Optimization of Zn2+-containing mobile phase for simultaneous determination of kynurenine,kynurenic acid and tryptophan in human plasma by high performance liquid chromatography

In the present work we have developed a standard-addition HPLC method using a mobile phase containing low concentration of ZnAc₂ to determine physiological level of kynurenine (KYN), kynurenic acid (KYNA) and tryptophan (TRP) in human plasma simultaneously. The method greatly improved the sensitivity of KYNA, the resolution of KYNA and TRP, and avoided clotting risk caused by high concentration of ZnAc₂ in mobile phase. Samples were deproteinized by addition of equal volume of 0.6 mol/L HClO₄. Analytes in supernatants were separated by an Agilent HC-C18 (2) analytical column; an aqueous mobile phase containing 20 mmol/L NaAc, 3 mmol/L ZnAc₂ and 7% acetonitrile at flow rate of 1.0 mL/min. Detections were performed by a variable wavelength detector at wavelength 365 nm for KYN and a fluorescence detector at wavelengths excitation 344 nm and emission 398 nm for KYNA and TRP. Good linear responses were found with r² > 0.999 for all analytes within the concentration range of physiological levels. The limit of detection of the developed method was 0.03 μmol/L, 0.9 nmol/L and 0.4 μmol/L for KYN, KYNA and TRP respectively. Recoveries from spiked human plasma were 95.4–99.7% for KYN, 98.9–104% for KYNA and 96.5–100.2% for TRP. All CVs for the repeatability and intermediate precision were less than 5%. We conclude that the developed method is helpful for the research investigations in KYN pathway of TRP metabolism.     

DNA damage induction and antiproliferative activity of vanadium(V) oxido monoperoxido complex containing two bidentate heteroligands

Several peroxidovanadium(V) complexes have been shown as a potent anticancer agents. The aim of this study was to investigate the interaction of monoperoxidovanadium(V) complex Pr₄N[VO(O₂)(ox)(phen)], (Vphen), [phen = 1,10-phenantroline, ox = oxalate(2?) and Pr₄N = tetra(n-propyl)ammonium(1+)] with DNA. UV–Vis spectrophotometry and the alkaline single-cell gel electrophoresis (SCGE, the comet assay) were used to examine the possibility of the vanadium(V) complex to induce changes in DNA. The interaction of Vphen with calf thymus DNA resulted in absorption hyperchromicity in DNA spectrum and shift of the absorption band of DNA to longer wavelengths for the [complex]/[DNA] concentration ratio equals to 4 and after 60 min of incubation. The rise in DNA absorption (by 34%) and bathochromic shift (Δλ_max = 6 nm) are indicative of the interaction between DNA and the complex molecules. DNA strand breaks in cellular DNA were investigated using the comet assay. The human lymphocytes were exposed to various concentrations of Vphen for 30 min. The results revealed that Vphen contributed to the DNA damage expressed as DNA strand breaks in concentration dependent manner. The used concentrations of Vphen (ranging from 0.1 to 100 μmol/L) caused higher DNA damage in lymphocytes compared to untreated cells (from 1.2 times for 0.1 μmol/L to 1.8 times for 100 μmol/L). Vphen was screened for its potential antitumor activity towards murine leukemia cell line L1210. Vphen exhibited significant antiproliferative activity depending on its concentration and time of exposure. The IC₅₀ values were 0.247 μg/mL (0.45 μmol/L) for 24 h, 0.671 μg/mL (1.21 μmol/L) for 48 h and 0.627 μg/mL (1.13 μmol/L) for 72 h.     

