Urine Analyzers

In addition to the automated devices and processing units that were developed primarily to automate chemistry and immunoassay that are described above, a variety of other instruments and processes have been automiated and used in the clinical laboratory. They include urine analyzers, flow cytometers, hematology cell counters, nucleic add analyzers, microtiter plate systems, point-of-care analyzers, and remotely located systems. 

Many of the same analytical principles are used for the quantitation of serum and urine constituents, but it is more difficult to automate testing of urine than serum, because the broad range of concentrations of many urine constituents requires a low limit of detection to measure low concentrations, and expanded linearity to permit measurements of high concentrations without dilution. This requirement, together with the relatively low demand for urine tests compared with that for serum tests, has restricted the development of analyzers designed specifically for urine constituents. Nevertheless, selected urine analyses are performed on the available analyzers in some institutions.  

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Equipment used to generate, measure, or assess data should undergo a validation process to ensure that such equipment is of appropriate design and adequate capacity and will consistently function as intended. Examples of such equipment include scales balances analytical equipment (HPLC, GC, etc.) hematology, blood chemistry, and urine analyzers computerized equipment for the direct capture of data and computers for the statistical analysis of data. Because the data generated, measured, or assessed by such equipment are the essence of a nonclinical laboratory study, the proper functioning of such equipment is essential to valid study results. 

It is plausible that analysis of urine samples may be used to monitor exposure to aromatic amines. In a model experiment, rats were treated with 2,4-diaminoanisole (2,4-DAA) and the urine analyzed for the amine and its most important metabolites (33). The results are shown in Table II. For the two doses used in this study, the major metabolite is 4-acetamido-2-aminoanisole, indicating that this compound may be used to monitor occupational exposure to 2,4-DAA. 

Magiera, S., Hejniak, J., Baranowski, J. Comparison of different sorbent materials for solid-phase extraction of selected drugs in human urine analyzed by UHPLC-UV. J. Chromatogr. B 958, 22-28 (2014)... 

Results. Excellent correlation between in vitro and in vivo release rates was observed (Figure 1). As in internal control, the inulln polymer squares were removed from the rats at the end of 450 hrs and rat urine analyzed 4.5 hrs later. As shown in Figure 1, the recovery rate of H-inulin dropped 300 fold. This control confirmed that H-inulin is rapidly cleared from the blood stream and excreted into the urine. Thus inulin is an excellent marker for directly assessing release rates from the polymer implant. 

Furthermore, as an internal control, inulin pellets were removed from several animals at 450 h and the urine analyzed 4.5 h later. Within that time, the inulin recovery rate had dropped by a factor of over 50 (Fig. 3). 

Aliquot of urine analyzed for drug concentration (Cv) Amount of drug excreted during collection interval i u) = Cu X Vu... 

Samples of urine are analyzed for riboflavin before and after taking a vitamin tablet containing riboflavin. Concentrations are determined using external standards or by the method of standard additions, fluorescence is monitored at 525 nm using an excitation wavelength of 280 nm. 

Most potentiometric electrodes are selective for only the free, uncomplexed analyte and do not respond to complexed forms of the analyte. Solution conditions, therefore, must be carefully controlled if the purpose of the analysis is to determine the analyte s total concentration. On the other hand, this selectivity provides a significant advantage over other quantitative methods of analysis when it is necessary to determine the concentration of free ions. For example, calcium is present in urine both as free Ca + ions and as protein-bound Ca + ions. If a urine sample is analyzed by atomic absorption spectroscopy, the signal is proportional to the total concentration of Ca +, since both free and bound calcium are atomized. Analysis with a Ca + ISE, however, gives a signal that is a function of only free Ca + ions since the protein-bound ions cannot interact with the electrode s membrane. 

Clinical Applications Perhaps the area in which ion-selective electrodes receive the widest use is in clinical analysis, where their selectivity for the analyte in a complex matrix provides a significant advantage over many other analytical methods. The most common analytes are electrolytes, such as Na+, K+, Ca +, H+, and Ch, and dissolved gases, such as CO2. For extracellular fluids, such as blood and urine, the analysis can be made in vitro with conventional electrodes, provided that sufficient sample is available. Some clinical analyzers place a series of ion-selective electrodes in a flow... 

Blood and urine are most often analyzed for alcohol by headspace gas chromatography (qv) using an internal standard, eg, 1-propanol. Assays are straightforward and lend themselves to automation (see Automated instrumentation). Urine samples are collected as a voided specimen, ie, subjects must void their bladders, wait about 20 minutes, and then provide the urine sample. Voided urine samples provide the most accurate deterrnination of blood alcohol concentrations. Voided urine alcohol concentrations are divided by a factor of 1.3 to determine the equivalent blood alcohol concentration. The 1.3 value is used because urine has approximately one-third more water in it than blood and, at equiUbrium, there is about one-third more alcohol in the urine as in the blood. 

The main advantages of the ms/ms systems are related to the sensitivity and selectivity they provide. Two mass analyzers in tandem significantly enhance selectivity. Thus samples in very complex matrices can be characterized quickly with Htde or no sample clean-up. Direct introduction of samples such as coca leaves or urine into an ms or even a gc/lc/ms system requires a clean-up step that is not needed in tandem mass spectrometry (28,29). Adding the sensitivity of the electron multiplier to this type of selectivity makes ms/ms a powerhil analytical tool, indeed. It should be noted that introduction of very complex materials increases the frequency of ion source cleaning compared to single-stage instmments where sample clean-up is done first. 

