Anja Mitschke

GC-MS determination of creatinine in human biological fluids as pentafluorobenzyl derivative in clinical studies and biomonitoring: Inter-laboratory comparison in urine with Jaffé, HPLC and enzymatic assays.

In consideration of its relatively constant urinary excretion rate, creatinine in urine is a useful biochemical parameter to correct the urinary excretion rate of endogenous and exogenous biomolecules. Assays based on the reaction of creatinine and picric acid first reported by Jaffé in 1886 still belong to the most frequently used laboratory approaches for creatinine measurement in urine. Further analytical methods for creatinine include HPLC-UV, GC-MS, and LC-MS and LC-MS/MS approaches. In the present article we report on the development, validation and biomedical application of a new GC-MS method for the reliable quantitative determination of creatinine in human urine, plasma and serum. This method is based on the derivatization of creatinine (d(0)-Crea) and the internal standard [methyl-trideutero]creatinine (d(3)-Crea) with pentafluorobenzyl (PFB) bromide in the biological sample directly or after dilution with phosphate buffered saline, extraction of the reaction products with toluene and quantification in 1-μl aliquots of the toluene extract by selected-ion monitoring of m/z 112 for d(0)-Crea-PFB and m/z 115 for d(3)-Crea-PFB in the electron-capture negative-ion chemical ionization mode. The limit of detection of the method is 100 amol of creatinine. In an inter-laboratory study on urine samples from 100 healthy subjects, the GC-MS method was used to test the reliability of currently used Jaffé, enzymatic and HPLC assays in clinical and occupational studies. The results of the inter-laboratory study indicate that all three tested methods allow for satisfactory quantification of creatinine in human urine. The GC-MS method is suitable for use as a reference method for urinary creatinine in humans. In serum, creatine was found to contribute to creatinine up to 20% when measured by the present GC-MS method. The application of the GC-MS method can be extended to other biological samples such as saliva.

Specific GC-MS/MS stable-isotope dilution methodology for free 9- and 10-nitro-oleic acid in human plasma challenges previous LC-MS/MS reports.

Nitrated unsaturated fatty acids including nitro-oleic acid (NO(2)-OA) have been measured in human blood samples in their free and esterified forms. Plasma concentrations in healthy subjects have been reported to be of the order of 600 nM for free NO(2)-OA and 300 nM for esterified NO(2)-OA, as measured by LC-MS/MS. In the present article we report a GC-MS/MS method for the specific and accurate quantification of two NO(2)-OA isomers, i.e., 9-NO(2)-OA and 10-NO(2)-OA, in human plasma using newly prepared, isolated, characterized and standardized (15)N-labeled analogs. This method involves SPE extraction of fatty acids from slightly acidified plasma samples (pH 5), conversion to their pentafluorobenzyl (PFB) esters, isolation by HPLC, solvent extraction from a single HPLC fraction and GC-MS/MS analysis in the electron capture negative-ion chemical ionization (ECNICI) mode. Quantification was performed by selected-reaction monitoring (SRM) of m/z 46 ([NO(2)](-)) and m/z 47 ([(15)NO(2)](-)) produced by collision-induced dissociation (CID) from the parent ions [M-PFB](-) at m/z 326 for endogenous 9-NO(2)-OA and 10-NO(2)-OA and m/z 327 for the internal standards 9-(15)NO(2)-OA and 10-(15)NO(2)-OA. We partially validated the GC-MS/MS method for 9-NO(2)-OA and 10-NO(2)-OA in human plasma and quantified these nitro-oleic species in plasma of 15 healthy volunteers. We identified two isomers, i.e., 9-NO(2)-OA and 10-NO(2)-OA, in human plasma under physiological conditions and found these nitrated fatty acids at a mean concentration of 1 nM each. This concentration is about 600 times lower than that reported by others using LC-MS/MS. Our GC-MS/MS studies on nitro-oleic acid and 3-nitrotyrosine suggest that the extent of nitration of biomolecules such as unsaturated fatty acids and tyrosine is very low in health. In this article we discuss analytical and biological ramifications potentially associated with measurement of nitrated biomolecules in biological systems.

