Concentration residual tests

Anhydrous ammonia is normally analy2ed for moisture, oil, and residue. The ammonia is first evaporated from the sample and the residue tested (86). In most instances, the amount of oil and sediment ia the samples are insignificant and the entire residue may be assumed to be water. For more accurate moisture determinations, the ammonia can be dissociated into nitrogen and hydrogen and the dewpoint of the dissociated gas obtained. This procedure works well where the concentration of water is in the ppm range. Where the amount of water is in the range of a few hundredths of a percent, acetic acid and methanol can be added to the residue and a Karl Fischer titration performed to an electrometricaHy detected end point (89—92). 

The Sarcina lutea test is the official US Food and Drug Administration (FDA) test for detecting penicillin residues in milk and dairy products (41). In this test, milk samples are placed in stainless steel cylinders on an agar plate seeded with Sarcina lutea ATCC 9341. As milk diffuses into the agar, inhibitors prevent the growth of the organism, causing a zone the width of which is a measure of the antibiotic concentration. The test is sensitive to about 0.006 g/ ml penicillin G, and confirmation of positive results can be performed by the addition of penicillinase. 

Freeze-drying. For a 7000-fold concentration, 70 L of drinking water was lyophilized in a Virtus Unitrap II. The dried residue was then divided into equal weights and packed into two columns (25 X 1.5 cm) with a sintered glass filter. The organic material was eluted consecutively with acetone, ether, and DMSO. The ether in the ether eluate was removed by rotary evaporation, and the dried residue was dissolved in DMSO. The DMSO concentrates were sterilized by filtration over a 0.2-/xm Teflon filter (Millipore). The acetone and DMSO concentrates were tested in the Ames test. 

Arsenical or mercury compounds are detected by evaporating a quarter of a litre of the ink and heating the extract with 1-2 c.c. of concentrated sulphuric acid and 5-10 c.c. of fuming nitric acid until nitrous vapours are eliminated, the addition of nitric acid and the heating being repeated until a perfectly colourless liquid is obtained (Rothe). The sulphuric add is then expelled and the residue tested for arsenic and mercury by the ordinary analytical methods. 

An alternative residual test solution can be made from a 10% solution of selenium toner concentrate. Use this solution in the same way as ST-1. Residual silver is indicated by a red stain. 

The development of veterinary products for cattle, pigs, sheep, poultry and other food producing animals also includes residue testing of all new pharmaceutical products, including adjuvants or other excipients in vaccines. Residue safety studies address the potential risk to humans due to the consumption of food from treated animals. Accordingly, the test substances in these toxicity studies are applied orally. Residue depletion studies (pharmacokinetic studies) in the target species must be carried out to define the occurence, concentration and elimination of the substance and its metabolites in edible tissues, milk and eggs. 

Determination of Assay Sensitivity with Replication and Statistics. Sensitivity can be defined as the ability of a test to discriminate between adjacent levels or concentrations of test analyte. There are other definitions of sensitivity, but the one specified is sufficiently general to serve several needs in residue analysis. For example, the definition recognizes that test sensitivity can vary with the point on the standard curve. If one of the points used is zero, then the sensitivity estimate can be either the level of smallest quantitation or the level of detectability of the method. The... 

Neither. These tell you about the linear relation between y and x, true, but in analytical chemistry you are rarely testing the linear model. The standard error of the regression (Sy/X) is a useful number to quote, or calculate 95% confidence intervals on parameters and estimated concentrations of test solutions. Plot residuals against concentration if you are concerned about curvature or heteroscedacity. (Sections 5.3.2, 5.5)... 

Cryopreserved human hepatocytes from three male and two female donors or freshly isolated male rat hepatocytes are analyzed for viabilities (75-85%) using the trypan blue exclusion methods. Incubations are performed by suspending the hepatocytes in Krebs-biocarbonate buffer followed by addition of a H-labeled compound in methanol. The specific radioactivity of the compounds is 100 Ci/ mol. The final concentration of test compound in the suspension is 10 xM in a final volume of 1 mL (1x10 cells/mL), and the final concentration of methanol does not exceed 0.2% (v/v). Incubations are allowed to proceed at 37 °C for 1 h, and are quenched with acetonitrile (5 mL). The remaining procedures are the same as described in Section 14.2.2 (the protocol for in vitro covalent protein binding in human or rat liver microsomes a test-tube method). Covalent protein binding values in pmol-equiv./mg protein are estimated based on the residual radioactivity in the protein pellets. 

