Deproteinization blood

Owing to the complexity of multi-residue methods for products of animal origin, it is not possible to outline a simple scheme however, readers should refer to methods described in two references for detailed guidance (Analytical Methods for Pesticides in Foodstuffs, Dutch method collection and European Norm EN 1528. ) There is no multi-method specifically designed for body fluids and tissues. The latter matrix can be partly covered by methods for products of animal origin. However, an approach published by Frenzel et al may be helpful (method principle whole blood is hemolyzed and then deproteinized. After extraction of the supernatant, the a.i. is determined by GC/MS. The LOQ is in the range 30-200 ag depending on the a.i.). 

Shaw et al. [64] described a (D)-penicillamine detection method in blood samples that had been treated with EDTA, deproteinized with trichloroacetic acid, and analyzed within 1 h. Penicillamine was detected at a vitreous-carbon electrode operated at +800 mV after HPLC separation. A linear calibration graph was obtained, and the method had a limit of detection equal to 5-20 ng. The method was useful in clinical and in pharmacokinetic studies. 

There is a great deal of interest in the determination of lead, particularly micromethods applicable to the analysis blood lead in children. Consequently, reports continue to appear on the atomic absorption determination of lead in blood and urine. Ninety percent of blood lead is found in the erythrocytes and, therefore, whole blood is analyzed rather than serum or plasma. Berman etal. 134) have described a procedure for determining normal lead levels in which only 250 fd of blood are taken. The blood is deproteinized with 1 ml of 10 % trichloroacetic acid and then the lead is extracted with APDC into 1 ml of MIBK, at pH 3.5. 

Blood should be deproteinized by some technique which leaves no extra salt, acid, or alkali in the supernatant Some suitable techniques are with tungstic acid, with ethanol (BIO), or with zinc sulphate and barium hydroxide (S21). The supernatant is desalted in the same way as urine and, if necessary, concentrated before applying to the paper. Subsequent technique is as for urine. 

The first fractionation of urinary ampholytes in this way was carried out by Boulanger et al. (BIO) in 1952 with the use of ion-exchange resins. They had designed this procedure previously for the fractionation of ampholytes in blood serum (B8). According to this method, deproteinized urine was subjected to a double initial procedure aiming at the separation of low-molecular weight substances from macro-molecular ones. One of the methods consisted of the fractionation of urinary constituents by means of dialysis, the second was based on the selective precipitation of urinary ampholytes with cadmium hydroxide, which, as had previously been demonstrated, permits separation of the bulk of amino acids from polypeptides precipitated under these circumstances. Three fractions, i.e., the undialyzable part of urine, the dialyzed fraction, and the so-called cadmium precipitate were analyzed subsequently. 

Vinyl alcohol copolymer gel is hydrophilic and has been developed for aqueous-phase size-exclusion liquid chromatography however, it is less polar than the polysaccharides. Its specificity permits the direct injection of a biological sample without deproteinization. For example, blood serum from a patient suffering from chronic nephritis has been injected directly as a measure of the degree of dialysis (Figure 3.17). Adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate in red blood cells have also been separated directly (Figure 3.18). Theophylline in blood serum has been... 

The UVV spectrum of 32a is pH dependent In the presence of Li+ ions the spectrum of 32b shows a specific shift that allows quantation, which is carried out at 490 nm, pH 13. Possible interference by traces of heavy and transition elements is masked witii [Mg-EDTA] added to the sample. The metiiod was tested for Li" " in seawater and deproteinized plasma. f Determinations of Li in drugs and blood serum are carried out at 601 nm in alkaline solution. Originally designed for colorimetric determination of thorium in trace amounts. Measurements of tile hthium complex can be carried out over a wide range of wavelengths Determination in pharmaceuticals at 468 nm, with linear range of 0.1 to 4.0 mg analysis of blood serum at... 

Sulfur-containing amino acids such as cystine and homocystine tend to bind to plasma proteins. This binding is irreversible hence, these amino acids will be severely underestimated unless the plasma is deproteinized immediately following its separation from red cells. Blood should be left standing for as short a time as possible to avoid binding of cystine to proteins and hemolysis. 

