Joachim Fauler

Analysis of cysteine and N-acetylcysteine in human plasma by high-performance liquid chromatography at the basal state and after oral administration of N-acetylcysteine

A high-performance liquid chromatographic method for the determination of free reduced cysteine and N-acetylcysteine in human plasma at the basal state and after oral administration of N-acetylcysteine is described. The method is based on acid-catalysed conversion of plasma thiols to the corresponding S-nitroso derivatives by excess of nitrite and their subsequent cation-pairing RP-HPLC with detection at 333 nm. Recovery rates of cysteine and N-acetylcysteine added to human plasma were 94.6 and 99.6%, respectively. Inter- and intra-day precision were below 6%. In healthy humans (n = 5), free reduced cysteine was determined to be (mean+/-S.E.) 10.0+/-0.96 microM. No N-acetylcysteine was detected in plasma of these subjects above the limit of detection (e.g. 170 nM). The method was successfully applied to a pharmacokinetic study on orally administered N-acetylcysteine to healthy volunteers.

Specific and rapid quantification of 8-iso-prostaglandin F2α in urine of healthy humans and patients with Zellweger syndrome by gas chromatography–tandem mass spectrometry

8-iso-Prostaglandin F2alpha (8-iso-PGF2alpha) is currently discussed as a potential index parameter of oxidative stress in vivo. We describe in this article a fully validated gas chromatographic-tandem mass spectrometric method for the quantitative determination of 8-iso-PGF2alpha in human urine. The method is highly specific and requires a single thin-layer chromatographic step for sample purification. Inter- and intraday imprecision were below 8%. Mean inaccuracy was 5.3% for added levels of 8-iso-PGF2alpha up to 2000 pg/ml of urine. We measured highly elevated excretion of 8-iso-PGF2alpha in the urine of children with peroxisomal beta-oxidation deficiency, i.e. Zellweger syndrome, (63.3+/-16.6 ng/mg creatinine) compared to that of healthy children (0.51+/-0.16 ng/mg creatinine) (mean+/-S.D., both n=5). The method could be useful for diagnosing Zellweger syndrome and for investigating the utility of 8-iso-PGF2alpha as a novel marker for oxidative stress in vivo in man.

Gas chromatographic—mass spectrometric determination of leukotriene E4 in human urine using deuterium-labelled leukotriene E4 standards

The utility of two deuterium-labelled leukotriene (LT) E4 analogs, e.g. [20,20,20-2H3]LTE4 and [14,15,17,17,18,18-2H6]LTE4, as internal standards for the determination of LTE4 in human urine by gas chromatography-mass spectrometry (GC-MS) was investigated. 2H-Exchange during hydrogenation occurred both in [20,20,20-2H3]LTE4 and [14,15,17,17,18,18-2H6]LTE4 in an extent of 9.4 +/- 0.5% and 67.3 +/- 0.6% (mean +/- S.D., n = 6), respectively. The lower extent of 2H-exchange in [20,20,20-2H3]LTE4 allowed a more accurate quantitation than the use of [14,15,17,17,18,18-2H6]LTE4. Applying [20,20,20-2H3]LTE4 as internal standard the coefficients of variation for the intra- and inter-assay determination of LTE4 in human urine were 5.7% and 6.2% (n = 4), respectively. The inter-assay coefficient of variation for [14,15,17,17,18,18-2H6]LTE4 was 15%. Using [20,20,20-2H3]LTE4 as internal standard and GC-MS, healthy volunteers were found to excrete 17 +/- 10 nmol LTE4 per mol creatinine (mean +/- S.D., n = 11). Similar excretion rates for LTE4 in urine of healthy volunteers were found using GC-tandem MS with [1,1-18O2]LTE4 as internal standard. Our results demonstrate that [20,20,20(-2)H3]LTE4 is a suitable internal standard for the GC-MS determination of urinary LTE4.

Determination of chloride in biological fluids as pentafluorobenzylchloride by reversed-phase high-performance liquid chromatography and UV detection

SummaryA reversed-phase high-performance liquid chromatographic method for the determination of chloride in plasma, urine, saliva, sweat and aqueous solution is described. Chloride, in solution in aqueous acetone, is converted by means of pentafluorobenzyl bromide into pentafluorobenzyl chloride. This derivative is separated on a ODS-5 μm reversed-phase column using isocratic elution with acctonitrile/water, 50/50, v/v, at a flow rate of 2.0 ml/min, and detected by a UV detector at 264 nm. The method is rapid, accurate and sufficiently sensitive for the determination of chloride in less than 10 μl sample volume of a biological fluid.

