Urine analysis catecholamines

Figure 4.10 Direct analysis of catecholamines in urine sample. Column, Asahipak ES-502C eluent, 75 mM succinic acid + 25 mM borate buffer (pH 6.10) containing 0.5 mM EDTA flow rate, 1.0 min-1 detection, fluorescence reaction detection Ex. 350 nm. Peaks-. 1, adrenaline-, 2, noradrenaline-, and 3, dopamine.

Chan EC, Ho PC. 2000. High-performance liquid chromatog-raphy/atmospheric pressure chemical ionization mass spec-trometric method for the analysis of catecholamines and metanephrines in human urine. Rapid Commun Mass Spectrom 14 1959. 

There are stability problems in urines stored for analysis. Fifty percent of delta-aminolevulinic acid was lost in specimens stored without preservative and exposed to light for 24 hours (V3). The loss increased to 80% in 48 hours, 85% in 72 hours, and 95% in 2 weeks. However, the same specimens acidified with tartaric acid and stored in the dark lost 2% of the aminolevulinic acid in 72 hours and 6% in 2 weeks (V3). The destruction of catecholamines collected in nonacidified urine specimens is well documented (Cll). Urinary acid phosphatase was destroyed on freezing (S15). The effect was related to increasing salt concentration during freezing and was prevented by the addition of albumin (S15). 

An important area of direct biochemical interference is that caused by fluorescent or fluorescent quenching materials in the blood or urine after the administration of a drug. These interferences may be observed during catecholamine analysis in urine from patients receiving -methyldopa, tetracyclines, chlortetracyclines, oxytetracycline, erythromycin, chlorpro-mazine, or quinidine (A2, G5). 

Norepinephrine and epinephrine can be metabolized by several enzymes, as shown in Figure 6-6. Because of the high activity of monoamine oxidase in the mitochondria of the nerve terminal, there is significant turnover of norepinephrine even in the resting terminal. Since the metabolic products are excreted in the urine, an estimate of catecholamine turnover can be obtained from laboratory analysis of total metabolites (sometimes... 

Kushnir MM, Urry FM, Frank EL, Roberts WL. Analysis of catecholamines in urine by positive-ion electrospray tandem mass spectrometry. Chn Chem 2002 48 323-31. 

Earlier fluorometric methods for analysis of urinary free catecholamines have been replaced by HPLC methods that allow selective quantitation of epinephrine, norepinephrine, and dopamine. Preliminary extraction of urine is stid required and numerous preanalytical cleanup techniques are available. An alumina extraction procedure is typically coupled with ion-exchange or adsorption chromatography. Alumina pretreatment usually involves a batch extraction technique in which catechols are first adsorbed at pH 8.6 and then eluted with boric acid, which forms a complex with cis-diol groups. Purification on boric acid affinity gels provides an alternative procedure for selective adsorption of catecholamines. 

Vanillylmandehc Acid (VMAj is a major catecholamine metabolite formed by the actions of catechol-0-methyl-transferase and MAO. It is excreted by the kidney and represents an average of 40% to 50% of the urinary excretion production of norepinephrine and epinephrine. Norepinephrine is the major source of VMA, with metabolism through MHPG as the major pathway. VA4A is not significantly conjugated and therefore is measured without a hydrolysis step. VMA was first isolated and identified in the urine of a patient with a pheochromocytoma, and its analysis is commonly performed to detect the presence of pheochromocytomas and neuroblastomas. 

Panholzer, T. J. Beyer, J. Lichtwald, K., Coupled-column liquid chromatographic analysis of catecholamines, serotonin, and metabolites in human urine, Clin. Chem. 1999, 45, 262-268... 

Figure 13.1 Analysis of catecholamines in urine (reproduced with permission of Bioanalytical Systems). Conditions stationary phase, octylsilane mobile phase, citrate-phosphate buffer (pH 4) containing 7% methanol and SOrngl" sodium octyl sulfate electrochemical detector, +700 mV for sample preparation see R.M. Riggin et a ., Anal. Chem., 49, 2109 (1977). Peaks (with concentrations in urine) 1 = norepinephrine (160ngml ) 2 = epinenphrine (31ngml ) 3 = dopamine (202ngmr ) IS —3,4-dihydroxybenzylamine (internal standard).

Only a few methods for urinary catecholamines have been published that do not require preextraction prior to analysis." " These methods minimized sample preparation by making use of different precolumn derivatization procedures. The selection of a suitable method for sample preparation prior to analysis by HPLC depends on a number of factors, such as the biological source, the type of column used and the selectivity of the detection method. In cases where the analyte concentration is very low and the analyte is present in a complex matrix (urine or plasma) with interfering compounds, an exhaustive pretreatment may be unavoidable. Sample pretreatment is also essential to ensure the sensitivity and specificity of the assay and protect the analytical column from contamination. 

Chan et developed an assay for the simultaneous determination of catecholamines and metanephrines using FMOC derivatization. The assay is convenient for the simultaneous analysis of NM, MN, E and DA in human urine sample without prior extraction procedures. In this study, urine was directly derivatized and subjected to a simple extraction step with... 

Catecholamines and amines of the histamine and tryptophan group are highly polar compounds and hence have low volatility. It is preferable, as is the case with amino acids, q.v., therefore, to form derivatives to obtain satisfactory gas-liquid chromatograms. The main problem in the analysis of urine for these amines lies not in the gas chromatographic aspect of the procedure, but rather in their isolation from the urine prior to GLC, where there may be as little as 1 /tg of amine per 100 ml of urine. It appears that this problem of extracting the amines into a sufficiently small volume of suitable solvent prior to GLC has not yet been solved satisfactorily. The fact that GLC can be used successfully to separate catecholamines through their derivatives when starting with pure substances should provide a stimulus for increased research activity in this field. 

Cholesterol, HMG-CoA reductase, triglycerides, LDL, HDL, HDL-apolipoprotein, cortisol, ACTH, testosterone, urine 17-OH-corticosteroids Acetylcholinesterase, MAOa, MAOb, ACTH, cGMP, phenylethylamine, catecholamines, neuroimaging (MRI, MR spectroscopy, magnetization transfer imaging, T2 relaxation analysis, SPECT, PET, fMRI)... 

Figure 10.341 Analysis of catecholamines and their metabolites in a urine sample. Separator column OmniPac PCX-100 eluent 0.75 mmol/L HCIO4 + 25 mmol/L NaCIO /MeCN (93.6 6.4 v/v) flow rate 03 mL/min detection DC...

These diseases are not detected via conventional screening methodology (i.e. organic acids, amino acids etc.), therefore diagnosis relies on the analysis of neurotransmitters and their metabolites in CSF, urine or plasma. In general, TH deficiency leads to low levels of catecholamines and their metabolites, AADC deficiency leads to decreased concentrations of catecholamines, serotonin and their metabolites. In AADC deficiency there is also an accumulation of neurotransmitter precursors, namely 5-hydroxytrypto-phan, levodopa and its methylated derivative, 3-0-methyldopa. D)9H deficiency leads to decreased norepinephrine and an increase in dopamine, and MAO-A deficiency to an increase in the biogenic amines and their 0-methylated catabolites, and to a decrease in concentration of their deami-nated catabolites. 

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