Thiamine- and pyridoxal-phoshates human blood/plasma deproteination PRP ion-pair PRP post-Flu 67... 

To run a patient sample, you will need to go through exactly the same deproteination, SFE cartridge extraction, IS addition, mobile phases dilution, and injection steps (Fig. 12.4f). From the peak heights relative to the IS height, we can now quantitate the amount of each drug in the patient s blood. To insure linearity, you may need to dilute our windowed plasma blank and spike it with different levels of each standard and plot calibration curves for each compound, but basically, our methods development is done. 

The most frequently studied clinically relevant material has been blood plasma. Although the estimated contributions of the main antioxidants obtained by different authors for various methods of assay differ to some extent, it is clear uric acid is the main determinant of TAC of human blood plasma (Table 6), the next being plasma protein (mainly thiol groups of the proteins). In deproteinized plasma, TAC showed a 99% correlation with urate (M14). It has been reported that about 30% of... 

In general, the material in blood to be tested must be obtained in a cell-free condition. Serum must be obtained free of cells and clots. Likewise, plasma either must be obtained without cells or possibly the whole blood may be deproteinized and the test substance may be determined... 

Nuttall et al. [231] reported the use of ICP-MS for routine determination of As, Bi, Cd, Pb, Sb, Te, and T1 in a clinical laboratory. Blood was deproteinized, centrifuged, and passed through a 2-jjim filter before analysis. Other specimens were diluted or digested with dilute mineral acids. Se, Zn, and As suffered from interferences that could be overcome by proper isotope selection or mathematical correction. The authors [231] concluded that Cr and Fe were too prone to interferences (due to spectral overlaps) to be practically measured by ICP-MS in clinical samples. 

For the COX-2 assay, fresh heparinized human whole blood is incubated with lipopolysaccharide from E. coli at 100 (ig/ml and with 2 pi of vehicle or a test compound for 24 h at 37 °C (Brideau et al. 1996). PGH2 levels in the plasma are measured using radioimmunoassay after deproteination. For the COX-1 assay, an aliquot of fresh blood is mixed with either DMSO or test compound and is allowed to clot for 1 h at 37 °C. TBX2 levels in the serum are measured using an enzyme immunoassay after deproteination. 

In considering the applications of AAS," the technique has made the biggest contribution to the determination of nonferrous metals and for the transition elements Cu and Ag, in particular, whose measmement is completely free from interferences due to Zn, Cd, Hg, Sn, Pb, Bi, or Sb. Blood sera and other body fluids are analyzed after deproteination using concentrated HCl, while for the analysis of plants, milk, and... 

Fig. 2B and D shows the typical chromatograms of deproteinized human serum and urine, respectively, which contain less glutathione than histidine. The high selectivity of the postcolumn detection made the chromatograms quite simple, where the peak due to histidine appeared as a sole peak. On the other hand, both glutathione and histidine were detected in human blood, which contains glutathione at a higher level than histidine (Fig. 2C). 

Dialysis can also be used as an on-line sample preparation technique for the deproteinization of biological samples prior to HPLC. Selecting the appropriate semipermeable membrane for the dialyzer can prevent interference from large macromolecules. Samples are introduced into the feed (or donor) chamber, and solvent is pumped through the lower acceptor (or recipient) chamber. The smaller molecules diffuse through the membrane to the acceptor chamber and are directed to an HPLC valve for injection. In case of low concentrations of compounds of interest, a trace enrichment step may be required this is accomplished with a column placed downstream of the dialyzer that will retain the analyte until the concentration is sufficient for detection. After this step, the analyte can be backflushed into the HPLC system. The technique is useful for blood studies as sampling can be achieved continuously without blood withdrawal. A commercial on-line system, such as Asted from Gilson, used for both cleanup and enrichment by a combination of dialysis with SPE, is shown in Fig. 7. 

Other than deficiencies of G6PD and possibly PK, RBC enzyme defects are rare therefore it is not practical to attempt to identify them in laboratories that perform such tests only rarely. Specimens should be shipped by mail to reference laboratories that specialize in performance of these assays. As a rule, whole-blood specimens anticoagulated with EDTA are suitable and the specimens can be shipped at room temperature. Exceptions are assays for phosphorylated sugar intermediates, 2,3-DPG, and nucleotide intermediates, which are unstable in freshly drawn blood and require immediate deproteinization in perchloric acid. ... 