Simultaneous determination of urinary tryptophan, tryptophan-related metabolites and creatinine by high performance liquid chromatography with ultraviolet and fluorimetric detection

A high performance liquid chromatography method with ultraviolet and fluorimetric detection has been developed for the simultaneous determination of urinary creatinine (Cr), tryptophan (Trp) and three Trp-related metabolites including kynurenine (Kyn), kynurenic acid (Kyna) and 5-hydroxyindole-3-acetic acid (5-HIAA). Samples were pretreated by centrifugation after a freeze-thaw cycle to remove protein and other precipitates. Separation was achieved by an Agilent HC-C18 (2) analytical column and a gradient elution program with a constant flow rate 1mL/min at an ambient temperature. Total run time was 30 min. Cr, Kyn and Kyna were measured by a variable wavelength detector at wavelengths 258 nm, 365 nm and 344 nm respectively. Trp and 5-HIAA were measured by a fluorescence detector with an excitation wavelength of 295 nm and an emission wavelength of 340 nm. This allowed the determination of Kyn/Cr, Kyna/Cr, Trp/Cr and 5-HIAA/Cr concentration ratios in a single run on the same urine sample. Good linear responses were found with correlation coefficient (r)>0.999 for all analytes within the concentration range of physiological level. The limit of detection of the developed method was: Cr, 0.0002 g/L; Kyn, 0.1 μmol/L; Kyna, 0.04 μmol/L; Trp, 0.02 μmol/L and 5-HIAA, 0.01 μmol/L. Recoveries from spiked human urine were: Cr, 93.0-106.4%; Kyn, 97.9-106.9%; Kyna, 98.5-105.6%; Trp, 96.7-105.2% and 5-HIAA, 96.1-99.7%. CVs of repeatability and intermediate precision of all analytes were less than 5%. This method has been applied to the analysis of urine samples from normal subjects.     

Omega-3 index determined by gas chromatography with electron impact mass spectrometry

Omega-3 index is a relatively new concept, defined as the sum of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) expressed as a percentage of the total fatty acids in red blood cell membranes. This index reflects medium to long-term intake of omega-3 polyunsaturated fatty acids and could be a useful tool in epidemiological studies. The standard technique used for fatty acid analysis and quantification has been gas chromatography (GC) with flame ionization detection. This method is robust and has good precision and sensitivity. However, a major disadvantage is inability to confirm spectrometrically the identity of fatty acids detected, which is important especially in complex biological samples. The current study measures omega-3 index in 12 healthy human volunteers using GC-mass spectrometry (MS). Both the intra-assay and day-to-day variations were well within 5% with linearity of response extending to a concentration of 250 μg/ml (830 μmol/L) of EPA. The limit of detection of EPA was 0.36 μg/ml (1.2 μmol/L). About 25 fatty acids were consistently detected in red blood cells from healthy volunteers including cis and trans isomers. The omega-3 index ranged from 2.4% to 6.2% among the 12 volunteers examined and there was no difference between samples taken in the fasting and postprandial states. EPA and DHA concentrations ranged from 3.53 to 105.89 μg/ml (11.7–350 μmol/L) and 12.19 to 214.42 μg/ml (37.1–652.7 μmol/L), respectively. Thus a GC–MS method has been developed for measuring the omega-3 index. Further studies are required to determine the role of this index as a predictor of disease.     

Simultaneous HPLC–UV analysis of rufinamide,zonisamide, lamotrigine,oxcarbazepine monohydroxy derivative and felbamate in deproteinized plasma of patients with epilepsy

