Internal standards phosphorous

Szathmary and Luhmann [50] described a sensitive and automated gas chromatographic method for the determination of miconazole in plasma samples. Plasma was mixed with internal standard l-[2,4-dichloro-2-(2,3,4-trichlorobenzyloxy) phenethyl]imidazole and 0.1 M sodium hydroxide and extracted with heptane-isoamyl alcohol (197 3) and the drug was back-extracted with 0.05 M sulfuric acid. The aqueous phase was adjusted to pH 10 and extracted with an identical organic phase, which was evaporated to dryness. The residue was dissolved in isopropanol and subjected to gas chromatography on a column (12 m x 0.2 mm) of OV-1 (0.1 pm) at 265 °C, with nitrogen phosphorous detection. Recovery of miconazole was 85% and the calibration graph was rectilinear for 0.25 250 ng/mL. 

Samples should be handled according to the complexity of the food matrix. This is described in Sec. I.C (14,16,29,36,37). Aspartame has also been extracted from different food samples through dialysis with 1% phosphoric acid (40,41). Adenine sulfate and theophyllin can be used as internal standard (31,44). 

P is an NMR active nucleus with a nuclear spin of 1/% Its sensitivity (relative to H) is only 0.066, but because of its 100% natural abundance, the overall relative sensitivity of 31P is about 375 times that of 13 C. Thus, acquisition of phosphorous NMR spectra is relatively easy. The internal standard generally used for measuring 31P chemical shifts is 85% H3PO4. 

A stereoselective GC method for determination of etodolac enantiomers in human plasma and urine was first reported as a preliminary method [35], and then as a validated method [36]. Sample preparation involved addition of (S)-(+)-naproxen (internal standard) and sodium hydroxide to diluted plasma or urine. The samples were washed with diethyl ether, acidified with hydrochloric acid, and extracted with toluene. ( )-(+)-naproxen was used as a derivatizing agent to form diastereomeric derivatives of etodolac. The gas chromatograph system used in this work was equipped with fused-silica capillary column (12 m x 0.2 mm i.d.) coated with high-performance cross-linked methylsilicone film (thickness 0.33 pm) and a nitrogen-phosphorous detector. The operating conditions were injector 250°C detector 300°C column 100-260°C (32 °C/min). 

We have also studied the use of paper as the source of cellulose (1) and we have learned that if the paper is pretreated with acid, yields of ca. 5% levoglucosenone (2) can be obtained. The paper was preacidified following the procedure of Shafizadeh and Chin.9 A quantity of 25 g of paper from a newspaper was shredded into strips which were then cut by hand into smaller pieces. To a 1000-mL single-neck round-bottom flask were added the shredded paper, 180 mL of water, and 0.8 g (3.2% of the weight of the paper) of 88% phosphoric acid. The mixture was heated for 3 hours at 60-70°C and then the water was removed by using a rotary evaporator (4 hr). The preacidified paper was then mixed with 25 g of soy oil and the mixture was pyrolyzed in a 500-mL round-bottom flask the yield as determined by GC using octyl alcohol as the internal standard was ca. 5%. 

Derivatization and Extraction. Modifications of the procedures of Ebeler et al. (49) were used for all aldehyde analyses. Briefly, 3.0 mL of wine were mixed with 60 pL of internal standard (10 mg 2,4,5-trimethylthiazole/mL in 10% aqueous ethanol) and 1 mL of 0.03 M aqueous cysteamine (pH 8.5) the pH was adjusted with HCl or NaOH (pH s from 2-10 were evaluated as discussed below). Following reaction at room temperature for 1 hour, the pH was re-adjusted to 8.5 and the solution was extracted two times with 1.5 mL of chloroform the chloroform layer was removed each time and then combined to give a total of 3.0 mL of extract. Samples were injected onto a gas chromatograph fitted with either a mass spectrometer or nitrogen phosphorous detector. Peak area ratios of the internal standard to the analyte were used for all quantitative calculations. 

Shah and Riegelman extracted theophylline from plasma and/or saliva samples (1 ml) with a mixture of diethyl ether, dichioromethane and isopropanol (6 4 1). Theophylline was removed from the organic solution by means of aqueous sodium hydroxide, the alkaline solution was acidified with phosphoric acid (pH 5) and theophylline was re-extracted with the organic mixture mentioned. The internal standard (thiobarbital or fluoranthene) was added, the solvent evaporated, and the residue dissolved in 25 ul of tetrapropylanmonium hydroxide and gas chromatographed on a packed column of 3 % 0V-17 on Gas Chrom Q at 190°C. In the injection heater (kept at 265°C) theophylline is quantitatively converted to its propyl derivative, which gives a symmetrical peak distinct from any other xanthines, or barbiturates. 

Column iiBondapak Phenyl (300x4 mm 101, mobile phase 0.0015 M aqueous phosphoric acid - acetonitrile (9 1), flow rate 2 ml/min, fluorescence detection (excitation 320 nm, emission 418 nm) Chromatogram A blank plasma chromatogram B plasma standard ( 10 umole/1 quinidine, 5 umole/ 1 3-hydroxyquinidine) chromatogram C plasma from patient on chronic oral quinidine therapy. Peaks 1, polar metabolites 2, 3-hydroxyquinidine 3, internal standard (cinchonidine) 4, unidentified metabolite 5, quinidine 6, dihydroquinidine. 