Ropero-Miller JD, Lambing MK, Winecker RE. Simultaneous quantitation of opioids in blood by GC-EI-MS analysis following deproteination, detau-tomerization of keto analytes, solid-phase extraction, and trimethylsilyl derivatization. J Anal Toxicol 2002 26 524-8. 

Farrelly and Watkins (F4) have used high voltage electrophoresis of unmodified urine or deproteinized serum for the rapid separation of fourteen amino acids in one direction on a thin-layer plate. Evered and Dando (E16) have employed low voltage electrophoresis for one way separation of amino acids on Whatman No. 1 paper using various buffer solutions. They stated that only the acidic and basic amino acids, p-amino acids, and cystine could be separated completely from a complex mixture such as blood or urine. Scherr (SIO) and Stevens (S52) have also used low voltage electrophoresis for the unidirectional separation of amino acid mixtures on cellulose acetate strips. 

The discrete systems of automatic analysis developed so far have not, in general, solved the problem of deproteinization in a way as satisfactory as the dialysis step adopted in the continuous-flow system. An earlier version of the Robot Chemist included a module in which precipitation of proteins followed by filtration was carried out, and the Mecolab system can incorporate a centrifugation step, following which a sample of supernatant can be automatically aspirated, but both these approaches are relatively cumbersome. Consequently attention has been directed to the development of analytical methods for blood serum or plasma that do not involve a deproteinization stage, but although alternative methods not involving the removal of protein may be feasible for many estimations, the responsibility for confirming the validity of such alternative procedures remains with the analyst. 

The normal lactate concentration in blood is between 1.2 and 2.7 mmol/1. For accurate lactate determination hemolysis of the sample is required to account for the (low) lactate content of erythrocytes. On the other hand, the glycolytic reactions in the sample have to be efficiently and rapidly inhibited in order to avoid lactate formation. Therefore the best-suited sample material is deproteinized blood however, the time period inevitably required for its preparation prevents rapid lactate assay. That is why the study of blood lactate sensors focuses not only on the sensor itself but also on the rapid pretreatment of blood samples. 

The first enzyme electrode-based lactate analyzer was developed in 1976 by La Roche (Switzerland) (see Table 23). It uses cytochrome b2 in a tiny reaction chamber on top of a platinum electrode polarized at +0.25-0.40V. The solution for blood sample pretreatment recommended by the manufacturer has been improved by Soutter et al. (1978) by addition of cetyltrimethylammonium bromide. This compound hemo-lyzes the sample, stabilizes the lactate content, and leads to a good correlation with the spectrophotometric reference method using deproteinized blood ... 

Other lactate analyzers use lactate oxidase (LOD). Clark et al. (1984b) use the enzyme in the YSI23L instrument (USA) as immobilized between a cellulose acetate membrane and a polycarbonate membrane, the latter serving to exclude high-molecular weight interferents. Lactate measurement in whole blood pipetted immediately after withdrawal into the phosphate buffer stream of the analyzer yielded the following correlation with values obtained with deproteinized blood (Weil et al., 1986) ... 

Our studies were carried out with HI-6, HS-6, HGG-12 and BDB-37 synthesized in Department of Military Toxicology, MMA. The reactivators were administrated i.m. to cats in dose O, 5.10-4 M/kg b.w. Serial blood samples were collected in heparinized tubes at different time intervals after injection. Plasma was deproteinized with 6 M trichloracetic acid (1 1) and centrifuged at 15 000 rpm for 10 min at 4°C. The supernatant was analysed by HPLC. The results are... 

Urine samples are unstable, and calcium phosphate precipitates out, entrapping metal ions or other substances of interest. Precipitation can be prevented by keeping the urine acidic (pH 4.5), usually by adding 1 or 2 mL glacial acetic acid per 100-mL sample. Store under refrigeration. Urine, as well as whole blood, serum, plasma, and tissue samples, can also be frozen for prolonged storage. Deproteinized blood samples are more stable than untreated samples. 

---