We present an implementation of a method we previously reported allowing the newer antiepileptic drugs (AEDs) rufinamide (RFN) and zonisamide (ZNS) to be simultaneously determined with lamotrigine (LTG), oxcarbazepine's (OXC) main active metabolite monohydroxycarbamazepine (MHD) and felbamate (FBM) in plasma of patients with epilepsy using high performance liquid chromatography (HPLC) with UV detection. Plasma samples (250 μL) were deproteinized by 1 mL acetonitrile spiked with citalopram as internal standard (I.S.). HPLC analysis was carried out on a Synergi 4 μm Hydro-RP, 250 mm × 4.6 mm I.D. column. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (50 mM, pH 4.5), acetonitrile and methanol (65:26.2:8.8, v/v/v) at an isocratic flow rate of 0.8 mL/min. The UV detector was set at 210 nm. The chromatographic run lasted 19 min. Commonly coprescribed AEDs did not interfere with the assay. Calibration curves were linear for both AEDs over a range of 2–40 μg/mL for RFN and 2–80 μg/mL for ZNS. The limit of quantitation was 2 μg/mL for both analytes and the absolute recovery ranged from 97% to 103% for RFN, ZNS and the I.S. Intra- and interassay precision and accuracy were lower than 10% at all tested concentrations. The present study describes the first simple and validated method for RFN determination in plasma of patients with epilepsy. By grouping different new AEDs in the same assay the method can be advantageous for therapeutic drug monitoring (TDM).     

New derivatives of salicylamides: Preparation and antimicrobial activity against various bacterial species

Three series of salicylanilides, esters of N-phenylsalicylamides and 2-hydroxy-N-[1-(2-hydroxyphenylamino)-1-oxoalkan-2-yl]benzamides, in total thirty target compounds were synthesized and characterized. The compounds were evaluated against seven bacterial and three mycobacterial strains. The antimicrobial activities of some compounds were comparable or higher than the standards ampicillin, ciprofloxacin or isoniazid. Derivatives 3f demonstrated high biological activity against Staphylococcus aureus (?0.03 μmol/L), Mycobacterium marinum (?0.40 μmol/L) and Mycobacterium kansasii (1.58 μmol/L), 3g shows activity against Clostridium perfringens (?0.03 μmol/L) and Bacillus cereus (0.09 μmol/L), 3h against Pasteurella multocida (?0.03 μmol/L) and M. kansasii (?0.43 μmol/L), 3i against methicillin-resistant S. aureus and B. cereus (?0.03 μmol/L). The structure–activity relationships are discussed for all the compounds.     

Determination and pharmacokinetic study of taxifolin in rabbit plasma by high-performance liquid chromatography

Taxifolin has been widely used in the treatment of cerebral infarction and sequelae, cerebral thrombus, coronary heart disease and angina pectoris. A reliable sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV detection for the pharmacokinetic study of taxifolin in rabbit plasma after enzymatic hydrolysis was developed and validated for the first time. Taxifolin, with biochanin A as the internal standard, was extracted from plasma samples by liquid/liquid extraction after hydrolysis with β-glucuronidase and sulfatase. Chromatographic separation was conducted on a Luna C18 column (4.6 mm×150 mm, 5 μm particle size) and pre-column (2.0 mm, the same sorbent). Two-step linear gradient elution with acetonitrile and 0.03% water solution of trifluoroacetic acid as mobile phase at a flow rate of 1.0 ml/min was used. The UV detector is set at 290 nm. The elution time for taxifolin and biochanin A was approximately 7.9 and 18.3 min, respectively. The calibration curve of taxifolin was linear (r>0.9997) over the range of 0.03–5.0 μg/ml in rabbit plasma. The limit of detection (LOD) and limit of quantification (LOQ) for taxifolin were 0.03 and 0.11 μg/ml, respectively. The present method was successfully applied for the estimation of the pharmacokinetic parameters of taxifolin following intravenous and oral administration of lipid solution to rabbits. The absolute bioavailability of taxifolin after oral administration of lipid solution was 36%.     

Novel liquid chromatographic determination of cystatin C in human urine

A rapid and sensitive method for quantitative determination of cystatin C (CC) protein in human urine via HPLC was developed and validated. Acetone has been used as a precipitating agent of CC protein from the urine biological matrix. Separation and detection were accomplished by ion pair liquid chromatography and UV detection. Gradient elution mode was utilized to elute CC with a UV detection of 224 nm. The analysis time was 14 min per sample using Ace C8 (150 × 4.6 mm i.d., 5 μm) as a chromatographic column with a flow rate of 1.0 mL/min. Calibration curve with good linearity (r² = 0.99) within the range from 0.390 to 0.001 mg/mL was obtained. Limits of detection and quantification were 0.001 and 0.002 mg/mL, respectively. Inter-assay and intra-assay variabilities were <15% for all levels and <20% at the limit of quantification level. Major advantages of the method: specific where no false positive results might be obtained and fast where sample pretreatment needs only 1 h.     

