Injection internal standards

Figure 14.8 Partial m/z 44 and m/z 45/44 traces obtained by GC C IRMS analysis of commonly occurring amino acids (as their triflouroacetyl isopropyl ester derivatives) together with a range ofco injected internal standards...

When the sample size is small (10-20 pL), it becomes possible to effectively inject most of the ions in the sample in less than 30 s at 5 kV. A small water plug injected hydrodynamically has been shown to further improve sensitivity [51], though this was not the case for the above-mentioned application. Since the sample conductivity severely affects the amount of solute injected, internal standards are important for this mode of stacking. For quantitative analysis, make only a single injection out of each vial, as the sample becomes ion-depleted. 

The problems generated using an open-ended injection system as shown by the Poiselle equation dictate that changes in the experimental conditions will result in variations of the amount of material injected. Internal standards are best used to compensate for some of the experimental variables. 

The criteria for acceptable linearity of least squares fit and zero intercept when plotting ratios of analyte to internal standard areas vs. concentration are similar to the case for external standard calibrations described earlier. More than one IS can be used, both for calculating RRTs to compensate for retention time variations as well as the RRFs for improving quantitation. The variations that a quantitation IS can compensate for depend upon the point at which it is introduced in the analysis. If it is put into the final extract prior to injection on the chromatograph, it can correct for concentration variations due to evaporative volume changes, variations in injection volume, and variations in detector response. This is called an injection internal standard. If the internal standard is put into the initial sample, and into calibration standards prepared in an equivalent matrix, it can additionally correct for variations in recovery during the sample preparation process. This is called a method internal standard. Combined use of separate compounds for each purpose can aid in determining the causes of peak area variability. 

The method is based on the international standard ISO 4053/IV. A small amount of the radioactive tracer is injected instantaneously into the flare gas flow through e.g. a valve, representing the only physical interference with the process. Radiation detectors are mounted outside the pipe and the variation of tracer concentration with time is recorded as the tracer moves with the gas stream and passes by the detectors. A control, supply and data registration unit including PC is used for on site data treatment... 

Caffeine is extracted from beverages by a solid-phase microextraction using an uncoated fused silica fiber. The fiber is suspended in the sample for 5 min and the sample stirred to assist the mass transfer of analyte to the fiber. Immediately after removing the fiber from the sample it is transferred to the gas chromatograph s injection port where the analyte is thermally desorbed. Quantitation is accomplished by using a C3 caffeine solution as an internal standard. 

As this example clearly shows, the variation in individual peak areas between injections is substantial. The use of an internal standard, however, corrects for these variations, providing a means for accurate and precise calibration. 

Precision The precision of a gas chromatographic analysis includes contributions from sampling, sample preparation, and the instrument. The relative standard deviation due to the gas chromatographic portion of the analysis is typically 1-5%, although it can be significantly higher. The principal limitations to precision are detector noise and the reproducibility of injection volumes. In quantitative work, the use of an internal standard compensates for any variability in injection volumes. 

An hplc assay was developed suitable for the analysis of enantiomers of ketoprofen (KT), a 2-arylpropionic acid nonsteroidal antiinflammatory dmg (NSAID), in plasma and urine (59). Following the addition of racemic fenprofen as internal standard (IS), plasma containing the KT enantiomers and IS was extracted by Hquid-Hquid extraction at an acidic pH. After evaporation of the organic layer, the dmg and IS were reconstituted in the mobile phase and injected onto the hplc column. The enantiomers were separated at ambient temperature on a commercially available 250 x 4.6 mm amylose carbamate-packed chiral column (chiral AD) with hexane—isopropyl alcohol—trifluoroacetic acid (80 19.9 0.1) as the mobile phase pumped at 1.0 mL/min. The enantiomers of KT were quantified by uv detection with the wavelength set at 254 nm. The assay allows direct quantitation of KT enantiomers in clinical studies in human plasma and urine after adrninistration of therapeutic doses. 

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.

Determine the response factors (r) for the detector relative to phenacetin ( = 1) as internal standard by carrying out three runs, using 1 /rL injection, and obtaining the average value of r. 

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). 

Quantitative analysis using the internal standard method. The height and area of chromatographic peaks are affected not only by the amount of sample but also by fluctuations of the carrier gas flow rate, the column and detector temperatures, etc., i.e. by variations of those factors which influence the sensitivity and response of the detector. The effect of such variations can be eliminated by use of the internal standard method in which a known amount of a reference substance is added to the sample to be analysed before injection into the column. The requirements for an effective internal standard (Section 4.5) may be summarised as follows ... 

If the detector response differs, make up by weight a 1 1 mixture of each of the separate components (I, II, and III) with compound (IV). Inject a 0.1 pL sample of each mixture, measure the corresponding peak area, and hence deduce the factors which will correct the peak areas of components (I), (II), and (III) with respect to the internal standard (IV). 

The use of a fused silica capillary column for the GC analysis of the neutral oil extract has provided the means for improving the resolution of components in a more inert system. The sultones are determined by temperature-programmed GC over CP-Sil-5 CB (methyl silicone fluid) in a 25 m x 0.2 mm fused silica capillary column using nonadecane as internal standard. A sample split ratio of 1 100 is recommended for a 3-pl injection. 

