women with affected pregnancies. CONCLUSIONS: Increased maternal oxidative stress and decreased methylation capacity may contribute to the occurrence of NTDs. Further analysis of relevant genetic and environmental factors is required to define the basis for these observed alterations.     

Induction of micronucleated erythrocytes by MEA,AET, WR-2721 and X-rays

The induction of micronucleated polychromatic erythrocytes (MNPCEs) was assessed in the bone marrow of adult male Swiss mice treated with MEA (cysteamine HCl), AET (2-aminoethylisothiouronium Br.HBr), or WR-2721 (S-2-(3-aminopropylamino)ethyl phosphorothioic acid), at a dose of 200 mg/kg body weight, and/or exposed to 6 Gy X-rays. MEA, AET, or WR-2721 was given alone or 15 min prior to X-ray exposure, and the frequency of MNPCEs was determined 24 h after the aminothiol treatment and X-irradiation of mice. A genotoxic effect was shown for MEA, AET, WR-2721, and X-rays, as well as a protective effect of the aminothiols against X-ray-induced genotoxicity in the mouse erythropoietic system. The aminothiol drugs given alone, without subsequent X-irradiation, elevated the frequency of MNPCEs, and WR-2721 appeared to be less toxic than AET and MEA. After exposure of mice to X-rays, the number of MNPCEs was distinctly increased. MEA, AET, or WR-2721 administration prior to X-irradiation resulted in a reduction of the X-ray-induced elevation of the frequency of micronuclei, but a stronger radioprotective effect was obtained following WR-2721 and AET treatment than after MEA application. So, the genotoxic and radioprotective effect of the aminothiols was dependent on the compound applied.     

Fully automated method for simultaneous determination of total cysteine,cysteinylglycine, glutathione and homocysteine in plasma by HPLC with UV absorbance detection

A fully automated HPLC method for the simultaneous determination of total thiols in plasma samples has been developed. The method involves reductive conversion of disulfides to their reduced counterparts with the use of tris(2-carboxyethyl)phosphine. After reduction the newly formed sulfhydryl groups are reacted with 2-chloro-1-methylquinolinium tetrafluoroborate to form 2-S-quinolinium derivatives followed by deproteinization by dialysis. The reaction products are separated by reversed-phase HPLC, detected and quantified by UV absorbance detection at 355 nm. The recommended HPLC procedure enables measurement of four main plasma aminothiols cysteine, cysteinylglycine, glutathione, and homocysteine with low imprecision (mean relative standard deviations within calibration range, 3.47%, 5.34%, 4.25% and 3.26%, respectively) and good sensitivity. Accuracy, expressed as the mean measured amount as percentage of added amount, was within 97.5–103.0%, 98.3–102.5%, 96.3–99.5% and 97.1–99.1%, respectively. The lower limit of quantification for all thiols was 0.5 μM. The whole unattended instrument acquisition time amounts 13 min.     

Determination of plasma aminothiols by high performance liquid chromatography after precolumn derivatization with N-(2-acridonyl)maleimide

Design, synthesis and properties of new derivatization reagent N-(2-acridonyl)-maleimide (MIAC) for thiol groups is presented. The reaction of MIAC with aminothiols is specific, very fast and yield highly fluorescent products. The HPLC method for determination of homocysteine, cysteine and glutathione based on utilization of MIAC is developed. A baseline separation of derivatives is achieved by isocratic elution on reverse phase column within 6 min. The method is linear in the range of 0.5-25 microM for homocysteine and glutathione, and in the range of 0.5-200 microM for cysteine. The limits of detection for homocysteine, cysteine and glutathione are 1.2, 1.4 and 2.0 pmol, respectively, per 20 microl injection. Within and between-run precision expressed as relative standard deviations are in the range of 1.35-4.38% and 0.89-4.13%, respectively.     