Enzymic preparation of dioxygen-18 labelled leukotriene E4 and its use in quantitative gas chromatography—mass spectrometry

A simple and rapid method is described for the preparation of a stable isotope oxygen-18 labelled leukotriene E4 (LTE4). Oxygen-18 labelling of LTE4 methyl ester in oxygen-18 water catalysed by a pig liver esterase resulted in the incorporation of two oxygen-18 atoms in the carboxylic group of LTE4 to the extent of 89.8% ([18O2]LTE4) and one oxygen-18 atom to the extent of 9.4% ([16O18O]LTE4), with only 0.7% remaining unchanged ([16O2]LTE4). [18O2]LTE4 was found not to back-exchange following incubation in acidified urine (pH 4.0) at 4 degrees C for up to 20 h. [18O2]LTE4 was demonstrated to be a useful internal standard in a method for the quantitative determination of LTE4 in human urine involving high-performance liquid chromatography and gas chromatography with negative-ion chemical ionization tandem mass spectrometry: the concentration of LTE4 in a 24-h urine sample of a healthy subject was determined to be 68.1 pg/ml.

Chemical synthesis of dioxygen-18 labelled ω-/β-oxidized cysteinyl leukotrienes: analysis by gas chromatography-mass spectrometry and gas chromatography-tandem mass spectrometry

Abstract Cysteinyl leukotrienes (LT) C 4 , LTD 4 , and LTE 4 are potent mediators of anaphylaxis and inflammatin. LTE 4 is extensively metabolized in man mainly by ω-oxidation followed by subsequent β-oxidation to more polar and biologically inactive metabolites. This paper describes a method for the synthesis of [1,20− 18 O 2 ]-carboxy-LTE 4 , [1,18− 18 O 2 ]-carboxy-dinor-LTE 4 , and [1,16− 18 O 2 ]-carboxy-14,15-dihydro-tetranor-LTE 4 starting from the unlabelled dimethyl esters of 20-carboxy-LTA 4 , 18-carboxy-dinor-LTA 4 and 16-carboxy-14,15-dihydro-tetranor-LTA 4 , respectively, by separate chemical conjugation with cysteine hydrochloride in H 2 18 O-methanol followed by alkaline hydrolysis with Li 18 OH. The isotopic purity of the isolated reaction products was 94% at 18 O for all three preparations while only 0.3% remained unlabelled as confirmed by negative-ion chemical-ionization gas chromatography-mass spectrometry (GC-NICI-MS) after their catalytical reduction/desulphurization and derivation. The 18 O 2 -labelled compounds are demonstrated to be suitable internal standards for quantification by GC-NICI-MS and GC-NICI-tandem MS. We found by GC-NICI-tandem MS that the excretion rate of 20-carboxy-LTE 4 is comparable to that of LTE 4 (both in nmol/mol creatinine, mean ± S.E.) in healthy children (26.7 ± 4.7 vs. 32.0 ± 6.0, n = 9) and adults (13.9 ± 1.1 vs. 27.2 ± 5.4, n = 3).

Capillary isotachophoretic determination of cysteinyl leukotrienes

The cysteinyl leukotrienes (LTs) C4, D4 and E4 are among the most potent lipid mediators of anaphylaxis and inflammation. A capillary isotachophoretic method is described for the determination of these cysteinyl LTs. The method is based on anionic separation and detection using UV (254 nm) and conductivity detectors. The total analysis time is of the order of 30 min. The limit of detection of the method was determined to be 0.5 nmol of LTE4. Despite of similar chemical structures, all three cysteinyl LTs can be determined simultaneously.

Analysis of leukotriene B4 as free acid and its δ-lactone by high-performance liquid chromatography: a study on the stability and formation of leukotriene B4 δ-lactone

SummaryReversed-phase high-performance liquid chromatography (RP-HPLC) with UV detection (270 nm) was applied to study the stability of LTB4 δ-lactone in aqueous solutions and its chemical formation from LTB4. LTB4 δ-lactone was found to be unstable in aqueous buffered solutions (pH range 4 to 8) and was converted in a pH and time depending manner mainly to LTB4 and most probably to an isomer of LTB4 δ-lactone. This compound was spontaneously converted to LTB4 δ-lactone when incubated in the buffers and exists in equilibrium with LTB4 δ-lactone at a molar ratio of about 1:1. Incubation of LTB4 in buffers (pH 4, 6 and 8) and human plasma did not result in formation of detectable amounts of LTB4 δ-lactone. Conversion of LTB4 into its δ-lactone form in a yield of 62±6% (mean ± SD, n=3) was achieved by treatment of LTB4 with a mixture of acetic acid, acetic acid anhydride and granular calcium chloride (1/5/3, v/v/w %) in ethylacetate. Evidence for LTB4δ-lactone formation from LTB4 under these conditions was demonstrated by gas chromatography-mass spectrometry.