The F test method for determining the optimum number of factors from a cross-validation PRESS analysis is also useful for determining the statistical significance of a sample s concentration residual with respect to the rest of the training set. In this case, the F ratio value is calculated by... 

Dissolve 1 g. of the secondary amine in 3-5 ml. of dilute hydrochloric acid or of alcohol (in the latter case, add 1 ml. of concentrated hydrochloric acid). Cool to about 5° and add 4-5 ml. of 10 per cent, sodium nitrite solution, and allow to stand for 5 minutes. Add 10 ml. of water, transfer to a small separatory funnel and extract the oil with about 20 ml. of ether. Wash the ethereal extract successively with water, dilute sodium hydroxide solution and water. Remove the ether on a previously warmed water bath no flames should be present in the vicinity. Apply Liebermann s nitroso reaction to the residual oil or solid thus. Place 1 drop or 0 01-0 02 g. of the nitroso compovmd in a dry test-tube, add 0 05 g. of phenol and warm together for 20 seconds cool, and add 1 ml. of concentrated sulphuric acid. An intense green (or greenish-blue) colouration will be developed, which changes to pale red upon pouring into 30-50 ml. of cold water the colour becomes deep blue or green upon adding excess of sodium hydroxide solution. 

Dissolve 1 0 g. of the compound in 5 ml. of dry chloroform in a dry test-tuhe, cool to 0°, and add dropwise 5g. (2-8 ml.) of redistilled chloro-sulphonic acid. When the evolution of hydrogen chloride subsides, allow the reaction mixture to stand at room temperature for 20 minutes. Pour the contents of the test-tube cautiously on to 25 g. of crushed ice contained in a small beaker. Separate the chloroform layer and wash it with a httle cold water. Add the chloroform layer, with stirring, to 10 ml. of concentrated ammonia solution. After 10 minutes, evaporate the chloroform on a water bath, cool the residue and treat it with 5 ml. of 10 per cent, sodium hydroxide solution the sulphonamide dissolves as the sodium derivative, RO.CgH4.SO,NHNa. Filter the solution to remove any insoluble matter (sulphone, etc.), acidify the filtrate with dilute hydrochloric acid, and cool in ice water. Collect the sulphonamide and recrystallise it from dilute alcohol. 

The hydrolysis by alkali is illustrated by the following experimental details for benzamido. Place 3 g. of benzamide and 50 ml. of 10 per cent, sodium hydroxide solution in a 150 ml. conical or round-bottomed flask equipped with a reflux condenser. Boil the mixture gently for 30 minutes ammonia is freely evolved. Detach the condenser and continue the boiling in the open flask for 3-4 minutes to expel the residual ammonia. Cool the solution in ice, and add concentrated hydrochloric acid until the mixture is strongly acidic benzoic acid separates immediately. Leave the mixture in ice until cold, filter at the pump, wash with a little cold water and drain well. RecrystaUise the benzoic acid from hot water. Determine the m.p., and confirm its identity by a mixed m.p. test. 

Step 3. The neutral components. The ethereal solution (E remaining after the acid extraction of Step 2 should contain only the neutral compounds of Solubility Groups V, VI and VII (see Table XI,5). Dry it with a little anhydrous magnesium sulphate, and distil off the ether. If a residue is obtained, neutral compounds are present in the mixture. Test a portion of this with respect to its solubility in concentrated sulphuric acid if it dissolves in the acid, pour the solution slowly and cautiously into ice water and note whether any compound is recovered. Examine the main residue for homogeneity and if it is a mixture devise procedures, based for example upon differences in volatility, solubility in inert solvents, reaction with hydrolytic and other reagents, to separate the components. 

Pesticides. Chlorinated hydrocarbon pesticides (qv) are often found in feed or water consumed by cows (19,20) subsequently, they may appear in the milk, where they are not permitted. Tests for pesticides are seldom carried out in the dairy plant, but are most often done in regulatory or private specialized laboratories. Examining milk for insecticide residues involves extraction of fat, because the insecticide is contained in the fat, partitioning with acetonitrile, cleanup (FlorisH [26686-77-1] column) and concentration, saponification if necessary, and determination by means of paper, thin-layer, microcoulometric gas, or electron capture gas chromatography (see Trace and residue analysis). 

---