Mobile phase injection

Clean-up and filter samples to prevent system and column damage. Dissolve or dilute the final sample solutions in mobile phase, if possible. If a final sample solvent is stronger than the mobile phase, inject smaller volumes to minimize peak distortion. 

Effect of Injected Solvent. It was eventually determined that the mobile phase injected from the first SEC (i.e., pure THF) affected the separation in the second SEC. This is dreunatically demonstrated in Figure 13 which shows the result of injecting a narrow molecular weight distribution polystyrene sample directly into the second SBC. Mobile phase was of constant composition throughout (63.8% n-heptane in THF). However, the solvent used to dissolve the polystyrene was varied from 0% n-heptane in THF to 50% and plotted on the abscissa versus peak retention time on the ordinate. Peak retention time varied from 915 seconds at 0% n-heptane to 960 seconds at 50%. 

Treatment Wash column into a beaker with strong solvent. Reequilibrate with mobile phase. Inject new sample. 

Using 50% water-saturated isooctane as the mobile phase, inject 10 /uL of the three-component mixture from Activity B-4. 

At 1 mL/min flow rate for the mobile phase, inject 10 jaL of the tablet solution (Activity B-2). 

Sample solvent incompatible with mobile phase. Inject samples in mobile phase. 

Run three unextracted internal standards (50 aL and 150 pL of mobile-phase inject 150 pL) at the start of each run. The mean peak area can then be used to correct the peak areas in the unknown samples. 

Figure 7-15 infusion chromatograms for hypothetical analyte. A, Mobile phase injection. B, Serum liquid-liquid extract injection.These profiles illustrate that ion suppression can be greater than 90%, a recovery time may exist, and that suppression is not limited to the solvent front region. For a comprehensive presentation of these types of effects, the reader is referred to references 7, 38, and 68. 

Sample preparation 100 p,L Plasma -f- 1 p,g p-anisamide -I- 1 mL 500 mM pH 7.4 Na2HP04/KH2P04 buffer + 1 mL ethyl acetate, shake for 10 min, centrifuge at 1300 g for 5 min. Evaporate the supernatant to dryness under reduced pressure, reconstitute the residue in 200 p,L mobile phase, inject a 100 p,L aliquot. 

Sample preparation 100 p.L Serum -l- 100 p,L buffer 1.5 mL IS in 5% isopropanol in chloroform, vortex for 30 s, centrifuge. Remove the organic layer and evaporate it to diyness under a stream of air at room temperature, reconstitute the residue in 100 p,L mobile phase, inject a 6-10 p,L aliquot. (Buffer was 13.6 g KH2PO4 in 90 mL water, pH adjusted to 6.8 with about 3 mL 10 M NaOH, made up to 100 mL.)... 

Sample preparation 500 p.L Saliva or plasma -i- 100 pL 100 pg/mL o-hydroxyacetanilide -I- 5 mL ethyl acetate, shake for 15 min, centrifuge at 2500 rpm for 5 min. Remove 4 mL of the organic layer and evaporate it to dryness under a stream of nitrogen at 50°, reconstitute the residue in 200 pL mobile phase, inject a 50 pL aliquot. 

Sample preparation Add one tablet to 10 mL MeOH and 80 mL dichloromethane, sonicate for 5 min, dilute to 100 mL with dichloromethane, dilute a 2 mL aliquot to 25 mL with mobile phase, inject a 10 p,L aliquot. 

Sample preparation Tissue. Homogenize brain with 4 volumes MeOH, stir for 15 min, centrifuge at 1300 g for 10 min, acidify supernatant with 300 p.L 1 M HCI, evaporate to dryness under reduced pressure, reconstitute with mobile phase, inject an aliquot. Plasma. Extract 1 mL plasma with 15 mL MeOH, stir for 15 min, centrifuge at 1300 g for 10 min, acidify supernatant with 300 p,L 1M HCI, evaporate to dryness imder reduced pressure, reconstitute with mobile phase, inject an aliquot. 

Sample preparation Condition a Bond Elut Si SPE cartridge by washing twice with 1 mL MeOH, twice with 1 mL water, and once with 1 mL 100 mM pH 9.2 K2HPO4. Add 1 mL plasma + 100 pL 500 ng/mL atenolol in water, wash twice with 1 mL water, centrifuge at 1000 g for 5 min, elute with 1 mL MeOH. Evaporate MeOH to dryness at 40° under a stream of air and dissolve residue in 200 xL mobile phase, inject an aliquot. 

Sample preparation 50 q,L Serum -i- 25 xL 0.5 p.g/mL demoxepam in water -i- 100 (jiL 1 M pH 9.0 borate buffer, mix well, add 2 mL diethyl ether, vortex for 40 s, centrifuge at 1100 g for 5 min. Remove ether layer and evaporate it at 40° under nitrogen. Take up residue in 50 pL mobile phase, inject an aliquot. 

Sample preparation 1 mL Serum -I- 25 p,L 1 pg/mL triazolam in toluene + 75 pL 0.1% ammonium hydroxide, vortex 30 s, add 5 mL methylene chloride -t- 5 mL toluene, shake 15 min, centrifuge at 177 g for 10 min. Remove aqueous layer and freeze residual aqueous layer in dry ice-acetone for 30 s. Decant organic layer, dry under nitrogen at 50°, vortex residue with 200 pL mobile phase, inject a 125 pL ahquot. 