Simultaneous HPLC–UV determination of rhein and aceclofenac in human plasma

Diacerein and aceclofenac are prescribed for reducing the symptoms associated with osteoarthritis. We present a simple HPLC method with UV detection for simultaneous determination of rhein (the immediate metabolite of diacerein) and aceclofenac from human plasma samples. Sample preparation was accomplished through liquid–liquid extraction with ethyl acetate and chromatographic separation was performed on a reversed-phase ODS column. Mobile phase consisted of a mixture of acetate buffer and acetonitrile run under gradient at flow rate of 1.0 ml/min. Wavelength was set at 258 nm. The method was validated for linearity, accuracy, precision and stability. The calibration was linear over the range of 0.1–7.0 μg/ml for rhein and 0.5–20 μg/ml for aceclofenac using 500 μl plasma samples. Extraction recoveries were 85% for rhein and 70% for aceclofenac. The method can easily be adopted for high-throughput clinical and pharmacokinetic studies of above two-drug fixed dose combination formulations.     

Simple and rapid liquid chromatography method for determination of moxifloxacin in plasma

A high performance liquid chromatographic method for determination of moxifloxacin in human plasma was developed. The method involved deproteinisation of the sample with perchloric acid and analysis of the supernatant using a reversed-phase C₁₈ column (150 mm) and fluorescence detection at an excitation wavelength of 290 nm and an emission wavelength of 460 nm. The assay was specific for moxifloxacin and linear from 0.125 to 10.0 μg/ml. The relative standard deviation of intra- and inter-day assays was lower than 10%. The average recovery of moxifloxacin from plasma was 101%. Due to its simplicity, the assay can be used for pharmacokinetic studies of moxifloxacin.     

Determination of asiatic acid in beagle dog plasma after oral administration of Centella asiatica extract by precolumn derivatization RP-HPLC

A novel precolumn derivatization reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV–vis detection for the quantitative determination of total concentration of asiatic acid (AA) in beagle dog plasma is described. AA was extracted with n-hexane-dichloromethane-2-propanol (20:10:1, v/v/v) from plasma, which had been hydrolyzed by acid and derivatized with p-Toluidine. Chromatographic separation was achieved on a C₁₈ column using gradient elution in a water–methanol system. Detection was set at UV wavelength of 248 nm. A calibration curve ranging from 0.01 to 1.5 μg/mL was shown to be linear, and the lower limit of quantification (LLOQ) was 0.01 μg/mL. The intra- and inter-day precisions which were determined by three different concentrations (0.05, 0.2 and 0.8 μg/mL) ranged from 4.4% to 13.1% and 4.6% to 14.2%, respectively. Mean extraction recoveries were no less than 65% for AA and ursolic acid (IS). Plasma samples containing asiatic acid were stable for 30 days at ?20 °C. The method was successfully applied to a pharmacokinetic study in beagle dogs after oral administration of Centella asiatica extract, and the main pharmacokinetic parameters obtained were: T_1/2, 4.29 h; T_max, 2.70 h; C_max, 0.74 μg/mL; AUC_0–t and AUC_0–∞, 3.74 and 3.82 μg h/mL, respectively.     

