Internal standard chromatography

Finally, other methods are used to obtain simulated distillation by gas phase chromatography for atmospheric or vacuum residues. For these cases, some of the sample components can not elute and an internal standard is added to the sample in order to obtain this quantity with precision. 

Blood and urine are most often analyzed for alcohol by headspace gas chromatography (qv) using an internal standard, eg, 1-propanol. Assays are straightforward and lend themselves to automation (see Automated instrumentation). Urine samples are collected as a voided specimen, ie, subjects must void their bladders, wait about 20 minutes, and then provide the urine sample. Voided urine samples provide the most accurate deterrnination of blood alcohol concentrations. Voided urine alcohol concentrations are divided by a factor of 1.3 to determine the equivalent blood alcohol concentration. The 1.3 value is used because urine has approximately one-third more water in it than blood and, at equiUbrium, there is about one-third more alcohol in the urine as in the blood. 

Content of prime - tertiary peroxide groups was measured by the quantity of products of complete decay, which were measured by chromatography. It is known that the main contents in products of the complete decay of Oct-MA-TBPMM samples are acetone and 2,2-dimethylpropanol, which arise in reactions of chain fragmentation of tert-butylperoxy radical or in reaction of chain transfer of this radical. In this case the sum of acetone and 2,2-dimethylpropanol molecules is equal to the quantity of peroxide groups in polymer. As an internal standard we used chloroform. 

Liquid chromatography was performed on symmetry 5 p.m (100 X 4.6 mm i.d) column at 40°C. The mobile phase consisted of acetronitrile 0.043 M H PO (36 63, v/v) adjusted to pH 6.7 with 5 M NaOH and pumped at a flow rate of 1.2 ml/min. Detection of clarithromycin and azithromycin as an internal standard (I.S) was monitored on an electrochemical detector operated at a potential of 0.85 Volt. Each analysis required no longer than 14 min. Quantitation over the range of 0.05 - 5.0 p.g/ml was made by correlating peak area ratio of the dmg to that of the I.S versus concentration. A linear relationship was verified as indicated by a correlation coefficient, r, better than 0.999. 

Figure 11.4 Chromatograms of plasma samples on a silica-chiralcel OJ coupled column system (a) plasma spiked with oxprenolol (internal standard) (b) plasma spiked with 040 p-g/ml metyrapone and 0.39 p-g/ml metyrapol (racemate) (c) plasma sample obtained after oral administration of 750 mg metaiypone. Peaks are as follows 1, metyrapone 2, metyrapol enantiomers 3, oxprenolol. Reprinted from Journal of Chromatography, 665, J. A. Chiarotto and I. W. Wainer, Determination of metyrapone and the enantiomers of its chfral metabolite metyrapol in human plasma and urine using coupled achfral-chfral liquid cltro-matography, pp. 147-154, copyright 1995, with permission from Elsevier Science.

Figure 15.5 Separation of Voriconazole and an internal standard by using SEC-HPLC. Adapted from Journal of Chromatography, B 691, D.A. Stopher and R. Gage, Determination of a new antifungal agent, voriconazole, by multidimensional high-perfomiance liquid chromatography with direct plasma injection onto a size exclusion column , pp. 441 -448, copyright 1997, with permission from Elsevier Science.

High performance liquid chromatography is used for the separation and quantitative analysis of a wide variety of mixtures, especially those in which the components are insufficiently volatile and/or thermally stable to be separated by gas chromatography. This is illustrated by the following method which may be used for the quantitative determination of aspirin and caffeine in the common analgesic tablets, using phenacetin as internal standard where APC tablets are available the phenacetin can also be determined by this procedure. 

It should be noted here that the difficulty of accurately injecting small quantities of liquids imposes a significant limitation on quantitative gas chromatography. For this reason, it is essential in quantitative GLC to use a procedure, such as the use of an internal standard, which allows for any variation in size of the sample and the effectiveness with which it is applied to the column (see Sections 9.4(5) and 9.7). 

Ethanol production in the fermentation process was detected with gas chromatography, HP 5890 series II (Hewlett-Packard, Avondale, PA, USA) equipped with a flame ionisation detector (FID) and GC column Porapak QS (Alltech Associates Inc., Deerfield, IL, USA) 100/120 mesh. The oven and detector temperature were 175 and 185 °C, respectively. Nitrogen gas was used as a carrier. Isopropanol was used as an internal standard. 

Ethanol concentration in the fermentation broth is determined by using gas chromatography (HP 5890 series II with HP Chemstation data processing software, Hewlett-Packard, Avondale, PA) with a Poropak Q Column, and a Hewlett-Packard model 3380A integrator. A flame ionisation detector (FID) is used to determine ethanol. The oven temperature is maintained at 180 °C, and the injector and detector temperature are maintained at 240 °C. The sample taken from the fermentation media has to be filtered and any internal standard must be added for analysis based on internal standard methods otherwise, the area under the peak must be compared with known standard samples for calculation based on external standard methods. 

Acetone and Ethanol (Grade AA only). Determine the acetone and ethanol content by the elution method of gas chromatography using internal standards... 

Figure 5.66 Molecular structures of Idoxifene and its deutrated internal standard ds-Idoxifene. Reprinted from J. Chromatogr., B, 757, Comparison between liqnid chromatography-time-of-flightmass spectrometry and selected-reaction monitoring liqnid chromatography-mass spectrometry for qnantitative determination of Idoxifene in hnman plasma , Zhang, H. and Henion, I., 151-159, Copyright (2001), with permission from Elsevier Science.

