Analytical sample drying

A solution of 85.8 g (0.2 moles) of 3/ -acetoxy-27-norchoIest-5-en-25-one in 500 ml of anhydrous thiophen-free benzene is added to a Grignard solution prepared from 24.3 g (1 g-atom) of magnesium and 149 g (1.05 moles) of freshly distilled methyl iodide in 575 ml of anhydrous ether. The mixture is refluxed for 3 hr and allowed to stand overnight. After cooling to 5° the complex is decomposed by the slow addition of 200 ml of ice water and 400 ml of 50% acetic acid solution, and steam distilled until no more oil passes over. The residual product is filtered, washed with water and dried at 80°. Crystallization from methanol gives 70 g (87%) of cholest-5-ene-3)5,25-diol mp 179.5-181°. The analytical sample melts at 181.5-182.5° [a]o —39° (CHCI3). 

A total of 50 ml (0.15 moles) of a 3 ethereal solution of methylmagnesium bromide is added slowly to a vigorously stirred solution of 5.8 g (12.5 mmoles) or 3,3 20,20-bisethylenedioxy-5a,6a-epoxy-5a-pregnane-ll/l,17a,21-triol in 400 ml of tetrahydrofuran. The solution is heated under reflux for 24 hr, cooled and treated with 32 ml of saturated ammonium chloride solution. The supernatant is decanted and the residue is washed with several portions of tetrahydrofuran. The combined supernatants are evaporated and extracted with ethyl acetate, washed with saturated salt solution, dried and concentrated to give 4,55 g (75%) of 3,3 20,20-bisethylenedioxy-6 -methyl-5a-pregnane-5a,ll, 17a,21-tetrol mp 170-172° after crystallisation from acetone-petroleum ether. The analytical sample is crystallized from acetone-petroleum ether mp 175-177° [aJo —11° (CHCI3). 

A total of 3 g (0.13 moles) of sodium hydride is added to a solution consisting of 10 g of 17 -hydroxy-5a-androstan-3-one (36 mmoles) in 200 ml of benzene and 10 ml of ethyl formate. The reaction mixture is allowed to stand under nitrogen for 3 days followed by dropwise addition of 10 ml of methanol to decompose the excess of sodium hydride. The solution is then diluted with 300 ml water and the layers are separated. The basic aqueous solution is extracted with ether to remove neutral material. The aqueous layer is acidified with 80 ml of 3 A hydrochloric acid and the hydroxymethylene steroid is extracted with benzene and ether. The combined organic extracts are washed with water and saturated sodium chloride solution and then dried over magnesium sulfate and concentrated. The residue, a reddish-yellow oil, crystallized from 10 ml of ether to yield 9.12 g (83%) of 17 -hydroxy-2-hydroxymethylene-5a-androstan-3-one mp 162-162.5°. Recrystallization from chloroform-ether gives an analytical sample mp 165-165.5° [a]o 53° (ethanol) 2 ° 252 mjj. (g 11,500), 307 m u (e 5,800). 

The above nitrite (0.93 g) is dissolved in 40 ml of dry benzene and irradiated for 1 hr at 0-5° in a nitrogen atmosphere with two 200 Watt mercury lamps. The resulting suspension is concentrated and filtered to give 0.59 g of essentially pure 20a-hydroxy-18-oximinopregn-4-en-3-one as a benzene solvate mp 110-125°. Recrystallization from acetone gives an analytical sample mp 184-186° [a] 149° (CHCI3). 

The above crude bromohydrin was mixed with 2.5 grams of potassium acetate and 60 cc of acetone and refluxed for 6 hours, at the end of which the acetone was distilled, water was added to the residue and the product was extracted with methylene chloride. The extract was washed with water, dried over anhydrous sodium sulfate and the solvent was evaporated. Recrystallization of the residue from methanol furnished 800 mg of the 16,21-diacetate of 6o-fluoro-9(3,11(3-oxido-A -pregnene-16o,l7a,2l-triol-3,20-dione with MP 120° to 124°C by chromatography of the mother liquors on silica gel there was obtained 180 milligrams more of the same compound with MP 117° to 119°C. The analytical sample was obtained by recrystallization from methanol it showed MP 125° to 127°C. 