Optimized method for the determination of itopride in human plasma by high-performance liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic method with fluorescence detection for the determination of itopride in human plasma is reported. The sample preparation was based on liquid–liquid extraction of itopride from plasma with t-butylmethylether and dichloromethane (70:30, v/v) mixture followed by a back extraction of the analyte to the phosphate buffer (pH 3.2). Liquid chromatography was performed on an octadecylsilica column (55 mm × 4 mm, 3 μm particles), the mobile phase consisted of acetonitrile–triethylamine–15 mM dihydrogenpotassium phosphate (14.5:0.5:85, v/v/v), pH of the mobile phase was adjusted to 4.8. The run time was 3 min. The fluorimetric detector was operated at 250/342 nm (excitation/emission wavelength). Naratriptan was used as the internal standard. The limit of quantitation was 9.5 ng/ml using 0.5 ml of plasma. The method precision and inaccuracy were less than 8%. The assay was applied to the analysis of samples from a bioequivalence study.     

Ethyl isovalerate synthesis using Candida rugosa lipase immobilized on silica nanoparticles prepared in nonionic reverse micelles

Uniform and monodispersed silica nanoparticles were synthesized with a mean diameter of 100 ± 20 nm as analyzed by Transmission Electron Microscopy (TEM). Glutaraldehyde was used as a coupling agent for efficient binding of the lipase onto the silica nanoparticles. For the hydrolysis of pNPP at pH 7.2, the activation energy within 25–40 °C for free and immobilized lipase was 7.8 and 1.25 KJ/mol, respectively. The V_max and K_m of immobilized lipase at 25 °C for pNPP hydrolysis were found to be 212 μmol/min/mg and 0.3 mM, whereas those for free lipase were 26.17 μmol/min and 1.427 mM, respectively. The lower activation energy of immobilized lipase in comparison to free lipase suggests a change in conformation of the enzyme leading to a requirement for lower energy on the surface of the nanoparticles. A better yield (7 fold higher) of ethyl isovalerate was observed using lipase immobilized onto silica nanoparticles in comparison to free lipase.     

Labeling of nucleosides with fluorescamine and detection by spectrofluorometer for End Stage Renal Disease

Nucleosides are characterized as biomarkers in AIDS, Alzheimer, tumor, breast cancer and various malignant diseases. In the present work a direct method for the detection of nucleosides (adenosine, cytidine, uridine and guanosine) from urine samples has been developed. Nucleosides represent the extent of damage in genetic material, analysis of nucleosides by ultrasonic assisted microextraction effectively eliminates the interfering constituent of urine. This has made it a highly selective and sensitive method to analyze the nucleosides with a lower limit of detection 0.220 μmol/L and Limit of quantitation 0.660 μmol/L. The method has been validated with good linearity and correlation of coefficients of the calibration curves was higher than 0.997. The coefficients were in the range of 0.11–16.92% (inter-day) and 0.38–16.43% (intra-day), respectively.     

Determination of phenothiazine derivatives in human urine by using ionic liquid-based dynamic liquid-phase microextraction coupled with liquid chromatography

A simple and rapid method for the determination of seven phenothiazines derivatives (chlorpromazine, promethazine, levomepromazine, prochlorperazine, trifluoperazine, fluphenazine and thioridazine) in human urine samples is presented. The analytes are extracted from the sample in 50 μL of the ionic liquid 1-butyl-3-methyl-imidazolium hexafluorophosphate working in an automatic flow system under dynamic conditions. The chemical affinity between the extractant and the analytes allows a good isolation of the drugs from the sample matrix achieving at the same time their preconcentration. The separation and detection of the extracted compounds is accomplished by liquid chromatography and UV detection. The proposed method is a valuable alternative for the analysis of these drugs in urine within the concentration range 0.07–10 μg mL^?1. Limits of detection were in the range from 21 ng mL^?1 (thioridazine) to 60 ng mL^?1 (levomepromazine). The repeatability of the proposed method expressed as RSD (n = 5) varied between 2.2% (levomepromazine) and 3.9% (chlorpromazine).     