Stable-isotope dilution GC―MS approach for nitrite quantification in human whole blood, erythrocytes, and plasma using pentafluorobenzyl bromide derivatization: Nitrite distribution in human blood

Previously, we reported on the usefulness of pentafluorobenzyl bromide (PFB-Br) for the simultaneous derivatization and quantitative determination of nitrite and nitrate in various biological fluids by GC-MS using their (15)N-labelled analogues as internal standards. As nitrite may be distributed unevenly in plasma and blood cells, its quantification in whole blood rather than in plasma or serum may be the most appropriate approach to determine nitrite concentration in the circulation. So far, GC-MS methods based on PFB-Br derivatization failed to measure nitrite in whole blood and erythrocytes because of rapid nitrite loss by oxidation and other unknown reactions during derivatization. The present article reports optimized and validated procedures for sample preparation and nitrite derivatization which allow for reliable quantification of nitrite in human whole blood and erythrocytes. Essential measures for stabilizing nitrite in these samples include sample cooling (0-4°C), hemoglobin (Hb) removal by precipitation with acetone and short derivatization of the Hb-free supernatant (5 min, 50°C). Potassium ferricyanide (K(3)Fe(CN)(6)) is useful in preventing Hb-caused nitrite loss, however, this chemical is not absolutely required in the present method. Our results show that accurate GC-MS quantification of nitrite as PFB derivative is feasible virtually in every biological matrix with similar accuracy and precision. In EDTA-anticoagulated venous blood of 10 healthy young volunteers, endogenous nitrite concentration was measured to be 486±280 nM in whole blood, 672±496 nM in plasma (C(P)), and 620±350 nM in erythrocytes (C(E)). The C(E)-to-C(P) ratio was 0.993±0.188 indicating almost even distribution of endogenous nitrite between plasma and erythrocytes. By contrast, the major fraction of nitrite added to whole blood remained in plasma. The present GC-MS method is useful to investigate distribution and metabolism of endogenous and exogenous nitrite in blood compartments under basal conditions and during hyperemia.

Gas Chromatography−Mass Spectrometry Analysis of Nitrite in Biological Fluids without Derivatization

We report on a gas chromatography-mass spectrometry (GC-MS) method for the quantification of nitrite in biological fluids without preceding derivatization. This method is based on the solvent extraction with ethyl acetate of nitrous acid (HONO, pK(a) = 3.29), i.e., HO(14)NO and (15)N-labeled nitrous acid (HO(15)NO) which was supplied as the sodium salt of (15)N-labeled nitrite and served as the internal standard. HO(14)NO and HO(15)NO react within the injector (at 300 degrees C) of the gas chromatograph with the solvent ethyl acetate to form presumably unlabeled and (15)N-labeled acetyl nitrite, respectively. Under negative ion chemical ionization (NICI) conditions with methane as the reagent gas, these species ionize to form O(14)NO(-) (m/z 46) and O(15)NO(-) (m/z 47), respectively. Quantification is performed by selected ion monitoring of m/z 46 for nitrite and m/z 47 for the internal standard. Nitrate at concentrations up to 20 mM does not interfere with nitrite analysis in this method. The GC-MS method was validated for the quantification of nitrite in aqueous buffer, human urine (1 mL, acidification) and saliva (0.1-1 mL, acidification), and hemolysates. The method was applied in studying reactions of nitrite (0-10 mM) with oxyhemoglobin ( approximately 6 mM) in lysed human erythrocytes (100 microL aliquots, no acidification).

Measurement of 3-Nitro-Tyrosine in Human Plasma and Urine by Gas Chromatography-Tandem Mass Spectrometry

Reaction of reactive nitrogen species (RNS), such as peroxynitrite and nitryl chloride with soluble tyrosine and tyrosine residues in proteins produces soluble 3-nitro-tyrosine and 3-nitro-tyrosino-proteins, respectively. Regular proteolysis of 3-nitro-tyrosino-proteins yields soluble 3-nitro-tyrosine. 3-Nitro-tyrosine circulates in plasma and is excreted in the urine. Both circulating and excretory 3-nitro-tyrosine are considered suitable biomarkers of nitrative stress. Tandem mass spectrometry coupled with gas chromatography (GC-MS/MS) or liquid chromatography (LC-MS/MS) is one of the most reliable analytical techniques to determine 3-nitro-tyrosine. Here, we describe protocols for the quantitative determination of soluble 3-nitro-tyrosine in human plasma and urine by GC-MS/MS.

Measurement of nitrite in urine by gas chromatography-mass spectrometry.