Recent advances in separation and detection methods for thiol compounds in biological samples

Biological aminothiols, such as cysteine, homocysteine, and glutathione, widely occur in animal tissues and fluids. The altered levels of the thiols (reduced forms) and their disulfides (oxidized forms) in physiological liquids have been linked to specific pathological conditions and closely associated with several human diseases. Therefore, it is well recognized that the determination of thiols and disufides is important in order to understand their physiological roles. The derivatization utilizing a suitable labeling reagent followed by chromatographic separation and detection is the most reliable means for sensitive and selective assays. Many reagents have typically been synthesized and successfully used for the determination of thiols and disulfides in biological specimens. The development of new reagents for highly sensitive detection is still continuing. This review describes the approaches for the separation assay of various thiol compounds, obtained through the analytical papers published in 2000–2008. The derivatization reagents are categorized with each type of chromophore and fluorophore and evaluated in terms of their reactivity, stability, detection wavelength, handling, sensitivity and selectivity. Application examples of the reagents for bioanalysis are also described in the text.     

Electrochemical determination of femtomole amounts of free reduced and oxidized glutathione: Application to human hair follicles

A simple and sensitive high-performance liquid chromatographic technique has been developed for the quantification of free reduced and free oxidized glutathione in biological samples. After acidic extraction and isocratic separation of the compounds of interest on a reversed-phase column, both forms of glutathione are quantified with a coulometric detector working in the oxidative mode. The limit of detection is 125 fmol for reduced glutathione and 400 fmol for the oxidized form (signal-to-noise ratio of 3). This sensitivity allows the measurement of the small amount of glutathione present in a single hair follicle. The technique is well adapted to microsamples, i.e. for non-invasive sampling technique (hair, skin, tears, etc.) and can be adapted to various cells or tissues.     

Effect of postmenopausal hormone replacement therapy on lipoprotein and homocysteine levels in Chinese women.

Epidemiological studies have revealed that postmenopausal estrogen replacement therapy results in a marked reduction in the risk for cardiovascular diseases. In the present study, we evaluated plasma lipoprotein profile as well as homocysteine levels in 145 postmenopausal and premenopausal Chinese women living in Hong Kong. We also investigated the effect of hormone-replacement therapy (HRT) with estrogen or estrogen combined with progestin on plasma lipoprotein profile and homocysteine concentrations in those individuals. Postmenopausal women displayed significantly higher plasma levels of total cholesterol, LDL-cholesterol and apoB as well as higher plasma homocysteine levels than that of premenopausal women. HRT with either estrogen (17beta-estradiol or conjugated equine estrogen) alone or estrogen combined with progestin for 3.5-4.5 years significantly improved the lipoprotein profile in postmenopausal women by decreasing the levels of total cholesterol (12-20% reduction), LDL-cholesterol (26-29% reduction) and apoB (21-25% reduction). In women treated with 17beta-estradiol or conjugated equine estrogens their plasma levels of apoAl were significantly elevated (18% elevation) as compared to non-users. HRT also reduced plasma concentrations of homocysteine (13-15% reduction). In conclusion, we found that long-term HRT was associated with improvement in plasma lipoprotein profile and a reduction in homocysteine concentration in postmenopausal women. These results support the notion that the improvement of lipoprotein profile and a reduction in homocysteine concentration may contribute to the beneficial effect of HRT on cardiovascular risk.     

Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma.

An assay that measures the reduced, oxidized, and protein-bound forms of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma is described. Oxidized and protein-bound thiols are converted to their reduced counterparts by the use of NaBH4, and, following derivatization with monobromobimane (mBrB), the thiol-bimane adducts are quantified by reversed-phase ion-pair liquid chromatography and fluorescence detection. The presence of 50 microM dithioerythritol provides linearity of the standard curves at very low thiol concentrations. Selective determination of the oxidized forms was accomplished by blocking free sulfhydryl groups with N-ethylmaleimide (NEM) and excess NEM is inactivated by the subsequent addition of NaBH4. The reduced forms of the thiols in plasma were trapped with minimal oxidation by derivatizing blood samples at the time of collection. This was attained by drawing blood directly into tubes containing isotonic solutions of mBrB or NEM. The assay is sufficiently sensitive (less than 2 pmol) to detect the various forms of the four thiol compounds in human plasma. The analytical recovery of cysteine, cysteinylglycine, homocysteine, and glutathione was close to 100%, and the within-day precision corresponded to a coefficient of variation of 7, 8, 6, and 7%, respectively. The assay has been used to determine the various forms of the four thiol compounds in human plasma.