One anabohc steroid, the presence of which has proven difficult to analyze, is stanozolol [10418-03-8] C2 H22N20. A metaboUte of the parent dmg, hydroxy stanozolol, detected in equine urine eight hours after ingestion of the parent dmg is actually identified, usually at very low levels. Analysis was done by Ic/ms/ms which had a shortened analysis time advantage over gc/ms procedures because of the elimination of the need for a derivatization step (33). 

One of the important problems in the diagnosis of different disease in their early stages is the determination of bio-active substances in biological fluids. We are currently interested in applying capillary electrophoresis (CE) as technique for the rapid and highly efficient separation of corticosteroids in semm and urine. Steroids can analyze by MEKC. 

A liquid chromatography-mass spectrometry (LC-MS) method that can quantitatively analyze urinar y normal and modified nucleosides in less than 30 min with a good resolution and sufficient sensitivity has been developed. Nineteen kinds of normal and modified nucleosides were determined in urine samples from 10 healthy persons and 18 breast cancer patients. Compounds were separ ated on a reverse phase Kromasil C18 column (2.1 mm I.D.) by isocratic elution mode using 20 mg/1 ammonium acetate - acetonitrile (97 3 % v/v) at 200 p.l/min. A higher sensitivity was obtained in positive atmospheric pressure chemical ionization mode APCI(-i-). 

A. Molecular Ion Because of low volatility, most steriods are derivatized before analysis by GC/MS. Molecular ions are usually observed for steriods sufficiently volatile to be analyzed underivatized by GC/MS (see Figure 31.1) Some important steroids in urine include estrone, estradiol, estriol, pregnanediol, and 17-ketosteroids. which can be analyzed by GC/MS as the TMS or the MO-TMS derivatives. The plant steroids, such as camposterol, ergosterol. stigmasterol, cholestanol, and sitosterol, are generally analyzed as the TMS derivatives. 

Mr. Elliott, age 42 years, had a UTI8 weeks ago. He failed to see his primary health care provider for a follow-up urine sample 2 weeks after completing his course of drug therapy. Mr. Elliot is in to see his primary health care provider because his symptoms of a UTI have recurred. The primary health care provider suspects that Mr. Elliott may not have followed instructions regarding treatment for his UTI. Analyze the situation to determine what points you would stress in a teaching plan for this patient. 

Methyl parathion was determined in dog and human serum using a benzene extraction procedure followed by GC/FID detection (Braeckman et al. 1980, 1983 DePotter et al. 1978). An alkali flame FID (nitrogen-phosphorus) detector increased the specificity of FID for the organophosphorus pesticides. The detection limit was in the low ppb (pg/L). In a comparison of rat blood and brain tissue samples analyzed by both GC/FPD and GC/FID, Gabica et al. (1971) found that GC/FPD provided better specificity. The minimum detectable level for both techniques was 3.0 ppb, but GC/FPD was more selective. The EPA-recommended method for analysis of low levels (<0.1 ppm) of methyl parathion in tissue, blood, and urine is GC/FPD for phosphorus (EPA 1980d). Methyl parathion is not thermally stable above 120 °C (Keith and Walters 1985). 

A new technique has been developed to analyze a- and (3-endosulfan concentrations in human urine (Vidal et al. 1998). Samples are mixed with a buffer solution and then passed through solid phase extraction cartridges for analysis using gas chromatography-tandem mass spectrometry (GC-MS-MS). 

GC/MS has been employed by Demeter et al. (1978) to quantitatively detect low-ppb levels of a- and P-endosulfan in human serum, urine, and liver. This technique could not separate a- and P-isomers, and limited sensitivity confined its use to toxicological analysis following exposures to high levels of endosulfan. More recently, Le Bel and Williams (1986) and Williams et al. (1988) employed GC/MS to confirm qualitatively the presence of a-endosulfan in adipose tissue previously analyzed quantitatively by GC/ECD. These studies indicate that GC/MS is not as sensitive as GC/ECD. Mariani et al. (1995) have used GC in conjunction with negative ion chemical ionization mass spectrometry to determine alpha- and beta-endosulfan in plasma and brain samples with limits of detection reported to be 5 ppb in each matrix. Details of commonly used analytical methods for several types of biological media are presented in Table 6-1. 

Radioactivity Analysis. Samples of urine, feces, and tissues were combusted to COo and analyzed for radioactivity (5). By using this method the recovery of radioactivity from samples spiked with C was 95 dt 5%. To determine the radioactivity expired as CO2, 5-ml aliquots of the solution used to trap the CO2 were added to 15 ml of a scintillation counting solution containing 4 grams 2,5-diphenyloxazole (PPO) and 0.1 grams l,4-bis-2(5-phenyloxazolyl)-benzene (POPOP) per liter of 1 1 toluene 2-methoxyethanol. Samples were counted for radioactivity in a Nuclear Chicago Mark II liquid scintillation counter. Counting eflSciency was corrected by the internal standard technique. 

The Beckman instrument suffers from certain faults. First, the standard for urea must contain the proper amount of saline or false values are obtained. Second, urine cannot be analyzed... 

Several methods are available for the analysis of trichloroethylene in biological media. The method of choice depends on the nature of the sample matrix cost of analysis required precision, accuracy, and detection limit and turnaround time of the method. The main analytical method used to analyze for the presence of trichloroethylene and its metabolites, trichloroethanol and TCA, in biological samples is separation by gas chromatography (GC) combined with detection by mass spectrometry (MS) or electron capture detection (ECD). Trichloroethylene and/or its metabolites have been detected in exhaled air, blood, urine, breast milk, and tissues. Details on sample preparation, analytical method, and sensitivity and accuracy of selected methods are provided in Table 6-1. 

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