Nitric oxide (NO) is enzymatically produced from L-arginine and has a variety of biological functions. Autoxidation of NO in aqueous media yields nitrite (O = N-O(-)). NO and nitrite are oxidized in erythrocytes by oxyhemoglobin to nitrate (NO(3)(-)). Nitrate reductases from bacteria reduce nitrate to nitrite. Nitrite and nitrate are ubiquitous in nature, they are present throughout the body and they are excreted in the urine. Nitrite in urine has been used for several decades as an indicator and measure of bacteriuria. Since the identification of nitrite as a metabolite of NO, circulating nitrite is also used as an indicator of NO synthesis and is considered an NO storage form. In contrast to plasma nitrite, the significance of nitrite in the urine beyond bacteriuria is poorly investigated and understood. This chapter describes a gas chromatography-mass spectrometry (GC-MS) protocol for the quantitative determination of nitrite in urine of humans. Although the method is useful for detection and quantification of bacteriuria, the procedures described herein are optimum for urinary nitrite in conditions other than urinary tract infection. The method uses [(15)N]nitrite as internal standard and pentafluorobenzyl bromide as the derivatization agent. Derivatization is -performed on 100-μL aliquots and quantification of toluene extracts by selected-ion monitoring of m/z 46 for urinary nitrite and m/z 47 for the internal standard in the electron-capture negative-ion chemical ionization mode.

18O-Labeled nitrous acid and nitrite: Synthesis, characterization, and oxyhemoglobin-catalyzed oxidation to 18O-labeled nitrate

We describe a simple laboratory method for specific labeling of nitrite with ¹⁸O for use in chemical and biochemical studies in the area of nitric oxide research. NaNO₂ (0.1 mmol) is diluted in H₂¹⁸O (45 μl) and acidified with HCl (1 μl, 5 M), and the solution is allowed to equilibrate. Subsequently, the sample is mixed by vortexing with ethyl acetate (500 μl), and the organic phase is dried over anhydrous Na₂SO(4). Ethyl acetate is evaporated to dryness, and the residue is reconstituted in phosphate-buffered saline. In human blood hemolysate, oxyhemoglobin (HbFe¹⁶O₂) was shown to oxidize N¹⁸O₂⁻ to ¹⁶ON¹⁸O₂⁻.

Evidence by gas chromatography–mass spectrometry of ex vivo nitrite and nitrate formation from air nitrogen oxides in human plasma, serum, and urine samples

Nitrite and nitrate in body fluids and tissues result from dietary source, endogenous nitric oxide (NO) production and from NO and its higher oxides (NO(x)) present as pollutants in the atmosphere. Nitrite and nitrate in human blood serum and plasma or urine are commonly used as biomarkers and measures of endogenous NO synthesis. In addition to dietary intake of nitrite and nitrate, our study indicates that NO(x) naturally present in the laboratory air may be an abundant source for nitrite and nitrate in human serum, plasma, and urine ex vivo. These artifacts can be effectively reduced by closing sample-containing vials during sample treatment.

Even and carbon dioxide independent distribution of nitrite between plasma and erythrocytes of healthy humans at rest

In the literature, the distribution of nitrite and nitrate, the major metabolites of nitric oxide (NO), between plasma and erythrocytes and its dependency on partial CO₂ pressure (pCO₂) in mammalian blood are uncertain. By means of a previously reported fully validated stable-isotope dilution gas chromatography-mass spectrometry (GC-MS) method, we measured nitrite and nitrate concentrations in heparinized plasma from venous, arterial and arterialized blood donated by five healthy non-exercising volunteers at three different time points (0, 15, 30 min). pCO₂, pH and oxygen saturation were measured by standard techniques. The nitrite and nitrate concentrations and the nitrite-to-nitrate ratio in plasma did not correlate with pCO₂ (r=-0.272, P=0.07). Nitrite was found to be almost evenly distributed between plasma and erythrocytes of another eleven healthy non-exercising subjects. In a rabbit model of ARDS, no differences were found in the plasma nitrite and nitrate concentrations comparing normoventilation with hypercapnia. Our studies suggest that the distribution of nitrite between plasma and erythrocytes at rest is largely even and independent of pCO₂ in blood of healthy humans and rabbits with ARDS.

Stable-isotope dilution GC-MS method for ethanol in vapour ethanol and microdialysis systems based on carbonate-catalyzed extractive pentafluorobenzoylation

Common ethanol detection methods are not applicable to cell culture media and microdialysates due to interference with medium constituents including amino acids and pH indicators. We present a novel GC-MS method for the accurate and precise analysis of ethanol in cell cultures and microdialysates. The method is based on the carbonate-catalyzed extractive pentafluorobenzoylation of ethanol and deuterium-labelled ethanol serving as the internal standard and on their GC-MS analysis in the electron-capture negative-ion chemical ionization mode. The method was used to optimize experimental conditions in a custom-made ethanol vapour system utilized for studies examining ethanol influences on neuronal cell lines and in microdialysis.