Molecular mechanisms of novel peptides from silkworm pupae that inhibit α-glucosidase

The objectives of this study were to identify peptides that inhibit α-glucosidase using a quantitative structure-activity relationship (QSAR) screening method and a database of silkworm peptides. This study compared the docking characteristics of several peptides with high inhibitory activity against α-glucosidase and summarized the molecular mechanisms by which the silkworm peptides affected α-glucosidase. Four peptides that strongly inhibited α-glucosidase were obtained: Gln-Pro-Gly-Arg with IC₅₀ at 65.8 μmol/L, Ser-Gln-Ser-Pro-Ala at 20 μmol/L, Gln-Pro-Pro-Thr at 560 μmol/L and Asn-Ser-Pro-Arg at 205 μmol/L. Studies docking the peptides to the active site of α-glucosidase (PDB ID: 2QMJ) showed that a common characteristic was Lys776 in 2QMJ, which could be a critical target for α-glucosidase trapping of inhibitory peptides. The results revealed that the four peptides, especially Ser-Gln-Ser-Pro-Ala, could be potential drugs for treating diabetes.     

Nitroxyl (HNO) suppresses vascular Nox2 oxidase activity

Nox2 oxidase activity underlies the oxidative stress and vascular dysfunction associated with several vascular-related diseases. We have reported that nitric oxide (NO) decreases reactive oxygen species production by endothelial Nox2. This study tested the hypothesis that nitroxyl (HNO), the redox sibling of NO, also suppresses vascular Nox2 oxidase activity. Specifically, we examined the influence of two well-characterized HNO donors, Angeli’s salt and isopropylamine NONOate (IPA/NO), on Nox2-dependent responses to angiotensin II (reactive oxygen species production and vasoconstriction) in mouse cerebral arteries. Angiotensin II (0.1 μmol/L)-stimulated superoxide (measured by lucigenin-enhanced chemiluminescence) and hydrogen peroxide (Amplex red fluorescence) levels in cerebral arteries (pooled basilar and middle cerebral (MCA)) from wild-type (WT) mice were ~60% lower (P<0.05) in the presence of either Angeli’s salt (1 μmol/L) or IPA/NO (1 μmol/L). Similarly, phorbyl 12,13-dibutyrate (10 μmol/L; Nox2 activator)-stimulated hydrogen peroxide levels were ~40% lower in the presence of IPA/NO (1 μmol/L; P<0.05). The ability of IPA/NO to decrease superoxide levels was reversible and abolished by the HNO scavenger l-cysteine (3 mmol/L; P<0.05), but was unaffected by hydroxocobalamin (100 μmol/L; NO scavenger), ODQ (10 μmol/L; soluble guanylyl cyclase (sGC) inhibitor), or Rp-8-pCPT-cGMPS (10 μmol/L; cyclic guanosine monophosphate (cGMP)-dependent protein kinase inhibitor). Angiotensin II-stimulated superoxide was substantially less in arteries from Nox2-deficient (Nox2^−/y) versus WT mice (P<0.05). In contrast to WT, IPA/NO (1 μmol/L) had no effect on superoxide levels in arteries from Nox2^−/y mice. Finally, angiotensin II (1–1000 μmol/L)-induced constriction of WT MCA was virtually abolished by IPA/NO (1 μmol/L), whereas constrictor responses to either the thromboxane A₂ mimetic U46619 (1–100 nmol/L) or high potassium (122.7 mmol/L) were unaffected. In conclusion, HNO suppresses vascular Nox2 oxidase activity via a sGC–cGMP-independent pathway. Thus, HNO donors might be useful therapeutic agents to limit and/or prevent Nox2-dependent vascular dysfunction.     