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. 

Figure 4.22. Correlation of assay values for components A and B, for three dosage levels of A, with 10 samples per group. The comer symbols indicate the 10% specification limits for each component. For manual injection (left panel) only relative standard deviations of 1-2% are found, but no correlation. Automatic injection (right panel) has a lower intrinsic relative standard deviation, but the data are smeared out along the proportionality line because no internal standard was used to correct for variability of the injected volume. The proportionality line does not go through the comers of the specification box because component B is either somewhat overdosed (2.4%). analytical bias, or because an interference results in too high area readings for B. The...

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). 

Radioisotope-labeled nitrosamines have proven valuable in development of analytical methods and for demonstrating efficiency of recovery of nitrosamines from tobacco products and smoke (37-39). The very high specific activity required for low part-per-billion determinations has discouraged most analysts from using this approach. Unless a radiochromatographic detector with adequate sensitivity is available, samples must be counted independently of the final chromatographic determination, and one of the advantages of internal standardization, correction for variation in volume injected, is lost. 

Flumetralin was extracted from tobacco using Soxhlet extraction. A 5-g amount of Florisil (5% deactivated) was transferred directly on to the filter disk of a Soxhlet extractor followed by another 5 g of Florisil mixed with 5 g of ground tobacco sample as an upper layer. A 60-mL volume of hexane and 3mL of a 4 agmL internal standard solution were placed in a 250-mL round-bottom flask prior to attaching the Soxhlet extractor. The unit was placed on a heating mantle and the hexane was refluxed through the extractor at the rate of about 250 mLh for 4.5 h. After cooling, 0.5 pL of the extract was injected directly into a GC/FCD or GC/MS system. 

The silica gel column eluates (Module Cl or C2) are injected, if necessary with the addition of an internal standard, into a gas chromatograph followed by ECD or NPD. The determinations can be performed with different gas chromatographs and fused-silica capillary columns. 

Combine a 0.5-mL aliquot of the Anal sample exAact or a 0.020 igmL azinphos-methyl standard solution in acetone-water (2 1 v/v) with 0.5 mL of a 0.040 ig mL deuterated internal standard solution in methanol-water (2 3 v/v) in an HPLC autosampler vial. Combination may be made using other volumes as long as the solutions are combined 1 1 (v/v). Inject 200 aL from the 0.020 and 0.040 igmL standard/internal standard soluAon. Inject 200 aL from each of the 10 sample exAact/ internal standard soluAons. Inject 200 p-L from anoAier 0.020 and 0.040 pgmL standard/internal standard solution. 

Water samples, received from the respective groundwater trials, are analyzed by direct aqueous injection (DAI) by LC/ESI-MS/MS. A 1-mL volume of the water is pipetted into a 1.8-mL autosampler vial. The internal standard solution is added (200 qL) and mixed. The vials are capped and analyzed by LC/ESI-MS/MS using the selected reaction monitoring (SRM) mode. 

The use of internal standards is somewhat controversial.115 There is agreement that an internal standard may be used as a correction for injection volume or to correct for pipetting errors. If an internal standard is included before sample hydrolysis or derivatization, it must be verified that the recovery of the internal standard peak is highly predictable. Ideally, the internal standard is unaffected by sample handling. Using an internal standard to correct for adsorptive or chemical losses is not generally approved, since the concentration of the standard may be altered by the conditions of sample preparation. An example of internal vs. external standards is given in Chapter 4. 

Sample preparation, injection, calibration, and data collection, must be automated for process analysis. Methods used for flow injection analysis (FLA) are also useful for reliable sampling for process LC systems.1 Dynamic dilution is a technique that is used extensively in FIA.13 In this technique, sample from a loop or slot of a valve is diluted as it is transferred to a HPLC injection valve for analysis. As the diluted sample plug passes through the HPLC valve it is switched and the sample is injected onto the HPLC column for separation. The sample transfer time typically is determined with a refractive index detector and valve switching, which can be controlled by an integrator or computer. The transfer time is very reproducible. Calibration is typically done by external standardization using normalization by response factor. Internal standardization has also been used. To detect upsets or for process optimization, absolute numbers are not always needed. An alternative to... 

An internal standard method gives more reliable results when elaborate sample preparation is required, as in extraction of a drug substance from biological fluids, or extraction of pesticides and herbicides from soil and plant matter. The addition of internal standard (IS) to the sample and standard acts as a marker to give accurate values of the recovery of the desired compound(s). Since the determination of wt% involves the ratio of the detector responses in the two chromatograms, the injection volume is not critical as in an external standard method. 

For the example of toluene given above, the external standard method can be converted into an internal standard method by adding anisole (an appropriate internal standard) to both standard and sample. The retention time of anisole is 4.5 minutes if analyzed by the method above. To calibrate the internal standard method for toluene, toluene standards of concentration 0.3 to 1.5 mg/ml containing 0.5 mg/ml anisole were prepared. The detector response as a function of the amount of sample injected is shown in Figure 4B. 

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