It is appropriate at this juncture to illustrate the power of chemiluminescence in an analytical assay by comparing the limits of sensitivity of the fluorescence-based and the chemllumlnescence-based detection for analytes in a biological matrix. The quantitation of norepinephrine and dopamine in urine samples will serve as an illustrative example. Dopamine, norepinephrine, and 3,4-dihydroxybenzy-lamine (an internal standard) were derivatized with NDA/CN, and chemiluminescence was used to monitor the chromatography and determine a calibration curve (Figure 15). The limits of detection were determined to be less than 1 fmol injected. A typical chromatogram is shown in Figure 16. 

Shatkay, A., Effect of Concentration on the Internal Standards Method in Gas-Liquid Chromatography, Ana/. Chem. 50, 1978, 1423-1429. 

Lipid Screening. The problems of lipid analysis in the newborn is difficult because of the fact that most methods for analysis for lipids require substantial amounts of serum, yet a total lipid determination is very important in various types of disease. This problem can be solved by thin-layer chromatography (59). Figure 38 shows a typical pattern obtained when an extract 7rom 10 microliters of serum is subjected to thin-layer chromatography. If these specimens are scanned, and an internal standard is run, one can obtain a rough approximation of the distribution of the various lipids in the serum. This is shown in Figure 39, in which a normal specimen is run in an adult. 

Figure 38, Patterns obtained from the extract of 10 fd of serum for lipid fraction by thin-layer chromatography. In sequence, starting from the bottom, phospholipids, pee cholesterol, cholesterol aniline as an internal standard, triglycerides, and cholesterol esters. The free fatty acids occur between cholesterol and the internal standard and are only barely visible in the print, on the extreme right. They are readily visible, normally, to the eye.

Successful use of modern liquid chromatography in the clinical laboratory requires an appreciation of the method s analytical characteristics. The quantitative reproducibility with respect to peak height or peak area is quite good. With a sample loop injector relative standard deviations better than 1% are to be expected. The variability of syringe injection (3-4% relative standard deviation) requires the use of an internal standard to reach the 1% level (2,27). 

This method requires about 40 g of tobacco which are extracted with ethyl acetate in the presence of ascorbic acid. A trace amount of C-NDELA is added as an internal standard for quantitative analytical work. The filtered extract is concentrated and NDELA is enriched by column chromatography of the concentrate on silica gel. The residues of fractions with p-activity are pooled and redissolved in acetonitrile. Initially, we attempted to separate NDELA on a 3% OV-225 Chromosorb W HP column at 210 C using a GC-TEA system with direct interface similar to the technique developed by Edwards a. for the analysis of NDELA in urine (18). We found this method satisfactory for reference compounds however, it was not useful for an optimal separation of NDELA from the crude concentrate of the tobacco extract (Figure 4). Therefore, we silylated the crude concentrate with BSTFA and an aliquot was analyzed by GC-TEA with direct interface. The chromatographic conditions were 6 ft glass column filled with 3% OV-... 

D2O = deutered water. HPLC = high performance liquid chromatography. IS = internal standard. MeOH = methanol. MS = mass spectrometry. NMR = nuclear magnetic resonance. PDA = photodiode array detector. TEA = triethylamine. MTBE = methyl tert-butyl ether. 

Reaction products were analyzed by on-line gas chromatography with a Shimadzu GC-14A gas chromatograph equipped with a 50 m CP Sil-5 fused silica capfllary column and a flame ionization detector. Reaction intermediates were identified by GC-MS. Samples were taken after 50 h on stream when the activity of the catalyst was stable, with n-nonane and n-dodecane as internal standards. Space time was defined as t = e Voat/vgas, where e is the void fraction of the... 

Characterization of various types of damage to DNA by oxygen-derived species can be achieved by the technique of gas chromatography-mass spectrometry (GC-MS), which may be applied to DNA itself or to DNA-protein complexes such as chromatin (Dizdaroglu, 1991). For GC-MS, the DNA or chromatin is hydrolysed (usually by heating with formic acid) and the products are converted to volatile derivatives, which are separated by gas chromatography and conclusively identified by the structural evidence provided by a mass spectrometer. Stable isotope-labelled bases may be used as internal standards... 

Residues of isoxaflutole, RPA 202248 and RPA 203328 are extracted from surface water or groundwater on to an RP-102 resin solid-phase extraction (SPE) cartridge, then eluted with an acetonitrile-methanol solvent mixture. Residues are determined by liquid chromatography/tandem mass spectrometry (LC/MS/MS) on a Cg column. Quantitation of results is based on a comparison of the ratio of analyte response to isotopically labeled internal standard response versus analyte response to internal standard response for calibration standards. 

Tebuconazole (provided by Bayer), Q -[2-(4-chlorophenyl)ethyl]-o -(l,l-dimethyl-ethyl)-li/-l,2,4-triazole-l-ethanol. Molar mass 307.8, (M- -H)+ ion observed at approximately m/z 308.1 [liquid chromatography/mass spectrometry (LC/MS)] Tebuconazole-fnflzoZe-i,2,4-- fV3 (provided in acetonitrile solution by Bayer), [ NsJtebuconazole stable-isotope internal standard, o -[2-(4-chlorophenyl)ethyl]-Q -(l,l-dimethylethyl)-li/- A3-l,2,4-triazole-l-ethanol. Molar mass 310.8, (M -I- H)+ ion observed at approximately m/z 311.1 (LC/MS)... 

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