After allowing the mixture to evaporate to dryness, water was added and the sulfonamide filtered, washed with water, and dried in vacuo. The crude product (32.5 grams, 96%) obtained melts at 163.5° to 165°C. One crystallization from ethanol chloroform yielded an analytical sample, MP 164.5 to 166.5°C. 

Undissolved substances were removed by filtration and the filtrate was concentrated on a steam bath to a volume of about 125 ml and cooled to effect crystallization. After 20 hours at room temperature the crystals that had formed were recovered, washed with isopropyl alcohol, and dried, yielding 15.61 grams (46.2%) of crystalline 5-[bis(2-hydroxy-ethyl)amino] uracil having a MP of 157° to 163°C. An analytical sample, obtained by several recrystallizations from isopropyl alcohol, melted at 166° to 168°C. 

To a solution of dibenzo[6,/][l, 4]diazocine-6,l (5H. 2H)-dione (7 1.7 g, 7.5 mmol) in morpholine (10 mL) was added dropwise a cooled solution of TiClj. (3.13 g, 16.5 mmol) in anisole (10 mL). The mixture was then heated at 125 C for 2.5 h. poured into ice and extracted with CHC13. The organic layer was dried (Na2S04), concentrated in vacuo, and the residue passed through a column of neutral alumina (CHC13/ MeOH 95 5) yield 1.92 g (68 %). An analytical sample was obtained by recrystallization (CH2Cl2/ petro-leum ether) as a gray solid nip 240-243 C. 

In the laboratory, soil samples collected in the held are mixed thoroughly and reduced in size to laboratory samples. The air-dried soils are passed through a 2-mm sieve in order to remove stones and roots, then the water content of the soil is calculated after drying at 105 °C for 5h. If the analytical samples cannot be analyzed immediately after drying and sieving, they should be stored at about —20 °C in glass or Teflon bottles fltted with screw-caps. 

The addition of water or the use of an aqueous solvent mixture is important for the extraction of other organic analytes from dry foodstuffs or dehydrated foods. It is particularly necessary in aiding the permeation of solvent through freeze-dried samples. 

The matrix compound is typically mixed together in an aqueous/ organic solution with the analyte, such that the relative concentration of matrix to analyte is on the order of 5000 or 10,000 to 1. The solution is applied to a surface that will be irradiated by the laser beam and the solvent is allowed to evaporate, leaving a solid, crystalline deposit of matrix and analyte. Many of the original applications described instruments in which the surface containing the dried matrix/ analyte sample was introduced into the vacuum housing of a mass spectrometer source housing for irradiation. However, recently it has been demonstrated that MALDI can be successfully carried out at atmospheric pressure (outside the vacuum chamber of the mass spectrometer), much in the same way as the ESI, APCI and APPI techniques. 

Time-resolved approaches for multi-analyte immunoassays have been described recently. Simultaneous determination of LH, follicle stimulating hormone (FSH), hCG, and prolactin (PRL) in a multisite manual strip format has been reported. 88 Four microtiter wells are attached to a plastic strip, two-by-two and back-to-back, such that the wells can be read on a microtiter plate reader. In a quadruple-label format, the simultaneous quantitative determination of four analytes in dried blood spots can be done using europium, samarium, dysprosium, and terbium. 89 In this approach, thyroid-stimulating hormone, 17-a-hydroxyprogesterone, immunoreactive trypsin, and creatine kinase MM (CK-MM) isoenzyme are determined from dried blood samples spotted on filter paper in a microtiter well coated with a mixture of antibodies. Dissociative fluorescence enhancement of the four ions using cofluorescence-based enhancement solutions enables the time-resolved fluorescence of each ion to be measured through four narrow-band interference filters. 

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