3-Penten-2-one,a novel aldehyde adduct,is a biomarker for increased acetaldehyde in urine

Diagnostic profiling of urine for volatile compounds of around 400 patients using headspace solid-phase microextraction (HS-SPME) in alkaline conditions identified 3-penten-2-one (approximately 1 to >6.3 μmol/L) in 26 patients. Five were in barbiturate coma. 3-Penten-2-one, previously of unknown origin, was shown to be formed by aldol condensation of acetaldehyde with acetone or acetoacetate during analysis. Semi-quantification of acetaldehyde using in-fibre derivatisation HS-SPME, showed high concentrations in five urine (33–348 μmol/L) and two plasma (17 and 43 μmol/L) samples. Hence, urinary 3-penten-2-one is a useful biomarker for increased accumulation of acetaldehyde during abnormal metabolic stress.     

HPLC analysis of human erythrocytic glutathione forms using OPA and N-acetyl-cysteine ethyl ester: Evidence for nitrite-induced GSH oxidation to GSSG

Glutathione exists in biological samples in the reduced form (GSH), as its disulfide (GSSG) and as a mixed disulfide (GSSR) with thiols (RSH). GSH is the most abundant low-molecular-mass thiol and plays important roles as a cofactor and as a main constituent of the intracellular redox status. Due to its own sulfhydryl (SH) group, GSH reacts readily with o-phthaldialdehyde (OPA) to form a highly stable and fluorescent isoindole derivative (GSH-OPA), which allows for sensitive and specific quantitative determination of GSH in biological systems by HPLC with fluorescence (FL) detection. In the present article we report on the utility of the novel, strongly disulfide bond-reducing thiol N-acetyl-cysteine ethyl ester (NACET) for the specific quantitative analysis of GSH and GSSG in the cytosol of red blood cells (RBC) as GSH-OPA derivative with FL (excitation/emission 338/458 nm) or UV absorbance (338 nm) detection. Unlike in aqueous solution, the derivatization of GSH in RBC cytosol yielded two closely related derivatives in the absence of NACET and only the GSH-OPA derivative in the presence of NACET. The HPLC method was optimized and validated for human RBC and applied to measure GSH and GSSG in RBC of healthy subjects. Basal GSH and GSSG concentrations were determined to be 2340 ± 350 μM and 11.4 ± 3.2 μM, respectively, in RBC of 12 healthy young volunteers (aged 23–38 years). The method was also applied to study the effects of nitrite on the glutathione status in intact and lysed human RBC. Nitrite at mM-concentrations caused instantaneous and considerable GSSG formation in lysed but much less pronounced in intact RBC. GSH externally added to lysed RBC inhibited nitrite-induced methemoglobin formation. Our findings suggest that nitric oxide/nitrite-related consumption rate of GSH, and presumably that of NADH and NADPH, could be of the order of 600 μmol/day in RBC of healthy subjects.     

Simultaneous determination of procaine,lidocaine, ropivacaine,tetracaine and bupivacaine in human plasma by high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method has been developed and validated for simultaneous quantification of five local anesthetics in human plasma: procaine, lidocaine, ropivacaine, tetracaine and bupivacaine. In an ice-water bath, 500 μL plasma sample, containing 100 μg/mL neostigmine methylsulfate as anticholinesterase, was spiked with carbamazepine as internal standard and alkalized by sodium hydroxide. Liquid–liquid extraction with ethyl ether was used for plasma sample preparation. The chromatographic separation was achieved on a Kromosil ODS C18 column with a mobile phase consisting of 30 mM potassium dihydrogen phosphate buffer (0.16% triethylamine, pH adjusted to 4.9 with phosphoric acid) and acetonitrile (63/37, v/v). The detection was performed simultaneously at wavelengths of 210 and 290 nm. The chromatographic analysis time was 13 min per sample. The calibration curves of all five analytes were linear between 0.05 and 5.0 μg/mL (r² ≥ 0.998). Precision ranged from 1.4% to 7.9% and accuracy was between 91.7% and 106.5%. The validated method is applicable for simultaneous determination of procaine, lidocaine, ropivacaine, tetracaine and bupivacaine for therapeutic drug monitoring and pharmacokinetic study.