Sample preparation Heat 5 mL urine + 1 mL temazepam in MeOH with 1 mL 3-glu-curonidase at 37° for 2.5 h, cool, adjust to pH 8.5 with saturated Na2C03, extract with 10 mL dichloromethane. Evaporate, take up the residue in 200 p.L mobile phase, inject an aliquot. 

Sample preparation 1 mL Serum -t- 3 pL 20 ng/mL clobazam in methanol + 1 mL saturated sodium borate (pH adjusted to 11 with 6 M NaOH) -1- 5 ml n-hexane, mix for 2 min, centrifuge at 3000 g for 10 min. Separate organic phase, evaporate to dryness under a stream of helium at 30°, reconstitute in 20 pL mobile phase, inject a 10 pL aliquot. 

Sample preparation Plasma. 1-5 mL Plasma + 1 mL 1 M NaOH, extract with mixed hexanes for 30 min, centrifuge. Remove a 9 mL aliquot of the hexane layer and evaporate it to dryness under a stream of nitrogen at 30°, dissolve residue in 100 pL mobile phase, inject a 50 pL aliquot. Whole blood. 10 mL Whole blood -I- 1 mL 1 M NaOH, extract with 15 mL mixed hexanes for 1 h. Remove an aliquot of the hexane layer and evaporate it to dryness, reconstitute the residue in 1 mL 100 mM HCl, extract with 5 mL chloroform by vortexing for 1 min, centrifuge. Remove a 4.5 mL aliquot of the chloroform layer, evaporate to dryness, dissolve in 10 pL mobile phase, inject an ediquot. 

Sample preparation Basify 500 pL perfusate or 1 mL microsomal incubation with 1 mL 200 mM pH 9.0 sodium borate buffer, extract into MTBE, evaporate extract to dryness under a stream of nitrogen, reconstitute the residue in 100 pL mobile phase, inject an aliquot. 

Sample preparation Blend tablets and capsules with water in a high-speed blender for 5 min, filter, dilute with mobile phase, inject a 20 pL aliquot. Dilute oral suspensions and injections with mobile phase, inject a 20 pL aliquot. 

Sample preparation Condition a Sep-Pak C18 SPE cartridge with 5 mL MeOH then 5 mL water. 1 mL Urine + 100 pL 0.5 M tetrabutylammonium bromide in water, vortex 30 s, add to SPE cartridge, elute with 9 mL MeCN buffer 3 97, make up to 10 mL with mobile phase, inject tui aliquot. (Buffer was 100 mL 0.5 M disodium hydrogen orthophosphate + 350 mL water adjusted to pH 4.85 with 1 M citric acid then made up to 500 mL.)... 

Sample preparation 500 p,L Serum -I- 1 mL 20% trichloroacetic acid, vortex for 15 s, centrifuge at 1000 g for 20 min. 1 mL Supernatant + 500 p,L 7% formaldehyde in 400 mM citric acid, vortex for 15 s, heat at 90° for 2 h, cool to room temperature. Either inject an aliquot of this solution directly or extract it twice with 3 mL portions of diethyl ether. Evaporate the extracts to diyness under reduced pressure, reconstitute with 100 jxL mobile phase, inject an aliquot. 

Sample preparation Condition a 500 mg Extract Clean silica SPE cartridge (Alltech stock no. 209250) with 2 mL hexane. Allow a solution of aspiiin in 10 mM sorbitan trioleate in CFC-11 to evaporate, dissolve the residue in 5 mL hexane. Add 1 mL to the SPE cartridge, elute with two 2 mL portions of mobile phase, make up eluate to 5 mL with mobile phase, inject a 20 p,L aliquot. 

Sample preparation Powder tablets, add 40 mg pyrimethamine, dissolve in 20 mL MeCN, add 40 mL mobile phase, filter (paper), wash filter with mobile phase, make up filtrate to 100 mL with mobile phase. Dilute a 5 mL aliquot to 50 mL with mobile phase, inject a 20 p.L aliquot. 

Sample preparation Condition a 3 mL cyano SPE cartridge (J.T.Baker) with 2 mL water. 400 p-L Serum + 20 pL practolol solution, vortex, add to the SPE cartridge, elute with 1 mL MeOH triethylamine 99 1. Evaporate the eluate to dryness under a stream of nitrogen at 37°, reconstitute the residue in 80 pL mobile phase, inject a 50 pL aliquot. 

Sample preparation Condition a 1 mL Bond-Elut CN SPE cartridge with 2 mL MeOH then 2 mL water. 400 p,L Plasma + 200 ng practolol, add to SPE cartridge, wash twice with 1 mL water, wash with 1 mL acetone, allow to go dry. Elute with three 200 xL aliquots of eluting solvent, combine the fractions, evaporate under nitrogen, suspend in 80 jxL mobile phase, inject a 50 j.L aliquot. (Eluting solvent was 10 mM acetic acid and 50 mM triethylamine in MeOH.)... 

Sample preparation Condition a Sep-Pak CIS SPE cartridge with water, MeOH, and 100 mM Eimmonium acetate. Add 200 p-L plasma to the SPE cartridge, wash with 100 mM ammonium acetate, elute with MeOH 100 mM ammonium acetate 3 1. Evaporate the eluate to dryness under reduced pressure, dissolve the residue in 200 p,L mobile phase, inject a 20 pL aliquot. 

Sample preparation Place 0.5 g powdered crude drug in 25 mL mobile phase, reflux 30 min, cool, centrifuge at 1600 g, decant wash residue twice with 10 mL portions of mobile phase, combine extracts and washings, make up to 50 mL with mobile phase, inject 10 fiL aliquot. 

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