Column Ultrasphere C8 (150x4.6 nm ID), mobile phase acetonitrile - methanol - tetrahydrofuran -0.01 M aqueous triethylamine (pH 2.5 with phosphoric acid)(5 5 3 87), flow rate 1.0 ml/min, fluorescence detection (excitation 245 nm, emission 435 nm). Peaks 1, quinidine-10,11-dihydro-diol 2, 3-hydroxyquinidine 3, quinidine N-oxide 4, quinidine 5, dihydroquinidine 6, 3-me-thyl-5-triazolophtalazine (internal standard) 7, 2 -quinidinone. (Reproduced with permission from ref. 65, by the courtesy of Clinical Chemistry)... 

Fig. 8.5. Separation of ellipticine and related alkaloids, isolated from mouse blood Column"yBondapak C18 (300x4.0 mm ID), protected with a precolumn (50x4.6 mm ID) packed with a 40 urn pellicular material, mobile phase acetonitrile - 0.01 M sodium dihydrogen phosphate (1 3)(pH 3.5 with 2 N phosphoric acid), flow rate 1.4 ml/min, detection UV 300 nm. Peaks 1, ellipticine 2, 9-hydroxyellipticine 3, 11-demethylellipticine (internal standard).

Addition of water and pentafluorophenyl-hydrazine in dilute phosphoric acid addition of a known amount of labeled formaldehyde as internal standard equilibration for 2 hours at 50 °C extraction with hcxanc/methy lenc chloride. 

Add internal standard (300 mg sodium sulfate, 100 uL 6M HC1) extract with diethyl ether dry and resuspend in phosphoric acid HPLC 25 ng/L >90% Lee et al. 1995... 

The most commonly applied methods for the analysis of polyamines in erythrocytes make use of amino acid analyzers and HPLC techniques. A capillary gas chromatographic method with nitrogen-phosphorous detection was applied to the simultaneous determination of 1,3-diaminopropane, putrescine, cadaverine (Cad), spermidine (Sd), and spermine (Sp) in human erythrocytes. Blood samples, collected by venipuncture into EDTA containing Venoject tubes, were subjected to the removal of plasma by centrifugation and erythrocytes were washed three times with two volumes of 0.9% NaCl. The stability of polyamines in erythrocyte suspensions was also investigated. Quantification of polyamines was done by comparing the peak-area ratio of each analyte and its internal standard with that of the standard. The polyamine samples were eluted with 0.1 M hydrochloric acid solutions. The eluate was evaporated to dryness at 120°C under a stream of air and 200 each of acetonitrile and heptafluorobutyric anhydride were added. The isolation of derivatives... 

D data processing consisted of zero-filling once and apodization by a 90°-shifted sine-square-bell function in both dimensions before Fourier transformation. Proton and carbon-13 chemical shifts are referenced to TSP as an internal standard. Phosphorus-31 chemical shifts are referenced to 85% phosphoric acid as an external standard. 

Figure 3.12 Analysis of paracetamol using the ESA Coulochem in redox mode (PGEs, Ej +0.25 V, E2 —0.15 V vs Pd). Column 150x4.6 (i.d) mm Ultrasphere ODS Eluent acetonitrile-aq. phosphoric acid (10 mmol L ) (7+93) Flow-rate 1 mL min Injection 50 pL supernatant obtained by mixing standard solution of paracetamol (5 mg L ) in neonatal calf plasma (100 pL) with aqueous trichloroacetic acid (5% v/v) containing N-propionyl-4-aminophenol (internal standard, 0.5 mg L ) (1 mL) Peaks 1 = paracetamol, 2 = N-propionyl-4-aminophenol.

Desacetylmetipranolol, the active metabolite of metipranolol (Figure 6.43), has been measured by HPLC-ED after LLE of plasma with dichloromethane. An ODS-modified silica column was used with methanol-phosphoric acid (50mmolL ) (4 + 6) containing disodium EDTA (lOmgL ) as eluent. The final pH was adjusted to 3.0 with aq. sodium hydroxide. Detection was at a GCE (+0.9 V V5 Ag/AgCl). Pindolol (Figure 6.41) was the internal standard. 

A Hewlett Packard Model 1090 fitted with an automatic injector and an HP 3385A integrator were used with a Shimadzu RF-530 fluorimeter as detector. The excitation and emission wavelengths were 274 and 300 nm, respectively. Separation was effected by reverse-phase chromatography, on a Brownlee 5 p Cyano column (100 x 4.6 mm) fitted with the appropriate guard column (30 x 4.6 mm) using isocratic elution with acetonitrile/ 0.6% phosphoric acid (40 60) at a flow rate of 1.5 mL/min and a temperature of 40 C. Under these conditions the retention time for betaxolol and metoprolol, the internal standard, were 6.3 and 7.1 minutes, respectively. 

Reactions were terminated by the addition of C M and either aqueous KCl or KCl in dilute phosphoric acid (4) so that C M aqueous = 1 1 1 v/v/v. Lipids were separated by TLC using either C Acet M AcH H20 50 20 10 10 5 by volume or pet ether-.diethyl ether formic acid 80 20 2. Lipids were quantified by GLC using an internal standard of heptadecanoic acid. Radioactivity was measured by liquid scintillation counting and counts were quench corrected when necessary using the channels ratio... 

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